Biology subjects

Hunker, A. C.

Publications and source records attributed to Hunker, A. C..

12 recordsLinked to original sources

bioRxiv2026

Morphoelectric Diversity and Specialization of Neuronal Cell Types in the Primate Striatum

The basal ganglia are evolutionary ancient subcortical nuclei that form interconnected loops with the neocortex and limbic system to regulate movement, learning, habit formation, emotion, and motivation. Their dysfunction contributes to major neurological and psychiatric disorders, yet most cellular-level insights derive from rodent studies, leaving knowledge gaps in humans and translationally relevant primate species. To address this, we generated multi-modal Patch-seq data linking transcriptomic identity with morphological and electrophysiological properties in macaque striatum, the input nucleus of the basal ganglia. We found underappreciated diversity among medium spiny neurons, including non-canonical types, and variation aligned with functional gradients. Interneurons also exhibited spatial variation and even greater morphoelectric diversity, highlighting their functional modularity. Despite broad evolutionary conservation, we identified primate-specific features and key differences from rodent striatal neurons. By integrating molecular classification with cellular properties that shape network function, our findings provide insights into the functional organization of the primate striatum.

neuroscience↗

bioRxiv2026

A Cross-Species Enhancer-AAV Toolkit for Cell Type-Specific Targeting Across the Basal Ganglia

The mammalian basal ganglia (BG) orchestrate motor, cognitive, and affective functions, yet cell type-specific genetic access remains limited, especially beyond rodents. Key structures implicated in movement and psychiatric disorders, including pallidum, subthalamic nucleus, and dopaminergic midbrain, lack scalable tools for cross-species targeting. Here, we present a comprehensive enhancer-AAV library enabling selective labeling and manipulation of major BG neuronal populations: striatal projection neuron subtypes, pallidal and subthalamic neurons, and midbrain dopaminergic and GABAergic populations. Using an evolutionarily informed discovery pipeline, we identified enhancers targeting canonical, non-canonical, and disease-relevant cell types, with validation demonstrating robust cross-species conservation of specificity between mouse and macaque. Computational modeling revealed sequence features predictive of in vivo performance, including motif grammar, chromatin accessibility, and evolutionary conservation, and identified distinct regulatory architectures across glial, projection, and interneuron lineages. This work establishes a comprehensive cross-species viral toolkit for the BG, unlocking previously inaccessible cell types for circuit dissection.

neuroscience↗

bioRxiv2025

Technical and biological sources of noise confound multiplexed enhancer AAV screening

Cis-acting regulatory enhancer elements are valuable tools for gaining cell type-specific genetic access. Leveraging large chromatin accessibility atlases, putative enhancer sequences can be identified and deployed in adeno-associated virus (AAV) delivery platforms. However, a significant bottleneck in enhancer AAV discovery is charting their detailed expression patterns in vivo, a process that currently requires gold-standard one-by-one testing. Here we present a barcoded multiplex strategy for screening enhancer AAVs at cell type resolution using single cell RNA sequencing and taxonomy mapping. We executed a proof-of-concept study using a small pool of validated enhancer AAVs expressing in a variety of neuronal and non-neuronal cell types across the mouse brain. Unexpectedly, we encountered substantial technical and biological noise including chimeric packaging products, necessitating development of novel techniques to accurately deconvolve enhancer expression patterns. These results underscore the need for improved methods to mitigate noise and highlight the complexity of enhancer AAV biology in vivo.

neuroscience↗

bioRxiv2024

Enhancer AAV toolbox for accessing and perturbing striatal cell types and circuits

We present an enhancer AAV toolbox for accessing and perturbing striatal cell types and circuits. Best-in-class vectors were curated for accessing major striatal neuron populations including medium spiny neurons (MSNs), direct and indirect pathway MSNs, as well as Sst-Chodl, Pvalb-Pthlh, and cholinergic interneurons. Specificity was evaluated by multiple modes of molecular validation, three different routes of virus delivery, and with diverse transgene cargos. Importantly, we provide detailed information necessary to achieve reliable cell type specific labeling under different experimental contexts. We demonstrate direct pathway circuit-selective optogenetic perturbation of behavior and multiplex labeling of striatal interneuron types for targeted analysis of cellular features. Lastly, we show conserved in vivo activity for exemplary MSN enhancers in rat and macaque. This collection of striatal enhancer AAVs offers greater versatility compared to available transgenic lines and can readily be applied for cell type and circuit studies in diverse mammalian species beyond the mouse model.

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bioRxiv2023

Dopamine D2 receptors in the bed nucleus of the stria terminalis modulate alcohol-related behaviors.

Dysregulation of the dopamine (DA) system is a hallmark of substance abuse disorders, including alcohol use disorder (AUD). Of the DA receptor subtypes, the DA D2 receptors (D2Rs) play a key role in the reinforcing effects of alcohol. D2Rs are expressed in numerous brain regions associated with the regulation of appetitive behaviors. One such region is the bed nucleus of the stria terminalis (BNST), which has been linked to the development and maintenance of AUD. Recently, we identified alcohol withdrawal-related neuroadaptations in the periaqueductal gray/dorsal raphe to BNST DA circuit in male mice. However, the role of D2R-expressing BNST neurons in voluntary alcohol consumption is not well characterized. In this study, we used a CRISPR-Cas9-based viral approach, to selectively reduce the expression of D2Rs in BNST VGAT neurons and interrogated the impact of BNST D2Rs in alcohol-related behaviors. In male mice, reduced D2R expression potentiated the stimulatory effects of alcohol and increased voluntary consumption of 20% w/v alcohol in a two-bottle choice intermittent access paradigm. This effect was not specific to alcohol, as D2R deletion also increased sucrose intake in male mice. Interestingly, cell-specific deletion of BNST D2Rs in female mice did not alter alcohol-related behaviors but lowered the threshold for mechanical pain sensitivity. Collectively, our findings suggest a role for postsynaptic BNST D2Rs in the modulation of sex-specific behavioral responses to alcohol and sucrose.

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bioRxiv2023

Novel genetically encoded tools for imaging or silencing neuropeptide release from presynaptic terminals in vivo

Neurons produce and release neuropeptides to communicate with one another. Despite their profound impact on critical brain functions, circuit-based mechanisms of peptidergic transmission are poorly understood, primarily due to the lack of tools for monitoring and manipulating neuropeptide release in vivo. Here, we report the development of two genetically encoded tools for investigating peptidergic transmission in behaving mice: a genetically encoded large dense core vesicle (LDCV) sensor that detects the neuropeptides release presynaptically, and a genetically encoded silencer that specifically degrades neuropeptides inside the LDCV. Monitoring and silencing peptidergic and glutamatergic transmissions from presynaptic terminals using our newly developed tools and existing genetic tools, respectively, reveal that neuropeptides, not glutamate, are the primary transmitter in encoding unconditioned stimulus during Pavlovian threat learning. These results show that our sensor and silencer for peptidergic transmission are reliable tools to investigate neuropeptidergic systems in awake behaving animals.

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bioRxiv2022

A Genetic Variant of Fatty Acid Amide Hydrolase (FAAH) Exacerbates Hormone- Mediated Orexigenic Feeding in Mice

Fatty acid amide hydrolase (FAAH) degrades the endocannabinoid anandamide. A polymorphism in FAAH (FAAH C385A) reduces FAAH expression, increases anandamide levels, and increases the risk of obesity. Nevertheless, some studies have found no association between FAAH C385A and obesity. We investigated whether the environmental context governs the impact of FAAH C385A on metabolic outcomes. Using a C385A knock-in mouse model, we found that under basal conditions, there was no effect of the FAAH C385A polymorphism on body weight or composition. By contrast, FAAH A/A mice are more susceptible to glucocorticoid-induced hyperphagia, weight gain, and activation of hypothalamic AMPK. AMPK inhibition occluded the amplified hyperphagic response to glucocorticoids in FAAH A/A mice. FAAH knockdown exclusively in AgRP neurons mimicked the exaggerated feeding response of FAAH A/A mice to glucocorticoids. FAAH A/A mice likewise presented exaggerated orexigenic responses to ghrelin, while FAAH knockdown in AgRP neurons blunted leptin anorectic responses. Together, the FAAH A/A genotype amplifies orexigenic responses and decreases anorexigenic responses, providing a putative mechanism explaining the diverging human findings.

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bioRxiv2022

Netrin-1 regulates the balance of glutamatergic connectivity in the adult ventral tegmental area

The axonal guidance cue netrin-1 serves a critical role in neural circuit development by promoting growth cone motility, axonal branching, and synaptogenesis. Within the adult mouse brain, expression of the gene encoding netrin-1 (Ntn1) is highly enriched in the ventral midbrain where it is expressed in both GABAergic and dopaminergic neurons, but its function in these cell types in the adult system remains largely unknown. To address this, we performed viral-mediated, cell-type specific CRISPR-Cas9 mutagenesis of Ntn1 in the ventral tegmental area (VTA) of adult mice. Ntn1 loss-of-function in either cell type resulted in a significant reduction in excitatory postsynaptic connectivity. In dopamine neurons, reduced excitatory tone had a minimal phenotypic behavioral outcome; however, reduced glutamatergic tone on VTA GABA neurons induced behaviors associated with a hyperdopaminergic phenotype. Loss of Ntn1 function in both cell types simultaneously largely rescued the phenotype observed in the GABA-only mutagenesis. These findings demonstrate an important role for netrin-1 in maintaining excitatory connectivity in the adult midbrain and that a balance in this connectivity within two of the major cell types of the VTA is critical for the proper functioning of the mesolimbic system.

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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

Intrinsic regulators of the action potential waveform control dopamine release to shape behavior

Despite the widely known role of dopamine in reinforcement learning, how the patterns of dopamine release that are critical to the acquisition, performance, and extinction of conditioned responses are generated is poorly resolved. Here, we demonstrate that the coordinated actions of two ion channels, Kv4.3 and BKCa1.1, control the pattern of dopamine neuron firing and dopamine release on different time scales to regulate separate phases of reinforced behavior in mice. Inactivation of Kv4.3 in VTA dopamine neurons increases ex vivo pacemaker activity and excitability that is associated with increased in vivo ramping dynamics prior to lever press in a learned instrumental response paradigm. Loss of Kv4.3 enhances performance of the learned response and facilitates extinction. In contrast, loss of BKCa1.1 increases burst firing and phasic dopamine release that enhances learning of an instrumental response. Inactivation of BKCa1.1 enhances extinction burst lever pressing in early extinction training that is associated with increased reward prediction error signals. These data demonstrate that temporally distinct patterns of dopamine release are governed by the intrinsic regulators of the cell to shape behavior. TeaserWe show that ion channels in midbrain dopamine neurons are critical for patterning action potential firing at the cell body and governing neurotransmitter release to regulate reinforcement learning.

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bioRxiv2020

CRISPR Knockdown of Kcnq3 Attenuates the M-current in NPY/AgRP Neurons

Arcuate nucleus Neuropeptide Y/Agouti-related peptide (NPY/AgRP) neurons drive ingestive behavior in response to the internal and external environment of an organism. NPY/AgRP neurons are adjacent to the median eminence, a circumventricular organ, and circulating metabolic factors and hormones communicate the energy state of the animal via these neurons by altering the excitability of NPY/AgRP neurons, which produces an appropriate change in behavior to maintain homeostasis. One example of this plasticity is seen in the M-current, a subthreshold, non-inactivating K+ current that acts to modulate excitability. Fasting decreases while estradiol increases the M-current through regulation of subunit mRNA expression of Kcnq 2, 3, & 5. KCNQ2/3 heteromers are thought to mediate the majority of the M-current. Here we used a recently developed single adeno-associated viral (AAV) vector containing a recombinase-dependent Staphylococcus aureus Cas9 (SaCas9) and a single guide RNA against Kcnq3 to selectively delete Kcnq3 in NPY/AgRP neurons to produce a loss of function in the M-current. We found that this virus was effective at knocking down Kcnq3 but not Kcnq2 expression. With the reduced KCNQ3 channel expression NPY/AgRP neurons were more depolarized, exhibited a higher input resistance, and the rheobase current needed to induce firing was significantly reduced, indicative of increased excitability. Although the resulting decrease in the M-current did not overtly alter ingestive behavior, it did significantly reduce the locomotor activity as measured in open field testing. Therefore, the SaCas9-sgKcnq3 is efficient to knock down Kcnq3 expression thereby reducing the M-current and increasing the excitability of NPY/AgRP neurons.

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bioRxiv2020

A midbrain dynorphin circuit promotes threat generalization

Discrimination between predictive and non-predictive threat stimuli decrease as threat intensity increases. The central mechanisms that mediate the transition from discriminatory to generalized threat responding remain poorly resolved. Here, we identify the stress- and dysphoria-associated kappa opioid receptor (KOR) and its ligand dynorphin (Dyn), acting in the ventral tegmental area (VTA), as a key substrate for regulating threat generalization. We identified several dynorphinergic inputs to the VTA and demonstrate that projections from the bed nucleus of the stria terminalis (BNST) and dorsal raphe nucleus (DRN) both contribute to anxiety-like behavior but differentially affect threat generalization. These data demonstrate that conditioned threat discrimination has an inverted U relationship with threat intensity and establish a role for KOR/Dyn signaling in the midbrain for promoting threat generalization.

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