BRAINA JOURNAL OF NEUROLOGY
Defective dentate nucleus GABA receptorsin essential tremorSarah Paris-Robidas,1,2,* Elodie Brochu,1,2,* Marion Sintes,1,2 Vincent Emond,1,2
Melanie Bousquet,1,2 Milene Vandal,1,2 Mireille Pilote,1,2 Cyntia Tremblay,1,2
Therese Di Paolo,1,2 Ali H. Rajput,3 Alex Rajput3 and Frederic Calon1,2
1 Faculty of Pharmacy, Laval University, Quebec, QC G1V 0A6, Canada
2 Centre Hospitalier de l’Universite Laval (CHUL) Research Centre, Quebec, QC G1V 4G2, Canada
3 Division of Neurology, Royal University Hospital, University of Saskatchewan, Saskatoon, SK S7N 0W8, Canada
*These authors contributed equally to this work.
Correspondence to: Dr Frederic Calon,
Centre Hospitalier de l’Universite Laval (CHUL) Research Centre,
Room T205, 2705 Laurier Blvd,
Quebec, QC G1V 4G2, Canada
E-mail: [email protected]
The development of new treatments for essential tremor, the most frequent movement disorder, is limited by a poor under-
standing of its pathophysiology and the relative paucity of clinicopathological studies. Here, we report a post-mortem decrease
in GABAA (35% reduction) and GABAB (22–31% reduction) receptors in the dentate nucleus of the cerebellum from individuals
with essential tremor, compared with controls or individuals with Parkinson’s disease, as assessed by receptor-binding auto-
radiography. Concentrations of GABAB receptors in the dentate nucleus were inversely correlated with the duration of essential
tremor symptoms (r2 = 0.44, P5 0.05), suggesting that the loss of GABAB receptors follows the progression of the disease. In
situ hybridization experiments also revealed a diminution of GABAB(1a + b) receptor messenger RNA in essential tremor (#27%).
In contrast, no significant changes of GABAA and GABAB receptors (protein and messenger RNA), GluN2B receptors, cytochrome
oxidase-1 or GABA concentrations were detected in molecular or granular layers of the cerebellar cortex. It is proposed that a
decrease in GABA receptors in the dentate nucleus results in disinhibition of cerebellar pacemaker output activity, propagating
along the cerebello-thalamo-cortical pathways to generate tremors. Correction of such defective cerebellar GABAergic drive
could have a therapeutic effect in essential tremor.
Keywords: essential tremor; cerebellum; GABA receptors; dentate nucleus; deep cerebellar nuclei
Abbreviations: GABA = �-aminobutyric acid
IntroductionMore than 10 million Americans suffer from essential tremor,
making it the most prevalent adult-onset movement disorder
(Moghal et al., 1994; Louis and Ferreira, 2010). Essential tremor
is characterized by a bilateral action tremor affecting predomin-
antly the arms, the head and/or the voice (Rajput et al., 2004;
Louis, 2005). Formerly regarded as benign, the symptoms of
essential tremor evolve gradually and can become very disturbing
for patients. Recent studies have emphasized the presence of
doi:10.1093/brain/awr301 Brain 2012: 135; 105–116 | 105
Received May 16, 2011. Revised September 2, 2011. Accepted September 12, 2011. Advance Access publication November 26, 2011
� The Author (2011). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: [email protected]
Dow
nloaded from https://academ
ic.oup.com/brain/article-abstract/135/1/105/325831 by guest on 21 N
ovember 2018
abnormalities within �-aminobutyric acid (GABA)-ergic Purkinje
cells in a significant proportion of patients with essential tremor
(Louis et al., 2007, 2009, 2010; Axelrad et al., 2008; Shill et al.,
2008). Several lines of evidence dating back to the early 1970s
suggest that cerebellar dysfunctions reverberating through the
cerebello-thalamo-cortical pathway play a key role in essential
tremor (Deuschl et al., 2000; McAuley and Marsden, 2000;
Pinto et al., 2003; Elble and Deuschl, 2009; Schnitzler et al.,
2009).
The standard pharmaceutical care of essential tremor has essen-
tially not changed in the last 40 years and still relies on primidone
and propranolol, which display moderate efficacy to decrease the
amplitude of tremor in less than half of patients (Lyons et al.,
2003; Louis et al., 2010; Deuschl et al., 2011). The small
number of post-mortem studies performed on essential tremor
subjects is likely linked to the limited therapeutic arsenal available
today to treat essential tremor (Louis, 2005, 2009).
The fact that drugs acting on GABAA receptors, such as primi-
done, benzodiazepines or ethanol, decrease tremor amplitude sug-
gests that altered GABAergic neurotransmission is involved in
essential tremor. Although the exact location of this impaired
GABAergic current is unknown, GABA dysfunction in the cerebel-
lum has been suggested as an aetiological factor in essential tremor
(Deuschl et al., 2000; Kralic et al., 2005; Louis, 2005; Elble and
Deuschl, 2009). More specifically, post-mortem studies indicate
that cerebellar GABAergic Purkinje cells show increased numbers
of axonal swellings and a reduction in numbers in a significant pro-
portion of subjects with essential tremor (Louis et al., 2007, 2009;
Axelrad et al., 2008; Shill et al., 2008). Low levels of GABA have
been reported in the CSF of patients with essential tremor com-
pared with controls (Mally and Baranyi, 1994; Mally et al., 1996).
Moreover, toxins such as aflatrem, penitrem A or harmaline have
been proposed to induce tremor in rodents by interacting with
GABA receptors (Cavanagh et al., 1998; Miwa, 2007). In addition,
GABAA receptor �1 subunit knockout mice exhibit postural and
kinetic tremors, partly reproducing some features of essential
tremor (Kralic et al., 2005). Finally, a recent PET study revealed
decreased binding of 11C-flumazenil at the benzodiazepine recep-
tor site of the GABAA receptor in several brain regions, supporting
the possibility of a general GABAergic dysfunction in essential
tremor (Boecker et al., 2010).
To characterize GABAergic neurotransmission within the cere-
bellum of patients with essential tremor, we have conducted post-
mortem investigations of GABAA and GABAB receptors by auto-
radiography and in situ hybridization on horizontal cerebellar
sections. Since the major GABAergic tract within the cerebellum
connects Purkinje cells to deep cerebellar nuclei and all outgoing
information leaving the cerebellum is funnelled through the deep
cerebellar nuclei, it was essential to measure GABA receptor in this
structure. However, deep cerebellar nuclei are both very small and
irregularly shaped, and it is therefore technically challenging to
measure their protein or messenger RNA content with available
neuroimaging approaches or techniques requiring tissue homogen-
ates. Autoradiography and in situ hybridization were thus selected
because these techniques allow the 2D resolution necessary to
quantify the expression of these receptors in the deep cerebellar
nuclei, notably the dentate nucleus—the largest and one of the
most important subparts of the deep cerebellar nuclei. A group of
patients with essential tremor (n = 10) was compared with control
(n = 16) and patients with Parkinson’s disease (n = 10), taking ad-
vantage of a clinicopathological study in which detailed clinical
variables have been prospectively recorded by the same neurolo-
gists (A.H.R. and A.R.).
Materials and methods
Clinicopathological assessment ofpatientsPatients with essential tremor and Parkinson’s disease controls were all
followed at the Movement Disorders Clinic Saskatchewan at 6- or
12-month intervals (Rajput et al., 2004, 2009). The Movement
Disorders Clinic Saskatchewan has operated uninterrupted since
1968. Autopsy was restricted to those patients who have been as-
sessed by the Movement Disorders Clinic Saskatchewan neurologists
(A.H.R. and A.R.). The clinical diagnosis of essential tremor was made
as described previously (Rajput et al., 2004). All cases with essential
tremor had postural and/or kinetic tremor for several years. All cases
with Parkinson’s disease had resting tremor: two were tremor-
dominant cases and eight were classical (mixed) with approximately
equal severity of bradykinesia, rigidity and tremor (Rajput et al., 2009).
For this study, three main criteria were used to select subjects with
essential tremor: the absence of neuropathologically confirmed
Parkinson’s disease; the presence of dentate nucleus in the tissue block;
and age and gender matching with the two other groups. The severity
of tremor was recorded at each visit and was based on the visual
assessment of tremor amplitude at any site [Unified Parkinson’s
Disease Rating Scale (UPDRS III)] and the history of its impact on
daily activities (Rajput et al., 2004). As essential tremor is often mis-
diagnosed as parkinsonian tremor (Rajput et al., 2004; Jain et al.,
2006; Shahed and Jankovic, 2007; Minen and Louis, 2008;
Benito-Leon et al., 2009; Adler et al., 2011), tissue from patients with
Parkinson’s disease were included to identify changes that are specific
for essential tremor (i.e. kinetic tremor versus resting tremor). Clinical
data including the age and mode of onset, severity of the disease,
duration of disease, response to treatment and adverse effects of
treatment, were recorded prospectively after each clinical assessment.
General information such as other diagnosis, sex, cause and age of
death were also documented. All autopsies were done within 25 h of
death. Table 1 shows a summary of relevant information available
regarding the subjects involved in the study and more detailed infor-
mation is provided in Supplementary Table 1. The maximal severity of
tremor scores is given in Table 1 and Supplementary Table 1.
Tissue handling and processingDetails of autopsy procedures have been described previously (Rajput
et al., 2004, 2009). One half of the brain was histologically examined
by a neuropathologist using representative sections including: the cere-
bral cortex, hippocampus, caudate, lentiform nucleus, thalamus,
mid-brain, pons, medulla and cerebellum with dentate nucleus.
Standard stains including silver stain and �-synuclein, ubiquitin and
tau immunostaining were used routinely as they became commercially
available (Rajput et al., 2009). Neuropathologists produced detailed
reports, which were provided to the family and discussed where
appropriate. In some cases with essential tremor, cerebellar Purkinje
106 | Brain 2012: 135; 105–116 S. Paris-Robidas et al.
Dow
nloaded from https://academ
ic.oup.com/brain/article-abstract/135/1/105/325831 by guest on 21 N
ovember 2018
cells counts were performed by a neuropathologist as reported previ-
ously (Rajput et al., 2011). The other half of the brain was frozen at
�80�C and was cut by hand in the frontal plane into 2–3-mm thick
slices. Coronal slices containing molecular layer of the cerebellar
cortex, granular layer of the cerebellar cortex and dentate nucleus
were cryostat-sectioned (20 mm), thaw-mounted onto SuperFrostPlus
75 � 50-mm slides (Brain Research Laboratories), desiccated overnight
at 4�C, and stored at �80�C until assayed. For homogenate-based
studies such as western immunoblotting or high-performance liquid
chromatography, cerebellar cortex extracts (�100 mg) were homoge-
nized and processed to generate a Tris-buffered saline-soluble fraction
containing extracellular and cytosolic proteins, a detergent-soluble frac-
tion containing membrane-bound proteins and a formic acid-soluble
fraction containing detergent-insoluble proteins, as described in detail
previously (Tremblay et al., 2007; Julien et al., 2008). Cerebellar pH
was measured in 10 mg of tissue diluted into 10 volumes of unbuf-
fered, deionized, distilled water, as described previously (Kingsbury
et al., 1995; Calon et al., 2003a; Julien et al., 2008). Indeed, tissue
pH has been identified as one of the best markers to assess the degree
of preservation of post-mortem brain tissue, as it is particularly sensi-
tive to ante-mortem agonal phase and is a good marker of messenger
RNA degradation (Kingsbury et al., 1995; Li et al., 2004; Catts et al.,
2005; Lipska et al., 2006; Mexal et al., 2006; Vawter et al., 2006; Atz
et al., 2007; Weis et al., 2007).
Receptor binding autoradiographyGABAA receptor density was assessed with 3H-flunitrazepam (GE-
Healthcare; 87 Ci/mmol) allosteric binding to the �1- and �2-interface
(benzodiazepine site) of the GABAA receptor on cerebellum sections,
using a previously reported autoradiography technique (Calon et al.,
2003a). Non-specific binding was determined in presence of 1-mM
clonazepam. GABAB receptor density was evaluated with 3H-CGP
54626 {3-N [1-(S)-(3,4-dichlorophenyl)-ethylamino]-2-(S)-hydroxypro-
pyl-P-(cyclo-hexylmethyl)-phosphinic acid; ANAWA; 40 Ci/mmol}
binding to the antagonist sites of the GABAB receptor using previously
published procedures (Calon et al., 2001, 2003a). Non-specific binding
was measured with 500mM (� )-baclofen (Tocris Bioscience).
N-methy-D-aspartic acid receptors containing GluN2B subunits
(GluR"2/NR2B) were quantified using a high-affinity selective antag-
onist 3H-Ro 25-6981 (F. Hoffmann-La Roche Ltd; 27.6 Ci/mmol;
Mutel et al., 1998; Calon et al., 2003b). Ro 25-6981 is an ifenprodil
derivative that has a high affinity for the GluN2B subunit (Mutel et al.,
1998; Calon et al., 2003b). Non-specific binding was determined by
adding 10 mM Ro 04-5595 hydrochloride (F. Hoffmann–La Roche Ltd)
to the incubation buffer. Slide-mounted tissue sections were exposed
to Kodak Biomax MR film (Carestream Health) along with standards
(3H-micro-scales, GE Healthcare) during 2, 6 and 12 weeks for3H-flunitrazepam, 3H-CGP 54626 and 3H-Ro 25-6981, respectively.
Macroscopic quantification of all autoradiograms was performed
on a KODAK Image Station 4000 MM Digital Imaging System
(Molecular Imaging Software version 5.0.0.90). Optical densities were
measured in the molecular layer of the cerebellar cortex, the granular
layer of the cerebellar cortex and the dentate nucleus of the deep
cerebellar nuclei.
Western immunoblottingFor western immunoblotting, protein concentration was determined
using bicinchoninic acid assays (Pierce). Equal amounts of protein per
sample (20mg of total protein per lane) were added to Laemmli’s
loading buffer, heated to 95�C for 5 min before loading, and subjected
to sodium dodecyl sulphate–polyacrylamide gel electrophoresis.
Proteins were electroblotted onto polyvinylidene fluoride membranes
(Immobilon, Millipore) before blocking in 5% non-fat dry milk and
0.5% bovine serum albumin (Invitrogen) in phosphate-buffered
saline containing 0.1% Tween for 1 h. Membranes were immuno-
blotted with appropriate primary and secondary antibodies followed
by chemiluminescence reagents (Lumiglo reserve, KPL Inc.). Optical
densities of bands were directly quantified using a KODAK Image
Station 4000 MM Digital Imaging System. The following antibodies
were used in this study for western immunoblotting: anti-GABAB
(NeuroMab), anti-cytochrome oxidase-1 (Santa Cruz Biotechnology)
and anti-actin (Sigma).
Table 1 Selected characteristics of study volunteers and comparison of tissue quality between groups
Characteristics Study group Statistical comparison
Control Parkinson’sdisease
Essentialtremor
Test Result
n 16 10 10 – –
Men, ratio 6/16 2/10 2/10 Contingency 0.51
Age at death, mean (SD) (years) 76 (6) 82 (8) 84 (11) ANOVA 0.08
Age of onset, mean (SD) (years) – 63 (12) 54 (20) – –
Disease duration, mean (SD) (years) – 19 (8) 30 (18) – –
Brain pH mean (SD) 6.14 (0.31) 6.46 (0.19) 6.36 (0.17) ANOVA 0.06
Post-mortem interval, mean(SD) (h) 19 (6) 18 (6) 15 (8) ANOVA 0.32
Brain weight, mean (SD) (g) 1249 (49) 1248 (150) 1178 (172) ANOVA 0.49
Hoehn and Yahr scale, mean (SD) – 3.5 (0.2) – – –
Tremor severity, UPDRS, mean (SD) – – 2.0 (0.3) – –
CO-1 messenger RNA DN-DCN, mean (SD) 525 (103) 562 (134) 584 (191) ANOVA 0.68
CO-1 messenger RNA MolCtx, mean (SD) 433 (114) 437 (122) 490 (102) ANOVA 0.51
CO-1 messenger RNA GraCtx, mean (SD) 872 (206) 931 (191) 1013 (159) ANOVA 0.31
Poly-T messenger RNA MolCtx, mean (SD) 124 (119) 86 (33) 105 (72) ANOVA 0.59
CO-1 = cytochrome oxydase-1; DN-DCN = dentate nucleus of the deep cerebellar nuclei; GraCtx = granular layer of the cerebellar cortex; MolCtx = molecular layer of the
cerebellar cortex; UPDRS = Unified Parkinson’s Disease Rating Scale.
Dentate nucleus GABA receptors in essential tremor Brain 2012: 135; 105–116 | 107
Dow
nloaded from https://academ
ic.oup.com/brain/article-abstract/135/1/105/325831 by guest on 21 N
ovember 2018
In situ hybridizationThe general methodology for in situ hybridization has been fully de-
scribed previously (Wisden and Morris, 1994; Calon et al., 2001; Julien
et al., 2008, 2009). GABAA receptors contain an intrinsic ligand-gated
chloride channel, formed by the pentameric assembly of multiple sub-
units. In situ hybridization, histochemistry and quantitative real-time
polymerase chain reaction data show that deep cerebellar nuclei neu-
rons mainly express the �1, �2, b2 and �2 subunit messenger RNAs
(Gambarana et al., 1993; Rotter et al., 2000; Linnemann et al., 2006).
Oligonucleotide probes for the human GABAA receptor subunits �1,
�2, b2 and �2 have thus been synthesized according to previous pub-
lications (Nicholson and Faull, 1996; Petri et al., 2002) and are sum-
marized in Supplementary Table 2.
Metabotropic GABAB receptors are heterodimers consisting of
GABAB1 and GABAB2 subunits (Jones et al., 1998). In situ hybridization
has been performed using oligonucleotide probes for GABAB(1a + b) and
GABAB(2) as described previously (Calon et al., 2001). Oligonucleotide
sequences were specific for the coding region of the GABAB(1a + b) re-
ceptor messenger RNA (NM_001470.2, 1807–1763 and 2933–2884),
whereas those specific to the GABAB(2) receptor (NM_005458.6,
557–513 and 1315–1271), as described (Calon et al., 2001;
Supplementary Table 2).
Subunit I of cytochrome c oxidase messenger RNA is encoded by
the mitochondrial genome and is a classical marker of neuronal activity
(Wong-Riley, 1989; Hirsch et al., 2000). Subunit I of cytochrome c
oxidase oligonucleotide probes were chosen for their coding sequence
within the human mitochondrial genome (NC_005089.1, 6268–6221,
6578–6534, 6854–6805 and 7441–7392) and are fully described in
Supplementary Table 2. To further assess total messenger RNA con-
tent, in situ hybridization using a poly-T probe was performed to label
poly-A tails, which are present on the vast majority of eukaryotic mes-
senger RNAs (Julien et al., 2009).
Oligonucleotides were labelled with 33P-dATP (PerkinElmer) using a
3-terminal deoxynucleotidyltransferase enzyme kit (New England
Biolabs). The reaction was carried out at 37�C for 60 min, and labelled
oligonucleotides were purified using a QIAquick� Nucleotide Removal
Kit (Qiagen). The purified probes were kept at �20�C until assayed
the next day. Pre-hybridization and hybridization conditions were
exactly as described (Julien et al., 2009). Slides were exposed to
Kodak Biomax MR film for 28, 28, 7 and 2 days for GABAA, GABAB,
poly-T and subunit I of cytochrome c oxidase probes, respectively,
and macroscopic optical density quantification was performed using
the KODAK Image Station. The hybridization signal value from a
single section was obtained after subtracting the labelling from
the white matter quantified in the same section. The final data
from each individual were from the mean of four to eight tissue sec-
tions. Non-specific hybridization to tissue sections was found to be neg-
ligible, as determined by adding a 100-fold excess of unlabelled probes.
Amine quantification byhigh-performance liquidchromatographyGABA was measured by high-performance liquid chromatography
(HPLC) coupled with UV detection. Supernatants from Tris-buffered
saline fraction extracts of cerebellum were directly derivated with the
reagent dansyl chloride (Sigma-Aldrich) based on previously published
methods with slight modifications (Saller and Czupryna, 1989; Calon
et al., 1999). Briefly, 50ml of dansyl chloride (1.2 mg/ml) and 50 ml of
sample (Tris-buffered saline extracts) or standard solution, also in
Tris-buffered saline, were mixed and then incubated for 30 min at
90�C. After a 10-min centrifugation at 9000 rpm (4�C), the super-
natant was immediately injected into the chromatograph consisting
of a Waters 717 plus autosampler automatic injector set at 6�C, a
Waters 1525 binary pump equipped with an Atlantis dC18 (3 ml;
3.9 � 150 mm) column, and a Waters 2487 Dual l Absorbance detect-
or (Waters limited). Absorbance was set at 337 nm and the sensitivity
at 0.5 absorbance units full scale. The mobile phase consisted of a
water-acetonitrile mixture (88.5–11.35% v/v) containing 0.15%
(v/v) of phosphoric acid and was delivered at a rate of 0.8 ml/min.
Samples were run in duplicates and peaks were identified and quanti-
fied using the Breeze software (Waters limited). HPLC concentration
values were normalized to tissue weight.
Data and statistical analysesStatistical comparisons of means between groups were performed
using ANOVA when the homogeneity of variance was confirmed
(P4 0.05 using Bartlett’s test). Log transformations of the data were
used when needed to equalize variance and to provide more normally
distributed measures. Post hoc tests were used to determine significant
difference between groups (ANOVA: Newman–Keuls). Adjustments
for age of death or cerebellar pH were performed using analysis of
covariance (ANCOVA), when needed. Using the least squares method,
coefficients of correlation and significance of the linear relationship
between parameters were determined with a simple regression
model. Adjustment for additional variables (age of death or cerebellar
pH) was performed using partial correlation analyses. Likelihood ratio
analysis of contingency tables using the Pearson’s method was used in
the investigation of categorical data such as sex and apoE "4 allele
status, the statistical result being distributed as chi-squared. All statis-
tical analyses were performed using JMP Statistical Analysis Software
(version 8.0.2) and P 5 0.05 were considered to be statistically
significant.
ResultsPrior to experiments, assessment of tissue quality was performed
by measuring tissue pH, cytochrome oxidase (subunit I of cyto-
chrome c oxidase) messenger RNA and total messenger RNA con-
tent in the cerebellum from our sample series. First, cerebellar pH
was comparable between groups suggesting equivalent tissue
quality between groups, although there was a trend for reduced
brain pH in controls (Table 1). Secondly, strong subunit I of cyto-
chrome c oxidase messenger RNA signal was detected in the deep
cerebellar nuclei and cerebellar cortex, showing relative distribu-
tion identical to a previous report in the monkey (Hevner and
Wong-Riley, 1991). No major change between groups was
detected indicating that groups were comparable in terms
of metabolic activity (Table 1). Thirdly, in situ hybridization of
poly-T oligonucleotide showed that total messenger RNA content
did not differ between experimental groups (Table 1). Overall,
these data confirm relative equivalent tissue quality between
groups.
Autoradiography of 3H-flunitrazepam to GABAA and 3H-CGP
54626 to GABAB receptors in the human cerebellum are displayed
in Figs 1 and 2. The general distribution of GABAA receptors
within the cerebellum was in agreement with earlier analyses in
human, monkey and rodent tissue sections (Bowery et al., 1987;
108 | Brain 2012: 135; 105–116 S. Paris-Robidas et al.
Dow
nloaded from https://academ
ic.oup.com/brain/article-abstract/135/1/105/325831 by guest on 21 N
ovember 2018
Dennis et al., 1988). Binding of 3H-CGP 54626 to GABAB recep-
tors was readily detectable in the dentate nucleus (Fig. 2), but
stronger in the cerebellar cortex, particularly in the molecular
layer, conforming with previous autoradiographic studies in
rodents, monkeys and humans (Bowery et al., 1987; Turgeon
and Albin, 1993; Billinton et al., 1999; Calon et al., 2000).
These patterns were also consistent with earlier immunohisto-
chemical evaluation of GABAB receptors in human and rodent
Figure 1 Decreased GABAA receptors in the dentate nucleus of individuals with essential tremor. (A) Representative autoradiograms of
human cerebellum sections showing 3H-flunitrazepam to the benzodiazepine site of GABAA receptors in the dentate nucleus and the
cerebellar cortex of individuals with essential tremor (ET) or Parkinson’s disease (PD), compared with controls (Ctrl). Specific binding of3H-flunitrazepam in the dentate nucleus (B), molecular layer (D) and granular layer (E) of the cerebellar cortex. Correlation analyses
between GABAA receptors and the duration of essential tremor symptoms showed a trend towards inverse relationship between the
duration of essential tremor and GABAA receptors in the dentate nucleus (C). Each point represents an individual and the horizontal bar is
the average (n = 9–15 per group). Statistical comparisons were performed using an ANOVA followed by Newman–Keuls post hoc test or
linear regression.
Dentate nucleus GABA receptors in essential tremor Brain 2012: 135; 105–116 | 109
Dow
nloaded from https://academ
ic.oup.com/brain/article-abstract/135/1/105/325831 by guest on 21 N
ovember 2018
cerebellum (Margeta-Mitrovic et al., 1999; Billinton et al., 2000;
Kulik et al., 2002).
The quantification of the specific binding of 3H-flunitrazepam
and 3H-CGP 54 626 revealed a significant decrease of GABAA
(35% versus controls and 35% versus Parkinson’s disease) and
GABAB (#22% versus controls and #31% versus Parkinson’s dis-
ease) receptors in the dentate nucleus of patients with essential
tremor, compared with the two other groups (Figs 1 and 2). The
effects remained statistically significant after adjustment for cov-
ariates age of death and cerebellar pH using ANCOVA.
Figure 2 Decreased GABAB receptors in the dentate nucleus of individuals with essential tremor. (A) Representative autoradiograms of
human cerebellum sections showing 3H-CGP 54626 to GABAB receptors in the dentate nucleus and the cerebellar cortex of individuals
with essential tremor (ET) or Parkinson’s disease (PD), compared with controls (Ctrl). Specific binding of 3H-CGP 54626 to GABAB
receptors in the dentate nucleus (B), molecular layer (D) and granular layer (E) of the cerebellar cortex. Correlation analyses between
GABAA receptors and the duration of essential tremor symptoms showed an inverse relationship between the duration of essential tremor
and GABAB receptors in the dentate nucleus (C). Each point represents an individual and the horizontal bar is the average (n = 9–15
per group). Statistical comparisons were performed using an ANOVA followed by Newman–Keuls post hoc test or linear regression.
110 | Brain 2012: 135; 105–116 S. Paris-Robidas et al.
Dow
nloaded from https://academ
ic.oup.com/brain/article-abstract/135/1/105/325831 by guest on 21 N
ovember 2018
Interestingly, the concentrations of GABAB receptors in the den-
tate nucleus were inversely correlated [Pearson correlation coeffi-
cient (pairwise detection method) = 0.66, r2 = 0.44, P = 0.0365]
with the duration of essential tremor symptoms, indicating that
the longer the clinical expression of the disease, the lower the
levels of GABAB receptors (Fig. 2C). Adjustment for age of
death or cerebellar pH using partial correlations had minimal
effect on the significance of the correlation. Controlling for both
age of death and cerebellar pH, a correlation of �0.757
(P = 0.0402) was obtained, as estimated by restricted maximum
likelihood method. In contrast, no significant differences between
groups were detected in either molecular or granular layers of the
cerebellar cortex (Figs 1D and E and 2D and E). The absence of
change in GABAB receptors in the cerebellar cortex was confirmed
by western immunoblot (Supplementary Fig. 1). No difference in3H-Ro 25-6981 specific binding to GluN2B subunits of N-methy-D-
aspartic acid receptors was detected in any subregion of the cere-
bellum, including the dentate nucleus, confirming that the de-
crease of GABAB receptor was specific (Supplementary Fig. 2).
We next ascertained whether the loss of GABA receptor in the
dentate nucleus was associated with reduced messenger RNA
transcription by performing in situ hybridization of subunits human
GABAA receptor subunit �1, �2, b2 and �2 (Supplementary Figs 3
and 4). Strong hybridization signals for �1-, b2- and �2 subunits
were detected in the cerebellar cortex, mostly in the granular layer
(Supplementary Fig. 3). The �2 subunit displayed a different ex-
pression pattern being almost exclusively expressed in the Purkinje
cells layer (Supplementary Figs 3 and 4). Overall, the distribution
of GABAA receptor subunit messenger RNA is in agreement with
previous work in the rat cerebellum (Laurie et al., 1992;
Gambarana et al., 1993; Rotter et al., 2000; Linnemann et al.,
2006). An increase of GABAA receptor subunit �1 and b2 was
detected in the cerebellar cortex of patients with Parkinson’s dis-
ease (Supplementary Fig. 4). However, the signal in the dentate
nucleus was too weak to allow reliable quantification, except for
�1 (Supplementary Fig. 4), but was not different between groups
(Supplementary Fig. 4).
In situ hybridization experiments targeting GABAB(1a + b) and
GABAB(2) messenger RNA were performed in the same series of
samples. The messenger RNA distribution of GABAB(1a + b) and
GABAB(2) messenger RNA was stronger in the molecular layer of
cerebellar cortex (Fig. 3A and B), as expected from previous stu-
dies in rodents (Billinton et al., 1999; Bischoff et al., 1999; Durkin
et al., 1999; Clark et al., 2000; Liang et al., 2000). However, only
GABAB(1a + b) messenger RNA was detectable in the dentate nu-
cleus (Fig. 3A). When comparing groups of patients, there was a
significant decrease in the density of messenger RNA for
GABAB(1a + b) in the dentate nucleus from individuals with essential
tremor, compared with controls without neurological impairment
(P50.01) and to individuals suffering from Parkinson’s disease
(P50.05; Fig. 3C). ANCOVA revealed that the changes of
GABAB(1a + b) messenger RNA remained significant after adjust-
ment for age of death and cerebellar pH. No difference in
GABAB(1a + b) or GABAB(2) subunit messenger RNA was seen in
the cerebellar cortex (Fig. 3D and E). Inverse relationships between
age of death and GABAB(1a + b) messenger RNA were detected in
the cerebellar cortex (r2 = 0.10, P50.05) and the dentate nucleus
(r2 = 0.15, P50.05).
Finally, to probe whether the changes in receptor were asso-
ciated with obvious changes in the production or release of the
neurotransmitter GABA, we determined the post-mortem concen-
tration of GABA in the cerebellar cortex by HPLC. However, no
significant difference was detected (Fig. 4).
DiscussionTo our knowledge, we report the first evidence of a statistically
significant neurochemical alteration within cerebellar nuclei that
distinguishes patients with essential tremor as a group from
Parkinson’s disease and/or age-matched controls. The decrease
in GABA receptors was present in patients who suffered tremors
for years and as such, may represent a long-term neurochemical
substrate of essential tremor. We propose that the disruption of
the GABAergic input into the deep cerebellar nuclei contributes to
the generation of oscillatory information conveyed to the thalamus
and the motor cortex, expressed clinically as action tremor
(Supplementary Fig. 5).
What may have caused a reduction ofGABA receptors?A first explanation is that the loss of GABA receptors is a conse-
quence of a neurodegenerative process in the dentate nucleus,
consistent with the correlation between the loss of GABAB recep-
tor and with the progression of the disease. However, our data
clearly show that the reduction of GABA receptor is specific: no
alteration of N-methy-D-aspartic acid receptors or subunit I of
cytochrome c oxidase messenger RNA has been observed in the
present sample series. Accordingly, post-mortem studies have not
reported frank evidence of cell death in the deep cerebellar
nuclei (Louis et al., 2007; Elble and Deuschl, 2009), although
degeneration of the deep cerebellar nuclei has been observed in
a subset of patients with essential tremor (Louis et al., 2006,
2007).
Secondly, a downregulation of dentate nucleus GABAA and
GABAB receptors may result from an exaggerated GABAergic
input from Purkinje cells, which project from the cerebellar cortex
to the deep cerebellar nuclei. In classical pharmacology, a decrease
in post-synaptic receptors is often a compensatory reaction to
pre-synaptic overactivity. Thus, the decrease in GABAA and
GABAB receptors could be a post-synaptic consequence of in-
creased GABA input from Purkinje cells. However, although
GABA concentration in the cerebellum rather displayed a trend
towards an increase, it was not statistically significant and thus
does not confirm this viewpoint. Alternatively, the decrease of
GABA receptors in the dentate nucleus could be a consequence
of a loss of GABA receptor located on Purkinje cells axons. This
hypothesis is consistent with reports of Purkinje cell loss, axonal
swellings (torpedoes) and other abnormalities that have been
observed in essential tremor (Louis et al., 2007; Axelrad et al.,
2008; Shill et al., 2008). However, while only GABAB receptors
are located pre-synaptically (see below), both GABAA and GABAB
Dentate nucleus GABA receptors in essential tremor Brain 2012: 135; 105–116 | 111
Dow
nloaded from https://academ
ic.oup.com/brain/article-abstract/135/1/105/325831 by guest on 21 N
ovember 2018
receptors were downregulated. In addition, the concomitant loss
of GABAB receptor messenger RNA transcripts rather suggests that
the loss of GABA receptors arises from a transcription decrease in
dentate nucleus cells.
A third possibility is that the GABAA and GABAB receptor reduc-
tion per se plays a causative role in essential tremor by restricting
the post-synaptic action of GABA released from Purkinje cell
axons, thereby disinhibiting deep cerebellar nuclei neurons. The
ensuing overactivity of deep cerebellar nuclei neurons would
spread up through the cerebellar-thalamo-cortical circuit, possibly
contributing to the generation of tremors (Supplementary Fig. 5).
How can a reduction of GABA receptorsin the dentate nucleus play a role inessential tremor?As mentioned in the ‘Introduction’ section, one of the most widely
accepted hypotheses proposes that essential tremor is caused by
central oscillators propagating within the cerebello-thalamo-
cortical network (Deuschl et al., 2000; McAuley and Marsden,
2000; Pinto et al., 2003; Elble and Deuschl, 2009; Schnitzler
et al., 2009). Virtually all output from the cerebellum originates
from deep cerebellar nuclei neurons, which project their axons to
various structures, including the ventral lateral nucleus of the thal-
amus forming a complex circuitry with cortico-thalamic afferents,
thalamo-cortical projection neurons and thalamic GABAergic inter-
neurons (Heck and Sultan, 2002; Ilinsky and Kultas-Ilinsky, 2002).
Given the key position of the deep cerebellar nuclei in this circuit-
ry, it is clear that the neurological information processed in the
cerebellum has to interact with GABA receptors in deep cerebellar
nuclei neurons before leaving the cerebellum. The deep cerebellar
nuclei have been known to play a crucial role in the generation of
tremor and experiments involving the local cooling or ablation of
dentate nucleus cells induces oscillations resembling intention tre-
mors in non-human primates (Growdon et al., 1967; Brooks et al.,
1973; Goldberger and Growdon, 1973; Cooke and Thomas,
1976).
Figure 3 Decreased GABAB receptor subunits messenger RNA in the dentate nucleus of individuals with essential tremor. The expression
of GABAB(1a + b) (A) and GABAB(2) (B) receptor subunits was determined using in situ hybridization and quantified in the dentate nucleus
(C) and the cerebellar cortex (E and F). Correlation analyses show an association between GABAB(1a + b) receptors messenger RNA and the
duration of essential tremor symptoms in the dentate nucleus (D). Each point represents an individual and the horizontal bar is the average
(n = 9–15 per group). Statistical comparisons were performed using an ANOVA followed by Newman–Keuls post hoc test (C, E and F) or
linear regression (D).
112 | Brain 2012: 135; 105–116 S. Paris-Robidas et al.
Dow
nloaded from https://academ
ic.oup.com/brain/article-abstract/135/1/105/325831 by guest on 21 N
ovember 2018
Electrophysiological studies show that all dentate nucleus cells
are sensitive to GABAergic input from Purkinje cells (Uusisaari and
Knopfel, 2008), whereas immunocytochemistry, autoradiography
and in situ hybridization experiments in rodent and human tissue
have detected both GABAA and GABAB receptors in the deep cere-
bellar nuclei (Kingsbury et al., 1980; Bowery et al., 1987; Chu
et al., 1990; Albin et al., 1991; Gambarana et al., 1993;
Turgeon and Albin, 1993). Electrophysiological recording of deep
cerebellar nuclei neurons confirms that both GABAA and GABAB
receptors mediate the inhibitory input coming from Purkinje cells
(Ito et al., 1970; Mouginot and Gahwiler, 1996; Chen et al.,
2005). In addition, GABAB receptors are present on the pre-
synaptic terminals of Purkinje cells into the deep cerebellar nuclei
(Mouginot and Gahwiler, 1996). Interestingly, electrophysiology
data also indicate that deep cerebellar nuclei neurons belong to
the family of single cell oscillators, with a pacemaker-like activity
(Jahnsen, 1986; Llinas, 1988; Llinas and Muhlethaler, 1988;
Mouginot and Gahwiler, 1996). Populations of neurons with an
inherent tendency for spontaneous rhythmic discharge (i.e. pace-
makers) have been identified in vertebrates and invertebrates
(Iles and Pearson, 1969) and have long been suspected to be a
driving force in essential tremor (DeLong, 1978). In the case of
deep cerebellar nuclei neurons, it has been shown that their
pacemaker-like activity is under tonic control by the GABAergic
input from Purkinje cells (Mouginot and Gahwiler, 1996). Even
more compelling is the demonstration that deep cerebellar nuclei
neurons can impose their rhythmicity to their thalamic target
neurons with highly regular patterns of activity (Pinault and
Deschenes, 1992).
Therefore, a decrease of deep cerebellar nuclei GABAA and
GABAB receptors in essential tremor, as reported here, is likely
to have a critical impact on cerebellar output, as schematized in
Supplementary Fig. 5. In this scheme, without the GABAergic in-
hibitory input from Purkinje cells, the pacemaker activity of
dentate nucleus neurons would be the origin of the oscillatory pat-
terns climbing the cerebello-thalamo-cortical pathways in essential
tremor (Supplementary Fig. 5). In the normal setting, GABA
released by axons originating from Purkinje cells interacts with
GABA receptor to restrain the pacemaker activity of deep cerebel-
lar nuclei neurons. It is suggested here that the lack of GABA
receptors in the dentate nucleus leads to incorrect response to
the GABAergic input from Purkinje cells. The ensuing disinhibition
of dentate nucleus neurons would then facilitate the transmission
of oscillatory discharge of information up to target neurons in the
thalamus. Such faulty tremogenic information would then be con-
veyed from the thalamus to the primary motor, pre-motor and
supplemental areas of the brain cortex (Supplementary Fig. 5).
Ensuing oscillation in the 10 Hz range reaching the motor cortex
could modulate descending motor pathways to limb muscle and
thus drive essential tremor (McAuley and Marsden, 2000).
Interestingly, one of the most efficient treatments of essential
tremor is deep brain stimulation of the ventral intermediate nu-
cleus of the thalamus (Zesiewicz et al., 2005; Pahwa et al., 2006;
Stani et al., 2009). The efficacy against tremor of the deep brain
stimulation technique has been attributed to its capacity to disrupt
pathological thalamic oscillatory activity, thereby stopping the
propagation of tremor-inducing signal climbing the cerebello-tha-
lamo-cortical system (Lozano et al., 2002; Kane et al., 2009).
Therefore, a defect of deep cerebellar nuclei GABAergic receptor
could play an upstream role in the generation of tremor.
The present results possibly explain the pharmacological efficacy
of GABAergic drugs in essential tremor, and further support the
hypothesis that GABAergic compounds specifically acting at the
deep cerebellar nuclei level could be even more efficient. There
is good evidence that tremor may be suppressed by substances
that facilitate inhibitory neurotransmission mediated by GABA
(Louis, 1999). For example, intrathecal pump releasing a GABAB
receptor agonist baclofen has already been successfully used to de-
crease tremor in a case report (Weiss et al., 2003). Intrathalamic
injection of GABAA receptor agonist led to tremor reduction
(Pahapill et al., 1999). A tremorlytic action of GABAA or GABAB
receptor agonists has also been reported in animal models (Tariq
et al., 2001; Paterson et al., 2009). Several clinical studies have
been performed with GABA analogues, such as gabapentin, preg-
abalin or benzodiazepines showing some efficacy (Louis, 2005;
Zesiewicz et al., 2007; Ferrara et al., 2009). An additional phase
IV multi-site, prospective, double-blind, randomized, placebo-
controlled, cross-over trial is currently being performed with preg-
abalin (ClinicalTrials.gov Identifier: NCT00584376). However, the
complexity of using GABAergic treatments stems from the diffi-
culty of selectively targeting one small nucleus like the deep cere-
bellar nuclei, which makes adverse effects practically unavoidable
with current therapies. However, the fact that the dentate nucleus
expresses GABAB(1a + b), but not GABAB(2) subunits, suggests that
GABAB receptors in the dentate nucleus may be specific to this
nucleus. Indeed, the vast majority of GABAB receptor heterodimers
contain both a GABAB(1a + b) and a GABAB(2) subunit, coexpressed
by the same cells (Benke et al., 1999; Billinton et al., 1999;
Bischoff et al., 1999; Durkin et al., 1999; Clark et al., 2000;
Liang et al., 2000; Bettler and Tiao, 2006). In structures where
only GABAB(1a + b) can be found, it has been proposed that an
Figure 4 GABA concentrations in the cerebellar cortex com-
paring individuals with essential tremor or Parkinson’s disease
versus controls. GABA concentrations were determined by high
performance liquid chromatography and expressed in ng/mg of
tissue. Each point represents an individual and the horizontal bar
is the average (n = 9–15 per group). Statistical comparisons were
performed using an ANOVA followed by Newman–Keuls post
hoc test.
Dentate nucleus GABA receptors in essential tremor Brain 2012: 135; 105–116 | 113
Dow
nloaded from https://academ
ic.oup.com/brain/article-abstract/135/1/105/325831 by guest on 21 N
ovember 2018
unidentified subunit may couple to GABAB(1a + b) to form a func-
tional receptor (Bowery et al., 2002; Bowery, 2010; Marshall and
Foord, 2010). If such is the case within the dentate nucleus, the
GABAB receptor assembly might be unique there, which opens the
door to tissue-specific pharmacological targeting. Then, hypothet-
ically, an agonist specific for GABAB receptor subspecies present in
the dentate nucleus could be used to re-establish the tonic inhib-
ition on dentate nucleus neurons and reduce tremor.
Although we had access to a wide range of clinical data, some
important information such as Braak (Alzheimer’s disease and
Parkinson’s disease) scores, CERAD (Consortium to Establish a
Registry for Alzheimer’s Disease) stages and ante-mortem cogni-
tive performance, were not documented. Therefore, the fact that
we were not able to link our observation on GABA receptors to
Alzheimer’s disease-related neurodegeneration and symptoms is a
limitation of our study. Finally, it is important to point out that the
decrease in GABA receptors correlated with the progression of
essential tremor, as patients who suffered for a longer time had
lower levels of GABA receptors. This suggests that this GABAergic
defect is not an acute phenomenon but is established progressively
during the evolution of the disease.
ConclusionOwing to the small number of clinicopathological studies, our
understanding of the pathophysiology of essential tremor has
lagged behind other CNS diseases. The present investigation is
probably the first to report a neurochemical difference in indi-
viduals with essential tremor, versus controls or patients with
Parkinson’s disease, using human brain tissue. Since GABAergic
input into deep cerebellar nuclei neurons is critical for the regula-
tion of its pacemaker activity extending through cerebello-
thalamo-cortical networks, a reduction of GABA receptors may
play an important role in the generation of tremor. Finally, our
results suggest that GABA receptors within the deep cerebellar
nuclei are potential drug targets in essential tremor.
AcknowledgementsThe authors wish to thank the patients and families who gener-
ously donated brain tissue to our research program; Novartis
(Basel, Switzerland) for the gift of 3H-CGP 54626; and F.
Hoffmann-La Roche Ltd (Basel, Switzerland) for the gift of3H-Ro 25-6981 and Ro 04-5595.
FundingThe International Essential Tremor Foundation (IETF); the Fonds
d’Enseignement et de Recherche of the Faculty of Pharmacy of
Laval University; and the Canada Foundation for Innovation
(10307). F.C. was supported by a New Investigator Award from
Canadian Institutes of Health Research (CAN-76833) and a salary
award from the Fonds de la recherche en sante du Quebec.
Supplementary materialSupplementary material is available at Brain online.
ReferencesAdler CH, Shill HA, Beach TG. Essential tremor and Parkinson’s disease:
lack of a link. Mov Disord 2011; 26: 372–7.
Albin RL, Price RH, Sakurai SY, Penney JB, Young AB. Excitatory and
inhibitory amino acid binding sites in human dentate nucleus. Brain
Res 1991; 560: 350–3.Atz M, Walsh D, Cartagena P, Li J, Evans S, Choudary P, et al.
Methodological considerations for gene expression profiling of
human brain. J Neurosci Methods 2007; 163: 295–309.
Axelrad JE, Louis ED, Honig LS, Flores I, Ross GW, Pahwa R, et al.
Reduced Purkinje cell number in essential tremor: a postmortem
study. Arch Neurol 2008; 65: 101–7.
Benito-Leon J, Louis ED, Bermejo-Pareja F. Risk of incident Parkinson’s
disease and parkinsonism in essential tremor: a population based
study. J Neurol Neurosurg Psychiatry 2009; 80: 423–5.Benke D, Honer M, Michel C, Bettler B, Mohler H. Gamma-aminobutyric
acid type B receptor splice variant proteins GBR1a and GBR1b are both
associated with GBR2 in situ and display differential regional and sub-
cellular distribution. J Biol Chem 1999; 274: 27323–30.
Bettler B, Tiao JY. Molecular diversity, trafficking and subcellular local-
ization of GABAB receptors. Pharmacol Ther 2006; 110: 533–43.
Billinton A, Ige AO, Wise A, White JH, Disney GH, Marshall FH, et al.
GABA(B) receptor heterodimer-component localisation in human brain.
Mol Brain Res 2000; 77: 111–24.Billinton A, Upton N, Bowery NG. GABA(B) receptor isoforms GBR1a and
GBR1b, appear to be associated with pre- and post-synaptic elements
respectively in rat and human cerebellum. Br J Pharmacol 1999; 126:
1387–92.
Bischoff S, Leonhard S, Reymann N, Schuler V, Shigemoto R,
Kaupmann K, et al. Spatial distribution of GABA(B)R1 receptor
mRNA and binding sites in the rat brain. J Comp Neurol 1999; 412:
1–16.
Boecker H, Weindl A, Brooks DJ, Ceballos-Baumann AO, Liedtke C,
Miederer M, et al. GABAergic dysfunction in essential tremor: an
11C-flumazenil PET study. J Nucl Med 2010; 51: 1030–5.
Bowery NG. Historical perspective and emergence of the GABAB recep-
tor. Adv Pharmacol 2010; 58: 1–18.
Bowery NG, Bettler B, Froestl W, Gallagher JP, Marshall F, Raiteri M,
et al. International union of pharmacology. XXXIII. Mammalian
gamma-aminobutyric acid(B) receptors: structure and function.
Pharmacol Rev 2002; 54: 247–64.
Bowery NG, Hudson AL, Price GW. GABAA and GABAB receptor site
distribution in the rat central nervous system. Neuroscience 1987; 20:
365–83.
Brooks VB, Kozlovskaya IB, Atkin A, Horvath FE, Uno M. Effects of
cooling dentate nucleus on tracking-task performance in monkeys. J
Neurophysiol 1973; 36: 974–95.Calon F, Lavertu N, Lemieux AM, Morissette M, Goulet M, Grondin R,
et al. Effect of MPTP-induced denervation on basal ganglia GABA(B)
receptors: correlation with dopamine concentrations and dopamine
transporter. Synapse 2001; 40: 225–34.Calon F, Morissette M, Goulet M, Grondin R, Blanchet PJ, Bedard PJ,
et al. Chronic D1 and D2 dopaminomimetic treatment of
MPTP-denervated monkeys: effects on basal ganglia GABA(A)/benzo-
diazepine receptor complex and GABA content. Neurochem Int 1999;
35: 81–91.Calon F, Morissette M, Goulet M, Grondin R, Blanchet PJ, Bedard PJ,
et al. 125I-CGP 64213 binding to GABA(B) receptors in the brain of
monkeys: effect of MPTP and dopaminomimetic treatments. Exp
Neurol 2000; 163: 191–9.
114 | Brain 2012: 135; 105–116 S. Paris-Robidas et al.
Dow
nloaded from https://academ
ic.oup.com/brain/article-abstract/135/1/105/325831 by guest on 21 N
ovember 2018
Calon F, Morissette M, Rajput AH, Hornykiewicz O, Bedard PJ, Di
Paolo T. Changes of GABA receptors and dopamine turnover in the
postmortem brains of parkinsonians with levodopa-induced motor
complications. Mov Disord 2003a; 18: 241–53.Calon F, Rajput AH, Hornykiewicz O, Bedard PJ, Di Paolo T.
Levodopa-induced motor complications are associated with alterations
of glutamate receptors in Parkinson disease. Neurobiol Dis 2003b; 14:
404–16.
Catts VS, Catts SV, Fernandez HR, Taylor JM, Coulson EJ, Lutze-
Mann LH. A microarray study of post-mortem mRNA degradation in
mouse brain tissue. Brain Res Mol Brain Res 2005; 138: 164–77.Cavanagh JB, Holton JL, Nolan CC, Ray DE, Naik JT, Mantle PG. The
effects of the tremorgenic mycotoxin penitrem A on the rat cerebel-
lum. Vet Pathol 1998; 35: 53–63.
Chen K, Li HZ, Ye N, Zhang J, Wang JJ. Role of GABAB receptors in
GABA and baclofen-induced inhibition of adult rat cerebellar interpo-
situs nucleus neurons in vitro. Brain Res Bull 2005; 67: 310–8.
Chu DC, Albin RL, Young AB, Penney JB. Distribution and kinetics of
GABAB binding sites in rat central nervous system: a quantitative auto-
radiographic study. Neuroscience 1990; 34: 341–57.
Clark JA, Mezey E, Lam AS, Bonner TI. Distribution of the GABA(B)
receptor subunit gb2 in rat CNS. Brain Res 2000; 860: 41–52.
Cooke JD, Thomas JS. Forearm oscillation during cooling of the den-
tate mucleus in the monkey. Can J Physiol Pharmacol 1976; 54:
430–6.
DeLong MR. Possible involvement of central pacemakers in clinical dis-
orders of movement. Fed Proc 1978; 37: 2171–5.
Dennis T, Dubois A, Benavides J, Scatton B. Distribution of central omega
1 (benzodiazepine1) and omega 2 (benzodiazepine2) receptor sub-
types in the monkey and human brain. An autoradiographic study
with [3H]flunitrazepam and the omega 1 selective ligand
[3H]zolpidem. J Pharmacol Exp Ther 1988; 247: 309–22.Deuschl G, Raethjen J, Hellriegel H, Elble R. Treatment of patients with
essential tremor. Lancet Neurol 2011; 10: 148–61.Deuschl G, Wenzelburger R, Loffler K, Raethjen J, Stolze H. Essential
tremor and cerebellar dysfunction clinical and kinematic analysis of
intention tremor. Brain 2000; 123: 1568–80.
Durkin MM, Gunwaldsen CA, Borowsky B, Jones KA, Branchek TA. An in
situ hybridization study of the distribution of the GABA(B2) protein
mRNA in the rat CNS. Brain Res Mol Brain Res 1999; 71: 185–200.
Elble RJ, Deuschl G. An update on essential tremor. Curr Neurol Neurosci
Rep 2009; 9: 273–7.
Ferrara JM, Kenney C, Davidson AL, Shinawi L, Kissel AM, Jankovic J.
Efficacy and tolerability of pregabalin in essential tremor: a rando-
mized, double-blind, placebo-controlled, crossover trial. J Neurol Sci
2009; 285: 195–7.
Gambarana C, Loria CJ, Siegel RE. GABAA receptor messenger RNA
expression in the deep cerebellar nuclei of Purkinje cell degeneration
mutants is maintained following the loss of innervating Purkinje neu-
rons. Neuroscience 1993; 52: 63–71.Goldberger ME, Growdon JH. Pattern of recovery following cerebellar
deep nuclear lesions in monkeys. Exp Neurol 1973; 39: 307–22.Growdon JH, Chambers WW, Liu CN. An experimental study of cere-
bellar dyskinesia in the rhesus monkey. Brain 1967; 90: 603–32.Heck D, Sultan F. Cerebellar structure and function: making sense of
parallel fibers. Hum Mov Sci 2002; 21: 411–21.Hevner RF, Wong-Riley MT. Neuronal expression of nuclear and mito-
chondrial genes for cytochrome oxidase (CO) subunits analyzed by in
situ hybridization: comparison with CO activity and protein. J Neurosci
1991; 11: 1942–58.
Hirsch EC, Perier C, Orieux G, Francois C, Feger J, Yelnik J, et al.
Metabolic effects of nigrostriatal denervation in basal ganglia. Trends
Neurosci 2000; 23: S78–85.Iles JF, Pearson KG. Central patterning of motoneuronal activity in the
cockroach. J Physiol 1969; 204: 54P–55P.Ilinsky IA, Kultas-Ilinsky K. Motor thalamic circuits in primates with em-
phasis on the area targeted in treatment of movement disorders. Mov
Disord 2002; 17 (Suppl. 3): S9–14.
Ito M, Yoshida M, Obata K, Kawai N, Udo M. Inhibitory control of
intracerebellar nuclei by the Purkinje cell axons. Exp Brain Res 1970;
10: 64–80.
Jahnsen H. Extracellular activation and membrane conductances of neur-
ones in the guinea-pig deep cerebellar nuclei in vitro. J Physiol 1986;
372: 149–68.
Jain S, Lo SE, Louis ED. Common misdiagnosis of a common neurological
disorder: how are we misdiagnosing essential tremor? Arch Neurol
2006; 63: 1100–4.Jones KA, Borowsky B, Tamm JA, Craig DA, Durkin MM, Dai M, et al.
GABA(B) receptors function as a heteromeric assembly of the subunits
GABA(B)R1 and GABA(B)R2. Nature 1998; 396: 674–9.
Julien C, Tremblay C, Bendjelloul F, Phivilay A, Coulombe MA, Emond V,
et al. Decreased drebrin mRNA expression in Alzheimer disease: cor-
relation with tau pathology. J Neurosci Res 2008; 86: 2292–302.
Julien C, Tremblay C, Emond V, Lebbadi M, Salem NJ, Bennett DA,
Calon F. Sirtuin 1 reduction parallels the accumulation of tau in
Alzheimer disease. J Neuropathol Exp Neurol 2009; 68: 48–58.
Kane A, Hutchison WD, Hodaie M, Lozano AM, Dostrovsky JO.
Enhanced synchronization of thalamic theta band local field
potentials in patients with essential tremor. Exp Neurol 2009; 217:
171–6.
Kingsbury AE, Foster OJ, Nisbet AP, Cairns N, Bray L, Eve DJ, et al.
Tissue pH as an indicator of mRNA preservation in human
post-mortem brain. Mol Brain Res 1995; 28: 311–8.
Kingsbury AE, Wilkin GP, Patel AJ, Balazs R. Distribution of GABA re-
ceptors in the rat cerebellum. J Neurochem 1980; 35: 739–42.
Kralic JE, Criswell HE, Osterman JL, O’Buckley TK, Wilkie ME,
Matthews DB, et al. Genetic essential tremor in gamma-aminobutyric
acidA receptor alpha1 subunit knockout mice. J Clin Invest 2005; 115:
774–9.Kulik A, Nakadate K, Nyiri G, Notomi T, Malitschek B, Bettler B, et al.
Distinct localization of GABA(B) receptors relative to synaptic sites in
the rat cerebellum and ventrobasal thalamus. Eur J Neurosci 2002; 15:
291–307.
Laurie DJ, Seeburg PH, Wisden W. The distribution of 13 GABAA recep-
tor subunit mRNAs in the rat brain. II. Olfactory bulb and cerebellum. J
Neurosci 1992; 12: 1063–76.
Li JZ, Vawter MP, Walsh DM, Tomita H, Evans SJ, Choudary PV, et al.
Systematic changes in gene expression in postmortem human brains
associated with tissue pH and terminal medical conditions. Hum Mol
Genet 2004; 13: 609–16.
Liang F, Hatanaka Y, Saito H, Yamamori T, Hashikawa T. Differential
expression of gamma-aminobutyric acid type B receptor-1a and -1b
mRNA variants in GABA and non-GABAergic neurons of the rat brain.
J Comp Neurol 2000; 416: 475–95.
Linnemann C, Schmeh I, Thier P, Schwarz C. Transient change in
GABA(A) receptor subunit mRNA expression in Lurcher cere-
bellar nuclei during Purkinje cell degeneration. BMC Neurosci 2006;
7: 59.
Lipska BK, Deep-Soboslay A, Weickert CS, Hyde TM, Martin CE,
Herman MM, et al. Critical factors in gene expression in postmortem
human brain: focus on studies in schizophrenia. Biol Psychiatry 2006;
60: 650–8.
Llinas RR. The intrinsic electrophysiological properties of mammalian
neurons: insights into central nervous system function. Science 1988;
242: 1654–64.Llinas R, Muhlethaler M. Electrophysiology of guinea-pig cerebellar nu-
clear cells in the in vitro brain stem-cerebellar preparation. J Physiol
1988; 404: 241–58.
Louis ED. Essential tremor. Lancet Neurol 2005; 4: 100–10.Louis ED. A new twist for stopping the shakes? Revisiting GABAergic
therapy for essential tremor. Arch Neurol 1999; 56: 807–8.Louis ED. Essential tremors: a family of neurodegenerative disorders?
Arch Neurol 2009; 66: 1202–8.Louis ED, Faust PL, Vonsattel JP, Honig LS, Rajput A, Rajput A, et al.
Torpedoes in Parkinson’s disease, Alzheimer’s disease, essential tremor,
and control brains. Mov Disord 2009; 24: 1600–5.
Dentate nucleus GABA receptors in essential tremor Brain 2012: 135; 105–116 | 115
Dow
nloaded from https://academ
ic.oup.com/brain/article-abstract/135/1/105/325831 by guest on 21 N
ovember 2018
Louis ED, Faust PL, Vonsattel JP, Honig LS, Rajput A, Robinson CA, et al.Neuropathological changes in essential tremor: 33 cases compared
with 21 controls. Brain 2007; 130: 3297–307.
Louis ED, Ferreira JJ. How common is the most common adult move-
ment disorder? Update on the worldwide prevalence of essentialtremor. Mov Disord 2010; 25: 534–41.
Louis ED, Rios E, Henchcliffe C. How are we doing with the treatment of
essential tremor (ET)? Eur J Neurol 2010; 17: 882–4.
Louis ED, Vonsattel JP, Honig LS, Lawton A, Moskowitz C, Ford B, et al.Essential tremor associated with pathologic changes in the cerebellum.
Arch Neurol 2006; 63: 1189–93.
Lozano AM, Dostrovsky J, Chen R, Ashby P. Deep brain stimulation forParkinson’s disease: disrupting the disruption. Lancet Neurol 2002; 1:
225–31.
Lyons KE, Pahwa R, Comella CL, Eisa MS, Elble RJ, Fahn S, et al. Benefits
and risks of pharmacological treatments for essential tremor. Drug Saf2003; 26: 461–81.
Mally J, Baranyi M. Change in the concentrations of amino acids in
cisternal CSF of patients with essential tremor. J Neurol Neurosurg
Psychiatry 1994; 57: 1012–3.Mally J, Baranyi M, Vizi ES. Change in the concentrations of amino acids
in CSF and serum of patients with essential tremor. J Neural Transm
1996; 103: 555–60.
Margeta-Mitrovic M, Mitrovic I, Riley RC, Jan LY, Basbaum AI.Immunohistochemical localization of GABA(B) receptors in the rat cen-
tral nervous system. J Comp Neurol 1999; 405: 299–321.
Marshall FH, Foord SM. Heterodimerization of the GABABreceptor-implications for GPCR signaling and drug discovery. Adv
Pharmacol 2010; 58: 63–91.
McAuley JH, Marsden CD. Physiological and pathological tremors and
rhythmic central motor control. Brain 2000; 123: 1545–67.Mexal S, Berger R, Adams CE, Ross RG, Freedman R, Leonard S. Brain
pH has a significant impact on human postmortem hippocampal gene
expression profiles. Brain Res 2006; 1106: 1–11.
Minen MT, Louis ED. Emergence of Parkinson’s disease in essentialtremor: a study of the clinical correlates in 53 patients. Mov Disord
2008; 23: 1602–5.
Miwa H. Rodent models of tremor. Cerebellum 2007; 6: 66–72.Moghal S, Rajput AH, D’Arcy C, Rajput R. Prevalence of movement
disorders in elderly community residents. Neuroepidemiology 1994;
13: 175–8.
Mouginot D, Gahwiler BH. Presynaptic GABAB receptors modulate IPSPsevoked in neurons of deep cerebellar nuclei in vitro. J Neurophysiol
1996; 75: 894–901.
Mutel V, Buchy D, Klingelschmidt A, Messer J, Bleuel Z, Kemp JA, et al.
In vitro binding properties in rat brain of [3H]Ro 25-6981, a potentand selective antagonist of NMDA receptors containing NR2B sub-
units. J Neurochem 1998; 70: 2147–55.
Nicholson LFB, Faull RLM. GABAA receptor subunit subtypes in thehuman putamen and globus pallidus in Huntington disease. In:
Ohye C, Kimura M, McKenzie J, editors. The Basal ganglia V. New
York: Plenum Press; 1996.
Pahapill PA, Levy R, Dostrovsky JO, Davis KD, Rezai AR, Tasker RR,et al. Tremor arrest with thalamic microinjections of muscimol in pa-
tients with essential tremor. Ann Neurol 1999; 46: 249–52.
Pahwa R, Lyons KE, Wilkinson SB, Simpson RKJ, Ondo WG, Tarsy D,
et al. Long-term evaluation of deep brain stimulation of the thalamus.J Neurosurg 2006; 104: 506–12.
Paterson NE, Malekiani SA, Foreman MM, Olivier B, Hanania T.
Pharmacological characterization of harmaline-induced tremor activity
in mice. Eur J Pharmacol 2009; 616: 73–80.Petri S, Krampfl K, Dengler R, Bufler J, Weindl A, Arzberger T. Human
GABA A receptors on dopaminergic neurons in the pars compacta of
the substantia nigra. J Comp Neurol 2002; 452: 360–6.Pinault D, Deschenes M. The origin of rhythmic fast subthreshold de-
polarizations in thalamic relay cells of rats under urethane anaesthesia.
Brain Res 1992; 595: 295–300.
Pinto AD, Lang AE, Chen R. The cerebellothalamocortical pathway in
essential tremor. Neurology 2003; 60: 1985–7.
Rajput A, Robinson CA, Rajput AH. Essential tremor course and disability:
a clinicopathologic study of 20 cases. Neurology 2004; 62: 932–6.Rajput AH, Robinson CA, Rajput ML, Rajput A. Cerebellar Purkinje cell
loss is not pathognomonic of essential tremor. Parkinsonism Relat
Disord 2011; 17: 16–21.Rajput AH, Voll A, Rajput ML, Robinson CA, Rajput A. Course in
Parkinson disease subtypes: a 39-year clinicopathologic study.
Neurology 2009; 73: 206–12.Rotter A, Rath S, Evans JE, Frostholm A. Modulation of GABA(A) recep-
tor subunit mRNA levels in olivocerebellar neurons of purkinje cell
degeneration and weaver mutant mice. J Neurochem 2000; 74:
2190–200.
Saller CF, Czupryna MJ. Gamma-Aminobutyric acid, glutamate, glycine
and taurine analysis using reversed-phase high-performance liquid
chromatography and ultraviolet detection of dansyl chloride deriva-
tives. J Chromatogr 1989; 487: 167–72.
Schnitzler A, Munks C, Butz M, Timmermann L, Gross J. Synchronized
brain network associated with essential tremor as revealed by magne-
toencephalography. Mov Disord 2009; 24: 1629–35.
Shahed J, Jankovic J. Exploring the relationship between essential tremor
and Parkinson’s disease. Parkinsonism Relat Disord 2007; 13: 67–76.Shill HA, Adler CH, Sabbagh MN, Connor DJ, Caviness JN, Hentz JG,
et al. Pathologic findings in prospectively ascertained essential tremor
subjects. Neurology 2008; 70: 1452–5.Stani TM, Burchiel KJ, Hart MJ, Lenar DP, Anderson VC. Effects of DBS
on precision grip abnormalities in essential tremor. Exp Brain Res 2009;
201: 331–8.Tariq M, Arshaduddin M, Biary N, Al Moutaery K, Al Deeb S. Baclofen
attenuates harmaline induced tremors in rats. Neurosci Lett 2001; 312:
79–82.Tremblay C, Pilote M, Phivilay A, Emond V, Bennett DA, Calon F.
Biochemical characterization of Abeta and tau pathologies in mild cog-
nitive impairment and Alzheimer’s disease. J Alzheimers Dis 2007; 12:
377–90.Turgeon SM, Albin RL. Pharmacology, distribution, cellular localization,
and development of GABAB binding in rodent cerebellum.
Neuroscience 1993; 55: 311–23.Uusisaari M, Knopfel T. GABAergic synaptic communication in the
GABAergic and non-GABAergic cells in the deep cerebellar nuclei.
Neuroscience 2008; 156: 537–49.Vawter MP, Tomita H, Meng F, Bolstad B, Li J, Evans S, et al.
Mitochondrial-related gene expression changes are sensitive to
agonal-pH state: implications for brain disorders. Mol Psychiatry
2006; 11: 615, 663–79.
Weis S, Llenos IC, Dulay JR, Elashoff M, Martinez-Murillo F, Miller CL.
Quality control for microarray analysis of human brain samples: the
impact of postmortem factors, RNA characteristics, and histopath-
ology. J Neurosci Methods 2007; 165: 198–209.
Weiss N, North RB, Ohara S, Lenz FA. Attenuation of cerebellar tremor
with implantation of an intrathecal baclofen pump: the role of
gamma-aminobutyric acidergic pathways. Case report. J Neurosurg
2003; 99: 768–71.
Wisden W, Morris BJ. In situ hybridization protocols for the brain. San
Diego: Academic Press; 1994.
Wong-Riley MT. Cytochrome oxidase: an endogenous metabolic marker
for neuronal activity. Trends Neurosci 1989; 12: 94–101.Zesiewicz TA, Elble R, Louis ED, Hauser RA, Sullivan KL, Dewey RBJ,
et al. Practice parameter: therapies for essential tremor: report of the
Quality Standards Subcommittee of the American Academy of
Neurology. Neurology 2005; 64: 2008–20.
Zesiewicz TA, Ward CL, Hauser RA, Pease Campbell JA, Sullivan KL.
Pregabalin (Lyrica) in the treatment of essential tremor. Mov Disord
2007; 22: 139–41.
116 | Brain 2012: 135; 105–116 S. Paris-Robidas et al.
Dow
nloaded from https://academ
ic.oup.com/brain/article-abstract/135/1/105/325831 by guest on 21 N
ovember 2018