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

National Institute On Alcohol Abuse And Alcoholism

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Abstract

Neuromodulator Dynamics in Striatum: Effects of Ethanol Neuromodulatory transmission is a crucial component of neural function, allowing for adjustments to synaptic transmission and cellular excitability as needed for adaptive circuit function. In the dorsal striatum, the neuromodulators acetylcholine (ACh), dopamine and endocannabinoids (eCBs) are all implicated in synaptic plasticity. Indeed, many forms of striatal plasticity involve interactions among these endogenous compounds. Striatal synaptic plasticity is also sensitive to ethanol at concentrations associated with intoxication, and these effects could be secondary to changes in neuromodulator release. Thus, we have focused on measuring extracellular dynamics of these neuromodulators as well as their effects on intracellular signaling using genetically encoded fluorescent sensors. We have also used fast scan cyclic voltammetry (FSCV) to measure striatal dopamine dynamics. Experiments in striatal brain slices using two different acetylcholine sensors, iAChSnFR and GRABACh revealed that ACh is readily released in response to single electrical stimuli or brief depolarization of striatal cholinergic interneurons (CINS) using optogenetic stimulation with the Chrimson opsin. Spontaneous increases in fluorescence are observed in slices expressing these sensors and are not observed with other sensors we have used, supporting the presence of spontaneous ACh release in this preparation. Additional experiments indicate that the increases in ACh are due to vesicular release mainly from CINs. Examination of the kinetics of stimulation-induced fluorescence increases with the two sensors indicated longer-duration responses measured with iAChSnFR (10s of sec) compared to GRABACh (1-2 sec). Subsequent experiments indicated that iAChSnFR reports both ACh and choline levels, while GRABACh is only sensitive to ACh. Thus, we have used GRABACh to examine the dynamics of ACh release and effects of, e.g., gestational ethanol exposure on this release (Barisella et al., Neuropsychopharmacology 2023). However, it is also important to understand choline dynamics in brain, as choline has been implicated in several brain functions and choline therapy may be useful for treatment of some symptoms of fetal alcohol spectrum disorder. Thus, we hope to take advantage of the choline-sensing capability of iAChSnFR in future studies. We have also used GRABACh in conjunction with FSCV to measure the relationship between ACh and dopamine release, given the large literature indicating interactions between the two modulators. As observed in previous studies, we found that blocking nicotinic ACh receptors (nAChRs) reduced dopamine release in response to a single electrical stimulus but enhanced responses to brief stimulus bursts. As mentioned above, single electrical stimuli produce robust ACh increases, but the amplitude of these increases diminishes in response to subsequent stimuli during short stimulus bursts. Blocking nAChRs does not alter ACh release. Thus, the change in effect of nAChR blockade on dopamine release during bursts is not due to stronger ACh release or suppressed ACh activation of dopaminergic axons. We hypothesized that the change in nAChR effects on dopamine release may be related to altered calcium signaling in dopaminergic terminals. To examine this possibility, we expressed the fluorescent calcium sensor GCaMP in dopaminergic neurons and measured presynaptic responses to electrical stimulation in striatum. We observed that nAChR blockade altered the calcium transients in a manner similar to the effect on dopamine release, i.e. inhibition of single stimulus-evoked responses and increased responses to stimulus bursts. Thus, the changes in presynaptic calcium likely explain the change in dopamine release. Considering recent studies that showed nAChR depolarization of striatal dopaminergic axons, we hypothesized that during stimulus bursts strong presynaptic depolarization, perhaps combined with calcium entry via presynaptic nAChRs, reduces action potential-induced calcium increases in dopaminergic axons. This would account for the increase in dopamine release observed when these nAChRs are blocked during burst stimulation. Recent work from the Cragg laboratory at Oxford suggests that a nAChR-mediated decrease in phasic dopamine release occurs in vivo and influences behavior. We are continuing to examine effects of acute and chronic alcohol on these ACh/dopamine interactions. Neuromodulation by the arachidonic acid-containing lipid metabolites known as endocannabinoids (eCBs) is also prominent in striatum. The development of fluorescent eCB sensors known as GRABeCBs has allowed us to measure the dynamics of release of these neuromodulators in striatum (Liput et al, Neuropharmacology, 2022). We have also observed that acute application of ethanol inhibits the release of eCBs from striatonigral medium spiny projection neurons (MSNs). The findings in these two studies indicated that the eCB 2-arachidonoyl glycerol (2-AG) is released in striatum following short bursts of electrical stimulation. However, previous studies indicated that the eCB arachidonoyl ethanolamide (AEA) plays a role in striatal synaptic plasticity. Thus, we have attempted to measure 2-AG and AEA separately using newly developed fluorecsent sensors specific for the two eCBs. Our findings to date with the 2-AG-specific sensor support our previous findings that 2-AG is released in response to burst stimulation. We are currently testing the newest generation of the AEA-specific sensor to determine if we can measure this eCB. Studies of acute and chronic ethanol effects on eCB release and synaptic plasticity will continue once we are confident as to which of the neuromodulators are being released. The three neuromodulators discussed above can impact intracellular levels of the second messenger cyclic adenosine monophosphate (cAMP), and this is an important component of their neuromodulatory actions. We have also developed a new fluorescent sensor to measure intracellular cAMP. This sensor is based on Forster Resonance Energy Transfer (FRET). We are currently examining how cAMP changes in striatal neurons in response to in vivo pharmacological challenges and during behavior. We are comparing these changes to the dopamine dynamics in these same paradigms. These studies will help us understand how modulation through this important intracellular signaling system leads to synaptic plasticity and long-term changes in behavior. Examination of ethanol effects on cAMP signaling will also be important in understanding the acute and long-term effects of the drug.