Biology subjects

Zweifel, L.

Publications and source records attributed to Zweifel, L..

16 recordsLinked to original sources

bioRxiv2026

Genetic Identification of Dopamine Neurons Required for Circadian Food Anticipatory Activity in Mice

Anticipating daily food availability is a conserved circadian behavior that persists even in animals lacking the suprachiasmatic nucleus, yet its neural substrates remain poorly defined. Previous studies implicated dopamine signaling in food anticipatory activity (FAA) but lacked the resolution to identify the responsible neuronal population. Here, we conditionally deleted tyrosine hydroxylase (Th) from molecularly defined dopamine neuron populations in mice. Broad deletion of Th in dopamine transporter-expressing neurons nearly abolished FAA, whereas restoration of Th in substantia nigra dopamine neurons rescued anticipatory locomotion. Surprisingly, deletion of Th from several large dopamine neuron populations had little effect on FAA. In contrast, deletion using Calb1Cre, targeting only [~]25% of substantia nigra dopamine neurons, produced a profound FAA deficit. Notably, these mice exhibited a profound loss of anticipatory locomotion while retaining substantial anticipatory food-seeking behavior. These findings identify a small Calbindin1+ dopamine population required for anticipatory locomotion and demonstrate that distinct behavioral components of food anticipation can be genetically dissociated.

neuroscience↗

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.

neuroscience↗

bioRxiv2025

Behavioral Decoding Reveals Cortical Endocannabinoid Potentiation during Δ9-THC Impairment

How {Delta}9-tetrahydrocannabinol (THC) impairs natural behaviors in mice remains unknown. We developed a video-monitored behavioral platform with machine learning classifiers to unravel discrete changes in natural mouse behaviors. THC infusion into the medial prefrontal cortex (mPFC) disrupted walking kinematic features characteristic of impairment responses. THC predominantly increased mPFC GABAergic activity preceding walk initiation shifting the mPFC excitatory/inhibitory (E/I) balance. Pose-defined closed loop photo-stimulation of mPFC GABAergic neurons demonstrated that THC exacerbates selected parameters of motor impairment. Surprisingly, THC also induced a time locked, movement-induced, transient potentiation of mPFC endocannabinoid (eCB) release and ensuing CB1R-mediated synaptic inhibition. Here we establish that THC-modifies mPFC E/I balance to excitation via dynamic changes in eCB release which acts to induce behavioral impairment.

neuroscience↗

bioRxiv2025

Melanocortin-responsive Kiss1 neurons of the arcuate nucleus drive energy expenditure through glutamatergic signaling to the dorsomedial hypothalamus.

Energy expenditure (EE) is essential for metabolic homeostasis, yet its central regulation remains poorly understood. Here, we identify arcuate Kiss1 neurons as key regulators of EE in male mice. Ablation of these neurons induced obesity, while their chemogenetic activation increased brown adipose tissue (BAT) thermogenesis without affecting food intake. This action is mediated by glutamatergic projections from Kiss1ARC neurons to CART/Lepr-expressing neurons in the dorsomedial hypothalamus, which activate the raphe pallidus-BAT pathway. CRISPR-mediated deletion of the vesicular glutamate transporter 2 (Vglut2) from Kiss1ARC neurons replicated the obesogenic effect. Furthermore, deletion of the melanocortin 4 receptor (MC4R) from Kiss1 neurons resulted in obesity, reduced energy expenditure and impaired thermogenesis. Optogenetic stimulation of pro-opiomelanocortin (POMC) fibers evoked inward currents in Kiss1 neurons, that were attenuated by MC4R antagonism. Our findings reveal a previously unrecognized neural circuit that mediates melanocortin action on energy expenditure, offering new insights into central mechanisms of metabolic control.

neuroscience↗

bioRxiv2025

Endocannabinoids facilitate transitory reward engagement through retrograde gain control

Neuromodulatory signaling is poised to serve as a neural mechanism for gain control, acting as a crucial tuning factor to influence neuronal activity by dynamically shaping excitatory and inhibitory fast neurotransmission. The endocannabinoid (eCB) signaling system, the most widely expressed neuromodulatory system in the mammalian brain, is known to filter excitatory and inhibitory inputs through retrograde, pre-synaptic action. However, whether eCBs exert retrograde gain control to ultimately facilitate reward-seeking behaviors in freely moving mammals is not established. Using a suite of in vivo physiological, imaging, genetic and machine learning-based approaches, we report a fundamental role for eCBs in controlling behavioral engagement in reward-seeking behavior through a defined thalamo-striatal circuit.

neuroscience↗

bioRxiv2024

Reductions of Grin2a in adolescent dopamine neurons confers aberrant salience and related psychosis phenotype

Psychosis is a hallmark of schizophrenia. It involves a collection of symptoms that are typically associated with disrupted dopamine signaling and emerges during adolescence or early adulthood. Most schizophrenia-associated genes, however, involve glutamatergic or other ubiquitous targets that do not explain the latent expression of psychosis or dopaminergic abnormalities. Here, we describe an etiologically relevant model for the adolescent onset of dopamine-related dysfunction in schizophrenia. We focused on GRIN2A, the gene encoding the GluN2A subunit of the NMDA receptor, as both the common loss-of-function variants and the rare missense variants in this gene are risk factors for schizophrenia. We find that GluN2A levels distinctly decline in dopamine neuron-containing regions throughout adolescence while remaining stable in other regions. This suggested that adolescent dopamine neurons may be particularly vulnerable to further reductions in GluN2A caused by a damaging variant of GRIN2A. Consistent with this idea, we find that selective knockout of Grin2a in adolescent rat dopamine neurons results in a psychosis-relevant behavioral phenotype. This manipulation also reduced dopamine release in response to unexpected outcomes in young adults, reflective of prediction error signaling abnormalities observed in the clinical population. These data provide mechanistic insight into how GRIN2A mutations may contribute to the delayed onset of dopamine-related symptoms and provide a model for identifying course altering treatments for schizophrenia.

neuroscience↗

bioRxiv2024

Dynorphin modulates motivation through a pallido-amygdala cholinergic circuit

The endogenous opioid peptide dynorphin and its receptor {kappa}-opioid receptor (KOR) have been implicated in divergent behaviors, but the underlying mechanisms remain elusive. Here we show that dynorphin released from nucleus accumbens dynorphinergic neurons exerts powerful modulation over a ventral pallidum (VP) disinhibitory circuit, thereby controlling cholinergic transmission to the amygdala and motivational drive in mice. On one hand, dynorphin acts postsynaptically via KORs on local GABAergic neurons in the VP to promote disinhibition of cholinergic neurons, which release acetylcholine into the amygdala to invigorate reward-seeking behaviors. On the other hand, dynorphin also acts presynaptically via KORs on dynorphinergic terminals to limit its own release. Such autoinhibition keeps cholinergic neurons from prolonged activation and release of acetylcholine, and prevents perseverant reward seeking. Our study reveals how dynorphin exquisitely modulate motivation through cholinergic system, and provides an explanation for why these neuromodulators are involved in motivational disorders, including depression and addiction.

neuroscience↗

bioRxiv2023

An arginine-rich nuclear localization signal (ArgiNLS) strategy for streamlined image segmentation of single-cells

High-throughput volumetric fluorescent microscopy pipelines can spatially integrate whole-brain structure and function at the foundational level of single-cells. However, conventional fluorescent protein (FP) modifications used to discriminate single-cells possess limited efficacy or are detrimental to cellular health. Here, we introduce a synthetic and non-deleterious nuclear localization signal (NLS) tag strategy, called Arginine-rich NLS (ArgiNLS), that optimizes genetic labeling and downstream image segmentation of single-cells by restricting FP localization near-exclusively in the nucleus through a poly-arginine mechanism. A single N-terminal ArgiNLS tag provides modular nuclear restriction consistently across spectrally separate FP variants. ArgiNLS performance in vivo displays functional conservation across major cortical cell classes, and in response to both local and systemic brain wide AAV administration. Crucially, the high signal-to-noise ratio afforded by ArgiNLS enhances ML-automated segmentation of single-cells due to rapid classifier training and enrichment of labeled cell detection within 2D brain sections or 3D volumetric whole-brain image datasets, derived from both staining-amplified and native signal. This genetic strategy provides a simple and flexible basis for precise image segmentation of genetically labeled single-cells at scale and paired with behavioral procedures. Significance StatementQuantifying labeled cells in fluorescent microscopy is a fundamental aspect of modern biology. Critically, the use of short nuclear localization sequences (NLS) is a key genetic modification for discriminating single-cells labeled with fluorescent proteins (FPs). However, mainstay NLS approaches typically localize proteins to the nucleus with limited efficacy, while alternative non-NLS tag strategies can enhance efficacy at the cost of cellular health. Thus, quantitative cell counting using FP labels remains suboptimal or not compatible with health and behavior. Here, we present a novel genetic tagging strategy - named ArgiNLS - that flexibly and safely achieves FP nuclear restriction across the brain to facilitate machine learning-based segmentation of single-cells at scale, delivering a timely update to the behavioral neuroscientists toolkit.

neuroscience↗

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.

neuroscience↗

bioRxiv2023

Pharmacological characterization of the endocannabinoid sensor GRABeCB2.0

IntroductionThe endocannabinoids (eCBs), 2-arachidonoylglycerol (2-AG) and arachidonoyl ethanolamine (AEA), are produced by separate enzymatic pathways, activate cannabinoid receptors with distinct pharmacology, and differentially regulate pathophysiological processes. The genetically encoded sensor, GRABeCB2.0, detects real-time changes in eCB levels in cells in culture and preclinical model systems; however, its activation by eCB analogues produced by cells and by phyto-cannabinoids remains uncharacterized, a current limitation when interpreting changes in its response. This information could provide additional utility for the tool in in vivo pharmacology studies of phyto-cannabinoid action. MethodsGRABeCB2.0 was expressed in cultured HEK293 cells. Live cell confocal microscopy and high-throughput fluorescent signal measurements. Results2-AG increased GRABeCB2.0 fluorescent signal (EC50 = 85 nM), and the cannabinoid 1 receptor (CB1R) antagonist, SR141617, decreased GRABeCB2.0 signal (SR1, IC50 = 3.3 nM), responses that mirror their known potencies at cannabinoid 1 receptors (CB1R). GRABeCB2.0 fluorescent signal also increased in response to AEA (EC50 = 815 nM), the eCB analogues 2-linoleoylglycerol and 2-oleoylglycerol (2-LG and 2-OG, EC50s = 1.5 and 1.0 M, respectively), {Delta}9-tetrahydrocannabinol ({Delta}9-THC) and{Delta} 8-THC (EC50s = 1.6 and 2.0 M, respectively), and the artificial CB1R agonist, CP55,940 (CP, EC50 = 82 nM); however their potencies were less than what has been described at CB1R. Cannabidiol (CBD) did not affect basal GRABeCB2.0 fluorescent signal and yet reduced the 2-AG stimulated GRABeCB2.0 responses (IC50 = 8.8 nM). Conclusions2-AG and SR1 modulate the GRABeCB2.0 fluorescent signal with EC50s that mirror their potencies at CB1R whereas AEA, eCB analogues, THC and CP increase GRABeCB2.0 fluorescent signal with EC50s significantly lower than their potencies at CB1R. CBD reduces the 2-AG response without affecting basal signal, suggesting that GRABeCB2.0 retains the negative allosteric modulator (NAM) property of CBD at CB1R. This study describes the pharmacological profile of GRABeCB2.0 to improve interpretation of changes in fluorescent signal in response to a series of known eCBs and CB1R ligands.

pharmacology and toxicology↗

bioRxiv2022

ABHD6 selectively controls Gq-dependent increases in 2-AG production

The most abundant endocannabinoid (eCB) in the brain, 2-arachidonoyl glycerol (2-AG), is hydrolyzed by /{beta}-hydrolase domain containing 6 (ABHD6); yet how ABHD6 controls stimuli-dependent increases in 2-AG production is unknown. To explore this question, we leveraged the recently developed 2-AG sensor, GRABeCB2.0, and found that stimulation of Neuro2a cells in culture with bradykinin (BK) acting at metabotropic B2K receptors and ATP acting at ionotropic P2X7 receptors led to differential increases in 2-AG levels. B2K triggered increases in 2-AG levels via diacylglycerol lipase (DAGL), and this mechanism was potentiated by increases in intracellular calcium and ABHD6 inhibition. By contrast, P2X7-triggered increases in 2-AG levels were dependent on DAGL and extracellular calcium but unaffected by ABHD6 inhibition. Thus, ABHD6 preferentially regulates metabotropic-dependent increases in 2-AG levels over ionotropic-dependent increases in 2-AG levels. Our study indicates that ABHD6 selectively controls stimuli-dependent increases in 2-AG production and emphasizes its specific role in eCB signaling.

cell biology↗

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.

neuroscience↗

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.

neuroscience↗

bioRxiv2021

Pubertal sex hormones control transcriptional trajectories in the medial preoptic area

Pubertal maturation aids development of emotion, cognition, and reproduction. We investigated transcriptional dynamics in the medial preoptic area (MPOA), a hypothalamic center for reproductive behaviors, in male and female mice at single-cell resolution (scRNAseq) during puberty. Defined subsets of neurons expressing Slc32a1 and Esr1 (Vgat+ Esr1+) were the most transcriptionally dynamic compared to other cell types throughout puberty. These cell type specific transcriptional progressions towards adulthood were bidirectionally controlled by the levels of circulating testosterone and estradiol. Selective deletion of Esr1 in Slc32a1-expressing cells in the MPOA prior to puberty arrested transcriptional progression and revealed a sexually dimorphic gene-regulatory network governed by Esr1. Deletion of Esr1 in Vgat+ cells prevented the development of mating behavior in both sexes. These analyses reveal both sexually common and dimorphic transcriptional progressions during puberty as well as their regulatory mechanisms, which have important implications towards understanding adaptative and maladaptive processes governing adolescent brain development.

neuroscience↗

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.

neuroscience↗

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.

neuroscience↗