Maturation of glutamatergic transmission onto dorsal raphe serotonergic neurons.

Serotonergic neurons in the dorsal raphe nucleus (DRN) play important roles early in postnatal development in the maturation and modulation of higher-order emotional, sensory, and cognitive circuitry. The pivotal functions of these cells in brain development make them a critical substrate by which early experience can be wired into the brain. In this study, we investigated the maturation of synapses onto dorsal raphe serotonergic neurons in typically developing male and female mice using whole cell patch-clamp recordings in ex vivo brain slices. We show that while inhibition of these neurons is relatively stable across development, glutamatergic synapses greatly increase in strength between postnatal day 6 (P6) and P21-23. In contrast to forebrain regions, where the components making up glutamatergic synapses are dynamic across early life, we find that DRN excitatory synapses maintain a very high ratio of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) to N-methyl-d-aspartate (NMDA) receptors and a rectifying component of the AMPA response until adulthood. Overall, these findings reveal that the development of serotonergic neurons is marked by a significant refinement of glutamatergic synapses during the first three postnatal weeks. This suggests this time is a sensitive period of heightened plasticity for the integration of information from upstream brain areas. Genetic and environmental insults during this period could lead to alterations in serotonergic output, impacting both the development of forebrain circuits and lifelong neuromodulatory actions.NEW & NOTEWORTHY Serotonergic neurons are regulators of both the development of and ongoing activity in neuronal circuits controlling affective, cognitive, and sensory processing. Here, we characterize the maturation of extrinsic synaptic inputs onto these cells, showing that the first three postnatal weeks are a period of synaptic refinement and a potential window for experience-dependent plasticity in response to both enrichment and adversity.

Sex-specific adaptations to VTA circuits following subchronic stress.

Dysregulation of the mesolimbic reward circuitry is implicated in the pathophysiology of stress-related illnesses such as depression and anxiety. These disorders are more frequently diagnosed in females, and sex differences in the response to stress are likely to be one factor that leads to enhanced vulnerability of females. In this study, we use subchronic variable stress (SCVS), a model in which females are uniquely vulnerable to behavioral disturbances, to investigate sexually divergent mechanisms of regulation of the ventral tegmental area by stress. Using slice electrophysiology, we find that female, but not male mice have a reduction in the ex vivo firing rate of VTA dopaminergic neurons following SCVS. Surprisingly, both male and female animals show an increase in inhibitory tone onto VTA dopaminergic neurons and an increase in the firing rate of VTA GABAergic neurons. In males, however, this is accompanied by a robust increase in excitatory synaptic tone onto VTA dopamine neurons. This supports a model by which SCVS recruits VTA GABA neurons to inhibit dopaminergic neurons in both male and female mice, but males are protected from diminished functioning of the dopaminergic system by a compensatory upregulation of excitatory synapses.

Ventral tegmental area glutamate neurons establish a mu-opioid receptor gated circuit to mesolimbic dopamine neurons and regulate opioid-seeking behavior.

A two-neuron model of ventral tegmental area (VTA) opioid function classically involves VTA GABA neuron regulation of VTA dopamine neurons via a mu-opioid receptor dependent inhibitory circuit. However, this model predates the discovery of a third major type of neuron in the VTA: glutamatergic neurons. We found that about one-quarter of VTA neurons expressing the mu-opioid receptor are glutamate neurons without molecular markers of GABA co-release. Glutamate-Mu opioid receptor neurons are largely distributed in the anterior VTA. The majority of remaining VTA mu-opioid receptor neurons are GABAergic neurons that are mostly within the posterior VTA and do not express molecular markers of glutamate co-release. Optogenetic stimulation of VTA glutamate neurons resulted in excitatory currents recorded from VTA dopamine neurons that were reduced by presynaptic activation of the mu-opioid receptor ex vivo, establishing a local mu-opioid receptor dependent excitatory circuit from VTA glutamate neurons to VTA dopamine neurons. This VTA glutamate to VTA dopamine pathway regulated dopamine release to the nucleus accumbens through mu-opioid receptor activity in vivo. Behaviorally, VTA glutamate calcium-related neuronal activity increased following oral oxycodone consumption during self-administration and response-contingent oxycodone-associated cues during abstinent reinstatement of drug-seeking behavior. Further, chemogenetic inhibition of VTA glutamate neurons reduced abstinent oral oxycodone-seeking behavior in male but not female mice. These results establish 1) a three-neuron model of VTA opioid function involving a mu-opioid receptor gated VTA glutamate neuron pathway to VTA dopamine neurons that controls dopamine release within the nucleus accumbens, and 2) that VTA glutamate neurons participate in opioid-seeking behavior.

Central amygdala angiotensin type 1 receptor (Agtr1) expressing neurons contribute to fear extinction.

The renin-angiotensin system (RAS) has been linked to the pathophysiology of posttraumatic stress disorder (PTSD) however, the underlying neurobiological mechanism(s) remain elusive. Here we utilized angiotensin II receptor type 1 (AT1R) transgenic mice combined with neuroanatomical, behavioral, and electrophysiological approaches, to examine the role of the central amygdala (CeA) expressing AT1R neurons in fear and anxiety-related behavior. Within the major amygdala subdivisions, AT1R+ neurons were localized to gamma-aminobutyric acid (GABA) expressing neurons in the lateral division of the central amygdala (CeL), and the majority of them were identified as protein kinase C-δ positive (PKCδ+) neurons. Following CeA-AT1R deletion using cre-expressing lentiviral delivery in AT1R-Flox mice, generalized anxiety and locomotor activity as well as the acquisition of conditioned fear were unaltered while the acquisition of extinction learning, as measured by percent freezing behavior, was significantly enhanced. During electrophysiological recordings of CeL-AT1R+ neurons, the application of angiotensin II (1 µm) increased the amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) and decreased the excitability of CeL-AT1R+ neurons. Overall, these findings demonstrate that CeL-AT1R-expressing neurons play a role in fear extinction, potentially through facilitated CeL-AT1R+ GABAergic inhibition. These results provide new evidence for mechanisms of angiotensinergic neuromodulation of the CeL and its role in fear extinction and may aid in further advancing targeted novel therapies for improving maladaptive fear learning processes associated with PTSD.

Maturation of glutamatergic transmission onto dorsal raphe serotonergic neurons.

Serotonergic neurons in the dorsal raphe nucleus (DRN) play important roles early in postnatal development in the maturation and modulation of higher order emotional, sensory, and cognitive circuitry. This unique position makes these cells a substrate by which early experience can be wired into brain. In this study, we have investigated the maturation of synapses onto dorsal raphe serotonergic neurons in typically developing male and female mice using whole-cell patch-clamp recordings in ex vivo brain slices. We show that while inhibition of these neurons is relatively stable across development, glutamatergic synapses greatly increase in strength between P6 and P21-23. In contrast to forebrain regions, where the components making up glutamatergic synapses are dynamic across early life, we find that the makeup of these synapses onto DRN serotonergic neurons is largely stable after P15. DRN excitatory synapses maintain a very high ratio of AMPA to NMDA receptors and a rectifying component of the AMPA response throughout the lifespan. Overall, these findings reveal that the development of serotonergic neurons is marked by a significant refinement of glutamatergic synapses during the first 3 postnatal weeks. This suggests this time as a sensitive period of heightened plasticity for integration of information from upstream brain areas and that genetic and environmental insults during this period could lead to alterations in serotonergic output, impacting both the development of forebrain circuits and lifelong neuromodulatory actions.

Patch-Clamp Proteomics of Single Neurons in Tissue Using Electrophysiology and Subcellular Capillary Electrophoresis Mass Spectrometry.

Understanding of the relationship between cellular function and molecular composition holds a key to next-generation therapeutics but requires measurement of all types of molecules in cells. Developments in sequencing enabled semiroutine measurement of single-cell genomes and transcriptomes, but analytical tools are scarce for detecting diverse proteins in tissue-embedded cells. To bridge this gap for neuroscience research, we report the integration of patch-clamp electrophysiology with subcellular shot-gun proteomics by high-resolution mass spectrometry (HRMS). Recording of electrical activity permitted identification of dopaminergic neurons in the substantia nigra pars compacta. Ca. 20-50% of the neuronal soma content, containing an estimated 100 pg of total protein, was aspirated into the patch pipette filled with ammonium bicarbonate. About 1 pg of somal protein, or ∼0.25% of the total cellular proteome, was analyzed on a custom-built capillary electrophoresis (CE) electrospray ionization platform using orbitrap HRMS for detection. A series of experiments were conducted to systematically enhance detection sensitivity through refinements in sample processing and detection, allowing us to quantify ∼275 different proteins from somal aspirate-equivalent protein digests from cultured neurons. From single neurons, patch-clamp proteomics of the soma quantified 91, 80, and 95 different proteins from three different dopaminergic neurons or 157 proteins in total. Quantification revealed detectable proteomic differences between the somal protein samples. Analysis of canonical knowledge predicted rich interaction networks between the observed proteins. The integration of patch-clamp electrophysiology with subcellular CE-HRMS proteomics expands the analytical toolbox of neuroscience.

A generation of junior faculty is at risk from the impacts of COVID-19.

For junior investigators starting their independent careers, the challenges of the Coronavirus Disease 2019 (COVID-19) pandemic extend beyond lost time and are career threatening. Without intervention, academic science could lose a generation of talent.

Neurochemical Signaling of Reward and Aversion to Ventral Tegmental Area Glutamate Neurons.

Ventral tegmental area (VTA) glutamate neurons signal and participate in reward and aversion-based behaviors. However, the neurochemical mechanisms that underlie how these neurons contribute to motivated behaviors is unknown. We used a combination of optical sensors to identify how distinct neurochemical inputs to VTA glutamate neurons participate in motivated behavior within female and male transgenic mice. Activity of glutamate inputs to VTA glutamate neurons increased for both reward-predicting and aversion-predicting cues and aversive outcomes, but subpopulations of glutamate inputs were increased or decreased by reward. For both reward and aversion-based cues and outcomes, activity of GABA inputs to VTA glutamate neurons mostly decreased. GCaMP recordings showed overall population increases in VTA glutamate neuron intracellular calcium during reward and aversion-based cues and outcomes. Electrophysiological recordings of VTA VGluT2 neurons showed that glutamate receptor activation increases firing while loss of excitation via glutamate receptor blockade decreases firing. GABA-A receptor activation decreased VTA glutamate neuron firing but GABA-A receptor blockade did not significantly change VTA glutamate neuron firing. Electrophysiological recordings in coordination with our sensor data suggest that glutamate inputs strongly regulate VTA glutamate neuron participation in diverse motivated behaviors.SIGNIFICANCE STATEMENT Glutamate and GABA are the primary excitatory and inhibitory neurotransmitters of the nervous system. However, identifying how these neurotransmitters regulate motivated behavior has remained challenging because of a lack of tools (1) capable of measuring neurotransmission at the temporal scale of motivated behaviors and (2) capable of capturing chemical signaling onto genetically-distinct neuronal populations. We have overcome these obstacles by implementing genetically-encoded fluorescent indicators to monitor both glutamate and GABA input dynamics exclusively to ventral tegmental area (VTA) glutamate neurons during reward and aversion-based behaviors. We identify that glutamate and GABA inputs to VTA glutamate neurons differentially and dynamically signal reward and aversion-based cues and outcomes. This research provides foundational evidence that links distinct neurotransmitters to motivated behaviors regulated by VTA glutamate neurons.

VTA GABA Neurons at the Interface of Stress and Reward.

The ventral tegmental area (VTA) is best known for its robust dopaminergic projections to forebrain regions and their critical role in regulating reward, motivation, cognition, and aversion. However, the VTA is not only made of dopamine (DA) cells, as approximately 30% of cells in the VTA are GABA neurons. These neurons play a dual role, as VTA GABA neurons provide both local inhibition of VTA DA neurons and long-range inhibition of several distal brain regions. VTA GABA neurons have increasingly been recognized as potent mediators of reward and aversion in their own right, as well as potential targets for the treatment of addiction, depression, and other stress-linked disorders. In this review article, we dissect the circuit architecture, physiology, and behavioral roles of VTA GABA neurons and suggest critical gaps to be addressed.

Two-Pronged Control of the Dorsal Raphe by the VTA.

In this issue of Neuron, Li et al. (2019) distinguish two separable GABAergic projections from the ventral tegmental area (VTA) to the dorsal raphe nucleus (DRN), with differential µ-opioid receptor regulation, each targeting different postsynaptic neurons and promoting opposing behavioral states.

Synaptic function and plasticity in identified inhibitory inputs onto VTA dopamine neurons.

Ventral tegmental area (VTA) dopaminergic neurons are key components of the reward pathway, and their activity is powerfully controlled by a diverse array of inhibitory GABAergic inputs. Two major sources of GABAergic nerve terminals within the VTA are local VTA interneurons and neurons in the rostromedial tegmental nucleus (RMTg). Here, using optogenetics, we compared synaptic properties of GABAergic synapses on VTA dopamine neurons using selective activation of afferents that originate from these two cell populations. We found little evidence of co-release of glutamate from either input, but RMTg-originating synaptic currents were reduced by strychnine, suggesting co-release of glycine and GABA. VTA-originating synapses displayed a lower initial release probability, and at higher frequency stimulation, short-term depression was more marked in VTA- but not RMTg-originating synapses. We previously reported that nitric oxide (NO)-induced potentiation of GABAergic synapses on VTA dopaminergic cells is lost after exposure to drugs of abuse or acute stress; in these experiments, multiple GABAergic afferents were simultaneously activated by electrical stimulation. Here we found that optogenetically-activated VTA-originating synapses on presumptive dopamine neurons also exhibited NO-induced potentiation, whereas RMTg-originating synapses did not. Despite providing a robust inhibitory input to the VTA, RMTg GABAergic synapses are most likely not those previously shown by our work to be persistently altered by addictive drugs and stress. Our work emphasises the idea that dopamine neuron excitability is controlled by diverse inhibitory inputs expected to exert varying degrees of inhibition and to participate differently in a range of behaviours.

Constitutive activation of kappa opioid receptors at ventral tegmental area inhibitory synapses following acute stress.

Stressful experiences potently activate kappa opioid receptors (κORs). κORs in the ventral tegmental area regulate multiple aspects of dopaminergic and non-dopaminergic cell function. Here we show that at GABAergic synapses on rat VTA dopamine neurons, a single exposure to a brief cold-water swim stress induces prolonged activation of κORs. This is mediated by activation of the receptor during the stressor followed by a persistent, ligand-independent constitutive activation of the κOR itself. This lasting change in function is not seen at κORs at neighboring excitatory synapses, suggesting distinct time courses and mechanisms of regulation of different subsets of κORs. We also provide evidence that constitutive activity of κORs governs the prolonged reinstatement to cocaine-seeking observed after cold water swim stress. Together, our studies indicate that stress-induced constitutive activation is a novel mechanism of κOR regulation that plays a critical role in reinstatement of drug seeking.

Yin and Yang: unsilencing synapses to control cocaine seeking.

In this issue of Neuron, Ma et al. (2014) show that long-term depression of two independent prefrontal cortical inputs to nucleus accumbens modifies behavioral responses controlling incubation of cocaine craving. Intriguingly, one input increases while the other attenuates behavioral responses, hinting that both "prorelapse" and "antirelapse" pathways are strengthened after cocaine self-administration.

Poststress block of kappa opioid receptors rescues long-term potentiation of inhibitory synapses and prevents reinstatement of cocaine seeking.

BACKGROUND: Dopaminergic neurons in the ventral tegmental area of the brain are an important site of convergence of drugs and stress. We previously identified a form of long-term potentiation of gamma-aminobutyric acid (GABA)ergic synapses on these neurons (LTPGABA). Our studies have shown that exposure to acute stress blocks this LTP and that reversal of the block of LTPGABA is correlated with prevention of stress-induced reinstatement of cocaine-seeking behavior. METHODS: Sprague-Dawley rats were subjected to cold-water swim stress. Midbrain slices were prepared following stress, and whole-cell patch clamp recordings of inhibitory postsynaptic currents were performed from ventral tegmental area dopamine neurons. Antagonists of glucocorticoid receptors and kappa opioid receptors (κORs) were administered at varying time points after stress. Additionally, the ability of a kappa antagonist administered following stress to block forced swim stress-induced reinstatement of cocaine self-administration was tested. RESULTS: We found that an acute stressor blocks LTPGABA for 5 days after stress through a transient activation of glucocorticoid receptors and more lasting contribution of κORs. Even pharmacological block of κORs beginning 4 days after stress has occurred reversed the block of LTPGABA. Administration of a κORs antagonist following stress prevents reinstatement of cocaine-seeking behavior. CONCLUSIONS: A brief stressor produces changes in the reward circuitry lasting several days. Our findings reveal roles for glucocorticoid receptors and κORs as mediators of the lasting effects of stress on synaptic plasticity. κORs antagonists reverse the neuroadaptations underlying stress-induced drug-seeking behavior and may be useful in the treatment of cocaine addiction.

Stress and VTA synapses: implications for addiction and depression.

While stressful experiences are a part of everyone's life, they can also exact a major toll on health. Stressful life experiences are associated with increased substance abuse, and there exists significant co-morbidity between mental illness and substance use disorders [N.D. Volkow & T.K. Li (2004) Nat. Rev. Neurosci., 5, 963-970; G. Koob & M.J. Kreek (2007) Am. J. Psych., 164, 1149-1159; R. Sinha (2008) Annals N.Y. Acad. Sci., 1141, 105-130]. The risk for development of mood or anxiety disorders after stress is positively associated with the risk for substance use disorders [R. Sinha (2008) Annals N.Y. Acad. Sci., 1141, 105-130], suggesting that there are common substrates for vulnerability to addictive and affective disorders. Understanding the molecular and physiological substrates of stress may lead to improved therapeutic interventions for the treatment of substance use disorders and mental illnesses.

Kappa opioid receptors regulate stress-induced cocaine seeking and synaptic plasticity.

Stress facilitates reinstatement of addictive drug seeking in animals and promotes relapse in humans. Acute stress has marked and long-lasting effects on plasticity at both inhibitory and excitatory synapses on dopamine neurons in the ventral tegmental area (VTA), a key region necessary for drug reinforcement. Stress blocks long-term potentiation at GABAergic synapses on dopamine neurons in the VTA (LTPGABA), potentially removing a normal brake on activity. Here we show that blocking kappa opioid receptors (KORs) prior to forced-swim stress rescues LTPGABA. In contrast, blocking KORs does not prevent stress-induced potentiation of excitatory synapses nor morphine-induced block of LTPGABA. Using a kappa receptor antagonist as a selective tool to test the role of LTPGABA in vivo, we find that blocking KORs within the VTA prior to forced-swim stress prevents reinstatement of cocaine seeking. These results suggest that KORs may represent a useful therapeutic target for treatment of stress-triggered relapse in substance abuse.

Functional significance of glycogen synthase kinase-3 regulation by serotonin.

Serotonin modulates brain physiology and behavior and has major roles in brain diseases involving abnormal mood and cognition. Enhancing brain serotonin has been found to regulate glycogen synthase Kinase-3 (GSK3), but the signaling mechanism and functional significance of this regulation remain to be determined. In this study, we tested the signaling mechanism mediating 5-HT1A receptor-regulated GSK3 in the hippocampus. Using mutant GSK3 knock-in mice, we also tested the role of GSK3 in the behavioral effects of 5-HT1A receptors and the serotonin reuptake inhibitor fluoxetine. The results showed that activation of 5-HT1A receptors by 8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) increased phosphorylation of the N-terminal serine of both GSK3α and GSK3ß in several areas of the hippocampus. The effect of 8-OH-DPAT was accompanied by an increase in the active phosphorylation of Akt, and was blocked by LY294002, an inhibitor of phosphoinositide 3-kinases (PI3K). Phosphorylation of GSK3ß, but not GSK3α, was necessary for 5-HT1A receptors to suppress the hippocampus-associated contextual fear learning. Furthermore, acute fluoxetine treatment up-regulated both phospho-Ser21-GSK3α and phospho-Ser9-GSK3ß in the hippocampus. Blocking phosphorylation of GSK3α and GSK3ß diminished the anti-immobility effect of fluoxetine treatment in the forced swim test, wherein the effect of GSK3ß was more prominent. These results together suggest that PI3K/Akt is a signaling mechanism mediating the GSK3-regulating effect of 5-HT1A receptors in the hippocampus, and regulation of GSK3 is an important intermediate signaling process in the behavioral functions of 5-HT1A receptors and fluoxetine.

Glycogen Synthase Kinase-3 is an Intermediate Modulator of Serotonin Neurotransmission.

Serotonin is a neurotransmitter with broad functions in brain development, neuronal activity, and behaviors; and serotonin is the prominent drug target in several major neuropsychiatric diseases. The multiple actions of serotonin are mediated by diverse serotonin receptor subtypes and associated signaling pathways. However, the key signaling components that mediate specific function of serotonin neurotransmission have not been fully identified. This review will provide evidence from biochemical, pharmacological, and animal behavioral studies showing that serotonin regulates the activation states of brain glycogen synthase kinase-3 (GSK3) via type 1 and type 2 serotonin receptors. In return, GSK3 directly interacts with serotonin receptors in a highly selective manner, with a prominent effect on modulating serotonin 1B receptor activity. Therefore, GSK3 acts as an intermediate modulator in the serotonin neurotransmission system, and balanced GSK3 activity is essential for serotonin-regulated brain function and behaviors. Particularly important, several classes of serotonin-modulating drugs, such as antidepressants and atypical antipsychotics, regulate GSK3 by inhibiting its activity in brain, which reinforces the importance of GSK3 as a potential therapeutic target in neuropsychiatric diseases associated with abnormal serotonin function.

Deficiency in the inhibitory serine-phosphorylation of glycogen synthase kinase-3 increases sensitivity to mood disturbances.

Bipolar disorder, characterized by extreme manic and depressive moods, is a prevalent debilitating disease of unknown etiology. Because mood stabilizers, antipsychotics, antidepressants, and mood-regulating neuromodulators increase the inhibitory serine-phosphorylation of glycogen synthase kinase-3 (GSK3), we hypothesized that deficient GSK3 serine-phosphorylation may increase vulnerability to mood-related behavioral disturbances. This was tested by measuring behavioral characteristics of GSK3 alpha/beta(21A/21A/9A/9A) knockin mice with serine-to-alanine mutations to block inhibitory serine-phosphorylation of GSK3. GSK3 knockin mice displayed increased susceptibility to amphetamine-induced hyperactivity and to stress-induced depressive-like behaviors. Furthermore, serine-phosphorylation of GSK3 was reduced during both mood-related behavioral responses in wild-type mouse brain and in blood cells from patients with bipolar disorder. Therefore, proper control of GSK3 by serine-phosphorylation, which is targeted by agents therapeutic for bipolar disorder, is an important mechanism that regulates mood stabilization, and mice with disabled GSK3 serine-phosphorylation may provide a valuable model to study bipolar disorder.

5-HT1A receptor-regulated signal transduction pathways in brain.

Serotonin is an influential monoamine neurotransmitter that signals through a number of receptors to modulate brain function. Among different serotonin receptors, the serotonin 1A (5-HT1A) receptors have been tied to a variety of physiological and pathological processes, notably in anxiety, mood, and cognition. 5-HT1A receptors couple not only to the classical inhibitory G protein-regulated signaling pathway, but also to signaling pathways traditionally regulated by growth factors. Despite the importance of 5-HT1A receptors in brain function, little is known about how these signaling mechanisms link 5-HT1A receptors to regulation of brain physiology and behavior. Following a brief summary of the known physiological and behavioral effects of 5-HT1A receptors, this article will review the signaling pathways regulated by 5-HT1A receptors, and discuss the potential implication of these signaling pathways in 5-HT1A receptor-regulated physiological processes and behaviors.