Modulation of CB1 cannabinoid receptor alters the electrophysiological properties of cerebellar Purkinje cells in harmaline-induced essential tremor

Abstract

Essential tremor (ET) is one of the most common motor disorders with debilitating effects on the affected individuals. The endocannabinoid system is widely involved in cerebellar signaling. Therefore, modulation of cannabinoid-1 receptors (CB1Rs) has emerged as a novel target for motor disorders. In this study, we aimed to assess whether modulation of cannabinoid receptors (CBRs) could alter the electrophysiological properties of Purkinje cells (PCs) in the harmaline-induced ET model. Male Wistar rats were assigned to control, harmaline (30 mg/kg), CBR agonist WIN 55,212–2 (WIN; 1 mg/kg), CB1R antagonists AM251 (1 mg/kg) and rimonabant (10 mg/kg). Spontaneous activity and positive and negative evoked potentials of PCs were evaluated using whole-cell patch clamp recording. Findings demonstrated that harmaline exposure induced alterations in the spontaneous and evoked firing behavior of PCs, as evidenced by a significant decrease in the mean number of spikes and half-width of action potential in spontaneous activity. WIN administration exacerbated the electrophysiological function of PCs, particularly in the spontaneous activity of PCs. However, CB1R antagonists provided protective effects against harmaline-induced electrophysiological changes in the spontaneous activity of PCs. Our findings reinforce the pivotal role of the endocannabinoid system in the underlying electrophysiological mechanisms of cerebellar disorders and suggest that antagonism of CB1R might provide therapeutic utility.

Full text

IBRO Neuroscience Reports 17 (2024) 196–206
Available online 22 August 2024
2667-2421/© 2024 The Author(s). Published by Elsevier Inc. on behalf of International Brain Research Organization. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Research paper
Modulation of CB1 cannabinoid receptor alters the electrophysiological
properties of cerebellar Purkinje cells in harmaline-induced
essential tremor
Hassan Abbassian
a,1
, Mehran Ilaghi
b,1
, Reza Saboori Amleshi
b
, Benjamin Jason Whalley
c,d
,
Mohammad Shabani
b,*,2
a
Mashhad Neuroscience Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
b
Kerman Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran
c
Department of Pharmacy, School of Chemistry, Food & Nutritional Sciences and Pharmacy, University of Reading, Whiteknights, Reading, Berkshire RG6 6AP, UK
d
Revelstone Consulting LLC, 1001 New Jersey Ave SE, Washington, DC, 20003
ARTICLE INFO
Keywords:
Essential tremor
Cannabinoid receptor
Cerebellum
Purkinje cell
Whole-cell recording
ABSTRACT
Essential tremor (ET) is one of the most common motor disorders with debilitating effects on the affected in-
dividuals. The endocannabinoid system is widely involved in cerebellar signaling. Therefore, modulation of
cannabinoid-1 receptors (CB1Rs) has emerged as a novel target for motor disorders. In this study, we aimed to
assess whether modulation of cannabinoid receptors (CBRs) could alter the electrophysiological properties of
Purkinje cells (PCs) in the harmaline-induced ET model. Male Wistar rats were assigned to control, harmaline
(30 mg/kg), CBR agonist WIN 55,212–2 (WIN; 1 mg/kg), CB1R antagonists AM251 (1 mg/kg) and rimonabant
(10 mg/kg). Spontaneous activity and positive and negative evoked potentials of PCs were evaluated using
whole-cell patch clamp recording. Findings demonstrated that harmaline exposure induced alterations in the
spontaneous and evoked ring behavior of PCs, as evidenced by a signicant decrease in the mean number of
spikes and half-width of action potential in spontaneous activity. WIN administration exacerbated the electro-
physiological function of PCs, particularly in the spontaneous activity of PCs. However, CB1R antagonists pro-
vided protective effects against harmaline-induced electrophysiological changes in the spontaneous activity of
PCs. Our ndings reinforce the pivotal role of the endocannabinoid system in the underlying electrophysiological
mechanisms of cerebellar disorders and suggest that antagonism of CB1R might provide therapeutic utility.
1. Introduction
Essential tremor (ET) is one of the most common movement disor-
ders in adults, affecting almost 1 % of the world’s population and more
than 4 % of individuals aged above sixty-ve (Louis and Ferreira, 2010).
ET is characterized by a 4–12 Hz action tremor that can potentially affect
all body parts (Deuschl and Elble, 2009); however, other manifestations,
including gait ataxia, intention tremor, and cognitive impairments have
also been reported (Louis and Faust, 2020). Although the exact mech-
anism of ET is not fully understood, post-mortem studies have demon-
strated that degeneration of cerebellar Purkinje cells (PCs) may
contribute to the pathogenesis (Louis et al., 2009; Louis and Vonsattel,
2008).
Animal models of ET have given researchers a useful tool for dis-
secting the potential mechanisms underlying ET. Harmaline, an alkaloid
metabolite of the plant Peganum harmala, is one of the tremor-inducing
agents that causes action-dependent tremor in animals, similar to what
is observed in humans, through disruption of the olivocerebellar
pathway. Accordingly, harmaline causes neuronal desynchronization
and arrhythmicity in the inferior olive activity (Elble, 1998), resulting in
an 8–16 Hz tremor in rodents that is associated with dysfunction and
degeneration of PCs (Miwa and K, 2011).
The endocannabinoid system has been shown to be involved in a
wide variety of physiological functions in the central nervous system
* Correspondence to: Institute of Neuropharmacology, Kerman Neuroscience Research Center, Kerman University of Medical Sciences, Kerman, Iran.
E-mail addresses: shabanimoh@yahoo.com, shabani@kmu.ac.ir (M. Shabani).
1
Hassan Abbassian and Mehran Ilaghi contributed equally as rst authors.
2
ORCID ID: 0000–0002-2082–5849
Contents lists available at ScienceDirect
IBRO Neuroscience Reports
journal homepage: www.sciencedirect.com/journal/IBRO-Neuroscience-Reports
https://doi.org/10.1016/j.ibneur.2024.08.005
Received 11 March 2024; Received in revised form 30 July 2024; Accepted 17 August 2024

IBRO Neuroscience Reports 17 (2024) 196–206
197
(CNS) and peripheral organs. It has been previously shown that
cannabinoid-1 receptors (CB
1
Rs) are densely distributed in areas of the
brain related to motor control, cognition, emotional responses, moti-
vated behavior, and homeostasis (Rodriguez de Fonesca et al., 2005).
Moreover, modulating the endocannabinoid system has been proposed
to possess therapeutic utility in a wide range of diseases, including mood
disorders (Tambaro and Bortolato, 2012), neuropathic pain (Rahn and
Hohmann, 2009), multiple sclerosis (de Lago et al., 2009), and spinal
cord injury (Pacher et al., 2006).
In the case of the cerebellum, as CB
1
Rs are highly expressed at the
interneuron inputs into PCs, and at the presynaptic terminals of parallel
and climbing bers that form synapses with PCs, it is conjectured that
CB
1
Rs are probably involved in the control of cerebellar motor function
(Kreitzer and Regehr, 2001; Stephens, 2016). Therefore, modulation of
CB
1
Rs has been proposed as a therapeutic target in cerebellar disorders.
Reinforcing this evidence, it has recently been shown that CB
1
R mod-
ulation had therapeutic efcacy in the rat ataxia model (Ranjbar et al.,
2022). Moreover, we previously demonstrated that CB
1
R antagonism
ameliorates the effects of harmaline in a rat model of ET (Abbassian
et al., 2016), while modulation of this receptor can also affect cognitive
alterations induced by harmaline in the animal model of ET (Abbassian
et al., 2016).
While our previous research highlights the role of CB
1
Rs in the
behavioral function of the animal model of harmaline-induced ET
(Abbassian et al., 2016; Arjmand et al., 2015), the electrophysiological
properties of cerebellar PCs in response to CBR modulation remain
elusive. Therefore, in this study, we aimed to expand our knowledge of
the underlying mechanism through which cannabinoid receptor (CBR)
modulation exerts such effects through a series of whole-cell patch
clamp recordings in the PCs of harmaline-treated rats. Based on the
previous ndings on the impact of CBR modulation in movement dis-
orders, we hypothesize that CB
1
R antagonism might mitigate the
adverse electrophysiological alterations induced by harmaline.
2. Materials and methods
2.1. Animals
Male Wistar Kyoto rats (aged 4 weeks and weighed 40–50 g), pro-
vided by the Kerman Neuroscience Research Center, were used in the
current study. Animals were kept in cages of three with access to food
and water ad libitum. A 12/12 h dark/light cycle was maintained. All the
procedures in this study were performed in accordance with the Na-
tional Institutes of Health (NIH) guidelines and were approved by the
Kerman University of Medical Sciences Ethical Committee (Ethics code:
EC/KNRC/92–63).
2.2. Drugs
Harmaline hydrochloride dihydrate (Sigma, USA, 30 mg/kg; i.p.)
was used to induce the ET model. AM251 (Sigma, 1 mg/kg; i.p.) and
rimonabant (Cayman, USA, 10 mg/kg; i.p.) were utilized as selective
CB
1
R antagonists, and WIN55,212–2 (WIN; Sigma, USA, 1 mg/kg; i.p.)
was used as the CBR agonist agent. Agonist and antagonist doses were
selected according to our previous studies (Abbassian et al., 2016;
Ranjbar et al., 2023). Harmaline hydrochloride dihydrate was dissolved
in saline and CBR agonist and antagonist agents were rst dissolved in
dimethylsulfoxide (DMSO) before further dilution in dH2O (maximum
DMSO concentration: 1 % v/v).
2.3. Experimental design
Animals were divided into ve groups (n =6 in each group): The
control group received CBR agonist/antagonist vehicle (i.p.; adminis-
tered 30 min before harmaline vehicle) plus harmaline vehicle (i.p.).
The harmaline group received CBR agonist/antagonist vehicle (i.p.;
administered 30 min before harmaline) plus harmaline (30 mg/kg; i.p.).
The WIN group received WIN55,212–2 (1 mg/kg; i.p.; administered
30 min before harmaline) plus harmaline (30 mg/kg; i.p.). The AM251
group received AM251 (1 mg/kg; i.p.; administered 30 min before
harmaline) plus harmaline (30 mg/kg; i.p.) and the rimonabant group
received rimonabant (10 mg/kg; i.p.; administered 30 min before har-
maline) plus harmaline (30 mg/kg; i.p.). Data regarding the electro-
physiological investigations of control groups receiving agonist and
antagonist agents individually are provided in our previous study else-
where (Ranjbar et al., 2023), therefore these experiments were not
repeated here due to ethical considerations. One hour after the harma-
line injection, animals were decapitated under deep anesthesia, brains
were removed instantly and placed in ice-cold articial cerebrospinal
uid (ACSF), containing (in mM) 25 NaHCO
3
, 124 NaCl, 10 d-glucose,
4.4 KCl, 2 MgCl
2
, 2 CaCl
2
and1.25 KH
2
PO
4
, which was bubbled with
95 % O
2
and 5 % CO
2
(pH: 7.3–7.4). The osmolarity was adjusted to
295–305 mOsm (Haghani et al., 2013). Parasagittal Vermis slices
(300 µm) were prepared by vibroslicer (Campden Instrument, NVSLM1,
Sarasota, FL, USA) . After one-hour recovery, slices were placed in a
whole-cell patch clamp recording chamber immersed in carbogenated
ACSF at room temperature. To investigate the intrinsic ring charac-
teristics of Purkinje neurons, 1 mM kynurenic acid and 100 µM picro-
toxin were added to the recording ACSF to block ionotropic glutamate
and GABA receptors, respectively Razavinasab et al. (2020); Shabani
et al. (2011); Shabani et al. (2014).
2.4. Whole-cell recording
Whole-cell recording (WCR) was performed on the soma of PCs in
cerebellar parasagittal vermis slices. From each animal, 1–2 cells were
investigated. The nal number of cells qualied for analysis in this study
was 48 (10 cells in each of the control, harmaline, WIN +harmaline, and
rimonabant+harmaline groups, and 8 cells in the AM251 +harmaline
group). The changes in voltage as well as the active and passive prop-
erties of membrane were recorded in current clamp conguration. Glass
microelectrodes (TW150F- Axon Instruments Inc USA) with 6–9 MΩ tip
resistance and amplier (Multiclamp 700 B, Axon Instruments Inc. USA)
were used. Electrodes were made by a vertical puller (Narishige PC10)
and lled with intracellular solution containing potassium gluconate
(135 mM), KCl (5 mM), HEPES (10 mM), EGTA (0.2 mM), MgCl2
(2 mM), Na2-ATP (2 mM), and Na2-GTP (0.4 mM) to pH 7.2 with KOH.
Purkinje neurons were identied using an IR-DIC upright microscope
with an objective lens (×40 magnication). After Gigaseal had been
formed, the potential was maintained at −60 mv to prevent signicant
changes in membrane potential due to perfusion of intracellular uid by
micropipette solution. The hyperpolarizing and depolarizing steps in a
square shape from −0.5 to +0.5 nA in 520 msec were injected and
evoked responses of membranes were recorded. In the case of sponta-
neous activity, the changes in membrane potentials without injection of
current was recorded. The recorded properties were analyzed in pClamp
10.1 ofine software (Molecular Devices, LLC, CA, USA).
Electrophysiological records were sampled at 10 kHz and ltered at
5 kHz. Data was acquired and digitized with Digidata 1440 A (Axon
instrument) and the evoked responses were recorded in addition to the
spontaneous activity, and the passive properties of PCs. The following
parameters were measured: spike frequency in spontaneous activity,
after-hyperpolarization amplitude (AHP), amplitudes of action poten-
tials, rst spike latency after hyperpolarizing and depolarizing currents,
and SAG ratio (i.e. the difference between steady state and minimum tip
voltage of action potentials), resting membrane potentials, input resis-
tance, and membrane capacitance of PC membranes.
Overall, three sets of experiments were performed. Experiment 1
assessed the passive properties of PC membranes and the effects of drug
agents on the properties of spontaneous activity in PCs. Experiments 2
and 3 assessed the effects of the aforementioned agents on the positive
and negative evoked response of the cells, respectively.
H. Abbassian et al.

IBRO Neuroscience Reports 17 (2024) 196–206
198
2.5. Statistical analysis
SPSS 16 (IBM, USA), Origin (OriginLab Co., MA, USA), and Graph
Pad Prism 8 (Graph Pad Software, USA) were used for statistical analysis
of the data and gure production. All data were rst assessed for normal
distribution using a Kolmogorov-Smirnov test. The variables that found
to be normally distributed were expressed as mean ±SEM and analyzed
using one-way ANOVA test. Where a main effect was seen in ANOVA
tests, pairwise comparisons between groups were then made using
Tukey’s post-hoc tests. The variables that were not normally distributed
were expressed as median and interquartile range and were analyzed
using Kruskal-Wallis test followed by the Dunn post-hoc test if neces-
sary. A p<0.05 was considered statistically signicant.
3. Results
3.1. Experiment 1: comparison of the passive properties and spontaneous
activity parameters of PCs
A total of 48 PCs were recorded. The passive properties of PCs, which
included membrane resting potential, input resistance, and membrane
capacitance, are summarized in Table 1 (Table 1). Our ndings suggest
that there were no signicant differences observed among the groups in
terms of the passive properties of the PC membranes. We used input
resistance (Rin) as a measure of how the cells would passively respond to
current inputs. We found no statistical differences in Rin between any of
the PCs in treatment groups. This indicates that exposure to Harmaline,
with or without cannabinoid agonist or antagonist, did not have any
effect on Rin.
Comparison of the spontaneous activity of PCs, showed that a sig-
nicant difference in action potential frequency was evident between
the groups (F (4, 43) =41.14; p=0.0001; Fig. 1A), where subsequent
pairwise comparisons revealed that harmaline-treated PCs had a
signicantly reduced frequency of action potentials compared to the
control group (p<0.001). Moreover, harmaline +WIN-treated PCs had a
signicantly higher frequency of action potentials as compared to the
control (p<0.001) and harmaline (p<0.001) groups. Both antagonist
agents could reverse the effect of harmaline, so that the neural ring
frequency of AM251- and rimonabant-treated groups was not signi-
cantly different from the control group.
Furthermore, signicant alterations of action potential half-width
existed between the groups (F (4, 43) =11.58; p<0.0001; Fig. 1B).
Accordingly, a reduction in the action potential half-width occurred in
the harmaline group compared to the control group (p<0.01). Similar
ndings were observed in groups treated with WIN (p<0.001) and
AM251 (p<0.01). However, the rimonabant-treated group did not show
any difference compared to the control group.
WIN signicantly reduced the time to peak amplitudes of action
potentials (H (4) =19.29; p=0.0007; Fig. 1C), in contrast to the control
(p<0.001) and harmaline (p<0.05) groups. Coefcient of variation
(CV), i.e. an index of the regularity of action potentials, was also
signicantly reduced in WIN group compared to the harmaline group
(p<0.01; Fig. 1D). Additionally, WIN (p<0.01) and rimonabant
(p<0.05) signicantly reduced AHP amplitudes of action potentials in
spontaneous activity of PCs compared to the harmaline group (H (4)
=15.66; p=0.0035; Fig. 1E). Comparison of the peak amplitudes of ac-
tion potentials in spontaneous activity of PCs showed a signicant
reduction by WIN (p<0.001) and rimonabant (p<0.01) in comparison
with the harmaline group (H (4) =18.95; p=0.0008; Fig. 1F).
Fig. 2 demonstrates a conventional whole-cell current clamp
recording of PCs with spontaneous ring in control and drug-treated
groups. As observed, PCs of harmaline-treated rats exposed to WIN
exhibited a regular, high-frequency ring activity (Fig. 1A, D and Fig. 2).
3.2. Experiment 2: the effects of CBR modulation on the positive evoked
response of PCs
The median number of action potentials in response to 0.1 nA posi-
tive current was signicantly reduced with WIN and both antagonist
agents compared to the control and harmaline groups (H (4) =37.04;
p<0.0001; Fig. 3A). Moreover, applying 0.3 nA positive current resulted
in a signicant reduction of action potentials in both antagonist-treated
groups compared to the control group (H (4) =16.16; p<0.0028;
Fig. 3B). Increasing the positive current to 0.5 nA also resulted in a
signicant decrease in the number of action potentials in WIN and
antagonist groups compared to the control group (H (4) =25.38;
p<0.0001; Fig. 3C).
Analyzing the rst spike latency revealed a signicantly increased
rst spike delay in the WIN group compared to the control and harma-
line groups in response to the 0.1 nA positive current (H (4) =33.67;
p<0.0001; Fig. 3D). Furthermore, a 0.3 nA positive current was also
accompanied by a signicant increase in the rst spike delay in the WIN
group compared to the control group (H (4) =32.42; p<0.0001;
Fig. 3E). However, no signicant changes were observed in the rst
spike delay between the groups by increasing the positive current to
0.5 nA (H (4) =25.38; p<0.0001; Fig. 3F). Fig. 4 illustrates the traces
exhibiting the action potential ring rate and rst spike delay in
response to 0.1 nA (left panel) and 0.5 nA (right panel) currents (Fig. 4).
3.3. Experiment 3: the effects of CBR modulation on the negative evoked
response of PCs
The number of rebound action potentials was signicantly reduced
by applying −0.1 nA negative current in both antagonists, as well as
WIN group compared to the control group (H (4) =30.07; p<0.0001;
Fig. 5A). Applying higher negative currents as −0.2 nA (H (4) =32.19;
p<0.0001), −0.3 nA (H (4) =35.53; p<0.0001), −0.4 nA (H (4) =
21.50; p<0.0003), and −0.5 nA (H (4) =21.13; p<0.0003; Fig. 5B) led
to signicant reductions in the number of rebound action potentials by
both antagonist groups compared to the control group.
The rst spike latency of PCs in response to negative −0.1 nA current
was reduced in the harmaline group, as well as the WIN group, when
compared to the controls (H (4) =23.04; p<0.0001; Fig. 5C). However,
no signicant changes were observed in antagonist agents. Moreover,
applying a −0.5 nA current was accompanied by a signicant reduction
of rst spike delay in the WIN and AM251 groups compared to the
control group (H (4) =20.93; p<0.0003; Fig. 5D).
Median SAG voltage was reduced in all groups by applying −0.1 nA
Table 1
Passive properties of Purkinje cell membranes.
Membrane Properties Control Harmaline WIN +Harmaline AM251 +Harmaline Rimonabant +Harmaline P-Value
RMP (mV) −62.4±0.8 −58.3±0.7 −57.4±0.7 −59.2±0.9 −58.6±0.7 NS
R
in
(MΩ) 90.3±7.5 85.1±5.9 80.8±5.5 85.2±7.5 81.1±6.5 NS
Cm (pf) 36.5±5.2 38.9±6.4 43.2±4.2 45.1±6.2 39.2±4.4 NS
The input resistance (Rin) was determined by measuring the change in membrane potential caused by hyperpolarizing current steps (ranging from 0 to 0.6 nA for
1000 ms, in increments of 0.1 nA), while the neuron was maintained in a hyperpolarized state using direct current to prevent spontaneous ring. RMP (mV): Resting
Membrane Potential (in millivolts), Rin (MΩ): Input Resistance (in megaohms), and Cm (pF): Membrane Capacitance (in picofarads). NS: Non-signicant (p>0.05).
The data is presented as Mean ±SEM.
H. Abbassian et al.

IBRO Neuroscience Reports 17 (2024) 196–206
199
negative current, and this reduction was more prominent in the WIN-
treated group (H (4) =25.90; p<0.0001; Fig. 5E). This reduction was
continuously seen by applying more negative currents, including a
−0.5 nA negative current (H (4) =39.72; p<0.0001; Fig. 5F). Traces
recorded of action potentials in response to negative current is depicted
in Fig. 6 (Fig. 6).
Applying −0.1 nA negative prepulse before 0.1 nA positive evoked
currents resulted in a signicantly different number of rebound action
potentials between the groups (H (4) =23.87; p<0.0001; Fig. 7A).
Pairwise comparisons revealed a signicant reduction in the number of
rebound action potentials in all groups compared to the control group,
while WIN-treated mice also exhibited a signicant decrease in the
number of rebound action potentials compared to the control group.
Similar ndings were observed in response to 0.5 nA positive evoked
currents (H (4) =21.84; p<0.0002; Fig. 7B), except that in the rimo-
nabant group, the number of rebound action potentials were normalized
as the control group.
4. Discussion
Accumulating evidence is suggestive of the vital role that the endo-
cannabinoid system plays in the functioning of the cerebellum
(G´
omez-Ruiz et al., 2019; Martinez et al., 2020; Ranjbar et al., 2022).
Modulation of CBRs has therefore recently come to attention to assess its
therapeutic utility in disorders affecting the cerebellum. While the
behavioral effects of CB
1
R modulation are well-studied (Lupica and
Riegel, 2005); Jeff M (Witkin et al., 2005); Jeffrey M (Witkin et al.,
2005), the modulation of this receptor in ET is less examined. Therefore,
in this study, we assessed the effects of CB
1
R modulation in an animal
model of ET using patch-clamp intracellular recordings. Overall, our
ndings provided evidence that harmaline induces robust alterations in
the intrinsic electrophysiological properties of rat cerebellar PCs, while
these properties could be altered following the agonism and antagonism
of CB
1
R. Our novel ndings indicate signicant changes in a number of
electrophysiological properties, including spontaneous ring frequency
and responses to positive and negative currents in response to CB
1
R
modulation in the animal model of ET.
Analyzing the electrophysiological properties of PCs in our study
demonstrated that harmaline induced several changes in the electro-
physiological function of these cells. The observed changes included a
signicant reduction of action potential frequency, decreased action
potential half-width, reduced number of rebound action potentials in
negative evoked response, decreased rst spike latency and sag voltage,
as well as reduced number of rebound action potential following
hyperpolarizing prepulse. To gain a better understanding of the effects
of harmaline on the electrophysiological function of PCs, it should be
taken into account that previous studies have mainly focused on the
inferior olive nucleus, as the main area of interest that is affected by
harmaline toxicity and the tremorgenic effects of harmaline have been
generally attributed to its action on the inferior olive climbing ber
system (Handforth, 2012; Loyola et al., 2021). Following harmaline
exposure, inferior olive neurons show increased rebound low threshold
(T-type) calcium spikes, so that each rebound is linked with bursts of
sodium action potentials (Handforth, 2012). Moreover, harmaline re-
sults in the attenuation of both low-voltage-activated and sustained
high-voltage-activated calcium currents in the inferior olive (Zhan and
Graf, 2012). These changes result in the hyperexcitability of the inferior
olive neurons. On the other hand, the afferent inputs from olivary
climbing bers to the PCs are known to be responsible for the generation
of complex spike waveforms (De Gruijl et al., 2012; Gibson et al., 2002).
It has previously been demonstrated that PCs generally show increased
complex spike activity and decreased simple spike activity following
harmaline administration (Lorden et al., 1988; Stratton et al., 1988).
Complex spike discharges are believed to inactivate PCs and might be
expected to decrease the overall simple spike rate, presumably via the
depression of parallel ber inputs (Najac and Raman, 2015; Stratton
et al., 1988). Taken together, it seems that harmaline induces an in-
crease in the rhythmicity and frequency of olivary neuronal activity that
is reected in the complex spike activity and diminished simple spike
activity of the PCs. Supporting this, Stratton et al. have shown that
harmaline increased the complex spike activity from 1 spike/s to 5
spikes/s, while simple spike activity was completely suppressed by this
agent (Stratton et al., 1988). Similarly, our results are indicative that the
tonic ring of PCs is negatively affected by harmaline, resulting in less
Fig. 1. : Whole-cell patch clamp recordings revealed that signicant changes were observed in the spontaneous activity of Purkinje neurons, including (A) action
potential frequency, (B) action potential half-width, (C), time to peak, (D) coefcient variation, (E) AHP amplitude, and (F) peak amplitude after harmaline and
cannabinoid receptor agonist and antagonist administration. **: (p <0.01), ***: (p <0.001) represent signicant differences as compared to the control group; #: (p
<0.05), ##: (p <0.01), ###: (p <0.001) represent signicant differences as compared to the harmaline-treated group.
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200
excitability as seen in the forms of decreased number of action potentials
as well as a reduced number of rebound action potentials in response to
negative evoked response and following hyperpolarizing prepulse.
Building on previous evidence, our ndings suggest that the simple spike
activity of PCs is reduced following harmaline administration, presum-
ably due to an increased ring of climbing bers that inactivate the
simple spike ring of PCs.
The ndings of our study further demonstrated that the modulation
of CB
1
Rs could differentially impact the electrophysiological properties
of PCs treated with harmaline. We demonstrated that administration of
WIN was accompanied by a signicantly increased frequency of spon-
taneous action potentials, reduced time to peak, reduced AHP and
decreased peak amplitude, overall suggesting a hyperexcitability of PCs
under spontaneous activity. In positive evoked responses, a signicant
reduction of rebound action potentials as well as increased spike delay in
WIN-treated groups suggested an adaptation of PCs. Considerably, in
some parameters, not only did WIN fail to reverse harmaline-induced
changes, but also exacerbated the electrophysiological function of PCs.
Our previous study has also shown that WIN (in the absence of other
agents) led to hyperexcitability of neurons as well (Ranjbar et al., 2023).
Moreover, it has also been demonstrated that the application of WIN
leads to increased spontaneous ring frequency in the Purkinje neurons
of cerebellar slices (Fisyunov et al., 2006), which is consistent with the
current study indicating that WIN signicantly increased the frequency
of spontaneous activity of PCs in comparison to the control and har-
maline groups. It seems that WIN could intrinsically result in the
hyperexcitability of PCs. Data from behavioral experiments support our
ndings. For instance, Ranjbar et al. have demonstrated that WIN failed
to exert any protective effects on ataxic symptoms in an ataxia rat
model, but also worsened the symptoms (Ranjbar et al., 2022). In
another study, consistent with our results , it has been reported that WIN
exacerbated the induced effects of 3-acetylpyridine on the cerebellum by
increasing the frequency of action potentials, reducing the rheobase, as
well as increasing the excitatory postsynaptic potential, exhibiting
increased excitability of PCs (Ranjbar et al., 2023). Other reports of
cerebellar dysfunction following the administration of CB
1
R agonists
have also been reported (DeSanty and Dar, 2001; Patel and Hillard,
2001).
Several mechanisms might be involved in the observed effects of CBR
agonism on the cerebellum. It is established that CB
1
Rs are widely
expressed in the cerebellum and regulate the synaptic signaling of PCs
(Barnes et al., 2020; Marcaggi, 2015). Endocannabinoids are primarily
released by postsynaptic neurons as a response to synaptic activity and
exert retrograde inuence on presynaptic terminals, altering the release
of neurotransmitters (Carey et al., 2011). The CB1Rs are expressed at
presynaptic terminals of parallel bers and climbing bers that synapse
onto PCs, as well as on inhibitory interneurons. Therefore, it is quite
possible that these changes in PCs activity could be mediated, at least in
part, by alterations in retrograde endocannabinoid signaling within
cerebellar circuits. However, this mechanism seems to be related to
presynaptic terminals impinging on PC rather than the expression of
CB1Rs on PCs themselves. The localization of CB
1
Rs on PCs has been
Fig. 2. : A sample of traces recorded in spontaneous activity demonstrating the effects of drug agents in each group. The arrows indicate a reduction in the time scale
for better observation of a large number of action potentials in seconds. The traces showing the action potential of each group are overlaid (superimposed) on an
enlarged timescale. The action potential duration is shorter in the Purkinje neuron treated with harmaline and exposed to WIN. Additionally, the group treated with
WIN has a smaller after-hyperpolarization amplitude.
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201
controversial. While some immunohistochemical and in situ hybridiza-
tion studies have not detected CB
1
R expression on PCs (Mailleux and
Vanderhaeghen, 1992; Su´
arez et al., 2008), CB
1
R immunoreactivity has
been reported in PC bodies, dendrites, and a few axons according to
another previous study (Moldrich and Wenger, 2000). Therefore, it is
generally more plausible that the presynaptic terminals on PCs
contribute the most to the observed cross-talk of the cannabinoid system
and PC function. Previous reports have indicated that CB
1
R agonism
might inhibit L-type calcium channel currents (Gebremedhin et al.,
1999), thereby promoting sodium conductance, which might justify why
we observed increased excitability of PCs in WIN-treated PCs. Moreover,
direct activation of voltage-gated potassium channels, including fast
transient (A-type) and large conductance calcium-activated potassium
channels by cannabinoids has also been reported in previous studies
(Matsuda, 1997). Furthermore, the shortening of time to peak could be
attributed to the enhancement of the K
V
3 channel function, which is
thought to play a pivotal role in terminating the action potential in PCs
(McKay and Turner, 2004; Southan and Robertson, 2000). Moreover, as
observed in our ndings, although the number of action potentials in
response to three levels of positive currents were not changed by har-
maline, these parameters were signicantly decreased in the
WIN-treated groups. Furthermore, median rst spike latency in response
to positive evoked current increased signicantly by WIN; however, in
response to three levels of negative currents, we observed a signicant
decrease in agonist-treated groups in addition to reduced sag voltages,
suggesting a possible enhancement of transient K outward channel
currents in PCs. Transient outward K channel current has a modication
effect on rst spike latencies that is critical for neuronal coding and
synaptic integration in cerebellar neurons (Molineux et al., 2005; Shi-
bata et al., 2000). Moreover, the reduction in the inward rectication
following hyperpolarization, as evidenced by a signicant reduction in
the sag ratio, suggests that the I
h
channel current underlying inward
rectication might be altered by cannabinoids.
Unlike the adverse effects of CBR agonism on the electrophysiolog-
ical function of harmaline-treated PCs, we observed protective effects of
CB
1
R antagonism on PCs functioning. Both AM251 and rimonabant
could prevent the effects of harmaline on reducing the action potential
frequency of PCs. Rimonabant could also reverse the action potential
half-width to an extent comparable to the control group. The time to
peak and CV in antagonist-treated groups were also comparable to that
of control groups. Moreover, protective effects against reduced rst
spike latency in negative evoked response were noted. On the other
hand, we observed some alterations that were parallel with what was
observed in the WIN-treated PCs. These ndings were mainly observed
in positive and negative evoked responses. In these experiments, a sig-
nicant reduction of rebound action potentials was seen in WIN-treated
groups, which were, for most parts, aligned with the ndings of antag-
onist groups. We speculate that these observations suggest an adaptation
of PCs towards evoked responses. In other words, following the hyper-
excitability of neurons upon WIN treatment, neurons might enter a burst
mode, leading to an altered adaptation index and fewer subsequent
rebound action potentials, which might justify why these ndings were
comparable to antagonist-treated groups. Our ndings generally indi-
cate that CB
1
R antagonists might provide protective effects in the
spontaneous activity of PCs in the animal model of ET; however, their
clinical utility under evoked response requires further investigations. In
line with our ndings, through a series of behavioral experiments on the
harmaline-induced essential tremor model, we have previously exhibi-
ted that antagonism of CB
1
R, as opposed to its agonism, resulted in
signicant improvement of motor symptoms (Abbassian et al., 2016).
Supporting our ndings, Ranjbar et al. have recently demonstrated that
pharmacological antagonism of CB
1
Rs improved the electrophysiolog-
ical changes in PCs in an animal model of ataxia (Ranjbar et al., 2023).
In another study, they reported that CB
1
R antagonism signicantly
improved abnormal gait and ataxic symptoms (Ranjbar et al., 2022).
Moreover, antagonizing the CB
1
R with AM251 has also been shown to
prevent PCs neuronal degeneration and promote locomotor activity
(Ranjbar et al., 2022). As a whole, these observations are suggestive that
CB
1
R antagonists might ameliorate the electrophysiological dysfunc-
tions seen in animal model of ET.
While our study provides preliminary novel insights into the mod-
ulation of CB
1
Rs on cerebellar PC electrophysiology in the context of
Fig. 3. : Whole-cell patch clamp recordings revealed that signicant changes were observed in response to the positive current evoked in the Purkinje neurons,
including action potential number by (A) 0.1 nA, (B) 0.3 nA, and (C) 0.5 nA positive currents. The same changes were seen in the rst spike latency in response to (D)
0.1 nA, (E) 0.3 nA, and (F) 0.5 nA currents after harmaline and cannabinoid receptor agonist and antagonist administration. *: (p <0.05), **: (p <0.01), ***: (p <
0.001) represent signicant differences as compared to the control group. #: (p <0.05), ###: (p <0.001) represent signicant differences as compared to the
harmaline-treated group.
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Fig. 4. : The recorded traces that showed differences in the action potential ring rate and rst spike latency in response to positive current injections. Left panel:
0.1 nA positive current and right panel: 0.5 nA positive current.
H. Abbassian et al.

IBRO Neuroscience Reports 17 (2024) 196–206
203
harmaline-induced ET, there are several limitations that should be
considered. Firstly, the complexity of the cerebellar circuitry, with
CB
1
Rs expressed on several neuron types, introduces challenges in
isolating the specic contributions of CB
1
R modulation to harmaline-
induced effects. While harmaline primarily affects the inferior olive
climbing ber system, CB
1
Rs are also expressed on parallel bers and
interneurons. Therefore, the observed effects are not solely dependent
on harmaline-induced changes in the inferior olive pathway but may
involve direct modulation of interneurons and parallel bers by CB
1
Rs.
Secondly, the selected doses for CB
1
R agonists and antagonists were
based on our previous behavioral ndings, but alternative dose adjust-
ments may yield different electrophysiological outcomes. Moreover, the
behavioral outcomes resulting from CB
1
R modulation cannot be solely
justied by the observed electrophysiological alterations, indicating the
potential involvement of multiple mechanisms. Future investigations
should therefore aim to unravel these intricate mechanisms, providing a
more holistic understanding of how CB
1
R modulation inuences cere-
bellar function in the context of ET.
5. Conclusions
Overall, the ndings of our study indicated that harmaline induced
electrophysiological alterations in the PCs which were signicantly
exacerbated by agonism of CBR and in part reversed by antagonism of
these receptors. These ndings reinforce the utility of the acute har-
maline model of ET, for evaluating the potential therapeutic effects of
pharmacological interventions on cerebellar PCs. More importantly, our
study reinforces the pivotal role of the endocannabinoid system in the
underlying electrophysiological mechanisms of motor disorders. The
data suggests that further exploration into the modulation of CB1R an-
tagonists could lead to the development of targeted therapies for ET.
Authors’ contributions
HA contributed to study conception and design, acquisition of animal
data, and writing the initial draft of the manuscript. MI, RSA and BJW
assisted with study conception and design, interpretation of the findings,
writing the manuscript, and critical revision . MS contributed to study
Fig. 5. : Whole-cell patch clamp recordings revealed that signicant changes were observed in response to the negative current in the Purkinje neurons, including
action potential number by (A) −0.1 nA and (B) −0.5 nA negative currents, changes in the rst spike latency in response to (C) −0.1 nA and (D) −0.5 nA negative
currents, Sag voltage in response to (E) −0.1 nA and (F) −0.5 nA negative currents. *: (p <0.05), **: (p <0.01), ***: (p <0.001) represent signicant differences as
compared to the control group. #: (p <0.05), ###: (p <0.001) represent signicant differences as compared to the harmaline-treated group.
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Fig. 6. : A sample of traces recorded of the response of PCs towards negative current. Left panel: −0.1 nA negative current and right panel: −0.5 nA negative current.
H. Abbassian et al.

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conception and design, analyzing data, interpretation of the findings,
and providinga critical revision of the manuscript. All authors critically
reviewed the content and approved the final version for publication.
Ethical statement
All experiments were done in accordance with the National Institutes
of Health Guide for the Care and Use of Laboratory Animals) NIH Pub-
lication No. 80–23, revised 1996). All procedures performed in this
study were in accordance with the ethical standards of the ethical
committee of Kerman University of Medical Sciences (Ethical approval
number EC/KNRC/92–63).
CRediT authorship contribution statement
Hassan Abbassian: Conceptualization, Data curation, Investigation,
Methodology, Software, Validation, Writing – original draft, Writing –
review & editing. Mohammad Shabani: Conceptualization, Data
curation, Formal analysis, Funding acquisition, Investigation, Method-
ology, Project administration, Supervision, Validation, Visualization,
Writing – original draft, Writing – review & editing. Benjamin Jason
Whalley: Conceptualization, Methodology, Writing – original draft.
Reza Saboori Amleshi: Data curation, Software, Writing – original
draft. Mehran Ilaghi: Conceptualization, Data curation, Investigation,
Methodology, Software, Validation, Writing – original draft.
Declaration of Competing Interest
The authors declare no conict of interest and declare that the
research was conducted in the absence of any commercial or nancial
relationships that could be construed as a potential conict of interest.
Data Availability
Data generated or analyzed during this study are available from the
corresponding author upon reasonable request.
Acknowledgements
Funding for this study was provided by Kerman University of
Medical Sciences as a grant (KNRC/92–63) for the PhD thesis conducted
by HA.
References
Abbassian, H., Esmaeili, P., Tahamtan, M., Aghaei, I., Vaziri, Z., Sheibani, V.,
Shabani, M., 2016. Cannabinoid receptor agonism suppresses tremor, cognition
disturbances and anxiety-like behaviors in a rat model of essential tremor. Physiol.
Behav. 164 (Pt A), 314–320. https://doi.org/10.1016/j.physbeh.2016.06.013.
Abbassian, H., Whalley, B.J., Sheibani, V., Shabani, M., 2016. Cannabinoid type 1
receptor antagonism ameliorates harmaline-induced essential tremor in rat. Br. J.
Pharm. 173 (22), 3196–3207. https://doi.org/10.1111/bph.13581.
Arjmand, S., Vaziri, Z., Behzadi, M., Abbassian, H., Stephens, G.J., Shabani, M., 2015.
Cannabinoids and tremor induced by motor-related disorders: friend or foe?
Neurotherapeutics 12 (4), 778–787. https://doi.org/10.1007/s13311-015-0367-5.
Barnes, J.L., Mohr, C., Ritchey, C.R., Erikson, C.M., Shiina, H., Rossi, D.J., 2020.
Developmentally transient CB1Rs on cerebellar afferents suppress afferent input,
downstream synaptic excitation, and signaling to migrating neurons. J. Neurosci. 40
(32), 6133–6145.
Carey, M.R., Myoga, M.H., McDaniels, K.R., Marsicano, G., Lutz, B., Mackie, K.,
Regehr, W.G., 2011. Presynaptic CB1 receptors regulate synaptic plasticity at
cerebellar parallel ber synapses. J. Neurophysiol. 105 (2), 958–963.
De Gruijl, J.R., Bazzigaluppi, P., de Jeu, M.T., De Zeeuw, C.I., 2012. Climbing ber burst
size and olivary sub-threshold oscillations in a network setting. PLoS Comput. Biol. 8
(12), e1002814.
de Lago, E., Gomez-Ruiz, M., Moreno-Martet, M., Fernandez-Ruiz, J., 2009.
Cannabinoids, multiple sclerosis and neuroprotection. Expert Rev. Clin. Pharm. 2
(6), 645–660. https://doi.org/10.1586/ecp.09.42.
DeSanty, K., Dar, M.S., 2001. Cannabinoid-induced motor incoordination through the
cerebellar CB1 receptor in mice. Pharmacol. Biochem. Behav. 69 (1-2), 251–259.
Deuschl, G., Elble, R., 2009. Essential tremor–neurodegenerative or nondegenerative
disease towards a working denition of ET. Mov. Disord. 24 (14), 2033–2041.
https://doi.org/10.1002/mds.22755.
Elble, R.J., 1998. Animal models of action tremor. Mov. Disord. 13 (Suppl 3), 35–39.
https://doi.org/10.1002/mds.870131306.
Fisyunov, A., Tsintsadze, V., Min, R., Burnashev, N., Lozovaya, N., 2006. Cannabinoids
modulate the P-type high-voltage-activated calcium currents in purkinje neurons.
J. Neurophysiol. 96 (3), 1267–1277. https://doi.org/10.1152/jn.01227.2005.
Gebremedhin, D., Lange, A.R., Campbell, W.B., Hillard, C.J., Harder, D.R., 1999.
Cannabinoid CB1 receptor of cat cerebral arterial muscle functions to inhibit L-type
Ca2+channel current. Am. J. Physiol. -Heart Circ. Physiol. 276 (6), H2085–H2093.
Gibson, A.R., Horn, K.M., Pong, M., 2002. Inhibitory control of olivary discharge. Ann. N.
Y. Acad. Sci. 978 (1), 219–231.
G´
omez-Ruiz, M., Rodríguez-Cueto, C., Luna-Pi˜
nel, E., Hern´
andez-G´
alvez, M., Fern´
andez-
Ruiz, J., 2019. Endocannabinoid system in spinocerebellar ataxia type-3 and other
autosomal-dominant cerebellar ataxias: potential role in pathogenesis and expected
relevance as neuroprotective targets. Front. Mol. Neurosci. 12, 94.
Haghani, M., Shabani, M., Moazzami, K., 2013. Maternal mobile phone exposure
adversely affects the electrophysiological properties of Purkinje neurons in rat
offspring. Neuroscience 250, 588–598.
Fig. 7. : Applying prepulse showed signicant changes in number of action potentials as seen in (A) prepulse −0.1 nA (0.1 nA test pulse) and (B) −0.1 nA prepulse
(0.5 nA test pulse) after harmaline and cannabinoid receptor agonist and antagonist administration. *: (p <0.05), **: (p <0.01) represent signicant differences as
compared to the control group. #: (p <0.05), ##: (p <0.01) represent signicant differences as compared to the harmaline-treated group.
H. Abbassian et al.

IBRO Neuroscience Reports 17 (2024) 196–206
206
Handforth, A., 2012. Harmaline tremor: underlying mechanisms in a potential animal
model of essential tremor. Tremor Other Hyperkinet Mov. 2. https://doi.org/
10.7916/d8td9w2p.
Kreitzer, A.C., Regehr, W.G., 2001. Retrograde inhibition of presynaptic calcium inux
by endogenous cannabinoids at excitatory synapses onto Purkinje cells. Neuron 29
(3), 717–727.
Lorden, J., Stratton, S., Mays, L., Oltmans, G., 1988. Purkinje cell activity in rats
following chronic treatment with harmaline. Neuroscience 27 (2), 465–472.
Louis, E.D., Faust, P.L., 2020. Essential tremor: the most common form of cerebellar
degeneration? Cerebellum Ataxias 7 (1), 1–10.
Louis, E.D., Faust, P.L., Vonsattel, J.-P.G., Honig, L.S., Henchcliffe, C., Pahwa, R.,
Lawton, A., 2009. Older onset essential tremor: more rapid progression and more
degenerative pathology. Mov. Disord. 24 (11), 1606–1612. https://doi.org/
10.1002/mds.22570.
Louis, E.D., Ferreira, J.J., 2010. How common is the most common adult movement
disorder? Update on the worldwide prevalence of essential tremor. Mov. Disord. 25
(5), 534–541.
Louis, E.D., Vonsattel, J.P.G., 2008. The emerging neuropathology of essential tremor.
Mov. Disord. 23 (2), 174–182. https://doi.org/10.1002/mds.21731.
Loyola, S., Bosman, L., De Gruijl, J., De Jeu, M., Negrello, M., Hoogland, T., De
Zeeuw, C., 2021. Inferior olive: all ins and outs Handbook of the Cerebellum and
Cerebellar Disorders. Springer, pp. 1137–1192.
Lupica, C.R., Riegel, A.C., 2005. Endocannabinoid release from midbrain dopamine
neurons: a potential substrate for cannabinoid receptor antagonist treatment of
addiction. Neuropharmacology 48 (8), 1105–1116.
Mailleux, P., Vanderhaeghen, J.-J., 1992. Distribution of neuronal cannabinoid receptor
in the adult rat brain: a comparative receptor binding radioautography and in situ
hybridization histochemistry. Neuroscience 48 (3), 655–668.
Marcaggi, P., 2015. Cerebellar endocannabinoids: retrograde signaling from purkinje
cells. Cerebellum 14, 341–353.
Martinez, L.R., Black, K.C., Webb, B.T., Bell, A., Baygani, S.K., Mier, T.J., Kalinovsky, A.,
2020. Components of endocannabinoid signaling system are expressed in the
perinatal mouse cerebellum and required for its normal development. Eneuro 7 (2).
Matsuda, L.A., 1997. Molecular aspects of cannabinoid receptors. Crit. Rev. Neurobiol.
11 (2-3), 143–166.
McKay, B.E., Turner, R.W., 2004. Kv3 K+channels enable burst output in rat cerebellar
Purkinje cells. Eur. J. Neurosci. 20 (3), 729–739. https://doi.org/10.1111/j.1460-
9568.2004.03539.x.
Miwa, H., K, T., 2011. T-type calcium channel as a new therapeutic target for tremor.
Cerebellum 10 (3), 7.
Moldrich, G., Wenger, T., 2000. Localization of the CB1 cannabinoid receptor in the rat
brain. An immunohistochemical study☆. Peptides 21 (11), 1735–1742.
Molineux, M.L., Fernandez, F.R., Mehaffey, W.H., Turner, R.W., 2005. A-type and T-type
currents interact to produce a novel spike latency-voltage relationship in cerebellar
stellate cells. J. Neurosci. 25 (47), 10863–10873. https://doi.org/10.1523/
jneurosci.3436-05.2005.
Najac, M., Raman, I.M., 2015. Integration of Purkinje cell inhibition by cerebellar
nucleo-olivary neurons. J. Neurosci. 35 (2), 544–549.
Pacher, P., B´
atkai, S., Kunos, G., 2006. The endocannabinoid system as an emerging
target of pharmacotherapy. Pharmacol. Rev. 58 (3), 389–462. https://doi.org/
10.1124/pr.58.3.2.
Patel, S., Hillard, C.J., 2001. Cannabinoid CB1 receptor agonists produce cerebellar
dysfunction in mice. J. Pharmacol. Exp. Ther. 297 (2), 629–637.
Rahn, E.J., Hohmann, A.G., 2009. Cannabinoids as pharmacotherapies for neuropathic
pain: from the bench to the bedside. Neurotherapeutics 6 (4), 713–737. https://doi.
org/10.1016/j.nurt.2009.08.002.
Ranjbar, H., Soti, M., Janahmadi, M., Kohlmeier, K.A., Sheibani, V., Ahmadi-
Zeidabadi, M., Shabani, M., 2022. Modulation of the CB1 cannabinoid receptor has
potential therapeutic utility in the 3-acetylpyridine cerebellar ataxia rat model. Exp.
Brain Res. 240 (9), 2339–2348.
Ranjbar, H., Soti, M., Kohlmeier, K.A., Janahmadi, M., Shabani, M., 2023. Pharmacologic
antagonism of CB1 receptors improves electrophysiological alterations in Purkinje
cells exposed to 3-AP. BMC Neurosci. 24 (1), 1–11.
Ranjbar, H., Soti, M., Razavinasab, M., Kohlmeier, K.A., Shabani, M., 2022. The
neglected role of endocannabinoid actions at TRPC channels in ataxia. Neurosci.
Biobehav. Rev., 104860
Razavinasab, M., Sheibani, V., Kohlmeier, K.A., Nazeri, M., Shabani, M., 2020.
Hyperexcitability of VTA dopaminergic neurons in male offspring exposed to
physical or psychological prenatal stress. Prog. Neuro-Psychopharmacol. Biol.
Psychiatry 101, 109923.
Rodriguez de Fonesca, F., Del Arco, I., Bermudez-Silva, F.J., Bilbao, A., Cippitelli, A.,
Navarro, M., 2005. The endocannabinoid system: physiology and pharmacology.
Alcohol. Alcohol. 40 (1), 2–14. https://doi.org/10.1093/alcalc/agh110.
Shabani, M., Hosseinmardi, N., Haghani, M., Shaibani, V., Janahmadi, M., 2011.
Maternal exposure to the CB1 cannabinoid agonist WIN 55212–2 produces robust
changes in motor function and intrinsic electrophysiological properties of cerebellar
Purkinje neurons in rat offspring. Neuroscience 172, 139–152.
Shabani, M., Mahnam, A., Sheibani, V., Janahmadi, M., 2014. Alterations in the intrinsic
burst activity of Purkinje neurons in offspring maternally exposed to the CB1
Cannabinoid agonist WIN 55212-2. J. Membr. Biol. 247, 63–72.
Shibata, R., Nakahira, K., Shibasaki, K., Wakazono, Y., Imoto, K., Ikenaka, K., 2000. A-
type K+current mediated by the Kv4 channel regulates the generation of action
potential in developing cerebellar granule cells. J. Neurosci. 20 (11), 4145–4155.
Southan, A.P., Robertson, B., 2000. Electrophysiological characterization of voltage-
gated K(+) currents in cerebellar basket and purkinje cells: Kv1 and Kv3 channel
subfamilies are present in basket cell nerve terminals. J. Neurosci. 20 (1), 114–122.
Stephens, G.J., 2016. Does modulation of the endocannabinoid system have potential
therapeutic utility in cerebellar ataxia? J. Physiol. https://doi.org/10.1113/
jp271106.
Stratton, S., Lorden, J., Mays, L., Oltmans, G., 1988. Spontaneous and harmaline-
stimulated Purkinje cell activity in rats with a genetic movement disorder.
J. Neurosci. 8 (9), 3327–3336.
Su´
arez, J., Bermúdez-Silva, F.J., Mackie, K., Ledent, C., Zimmer, A., Cravatt, B.F., de
Fonseca, F.R., 2008. Immunohistochemical description of the endogenous
cannabinoid system in the rat cerebellum and functionally related nuclei. J. Comp.
Neurol. 509 (4), 400–421.
Tambaro, S., Bortolato, M., 2012. Cannabinoid-related agents in the treatment of anxiety
disorders: current knowledge and future perspectives. Recent Pat. CNS Drug Discov.
7 (1), 25–40.
Witkin, J.M., Tzavara, E., Nomikos, G., 2005. A role for cannabinoid CB1 receptors in
mood and anxiety disorders. Behav. Pharmacol. 16 (5-6), 315–331.
Witkin, J.M., Tzavara, E.T., Davis, R.J., Li, X., Nomikos, G.G., 2005. A therapeutic role
for cannabinoid CB1 receptor antagonists in major depressive disorders. Trends
Pharmacol. Sci. 26 (12), 609–617.
Zhan, X., Graf, W.M., 2012. Harmaline attenuates voltage-sensitive Ca2+currents in
neurons of the inferior olive. J. Pharm. Pharm. Sci. 15 (5), 657–668.
H. Abbassian et al.