Biology subjects

Hnasko, T. S.

Publications and source records attributed to Hnasko, T. S..

15 recordsLinked to original sources

bioRxiv2025

GABA-glutamate corelease is a mechanism for state-dependent neurotransmission

Ventral tegmental area neurons projecting to lateral habenula (LHb) corelease the main inhibitory and excitatory transmitters, GABA and glutamate (VTAGG). Yet the functional role of this synchronous signal remains unclear. We hypothesized that the sign of VTAGG action depends on postsynaptic state in LHb. Ex vivo, activating VTAGG terminals evoked excitatory and inhibitory responses in LHb that varied with postsynaptic membrane potential. In vivo, VTAGG inputs drove net inhibition and supported positive reinforcement that was dependent on GABA, but not glutamate, release. Using chemogenetics to bidirectionally modulate LHb, we found that LHb hyperpolarization shifted VTAGG effects toward excitation, abolishing positive reinforcement, whereas LHb depolarization enhanced net inhibition and positive reinforcement. Thus, the activity state of LHb neurons dictates whether GABA-glutamate corelease is functionally inhibitory or excitatory and can reverse the motivational valence of VTAGG input, supporting a homeostatic role for GABA-glutamate cotransmission with broad implications for disorders of imbalanced motivation.

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bioRxiv2025

Parkinson's disease-vulnerable and -resilient dopamine neurons display opposite responses to excitatory input

Dopamine (DA) neurons of the substantia nigra (SN) are essential for motor control and selectively degenerate in Parkinsons disease (PD). However, DA neurons are molecularly heterogeneous, with some showing greater vulnerability and others resilience. Here, we show that the DA subtype marker Anxa1, identified in mice, labels PD-vulnerable DA neurons in human SN. Using mice, we found that excitatory inputs from subthalamic (STN) and pedunculopontine (PPN) nuclei evoked frequency-dependent excitation in SN GABA neurons, but complex multiphasic DA neuron responses, suggesting heterogeneous DA subtype responses. Indeed, excitatory inputs evoked differential DA responses in striatal subregions, an increase in caudal striatum, but inhibition followed by rebound in dorsolateral striatum. Additionally, PD-resilient Vglut2+ DA neurons were excited by STN/PPN input, while vulnerable Anxa1+ DA neurons were inhibited. These findings demonstrate that DA subtypes are embedded in distinct functional networks, suggesting that some therapeutic interventions may differentially impact vulnerable and resilient DA subtypes.

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bioRxiv2024

Mu-opioid receptor activation potentiates excitatory transmission at the habenulo-peduncular synapse

The continuing opioid epidemic poses a huge burden on public health. Identifying the neurocircuitry involved and how opioids modulate their signaling is essential for developing new therapeutic strategies. The medial habenula (MHb) is a small epithalamic structure that projects predominantly to the interpeduncular nucleus (IPN) and represents a mu-opioid receptor (MOR) hotspot. This habenulo-peduncular (HP) circuit can regulate nicotine and opioid withdrawal; however, little is known about the physiological impact of MOR on its function. Using MOR-reporter mice, we observed that MORs are expressed in a subset of MHb and IPN cells. Patch-clamp recordings revealed that MOR activation inhibited action potential firing in MOR+ MHb neurons and induced an inhibitory outward current in IPN neurons, consistent with canonical inhibitory effects of MOR. We next used optogenetics to stimulate MOR+ MHb axons to investigate the effects of MOR activation on excitatory transmission at the HP synapse. In contrast to its inhibitory effects elsewhere, MOR activation significantly potentiated evoked glutamatergic transmission to IPN. The facilitatory effects of MOR activation on glutamate co-release was also observed from cholinergic-defined HP synapses. The potentiation of excitatory transmission mediated by MOR activation persisted in the presence of blockers of GABA receptors or voltage-gated sodium channels, suggesting a monosynaptic mechanism. Finally, disruption of MOR in the MHb abolished the faciliatory action of DAMGO, indicating that this non-canonical effect of MOR activation on excitatory neurotransmission at the HP synapse is dependent on pre-synaptic MOR expression. Our study demonstrates canonical inhibitory effects of MOR activation in somatodendritic compartments, but non-canonical faciliatory effects on evoked glutamate transmission at the HP synapse, establishing a new mode by which MOR can modulate neuronal function.

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bioRxiv2024

Evidence for low affinity of GABA at the vesicular monoamine transporter VMAT2. Implications for transmitter co-release from dopamine neurons

Background and PurposeMidbrain dopamine (DA) neurons comprise a heterogeneous population of cells. For instance, some DA neurons express the vesicular glutamate transporter VGLUT2 allowing these cells to co-release DA and glutamate. Additionally, GABA may be co-released from DA neurons. However, most cells do not express the canonical machinery to synthesize GABA or the vesicular GABA transporter VGAT. Instead, GABA seems to be taken up into DA neurons by a plasmalemmal GABA transporter (GAT1) and stored in synaptic vesicles via the vesicular monoamine transporter VMAT2. Yet, it remains unclear whether GABA indeed interacts with VMAT2, or whether another transmitter could be responsible for the observed inhibitory effects attributed to GABA. Experimental ApproachWe used radiotracer flux measurements in VMAT2 expressing HEK-293 cells and synaptic vesicles from rodents to determine whether GABA qualifies as substrate at VMAT2. mRNA in situ hybridization was employed to determine expression of VMAT2 and GAT1 transcripts in DA neurons of mouse and in human midbrains. Key ResultsWe found that GABA reduced uptake of VMAT2 substrates in rodent synaptic vesicle preparations from striatum and cerebellum at millimolar concentrations but had no effect in VMAT2-expressing cells indicating that key components are missing in a non-neuronal system. Roughly 60 % of murine and human DA neurons in the substantia nigra express VMAT2 and GAT1 suggesting that many may be capable of co-releasing DA and GABA. Conclusion and ImplicationOur experiments suggest that GABA is a low-affinity substrate at VMAT2 with potential implications for basal ganglia physiology and disease. Bullet point summaryO_ST_ABSWhat is already knownC_ST_ABSO_LISubpopulations of dopamine neurons co-release glutamate and/or GABA. C_LIO_LIWhile glutamate is loaded into vesicles by VGLUT2, GABA co-release depends on GAT1 and VMAT2. C_LI What this study addsO_LIThe relative affinity of GABA at VMAT2 was found to be in the millimolar range. C_LIO_LIHuman midbrain dopamine neurons express GAT1. C_LI Clinical significanceO_LIGABA co-release from midbrain dopamine neurons may also occur in humans. C_LIO_LIGABA co-release from dopamine neurons may play a role in neuropsychiatric diseases C_LI

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bioRxiv2024

Ventral tegmental area interneurons revisited: GABA and glutamate projection neurons make local synapses

The ventral tegmental area (VTA) contains projection neurons that release the neurotransmitters dopamine, GABA, and/or glutamate from distal synapses. VTA also contains GABA neurons that synapse locally on to dopamine neurons, synapses widely credited to a population of so-called VTA interneurons. Interneurons in cortex, striatum, and elsewhere have well-defined morphological features, physiological properties, and molecular markers, but such features have not been clearly described in VTA. Indeed, there is scant evidence that local and distal synapses originate from separate populations of VTA GABA neurons. In this study we tested whether several markers expressed in non-dopamine VTA neurons are selective markers of interneurons, defined as neurons that synapse locally but not distally. Challenging previous assumptions, we found that VTA neurons genetically defined by expression of parvalbumin, somatostatin, neurotensin, or mu-opioid receptor project to known VTA targets including nucleus accumbens, ventral pallidum, lateral habenula, and prefrontal cortex. Moreover, we provide evidence that VTA GABA and glutamate projection neurons make functional inhibitory or excitatory synapses locally within VTA. These findings suggest that local collaterals of VTA projection neurons could mediate functions prior attributed to VTA interneurons. This study underscores the need for a refined understanding of VTA connectivity to explain how heterogeneous VTA circuits mediate diverse functions related to reward, motivation, or addiction. Significance statementGABA neurons in VTA are key regulators of VTA dopamine neurons and considered central to the mechanisms by which opioids and other drugs of abuse can induce addiction. Conventionally, these VTA GABA neurons are considered interneurons, but GABA projection neurons are also abundant in VTA, and it is unclear if these represent separate populations. We found that several markers enriched in non-dopamine neurons of VTA, including Mu-opioid receptor, are also expressed in projection neurons, and thus are not selective interneuron markers. Moreover, we found that VTA GABA and glutamate projection neurons collateralize within VTA where they make local synapses. These data challenge the notion of a VTA interneuron that synapses only within VTA and suggest that inhibitory projection neurons can serve functions previously attributed to VTA interneurons.

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bioRxiv2024

Viral overexpression of human alpha-synuclein in mouse substantia nigra dopamine neurons results in hyperdopaminergia but no neurodegeneration

Loss of select neuronal populations such as midbrain dopamine (DA) neurons is a pathological hallmark of Parkinsons disease (PD). The small neuronal protein -synuclein has been related both genetically and neuropathologically to PD, yet how it contributes to selective vulnerability remains elusive. Here, we describe the generation of a novel adeno-associated viral vector (AAV) for Cre-dependent overexpression of wild-type human -synuclein. Our strategy allows us to restrict -synuclein to select neuronal populations and hence investigate the cell-autonomous effects of elevated -synuclein in genetically-defined cell types. Since DA neurons in the substantia nigra pars compacta (SNc) are particularly vulnerable in PD, we investigated in more detail the effects of increased -synuclein in these cells. AAV-mediated overexpression of wildtype human -synuclein in SNc DA neurons increased the levels of -synuclein within these cells and augmented phosphorylation of -synuclein at serine-129, which is considered a pathological feature of PD and other synucleinopathies. However, despite abundant -synuclein overexpression and hyperphosphorylation we did not observe any DA neurodegeneration up to 90 days post virus infusion. In contrast, we noticed that overexpression of -synuclein resulted in increased locomotor activity and elevated striatal DA levels suggesting that -synuclein enhanced dopaminergic activity. We therefore conclude that cell-autonomous effects of elevated -synuclein are not sufficient to trigger acute DA neurodegeneration.

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bioRxiv2024

Proportion and distribution of neurotransmitter-defined cell types in the ventral tegmental area and substantia nigra pars compacta

Most studies on the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) have focused on dopamine neurons and their role in processes such as motivation, learning, movement, and associated disorders such as addiction and Parkinsons disease. However there has been increasing attention on other VTA and SNc cell types that release GABA, glutamate, or a combination of neurotransmitters. Yet the relative distributions and proportions of neurotransmitter-defined cell types across VTA and SNc has remained unclear. Here, we used fluorescent in situ hybridization in male and female mice to label VTA and SNc neurons that expressed mRNA encoding the canonical vesicular transporters for dopamine, GABA, or glutamate: vesicular monoamine transporter (VMAT2), vesicular GABA transporter (VGAT), and vesicular glutamate transporter (VGLUT2). Within VTA, we found that no one type was particularly more abundant, instead we observed similar numbers of VMAT2+ (44%), VGAT+ (37%) and VGLUT2+ (41%) neurons. In SNc we found that a slight majority of neurons expressed VMAT2 (54%), fewer were VGAT+ (42%), and VGLUT2+ neurons were least abundant (16%). Moreover, 20% of VTA neurons and 10% of SNc neurons expressed more than one vesicular transporter, including 45% of VGLUT2+ neurons. We also assessed within VTA and SNc subregions and found remarkable heterogeneity in cell-type composition. And by quantifying density across both anterior-posterior and medial-lateral axes we generated heatmaps to visualize the distribution of each cell type. Our data complement recent single-cell RNAseq studies and support a more diverse landscape of neurotransmitter-defined cell types in VTA and SNc than is typically appreciated.

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bioRxiv2023

Sexually dimorphic mechanisms of VGLUT-mediated protection from dopaminergic neurodegeneration

Parkinsons disease (PD) targets some dopamine (DA) neurons more than others. Sex differences offer insights, with females more protected from DA neurodegeneration. The mammalian vesicular glutamate transporter VGLUT2 and Drosophila ortholog dVGLUT have been implicated as modulators of DA neuron resilience. However, the mechanisms by which VGLUT2/dVGLUT protects DA neurons remain unknown. We discovered DA neuron dVGLUT knockdown increased mitochondrial reactive oxygen species in a sexually dimorphic manner in response to depolarization or paraquat-induced stress, males being especially affected. DA neuron dVGLUT also reduced ATP biosynthetic burden during depolarization. RNA sequencing of VGLUT+ DA neurons in mice and flies identified candidate genes that we functionally screened to further dissect VGLUT-mediated DA neuron resilience across PD models. We discovered transcription factors modulating dVGLUT-dependent DA neuroprotection and identified dj-1{beta} as a regulator of sex-specific DA neuron dVGLUT expression. Overall, VGLUT protects DA neurons from PD-associated degeneration by maintaining mitochondrial health.

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bioRxiv2023

Ventral pallidum GABA and glutamate neurons drive approach and avoidance through distinct modulation of VTA cell types

The ventral pallidum (VP) contains GABA and glutamate (Glut) neurons projecting to ventral tegmental area (VTA) whose stimulation drives approach and avoidance, respectively. Yet little is known about the cell-type-specific mechanisms by which VP projections to VTA drive behavior. Here, we found that both VP GABA and Glut neurons were activated during approach to reward or delivery of an aversive stimulus. Stimulation of VP GABA neurons inhibited VTA GABA, but activated dopamine (DA) and glutamate neurons. Remarkably, this cell-type-specific recruitment was behavior-contingent such that VTA recruitment was inhibited when evoked by the subjects own action. Conversely, VP Glut neurons activated VTA GABA, as well as DA and Glut neurons, despite driving aversion. However, VP Glut neurons evoked DA in reward-associated ventromedial nucleus accumbens (NAc), but reduced DA in aversion-associated dorsomedial NAc. These findings show how heterogeneous VP cell types can engage VTA cell types to shape approach and avoidance behaviors. HIGHLIGHTSO_LIVP GABA and Glut neurons are activated by approach to reward and aversive stimuli C_LIO_LIVP GABA and Glut neurons oppositely affect VTA GABA, both activate VTA DA and Glut C_LIO_LIVP Glut neurons activate DA release in ventral NAc but inhibit DA from dorsal NAc C_LIO_LIVTA DA responses to VP GABA activity are inhibited by the subjects own action C_LI

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bioRxiv2022

Mesoaccumbal glutamate neurons drive reward via glutamate release, but aversion via dopamine co-release

Ventral tegmental area (VTA) projections to the nucleus accumbens medial shell (NAc) drive reward-related motivation. Although dopamine neurons are predominant, a substantial glutamatergic projection is also present, and a subset of these populations can release both dopamine and glutamate. Optogenetic stimulation of VTA glutamate neurons supports self-stimulation, but can also induce place avoidance, even in the same assay. Here, we parsed the selective contribution of glutamate or dopamine co-release from VTA glutamate neurons to reinforcement and avoidance. We expressed Channelrhodopsin (ChR2) in VTA glutamate neurons, in combination with CRISPR/Cas9 to disrupt either the gene encoding vesicular glutamate transporter 2 (VGLUT2) or Tyrosine hydroxylase (Th). Selective disruption of VGLUT2 abolished optogenetic self-stimulation, but left real-time place avoidance intact, while CRISPR/Cas9 deletion of Th preserved optogenetic self-stimulation but abolished place avoidance. Our results demonstrate that glutamate release from VTA glutamate neurons is positively reinforcing, but that dopamine release from these same neurons can induce avoidance behavior.

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bioRxiv2021

Disruption of VGLUT1 in cholinergic medial habenula projections increases nicotine self-administration

Cholinergic projections from the medial habenula (MHb) to the interpeduncular nucleus (IPN) have been studied for their complex contributions to nicotine addiction and have been implicated in nicotine reinforcement, aversion, and withdrawal. While it has been established that MHb cholinergic projections co-release glutamate, no direct evidence has demonstrated a role for this specific glutamate projection in nicotine consumption. In the present study, a novel floxed Slc17a7 (VGLUT1) mouse was generated and used to create conditional knockout (cKO) mice that lack VGLUT1 in MHb cholinergic neurons. Histochemical approaches and optogenetics-assisted electrophysiology were used to validate the disruption of VGLUT1 from cholinergic MHb to IPN projections. The mice displayed no gross phenotypic abnormalities and exhibited normal exploratory and locomotor behavior in the open-field assay. However, the loss of VGLUT1-mediated glutamate co-release led to increased nicotine self-administration. These findings indicate that glutamate co-release from ventral MHb cholinergic neurons opposes nicotine consumption and provide additional support for targeting this synapse to develop potential treatments to nicotine addiction.

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bioRxiv2020

Mechanism for differential recruitment of orbitostriatal transmission during outcomes and actions in alcohol dependence

Psychiatric disease often produces symptoms that have divergent effects on neural activity. For example, in drug dependence, dysfunctional value-based decision-making and compulsive-like actions have been linked to hypo- and hyper-activity of orbital frontal cortex (OFC)-basal ganglia circuits, respectively, however, the underlying mechanisms are unknown. Here we show that alcohol dependence enhanced activity in OFC terminals in dorsal striatum (OFC-DS) associated with actions, but reduced activity of the same terminals during periods of outcome retrieval, corresponding with a loss of outcome control over decision-making. Disrupted OFC-DS terminal activity was due to a dysfunction of dopamine-type 1 receptors on spiny projection neurons (D1R SPNs) that resulted in increased retrograde endocannabinoid (eCB) signaling at OFC-D1R SPN synapses reducing OFC-DS transmission. Blocking CB1 receptors restored OFC-DS activity in vivo and rescued outcome-based control over decision-making. These findings demonstrate a circuit-, synapse-, and computation specific mechanism gating OFC activity following the induction of alcohol dependence.

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bioRxiv2020

VGLUT modulates sex differences in dopamine neuron vulnerability to age-related neurodegeneration

Age is the greatest risk factor for Parkinsons disease (PD) which causes progressive loss of dopamine (DA) neurons, with males at greater risk than females. We found that vesicular glutamate transporter (VGLUT) expression mediates vulnerability to age-related DA neurodegeneration in a sex-dependent manner, providing a new mechanism for sex differences in selective DA neuron vulnerability. These findings lay the groundwork for novel therapeutic strategies to boost neuronal resilience throughout aging.

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bioRxiv2020

Ventral Pallidum GABA Neurons Mediate Motivation Underlying Risky Choice

Pursuing rewards while avoiding danger is an essential function of any nervous system. Here, we examine a new mechanism helping rats negotiate the balance between risk and reward when making high-stakes decisions. Specifically, we focus on GABA neurons within an emerging mesolimbic circuit nexus--the ventral pallidum (VP). These neurons play a distinct role from other VP neurons in simple motivated behaviors in mice, but their roles in more complex motivated behaviors is unknown. Here, we interrogate the behavioral functions of VPGABA neurons in male and female transgenic GAD1:Cre rats (and wildtype littermates), using a reversible chemogenetic inhibition approach. Employing a behavioral assay of risky decision making, and of the food-seeking and shock-avoidance components of this task, we show that engaging inhibitory Gi/o signaling specifically in VPGABA neurons suppresses motivation to pursue highly salient palatable foods, and possibly also motivation to avoid being shocked. In contrast, inhibiting these neurons did not affect seeking of low-value food, free consumption of palatable food, or unconditioned affective responses to shock. Accordingly, when rats considered whether to pursue food despite potential for shock in a risky decision-making task, inhibiting VPGABA neurons caused them to more readily select a small but safe reward over a large but dangerous one--an effect not seen in the absence of shock threat. Together, results indicate that VPGABA neurons are critical for high-stakes adaptive responding that is necessary for survival, but which may also malfunction in psychiatric disorders. Significance StatementIn a dynamic world, it is essential to implement appropriate behaviors under circumstances involving rewards, threats, or both. Here, we demonstrate a crucial role for VPGABA neurons in high-stakes motivated behavior of several types. We show that this VPGABA role in motivation impacts decision making, as inhibiting these neurons yields a conservative, risk-averse strategy not seen when the task is performed without threat of shock. These new roles for VPGABA neurons in behavior may inform future strategies for treating addiction, and other disorders of maladaptive decision making.

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bioRxiv2019

Ventral Pallidum is Essential for Cocaine Reinstatement After Voluntary Abstinence

Addiction is a chronic relapsing disorder, and during recovery many people experience several relapse events as they attempt to voluntarily abstain from drug. New preclinical relapse models have emerged which capture this common human experience of relapse after voluntary abstinence, and mounting evidence indicates that reinstatement of drug seeking after voluntary abstinence recruits neural circuits distinct from reinstatement following experimenter-imposed abstinence, or abstinence due to extinction training. Ventral pallidum (VP), a key limbic node involved in drug seeking, has well-established roles in conventional reinstatement models tested following extinction training, but it is unclear whether this region also participates in more translationally-relevant models of relapse. Here we show that chemogenetic inhibition of VP neurons strongly attenuates cocaine-, context-, and cue-induced reinstatement tested after voluntary, punishment-induced abstinence. This effect was strongest in the most compulsive, punishment-resistant rats, and reinstatement was associated with neural activity in anatomically-defined VP subregions. VP inhibition also attenuated the propensity of rats to display hesitations, a risk assessment behavior seen during punished drug taking that is likely due to concurrent approach and avoidance motivations. These results indicate that VP, unlike other connected limbic brain regions, is essential for reinstatement of drug seeking after voluntary abstinence. Since VP inhibition effects were strongest in the most compulsively cocaine-seeking individuals, this could indicate that VP plays a particularly important role in the most pathological, addiction-like behavior, making it an attractive target for future therapeutic interventions.

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