Fast-scan cyclic voltammetry (FSCV) is an effective tool for measuring dopamine release and clearance throughout the brain, especially the striatum where dopamine terminals are abundant and signals are heavily regulated by release machinery and the dopamine transporter (DAT). Peak height measurement is perhaps the most common method for measuring dopamine release, but it is influenced by changes in clearance. Michaelis–Menten-based modeling has been a standard in measuring dopamine clearance, but it is problematic in that it requires experimenter fitted modeling subject to experimenter bias. This study presents the use of the first derivative (velocity) of evoked dopamine signals as an alternative approach for measuring and distinguishing dopamine release from clearance. Maximal upward velocity predicts reductions in dopamine peak height due to D2 and GABAB receptor stimulation and by alterations in calcium concentrations. The Michaelis–Menten maximal velocity (V max) measure, an approximation for DAT levels, predicts maximal downward velocity in slices and in vivo. Dopamine peak height and upward velocity were similar between wild-type and DAT knock-out (DATKO) mice. In contrast, downward velocity was lower and exponential decay (tau) was higher in DATKO mice, supporting the use of both measures for extreme changes in DAT activity. In slices, the competitive DAT inhibitors cocaine, PTT, and WF23 increased peak height and upward velocity differentially across increasing concentrations, with PTT and cocaine reducing these measures at high concentrations. Downward velocity and tau values decreased and increased respectively across concentrations, with greater potency and efficacy observed with WF23 and PTT. In vivo recordings demonstrated similar effects of WF23, PTT, and cocaine on measures of release and clearance. Tau was a more sensitive measure at low concentrations, supporting its use as a surrogate for the Michaelis–Menten measure of apparent affinity (K m). Together, these results inform on the use of these various measures for dopamine release and clearance.
Biological sex has been pinpointed as significant biological factor impacting the prevalence and prognosis of Substance Use Disorder (SUD). Despite the overwhelming utilization of male subjects in SUD research, epidemiological evidence shows that women are the most susceptible population. At the hub of female vulnerability to SUD is significant dysfunction in the mesolimbic dopamine system connecting the ventral tegmental area (VTA) to the nucleus accumbens (NAc). Importantly, an important characteristic of dopamine release from axon terminals in the NAc is that it is rapidly modulated by local regulatory microcircuit mechanisms independent of somatic activity in the VTA. In the NAc, dopamine is released in tonic (slow and regular) and phasic (short, burst/spikes) patterns that are subject to heavy modulation by cholinergic (ChAT) interneurons signaling through α4β2*‐containing nicotinic acetylcholine receptors (nAChRs) located directly on dopamine terminals. Previous work suggests that ChAT regulation of dopamine release through nAChRs is fundamentally different between males and females, yet the processes and mechanisms that underlie these sex differences are largely unknown. In this project, we took a multifaceted approach to determine sex‐dependent neurochemical mechanisms that underlie ChAT regulation of dopamine release through nAChRs in male, naturally cycling and ovariectomized (OVX) female mice. Using ex vivo fast scan cyclic voltammetry (FSCV) paired with pharmacological applications, we found that ChAT regulation of dopamine release through α4β2*‐nAChRs is not present in female mice under most conditions. Deficits in nAChR modulation of dopamine release in intact females were not affected by the estrous cycle; however, they were rescued by ovariectomy – indicating that ovarian hormones play significant a role in this process. Critically, we find that 17β−Estradiol (E2) increases dopamine release acutely, an effect that is blocked by antagonism of α4β2*‐nAChRs. Additionally, we observed reduced nAChR agonist effects on dopamine release in females; which is what would be expected for desensitized receptors. Finally, Gq‐DREADD behavior studies revealed that male mice learned at a faster rate than intact females when ChAT interneurons were activated. Overall, we show that circulating ovarian hormones, regardless of the hormone cycle, alter the ability of α4β2*‐nAChRs on dopamine terminals to modulate dopamine release in the NAc. This suggests that sex differences in ChAT regulation of dopamine neurotransmission underlies sex‐dependent differentiation in reward learning. Moving forward, it will be critical to directly link these sex differences to reward learning for the development of sex‐specific pharmacotherapies to treat SUD.
Basal forebrain cholinergic neurons are thought to modulate how organisms process and respond to environmental stimuli through impacts on arousal, attention, memory, and motivated behavior. We questioned whether basal forebrain cholinergic neurons are directly involved in conditioned behavior, independent of ancillary roles in stimulus processing. We found that cholinergic neurons are active during behavioral responding for a reward - even in the absence of reward or discrete stimuli. Photostimulation of cholinergic neurons in the basal forebrain or their terminals in the basolateral amygdala (BLA) selectively drove conditioned responding (licking), but not unconditioned licking nor innate motor outputs. In vivo electrophysiological recordings revealed reward-contingency-dependent-gating of cholinergic suppression of BLA neural activity during cholinergic photostimulation, but not dorsomedial prefrontal cortex (dmPFC). Finally, cholinergic terminals suppressed BLA projection neuron activity via monosynaptic muscarinic receptor signaling and facilitation of firing in GABAergic interneurons. Taken together, we show that cholinergic effects are modulated by reward contingency in a target-specific manner to promote conditioned responding. Given that the effects cholinergic photostimulation were modulated by rewards, our results constrain clinical goals of augmenting cholinergic function to improve neuropsychiatric symptoms.
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