
==== Front
IBRO Neurosci Rep
IBRO Neurosci Rep
IBRO Neuroscience Reports
2667-2421
Elsevier

S2667-2421(24)00075-7
10.1016/j.ibneur.2024.08.005
Research Paper
Modulation of CB1 cannabinoid receptor alters the electrophysiological properties of cerebellar Purkinje cells in harmaline-induced essential tremor
Abbassian Hassan a1
Ilaghi Mehran b1
Amleshi Reza Saboori b
Whalley Benjamin Jason cd
Shabani Mohammad shabanimoh@yahoo.com
shabani@kmu.ac.ir
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
⁎ Correspondence to: Institute of Neuropharmacology, Kerman Neuroscience Research Center, Kerman University of Medical Sciences, Kerman, Iran. shabanimoh@yahoo.comshabani@kmu.ac.ir
1 Hassan Abbassian and Mehran Ilaghi contributed equally as first authors.

2 ORCID ID: 0000–0002-2082–5849

22 8 2024
12 2024
22 8 2024
17 196206
11 3 2024
30 7 2024
17 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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.

Highlights

• Harmaline exposure caused changes in the firing of Purkinje cells (PCs).

• Administration of WIN worsened the electrophysiological function of PCs.

• CB1R antagonists had protective effects against harmaline-induced changes.

• Antagonism of CB1R may have therapeutic benefits.

Keywords

Essential tremor
Cannabinoid receptor
Cerebellum
Purkinje cell
Whole-cell recording
==== Body
pmc1 Introduction

Essential tremor (ET) is one of the most common movement disorders in adults, affecting almost 1 % of the world's population and more than 4 % of individuals aged above sixty-five (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 mechanism of ET is not fully understood, post-mortem studies have demonstrated 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 dissecting 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 (CNS) and peripheral organs. It has been previously shown that cannabinoid-1 receptors (CB1Rs) are densely distributed in areas of the brain related to motor control, cognition, emotional responses, motivated 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 CB1Rs are highly expressed at the interneuron inputs into PCs, and at the presynaptic terminals of parallel and climbing fibers that form synapses with PCs, it is conjectured that CB1Rs are probably involved in the control of cerebellar motor function (Kreitzer and Regehr, 2001, Stephens, 2016). Therefore, modulation of CB1Rs has been proposed as a therapeutic target in cerebellar disorders. Reinforcing this evidence, it has recently been shown that CB1R modulation had therapeutic efficacy in the rat ataxia model (Ranjbar et al., 2022). Moreover, we previously demonstrated that CB1R 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 CB1Rs in the behavioral function of the animal model of harmaline-induced ET (Abbassian et al., 2016; 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 findings on the impact of CBR modulation in movement disorders, we hypothesize that CB1R 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), provided 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 National 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 CB1R 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 first dissolved in dimethylsulfoxide (DMSO) before further dilution in dH2O (maximum DMSO concentration: 1 % v/v).

2.3 Experimental design

Animals were divided into five groups (n = 6 in each group): The control group received CBR agonist/antagonist vehicle (i.p.; administered 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 harmaline) plus harmaline (30 mg/kg; i.p.). Data regarding the electrophysiological investigations of control groups receiving agonist and antagonist agents individually are provided in our previous study elsewhere (Ranjbar et al., 2023), therefore these experiments were not repeated here due to ethical considerations. One hour after the harmaline injection, animals were decapitated under deep anesthesia, brains were removed instantly and placed in ice-cold artificial cerebrospinal fluid (ACSF), containing (in mM) 25 NaHCO3, 124 NaCl, 10 d-glucose, 4.4 KCl, 2 MgCl2, 2 CaCl2 and1.25 KH2PO4, which was bubbled with 95 % O2 and 5 % CO2 (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 firing characteristics of Purkinje neurons, 1 mM kynurenic acid and 100 µM picrotoxin 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 final number of cells qualified 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 properties of membrane were recorded in current clamp configuration. Glass microelectrodes (TW150F- Axon Instruments Inc USA) with 6–9 MΩ tip resistance and amplifier (Multiclamp 700 B, Axon Instruments Inc. USA) were used. Electrodes were made by a vertical puller (Narishige PC10) and filled 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 identified using an IR-DIC upright microscope with an objective lens (×40 magnification). After Gigaseal had been formed, the potential was maintained at −60 mv to prevent significant changes in membrane potential due to perfusion of intracellular fluid 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 spontaneous activity, the changes in membrane potentials without injection of current was recorded. The recorded properties were analyzed in pClamp 10.1 offline software (Molecular Devices, LLC, CA, USA).

Electrophysiological records were sampled at 10 kHz and filtered 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 potentials, first 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 resistance, 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.

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 figure production. All data were first 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 necessary. A p<0.05 was considered statistically significant.

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 findings suggest that there were no significant 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.Table 1 Passive properties of Purkinje cell membranes.

Table 1Membrane 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	
Rin (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 firing. RMP (mV): Resting Membrane Potential (in millivolts), Rin (MΩ): Input Resistance (in megaohms), and Cm (pF): Membrane Capacitance (in picofarads). NS: Non-significant (p>0.05). The data is presented as Mean ± SEM.

Comparison of the spontaneous activity of PCs, showed that a significant 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 significantly reduced frequency of action potentials compared to the control group (p<0.001). Moreover, harmaline + WIN-treated PCs had a significantly 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 firing frequency of AM251- and rimonabant-treated groups was not significantly different from the control group.Fig. 1 : Whole-cell patch clamp recordings revealed that significant 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) coefficient variation, (E) AHP amplitude, and (F) peak amplitude after harmaline and cannabinoid receptor agonist and antagonist administration. **: (p < 0.01), ***: (p < 0.001) represent significant differences as compared to the control group; #: (p < 0.05), ##: (p < 0.01), ###: (p < 0.001) represent significant differences as compared to the harmaline-treated group.

Fig. 1

Furthermore, significant 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 findings 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 significantly 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. Coefficient of variation (CV), i.e. an index of the regularity of action potentials, was also significantly reduced in WIN group compared to the harmaline group (p<0.01; Fig. 1D). Additionally, WIN (p<0.01) and rimonabant (p<0.05) significantly 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 action potentials in spontaneous activity of PCs showed a significant 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 firing in control and drug-treated groups. As observed, PCs of harmaline-treated rats exposed to WIN exhibited a regular, high-frequency firing activity (Fig. 1A, D and Fig. 2).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.

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 positive current was significantly 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 significant 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 significant 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).Fig. 3 : Whole-cell patch clamp recordings revealed that significant 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 first 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 significant differences as compared to the control group. #: (p < 0.05), ###: (p < 0.001) represent significant differences as compared to the harmaline-treated group.

Fig. 3

Analyzing the first spike latency revealed a significantly increased first spike delay in the WIN group compared to the control and harmaline 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 significant increase in the first spike delay in the WIN group compared to the control group (H (4) = 32.42; p<0.0001; Fig. 3E). However, no significant changes were observed in the first 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 firing rate and first spike delay in response to 0.1 nA (left panel) and 0.5 nA (right panel) currents (Fig. 4).Fig. 4 : The recorded traces that showed differences in the action potential firing rate and first spike latency in response to positive current injections. Left panel: 0.1 nA positive current and right panel: 0.5 nA positive current.

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 significantly 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 significant reductions in the number of rebound action potentials by both antagonist groups compared to the control group.Fig. 5 : Whole-cell patch clamp recordings revealed that significant 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 first 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 significant differences as compared to the control group. #: (p < 0.05), ###: (p < 0.001) represent significant differences as compared to the harmaline-treated group.

Fig. 5

The first 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 significant changes were observed in antagonist agents. Moreover, applying a −0.5 nA current was accompanied by a significant reduction of first 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 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).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.

Fig. 6

Applying −0.1 nA negative prepulse before 0.1 nA positive evoked currents resulted in a significantly different number of rebound action potentials between the groups (H (4) = 23.87; p<0.0001; Fig. 7A). Pairwise comparisons revealed a significant reduction in the number of rebound action potentials in all groups compared to the control group, while WIN-treated mice also exhibited a significant decrease in the number of rebound action potentials compared to the control group. Similar findings were observed in response to 0.5 nA positive evoked currents (H (4) = 21.84; p<0.0002; Fig. 7B), except that in the rimonabant group, the number of rebound action potentials were normalized as the control group.Fig. 7 : Applying prepulse showed significant 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 significant differences as compared to the control group. #: (p < 0.05), ##: (p < 0.01) represent significant differences as compared to the harmaline-treated group.

Fig. 7

4 Discussion

Accumulating evidence is suggestive of the vital role that the endocannabinoid system plays in the functioning of the cerebellum (Gómez-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 CB1R 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 CB1R modulation in an animal model of ET using patch-clamp intracellular recordings. Overall, our findings 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 CB1R. Our novel findings indicate significant changes in a number of electrophysiological properties, including spontaneous firing frequency and responses to positive and negative currents in response to CB1R modulation in the animal model of ET.

Analyzing the electrophysiological properties of PCs in our study demonstrated that harmaline induced several changes in the electrophysiological function of these cells. The observed changes included a significant reduction of action potential frequency, decreased action potential half-width, reduced number of rebound action potentials in negative evoked response, decreased first 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 fiber 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 results 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 fibers 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 fiber inputs (Najac and Raman, 2015, Stratton et al., 1988). Taken together, it seems that harmaline induces an increase in the rhythmicity and frequency of olivary neuronal activity that is reflected 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 firing of PCs is negatively affected by harmaline, resulting in less 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 findings suggest that the simple spike activity of PCs is reduced following harmaline administration, presumably due to an increased firing of climbing fibers that inactivate the simple spike firing of PCs.

The findings of our study further demonstrated that the modulation of CB1Rs could differentially impact the electrophysiological properties of PCs treated with harmaline. We demonstrated that administration of WIN was accompanied by a significantly increased frequency of spontaneous 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 significant 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 firing frequency in the Purkinje neurons of cerebellar slices (Fisyunov et al., 2006), which is consistent with the current study indicating that WIN significantly increased the frequency of spontaneous activity of PCs in comparison to the control and harmaline groups. It seems that WIN could intrinsically result in the hyperexcitability of PCs. Data from behavioral experiments support our findings. 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 CB1R 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 CB1Rs 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 influence on presynaptic terminals, altering the release of neurotransmitters (Carey et al., 2011). The CB1Rs are expressed at presynaptic terminals of parallel fibers and climbing fibers 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 CB1Rs on PCs has been controversial. While some immunohistochemical and in situ hybridization studies have not detected CB1R expression on PCs (Mailleux and Vanderhaeghen, 1992, Suárez et al., 2008), CB1R 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 CB1R 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 KV3 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 findings, although the number of action potentials in response to three levels of positive currents were not changed by harmaline, these parameters were significantly decreased in the WIN-treated groups. Furthermore, median first spike latency in response to positive evoked current increased significantly by WIN; however, in response to three levels of negative currents, we observed a significant 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 modification effect on first spike latencies that is critical for neuronal coding and synaptic integration in cerebellar neurons (Molineux et al., 2005, Shibata et al., 2000). Moreover, the reduction in the inward rectification following hyperpolarization, as evidenced by a significant reduction in the sag ratio, suggests that the Ih channel current underlying inward rectification might be altered by cannabinoids.

Unlike the adverse effects of CBR agonism on the electrophysiological function of harmaline-treated PCs, we observed protective effects of CB1R 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 first 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 findings were mainly observed in positive and negative evoked responses. In these experiments, a significant reduction of rebound action potentials was seen in WIN-treated groups, which were, for most parts, aligned with the findings of antagonist groups. We speculate that these observations suggest an adaptation of PCs towards evoked responses. In other words, following the hyperexcitability 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 findings were comparable to antagonist-treated groups. Our findings generally indicate that CB1R 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 findings, through a series of behavioral experiments on the harmaline-induced essential tremor model, we have previously exhibited that antagonism of CB1R, as opposed to its agonism, resulted in significant improvement of motor symptoms (Abbassian et al., 2016). Supporting our findings, Ranjbar et al. have recently demonstrated that pharmacological antagonism of CB1Rs improved the electrophysiological changes in PCs in an animal model of ataxia (Ranjbar et al., 2023). In another study, they reported that CB1R antagonism significantly improved abnormal gait and ataxic symptoms (Ranjbar et al., 2022). Moreover, antagonizing the CB1R 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 CB1R antagonists might ameliorate the electrophysiological dysfunctions seen in animal model of ET.

While our study provides preliminary novel insights into the modulation of CB1Rs on cerebellar PC electrophysiology in the context of harmaline-induced ET, there are several limitations that should be considered. Firstly, the complexity of the cerebellar circuitry, with CB1Rs expressed on several neuron types, introduces challenges in isolating the specific contributions of CB1R modulation to harmaline-induced effects. While harmaline primarily affects the inferior olive climbing fiber system, CB1Rs are also expressed on parallel fibers 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 fibers by CB1Rs. Secondly, the selected doses for CB1R agonists and antagonists were based on our previous behavioral findings, but alternative dose adjustments may yield different electrophysiological outcomes. Moreover, the behavioral outcomes resulting from CB1R modulation cannot be solely justified 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 CB1R modulation influences cerebellar function in the context of ET.

5 Conclusions

Overall, the findings of our study indicated that harmaline induced electrophysiological alterations in the PCs which were significantly exacerbated by agonism of CBR and in part reversed by antagonism of these receptors. These findings reinforce the utility of the acute harmaline 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 antagonists 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 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 Publication 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, Methodology, 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 conflict of interest and declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict 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 10.13039/501100004621 Kerman University of Medical Sciences as a grant (KNRC/92–63 ) for the PhD thesis conducted by HA.
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