Unexpected role of interferon-γ in regulating neuronal connectivity and social behaviour.

Immune dysfunction is commonly associated with several neurological and mental disorders. Although the mechanisms by which peripheral immunity may influence neuronal function are largely unknown, recent findings implicate meningeal immunity influencing behaviour, such as spatial learning and memory. Here we show that meningeal immunity is also critical for social behaviour; mice deficient in adaptive immunity exhibit social deficits and hyper-connectivity of fronto-cortical brain regions. Associations between rodent transcriptomes from brain and cellular transcriptomes in response to T-cell-derived cytokines suggest a strong interaction between social behaviour and interferon-γ (IFN-γ)-driven responses. Concordantly, we demonstrate that inhibitory neurons respond to IFN-γ and increase GABAergic (γ-aminobutyric-acid) currents in projection neurons, suggesting that IFN-γ is a molecular link between meningeal immunity and neural circuits recruited for social behaviour. Meta-analysis of the transcriptomes of a range of organisms reveals that rodents, fish, and flies elevate IFN-γ/JAK-STAT-dependent gene signatures in a social context, suggesting that the IFN-γ signalling pathway could mediate a co-evolutionary link between social/aggregation behaviour and an efficient anti-pathogen response. This study implicates adaptive immune dysfunction, in particular IFN-γ, in disorders characterized by social dysfunction and suggests a co-evolutionary link between social behaviour and an anti-pathogen immune response driven by IFN-γ signalling.

Pharmacological rescue of Ras signaling, GluA1-dependent synaptic plasticity, and learning deficits in a fragile X model.

Fragile X syndrome, caused by the loss of Fmr1 gene function, is the most common form of inherited mental retardation, with no effective treatment. Using a tractable animal model, we investigated mechanisms of action of a few FDA-approved psychoactive drugs that modestly benefit the cognitive performance in fragile X patients. Here we report that compounds activating serotonin (5HT) subtype 2B receptors (5HT2B-Rs) or dopamine (DA) subtype 1-like receptors (D1-Rs) and/or those inhibiting 5HT2A-Rs or D2-Rs moderately enhance Ras-PI3K/PKB signaling input, GluA1-dependent synaptic plasticity, and learning in Fmr1 knockout mice. Unexpectedly, combinations of these 5HT and DA compounds at low doses synergistically stimulate Ras-PI3K/PKB signal transduction and GluA1-dependent synaptic plasticity and remarkably restore normal learning in Fmr1 knockout mice without causing anxiety-related side effects. These findings suggest that properly dosed and combined FDA-approved psychoactive drugs may effectively treat the cognitive impairment associated with fragile X syndrome.

Phosphosite T674A mutation in kinesin family member 3A fails to reproduce tissue and ciliary defects characteristic of CILK1 loss of function.

BACKGROUND: Kinesin family member 3A (KIF3A) is a molecular motor protein in the heterotrimeric kinesin-2 complex that drives anterograde intraflagellar transport. This process plays a pivotal role in both biogenesis and maintenance of the primary cilium that supports tissue development. Ciliogenesis associated kinase 1 (CILK1) phosphorylates human KIF3A at Thr672. CILK1 loss of function causes ciliopathies that manifest profound and multiplex developmental defects, including hydrocephalus, polydactyly, shortened and hypoplastic bones and alveoli airspace deficiency, leading to perinatal lethality. Prior studies have raised the hypothesis that CILK1 phosphorylation of KIF3A is critical for its regulation of organ development. RESULTS: We produced a mouse model with phosphorylation site Thr674 in mouse Kif3a mutated to Ala. Kif3a T674A homozygotes are viable and exhibit no skeletal and brain abnormalities, and only mildly reduced airspace in alveoli. Mouse embryonic fibroblasts carrying Kif3a T674A mutation show a normal rate of ciliation and a moderate increase in cilia length. CONCLUSION: These results indicate that eliminating Kif3a Thr674 phosphorylation by Cilk1 is insufficient to reproduce the severe developmental defects in ciliopathies caused by Cilk1 loss of function. This suggests KIF3A-Thr672 phosphorylation by CILK1 is not essential for tissue development and other substrates are involved in CILK1 ciliopathies.

Impact of Menthol on Oral Nicotine Consumption in Female and Male Sprague Dawley Rats.

INTRODUCTION: One of the preferable flavors in oral nicotine delivery systems is menthol which masks the harshness of tobacco. However, possible interactions between oral menthol and nicotine on intake and preference remain unclear. Therefore, we aimed to determine the impact of menthol on oral nicotine consumption. METHODS: Adult Sprague Dawley female and male rats (n = 8 per group) were given a choice of water or drug solution by using two-bottle free choice paradigm for 2 weeks: vehicle (5% ethanol), nicotine (20 mg/L), menthol (1 g/L) and mentholated nicotine groups. At the end of the study, plasma nicotine levels were determined. RESULTS: When rats were given a choice of nicotine or water, nicotine intake was similar between female and male rats. Menthol addition to nicotine solution significantly increased nicotine intake and preference in male but not female rats without a considerable effect on total fluid intake and body weight change in either sex. The average nicotine intake in male rats was 0.5 ± 0.05 and 1.4 ± 0.12 mg/kg/day for nicotine and menthol-nicotine combination (p < .05), respectively. The average nicotine intake in female rats was 0.6 ± 0.05 and 0.6 ± 0.03 mg/kg/day for nicotine and menthol-nicotine combination (p > .05), respectively. Plasma nicotine levels were not significantly different between the groups in either male (nicotine group: 20.8 ± 4.9, mentholated nicotine group: 31.9 ± 3.2 ng/mL) or female (nicotine group: 24.0 ± 3.3, mentholated nicotine group: 17.8 ± 2.9 ng/mL) rats (p > .05). CONCLUSIONS: Menthol increases oral nicotine consumption in male, but not female, rats. IMPLICATIONS: This study may provide data on the co-use of menthol and nicotine in smokeless tobacco, particularly oral dissolvable tobacco products.

Weight-independent effects of roux-en-Y gastric bypass on glucose homeostasis via melanocortin-4 receptors in mice and humans.

BACKGROUND & AIMS: Roux-en-Y gastric bypass (RYGB) improves glucose homeostasis independently of changes in body weight by unknown mechanisms. Melanocortin-4 receptors (MC4R) have weight-independent effects on glucose homeostasis, via autonomic neurons, and also might contribute to weight loss after RYGB. We investigated whether MC4Rs mediate effects of RYGB, such as its weight-independent effects on glucose homeostasis, in mice and humans. METHODS: We studied C57BL/6 mice with diet-induced obesity, MC4R-deficient mice, and mice that re-express MC4R specifically in autonomic neurons after RYGB or sham surgeries. We also sequenced the MC4R locus in patients undergoing RYGB to investigate diabetes resolution in carriers of rare MC4R variants. RESULTS: MC4Rs in autonomic brainstem neurons (including the parasympathetic dorsal motor vagus) mediated improved glucose homeostasis independent of changes in body weight. In contrast, MC4Rs in cholinergic preganglionic motor neurons (sympathetic and parasympathetic) mediated RYGB-induced increased energy expenditure and weight loss. Increased energy expenditure after RYGB is the predominant mechanism of weight loss and confers resistance to weight gain from a high-fat diet, the effects of which are MC4R-dependent. MC4R-dependent effects of RYGB still occurred in mice with Mc4r haplosufficiency, and early stage diabetes resolved at a similar rate in patients with rare variants of MC4R and noncarriers. However, carriers of MC4R (I251L), a rare variant associated with increased weight loss after RYGB and increased basal activity in vitro, were more likely to have early and weight-independent resolution of diabetes than noncarriers, indicating a role for MC4Rs in the effects of RYGB. CONCLUSIONS: MC4Rs in autonomic neurons mediate beneficial effects of RYGB, including weight-independent improved glucose homeostasis, in mice and humans.

Selective ablation of VIP interneurons in the rodent prefrontal cortex results in increased impulsivity.

It has been well-established that novelty-seeking and impulsivity are significant risk factors for the development of psychological disorders, including substance use disorder and behavioral addictions. While dysfunction in the prefrontal cortex is at the crux of these disorders, little is known at the cellular level about how alterations in neuron activity can drive changes in impulsivity and novelty seeking. We harnessed a cre-dependent caspase-3 ablation in both male and female mice to selectively ablate vasoactive intestinal peptide (VIP)-expressing interneurons in the prefrontal cortex to better explore how this microcircuit functions during specific behavioral tasks. Caspase-ablated animals had no changes in anxiety-like behaviors or hedonic food intake but had a specific increase in impulsive responding during longer trials in the three-choice serial reaction time test. Together, these data suggest a circuit-level mechanism in which VIP interneurons function as a gate to selectively respond during periods of high expectation.

Redefining the serotonergic system by genetic lineage.

Central serotonin-producing neurons are heterogeneous-differing in location, morphology, neurotoxin sensitivity and associated clinical disorders-but the underpinnings of this heterogeneity are largely unknown, as are the markers that distinguish physiological subtypes of serotonergic neurons. Here we redefined serotonergic subtypes on the basis of genetic programs that are differentially enacted in progenitor cells. We uncovered a molecular framework for the serotonergic system that, having genetic lineages as its basis, is likely to have physiological relevance and will permit access to genetically defined subtypes for manipulation.

Targeting oxidized phospholipids by AAV-based gene therapy in mice with established hepatic steatosis prevents progression to fibrosis.

Oxidized phosphatidylcholines (OxPCs) are implicated in chronic tissue damage. Hyperlipidemic LDL-R--deficient mice transgenic for an OxPC-recognizing IgM fragment (scFv-E06) are protected against nonalcoholic fatty liver disease (NAFLD). To examine the effect of OxPC elimination at different stages of NAFLD progression, we used cre-dependent, adeno-associated virus serotype 8-mediated expression of the single-chain variable fragment of E06 (AAV8-scFv-E06) in hepatocytes of albumin-cre mice. AAV8-induced expression of scFv-E06 at the start of FPC diet protected mice from developing hepatic steatosis. Independently, expression of scFv-E06 in mice with established steatosis prevented the progression to hepatic fibrosis. Mass spectrometry-based oxophospho-lipidomics identified individual OxPC species that were reduced by scFv-E06 expression. In vitro, identified OxPC species dysregulated mitochondrial metabolism and gene expression in hepatocytes and hepatic stellate cells. We demonstrate that individual OxPC species independently affect disease initiation and progression from hepatic steatosis to steatohepatitis, and that AAV-mediated expression of scFv-E06 is an effective therapeutic intervention.

Glucose-sensing glucagon-like peptide-1 receptor neurons in the dorsomedial hypothalamus regulate glucose metabolism.

Glucagon-like peptide-1 (GLP-1) regulates energy homeostasis via activation of the GLP-1 receptors (GLP-1Rs) in the central nervous system. However, the mechanism by which the central GLP-1 signal controls blood glucose levels, especially in different nutrient states, remains unclear. Here, we defined a population of glucose-sensing GLP-1R neurons in the dorsomedial hypothalamic nucleus (DMH), by which endogenous GLP-1 decreases glucose levels via the cross-talk between the hypothalamus and pancreas. Specifically, we illustrated the sufficiency and necessity of DMHGLP-1R in glucose regulation. The activation of the DMHGLP-1R neurons is mediated by a cAMP-PKA-dependent inhibition of a delayed rectifier potassium current. We also dissected a descending control of DMHGLP-1R -dorsal motor nucleus of the vagus nerve (DMV)-pancreas activity that can regulate glucose levels by increasing insulin release. Thus, our results illustrate how central GLP-1 action in the DMH can induce a nutrient state-dependent reduction in blood glucose level.

Segregation of acute leptin and insulin effects in distinct populations of arcuate proopiomelanocortin neurons.

Acute leptin administration results in a depolarization and concomitant increase in the firing rate of a subpopulation of arcuate proopiomelanocortin (POMC) cells. This rapid activation of POMC cells has been implicated as a cellular correlate of leptin effects on energy balance. In contrast to leptin, insulin inhibits the activity of some POMC neurons. Several studies have described a "cross talk" between leptin and insulin within the mediobasal hypothalamus via the intracellular enzyme, phosphoinositol-3-kinase (PI3K). Interestingly, both insulin and leptin regulate POMC cellular activity by activation of PI3K; however, it is unclear whether leptin and insulin effects are observed in similar or distinct populations of POMC cells. We therefore used dual label immunohistochemistry/in situ hybridization and whole-cell patch-clamp electrophysiology to map insulin and leptin responsive arcuate POMC neurons. Leptin-induced Fos activity within arcuate POMC neurons was localized separate from POMC neurons that express insulin receptor. Moreover, acute responses to leptin and insulin were largely segregated in distinct subpopulations of POMC cells. Collectively, these data suggest that cross talk between leptin and insulin occurs within a network of cells rather than within individual POMC neurons.

Networking of glucagon-like peptide-1 axons with GnRH neurons in the basal forebrain of male mice revealed by 3DISCO-based immunocytochemistry and optogenetics.

Glucagon-like peptide-1 (GLP-1) regulates reproduction centrally, although, the neuroanatomical basis of the process is unknown. Therefore, the putative networking of the central GLP-1 and gonadotropin-releasing hormone (GnRH) systems was addressed in male mice using whole mount immunocytochemistry and optogenetics. Enhanced antibody penetration and optical clearing procedures applied to 500-1000 µm thick basal forebrain slices allowed the simultaneous visualization of the two distinct systems in the basal forebrain. Beaded GLP-1-IR axons innervated about a quarter of GnRH neurons (23.2 ± 1.4%) forming either single or multiple contacts. GnRH dendrites received a more intense GLP-1 innervation (64.6 ± 0.03%) than perikarya (35.4 ± 0.03%). The physiological significance of the innervation was examined by optogenetic activation of channelrhodopsin-2 (ChR2)-expressing axons of preproglucagon (GCG) neurons upon the firing of GnRH neurons by patch clamp electrophysiology in acute brain slices of triple transgenic mice (Gcg-cre/ChR2/GFP-GnRH). High-frequency laser beam stimulation (20 Hz, 10 ms pulse width, 3 mW laser power) of ChR2-expressing GCG axons in the mPOA increased the firing rate of GnRH neurons (by 75 ± 17.3%, p = 0.0007). Application of the GLP-1 receptor antagonist, Exendin-3-(9-39) (1 µM), prior to the photo-stimulation, abolished the facilitatory effect. In contrast, low-frequency trains of laser pulses (0.2 Hz, 60 pulses) had no effect on the spontaneous postsynaptic currents of GnRH neurons. The findings indicate a direct wiring of GLP-1 neurons with GnRH cells which route is excitatory for the GnRH system. The pathway may relay metabolic signals to GnRH neurons and synchronize metabolism with reproduction.

Orexin neurons and the TASK of glucosensing.

Relatively little is known about the mechanisms that link changing levels of glucose and neuronal activity. A paper in the current issue of Neuron by Burdakov et al. demonstrates that orexin/hypocretin neurons are inhibited by rising glucose in part due to membrane potential effects mediated by tandem-pore K(+) (K(2P)) channels. The findings may shed light on the mechanisms that link hypoglycemia and coordinated arousal and autonomic responses.

Leptin suppresses development of GLP-1 inputs to the paraventricular nucleus of the hypothalamus.

The nucleus of the solitary tract (NTS) is critical for the central integration of signals from visceral organs and contains preproglucagon (PPG) neurons, which express leptin receptors in the mouse and send direct projections to the paraventricular nucleus of the hypothalamus (PVH). Here, we visualized projections of PPG neurons in leptin-deficient Lepob/ob mice and found that projections from PPG neurons are elevated compared with controls, and PPG projections were normalized by targeted rescue of leptin receptors in LepRbTB/TB mice, which lack functional neuronal leptin receptors. Moreover, Lepob/ob and LepRbTB/TB mice displayed increased levels of neuronal activation in the PVH following vagal stimulation, and whole-cell patch recordings of GLP-1 receptor-expressing PVH neurons revealed enhanced excitatory neurotransmission, suggesting that leptin acts cell autonomously to suppress representation of excitatory afferents from PPG neurons, thereby diminishing the impact of visceral sensory information on GLP-1 receptor-expressing neurons in the PVH.

Publisher Correction: Genetic variation affects binge feeding behavior in female inbred mouse strains.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Dopamine Signaling in the Suprachiasmatic Nucleus Enables Weight Gain Associated with Hedonic Feeding.

The widespread availability of energy-dense, rewarding foods is correlated with the increased incidence of obesity across the globe. Overeating during mealtimes and unscheduled snacking disrupts timed metabolic processes, which further contribute to weight gain. The neuronal mechanism by which the consumption of energy-dense food restructures the timing of feeding is poorly understood. Here, we demonstrate that dopaminergic signaling within the suprachiasmatic nucleus (SCN), the central circadian pacemaker, disrupts the timing of feeding, resulting in overconsumption of food. D1 dopamine receptor (Drd1)-null mice are resistant to diet-induced obesity, metabolic disease, and circadian disruption associated with energy-dense diets. Conversely, genetic rescue of Drd1 expression within the SCN restores diet-induced overconsumption, weight gain, and obesogenic symptoms. Access to rewarding food increases SCN dopamine turnover, and elevated Drd1-signaling decreases SCN neuronal activity, which we posit disinhibits downstream orexigenic responses. These findings define a connection between the reward and circadian pathways in the regulation of pathological calorie consumption.

Shared long-range regulatory elements coordinate expression of a gene cluster encoding nicotinic receptor heteromeric subtypes.

The nicotinic acetylcholine receptor (nAChR) beta4/alpha3/alpha5 gene cluster encodes several heteromeric transmitter receptor subtypes that are essential for cholinergic synaptic transmission in adrenal gland, autonomic ganglia, pineal gland, and several nuclei in the central nervous system. However, the transcriptional mechanisms coordinating expression of these subunit genes in different cell populations are unknown. Here, we used transgenic methods to investigate long-range transcriptional control of the cluster. A 132-kb P1-derived artificial chromosome (PAC) encoding the rat cluster recapitulated the neurally- and endocrine-restricted expression patterns of the endogenous beta4/alpha3/alpha5 genes. Mutation of ETS factor binding sites in an enhancer, beta43', embedded in the beta4 3'-untranslated exon resulted in greatly diminished beta4, alpha3, and alpha5 expression in adrenal gland and to a lesser extent in the superior cervical ganglion (SCG) but not in other tissues. Phylogenetic sequence analyses revealed several conserved noncoding regions (CNRs) upstream of beta4 and alpha5. Deletion of one of them (CNR4) located 20 kb upstream of beta4 resulted in a dramatic decrease in beta4 and alpha3 expression in the pineal gland and SCG. CNR4 was sufficient to direct LacZ transgene expression to SCG neurons, which express the endogenous beta4alpha3alpha5 subunits, and pineal cells, which express the endogenous beta4alpha3 combination. Finally, CNR4 was able to direct transgene expression to major sites of expression of the endogenous cluster in the brain. Together, our findings support a model in which cell type-specific shared long-range regulatory elements are required for coordinate expression of clustered nAChR genes.

Genetic variation affects binge feeding behavior in female inbred mouse strains.

Identifying genetic variants that regulate binge eating (BE) is critical for understanding the factors that control this behavior and for the development of pharmacological treatment strategies. Although several studies have revealed specific genes capable of affecting BE behavior, less is known about how genetic variation modulates BE. Thus, through a paradigm that promoted binge-like food intake through intermittent access to high calorie diet (HCD), we quantified food-intake in four inbred mouse strains: C57Bl/6J (B6), NOD/LtJ (NOD), 129S1/SvlmJ (S1), and A/J (AJ). We report that genetic variation likely influences the chronic regulation of food intake and the binge-like consumption of a palatable HCD. AJ mice consumed more of both standard chow and HCD than the other three strains tested when both diets were available ad libitum, while S1 mice consumed significantly less HCD than other strains during intermittent HCD access. Behavioral differences were also associated with differential changes in c-Fos immunohistochemistry in brain regions traditionally associated with appetite regulation. Our results identify 129S1/SvlmJ as a strain that exhibits low levels of binge feeding behavior and suggests that this strain could be useful in the investigation of the influence of genetic variation in the control of binge food intake.

VIPergic neurons of the infralimbic and prelimbic cortices control palatable food intake through separate cognitive pathways.

The prefrontal cortex controls food reward seeking and ingestion, playing important roles in directing attention, regulating motivation towards reward pursuit, and the assignment of reward salience and value. The cell types that mediate these behavioral functions, however, are not well described. We report here that optogenetic activation of vasoactive peptide expressing (VIP) interneurons in both the infralimbic (IL) and prelimbic (PL) divisions of the medial prefrontal cortex in mice is sufficient to reduce acute, binge-like intake of high calorie palatable food in the absence of any effect on low calorie rodent chow intake in the sated animal. In addition, we discovered that the behavioral mechanisms associated with these changes in feeding differed between animals that underwent either IL or PL VIPergic stimulation. While IL VIP neurons showed the ability to reduce palatable food intake, this effect was dependent upon the novelty and relative value of the food source. In addition, IL VIP neuron activation significantly reduced novel object- and novel social investigative behavior. Activation of PL VIP neurons, however, produced a reduction in high calorie palatable food intake that was independent of food novelty. Neither IL nor PL VIP excitation changed motivation to obtain food reward. Our data show how neurochemically-defined populations of cortical interneurons can regulate specific aspects of food reward-driven behavior, resulting in a selective reduction in intake of highly valued food.

Enhanced AMPA Receptor Trafficking Mediates the Anorexigenic Effect of Endogenous Glucagon-like Peptide-1 in the Paraventricular Hypothalamus.

Glucagon-like Peptide 1 (GLP-1)-expressing neurons in the hindbrain send robust projections to the paraventricular nucleus of the hypothalamus (PVN), which is involved in the regulation of food intake. Here, we describe that stimulation of GLP-1 afferent fibers within the PVN is sufficient to suppress food intake independent of glutamate release. We also show that GLP-1 receptor (GLP-1R) activation augments excitatory synaptic strength in PVN corticotropin-releasing hormone (CRH) neurons, with GLP-1R activation promoting a protein kinase A (PKA)-dependent signaling cascade leading to phosphorylation of serine S845 on GluA1 AMPA receptors and their trafficking to the plasma membrane. Finally, we show that postnatal depletion of GLP-1R in the PVN increases food intake and causes obesity. This study provides a comprehensive multi-level (circuit, synaptic, and molecular) explanation of how food intake behavior and body weight are regulated by endogenous central GLP-1. VIDEO ABSTRACT.

Activation of murine pre-proglucagon-producing neurons reduces food intake and body weight.

Peptides derived from pre-proglucagon (GCG peptides) act in both the periphery and the CNS to change food intake, glucose homeostasis, and metabolic rate while playing a role in anxiety behaviors and physiological responses to stress. Although the actions of GCG peptides produced in the gut and pancreas are well described, the role of glutamatergic GGC peptide-secreting hindbrain neurons in regulating metabolic homeostasis has not been investigated. Here, we have shown that chemogenetic stimulation of GCG-producing neurons reduces metabolic rate and food intake in fed and fasted states and suppresses glucose production without an effect on glucose uptake. Stimulation of GCG neurons had no effect on corticosterone secretion, body weight, or conditioned taste aversion. In the diet-induced obese state, the effects of GCG neuronal stimulation on gluconeogenesis were lost, while the food intake-lowering effects remained, resulting in reductions in body weight and adiposity. Our work suggests that GCG peptide-expressing neurons can alter feeding, metabolic rate, and glucose production independent of their effects on hypothalamic pituitary-adrenal (HPA) axis activation, aversive conditioning, or insulin secretion. We conclude that GCG neurons likely stimulate separate populations of downstream cells to produce a change in food intake and glucose homeostasis and that these effects depend on the metabolic state of the animal.