Williams BM et al. (2024), Catharanthine Modulates Mesolimbic Dopamine Tra...

XB-ART-60731

ACS Chem Neurosci 2024 Apr 01;159:1738-1754. doi: 10.1021/acschemneuro.3c00478.

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Catharanthine Modulates Mesolimbic Dopamine Transmission and Nicotine Psychomotor Effects via Inhibition of α6-Nicotinic Receptors and Dopamine Transporters.

Williams BM , Steed ND , Woolley JT , Moedl AA , Nelson CA , Jones GC , Burris MD , Arias HR , Kim OH , Jang EY , Hone AJ , McIntosh JM , Yorgason JT , Steffensen SC .

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Iboga alkaloids, also known as coronaridine congeners, have shown promise in the treatment of alcohol and opioid use disorders. The objective of this study was to evaluate the effects of catharanthine and 18-methoxycoronaridine (18-MC) on dopamine (DA) transmission and cholinergic interneurons in the mesolimbic DA system, nicotine-induced locomotor activity, and nicotine-taking behavior. Utilizing ex vivo fast-scan cyclic voltammetry (FSCV) in the nucleus accumbens core of male mice, we found that catharanthine or 18-MC differentially inhibited evoked DA release. Catharanthine inhibition of evoked DA release was significantly reduced by both α4 and α6 nicotinic acetylcholine receptors (nAChRs) antagonists. Additionally, catharanthine substantially increased DA release more than vehicle during high-frequency stimulation, although less potently than an α4 nAChR antagonist, which confirms previous work with nAChR antagonists. Interestingly, while catharanthine slowed DA reuptake measured via FSCV ex vivo, it also increased extracellular DA in striatal dialysate from anesthetized mice in vivo in a dose-dependent manner. Superfusion of catharanthine or 18-MC inhibited the firing rate of striatal cholinergic interneurons in a concentration dependent manner, which are known to potently modulate presynaptic DA release. Catharanthine or 18-MC suppressed acetylcholine currents in oocytes expressing recombinant rat α6/α3β2β3 or α6/α3β4 nAChRs. In behavioral experiments using male Sprague-Dawley rats, systemic administration of catharanthine or 18-MC blocked nicotine enhancement of locomotor activity. Importantly, catharanthine attenuated nicotine self-administration in a dose-dependent manner while having no effect on food reinforcement. Lastly, administration of catharanthine and nicotine together greatly increased head twitch responses, indicating a potential synergistic hallucinogenic effect. These findings demonstrate that catharanthine and 18-MC have similar, but not identical effects on striatal DA dynamics, striatal cholinergic interneuron activity and nicotine psychomotor effects.

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Species referenced: Xenopus laevis
Genes referenced: gabarap nlrp1
GO keywords: regulation of synaptic transmission, cholinergic [+]

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	Summary Image
	Figure 1. Pharmacology of iboga alkaloids 18-methoxycoronaridine (18-MC), catharanthine base, and catharanthine sulfate on single pulse evoked DA release in the NAcc ex vivo. (A) Example current trace (Ivst) and voltammograms (inset) demonstrating DA release with superimposed effect of catharanthine sulfate (20 μM). (B) Corresponding three-dimensional color plots (CVvst) demonstrating DA signal and effect of catharanthine sulfate (20 μM). (C, D) Comparison of 18-MC, catharanthine base (cath base) and catharanthine sulfate (cath sulfate) on evoked DA peak height. (D) In depth comparison at 30 μM. (E–M) Representative superimposed current traces and summary of pharmacology of catharanthine inhibition of evoked DA release in the NAcc including nicotinic acetylcholine receptor (nAChR) antagonists 300 nM and 1 μM DhβE (E, F), α-conotoxin 500 nM MII [H9A, L15b] (G), 2 and 10 μM mecamylamine (Meca) (H, I). Other receptors tested include DA D2 receptor antagonist 1 μM eticlopride (J), voltage-gated calcium channel blocker 10 μM cadmium (K), GABAA receptor antagonist 50 μM picrotoxin (L), and μ-opioid receptor antagonist 1 μM naltrexone (M). Asterisks , , ** indicate significance levels p < 0.05, p < 0.01, and p < 0.001, respectively, for effects of antagonists on evoked DA release. Hashtags # and ## indicate significance levels p < 0.05 and p < 0.01 for catharanthine sulfate in the presence of each antagonist. DA release more than 18-MC at 30 μM (Figure 1D). Additionally, catharanthine sulfate also inhibited DA release significantly more than catharanthine base at 30 μM (Tukey, p = 0.005 55), indicating that catharanthine sulfate is more effective at lower concentrations.
	Figure 2. (A, B) Example 5 pulse, 20 Hz stimulation on evoked DA release. (C) Frequency response of catharanthine base and catharanthine sulfate compared to 1 μM DhβE. Concentrations of catharanthine (cath) base and sulfate were their IC50 concentrations. (D) Comparison of catharanthine base, catharanthine (SO4) and 1 μM DhβE at a stimulation frequency of 20 Hz. Asterisks and * indicate significance levels p < 0.01 and p < 0.001, respectively.
	Figure 3. Catharanthine, but not 18-MC, slows DA reuptake. (1) Effect of catharanthine base, catharanthine sulfate, and 18-MC on DA reuptake of electrically evoked DA release in the NAc. (A, C) Dose–response for catharanthine and 18-MC on evoked DA reuptake time constant (tau) and downward velocity, with comparison at 100 μM in (B) and (D). (2) Effect of catharanthine and 18-MC on reuptake of DA delivered via iontophoresis. (E, G) Representative current traces demonstrating iontophoretically applied DA in the presence of 100 μM catharanthine or 18-MC. (F, H) Corresponding three-dimensional color plots (Ivst) demonstrating DA signal and effect of catharanthine sulfate or 18-MC. (I, K) Effect of catharanthine or 18-MC on tau of iontophoretic DA in the NAc, with comparison at 100 μM (J, L). Asterisks , , and ** indicate significance levels p < 0.05, p < 0.01, and p < 0.001, respectively.
	Figure 4. (A) Illustration demonstrating in vivo measurements of DA via microdialysis. (B) Effect of catharanthine sulfate vs 18-MC on extracellular DA levels in the NAcc in vivo. Catharanthine increased while 18-MC decreased extracellular DA levels. (C) Comparison catharanthine sulfate and 18-MC at 50 mg/kg. Asterisks , , and ** indicate significance levels p < 0.05, p < 0.01, and p < 0.001, respectively.
	Figure 5. Catharanthine and 18-MC inhibit striatal cholinergic interneurons through antagonism of α6-nAChRs. (A) Illustration showing the experimental framework wherein Cre-dependent AAV-DIO-mCherry was injected into the VTA in VGAT-Cre/GAD67-GFP mice. (B) Immunohistochemical panel showing that CINs in VGAT-Cre/GAD67-GFP mice are innervated by VTA-NAcc GABAergic projections expressing α6-nAChRs, as assessed with MII-biotin labeling. White arrow points to a medium spiny neuron, and yellow arrow points to a putative CIN. Note that α6-nAChRs are expressed on VTA GABA neuron terminals to both neuron types. Imaged using oil immersion 40× objective (Olympus, UPlanFLN 1.30 numerical aperture; ac represents anterior commissure as a landmark for the NAc). (C, D) Firing rate recordings were obtained via patch clamp electrophysiology of cholinergic interneurons (CINs) in the NAcc of the slice preparation. Representative ratemeter recordings showing dose-dependent inhibition of CIN firing rate with catharanthine sulfate and 18-MC at 4 concentrations (1.0–30.0 μM). Insets are 6 s spike recordings before (a) and following increasing concentrations of superfused catharanthine or 18-MC (b, c) at the times indicated on the representative ratemeters below. (E) Summary of catharanthine and 18-MC inhibition of CIN firing rate in the NAcc slice. (F) Representative recordings of current traces from Xenopus laevis oocytes expressing recombinant rat α6/α3β4 or α6/α3β2β3 nAChRs demonstrating suppression of ACh currents by catharanthine and 18-MC. (G) Concentration–response curves for catharanthine and 18-MC on α6/α3β4 nAChRs and α6/α3β2β3 nAChRs with best of fit curves. The error bars indicate SDs from four independent IC50 determinations; values in parentheses indicate 95% CI. Asterisks and *** indicate significance levels p < 0.05 and p < 0.001, respectively.
	Figure 6. Catharanthine and 18-MC reduce NIC-mediated hyperlocomotion in rats. (A–C) Effect of systemic administration of catharanthine on locomotor activity in rats. (A) Representative open field tracking plots. (B) Effect of catharanthine (CATH) or vehicle (VEH) on NIC-induced hyperlocomotion over time. Nicotine markedly increased locomotor activity compared to saline (SAL), and catharanthine significantly reduced NIC hyperlocomotor behavior. (C) Summary of the effects of catharanthine on hyperlocomotion across doses 10–40 mg/kg. (D–F) Effect of systemic administration of 18-MC on locomotor activity in rats. (D) Example open field tracking plots. (E) Effect of 18-MC on NIC-induced hyperlocomotion over time. 18-MC significantly reduced NIH hyperlocomotor behavior. (F) Summary of the effects of 18-MC on NIC-induced hyperlocomotion. Asterisks and * indicate significance levels p < 0.01 and p < 0.001 compared to saline. Hashtags # and ### indicate significance levels p < 0.05 and p < 0.001 compared to NIC.
	Figure 7. Catharanthine inhibits NIC self-administration behavior and hallucinogenic properties. (A) Timeline for NIC self-administration procedure (see Methods). (B) Effects of catharanthine (10–40 mg/kg) on baseline NIC infusions 3 days before catharanthine or vehicle (VEH) injections. There was no significant difference in responding preinjection. (C) Catharanthine (CATH) significantly reduced NIC infusions but only at the 40 mg/kg dose level. (D) Catharanthine also significantly reduced active lever responses for NIC but only at 40 mg/kg. (E) Catharanthine reduced inactive lever pressing but with no significant dose in post hoc analysis. (F) Catharanthine did not significantly affect lever presses for food reinforcement. (G) Catharanthine + NIC significantly increased head twitch responses. Asterisks and * indicate significance levels p < 0.01 and p < 0.001, respectively.
	Supplemental Figure 1: Example evoked DA currents for iboga alkaloids catharanthine base and 18-methoxycoronaridine. (A-B) Current vs time and color plots of the effect of catharanthine base (30 uM) on evoked DA release in the NAcc. (C-D) Current vs time and color plots of the effect of 18-methoxycoronaridine (30 uM) on evoked DA release in the NAcc.
	Supplemental Figure 2: Changes in DA release following catharanthine administration has no effect on DA uptake. (A) DA release as measured using peak height is not correlated with Tau, a measure of DA uptake. (A) DA release as measured using peak height is also not correlated with the downward velocity, another measure of DA uptake.

	Summary Image
	Figure 1. Pharmacology of iboga alkaloids 18-methoxycoronaridine (18-MC), catharanthine base, and catharanthine sulfate on single pulse evoked DA release in the NAcc ex vivo. (A) Example current trace (Ivst) and voltammograms (inset) demonstrating DA release with superimposed effect of catharanthine sulfate (20 μM). (B) Corresponding three-dimensional color plots (CVvst) demonstrating DA signal and effect of catharanthine sulfate (20 μM). (C, D) Comparison of 18-MC, catharanthine base (cath base) and catharanthine sulfate (cath sulfate) on evoked DA peak height. (D) In depth comparison at 30 μM. (E–M) Representative superimposed current traces and summary of pharmacology of catharanthine inhibition of evoked DA release in the NAcc including nicotinic acetylcholine receptor (nAChR) antagonists 300 nM and 1 μM DhβE (E, F), α-conotoxin 500 nM MII [H9A, L15b] (G), 2 and 10 μM mecamylamine (Meca) (H, I). Other receptors tested include DA D2 receptor antagonist 1 μM eticlopride (J), voltage-gated calcium channel blocker 10 μM cadmium (K), GABAA receptor antagonist 50 μM picrotoxin (L), and μ-opioid receptor antagonist 1 μM naltrexone (M). Asterisks , , ** indicate significance levels p < 0.05, p < 0.01, and p < 0.001, respectively, for effects of antagonists on evoked DA release. Hashtags # and ## indicate significance levels p < 0.05 and p < 0.01 for catharanthine sulfate in the presence of each antagonist. DA release more than 18-MC at 30 μM (Figure 1D). Additionally, catharanthine sulfate also inhibited DA release significantly more than catharanthine base at 30 μM (Tukey, p = 0.005 55), indicating that catharanthine sulfate is more effective at lower concentrations.
	Figure 2. (A, B) Example 5 pulse, 20 Hz stimulation on evoked DA release. (C) Frequency response of catharanthine base and catharanthine sulfate compared to 1 μM DhβE. Concentrations of catharanthine (cath) base and sulfate were their IC50 concentrations. (D) Comparison of catharanthine base, catharanthine (SO4) and 1 μM DhβE at a stimulation frequency of 20 Hz. Asterisks and * indicate significance levels p < 0.01 and p < 0.001, respectively.
	Figure 3. Catharanthine, but not 18-MC, slows DA reuptake. (1) Effect of catharanthine base, catharanthine sulfate, and 18-MC on DA reuptake of electrically evoked DA release in the NAc. (A, C) Dose–response for catharanthine and 18-MC on evoked DA reuptake time constant (tau) and downward velocity, with comparison at 100 μM in (B) and (D). (2) Effect of catharanthine and 18-MC on reuptake of DA delivered via iontophoresis. (E, G) Representative current traces demonstrating iontophoretically applied DA in the presence of 100 μM catharanthine or 18-MC. (F, H) Corresponding three-dimensional color plots (Ivst) demonstrating DA signal and effect of catharanthine sulfate or 18-MC. (I, K) Effect of catharanthine or 18-MC on tau of iontophoretic DA in the NAc, with comparison at 100 μM (J, L). Asterisks , , and ** indicate significance levels p < 0.05, p < 0.01, and p < 0.001, respectively.
	Figure 4. (A) Illustration demonstrating in vivo measurements of DA via microdialysis. (B) Effect of catharanthine sulfate vs 18-MC on extracellular DA levels in the NAcc in vivo. Catharanthine increased while 18-MC decreased extracellular DA levels. (C) Comparison catharanthine sulfate and 18-MC at 50 mg/kg. Asterisks , , and ** indicate significance levels p < 0.05, p < 0.01, and p < 0.001, respectively.
	Figure 5. Catharanthine and 18-MC inhibit striatal cholinergic interneurons through antagonism of α6-nAChRs. (A) Illustration showing the experimental framework wherein Cre-dependent AAV-DIO-mCherry was injected into the VTA in VGAT-Cre/GAD67-GFP mice. (B) Immunohistochemical panel showing that CINs in VGAT-Cre/GAD67-GFP mice are innervated by VTA-NAcc GABAergic projections expressing α6-nAChRs, as assessed with MII-biotin labeling. White arrow points to a medium spiny neuron, and yellow arrow points to a putative CIN. Note that α6-nAChRs are expressed on VTA GABA neuron terminals to both neuron types. Imaged using oil immersion 40× objective (Olympus, UPlanFLN 1.30 numerical aperture; ac represents anterior commissure as a landmark for the NAc). (C, D) Firing rate recordings were obtained via patch clamp electrophysiology of cholinergic interneurons (CINs) in the NAcc of the slice preparation. Representative ratemeter recordings showing dose-dependent inhibition of CIN firing rate with catharanthine sulfate and 18-MC at 4 concentrations (1.0–30.0 μM). Insets are 6 s spike recordings before (a) and following increasing concentrations of superfused catharanthine or 18-MC (b, c) at the times indicated on the representative ratemeters below. (E) Summary of catharanthine and 18-MC inhibition of CIN firing rate in the NAcc slice. (F) Representative recordings of current traces from Xenopus laevis oocytes expressing recombinant rat α6/α3β4 or α6/α3β2β3 nAChRs demonstrating suppression of ACh currents by catharanthine and 18-MC. (G) Concentration–response curves for catharanthine and 18-MC on α6/α3β4 nAChRs and α6/α3β2β3 nAChRs with best of fit curves. The error bars indicate SDs from four independent IC50 determinations; values in parentheses indicate 95% CI. Asterisks and *** indicate significance levels p < 0.05 and p < 0.001, respectively.
	Figure 6. Catharanthine and 18-MC reduce NIC-mediated hyperlocomotion in rats. (A–C) Effect of systemic administration of catharanthine on locomotor activity in rats. (A) Representative open field tracking plots. (B) Effect of catharanthine (CATH) or vehicle (VEH) on NIC-induced hyperlocomotion over time. Nicotine markedly increased locomotor activity compared to saline (SAL), and catharanthine significantly reduced NIC hyperlocomotor behavior. (C) Summary of the effects of catharanthine on hyperlocomotion across doses 10–40 mg/kg. (D–F) Effect of systemic administration of 18-MC on locomotor activity in rats. (D) Example open field tracking plots. (E) Effect of 18-MC on NIC-induced hyperlocomotion over time. 18-MC significantly reduced NIH hyperlocomotor behavior. (F) Summary of the effects of 18-MC on NIC-induced hyperlocomotion. Asterisks and * indicate significance levels p < 0.01 and p < 0.001 compared to saline. Hashtags # and ### indicate significance levels p < 0.05 and p < 0.001 compared to NIC.
	Figure 7. Catharanthine inhibits NIC self-administration behavior and hallucinogenic properties. (A) Timeline for NIC self-administration procedure (see Methods). (B) Effects of catharanthine (10–40 mg/kg) on baseline NIC infusions 3 days before catharanthine or vehicle (VEH) injections. There was no significant difference in responding preinjection. (C) Catharanthine (CATH) significantly reduced NIC infusions but only at the 40 mg/kg dose level. (D) Catharanthine also significantly reduced active lever responses for NIC but only at 40 mg/kg. (E) Catharanthine reduced inactive lever pressing but with no significant dose in post hoc analysis. (F) Catharanthine did not significantly affect lever presses for food reinforcement. (G) Catharanthine + NIC significantly increased head twitch responses. Asterisks and * indicate significance levels p < 0.01 and p < 0.001, respectively.
	Supplemental Figure 1: Example evoked DA currents for iboga alkaloids catharanthine base and 18-methoxycoronaridine. (A-B) Current vs time and color plots of the effect of catharanthine base (30 uM) on evoked DA release in the NAcc. (C-D) Current vs time and color plots of the effect of 18-methoxycoronaridine (30 uM) on evoked DA release in the NAcc.
	Supplemental Figure 2: Changes in DA release following catharanthine administration has no effect on DA uptake. (A) DA release as measured using peak height is not correlated with Tau, a measure of DA uptake. (A) DA release as measured using peak height is also not correlated with the downward velocity, another measure of DA uptake.