Fiber Photometry publications

Thermoregulatory behavior is a basic motivated behavior for body temperature homeostasis. Despite its fundamental importance, a forebrain region or defined neural population required for this process has yet to be established. Here, we show that Vgat-expressing neurons in the lateral hypothalamus (LHVgat neurons) are required for diverse thermoregulatory behaviors. The population activity of LHVgat neurons is increased during thermoregulatory behavior and bidirectionally encodes thermal punishment and reward (P&R). Although this population also regulates feeding and caloric reward, inhibition of parabrachial inputs selectively impaired thermoregulatory behaviors and encoding of thermal stimulus by LHVgat neurons. Furthermore, two-photon calcium imaging revealed a subpopulation of LHVgat neurons bidirectionally encoding thermal P&R, which is engaged during thermoregulatory behavior, but is largely distinct from caloric reward-encoding LHVgat neurons. Our data establish LHVgat neurons as a required neural substrate for behavioral thermoregulation and point to the key role of the thermal P&R-encoding LHVgat subpopulation in thermoregulatory behavior.

Insulin acts on neurons and glial cells to regulate systemic glucose metabolism and feeding. However, the mechanisms of insulin access in discrete brain regions are incompletely defined. Here we show that insulin receptors in tanycytes, but not in brain endothelial cells, are required to regulate insulin access to the hypothalamic arcuate nucleus. Mice lacking insulin receptors in tanycytes (IR ∆Tan mice) exhibit systemic insulin resistance, while displaying normal food intake and energy expenditure. Tanycytic insulin receptors are also necessary for the orexigenic effects of ghrelin, but not for the anorexic effects of leptin. IR ∆Tan mice exhibit increased agouti-related peptide (AgRP) neuronal activity, while displaying blunted AgRP neuronal adaptations to feeding-related stimuli. Lastly, a highly palatable food decreases tanycytic and arcuate nucleus insulin signalling to levels comparable to those seen in IR ∆Tan mice. These changes are rooted in modifications of cellular stress responses and of mitochondrial protein quality control in tanycytes. Conclusively, we reveal a critical role of tanycyte insulin receptors in gating feeding-state-dependent regulation of AgRP neurons and systemic insulin sensitivity, and show that insulin resistance in tanycytes contributes to the pleiotropic manifestations of obesity-associated insulin resistance.

Fiber photometry (FP) is an adaptable method for recording in vivo neural activity in freely behaving animals. It has become a popular tool in neuroscience due to its ease of use, low cost, the ability to combine FP with freely moving behavior, among other advantages. However, analysis of FP data can be challenging for new users, especially those with a limited programming background. Here, we present Guided Photometry Analysis in Python (GuPPy), a free and open-source FP analysis tool. GuPPy is designed to operate across computing platforms and can accept data from a variety of FP data acquisition systems. The program presents users with a set of graphic user interfaces (GUIs) to load data and provide input parameters. Graphs are produced that can be easily exported for integration into scientific figures. As an open-source tool, GuPPy can be modified by users with knowledge of Python to fit their specific needs.

Significance Impulsive overeating is a common, disabling feature of eating disorders. Calcium imaging using fiber photometry has emerged as an in vivo methodology to measure neuronal population activity immune to electrical stimulation artifact from deep brain stimulation (DBS). Thus, when used simultaneously, calcium imaging can elucidate poorly understood DBS mechanisms. We show that nucleus accumbens D1 medial spiny calcium signaling increases in preparation of hedonic feeding of high-fat food. Further, responsive, over continuous, DBS strategies effectively disrupt this activity leading to decreased consumption. Implementation of this methodology to better understand mechanisms of these and other forms of neuromodulation for various indications may help advance the field to identify novel therapeutic targets with applications extending beyond obesity.

In the striatum, acetylcholine (ACh) neuron activity is modulated co-incident with dopamine (DA) release in response to unpredicted rewards and reward predicting cues and both neuromodulators are thought to regulate each other. While this co-regulation has been studied using stimulation studies, the existence of this mutual regulation in vivo during natural behavior is still largely unexplored. One long-standing controversy has been whether striatal DA is responsible for the induction of the cholinergic pause or whether D2R modulate a pause that is induced by other mechanisms. Here, we used genetically encoded sensors in combination with pharmacological and genetic inactivation of D2Rs from cholinergic interneurons (CINs) to simultaneously measure ACh and DA levels after CIN D2R inactivation. We found that CIN D2Rs are not necessary for the induction of cue induced dips in ACh levels but regulate dip lengths and rebound ACh levels. Importantly, D2R inactivation strongly decreased the temporal correlation between DA and Ach signals not only at cue presentation but also during the intertrial interval. This points to a general mechanism by which D2Rs coordinate both signals. At the behavioral level D2R antagonism increased the latency to lever press, which was not observed in CIN-selective D2R knock out mice. This latency correlated with the cue evoked dip length supporting a role of the ACh dip and it’s regulation by D2Rs in motivated behavior. Overall, our data indicate that striatal DA coordinate phasic ACh and DA signals via CIN D2Rs which is important for the regulation of motivated behavior.

Tobacco use is the leading cause of preventable death worldwide, and relapse during abstinence remains the key barrier to successful treatment of tobacco addiction. During abstinence, environmental contexts associated with nicotine use can induce craving and contribute to relapse. The insular cortex (IC) is thought to be a critical substrate of nicotine addiction and relapse. However, its specific role in context-induced relapse of nicotine-seeking is not fully known. In this study, we report a novel rodent model of context-induced relapse to nicotine-seeking after punishment-imposed abstinence, which models self-imposed abstinence through increasing negative consequences of excessive drug use. Using the neuronal activity marker Fos we find that the anterior (aIC), but not the middle or posterior IC, shows increased activity during context-induced relapse. Combining Fos with retrograde labelling of aIC inputs, we show projections to aIC from contralateral aIC and basolateral amygdala exhibit increased activity during context-induced relapse. Next, we used fiber photometry in aIC and observed phasic increases in aIC activity around nicotine-seeking responses during self-administration, punishment, and the context-induced relapse tests. Next, we used chemogenetic inhibition in both male and female rats to determine whether activity in aIC is necessary for context-induced relapse. We found that chemogenetic inhibition of aIC decreased context-induced nicotine-seeking after either punishment- or extinction-imposed abstinence. These findings highlight the critical role nicotine-associated contexts play in promoting relapse, and they show that aIC activity is critical for this context-induced relapse following both punishment and extinction imposed abstinence.

Objective: Long-acting glucagon-like peptide-1 receptor agonists (GLP-1RAs), like liraglutide and semaglutide, are viable treatments for diabetes and obesity. Liraglutide directly activates hypothalamic proopiomelanocortin (POMC) neurons while indirectly inhibiting Neuropeptide Y/Agouti-related peptide (NPY/AgRP) neurons ex vivo. While temporal control of GLP-1R agonist concentration as well as accessibility to tissues/cells can be achieved with relative ease ex vivo, in vivo this is dependent upon the pharmacokinetics of these agonists and relative penetration into structures of interest. Thus, whether liraglutide or semaglutide modifies the activity of POMC and NPY/AgRP neurons in vivo as well as mechanisms required for any changes in cellular activity remains undefined. Methods: In order to resolve this issue, we utilized neuron-specific transgenic mouse models to examine changes in the activity of POMC and NPY/AgRP neurons after injection of either liraglutide or semaglutide (intraperitoneal - I.P. and subcutaneous - S·C.). POMC and NPY/AgRP neurons were targeted for patch-clamp electrophysiology as well as in vivo fiber photometry. Results: We found that liraglutide and semaglutide directly activate and increase excitatory tone to POMC neurons in a time-dependent manner. This increased activity of POMC neurons required GLP-1Rs in POMC neurons as well as a downstream mixed cation channel comprised of TRPC5 subunits. We also observed an indirect upregulation of excitatory input to POMC neurons originating from glutamatergic cells that also required TRPC5 subunits. Conversely, GLP-1Ra's decreased excitatory input to and indirectly inhibited NPY/AgRP neurons through activation of K-ATP and TRPC5 channels in GABAergic neurons. Notably, the temporal activation of POMC and inhibition of NPY/AgRP neuronal activity after liraglutide or semaglutide was injected [either intraperitoneal (I.P.) or subcutaneous (S·C.)] was dependent upon the nutritional state of the animals (fed vs food-deprived). Conclusions: Our results support a mechanism of liraglutide and semaglutide in vivo to activate POMC while inhibiting NPY/AgRP neurons, which depends upon metabolic state and mirrors the pharmacokinetic profile of these compounds in vivo.

Basal forebrain cholinergic neurons (BFCNs) project throughout the cortex to regulate arousal, stimulus salience, plasticity, and learning. Although often treated as a monolithic structure, the basal forebrain features distinct connectivity along its rostrocaudal axis that could impart regional differences in BFCN processing. Here, we performed simultaneous bulk calcium imaging from rostral and caudal BFCNs over a 1-month period of variable reinforcement learning in mice. BFCNs in both regions showed equivalently weak responses to unconditioned visual stimuli and anticipated rewards. Rostral BFCNs in the horizontal limb of the diagonal band were more responsive to reward omission, more accurately classified behavioral outcomes, and more closely tracked fluctuations in pupil-indexed global brain state. Caudal tail BFCNs in globus pallidus and substantia innominata were more responsive to unconditioned auditory stimuli, orofacial movements, aversive reinforcement, and showed robust associative plasticity for punishment-predicting cues. These results identify a functional topography that diversifies cholinergic modulatory signals broadcast to downstream brain regions.

Decisions to act while pursuing goals in the presence of danger must be made quickly but safely. Premature decisions risk injury or death whereas postponing decisions risk goal loss. Here we show how mice resolve these competing demands. Using microstructural behavioral analyses, we identified the spatiotemporal dynamics of approach-avoidance decisions under motivational conflict. Then we used cognitive modelling to show that these dynamics reflect the speeded decision-making mechanisms used by humans and non-human primates, with mice trading off decision speed for safety of choice when danger loomed. Using calcium imaging and functional circuit analyses, we show that this speed-safety trade off occurs because increases in paraventricular thalamus (PVT) activity increase decision caution, thereby increasing approach-avoid decision times in the presence of danger. Our findings demonstrate that a discrete brain circuit involving the PVT and its prefrontal cortical input dynamically adjusts decision caution during motivational conflict, trading off decision speed for decision safety when danger is close. They identify the corticothalamic pathway as central to cognitive control during decision-making under conflict.

The striatum comprises multiple subdivisions and neural circuits that differentially control motor output. The islands of Calleja (IC) contain clusters of densely packed granule cells situated in the ventral striatum, predominantly in the olfactory tubercle (OT). Characterized by expression of the D3 dopamine receptor, the IC are evolutionally conserved, but have undefined functions. Here, we show that optogenetic activation of OT D3 neurons robustly initiates self-grooming in mice while suppressing other ongoing behaviors. Conversely, optogenetic inhibition of these neurons halts ongoing grooming, and genetic ablation reduces spontaneous grooming. Furthermore, OT D3 neurons show increased activity before and during grooming and influence local striatal output via synaptic connections with neighboring OT neurons (primarily spiny projection neurons), whose firing rates display grooming-related modulation. Our study uncovers a new role of the ventral striatum's IC in regulating motor output and has important implications for the neural control of grooming.

Hypothalamic oxytocin (OXT) and arginine-vasopressin (AVP) neurons have been at the center of several physiological and behavioral studies. Advances in viral vector biology and the development of transgenic rodent models have allowed for targeted gene expression to study the functions of specific cell populations and brain circuits. In this study, we compared the efficiency of various adeno-associated viral vectors in these cell populations and demonstrated that none of the widely used promoters were, on their own, effective at driving expression of a down-stream fluorescent protein in OXT or AVP neurons. As anticipated, the OXT promoter could efficiently drive gene expression in OXT neurons and this efficiency is solely attributed to the promoter and not the viral serotype. We also report that a dual virus approach using an OXT promoter driven Cre recombinase significantly improved the efficiency of viral transduction in OXT neurons. Finally, we demonstrate the utility of the OXT promoter for conducting functional studies on OXT neurons by using an OXT specific viral system to record neural activity of OXT neurons in lactating female rats across time. We conclude that extreme caution is needed when employing non-neuron-specific viral approaches/promoters to study neural populations within the paraventricular nucleus of the hypothalamus.

Although fear memory formation is essential for survival and fear-related mental disorders, the neural circuitry and mechanism are incompletely understood. Here, we utilized trace fear conditioning to study the formation of trace fear memory in mice. We identified the entorhinal cortex (EC) as a critical component of sensory signaling to the amygdala. We adopted both loss-of-function and gain-of-function experiments to demonstrate that release of the cholecystokinin (CCK) from the EC is required for trace fear memory formation. We discovered that CCK-positive neurons project from the EC to the lateral nuclei of the amygdala (LA), and inhibition of CCK-dependent signaling in the EC prevented long-term potentiation of the auditory response in the LA and formation of trace fear memory. In summary, high-frequency activation of EC neurons triggers the release of CCK in their projection terminals in the LA, potentiating auditory response in LA neurons. The neural plasticity in the LA leads to trace fear memory formation.

While midbrain dopamine (DA) neuronal circuits are central to motivated behaviors, much remains unknown about our knowledge of how these circuits are modified over time by experience to facilitate selective aspects of experience-dependent plasticity. Most studies of the DA system in drug addiction focus on the role of the mesolimbic DA pathway from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) in facilitating drug-associated reward. In contrast, less is known about how midbrain DA cells and associated circuits contribute to negative affective states including anxiety that emerge during protracted withdrawal from drug administration. Here, we demonstrate the selective role of a midbrain DA projection to the amygdala (VTA DA →Amygdala) for anxiety that develops during protracted withdrawal from cocaine administration but does not participate in cocaine reward or sensitization. Our rabies virus-mediated circuit mapping approach revealed a persistent elevation in spontaneous and task-related activity of GABAergic cells from the bed nucleus of the stria terminals (BNST) and downstream VTA DA →Amygdala cells that could be detected even after a single cocaine exposure. Activity in BNST GABA cells was related to cocaine-induced anxiety but not reward or sensitization, and silencing the projection from these cells to the midbrain was sufficient to prevent the development of anxiety during protracted withdrawal following cocaine administration. We observed that VTA DA →Amygdala cells, but not other midbrain DA cells, were strongly activated after a challenge exposure to cocaine, and found that activity in these cells was necessary for the expression of reinstatement of cocaine place preference. Lastly, the importance of activity in VTA DA →Amygdala cells extends beyond cocaine, as these cells mediate the development of anxiety states triggered by morphine and a predator odor. Our results provide an exemplar for how to identify key circuit substrates that contribute to behavioral adaptations and reveal a critical role for BNST GABA →VTA DA →Amygdala pathway in anxiety states induced by drugs of abuse or natural experiences as well as cocaine-primed reinstatement of conditioned place preference.

A large body of work has aimed to define the precise information encoded by dopaminergic projections innervating the nucleus accumbens (NAc). Prevailing models are based on reward prediction error (RPE) theory, in which dopamine updates associations between rewards and predictive cues by encoding perceived errors between predictions and outcomes. However, RPE cannot describe multiple phenomena to which dopamine is inextricably linked, such as behavior driven by aversive and neutral stimuli. We combined a series of behavioral tasks with direct, subsecond dopamine monitoring in the NAc of mice, machine learning, computational modeling, and optogenetic manipulations to describe behavior and related dopamine release patterns across multiple contingencies reinforced by differentially valenced outcomes. We show that dopamine release only conforms to RPE predictions in a subset of learning scenarios but fits valence-independent perceived saliency encoding across conditions. Here, we provide an extended, comprehensive framework for accumbal dopamine release in behavioral control.

Oxytocin is a neuropeptide important for maternal physiology and childcare, including parturition and milk ejection during nursing1-8. Suckling triggers oxytocin release, but other sensory cues- specifically infant cries- can elevate oxytocin levels in new human mothers9-11 indicating that cries can activate hypothalamic oxytocin neurons. Here we describe a neural circuit routing auditory information about infant vocalizations to mouse oxytocin neurons. We performed in vivo electrophysiological recordings and photometry from identified oxytocin neurons in awake maternal mice presented with pup calls. We found that oxytocin neurons responded to pup vocalizations, but not pure tones, via input from the posterior intralaminar thalamus, and repetitive thalamic stimulation induced lasting disinhibition of oxytocin neurons. This circuit gates central oxytocin release and maternal behavior in response to calls, providing a mechanism for the integration of sensory cues from the offspring in maternal endocrine networks to ensure modulation of brain state for successful parenting.

Locomotor sensitization (LS) is an early behavioral adaptation to addictive drugs, driven by the increase of dopamine in the Nucleus Accumbens (NAc). However, the effect on accumbal population activity remains elusive. Here, we used single-cell calcium imaging in mice to record the activity of dopamine-1-receptor (D1R) and dopamine-2-receptor (D2R) expressing spiny projection neurons (SPNs) during cocaine LS. Acute exposure to cocaine elevated D1R SPN activity and reduced D2R SPN activity, albeit with high variability between neurons. During LS, the number of D1R and D2R neurons responding in opposite directions increased. Moreover, preventing LS by inhibition of the ERK signaling pathway decreased the number of cocaine responsive D1R SPNs, but had little effect on D2R SPNs. These results indicate that accumbal population dichotomy is dynamic and contains a subgroup of D1R SPNs that eventually drives LS. Insights into the drug-related activity dynamics provides a foundation for understanding the circuit-level addiction pathogenesis.

Amygdala abnormalities characterize several psychiatric disorders with prominent social deficits and often emerge during adolescence. The basolateral amygdala (BLA) bidirectionally modulates social behavior and has increased sensitivity during adolescence. We tested how an environmentally-driven social state is regulated by the BLA in adults and adolescent male rats. We found that a high social drive state caused by brief social isolation increases age-specific social behaviors and increased BLA neuronal activity. Chemogenetic inactivation of BLA decreased the effect of high social drive on social engagement. High social drive preferentially enhanced BLA activity during social engagement; however, the effect of social opportunity on BLA activity was greater during adolescence. While this identifies a substrate underlying age differences in social drive, we then determined that high social drive increased BLA NMDA GluN2B expression and sensitivity to antagonism increased with age. Further, the effect of a high social drive state on BLA activity during social engagement was diminished by GluN2B blockade in an age-dependent manner. These results demonstrate the necessity of the BLA for environmentally driven social behavior, its sensitivity to social opportunity, and uncover a maturing role for BLA and its GluN2B receptors in social engagement. SIGNIFICANCE STATEMENT Social engagement during adolescence is a key component of healthy development. Social drive provides the impetus for social engagement and abnormalities underlie social symptoms of depression and anxiety. While adolescence is characterized by transitions in social drive and social environment sensitivity, little is known about the neural basis for these changes. We found that amygdala activity is uniquely sensitive to social environment during adolescence compared with adulthood, and is required for expression of heightened social drive. In addition, the neural substrates shift toward NMDA dependence in adulthood. These results are the first to demonstrate a unique neural signature of higher social drive and begin to uncover the underlying factors that heighten social engagement during adolescence.

Cannabis use disorder (CUD) has doubled in prevalence over the past decade as a nation-wide trend toward legalization allows for increased drug accessibility. As a result, marijuana has become the most commonly used illicit drug in the United States particularly among the adolescent population. This is especially concerning since there is greater risk for the harmful side effects of drug use during this developmental period due to ongoing brain maturation. Increasing evidence indicates that CUD often occurs along with other debilitating conditions including both alcohol use disorder (AUD) and anxiety disorders such post-traumatic stress disorder (PTSD). Additionally, exposure to cannabis, alcohol, and stress can induce alterations in glutamate regulation and homeostasis in the prefrontal cortex (PFC) that may lead to impairments in neuronal functioning and cognition. Therefore, in order to study the relationship between drug exposure and the development of PTSD, these studies utilized rodent models to determine the impact of adolescent exposure to delta-9-tetrahydrocannabinol (THC) and ethanol on responses to fear stimuli during fear conditioning and used calcium imaging to measure glutamate activity in the prelimbic cortex during this behavioral paradigm. The results from these experiments indicate that adolescent exposure to THC and ethanol leads to enhanced sensitivity to fear stimuli both behaviorally and neuronally. Additionally, these effects were attenuated when animals were treated with the glutamatergic modulator N-acetylcysteine (NAC). In summary, these studies support the hypothesis that adolescent exposure to THC and ethanol leads to alterations in fear stimuli processing through glutamatergic reliant modifications in PFC signaling.

The basolateral amygdala (BLA) is obligatory for fear learning. This learning is linked to BLA excitatory projection neurons whose activity is regulated by complex networks of inhibitory interneurons, dominated by parvalbumin (PV)-expressing GABAergic neurons. The roles of these GABAergic interneurons in learning to fear and learning not to fear, activity profiles of these interneurons across the course of fear learning, and whether or how these change across the course of learning all remain poorly understood. Here, we used PV cell-type-specific recording and manipulation approaches in male transgenic PV-Cre rats during pavlovian fear conditioning to address these issues. We show that activity of BLA PV neurons during the moments of aversive reinforcement controls fear learning about aversive events, but activity during moments of nonreinforcement does not control fear extinction learning. Furthermore, we show expectation-modulation of BLA PV neurons during fear learning, with greater activity to an unexpected than expected aversive unconditioned stimulus (US). This expectation-modulation was specifically because of BLA PV neuron sensitivity to aversive prediction error. Finally, we show that BLA PV neuron function in fear learning is conserved across these variations in prediction error. We suggest that aversive prediction-error modulation of PV neurons could enable BLA fear-learning circuits to retain selectivity for specific sensory features of aversive USs despite variations in the strength of US inputs, thereby permitting the rapid updating of fear associations when these sensory features change. SIGNIFICANCE STATEMENT The capacity to learn about sources of danger in the environment is essential for survival. This learning depends on complex microcircuitries of inhibitory interneurons in the basolateral amygdala. Here, we show that parvalbumin-positive GABAergic interneurons in the rat basolateral amygdala are important for fear learning during moments of danger, but not for extinction learning during moments of safety, and that the activity of these neurons is modulated by expectation of danger. This may enable fear-learning circuits to retain selectivity for specific aversive events across variations in expectation, permitting the rapid updating of learning when aversive events change.

Hair cells in the mammalian inner ear sensory epithelia are surrounded by supporting cells which are essential for function of cochlear and vestibular systems. In mice, support cells exhibit spontaneous intracellular Ca 2+ transients in both auditory and vestibular organs during the first postnatal week before the onset of hearing. We recorded long lasting (>200 ms) Ca 2+ transients in cochlear and vestibular support cells in neonatal mice using the genetic calcium indicator GCaMP5. Both cochlear and vestibular support cells exhibited spontaneous intracellular Ca 2+ transients (GCaMP5 ΔF/F), in some cases propagating as waves from the apical (endolymph facing) to the basolateral surface with a speed of ∼25 μm per second, consistent with inositol trisphosphate dependent calcium induced calcium release (CICR). Acetylcholine evoked Ca 2+ transients were observed in both inner border cells in the cochlea and vestibular support cells, with a larger change in GCaMP5 fluorescence in the vestibular support cells. Adenosine triphosphate evoked robust Ca 2+ transients predominantly in the cochlear support cells that included Hensen’s cells, Deiters’ cells, inner hair cells, inner phalangeal cells and inner border cells. A Ca 2+ event initiated in one inner border cells propagated in some instances longitudinally to neighboring inner border cells with an intercellular speed of ∼2 μm per second, and decayed after propagating along ∼3 cells. Similar intercellular propagation was not observed in the radial direction from inner border cell to inner sulcus cells, and was not observed between adjacent vestibular support cells.