Protein Appetite Drives Macronutrient-Related Differences in Ventral Tegmental Area Neural Activity.

Control of protein intake is essential for numerous biological processes as several amino acids cannot be synthesized de novo, however, its neurobiological substrates are still poorly understood. In the present study, we combined in vivo fiber photometry with nutrient-conditioned flavor in a rat model of protein appetite to record neuronal activity in the VTA, a central brain region for the control of food-related processes. In adult male rats, protein restriction increased preference for casein (protein) over maltodextrin (carbohydrate). Moreover, protein consumption was associated with a greater VTA response, relative to carbohydrate. After initial nutrient preference, a switch from a normal balanced diet to protein restriction induced rapid development of protein preference but required extensive exposure to macronutrient solutions to induce elevated VTA responses to casein. Furthermore, prior protein restriction induced long-lasting food preference and VTA responses. This study reveals that VTA circuits are involved in protein appetite in times of need, a crucial process for animals to acquire an adequate amount of protein in their diet.SIGNIFICANCE STATEMENT Acquiring insufficient protein in one's diet has severe consequences for health and ultimately will lead to death. In addition, a low level of dietary protein has been proposed as a driver of obesity as it can leverage up intake of fat and carbohydrate. However, much remains unknown about the role of the brain in ensuring adequate intake of protein. Here, we show that in a state of protein restriction a key node in brain reward circuitry, the VTA, is activated more strongly during consumption of protein than carbohydrate. Moreover, although rats' behavior changed to reflect new protein status, patterns of neural activity were more persistent and only loosely linked to protein status.

Cell Class-Dependent Intracortical Connectivity and Output Dynamics of Layer 6 Projection Neurons of the Rat Primary Visual Cortex.

Neocortical information processing is powerfully influenced by the activity of layer 6 projection neurons through control of local intracortical and subcortical circuitry. Morphologically distinct classes of layer 6 projection neuron have been identified in the mammalian visual cortex, which exhibit contrasting receptive field properties, but little information is available on their functional specificity. To address this we combined anatomical tracing techniques with high-resolution patch-clamp recording to identify morphological and functional distinct classes of layer 6 projection neurons in the rat primary visual cortex, which innervated separable subcortical territories. Multisite whole-cell recordings in brain slices revealed that corticoclaustral and corticothalamic layer 6 projection neurons exhibited similar somatically recorded electrophysiological properties. These classes of layer 6 projection neurons were sparsely and reciprocally synaptically interconnected, but could be differentiated by cell-class, but not target-cell-dependent rules of use-dependent depression and facilitation of unitary excitatory synaptic output. Corticoclaustral and corticothalamic layer 6 projection neurons were differentially innervated by columnar excitatory circuitry, with corticoclaustral, but not corticothalamic, neurons powerfully driven by layer 4 pyramidal neurons, and long-range pathways conveyed in neocortical layer 1. Our results therefore reveal projection target-specific, functionally distinct, streams of layer 6 output in the rodent neocortex.

Left-right dissociation of hippocampal memory processes in mice.

Left-right asymmetries have likely evolved to make optimal use of bilaterian nervous systems; however, little is known about the synaptic and circuit mechanisms that support divergence of function between equivalent structures in each hemisphere. Here we examined whether lateralized hippocampal memory processing is present in mice, where hemispheric asymmetry at the CA3-CA1 pyramidal neuron synapse has recently been demonstrated, with different spine morphology, glutamate receptor content, and synaptic plasticity, depending on whether afferents originate in the left or right CA3. To address this question, we used optogenetics to acutely silence CA3 pyramidal neurons in either the left or right dorsal hippocampus while mice performed hippocampus-dependent memory tasks. We found that unilateral silencing of either the left or right CA3 was sufficient to impair short-term memory. However, a striking asymmetry emerged in long-term memory, wherein only left CA3 silencing impaired performance on an associative spatial long-term memory task, whereas right CA3 silencing had no effect. To explore whether synaptic properties intrinsic to the hippocampus might contribute to this left-right behavioral asymmetry, we investigated the expression of hippocampal long-term potentiation. Following the induction of long-term potentiation by high-frequency electrical stimulation, synapses between CA3 and CA1 pyramidal neurons were strengthened only when presynaptic input originated in the left CA3, confirming an asymmetry in synaptic properties. The dissociation of hippocampal long-term memory function between hemispheres suggests that memory is routed via distinct left-right pathways within the mouse hippocampus, and provides a promising approach to help elucidate the synaptic basis of long-term memory.

Optogenetic evidence for inhibitory signaling from orexin to MCH neurons via local microcircuits.

The lateral hypothalamus (LH) is a key regulator of multiple vital behaviors. The firing of brain-wide-projecting LH neurons releases neuropeptides promoting wakefulness (orexin/hypocretin; OH), or sleep (melanin-concentrating hormone; MCH). OH neurons, which coexpress glutamate and dynorphin, have been proposed to excite their neighbors, including MCH neurons, suggesting that LH may sometimes coengage its antagonistic outputs. However, it remains unclear if, when, and how OH actions promote temporal separation of the sleep and wake signals, a process that fails in narcolepsy caused by OH loss. To explore this directly, we paired optogenetic stimulation of OH cells (at rates that promoted awakening in vivo) with electrical monitoring of MCH cells in mouse brain slices. Membrane potential recordings showed that OH cell firing inhibited action potential firing in most MCH neurons, an effect that required GABAA but not dynorphin receptors. Membrane current analysis showed that OH cell firing increased the frequency of fast GABAergic currents in MCH cells, an effect blocked by antagonists of OH but not dynorphin or glutamate receptors, and mimicked by bath-applied OH peptide. In turn, neural network imaging with a calcium indicator genetically targeted to MCH neurons showed that excitation by bath-applied OH peptides occurs in a minority of MCH cells. Collectively, our data provide functional microcircuit evidence that intra-LH feedforward loops may facilitate appropriate switching between sleep and wake signals, potentially preventing sleep disorders.

Cellular activation of hypothalamic hypocretin/orexin neurons facilitates short-term spatial memory in mice.

The hypothalamic hypocretin/orexin (HO) system holds a central role in the regulation of several physiological functions critical for food-seeking behavior including mnemonic processes for effective foraging behavior. It is unclear however whether physiological increases in HO neuronal activity can support such processes. Using a designer rM3Ds receptor activation approach increasing HO neuronal activity resulted in improved short-term memory for novel locations. When tested on a non-spatial novelty object recognition task no significant difference was detected between groups indicating that hypothalamic HO neuronal activation can selectively facilitate short-term spatial memory for potentially supporting memory for locations during active exploration.

Amygdala intercalated neurons are required for expression of fear extinction.

Congruent findings from studies of fear learning in animals and humans indicate that research on the circuits mediating fear constitutes our best hope of understanding human anxiety disorders. In mammals, repeated presentations of a conditioned stimulus that was previously paired to a noxious stimulus leads to the gradual disappearance of conditioned fear responses. Although much evidence suggests that this extinction process depends on plastic events in the amygdala, the underlying mechanisms remain unclear. Intercalated (ITC) amygdala neurons constitute probable mediators of extinction because they receive information about the conditioned stimulus from the basolateral amygdala (BLA), and contribute inhibitory projections to the central nucleus (CEA), the main output station of the amygdala for conditioned fear responses. Thus, after extinction training, ITC cells could reduce the impact of conditioned-stimulus-related BLA inputs to the CEA by means of feed-forward inhibition. Here we test the hypothesis that ITC neurons mediate extinction by lesioning them with a toxin that selectively targets cells expressing micro-opioid receptors (microORs). Electron microscopic observations revealed that the incidence of microOR-immunoreactive synapses is much higher in ITC cell clusters than in the BLA or CEA and that microORs typically have a post-synaptic location in ITC cells. In keeping with this, bilateral infusions of the microOR agonist dermorphin conjugated to the toxin saporin in the vicinity of ITC neurons caused a 34% reduction in the number of ITC cells but no significant cell loss in surrounding nuclei. Moreover, ITC lesions caused a marked deficit in the expression of extinction that correlated negatively with the number of surviving ITC neurons but not CEA cells. Because ITC cells exhibit an unusual pattern of receptor expression, these findings open new avenues for the treatment of anxiety disorders.

Optogenetic probing of fast glutamatergic transmission from hypocretin/orexin to histamine neurons in situ.

Hypothalamic hypocretin/orexin (hcrt/orx) neurons coordinate sleep-wake cycles, reward seeking, and body energy balance. Neurochemical data suggest that hcrt/orx cells contain several transmitters, but what hcrt/orx cells release onto their projection targets is unknown. A major pathway by which hcrt/orx neurons are thought to promote arousal is through projections to tuberomammillary histamine (HA) neurons. To study the impact of the electrical activity in hcrt/orx cells on HA neurons, we genetically targeted the light-activated excitatory ion channel channelrhodopsin-2 (ChR2) to the plasma membrane of hcrt/orx cells, and performed patch-clamp recordings from HA cells in acute mouse brain slices. Stimulation of ChR2-containing fibers with millisecond flashes of blue light produced fast postsynaptic currents in HA neurons, with a high connection probability (≈60% of HA cells were connected to ≈40% of hcrt/orx cells expressing ChR2). These inputs depended on tetrodotoxin-sensitive action potentials, had kinetics typical of glutamatergic responses mediated by AMPA receptors, were blocked by the AMPA receptor blocker CNQX, and displayed multiple forms of short-term plasticity (depression in ≈70% trials, facilitation in ≈30% trials, both often in the same cell). Furthermore, stimulation of hcrt/orx axons at physiological frequencies rapidly and reversibly increased action potential firing in HA cells, an effect that was abolished by blockade of AMPA receptors. These results provide the first functional evidence that hcrt/orx neurons are capable of fast glutamatergic control of their projection targets, and suggest that variations in electrical activity of hcrt/orx axons can induce rapid changes in long-range signals generated by HA neurons.

Associative properties of the perirhinal network.

The perirhinal area is a rostrocaudally oriented cortical region involved in recognition and associative memory. It receives topographically organized transverse projections from high-order neocortical areas and is endowed with intrinsic longitudinal connections that distribute neocortical inputs rostrocaudally. Earlier work has revealed that neocortical inputs strongly recruit perirhinal interneurons located at the same transverse level, limiting the depolarization of principal cells. In contrast, at a distance, neocortical stimuli only evoke excitation because longitudinal perirhinal pathways do not engage interneurons. This raises the possibility that the perirhinal network allows for Hebbian-like associative interactions between coincident and spatially distributed inputs. To test this, we analyzed the effects of theta-frequency neocortical stimulation using simultaneous field potential recordings and optical imaging in the whole guinea pig brain in vitro. Theta-frequency stimulation (TFS) at one neocortical site resulted in a prolonged input-specific response depression at all perirhinal levels. In contrast, paired TFS of 2 distant neocortical sites resulted in a prolonged response potentiation to the paired inputs, suggesting that longitudinal perirhinal connections can support associative interactions between coincident but spatially distributed inputs. Moreover, we found that induction of these 2 forms of plasticity depended on the competing influence of glutamate group I metabotropic and NMDA receptors, respectively.

Involvement of A13 dopaminergic neurons in prehensile movements but not reward in the rat.

Tyrosine hydroxylase (TH)-containing neurons of the dopamine (DA) cell group A13 are well positioned to impact known DA-related functions as their descending projections innervate target regions that regulate vigilance, sensory integration, and motor execution. Despite this connectivity, little is known regarding the functionality of A13-DA circuits. Using TH-specific loss-of-function methodology and techniques to monitor population activity in transgenic rats in vivo, we investigated the contribution of A13-DA neurons in reward and movement-related actions. Our work demonstrates a role for A13-DA neurons in grasping and handling of objects but not reward. A13-DA neurons responded strongly when animals grab and manipulate food items, whereas their inactivation or degeneration prevented animals from successfully doing so-a deficit partially attributed to a reduction in grip strength. By contrast, there was no relation between A13-DA activity and food-seeking behavior when animals were tested on a reward-based task that did not include a reaching/grasping response. Motivation for food was unaffected, as goal-directed behavior for food items was in general intact following A13 neuronal inactivation/degeneration. An anatomical investigation confirmed that A13-DA neurons project to the superior colliculus (SC) and also demonstrated a novel A13-DA projection to the reticular formation (RF). These results establish a functional role for A13-DA neurons in prehensile actions that are uncoupled from the motivational factors that contribute to the initiation of forelimb movements and help position A13-DA circuits into the functional framework regarding centrally located DA populations and their ability to coordinate movement.

Restriction of dietary protein in rats increases progressive-ratio motivation for protein.

Low-protein diets can impact food intake and appetite, but it is not known if motivation for food is changed. In the present study, we used an operant behavioral task - the progressive ratio test - to assess whether motivation for different foods was affected when rats were maintained on a protein-restricted diet (REST, 5% protein diet) compared to non-restricted control rats (CON, 18% protein). Rats were tested either with nutritionally-balanced pellets (18.7% protein, Experiment 1) or protein-rich pellets (35% protein, Experiment 2) as reinforcers. Protein restriction increased breakpoint for protein-rich pellets, relative to CON rats, whereas no difference in breakpoint for nutritionally-balanced pellets was observed between groups. When given free access to either nutritionally-balanced pellets or protein-rich pellets, REST and CON rats did not differ in their intake. We also tested whether a previous history of protein restriction might affect present motivation for different types of food by assessing breakpoint of previously REST animals that were subsequently put on standard maintenance chow (protein-repleted rats, REPL, Experiment 2). REPL rats did not show increased breakpoint, relative to their initial encounter with protein-rich pellets while they were protein-restricted. This study demonstrates that restriction of dietary protein induces a selective increased motivation for protein-rich food, a behavior that disappears once rats are not in need of protein.

Predictive and motivational factors influencing anticipatory contrast: A comparison of contextual and gustatory predictors in food restricted and free-fed rats.

In anticipation of palatable food, rats can learn to restrict consumption of a less rewarding food type resulting in an increased consumption of the preferred food when it is made available. This construct is known as anticipatory negative contrast (ANC) and can help elucidate the processes that underlie binge-like behavior as well as self-control in rodent motivation models. In the current investigation we aimed to shed light on the ability of distinct predictors of a preferred food choice to generate contrast effects and the motivational processes that underlie this behavior. Using a novel set of rewarding solutions, we directly compared contextual and gustatory ANC predictors in both food restricted and free-fed Sprague-Dawley rats. Our results indicate that, despite being food restricted, rats are selective in their eating behavior and show strong contextually-driven ANC similar to free-fed animals. These differences mirrored changes in palatability for the less preferred solution across the different sessions as measured by lick microstructure analysis. In contrast to previous research, predictive cues in both food restricted and free-fed rats were sufficient for ANC to develop although flavor-driven ANC did not relate to a corresponding change in lick patterning. These differences in the lick microstructure between context- and flavor-driven ANC indicate that the motivational processes underlying ANC generated by the two predictor types are distinct. Moreover, an increase in premature port entries to the unavailable sipper - a second measure of ANC - in all groups reveals a direct influence of response competition on ANC development.

Methods for predicting cortical UP and DOWN states from the phase of deep layer local field potentials.

During anesthesia, slow-wave sleep and quiet wakefulness, neuronal membrane potentials collectively switch between de- and hyperpolarized levels, the cortical UP and DOWN states. Previous studies have shown that these cortical UP/DOWN states affect the excitability of individual neurons in response to sensory stimuli, indicating that a significant amount of the trial-to-trial variability in neuronal responses can be attributed to ongoing fluctuations in network activity. However, as intracellular recordings are frequently not available, it is important to be able to estimate their occurrence purely from extracellular data. Here, we combine in vivo whole cell recordings from single neurons with multi-site extracellular microelectrode recordings, to quantify the performance of various approaches to predicting UP/DOWN states from the deep-layer local field potential (LFP). We find that UP/DOWN states in deep cortical layers of rat primary auditory cortex (A1) are predictable from the phase of LFP at low frequencies (< 4 Hz), and that the likelihood of a given state varies sinusoidally with the phase of LFP at these frequencies. We introduce a novel method of detecting cortical state by combining information concerning the phase of the LFP and ongoing multi-unit activity.

Muscarinic control of long-range GABAergic inhibition within the rhinal cortices.

The perirhinal cortex plays a critical role in memory formation, in part because it forms reciprocal connections with the neocortex and entorhinal cortex and is thus in a position to integrate and transfer higher-order information to and from the hippocampus. However, for reasons that remain unclear, perirhinal transfer of neocortical inputs to the entorhinal cortex occurs with a low probability. Using patch recordings in vitro and tract-tracing combined with GAD-67 immunohistochemistry, we show that the perirhinal cortex contains GABAergic neurons with long-range projections to superficial entorhinal cells. This finding challenges the traditional model of cortical inhibition in which all trans-areal inhibition is thought to be disynaptic because the axons of GABAergic interneurons are assumed to be confined within the area in which their somata are located. Moreover, consistent with recent studies indicating that the formation of perirhinal-dependent memories requires activation of muscarinic receptors, long-range IPSPs were presynaptically inhibited by M2 receptor activation. Overall, these results suggest that long-range feedforward inhibition regulates perirhinal transfer of neocortical inputs to the entorhinal cortex, but that cholinergic inputs can presynaptically adjust the impact of this control mechanism as a function of environmental contingencies.

General Principles of Neuronal Co-transmission: Insights From Multiple Model Systems.

It is now accepted that neurons contain and release multiple transmitter substances. However, we still have only limited insight into the regulation and functional effects of this co-transmission. Given that there are 200 or more neurotransmitters, the chemical complexity of the nervous system is daunting. This is made more-so by the fact that their interacting effects can generate diverse non-linear and novel consequences. The relatively poor history of pharmacological approaches likely reflects the fact that manipulating a transmitter system will not necessarily mimic its roles within the normal chemical environment of the nervous system (e.g., when it acts in parallel with co-transmitters). In this article, co-transmission is discussed in a range of systems [from invertebrate and lower vertebrate models, up to the mammalian peripheral and central nervous system (CNS)] to highlight approaches used, degree of understanding, and open questions and future directions. Finally, we offer some outlines of what we consider to be the general principles of co-transmission, as well as what we think are the most pressing general aspects that need to be addressed to move forward in our understanding of co-transmission.

Basal Forebrain and Brainstem Cholinergic Neurons Differentially Impact Amygdala Circuits and Learning-Related Behavior.

The central cholinergic system and the amygdala are important for motivation and mnemonic processes. Different cholinergic populations innervate the amygdala, but it is unclear how these projections impact amygdala processes. Using optogenetic circuit-mapping strategies in choline acetyltransferase (ChAT)-cre mice, we demonstrate that amygdala-projecting basal forebrain and brainstem ChAT-containing neurons can differentially affect amygdala circuits and behavior. Photo-activating ChAT terminals in vitro revealed the underlying synaptic impact of brainstem inputs to the central lateral division to be excitatory, mediated via the synergistic glutamatergic activation of AMPA and NMDA receptors. In contrast, stimulating basal forebrain inputs to the basal nucleus resulted in endogenous acetylcholine (ACh) release, resulting in biphasic inhibition-excitation responses onto principal neurons. Such response profiles are physiological hallmarks of neural oscillations and could thus form the basis of ACh-mediated rhythmicity in amygdala networks. Consistent with this, in vivo basal forebrain ChAT+ activation strengthened amygdala basal nucleus theta and gamma frequency rhythmicity, both of which continued for seconds after stimulation and were dependent on local muscarinic and nicotinic receptor activation, respectively. Activation of brainstem ChAT-containing neurons, however, resulted in a transient increase in central lateral amygdala activity that was independent of cholinergic receptors. In addition, driving these respective inputs in behaving animals induced opposing appetitive and defensive learning-related behavioral changes. Because learning and memory are supported by both cellular and network-level processes in central cholinergic and amygdala networks, these results provide a route by which distinct cholinergic inputs can convey salient information to the amygdala and promote associative biophysical changes that underlie emotional memories.

Accumbal Cholinergic Interneurons Differentially Influence Motivation Related to Satiety Signaling.

Satiety, rather than all or none, can instead be viewed as a cumulative decrease in the drive to eat that develops over the course of a meal. The nucleus accumbens (NAc) is known to play a critical role in this type of value reappraisal, but the underlying circuits that influence such processes are unclear. Although NAc cholinergic interneurons (CINs) comprise only a small proportion of NAc neurons, their local impact on reward-based processes provides a candidate cell population for investigating the neural underpinnings of satiety. The present research therefore aimed to determine the role of NAc-CINs in motivation for food reinforcers in relation to satiety signaling. Through bidirectional control of CIN activity in mice, we show that when motivated by food restriction, increasing CIN activity led to a reduction in palatable food consumption while reducing CIN excitability enhanced food intake. These activity-dependent changes developed only late in the session and were unlikely to be driven by the innate reinforcer strength, suggesting that CIN modulation was instead impacting the cumulative change in motivation underlying satiety signaling. We propose that on a circuit level, an overall increase in inhibitory tone onto NAc output neurons played a role in the behavioral results, as activating NAc-CINs led to an inhibition of medium spiny neurons that was dependent on nicotinic receptor activation. Our results reveal an important role for NAc-CINs in controlling motivation for food intake and additionally provide a circuit-level framework for investigating the endogenous cholinergic circuits that signal satiety.

Sex difference in physical activity, energy expenditure and obesity driven by a subpopulation of hypothalamic POMC neurons.

OBJECTIVE: Obesity is one of the primary healthcare challenges of the 21st century. Signals relaying information regarding energy needs are integrated within the brain to influence body weight. Central among these integration nodes are the brain pro-opiomelanocortin (POMC) peptides, perturbations of which disrupt energy balance and promote severe obesity. However, POMC neurons are neurochemically diverse and the crucial source of POMC peptides that regulate energy homeostasis and body weight remains to be fully clarified. METHODS: Given that a 5-hydroxytryptamine 2c receptor (5-HT2CR) agonist is a current obesity medication and 5-HT2CR agonist's effects on appetite are primarily mediated via POMC neurons, we hypothesized that a critical source of POMC regulating food intake and body weight is specifically synthesized in cells containing 5-HT2CRs. To exclusively manipulate Pomc synthesis only within 5-HT2CR containing cells, we generated a novel 5-HT 2C R (CRE) mouse line and intercrossed it with Cre recombinase-dependent and hypothalamic specific reactivatable Pomc (NEO) mice to restrict Pomc synthesis to the subset of hypothalamic cells containing 5-HT2CRs. This provided a means to clarify the specific contribution of a defined subgroup of POMC peptides in energy balance and body weight. RESULTS: Here we transform genetically programed obese and hyperinsulinemic male mice lacking hypothalamic Pomc with increased appetite, reduced physical activity and compromised brown adipose tissue (BAT) into lean, healthy mice via targeted restoration of Pomc function only within 5-HT2CR expressing cells. Remarkably, the same metabolic transformation does not occur in females, who despite corrected feeding behavior and normalized insulin levels remain physically inactive, have lower energy expenditure, compromised BAT and develop obesity. CONCLUSIONS: These data provide support for the functional heterogeneity of hypothalamic POMC neurons, revealing that Pomc expression within 5-HT2CR expressing neurons is sufficient to regulate energy intake and insulin sensitivity in male and female mice. However, an unexpected sex difference in the function of this subset of POMC neurons was identified with regard to energy expenditure. We reveal that a large sex difference in physical activity, energy expenditure and the development of obesity is driven by this subpopulation, which constitutes approximately 40% of all POMC neurons in the hypothalamic arcuate nucleus. This may have broad implications for strategies utilized to combat obesity, which at present largely ignore the sex of the obese individual.

Nicotinic α4 Receptor-Mediated Cholinergic Influences on Food Intake and Activity Patterns in Hypothalamic Circuits.

Nicotinic acetylcholine receptors (nAChRs) play an important role in regulating appetite and have been shown to do so by influencing neural activity in the hypothalamus. To shed light on the hypothalamic circuits governing acetylcholine's (ACh) regulation of appetite this study investigated the influence of hypothalamic nAChRs expressing the α4 subunit. We found that antagonizing the α4ß2 nAChR locally in the lateral hypothalamus with di-hydro-ß-erythroidine (DHßE), an α4 nAChR antagonist with moderate affinity, caused an increase in food intake following free access to food after a 12 hour fast, compared to saline-infused animals. Immunocytochemical analysis revealed that orexin/hypocretin (HO), oxytocin, and tyrosine hydroxylase (TH)-containing neurons in the A13 and A12 of the hypothalamus expressed the nAChR α4 subunit in varying amounts (34%, 42%, 50%, and 51%, respectively) whereas melanin concentrating hormone (MCH) neurons did not, suggesting that DHßE-mediated increases in food intake may be due to a direct activation of specific hypothalamic circuits. Systemic DHßE (2 mg/kg) administration similarly increased food intake following a 12 hour fast. In these animals a subpopulation of orexin/hypocretin neurons showed elevated activity compared to control animals and MCH neuronal activity was overall lower as measured by expression of the immediate early gene marker for neuronal activity cFos. However, oxytocin neurons in the paraventricular hypothalamus and TH-containing neurons in the A13 and A12 did not show differential activity patterns. These results indicate that various neurochemically distinct hypothalamic populations are under the influence of α4ß2 nAChRs and that cholinergic inputs to the lateral hypothalamus can affect satiety signals through activation of local α4ß2 nAChR-mediated transmission.

Coreleased orexin and glutamate evoke nonredundant spike outputs and computations in histamine neurons.

Stable wakefulness requires orexin/hypocretin neurons (OHNs) and OHR2 receptors. OHNs sense diverse environmental cues and control arousal accordingly. For unknown reasons, OHNs contain multiple excitatory transmitters, including OH peptides and glutamate. To analyze their cotransmission within computational frameworks for control, we optogenetically stimulated OHNs and examined resulting outputs (spike patterns) in a downstream arousal regulator, the histamine neurons (HANs). OHR2s were essential for sustained HAN outputs. OHR2-dependent HAN output increased linearly during constant OHN input, suggesting that the OHN→HAN(OHR2) module may function as an integral controller. OHN stimulation evoked OHR2-dependent slow postsynaptic currents, similar to midnanomolar OH concentrations. Conversely, glutamate-dependent output transiently communicated OHN input onset, peaking rapidly then decaying alongside OHN→HAN glutamate currents. Blocking glutamate-driven spiking did not affect OH-driven spiking and vice versa, suggesting isolation (low cross-modulation) of outputs. Therefore, in arousal regulators, cotransmitters may translate distinct features of OHN activity into parallel, nonredundant control signals for downstream effectors.