Access the full text.
Sign up today, get DeepDyve free for 14 days.
Loading next page...
/lp/springer-journals/pharmacologic-antagonism-of-cb1-receptors-improves-kxMQ88IVLZ
- Publisher
- Springer Journals
- Copyright
- Copyright © The Author(s) 2023
- eISSN
- 1471-2202
- DOI
- 10.1186/s12868-023-00786-4
- Publisher site
- See Article on Publisher Site
Abstract
Introduction Although ataxia is associated with cerebellar dysfunction, little is known about the effects of 3-AP exposure on Purkinje cell electrophysiological properties. Here, we evaluated these parameters in cerebellar vermis brain slices. Methods Purkinje cells were exposed to artificial cerebrospinal fluid (aCSF) (control) or to 1 mM 3-acetylpyridine (3-AP) in the recording chamber. The effects of a cannabinoid agonist ( WIN; 7.5 nmol) and a cannabinoid antagonist (AM; 20 nmol) were evaluated under both conditions. Results Exposure to 3-AP induced dramatic changes in cellular excitability that likely would affect Purkinje cell output. In whole-cell current clamp recordings, 3-AP-exposed Purkinje cells demonstrated a significantly higher frequency of action potentials, a larger afterhyperpolarization (AHP), and a larger rebound of action potentials. In addi- tion, 3-AP caused a significant decrease in the interspike interval (ISI), half-width, and first spike latency. Remarkably, the action potential frequency, AHP amplitude, rebound, ISI, action potential halfwidth, and first spike latency were no longer different from controls in 3-AP cells treated with AM. Sag percentage, on the other hand, showed no signifi- cant difference under any treatment condition, indicating that cannabinoids’ actions on 3-AP-mediated Purkinje cell changes may not include effects on neuronal excitability through changes of Ih. Conclusions These data show that cannabinoid antagonists reduce the excitability of Purkinje cells following expo- sure to 3-AP and suggest their potential as therapeutics in cerebellar dysfunctions. Highlights • Purkinje cell excitability dramatically increased after exposure to 3-AP. • 3-AP caused a significant decrease in ISI, half-width, and first spike latency. • CB1R antagonist attenuated the cellular effects of 3-AP. • AM251 reduced the excitability of Purkinje cells exposed to 3-AP. Keywords Ataxia, Cerebellum, Purkinje cell, Cannabinoid, CB1R *Correspondence: Mohammad Shabani shabanimoh@yahoo.com; shabani@kmu.ac.ir Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ranjbar et al. BMC Neuroscience (2023) 24:18 Page 2 of 11 patients suffering from SCA have also demonstrated ele - Introduction vated levels of CB1Rs in Purkinje neurons, with a similar Cerebellar ataxia is considered a diverse group of neu- profile found for endocannabinoid hydrolyzing enzymes rological disorders characterized by loss of balance and [13, 15]. When taken together, these studies and others motor coordination caused by abnormal neuronal func- suggest that activating CB1Rs and/or inhibiting these tioning in the cerebellum [1]. In addition to acquired enzymes could serve to develop cannabinoid-based neu- causes (such as brain trauma, infections, ischemia, roprotective therapies. Therefore, the endocannabinoid alcohol misuse, or other factors) [2], ataxias can also be system is a current focus of studies examining treatment inherited [3]. The cerebellar Purkinje cells are part of a approaches for ataxia [15–18]. complex circuit that integrates information from many Changes in endocannabinoid transmission could affect sources and provides the sole output from the cerebellar the firing of Purkinje cells prior to their death in ataxia. cortex. Like other neuronal types, the activity of Purkinje However, as studies of Purkinje cell electrical activity cells is regulated by synaptic inputs, which include excit- associated with ataxia have been conducted with extra- atory inputs from parallel and climbing fibers and inhibi - cellular recordings, information is lacking regarding the tory inputs from basket and stellate cells [4]. effect(s) of ataxia on the cellular electrophysiology of Animal models of ataxia include genetic mutations, cerebellar neurons prior to death. Therefore, since the but 3-acetyl pyridine (3-AP) administration to rats can 3-AP model is useful for studying mechanisms under- also produce symptoms of ataxia, such as severe motor lying changes in Purkinje cells activity associated with coordination and locomotor activity impairment [5, 6]. ataxia, the role played by endocannabinoid transmission The inferior olive is the only target of climbing fiber affer - in dysfunctions involving changes in Purkinje cell prop- ents to the cerebellum, and 3-AP affects the inferior olive erties can be studied in this model. Further, the effects through impairing electron transport [7]. As a result, of administration of agonist/antagonist of cannabinoid because climbing fibers modulate Purkinje cell responses, receptors have not been shown in this model. Accord- impaired climbing fibers alter Purkinje cell activity [8, 9]. ingly, this study characterized changes in the electro- Since Purkinje cells are the cerebellar cortex’s only output physiological activity of cerebellar Purkinje cell exposed neuron, under conditions of ataxia that lead to Purkinje to 3-AP prior to their inevitable death. In addition, the cell degeneration, it is important to understand the effects of agonist/antagonists of cannabinoid receptors impact of the impaired climbing fibers on intrinsic activ - on changes in Purkinje cell function exposed to 3-AP ity of Purkinje cells prior to the death of these cells [7]. In were examined to evaluate whether cannabinoids could addition to previously recognized effects on the inferior offer a potential therapy for cerebellar dysfunction. olive, the 3-AP has direct effects on Purkinje cells, which change electrophysiological properties and ultimately Method and material lead to Purkinje cell death [10]. Animals The endocannabinoid system is comprised of recep - Experiments were performed on tissue obtained from 36 tors, endogenous agonists, and related biochemical male Wistar rats (weighing 30–60 g) aged 4–6 weeks at mechanisms that synthesize and terminate the actions the start of the protocol, obtained from the Kerman Uni- of endogenous agonists. CB1 and CB2 receptors were versity of Medical Sciences, and housed under controlled named after the receptors activated by cannabinoids in photoperiod (lights on: 07:00–19:00 h), at 22 ± 1 °C the order of discovery [11]. CB1 receptors (CB1Rs) are with food and water available. All protocols and proce- mainly expressed in the CNS, and their density is par- dures using animals were approved by the Kerman Uni- ticularly high in the cerebellum, suggesting that they play versity of Medical Sciences Ethic Committee (IR.KMU. a significant role in cerebellum function, which is consist - REC.1399.254). Male rats (n = 6 cells/group; one cell ent with findings that Purkinje cell synaptic transmission from each rat) were divided randomly into six groups: is modulated by CB1R activation at excitatory and inhibi- control (Co), cannabinoid agonist (WIN; 7.5 nmol) [19], tory synapses [12]. cannabinoid antagonist (AM; 20 nmol) [19], 3 acetyl pyri- In spinocerebellar ataxias (SCAs), the endocannabinoid dine (3-AP; 1 mM) [20], 3-AP + WIN, and 3-AP + AM. system becomes dysregulated in the cerebellum and in other parts of the central nervous system (e.g., brainstem, Preparation of cerebellar slices basal ganglia), which contributes to the progression of Rats were euthanized by cervical dislocation, and the pathogenic events. Based on the results of studies con- posterior skull was removed allowing extraction of the ducted in rodent models and postmortem tissue analysis, cerebellum in the coronal plane. The brain was sliced changes in CB1Rs appear to be responsible for acutely into sections of a 250 μm thickness using a Vibro- modulating motor incoordination in cerebellar ataxias slicer (Campden Instrument, NVSLM1, Sarasota, FL) [13, 14]. Additional studies in post-mortem tissues from R anjbar et al. BMC Neuroscience (2023) 24:18 Page 3 of 11 in artificial cerebrospinal fluid (aCSF) containing (in Drug application mM) 124 NaCl, 25 NaHCO3, 10 D-glucose, 4.4 KCl, 2 Drugs were applied in aCSF from a separate reservoir MgCl2, 1.25 NaH2PO4, and 2 CaCl2, which was bub- (also gassed with carbogen) to the recording chamber. bled with 95% O2–5% CO2 (pH 7.4 ± 0.05 and osmo- 3-AP (Sigma, USA) was dissolved in deionized water larity was adjusted to 300 ± 10 mOsm). The slices were as a × 100 stock solution containing 0.4% ascorbic acid. immediately incubated in aCSF at 35 ± 2 °C for 30 min Purkinje cells were exposed to aCSF (control) or 1 mM before being recorded within 6 h at room temperature 3-AP (for at least 20 min). WIN (7.5 nmol) and AM (20 (22 °C). nmol) were added to the bath [23]. Statistical analysis Whole cell patch clamp Data are presented as mean ± SEM using Graph Pad A Multiclamp 700B amplifier from Axon Instruments was Prism 9 (Graph Pad Software, USA). Normally distrib- used to conduct whole-cell patch clamp recordings from uted data were compared by a two-way ANOVA, and the Purkinje cells in current clamp and voltage clamp modes. Tukey post hoc analysis was used for multiple compari- Purkinje cells were identified using the established crite - sons between multiple groups. Differences were consid - ria listed below. Signals were digitized using a 1440 A/D ered statistically significant when P < 0.05. converter (Axon Instruments). Electrophysiological data were sampled at 10 kHz and filtered at 20 kHz [21]. Patch Result pipettes had a resistance of 5–9 MΩ when filled with an The effect of CB1R agonist/antagonist internal solution comprising (in mM) 140 potassium glu- on the electrophysiological activities of Purkinje cells conate, 5 KCl, 10 HEPES, 2 MgCl2, 0.2 EGTA, 2 Na2ATP, following exposure of 3‑AP in cerebellum slices and 0.4 Na2GTP. The internal solution’s pH and osmo - In CB1R agonist/antagonist and 3-AP-exposed slices, a larity were adjusted to 7.3 (by KOH) and 290 mOsm, total of 30 Purkinje cells were recorded. The passive and respectively. The perfusion rate of the recorded slides active properties of 6 cells were examined for each group. was 1.9–2 ml/min. Following the formation of a giga seal Our results indicated that cells in the 3-AP (P < 0.001), on the cell membrane, a brief suction was used to rup- 3-AP + WIN (P < 0.01), and 3-AP + AM (P < 0.01) treated ture the membrane, allowing the cell to be clamped to groups exhibited a significantly lower resting membrane a holding voltage of -60 mV. The test seal function was potential (RMP) (F (2, 24) = 1.446, P = 0.2554) com- continuously used throughout the recording to make pared to cells in the Co, WIN, and AM groups, respec- sure the seal was stable. The number of action potentials tively (Fig. 1A). We measured R as an index of how the in generated after negative current injection when the cell cells would passively respond to current inputs. There rebounded to the resting membrane potential, and the were no statistical differences in R (F (2, 23) = 0.9682, in spike latency of the first action potential were measured. P = 0.3947) between any of the Purkinje cell treatment In spontaneously firing neurons, action potential param - groups, indicating that exposure to 3-AP whether in the eters including action potential (AP) half-width, AP fre- presence or absence of cannabinoid agonists or antago- quency, AP amplitude, AHP amplitude, voltage threshold nists, had no effect on R (Fig. 1B). in (the membrane potential from the base and start of the Purkinje cells in 3-AP (P < 0.001), 3-AP + WIN action potential), interspike interval (ISI), and coefficient (P < 0.05) and 3-AP + A M (P < 0.001) exposed slices of variation (CV) (regularity of action potential and dis- demonstrated a higher spontaneous firing frequency (F persion of a probability/frequency) were measured [22]. (2, 24) = 12.41, P = 0.0002) when compared to that in The sag voltage in response to hyperpolarizing cur - the Co, WIN, and AM groups, respectively; whereas, rent pulses (amplitude of -100 pA to -500 pA) was cal- Purkinje cells in the 3-AP + A M (P < 0.01) group dem- culated. The peak voltage deviation was divided by the onstrated a significantly lower firing frequency when amplitude of the steady-state voltage deviation using compared to that in 3-AP-treated cells (Fig. 2A). the following formula: Besides firing rate, another important parameter of Sag voltage = Vpeak − Vsteady state. the Purkinje cell’s function is the interspike interval Furthermore, the sag was calculated as [(peak (ISI). We examined this parameter by measuring the response—steady state response) / peak response] 100. spike intervals in Purkinje cells that were spiking spon- To examine the effect of a CB1R agonist/antagonist on taneously. This analysis indicated that cells exposed Ih in Purkinje cells, I-V activation curves were obtained to 3-AP (P < 0.001) demonstrated a lower ISI (F (2, in voltage clamp mode using 520 ms of hyperpolarizing 24) = 4.817, P = 0.0174) compared to the Co and AM steps (50 to 140 mV in increments of 10 mV). (P < 0.05) treated groups (Fig. 2B). We also found that Purkinje cells in the AM and 3-AP + A M (P < 0.05) Ranjbar et al. BMC Neuroscience (2023) 24:18 Page 4 of 11 Fig. 1 Changes in electrophysiological properties of Purkinje cells in vermis slices treated with CB1R agonist/antagonist and 3-AP. A 3-AP alone and in combination with CB1R agonist/antagonist resulted in a lower RMP compared to control, WIN, and AM respectively. B No significant differences were observed in Rin. n = 6 cells/group; one cell from each rat. The data are expressed as mean ± SEM. **(P < 0.01) and ***(P < 0.001) represent a significant difference with the counterpart group width, and threshold were lower in the 3-AP groups groups had significantly broader AP half-widths (Fig. 3). (P < 0.05) compared to those seen in the 3-AP group To determine if 3-AP affects the excitability of cer (Fig. 2C). The amplitude of the action potential was - significantly larger in Purkinje cells in the 3-AP group ebellar Purkinje cells, we first measured the rheobase. compared to cells in the Co group (P < 0.05). However, Rheobase is operationally defined as the minimal our data showed a lower amplitude of the action poten- amount of electrical current required to trigger an tial in cells from the WIN (P < 0.01), AM (P < 0.001), action potential when current is injected into a cell. In and 3-AP + AM (P < 0.001) groups compared to the current clamp mode, Purkinje cells exposed to 3-AP amplitude in Purkinje cells of the 3-AP group (Fig. 2D). (P < 0.05) and 3-AP + WIN (P < 0.05) showed a signifi - Purkinje cells in 3-AP (P < 0.001) exposed cell groups cantly lower rheobase (F (2, 24) = 2.056, P = 0.1499), showed a lower CV (F (2, 24) = 12.67, P = 0.0002) indicating a heightened excitability compared to cells in compared to cells in the Co. In addition, 3-AP + A M the Co and WIN groups, respectively (Fig. 4A). How- (P < 0.001) cells showed lower CV compared to that ever, while the 3-AP and 3-AP + WIN groups required seen in cells in the AM group (Fig. 2E). We examined less current to stimulate an action potential, there was the voltage threshold (F (2, 24) = 1.112, P = 0.3454) not a significant difference noted in the threshold volt - and kinetics of action potentials to determine if 3-AP age noted in the RAMP protocol, which indicates the affects these parameters. Purkinje cells from 3-AP- current at which an action potential was triggered (F (2, exposed (P < 0.01) cells demonstrated a significantly 24) = 1.615, P = 0.2198) (Fig . 4B). lower voltage threshold compared to cells in the Co We next examined the response of Purkinje cells to group (Fig. 2F). The prominent AHP seen in Purkinje the effects of CB1R agonist/antagonist on spontaneous cells following an action potential reflects conductance excitatory synaptic activity following exposure to 3-AP. activated during the spike. Amplitudes of the AHP (F Purkinje cells in the 3-AP (P < 0.05) and 3-AP + WIN (2, 24) = 16.49, P < 0.0001) in the Purkinje cells of the (P < 0.05) groups showed smaller intervals of sEP- 3-AP (P < 0.01) and 3-AP + WIN groups (P < 0.001) SCs (F (2, 24) = 1.926, P = 0.1676) when compared to were larger in amplitude compared to amplitudes the sEPSCs intervals seen in the Co and WIN groups, seen in Co and WIN cells, respectively. Purkinje cells respectively (Fig. 5A). Consistent with a heightened in the AM (P < 0.05) and 3-AP + A M (P < 0.05) groups increase in excitability, the amplitude of sEPSCs (F (2, displayed a significantly lower AHP compared to that 24) = 1.717, P = 0.2009) in the Purkinje cells of the 3-AP seen in the 3-AP group (Fig. 2G). Furthermore, traces group (P < 0.01) was increased. In addition, the combi- of spontaneous activity can be seen in Fig. 3, where nation of 3-AP and WIN (P < 0.01) amplified the action two action potentials from each group have been of 3-AP in enhancing sEPSP amplitudes. In Purkinje selected and then superimposed. Compared to the Co cells in the WIN (P < 0.001) and AM (P < 0.01) groups, and 3-AP + AM groups, the AP amplitude, AHP, and there was a significantly lower amplitude of sEPSCs AP frequency were significantly higher, and the half when compared to amplitudes seen in 3-AP cells. R anjbar et al. BMC Neuroscience (2023) 24:18 Page 5 of 11 Fig. 2 The effect of CB1R agonist/antagonist on the spontaneous firing properties of Purkinje cells in control and 3-AP treated slices. A Electrophysiological properties shown are action potential frequency, B interspike interval, C action potential half-width, D action potential amplitude, E coefficient variant, F action potential threshold, and G AHP amplitude. n = 6 cells/group; one cell from each rat. The data are expressed as mean ± SEM. *(P < 0.05), **(P < 0.01), and ***(P < 0.001) represent a significant difference with the counterpart group. #(P < 0.05), ## (P < 0.01), and ###(P < 0.001) represent a significant difference versus 3-AP Administration of AM failed to significantly alter the treated with AM (P < 0.05), conversely, a lower number of effects of 3-AP on sEPSP amplitudes (Fig. 5 B). rebound action potentials was noted (Fig. 6A, B) as well Rebound is defined as membrane depolarization occur - as an enhanced first spike latency (Fig. 7A, B) at both ring at the offset of a hyperpolarizing stimulus and is one -100 pA (P < 0.01) and -500 pA (P < 0.05) compared to of several intrinsic properties that may promote rhythmic 3-AP Purkinje cells. electrical activity. Purkinje cells from 3-AP (P < 0.001) The rebound of action potentials was significantly and 3-AP + WIN (P < 0.001) exposed brain slices dis- higher in Purkinje cells from the 3-AP and 3-AP + WIN played a significantly higher number of rebound action groups when compared to that seen in the Co and WIN potentials (F (2, 24) = 8.932, P = 0.0013) appearing fol- groups at both -100 pA (P < 0.001) and -200 pA (P < 0.05). lowing presence of a hyperpolarizing conditioning pulse Furthermore, there was a significantly lower rebound in (Fig. 6A) and a reduction in the first spike latency (F (2, Purkinje cells in the 3-AP + AM group at -200 pA and 24) = 10.71, P = 0.0005), (Fig. 7A) at -100 pA (P < 0.01) -500 pA compared to 3-AP cells. Therefore, exposure to AM was capable of reducing the heightened excitability compared to averages for these same parameters in of Purkinje cells associated with 3-AP treatment. Purkinje cells from Co and WIN groups, respectively. In Purkinje cells in the 3-AP treatment group, which were Ranjbar et al. BMC Neuroscience (2023) 24:18 Page 6 of 11 Fig. 3 Representative traces of spontaneous firing from control and CB1R agonist/antagonist are depicted Fig. 4 3-AP causes a reduction in rheobase in cerebellar Purkinje cells in current clamp recordings. A Rheobase (current clamp), B RAMP threshold. n = 6 cells/group; one cell from each rat. The data are expressed as mean ± SEM. *(P < 0.05) represent a significant difference with the counterpart group R anjbar et al. BMC Neuroscience (2023) 24:18 Page 7 of 11 Fig. 5 EPSPCs of Purkinje cells in slices exposed to CB1R agonist/antagonist in control and 3-AP treated slices. A EPSPCs interspike interval, B EPSCs amplitude is shown. n = 6 cells/group; one cell from each rat. The data are expressed as mean ± SEM. *(P < 0.05) and **(P < 0.01) represent significant differences with the same group with Vehicle (DMSO). #(P < 0.05) represents a significant difference versus 3-AP Fig. 6 The effect of CB1R agonist/antagonist on the rebound of action potential at A -100pA and B -500 pA. n = 6 cells/group; one cell from each rat. The data are expressed as mean ± SEM. ***(P < 0.001) represents the significant difference with the same group with Vehicle (DMSO). #(P < 0.05) represents the significant difference versus 3-AP Exposing Purkinje cells to 3-AP led to an increase in spikes. Thus, our data shows that evoked and spontane - rebound spikes, which is a sign of heightened excitabil- ous activities can be reversed by AM. ity, and treating them with AM decreased the rebound Ranjbar et al. BMC Neuroscience (2023) 24:18 Page 8 of 11 Fig. 7 Antagonist/agonist of CB1R effects on 3-AP-exposed cells’ first spike latency at A -100 pA and B -500 pA. n = 6 cells/group; one cell from each rat. The data are expressed as mean ± SEM. *(P < 0.05), and **(P < 0.01) represent the significant difference with the same group with Vehicle (DMSO). #(P < 0.05), ##(P < 0.01) represents the significant difference versus 3-AP Furthermore, the prolonged, time-dependent changes evident in the spike rate/pulse of action potential after the increase in the current step from 0.1 nA to 0.5 nA indicate that an adaptation occurs in the action potential in all groups. Particularly, we did note a significant differ - ence in the adaptation seen in the 3-AP groups (Fig. 8). The sag voltage was studied to determine the effects of CB1R modulation on Ih currents (hyperpolarization- activated currents) in Co and 3-AP treated slices. Sag is considered an indicator of the kinetics and amplitude of Ih relative to the membrane time constant. After the application of hyperpolarizing current pulses (-100 Fig. 8 Population data representative of the effects on spike rate/ pA (F (2, 24) = 0.3777, P = 0.6894) and -500 pA (F (2, pulse (0.1 nA–0.5 nA). n = 6 cells/group; one cell from each rat. The 24) = 1.931, P = 0.1668)) the %sag voltage showed no sig- data are expressed as mean ± SEM. *(P < 0.05) and ***(P < 0.001) nificant difference in any group (Fig. 9). This finding sug - represent the significant differences with the same group with Vehicle (DMSO). ###(P < 0.001) represents the significant difference gests that the effects of cannabinoids on ataxia might not versus 3-AP involve actions at Ih. Fig. 9 The percentage of the sag potential was determined at A -100pA and B -500pA. n = 6 cells/group; one cell from each rat. The data are expressed as mean ± SEM R anjbar et al. BMC Neuroscience (2023) 24:18 Page 9 of 11 Discussion hyperpolarization, the slower the cell will reach the firing The sequence of events that occur during 3-AP expo - threshold, which is reflected as a decrease in excitability, sure that result in permanent damage to the Purkinje whereas if the AHP is smaller, the cell can reach the fir - cells is poorly understood. Here, we utilized 3-AP-expo- ing threshold more quickly, which indicates an increase sure, which is a valid model for inducing [7, 20] damage in excitability. However, we observed an enhancement in to rat Purkinje cells associated with behavioral signs of AHP in 3-AP Purkinje cells with an increase in firing rate, ataxia, in order to understand alterations in the electro- which does not suggest such a direct relationship. We physiological properties of cerebellar Purkinje cells. 3-AP interpret our data to suggest that the greater amplitude caused marked changes to the activity of Purkinje cells, AHP increases the availability of N a channels through consistent with previous studies showing that 3-AP can more rapid removal of their inactive phase, which facili- regulate neuronal excitability [4, 24]. In some aspects, the tates the generation of burst discharge and thus increases electrophysiological properties of 3-AP-exposed Purkinje excitability [27]. cells were similar to those seen in controls, as we saw AHP is a principal feedback mechanism for the con- no differences in Rin, rebound (-500 pA), or %Sag volt - trol of the frequency and pattern of neuronal firing [28]. age (-100 pA and -500 pA). However, there were differ - At high firing frequencies, AHP amplitudes and thresh - ences, as Purkinje cells from the 3-AP group exhibited a olds would be expected to decrease, but our results higher firing frequency and greater AP amplitude. These showed no decrease in AHP amplitudes. One potential electrophysiological changes were accompanied by an explanation for our data is that the combination of the increase in putative excitatory spontaneous synaptic decrease in RMP (more depolarized Purkinje cells) with activity. While we did not identify the synaptic activity the increase in AHP in 3-AP-exposed rats prevented the as glutamatergic, with the recording conditions we uti- threshold from changing. Furthermore, the decrease in lized synaptic events indicative of inward currents were ISI indicates that the kinetics of the N a channels have unlikely to be GABA mediated, and the kinetic profiles of changed, leading to a more rapid opening and closing of these events suggested involvement of an AMPA recep- these channels and contributing to the decrease in ISI. tor. Further, the lower rheobase suggested heightened The AHP amplitude was significantly larger in neurons excitability in Purkinje cells exposed to 3-AP. Height- from 3-AP-exposed animals, but other parameters such ened excitability was also seen in 3-AP-exposed cells, as as the Rin during the rising or decay phase of the action hyperpolarization was strongly and positively associated potential were not different between groups. Therefore, with increased spike probability. The effects of 3-AP on it seems likely that voltage-gated Na channels are key the frequency, AP amplitude, ISI, rebound spikes, and regulators of the alteration in AHP. The involvement of rheobase were reversed by the administration of AM251. Na channels could suggest that the fast/medium AHP Therefore, using 3-AP-exposed neurons in the current might be involved; however, our data argue against a role study, we found that Purkinje cells had a higher excitabil- for the medium AHP. The slow component of the AHP is ity, which was modulated by blocking CB1Rs. mediated by Ih currents [29], and as our analysis showed WIN, a cannabinoid agonist, exacerbated the 3-AP- that there were no significant differences in %sag, this induced effects by further increasing the frequency and suggests not only that Ih currents might not contribute reducing the rheobase as well as increasing the EPSCs to effects of 3-AP exposure but also provides indirect evi - amplitudes exhibiting hyperexcitability. Previous stud- dence that the medium AHP is not involved in the dys- ies have shown that CB1Rs are strongly expressed in the function induced by 3-AP. cerebellum and regulate Purkinje cell synaptic signaling A possible mechanism could be alterations in the 3-AP [25, 26]. Accordingly, CB1Rs modulate GABA, gluta- model of fast AHP (fAHP), which is mediated by the BK mate, and other neurotransmitters throughout the cer- channel. BK channels are activated at negative membrane ebellum, thereby controlling movement and contributing potentials, and their large conductivities depend on high 2+ to some movement disorders, such as ataxia [13–15]. Ca levels, which affect the local membrane excitability. 2+ From the reports above, our findings demonstrated a AHP and the regulation of C a spikes occur in cerebellar higher excitability in the CB1R agonist exposure that Purkinje cells through activity of BK channels expressed suggests alterations in glutamate/GABA might play a in dendrites, soma, and myelinated axons [30]. However, role in CB1R-associated hyperexcitability, and it might BK channels should not be considered strictly excita- be possible to modulate these changes by blocking these tory or inhibitory, as they can be inhibited or activated receptors. by pharmacological or genetic inhibition. In other words, The amplitude of the AHP can regulate the ISI and BK channels can amplify the firing of quiet neurons and thus determine the pace of spontaneously firing neu - reduce the firing of overly active ones [31, 32]. Further- rons. The larger the AHP or the greater the degree of more, recent studies indicated that the BK channel could Ranjbar et al. BMC Neuroscience (2023) 24:18 Page 10 of 11 Funding represent an outstanding therapeutic target for the man- Funding for this study was provided by Kerman University of Medical Sciences agement of ataxia [33], as BK channel mutations cause as a grant (98001230) for the PhD thesis conducted by Hoda Ranjbar. ataxia [34]. Overall, based on previous studies and our Availability of data and materials findings, it appears that BK channels are possible media - The datasets used or analyzed during the current study are available from the tors of the effects of 3-AP on AHP and could be involved corresponding author on reasonable request. in mechanisms underlying ataxia. Therefore, future Code availability experiments should examine whether impairment of BK All software applications used are included in this article. channels leads to changes in the excitability of ataxic Purkinje cells to determine the role played by these chan- Declarations nels in ataxia. The electrophysiological alterations described above Ethics approval for consent to participate All experiments were done in accordance with the ARRIVE guidelines and were absent in Purkinje cells from rats that were treated National Institutes of Health Guide for the Care and Use of Laboratory Ani- with AM251. A potential mechanism might be the inhibi- mals) NIH Publication No. 80–23, revised 1996). All procedures were approved tion of Na channels by AM251, which reduces neuronal by the Research and Ethics Committee of Kerman Universities of Medical Sciences, Kerman, Iran. excitability through blockade of voltage-sensitive Na channels in the brain [35]. Another non mutually exclu- Consent for publication sive possibility is that effects could be due to (endo)-can - Not applicable. nabinoid-mediated modulation of BK channels as it has Competing interests been reported that (endo)-cannabinoids directly interact The authors declare that they have no known competing financial interests with and can modify BK channel activity [36]. As a result, or personal relationships that could have appeared to influence the work reported in this paper. AM251’s effects on modulating excitability changes seen in the 3-AP Purkinje cells could be via modulation of BK Author details function. Neuroscience Research Center, Neuropharmacology Institute, Kerman Uni- versity of Medical Sciences, Kerman 76198-13159, Iran. Department of Drug In conclusion, dysfunction of Purkinje cells in the cer- Design and Pharmacology, Faculty of Health Sciences, University of Copenha- ebellum contributes to the clinical signs and symptoms gen, Copenhagen, Denmark. Department of Physiology, School of Medicine, of ataxia. Based on our findings, we conclude that effects Shahid Beheshti University of Medical Sciences, Tehran, Iran. of 3-AP exposure alter functioning of Purkinje cells. One Received: 19 December 2022 Accepted: 23 February 2023 caveat of our study was that we were not able to deter- mine whether dysfunctions of Purkinje cells associated with 3-AP were mediated through direct effects on the Purkinje cells or on inputs directed to these cells. How- References ever, as our intention was to determine whether the excit- 1. Rossi M, Perez-Lloret S, Doldan L, Cerquetti D, Balej J, Millar Vernetti P, et al. Autosomal dominant cerebellar ataxias: a systematic review of ability of Purkinje cells was altered by 3-AP treatment, clinical features. Eur J Neurol. 2014;21(4):607–15. https:// doi. org/ 10. whether effects were direct or indirect awaits further 1111/ ene. 12350. experiments. Our data also suggest that an antagonist of 2. Klockgether T. Update on degenerative ataxias. Curr Opin Neurol. 2011;24(4):339–45. https:// doi. org/ 10. 1097/ WCO. 0b013 e3283 4875ba. CB1R could be effective in modulating the devastating 3. Gómez-Ruiz M, Rodríguez-Cueto C, Luna-Piñel E, Hernández-Gálvez M, cellular effects of 3-AP. Although the mechanism respon - Fernández-Ruiz J. Endocannabinoid system in spinocerebellar ataxia sible for this protection is unknown, findings with CB1R type-3 and other autosomal-dominant cerebellar ataxias: potential role in pathogenesis and expected relevance as neuroprotective targets. antagonists in this study could be relevant for future Front Mol Neurosci. 2019;12:94. https:// doi. org/ 10. 3389/ fnmol. 2019. therapies for ataxia. 4. Morton SM, Bastian AJ. Cerebellar control of balance and locomotion. Acknowledgements Neurosci Review J Bringing Neurobiol Neurol Psychiatry. 2004;10(3):247– This study was made possible through the supports received from Neurosci- 59. https:// doi. org/ 10. 1177/ 10738 58404 263517. ence Research Center, Kerman University of Medical Sciences, Kerman, Iran. 5. Ranjbar H, Soti M, Janahmadi M, Kohlmeier KA, Sheibani V, Ahmadi- Zeidabadi M, et al. Modulation of the CB1 cannabinoid receptor has Author contributions potential therapeutic utility in the 3-acetylpyridine cerebellar ataxia rat HR contributed to study conception and design, acquisition of animal data, model. Exp Brain Res. 2022;240(9):2339–48. https:// doi. org/ 10. 1007/ analyzing data, interpretation of the findings and writing the manuscript. s00221- 022- 06415-6. MS, KAK, and MJ assisted with study conception and design, data analysis, 6. Ranjbar H, Soti M, Kohlmeier KA, Sheibani V, Ahmadi-Zeidabadi M, interpretation of the findings and critical revision of the manuscript. MSh Rafiepour K, et al. The cannabinoid antagonist, AM251 attenuates ataxia contributed to study conception and design, analyzing data, interpretation of related deficiencies in a cerebellar ataxic model. Int J Neurosci. 2022. the findings and writing the manuscript, and provided a critical revision of the https:// doi. org/ 10. 1080/ 00207 454. 2022. 21267 71. manuscript for important intellectual content. All authors critically reviewed 7. Smeets CJ, Verbeek DS. Climbing fibers in spinocerebellar ataxia: a content and approved final version for publication. All authors read and mechanism for the loss of motor control. Neurobiol Dis. 2016;88:96–106. approved the final manuscript. https:// doi. org/ 10. 1016/j. nbd. 2016. 01. 009. R anjbar et al. BMC Neuroscience (2023) 24:18 Page 11 of 11 8. Medina JF. The multiple roles of Purkinje cells in sensori-motor calibration: downstream synaptic excitation, and signaling to migrating neurons. J to predict, teach and command. Curr Opin Neurobiol. 2011;21(4):616–22. Neurosci. 2020;40(32):6133–45. https:// doi. org/ 10. 1523/ jneur osci. 1931- https:// doi. org/ 10. 1016/j. conb. 2011. 05. 025.19. 2020. 9. Duvick L, Barnes J, Ebner B, Agrawal S, Andresen M, Lim J, et al. SCA1-like 27. Fourcaud-Trocmé N, Zbili M, Duchamp-Viret P, Kuczewski N. Afterhy- disease in mice expressing wild-type ataxin-1 with a serine to aspartic perpolarization Promotes the Firing of Mitral Cells through a Voltage- acid replacement at residue 776. Neuron. 2010;67(6):929–35. https:// doi. Dependent Modification of Action Potential Threshold. eNeuro. 2022. org/ 10. 1016/j. neuron. 2010. 08. 022.https:// doi. org/ 10. 1523/ eneuro. 0401- 21. 2021. 10. Cao BB, Zhang XX, Du CY, Liu Z, Qiu YH, Peng YP. TGF-β1 provides 28. Cloues RK, Sather WA. Afterhyperpolarization regulates firing rate in neuroprotection via inhibition of microglial activation in 3-acetylpyridine- neurons of the suprachiasmatic nucleus. J Neurosci. 2003;23(5):1593–604. induced cerebellar ataxia model rats. Front Neurosci. 2020;14:187. https:// https:// doi. org/ 10. 1523/ JNEUR OSCI. 23- 05- 01593. 2003. doi. org/ 10. 3389/ fnins. 2020. 00187. 29. Velumian AA, Carlen PL. Differential control of three after-hyperpolariza- 11. Saito VM, Wotjak CT, Moreira FA. Pharmacological exploitation of the tions in rat hippocampal neurones by intracellular calcium buffering. J endocannabinoid system: new perspectives for the treatment of depres- Physiol. 1999;517(pt 1):201–16. https:// doi. org/ 10. 1111/j. 1469- 7793. 1999. sion and anxiety disorders? Revista Brasileira de Psiquiatria. 2010;32(Suppl 0201z.x. 1):S7-14. 30. Hirono M, Ogawa Y, Misono K, Zollinger DR, Trimmer JS, Rasband MN, 12. DeSanty KP, Dar MS. Cannabinoid-induced motor incoordination through et al. BK channels localize to the Paranodal junction and regulate action the cerebellar CB(1) receptor in mice. Pharmacol Biochem Behav. potentials in myelinated axons of cerebellar Purkinje cells. J Neurosci. 2001;69(1–2):251–9. https:// doi. org/ 10. 1016/ s0091- 3057(01) 00539-1. 2015;35(18):7082–94. https:// doi. org/ 10. 1523/ JNEUR OSCI. 3778- 14. 2015. 13. Rodríguez-Cueto C, Benito C, Fernández-Ruiz J, Romero J, Hernández- 31. Contet C, Goulding SP, Kuljis DA, Barth AL. BK channels in the central Gálvez M, Gómez-Ruiz M. Changes in CB(1) and CB(2) receptors in the nervous system. Int Rev Neurobiol. 2016;128:281–342. https:// doi. org/ 10. post-mortem cerebellum of humans affected by spinocerebellar ataxias. 1016/ bs. irn. 2016. 04. 001. Br J Pharmacol. 2014;171(6):1472–89. https:// doi. org/ 10. 1111/ bph. 12283. 32. Matthews EA, Weible AP, Shah S, Disterhoft JF. The BK-mediated fAHP is 14. Wang X, Whalley BJ, Stephens GJ. The du(2J ) mouse model of ataxia modulated by learning a hippocampus-dependent task. Proc Natl Acad and absence epilepsy has deficient cannabinoid CB receptor-mediated Sci USA. 2008;105(39):15154–9. https:// doi. org/ 10. 1073/ pnas. 08058 signalling. J Physiol. 2013;591(16):3919–33. https:// doi. org/ 10. 1113/ jphys 55105. iol. 2012. 244947. 33. Du X, Carvalho-de-Souza JL, Wei C, Carrasquel-Ursulaez W, Lorenzo Y, 15. Stephens GJ. Does modulation of the endocannabinoid system Gonzalez N, et al. Loss-of-function BK channel mutation causes impaired have potential therapeutic utility in cerebellar ataxia? J Physiol. mitochondria and progressive cerebellar ataxia. Proc Natl Acad Sci USA. 2016;594(16):4631–41. https:// doi. org/ 10. 1113/ jp271 106. 2020;117(11):6023–34. https:// doi. org/ 10. 1073/ pnas. 19200 08117. 16. Di Marzo V, Stella N, Zimmer A. Endocannabinoid signalling and the 34. Srinivasan SR, Huang H, Chang WC, Nasburg JA, Nguyen HM, Strassmaier deteriorating brain. Nat Rev Neurosci. 2015;16(1):30–42. https:// doi. org/ T, et al. Discovery of novel activators of large-conductance calcium- 10. 1038/ nrn38 76. activated potassium channels for the treatment of cerebellar ataxia. Mol 17. Rodríguez-Cueto C, Hernández-Gálvez M, Hillard CJ, Maciel P, García- Pharmacol. 2022;102(1):438–49. https:// doi. org/ 10. 1124/ molph arm. 121. García L, Valdeolivas S, et al. Dysregulation of the endocannabinoid 000478. signaling system in the cerebellum and brainstem in a transgenic mouse 35. Liao C, Zheng J, David LS, Nicholson RA. Inhibition of voltage-sensitive model of spinocerebellar ataxia type-3. Neuroscience. 2016;339:191–209. sodium channels by the cannabinoid 1 receptor antagonist AM 251 in https:// doi. org/ 10. 1016/j. neuro scien ce. 2016. 09. 046. mammalian brain. Basic Clin Pharmacol Toxicol. 2004;94(2):73–8. https:// 18. Ranjbar H, Soti M, Razavinasab M, Kohlmeier KA, Shabani M. The doi. org/ 10. 1111/j. 1742- 7843. 2004. pto94 0204.x. neglected role of endocannabinoid actions at TRPC channels in ataxia. 36. Lin Y-F. Potassium channels as molecular targets of endocannabinoids. Neurosci Biobehav Rev. 2022;141:104860. https:// doi. org/ 10. 1016/j. neubi Channels. 2021;15(1):408–23. https:// doi. org/ 10. 1080/ 19336 950. 2021. orev. 2022. 104860.19104 61. 19. Zheng T, Zhang R, Zhang T, Zhang M-N, Xu B, Song J-j, et al. CB1 cannabinoid receptor agonist mouse VD-hemopressin (α) produced Publisher’s Note supraspinal analgesic activity in the preclinical models of pain. Brain Res. Springer Nature remains neutral with regard to jurisdictional claims in pub- 2018;1680:155–64. lished maps and institutional affiliations. 20. Wüllner U, Weller M, Groscurth P, Löschmann PA, Schulz JB, Müller I, et al. Evidence for an active type of cell death with ultrastructural features distinct from apoptosis: the effects of 3-acetylpyridine neurotoxicity. Neuroscience. 1997;81(3):721–34. https:// doi. org/ 10. 1016/ s0306- 4522(97) 00181-4. 21. Shabani M, Hosseinmardi N, Haghani M, Shaibani V, Janahmadi M. Mater- nal 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. 2011;172:139–52. https:// doi. org/ 10. 1016/j. neuro scien ce. 2010. 10. 031. 22. Haghani M, Shabani M, Javan M, Motamedi F, Janahmadi M. CB1 can- nabinoid receptor activation rescues amyloid β-induced alterations in Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : behaviour and intrinsic electrophysiological properties of rat hippocam- pal CA1 pyramidal neurones. Cell Physiol Biochem. 2012;29(3–4):391–406. fast, convenient online submission 23. Hajos N, Freund T. Pharmacological separation of cannabinoid sensitive thorough peer review by experienced researchers in your field receptors on hippocampal excitatory and inhibitory fibers. Neurophar - macology. 2002;43(4):503–10. rapid publication on acceptance 24. Llinás R, Sugimori M. Electrophysiological properties of in vitro Purkinje support for research data, including large and complex data types cell somata in mammalian cerebellar slices. J Physiol. 1980;305:171–95. • gold Open Access which fosters wider collaboration and increased citations https:// doi. org/ 10. 1113/ jphys iol. 1980. sp013 357. maximum visibility for your research: over 100M website views per year 25. Yoshida T, Hashimoto K, Zimmer A, Maejima T, Araishi K, Kano M. The • cannabinoid CB1 receptor mediates retrograde signals for depolarization- induced suppression of inhibition in cerebellar Purkinje cells. J Neurosci. At BMC, research is always in progress. 2002;22(5):1690–7. https:// doi. org/ 10. 1523/ JNEUR OSCI. 22- 05- 01690. 2002. Learn more biomedcentral.com/submissions 26. Barnes JL, Mohr C, Ritchey CR, Erikson CM, Shiina H, Rossi DJ. Develop- mentally transient CB1Rs on cerebellar afferents suppress afferent input,
Journal
BMC Neuroscience – Springer Journals
Published: Mar 3, 2023
Keywords: Ataxia; Cerebellum; Purkinje cell; Cannabinoid; CB1R