Distinct sub-second dopamine signaling in dorsolateral striatum measured by a genetically-encoded fluorescent sensor.

The development of genetically encoded dopamine sensors such as dLight has provided a new approach to measuring slow and fast dopamine dynamics both in brain slices and in vivo, possibly enabling dopamine measurements in areas like the dorsolateral striatum (DLS) where previously such recordings with fast-scan cyclic voltammetry (FSCV) were difficult. To test this, we first evaluated dLight photometry in mouse brain slices with simultaneous FSCV and found that both techniques yielded comparable results, but notable differences in responses to dopamine transporter inhibitors, including cocaine. We then used in vivo fiber photometry with dLight in mice to examine responses to cocaine in DLS. We also compared dopamine responses during Pavlovian conditioning across the striatum. We show that dopamine increases were readily detectable in DLS and describe transient dopamine kinetics, as well as slowly developing signals during conditioning. Overall, our findings indicate that dLight photometry is well suited to measuring dopamine dynamics in DLS.

Chronic Ethanol Consumption Alters Presynaptic Regulation of Dorsal Striatal Dopamine Release in C57BL/6J Mice.

Alcohol use disorder (AUD) is characterized by escalating alcohol consumption, preoccupation with alcohol, and continued alcohol consumption despite adverse consequences. Dopamine has been implicated in neural and behavioral processes involved in reward and reinforcement and is a critical neurotransmitter in AUD. Clinical and preclinical research has shown that long-term ethanol exposure can alter dopamine release, though most of this work has focused on nucleus accumbens (NAc). Like the NAc, the dorsal striatum (DS) is implicated in neural and behavioral processes in AUD. However, little work has examined chronic ethanol effects on DS dopamine dynamics. Therefore, we examined the effect of ethanol consumption and withdrawal on dopamine release and its presynaptic regulation with fast-scan cyclic voltammetry in C57BL/6J mice. We found that one month of ethanol consumption did not alter maximal dopamine release or dopamine tissue content. However, we did find that D2 dopamine autoreceptors were sensitized. We also found a decrease in cholinergic control of dopamine release via ß2-containing nAChRs on dopamine axons. Interestingly, both effects were reversed following withdrawal, raising the possibility that some of the neuroadaptations in AUD might be reversible in abstinence. Altogether, this work elucidates some of the chronic alcohol-induced neurobiological dysfunctions in the dopamine system.

Deficiency in endocannabinoid synthase DAGLB contributes to early onset Parkinsonism and murine nigral dopaminergic neuron dysfunction.

Endocannabinoid (eCB), 2-arachidonoyl-glycerol (2-AG), the most abundant eCB in the brain, regulates diverse neural functions. Here we linked multiple homozygous loss-of-function mutations in 2-AG synthase diacylglycerol lipase ß (DAGLB) to an early onset autosomal recessive Parkinsonism. DAGLB is the main 2-AG synthase in human and mouse substantia nigra (SN) dopaminergic neurons (DANs). In mice, the SN 2-AG levels were markedly correlated with motor performance during locomotor skill acquisition. Genetic knockdown of Daglb in nigral DANs substantially reduced SN 2-AG levels and impaired locomotor skill learning, particularly the across-session learning. Conversely, pharmacological inhibition of 2-AG degradation increased nigral 2-AG levels, DAN activity and dopamine release and rescued the locomotor skill learning deficits. Together, we demonstrate that DAGLB-deficiency contributes to the pathogenesis of Parkinsonism, reveal the importance of DAGLB-mediated 2-AG biosynthesis in nigral DANs in regulating neuronal activity and dopamine release, and suggest potential benefits of 2-AG augmentation in alleviating Parkinsonism.

Impact of α-synuclein spreading on the nigrostriatal dopaminergic pathway depends on the onset of the pathology.

Misfolded α-synuclein spreads along anatomically connected areas through the brain, prompting progressive neurodegeneration of the nigrostriatal pathway in Parkinson's disease. To investigate the impact of early stage seeding and spreading of misfolded α-synuclein along with the nigrostriatal pathway, we studied the pathophysiologic effect induced by a single acute α-synuclein preformed fibrils (PFFs) inoculation into the midbrain. Further, to model the progressive vulnerability that characterizes the dopamine (DA) neuron life span, we used two cohorts of mice with different ages: 2-month-old (young) and 5-month-old (adult) mice. Two months after α-synuclein PFFs injection, we found that striatal DA release decreased exclusively in adult mice. Adult DA neurons showed an increased level of pathology spreading along with the nigrostriatal pathway accompanied with a lower volume of α-synuclein deposition in the midbrain, impaired neurotransmission, rigid DA terminal composition, and less microglial reactivity compared with young neurons. Notably, preserved DA release and increased microglial coverage in the PFFs-seeded hemisphere coexist with decreased large-sized terminal density in young DA neurons. This suggests the presence of a targeted pruning mechanism that limits the detrimental effect of α-synuclein early spreading. This study suggests that the impact of the pathophysiology caused by misfolded α-synuclein spreading along the nigrostriatal pathway depends on the age of the DA network, reducing striatal DA release specifically in adult mice.

Long-term alcohol consumption alters dorsal striatal dopamine release and regulation by D2 dopamine receptors in rhesus macaques.

The dorsal striatum (DS) is implicated in behavioral and neural processes including action control and reinforcement. Alcohol alters these processes in rodents, and it is believed that the development of alcohol use disorder involves changes in DS dopamine signaling. In nonhuman primates, the DS can be divided into caudate and putamen subregions. As part of a collaborative effort examining the effects of long-term alcohol self-administration in rhesus macaques, we examined DS dopamine signaling using fast-scan cyclic voltammetry. We found that chronic alcohol self-administration resulted in several dopamine system adaptations. Most notably, dopamine release was altered in a sex- and region-dependent manner. Following long-term alcohol consumption, male macaques, regardless of abstinence status, had reduced dopamine release in putamen, while only male macaques in abstinence had reduced dopamine release in caudate. In contrast, female macaques had enhanced dopamine release in the caudate, but not putamen. Dopamine uptake was also enhanced in females, but not males (regardless of abstinence state). We also found that dopamine D2/3 autoreceptor function was reduced in male, but not female, alcohol drinkers relative to control groups. Finally, we found that blockade of nicotinic acetylcholine receptors inhibited evoked dopamine release in nonhuman primates. Altogether, our findings demonstrate that long-term alcohol consumption can sex-dependently alter dopamine release, as well as its feedback control mechanisms in both DS subregions.

Molecular mechanisms underlying striatal synaptic plasticity: relevance to chronic alcohol consumption and seeking.

The striatum, the input structure of the basal ganglia, is a major site of learning and memory for goal-directed actions and habit formation. Spiny projection neurons of the striatum integrate cortical, thalamic, and nigral inputs to learn associations, with cortico-striatal synaptic plasticity as a learning mechanism. Signaling molecules implicated in synaptic plasticity are altered in alcohol withdrawal, which may contribute to overly strong learning and increased alcohol seeking and consumption. To understand how interactions among signaling molecules produce synaptic plasticity, we implemented a mechanistic model of signaling pathways activated by dopamine D1 receptors, acetylcholine receptors, and glutamate. We use our novel, computationally efficient simulator, NeuroRD, to simulate stochastic interactions both within and between dendritic spines. Dopamine release during theta burst and 20-Hz stimulation was extrapolated from fast-scan cyclic voltammetry data collected in mouse striatal slices. Our results show that the combined activity of several key plasticity molecules correctly predicts the occurrence of either LTP, LTD, or no plasticity for numerous experimental protocols. To investigate spatial interactions, we stimulate two spines, either adjacent or separated on a 20-µm dendritic segment. Our results show that molecules underlying LTP exhibit spatial specificity, whereas 2-arachidonoylglycerol exhibits a spatially diffuse elevation. We also implement changes in NMDA receptors, adenylyl cyclase, and G protein signaling that have been measured following chronic alcohol treatment. Simulations under these conditions suggest that the molecular changes can predict changes in synaptic plasticity, thereby accounting for some aspects of alcohol use disorder.

Alcohol and the Brain: Neuronal Molecular Targets, Synapses, and Circuits.

Ethanol is one of the most commonly abused drugs. Although environmental and genetic factors contribute to the etiology of alcohol use disorders, it is ethanol's actions in the brain that explain (1) acute ethanol-related behavioral changes, such as stimulant followed by depressant effects, and (2) chronic changes in behavior, including escalated use, tolerance, compulsive seeking, and dependence. Our knowledge of ethanol use and abuse thus relies on understanding its effects on the brain. Scientists have employed both bottom-up and top-down approaches, building from molecular targets to behavioral analyses and vice versa, respectively. This review highlights current progress in the field, focusing on recent and emerging molecular, cellular, and circuit effects of the drug that impact ethanol-related behaviors. The focus of the field is now on pinpointing which molecular effects in specific neurons within a brain region contribute to behavioral changes across the course of acute and chronic ethanol exposure.

Aldehyde dehydrogenase 1-positive nigrostriatal dopaminergic fibers exhibit distinct projection pattern and dopamine release dynamics at mouse dorsal striatum.

Aldehyde dehydrogenase 1 (ALDH1A1)-positive dopaminergic (DA) neurons at the ventral substantia nigra pars compacta (SNpc) preferentially degenerate in Parkinson's disease (PD). Their projection pattern and dopamine release properties, however, remains uncharacterized. Here we show that ALDH1A1-positive axons project predominantly to the rostral two-thirds of dorsal striatum. A portion of these axons converge on a small fraction of striosome compartments restricted to the dorsolateral striatum (DLS), where less dopamine release was measured compared to the adjacent matrix enriched with the ALDH1A1-negative axons. Genetic ablation of Aldh1a1 substantially increases the dopamine release in striosomes, but not in matrix. Additionally, the presence of PD-related human α-synuclein A53T mutant or dopamine transporter (DAT) blockers also differentially affects the dopamine output in striosomes and matrix. Together, these results demonstrate distinct dopamine release characteristics of ALDH1A1-positive DA fibers, supporting a regional specific function of ALDH1A1 in regulating dopamine availability/release in striatum.

Long-term plasticity of corticostriatal synapses is modulated by pathway-specific co-release of opioids through κ-opioid receptors.

KEY POINTS: Both endogenous opioids and opiate drugs of abuse modulate learning of habitual and goal-directed actions, and can also modify long-term plasticity of corticostriatal synapses. Striatal projection neurons of the direct pathway co-release the opioid neuropeptide dynorphin which can inhibit dopamine release via κ-opioid receptors. Theta-burst stimulation of corticostriatal fibres produces long-term potentiation (LTP) in striatal projection neurons when measured using whole-cell patch recording. Optogenetic activation of direct pathway striatal projection neurons inhibits LTP while reducing dopamine release. Because the endogenous release of opioids is activity dependent, this modulation of synaptic plasticity represents a negative feedback mechanism that may limit runaway enhancement of striatal neuron activity in response to drugs of abuse. ABSTRACT: Synaptic plasticity in the striatum adjusts behaviour adaptively during skill learning, or maladaptively in the case of addiction. Just as dopamine plays a critical role in synaptic plasticity underlying normal skill learning and addiction, endogenous and exogenous opiates also modulate learning and addiction-related striatal plasticity. Though the role of opioid receptors in long-term depression in striatum has been characterized, their effect on long-term potentiation (LTP) remains unknown. In particular, direct pathway (dopamine D1 receptor-containing; D1R-) spiny projection neurons (SPNs) co-release the opioid neuropeptide dynorphin, which acts at presynaptic κ-opioid receptors (KORs) on dopaminergic afferents and can negatively regulate dopamine release. Therefore, we evaluated the interaction of co-released dynorphin and KOR on striatal LTP. We optogenetically facilitate the release of endogenous dynorphin from D1R-SPNs in brain slice while using whole-cell patch recording to measure changes in the synaptic response of SPNs following theta-burst stimulation (TBS) of cortical afferents. Our results demonstrate that TBS evokes corticostriatal LTP, and that optogenetic activation of D1R-SPNs during induction impairs LTP. Additional experiments demonstrate that optogenetic activation of D1R-SPNs reduces stimulation-evoked dopamine release and that bath application of a KOR antagonist provides full rescue of both LTP induction and dopamine release during optogenetic activation of D1R-SPNs. These results suggest that an increase in the opioid neuropeptide dynorphin is responsible for reduced TBS LTP and illustrate a physiological phenomenon whereby heightened D1R-SPN activity can regulate corticostriatal plasticity. Our findings have important implications for learning in addictive states marked by elevated direct pathway activation.

Dopamine dynamics and cocaine sensitivity differ between striosome and matrix compartments of the striatum.

The striatum is typically classified according to its major output pathways, which consist of dopamine D1 and D2 receptor-expressing neurons. The striatum is also divided into striosome and matrix compartments, based on the differential expression of a number of proteins, including the mu opioid receptor, dopamine transporter (DAT), and Nr4a1 (nuclear receptor subfamily 4, group A, member 1). Numerous functional differences between the striosome and matrix compartments are implicated in dopamine-related neurological disorders including Parkinson's disease and addiction. Using Nr4a1-eGFP mice, we provide evidence that electrically evoked dopamine release differs between the striosome and matrix compartments in a regionally-distinct manner. We further demonstrate that this difference is not due to differences in inhibition of dopamine release by dopamine autoreceptors or nicotinic acetylcholine receptors. Furthermore, cocaine enhanced extracellular dopamine in striosomes to a greater degree than in the matrix and concomitantly inhibited dopamine uptake in the matrix to a greater degree than in striosomes. Importantly, these compartment differences in cocaine sensitivity were limited to the dorsal striatum. These findings demonstrate a level of exquisite microanatomical regulation of dopamine by the DAT in striosomes relative to the matrix.

Reduced ethanol consumption and preference in cocaine- and amphetamine-regulated transcript (CART) knockout mice.

Cocaine- and amphetamine-regulated transcript (CART) is a neuropeptide implicated in addiction to drugs of abuse. Several studies have characterized the role of CART in addiction to psychostimulants, but few have examined the role of CART in alcohol use disorders including alcoholism. The current study utilized a CART knockout (KO) mouse model to investigate the role of CART in ethanol appetitive behaviors. A two-bottle choice, unlimited-access paradigm was used to compare ethanol appetitive behaviors between CART wild type (WT) and KO mice. The mice were presented with an ethanol solution (3%-21%) and water, each concentration for 4 days, and their consumption was measured daily. Consumption of quinine (bitter) and saccharin (sweet) solutions was measured following the ethanol preference tests. In addition, ethanol metabolism rates and ethanol sensitivity were compared between genotypes. CART KO mice consumed and preferred ethanol less than their WT counterparts in both sexes. This genotype effect could not be attributed to differences in bitter or sweet taste perception or ethanol metabolism rates. There was also no difference in ethanol sensitivity in male mice; however, CART KO female mice showed a greater ethanol sensitivity than the WT females. Taken together, these data demonstrate a role for CART in ethanol appetitive behaviors and as a possible therapeutic drug target for alcoholism and abstinence enhancement.

Aberrant synaptic activation of N-methyl-D-aspartate receptors underlies ethanol withdrawal hyperexcitability.

Chronic ethanol exposure may induce neuroadaptive responses in N-methyl-d-aspartate (NMDA) receptors, which are thought to underlie a variety of alcohol-related brain disorders. Here, we demonstrate that hyperexcitability triggered by withdrawal from chronic ethanol exposure is associated with increases in both synaptic NMDA receptor expression and activation. Withdrawal from chronic ethanol exposure (75 mM ethanol, 5-9 days) elicited robust and prolonged epileptiform activity in CA1 pyramidal neurons from hippocampal explants, which was absolutely dependent upon NMDA receptor activation but independent of chronic inhibition of protein kinase A (PKA). Analysis of Sr(2+)-supported asynchronous NMDA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) was employed to assess changes in NMDA neurotransmission. After chronic exposure, ethanol withdrawal was associated with an increase in mEPSC amplitude 3.38-fold over that after withdrawal from acute ethanol exposure. Analysis of paired evoked alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid EPSCs and spontaneous mEPSCs indicated that withdrawal after chronic exposure was also associated with a selective increase in action potential evoked but not spontaneous transmitter release probability. Immunoblot analysis revealed significant increases in total NR1, NR2A, and NR2B subunit expression after chronic exposure and unaffected by PKA-inhibition manner. Confocal imaging studies indicate that increased NR1 subunit expression was associated with increased density of NR1 expression on dendrites in parallel with a selective increase in the size of NR1 puncta on dendritic spines. Therefore, neuroadaptation to chronic ethanol exposure in NMDA synaptic transmission is responsible for aberrant network excitability after withdrawal and results from changes in both postsynaptic function as well as presynaptic release.