Pituitary Adenylate Cylase-Activating Polypeptide Receptor: Multiple Signaling Pathways Involved in Energy Homeostasis

Pituitary adenylate cyclase activating polypeptide (PACAP) exerts pleiotropic effects on ventromedial nuclei (VMN) of the hypothalamus and its control of feeding and energy expenditure through the Type I PAC1 receptor (PAC1R). However, the endogenous role of PAC1R’s in the VMN and the downstream signaling responsible for PACAP’s effects are unknown.To determine the endogenous role of PAC1Rs and signaling that may explain PACAP’s pleiotropic effects, we knocked down VMN PAC1Rs and pharmacologically inhibited PKA, PKC and PAC1R trafficking. Knocking down PAC1Rs increased meal sizes, reduced total number of meals, and induced body weight gain. Inhibition of either PKA or PKC alone in awake male Sprague Dawley rats, attenuated PACAP’s anorectic effects during the dark phase. However, PKA or PKC inhibition resulted in a potentiation of PACAP’s hypophagic effects during the light phase. Additionally, PKA or PKC inhibition augmented PACAP’s thermogenic effects during the light phase, whereas only PKA inhibition augmented PACAP’s locomotor effects given that PKC inhibition had no effect. PACAP administration in the VMN induces PAC1R surface trafficking into the cytosol which was blocked by endocytosis inhibitors. Subsequently, inhibition of PAC1R trafficking into the cytosol attenuated PACAP-induced hypophagia. PACAP signaling replicates the effects of leptin administration in the VMN and appears to enable leptin regulation of energy homeostasis. However, the manner in which PACAP influences leptin signaling is unknown. We used co-immunoprecipitation to show that VMN PAC1 and leptin receptors are found in the same cell, and they form an immunocomplex. Inhibiting downstream effectors of PACAP signaling, such as PKA and PKC, enhanced or prevented leptin signaling respectively. The current findings revealed that endogenous PACAP signaling in the VMN has a potent regulatory influence over both energy intake in the form of feeding, and energy output via thermogenesis and locomotor activity. Moreover, PACAP actions in the VMN share a nearly identical sequelae to leptin administration in the same brain region suggesting that these two neuropeptides could functionally intersect. These experiments explored VMN PAC1Rs dependence on PKA, PKC, and receptor trafficking to mediate PACAP’s pleiotropic effects on feeding and metabolism as well as potential intersecting points with leptin receptor signaling

Inhibition of Food Intake by PACAP in the Hypothalamic Ventromedial Nuclei is Mediated by NMDA Receptors

Central injections of pituitary adenylate cyclase-activating polypeptide (PACAP) into the ventromedial nuclei (VMN) of the hypothalamus produce hypophagia that is dependent upon the PAC1 receptor; however, the signaling downstream of this receptor in the VMN is unknown. Though PACAP signaling has many targets, this neuropeptide has been shown to influence glutamate signaling in several brain regions through mechanisms involving NMDA receptor potentiation via activation of the Src family of protein tyrosine kinases. With this in mind, we examined the Src-NMDA receptor signaling pathway as a target for PACAP signaling in the VMN that may mediate its effects on feeding behavior. Under nocturnal feeding conditions, NMDA receptor antagonism prior to PACAP administration into the VMN attenuated PACAP-mediated decreases in feeding suggesting that glutamatergic signaling via NMDA receptors is necessary for PACAP-induced hypophagia. Furthermore, PACAP administration into the VMN resulted in increased tyrosine phosphorylation of the GluN2B subunit of the NMDA receptor, and inhibition of Src kinase activity also blocked the effects of PACAP administration into the VMN on feeding behavior. These results indicate that PACAP neurotransmission in the VMN likely augments glutamate signaling by potentiating NMDA receptors activity through the tyrosine phosphorylation events mediated by the Src kinase family, and modulation of NMDA receptor activity by PACAP in the hypothalamus may be a primary mechanism for its regulation of food intake

Global Connectivity and Function of Descending Spinal Input Revealed by 3D Microscopy and Retrograde Transduction

The brain communicates with the spinal cord through numerous axon tracts that arise from discrete nuclei, transmit distinct functions, and often collateralize to facilitate the coordination of descending commands. This complexity presents a major challenge to interpreting functional outcomes from therapies that target supraspinal connectivity after injury or disease, while the wide distribution of supraspinal nuclei complicates the delivery of therapeutics. Here we harness retrograde viral vectors to overcome these challenges. We demonstrate that injection of AAV2-Retro to the cervical spinal cord of adult female mice results in highly efficient transduction of supraspinal populations throughout the brainstem, midbrain, and cortex. Some supraspinal populations, including corticospinal and rubrospinal neurons, were transduced with \u3e90% efficiency, with robust transgene expression within 3 d of injection. In contrast, propriospinal and raphe spinal neurons showed much lower rates of retrograde transduction. Using tissue clearing and light-sheet microscopy we present detailed visualizations of descending axons tracts and create a mesoscopic projectome for the spinal cord. Moreover, chemogenetic silencing of supraspinal neurons with retrograde vectors resulted in complete and reversible forelimb paralysis, illustrating effective modulation of supraspinal function. Retrograde vectors were also highly efficient when injected after spinal injury, highlighting therapeutic potential. These data provide a global view of supraspinal connectivity and illustrate the potential of retrograde vectors to parse the functional contributions of supraspinal inputs

The Application of CRISPR Technology to High Content Screening in Primary Neurons

Axon growth is coordinated by multiple interacting proteins that remain incompletely characterized. High content screening (HCS), in which manipulation of candidate genes is combined with rapid image analysis of phenotypic effects, has emerged as a powerful technique to identify key regulators of axon outgrowth. Here we explore the utility of a genome editingapproach referred to as CRISPR (Clustered Regularly Interspersed Palindromic Repeats) for knockout screening in primary neurons. In the CRISPR approach a DNA-cleaving Cas enzyme is guided to genomic target sequences by user-created guide RNA (sgRNA), where it initiates a double-stranded break that ultimately results in frameshift mutation and loss of protein production. Using electroporation of plasmid DNA that co-expresses Cas9enzyme and sgRNA, we first verified the ability of CRISPR targeting to achieve protein-level knockdown in cultured postnatal cortical neurons. Targeted proteins included NeuN (RbFox3) and PTEN, a well-studied regulator of axon growth. Effective knockdown lagged at least four days behind transfection, but targeted proteins were eventually undetectable by immunohistochemistry in \u3e 80% of transfected cells. Consistent with this, anti-PTEN sgRNA produced no changes in neurite outgrowth when assessed three days post-transfection. When week-long cultures were replated, however, PTEN knockdown consistently increased neurite lengths. These CRISPR-mediated PTEN effects were achieved using multi-well transfection and automated phenotypic analysis, indicating the suitability of PTEN as a positive control for future CRISPR-based screening efforts. Combined, these data establish an example of CRISPR-mediated protein knockdown in primary cortical neurons and its compatibility with HCS workflows

\u3cem\u3eN\u3c/em\u3e-acetylcysteine Decreases Binge Eating in a Rodent Model

Binge-eating behavior involves rapid consumption of highly palatable foods leading to increased weight gain. Feeding in binge disorders resembles other compulsive behaviors, many of which are responsive to N-acetylcysteine (NAC), which is a cysteine prodrug often used to promote non-vesicular glutamate release by a cystine–glutamate antiporter. To examine the potential for NAC to alter a form of compulsive eating, we examined the impact of NAC on binge eating in a rodent model. Specifically, we monitored consumption of standard chow and a high-fat, high carbohydrate western diet (WD) in a rodent limited-access binge paradigm. Before each session, rats received either a systemic or intraventricular injection of NAC. Both systemic and central administration of NAC resulted in significant reductions of binge eating the WD without decreasing standard chow consumption. The reduction in WD was not attributable to general malaise as NAC did not produce condition taste aversion. These results are consistent with the clinical evidence of NAC to reduce or reverse compulsive behaviors, such as, drug addiction, skin picking and hair pulling

KLF6 and STAT3 Co-Occupy Regulatory DNA and Functionally Synergize to Promote Axon Growth in CNS Neurons

The failure of axon regeneration in the CNS limits recovery from damage and disease. Members of the KLF family of transcription factors can exert both positive and negative effects on axon regeneration, but the underlying mechanisms are unclear. Here we show that forced expression of KLF6 promotes axon regeneration by corticospinal tract neurons in the injured spinal cord. RNA sequencing identified 454 genes whose expression changed upon forced KLF6 expression in vitro, including sub-networks that were highly enriched for functions relevant to axon extension including cytoskeleton remodeling, lipid synthesis, and bioenergetics. In addition, promoter analysis predicted a functional interaction between KLF6 and a second transcription factor, STAT3, and genome-wide footprinting using ATAC-Seq data confirmed frequent co-occupancy. Co-expression of the two factors yielded a synergistic elevation of neurite growth in vitro. These data clarify the transcriptional control of axon growth and point the way toward novel interventions to promote CNS regeneration

Expression Patterns of BDNF with Central Anorexigenic Signaling Pathways Involving PACAP in the Hypothalamic Ventromedial Nuclei

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid polypeptide belonging to the secretin super family of peptides. PACAP binds to its type 1 receptor (PAC1R) with greater affinity than for the receptors for vasoactive intestinal polypeptides (VIP), VPAC1 and VPAC2. Although mRNA for PACAP and its receptor PAC1R are found throughout the central nervous system, they are abundantly expressed in the hypothalamic ventromedial nuclei (VMN). In male Sprague Dawley rats, infusions of PACAP into the VMN produce a robust decrease in food intake with concomitant increased energy expenditure, decreased body weight, and significantly elevated brain-derived neurotrophic factor (BDNF) mRNA expression in the VMN. This latter effect of PACAP on BDNF mRNA expression has been shown to occur in other brain regions. Exogenous BDNF in the VMN regulates energy homeostasis in a manner similar to that of PACAP with decreased feeding and increased metabolism. Although the physiological responses to individual PACAP and BDNF infusions in the VMN lead to decreased feeding behavior and body weight loss, the anatomical distribution of these two cell signals in the VMN has not been established. PACAP-induced changes in BDNF mRNA expression in the VMN may reveal an important interaction with PACAP signaling in the control of feeding behavior. In the present study, we have employed double-labeled fluorescent in-situ hybridization (FISH) to examine the expression patterns of PACAP, PAC1R and BDNF mRNA containing neuronal cells. In the VMN, PACAP mRNA expressing cells co-express BDNF, PAC1R, and VGLUT2. BDNF mRNA expressing cells co-express PAC1R and PACAP. Coupled with previous behavioral data demonstrating PACAP- and BDNF-induced changes in feeding behavior, the co-expression of BDNF with PACAP and PAC1R mRNA in the VMN suggest a potential functional relationship between the two signaling peptides in the regulation of energy homeostasis. The specific and integrated contributions of PACAP and BDNF in the VMN towards regulating energy homeostasis and feeding behavior still remain to be studied

Pituitary Adenylate-Cyclase Activating Polypeptide Regulates Hunger- and Palatability-Induced Binge Eating

While pituitary adenylate cyclase activating polypeptide (PACAP) signaling in the hypothalamic ventromedial nuclei (VMN) has been shown to regulate feeding, a challenge in unmasking a role for this peptide in obesity is that excess feeding can involve numerous mechanisms including homeostatic (hunger) and hedonic-related (palatability) drives. In these studies, we first isolated distinct feeding drives by developing a novel model of binge behavior in which homeostatic-driven feeding was temporally separated from feeding driven by food palatability. We found that stimulation of the VMN, achieved by local microinjections of AMPA, decreased standard chow consumption in food-restricted rats (e.g., homeostatic feeding); surprisingly, this manipulation failed to alter palatable food consumption in satiated rats (e.g., hedonic feeding). In contrast, inhibition of the nucleus accumbens (NAc), through local microinjections of GABA receptor agonists baclofen and muscimol, decreased hedonic feeding without altering homeostatic feeding. PACAP microinjections produced the site-specific changes in synaptic transmission needed to decrease feeding via VMN or NAc circuitry. PACAP into the NAc mimicked the actions of GABA agonists by reducing hedonic feeding without altering homeostatic feeding. In contrast, PACAP into the VMN mimicked the actions of AMPA by decreasing homeostatic feeding without affecting hedonic feeding. Slice electrophysiology recordings verified PACAP excitation of VMN neurons and inhibition of NAc neurons. These data suggest that the VMN and NAc regulate distinct circuits giving rise to unique feeding drives, but that both can be regulated by the neuropeptide PACAP to potentially curb excessive eating stemming from either drive

PACAP and Cocaine Reinstatement: A Neuropeptide Expressed by Corticostriatal Neurons that Regulates Nucleus Accumbens Astrocytes

Drug addiction involves heightened relapse vulnerability arising from persistent drug-induced neuro-adaptations, including a) hypofrontality which is thought to reflect reduced firing of cortical afferents to the nucleus accumbens (NAcc) and b) altered glutamate homeostasis in NAcc that likely involves reduced glutamate release and uptake by astrocytes. An important question is whether these forms of pathological plasticity are functionally linked such that reduced corticostriatal firing may result in aberrant regulation of astrocytes in the NAcc. To begin to evaluate this possibility, we first determined whether neurons regulate system xc- (Sxc) activity, a mechanism of non-vesicular glutamate release by astrocytes. We found that the rate of Sxc activity in astrocyte cultures was significantly increased in cells exposed to neuronal conditioned media achieved using neuronal inserts. These experiments demonstrate that releasable neuronal factors significantly upregulate Sxc activity. We hypothesize that the pituitary adenylyl cyclase activating peptide (PACAP) may be the neuronal factor regulating glutamate release by astrocytes involving Sxc. First we determined that PACAP mimics a neuronal insert in that it significantly upregulates Sxc activity in astrocytes. Next, we verified the expression of PACAP in neurons from the prefrontal cortex (PFC) projecting to NAcc. Together, these data support the hypothesis that reduced corticostriatal firing may result in decreased PACAP release in NAcc which could potentially blunt Sxc activity in NAcc astrocytes. To determine whether this would impact relapse vulnerability, we microinjected PACAP into the NAcc and found that this significantly reduced cocaine-primed reinstatement, suggesting that increased PACAP signaling, consistent with other approaches capable of increasing Sxc activity, may blunt relapse vulnerability. In order to determine whether reduced PACAP signaling is sufficient to increase relapse vulnerability, we microinjected the PAC1R inhibitor PACAP6-38 into the NAcc. Preliminary data indicate that this is sufficient to produce an increase in cocaine reinstatement. Collectively, these studies demonstrate that neuropeptide PACAP is a powerful regulator of cocaine-related behaviors, likely through the modulation of glutamate homeostasis as maintained by astrocytes. As a result, an unrecognized consequence of hypofrontality may be impairing neuron-astrocyte interactions in a manner that determines the magnitude of relapse vulnerability

A New Obesity Model Reveals the Hypophagic Properties of PACAP Involve the Regulation of Homeostatic Feeding in the Ventromedial Hypothalamic Nucleus and Hedonic Feeding in the Nucleus Accumbens

Binge eating in humans is a complex disorder that often involves discrete, compulsive feeding sessions of highly palatable foods even in the absence of a deprivation state or hunger. Binging can be effectively modeled in rodents by providing subjects with limited access to a palatable food source (Western Diet; WD) as an adjunct to ad lib access to normal chow (Standard Chow; SC). Although this design recapitulates several fundamental characteristics observed in binge eating disorder, the binge eating observed in this paradigm is likely a product of both hedonic and homeostatic drives with the need to balance energy stores still present. To isolate these feeding drives, we have developed a novel feeding regimen that modifies the classic limited access binge model to effectively delineate and separate homeostatic feeding from motivational feeding. This is achieved by entraining male Sprague-Dawley rats to a restricted feeding schedule (two hours per day) of SC followed by a short 15 minute limited access meal of either SC or WD (Restrict Fed-Limited Access; RFLA). The RFLA paradigm allows for the examination of pituitary adenylate-cyclase activating polypeptide (PACAP) on palatable food consumption in a fully satiated subject. PACAP has previously been shown to suppress feeding behavior when injected into the hypothalamus. In the current study, PACAP injected into the ventromedial hypothalamic nuclei (VMN) suppressed the two hour homeostatic SC meal, but not the subsequent 15 minute limited access meal of WD. By contrast, PACAP bilaterally administered into the nucleus accumbens (NAc) produced the opposite effect with PACAP suppressing the consumption of WD but not SC. Thus, PACAP mediated signaling in the VMN appears to be involved in homeostatic regulation of energy stores, whereas PACAP signaling in the NAc regulates feeding driven by palatability or hedonic qualities