Leptin action through hypothalamic nitric oxide synthase-1-expressing neurons controls energy balance

Few effective measures exist to combat the worldwide obesity epidemic(1), and the identification of potential therapeutic targets requires a deeper understanding of the mechanisms that control energy balance. Leptin, an adipocyte-derived hormone that signals the long-term status of bodily energy stores, acts through multiple types of leptin receptor long isoform (LepRb)-expressing neurons (called here LepRb neurons) in the brain to control feeding, energy expenditure and endocrine function(2-4). The modest contributions to energy balance that are attributable to leptin action in many LepRb populations(5-9) suggest that other previously unidentified hypothalamic LepRb neurons have key roles in energy balance. Here we examine the role of LepRb in neuronal nitric oxide synthase (NOS1)-expressing LebRb (LepRb(NOS1)) neurons that comprise approximately 20% of the total hypothalamic LepRb neurons. Nos1(cre)-mediated genetic ablation of LepRb (Lepr(Nos1KO)) in mice produces hyperphagic obesity, decreased energy expenditure and hyperglycemia approaching that seen in whole-body LepRb-null mice. In contrast, the endocrine functions in Lepr(Nos1KO) mice are only modestly affected by the genetic ablation of LepRb in these neurons. Thus, hypothalamic LepRb(NOS1) neurons are a key site of action of the leptin-mediated control of systemic energy balance.     

Endogenous leptin receptor signaling in the medial nucleus tractus solitarius affects meal size and potentiates intestinal satiation signals

Leptin receptor (LepRb) signaling in the hindbrain is required for energy balance control. Yet the specific hindbrain neurons and the behavioral processes mediating energy balance control by hindbrain leptin signaling are unknown. Studies here employ genetic [adeno-associated virally mediated RNA interference (AAV-RNAi)] and pharmacological methodologies to specify the neurons and the mechanisms through which hindbrain LepRb signaling contributes to the control of food intake. Results show that AAV-RNAi-mediated LepRb knockdown targeting a region encompassing the mNTS and area postrema (AP) (mNTS/AP LepRbKD) increases overall cumulative food intake by increasing the size of spontaneous meals. Other results show that pharmacological hindbrain leptin delivery and RNAi-mediated mNTS/AP LepRb knockdown increased and decreased the intake-suppressive effects of intraduodenal nutrient infusion, respectively. These meal size and intestinally derived signal amplification effects are likely mediated by LepRb signaling in the mNTS and not the AP, since 4th icv and mNTS parenchymal leptin (0.5 μg) administration reduced food intake, whereas this dose did not influence food intake when injected into the AP. Overall, these findings deepen the understanding of the distributed neuronal systems and behavioral mechanisms that mediate the effects of leptin receptor signaling on the control of food intake.     

Leptin action via neurotensin neurons controls orexin, the mesolimbic dopamine system and energy balance

Leptin acts on leptin receptor (LepRb)-expressing neurons throughout the brain, but the roles for many populations of LepRb neurons in modulating energy balance and behavior remain unclear. We found that the majority of LepRb neurons in the lateral hypothalamic area (LHA) contain neurotensin (Nts). To investigate the physiologic role for leptin action via these LepRb(Nts) neurons, we generated mice null for LepRb specifically in Nts neurons (Nts-LepRbKO mice). Nts-LepRbKO mice demonstrate early-onset obesity, modestly increased feeding, and decreased locomotor activity. Furthermore, consistent with the connection of LepRb(Nts) neurons with local orexin (OX) neurons and the ventral tegmental area (VTA), Nts-LepRbKO mice exhibit altered regulation of OX neurons and the mesolimbic DA system. Thus, LHA LepRb(Nts) neurons mediate physiologic leptin action on OX neurons and the mesolimbic DA system, and contribute importantly to the control of energy balance.     

Somato-Dendritic Localization and Signaling by Leptin Receptors in Hypothalamic POMC and AgRP Neurons

Leptin acts via neuronal leptin receptors to control energy balance. Hypothalamic pro-opiomelanocortin (POMC) and agouti-related peptide (AgRP)/Neuropeptide Y (NPY)/GABA neurons produce anorexigenic and orexigenic neuropeptides and neurotransmitters, and express the long signaling form of the leptin receptor (LepRb). Despite progress in the understanding of LepRb signaling and function, the sub-cellular localization of LepRb in target neurons has not been determined, primarily due to lack of sensitive anti-LepRb antibodies. Here we applied light microscopy (LM), confocal-laser scanning microscopy (CLSM), and electron microscopy (EM) to investigate LepRb localization and signaling in mice expressing a HA-tagged LepRb selectively in POMC or AgRP/NPY/GABA neurons. We report that LepRb receptors exhibit a somato-dendritic expression pattern. We further show that LepRb activates STAT3 phosphorylation in neuronal fibers within several hypothalamic and hindbrain nuclei of wild-type mice and rats, and specifically in dendrites of arcuate POMC and AgRP/NPY/GABA neurons of Leprb ^+/+ mice and in Leprb ^db/db mice expressing HA-LepRb in a neuron specific manner. We did not find evidence of LepRb localization or STAT3-signaling in axon-fibers or nerve-terminals of POMC and AgRP/NPY/GABA neurons. Three-dimensional serial EM-reconstruction of dendritic segments from POMC and AgRP/NPY/GABA neurons indicates a high density of shaft synapses. In addition, we found that the leptin activates STAT3 signaling in proximity to synapses on POMC and AgRP/NPY/GABA dendritic shafts. Taken together, these data suggest that the signaling-form of the leptin receptor exhibits a somato-dendritic expression pattern in POMC and AgRP/NPY/GABA neurons. Dendritic LepRb signaling may therefore play an important role in leptin’s central effects on energy balance, possibly through modulation of synaptic activity via post-synaptic mechanisms.     

Leptin Acts via Leptin Receptor-Expressing Lateral Hypothalamic Neurons to Modulate the Mesolimbic Dopamine System and Suppress Feeding

The lateral hypothalamic area (LHA) acts in concert with the ventral tegmental area (VTA) and other components of the mesolimbic dopamine (DA) system to control motivation, including the incentive to feed. The anorexigenic hormone leptin modulates the mesolimbic DA system, although the mechanisms underlying this control have remained incompletely understood. We show that leptin directly regulates a population of leptin receptor (LepRb)-expressing inhibitory neurons in the LHA and that leptin action via these LHA LepRb neurons decreases feeding and body weight. Furthermore, these LHA LepRb neurons innervate the VTA, and leptin action on these neurons restores VTA expression of the rate-limiting enzyme in DA production along with mesolimbic DA content in leptin-deficient animals. Thus, these findings reveal that LHA LepRb neurons link anorexic leptin action to the mesolimbic DA system.     

Leptin excites basolateral amygdala principal neurons and reduces food intake by LepRb-JAK2-PI3K-dependent depression of GIRK channels

Leptin is an adipocyte-derived hormone that modulates food intake, energy balance, neuroendocrine status, thermogenesis, and cognition. Whereas a high density of leptin receptors has been detected in the basolateral amygdala (BLA) neurons, the physiological functions of leptin in the BLA have not been determined yet. We found that application of leptin excited BLA principal neurons by activation of the long form leptin receptor, LepRb. The LepRb-elicited excitation of BLA neurons was mediated by depression of the G protein-activated inwardly rectifying potassium (GIRK) channels. Janus Kinase 2 (JAK2) and phosphoinositide 3-kinase (PI3K) were required for leptin-induced excitation of BLA neurons and depression of GIRK channels. Microinjection of leptin into the BLA reduced food intake via activation of LepRb, JAK2, and PI3K. Our results may provide a cellular and molecular mechanism to explain the physiological roles of leptin in vivo.     

The role of 5-HT1B receptors in the regulation of serotonin cell firing and release in the rat brain

The release of 5-HT in terminal areas of the rodent brain is regulated by 5-HT1B receptors. Here we examined the role of 5-HT1B receptors in the control of 5-HT output and firing in the dorsal raphe nucleus (DR), median raphe nucleus (MnR) and forebrain of the rat in vivo. The local perfusion (30-300 microM) of the selective 5-HT1B receptor agonist CP-93,129 to freely moving rats decreased 5-HT release in the DR and more markedly in the MnR. Likewise, 300 microM CP-93,129 reduced 5-HT output in substantia nigra pars reticulata, ventral pallidum, lateral habenula and the suprachiasmatic nucleus. The effect of CP-93,129 was prevented by SB-224289, but not by WAY-100635, selective 5-HT1B and 5-HT1A receptor antagonists, respectively. SB-224289 did not alter dialysate 5-HT in any raphe nuclei. The intravenous administration of the brain-penetrant selective 5-HT1B receptor agonist CP-94,253 (0.5-2.0 mg/kg) to anesthetized rats decreased dialysate 5-HT in dorsal hippocampus and globus pallidus, increased it in MnR and left it unaltered in the DR and medial prefrontal cortex. SB-224289, at a dose known to block 5-HT1B autoreceptor-mediated effects (5 mg/kg), did not prevent the effect of CP-94,253 on MnR 5-HT. The intravenous administration of CP-94,253 (0.05-1.6 mg/kg) to anesthetized rats increased the firing rate of MnR, but not DR-5-HT neurons. The local perfusion of CP-94,253 in the MnR showed a biphasic effect, with 5-HT reductions at 0.3-3 microM and increase at 300 microM. These results suggest that 5-HT cell firing and release in midbrain raphe nuclei (particularly in the MnR) are under control of 5-HT1B receptors. The activation of 5-HT1B autoreceptors (possibly located on 5-HT nerve endings and/or varicosities within DR and MnR) reduces 5-HT release. The effects of higher concentrations of 5-HT1B receptor agonists seem more compatible with the activation of 5-HT1B heteroreceptors on inhibitory neurons.     

Serotonin 2C receptor activates a distinct population of arcuate pro-opiomelanocortin neurons via TRPC channels

Serotonin 2C receptors (5-HT(2C)Rs) expressed by pro-opiomelanocortin (POMC) neurons of hypothalamic arcuate nucleus regulate food intake, energy homeostasis and glucose metabolism. However, the cellular mechanisms underlying the effects of 5-HT to regulate POMC neuronal activity via 5-HT(2C)Rs have not yet been identified. In the present study, we found the putative transient receptor potential C (TRPC) channels mediate the activation of a subpopulation of POMC neurons by mCPP (a?5-HT(2C)R agonist). Interestingly, mCPP-activated POMC neurons were found to be a distinct population from those activated by leptin. Together, our data suggest that 5-HT(2C)R and leptin receptors are expressed by distinct subpopulations of arcuate POMC neurons and that both 5-HT and leptin exert their actions in POMC neurons via TRPC channels. VIDEO ABSTRACT:     

Chronic Alterations in Monoaminergic Cells in the Locus Coeruleus in Orexin Neuron-Ablated Narcoleptic Mice

Narcolepsy patients often suffer from insomnia in addition to excessive daytime sleepiness. Narcoleptic animals also show behavioral instability characterized by frequent transitions between all vigilance states, exhibiting very short bouts of NREM sleep as well as wakefulness. The instability of wakefulness states in narcolepsy is thought to be due to deficiency of orexins, neuropeptides produced in the lateral hypothalamic neurons, which play a highly important role in maintaining wakefulness. However, the mechanism responsible for sleep instability in this disorder remains to be elucidated. Because firing of orexin neurons ceases during sleep in healthy animals, deficiency of orexins does not explain the abnormality of sleep. We hypothesized that chronic compensatory changes in the neurophysiologica activity of the locus coeruleus (LC) and dorsal raphe (DR) nucleus in response to the progressive loss of endogenous orexin tone underlie the pathological regulation of sleep/wake states. To evaluate this hypothesis, we examined firing patterns of serotonergic (5-HT) neurons and noradrenergic (NA) neurons in the brain stem, two important neuronal populations in the regulation of sleep/wakefulness states. We recorded single-unit activities of 5-HT neurons and NA neurons in the DR nucleus and LC of orexin neuron-ablated narcoleptic mice. We found that while the firing pattern of 5-HT neurons in narcoleptic mice was similar to that in wildtype mice, that of NA neurons was significantly different from that in wildtype mice. In narcoleptic mice, NA neurons showed a higher firing frequency during both wakefulness and NREM sleep as compared with wildtype mice. In vitro patch-clamp study of NA neurons of narcoleptic mice suggested a functional decrease of GABAergic input to these neurons. These alterations might play roles in the sleep abnormality in narcolepsy.     

Hyperleptinemia elicited by the 5-HT precursor, 5-hydroxytryptophan in mice: involvement of insulin

Mechanisms for hyperleptinemia elicited by a serotonin (5-hydroxytryptamine, 5-HT) precursor, 5-hydroxytryptophan (5-HTP), were investigated. 5-HTP elicited apparent increases in serum leptin levels of mice. Administration of 5-HTP did not alter expression of leptin mRNA in white adipose tissues. Furthermore, neither 5-HTP nor 5-HT increased leptin secretion from isolated fat pads of mice. Since insulin is known to enhance leptin release, involvement of insulin in 5-HTP-induced hyperleptinemia was examined. 5-HTP significantly elevated serum insulin levels. In mice treated with streptozotocin, which depletes insulin, 5-HTP did not increase serum leptin levels. These results suggest that hyperinsulinemia participates the elevation of serum leptin levels elicited by 5-HTP.     

Activation of 5-HT1A receptors in raphe pallidus inhibits leptin-evoked increases in brown adipose tissue thermogenesis

To elucidate the central neural pathways contributing to the thermogenic component of the autonomic response to intravenous administration of leptin, experiments were conducted in urethane-chloralose-anesthetized, ventilated rats to address 1) the role of neurons in the rostral ventromedial medulla, including raphe pallidus (RPa), in the leptin-evoked stimulation of brown adipose tissue (BAT) sympathetic nerve activity (SNA); and 2) the potential thermolytic effect of 5-hydroxytryptamine(1A) (5-HT(1A)) receptors on RPa neurons that influence BAT thermogenesis. Leptin (1 mg/kg) administration increased BAT SNA by 1,219% of control, BAT temperature by 2.8 degrees C, expired CO(2) by 1.8%, heart rate by 90 beats/min, and mean arterial pressure by 12 mmHg. Microinjection of the 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) into RPa resulted in a prompt and sustained reversal of the leptin-evoked stimulation of BAT SNA, BAT thermogenesis, and heart rate, with these variables returning to their pre-leptin control levels. Subsequent microinjection of the selective 5-HT(1A) receptor antagonist WAY-100635 into RPa reversed the BAT thermolytic effects of 8-OH-DPAT, returning BAT SNA and BAT temperature to the elevated levels after leptin. In conclusion, activation of neurons in RPa, possibly BAT sympathetic premotor neurons, is essential for the increases in BAT SNA, BAT thermogenesis, and heart rate stimulated by intravenous administration of leptin. Neurons in RPa express 5-HT(1A) receptors whose activation leads to reversal of the BAT thermogenic and the cardiovascular responses to intravenous leptin, possibly through hyperpolarization of local sympathetic premotor neurons. These results contribute to our understanding of central neural substrates for the augmented energy expenditure stimulated by leptin.     

Antibodies as molecular probes in neurobiology

Immunocytochemical localization of 5-hydroxytryptamine (5-HT) in the nervous system and aggregate tissue cultures was performed employing an antibody to 6-OH-1,2,3,4-tetrahydro-beta-carboline. A number of immunochemical and biochemical tests with the antigen and the antibody and some procedural changes in the methodology applied for immunolocalization revealed the anti-5-HT-like affinity of the antibody, if applied in paraformaldehyde-fixed tissues. Studies in the hypothalamus, striatum, brainstem, spinal cord, and pineal gland show the complexities of the serotoninergic system. Ultrastructural immunocytochemistry with the preembedding technique reveals that 5-HT synapses are of the asymmetric type. The presynaptic element contains clear, round, small vesicles, with some large dense-core vesicles. The contacts are made with the somata and primary, secondary dendrites or with spines of non-5-HT neurons. Presynaptic dendrites are found in the n. raphe dorsalis, contacting non-5-HT dendrites. Double immunocytochemical methods demonstrated contacts of 5-HT fibers on enkephalin containing neurons of the spinal trigeminal nucleus and on somatostatin containing neurons of the medullary reticular formation. In vitro studies of cultured mesencephalic neurons were performed with the method of aggregating cultures. Such development of a miniature organized nerve tissue was followed up to 35 d in culture. Organization of the neuropil and synaptogenesis was studied using standard electron microscopy. The differentiation of neurons and astrocytes was studied using antibodies to 5-HT and GFAP. Serotonin immunoreactivity could be observed in neuronal bodies and processes at light microscope level as early as the fourth day of culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective serotonin reuptake inhibitors and neuroendocrine function.

Selective serotonin (5-HT) reuptake inhibitors (SSRIs) are effective drugs for the treatment of several neuropsychiatric disorders associated with reduced serotonergic function. Serotonergic neurons play an important role in the regulation of neuroendocrine function. This review will discuss the acute and chronic effects of SSRIs on neuroendocrine function. Acute administration of SSRIs increases the secretion of several hormones, but chronic treatment with SSRIs does not alter basal blood levels of hormones. However, adaptive changes are induced by long-term treatment with SSRIs in serotonergic, noradrenergic and peptidergic neural function. These adaptive changes, particularly in the function of specific post-synaptic receptor systems, can be examined from altered adrenocorticotrophic hormone (ACTH), cortisol, oxytocin, vasopressin, prolactin, growth hormone (GH) and renin responses to challenges with specific agonists. Neuroendocrine challenge tests both in experimental animals and in humans indicate that chronic SSRIs produce an increase in serotonergic terminal function, accompanied by desensitization of post-synaptic 5-HT1A receptor-mediated ACTH, cortisol, GH and oxytocin responses, and by supersensitivity of post-synaptic 5-HT2A (and/or 5-HT2C) receptor-mediated secretion of hormones. Chronic exposure to SSRIs does not alter the neuroendocrine stress-response and produces inconsistent changes in alpha2 adrenoceptor-mediated GH secretion. Overall, the effects of SSRIs on neuroendocrine function are dependent on adaptive changes in specific neurotransmitter systems that regulate the secretion of specific hormones.     

Serotonin 5-HT2C receptor-mediated inhibition of the M-current in hypothalamic POMC neurons

Hypothalamic proopiomelanocortin (POMC) neurons are controlled by many central signals, including serotonin. Serotonin increases POMC activity and reduces feeding behavior via serotonion [5-hydroxytryptamine (5-HT)] receptors by modulating K(+) currents. A potential K(+) current is the M-current, a noninactivating, subthreshold outward K(+) current. Previously, we found that M-current activity was highly reduced in fasted vs. fed states in neuropeptide Y neurons. Because POMC neurons also respond to energy states, we hypothesized that fasting may alter the M-current and/or its modulation by serotonergic input to POMC neurons. Using visualized-patch recording in neurons from fed male enhanced green fluorescent protein-POMC transgenic mice, we established that POMC neurons expressed a robust M-current (102.1 ± 6.7 pA) that was antagonized by the selective KCNQ channel blocker XE-991 (40 μM). However, the XE-991-sensitive current in POMC neurons did not differ between fed and fasted states. To determine if serotonin suppresses the M-current via the 5-HT(2C) receptor, we examined the effects of the 5-HT(2A)/5-HT(2C) receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) on the M-current. Indeed, DOI attenuated the M-current by 34.5 ± 6.9% and 42.0 ± 5.3% in POMC neurons from fed and fasted male mice, respectively. In addition, the 5-HT(1B)/5-HT(2C) receptor agonist m-chlorophenylpiperazine attenuated the M-current by 42.4 ± 5.4% in POMC neurons from fed male mice. Moreover, the selective 5-HT(2C) receptor antagonist RS-102221 abrogated the actions of DOI in suppressing the M-current. Collectively, these data suggest that although M-current expression does not differ between fed and fasted states in POMC neurons, serotonin inhibits the M-current via activation of 5-HT(2C) receptors to increase POMC neuronal excitability and, subsequently, reduce food intake.