Biology subjects

Steinkellner, T.

Publications and source records attributed to Steinkellner, T..

7 recordsLinked to original sources

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

neuroscience↗

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.

neuroscience↗

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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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.

neuroscience↗

bioRxiv2020

A genetic probe for visualizing glutamatergic synapses and vesicles by 3D electron microscopy

Communication between neurons relies on the release of diverse neurotransmitters, which represent a key-defining feature of a neurons chemical and functional identity. Neurotransmitters are packaged into vesicles by specific vesicular transporters. However, tools for labeling and imaging synapses and synaptic vesicles based on their neurochemical identity remain limited. We developed a genetically encoded probe to identify glutamatergic synaptic vesicles at the levels of both light and electron microscopy (EM) by fusing the mini singlet oxygen generator (miniSOG) probe to an intra-lumenal loop of the vesicular glutamate transporter-2. We then used a 3D imaging method, serial block face scanning EM, combined with a deep learning approach for automatic segmentation of labeled synaptic vesicles to assess the subcellular distribution of transporter-defined vesicles at nanometer scale. These tools represent a new resource for accessing the subcellular structure and molecular machinery of neurotransmission and for transmitter-defined tracing of neuronal connectivity.

neuroscience↗