Synaptic Transmission: Modulation, Plasticity And Effects Of Drugs Of Abuse

National Institute On Alcohol Abuse And Alcoholism

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Abstract

Differential Mechanisms of Synaptic Modulation at Synapses made by Striatal Efferents The basal ganglia (BG), including the striatum and connected regions, have critical roles in action learning, selection, and production, as well as responses to psychoactive drugs. The medium spiny projection neurons (MSNs) of the dorsal striatum (DS) innervate two BG subregions, the Globus pallidus external segment (GPe) and the Substantia nigra pars reticulata (SNr). The MSNs that express D2 dopamine and A2A adenosine receptors project predominantly to GPe (the indirect pathway), while those that express D1 dopamine receptors primarily innervate the SNr (the direct pathway) and send collateral projections to GPe. The balance between the activity of the different MSN projections is crucial for performance, structure and timing of new and learned behaviors. Presynaptic modulation that regulates release of Gamma-aminobutyric acid (GABA) from the MSN terminals in GPe and SNr helps to shape activity in these two regions. The GABAB and cannabinoid type 1 (CB1) G protein-coupled receptors (GPCRs) are highly expressed in GPe and SNr, where they activate Gi/o-type G proteins to reduce synaptic transmission. Thus, we have examined how these two GPCRs modulate GABAergic striatopallidal and striatonigral transmission using optogenetic and electrophysiological techniques. Expressing channelrhodopsin2 (ChR2) in A2A- or D1-expressing MSNs was achieved by injection of a Cre-dependent, ChR2-coding virus into the DS of A2A-Cre or D1-Cre mice. Whole-cell recordings in GPe revealed that optical activation of indirect pathway afferents produced GABAergic inhibitory postsynaptic currents (oIPSCs) in GPe neurons, and similar synaptic responses were also produced by stimulation of collateral afferents made by D1-MSNs in GPe. Recordings in SNr neurons showed that optogenetic activation of direct pathway MSNs produced GABAergic IPSCs, as expected from previous studies. We next determined if GABAB or CB1 agonists altered oIPSCs. Agonists for both receptors reduced oIPSC amplitude at both SNr direct pathway synapses and GPe indirect pathway synapses. The synaptic depression induced by the GABAB agonist was generally larger in magnitude than that induced by the CB1 agonist. Agonists for both receptors decreased the frequency of spontaneous IPSCs in both regions. By substituting strontium for most of the extracellular calcium we were able to measure optically-evoked asynchronous miniature IPSCs. The frequency of these events was reduced by the CB1 agonist. These findings support the presynaptic location of GABAB and CB1 synaptic modulation at both sets of afferents. Inhibition of voltage-gated calcium channels (VGCCs) leading to reduction in presynaptic calcium influx is a prominent mechanism through which Gi/o-coupled GPCRs reduce excitation-secretion coupling. To assess if GABAB and CB1 receptors reduce presynaptic calcium we used the genetically-encoded calcium indicator GCaMP6 to examine changes in presynaptic calcium in direct and indirect pathway afferents. In these experiments, a virus coding for GCaMP6f was injected into the dorsal striatum (DS) of either A2A- or D1-Cre mice. Brain slices were prepared, and electrical stimulation was applied in the GPe or SNr. Fluorescence was detected in DS and GPe in slices from GCaMP-injected A2A-Cre mice, while fluorescence was present in DS, GPe and SNr in slices from injected D1-Cre mice. The fluorescence in GPe and SNr was present in processes that resembled axons and axon terminals. Electrical stimulation evoked fluorescence increases, measured with slice photometry, in GPe in the indirect pathway-expressing slices and in SNr in the direct pathway-expressing slices. The majority or pharmacological experiments were performed using 4 pulse, 10 Hz stimulus bursts that reliably evoked stable fluorescence increases of 6-10% df/f. The stimulus burst-induced increases in presynaptic calcium were eliminated by application of the sodium channel blocker tetrodotoxin, and inhibited by reducing extracellular calcium, indicating that the increases were dependent on neuronal activation and calcium entry. We next examined the role of different VGCCs in the presynaptic calcium increases by applying blockers of the L, N, and P/Q-type VGCCs to slices. In both indirect pathway afferents to GPe and direct pathway afferents to SNr, burst-induced calcium increases were reduced by blockers of both N and P/Q-type VGCCs. Blockers of L-type VGCCs had no effect in either brain region. These findings support the idea that the VGCCs involved in calcium entry in these afferents are subtypes that can be inhibited by Gi/o-coupled GPCRs. Similar results were obtained in electrophysiological experiments that examined IPSCs, indicating that the N and P/Q-type VGCCs are involved in excitation-secretion coupling. To assess if reduced presynaptic calcium is associated with synaptic modulation, we examined effects of GABAB and CB1 receptor agonists on the burst-induced presynaptic calcium increases measured in GPe and SNr with GCaMP6f and slice photometry. In both indirect pathway GPe and direct pathway SNr afferents we observed that GABAB agonist application reduced burst-induced calcium transients by 50%, while there was no discernable effect of CB1 agonist application. These findings indicate that inhibition of voltage-dependent calcium increases (most likely due to VGCC inhibition) is likely a prominent mechanism underlying modulation of GABA release at these synapses. This finding is not surprising given the role of decreased calcium entry in Gi/o-coupled GPCR presynaptic modulation at many synapses. The lack of CB1 agonist effect on presynaptic calcium transients despite a strong effect on transmission indicates that mechanisms other than VGCC modulation may play a role at striatonigral and striatopallidal synapses. Thus, we examined CB1 agonist effects on miniature IPSCs (mIPSCs) that are independent of action potentials and VGCCs. We observed that CB1 activation reduced the frequency and amplitude of mIPSCs at synapses in the GPe, supporting the idea that CB1 can inhibit neurotransmitter release at a site downstream from VGCCs. The CB1-mediated presynaptic modulation appears to involve direct effects on vesicle fusion mechanisms, as has been reported for Gi/o-coupled GPCRs at other synapses. We are currently exploring potential roles of vesicle-associated proteins in this CB1 action. Our findings indicate that CB1 and GABAB receptors produce strong presynaptic modulation of GABA release at striatonigral and striatopallidal synapses. The GABAB receptors appear to modulate neurotransmitter release via effects on VGCCs, while CB1 receptor actions appear to involve more direct inhibition of mechanisms downstream of these channels. It will be interesting to determine how this modulation shapes activity of spontaneously-firing GPe and SNr neurons. The source of endocannabinoid actions on CB1 receptors in these terminals is also of interest, as are the roles of these Gi/o-coupled GPCRs in behavior. Indeed, we are currently examining the effects on action control and drug misuse-related behaviors of CB1 receptors expressed by striatal MSNs in collaboration with the Stella laboratory at the University of Washington. We are also examining effects of acute ethanol exposure on synapses made by striatal efferents in both GPe and SNr, and we have initiated experiments examining effects of chronic intermittent ethanol exposure on striatonigral synaptic transmission. We will also use chemo- and optogenetic approaches to assess the roles of striatonigral synapses in ethanol effects on reversal learning and alcohol drinking.