Canonical parasympathetic innervation of tissues/organs is accomplished by two in-series neurons, often called pre- and postganglionic neurons. Such two-neuron in-series organization has recently been challenged by claiming direct vagal preganglionic innervation of Brunner's glands (BGs) in the duodenal bulb, as well as the spleen, and the liver. To resolve this controversial issue, we labeled vagal preganglionic neurons in the dorsal motor nucleus of the vagus (DMV) by injecting a Cre-dependent AAV construct to express tdTomato in ChAT-IRES-Cre transgenic mice and analyzed the distribution of labeled fibers in the duodenal bulb. We found that labeled vagal preganglionic fibers terminate exclusively in myenteric ganglia but not in or between BGs. This lack of direct BG innervation is unlikely to result from incomplete labeling of vagal preganglionic neurons, as the entire extent of the DMV was well covered by bilateral injections, and there were abundant vagal axons and terminals in the gastric myenteric plexus and other vagal targets. BGs were innervated by a consistent supply of cholinergic (VAChT-positive) and sparsely by adrenergic (TH-positive) nerve fibers. There was also a moderate supply of vagal afferents anterogradely traced from the nodose ganglia and CGRP-positive fibers. We conclude that BGs in the mouse are not directly innervated by vagal preganglionic neurons of DMV origin but receive cholinergic innervation from myenteric plexus neurons serving as postganglionic neurons. Further experiments will be necessary to clarify the issue for other vagal targets.

AgRP neurons cause hunger, the drive to seek and consume food. Their activation by fasting is key for survival and is thought to be triggered by feedback when energy stores are low. However, we know that environmental cues can also regulate AgRP neurons since cues that predict future food intake rapidly inhibit AgRP neurons, but is the converse true: can the prediction of future fasting rapidly activate AgRP neurons? Here, we show in mice that such rapid fasting activation of AgRP neurons does occur. This rapid activation is driven by excitatory input from paraventricular hypothalamic (PVH) neurons expressing Sim2, which are bidirectionally sensitive to predictions of future energy state. Thus, cognitively processed contextual information conveyed by PVHSim2 neurons strongly activates AgRP neurons. Lastly, chronic silencing of PVHSim2 neurons causes persistent hypophagia. This PVHSim2-to-AgRP-neuron circuit, by anticipating and preventing negative energy balance, provides an important new dimension of hunger regulation.

The canonical autonomic nervous system pathway to peripheral targets consists of two neurons in series, i.e., preganglionic and postganglionic. However, several recent reports claim a direct innervation of some sub-diaphragmatic organs by neurons of the vagal dorsal motor nucleus without intercalated postganglionic neurons. The aim of this minireview is to summarize these studies e.g. on direct vagal preganglionic innervation of Brunner's glands, liver and spleen and to critically discuss their unorthodox findings and discrepancies with previous literature. We conclude that rigorous re-evaluation by complementary state-of-the-art strategies is obligatory.

Neuropeptides have important roles in neural plasticity, spiking and behaviour1. Yet, many fundamental questions remain regarding their spatiotemporal transmission, integration and functions in the awake brain. Here we examined how MC4R-expressing neurons in the paraventricular nucleus of the hypothalamus (PVHMC4R) integrate neuropeptide signals to modulate feeding-related fast synaptic transmission and titrate the transition to satiety2-6. We show that hunger-promoting AgRP axons release the neuropeptide NPY to decrease the second messenger cAMP in PVHMC4R neurons, while satiety-promoting POMC axons release the neuropeptide αMSH to increase cAMP. Each release event is all-or-none, stochastic and can impact multiple neurons within an approximately 100-µm-diameter region. After release, NPY and αMSH peptides compete to control cAMP-the amplitude and persistence of NPY signalling is blunted by high αMSH in the fed state, while αMSH signalling is blunted by high NPY in the fasted state. Feeding resolves this competition by simultaneously elevating αMSH release and suppressing NPY release7,8, thereby sustaining elevated cAMP in PVHMC4R neurons throughout a meal. In turn, elevated cAMP facilitates potentiation of feeding-related excitatory inputs with each bite to gradually promote satiation across many minutes. Our findings highlight biochemical modes of peptide signal integration and information accumulation to guide behavioural state transitions.

Reward-seeking involves the engagement and computation of multiple physiological and motivational parameters. The lateral hypothalamus (LH) is a necessary node in the circuits that control food-seeking and motivation. One group of cells that plays an important yet incompletely understood role in these processes are the orexin/hypocretin (OX/HT) neurons. OX/HT cells are located exclusively within the LH and are implicated in feeding, arousal, and reward-seeking behavior. Importantly, the role of OX/HT neurons in consummatory versus reward-seeking actions is not fully defined, nor are the circuits that control the activity of these neurons under different behavioral states. Here we show that OX/HT neurons respond in real time to food presentation and that this response is modulated by differences in metabolic state. We observed increased OX/HT neuron activity on approach to food, but this activity trended towards pre-approach levels by the start of the consummatory phase. Next, we studied ventrostriatopallidal (VSP) inputs to the OX/HT neurons. Using optogenetics and cell type-specific electrophysiology, we found that ventral pallidum inputs onto OX/HT neurons exert strong inhibitory (and weak excitatory) effects whereas the lateral nucleus accumbens shell provides weaker direct inhibitory connections with OX/HT neurons. These findings reveal that the activity of OX/HT neurons is strongly modulated by metabolic and hedonic state. Further, OX/HT neurons is primarily associated with food approach and that the effect of VSP-terminal output is to suppress OX/HT activity.

When food is freely available, eating occurs without energy deficit. While agouti-related peptide (AgRP) neurons are likely involved, their activation is thought to require negative energy balance. To investigate this, we implemented long-term, continuous in vivo fiber-photometry recordings in mice. We discovered new forms of AgRP neuron regulation, including fast pre-ingestive decreases in activity and unexpectedly rapid activation by fasting. Furthermore, AgRP neuron activity has a circadian rhythm that peaks concurrent with the daily feeding onset. Importantly, this rhythm persists when nutrition is provided via constant-rate gastric infusions. Hence, it is not secondary to a circadian feeding rhythm. The AgRP neuron rhythm is driven by the circadian clock, the suprachiasmatic nucleus (SCN), as SCN ablation abolishes the circadian rhythm in AgRP neuron activity and feeding. The SCN activates AgRP neurons via excitatory afferents from thyrotrophin-releasing hormone-expressing neurons in the dorsomedial hypothalamus (DMHTrh neurons) to drive daily feeding rhythms.

Astrocytes are multi-functional glial cells in the central nervous system that play critical roles in modulation of metabolism, extracellular ion and neurotransmitter levels, and synaptic plasticity. Astrocyte-derived signaling molecules mediate many of these modulatory functions of astrocytes, including vesicular release of ATP. In the present study, we used a unique genetic mouse model to investigate the functional significance of astrocytic exocytosis of ATP. Using primary cultured astrocytes, we show that loss of vesicular nucleotide transporter (Vnut), a primary transporter responsible for loading cytosolic ATP into the secretory vesicles, dramatically reduces ATP loading into secretory lysosomes and ATP release, without any change in the molecular machinery of exocytosis or total intracellular ATP content. Deletion of astrocytic Vnut in adult mice leads to increased anxiety, depressive-like behaviors, and decreased motivation for reward, especially in females, without significant impact on food intake, systemic glucose metabolism, cognition, or sociability. These behavioral alterations are associated with significant decreases in the basal extracellular dopamine levels in the nucleus accumbens. Likewise, ex vivo brain slices from these mice show a strong trend toward a reduction in evoked dopamine release in the nucleus accumbens. Mechanistically, the reduced dopamine signaling we observed is likely due to an increased expression of monoamine oxidases. Together, these data demonstrate a key modulatory role of astrocytic exocytosis of ATP in anxiety, depressive-like behavior, and motivation for reward, by regulating the mesolimbic dopamine circuitry.

Fasting initiates a multitude of adaptations to allow survival. Activation of the hypothalamic-pituitary-adrenal (HPA) axis and subsequent release of glucocorticoid hormones is a key response that mobilizes fuel stores to meet energy demands. Despite the importance of the HPA axis response, the neural mechanisms that drive its activation during energy deficit are unknown. Here, we show that hypothalamic agouti-related peptide (AgRP)-expressing neurons trigger and are essential for fasting-induced HPA axis activation. AgRP neurons stimulate activity of the HPA axis via projections to the paraventricular hypothalamus (PVH), where, in a mechanism not previously described for AgRP neurons, they presynaptically inhibit the terminals of tonically active GABAergic afferents from the bed nucleus of the stria terminalis (BNST) that otherwise restrain activity of corticotrophin-releasing hormone (CRH)-expressing neurons. This disinhibition of PVH-Crh neurons requires GABA (γ-aminobutyric acid)/GABA-B receptor signaling and potently activates the HPA axis. Importantly, stimulation of the HPA axis by AgRP neurons is independent of their induction of hunger, showing that these canonical ‘hunger neurons’ drive multiple parallel adaptations to the fasted state. Together our findings identify the neural basis for fasting-induced HPA axis activation and uncover a unique means by which AgRP neurons activate downstream neurons – via presynaptic inhibition of GABAergic afferents. Given the potency of this disinhibition of tonically active BNST afferents, other activators of the HPA axis such as psychological stress may work via a similar mechanism. NIH R01DK096010, NIH R00HL144923, AHA 935362 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Combining the use of ex vivo and in vivo optogenetics, viral tracing, electrophysiology and behavioral testing, we show that the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) gates anxiety-controlling circuits by differentially affecting synaptic efficacy at projections from the basolateral amygdala (BLA) to two different subdivisions of the dorsal subdivision of the bed nucleus of the stria terminalis (BNST), modifying the signal flow in BLA-ovBNST-adBNST circuits in such a way that adBNST is inhibited. Inhibition of adBNST is translated into the reduced firing probability of adBNST neurons during afferent activation, explaining the anxiety-triggering actions of PACAP in BNST, as inhibition of adBNST is anxiogenic. Our results reveal how innate, fear-related behavioral mechanisms may be controlled by neuropeptides, PACAP specifically, at the level of underlying neural circuits by inducing long-lasting plastic changes in functional interactions between their different structural components.

In addition to its motor functions, the cerebellum is involved in emotional regulation, anxiety and affect. We found that suppressing the firing of cerebellar Purkinje cells (PCs) rapidly excites forebrain areas that contribute to such functions (including the amygdala, basal forebrain and septum), but that the classic cerebellar outputs, the deep cerebellar nuclei, do not directly project there. We show that PCs directly inhibit parabrachial nuclei (PBN) neurons that project to numerous forebrain regions. Suppressing the PC-PBN pathway influences many regions in the forebrain and is aversive. Molecular profiling shows that PCs directly inhibit numerous types of PBN neurons that control diverse behaviors that are not involved in motor control. Therefore, the PC-PBN pathway allows the cerebellum to directly regulate activity in the forebrain, and may be an important substrate for cerebellar disorders arising from damage to the posterior vermis.

Obesity is increasing at an alarming rate. The effectiveness of currently available strategies for the treatment of obesity (including pharmacologic, surgical, and behavioral interventions) is limited. Understanding the neurobiology of appetite and the important drivers of energy intake (EI) can lead to the development of more effective strategies for the prevention and treatment of obesity. Appetite regulation is complex and is influenced by genetic, social, and environmental factors. It is intricately regulated by a complex interplay of endocrine, gastrointestinal, and neural systems. Hormonal and neural signals generated in response to the energy state of the organism and the quality of food eaten are communicated by paracrine, endocrine, and gastrointestinal signals to the nervous system. The central nervous system integrates homeostatic and hedonic signals to regulate appetite. Although there has been an enormous amount of research over many decades regarding the regulation of EI and body weight, research is only now yielding potentially effective treatment strategies for obesity. The purpose of this article is to summarize the key findings presented in June 2022 at the 23rd annual Harvard Nutrition Obesity Symposium entitled “The Neurobiology of Eating Behavior in Obesity: Mechanisms and Therapeutic Targets.” Findings presented at the symposium, sponsored by NIH P30 Nutrition Obesity Research Center at Harvard, enhance our current understanding of appetite biology, including innovative techniques used to assess and systematically manipulate critical hedonic processes, which will shape future research and the development of therapeutics for obesity prevention and treatment.

We investigated how transmission of hunger- and satiety-promoting neuropeptides, NPY and αMSH, is integrated at the level of intracellular signaling to control feeding. Receptors for these peptides use the second messenger cAMP, but the messenger's spatiotemporal dynamics and role in energy balance are controversial. We show that AgRP axon stimulation in the paraventricular hypothalamus evokes probabilistic and spatially restricted NPY release that triggers stochastic cAMP decrements in downstream MC4R-expressing neurons (PVH MC4R ). Meanwhile, POMC axon stimulation triggers stochastic, αMSH-dependent cAMP increments. NPY and αMSH competitively control cAMP, as reflected by hunger-state-dependent differences in the amplitude and persistence of cAMP transients evoked by each peptide. During feeding bouts, elevated αMSH release and suppressed NPY release cooperatively sustain elevated cAMP in PVH MC4R neurons, thereby potentiating feeding-related excitatory inputs and promoting satiation across minutes. Our findings highlight how state-dependent integration of opposing, quantal peptidergic events by a common biochemical target calibrates energy intake.

Restricting caloric intake effectively reduces body weight, but most dieters fail long-term adherence to caloric deficit and eventually regain lost weight. Hypothalamic circuits that control hunger drive critically determine body weight; yet, how weight loss sculpts these circuits to motivate food consumption until lost weight is regained remains unclear. Here, we probe the contribution of synaptic plasticity in discrete excitatory afferents on hunger-promoting AgRP neurons. We reveal a crucial role for activity-dependent, remarkably long-lasting amplification of synaptic activity originating from paraventricular hypothalamus thyrotropin-releasing (PVHTRH) neurons in long-term body weight control. Silencing PVHTRH neurons inhibits the potentiation of excitatory input to AgRP neurons and diminishes concomitant regain of lost weight. Brief stimulation of the pathway is sufficient to enduringly potentiate this glutamatergic hunger synapse and triggers an NMDAR-dependent gaining of body weight that enduringly persists. Identification of this activity-dependent synaptic amplifier provides a previously unrecognized target to combat regain of lost weight.

The "dorsal pons", or "dorsal pontine tegmentum" (dPnTg), is part of the brainstem. It is a complex, densely packed region whose nuclei are involved in regulating many vital functions. Notable among them are the parabrachial nucleus, the Kölliker Fuse, the Barrington nucleus, the locus coeruleus, and the dorsal, laterodorsal, and ventral tegmental nuclei. In this study, we applied single-nucleus RNA-seq (snRNA-seq) to resolve neuronal subtypes based on their unique transcriptional profiles and then used multiplexed error robust fluorescence in situ hybridization (MERFISH) to map them spatially. We sampled ~1 million cells across the dPnTg and defined the spatial distribution of over 120 neuronal subtypes. Our analysis identified an unpredicted high transcriptional diversity in this region and pinpointed the unique marker genes of many neuronal subtypes. We also demonstrated that many neuronal subtypes are transcriptionally similar between humans and mice, enhancing this study's translational value. Finally, we developed a freely accessible, GPU and CPU-powered dashboard ( http://harvard.heavy.ai:6273/ ) that combines interactive visual analytics and hardware-accelerated SQL into a data science framework to allow the scientific community to query and gain insights into the data.

Obesity is a prevalent disorder that often leads to diabetes and cardiovascular disease. The development of obesity is thought to represent the dysregulation of various homeostatic mechanisms which normally function to maintain body weight. Transgenics and gene targeting are also contributing importantly to obesity research. Initially, obesity occurred in the absence of hyperphagia indicating a reduction in energy expenditure. Kozak et al. has generated transgenic mice which overexpress cytoplasmic glycerol 3-phosphate dehydrogenase by approximately 50–200 fold. Transgenic mice bearing adipocyte fatty acid binding protein (ALBP) promoter/enhancer – reporter gene constructs have been generated and a region required for fat-specific expression of ALBP in vivo has been identified. The mutations responsible for causing a number of single gene obesity syndromes in rodents have recently been identified. Transgenic and gene knockout mice are being used in pharmaceutical drug development as a means of learning more about potential durg targets, or to create mice which have human but not rodent drug targets.

Cell death-inducing DNA fragmentation factor-like effector C (CIDEC) expression in adipose tissue positively correlates with insulin sensitivity in obese humans. Further, E186X, a single-nucleotide CIDEC variant is associated with lipodystrophy, hypertriglyceridemia, and insulin resistance. To establish the unknown mechanistic link between CIDEC and maintenance of systemic glucose homeostasis, we generated transgenic mouse models expressing CIDEC (Ad-CIDECtg) and CIDEC E186X variant (Ad-CIDECmut) transgene specifically in the adipose tissue. We found that Ad-CIDECtg but not Ad-CIDECmut mice were protected against high-fat diet-induced glucose intolerance. Furthermore, we revealed the role of CIDEC in lipid metabolism using transcriptomics and lipidomics. Serum triglycerides, cholesterol, and low-density lipoproteins were lower in high-fat diet-fed Ad-CIDECtg mice compared to their littermate controls. Mechanistically, we demonstrated that CIDEC regulates the enzymatic activity of adipose triglyceride lipase via interacting with its activator, CGI-58, to reduce free fatty acid release and lipotoxicity. In addition, we confirmed that CIDEC is indeed a vital regulator of lipolysis in adipose tissue of obese humans, and treatment with recombinant CIDEC decreased triglyceride breakdown in visceral human adipose tissue. Our study unravels a central pathway whereby adipocyte-specific CIDEC plays a pivotal role in regulating adipose lipid metabolism and whole-body glucose homeostasis. In summary, our findings identify human CIDEC as a potential 'drug' or a 'druggable' target to reverse obesity-induced lipotoxicity and glucose intolerance.

Humans and animals lacking orexin neurons exhibit daytime sleepiness, sleep attacks, and state instability. While the circuit basis by which orexin neurons contribute to consolidated wakefulness remains unclear, existing models posit that orexin neurons provide their wake-stabilizing influence by exerting excitatory tone on other brain arousal nodes. Here we show using in vivo optogenetics, in vitro optogenetic-based circuit mapping, and single-cell transcriptomics that orexin neurons also contribute to arousal maintenance through indirect inhibition of sleep-promoting neurons of the ventrolateral preoptic nucleus. Activation of this subcortical circuit rapidly drives wakefulness from sleep by differentially modulating the activity of ventrolateral preoptic neurons. We further identify and characterize a feedforward circuit through which orexin (and co-released glutamate) acts to indirectly target and inhibit sleep-promoting ventrolateral preoptic neurons to produce arousal. This revealed circuitry provides an alternate framework for understanding how orexin neurons contribute to the maintenance of consolidated wakefulness and stabilize behavioral state.

Agouti-related peptide (AGRP)-expressing neurons are activated by fasting—this causes hunger 1 – 4 , an aversive state that motivates the seeking and consumption of food 5 , 6 . Eating returns AGRP neuron activity towards baseline on three distinct timescales: rapidly and transiently following sensory detection of food cues 6 – 8 , slowly and longer-lasting in response to nutrients in the gut 9 , 10 , and even more slowly and permanently with restoration of energy balance 9 , 11 . The rapid regulation by food cues is of particular interest as its neurobiological basis and purpose are unknown. Given that AGRP neuron activity is aversive 6 , the sensory cue-linked reductions in activity could function to guide behaviour. To evaluate this, we first identified the circuit mediating sensory cue inhibition and then selectively perturbed it to determine function. Here, we show that a lateral hypothalamic glutamatergic → dorsomedial hypothalamic GABAergic (γ-aminobutyric acid-producing) 12 → AGRP neuron circuit mediates this regulation. Interference with this circuit impairs food cue inhibition of AGRP neurons and, notably, greatly impairs learning of a sensory cue-initiated food-acquisition task. This is specific for food, as learning of an identical water-acquisition task is unaffected. We propose that decreases in aversive AGRP neuron activity 6 mediated by this food-specific circuit increases the incentive salience 13 of food cues, and thus facilitates the learning of food-acquisition tasks.

Body energy homeostasis results from balancing energy intake and energy expenditure. Central nervous system administration of pituitary adenylate cyclase activating polypeptide (PACAP) dramatically alters metabolic function, but the physiologic mechanism of this neuropeptide remains poorly defined. PACAP is expressed in the mediobasal hypothalamus (MBH), a brain area essential for energy balance. Ventromedial hypothalamic nucleus (VMN) neurons contain, by far, the largest and most dense population of PACAP in the medial hypothalamus. This region is involved in coordinating the sympathetic nervous system in response to metabolic cues in order to re-establish energy homeostasis. Additionally, the metabolic cue of leptin signaling in the VMN regulates PACAP expression. We hypothesized that PACAP may play a role in the various effector systems of energy homeostasis, and tested its role by using VMN-directed, but MBH encompassing, adeno-associated virus (AAVCre) injections to ablate Adcyap1 (gene coding for PACAP) in mice (Adcyap1MBHKO mice). Adcyap1MBHKO mice rapidly gained body weight and adiposity, becoming hyperinsulinemic and hyperglycemic. Adcyap1MBHKO mice exhibited decreased oxygen consumption (VO2), without changes in activity. These effects appear to be due at least in part to brown adipose tissue (BAT) dysfunction, and we show that PACAP-expressing cells in the MBH can stimulate BAT thermogenesis. While we observed disruption of glucose clearance during hyperinsulinemic/euglycemic clamp studies in obese Adcyap1MBHKO mice, these parameters were normal prior to the onset of obesity. Thus, MBH PACAP plays important roles in the regulation of metabolic rate and energy balance through multiple effector systems on multiple time scales, which highlight the diverse set of functions for PACAP in overall energy homeostasis.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract While Cre-dependent viral systems permit the manipulation of many neuron types, some cell populations cannot be targeted by a single DNA recombinase. Although the combined use of Flp and Cre recombinases can overcome this limitation, insufficient recombinase activity can reduce the efficacy of existing Cre+Flp-dependent viral systems. We developed a sensitive dual recombinase-activated viral approach: tTA-driven Recombinase-Guided Intersectional Targeting (tTARGIT) adeno-associated viruses (AAVs). tTARGIT AAVs utilize a Flp-dependent tetracycline transactivator (tTA) ‘Driver’ AAV and a tetracycline response element-driven, Cre-dependent ‘Payload’ AAV to express the transgene of interest. We employed this system in Slc17a6FlpO;LeprCre mice to manipulate LepRb neurons of the ventromedial hypothalamus (VMH; LepRbVMH neurons) while omitting neighboring LepRb populations. We defined the circuitry of LepRbVMH neurons and roles for these cells in the control of food intake and energy expenditure. Thus, the tTARGIT system mediates robust recombinase-sensitive transgene expression, permitting the precise manipulation of previously intractable neural populations. eLife digest The brain contains hundreds of types of neurons, which differ in size, shape and behavior. But neuroscientists often wish to study individual neuronal types in isolation. They are able to do this with the aid of a toolkit made up of two parts: viral vectors and genetically modified mice. Viral vectors are viruses that have been modified so that they are no longer harmful and can instead be used to introduce genetic material into cells on demand. To create a viral vector, the virus’ own genetic material is replaced with a ‘cargo’ gene, such as the gene for a fluorescent protein. The virus is then introduced into a new host such as a mouse. Importantly, the virus only produces the protein encoded by its ‘cargo’ gene if it is inside a cell that also contains one of two specific enzymes. These enzymes are called Cre and Flp. This is where the second part of the toolkit comes in. Mice can be genetically engineered to produce either Cre or Flp exclusively in specific cell types. By introducing a viral vector into mice that produce either Cre or Flp only in one particular type of neuron, researchers can limit the activity of the cargo gene to that neuronal type. But sometimes even this approach is not selective enough. Researchers may wish to limit the activity of the cargo gene to a subpopulation of cells that produce Cre or Flp. Or they may wish to target only Cre- or Flp-producing cells in a small area of the brain, while leaving cells in neighboring areas unaffected. Sabatini et al. have now overcome this limitation by developing and testing a new set of viral vectors that are active only in neurons that produce both Cre and Flp. The vectors are called tTARGIT AAVs and allow researchers to target cells more precisely than was possible with the previous version of the toolkit. Sabatini et al. show tTARGIT AAVs in action by using them to identify a group of neurons that control how much energy mice use and how much food they eat. As well as applying the vectors to their own research on obesity, Sabatini et al. have also made them freely available for other researchers to use in their own projects. Introduction The molecular heterogeneity of the nervous system requires a rich toolkit for precise study of distinct cell populations. Together with Cre recombinase-expressing mice, the available suite of Cre-dependent viral vectors permits the manipulation of genetically identified neural types. However, this approach neither permits the study of subpopulations of cells expressing a particular Cre allele (Hodge et al., 2019; Mickelsen et al., 2019) nor permits the study of Cre-expressing cells within a defined Central nervous system (CNS) site while excluding the Cre-expressing cells in closely apposed brain regions. Restricting transgene expression to cells that express two marker genes can overcome this challenge (Luan and White, 2007). Early attempts at this approach utilized gene-specific, Cre-sensitive transgene-expressing alleles in combination with Cre alleles driven by distinct genes (Chen et al., 2011). Another solution involves expressing two Cre fragments across different transgenes with distinct promoters, reconstituting active Cre only in cells that express both pieces (Hirrlinger et al., 2009). These approaches generally involve substantial investments of time and resources, however, as they require generating and interbreeding multiple new gene- and experiment-specific alleles. Transgene expression can also be directed to cell types defined by two marker genes through the use of recombinase-dependent alleles (often inserted into the Rosa26 Locus) containing a ubiquitous promoter and tandem STOP cassettes that are each excised by distinct recombinases (Daigle et al., 2018; Sciolino et al., 2016). Neuroscience research often requires a higher degree of spatial specificity than afforded by the intersectional genetic models outlined above. Stereotaxic injections of recombinase-sensitive viral vectors can restrict transgene expression to a narrow anatomical region. Adeno-associated viruses (AAVs) generally represent the preferred viral system for Cre-dependent transgene expression, given their minimal toxicity and the speed and ease of their generation. Developing AAVs sensitive to multiple recombinases has been challenging because of the limited AAV genome size, which precludes the use of multiple recombinase-sensitive STOP cassettes (Wu et al., 2010). The INTRSECT system overcomes this limitation by utilizing a single AAV vector that flanks the transgene coding sequence with lox and FRT sites in such a way that combinatorial expression of Cre and Flp permits expression of a functional pre-mRNA that can then be spliced to produce a mature coding sequence (Fenno et al., 2020; Fenno et al., 2014). The multiple inversion and splicing steps involved in this system can limit transgene expression, however, perhaps due to the relatively poor recombinase activity of Flp (and even optimized FlpO). Furthermore, generating INTRSECT viruses that express new transgenes requires a relatively complex and labor-intensive design and optimization process (Fenno et al., 2020; Fenno et al., 2017). Seeking a dual recombinase-activated AAV system to overcome these limitations and that could be modified quickly and easily, we generated tetracycline transactivator (tTA)-driven Recombinase-Guided Intersectional Targeting (tTARGIT) AAVs composed of a Flp-dependent tetracycline transactivator (tTA) ‘Driver’ AAV and a tetracycline response element (TRE)-driven Cre-dependent ‘Payload’ AAV to express the transgene of interest. We applied tTARGIT AAVs to the study of ventromedial hypothalamic LepRb (VMH; LepRbVMH) neurons, which modulate metabolic adaptations to obesogenic diets (Bingham et al., 2008; Dhillon et al., 2006) but have proven difficult to study directly due to the density and proximity of neighboring LepRb populations. Together with LeprCre and Slc16a7FlpO, tTARGIT AAVs allowed us to overcome these challenges and reveal a specific role for LepRbVMH neurons in suppressing food intake and increasing energy expenditure to promote weight loss. Results The density of LepRb neurons in the adjacent arcuate nucleus (ARC), dorsomedial hypothalamus, and lateral hypothalamic area complicated our initial attempts to study LepRbVMH neurons using LeprCre mice and Cre-dependent vectors; viruses targeted to the VMH spread to other nearby LeprCre neurons, confounding the interpretation of our results (data not shown). Because Slc17a6, encoding the vesicular glutamate transporter 2 (vGLUT2) protein, expression is largely restricted to LepRbVMH neurons and absent from most surrounding LepRb cells (Vong et al., 2011), we generated a Slc17a6FlpO strain and crossed it with LeprCre and a novel Flp- and Cre-dependent reporter (Rosa26RCFL-eGFP-L10a). We then tested the potential ability of this combination of Cre and Flp alleles to specify LepRbVMH neurons in the mediobasal hypothalamus. Although these reporter mice identified Cre- and Flp-co-expressing LepRbSlc17a6 neurons elsewhere in the brain, within the mediobasal hypothalamus this approach largely limited eGFP expression to VMH cells (Figure 1—figure supplement 1). We thus sought to use Flp- and Cre-dependent AAVs to target LepRbVMH neurons and omit manipulation of non-VMH LepRbSlc17a6 cells. We injected the INTRSECT AAV system (Fenno et al., 2014) into the VMH of Slc17a6FlpO;LeprCre mice in an attempt to express channelrhodopsin (ChR2) in LepRbVMH cells. However, this approach resulted in detectable ChR2 expression in one or fewer cells per section (Figure 1—figure supplement 2). We surmised that while INTRSECT works well in systems with robust Flp and Cre activities, the poor recombinase activity of Flp and the more moderate Flp and Cre expression mediated by Slc17a6FlpO and LeprCre, respectively, might limit INTRSECT-mediated transgene expression in LepRbVMH cells. We therefore set out to develop a more sensitive AAV system to drive Cre+Flp-dependent transgene expression, using as our framework a previously described inducible gene expression system based on recombinase-dependent expression of the tetracycline transactivator (tTA) in combination with a TRE-driven transgene-expressing allele (Chan et al., 2017; He et al., 2016). We packaged this system into two viral vectors, hereafter ‘tTA-driven Recombinase-Guided Intersectional Targeting’ (tTARGIT) AAVs. Our tTARGIT system utilizes ‘Driver’ and ‘Payload’ AAVs. The Driver (AAV-hSYN1-fDIO-tTA) utilizes Flp-dependent Double-Floxed Inverted Open reading frame (fDIO) cassette (Fenno et al., 2014) to Flp-dependently invert tTA, permitting its expression by a human synapsin I (hSYN1) promoter. This virus also contains two tetracycline operators (TetO) to drive a positive feedback loop and increase tTA expression (Chan et al., 2017). The Payload AAV mediates tTA/TRE-dependent transgene expression following its Cre-mediated inversion into the sense orientation. Hence, only cells that contain both recombinases express the transgene (Figure 1a). Tetracycline inhibits tTA-dependent gene expression (Das et al., 2016), so this system mediates constitutive payload expression in target cells in the absence of tetracycline. Figure 1 with 5 supplements see all Download asset Open asset tTARGIT AAVs: a dual virus system to target intersectional populations. (a) The tTARGIT system employs the combination of ‘Driver’ (AAV-hSYN-fDIO-tTA) and ‘Payload’ (AAV-TRE-DIO-Payload) AAVs. The Driver virus encodes a Flp-dependent tetracycline transactivator (tTA) under control of the human synapsin I (hSYN1) promoter and two tetracycline operators (TetO). The Payload virus encodes a Cre-dependent Payload transgene under control of the tetracycline response element (TRE). (b,c) Experimental scheme (b) and representative images (c) showing the detection of TdTomato (DSRed-IR, red) following the co-injection of AAV-hSYN-fDIO-tTA and AAV-TRE-DIO-ChR2-TdT into the VMH of (from left to right, as labeled) wild-type (WT), Slc17a6FlpO, LeprCre, or Slc17a6FlpO;LeprCre mice. (d,e) Experimental schematic (d) and representative images (e) showing the detection of TdTomato (DSRed-IR, red) following the injection of (from left to right, as labeled): (1) AAV-hSYN-fDIO-tTA, (2) AAV-TRE-DIO-ChR2-TdT, or the two viruses combined (1+2) into the VMH of Slc17a6FlpO;LeprCre mice. Scale bars = 100 μm. To test the recombinase dependence of this system, we combined the Flp-dependent Driver AAV and a Payload AAV that permits the tTA/TRE-mediated Cre-dependent expression of a ChR2-TdTomato fusion protein (ChR2-TdT; AAV-TRE-DIO-ChR2-TdT). We co-injected these viruses into the VMH of wild-type mice, mice that expressed either Slc17a6FlpO or LeprCre only, or Slc17a6FlpO;LeprCre mice (Figure 1b,c). We detected no TdT (DSRed-immunoreactivity [IR]) in the VMH of wild-type or Slc17a6FlpO animals and minimal expression in LeprCre mice (Figure 1—figure supplement 3). In contrast, the Driver+Payload combination mediated robust DSRed-IR in the VMH of Slc17a6FlpO;LeprCre mice (Figure 1c); furthermore, VMH photostimulation in these mice promoted robust colocalization of DSRed- and FOS-IR, consistent with the ability of this system to activate transduced Flp+Cre-expressing cells (Figure 1—figure supplement 4). We also tested the requirement for both the Driver and Payload viruses in this system by injecting each virus alone, or both viruses together, into the VMH of Slc17a6FlpO;LeprCre animals (Figure 1d). As expected, injecting either virus alone produced minimal or no detectable DSRed-IR, while co-injection of the two viruses yielded robust DSRed-IR (Figure 1e). Thus, robust transgene expression by the tTARGIT system requires injection of both the Driver and Payload AAVs, as well as the presence of both Flp and Cre recombinases. The tTARGIT approach can also be modified from a Flp-ON/Cre-ON system, requiring both Flp and Cre recombinases for payload expression, to a Flp-ON/Cre-OFF system, mediating transgene expression in all Flp-expressing cells that do not contain Cre (Figure 1—figure supplement 5; Table 1). Placing the payload transgene in the forward orientation within the DIO cassette permits tTA-driven transgene expression in all Flp-expressing cells that do not contain Cre because Cre inverts the Payload transgene into the antisense orientation. We tested this system with a novel Cre-inactivated Payload virus expressing a hM3Dq designer receptor exclusively activated by designer drugs (DREADD)-mCherry transgene. We co-injected this Cre-OFF Payload and the Flp-dependent Driver AAV into the VMH of Slc17a6FlpO;LeprCre animals on the Cre-dependent Rosa26LSL-GFP-L10a background (Figure 1—figure supplement 5b). As expected, this modified tTARGIT system drove hM3Dq-mCherry expression almost exclusively in cells that did not express the Cre-dependent GFP (Figure 1—figure supplement 5c). We surmise the few GFP-IR neurons with detectable mCherry, constituting 1–7% of all mCherry cells, might result from the low Cre expression mediated by LeprCre (Patterson et al., 2011) and predict that the Flp-On, Cre-Off tTARGIT system should demonstrate complete Payload inactivation when used in conjunction with a more robustly expressing Cre allele or require longer incubation periods to fully inactivate the payload transgene. Table 1 Available tTARGIT Vectors. Driver virusesNotesAAV-hSYN1-fDIO-tTADrives expression in the absence of DOXAAV-hSYN1-fDIO-rtTADrives expression in the presence of DOX; not testedPayload virusesFlp-ON/Cre-ON (Payload inverted)AAV-TRE-DIO-hM3Dq-mCherryTestedAAV-TRE-DIO-ChR2-TdTomatoTestedAAV-TRE-DIO-Cas9Not testedAAV-TRE-DIO-TVA-oG-mCherryNot testedAAV-TRE-DIO-eGFP-2A-TetanusToxinNot testedAAV-TRE-DIO-SwiChR-eYFPNot testedAAV-TRE-DIO-eGFP-L10aNot testedAAV-TRE-DIO-GCaMP6sNot testedAAV-TRE-DIO- proCaspase-3-TEVpNot testedAAV-TRE-DIO-GFP-2A-SynmRubyTested; GFP expression permits tracing; limited SynmRuby expressionFlp ON/Cre-OFF (Payload Sense orientation)AAV-TRE-DIO-hM3Dq-mCherryTested; may require strong Cre driver for complete inactivation in Cre neuronsAAV-TRE-DIO-ChR2-TdTomatoAAV-TRE-DIO-eGFP-2A-TetanusToxin To define the projection targets of LepRbVMH neurons, we developed a payload virus (AAV-TRE-DIO-GFP-2A-SynmRuby) that encodes GFP plus a cotranslationally expressed synaptophysin-mRuby transgene (Figure 2a). Co-injection of this tTARGIT Payload AAV and the Driver virus into the VMH of Slc17a6FlpO;LeprCre mice promoted robust VMH-restricted GFP-IR (Figure 2b). mRuby detection (DSRed-IR) for this virus was much lower than for GFP, however (data not shown); thus, we used GFP-IR to detect projections from LepRbVMH cells. Assessing the entire CNS for the presence of GFP-IR revealed terminals in the periaqueductal gray, the arcuate, the periventricular thalamic nucleus, the periventricular hypothalamic nucleus, the bed nucleus of the stria terminalis, and the preoptic area (Figure 2c,d). These are consistent with the known projections of the VMH (Canteras et al., 1994; Meek et al., 2016; Zhang et al., 2020). Figure 2 Download asset Open asset Defining the downstream projections of LepRbVMH neurons. (a) Experimental schematic showing the injection of the Driver AAV with the Payload AAV encoding a GFP-2A-SynmRuby transgene into the VMH of Slc17a6FlpO;LeprCre animals. (b,c) Representative image of GFP-IR (green) showing viral transduction in the VMH (b) and projections (c) in the periaqueductal gray (PAG), arcuate nucleus (ARC), paraventricular hypothalamic nucleus (PVN), bed nucleus of the stria terminalis (BNST), and preoptic area (POA). (d) Cartoon showing the projection targets of LepRbVMH neurons. Ablating Lepr expression from the VMH (Nr5a1Cre-mediated) promotes obesity associated with decreased energy expenditure in high-fat-diet-fed animals, suggesting a specific role for LepRb signaling in LepRbVMH cells in the control of energy balance via the dietary modulation of energy utilization (Bingham et al., 2008; Dhillon et al., 2006). To define the function of LepRbVMH cells, rather than the function of LepRb in these cells, we developed a Payload virus containing an inverted hM3Dq-mCherry transgene. We co-injected the Driver AAV and this AAV-TRE-DIO-hM3Dq-mCherry Payload virus into the VMH of Slc17a6FlpO;LeprCre animals (LepRbVMH-Dq mice; Figure 3a). This approach promoted robust VMH-restricted expression of functional hM3Dq-mCherry as administration of the DREADD activator (Pei et al., 2008), clozapine-N-oxide (CNO), stimulated colocalization of FOS with DSRed-IR cells (Figure 3b–d). Figure 3 Download asset Open asset Activation of LepRbVMH neurons decreases food intake in addition to increasing brown adipose tissue (BAT) thermogenesis and energy expenditure. (a) Approach for activating LepRbVMH neurons by co-injecting the AAV-hSYN-fDIO-tTA Driver and the AAV-TRE-DIO-hM3Dq-mCherry Payload viruses into the VMH of Slc17a6FlpO;LeprCre animals. (b–d) Representative images showing mCherry detection (DSRed-IR, red) and FOS-IR (cyan) in LepRbVMH-Dq mice administered Vehicle (b) or CNO (1 mg/kg) (c,d). The right-hand panel of (c) shows a digital zoom of the boxed region in the left panel. Scale bars = 100 μm. (e–j) Results from metabolic cage analysis of LepRbVMH-Dq mice (n = 6) treated with either vehicle (gray) or 1 mg/kg CNO (red) at 9:30 and 16:30 (dotted lines). Lines in left panels denotes mean value; shading denotes SEM. Each animal was treated with vehicle for 2 days followed by CNO for 2 days to allow pairing. Bar graphs to the right show the average for each mouse at each time point across 24 hr and separated by time in light cycle (day = light, night = dark). (k, l) Changes to intrascapular temperatures over 120 min in LepRbVMH-Dq mice (n = 8) or GFP-injected (n = 7) controls following vehicle (gray) or CNO (red; 1 mg/kg) administration at 30°C. (l) Shows area under the curve (AUC) for each treatment condition in (k). For metabolic cage studies, statistical significance was determined using either a paired t-test (full-day data) or a linear mixed model for effects by time of day. For interscapular temperature measurements, significance was determined by paired t-test. *p<0.05, **p<0.01, ***p<0.001. Figure 3—source data 1 Raw Data from metabolic cages. https://cdn.elifesciences.org/articles/66835/elife-66835-fig3-data1-v2.xlsx Download elife-66835-fig3-data1-v2.xlsx To determine the potential modulation of energy expenditure, activity, and food intake by LepRbVMH neuron activation, we placed LepRbVMH-Dq animals in metabolic cages and administered either vehicle or CNO twice daily (Figure 3e–j). Compared to vehicle administration, activating LepRbVMH neurons significantly increased 24 hr oxygen consumption (VO2) and energy expenditure, both primarily due to effects during the light phase, despite decreasing ambulatory activity over 24 hr (primarily due to effects during the dark phase) (Figure 3g,h). Additionally, the hM3Dq-mediated activation of LepRbVMH neurons also suppressed 24 hr food intake, primarily due to decreased light-phase feeding, revealing a previously unsuspected role for these cells in the suppression of feeding. CNO also decreased the respiratory exchange ratio during the light phase, consistent with the increased metabolism of fat stores due to the combination of increased energy expenditure and decreased food intake. To understand whether effects on brown adipose tissue (BAT) might contribute to the increased energy expenditure during LepRbVMH neuron activation, we placed temperature probes in the interscapular space of LepRbVMH-Dq animals to monitor BAT thermogenesis. Compared to controls, CNO significantly increased intrascapular temperatures in LepRbVMH-Dq animals, suggesting LepRbVMH neurons promote energy expenditure at least in part by augmenting BAT thermogenesis (Figure 3k–l). As activating LepRbVMH neurons increased energy expenditure and decreased food intake, we surmised that these neurons should promote weight loss. We thus administered CNO in drinking water to LepRbVMH-Dq mice or LeprCre-only control animals (lacking any Flp expression) injected with the tTARGIT hM3Dq for 3 days. During this time, the body weight of LepRbVMH-Dq mice decreased by approximately 10% (Figure 4a), returning to baseline following the cessation of CNO exposure. Importantly, body weight of LeprCre-only controls remained stable. While CNO treatment decreased food consumption (largely during the second day of treatment) and water intake (Figure 4b–d),the magnitude and timing of these ingestive effects dictates that neither could account for the decreased body weight mediated by CNO, consistent with the notion that increased energy expenditure mediates the major effect of LepRbVMH cells on body weight. Figure 4 Download asset Open asset Chronically activating LepRbVMH neurons promotes negative energy balance. (a–c) Body weight, food intake, and water intake of chow-fed LeprCre-only controls injected with AAV-hSYN1-fDIO-tTA and AAV-TRE-DIO-hM3Dq-mCherry (black lines; VEH n = 5; CNO n = 8) and LepRbVMH-Dq mice (red lines n = 8 both conditions) receiving 2 days normal drinking water (days 0–2), followed by 3 days exposure to CNO-laced drinking water (days 2–5), followed by an additional 2 days of normal drinking water. Onset and termination of CNO treatment are denoted by vertical lines in each panel. Vehicle controls (dashed red and black lines) did not receive CNO on days 2–5. (d) Comparisons between body weight (closed black circles) and food intake (gray squares) over time in LepRbVMH-Dq animals. Data is presented as mean ± SEM. Significance was determined by linear mixed model; ***p<0.001 between CNO and VEH LepRbVMH-Dq animals. #p<0.05, ###p<0.001 between CNO and VEH LeprCre-only Control animals. Figure 4—source data 1 Raw data from chronic CNO dosing. https://cdn.elifesciences.org/articles/66835/elife-66835-fig4-data1-v2.xlsx Download elife-66835-fig4-data1-v2.xlsx Hence, the use of our dual recombinase-dependent tTARGIT AAV system permitted us to determine that LepRbVMH neuron activation increased energy expenditure and decreased food intake during the inactive phase, suggesting the diurnal control of energy balance by LepRbVMH neurons. Discussion The use of sequence-specific DNA recombinases (Cre, Flp, and others) in conjunction with recombinase-dependent genetic alleles and viral vectors has revolutionized our ability to manipulate specific circuits and understand the central nervous system. The lack of robust viral systems to manipulate cell populations defined by the expression of multiple genes has impeded the study of more refined neural populations, however, including those identified by single cell RNA-sequencing (Campbell et al., 2017). Our tTARGIT AAV system addresses many shortcomings of previous intersectional tools, including limitations to transgene expression and the difficulty of incorporating novel transgenes into the AAV plasmids. As the tTARGIT system is based on the use of dual AAVs, it is possible that the two viruses could compete with each other for the surface receptors required for viral internalization and/or for the cellular machinery required for transgene production, thereby reducing expression efficiency from both AAVs. Based upon our results, and since dual AAVs have been used successfully previously (Akil et al., 2019; Xu et al., 2018; Yang et al., 2016) and transduce the same cells in vivo (Vardy et al., 2015), we surmise that if such competition exists with tTARGIT AAVs, it is minor. To further facilitate the study of intersectional neural populations, we developed a suite of Cre-dependent tTARGIT Payload plasmids (Table 1). While we have not yet tested the function of all of these, our experience with the transgenes that we have tested predicts similarly robust expression of the various Payload AAV transgenes. The limitations of these Payload vectors are likely to mirror those of standard viruses with Cre-dependent transgenes, including the requirement for stoichiometric transduction of/recombination in the cell type of interest to observe the effects of interfering with neuronal function. Additionally, the tTARGIT system displays some detectable payload expression, albeit at low levels and in a small number of cells, in LeprCre-only mice co-injected with Driver and Payload AAVs, as well as in Slc17a6FlpO;LeprCre mice injected with the Payload AAV alone. We surmise this results from the previously noted tTA-independent activity of the TRE (Das et al., 2016). We expect this tTA-independent Payload transgene expression to predominately occur in Cre-expressing cells, which have inverted the transgene into the correct orientation downstream of the TRE. In our studies, leaky payload expression had little impact as the expression levels were low and failed to produce a metabolic response to CNO in drinking water. Nevertheless, the possibility of tTA-independent payload expression represents an important consideration for experimental design and requires the inclusion of appropriate controls. In addition to Cre-ON/Flp-ON tTARGIT plasmids, we also generated Cre-inactivated Payload vectors to specifically mark Cre-negative Flp-expressing cells within the injection field. Within our model, we found up to 7% of mCherry-IR cells were co-labeled with the Cre-dependent GFP reporter. We posit that increasing post-injection incubation times prior to study or using alleles with higher Cre expression than observed in LeprCre may further reduce the number of Cre-labeled cells expressing the Cre-OFF tTARGIT payload. Hence, inclusion of appropriate control will be required for studies employing tTARGIT AAVs. While the tTARGIT AAV system as we have built it is designed to constitutively express transgenes in Cre- and Flp-expressing cells without the use of tetracycline-like compounds (usually doxycycline), it should be possible to decrease transgene expression from the tTARGIT AAVs by doxycycline treatment. Indeed, we built a Flp-dependent rtTA Driver virus that is predicted to require doxycycline treatment to mediate strong transgene expression. The rtTA-based system can drive low-level transcription independent of doxycycline (Zhu et al., 2001), however, and we have not yet tested this system. The use of tTARGIT AAVs permitted us to target robust transgene expression to LepRbVMH neurons specifically, in isolation from LepRb neurons in adjacent hypothalamic nuclei. While deleting Lepr from the VMH of chow-fed mice or restoring VMH Lepr expression on an otherwise LepRb-deficient background minimally (if at all) alters energy balance, knockout mice fail to increase energy expenditure on high-fat chow and become more obese than controls (Bingham et al., 2008; Dhillon et al., 2006; Gonçalves et al., 2014; Senn et al., 2019). These studies thus suggest a role for leptin action on LepRbVMH cells in the control of energy expenditure, but were unable to study LepRbVMH cells more broadly. In contrast, our use of the tTARGIT system, together with Slc17a6FlpO;LeprCre mice, identified the projections of LepRbVMH cells, demonstrated their ability to acutely suppress food intake as well as promoting energy expenditure (identifying BAT thermogenesis as a target for these cells), and revealed the diurnal nature of LepRbVMH neuron-mediated control of energy balance. Presumably, the finding that LepRbVMH neuron activation alters food intake and energy expenditure specifically during the light cycle suggests that these neurons may decrease in activity during the inactive/light phase, permitting us to observe the effects of artificial neuron activation during this time. In summary, we have developed a suite of dyad AAV vectors for the study of intersectional neural populations marked by the co-expression of Flp and Cre or marked by expression of Flp in the absence of Cre. This tTARGIT system yields robust dual recombinase-sensitive expression of the desired payload in vivo. With this approach, we defined the neural circuitry and functional capacity of LepRbVMH neurons. These intersectional genetic tools will facilitate the study of a broad range of dual gene-defined cell populations across the central nervous system. Materials and methods Key resources table Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional inf