Contribution of NPY Y5 Receptors to the Reversible Structural Remodeling of Basolateral Amygdala Dendrites in Male Rats Associated with NPY-Mediated Stress Resilience.

Endogenous neuropeptide Y (NPY) and corticotrophin-releasing factor (CRF) modulate the responses of the basolateral amygdala (BLA) to stress and are associated with the development of stress resilience and vulnerability, respectively. We characterized persistent effects of repeated NPY and CRF treatment on the structure and function of BLA principal neurons in a novel organotypic slice culture (OTC) model of male rat BLA, and examined the contributions of specific NPY receptor subtypes to these neural and behavioral effects. In BLA principal neurons within the OTCs, repeated NPY treatment caused persistent attenuation of excitatory input and induced dendritic hypotrophy via Y5 receptor activation; conversely, CRF increased excitatory input and induced hypertrophy of BLA principal neurons. Repeated treatment of OTCs with NPY followed by an identical treatment with CRF, or vice versa, inhibited or reversed all structural changes in OTCs. These structural responses to NPY or CRF required calcineurin or CaMKII, respectively. Finally, repeated intra-BLA injections of NPY or a Y5 receptor agonist increased social interaction, a validated behavior for anxiety, and recapitulated structural changes in BLA neurons seen in OTCs, while a Y5 receptor antagonist prevented NPY's effects both on behavior and on structure. These results implicate the Y5 receptor in the long-term, anxiolytic-like effects of NPY in the BLA, consistent with an intrinsic role in stress buffering, and highlight a remarkable mechanism by which BLA neurons may adapt to different levels of stress. Moreover, BLA OTCs offer a robust model to study mechanisms associated with resilience and vulnerability to stress in BLA.SIGNIFICANCE STATEMENT Within the basolateral amygdala (BLA), neuropeptide Y (NPY) is associated with buffering the neural stress response induced by corticotropin releasing factor, and promoting stress resilience. We used a novel organotypic slice culture model of BLA, complemented with in vivo studies, to examine the cellular mechanisms associated with the actions of NPY. In organotypic slice cultures, repeated NPY treatment reduces the complexity of the dendritic extent of anxiogenic BLA principal neurons, making them less excitable. NPY, via activation of Y5 receptors, additionally inhibits and reverses the increases in dendritic extent and excitability induced by the stress hormone, corticotropin releasing factor. This NPY-mediated neuroplasticity indicates that resilience or vulnerability to stress may thus involve neuropeptide-mediated dendritic remodeling in BLA principal neurons.

NPY2 Receptors Reduce Tonic Action Potential-Independent GABAB Currents in the Basolateral Amygdala.

Although NPY has potent anxiolytic actions within the BLA, selective activation of BLA NPY Y2 receptors (Y2Rs) acutely increases anxiety by an unknown mechanism. Using ex vivo male rat brain slice electrophysiology, we show that the selective Y2R agonist, [ahx5-24]NPY, reduced the frequency of GABAA-mediated mIPSCs in BLA principal neurons (PNs). [ahx5-24]NPY also reduced tonic activation of GABAB receptors (GABABR), which increased PN excitability through inhibition of a tonic, inwardly rectifying potassium current (KIR ). Surprisingly, Y2R-sensitive GABABR currents were action potential-independent, persisting after treatment with TTX. Additionally, the Ca2+-dependent, slow afterhyperpolarizing K+ current (IsAHP ) was enhanced in approximately half of the Y2R-sensitive PNs, possibly from enhanced Ca2+ influx, permitted by reduced GABABR tone. In male and female mice expressing tdTomato in Y2R-mRNA cells (tdT-Y2R mice), immunohistochemistry revealed that BLA somatostatin interneurons express Y2Rs, as do a significant subset of BLA PNs. In tdT-Y2R mice, [ahx5-24]NPY increased excitability and suppressed the KIR in nearly all BLA PNs independent of tdT-Y2R fluorescence, consistent with presynaptic Y2Rs on somatostatin interneurons mediating the above effects. However, only tdT-Y2R-expressing PNs responded to [ahx5-24]NPY with an enhancement of the IsAHP Ultimately, increased PN excitability via acute Y2R activation likely correlates with enhanced BLA output, consistent with reported Y2R-mediated anxiogenesis. Furthermore, we demonstrate the following: (1) a novel mechanism whereby activity-independent GABA release can powerfully dampen BLA neuronal excitability via postsynaptic GABABRs; and (2) that this tonic inhibition can be interrupted by neuromodulation, here by NPY via Y2Rs.SIGNIFICANCE STATEMENT Within the BLA, NPY is potently anxiolytic. However, selective activation of NPY2 receptors (Y2Rs) increases anxiety by an unknown mechanism. We show that activation of BLA Y2Rs decreases tonic GABA release onto BLA principal neurons, probably from Y2R-expressing somatostatin interneurons, some of which coexpress NPY. This increases principal neuron excitability by reducing GABAB receptor (GABABR)-mediated activation of G-protein-coupled, inwardly rectifying K+ currents. Tonic, Y2R-sensitive GABABR currents unexpectedly persisted in the absence of action potential firing, revealing, to our knowledge, the first report of substantial, activity-independent GABABR activation. Ultimately, we provide a plausible explanation for Y2R-mediated anxiogenesis in vivo and describe a novel and modulatable means of damping neuronal excitability.

Integration of energy homeostasis and stress by parvocellular neurons in rat hypothalamic paraventricular nucleus.

KEY POINTS: Central regulation of energy homeostasis and stress are believed to be reciprocally regulated, i.e. excessive food intake suppresses, while prolonged hunger exacerbates, stress responses in vivo. This relationship may be mediated by neuroendocrine parvocellular corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus that receive both stress- and feeding-related input. We find that hunger strongly and selectively potentiates, while re-feeding suppresses, a cellular analogue of a stress response induced by acute glucopenia in CRH neurons in rat hypothalamic slices. Neuronal activation in response to glucopenia was mediated synaptically, via the relative enhancement of glutamate over GABA input. These results illustrate how acute stress responses may be initiated in vivo and show that it is reciprocally integrated with energy balance via local hypothalamic mechanisms acting at the level of CRH neurons and their afferent terminals. ABSTRACT: Increased food intake is a common response to help cope with stress, implying the existence of a previously postulated but imperfectly understood, inverse relationship between the regulation of feeding and stress. We have identified components of the neural circuitry that can integrate these homeostatic responses. Prior fasting (∼24 h) potentiates, and re-feeding suppresses, excitatory responses to acute glucopenia in about half of the corticotropin releasing hormone (CRH)-expressing, putatively neurosecretory, stress-related neurons in the paraventricular nucleus of the hypothalamus studied. Glucoprivation stress ex vivo resulted from a preferential relative increase in excitatory (glutamatergic) over inhibitory (GABAergic) inputs. Putative preautonomic cells were less sensitive to fasting, and showed a predominant inhibition to acute glucopenia. We conclude that hunger may sensitize hypothalamic stress responses by acting via local mechanisms, at the level of CRH neurons and their presynaptic inputs. Those mechanisms involve neither presynaptic ATP-sensitive potassium channels nor postsynaptic ATP levels.

NPY Induces Stress Resilience via Downregulation of Ih in Principal Neurons of Rat Basolateral Amygdala.

Neuropeptide Y (NPY) expression is tightly linked with the development of stress resilience in rodents and humans. Local NPY injections targeting the basolateral amygdala (BLA) produce long-term behavioral stress resilience in male rats via an unknown mechanism. Previously, we showed that activation of NPY Y1 receptors hyperpolarizes BLA principal neurons (PNs) through inhibition of the hyperpolarization-activated, depolarizing H-current, Ih The present studies tested whether NPY treatment induces stress resilience by modulating Ih NPY (10 pmol) was delivered daily for 5 d bilaterally into the BLA to induce resilience; thereafter, the electrophysiological properties of PNs and the expression of Ih in the BLA were characterized. As reported previously, increases in social interaction (SI) times persisted weeks after completion of NPY administration. In vitro intracellular recordings showed that repeated intra-BLA NPY injections resulted in hyperpolarization of BLA PNs at 2 weeks (2W) and 4 weeks (4W) after NPY treatment. At 2W, spontaneous IPSC frequencies were increased, whereas at 4W, resting Ih was markedly reduced and accompanied by decreased levels of HCN1 mRNA and protein expression in BLA. Knock-down of HCN1 channels in the BLA with targeted delivery of lentivirus containing HCN1-shRNA increased SI beginning 2W after injection and induced stress resilience. NPY treatment induced sequential, complementary changes in the inputs to BLA PNs and their postsynaptic properties that reduce excitability, a mechanism that contributes to less anxious behavior. Furthermore, HCN1 knock-down mimicked the increases in SI and stress resilience observed with NPY, indicating the importance of Ih in stress-related behavior.SIGNIFICANCE STATEMENT Resilience improves mental health outcomes in response to adverse situations. Neuropeptide Y (NPY) is associated with decreased stress responses and the expression of resilience in rodents and humans. Single or repeated injections of NPY into the basolateral amygdala (BLA) buffer negative behavioral effects of stress and induce resilience in rats, respectively. Here, we demonstrate that repeated administration of NPY into the BLA unfolds several cellular mechanisms that decrease the activity of pyramidal output neurons. One key mechanism is a reduction in levels of the excitatory ion channel HCN1. Moreover, shRNA knock-down of HCN1 expression in BLA recapitulates some of the actions of NPY and causes potent resilience to stress, indicating that this channel may be a possible target for therapy.

Progressive postnatal decline in leptin sensitivity of arcuate hypothalamic neurons in the Magel2-null mouse model of Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a multigene disorder associated with neonatal failure to thrive, developmental delay and endocrine abnormalities suggestive of hypothalamic dysfunction. Children with PWS typically develop overt hyperphagia and obesity ∼8 years of age, later than children with other genetic forms of obesity. This suggests a postnatal developmental or degenerative component to PWS-associated obesity. De novo inactivating mutations in one PWS candidate gene, MAGEL2, have been identified in children with features of PWS. Adult mice lacking Magel2 are insensitive to the anorexic effect of leptin treatment, and their hypothalamic pro-opiomelanocortin (POMC) neurons fail to depolarize in response to leptin. However, it is unclear whether this leptin insensitivity is congenital, or whether normal leptin sensitivity in neonatal Magel2-null mice is lost postnatally. We used in vitro cytosolic calcium imaging to follow the postnatal development of leptin responses in POMC neurons in these mice. Leptin caused an activation of POMC neurons in wild-type acute hypothalamic slice preparations at all ages, reflecting their normal leptin-invoked depolarization. Normal leptin responses were found in Magel2-null mice up to 4 weeks of age, but the proportion of leptin-responsive POMC neurons was reduced in 6-week-old Magel2-null mice. The number of α-melanocyte-stimulating hormone immunoreactive fibers in the paraventricular hypothalamic nucleus was also reduced in mutant mice at 6 weeks of age. A similar progressive loss of leptin sensitivity caused by loss of MAGEL2 in children with PWS could explain the delayed onset of increased appetite and weight gain in this complex disorder.

Magel2 is required for leptin-mediated depolarization of POMC neurons in the hypothalamic arcuate nucleus in mice.

Prader-Willi Syndrome is the most common syndromic form of human obesity and is caused by the loss of function of several genes, including MAGEL2. Mice lacking Magel2 display increased weight gain with excess adiposity and other defects suggestive of hypothalamic deficiency. We demonstrate Magel2-null mice are insensitive to the anorexic effect of peripherally administered leptin. Although their excessive adiposity and hyperleptinemia likely contribute to this physiological leptin resistance, we hypothesized that Magel2 may also have an essential role in intracellular leptin responses in hypothalamic neurons. We therefore measured neuronal activation by immunohistochemistry on brain sections from leptin-injected mice and found a reduced number of arcuate nucleus neurons activated after leptin injection in the Magel2-null animals, suggesting that most but not all leptin receptor-expressing neurons retain leptin sensitivity despite hyperleptinemia. Electrophysiological measurements of arcuate nucleus neurons expressing the leptin receptor demonstrated that although neurons exhibiting hyperpolarizing responses to leptin are present in normal numbers, there were no neurons exhibiting depolarizing responses to leptin in the mutant mice. Additional studies demonstrate that arcuate nucleus pro-opiomelanocortin (POMC) expressing neurons are unresponsive to leptin. Interestingly, Magel2-null mice are hypersensitive to the anorexigenic effects of the melanocortin receptor agonist MT-II. In Prader-Willi Syndrome, loss of MAGEL2 may likewise abolish leptin responses in POMC hypothalamic neurons. This neural defect, together with increased fat mass, blunted circadian rhythm, and growth hormone response pathway defects that are also linked to loss of MAGEL2, could contribute to the hyperphagia and obesity that are hallmarks of this disorder.

Modulation of distal calcium electrogenesis by neuropeptide Y1 receptors inhibits neocortical long-term depression.

In layer 5 neocortical pyramidal neurons, backpropagating action potentials (bAPs) firing at rates above a critical frequency (CF) induce supralinear Ca²âº influx and regenerative potentials in apical dendrites. Paired temporally with an EPSP, this Ca²âº influx can result in synaptic plasticity. We studied the actions of neuropeptide Y (NPY), an abundant neocortical neuropeptide, on Ca²âº influx in layer 5 pyramidal neurons of somatosensory neocortex in Sprague Dawley and Wistar rats, using a combination of somatic and dendritic intracellular recordings and simultaneous Ca²âº imaging. Ca²âº influx induced by trains of bAPs above a neuron's CF was inhibited by NPY, acting only at the distal dendrite, via Y1 receptors. NPY does not affect evoked synaptic glutamate release, paired synaptic facilitation, or synaptic rundown in longer trains. Extracellular Cs⁺ did not prevent NPY's postsynaptic effects, suggesting it does not act via either G-protein-activated inwardly rectifying K⁺ conductance (G(IRK)) or hyperpolarization-activated, cyclic nucleotide-gated channels. NPY application suppresses the induction of the long-term depression (LTD) normally caused by pairing 100 EPSPs with bursts of 2 bAPs evoked at a supracritical frequency. These findings suggest that distal dendritic Ca²âº influx is necessary for LTD induction, and selective inhibition of this distal dendritic Ca²âº influx by NPY can thus regulate synaptic plasticity in layer 5 pyramidal neurons.

Dopamine modulates synaptic plasticity in dendrites of rat and human dentate granule cells.

The mechanisms underlying memory formation in the hippocampal network remain a major unanswered aspect of neuroscience. Although high-frequency activity appears essential for plasticity, salience for memory formation is also provided by activity in ventral tegmental area (VTA) dopamine projections. Here, we report that activation of dopamine D1 receptors in dentate granule cells (DGCs) can preferentially increase dendritic excitability to both high-frequency afferent activity and high-frequency trains of backpropagating action potentials. Using whole-cell patch clamp recordings, calcium imaging, and neuropeptide Y to inhibit postsynaptic calcium influx, we found that activation of dendritic voltage-dependent calcium channels (VDCCs) is essential for dopamine-induced long-term potentiation (LTP), both in rat and human dentate gyrus (DG). Moreover, we demonstrate previously unreported spike-timing-dependent plasticity in the human hippocampus. These results suggest that when dopamine is released in the dentate gyrus with concurrent high-frequency activity there is an increased probability that synapses will be strengthened and reward-associated spatial memories will be formed.

Long-term actions of BDNF on inhibitory synaptic transmission in identified neurons of the rat substantia gelatinosa.

Peripheral nerve injury promotes the release of brain-derived neurotrophic factor (BDNF) from spinal microglial cells and primary afferent terminals. This induces an increase in dorsal horn excitability that contributes to "central sensitization" and to the onset of neuropathic pain. Although it is accepted that impairment of GABAergic and/or glycinergic inhibition contributes to this process, certain lines of evidence suggest that GABA release in the dorsal horn may increase after nerve injury. To resolve these contradictory findings, we exposed rat spinal cord neurons in defined-medium organotypic culture to 200 ng/ml BDNF for 6 days to mimic the change in spinal BDNF levels that accompanies peripheral nerve injury. Morphological and electrophysiological criteria and glutamic acid decarboxylase (GAD) immunohistochemistry were used to distinguish putative inhibitory tonic-islet-central neurons from putative excitatory delay-radial neurons. Whole cell recording in the presence of 1 µM tetrodotoxin showed that BDNF increased the amplitude of GABAergic and glycinergic miniature inhibitory postsynaptic currents (mIPSCs) in both cell types. It also increased the amplitude and frequency of spontaneous, action potential-dependent IPSCs (sIPSCs) in putative excitatory neurons. By contrast, BDNF reduced sIPSC amplitude in inhibitory neurons but frequency was unchanged. This increase in inhibitory drive to excitatory neurons and decreased inhibitory drive to inhibitory neurons seems inconsistent with the observation that BDNF increases overall dorsal horn excitability. One of several explanations for this discrepancy is that the action of BDNF in the substantia gelatinosa is dominated by previously documented increases in excitatory synaptic transmission rather than by impediment of inhibitory transmission.

The role of NPY in hypothalamic mediated food intake.

Neuropeptide Y (NPY) is a highly conserved neuropeptide with orexigenic actions in discrete hypothalamic nuclei that plays a role in regulating energy homeostasis. NPY signals via a family of high affinity receptors that mediate the widespread actions of NPY in all hypothalamic nuclei. These actions are also subject to tight, intricate regulation by numerous peripheral and central energy balance signals. The NPY system is embedded within a densely-redundant network designed to ensure stable energy homeostasis. This redundancy may underlie compensation for the loss of NPY or its receptors in germline knockouts, explaining why conventional knockouts of NPY or its receptors rarely yield a marked phenotypic change. We discuss insights into the hypothalamic role of NPY from studies of its physiological actions, responses to genetic manipulations and interactions with other energy balance signals. We conclude that numerous approaches must be employed to effectively study different aspects of NPY action.

Developmental switch in neuropeptide Y and melanocortin effects in the paraventricular nucleus of the hypothalamus.

Homeostatic regulation of energy balance in rodents changes dramatically during the first 3 postnatal weeks. Neuropeptide Y (NPY) and melanocortin neurons in the arcuate nucleus, a primary energy homeostatic center in adults, do not fully innervate the paraventricular nucleus (PVN) until the third postnatal week. We have identified two classes of PVN neurons responsive to these neuropeptides, tonically firing neurosecretory (NS) and burst-firing preautonomic (PA) cells. In neonates, NPY could inhibit GABAergic inputs to nearly all NS and PA neurons, while melanocortin regulation was minimal. However, there was a dramatic, age-dependent decrease in NPY responses specifically in the PA neurons, and a 3-fold increase in melanocortin responses in NS cells. These age-dependent changes were accompanied by changes in spontaneous GABAergic currents onto these neurons. This primarily NPYergic regulation in the neonates likely promotes the positive energy balance necessary for growth, while the developmental switch correlates with maturation of homeostatic regulation of energy balance.

Countervailing modulation of Ih by neuropeptide Y and corticotrophin-releasing factor in basolateral amygdala as a possible mechanism for their effects on stress-related behaviors.

Stress and anxiety-related behaviors controlled by the basolateral amygdala (BLA) are regulated in vivo by neuropeptide Y (NPY) and corticotrophin-releasing factor (CRF): NPY produces anxiolytic effects, whereas CRF produces anxiogenic effects. These opposing actions are likely mediated via regulation of excitatory output from the BLA to afferent targets. In these studies, we examined mechanisms underlying the effects of NPY and CRF in the BLA using whole-cell patch-clamp electrophysiology in rat brain slices. NPY, even with tetrodotoxin present, caused a dose-dependent membrane hyperpolarization in BLA pyramidal neurons. The hyperpolarization resulted in the inhibition of pyramidal cells, despite arising from a reduction in a voltage-dependent membrane conductance. The Y(1) receptor agonist, F(7)P(34) NPY, produced a similar membrane hyperpolarization, whereas the Y(1) antagonist, BIBO3304 [(R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N(2)-(diphenylacetyl)-argininamide trifluoroacetate], blocked the effect of NPY. The NPY-inhibited current was identified as I(h), which is active at and hyperpolarized to rest. Responses to NPY were occluded by either Cs(+) or ZD7288 (4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride), but unaffected by the G(IRK)-preferring blockers Ba(2+) and SCH23390 [(R)-(+)-7-chloro-8-hydroxy-3-methyl-l-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride]. Application of CRF, with or without TTX present, depolarized NPY-sensitive BLA pyramidal neurons, resulting from an increase in I(h). Electrophysiological and immunocytochemical data were consistent with a major role for the HCN1 subunit. Our results indicate that NPY, via Y(1) receptors, directly inhibits BLA pyramidal neurons by suppressing a postsynaptic I(h), whereas CRF enhances resting I(h), causing an increased excitability of BLA pyramidal neurons. The opposing actions of these two peptides on the excitability of BLA output cells are consistent with the observed behavioral actions of NPY and CRF in the BLA.

Neuropeptide Y suppresses anorexigenic output from the ventromedial nucleus of the hypothalamus.

Output from the hypothalamic ventromedial nucleus (VMN) is anorexigenic and is supported by the excitatory actions of leptin. The VMN is also highly sensitive to the orexigenic actions of Neuropeptide Y (NPY). We report that NPY robustly inhibits VMN neurons by hyperpolarizing them and decreasing their ability to fire action potentials. This action was mediated by Y(1) receptors coupled to the activation of GIRKs (G-protein-coupled inwardly rectifying potassium channels). Approximately 80% of VMN neurons expressing leptin receptors were sensitive to the actions of NPY, whereas 75% of NPY-sensitive neurons in VMN also responded to glucose by being uniformly inhibited by elevations in glucose. Interestingly, only approximately 36% of NPY-sensitive, leptin receptor b-expressing neurons were also glucosensitive. We suggest that NPY inhibits VMN neurons that are excited by leptin, thereby arresting the anorexigenic tone exerted by VMN neurons. The results further suggest a dynamic interplay between anorexigenic and orexigenic neuromodulators within the VMN to directly affect energy balance.

Nociceptin/orphanin FQ suppresses the excitability of neurons in the ventromedial nucleus of the hypothalamus.

Nociceptin or orphanin FQ (N/OFQ) stimulates food intake when injected into the ventromedial nucleus of the hypothalamus (VMN). The VMN negatively regulates energy balance in part by tonically activating proopiomelanocortin arcuate neurons, thereby suppressing food intake. However, it is not clear how orexigenic neurotransmission within the VMN can stimulate food intake. We tested the hypothesis that the orexigenic action of N/OFQ results from its inhibition of anorexigenic VMN neurons. We studied the effects of N/OFQ on the electrical properties of anorexigenic VMN neurons in acute brain slices. Ionic mechanisms underlying the actions of N/OFQ were studied using whole cell patch-clamp recordings from VMN neurons expressing the anorexigenic leptin receptor (LepRb). Bath application of N/OFQ to LepRb-expressing VMN neurons elicited a robust, reversible membrane hyperpolarization that suppressed neuronal excitability by raising the action potential firing threshold and cell rheobase. N/OFQ activated a postsynaptic, G-protein coupled, inwardly rectifying potassium (GIRK) current that was sensitive to G-protein inactivation, blocked by the GIRK blocker SCH23390, and occluded by the GABAB agonist and potent GIRK activator, baclofen. Application of the selective N/OFQ receptor antagonist SB-612111 blocked the inhibitory effects of N/OFQ. We concluded that N/OFQ directly inhibited VMN neurons by activating a GIRK. These results implicate the site-specific contributions of orexigenic neuropeptides at VMN neurons to suppress anorexigenic output. This study thus advances our understanding regarding the contributions of the VMN to hypothalamic regulation of energy balance.

Glucosensing in parvocellular neurons of the rat hypothalamic paraventricular nucleus.

Specialized hypothalamic neurons responding to rising extracellular glucose via increases or decreases in their electrical activity [glucose-excited (GE) and glucose-inhibited (GI) cells, respectively] have been reported in the hypothalamic arcuate, ventromedial and lateral nuclei. The hypothalamic paraventricular nucleus (PVN) is an important neurosecretory and preautonomic output nucleus. We tested whether parvocellular PVN neurons also possess glucosensing properties, using patch-clamp recording and immunocytochemistry. Putative neurosecretory (p-NS) and preautonomic (p-PA) cells were identified electrophysiologically. Although parvocellular neurons were insensitive to transitions from 10 to 2.5 mm glucose, approximately 68% of p-PA cells responded directly to glucopenia (mimicked by a step to 0.2 mm glucose) with an increased membrane conductance. Of these, approximately 24% hyperpolarized (accompanied by an outward current) and thus were GE, approximately 26% depolarized (with an inward current, thus GI) and approximately 18% did not change membrane potential. The concentration dependence of the glucose response was similar for both GE and GI cells (EC(50) of 0.67-0.7 mm), but was steep, with Hill slopes of 3-4. The K(ATP) channel blockers glibenclamide and tolbutamide did not prevent, while the K(ATP) channel opener diazoxide did not mimic, the effects of low glucose on GE neurons. Moreover, the K(ATP) sulfonylurea receptor SUR1 was not detected in glucosensitive neurons. We conclude that the PVN contains previously unknown GE and GI cells that could participate in regulation of autonomic functions. GE neurons in the PVN sense ambient glucose via a unique mechanism, probably independent of K(ATP) channels, in contrast to neurons in other hypothalamic nuclei.

Long-Lived Organotypic Slice Culture Model of the Rat Basolateral Amygdala.

Organotypic slice cultures (OTCs) have been employed in the laboratory since the early 1980s and have proved to be useful for the study of a number of neural systems. Our recent work focuses on the development of behavioral stress resilience induced by repeated daily injections of neuropeptide Y into the basolateral amygdala (BLA). Resilience develops over weeks, persisting to 8 weeks. To unravel the cellular mechanisms underlying neuropeptide Y-induced stress resilience we developed in vitro OTCs of the BLA. Here, we provide an optimized protocol that consistently yields viable and healthy OTCs containing the BLA and surrounding tissue using the interface method, prepared with slices taken from postnatal (P) day 14 rats. We explain key points to optimizing tissue viability and discuss mitigation or avoidance of pitfalls that can arise to aid in successful implementation of this technique. We show that principal neurons in BLA OTCs (8 weeks in vitro = equivalent postnatal day 70) develop into networks that are electrophysiologically very similar to those from acute slices obtained from older rats (P70) and respond to pharmacological treatments in a comparable way. Furthermore, we highlight how these cultures be used to further understand the molecular, cellular, and circuit-level neuropathophysiological changes underlying stress disorders. BLA OTCs provide long-term physiological and pharmacological results whose predictions were borne out in vivo, supporting the validity of the BLA OTC as a model to unravel BLA neurocircuitry. Recent preliminary results also support the successful application of this approach to preparing long-lived OTCs of BLA and neocortex from mice. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Organotypic slice culture Support Protocol 1: Changing medium Support Protocol 2: Drug incubations Basic Protocol 2: Excision of OTC slices from inserts Support Protocol 3: Fixation of slices.

PYY3-36 inhibits the action potential firing activity of POMC neurons of arcuate nucleus through postsynaptic Y2 receptors.

Intracerebroventricular administration of gut peptide PYY3-36 stimulates food intake. In contrast, peripheral administration inhibits food intake, suggesting that the peptide has the opposite effect by virtue of accessing a unique subset of brain sites. A previous study suggested that peripheral PYY3-36 activates anorexigenic POMC neurons in the arcuate nucleus, and this was proposed to be the mechanism underlying the peptide's anorexigenic activity. Here, we demonstrate in an electrophysiological slice preparation that, in contrast to the original model, PYY3-36 potently and reversibly inhibits POMC neurons via postsynaptic Y2 receptors. These data show a complex role for Y2 receptors in regulation of the NPY/POMC circuitry, as they are present as inhibitory receptors on both the orexigenic NPY neurons as well as the anorexigenic POMC neurons. Secondly, these data argue against a direct role of POMC neurons in mediating the anorexigenic response to administration of peripheral PYY3-36.

Brain-derived neurotrophic factor drives the changes in excitatory synaptic transmission in the rat superficial dorsal horn that follow sciatic nerve injury.

Peripheral nerve injury can promote neuropathic pain. The basis of the 'central sensitization' that underlies this often intractable condition was investigated using 14-20-day chronic constriction injury (CCI) of the sciatic nerve of 20-day-old rats followed by electrophysiological analysis of acutely isolated spinal cord slices. In addition, defined-medium organotypic spinal cord slice cultures were exposed for 5-6 days to brain-derived neurotrophic factor (BDNF, 200 ng ml(-1)) or to medium conditioned with activated microglia (aMCM). Since microglial activation is an early consequence of CCI, the latter manipulation allowed us to model the effect of peripheral nerve injury on the dorsal horn in vitro. Using whole-cell recording from superficial dorsal horn neurons, we found that both BDNF and CCI increased excitatory synaptic drive to putative excitatory 'radial delay' neurons and decreased synaptic excitation of inhibitory 'tonic islet/central' neurons. BDNF also attenuated synaptic excitation of putative GABAergic neurons identified by glutamic acid decarboxylase (GAD) immunoreactivity. Intrinsic neuronal properties (rheobase, input resistance and action potential discharge rates) were unaffected. Exposure of organotypic cultures to either BDNF or aMCM increased overall excitability of the dorsal horn, as seen by increased cytoplasmic Ca(2+) responses to 35 mm K(+) as monitored by confocal Fluo-4AM imaging. The effect of aMCM was attenuated by the recombinant BDNF binding protein TrkBd5 and the effect of BDNF persisted when GABAergic inhibition was blocked with SR95531. These findings suggest that CCI enhances excitatory synaptic drive to excitatory neurons but decreases that to inhibitory neurons. Both effects are mediated by nerve injury-induced release of BDNF from microglia.

Convergent neuronal projections from paraventricular nucleus, parabrachial nucleus, and brainstem onto gastrocnemius muscle, white and brown adipose tissue in male rats.

When energy balance is altered by aerobic exercise, starvation, and cold exposure, for example, there appears to be coordination of the responses of skeletal muscle, white adipose (WAT), and brown adipose (BAT) tissues. We hypothesized that WAT, BAT, and skeletal muscle may share an integrated regulation by the central nervous system (CNS); specifically, that neurons in brain regions associated with energy balance would possess neuroanatomical connections to permit coordination of multiple, complementary responses in these downstream tissues. To study this, we used trans-neuronal viral retrograde tract tracing, using isogenic strains of pseudorabies virus (PRV) with distinct fluorescent reporters (either eGFP or mRFP), injected pairwise into male rat gastrocnemius, subcutaneous WAT and interscapular BAT, coupled with neurochemical characterization of specific cell populations for cocaine- and amphetamine-related transcript (CART), oxytocin (OX), corticotrophin releasing hormone (CRH) and calcitonin gene-related peptide (CGRP). Cells in the paraventricular (PVN) and parabrachial (PBN) nuclei and brainstem showed dual projections to muscle + WAT, muscle + BAT, and WAT + BAT. Dual PRV-labeled cells were found in parvocellular, magnocellular and descending/pre-autonomic regions of the PVN, and multiple structural divisions of the PBN and brainstem. In most PBN subdivisions, more than 50% of CGRP cells dually projected to muscle + WAT and muscle + BAT. Similarly, 31-68% of CGRP cells projected both to WAT + BAT. However, dual PRV-labeled cells in PVN only occasionally expressed OX or CRH but not CART. These studies reveal for the first time both separate and shared outflow circuitries among skeletal muscle and subcutaneous WAT and BAT.

The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis.

The gastrointestinal peptide hormone ghrelin stimulates appetite in rodents and humans via hypothalamic actions. We discovered expression of ghrelin in a previously uncharacterized group of neurons adjacent to the third ventricle between the dorsal, ventral, paraventricular, and arcuate hypothalamic nuclei. These neurons send efferents onto key hypothalamic circuits, including those producing neuropeptide Y (NPY), Agouti-related protein (AGRP), proopiomelanocortin (POMC) products, and corticotropin-releasing hormone (CRH). Within the hypothalamus, ghrelin bound mostly on presynaptic terminals of NPY neurons. Using electrophysiological recordings, we found that ghrelin stimulated the activity of arcuate NPY neurons and mimicked the effect of NPY in the paraventricular nucleus of the hypothalamus (PVH). We propose that at these sites, release of ghrelin may stimulate the release of orexigenic peptides and neurotransmitters, thus representing a novel regulatory circuit controlling energy homeostasis.