Cannabidiol Reduces D1 and D2 Medium Spiny Neuron Excitability in the Nucleus Accumbens Core

Abstract

Cannabidiol (CBD) is a non-psychotomimetic phytocannabinoid constituent of the cannabis plant that shows promise for the treatment of a variety of neuropsychiatric disorders such as anxiety disorders, post-traumatic stress disorder, and substance use disorders. The nucleus accumbens (NAc) is a key brain region in the etiologies of these disorders and is actively modulated by CBD. Prior research has established that CBD alters the molecular composition of the NAc, but none have assessed how CBD affects NAc neuronal function. In this study, we demonstrate that CBD significantly decreases D1 and D2 medium spiny neuron membrane excitability, broadens action potentials, and has no effect on spontaneous excitatory synaptic transmission in the NAc core. These data enhance our understanding of CBD’s physiological effects and provide mechanistic insight into its therapeutic potential.

Introduction

Cannabidiol (CBD) is a major non-psychotomimetic phytocannabinoid constituent of the cannabis plant that has seen significant increases in commercialization and recreational use. In 2022, 20% of adults in the U.S. reported using CBD/hemp-derived products¹. However, there is a paucity of knowledge of the mechanisms behind CBD’s putative therapeutic effects. CBD has been FDA approved for the treatment of certain pediatric-onset epilepsies and shows promise for the treatment of a variety of neuropsychiatric disorders such as anxiety disorders, post-traumatic stress disorder, and substance use disorders^2–4. The nucleus accumbens (NAc) is a key brain region in the etiologies of these disorders and is actively modulated by CBD^5–11. For example, CBD decreases alcohol craving and cue-induced NAc activation in human subjects with alcohol use disorder¹². Preclinical work has correlated improvements in constructs of neuropsychiatric disorders with changes in the molecular composition of the NAc^13–17. However, there is a gap in knowledge of the functional consequences of CBD on NAc neuronal properties.

95% of NAc neurons are medium spiny projection neurons (MSNs). MSNs can be subdivided into two types: dopamine receptor type 1 and type 2-expressing (D1 and D2 MSNs). NAc D1 and D2 MSNs serve as a key node in the integration of information from a variety of brain regions involved in neuropsychiatric disorders such as the hippocampus, amygdala, prefrontal cortex, and thalamus¹⁰. Therefore, assessing how CBD affects MSN function may elucidate mechanisms by which CBD exerts its therapeutic effects. In this study, we utilized whole-cell patch-clamp electrophysiology in acute brain slices from transgenic mice to assess the functional effects of CBD on D1 and D2 MSNs within the NAc core (NAcC). We found that CBD significantly decreases D1 and D2 MSN membrane excitability, broadens action potentials, increases afterhyperpolarization, and has no effect on spontaneous excitatory synaptic transmission. These results enhance our understanding of CBD’s effects on NAc physiology and provide mechanistic insight into its therapeutic potential.

Methods

Subjects.

Experiments were performed adhering to the National Institutes of Health guidelines for the Care and Use of Laboratory Animals and were approved by the Vanderbilt University Institutional Animal Care and Use Committee. All experiments used male and female D1^tdtomato (Jackson Laboratories, Cat. #016204) and/or D2^GFP mice (Tg(Drd2-EGPF)S118Gsat) aged 8–12 weeks.

Ex Vivo Electrophysiology.

To collect acute ex vivo brain slices containing NAcC, mice were anesthetized with isoflurane and rapidly decapitated. Dissected tissue was placed into ice-cold oxygenated dissecting solution and sectioned at 250 μM for sagittal slices of NAcC using the Leica VT1200S Vibratome. The dissecting solution consisted of (in mM): 93 N-methyl-D-glucamine (NMDG), 2.5 KCl, 20 HEPES, 10 MgCl₂, 1.2 NaH₂PO₄·H₂O, 30 NaHCO₃, 0.5 CaCl₂· 2H₂0, 25 glucose, 3 Na-pyruvate, 5 ascorbic acid. The aCSF consisted of (in mM): 118.9 NaCl, 2.5 KCl, 1.3 MgCl₂·6H₂O, 2.5 CaCl₂, 1 NaH₂PO₄, 26.2 NaHCO₃, 11 glucose. Cannabidiol (Cayman Chemical, Cat. #90080) was dissolved in DMSO at a stock concentration of 20 mM. For incubation experiments, slices were incubated for a minimum of 1 hr with aCSF containing either 20 μM CBD or equivalent dimethyl sulfoxide (DMSO). For acute application experiments, baseline was obtained with aCSF prior to 10 min perfusion of 20 μM CBD or DMSO. Slices were acclimated to the rig for 30 min prior to recording, perfused at a flow rate of 2–3 mL/min, and maintained at 29–31 °C.

Whole-cell patch clamp electrophysiology was performed using a CV-7B headstage, Multiclamp 700B Amplifier, and Axopatch Digidata 1550 digitizer. Cells were visualized at 40X magnification using an upright microscope (Scientifica) that allowed for both infrared-differential interference contrast and fluorescence optics. D1 MSNs were identified as fluorescent cells at 550nM in D1^tdtomato mice and non-fluorescent cells at 470nM in D2^GFP mice. D2 MSNs were identified as fluorescent cells at 470nM in D2^GFP mice and non-fluorescent cells at 550nM in D1^tdtomato mice. 3–5 MΩ glass recording micropipettes (Sutter P1000 Micropipette Puller) filled with potassium gluconate internal solution were used for patching. The internal solution consisted of (in mM): 135 K-gluconate, 5 NaCl, 2 MgCl₂, 10 HEPES, 0.2 EGTA, 2.5 Mg₂-ATP, 0.2 Na₂-GTP. All cells were voltage clamped at −70 mV for 5 min of dialysis prior to recording. Current clamp experiments were performed by clamping cells at a baseline holding current to rest at −70 mV. The excitability protocol consisted of 800 ms square wave current every 5 sec from −50 pA to 400 pA in 25 pA increments. Voltage clamp experiments were performed by voltage clamping cells at −70 mV for 5 min of gap free recording. Cells were excluded from analysis if they had a baseline series resistance ≥ 25 MOhm or change in series resistance ≥ 20%.

Quantification and Statistical Analysis.

Electrophysiology data were initially processed in Clampfit 10 software (Molecular Devices) and quantified in Microsoft Excel. Data were analyzed and graphs created with GraphPad Prism 10 software. The significance threshold for all analyses was p < 0.05. Data are shown as mean ± SEM.

Results

Cannabidiol incubation reduces NAcC D1 and D2 MSN membrane excitability.

CBD interacts with various substrates that can influence membrane excitability and has been shown to inhibit action potential generation in other brain regions¹⁸. To determine if CBD affects MSN excitability in the NAcC, we incubated acute brain slices containing NAcC in 20 μM CBD or vehicle solution (aCSF with proportional DMSO) for a minimum of 1 hr and recorded action potential frequency in response to square wave current injection. Current injections for each cell ranged from −50 to 400 pA in 25 pA increments. D1 and D2 MSNs incubated in CBD showed significantly decreased action potential firing frequency compared to vehicle control (Figures 1, 2 A). Next, we assessed action potential properties in response to CBD. We analyzed the half-width, maximum decay slope, afterhyperpolarization peak, and maximum rise slope of the first action potential generated under 200 pA current injection. For D1 and D2 MSNs, 20 μM CBD significantly increased the half-width, decreased the maximum decay slope, decreased the afterhyperpolarization peak, and had no effect on maximum rise slope (Figures 1, 2 B–E). These results implicate potassium channels as a CBD target, as half-width, afterhyperpolarization peak, and maximum decay slope are indicators of potassium channel activity.

Figure 1.

20 uM CBD incubation decreases NAcC D1 MSN excitability. (A) Effect of 20 μM CBD incubation on D1 MSN excitability. (B) Representative traces of D1 MSNs incubated in either DMSO or CBD (scale = 100 ms, 50 mV). For C-F, the first action potential generated at 200 pA is analyzed. (C-F) Effect of 20 μM CBD incubation on D1 MSN (C) half-width, (D) maximum decay slope, (E) afterhyperpolarization peak, (F) maximum rise slope. Black and white points denote male and female mice, respectively. Data analyzed by (A) two-way repeated measures ANOVA or (C-F) independent two-tailed t-test (**p < .01).

Figure 2.

20 uM CBD incubation decreases NAcC D2 MSN excitability. (A) Effect of 20 μM CBD incubation on D2 MSN excitability. (B) Representative traces of D2 MSNs incubated in either DMSO or CBD (scale = 100 ms, 50 mV). For C-F, the first action potential generated at 200 pA is analyzed. (C-F) Effect of 20 μM CBD incubation on D2 MSN (C) half-width, (D) maximum decay slope, (E) afterhyperpolarization peak, (F) maximum rise slope. Black and white points denote male and female mice, respectively. Data analyzed by (A) two-way repeated measures ANOVA or (C-F) independent two-tailed t-test (**p < .01, ****p < .0001).

Acute cannabidiol exposure decreases NAcC D1 and D2 MSN membrane excitability at high levels of current.

To evaluate the time course of CBD’s effects on MSN excitability, we compared action potential frequency pre and post 10 min 20 μM CBD or DMSO bath application. D1 and D2 MSNs showed a significant decrease in frequency at high levels of current injection with CBD but not DMSO (Figures 3, 4 A–C). D1 MSNs displayed no change in half-width or maximum decay slope with CBD or DMSO, a significant decrease in afterhyperpolarization peak with CBD but not DMSO, and a significant decrease in maximum rise slope with both CBD and DMSO (Figure 3, D–G, K–N). D2 MSNs displayed no change in half-width with CBD or DMSO, a significant decrease in maximum decay slope with CBD but not DMSO, a significant decrease in afterhyperpolarization peak with both CBD and DMSO, and no change in maximum rise slope with CBD or DMSO (Figure 4, D–G, K–N). These results suggest that CBD may have low potency at its target or activates a signaling cascade that regulates ion channel function on a slower time scale.

Figure 3.

Acute 20 uM CBD decreases NAcC D1 MSN excitability at high levels of current. Acute 10 min application of 20 μM CBD or equivalent DMSO (A) Effect of acute DMSO on D1 MSN excitability. (B) Representative traces of D1 MSNs pre and post DMSO (scale = 100 ms, 50 mV). (C) Action potential frequency at 400pA current injection pre and post DMSO. (D-G) Effect of acute DMSO on D1 MSN (D) half-width, (E) maximum decay slope, (F) afterhyperpolarization peak, (G) maximum rise slope. (H-N) same as (A-G) for CBD. Black and white lines denote male and female mice, respectively. Data analyzed by (A, H) two-way repeated measures ANOVA with Holm-Šídák multiple comparisons, (C, J) Holm-Šídák multiple comparison from (A, H), (D-G, K-N) paired two-tailed t-test (*p < .05, **p < .01).

Figure 4.

Acute 20 uM CBD decreases NAcC D2 MSN excitability at high levels of current. Acute 10 min application of 20 μM CBD or equivalent DMSO (A) Effect of acute DMSO on D2 MSN excitability. (B) Representative traces of D1 MSNs pre and post DMSO (scale = 100 ms, 50 mV). (C) Action potential frequency at 400pA current injection pre and post DMSO. (D-G) Effect of acute DMSO on D1 MSN (D) half-width, (E) maximum decay slope, (F) afterhyperpolarization peak, (G) maximum rise slope. (H-N) same as (A-G) for CBD. Black and white lines denote male and female mice, respectively. Data analyzed by (A, H) two-way repeated measures ANOVA with Holm-Šídák multiple comparisons, (C, J) Holm-Šídák multiple comparison from (A, H), (D-G, K-N) paired two-tailed t-test (*p < .05, **p < .01, #p < .001, $p < .0001).

Cannabidiol incubation has no effect on NAcC D1 and D2 MSN spontaneous excitatory neurotransmission.

Prior research has shown that CBD can alter spontaneous excitatory post synaptic currents (sEPSCs) in multiple brain regions^18–20. To determine if CBD affects sEPSCs in the NAcC, we incubated acute brain slices containing NAcC in 20 μM CBD or DMSO and recorded sEPSCs. We found no significant difference in sEPSC frequency or amplitude for D1 and D2 MSNs, suggesting that 20 μM CBD does not affect glutamatergic presynaptic release probability or postsynaptic AMPA receptor function in NAcC MSNs (Figure 5).

Figure 5.

20 uM CBD incubation has no effect on NAcC D1 or D2 MSN spontaneous excitatory transmission. (A) Effect of 20 μM CBD or DMSO on sEPSC frequency in D1 MSNs. (B) Effect of 20 μM CBD or DMSO on sEPSC amplitude in D1 MSNs. (C) Representative traces of CBD and DMSO-incubated D1 MSN sEPSCs (scale = 250 ms, 20 pA). (D-F) Same as A-C but for D2 MSNs. Black and white points denote male and female mice, respectively. Data analyzed by independent two-tailed t-test.

Discussion

This study is the first to investigate the consequences of CBD exposure on electrophysiological properties of NAcC MSNs. We establish that CBD reduces NAcC D1 and D2 MSN membrane excitability, broadens action potentials, and decreases afterhyperpolarization while having no detectable effects on spontaneous excitatory synaptic transmission. Because CBD incubation resulted in a robust decrease in excitability while acute application only showed effects at high levels of current injection, CBD may have low potency at its target or activate a signaling cascade that regulates ion channel function on a slower time scale (Figures 1–4).

Our finding that CBD causes reduced membrane excitability in D1 and D2 MSNs (Figures 1, 2) is consistent with results from striatal cultures²¹. Additionally, CBD has been shown to reduce excitability of neurons in the central and basolateral amygdala, hippocampus, dentate gyrus, and dorsal root ganglia^{18,20,22–24}. The molecular mechanism(s) underlying CBD’s inhibition of neuronal excitability are unclear. One potential site of action is voltage gated sodium channel 1.6 (NaV1.6). NaV1.6 regulates MSN excitability in the NAc, and CBD inhibits NaV1.6 resurgent and persistent current in striatal cultures^21,25,26. While our analysis found no effect of CBD on maximum rise slope, this metric is an indicator of total sodium current during the depolarization phase and is thus not able to detect a change in resurgent or persistent current. Indeed, CBD has no effect on NaV1.6 peak transient current²¹. Our action potential analysis suggests that CBD targets potassium channels in the NAcC, as half-width, maximum decay slope, and afterhyperpolarization peak are potassium channel dependent (Figures 1, 2). In vitro data suggests multiple potassium channels as CBD targets. For example, Kv7.2/7.3-mediated low-voltage activated M-current is enhanced by CBD in vitro, Kv7.2/7.3 enhancement decreases excitability, and these channels are expressed on NAc MSNs^27–32. Thus, CBD-enhanced M-current may be responsible for our results. However, our finding of increased half-width and decreased maximum decay slope and afterhyperpolarization peak would suggest inhibited M-current (Figures 1, 2). Still, other potassium channels may mediate these action potential properties while CBD decreases MSN excitability by enhancing Kv7.2/7.3. Additional potassium channels expressed on NAc MSNs that are targeted by CBD include Trek-1 and BK channels^33–38. CBD inhibits both mechanosensitive Trek-1 and calcium-activated BK channels in vitro and their respective knockouts lead to increased half-width^33,36,39,40. However, Trek-1 and BK channel activation has been shown to decrease neuronal excitability in other brain regions^40,41. Thus, one would predict increased excitability if CBD were inhibiting these channels in MSNs. Another ion channel targeted by CBD that is expressed by NAc MSNs is the non-selective cation channel transient receptor potential vanilloid 1 (TRPV1)⁴². CBD is a TRPV1 agonist and altered conductance by TRPV1 activation may contribute to the phenotypes we uncovered^42–44. We have discussed a myriad of previously identified membrane effector systems linked to CBD action. However, it is possible that a unique signaling mechanism is recruited by CBD in NAcC MSNs.

Acute 10 min CBD application led to a similar decrease in NAcC D1 and D2 MSN excitability as incubation, but only at high levels of current injection. Most action potential properties were unaffected by acute CBD (either no effect with CBD or DMSO or a change in both). However, D1 MSNs showed a significant decrease in afterhyperpolarization peak and D2 MSNs showed a significant decrease in maximum decay slope with acute CBD but not DMSO. These results are in the same direction as with incubation. However, most action potential properties were still unaffected by acute CBD and the two that showed effects were distinct between D1 and D2 MSNs (Figures 4, 5). We believe that the dichotomy between incubation and acute exposure in terms of excitability and action potential properties suggests that distinct mechanisms may be responsible for lowering excitability and altering action potential kinetics. Conversely, action potential broadening/increased afterhyperpolarization may be a consequence of reducing excitability that only emerges with longer incubation. The fact that CBD requires incubation to reach its full effects suggests that it may have low potency at its target (e.g. sodium/potassium channels) or work on a longer time scale through second messenger pathways. Beyond ion channels, CBD can act as an: inhibitor of the reuptake/metabolism of anandamide (cannabinoid receptor 1 [CB1] endogenous agonist), CB1 negative allosteric modulator, G-protein-coupled receptor 55 (GPR55) antagonist, 5-HT_1A agonist, peroxisome proliferator-activated receptor (PPAR) gamma agonist, mu and delta opioid receptor negative allosteric modulator, adenosine 1 receptor (A1) agonist and reuptake inhibitor, and more^45–47. With such a wide array of targets, some expressed on NAc MSNs (e.g. GPR55, A1) and some not (e.g. 5-HT_1A, PPAR), there is a high likelihood of complex synergistic effects of multiple receptor systems to lead to our results. Suggesting the complexity of CBD-mediated altered excitability, CBD has an inhibitory effect on action potential firing frequency in almost every cell type in which it has been assessed except parvalbumin-expressing fast-spiking interneurons, in which it conversely leads to increased membrane excitability^18,20,22,23.

We found that CBD caused no change in spontaneous excitatory synaptic transmission in D1 and D2 MSNs in the NAcC. Prior literature has established that CBD affects synaptic transmission in a brain region and cell-type specific manner^18,20,23. Our work suggests that CBD’s effect on NAcC MSNs may be constrained to membrane excitability with no effect on synaptic transmission. However, we only assessed sEPSCs. There are cell types such as somatostatin neurons in the central amygdala that show a reduction in spontaneous inhibitory postsynaptic currents (sIPSCs) but no change in sEPSCs with CBD incubation¹⁸. Future work should assess the effects of CBD on sIPSCs in NAcC MSNs. Additionally, there is the possibility of CBD acting as a depotentiator of synaptic plasticity. CBD has been shown to normalize increases in sEPSC and decreases in sIPSC frequency caused by the endogenous membrane phospholipid lysophosphatidylinositol in CA1 pyramidal neurons with no effect by itself¹⁹. Therefore, CBD may be able to normalize plastic changes in spontaneous synaptic transmission in NAc MSNs as can be caused by chronic cocaine or morphine exposure^48,49.

The ability of CBD to decrease NAc MSN excitability shows therapeutic promise for the treatment of a variety of disease states. For example, increased NAc MSN (core and/or shell) excitability has been found in response to exposure/withdrawal from drugs of abuse, acute restraint stress, juvenile social isolation, fear conditioning, and obesity^50–56. Indeed, CBD has shown promise as a treatment for anxiety disorders, post-traumatic stress disorder, and substance use disorders^2,3. Clinically, CBD has been shown to reduce alcohol craving and decrease functional NAc activity in response to alcohol-paired cues in subjects with alcohol use disorder¹². Our finding of decreased NAcC MSN excitability provides a physiological mechanism for decreased alcohol-paired cue-induced NAc activation.

In conclusion, this work is the first to investigate the functional physiological effects of CBD in the NAc. We establish that CBD massively inhibits D1 and D2 MSN membrane excitability in the NAcC and alters potassium channel-dependent action potential kinetics. Future work should further evaluate the molecular mechanisms underlying these effects and assess whether inhibition of NAc excitability by CBD can improve preclinical disease models.

Funding Statement

This work was supported by NIDA R01DA040630 (BAG) NIMH T32 GM149363 (ZEJ)