Identification of a stress-responsive subregion of the basolateral amygdala in male rats.

The basolateral amygdala (BLA) is reliably activated by psychological stress and hyperactive in conditions of pathological stress or trauma; however, subsets of BLA neurons are also readily activated by rewarding stimuli and can suppress fear and avoidance behaviours. The BLA is highly heterogeneous anatomically, exhibiting continuous molecular and connectivity gradients throughout the entire structure. A critical gap remains in understanding the anatomical specificity of amygdala subregions, circuits, and cell types explicitly activated by acute stress and how they are dynamically activated throughout stimulus exposure. Using a combination of topographical mapping for the activity-responsive protein FOS and fiber photometry to measure calcium transients in real-time, we sought to characterize the spatial and temporal patterns of BLA activation in response to a range of novel stressors (shock, swim, restraint, predator odour) and non-aversive, but novel stimuli (crackers, citral odour). We report four main findings: (1) the BLA exhibits clear spatial activation gradients in response to novel stimuli throughout the medial-lateral and dorsal-ventral axes, with aversive stimuli strongly biasing activation towards medial aspects of the BLA; (2) novel stimuli elicit distinct temporal activation patterns, with stressful stimuli exhibiting particularly enhanced or prolonged temporal activation patterns; (3) changes in BLA activity are associated with changes in behavioural state; and (4) norepinephrine enhances stress-induced activation of BLA neurons via the ß-noradrenergic receptor. Moving forward, it will be imperative to combine our understanding of activation gradients with molecular and circuit-specificity.

Dynorphin modulates motivation through a pallido-amygdala cholinergic circuit.

The endogenous opioid peptide dynorphin and its receptor κ-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.

Analgesic properties of next generation modulators of endocannabinoid signaling: leveraging modern tools for the development of novel therapeutics.

Targeting the endocannabinoid (eCB) signaling system for pain relief is an important treatment option that is only now beginning to be mechanistically explored. In this review, we focus on two recently appreciated cannabinoid-based targeting strategies, treatments with cannabidiol (CBD) and a/b-hydrolase domain containing 6 (ABHD6) inhibitors, which have the exciting potential to produce pain relief through distinct mechanisms of action (MOA) and without intoxication. We review evidence on plant-derived cannabinoids for pain, with an emphasis on CBD and its multiple molecular targets expressed in pain pathways. We also discuss the function of eCB signaling in regulating pain responses and the therapeutic promises of inhibitors targeting ABHD6, a 2-arachidonoylglycerol (2-AG) hydrolyzing enzyme. Finally, we discuss how the novel cannabinoid biosensor, GRABeCB2.0, may be leveraged to enable the discovery of targets modulated by cannabinoids at a circuit-specific level. Significance Statement Cannabis has been used by humans as an effective medicine for millennia, including for pain management. Recent evidence emphasizes the therapeutic potential of compounds that modulate endocannabinoid signaling. Specifically, cannabidiol and inhibitors of the enzyme ABHD6 represent promising strategies to achieve pain relief by modulating endocannabinoid signaling in pain pathways via distinct, non-intoxicating, mechanisms of action.

Noradrenergic tuning of arousal is coupled to coordinated movements.

Matching arousal level to the motor activity of an animal is important for efficiently allocating cognitive resources and metabolic supply in response to behavioral demands, but how the brain coordinates changes in arousal and wakefulness in response to motor activity remains an unclear phenomenon. We hypothesized that the locus coeruleus (LC), as the primary source of cortical norepinephrine (NE) and promoter of cortical and sympathetic arousal, is well-positioned to mediate movement-arousal coupling. Here, using a combination of physiological recordings, fiber photometry, optogenetics, and behavioral tracking, we show that the LCNE activation is tightly coupled to the return of organized movements during waking from an anesthetized state. Moreover, in an awake animal, movement initiations are coupled to LCNE activation, while movement arrests, to LCNE deactivation. We also report that LCNE activity covaries with the depth of anesthesia and that LCNE photoactivation leads to sympathetic activation, consistent with its role in mediating increased arousal. Together, these studies reveal a more nuanced, modulatory role that LCNE plays in coordinating movement and arousal.

Unlocking opioid neuropeptide dynamics with genetically encoded biosensors.

Neuropeptides are ubiquitous in the nervous system. Research into neuropeptides has been limited by a lack of experimental tools that allow for the precise dissection of their complex and diverse dynamics in a circuit-specific manner. Opioid peptides modulate pain, reward and aversion and as such have high clinical relevance. To illuminate the spatiotemporal dynamics of endogenous opioid signaling in the brain, we developed a class of genetically encoded fluorescence sensors based on kappa, delta and mu opioid receptors: κLight, δLight and µLight, respectively. We characterized the pharmacological profiles of these sensors in mammalian cells and in dissociated neurons. We used κLight to identify electrical stimulation parameters that trigger endogenous opioid release and the spatiotemporal scale of dynorphin volume transmission in brain slices. Using in vivo fiber photometry in mice, we demonstrated the utility of these sensors in detecting optogenetically driven opioid release and observed differential opioid release dynamics in response to fearful and rewarding conditions.

Endogenous opioids facilitate stress-induced binge eating via an insular cortex-claustrum pathway.

Stress has been shown to promote the development and persistence of binge eating behaviors. However, the neural circuit mechanisms for stress-induced binge-eating behaviors are largely unreported. The endogenous dynorphin (dyn)/kappa opioid receptor (KOR) opioid neuropeptide system has been well established to be a crucial mediator of the anhedonic component of stress. Here, we aimed to dissect the basis of dynorphinergic control of stress-induced binge-like eating behavior. We first established a mouse behavioral model for stress-induced binge-like eating behaviors. We found that mice exposed to stress increased their food intake of familiar palatable food (high fat, high sugar, HPD) compared to non-stressed mice. Following a brain-wide analysis, we isolated robust cFos-positive cells in the Claustrum (CLA), a subcortical structure with highly abundant KOR expression, following stress-induced binge-eating behavior. We report that KOR signaling in CLA is necessary for this elevated stress-induced binge eating behavior using local pharmacology and local deletion of KOR. In vivo calcium recordings using fiber photometry revealed a disinhibition circuit structure in the CLA during the initiation of HPD feeding bouts. We further established the dynamics of endogenous dynorphinergic control of this behavior using a genetically encoded dynorphin biosensor, Klight. Combined with 1-photon single-cell calcium imaging, we report significant heterogeneity with the CLA population during stress-induced binge eating and such behavior attenuates local dynorphin tone. Furthermore, we isolate the anterior Insular cortex (aIC) as the potential source of endogenous dynorphin afferents in the CLA. By characterizing neural circuits and peptidergic mechanisms within the CLA, we uncover a pathway that implicates endogenous opioid regulation stress-induced binge eating.

Development of a genetically encoded sensor for probing endogenous nociceptin opioid peptide release.

Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fibre photometry enabled direct recording of NOPLight binding to exogenous N/OFQ receptor ligands, as well as detection of endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA) during natural behaviors and chemogenetic activation of PNOC neurons. In summary, we show here that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely behaving animals.

Glutamatergic supramammillary nucleus neurons respond to threatening stressors and promote active coping.

Threat-response neural circuits are conserved across species and play roles in normal behavior and psychiatric diseases. Maladaptive changes in these neural circuits contribute to stress, mood, and anxiety disorders. Active coping in response to stressors is a psychosocial factor associated with resilience against stress-induced mood and anxiety disorders. The neural circuitry underlying active coping is poorly understood, but the functioning of these circuits could be key for overcoming anxiety and related disorders. The supramammillary nucleus (SuM) has been suggested to be engaged by threat. SuM has many projections and a poorly understood diversity of neural populations. In studies using mice, we identified a unique population of glutamatergic SuM neurons (SuMVGLUT2+::POA) based on projection to the preoptic area of the hypothalamus (POA) and found SuMVGLUT2+::POA neurons have extensive arborizations. SuMVGLUT2+::POA neurons project to brain areas that mediate features of the stress and threat responses including the paraventricular nucleus thalamus (PVT), periaqueductal gray (PAG), and habenula (Hb). Thus, SuMVGLUT2+::POA neurons are positioned as a hub, connecting to areas implicated in regulating stress responses. Here we report SuMVGLUT2+::POA neurons are recruited by diverse threatening stressors, and recruitment correlated with active coping behaviors. We found that selective photoactivation of the SuMVGLUT2+::POA population drove aversion but not anxiety like behaviors. Activation of SuMVGLUT2+::POA neurons in the absence of acute stressors evoked active coping like behaviors and drove instrumental behavior. Also, activation of SuMVGLUT2+::POA neurons was sufficient to convert passive coping strategies to active behaviors during acute stress. In contrast, we found activation of GABAergic (VGAT+) SuM neurons (SuMVGAT+) neurons did not alter drive aversion or active coping, but termination of photostimulation was followed by increased mobility in the forced swim test. These findings establish a new node in stress response circuitry that has projections to many brain areas and evokes flexible active coping behaviors.

A bistable inhibitory optoGPCR for multiplexed optogenetic control of neural circuits.

Information is transmitted between brain regions through the release of neurotransmitters from long-range projecting axons. Understanding how the activity of such long-range connections contributes to behavior requires efficient methods for reversibly manipulating their function. Chemogenetic and optogenetic tools, acting through endogenous G-protein-coupled receptor pathways, can be used to modulate synaptic transmission, but existing tools are limited in sensitivity, spatiotemporal precision or spectral multiplexing capabilities. Here we systematically evaluated multiple bistable opsins for optogenetic applications and found that the Platynereis dumerilii ciliary opsin (PdCO) is an efficient, versatile, light-activated bistable G-protein-coupled receptor that can suppress synaptic transmission in mammalian neurons with high temporal precision in vivo. PdCO has useful biophysical properties that enable spectral multiplexing with other optogenetic actuators and reporters. We demonstrate that PdCO can be used to conduct reversible loss-of-function experiments in long-range projections of behaving animals, thereby enabling detailed synapse-specific functional circuit mapping.

Developmental and adult striatal patterning of nociceptin ligand marks striosomal population with direct dopamine projections.

Circuit influences on the midbrain dopamine system are crucial to adaptive behavior and cognition. Recent developments in the study of neuropeptide systems have enabled high-resolution investigations of the intersection of neuromodulatory signals with basal ganglia circuitry, identifying the nociceptin/orphanin FQ (N/OFQ) endogenous opioid peptide system as a prospective regulator of striatal dopamine signaling. Using a prepronociceptin-Cre reporter mouse line, we characterized highly selective striosomal patterning of Pnoc mRNA expression in mouse dorsal striatum, reflecting early developmental expression of Pnoc . In the ventral striatum, Pnoc expression was was clustered across the nucleus accumbens core and medial shell, including in adult striatum. We found that Pnoc tdTomato reporter cells largely comprise a population of dopamine receptor D1 ( Drd1 ) expressing medium spiny projection neurons localized in dorsal striosomes, known to be unique among striatal projections neurons for their direct innervation of midbrain dopamine neurons. These findings provide new understanding of the intersection of the N/OFQ system among basal ganglia circuits with particular implications for developmental regulation or wiring of striatal-nigral circuits.

An integrated microfluidic and fluorescence platform for probing in vivo neuropharmacology.

Neurotechnologies and genetic tools for dissecting neural circuit functions have advanced rapidly over the past decade, although the development of complementary pharmacological method-ologies has comparatively lagged. Understanding the precise pharmacological mechanisms of neuroactive compounds is critical for advancing basic neurobiology and neuropharmacology, as well as for developing more effective treatments for neurological and neuropsychiatric disorders. However, integrating modern tools for assessing neural activity in large-scale neural networks with spatially localized drug delivery remains a major challenge. Here, we present a dual microfluidic-photometry platform that enables simultaneous intracranial drug delivery with neural dynamics monitoring in the rodent brain. The integrated platform combines a wireless, battery-free, miniaturized fluidic microsystem with optical probes, allowing for spatially and temporally specific drug delivery while recording activity-dependent fluorescence using genetically encoded calcium indicators (GECIs), neurotransmitter sensors GRAB NE and GRAB DA , and neuropeptide sensors. We demonstrate the performance this platform for investigating neuropharmacological mechanisms in vivo and characterize its efficacy in probing precise mechanistic actions of neuroactive compounds across several rapidly evolving neuroscience domains.

P2X7 receptor-dependent increase in endocannabinoid 2-arachidonoyl glycerol production by neuronal cells in culture: Dynamics and mechanism.

BACKGROUND AND PURPOSE: Neurotransmission and neuroinflammation are controlled by local increases in both extracellular ATP and the endocannabinoid 2-arachidonoyl glycerol (2-AG). While it is known that extracellular ATP stimulates 2-AG production in cells in culture, the dynamics and molecular mechanisms that underlie this response remain poorly understood. Detection of real-time changes in eCB levels with the genetically encoded sensor, GRABeCB2.0, can address this shortfall. EXPERIMENTAL APPROACH: 2-AG and arachidonoylethanolamide (AEA) levels in Neuro2a (N2a) cells were measured by LC-MS, and GRABeCB2.0 fluorescence changes were detected using live-cell confocal microscopy and a 96-well fluorescence plate reader. KEY RESULTS: 2-AG and AEA increased GRABeCB2.0 fluorescence in N2a cells with EC50 values of 81 and 58 nM, respectively; both responses were reduced by the cannabinoid receptor type 1 (CB1R) antagonist SR141617 and absent in cells expressing the mutant-GRABeCB2.0. ATP increased only 2-AG levels in N2a cells, as measured by LC-MS, and induced a transient increase in the GRABeCB2.0 signal within minutes primarily via activation of P2X7 receptors (P2X7R). This response was dependent on diacylglycerol lipase ß activity, partially dependent on extracellular calcium and phospholipase C activity, but not controlled by the 2-AG hydrolysing enzyme, α/ß-hydrolase domain containing 6 (ABHD6). CONCLUSIONS AND IMPLICATIONS: Considering that P2X7R activation increases 2-AG levels within minutes, our results show how these molecular components are mechanistically linked. The specific molecular components in these signalling systems represent potential therapeutic targets for the treatment of neurological diseases, such as chronic pain, that involve dysregulated neurotransmission and neuroinflammation.

A preclinical model of THC edibles that produces high-dose cannabimimetic responses.

No preclinical experimental approach enables the study of voluntary oral consumption of high-concentration Δ9-tetrahydrocannabinol (THC) and its intoxicating effects, mainly owing to the aversive response of rodents to THC that limits intake. Here, we developed a palatable THC formulation and an optimized access paradigm in mice to drive voluntary consumption. THC was formulated in chocolate gelatin (THC-E-gel). Adult male and female mice were allowed ad libitum access for 1 and 2 hr. Cannabimimetic responses (hypolocomotion, analgesia, and hypothermia) were measured following access. Levels of THC and its metabolites were measured in blood and brain tissue. Acute acoustic startle responses were measured to investigate THC-induced psychotomimetic behavior. When allowed access for 2 hr to THC-E-gel on the second day of a 3-day exposure paradigm, adult mice consumed up to ≈30 mg/kg over 2 hr, which resulted in robust cannabimimetic behavioral responses (hypolocomotion, analgesia, and hypothermia). Consumption of the same gelatin decreased on the following third day of exposure. Pharmacokinetic analysis shows that THC-E-gel consumption led to parallel accumulation of THC and its psychoactive metabolite, 11-OH-THC, in the brain, a profile that contrasts with the known rapid decline in brain 11-OH-THC levels following THC intraperitoneal (i.p.) injections. THC-E-gel consumption increased the acoustic startle response in males but not in females, demonstrating a sex-dependent effect of consumption. Thus, while voluntary consumption of THC-E-gel triggered equivalent cannabimimetic responses in male and female mice, it potentiated acoustic startle responses preferentially in males. We built a dose-prediction model that included cannabimimetic behavioral responses elicited by i.p. versus THC-E-gel to test the accuracy and generalizability of this experimental approach and found that it closely predicted the measured acoustic startle results in males and females. In summary, THC-E-gel offers a robust preclinical experimental approach to study cannabimimetic responses triggered by voluntary consumption in mice, including sex-dependent psychotomimetic responses.

Holographic stimulation of opposing amygdala ensembles bidirectionally modulates valence-specific behavior via mutual inhibition.

The basolateral amygdala (BLA) is an evolutionarily conserved brain region, well known for valence processing. Despite this central role, the relationship between activity of BLA neuronal ensembles in response to appetitive and aversive stimuli and the subsequent expression of valence-specific behavior has remained elusive. Here, we leverage two-photon calcium imaging combined with single-cell holographic photostimulation through an endoscopic lens to demonstrate a direct causal role for opposing ensembles of BLA neurons in the control of oppositely valenced behavior in mice. We report that targeted photostimulation of either appetitive or aversive BLA ensembles results in mutual inhibition and shifts behavioral responses to promote consumption of an aversive tastant or reduce consumption of an appetitive tastant, respectively. Here, we identify that neuronal encoding of valence in the BLA is graded and relies on the relative proportion of individual BLA neurons recruited in a stable appetitive or quinine ensemble.

Targeting Nociceptin/Orphanin FQ receptor to rescue cognitive symptoms in a mouse neuroendocrine model of chronic stress.

Chronic stress causes cognitive deficits, such as impairments in episodic-like hippocampus-dependent memory. Stress regulates an opioid-related neuropeptide named Nociceptin/Orphanin FQ (N/OFQ), the ligand of the G protein-coupled receptor NOP. Since this peptide has deleterious effects on memory, we hypothesized that the N/OFQ system could be a mediator of the negative effects of stress on memory. Chronic stress was mimicked by chronic exposure to corticosterone (CORT). The NOP receptor was either acutely blocked using selective antagonists, or knocked-down specifically in the hippocampus using genetic tools. Long-term memory was assessed in the object recognition (OR) and object location (OL) paradigms. Acute injection of NOP antagonists before learning had a negative impact on memory in naive mice whereas it restored memory performances in the chronic stress model. This rescue was associated with a normalization of neuronal cell activity in the CA3 part of the hippocampus. Chronic CORT induced an upregulation of the N/OFQ precursor in the hippocampus. Knock-down of the NOP receptor in the CA3/Dentate Gyrus region prevented memory deficits in the CORT model. These data demonstrate that blocking the N/OFQ system can be beneficial for long-term memory in a neuroendocrine model of chronic stress. We therefore suggest that NOP antagonists could be useful for the treatment of memory deficits in stress-related disorders.

Pharmacological Characterization of the Endocannabinoid Sensor GRABeCB2.0.

Introduction: The endocannabinoids (eCBs), 2-arachidonoylglycerol (2-AG) and arachidonoyl ethanolamine (AEA), are produced by separate enzymatic pathways, activate cannabinoid (CB) receptors with distinct pharmacological profiles, 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-CBs 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-CB action. Materials and Methods: GRABeCB2.0 was expressed in cultured HEK293 cells. Live cell confocal microscopy and high-throughput fluorescent signal measurements. Results: 2-AG increased GRABeCB2.0 fluorescent signal (EC50=85 nM), and the cannabinoid 1 receptor (CB1R) antagonist, SR141716 (SR1), decreased GRABeCB2.0 signal (IC50=3.3 nM), responses that mirror their known potencies at the CB1R. GRABeCB2.0 fluorescent signal also increased in response to AEA (EC50=815 nM), the eCB analogues 2-linoleoylglycerol and 2-oleoylglycerol (EC50=632 and 868 nM, respectively), Δ9-tetrahydrocannabinol (Δ9-THC), and Δ8-THC (EC50=1.6 and 2.0 µM, 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=9.7 nM). Conclusions: 2-AG and SR1 modulate the GRABeCB2.0 fluorescent signal with EC50 values that mirror their potencies at CB1R, whereas AEA, eCB analogues, THC, and CP increase GRABeCB2.0 fluorescent signal with EC50 values 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.

A cluster of neuropeptide S neurons regulates breathing and arousal.

Neuropeptide S (NPS) is a highly conserved peptide found in all tetrapods that functions in the brain to promote heightened arousal; however, the subpopulations mediating these phenomena remain unknown. We generated mice expressing Cre recombinase from the Nps gene locus (NpsCre) and examined populations of NPS+ neurons in the lateral parabrachial area (LPBA), the peri-locus coeruleus (peri-LC) region of the pons, and the dorsomedial thalamus (DMT). We performed brain-wide mapping of input and output regions of NPS+ clusters and characterized expression patterns of the NPS receptor 1 (NPSR1). While the activity of all three NPS+ subpopulations tracked with vigilance state, only NPS+ neurons of the LPBA exhibited both increased activity prior to wakefulness and decreased activity during REM sleep, similar to the behavioral phenotype observed upon NPSR1 activation. Accordingly, we found that activation of the LPBA but not the peri-LC NPS+ neurons increased wake and reduced REM sleep. Furthermore, given the extended role of the LPBA in respiration and the link between behavioral arousal and breathing rate, we demonstrated that the LPBA but not the peri-LC NPS+ neuronal activation increased respiratory rate. Together, our data suggest that NPS+ neurons of the LPBA represent an unexplored subpopulation regulating breathing, and they are sufficient to recapitulate the sleep/wake phenotypes observed with broad NPS system activation.

Activity-dependent constraints on catecholamine signaling.

Catecholamine signaling is thought to modulate cognition in an inverted-U relationship, but the mechanisms are unclear. We measured norepinephrine and dopamine release, postsynaptic calcium responses, and interactions between tonic and phasic firing modes under various stimuli and conditions. High tonic activity in vivo depleted catecholamine stores, desensitized postsynaptic responses, and decreased phasic transmission. Together, these findings provide a more complete understanding of the inverted-U relationship, offering insights into psychiatric disorders and neurodegenerative diseases with impaired catecholamine signaling.

High-fat diet feeding disrupts the coupling of thermoregulation to energy homeostasis.

OBJECTIVE: Preserving core body temperature across a wide range of ambient temperatures requires adaptive changes of thermogenesis that must be offset by corresponding changes of energy intake if body fat stores are also to be preserved. Among neurons implicated in the integration of thermoregulation with energy homeostasis are those that express both neuropeptide Y (NPY) and agouti-related protein (AgRP) (referred to herein as AgRP neurons). Specifically, cold-induced activation of AgRP neurons was recently shown to be required for cold exposure to increase food intake in mice. Here, we investigated how consuming a high-fat diet (HFD) impacts various adaptive responses to cold exposure as well as the responsiveness of AgRP neurons to cold. METHODS: To test this, we used immunohistochemistry, in vivo fiber photometry and indirect calorimetry for continuous measures of core temperature, energy expenditure, and energy intake in both chow- and HFD-fed mice housed at different ambient temperatures. RESULTS: We show that while both core temperature and the thermogenic response to cold are maintained normally in HFD-fed mice, the increase of energy intake needed to preserve body fat stores is blunted, resulting in weight loss. Using both immunohistochemistry and in vivo fiber photometry, we show that although cold-induced AgRP neuron activation is detected regardless of diet, the number of cold-responsive neurons appears to be blunted in HFD-fed mice. CONCLUSIONS: We conclude that HFD-feeding disrupts the integration of systems governing thermoregulation and energy homeostasis that protect body fat mass during cold exposure.

Long-Term In Vivo Molecular Monitoring Using Aptamer-Graphene Microtransistors.

Long-term, real-time molecular monitoring in complex biological environments is critical for our ability to understand, prevent, diagnose, and manage human diseases. Aptamer-based electrochemical biosensors possess the promise due to their generalizability and a high degree of selectivity. Nevertheless, the operation of existing aptamer-based biosensors in vivo is limited to a few hours. Here, we report a first-generation long-term in vivo molecular monitoring platform, named aptamer-graphene microtransistors (AGMs). The AGM incorporates a layer of pyrene-(polyethylene glycol)5-alcohol and DNase inhibitor-doped polyacrylamide hydrogel coating to reduce biofouling and aptamer degradation. As a demonstration of function and generalizability, the AGM achieves the detection of biomolecules such as dopamine and serotonin in undiluted whole blood at 37 °C for 11 days. Furthermore, the AGM successfully captures optically evoked dopamine release in vivo in mice for over one week and demonstrates the capability to monitor behaviorally-induced endogenous dopamine release even after eight days of implantation in freely moving mice. The results reported in this work establish the potential for chronic aptamer-based molecular monitoring platforms, and thus serve as a new benchmark for molecular monitoring using aptamer-based technology.