University of Veterinary Medicine Hannover
Department of Neurosurgery, Hannover Medical School
Center for Systems Neuroscience, Hannover
Experimental models of Parkinson’s disease with
levodopa-induced dyskinesias and gait dysfunction:
electrophysiological and behavioural measures in rats
THESIS
Submitted in partial fulfilment of the requirements for the degree
DOCTOR OF PHILOSOPHY
(PhD)
awarded by the University of Veterinary Medicine Hannover
by
Xingxing Jin
Zhejiang, China
Hannover, Germany 2015
Supervisor:
Prof. Dr. Joachim K. Krauss
Co-Supervisor:
Prof. Dr. Kerstin Schwabe
Supervision Group:
Prof. Dr. Joachim K. Krauss
Prof. Dr. Claudia Grothe
PD Dr. Florian Wegner
1st Evaluation:
Prof. Dr. Joachim K. Krauss (Department of Neurosurgery, Hannover Medical School)
Prof. Dr. Claudia Grothe (Institute of Neuroanatomy,
Hannover Medical School)
PD Dr. Florian Wegner (Department of Neurology,
Hannover Medical School)
2nd Evaluation:
Prof. Dr. Michael Koch (Brain Research Institute, Department of Neuropharmacology, Center for Cognitive
Sciences, Bremen)
Date of final exam:
06-11-2015
Sponsorship:
China Scholarship Console
To my family and friends
Contents
1 Introduction
1
2 Levodopa-induced dyskinesias and gait disturbances in Parkinson’s disease
3
2.1
Parkinson’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
2.2
Levodopa-induced dyskinesias . . . . . . . . . . . . . . . . . . . . . . . . .
4
2.3
Gait disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7
3 The basal ganglia
11
3.1
Anatomy and functional circuitry . . . . . . . . . . . . . . . . . . . . . . . 11
3.2
Models of basal ganglia signaling in Parkinson’s disease . . . . . . . . . . . 12
4 The pedunculopontine nucleus
17
5 Animal models
19
5.1
The 6-hydroxydopamine animal model of Parkinson’s disease and levodopainduced dyskinesias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
5.2
Ethylcholine mustard aziridinium ion-induced pedunculopontine nucleus
cholinergic lesion and evaluation of motor function in rats . . . . . . . . . . 20
6 Objectives
23
7 Manuscript one
Coherence of neuronal firing of the entopeduncular nucleus with motor
cortex oscillatory activity in the 6-OHDA rat model of Parkinson’s
disease with levodopa-induced dyskinesias
25
8 Manuscript two
Cholinergic lesion in the anterior and posterior pedunculopontine tegmental nucleus: behaviour and neuronal activity in the cuneiform and
entopeduncular nuclei
47
i
9 Discussion
75
10 Summary
79
11 Zusammenfassung
81
References
83
Acknowledgements
97
ii
Abbreviations
6-OHDA
6-hydroxydopamine
AF64A
ethylcholine mustard aziridinium ion
AI
asymmetry index
AIMs
abnormal involuntary movements
ANOVA
analysis of variance
AP
anterior-posterior
aPPTg
anterior pedunculopontine tegmental nucleus
BG
basal ganglia
ChAT
choline-acetyltransferase
CnF
cuneiform nucleus
CV
coefficient of variation
DBS
deep brain stimulation
ECG
electrocardiography
ECoG
electrocorticogram
EEG
electroencephalography
EPN
entopeduncular nucleus
FFT
fast fourier transform
FIR
finite impulse response
fMRI
functional magnetic resonance imaging
FRA
Fos-related proteins
GABA
γ-aminobutyric acid
GPe
external segment of globus pallidus
GPi
internal segment of globus pallidus
HP
hemiparkinsonian
iii
HP-LID
hemiparkinsonian with levodopa-induced diskinesia
ISI
inter-spike interval
L
lateral
L-DOPA
levodopa
LFP
local field potential
LIDs
levodopa-induced dyskinesias
MCx
motor cortex
MFB
medial forebrain bundle
MPTP
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
NMDA
N-methyl-D-aspartate
PBS
phosphate-buffered saline
PD
Parkinson’s disease
PDFs
probability density functions
PFA
paraformaldehyde
PPN
pedunculopontine nucleus
PPNc
pedunculopontine nucleus pars compacta
PPNd
pedunculopontine nucleus pars dissipata
pPPTg
posterior pedunculopontine tegmental nucleus
PPTg
pedunculopontine tegmental nucleus
RPM
round per minute
s.c.
injected subcutaneously
SEM
standard error of mean
SNc
substantia nigra pars compacta
SNr
substantia nigra pars reticulata
STN
subthalamic nucleus
STWA
spike-triggered waveform average
SU
single unit
V
ventral
iv
List of Figures
2.1
Symptoms of Parkinson’s disease . . . . . . . . . . . . . . . . . . . . . . .
4
2.2
Phenotypes of levodopa-induced dyskinesias . . . . . . . . . . . . . . . . .
6
2.3
Schematic representation of sequence of events leading to levodopa-induced
dyskinesias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7
3.1
Anatomy of the basal ganglia . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.2
Simplified illustration of the basal ganglia motor circuit in normal and
parkinsonian states . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
5.1
Subtypes of levodopa-induced abnormal involuntary movements in the unilateral 6-OHDA rat model of Parkinson’s disease . . . . . . . . . . . . . . . 21
7.1
Recording trajectories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
7.2
Firing rate and firing pattern . . . . . . . . . . . . . . . . . . . . . . . . . 34
7.3
Coherence of EPN-spikes and MCx-ECoG spectral power . . . . . . . . . . 36
7.4
Phase-lock ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
8.1
Recording trajectories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
8.2
Cholinergic lesion effect in the PPTg . . . . . . . . . . . . . . . . . . . . . 58
8.3
Motor impairment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
8.4
Neuronal firing rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
8.5
Distribution of cellular firing patterns . . . . . . . . . . . . . . . . . . . . . 63
8.6
Coherence of LFPs and MCx-ECoG . . . . . . . . . . . . . . . . . . . . . . 65
v
List of Tables
8.1
Coefficient variation of Interspike intervals . . . . . . . . . . . . . . . . . . 59
8.2
Burst parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
vi
1
Introduction
Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by
the loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). With
the progression of disease, severe motor and non-motor dysfunctions take place, such as
tremor, rigidity, bradykinesia, postural instability, but also cognitive and behavioural impairments. While the mechanism underlying the neurodegeneration remains unknown,
most of the current pharmacological and surgery therapies focus on relieve of clinical
symptoms. The dopaminergic precursor, levodopa, remains the mainstay of therapy since
the 1970s.
Chronic use of levodopa, however, often leads to therapy-related motor complications such
as motor fluctuation or the “on-off” phenomenon and abnormal involuntary movements,
termed levodopa-induced dyskinesias (LIDs). Although consensus has been reached that
both progressive nigral denervation in the basal ganglia (BG) and pulsatile dopamine
stimulation contribute to the development of LIDs (Nadjar et al., 2009), the underlying
pathophysiology remain elusive. In PD patients, electrophysiology studies have reported
reduced oscillatory beta band activity and enhanced theta band activity in the BG during
expression of LIDs (Alonso-Frech et al., 2006; Lozano et al., 2000; Obeso et al., 2000).
However, little is known with regard to the differences of neuronal single units and oscillatory activity in patients with advanced PD with or without peak-dose dyskinesias. In
the first project we were interested in the neuronal firing activity of the entopeduncular
nucleus (EPN, the analogue to the major output site of the BG motor loop in human,
the internal segment of globus pallidus “GPi”), and its coherence with the motor cortex
(MCx) field potentials in the 6-hydroxydopamine (6-OHDA) lesioned rat model of PD
with or without established LIDs before and after levodopa-injection, i.e., a model for
advanced PD with peak-dose dyskinesias on/off levodopa.
Gait and postural dysfunctions in advanced PD are another troublesome but frequently
occurring problem, which often does not respond to either levodopa or electrical stimulation of the subthalamic nucleus (STN) or the GPi. Post mortem studies in patients
with PD and non-human primate models have shown that cholinergic neurons in the pedunculopontine nucleus (PPN), which together with the cuneiform nucleus (CnF) forms
the mesencephalic locomotor region (MLR), degenerate in parallel to dopaminergic neu1
rons in the SNc (Zweig et al., 1989; Jellinger, 1988). This is considered important for the
pathophysiological mechanisms leading to these symptoms. Electrical stimulation of the
PPN has been tested for treating these symptoms, using stimulation parameters thought
to stimulate the remaining neurons, however, with variable results and with substantial
controversy, where exactly the optimal site for stimulation is located (Ferraye et al., 2010;
Mazzone et al., 2005; Moro et al., 2010; Plaha and Gill, 2005; Stefani et al., 2007). The
PPN is heavily interconnected with the BG motor loop, and also act as an information
relay site to lower motor regions in the brainstem and spinal cord (Alam et al., 2011).
One recent study using a rat model observed a reduction in locomotion after lesioning
of a restricted portion of the anterior but not the posterior part of the pedunculopontine tegmental nucleus (PPTg, analogue to the PPN in primates; Alderson et al., 2008).
Whether the effects of the anterior PPTg (aPPTg) lesions are achieved through the effects of cholinergic neurons on descending motor projections, or through effects on the
BG motor loop, possibly via the CnF as suggested by Alam et al., (2012), has not been
investigated. In the second project, we examined the effects of specific cholinergic lesions
of either the aPPTg or the posterior PPTg (pPPTg) on rodent gait-related behaviour and
extracellular neuronal activity of the unlesioned part of the PPTg, as well as on the CnF
and the EPN.
Together, these investigations utilizing electrophysiology and behaviour approaches will
help us to extend our knowledge regarding the neuronal mechanisms involved in symptoms
that develop in advanced PD.
2
2
Levodopa-induced dyskinesias and gait disturbances in Parkinson’s disease
2.1
Parkinson’s disease
PD is one of the most common neurodegenerative disorders secondly only to Alzheimer’s
disease in industrialized society, with a prevalence of about 1% in the population over
60 (de Lau and Breteler, 2006). It is characterized by a number of disturbances of motor
function including tremor at rest, rigidity, akinesia (or bradykinesia) and postural instability (see Fig. 2.1). These cardinal features of PD (Jankovic, 2008) are accompanied by
manifestations of symptoms of different kinds and variable severity, such as autonomic
disturbances, sensory alterations, sleep dysfunction, cognitive impairment. Diagnosis of
PD is mainly based on the typical neurological findings, their evolution over the course
of the disease and responsiveness to levodopa.
The major pathological hallmarks of PD is the presence of Lewy bodies and the loss of
dopaminergic neurons in the SNc leading to dopamine depletion in the nigrostriatal pathway, which triggers a cascade of functional changes affecting the whole BG network. The
dopamine depletion in the SNc and subsequent changes in the neuronal activity within
the BG motor loop would then result in the aforementioned alterations in motor functions (Alves et al., 2008; Galvan and Wichmann, 2008). Electrophysiological studies have
reported abnormal neuronal activities in the BG in both animal models and patients with
PD, specifically a greater tendency to discharge in bursts and with a higher degree of
synchronized oscillatory beta band activity (13-30 Hz; Hashimoto et al., 2003; Wichmann
and DeLong, 2006). However, aside the neuronal loss in the SNc, the neurodegenerative
effects in PD affects several other nuclei as well, such as the PPN, amygdala, ventral
tegmental area, locus coeruleus, raphe nuclei and the vagal dorsal motor nucleus (Dauer
and Przedborski, 2003; Braak et al., 2003; Lang and Lozano, 1998). This implies that
other neurotransmitter, such as cholinergic, adrenergic, and serotonergic systems, are
also involved in the various clinical symptoms in PD.
As the mechanism underlying this neurodegeneration remains unknown, PD is basically
incurable at present. A number of symptomatic therapies have been developed for the
3
Figure 2.1: Symptoms of Parkinson’s disease (adapted from http://parkinsons.ie/
Professionals_What_Is_Parkinsons)
improvement of patient’s quality of life, among which, levodopa remains the mainstay
since its first introduction into the disease. Other medications include dopamine agonists
that act on the nigra-striatal dopamine pathway similar to levodopa, i.e., monoamine
oxidase type B inhibitor that slows down the breakdown of dopamine in the BG, and a
number of non-dopaminergic agents that act on other neurotransmitter systems involved
in PD as mentioned above (Rascol et al., 2003). Deep brain stimulation (DBS), i.e., stimulation of specific brain regions using electrical impulses through implanted electrodes, is
frequently been used as a neurosurgical procedure for otherwise intractable cases of PD.
Other neurosurgical procedures include stereotactic ablative surgeries of certain targets
like the motor thalamus and the GPi (Fasano et al., 2015; Martinez-Ramirez et al., 2015).
2.2
Levodopa-induced dyskinesias
Chronic treatment with levodopa is associated with the emergence of LIDs, defined as
abnormal involuntary dyskinetic movements induced by levodopa administration. LIDs
are common in late stage of PD, especially in patients with early onset of disease. Clinical
studies have observed that about 53% of younger onset patients (onset age 50-59 years)
develop dyskinesias at 5 years as compared to 16% with the age of onset at 70-79 years
(Kumar et al., 2005). Certain mutations such as the PARK2 (parkin), PARK6 (pink-1)
4
and PARK7 (DJ-1) have been associated with a higher risk of levodopa-related motor
complications (Penney et al., 1996; Shoulson et al., 1996; Schrag and Schott, 2006). It
remains unclear whether these genetic abnormalities have a direct effect on the risk of
developing LIDs or via other mechanisms consistent with the earlier age at onset. Other
risk factors include female gender (Lyons et al., 1998; Zappia et al., 2005), lower body
weight (Sharma et al., 2006) and history of non-smoking (Zappia et al., 2005). Besides, a
negative association of resting tremor as a first sign of PD and the development of LIDs
has also been reported more recently (Kipfer et al., 2011).
Clinical Features
The clinical manifestation of LIDs covers a broad clinical spectrum of different types
of involuntary movements ranging from chorea affecting the limbs and trunk, slow dystonic movements, fixed dystonic postures or, more rarely, myoclonus or ballism (Fig. 2.2;
Hametner et al., 2010). The most common phenotype is the “On” state LID or “Peak
dose” dyskinesia, which occurs around the peak level of levodopa-derived dopamine in the
brain in parallel with the maximal anti-parkinsonian benefit. It is usually generalized,
manifesting as chorea-like movements involving the head, trunk and limbs, and sometimes
even respiratory muscles (Thanvi et al., 2007). These are often exaggerated by stress or
activity and are typically asymmetric (Nutt, 1990; Mones et al., 1969; Murphy, 1978;
Marconi et al., 1994).
Other phenotypes of LIDs include the “Off” state LID, which is usually manifested by
dystonia-like movements occurring when plasma levodopa levels are low, and the “biphasic” dyskinesia, which is characterized by stereotyped repetitive slow (< 4 Hz) movements
appearing at the onset and offset of the levodopa effect.
Pathophysiology
The pathogenesis of LIDs remains incompletely understood. Consensus has been reached
that progressive nigral denervation and chronic pulsatile dopaminergic stimulation play
a critical role. A chronic dopaminergic stimulation on a denervated substantia nigra
induces a process of sensitization such that each following administration modifies the
response to subsequent dopaminergic treatments, which is referred to as the “priming”
process (Tambasco et al., 2012). Increased responsiveness of postsynaptic dopamine receptor (possibly D1) and glutamate receptor N-methyl-D-aspartate (NMDA) have been
observed in the striatum (Gerfen et al., 1990; Nash and Brotchie, 2000), which could be
involved in the priming process. Both receptors are expressed along the dendritic spines
of the medium size γ-aminobutyric acid (GABA)-ergic neurons. Enhanced glutamatergic
5
Figure 2.2: The different types of dyskinesias present following an effective levodopa dose.
“Peak dose” dyskinesia, “biphasic” dyskinesia and ‘Off” state LID are indicated as the
overlap parts of the concentration-time curve of plasmic levodopa (red curve) and three
color stripes corresponding to different concentration levels (adapted from Stefani et al.,
2010
input and altered dopamine responsiveness further leads to decreased neuronal activity
in the GPi and eventually to the disinhibition of the thalamus and motor cortex (Thanvi
et al., 2007). Involvement of other non-dopaminergic systems, such as α2 adrenergic,
serotonergic, cannabinoid and opioid have also been reported (Brotchie, 2005). Further,
down-stream changes in the genes and protein synthesis, which could be involved in the
neuronal plasticity during development of LIDs, are discussed (Fig. 2.3; Calon et al.,
2003).
Management
As the pulsatile dopaminergic stimulation is considered to be important in the genesis
of LIDs, it is anticipated that any strategies with a “dopa-sparing” technique or one
that can produce smooth dopaminergic stimulations may prevent or treat LIDs. These
mainly include the use of controlled-release preparations of levodopa, continuous delivery
of levodopa via a duodenal infusion pump, use of dopamine receptor agonists or other
medications acting on non-dopaminergic systems such as NMDA or serotonergic receptors, and also functional surgery (Thanvi et al., 2007; Manson et al., 2011; Loher et al.,
2002; Jankovic et al., 1999).
In routine clinical practice, younger and biologically fit older patients are usually given a
6
Figure 2.3: Schematic representation of sequence of events leading to levodopa-induced
dyskinesias (LIDs). FRA, Fos-related proteins; NMDA, N-methyl-D-aspartate (adapted
from Thanvi et al., 2007)
dopamine receptor agonist as the initial monotherapy for the control of PD motor symptoms, in order to delay the priming of LIDs. In patients with late onset of PD, levodopa
is usually not withheld since the risk of LIDs is substantially low in these patients. Once
LIDs are established, levodopa dose reduction combined with adjunctive dopamine receptor agonist can be used as a strategy to reduce the use of levodopa. When this fails,
amantadine, or low dose clozapine with close hematological monitoring can be the next
strategy. Continuous subcutaneous infusion of apomorphine can be used as an alternative
strategy for the treatment of difficult LIDs.
DBS of the STN or the GPi is also successfully used to relieve dyskinesias in addition
to treating the cardinal motor symptoms of PD. The antidyskinetic effect depends to
some extent on the target. Stimulation of the GPi has a direct anti-dyskinetic effect,
i.e., dyskinesias are improved while the need for levodopa remains unchanged. On the
other hand, STN stimulation allows reduction of levodopa, hence relieving dyskinesias,
but some studies also suggested a direct anti-dyskinetic effect upon chronic stimulation
(Oyama et al., 2012; Follett, 2004; Krack et al., 2002).
2.3
Gait disorders
Gait disturbances form part of the axial symptoms observed in PD and can significantly
impact the quality of life for patients. These comprise the typical “Parkinsonian gait”
with small shuffling steps, reduction of gait speed and a forward-leaning stance, which
7
is considered as one of the diagnostic criteria of PD, and the so called “freezing of gait”
and postural instability, which frequently occurs in advanced PD and represents a major
therapeutic challenge since it often does not respond to levodopa and DBS of the STN or
GPi.
Clinical features
In the early stage of PD, the gait alterations are usually of moderate extent, characterized
by a reduction of stride length (Stolze et al., 2001) and an unchanged or slightly increased
cadence. Studies using imposed gait speed showed that patients were able to increase their
cadence. The stride length, however, remained the same, which implies that the gait hypokinesia is mainly associated with the internal generation of adapted stride length, and
that the increase of cadence could be a compensatory effect (Morris et al., 1994). After a
few years of chronic levodopa medication, some patients report fluctuations of the ability
to walk, which are part of the motor fluctuations related to levodopa therapy.
In the late stage of PD, severe gait disturbances, together with postural instability and
postural abnormalities, constitutes the most characteristic axial motor symptoms in patients. These symptoms are very common and closely associated with increased risk of
falls that could significantly impact a person’s mobility and quality of life. Freezing of
gait is a special clinical phenomenon of the gait disturbance in advanced PD, which is
defined as “a brief, episodic absence or a marked reduction of forward progression of the
feet despite the intention to walk” (Bloem et al., 2004; Giladi and Nieuwboer, 2008). By
its definition it includes episodes in which the patient cannot initiate gait and arrests in
forward progression during walking (freezing episodes), as well as episodes of shuffling
forward with steps that are millimeters to a couple of centimeters in length (Nutt et al.,
2011). The freezing episode usually lasts a couple of seconds, but in rare cases it appears
almost continuous and the patients experience complete akinesia with no limb or trunk
movement. Clinical features accompanying freezing of gait include: (1) alternating knee
trembling at the frequency of 3-8 Hz (knee trembling, Yanagisawa et al., 2001; Hausdorff
et al., 2003); (2) hastening or an increased cadence with shuffling small steps (Nieuwboer
et al., 2001); (3) can be relieved by attention focusing or external stimuli (cues); and
(4) can be asymmetric, affecting only one foot or being elicit more easily by turning one
direction.
Pathophysiology
The hypokinetic gait in the early stage of PD and the motor fluctuation appear to be
related to the dopamine depletion following the loss of dopaminergic neurons in the SNc,
8
since dopaminergic therapy is effective in both conditions. However, with regard to the
gait disturbances and postural instability in the late stage of PD, traditional treatments
for PD such as the dopamine replacement therapy and physiotherapy often provide only
partial relief of the symptoms. Effects of DBS therapy targeting the STN or GPi also
remains unclear (Fasano et al., 2015). These suggest that the underlying pathophysiology
of gait disturbances in advanced PD is more complex than just dopamine depletion in
BG.
It has long been recognized that the BG are integral to the production and maintenance
of automatic motor functions. In PD, disruption of the “BG to motor supplementary
motor area” circuit impairs the central driving and the automatic “updating” of motor
programs for skilled movements such as gait (Iansek et al., 2006). One evidence is the
“sequence effect”, which describes the progressive reduction of step length, which in PD
gait eventually disintegrates into a freezing episode (Chee et al., 2009; Iansek et al., 2006).
Aside from the BG, the central pattern generator of the spinal cord could also be involved
in the impairment of automaticity (Okuma, 2014).
The impairment of central drive and automaticity put more stress on voluntary mechanisms and thus increase cognitive load. It has been proposed that patients with freezing
of gait may have a frontal lobe dysfunction or a disconnection between the frontal lobe
and the BG (Okuma, 2014). Several studies have reported the induction of freezing of
gait using dual-task paradigms, where patients are required to perform cognitive tasks
while walking (Almeida, 2009; Yogev-Seligmann et al., 2008). Recent studies have reported
that increased freezing behaviour occurs when patients are denied adequate proprioceptive feedback, which led to a hypothesis of the impairment of the integration of visual and
proprioceptive inputs with motor output in patients with freezing of gait (Almeida et al.,
2005).
Disturbed gait has also been related to degeneration of cholinergic neurons in the PPN,
which, together with the CnF, forms the MLR. Postmortem studies in patient with PD
have shown that cholinergic neurons in the PPN degenerate in parallel to dopaminergic
neurons in the SNc (Hirsch et al., 1987; Jellinger, 1988; Zweig et al., 1989). The key roles
of the PPN in the control of gait and posture (Pahapill and Lozano, 2000), in cognition
(notably attention; Mena-Segovia et al., 2004) as well as in sensorimotor gating processes
(Diederich and Koch, 2005) have been identified. As mentioned above, these evidences
strongly suggest that the PPN may be crucially involved in the pathophysiology of gait
disturbances and postural instability in advanced PD.
9
Management
Clinical options for the treatment of gait disturbances in late stage of PD are limited.
Dopamine replacement therapy has, at best, only a partial relieve effect. The freezing
of gait is generally considered to be dopamine-resistant. Aside from that, only a few
trials have tested drugs targeting extra-dopaminergic systems, such as methylphenidate
(Devos et al., 2007). But the results remain controversial and some were reported to even
worsen PD symptoms (Espay et al., 2011). Rehabilitation targeting gait and balance has
been widely used in clinics for gait disturbances, although no consensus has been reached
concerning the optimal program. Various rehabilitation approaches were evaluated in
PD, and almost all types of programs showed a beneficial effect compared with nonintervention (Grabli et al., 2012).
High frequency pallidal DBS showed mild improvement of the dopa-responsive postural
deficit and freezing of gait, however this effect only lasts for 3-4 years (Houeto et al.,
2000). Low frequency (60 Hz) stimulation of the STN has been shown to significantly
improve freezing of gait (Moreau et al., 2008), but it was less effective for the cardinal
symptoms in PD than stimulation with 130 Hz, which is usually used.
The PPN has been proposed as a novel target for the treatment of PD, especially for
the gait and postural disturbances in advanced stage of the disease (Fasano et al., 2015).
Low frequency (5-10 Hz) electrical stimulation of the PPN in monkey model of PD has
been reported to be effective in reversing akinesia symptoms (Jenkinson et al., 2004;
Jenkinson et al., 2006; Mazzone et al., 2005), probably by driving the cholinergic and
glutamatergic neurons in the PPN, which are probably inhibited by the altered BG output
in PD (Jenkinson et al., 2004). These findings have been swiftly transferred to the clinic
by two different groups in 2005. The results seemed to be promising with a significant
improvement of akinesia, gait and postural disturbances and even the frequency of falls,
which has not been affected by stimulation in traditional targets like the STN and GPi
(Mazzone et al., 2005; Plaha and Gill, 2005). However later studies showed mixed results,
and raised a fierce controversy about where exactly the optimal site for stimulation is
located (Plaha and Gill, 2005; Stefani et al., 2007; Zrinzo et al., 2007). PPN DBS in
patient with PD and monkey models of PD showed an additive effect to any benefits from
dopaminergic therapy, suggesting that the effect was mediated via a non-dopaminergic
pathway (Jenkinson et al., 2006; Plaha and Gill, 2005).
10
3
The basal ganglia
3.1
Anatomy and functional circuitry
The BG are a richly interconnected set of nuclei that form cortico-subcortical circuitries.
The cortico-BG motor circuitry is considered to play a central role in the pathophysiology
of PD (Fig. 3.1). The BG comprise two principal input nuclei, the striatum and the STN,
and two principal output nuclei, the GPi and the substantia nigra pars reticulata (SNr).
The external segment of globus pallidus (GPe) is an intrinsic structure that interconnects
with other BG nuclei. Finally, the SNc provide the striatum with important modulatory
signals.
In the classical scheme of the organization of the BG motor circuitry, signals originating
in the cerebral cortex are sent to the striatum via glutamatergic projections in a topographic manner. The information is then distributed to the two intrinsic populations
of striatal GABAergic projecting neurons. The neurons that express D1-type dopamine
receptors contact directly with the BG output nuclei—the “direct pathway”, while the
neurons that express D2-type dopamine receptors contact indirectly with the BG output
nuclei via relays in the GABAergic GPe and glutamatergic STN—the “indirect pathway”.
GABAergic neurons in the BG output nuclei, the GPi/SNr, project back to the cerebral
cortex via glutamatergic neurons in the motor thalamus.
In addition, cortical areas that project to the striatum also send parallel glutamatergic
input to the STN, which contact directly with the GPi/SNr via glutamatergic projection.
This third pathway allows information to bypass the striatum and reach the BG output
nuclei in a shorter latency compared to both the “direct” and “indirect” pathway (approximately 5-8 ms vs. 15-20 ms), and is thus named the “hyperdirect pathway” (Nambu
et al., 2002; Nambu, 2005; von Monakow et al., 1978; Kitai and Deniau, 1981; Olszewski
and Baxter, 1982).
11
Figure 3.1:
Anatomy of the basal ganglia (adapted
kin450-neurophysiology.wikispaces.com/Basal+Ganglia+II)
3.2
from
https://
Models of basal ganglia signaling in Parkinson’s
disease
Although loss of dopaminergic neurons in the nigro-striatal system has been identified
early in the 1960s, how this neurodegenerative change eventually triggers motor dysfunctions in PD is still discussed. Two major hypotheses have been developed to explain the
pathophysiology of PD, i.e., the “firing rate” model and the “non-stationary oscillatory”
model.
Firing rate model
The “firing rate model”, sometimes also referred to as the “classic Albin/DeLong model”,
explains the pathophysiology of PD as follows (Albin et al., 1989; DeLong, 1990, see Figure 3.2): Loss of dopaminergic neurons in the SNc lead to the dopamine depletion in the
striatum, which decreases the firing rate of the striatal neurons that express dopamine D1
receptor. This result in direct disinhibition of the neuronal activities in the BG output
nuclei, the GPi/SNr, and cause an enhanced inhibitory input to the thalamus and cortical
motor area. Further, striatal dopamine depletion increases the firing rate of the striatal
neurons that express dopamine D2 receptor. This excites the GPi/SNr via the “indirect
pathway”, which consists of a GABAergic GPe and a glutamatergic STN, and eventually
leads to the inhibition of thalamus and cortex as well. Increased tonic neuronal discharging rate in the GPi and SNr as well as in the STN have been confirmed by many clinical
12
Figure 3.2: Simplified illustration of the basal ganglia motor circuit in normal and parkinsonian states. Red and blue arrows indicate inhibitory and excitatory projections, respectively. The changes in the thickness of the arrows in the parkinsonian state indicate the
proposed increase (larger arrow) or decrease (thinner arrow) in the firing rate of specific
connections. The dashed arrows used to label the dopaminergic projection from the SNc
to the striatum in parkinsonism indicate partial lesion of that system in this condition.
CM, centromedian nucleus; CMA, cingulate motor area; GPe, globus pallidus, external
segment; GPi, globus pallidus, internal segment; M1, primary motor cortex; PMC, premotor cortex; PPN, pedunculopontine nucleus; SMA, supplementary motor area; SNc,
substantia nigra pars compacta; SNr, substantia nigra pars reticulata; STN, subthalamic nucleus; VA/VL, ventral anterior/ventral lateral nucleus (adapted from Smith et al.,
2012)
and experimental studies, and a decreased rate of discharge has also been reported in the
GPe (Tang et al., 2007; Starr et al., 2005; Bergman et al., 1994; Filion and Tremblay, 1991;
Soares et al., 2004; Mallet et al., 2008). Further, lesions in the GPe have been associated
with increased inhibition of the thalamus and worsening of motor symptoms in monkey
models and patients with PD (Bucher et al., 1996; Zhang et al., 2006).
This model also seems to be applicable to hyperkinesia conditions such as LIDs when
the opposite effect takes place. Long-term plasticity triggered by chronic dopamine depletion may lead to increased dopamine receptor sensitivity in the striatum. When given
dopamine agonist, the BG output nuclei receive an increased inhibitory influence via the
direct pathway and a decreased excitatory influence from the indirect pathway. Both
changes lead to the disinhibition of the thalamus and cortex and eventually cause hyperkinetic movements (Nambu et al., 2014).
Although the classic firing rate model has driven the field of basic and clinical BG research
13
for the past decades, it is still over simplified and faces many criticisms: First, some studies failed to find expected firing rate changes in the GPi or GPe, but even found opposite
changes to those predicted by the model (Wichmann and Soares, 2006; Leblois et al., 2007;
Galvan et al., 2010; Tachibana et al., 2011). Second, the model predicts that GPi lesions
should improve akinesia by removing excessive inhibition of the motor thalamus at the
expense of introducing more involuntary movements. However, GPi lesion studies using
normal monkeys failed to induce involuntary movements (Inase et al., 1996; Desmurget
and Turner, 2008), while pallidotomy in patients with PD showed improved LIDs (Baron
et al., 1996). Third, sequential activity changes along these pathways leading to increased
GPi firing rate has yet to be directly demonstrated (Nambu et al., 2014). Fourth, there
are more internal connections between the components of the BG that could be involved
in the pathophysiology of PD, but have not been included in this model (Bolam et al.,
2004a; Bolam et al., 2004b).
Non-stationary oscillatory model
The neuronal oscillatory activity has received increasing interest more recently. The term
typically refers to rhythmic amplitude fluctuations in the field potentials recorded either
directly from the neural ensembles by invasive method (local field potential or LFP), or
indirectly from the scalp using electroencephalography (EEG). Underlying the oscillatory
appearance of the field potential are the synchronized transmembrane currents in large
populations of neurons. Extremely well conserved across the evolution of mammalian
brains, the temporal modulation of neuronal activity in different frequency ranges may
have important functions in brain information processes rather than being just epiphenomenal (Buzsáki et al., 2013). Further, recording of field potential is considered more
important than simply single unit activity. Studies comparing single unit and LFP recordings to blood-oxygen-level-dependent activations in functional magnetic resonance (fMRI)
imaging showed that local blood flow is driven much more by LFP activity, which corresponds to local synaptic activity, than by single unit firing rates (Logothetis et al., 2001).
There is increasing evidence that certain brain oscillatory rhythms play critical roles in
processes such as perception, motor action and conscious experience. With respect to
movement disorders, various abnormal oscillatory activities have been associated with
specific motor symptoms throughout the motor networks, especially in the STN, the GPi
and the motor thalamus (Hammond et al., 2007).
To that end, many efforts have been made in recent years focusing on the dynamic and
non-stationary features of neuronal activity changes in PD, such as oscillatory bursting
and synchronization of discharge among BG nuclei. The basic idea of the “non-stationary
oscillatory model” can be better described with the “noise” hypothesis first proposed
14
by Marsden, who hypothesized that the damaged BG in PD generates uninterpretable
“noise” and causes the movement disorders. Later the work of his student, Brown and
others further developed the “noise” concept, and explained it is the over synchronization of BG neurons at wrong frequencies caused by uncontrolled spontaneous oscillations
(Marsden et al., 2001). This hypothesis implies that the motor symptoms could be treated
by drowning out the uncontrolled oscillatory activity or by replacing it with an oscillation
at desirable frequency band. These could be the possible mechanisms underlying the DBS
therapies in use.
Beta oscillation and akinesia
Electrophysiological studies in 6-OHDA rodent model of PD revealed prominent oscillatory activity in the β band (10-30 Hz) at multiple levels of the BG cortical loops (Mallet
et al., 2008; Sharott et al., 2005; Hammond et al., 2007). Similar results have been reported in PD patients as well (Brown et al., 2001; Obeso et al., 2000). Later on, Brown
and his colleagues succeeded to demonstrate that maintained oscillations in the β band
in the STN and sensorimotor cortex are associated with akinesia and that driving the
STN at β frequency (20 Hz) actually made akinesia worse (Kühn et al., 2004). Studies in
patient with successfully improved akinesia after use of levodopa showed a replacement
of these β oscillations by a µ rhythm (10 Hz) that precedes voluntary movements (Aziz
and Stein, 2008).
Theta oscillation and dyskinesia
Analysis of the unit oscillatory activities and LFPs recorded in patients during surgery
showed that LIDs are characterized by an enhanced θ frequency range oscillatory activity
in the STN and GPi (Alonso-Frech et al., 2006; Merello et al., 1999; Papa et al., 1999;
Lozano et al., 2000; Vitek and Giroux, 2000; Levy et al., 2001; Neumann et al., 2012;
Liu et al., 2008). In a study carried out by our lab using free-moving 6-OHDA-induced
parkinsonian rats, we also observed a significantly increased θ band (4-8 Hz) oscillatory
activity in rats with LIDs (Alam et al., 2014).
Another significant oscillatory activity is the 4-9 Hz (typically 5 Hz) oscillation, which is
associated with tremor symptoms, and is therefore named the “tremor frequency activity”.
The “non-stationary oscillatory model” is not as “straightforward” as the classical firing
rate model (Nambu et al., 2014). The causal relationship between the abnormal spontaneous oscillations and the motor symptoms remains unclear, so that both models are
relevant for understanding the pathophysiology of PD. Further investigation in this field
is necessary.
15
16
4
The pedunculopontine nucleus
The PPN, or often referred to as the PPTg in rodents, has been considered important for
the pathophysiology of gait disturbances in late stage PD. In the human brain, the PPN
is bounded on its lateral side by fibers of the medial lemniscus and on its medial side
by fibers of the superior cerebellar peduncle and its decussation. Rostrally, the anterior
aspect of the PPN contacts the dorsomedial aspect of the posterolateral portion of the
substantia nigra, while the retrorubral fields border it dorsally. The most dorsal aspect
of the PPN is bounded caudally by the pontine cuneiform and subcuneiform nuclei and
ventrally by the pontine reticular formation. The most caudal pole of the PPN is adjacent
to neurons of the locus coeruleus (Olszewski and Baxter, 1982).
The PPN consists of two regions on the basis of cell density, a pars compacta (PPNc)
located within the caudal half and a more anterior pars dissipata (PPNd; Mena-Segovia
et al., 2004). The former is reported to contain > 90% of cholinergic neurons, probably
with a few dopaminergic neurons intermixed (Pahapill and Lozano, 2000), while the latter contains a considerable number of glutamatergic neurons and less cholinergic neurons
(Lavoie and Parent, 1994a; Mesulam et al., 1989). Both regions contain GABAergic interneurons (Stein, 2009).
The PPN has diverse synaptic connections with many areas in the brain and the spinal
cord, including the BG, i.e., almost all thalamic nuclei, the limbic system (amygdala, hypothalamus, zona incerta), the ascending reticular activating system (raphe nuclei, locus
coeruleus, laterodosal tegmental nucleus), and cortical motor areas (von Monakow et al.,
1979; Edley and Graybiel, 1983). It is involved in many functions such as control of the
sleep-wake cycle, locomotor activity, muscle tone, incentive motivation, biting and gnawing, antinociception, gating of the startle reflex, and cognitive and auditory processing
(Garcia-Rill, 1986; Inglis and Winn, 1995; Takakusaki et al., 2004; Benarroch, 2013).
The BG is considered more highly interconnected with the PPN than any other brain region (Mena-Segovia et al., 2004). Although it is still under debate whether the PPN should
be considered part of the BG, it is obviously a significant outpost of the BG. A pedunculostriatal projection has been reported in monkeys (Lavoie and Parent, 1994b) and the
STN provides glutamatergic innervation of the PPN which, in turn, sends both cholinergic and non-cholinergic and probably excitatory projections back to the STN (Hammond
17
et al., 1983; Bevan and Bolam, 1995). Further the pallidum and the SNr send GABAergic inhibitory projection to the PPN (Noda and Oka, 1986; Granata and Kitai, 1991),
terminating preferentially on the non-cholinergic cells of the PPNd and largely avoid the
cholinergic neurons of the PPNc and PPNd (Rye et al., 1995; Shink et al., 1997; Kang and
Kitai, 1990; Spann and Grofova, 1991). Anatomical studies using monkeys have shown
that > 80% of GPi neurons send axons collaterally to both the ventrolateral nucleus of
the thalamus and the PPN (Harnois and Filion, 1982). In turn, the PPN sends back a
mixed cholinergic and glutamatergic projection to the SNr and GPi, as well as to the SNc
and GPe.
18
5
5.1
Animal models
The 6-hydroxydopamine animal model of Parkinson’s disease and levodopa-induced dyskinesias
The 6-OHDA rat model is one of the oldest and most widely used rodent models for
PD (Ungerstedt, 1968). 6-OHDA is a synthetic neurotoxin and a structural analogue of
dopamine neurotransmitter carried on by dopamine transporter. This toxin does not cross
the blood brain barrier, and therefore it is stereotaxically injected locally into certain brain
regions. In preclinical research 6-OHDA is most commonly injected unilaterally into the
SNc, medial forebrain bundle, or striatum. When injected into the SNc it induces a fast
and specific degeneration of the dopaminergic neurons. After striatal injection lesions
are generated via retrograde transport of the neurotoxin to the SNc cell bodies and tend
to form a more progressive partial lesion. The efficacy of the unilateral lesion can then
be easily assessed by drug-induced rotation tests, usually with injection of the dopamine
receptor agonist apomorphin (Jerussi and Glick, 1975; Dunnett and Lelos, 2010). An
alternative are drug-free behavioural tests such as cylinder test (Schallert et al., 2000;
Glajch et al., 2012). Electrophysiological studies in this model showed, in general, similar
findings with regard to the firing rate (Mallet et al., 2006; Kita and Kita, 2011), burstfiring (Bergman et al., 1994; Soares et al., 2004; Tachibana et al., 2011; Wichmann and
Soares, 2006; Mallet et al., 2008) and oscillatory activities (Jenkinson and Brown, 2011;
Kühn et al., 2009) as compared to patient with PD and non-human primate. However,
inter-species differences still need to be cautiously taken into account. Despite the fact
that the 6-OHDA model cannot mimic all stages of PD (Papa et al., 1994) and that the
acute nature of lesion effect is different from the insidious progression of PD observed in
patients, this model has been proved a good tool for studying PD and remains popular
after decades since its first introduction.
Chronic treatment of levodopa in this model of PD has been reported to induce LIDlike movements, such as movements with dystonic or hyperkinetic features, which were
observed in axial and orofacial muscles (Andersson et al., 1999; Cenci et al., 1998). In order
to model the LIDs and evaluate its severity, a special rating scale has been developed by
19
Cenci et al. in 1998 to quantify the abnormal involuntary movements (AIMs) induced
by levodopa treatment. The AIMs rating scale, which is currently still in use, evaluates
four aspects of the movements in rats following administration of levodopa, including
locomotion, axial dyskinesia, orolingual dyskinesia and limb AIMs (Fig. 5.1). The rating
is based on both amplitude and time, which give the AIMs test a large dynamic range
and allows for precise evaluation.
5.2
Ethylcholine mustard aziridinium ion-induced pedunculopontine nucleus cholinergic lesion and evaluation of motor function in rats
The ethylcholine mustard aziridinium ion (AF64-A) has been used as a selective presynaptic cholinergic neurotoxin since about three decades ago (Fisher and Hanin, 1980). It acts
by inhibitory irreversible alkylation of the choline uptake system and different cholinerelated enzymes (Fisher et al., 1982; Leventer et al., 1985a; Leventer et al., 1985b) and is
considered as a potent and remarkably selective cholinergic neurotoxin for the PPN in a
dose and site-dependent manner (Hanin, 1996; Kása and Hanin, 1985; Lança et al., 2000).
Alterations of the motor function following PPN lesion can be evaluated via different behavioural tests. The traditional open field test can be used to quantify the general locomotor activity level of the rat by placing it in an open field arena, such as a 60×60×30cm3
black box. The Rotarod test can be used to assay the motor coordination of the animal
by placing it on a suspended rotating rod (namely rotarod) and measuring how long the
rat is able to maintain its balance on the rotarod. Utilizing high speed digital camera,
the motion of a rat walking on a treadmill can be captured and different gait parameters
can thus be measured for further analyses.
20
Figure 5.1: Subtypes of levodopa-induced abnormal involuntary movements in the unilateral 6-OHDA rat model of Parkinson’s disease. After injection of levodopa, rat was
affected by locomotive (A), axial (B) orolingual (C), and forelimb AIMs (D; adapted from
Winkler et al., 2002)
21
22
6
Objectives
Both LIDs and gait disturbances frequently occur in patients with advanced PD. These
conditions not only severely impact the quality of life and increase the financial burden
for patients and social healthcare systems, but also bring major therapeutic challenges
to clinics. In-depth studies are needed in order to enhance our understanding of the
pathophysiology underlying these conditions.
Project one
We aimed to investigate the neuronal firing characteristics of the EPN, the rat
equivalent of the human GPi and output nucleus of the BG, and its coherence with
the motor cortex field potentials in the 6-OHDA rat model of PD with and without
LIDs.
Project two
We aimed to investigate the effect of anterior or posterior cholinergic lesions of the
PPTg (equivalent to the PPN in primates) on gait-related motor behaviour, and on
neuronal network activity of the PPTg area and BG motor loop in rats.
23
24
7
Manuscript one
Title:
Coherence of neuronal firing of the entopeduncular nucleus with motor cortex oscillatory
activity in the 6-OHDA rat model of Parkinson’s disease with levodopa-induced dyskinesias
Order of Authors:
Xingxing Jin, Kerstin Schwabe, Joachim K. Krauss, Mesbah Alam
Contribution:
Authors Jin, Alam and Schwabe designed the study and wrote the protocol. Experiments were performed by author Xingxing. Authors Mesbah and Xingxing undertook the
statistical analysis of the data and wrote the first draft of the manuscript. All authors
contributed to and have approved the final version of the manuscript. Critical revision
was done by authors Schwabe and Krauss.
25
Coherence of neuronal firing of the entopeduncular nucleus with motor cortex oscillatory activity in the 6-OHDA rat model of Parkinson’s disease with
levodopa-induced dyskinesias
Xingxing Jin, Kerstin Schwabe, Joachim K. Krauss, Mesbah Alam
Department of Neurosurgery, Medical University Hannover, Carl-Neuberg-Str. 1, D-30625
Hannover, Germany
Abstract
Objective: The pathophysiological mechanisms leading to dyskinesias in Parkinson’s
disease (PD) after long-term treatment with levodopa remain unclear. This study investigates the neuronal firing characteristics of the entopeduncular nucleus (EPN), the rat
equivalent of the human globus pallidus internus and output nucleus of the basal ganglia,
and its coherence with the motor cortex (MCx) field potentials in the unilateral 6-OHDA
rat model of PD with and without levodopa-induced dyskinesias (LID).
Methods: 6-hydroxydopamine lesioned hemiparkinsonian (HP) rats, 6-OHDA lesioned
HP rats with LID (HP-LID) rats, and naı̈ve controls were used for recording of single unit
activity under urethane (1.4 g/kg, i.p.) anesthesia in the EPN “on” and “off” levodopa.
Over the MCx, the electrocorticogram (ECoG) was recorded.
Results: Analysis of single unit activity in the EPN showed enhanced firing rates, burst
activity and irregularity compared to naı̈ve controls, which did not differ between drugnaı̈ve HP and HP-LID rats. Analysis of EPN spike coherence and phase locked ratio with
MCx field potentials showed a shift of low (12-19Hz) and high (19-30Hz) beta oscillatory activity between HP and HP-LID groups. EPN theta phase locked ratio was only
enhanced in HP-LID compared to HP rats. Overall, levodopa injection had no stronger
effect in HP-LID rats than in HP rats.
Conclusions: Altered coherence and changes in the phase lock ratio of spike and local
field potentials in the beta range may play a role for the development of LID.
Keywords: Entopeduncular nucleus, Motor cortex, Parkinson’s disease, Neuronal coherence, Phase locking
26
Indroduction
The degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc),
which leads to the depletion of dopamine in the striatum, the entrance region of the
basal ganglia (BG) motor loop, is one of the pathophysiological hallmarks of Parkinson’s
disease (PD). Chronic replacement therapy with levodopa relieves symptoms, however,
eventually may lead to abnormal involuntary movements, termed dyskinesias, which become treatment-limiting. It has been thought that levodopa-induced dyskinesias (LIDs)
develop as a consequence of pulsatile stimulation of dopamine receptors, with consequent
dysregulation in downstream neurons resulting in changes in neuronal firing patterns
(Obeso et al., 2000).
In patients with PD abnormal neuronal activity has been found in the globus pallidus
internus (GPi) and the subthalamic nucleus (STN), especially an increase in synchronized
oscillatory beta band activity (13-30 Hz) has been noted, along with enhanced neuronal
firing rates and burst activity (Brown, 2003; Obeso et al., 2006; Wichmann and Dostrovsky, 2011; Weinberger et al., 2012). Recordings from patients undergoing pallidotomy or
deep brain stimulation have shown that dyskinesias after chronic levodopa treatment are
accompanied by reduced oscillatory beta band activity and enhanced theta band activity
(4-10 Hz), together with an extensive decrease in firing rates and abnormal firing patterns
(Lozano et al., 2000; Obeso et al., 2000; Alonso-Frech et al., 2006). These studies, however,
did not address differences of neuronal single units and oscillatory activity in patients with
drug naı̈ve advanced PD with or without peak-dose dyskinesias, since almost all patients
undergoing neurosurgical treatment have received chronic treatment with levodopa and
therefore developed levodopa-induced dyskinesia (LID) at least to some extent at the time
of surgery.
Injection of 6-hydroxydopamine (6-OHDA) into the rat nigrostriatal system leads to degeneration of dopaminergic neurons in the SNc together with concomitant abnormal neuronal activity in the BG, which closely parallels the findings in PD patients. When chronically treated with levodopa, 6-OHDA lesioned rats exhibit a broad range of behavioural,
physiological, and biochemical features that are similar to LIDs in human patients (Lundblad et al., 2002; Picconi et al., 2005; Marin et al., 2008; Marin et al., 2009; Alam et al.,
2014). The oscillatory theta band activity recorded in different basal ganglia regions was
significantly more pronounced in 6-OHDA lesioned animals with LIDs than in drug-naı̈ve
6-OHDA lesioned rats (Alam et al., 2014; Meissner et al., 2006).
In order to better understand the neuronal mechanisms involved in the development of
LIDs, we investigated the neuronal firing activity of the entopeduncular nucleus (EPN),
the rat equivalent to the human GPi, and its coherence with the motor cortex (MCx) field
27
potentials in 6-OHDA lesioned hemiparkinsonian (HP) rats with LIDs on/off levodopa,
i.e., a model for advanced PD with peak-dose dyskinesias. Measures were compared with
the neuronal activity of HP rats without dyskinesias and naı̈ve rats, which served as
controls.
Material and methods
Animals
Thirty eight adult male Sprague Dawley rats (Charles River Laboratories, Germany)
were used in this study. They were housed in groups of three to four animals per cage
(Macrolon Type IV) and kept under controlled environmental conditions (temperature
22◦ C, relative humidity 45-55%, 14/10 h light/dark cycle) and fed with laboratory rat
chow and water ad libitum. All animal procedures were in accordance with the European
Council Directive of November 24, 1986 (86/609/EEC) and were approved by the local
animal ethic committee. All efforts were made to minimize the number of animals used
and their suffering.
Thirty two rats were rendered hemiparkinsonian by unilateral injection of 6-OHDA in the
medial forebrain bundle (MFB). Subsequently, these HP rats were divided into two groups.
One group (n=24) were rendered dyskinetic by long-term injections of levodopa, in the
following termed HP-LID rats, while the other HP group (n=8) received no levodopa
injection. Another group of rats (n=6) without surgery served as naı̈ve controls.
6-OHDA lesion
For surgery, rats were anaesthetized with 3.6% chloral hydrate (1ml/100g body weight,
i.p., Sigma, Germany) and placed in a stereotaxic frame (Stoelting, Wood Dale, Illinois,
USA). Two holes were drilled over the targets above the right medial forebrain bundle and
the dura was exposed. 6-OHDA was dissolved in 0.02% ascorbate saline at a concentration of 3.6µg/µl and was injected (1µl/min) in two deposits (2.5µl and 3µl, respectively)
at the following coordinates in mm relative to bregma and to the surface of the dura
mater: anterior-posterior (AP) = 4.0; lateral (L) = ±0.8; ventral (V) = −8.0; tooth bar
at +3.4 and AP = 4.4; L = ±1.2; V = −7.8; tooth bar at −2.4, respectively. Sham
lesioned rats received only the vehicle (0.02% ascorbic acid in physiological saline) at the
same coordinates. After infusion, the incision was closed by stitches and the animals were
returned to their home cages for recovery.
The efficacy of the 6-OHDA-induced lesion was validated 3 weeks after surgery by injection of apomorphine (0.05mg/kg, s.c.; Sigma) as previously described (Alam et al., 2014).
28
The lesion was considered successful in those animals that made more than 80 net contraversive rotations in 20min. To induce dyskinesias the rats were treated for four weeks
with 6mg/kg L-DOPA methylester (Simga-Aldrich, Germany) plus 12mg/kg benserazidHCl. Both drugs were dissolved in physiological saline and injected subcutanously (s.c.)
with a volume of 1ml/kg body.
Dyskinesias were scored by the Abnormal Involuntary Movements (AIMs) scale as described earlier (Alam et al., 2014). The different subtypes of AIMs: orolingual, forelimb,
and axial dyskinesias were scored separately for 2 h after levodopa injection on an ordinal
scale from 0 to 4, respectively, for 1 min every 10 min (i.e., 12 monitoring periods from
10 to 120 min postinjection). The mean value of these measures was used for further
analysis. Only rats with total AIMs scores higher than 4 were included in HP-LID group.
Electrophysiology
Neuronal activity was recorded in the EPN in naı̈ve controls, HP and HP-LID rat groups
before and after levodopa injections. Recordings were done under urethane anesthesia
(1.4g/kg i.p. with additional 25% doses as needed) as described previously (Alam et al.,
2012). The temperature of the anesthetized animals was constantly controlled with a rectal probe and maintained at 37.2 to 37.6◦ C with a heating pad (Harvard Apparatus).
Electrocardiographic (ECG) activity was monitored constantly to ensure the animals’
wellbeing. A drop of Silicon oil was applied to all areas of the exposed cortex to prevent
dehydration. Depth of anesthesia was monitored by examination of the reflex answer to
a toe pinch.
The recordings of extra cellular single unit (SU) activity were performed in the EPN
(coordinates relative to bregma AP: -2.3 to -2.8 mm posterior to bregma: L: -2.6 to -3.0
mm from the midline; V: 7.5 to 8.0 mm from the dura, tooth bar at -3.3 mm. Spike train
recordings from the EPN were paired with simultaneous recordings of the MCx-ECoG.
Extracellular SU recordings were taken by quartz coated pulled and ground platinumtungsten alloy core (95%-5%) micro electrode with a diameter of 80 µm, and an impedance
of 1-2 MΩ at 1 kHz. The electrode was advanced using a microdrive (Thomas Recording
GmbH, Giessen, Germany) in the ipsilateral EPN. The SU signals were digitized at a
sampling rate of 25 kHz with 0.5 kHz-5 kHz band-pass filter and amplification of signals from ×9, 500 to ×19, 000. Additionally, the MCx-ECoG was recorded via a 1 mm
diameter jeweller’s screw, which was positioned on the dura mater above the frontal cortex ipsilateral to the lesioned or sham-lesioned hemisphere (AP, +2.7 mm; L, 2.0 mm;
which corresponds to the primary motor cortex region). Two additional screws, serving
as MCx-ECoG reference and ground, were placed over the parietal lobe and cerebellum
and band pass filtered (0.5 Hz to 100 Hz) with a sampling rate of 1 kHz (Alam et al.,
29
2012). All signals were digitized with a CED 1401 (Cambridge Electronic Design (CED),
Cambridge, UK). The firing of each neuron was recorded for 8 to 10 min after signal stabilization. After termination of the experiment, electrical lesions were made at the recording
sites (10 µA for 10s; both negative and positive polarity) and the rat was perfused with
4% paraformaldehyde. Each brain was then cut into 20 µm sections and stained with a
standard HE protocol to verify the position of each electrode.
Analysis of electrophysiological data
One epoch of 300 sec recordings was analysed and sorted on the base of a 3:1 signal
to noise ratio. Neuronal firing activity arising from a single neuron was discriminated
by threshold spike detection and template matching, controlled by cluster analysis with
principal component analysis and final visual inspection by using the template-matching
function of the spike-sorting software (Spike2; Cambridge Electronic Design, Cambridge,
UK).
The firing rate was calculated with the firing rate histograms generated in NeuroExplorer
version 4 (NEX Technologies, NC). The coefficient of variation (CV) of the spike interspike interval sequence was computed for each recording as a measure of the regularity
of the spike firing. CV is a measure of spike train irregularity defined as the standard
deviation divided by the mean interspike interval. Exponential distributions have a CV
of 1, i.e., describe more irregular discharge patterns, whereas distributions derived from
more regular ISIs have CV values below 1.
An asymmetry index was computed as the mode inter-spike interval divided by the mean
inter-spike interval. It provides information on the shape of the ISI histogram and the
regularity of the discharge pattern. An asymmetry index close to 1 reveals a relatively
regular firing pattern, whereas the more the index differs from unity, the more irregular
the spike trains. A ratio of less than 1 reflects an asymmetrical shape, indicating a larger
fraction of short interspike intervals (positively skewed), as is expected when there is
bursting activity.
Firing patterns of spikes events
The analysis classified discharge patterns into 1 of 3 basic categories, i.e., regular, irregular,
and bursty firing. Its discharge density histogram was estimated on the base of three
reference probability density functions (PDFs) as proposed by Labarre et al. (2008).
This method is a comparison of the density histogram d(λ) to a reference density function
px (λ). For the reference functions (1) a Gaussian PDF with mean 1 and variance 0.5, (2)
a Poisson PDF with mean 1 and (3) a Poisson PDF with mean 0.8 were used to represent
30
regular, irregular and bursting activity, respectively (Lourens et al., 2013). The smallest
distance of the estimated discharge density histogram of the neuron to the three reference
PDFs determined the type of neuron.
Coherence and phase lock of EPN spikes with MCx-ECoG
The duration of 300 sec simultaneous recorded EPN neuronal spikes and MCx-ECoG
signals were used to determine coherence between a point process and a field potential
using the neurospec toolkit (version 2.0) in MATLAB, as described in Halliday et al.
(1995). ECoG signals were notch filtered to eliminate the 50 Hz noise with a finite
impulse response (FIR) notch filter prior to analysis. Autospectra of ECoG necessary for
the calculation of coherence were derived by discrete Fourier transformation with blocks of
1024 samples using a Welch periodogram. Mean coherences were calculated for the theta
(4-8 Hz) and beta (12-30 Hz) frequency ranges. Since several studies in human PD patients
have suggested that low- and high-beta activities may have a different functional signalling
(Priori et al., 2004; Marceglia et al., 2006; Marceglia et al., 2007; Lopez-Azcarate et al.,
2010), we additionally analyzed low (12-19 Hz) and high (19-30 Hz) beta band coherence
in order to determine any possible changes within the beta frequency range.
Additionally, phase relationships between spikes and MCx-ECoG field potentials were
assessed using spike-triggered waveform averages (STWA). The ECoG channels were band
pass filtered at different bands with an ideal (noncausal) filter to prevent phase distortions.
STWAs were calculated for 150 ms before and after the spike trigger over a 300 s epoch.
Spike trains of each neuron were shuffled 20 times to create a null hypothesis for a non
phase locked spike train with the same first order statistics as the original spike train.
The phase-locked ratio was obtained by dividing the peak-to-through amplitude of the
unshuffled spike trains STWA by the mean of the shuffled distribution. A comparison
of the mean ratios was analyzed for the EPN single unit firing neuron referenced to the
MCx-ECoG filtered in theta (4-8Hz) and beta (12-30Hz) frequency ranges, beta activity
was further divided into low (12-19Hz) and high beta (19-30Hz).
Statistical analysis
Two-way analysis of variance (2-way ANOVA) was used to test for significant differences
among the groups followed by post hoc Tukey Test for multiple comparisons between
groups for detection of significance (P value less than 0.05). Pearson’s chi-square (Chi2 )
test was used to determine differences in the distribution of firing patterns. All data are
expressed as the mean ± SEM .
31
Figure 7.1: Histological pictures showing examples of recording trajectories in the EPN
magnified from 10× and 50× (A and B), and corresponding schematic reconstructions
from Paxinos and Watson (1998; C). White arrows indicate the 200µm distanced electrolytic coagulations (10µA bipolar current for 10 s) along the recording trajectories.
Results
Two of the 24 animals in the HP-LID group were euthanized because of severe and continuous loss of body weight after surgery for unilateral 6-OHDA lesions. All remaining
6-OHDA lesioned rats showed more than 80 contraversive rotations during the apomorphine challenge and were thus considered suitable for the experiments. After four weeks
of chronic levodopa injection, 12 of the 22 animals in the HP-LID group showed dyskinesias as determined by the AIM (mean score of 7.53 ± 0.71, range 4.33 − 11.5), and were
thus used for the electrophysiological recordings. All rats operated for the HP group had
appropriate 6-OHDA lesions, i.e., showed more than 80 contraversive rotations during the
apomorphine challenge, and were used for electrophysiological recordings.
The neuronal activity of 307 single units was recorded before and of 307 single units after
levodopa injection. The average units number (mean and SEM) recorded per individual
rat was 21.83 ± 1.90. All recording sites were localized in the EPN (see Fig. 7.1 for
examples).
Firing rate
In the EPN the firing rate was higher in 6-OHDA lesioned than in naı̈ve rats, long-term
treatment with levodopa, however, had no additional effect. Acute levodopa injection
reduced firing rates in HP and HP-LID rats without difference, but increased this measure
in naı̈ve rats (Fig. 7.2 A). Statistical analysis with two-way ANOVA showed an effect for
32
the factor drug (F2,523 = 16.12, P < 0.001), and an interaction between the factors drug
and group (F2,523 = 17.72, P < 0.001), but no effect for the factor group (F2,523 = 1.68,
P = 0.19). Post-hoc testing showed that the mean firing rates were enhanced in HP
and HP-LID rats as compared to naı̈ve controls without difference (all p-values < 0.05).
Injection of levodopa increased the firing rates in naı̈ve controls, but reduced this measure
in HP and HP-LID rats without difference (all p-values < 0.05).
CV and asymmetry index
The CV of HP and HP-LID groups was higher as compared to naı̈ve controls. Levodopa
injection, further enhanced this measure in both groups. Statistical analysis with ANOVA
revealed a significant effect for the factor group (F5,523 = 34.48, P < 0.001), the factor drug
(F2,523 = 27.23, P < 0.001) and the interaction between factors (F2,523 = 5.21, P < 0.006).
Post-hoc analysis revealed an enhanced CV in HP and HP-LID rats (P < 0.05) without
difference between these groups. Levodopa significantly increased CV in HP and HP-LID
groups. This enhancement was less in HP-LID rats, leading to a significant lower CV in
HP-LID rats after levodopa injection compared to HP rats (P < 0.05; Fig. 7.2 B).
The asymmetry index of HP and HP-LID groups was lower as compared to naı̈ve controls.
Levodopa injection further decreased this measure in both groups. Two way ANOVA
revealed significant effects on asymmetry index for the factor group (F2,523 = 40.409,
P < 0.001), the factor drug (F2,511 = 29.968, P < 0.001) and interaction between factors
(F2,511 = 7.609, P < 0.001). Both HP and HP-LID rats showed a lower asymmetry
index compared to naı̈ve controls (P < 0.001), but without difference between groups.
Injection of levodopa significantly reduced the asymmetry index in both groups, but to
a lesser extent in HP-LID rats, leading to a significant lower asymmetry index in HP
compared to HP-LID rats (P < 0.05; Fig. 7.2 C).
Firing patterns
The percentage of bursty pattern neurons was higher and the percentage of regular pattern
neurons was lower in HP and HP-LID rats as compared to naı̈ve control rats (P¡0.01).
Administration of levodopa increased the number of bursty neurons only in HP rats (Fig.
7.2 D).
Coherence of EPN-spikes and MCx-ECoG
Analysis of the coherence of theta band activity between EPN spikes and MCx-ECoG
showed enhanced coherences in HP and HP-LID rats compared to naı̈ve controls, but no
difference between groups. Levodopa injection reduced this measure only in HP-LID rats,
33
Figure 7.2: Neuronal firing rates, (A) coefficient variation of inter-spike intervals (CV;
B), asymmetry index (AI; C) and the percentage of three different discharge patterns
(burst, irregular, and regular; D) of the EPN neuronal activity. Significant differences in
comparison with naı̈ve control group is indicated by asterisks (∗), differences within group
after treatment of L-DOPA with ($) and differences between HP and HP-LID comparisons
by (#; P < 0.05; two-way ANOVA and post hoc Tukey test for the neuronal firing rate;
Chi-square test with Bonferroni adjustment for the distributions of discharge patterns).
34
without affecting HP rats. The statistical analysis with ANOVA of EPN spikes and MCxECoG coherence of theta band showed a significant effect for the factor group (F2,523 =
7.35, P < 0.001) and for the interaction for the factors group and drug (F2,523 = 6.14,
P < 0.01), but no statistical difference for the factor drug (F1,523 = 1.16, P = 0.28). Post
hoc tests confirmed that compared to controls the theta frequency band coherence was
higher in the HP rats (P < 0.05) and in the HP-LID rats (P < 0.001), without differences
between groups. Injection of levodopa decreased theta band coherence only in HP-LID
rats (P < 0.001; Fig. 7.3 B).
Analysis of the coherence of beta band activity between EPN spikes and MCx-ECoG
showed that beta band activity was more enhanced in HP than in HP-LID rats. Injection
of levodopa decreased beta band coherence in both groups. Statistical analysis with
ANOVA showed a significant effect for the factor group (F2,523 = 6.57, P < 0.002), for
the factor drug (F1,523 = 17.70, P < 0.001), and for the interaction between factors
(F2,523 = 4.82, P < 0.008). The beta modulating spikes coherence with the MCx was
significantly higher in HP rats compared to both naı̈ve and HP-LID rats (P < 0.001).
Levodopa injection decreased beta coherence in HP rats (P < 0.001), while this effect it
did not reach the level of significance in HP-LID rats (P = 0.25).
Adspection of the coherence spectrum showed that two frequency peaks dominated the
spectrum of the beta range, one in the low-beta (12-19 Hz) and another in the highbeta range (19-30 Hz), i.e., the EPN-spikes and motor cortex ECoG coherence showed
a shift in the beta spectrum between HP and HP-LID rats. Statistical analysis of the
low and high beta bands with ANOVA showed a significant effect for the factor group
(low: F2,523 = 7.05, P < 0.001; high: F2,523 = 7.80, P < 0.001), the factor drug (low:
F1,523 = 18.98, P < 0.001; high: F1,523 = 10.28, P < 0.001), and the interaction between
factors (low: F2,523 = 6.14, P < 0.01; high: F2,523 = 5.67, P < 0.01). Post hoc analysis
revealed that the low beta range frequency modulating spikes coherence was higher in
both HP and HP-LID groups as compared to naı̈ve control rats (P < 0.05; P < 0.001),
but also higher in HP-LID rats compared to HP rats (P < 0.05). Levodopa injection
decreased the low beta coherence in both HP (P < 0.05) and HP-LID rats (P < 0.001;
Fig. 7.3 D). The high beta frequency (19-30Hz) spikes coherence was higher in HP rats
as compared to HP-LID rats (P < 0.001), which did not differ from naı̈ve control rats.
Treatment with levodopa significantly decreased the high beta coherence only in HP rats
(P < 0.001; Fig. 7.3 E).
EPN spikes and MCx-ECoG phase relation
The EPN spikes and MCx phase locked ratio of theta band activity was only enhanced
in HP-LID rats. Injection with levodopa had a different effect on HP and HP-LID rats.
35
Figure 7.3: Coherence of EPN-spikes and MCx-ECoG spectral power as shown within the
frequency range of 1-40 Hz (A). The bar plot shows the mean ± SEM ratio transformed
coherence of the theta (B) and beta oscillatory coherence (C), as well as for the low
and high beta oscillatory coherence across experimental groups (D and E) respectively.
Significant differences in comparison with naı̈ve control group is indicated by asterisks
(∗), differences within group after treatment of L-DOPA with ($) and differences between
HP and HP-LID comparisons by (#; P < 0.05; two-way ANOVA and post hoc Tukey
test).
36
Figure 7.4: The bar graphs show a comparison of the mean ratios between peak to trough
amplitudes of the original STWA and the mean of 20 shuffled STWAs for MCx-ECoG
frequency ranges for the theta (A) and beta (B) for EPN neuronal firing activity.
While it reduced the phase locked ratio in HP-LID rats, this measure was enhanced
in HP rats. Statistical analysis with ANOVA showed a significant effect for the factor
group (F2,523 = 3.74, P < 0.05) and for the interaction between factors (F2,523 = 8.59,
P < 0.001), while the factor drug had no effect (F2,523 = 0.27, P = 0.61). Post hoc testing
showed that the theta phase locked ratio was significantly enhanced only in the HP-LID
group (P < 0.001; Fig. 7.4 A). Treatment with levodopa increased the theta phase locked
ratio in the HP group, but decreased this measure in HP-LID rats (P < 0.001).
The EPN spikes and MCx phase locked ratio of beta oscillatory activity was enhanced
in both HP and HP-LID rats without difference, and injection of levodopa reduced this
measure in both groups. Statistical analysis with ANOVA showed a significant effect for
the factor group (F2,523 = 8.58, P < 0.001), the factor drug (F1,523 = 17.75, P < 0.001)
and interaction between factors (F2,523 = 4.58, P < 0.05). Post hoc analysis showed that
the beta phase locked ratios were higher in both HP and LID groups without difference,
and treatment with levodopa decreased beta phase locked ratio in both groups (P < 0.001;
37
Fig. 7.4 B).
Similar to the coherence of EPN spike and MCx-ECoG, two-way ANOVA of the low
and high beta band showed a significant effect for the factor group (low: F2,523 = 11.24,
P < 0.001; high: F2,523 = 8.47, P < 0.001), the factor drug (low: F1,523 = 27.24,
P < 0.001; high: F1,523 = 7.453, P < 0.01), and the interaction between factors (low:
F2,523 = 6.53, P < 0.01; high: F2,523 = 4.73, P < 0.01). Post hoc analysis showed a
higher spike-ECoG phase locked ratio at low beta band in both HP and HP-LID groups
compared to naı̈ve controls (P < 0.001), but also higher activity in HP-LID rats compared
to the HP group (P < 0.01). Levodopa injection significantly decreased the phase locked
ratios in both HP and HP-LID groups (P < 0.001). EPN spike-ECoG phase locked ratio
at high beta frequency band was significantly higher in the HP group compared to both
naı̈ve (P < 0.001) and HP-LID rats (P < 0.01), however the HP-LID rats did not differ
from naı̈ve controls. Levodopa injection significantly reduced the phase locked ratio only
in the HP group (P < 0.001).
Discussion
Analysis of single unit activity in the EPN of 6-OHDA lesioned rats showed enhanced
neuronal firing rates, which were reduced by levodopa. This is in line with the classical
rate-coding model of PD, which predicts that the loss of nigrostriatal dopamine leads to
disinhibition of the STN, which subsequently results in overactivity of the GPi. Dopamine
replacement therapy normalizes GPi activity by its action through the direct and indirect
striatal output pathway. Consistent with this concept, enhanced neuronal firing rates
have been found in the STN and GPi of patients with PD (Rodriguez-Oroz et al., 2001;
Brown, 2003; Obeso et al., 2006; Wichmann and Dostrovsky, 2011; Weinberger et al.,
2012) and in experimental studies using the 6-OHDA rat model and the MPTP monkey
model of PD (Hollerman and Grace, 1992; Wichmann et al., 1994; Hassani et al., 1996;
Ni et al., 2001). Further, neurophysiological studies in parkinsonian monkeys (Filion and
Tremblay, 1991; Boraud et al., 1998) and patients during pallidotomy (Hutchison et al.,
1997) have shown a reduction of the firing rate in the GPi after application of dopamine
agonists. In PD patients with LID neuronal activity of the STN and GPi also shift from
increased neuronal firing in the parkinsonian state to marked hypoactivity during expression of dyskinesias (Merello et al., 1999; Lozano et al., 2000). This corroborates findings
of Papa et al., (1999), showing that the firing rates in monkeys expressing dyskinesias after administration of levodopa were substantially more reduced than in monkeys without
expression of dyskinesias (Papa et al., 1999). These studies used a low dose of levodopa
to examine the “on” state without dyskinesias versus the “dyskinesia” state after high
38
dose of levodopa, but did not differentiate between drug-naı̈ve PD and PD with LID after
longterm treatment with levodopa. In contrast, in the present study, we measured neuronal activity in drug-naı̈ve HP rats compared to HP-LID rats “off” and “on” levodopa.
With this study design, in the “off” state the EPN firing rate was enhanced in both
drug-naı̈ve HP and HP-LID rats without difference. Further, the firing rate was reduced
in the “on” levodopa state in both groups without difference, i.e., levodopa did not have
a stronger effect on the firing rate in HP-LID rats than on drug-naı̈ve HP rats.
The induction of LIDs by dopamine agonists has not only been associated with a mean
reduction in the firing rate, but also with an increase of burst activity in the STN and GPi
when patients express dyskinesias (Lozano et al., 2000; Obeso et al., 2000; Levy et al.,
2001). In the present study, we found enhanced bursty pattern neurons in HP and HP-LID
rats, which did not differ between groups. Treatment with levodopa, however, increased
the bursty pattern neurons only in HP rats. Likewise, in both HP and HP-LID rats the
irregularity of firing activity, as measured by the CV, was increased with no difference
between HP and HP-LID groups, and the effect of levodopa injection was more severe in
HP than in HP-LID rats.
Overall, using the 6-OHDA rat model with LID we did not find differences between HP
and HP-LID rats, and acute levodopa injection did not have a stronger effect on neuronal
activity in HP-LID rats. The effects seen in the human and monkey studies may therefore be more related to different dosages of dopamine agonists, than to the underlying
neurophysiological changes induced by longterm application of levodopa.
Besides the classical rate model, the oscillatory activity in different spectral bands and
their synchronization provide information about brain network activity. Changes in oscillatory activity are thought to be correlates of abnormal neuronal processing in movement
disorders. Further, neural spiking activity is likely transiently coupled to the field potentials in a rhythmic or non-oscillatory fashion, and allow insight into how altered firing
pattern in different nuclei relate to changes in oscillatory activity throughout the basal
ganglia network (Fries et al., 2007; Kayser et al., 2009). With that regard, beta oscillations
are enhanced in akinetic PD and possibly contribute to bradykinesia and rigidity (Brown,
2003; Kühn et al., 2006; Chen et al., 2007; Kühn et al., 2008; Ray et al., 2008). Injection
of dopamine agonists have been reported to reduce beta band activity in patients with
PD (Kühn et al., 2006; Weinberger et al., 2006; Ray et al., 2008) and in patients expressing dyskinesias (Alonso-Frech et al., 2006). In the present study, the coherence of EPN
spike and MCx field potentials showed enhanced beta activity in both HP and HP-LID
rats when using the whole range of beta frequency band (12-30Hz), however, without
difference between groups. This differ from our previous studies using the 6-OHDA rat
model of PD, where we observed a higher beta band activity in HP rats than in HP-LID
39
rats (Alam et al., 2014). Interestingly, however, we observed a shift of high (19-30Hz)
to low (12-19Hz) range of beta in the EPN spikes and MCx coherence between HP and
HP-LID groups, which was also observed for the phase relation analysis. High beta range
oscillatory activity in the EPN may therefore be associated with drug-naı̈ve PD, whereas
enhanced low beta range activity may be a correlate of changes in neuronal activity that
parallels the behavioural development of dyskinesias after chronic injection of levodopa.
The role of high versus low oscillatory beta band activity is not entirely clear. In humans it has been suggested that low-beta modulations are specific for action observation,
whereas high-beta modulations have been related to the action scene (Marceglia et al.,
2009). The rigid-akinetic parkinsonian state has been associated with an increase of high
range beta power (20-30Hz) in M1 motor cortex or STN (Crowell et al., 2012; Shimamoto
et al., 2013). In line with this, the power and coherence of high beta (22-32 Hz) oscillations
in the cerebral cortex and STN of awake free moving rats was enhanced in the 6-OHDA
rat model of PD (Sharott et al., 2005; Li et al., 2012). On the other hand, several clinical
studies described a predominant peak within the low-beta band (12-20 Hz) in the off
levodopa state, together with a smaller peak in the high-beta (20-30 Hz) band (LopezAzcarate et al., 2010; Rodriguez-Oroz et al., 2011; Thompson et al., 2014). The low-beta
peak disappeared after levodopa application or was greatly reduced during movement,
whereas the high-beta peak remained at similar power (Thompson et al., 2014).
In patients with PD it has been shown that oscillatory activity in the theta frequency band
was higher after administration of levodopa, i.e., in the “On” dyskinesia condition, than
in patients in the “Off” medication state (Alonso-Frech et al., 2006). In line with this, the
theta oscillatory activity recorded in different basal ganglia regions was significantly more
pronounced in 6-OHDA lesioned animals with LIDs than in drug-naı̈ve 6-OHDA lesioned
rats (Alam et al., 2014; Meissner et al., 2006). In our present study, however, analysis of
the coherence and phase lock ratio of theta frequency spike trains of MCx-ECoG showed
that although oscillatory theta band activity was somewhat higher in HP-LID rats than
in drug-naı̈ve HP rats, levodopa injection only enhanced theta band activity in HP rats,
but reduced this measure in HP-LID rats.
It should be noted that all recordings in the present study were made in the urethane
anaesthetized condition, therefore, the genesis of increased theta spike-MCx coherence or
phase locked ratio after treatment of levodopa occurred without the sensory motor feedback of involuntary movements. Especially theta band activity has been shown to depend
on the presence or absence of the abnormal dystonic posture or the phasic movements
and may therefore be more pronounced in the non-anesthetized state (Brazhnik et al.,
2012; Lemaire et al., 2012).
The pathophysiological mechanisms underlying the development of dyskinesias still need
40
further clarification. While we did not find differences of single unit activity in the EPN
of drug naı̈ve HP and HP-LID rats, the coupling of neural spiking activity to the MCx
oscillatory activity differed between HP and HP-LID. Further research is needed to investigate whether the interaction between BG activity and cortical processing would indeed
be relevant for the occurrence of dyskinesias.
41
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46
8
Manuscript two
Title:
Cholinergic lesion in the anterior and posterior pedunculopontine tegmental nucleus: behaviour and neuronal activity in the cuneiform and entopeduncular nuclei
Order of Authors:
Xingxing Jin, Kerstin Schwabe, Joachim K. Krauss, Mesbah Alam
Contribution:
Authors Jin, Alam and Schwabe designed the study and wrote the protocol. Experiments were performed by author Xingxing. Authors Mesbah and Xingxing undertook the
statistical analysis of the data and wrote the first draft of the manuscript. All authors
contributed to and have approved the final version of the manuscript. Critical revision
was done by authors Schwabe and Krauss.
47
Cholinergic lesion in the anterior and posterior pedunculopontine tegmental nucleus: behaviour and neuronal activity in the cuneiform and entopeduncular nuclei
Xingxing Jin, Kerstin Schwabe, Joachim K. Krauss, Mesbah Alam
Department of Neurosurgery, Medical University Hannover, Carl-Neuberg-Str. 1, D-30625
Hannover, Germany
Abstract
Objective: Loss of cholinergic neurons in the mesencephalic locomotor region, comprising the pedunculopontine nucleus (PPN) and the cuneiform nucleus (CnF), are related
to gait disturbances in late stage Parkinson’s disease (PD). We investigate the effect of
anterior or posterior cholinergic lesions of the PPN on gait-related motor behaviour, and
on neuronal network activity of the PPN area and basal ganglia (BG) motor loop in rats.
Methods: Anterior PPN lesions, posterior PPN lesions or sham lesions were induced by
stereotaxic microinjection of the cholinergic toxin AF64-A or vehicle in male Sprague Dawley rats. First, locomotor activity (open field), postural disturbances (Rotarod) and gait
asymmetry (treatmill test) were assessed. Thereafter, single unit and oscillatory activity
were measured in the non-lesioned area of the PPN, the CnF and in the entopeduncular
nucleus (EPN), the BG output region, with microelectrodes under urethane anaesthesia.
Additionally, ECoG was recorded in the motor cortex.
Results: Injection of AF64-A into the anterior and posterior PPN decreased cholinergic
cell counts as compared to naı̈ve controls (P < 0.001). Only anterior PPN lesions decreased the front limb swing time of gait in the treadmill test, while not affecting other
gait related parameters tested. Main electrophysiological findings were that anterior PPN
lesions increased the firing activity in the CnF (P < 0.001). Further, lesions of either PPN
region decreased the coherence of alpha (8-12Hz) band between CnF and MCx, and increased the beta (12-30Hz) oscillatory synchronization between EPN and the MCx.
Conclusion: Cholinergic lesions of the PPN in rats had complex effects on oscillatory
neuronal activity of the CnF and the BG network, which may contribute to the understanding of the pathophysiology of gait disturbances in PD.
Keywords:
48
Indroduction
The late stages of Parkinson’s disease (PD) and progressive supranuclear palsy are characterized by postural instability and gait disturbances, which contribute to marked disability. These may be refractory to dopaminergic medication, but also to deep brain
stimulation (DBS) of the subthalamic nucleus (STN) or the globus pallidus internus
(Fasano et al., 2015; Kasashima and Oda, 2003; Schrader et al., 2013). Disturbed gait
has been related to degeneration of cholinergic neurons in the pedunculopontine nucleus
(PPN; Hirsch et al., 1987; Jellinger, 1988; Zweig et al., 1989), which, together with the
cuneiform nucleus (CnF), forms the mesencephalic locomotor region (MLR). Deep brain
stimulation (DBS) of the PPN has been tested for treating these symptoms, using stimulation parameters thought to stimulate the remaining neurons, however, with variable
results and with substantial controversy, where exactly the optimal site for stimulation is
located (Ferraye et al., 2010; Moro et al., 2010; Nosko et al., 2015; Plaha and Gill, 2005;
Schrader et al., 2013).
In primates, the PPN consists of a compact part (PPNc) with a higher density of cholinergic neurons, and a pars dissipata (PPNd), with glutamatergic, GABAergic, and cholinergic
neurons (Inglis and Winn, 1995; Martinez-Gonzalez et al., 2011; Ros et al., 2010). The
PPN interacts with the basal ganglia (BG) motor loop, but also relays information to the
brainstem and spinal cord relevant for postural muscle tone (Alam et al., 2011). Lesions of
cholinergic PPN neurons in monkeys induce akinesia, gait and postural changes resistant
to dopaminergic agents, which closely parallels findings in patients with late stage PD
(Karachi et al., 2010; Kojima et al., 1997; Matsumura and Kojima, 2001). In rodents,
however, the pedunculopontine tegmental nucleus (PPTg, the equivalent to the PPN in
primates) has primarily been related to non-motor behaviour, such as cognition and sensorimotor gating (see Winn, 2006 for review). Bilateral excitotoxic lesions of the PPTg
did not affect spontaneous locomotor activity stability, speed, stride or coordination (Inglis et al., 1994; Olmstead and Franklin, 1994; Winn, 2006). Nevertheless, more recently,
Alderson et al., (2008) have observed a reduction in locomotion after lesioning a restricted
portion of the anterior but not of the posterior part of the PPTg. These results are consistent with the hypothesis that in rats the anterior PPTg (aPPTg), which is thought to
resemble the PPNd, has functions and anatomical connections related to motor processes
(Honda and Semba, 1995; Rye et al., 1987), while the posterior PPTg (pPPTg), which is
regarded the PPNc because of a high density of cholinergic neurons, has stronger anatomical connections to associative-limbic structures (Mena-Segovia et al., 2008; Manaye et al.,
1999; Olszewski and Baxter, 1954). Whether the effects of the aPPTg lesions are achieved
through the effects on cholinergic neurons on descending motor projections, or through
49
effects on the basal ganglia motor loop, possibly via the CnF as suggested by Alam et al.,
(2012), has not been investigated.
Ethylcholine mustard aziridinium ion (AF64-A) is an irreversible inhibitor of the choline
uptake system and choline-related enzymes (Fisher and Hanin, 1980) and acts as a potent
and selective cholinergic neurotoxin for the PPN in a dose- and site-dependent manner
(Hanin, 1996; Kása and Hanin, 1985; Lança et al., 2000). We here tested the effects of
specific AF64-A-induced cholinergic lesions of either the aPPTg or the pPPTg on rodent
gait-related behaviour and extracellular neuronal activity of the unlesioned part of the
PPTg, as well as on the CnF and the entopeduncular nucleus (EPN), which is regarded
the rat equivalent of the human GPi and the output nucleus of the basal ganglia motor
loop.
Materials and Methods
Animals
Male Sprague-Dawley rats (N = 20; weighing 220-230 g; Charles River Laboratories,
Germany) were randomly divided into three groups, including naı̈ve controls (N = 8),
aPPTg lesion (N = 6) and pPPTg lesion (N = 6) animals. Rats were housed in groups
of four in standard Macrolon Type IV cages (Techniplast, Hohenpeissenberg, Germany)
under a 14 h/10 h light-dark cycle with light on at 07:00 h at a room temperature of
22 ± 2◦ C, and with food and water available at all times. All animal procedures were
in accordance with the European Council Directive of November 24, 1986 (86/609/EEC)
and were approved by the local animal ethic committee. All efforts were made to minimize
the number of animals used and their suffering.
PPTg cholinergic lesion
The cholinergic neurotoxin AF64-A solution was prepared from acetylethylcholine mustard HCl (Sigma, Germany) as described previously by Fisher et al. (1982). The solution
was freshly prepared in 1mg/mL aqueous solution, adjusted with 10N NaOH to a pH of
11.5 − 11.7 and kept in room temperature for 30 min with continuous stirring. Thereafter, the pH was adjusted to 7.4 using 6N HCl and NaOH. The AF64-A solution was
further diluted with saline (0.9% NaCl) for a final concentration of 13.5ng/µL and stored
at 4◦ C until microinjection within 6h. In preliminary studies this dosage has been found
to induce selective lesions of cholinergic neurons within the aPPTg or pPPTg without
damaging the tissue.
The surgical procedure was adapted according to a protocol for PPTg lesion in rats
50
(Rodriguez et al., 1998). Animals were anesthetized with 3.6% chloral hydrate solution
(1mL/100g body weight, i.p., Sigma, Germany) and placed in a rodent stereotaxic frame
(Stoelting, Wood Dale, Illinois, USA). After making a small incision to expose the scalp,
a bone scraper was used to clean the skull above bregma and lambda and a small craniotomy was made with a dental drill above the PPTg area of each hemisphere. The aPPTg
(AP: −7.3mm posterior to bregma; L: ±1.8mm and V: −7.3mm; according to the atlas
of Paxinos and Watson, 1997) and the pPPTg (AP: −8.3mm posterior to bregma; L:
±1.8mm and V: −7.0mm) were lesioned bilaterally by microinjection of a total volume of
0.5µl of AF64-A (6.75ng) that was injected with a rate of 0.1µl/min into each PPTg region. Controls received microinjection of vehicle only. The tooth bar was set at −3.3mm
for all coordinates.
Behaviour
Three weeks after surgery behavioural testing started. All tests were performed after
30-60 min habituation to the testing room during the day light cycle under artificial light
with a fixed intensity and acoustic exposure to a masking noise (playing radio).
Activity box test: To assess spontaneous locomotion, the animals were placed in a black
plastic open field (60 × 60 × 30cm3 ). A video of the animals was recorded for 10 minutes
by a camera installed above the box and the total distance travelled was automatically
calculated by a video tracking system using the same settings for all rats (TopScan 1.0,
Clever Sys. Inc., Reston, VA, USA).
Rotarod testing: To assess the motor coordination of the animals, an accelerating Rotarod
(IITC Life Science, Woodland Hills, CA) was used. The Rotarod consisted of a suspended
rod, accelerating for 60 seconds from 1 round per minute (RPM) to 12 RPM, thereafter
continuing at that speed for another 60 seconds. A trial was stopped when the rat fell
off the Rotarod or after 120 seconds. Three consecutive trials were performed with a
rest period of 5 min in between, and the mean duration of rat staying on the Rotarod
was calculated. Prior to surgery, the rats were trained for five days to achieve a stable
performance.
Automated treadmill gait test: Treadmill gait assessment was performed with the TreadScan system (CleverSys, Inc., Reston, VA, USA). Rats were placed on a motorized treadmill within a plexiglass compartment (∼ 25cm long and ∼ 5cm wide). Digital video
images were acquired at a rate of 100 frames per second by a camera mounted underneath the treadmill to visualize paw contacts on the treadmill belt. The treadmill was
set at a fixed speed of 17 cm/sec at which most animals were able to move continuously.
The videos were analysed by the TreadScan software, which automatically identifies the
paw footprints. Manual adjustments of the contrast of the images were made, if neces51
sary, to properly distinguish the footprints from the background. The images were then
automatically processed by the software to calculate values for gait parameters, including
stance time and swing time for the front and hind paw. The stance phase is the part of
the gait cycle that begins as soon as the paw contacts the ground and terminates when
the paw starts its forward movement. The swing phase is defined as the period following
the stance phase, when the foot is off the ground.
Electrophysiological recordings
After behavioural testing, electrophysiological recordings were taken in the aPPTg in rats
with pPPTg lesions, and in the pPPTg in rats with aPPTg lesions, i.e., in the non-lesioned
PPTg area. In controls, recordings were taken both in aPPTg and pPPTg. In all groups,
additional recordings were taken in the CnF and the EPN. For recording, the rats were
anesthetized with urethane (1.4g/kg, i.p.; ethyl carbamate, Sigma, St. Louis, MO) and
placed in a stereotaxic frame. The body temperature was maintained at 37 ± 0.5◦ C by
a heating device (FHC, Bowdoinham, ME). Surface ECG was recorded to monitor and
ensure constant physiological conditions and wellbeing during recording. The ECoG electrodes were placed in the axilla and pelvic region (Lead II, CED1902 isolated amplifier,
Cambridge Electronic Design, Cambridge, UK).
Small craniotomies were made over the target coordinates for the aPPTg and/or the
pPPTg, the CnF and the EPN in both hemispheres. A single microelectrode for extracellular recordings (quartz coated pulled with a ground platinum-tungsten alloy core
(95%-5%), diameter 80µm, impedance 1−2M Ω) was connected to the Mini Matrix 2 channel version drives headstage (Thomas Recording, Germany) and stereotaxically guided
through the skull burr holes to the target coordinates in the aPPTg (A: −7.3 to −7.8mm;
L: ±1.8mm; V: −7.0 to −7.8mm), the pPPTg (A: −8.3 to −8.5mm; L: ±2.0mm; V:
−6.8 to −7.2mm), the CnF (A: −8.3 to −8.5mm; L: ±2.0mm; V: −6.0 to −6.4mm) and
the EPN (A: −2.3 to −2.8mm; L: ±2.6 to ±3.0mm; V: −7.5 to −8.0mm). Regions were
recorded in randomized order. The recorded signals were split into two signals, which
allowed single unit (SU) activity and local field potentials (LFPs) to be analysed from
the same electrode: (1) SU activity was recorded with a 0.5 to 5 kHz band pass filter
at a sampling rate of 25 kHz and ×9500 to ×19, 000 amplification, and (2) LFPs were
recorded with a 0.5 to 100 Hz band pass filter at a sampling rate of 1 kHz, as described by
Alam et al. (2012). Additionally, an electrocorticogram (ECoG) was recorded via a 1 mm
diameter jeweler screw, which was positioned on the dura mater above the primary motor
cortex (MCx) ipsilateral to the recording site (AP: +2.7mm; L: ±2.5mm). The ECoG
reference and ground was positioned in a skin pocket over the neck muscle. All signals
were digitized with a CED 1401 (Cambridge Electronic Design, UK) and recorded for 10
52
to 12 min after signal stabilization with Spike2 analysis software (Cambridge Electronic
Design, Cambridge, UK). Recordings of 300 s were analysed and sorted on the base of a
3:1 signal to noise ratio. After termination of the experiment, electrical lesions were made
at the proximal and the distal site of the trajectory to allow histological verification of
the recording site (10µA for 10 seconds; both negative and positive polarities; Fig. 8.1).
8.1 for examples).
Data analysis
Action potentials arising from a single neuron were discriminated by the template-matching
function of the spike-sorting software (Spike2; Cambridge Electronic Design, Cambridge,
UK). Only well isolated single unit activities were included in the analysis, which was determined by the homogeneity of spike waveforms, the separation of the projections of spike
waveforms onto principal components during spike sorting, and clear refractory periods in
inter-spike interval (ISI) histograms. All analyses were performed using custom-written
Matlab (Mathworks, Natick, MA) functions unless otherwise noted.
Firing rates were calculated by taking the reciprocal value of the mean ISI for the whole
300 seconds of recording. The coefficient of variation (CV) of the ISI sequence is a measure of spike train irregularity defined as the standard deviation divided by the mean
ISI. An exponential distribution has a CV of 1, which describes more irregular discharge
patterns; whereas distributions derived from more regular ISIs have CV values below 1.
Firing patterns of all spike trains were classified into one of the 3 basic categories (regular,
irregular, and bursty firing) using the method described by Labarre et al. (2008). The
discharge density histogram d(λ) of each spike train was compared to reference density
functions (PDF) px (λ). For the reference functions (1) a Gaussian PDF with mean 1 and
variance 0.5, (2) a Poisson PDF with mean 1 and (3) a Poisson PDF with mean 0.8 were
used to represent regular, irregular and bursting activity, respectively (Lourens et al.,
2013).
The bursting characteristics of neuronal activities were analysed using traditional maximum interval method with NeuroExplorer version 4 (Nex Technologies). A burst was
defined as at least two spikes with an inter-spike interval equal to or less than 80 ms, with
burst termination defined as a subsequent inter-spike interval more than 160 ms (Grace
and Bunney, 1984). Additionally, the minimum duration of a burst was set at 10 ms,
the minimum number of spikes in a burst was n=3, and the minimum interval between
bursts was set at 300 ms. Following the burst detection, a set of burst parameters were
determined, including the number of bursts per minute, the percentage of spikes in bursts
and the mean firing frequency in bursts.
Representative epochs of 300 seconds without major artefacts were used for the fre53
Figure 8.1: Schematic drawings of the rat coronal brain slices (adapted from Paxinos
and Watson, 1986) of the aPPTg (a), the pPPTg and CnF region (b), and the EPN
(c). Photographs show the histological verification of the recording electrode by electric
coagulation (black arrows) in hematoxylin-eosin (HE) stained coronal sections in the anterior and posterior part of the PPTg, the CnF and the EPN with ×10 and ×50 times
magnification.
54
quency domain signal processing for LFPs and ECoGs. After eliminating the 50 Hz
power line artefacts using a finite impulse response (FIR) notch filter, data was normalized by subtracting the mean amplitude and dividing the standard deviation, which
allowed the frequency domain signals to be pooled and compared with less influences
from individual/non-specific differences. Frequency domain transformation was applied
by computing the Fast Fourier Transform (FFT) spectra from blocks of 1024 samples,
which resulted in a frequency resolution of 0.9766 Hz. Hanning’s window function was
applied to overcome spectral leakage phenomena. Functional relationships between the
MCx and LFPs were estimated by means of coherence using traditional methods described
by Halliday et al. (1995). Coherence of oscillatory signals provides a frequency-domain
measure of the linear phase and amplitude relationships between signals. It is a finite
measure of values from 0 to 1, where 0 indicates that there is no linear association and 1
indicates a perfect linear association.
Choline acetyltransferase staining and quantification
After electrophysiological recordings, rats were transcardially perfused with 150mL 0.1M
phosphate-buffered saline (PBS), followed by 200mL 4% paraformaldehyde (PFA) in PBS.
Following perfusion, the brains were collected and post fixed with PFA overnight at room
temperature, thereafter transferred to a 30% sucrose solution in PBS and stored at 4◦ C before cutting.
The brains were cut on a freezing microtome in the coronal plane with a section width
of 30µm. Every second section that contained the PPTg or the CnF was processed for
free-floating choline-acetyltransferase immunohistochemistry (ChAT; primary goat polyclonal anti-ChAT antibody, 1:200 dilution; Millipore). The sections were incubated with
secondary antibody solution (1:200 dilution) with biotinylated IgG donkey anti-goat (Millipore). Staining was performed using the ABC-Standard-Kit (1:1,000; 1µL Avidin-H
+ 1µL biotinyl-peroxidase in 1 mL PBS; ABC-Kit, Vector Laboratories, Burlingame,
CA) and 3.3’-diaminobenzidin (DakoCytomation, Glostrup, Denmark). Sections were
mounted on gelatine coated microscope slides, dehydrated in ascending concentrations of
alcohol, cleared in xylene and cover slipped using Vecta Mount (Vector Laboratories, Inc.,
Burlingame, CA).
Every first section and additional coronal sections of a width of 10µm that contained the
EPN were mounted on glass slides (SuperFrost, Thermo Scientific, Germany), and stained
with hematoxylin-eosin (HE), dehydrated in alcohol, and coverslipped with Vitro Clud
(Langenbrinck, Emmendingen, Germany), in order to visualize the electrolytic coagulations along the microelectrode recording tracks.
To evaluate the cholinergic lesion effects of the aPPTg and pPPTg, ChAT-positive (ChAT+)
55
cells were bilaterally counted in four sections per animal at 400 times magnification. Two
sections were located approximately between −7.2 to −7.4mm posterior to the bregma,
which corresponds to the aPPTg, whereas the other two were located between −8.2 to
8.4mm, which corresponds to the pPPTg. The transition from the superior colliculus to
the inferior colliculus, which was located approximately at −8.0mm posterior to bregma,
was used as an anatomical landmark to distinguish between aPPTg and pPPTg. Cell
counts were then averaged for each PPTg subregion for each animal. The mean counts of
ChAT+ cells in the aPPTg and pPPTg in naı̈ve controls were taken as 100%. Percentages
of the averaged counts in the aPPTg and pPPTg were calculated over the mean counts
in naı̈ve controls.
Statistics
The number of ChAT positive neurons was compared between groups by t test. Behavioural data were analysed using one way analysis of variances (ANOVA) followed by
Tukey post hoc comparisons (Sigma Stat 3.5, Software, Inc., USA). For the SU activity
data nonparametric Kruskal-Wallis ANOVA tests were used due to the significant deviation from normality and a lack of homogeneous variances that existed in most extracellular
SU spike data. Statistically significant differences between groups were assessed by using post hoc pairwise multiple comparisons with Dunn’s method if the Kruskal-Wallis
ANOVA showed significant differences (P < 0.05). Distributions of the firing patterns
were compared using chi-square test. Further, z-test was applied between percentages of
patterns by adjusting Bonferroni correction to determine significant changes of P values
for individual (regular, irregular and burst) observations. Statistics were performed with
Sigma Stat Software. For all tests, results were considered statistically significant with a
P-value < 0.05. All results are shown as mean ± standard error unless noted otherwise.
Results
One animal from the aPPTg lesion group died during urethane injection prior to the
electrophysiological recording. Therefore in the electrophysiology results only five animals
were included in the aPPTg lesion group.
In the aPPTg lesion group and the naı̈ve control group a total of 211 SU activities were
recorded in the pPPTg region; similarly in the pPPTg lesion group and the naı̈ve control
group a total of 143 SU activities were recorded in the aPPTg area. In all three groups
of animals (naı̈ve controls, rats with aPPTg lesions and rats with pPPTg lesions), a total
of 207 and 154 single neuronal activities were recorded in the EPN and in the CnF,
respectively. The average counts of neurons recorded per animal were 15.07 ± 1.46 in the
56
aPPTg, 11.00 ± 1.72 in the pPPTg, 10.89 ± 1.53 in the EPN, and 8.11 ± 1.31 in the CnF.
All recording sites marked with electrolytic lesions were verified in the different regions
(see Fig. 8.1 for examples).
Lesion effect
The mean percentage of surviving neurons after AF64-A injection in the aPPTg and
pPPTg area were 38.83 ± 11.76% and 38.12 ± 9.34%, which significantly differed from the
cell-count of naı̈ve controls. Injection of AF64-A into the aPPTg only marginally affected
the ChAT-positive cell count in the pPPTg (88.18 ± 8.58%). Likewise, injection of AF64A into the pPPTg only marginally affected the number of ChAT-positive neurons in the
aPPTg (73.18 ± 5.94%; see Fig. 8.2).
Motor impairment
Statistical analysis with ANOVA showed that neither aPPTg lesions nor pPPTg lesions
changed measures in the Rotarod test or spontaneous locomotion in the activity box (both
F-values > 0.256; both P-values > 0.7; Fig. 8.3 A and B).
In the treadmill gait test, the front limb swing time was reduced in aPPTg-lesioned rats
(P = 0.041 after significant ANOVA: F2,18 = 3.816; P = 0.04), while the swing time
of rats with pPPTg did not differ from naı̈ve controls and rats with aPPTg lesions. No
differences in the front limb stance time were observed (Fig. 8.3 C and D). All other
measures did not differ between groups.
Neuronal firing rate and CV
Neither the aPPTg nor the pPPTg lesions altered the firing rate in the non-lesioned part
of the PPTg (Fig. 8.4 A and B), but the CV in the aPPTg area was significantly enhanced
after pPPTg lesions as compared to that of naı̈ve controls (0.68 ± 0.06 vs. 0.49 ± 0.05;
P < 0.05; Table. 8.1).
After aPPTg lesions the firing rate was significantly enhanced in the CnF compared to
controls (P < 0.05 after significant ANOVA: χ2 = 9.043; P = 0.011), while the CnF firing
rate did not differ between naı̈ve controls and rats with aPPTg lesions (Fig. 8.4 C). In
the EPN, the firing rate did not differ between groups (see Fig. 8.4 D). Also, the CV of
the CnF and the EPN did not differ between groups.
57
Figure 8.2: Histological micrographs showing the choline acetyl transferase immunepositive (ChAT+) neurons in the naı̈ve (A) and lesioned (B) anterior PPTg and the
naı̈ve (C) and lesioned (D) posterior PPTg. Schematic diagrams illustrating the lesioned
areas in the aPPTg and pPPTg (E; adapted from Paxinos and Watson, 1986). The bar
histograms show the mean percentage (±SEM ) of ChAT+ neurons in the aPPTg and
the pPPTg after aPPTg lesions (F) and after pPPTg lesion (G). Significant differences
between the aPPTg and the pPPTg are indicated by asterisk (∗; P < 0.05; t test).
58
Figure 8.3: Time spent on the rotarod (A), total distance travelled in the activity box (B),
average swing time (C) and stance time (D) of the front limb measured using treadmill.
Bars show the mean ± SEM of naı̈ve, aPPTg lesioned and pPPTG lesioned rats. A
significant difference to naı̈ve controls is indicated by asterisks (∗; P < 0.05; one-way
ANOVA and post hoc Tukey test).
M edian
[25% − 75%]
naı̈ve
n
EPN
65
CnF
49
aPPTg
89
pPPTg
59
CV
0.41
[0.33 − 0.56]
1.02
[0.78 − 1.54]
0.40
[0.31 − 0.55]
0.77
[0.40 − 1.12]
aPPTg lesion
n
65
52
CV
0.36
[0.26 − 0.65]
1.20
[0.65 − 1.73]
pPPTg lesion
n
77
53
-
-
122
84
0.59
[0.42 − 1.03]
-
CV
0.45
[0.34 − 0.63]
1.04
[0.701.39]
0.49∗
[0.34 − 0.73]
-
Table 8.1: Coefficient variation of ISIs (CV; all values presented as median and its 25th
and 75th percentile). A comparison with naı̈ve control has been indicated by (∗) asterisks
(P < 0.05; one-way ANOVA and post hoc Tukey test).
59
Figure 8.4: The neuronal firing rates of the aPPTg (A) and pPPTg (B), the CnF (C) and
EPN (D). Bars show the mean ± SEM of naı̈ve, aPPTg lesioned and pPPTG lesioned
rats. A significant difference to naı̈ve controls is indicated by asterisks (∗; P < 0.05;
one-way ANOVA and post hoc Tukey test).
60
Burst parameters
Neither aPPTg nor pPPTg lesions affected the burst activity in the non-lesioned part of
the PPTg. In the CnF, statistical analysis with ANOVA revealed significant differences
in the number of bursts per minute, the percentage of spikes in burst and the mean frequency of bursts (all χ2 -values > 12; all p-values < 0.002). Post hoc testing showed that
the pPPTg lesions decreased the burst per minute compared to both naı̈ve controls and
the aPPTg lesioned group (P < 0.05). The aPPTg lesions did not alter the number of
bursts per minute in the CnF nucleus but increased the percentage of spikes in burst
parameter compared to both naı̈ve control and the pPPTg lesioned group (P < 0.05). No
significant changes were observed after pPPTg lesions in the CnF nucleus.
Additionally, aPPTg lesions increased the mean frequency of bursts compared to both
naı̈ve controls and the pPPTg lesioned group in the CnF nucleus (P < 0.05; Table.
8.2). All other parameters did not differ between groups. In the EPN, statistical analysis
showed that only pPPTg lesion significantly increased the number of bursts per minute
compared to naı̈ve controls (P < 0.05 after significant ANOVA: χ2 = 6.678; P = 0.035)
but no differences were determined for the percentage of spikes in bursts and mean frequency of bursts.
Firing patterns
Chi-square test showed that pPPTg lesions significantly affected the neuronal firing pattern distribution in the aPPTg (χ2 = 7.413, df = 2; P = 0.025), while aPPTg lesions had
no effect on the pPPTg area (χ2 = 1.691, df = 2; P = 0.429). Post hoc test showed that
after pPPTg lesions the irregular firing patterns were enhanced in the aPPTg (31.15% vs.
15.73%; P = 0.02) and the regular firing patterns were decreased (66.39% vs. 83.15%;
P = 0.01) as compared with the naı̈ve controls, but no differences in the burst patterns
were detected (Fig. 8.5 A and B).
Chi-square test also showed that the percentage of bursts in the CnF nucleus were
higher after aPPTg lesions than after pPPTg lesions (38.5% and 15.1%; P < 0.01; after
χ2 = 8.821, df = 4; P = 0.06; Fig. 8.5 C). PPTg lesions had no effect on the neuronal
firing pattern distribution in the EPN (χ2 = 3.198, df = 4; P = 0.525; Fig. 8.5 D).
Coherence of LFPs and MCx-ECoG
Analysis of the coherence of PPTg LFPs and MCx-ECoG in the alpha (8-12 Hz) and in
the beta (12-30 Hz) band showed that both aPPTg and pPPTg lesions did not affect the
non-lesioned PPTg-LFP and MCx coherence.
61
naı̈ve
%of spikes
inburst
f requency
inburst
n
Burst
permin
aPPTg lesion
%of spikes
inburst
f requency
inburst
n
Burst
permin
pPPTg lesion
%of spikes
inburst
f requency
inburst
6.60
95.99
13.16
12.60
93.43
11.97
17.00∗
82.95
12.94
25
27
[2.29 − 12.00] [32.69 − 99.42] [12.02 − 16.81]
[3.00 − 24.10] [42.49 − 98.07] [11.34 − 15.97]
[7.20 − 30.20] [56.89 − 97.60] [11.60 − 15.45]
Burst
permin
25
19.40
79.60
15.31
22.40
93.42 ∗ #
22.26 ∗ #
7.60 ∗ #
70.08
14.82
41
44
[9.80 − 30.60] [55.47 − 90.37] [13.36 − 17.98]
[9.60 − 46.70] [82.95 − 98.51] [16.34 − 30.68]
[5.70 − 17.95] [35.51 − 98.40] [12.21 − 21.92]
M edian
[25% − 75%] n
EPN
39
-
-
-
7.80
89.75
12.21
[3.40 − 15.40] [47.96 − 97.59] [11.09 − 13.68]
CnF
86
9.40
85.02
11.25
[2.87 − 17.80] [53.02 − 94.78] [10.78 − 13.58]
-
53
9.20
68.12
12.44
12.20
64.05
13.06
65
[4.02 − 15.37] [26.20 − 96.90] [11.39 − 15.71]
[4.90 − 20.40] [34.59 − 91.60] [11.35 − 18.14]
-
aPPTg
49
-
pPPTg
Table 8.2: Burst parameters including the bursts per minute, the percentage of spikes in bursts and the mean firing frequency in bursts
(all values presented as median and its 25th and 75th percentile). A comparison with naı̈ve control has been indicated by (∗) asterisks,
differences between aPPTg lesion and pPPTg lesion with (#; P < 0.05; one-way ANOVA and post hoc Tukey test).
62
Figure 8.5: Distribution of cellular firing patterns (burst, irregular and regular) in the
aPPTg (A), the pPPTg (B), the CnF (C) and the EPN (D). Significant differences to
naı̈ve controls are indicated by asterisks (∗; P < 0.05; Chi-square test with Bonferroni
adjustment for the distributions of discharge patterns).
63
In the CnF, one-way ANOVA showed significant effects of PPTg lesion on the LFPECoG coherence of the alpha (F2,146 = 12.339; P < 0.001; Fig. 8.6) and beta frequency
bands (F2,146 = 4.038; P = 0.020). CnF-LFP and MCx coherence in the alpha band
was significantly decreased after aPPTg lesions (P < 0.001), but to a lesser extent after
pPPTg lesions (P < 0.05). The difference between aPPTg lesion and pPPTg lesions
was significant (P < 0.05). The beta band coherence were also decreased after pPPTg
lesions (P < 0.05), however, tendency for a decrease was also found after aPPTg lesions
(P = 0.058).
In the EPN, one-way ANOVA showed significant effects of PPTg lesion on the LFP-ECoG
coherence of the beta frequency band (F2,200 = 5.012; P < 0.01; Fig. 8.6) but not the
alpha frequency band (F2,200 = 0.289; P = 0.749). Post hoc test showed a significantly
enhanced beta coherence after aPPTg lesions (P < 0.01), however, a tendency for an
increase was also observed after pPPTg lesion (P = 0.074).
Discussion
Cholinergic lesions in the aPPTg marginally affected gait, i.e., reduced the front swing
duration of the gait cycle, while all other gait related measures were not affected by lesions of either PPTg subregion. Our results are in line with the report of Alderson et al.,
(2008), who found a small reduction in locomotion after lesioning the aPPTg but not the
pPPTg. Nevertheless, most previous studies have not seen changes in locomotion after
full or partial lesions of the PPTg in rodents (Winn, 2006), while in primates PPN lesions
lead to gait dysfunction and akinesia (Kojima et al., 1997; Aziz et al., 1998; Matsumura
and Kojima, 2001; Karachi et al., 2010). With that regard, species dependent adaptive
compensations of gait related problems must be considered, especially when comparing
the quadruped locomotion in rodents with the biped locomotion in non-humane primates
and humans. Interesting with that regard is that gait deficits after cholinergic PPN lesions
were only found in adult monkeys (Aziz et al., 1998), while in young monkeys balance
deficits and falls were only observed with the combination of nigrostriatal dopaminergic
and PPN lesions (Karachi et al., 2010). Our finding about the effect of aPPTg lesions on
gait is supported by the anatomic connection of this region to the basal ganglia. In rats
the aPPTg projects to the SNc, while the pPPTg projects to the ventral tegmental area
(Oakman et al., 1995; Alderson et al., 2008). Further, cholinergic neurons of the aPPTg
preferentially innervates the motor associated dorsolateral striatum, while the pPPTg innervates the associative-limbic medial striatum and the nucleus accumbens shell (Alderson
et al., 2008; Winn, 2008).
Because of the strong anatomical input to different BG regions we hypothesized that
64
Figure 8.6: Mean coherence of LFPs with the MCx-ECoG in different nuclei for the alpha
(8-12 Hz) and beta (12-30 Hz) frequency range. Bars show the mean ± SEM of naı̈ve,
aPPTg lesioned and pPPTg lesioned rats. A significant difference to naı̈ve controls is
indicated by asterisks (∗), a differences between aPPTg lesion and pPPTg lesion with (#;
P < 0.05; one-way ANOVA and post hoc Tukey test).
65
cholinergic PPTg lesions would affect neuronal activity in the EPN, the output region of
the BG. With that regard, both aPPTg and pPPTg cholinergic lesions lead to enhanced
coherence of EPN beta band activity with MCx. Beta oscillations are enhanced in PD
and have been associated with bradykinesia and rigidity since treatment with dopaminergic medication reduces beta band activity in parallel to improvement of symptoms in PD
(Brown, 2003; Weinberger et al., 2006; Chen et al., 2007; Kühn et al., 2008; George et al.,
2013; Connolly et al., 2015). Our findings implicate that the loss of cholinergic neurons in
the PPN area may contribute to increased beta oscillatory activity, which has been observed after loss of nigrostriatal dopamine. Interesting with that regard is that bilateral
lesions of the PPN in normal monkeys induce akinesia and bradykinesia that look like
PD (Aziz et al., 1998). Also, unilateral excitotoxic lesions of the PPN with kainic acid
injections induced mild levels of flexed posture and hypokinesia contralateral to the lesion
(Kojima et al., 1997).
Nevertheless, except enhanced burst activity after pPPTg lesions, single unit activity of
the EPN was not altered after cholinergic lesions of either PPTg subregion. It has been reported that rodents have less pallidal projection neurons to the PPN than monkeys (Alam
et al., 2011), and some authors have even suggested that the EPN is not linked with the
PPN, but rather with a brainstem region located just medial to it, which they referred to
as the ‘midbrain extrapyramidal area’ (Rye et al., 1987; Lee et al., 1988; Steininger et al.,
1992). Still, electrical stimulation of the rat PPTg increases the firing rates of neurons in
the EPN (Scarnati et al., 1988), suggesting a strong functional connection to this nucleus.
The PPN not only interacts with the BG motor loop, but also relays information to neural
structures located in more caudal areas of the brainstem, which play a role in postural
muscle tone (Alam et al., 2013). Cholinergic lesions of the aPPTg enhanced the firing
rate and burst parameters in the CnF, whereas after pPPTg lesions only the number
of bursts was reduced in the CnF. Since the majority of CnF neurons are GABAergic,
enhanced CnF activity after aPPTg lesions may provide excess inhibitory output to the
muscle tone via descending projections to the lower brainstem and spinal cord, resulting
in subtle alteration in gait such as seen in our study (Kerr, 1975; Menetrey et al., 1982;
Bjorkeland and Boivie, 1984). Interestingly, functional MRI studies during fast imagined
gait in healthy humans showed activation of the region comprising the PPN and the CnF
(Karachi et al., 2010). With that regard, boundaries between the posterior PPN and the
CnF are indistinct — potentially confounding precise determination of the source of neuronal recordings and of the structures responsible for clinical effects. Interestingly, PPN
electrodes in PD patients were most effective when located slightly posterior to the PPNc,
which corresponds to the ventral part of the CnF (Ferraye et al., 2010).
Cholinergic lesions of both PPTg subregions decreased the alpha (8-12Hz) oscillatory co-
66
herence of the CnF and MCx-ECoG compared to naı̈ve controls, an effect that was even
stronger after aPPN lesion. Further, beta synchronization in the CnF and motor cortex
was decreased after cholinergic lesions of either PPTg region. A clinical study in PD
patients has demonstrated a correlation between alpha oscillations in the PPN and gait
performance, which was particularly strong in the posterior PPN region, while beta oscillatory activity in the PPN area did not correlate with gait measures (Thevathasan et al.,
2012). Gait freezing was associated with attenuation of alpha activity, whilst increases
in PPN alpha power correlated with improved gait. Further, treatment with levodopa
strongly enhanced alpha oscillatory activity in the PPN, suggesting that attenuated alpha activity could be pathologically associated with gait in PD (Androulidakis et al.,
2008a; Androulidakis et al., 2008b). In their manuscript, the authors already raised the
question, whether their findings would be specific for the posterior PPN, but may rather
relate to the CnF, since the boundaries between the posterior PPN and the CnF are indistinct. Nevertheless, the authors discarded this thought, since in PD loss of cholinergic
neurons is refined to the PPN, whereas no cellular degeneration of neurons has been found
in the CnF. However, in the present study we showed that aPPTg and pPPTg cholinergic lesions led to reduced alpha power in the CnF, either by direct input or by indirect
anatomical connections via the BG.
Oscillatory activity in the alpha frequency band has been shown to correlate with performance of cognitive tasks by active suppression of task irrelevant processes (Jensen and
Mazaheri, 2010; Thevathasan et al., 2012). With that regard, in PD attentional deficits
have been described, together with impaired automaticity of movement so that processing
demands are higher in these patients (Wu and Hallett, 2005; Wu and Hallett, 2008). In
line with this, PD patients with gait freezing seem to have even more attentional deficits
than those without freezing of gait (Amboni et al., 2008; Yogev-Seligmann et al., 2008).
Further, dual tasking, which is thought to ‘distract’ attention away from gait, can worsen
gait freezing (Giladi and Hausdorff, 2006).
Within the PPTg, only cholinergic lesions in the pPPTg lesions led to enhanced irregular
firing in the aPPTg, while lesions of the aPPTg had no effect. Further, the coherence of
PPTg LFPs and MCx-ECoG oscillatory activity was not affected by cholinergic lesions of
either region. The PPTg is formed by a cluster of intermingled cholinergic, glutamatergic
and GABAergic neurons. Little is known, however, about its internal connections, but
our finding may indicate that there is no strong interaction between subregion. One explanation for the enhanced irregular firing in the aPPTg may be that lesions in the pPPTg
led to more than 20% reduction of aPPTg cholinergic neurons, while aPPTg lesions only
marginally affected cholinergic neurons in the pPPTg (about 10%).
67
Conclusion
The pathophysiological mechanisms underlying gait disturbances after loss of cholinergic
neurons in the PPTg still need further clarification. Using a rat model we showed that
enhanced firing activity in the CnF after loss of cholinergic neurons in the aPPTg may
provide excess inhibitory output to the muscle tone via descending projections to the lower
brainstem and spinal cord. Additionally, reduced oscillatory alpha band activity in the
CnF after cholinergic lesions of either PPTg subregion may contribute to gait disturbances
via disturbed attention. Further, enhanced beta activity in the output nucleus EPN
may contribute to altered gait behaviour. Together, these findings will contribute to the
understanding of how the PPTg affects BG and MLR function that may be relevant for
postural and gait abnormalities.
68
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9
Discussion
Neuronal activities of the entopeduncular nucleus in
Parkinson’s disease and dyskinesia
The major difference between 6-OHDA lesioned rats and those with LIDs in our study
existed in the oscillatory activity, where we observed a remarked shift of EPN spike-MCx
ECoG oscillatory coherence from high-beta (19-30 Hz) band in 6-OHDA lesioned rats to
low-beta (12-19 Hz) band in those with LIDs. The EPN spike-MCx ECoG phase locked
ratio, which is also a measurement of synchronization, showed similar result. Enhanced
beta oscillations were consistently found in patients with PD (Brown, 2003; Kühn et al.,
2006; Chen et al., 2007; Kühn et al., 2008; Ray et al., 2008) and animal models (Devergnas
et al., 2014; von Wrangel et al., 2015; Nambu and Tachibana, 2014) and were considered
to play an anti-kinetic role in the pathophysiology. Low- and high-beta oscillations have
been reported in the PD patients undergoing DBS implantation in the STN (Priori et al.,
2004; Marceglia et al., 2006; Marceglia et al., 2007; Lopez-Azcarate et al., 2010), and
were thought to be differently modulated during motor tasks depending on the context
(Marceglia et al., 2009). However, the role of these oscillations is still not well known.
Another difference we have observed is that levodopa injection seems to have less effect of
reversing the enhanced firing irregularity in rats with LIDs as compared to those with out
LIDs, as shown in the coefficient variation of inter-spike interval, the asymmetry index
and distribution of firing patterns. An earlier study by Boraud et al. (2001) using a PD
monkey model reported that both D1 and D2 dopamine receptor agonists induced similar
enhanced firing irregularity during the onset of LIDs. However, the use of apomorphine,
which is a mixed D1/D2 agonist, failed to show similar enhancement in the irregularity.
This indicates that the underlying neuronal mechanisms during LIDs are more complex
than simply irregular firing (Bezard et al., 2001).
In addition, we found significantly increased firing rate, higher divergence of the neuronal
firing activity and more bursting neurons in 6-OHDA lesioned rats with and without
LIDs, which is in line with many clinical and experimental findings (Tang et al., 2007;
Starr et al., 2005; Bergman et al., 1994; Filion and Tremblay, 1991; Soares et al., 2004;
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Mallet et al., 2008) and the “firing rate” model that predicts enhanced cortical inhibition
from the BG output nuclei in PD.
Nevertheless, we failed to find significant differences between 6-OHDA lesioned rats and
those with LIDs with regard to firing rate. As predicted by the “firing rate” model,
decreased neuronal activity in the BG output nuclei is responsible for the hyperactive
motor cortex that results in LIDs. Previous studies in patient with PD also showed lower
firing rate during the expression of LIDs after application of different doses of levodopa to
examine the “on” state without LIDs versus with LIDs (Lozano et al., 2000; Merello et al.,
1999; Papa et al., 1999). One possible explanation for the inconsistency could be that these
effects were more related to the doses applied, than to the underlying pathophysiology of
LIDs.
Another inconsistency comes from the theta (4-8 Hz) band oscillatory activity, which
is considered to be associated with the presence of abnormal dystonic posture or phasic
movements in LIDs (Brazhnik et al., 2012; Lemaire et al., 2012). Although our EPN spikeMCx ECoG coherence showed somewhat higher theta (4-8 Hz) band synchronization, the
theta band activity was reduced rather than increased after injection of levodopa. This
does not match results from other studies (Alonso-Frech et al., 2006; Meissner et al.,
2006). One previous study carried out in our lab also found enhanced theta oscillatory
activity in the same 6-OHDA rat model of PD during the expression of LIDs, however the
electrophysiology recordings of rat were performed in the free-moving state (Alam et al.,
2014). With that regard, this inconsistent result of theta oscillatory activity, as well as the
lack of differences in firing rate, could be due to the effect of anesthesia, which abolished
the sensory motor feedback of involuntary movements after treatment of levodopa that
may be involved in the genesis of theta spike-MCx coherence. This is also the major
limitation in our study.
In the near future, techniques of electrophysiological recording in free-moving rat will be
applied in the EPN and also other BG nuclei in order to eliminate the influence from
anesthesia. In a previous study we found that DBS of the EPN reduced the differences
of neuronal oscillatory activity in the striatum found between 6-OHDA lesioned rats with
and without LIDs (Alam et al., 2014). We therefore plan to apply experimental DBS
therapy and test its effect on single unit activity in different BG nuclei.
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Motor function and neuronal activities following cholinergic lesions of anterior and posterior pedunculopontine tegmental nucleus
Evaluation of the motor functions following PPTg lesion only marginally affected swing
duration of the front paws of the rats after cholinergic lesion in the aPPTg, whereas posture stability in the rotarod test, the spontaneous locomotor activity in the activity box,
and other gait parameters were not altered. Lack of changes on spontaneous locomotor
activity after lesion of PPTg has been reported in a recent study by MacLaren et al.,
(2014). In this study they also reported significant impairment of rat performance on
the accelerating rotarod, but not on the rotarod with fixed speed. The reason why we
did not find similar result in the rotarod test in the present study could be the different
parameters we applied for the test (smooth accelerating 0-12 RPM over 60 s v.s. 0-40
RPM over 180 s in MacLaren’s study). Alterations in the front paw swing time aPPTg
lesion are somewhat in line with the findings by Alderson et al., (2008), where a small
reduction in locomotion has been observed after lesioning the aPPTg but not the pPPTg.
These findings are at least to some extent supported by the anatomic connection of this
region. The aPPTg projects to the SNc, which is a critical part of the BG motor loop,
while the pPPTg projects to the ventral tegmental area, which is more related with limbic
system (Alderson et al., 2008; Oakman et al., 1995).
One of the main findings in our study is the significantly increased firing rate and enhanced
bursting in the single neuronal activities in the CnF following aPPTg lesion, whereas no
significant alterations on single unit activity have been observed in the major BG output
nuclei, the EPN. Studies of PD patients during an imagined gait task also showed significant associations with the subcuneiform region dorsal to the PPN (Piallat et al., 2009) and
with the CnF (Karachi et al., 2010). As one of the major components of the MLR beside
the PPTg, the CnF, which consists of mainly inhibitory GABAergic neurons, may exert
an enhanced inhibitory influence on more caudal areas of the brainstem and mediate the
suppression of muscle tone and subtle alteration in gait after aPPTg lesion (Bjorkeland
and Boivie, 1984; Oakman et al., 1995; Menetrey et al., 1982). Combined with the results
in the EPN, these may to some extent answer our question in the beginning about how
the motor effects of the aPPTg lesions are achieved.
However, all of our findings could be epiphenomenal and need further investigation, especially with regard to interspecies differences of human and rodents. For instance, little is
known regarding the interconnections between CnF and BG nuclei. Also, it is not known
how well the EPN in rodent represents the human GPi. Studies have actually shown less
77
pallidal projection neurons in the PPTg in rodents compared to monkey (Alam et al.,
2011). Some authors have even suggested that the EPN is not linked with the PPTg,
but rather with the “midbrain extrapyramidal area” located just medial to it (Rye et al.,
1987; Lee et al., 1988; Steininger et al., 1992).
We also found decreased alpha (8-12 Hz) oscillatory coherence of the CnF-LFP and MCxECoG after both aPPTg and pPPTg lesion. Alpha oscillations are thought to play an
important role in attention processes, particularly when these tend to occur over the
lower frequency band in the alpha range (Klimesch, 1999; Palva and Palva, 2007). Studies
in rodents have also reported significant impact on sustained attention as well as other
cognitive functions (Cyr et al., 2015; Ivlieva and Timofeeva, 2003; Rostron et al., 2008).
A clinical study in PD patients has demonstrated a correlation between alpha oscillations in the PPTg and gait performance. Gait freezing was associated with attenuation
of alpha activity, whilst increases in PPTg alpha power correlated with improved gait
(Thevathasan et al., 2012). Our results of the reduction of alpha synchronization in the
CnF and motor cortical region suggest that the CnF area may have a function for modulating the network internal attention, which is considered to play a critical role in the
pathophysiology in the gait disturbances in advanced PD.
In addition, enhanced beta (12-30 Hz) oscillatory coherence of EPN-LFP and MCx-ECoG
were found after both aPPTg and pPPTg lesion. As usually considered to play an antikinetic role in PD (Brown et al., 2001), this enhanced beta activity by PPTg lesion may
contribute to the akinesia independent of the nigrostriatal dopamine system.
In future, recordings in other BG nuclei such as the STN could help to further understand
the pathophysiology underlying cholinergic lesions of the PPTg. We will also combine the
6-OHDA rodent model of PD with the cholinergic lesions of the PPTg, as the traditional
6-OHDA model does not lead to lesion of the PPN cholinergic system, which is involved
in advanced PD. Further, experimental tests of DBS targeting the PPTg area will be
applied in this combined model.
Overall, our data revealed remarkable changes in the oscillatory synchronization between
BG network and the motor cortex, which may play a role for the development of LID.
And the significant alterations in the single unit activity and oscillatory activity in the
CnF following aPPTg lesion may contribute the gait disturbances in late stage of PD
via its descending projections to lower motor regions and disturbed attention. However,
further investigations are still necessary.
78
10
Summary
Experimental models of Parkinson’s disease with levodopa-induced dyskinesias and gait dysfunction: electrophysiological and behavioural measures in
rats
Xingxing Jin
Levodopa-induced dyskinesias (LIDs) and gait disturbances are two troublesome conditions frequently occurring in advanced stage of Parkinson’s disease (PD), which also
represents major therapeutic challenges in clinical practice. Although progressive nigral
dopamine denervation and pulsatile dopamine stimulation are considered to play an important role in LIDs, the pathophysiology remains unclear. Further, the pedunculopontine
nucleus (PPN), as a novel target for treatment of gait and posture instability in advanced
PD, is considered critical for the pathophysiological mechanisms leading to these symptoms. However, to date the results of therapies targeting this area have been mixed, and
the high heterogeneity of the neuronal discharge properties and neurochemical nature of
the PPN area make it difficult to understand its exact role in the pathophysiology. Electrophysiological recording will shed light on the understanding of the pathophysiology
and mechanisms underlying these conditions.
In the first project, we investigated the neuronal firing characteristics of the entopeduncular nucleus (EPN, equivalent to the internal segment of globus pallidus or GPi in human) and its coherence with the motor cortex field potentials in the 6-hydroxydopamine
(6-OHDA) rat model of PD with and without LIDs. Our results showed significantly
increased firing rate, higher divergence of the neuronal firing activity and more bursting
neurons in the EPN in PD and LIDs with little differences in between. A shift from high
(19-30 Hz) to low (12-19 Hz) beta oscillatory activity in the EPN spikes and motor cortex
(MCx) coherence were found in rats with LIDs. We conclude that altered coherence and
phase lock ratio of spike and local field potentials in the beta range may play a role in
the pathophysiology of LIDs.
In the second project, we compared the effects of cholinergic lesions in the anterior or
79
posterior part of pedunculopontine tegmental nucleus (PPTg, equivalent to the PPN in
primates) on gait-related motor behaviour and electrophysiological alterations of the EPN
and the cuneiform nucleus (CnF), another mesencephalic motor nucleus adjacent to the
PPTg in rats. Cholinergic lesions in the PPTg area showed no differences in posture on
the rotarod and spontaneous movement in open field box. Only a decreased front limb
swing time of gait in the treadmill test was found after anterior PPTg lesion. Changes of
firing rate were only found in the CnF, which was increased after anterior PPTg lesion.
The alpha (8-12 Hz) band CnF coherence with MCx field potentials were decreased after
anterior or posterior PPTg lesions, especially after lesion of anterior PPTg, whereas, beta
(12-30 Hz) band oscillatory coherence was decreased by posterior PPTg lesion. Anterior
PPTg lesion also increased the beta (12-30 Hz) oscillatory synchronization in the EPN
and the MCx field potentials coherence. Considering the results in this study, we can
conclude that cholinergic lesions of the PPTg in rats had complex effects on oscillatory
neuronal activity of the CnF and the basal ganglia (BG) network, and that the CnF may
contribute to gait disturbances after loss of cholinergic neurons of either PPN subregion
in late stage of PD.
Together, these data of the electrophysiology and behaviour alterations in rats will shed
light on the understanding of the modulation of BG motor circuitry and pathophysiology
in advanced PD.
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11
Zusammenfassung
Experimentelle Modelle für die Parkinsonerkrankung mit Levodopa-induzierten
Dyskinesien und Gangstörungen: elektrophysiologische Messungen und Verhaltensuntersuchen in der Ratte
Xingxing Jin
Levodopa-induzierte Dyskinesien (LIDs) und Gangstörungen sind im fortgeschrittenen
Stadium der Parkinsonerkrankung (PD) häufig auftretende und stark beeinträchtigende
Symptome, die große therapeutische Herausforderungen in der klinischen Praxis darstellen.
Auch wenn die fortschreitende nigrale dopaminerge Denervation und die schubweise Dopaminstimulation bei der Behandlung eine wichtige Rolle bei LIDs spielen, bleibt die Pathophysiologie unklar. Zudem wird der Nucleus pedunculopontinus (PPN), ein neues Ziel zur
Behandlung von Gang- und Haltungsinstabilität bei fortgeschrittener PD, als bedeutend
für die pathophysiologischen Mechanismen, die zu diesen Symptomen führen, angesehen. Allerdings sind bis heute die Ergebnisse der Therapien, die auf dieses Areal abzielen,
heterogen. Die große Heterogenität der neuronalen Aktivität und neurochemischen Eigenschaften der PPN Areale machen es schwierig, seine exakte Rolle in der Pathophysiologie
zu verstehen. Elektrophysiologische Aufnahmen werden das Verständnis der Pathophysiologie und die diesen Beschwerden zu Grunde liegenden Mechanismen beleuchten.
Im ersten Projekt untersuchten wir die neuronale Aktivität des entopenunkulären Nucleus (EPN, Äquivalent zum internalen Segment des Globus pallidus beim Menschen)
und sein Zusammenspiel mit Feldpotentialen des Motorkortex beim 6-Hydroxydopamin
Ratten-Modell für PD mit und ohne LIDs. Unsere Ergebnisse zeigten eine signifikant
erhöhte Feuerrate, höhere Divergenz bei der neuronalen Feueraktivität und mehr in Bursts
feuernde Neurone im EPN beim 6-OHDA Rattenmodell der PD mit und ohne LIDs mit
geringen Unterschieden zwischen den Gruppen. Eine Verschiebung von hoher (19-30 Hz)
zu geringer (12-19 Hz) beta-oszillatorischer Aktivität bei den EPN Aktionspotentialen
und deren Zusammenspiel mit dem Motorkortex (MCx) wurde bei Ratten mit LIDs gefunden. Wir schlussfolgern, dass das veränderte Zusammenspiel und das Ausmaß des
81
Phasenbezugs (phase lock ratio) von Aktionspotentialen und lokalen Feldpotentialen im
Beta-Spektrum eine Rolle bei der Pathophysiologie der LIDs spielen könnte.
Im zweiten Projekt verglichen wir die Effekte von cholinergen Läsionen im anterioren und
posterioren Anteil des pedunkulopontinen tegmentalen Nukleus (PPTg, Äquivalent zum
PPN bei Primaten) auf gangspezifische Motorik und elektrophysiologische Veränderungen
des EPN und des cuneiformen Nukleus (CnF), eines anderen mesenzephalen motorischen
Kerns, der bei Ratten dem PPTg benachbart liegt. Cholinerge Läsionen im PPTg Areal
führten zu keinen Veränderungen in der Haltung auf dem Rotarod, sowie der spontanen
Bewegung in der Open Field Box. Einzig eine verminderte Schwunggeschwindigkeit der
Vorderpfoten beim Gang wurde beim Treadmill-Test nach anteriorer PPTg-Läsion festgestellt. Veränderungen in der Feuerrate wurden nur im CnF gefunden, sie war nach
anteriorer PPTg-Läsion erhöht. Die Alpha-Band (8-12 Hz) Kohärenzen mit den MCx
Feldpotentialen waren vermindert nach anteriorer und posteriorer PPTg Läsionen, insbesondere nach Läsionen des anterioren PPTg, während die oszilatorische Kohärenz im
beta-Band durch posteriore PPTg-Läsionen vermindert war. Anteriore PPTg-Läsionen
verstärkten auch die beta-oszillatorische (12-30 Hz) Synchronisation zwischen dem EPN
und den MCx-Feldpotentialen. In Anbetracht der Ergebnisse dieser Studie können wir
schließen, dass cholinerge Läsionen im PPTg bei Ratten komplexe Effekte auf die oszillatorische neuronale Aktivität des CnF und des Basalgangliennetzwerkes (BG) hatten und
dass der CnF möglicherweise zu den Gangstörungen nach Verlust von cholinergen Neuronen in einer der Subregionen des PPN beiträgt.
Insgesamt tragen die gezeigten elektrophysiologischen Daten und Verhaltensveränderungen bei Ratten zum Verständnis der Modulation des BG Motor-Schaltkreises und der
Pathophysiologie bei fortgeschrittenem PD bei.
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Acknowledgements
Immeasurable appreciation and deepest gratitude for the help and support are extended
to the following persons who in one way or another have contributed in making this study
possible.
Prof. Dr. Joachim K. Krauss, for giving me the opportunity to perform my PhD
thesis in the Clinic of Neurosurgery, Hannover Medical School, and for his continued support, scientific advice.
Dr. Mesbah Alam, for his unwavering enthusiasm for neuroscience and electrophysiology that kept me constantly engaged with my research, and for his expert guidance
that lead me into the field of electrophysiology and helped me went through my entire
graduate education.
Prof. Dr. Kerstin Schwabe, for her patient guidance and lots of academic advice
throughout my time as her student. Without her kind help, my life as a foreign student
in Germany would be a mess in the first place.
Prof. Dr. Claudia Grothe and PD Dr. Florian Wegner, as my co-supervisors for
their kind support and scientific comments.
Dr. Christof v. Wrangel, his work help me recognize how great programming skills
and MATLAB can help in the research of electrophysiology.
Mr. Juergen Wittek, for his patient guidance in the histological staining and use of
microscope.
Mrs. Monika van Iterson and Mrs. Heike Achilles, for their technical assistance.
Mrs. Linda Armstrong, for her untiring support and patience in the English correction
of this thesis and other manuscripts.
To all students and members of the Department of Neurosurgery, for pleasurable working conditions and for giving me a wonderful life experience.
To all PhD students I worked with, for being a great group.
To my parents and my wife, without whose constant support and encouragement
nothing is possible.
And also To the China Scholarship Council, who financially supported my PhD study
in Germany.
97

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