Perifornical Lateral Hypothalamic Area Research Articles

Estrous Cycle Modulation of Feeding and Relaxin-3/Rxfp3 mRNA Expression: Implications for Estradiol Action

Introduction: Food intake varies during the ovarian hormone/estrous cycle in humans and rodents, an effect mediated mainly by estradiol. A potential mediator of the central anorectic effects of estradiol is the neuropeptide relaxin-3 (RLN3) synthetized in the nucleus incertus (NI) and acting via the relaxin family peptide-3 receptor (RXFP3). Methods: We investigated the relationship between RLN3/RXFP3 signaling and feeding behavior across the female rat estrous cycle. We used in situ hybridization to investigate expression patterns of Rln3 mRNA in NI and Rxfp3 mRNA in the hypothalamic paraventricular nucleus (PVN), lateral hypothalamic area (LHA), medial preoptic area (MPA), and bed nucleus of the stria terminalis (BNST), across the estrous cycle. We identified expression of estrogen receptors (ERs) in the NI using droplet digital PCR and assessed the electrophysiological responsiveness of NI neurons to estradiol in brain slices. Results: Rln3 mRNA reached the lowest levels in the NI pars compacta during proestrus. Rxfp3 mRNA levels varied across the estrous cycle in a region-specific manner, with changes observed in the perifornical LHA, magnocellular PVN, dorsal BNST, and MPA, but not in the parvocellular PVN or lateral LHA. G protein-coupled estrogen receptor 1 (Gper1) mRNA was the most abundant ER transcript in the NI. Estradiol inhibited 33% of type 1 NI neurons, including RLN3-positive cells. Conclusion: These findings demonstrate that the RLN3/RXFP3 system is modulated by the estrous cycle, and although further studies are required to better elucidate the cellular and molecular mechanisms of estradiol signaling, current results implicate the involvement of the RLN3/RXFP3 system in food intake fluctuations observed across the estrous cycle in female rats.

Homeostatic state of microglia in a rat model of chronic sleep restriction.

Chronic sleep restriction (CSR) negatively impacts brain functions. Whether microglia, the brain's resident immune cells, play any role is unknown. We studied microglia responses to CSR using a rat model featuring slowly rotating wheels (3 h on/1 h off), which was previously shown to induce both homeostatic and adaptive responses in sleep and attention. Adult male rats were sleep restricted for 27 or 99 h. Control rats were housed in locked wheels. After 27 and/or 99 h of CSR, the number of cells immunoreactive for the microglia marker ionized calcium-binding adaptor molecule-1 (Iba1) and the density of Iba1 immunoreactivity were increased in 4/10 brain regions involved in sleep/wake regulation and cognition, including the prelimbic cortex, central amygdala, perifornical lateral hypothalamic area, and dorsal raphe nucleus. CSR neither induced mitosis in microglia (assessed with bromodeoxyuridine) nor impaired blood-brain barrier permeability (assessed with Evans Blue). Microglia appeared ramified in all treatment groups and, when examined quantitatively in the prelimbic cortex, their morphology was not affected by CSR. After 27 h, but not 99 h, of CSR, mRNA levels of the anti-inflammatory cytokine interleukin-10 were increased in the frontal cortex. Pro-inflammatory cytokine mRNA levels (tumor necrosis factor-α, interleukin-1β, and interleukin-6) were unchanged. Furthermore, cortical microglia were not immunoreactive for several pro- and anti-inflammatory markers tested, but were immunoreactive for the purinergic P2Y12 receptor. These results suggest that microglia respond to CSR while remaining in a physiological state and may contribute to the previously reported homeostatic and adaptive responses to CSR.

Adenosine in the lateral hypothalamus/perifornical area does not participate on the CO2 chemoreflex

The Lateral Hypothalamus/Perifornical Area (LH/PFA) has been shown to be involved with the hypercapnic ventilatory response, in a state-dependent manner. We have demonstrated that purinergic signaling through ATP in the LH/PFA has an excitatory effect in ventilatory response to CO2 in awake rats in the dark phase of the diurnal cycle, but it is unknown whether the ATP metabolite adenosine, acting in the LH/PFA, modulates the ventilatory responses to hypercapnia. Here, we studied the effects of the microdialysis of adenosine (A1/A2 adenosine receptors agonist; 17 mM) and an A1 receptor antagonist (DPCPX; 0.1 mM) into the LH/PFA of conscious rats on ventilation in room air and in 7% CO2 during the light and the dark phases of the diurnal cycle. The microdialysis of adenosine and DPCPX caused no change in the CO2 ventilatory responses of rats during wakefulness or NREM sleep in either the dark or light period. Our data suggest that adenosine in the LH/PFA does not contribute to the hypercapnic ventilatory response in conscious rats.

Melanocortin regulation of histaminergic neurons via perifornical lateral hypothalamic melanocortin 4 receptors

ObjectiveHistaminergic neurons of the tuberomammillary nucleus (TMN) are wake-promoting and contribute to the regulation of energy homeostasis. Evidence indicates that melanocortin 4 receptors (MC4R) are expressed within the TMN. However, whether the melanocortin system influences the activity and function of TMN neurons expressing histidine decarboxylase (HDC), the enzyme required for histamine synthesis, remains undefined. MethodsWe utilized Hdc-Cre mice in combination with whole-cell patch-clamp electrophysiology and in vivo chemogenetic techniques to determine whether HDC neurons receive metabolically relevant information via the melanocortin system. ResultsWe found that subsets of HDC-expressing neurons were excited by melanotan II (MTII), a non-selective melanocortin receptor agonist. Use of melanocortin receptor selective agonists (THIQ, [D-Trp8]-γ-MSH) and inhibitors of synaptic transmission (TTX, CNQX, AP5) indicated that the effect was mediated specifically by MC4Rs and involved a glutamatergic dependent presynaptic mechanism. MTII enhanced evoked excitatory post-synaptic currents (EPSCs) originating from electrical stimulation of the perifornical lateral hypothalamic area (PeFLH), supportive of melanocortin effects on the glutamatergic PeFLH projection to the TMN. Finally, in vivo chemogenetic inhibition of HDC neurons strikingly enhanced the anorexigenic effects of intracerebroventricular administration of MTII, suggesting that MC4R activation of histaminergic neurons may restrain the anorexigenic effects of melanocortin system activation. ConclusionsThese experiments identify a functional interaction between the melanocortin and histaminergic systems and suggest that HDC neurons act naturally to restrain the anorexigenic effect of melanocortin system activation. These findings may have implications for the control of arousal and metabolic homeostasis, especially in the context of obesity, in which both processes are subjected to alterations.

Glutamate metabotropic receptors in the lateral hypothalamus/perifornical area reduce the CO2 chemoreflex

It has been shown that the lateral hypothalamus/perifornical area (LH/PFA) exerts an important role on arousal-state variations of the central chemoreflex, but the mechanisms that underlie LH/PFA chemoreception are poorly understood. Here we asked whether glutamate inputs on metabotropic receptors in the LH/PFA modulate the hypercapnic ventilatory response. We studied the effects of microinjection of a glutamate metabotropic receptor (mGluR) antagonist ((+)-α-Methyl-4-carboxyphenylglycine; MCPG; 100 mM) and a selective Group II/III mGluR antagonist ((2S)-2-Amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid; LY341495; 5 mM) into the LH/PFA of conscious rats on ventilation in room air and in 7% CO2, during wakefulness and sleep, in the dark and light periods of the diurnal cycle. Microinjection of MCPG and LY341495 increased the hypercapnic ventilatory response in both the light and the dark period during wakefulness, but not during sleep, (p < 0.001). Our data suggest that glutamate, acting on Group II/III metabotropic receptors in the LH/PFA, exerts an inhibitory modulation of the hypercapnic ventilatory response in awake rats.

ATP in the lateral hypothalamus/perifornical area enhances the CO2 chemoreflex control of breathing.

What is the central question of this study? ATP is known to modulate the chemosensitivity of some brain areas. However, whether the ATP contributes specifically to the mechanism of chemoreception in the lateral hypothalamus/perifornical area (LH/PFA) remains to be determined. What is the main finding and its importance? ATP, acting on the LH/PFA, enhances the hypercapnic ventilatory response in rats during wakefulness, in the dark period. Our results highlight the importance of ATP as a modulator of central chemoreception and provide new insight regarding the mechanisms involved in LH/PFA chemosensitivity and the sleep-wake differences in the CO2 /H+ -dependent drive to breathe. The lateral hypothalamus/perifornical area (LH/PFA) is a central chemoreceptor site, which acts in an arousal state-dependent manner. It has been shown that purinergic signalling through ATP influences the CO2 /H+ responsiveness of other chemosensitive regions, but it is unknown whether ATP is also involved in the mechanisms that underlie LH/PFA chemoreception. Here, we studied the effects of microdialysis of a P2X-receptor agonist [α,β-methylene ATP (α,β-meATP), 10mm] and a non-selective P2-receptor antagonist [pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), 1mm] into the LH/PFA of conscious rats on ventilation in room air and in 7% CO2 . In the dark (active) phase, but not in the light, microdialysis of α,β-meATP caused an augmented hypercapnic ventilatory response during wakefulness, but not during non-REM sleep (P< 0.001). PPADS caused no change in CO2 ventilatory responses in either the dark period or the light period. Our data suggest that ATP in LH/PFA contributes to the hypercapnic ventilatory response in conscious rats during wakefulness in the dark phase of the diurnal cycle.

ATP in the Lateral Hypothalamus/Perifornical area (LH/PFA) Increases CO2 Chemoreflex.

The Lateral Hypothalamus/Perifornical area (LH/PFA) is a chemosensitive site and participates in the CO2 ventilatoy response in a vigilance‐state‐dependent manner. Studies provide evidence of a physiological role for ATP in central chemoreception. We tested the hypothesis that ATP acting on LH/PFA contributes to the CO2 ventilatory response. To this end, ventilation (VE) was recorded in unanesthetized rats by whole body plethysmograph, together with EEG and EMG. We dialyzed into the LH/PFA vehicle and then α,β‐methyleneATP (selective P2X receptor agonist; 10 mM) or PPADS (purinergic antagonist; 1 mM), and studied its effects on VE in air and hypercapnia (7% CO2) during the light and dark period of the diurnal cycle. During wakefulness, but not during sleep, α,β‐methyleneATP microdialysis augmented the hypercapnic ventilatory response by 25% during the dark‐active period (2288 ± 80 mL/kg/min) compared with its vehicle (1786 ± 161 mL/kg/min) (P&lt; 0.01), but not during the light. This effect was due to an increased tidal volume. No effect was observed in normocapnia or in a separate group of animals who had the microdialysis probe misplaced (peri‐LH/PFA). PPADS did not change the ventilatory parameters in all groups. These results suggest that ATP exerts, in the LH/PFA, an excitatory modulation in the hypercapnic chemoreflex during wakefulness in the dark‐active period of the diurnal cycle.Support or Funding InformationFAPESPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential effects of relaxin-3 and a selective relaxin-3 receptor agonist on food and water intake and hypothalamic neuronal activity in rats

The neuropeptide relaxin-3 (RLN3) binds with high affinity to its cognate receptor, relaxin-family peptide receptor 3 (RXFP3), and with lower affinity to RXFP1, the cognate receptor for relaxin. Intracerebroventricular (icv) administration of RLN3 in rats strongly increases food and water intake and alters the activity of the hypothalamic-pituitary-adrenal (HPA) and gonadal (HPG) axes, but the relative involvement of RXFP3 and RXFP1 in these effects is not known. Therefore, the effects of icv administration of equimolar (1.1 nmol) amounts of RLN3 and the RXFP3-selective agonist RXFP3-A2 on food and water intake, plasma levels of corticosterone, testosterone, and oxytocin and c-fos mRNA expression in key hypothalamic regions in male rats were compared. Food intake was increased by both RLN3 and RXFP3-A2, but the orexigenic effects of RXFP3-A2 were significantly stronger than RLN3, 30 and 60min after injection. Water intake and plasma corticosterone and testosterone levels were significantly increased by RLN3, but not by RXFP3-A2. Conversely, RXFP3-A2 but not RLN3 decreased oxytocin plasma levels. RLN3, but not RXFP3-A2, increased c-fos mRNA levels in the parvocellular (PVNp) and magnocellular (PVNm) paraventricular and supraoptic (SON) hypothalamic nuclei, in the ventral medial preoptic area (MPAv), and in the organum vasculosum of the lamina terminalis (OVLT). A significant increase in c-fos mRNA expression was induced in the perifornical lateral hypothalamic area (LHApf) by RLN3 and RXFP3-A2. These results suggest that RXFP1 is involved in the RLN3 stimulation of water intake and activation of the HPA and HPG axes. The reduced food intake stimulation by RLN3 compared to RXFP3-A2 may relate to activation of both orexigenic and anorexigenic circuits by RLN3.

Lack of Hypocretin Attenuates Behavioral Changes Produced by Glutamatergic Activation of the Perifornical-Lateral Hypothalamic Area

The hypocretins (HCRTs) are two hypothalamic peptides predominantly localized to neurons in the perifornical, dorsomedial, and lateral hypothalamic area (PF-LHA). Evidence suggests that HCRT signaling is critical for the promotion and stabilization of active-arousal and its loss or malfunction leads to symptoms of narcolepsy. In the PF-LHA, HCRT neurons are intermingled with glutamate-expressing neurons and also co-express glutamate. Evidence suggests that HCRT-glutamate interactions within the PF-LHA may play a critical role in maintaining behavioral arousal. However, the relative contributions of the glutamate and HCRT in sleep-wake regulation are not known. We determined whether a lack of HCRT signaling in the prepro-orexin-knockout (HCRT-KO) mouse attenuates/compromises the wake-promoting ability of glutamatergic activation of the PF-LHA region. We used reverse microdialysis to deliver N-methyl-D-aspartate (NMDA) into the HCRT zone of the PF-LHA in HCRT-KO and wild-type (WT) mice to evaluate the contributions of glutamatergic vs. HCRT signaling in sleep-wake regulation. As compared to respective controls, local perfusion of NMDA into the PF-LHA, dose-dependently increased active-waking with concomitant reductions in nonREM and REM sleep in spontaneously sleeping WT as well as HCRT-KO mice. However, compared to WT, the NMDA-induced behavioral changes in HCRT-KO mice were significantly attenuated, as evidenced by the higher dose of NMDA needed and lower magnitude of changes induced in sleep-wake parameters. Although not observed in WT mice, the number of cataplectic events increased significantly during NMDA-induced behavioral arousal in HCRT-KO mice. The findings of this study are consistent with a hypothesis that synergistic interactions between hypocretin and glutamatergic mechanisms within the perifornical, dorsomedial, and lateral hypothalamic area are critical for maintaining behavioral arousal, especially arousal involving elevated muscle tone.

Sleep-wake and diurnal modulation of nitric oxide in the perifornical-lateral hypothalamic area: Real-time detection in freely behaving rats

Nitric oxide (NO) has been implicated in the regulation of sleep. The perifornical-lateral hypothalamic area (PF-LHA) is a key wake-promoting region and contains neurons that are active during behavioral or cortical activation. Recently, we found higher levels of NO metabolites (NOx), an indirect measure of NO levels, in the PF-LHA during prolonged waking (SD). However, NO is highly reactive and diffuses rapidly and the NOx assay is not sensitive enough to detect rapid-changes in NO levels across spontaneous sleep-waking states. We used a novel Nafion®-modified Platinum (NF-PT) electrode for real-time detection of NO levels in the PF-LHA across sleep–wake cycles, dark–light phases, and during SD. Sprague–Dawley male rats were surgically prepared for chronic sleep–wake recording and implantation of NF-PT electrode into the PF-LHA. Electroencephalogram (EEG), electromyogram (EMG), and electrochemical current generated by NF-PT electrode were continuously acquired for 5–7days including one day with 3h of SD. In the PF-LHA, NO levels exhibited a waking>rapid eye movement (REM)>non-rapid eye movement (nonREM) sleep pattern (0.56±0.03μM>0.47±0.02μM>0.42±0.02μM; p<0.01). NO levels were also higher during the dark- as compared to the light-phase (0.53±0.03μM vs. 0.44±0.02μM; p<0.01). NO levels increased during 3h of SD as compared to undisturbed control (0.58±0.04μM vs. 0.47±0.01μM; p<0.05). The findings indicate that in the PF-LHA, NO is produced during behavioral or cortical activation. Since elevated levels of NO inhibits most of the PF-LHA neurons that are active during cortical activation, these findings support a hypothesis that NO produced in conjunction with the activation of PF-LHA neurons during waking/SD, inhibits the same neuronal population to promote sleep.

Efferent projections of neuropeptide Y‐expressing neurons of the dorsomedial hypothalamus in chronic hyperphagic models

The dorsomedial hypothalamus (DMH) has long been implicated in feeding behavior and thermogenesis. The DMH contains orexigenic neuropeptide Y (NPY) neurons, but the role of these neurons in the control of energy homeostasis is not well understood. NPY expression in the DMH is low under normal conditions in adult rodents but is significantly increased during chronic hyperphagic conditions such as lactation and diet-induced obesity (DIO). To understand better the role of DMH-NPY neurons, we characterized the efferent projections of DMH-NPY neurons using the anterograde tracer biotinylated dextran amine (BDA) in lactating rats and DIO mice. In both models, BDA- and NPY-colabeled fibers were limited mainly to the hypothalamus, including the paraventricular nucleus of the hypothalamus (PVH), lateral hypothalamus/perifornical area (LH/PFA), and anteroventral periventricular nucleus (AVPV). Specifically in lactating rats, BDA-and NPY-colabeled axonal swellings were in close apposition to cocaine- and amphetamine-regulated transcript (CART)-expressing neurons in the PVH and AVPV. Although the DMH neurons project to the rostral raphe pallidus (rRPa), these projections did not contain NPY immunoreactivity in either the lactating rat or the DIO mouse. Instead, the majority of BDA-labeled fibers in the rRPa were orexin positive. Furthermore, DMH-NPY projections were not observed within the nucleus of the solitary tract (NTS), another brainstem site critical for the regulation of sympathetic outflow. The present data suggest that NPY expression in the DMH during chronic hyperphagic conditions plays important roles in feeding behavior and thermogenesis by modulating neuronal functions within the hypothalamus, but not in the brainstem.

Synaptic interactions between perifornical lateral hypothalamic area, locus coeruleus nucleus and the oral pontine reticular nucleus are implicated in the stage succession during sleep-wakefulness cycle

The perifornical area in the posterior lateral hypothalamus (PeFLH) has been implicated in several physiological functions including the sleep-wakefulness regulation. The PeFLH area contains several cell types including those expressing orexins (Orx; also known as hypocretins), mainly located in the PeF nucleus. The aim of the present study was to elucidate the synaptic interactions between Orx neurons located in the PeFLH area and different brainstem neurons involved in the generation of wakefulness and sleep stages such as the locus coeruleus (LC) nucleus (contributing to wakefulness) and the oral pontine reticular nucleus (PnO) nucleus (contributing to REM sleep). Anatomical data demonstrated the existence of a neuronal network involving the PeFLH area, LC, and the PnO nuclei that would control the sleep-wake cycle. Electrophysiological experiments indicated that PeFLH area had an excitatory effect on LC neurons. PeFLH stimulation increased the firing rate of LC neurons and induced an activation of the EEG. The excitatory effect evoked by PeFLH stimulation in LC neurons was blocked by the injection of the Orx-1 receptor antagonist SB-334867 into the LC. Similar electrical stimulation of the PeFLH area evoked an inhibition of PnO neurons by activation of GABAergic receptors because the effect was blocked by bicuculline application into the PnO. Our data also revealed that the LC and PnO nuclei exerted a feedback control on neuronal activity of PeFLH area. Electrical stimulation of LC facilitated firing activity of PeFLH neurons by activation of catecholaminergic receptors whereas PnO stimulation inhibited PeFLH neurons by activation of GABAergic receptors. In conclusion, Orx neurons of the PeFLH area seem to be an important organizer of the wakefulness and sleep stages in order to maintain a normal succession of stages during the sleep-wakefulness cycle.

Focal microdialysis of CO2 in the perifornical-hypothalamic area increases ventilation during wakefulness but not NREM sleep

We investigated whether the perifornical-lateral hypothalamic area (PF-LHA), where the orexin neurons reside, is a central chemoreceptor site by microdialysis of artificial cerebrospinal fluid (aCSF) equilibrated with 25% CO2 into PF-LHA in conscious rats. This treatment is known to produce a focal tissue acidification like that associated with a 6–7mm Hg increase in arterial PCO2. Such focal acidification in the PF-LHA significantly increased ventilation up to 15% compared with microdialysis of normal aCSF equilibrated with 5% CO2 only in wakefulness but not in sleep in both the dark (P=0.004) and light (P<0.001) phases of the diurnal cycle. This response was predominantly due to a significant increase in respiratory frequency (11%, P<0.001). There were no significant effects on ventilation in the group with probes misplaced outside the PF-LHA. These results suggest that PF-LHA functions as a central chemoreceptor site in the central nervous system in a vigilant state dependent manner with predominant effects in wakefulness.

Nociceptin induces hypophagia in the perifornical and lateral hypothalamic area.

Nociceptin/orphanin FQ (N/OFQ) is known to induce food intake when administered into the lateral ventricle or certain brain areas. This is somewhat contradictory to its reward-suppressing role, as food is a strong rewarding stimulus. This discrepancy may be due to the functional diversity of N/OFQ’s target brain areas. N/OFQ has been shown to inhibit orexin and melanin-concentrating hormone (MCH) neurons, both of which are appetite-inducing cells. As the expression of these neurons is largely confined to the lateral hypothalamus/perifornical area (LH/PFA), we hypothesized that N/OFQ inhibits food intake by acting in this area. To test this hypothesis, we examined the effect of local N/OFQ infusion within the LH/PFA on food intake in the rat and found that N/OFQ decreased sugar pellet as well as chow intake. This effect was not seen when the injection site was outside of the LH/PFA, suggesting a site-specific effect. Next, to determine a possible cellular mechanism of N/OFQ action on food intake, whole cell patch clamp recordings were performed on rat orexin neurons. As previously reported in mice, N/OFQ induced a strong and long lasting hyperpolarization. Pharmacological study indicated that N/OFQ directly inhibited orexin neurons by activating ATP-sensitive potassium (KATP) channels. This effect was partially but significantly attenuated by the inhibitors of PI3K, PKC and PKA, suggesting that the N/OFQ signaling is mediated by these protein kinases. In summary, our results demonstrate a KATP channel-dependent N/OFQ signaling and that N/OFQ is a site-specific anorexic peptide.

Cocaine potentiates excitatory drive in the perifornical/lateral hypothalamus

The hypothalamus is a critical controller of homeostatic responses and plays a fundamental role in reward-seeking behaviour. Recently, hypothalamic neurones in the perifornical/lateral hypothalamic area (PF/LHA) have also been implicated in drug-seeking behaviour through projections to extra-hypothalamic sites such as the ventral tegmental area. For example, a population of neurones that expresses the peptide orexin has been strongly implicated in addiction-relevant behaviours. To date, the effect of addictive drugs on synaptic properties in the hypothalamus remains largely unexplored. Previous studies focusing on the PF/LHA neurones, however, have shown that the orexin system exhibits significant plasticity in response to food or sleep restriction. This neuroadaptive ability suggests that PF/LHA neurones could be highly susceptible to modifications by drug exposure. Here, we sought to determine whether cocaine produces synaptic plasticity in PF/LHA neurones. Whole-cell patch-clamp techniques were used to examine the effects of experimenter-administered (passive) or self-administered (SA) cocaine on glutamatergic synaptic transmission in PF/LHA neurones. These experiments demonstrate that both passive and SA cocaine exposure increases miniature excitatory postsynaptic current (mEPSC) frequency in PF/LHA neurones. In addition, SA cocaine reduced the paired-pulse ratio but the AMPA/NMDA ratio of evoked excitatory inputs was unchanged, indicative of a presynaptic locus for synaptic plasticity. Dual-labelling for orexin and excitatory inputs using the vesicular glutamate transporter (VGLUT2), showed that passive cocaine exposure increased VGLUT2-positive appositions onto orexin neurones. Further, a population of recorded neurones that were filled with neurobiotin and immunolabelled for orexin confirmed that increased excitatory drive occurs in this PF/LHA population. Given the importance of the PF/LHA and the orexin system in modulating drug addiction, we suggest that these cocaine-induced excitatory synapse-remodelling events within the hypothalamus may contribute to persistence in drug-seeking behaviour and relapse.

Orexinergic signaling mediates light-induced neuronal activation in the dorsal raphe nucleus

Seasonal affective disorder (SAD), a major depressive disorder recurring in the fall and winter, is caused by the reduction of light in the environment, and its depressive symptoms can be alleviated by bright light therapy. Both circadian and monoaminergic systems have been implicated in the etiology of SAD. However, the underlying neural pathways through which light regulates mood are not well understood. The present study utilized a diurnal rodent model, Arvicanthis niloticus, to explore the neural pathways mediating the effects of light on brain regions involved in mood regulation. Animals kept in constant darkness received light exposure in early subjective day, the time when light therapy is usually applied. The time course of neural activity following light exposure was assessed using Fos protein as a marker in the following brain regions/cells: the suprachiasmatic nucleus (SCN), orexin neurons in the perifornical-lateral hypothalamic area (PF-LHA) and the dorsal raphe nucleus (DRN). A light-induced increase in Fos expression was observed in orexin neurons and the DRN, but not in the SCN. As the DRN is densely innervated by orexinergic inputs, the involvement of orexinergic signaling in mediating the effects of light on the DRN was tested in the second experiment. The animals were injected with the selective orexin receptor type 1 (OXR1) antagonist SB-334867 prior to the light exposure. The treatment of SB-334867 significantly inhibited the Fos induction in the DRN. The results collectively point to the role of orexin neurons in mediating the effects of light on the mood-regulating monoaminergic areas, suggesting an orexinergic pathway that underlies light-dependent mood fluctuation and the beneficial effects of light therapy.

Mechanisms mediating effects of nitric oxide on perifornical lateral hypothalamic neurons

The perifornical-lateral hypothalamic area (PF-LHA) is a major wake-promoting structure. It predominantly contains neurons that are active during behavioral and cortical activation. Nitric oxide (NO) is a gaseous neurotransmitter that has been implicated in the regulation of sleep. Recently we found that NO levels in the PF-LHA are higher during sustained waking and that NO exerts predominantly inhibitory effects, especially on PF-LHA neurons excited by tactile stimulation. The mechanisms underlying this NO-evoked inhibitory action on the PF-LHA neurons were assessed in the present study.We investigated the contributions of adenosinergic, GABAergic, and sGC-cGMP signaling mechanisms in mediating nitrergic influences on the PF-LHA neurons. The extracellular discharge activity of PF-LHA neurons was recorded in combination with microdialytic delivery of pharmacological agents adjacent to the recorded neurons in urethane-anesthetized rats. First, we quantified changes in the discharge activity of the PF-LHA neurons during the blockade of the adenosine A1 receptor, GABAA receptor, and sGC-cGMP pathway. Then, we determined the efficacy of blocking adenosine A1 receptor, GABAA receptor, and sGC signaling mechanisms in attenuating the inhibitory influences of 3,3-bis(aminoethyl)-1-hydroxy-1-oxo-1-triazene (a NO donor) (NOC-18), a NO donor, on the discharge activity of the PF-LHA neurons.We found that NOC-18-induced suppression in the discharge activity of PF-LHA neurons was significantly attenuated during the blockade of adenosine A1 receptor-, GABAA receptor-, and sGC-cGMP-mediated signaling. These findings suggest that NO-evoked inhibition of PF-LHA neurons involves a complex mechanism including, but may not be limited to, adenosinergic, GABAergic and sGC-cGMP signaling pathways. The findings are consistent with a generalized sleep-promoting role of NO within the PF-LHA and, given the sleep-promoting roles of adenosinergic and GABAergic systems in this area, further suggest that this effect may be mediated through nitrergic interactions with other neurotransmitters and neuromodulators.

Behavioural and neuronal activation after microinjections of AMPA and NMDA into the perifornical lateral hypothalamus in rats

The perifornical lateral hypothalamic area (PeFLH), which houses orexin/hypocretin (OX) neurons, is thought to play an important role in arousal, feeding, and locomotor activity. The present study examined behavioural effects of activating PeFLH neurons with microinjections of ionotropic glutamate receptor agonists. Three separate unilateral microinjections of either (1) AMPA (1 and 2 mM in 0.1 μL artificial cerebrospinal fluid, ACSF) and ACSF, or (2) NMDA (1 and 10 mM in 0.1 μL ACSF), and ACSF were made into the PeFLH of adult male rats. Following each injection, the rats were placed into an open field for behavioural scoring for 45 min. Rats were perfused after the third injection for immunohistochemistry for c-Fos and OX to assess the level of activation of OX neurons. Behavioural analyses showed that, as compared to ACSF conditions, AMPA injections produced a dose-dependent increase in locomotion and rearing that persisted throughout the 45 min recording period, and an increase in drinking. Injection of NMDA at 10 mM, but not 1 mM, induced a transient increase in locomotion and an increase in feeding. Histological analyses showed that while both agonists increased the number of neurons immunoreactive for c-Fos in the PeFLH, only AMPA increased the number of neurons immunoreactive for both c-Fos and OX. There were positive correlations between the number of c-Fos/OX-immunoreactive neurons and the amounts of locomotion, rearing, and drinking. These results support the role of ionotropic glutamate receptors on OX and other neurons in the PeFLH in the regulation of locomotor and ingestive behaviours.

Nitric oxide production in the perifornical-lateral hypothalamic area and its influences on the modulation of perifornical-lateral hypothalamic area neurons

The perifornical-lateral hypothalamic area (PF-LHA) is a major wake-promoting structure. It predominantly contains neurons that are active during behavioral and cortical activation. PF-LHA stimulation produces arousal and PF-LHA lesions produce somnolence. Nitric oxide (NO) is a gaseous neurotransmitter that has been implicated in the regulation of multiple pathological and physiological processes including the regulation of sleep. NO levels are higher in the cortex and in the basal forebrain (BF) during arousal. In this study we determined whether NO levels increase in the PF-LHA during prolonged arousal and whether increased NO modulates the discharge activity of PF-LHA neurons. Experiments were conducted during lights-on phase between 8.00 and 20.00 h (lights-on at 8.00 h). First, we quantified levels of NO metabolites, NO 2 − and NO 3 − (collectively called NOx −) in the microdialysis dialysates collected from the PF-LHA during baseline (undisturbed rats), 6 h of sleep deprivation (SD), and recovery after SD. We further determined the effects of a NO donor, NOC-18, on the discharge activity of PF-LHA neurons in urethane-anesthetized rats. Overall, SD significantly affected NOx − production in the PF-LHA (one way repeated measures ANOVA, F=7.827, P=0.004). The levels of NOx − increased progressively in animals that were subjected to prolonged arousal as compared to the undisturbed predominantly sleeping animals and decreased during the recovery period. Local application of NOC-18 significantly suppressed the discharge of PF-LHA neurons including a majority of stimulus-on neurons or neurons exhibiting activation during electroencephalogram (EEG) desynchronization. The findings of this study suggest that in the PF-LHA, NO production is elevated during prolonged waking and that NO exerts predominantly inhibitory effects on PF-LHA neurons, especially on those neurons that are active during cortical activation. These findings are consistent with a hypothesis that NO in the PF-LHA plays a role in sleep regulation by inhibiting its neurons.

GABAergic regulation of the perifornical–lateral hypothalamic neurons during non-rapid eye movement sleep in rats

The perifornical–lateral hypothalamic area (PF–LHA) has been implicated in the regulation of behavioral arousal. The PF–LHA predominantly contains neurons that are active during behavioral and cortical activation and quiescent during non-rapid eye movement (nonREM) sleep, that is, are nonREM-off neurons. Some in vitro and in vivo studies indicate that PF–LHA neurons, including hypocretin-expressing neurons, are under GABAergic control. However, a role of GABA in suppressing the discharge of PF–LHA neurons during spontaneous nonREM sleep has not been confirmed. We recorded the sleep–wake discharge profiles of PF–LHA neurons and simultaneously assessed the contributions of local GABA A receptor activation and blockade on their wake- and nonREM sleep-related discharge activities by delivering GABA A receptor agonist, muscimol (500 nm, 5 μM, and 10 μM) and its antagonist, bicuculline (5 μM, 10 μM, and 20 μM), adjacent to the recorded neurons via reverse microdialysis. Muscimol dose-dependently decreased the discharge of PF–LHA neurons including nonREM-off neurons. Muscimol-induced suppression of discharge during nonREM sleep was significantly weaker than the suppression produced during waking. In the presence of bicuculline, PF–LHA neurons, including nonREM-off neurons, exhibited elevated discharge, which was dose-dependent and was significantly higher during nonREM sleep, compared to waking. These results suggest that GABA A receptor mediated increased GABAergic tone contributes to the suppression of PF–LHA neurons, including nonREM-off neurons, during spontaneous nonREM sleep.