Lassa-VSV chimeric virus targets and destroys human and mouse ovarian cancer by direct oncolytic action and by initiating an anti-tumor response.

Ovarian cancer is the third most common female cancer, with poor survival in later stages of metastatic spread. We test a chimeric virus consisting of genes from Lassa and vesicular stomatitis viruses, LASV-VSV; the native VSV glycoprotein is replaced by the Lassa glycoprotein, greatly reducing neurotropism. Human ovarian cancer cells in immunocompromised nude mice were lethal in controls. Chemotherapeutic paclitaxel and cisplatin showed modest cancer inhibition and survival extension. In contrast, a single intraperitoneal injection of LASV-VSV selectively infected and killed ovarian cancer cells, generating long-term survival. Mice with human ovarian cancer cells in brain showed rapid deterioration; LASV-VSV microinjection into brain blocked cancer growth, and generated long-term survival. Treatment of immunocompetent mice with infected mouse ovarian cancer cells blocked growth of non-infected ovarian cancer cells peritoneally and in brain. These results suggest LASV-VSV is a viable candidate for further study and may be of use in the treatment of ovarian cancer.

Weighing the role of hypothalamic feeding neurotransmitters.

Growing health problems related to obesity have focused considerable attention on a number of neurotransmitters, particularly hypothalamic neuropeptides, involved in regulating energy homeostasis and food intake. As the fast-acting transmitters GABA and glutamate underlie the majority of fast synaptic activity in the hypothalamus, understanding neuropeptide modulation of amino acid transmitter actions may be key to a full appreciation of how the brain controls caloric balances.

Selective enhancement of excitatory synaptic activity in the rat nucleus tractus solitarius by hypocretin 2.

Hypocretin 2 (orexin B) is a hypothalamic neuropeptide thought to be involved in regulating energy homeostasis, autonomic function, arousal, and sensory processing. Neural circuits in the caudal nucleus tractus solitarius (NTS) integrate viscerosensory inputs, and are therefore implicated in aspects of all these functions. We tested the hypothesis that hypocretin 2 modulates fast synaptic activity in caudal NTS areas that are generally associated with visceral sensation from cardiorespiratory and gastrointestinal systems. Hypocretin 2-immunoreactive fibers were observed throughout the caudal NTS. In whole-cell recordings from neurons in acute slices, hypocretin 2 depolarized 48% and hyperpolarized 10% of caudal NTS neurons, effects that were not observed when Cs(+) was used as the primary cation carrier. Hypocretin 2 also increased the amplitude of tractus solitarius-evoked excitatory postsynaptic currents (EPSCs) in 36% of neurons and significantly enhanced the frequency of spontaneous EPSCs in most (59%) neurons. Spontaneous inhibitory postsynaptic currents (IPSCs) were relatively unaffected by the peptide. The increase in EPSC frequency persisted in the presence of tetrodotoxin, suggesting a role for the peptide in regulating glutamate release in the NTS by acting at presynaptic terminals. These data suggest that hypocretin 2 modulates excitatory, but not inhibitory, synapses in caudal NTS neurons, including viscerosensory inputs. The selective nature of the effect supports the hypothesis that hypocretin 2 plays a role in modulating autonomic sensory signaling in the NTS.

Membrane properties underlying patterns of GABA-dependent action potentials in developing mouse hypothalamic neurons.

Spikes may play an important role in modulating a number of aspects of brain development. In early hypothalamic development, GABA can either evoke action potentials, or it can shunt other excitatory activity. In both slices and cultures of the mouse hypothalamus, we observed a heterogeneity of spike patterns and frequency in response to GABA. To examine the mechanisms underlying patterns and frequency of GABA-evoked spikes, we used conventional whole cell and gramicidin perforation recordings of neurons (n = 282) in slices and cultures of developing mouse hypothalamus. Recorded with gramicidin pipettes, GABA application evoked action potentials in hypothalamic neurons in brain slices of postnatal day 2-9 (P2-9) mice. With conventional patch pipettes (containing 29 mM Cl-), action potentials were also elicited by GABA from neurons of 2-13 days in vitro (2-13 DIV) embryonic hypothalamic cultures. Depolarizing responses to GABA could be generally classified into three types: depolarization with no spike, a single spike, or complex patterns of multiple spikes. In parallel experiments in slices, electrical stimulation of GABAergic mediobasal hypothalamic neurons in the presence of glutamate receptor antagonists [10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 100 microM 2-amino-5-phosphonopentanoic acid (AP5)] resulted in the occurrence of spikes that were blocked by bicuculline (20 microM). Blocking ionotropic glutamate receptors with AP5 and CNQX did not block GABA-mediated multiple spikes. Similarly, when synaptic transmission was blocked with Cd(2+) (200 microM) and Ni(2+) (300 microM), GABA still induced multiple spikes, suggesting that the multiple spikes can be an intrinsic membrane property of GABA excitation and were not based on local interneurons. When the pipette [Cl-] was 29 or 45 mM, GABA evoked multiple spikes. In contrast, spikes were not detected with 2 or 10 mM intracellular [Cl-]. With gramicidin pipettes, we found that the mean reversal potential of GABA-evoked current (E(GABA)) was positive to the resting membrane potential, suggesting a high intracellular [Cl-] in developing mouse neurons. Varying the holding potential from -80 to 0 mV revealed an inverted U-shaped effect on spike probability. Blocking voltage-dependent Na+ channels with tetrodotoxin eliminated GABA-evoked spikes, but not the GABA-evoked depolarization. Removing Ca(2+) from the extracellular solution did not block spikes, indicating GABA-evoked Na+ -based spikes. Although E(GABA) was more positive within 2-5 days in culture, the probability of GABA-evoked spikes was greater in 6- to 9-day cells. Mechanistically, this appears to be due to a greater Na+ current found in the older cells during a period when the E(GABA) is still positive to the resting membrane potential. GABA evoked similar spike patterns in HEPES and bicarbonate buffers, suggesting that Cl-, not bicarbonate, was primarily responsible for generating multiple spikes. GABA evoked either single or multiple spikes; neurons with multiple spikes had a greater Na+ current, a lower conductance, a more negative spike threshold, and a greater difference between the peak of depolarization and the spike threshold. Taken together, the present results indicate that the patterns of multiple action potentials evoked by GABA are an inherent property of the developing hypothalamic neuron.

Melanin concentrating hormone depresses synaptic activity of glutamate and GABA neurons from rat lateral hypothalamus.

The neuropeptide melanin concentrating hormone (MCH) is synthesised only by neurons of the lateral hypothalamic (LH) area in the CNS. MCH cells project widely throughout the brain. Despite the growing interest in this peptide, in part related to its role in feeding, little has been done to characterise its physiological effects in neurons. Using whole-cell recording with current and voltage clamp, we examined the cellular actions in neurons from the LH. MCH induced a consistent decrease in the frequency of action potentials and reduced synaptic activity. Most fast synaptic activity in the hypothalamus is mediated by GABA or glutamate. MCH inhibited the synaptic activity of both glutamatergic and GABAergic LH neurons, each tested independently. MCH reduced the amplitude of glutamate-evoked currents and reduced the amplitude of miniature excitatory currents, indicating an inhibitory modulation of postsynaptic glutamate receptors. In the presence of tetrodotoxin to block action potentials, MCH caused a depression in the frequency of miniature glutamate-mediated postsynaptic currents, suggesting a presynaptic site of receptor expression. In voltage clamp experiments, MCH depressed the amplitude of calcium currents, suggesting that a mechanism of inhibition may involve a reduced calcium-dependent release of amino acid transmitter. Previous reports have suggested that MCH activated potassium channels in non-neuronal cells transfected with the MCH receptor gene. We found no effect of MCH on voltage-dependent potassium channels in LH neurons. Baclofen, a GABAB receptor agonist, activated G-protein gated inwardly rectifying potassium (GIRK)-type channels; in the same neurons, MCH had no effect on GIRK channels. MCH showed no modulation of sodium currents. Blockade of the Gi/Go protein with pertussis toxin eliminated the actions of MCH. The inhibitory actions of MCH on both excitatory and inhibitory synaptic events, coupled with opposing excitatory actions of hypocretin, another LH peptide that projects to many of the same loci, suggest a substantial level of complexity in neuropeptide modulation of LH actions.

Distribution of hypocretin-(orexin) immunoreactivity in the central nervous system of Syrian hamsters (Mesocricetus auratus).

The hypocretins are peptides synthesized in neurons of the hypothalamus. Recent studies have suggested a role for these peptides in the regulation of sleep, feeding, and endocrine regulation. The distribution of hypocretin-immunoreactive cell bodies and fibers has been extensively described in rats, but not in other species. This study was designed to examine the distribution of hypocretin immunoreactivity in Syrian hamsters, as important differences in neuropeptide distribution between rats and hamsters have previously been demonstrated. Immunoreactive cell bodies were found primarily in the lateral hypothalamic area and the perifornical area, although a few hypocretin-positive cells were also located in the dorsomedial hypothalamus and the retrochiasmatic area. Fibers were distributed throughout the brain in a pattern similar to that seen in rats. The densest projections were found in the paraventricular nucleus of the thalamus, locus coeruleus, dorsal raphe, and lateroanterior hypothalamus. The innervation of the anterior hypothalamus may be of particular interest as similar cluster of immunoreactivity does not appear to be present in rats. Moderate levels of immunoreactivity could be seen throughout the hypothalamus, the lateral septum, bed nucleus of the stria terminalis, A5 noradrenergic area, and the midline thalamic nuclei. Hypocretin-immunoreactive fibers are present in all lamina of the spinal cord, with the greatest axon densities in lamina 1 and 10. The widespread distribution of hypocretin suggests its involvement in a wide variety of physiological and behavioral processes. Our results in hamsters indicate that the organization of the hypocretin system is strongly conserved across species, suggesting an important role for the peptide and its projections.

Lateral hypothalamus: early developmental expression and response to hypocretin (orexin).

Hypocretin is a recently discovered peptide that is synthesized by neurons in the lateral hypothalamic area (LH) and is believed to play a role in sleep regulation, arousal, endocrine control, and food intake. These functions are critical for the development of independent survival. We investigated the developmental profile of the hypocretin system in rats. Northern blot analysis showed that the expression of hypocretin mRNA increased from postnatal day 1 to adulthood. Both of the identified hypocretin receptor mRNAs were strongly expressed very early in hypothalamic development, and expression subsequently decreased in the mature brain. Immunocytochemistry revealed hypocretin-2 peptide expression in the cell bodies of LH neurons and in axons in the brain and spinal cord as early as embryonic day 19. Whole-cell patch clamp recordings from postnatal P1-P14 LH slices demonstrated a robust increase in synaptic activity in all LH neurons tested (n = 20) with a 383% increase in the frequency of spontaneous activity upon hypocretin-2 (1.5 microM) application. A similar increase in activity was found with hypocretin-1 application to LH slices. Hypocretin-2 evoked a robust increase in synaptic activity even on the earliest day tested, the day of birth. Furthermore, voltage-clamp recordings and calcium digital imaging experiments using cultured LH cells revealed that both hypocretin-1 and -2 induced enhancement of neuronal activity occurred as early as synaptic activity was detected. Thus, as in the adult central nervous system, hypocretin exerts a profound excitatory influence on neuronal activity early in development, which might contribute to the development of arousal, sleep regulation, feeding, and endocrine control.

Immunohistochemical localization and biochemical characterization of hypocretin/orexin-related peptides in the central nervous system of the frog Rana ridibunda.

In the present study, we have investigated the distribution and biochemical characteristics of hypocretin (hcrt) -like immunoreactivity in the central nervous system (CNS) of the frog Rana ridibunda by using an antiserum directed against rat hcrt2. Immunoreactive cell bodies were only detected in four diencephalic nuclei, including the anterior preoptic area and the suprachiasmatic, magnocellular, and ventral hypothalamic nuclei. In contrast, hcrt2-immunoreactive fibers were widely distributed throughout the frog CNS. In particular, a high density of hcrt-positive fibers was detected in several areas of the telencephalon, including the olfactory bulb, the nucleus of the diagonal band of Broca, and the amygdala. A dense network of hcrt-containing fibers was observed in all thalamic and hypothalamic nuclei. A low to moderate density of immunoreactive fibers was also found in the mesencephalon, rhombencephalon, and spinal cord. Reversed-phase high performance liquid chromatography analysis of frog brain extracts revealed that hcrt2-immunoreactive material eluted as two peaks, the major one exhibiting the same retention time as synthetic rat hcrt2. The present data provide the first detailed mapping of the hcrt neuronal system in the CNS of a nonmammalian vertebrate. The occurrence of hcrt-containing cell bodies in the hypothalamus and the widespread distribution of hcrt-immunoreactive fibers throughout the brain and spinal cord suggest that, in amphibians, hcrts may exert neuroendocrine, neurotransmitter, and/or neuromodulator activities.

Mouse cytomegalovirus in developing brain tissue: analysis of 11 species with GFP-expressing recombinant virus.

Cytomegaloviruses (CMVs) are species-specific large double-stranded DNA viruses. Mouse and human CMVs have a similar morphology, similar gene sequence, and exert similar cellular effects, but the replication of the virus outside its primary host species is limited. This may confer upon CMV certain advantages for expression of foreign genes or cellular labels in brain cells of nonhost species. We examined the ability of recombinant mouse (m)CMV expressing green fluorescent protein (GFP) to serve as a vector for transgene expression in developing neurons and glia outside the normal host species. For comparative purposes, 11 species were examined. Mouse CMV reporter gene expression was particularly strong in the developing brain of its normal host species, mouse, where it replicated in cultures and brain slices, leading to cell death. All mammalian species tested (human, rat, gerbil, hamster, mouse) showed reporter gene expression after mCMV infection. High levels of mCMV infection were also found in chicken central nervous system cells in vitro, and a low level of mCMV expression was found after an initial delay in turtle neurons and glia. No mCMV reporter gene expression was found in frog cells or aplysia neurons or glia or in drosophila or fungal cells. Infection of nonmouse neurons by low concentrations of mCMV led to strong expression of GFP in dendrites and axons with normal morphology. Despite the lack of replication, high doses of mCMV induced morphologic changes in neurons and glia from hamster and rat brain slices, leading to cells rounding up, and to the formation of giant cells consisting of an aggregate of many cells fused together into a syncytium. In contrast, in human hippocampal slices, GFP-expressing cells infected with mCMV had a relatively normal appearance 12 days after inoculation. To determine whether a CMV from another species could serve as a vector for gene transfer, a recombinant human CMV-expressing GFP was used for transgene expression in rat brain cells in vitro. Cytomegaloviruses thus have potential as useful vectors for gene transfer and labeling central nervous system cells, with the actions of CMV being dependent on a number of factors.

GABA release from mouse axonal growth cones.

1. Using developing hypothalamic neurons from transgenic mice that express high levels of green fluorescent protein in growing axons, and an outside-out patch from mature neuronal membranes that contain neurotransmitter receptors as a sensitive detector, we found that GABA is released by a vesicular mechanism from the growth cones of developing axons prior to synapse formation. 2. A low level of GABA release occurs spontaneously from the growth cone, and this is substantially increased by evoked action potentials. 3. Neurotransmitters such as acetylcholine can enhance protein kinase C (PKC) activity even prior to synapse formation; PKC activation caused a substantial increase in spontaneous GABA release from the growth cone, probably acting at the axon terminal. 4. These data indicate that GABA is secreted from axons during a stage of neuronal development when GABA is excitatory, and that neuromodulators could alter GABA release from the growing axon, potentially enabling other developing neurons of different transmitter phenotype to modulate the early actions of GABA.

Differential neurite growth on astrocyte substrates: interspecies facilitation in green fluorescent protein-transfected rat and human neurons.

In the present study, we used co-culture of astrocytes from one species with neurons from a different species to examine neuritic outgrowth. We include a focus on human cells. Three types of neuron were used, including rat hippocampal dentate granule cells, rat hypothalamic neurons and human cortical neurons. To visualize neuronal processes, neurons were either immunostained with GABA antiserum or transfected with the jellyfish green fluorescent protein gene. The entire axonal and dendritic fields of single neurons could be quantitatively analysed based on their strong green fluorescent protein label. Astrocytes were obtained from rat hippocampus or hypothalamus, chicken cortex, normal human cortex, human cortex lesion, and from the sclerotic human hippocampus after surgery for intractable temporal lobe epilepsy. In the absence of astrocytes, isolated neurons died within three to four days. In contrast, neurons from both rat and human brains survived and extended dendrites and axons on rat, chicken and human astrocytes or in their conditioned medium. Astrocytes from interspecies cultures were not only capable of enhancing the survival of neuron co-cultures, but neuronal neurite extension in some cases was even greater on heterospecific astrocytes than on homospecific astrocytes. To support the hypothesis that synaptogenesis of rat hippocampal neurons was accelerated by a substrate of human astrocytes, we used a functional assay based on time-lapse confocal laser or digital imaging of calcium responses to transmitter release; synaptic responses were found earlier when rat neurons were grown on rat or human astrocytes than in the absence of these astrocytes. These data indicate that rodent glial cells enhance human neurite extension, and that rat neurite outgrowth can be used as a type of bioassay for the neurite promoting capacity of different derivations of human glia.

Cytomegalovirus cell tropism, replication, and gene transfer in brain.

Cytomegalovirus (CMV) infects a majority of adult humans. During early development and in the immunocompromised adult, CMV causes neurological deficits. We used recombinant murine cytomegalovirus (mCMV) expressing either green fluorescent protein (GFP) or beta-galactosidase under control of human elongation factor 1 promoter or CMV immediate early-1 promoter as reporter genes for infected brain cells. In vivo and in vitro studies revealed that neurons and glial cells supported strong reporter gene expression after CMV exposure. Brain cultures selectively enriched in either glia or neurons supported viral replication, leading to process degeneration and cell death within 2 d of viral exposure. In addition, endothelial cells, tanycytes, radial glia, ependymal cells, microglia, and cells from the meninges and choroid were infected. Although mCMV showed no absolute brain cell preference, relative cell preferences were detected. Radial glia cells play an important role in guiding migrating neurons; these were viral targets in the developing brain, suggesting that cortical problems including microgyria that are a consequence of CMV may be caused by compromised radial glia. Although CMV is a species-specific virus, recombinant mCMV entered and expressed reporter genes in both rat and human brain cells, suggesting that mCMV might serve as a vector for gene transfer into brain cells of non-murine species. GFP expression was sufficiently strong that long axons, dendrites, and their associated spines were readily detected in both living and fixed tissue, indicating that mCMV reporter gene constructs may be useful for labeling neurons and their pathways.

Hypocretin (orexin) activation and synaptic innervation of the locus coeruleus noradrenergic system.

Hypocretin has been identified as a regulator of metabolic and endocrine systems. Several brain regions involved in the central regulation of autonomic and endocrine processes or attention are targets of extensive hypocretin projections. The most dense arborization of hypocretin axons in the brainstem was detected in the locus coeruleus (LC). Multiple labeling immunocytochemistry revealed a massive synaptic innervation of catecholaminergic LC cells by hypocretin axon terminals in rats and monkeys. In both species, all tyrosine hydroxylase-immunopositive cells in the LC examined by electron microscopy were found to receive asymmetrical (excitatory) synaptic contacts from multiple axons containing hypocretin. In parallel electrophysiological studies with slices of rat brain, all LC cells showed excitatory responses to the hypocretin-2 peptide. Hypocretin-2 uniformly increased the frequency of action potentials in these cells, even in the presence of tetrodotoxin, indicating that receptors responding to hypocretin were expressed in LC neurons. Two mechanisms for the increased firing rate appeared to be a reduction in the slow component of the afterhyperpolarization (AHP) and a modest depolarization. Catecholamine systems in other parts of the brain, including those found in the medulla, zona incerta, substantia nigra or olfactory bulb, received significantly less hypocretin input. Comparative analysis of lateral hypothalamic input to the LC revealed that hypocretin-containing axon terminals were substantially more abundant than those containing melanin-concentrating hormone. The present results provide evidence for direct action of hypothalamic hypocretin cells on the LC noradrenergic system in rats and monkeys. Our observations suggest a signaling pathway via which signals acting on the lateral hypothalamus may influence the activity of the LC and thereby a variety of CNSfunctions related to noradrenergic innervation, including vigilance, attention, learning, and memory. Thus, the hypocretin innervation of the LC may serve to focus cognitive processes to compliment hypocretin-mediated activation of autonomic centers already described.

Kainate acts at presynaptic receptors to increase GABA release from hypothalamic neurons.

Recent reports suggest that kainate acting at presynaptic receptors reduces the release of the inhibitory transmitter GABA from hippocampal neurons. In contrast, in the hypothalamus in the presence of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor antagonists [1-(4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466) and D,L-2-amino-5-phosphonopentanoic acid (AP5)], kainate increased GABA release. In the presence of tetrodotoxin, the frequency, but not the amplitude, of GABA-mediated miniature inhibitory postsynaptic currents (IPSCs) was enhanced by kainate, consistent with a presynaptic site of action. Postsynaptic activation of kainate receptors on cell bodies/dendrites was also found. In contrast to the hippocampus where kainate increases excitability by reducing GABA release, in the hypothalamus where a much higher number of GABAergic cells exist, kainate-mediated activation of transmitter release from inhibitory neurons may reduce the level of neuronal activity in the postsynaptic cell.

GABAB receptor-mediated regulation of glutamate-activated calcium transients in hypothalamic and cortical neuron development.

In the mature nervous system excitatory neurotransmission mediated by glutamate is balanced by the inhibitory actions of GABA. However, during early development, GABA acting at the ligand-gated GABAA Cl- channel also exerts excitatory actions. This raises a question as to whether GABA can exert inhibitory activity during early development, possibly by a mechanism that involves activation of the G protein-coupled GABAB receptor. To address this question we used Ca2+ digital imaging to assess the modulatory role of GABAB receptor signaling in relation to the excitatory effects of glutamate during hypothalamic and cortical neuron development. Ca2+ transients mediated by synaptic glutamate release in neurons cultured from embryonic rat were dramatically depressed by the administration of the GABAB receptor agonist baclofen in a dose-dependent manner. The inhibitory effects of GABAB receptor activation persisted for the duration of baclofen administration (>10 min). Preincubation with the Gi protein inhibitor pertussis toxin resulted in a substantial decrease in the inhibitory actions of baclofen, confirming that a Gi-dependent mechanism mediated the effects of the GABAB receptor. Co-administration of the GABAB receptor antagonist 2-hydroxy-saclofen eliminated the inhibitory action of baclofen. Alone, GABAB antagonist application elicited a marked potentiation of Ca2+ transients mediated by glutamatergic neurotransmission, suggesting that tonic synaptic GABA release exerts an inhibitory tone on glutamate receptor-mediated Ca2+ transients via GABAB receptor activation. In the presence of TTX to block action potential-mediated neurotransmitter release, stimulation with exogenously applied glutamate triggered a robust postsynaptic Ca2+ rise that was dramatically depressed (>70% in cortical neurons, >40% in hypothalamic neurons) by baclofen. Together these data suggest both a pre- and postsynaptic component for the modulatory actions of the GABAB receptor. These results indicate a potentially important role for the GABAB receptor as a modulator of the excitatory actions of glutamate in developing neurons.

NPY inhibits glutamatergic excitation in the epileptic human dentate gyrus.

Neuropeptide Y (NPY) has been shown to depress hyperexcitable activity that has been acutely induced in the normal rat brain. To test the hypothesis that NPY can also reduce excitability in the chronically epileptic human brain, we recorded intracellularly from dentate granule cells in hippocampal slices from patients with hippocampal seizure onset. NPY had a potent and long-lasting inhibitory action on perforant path-evoked excitatory responses. In comparison, the group 3 metabotropic glutamate receptor agonist L-2-amino-4-phosphonobutyric acid (L-AP4) evoked a mild and transient decrease. NPY-containing axons were found throughout the hippocampus, and in many epileptic patients were reorganized, particularly in the dentate molecular layer. NPY may therefore play a beneficial role in reducing granule cell excitability in chronically epileptic human tissue, and subsequently limit seizure severity.

Neurotrophin-3 potentiates excitatory GABAergic synaptic transmission in cultured developing hypothalamic neurones of the rat.

1. Neurotrophin-3 (NT-3) supports the survival and differentiation of neurones in the central and peripheral nervous systems through a number of mechanisms that occur in a matter of hours or days. NT-3 may also have a more rapid mode of action that influences synaptic activity in mature neurones. In the present study, the effect of NT-3 on developing GABAergic synapses was investigated in 3- to 7-day-old cultures of rat hypothalamic neurones with whole-cell patch-clamp recording. 2. NT-3 induced a substantial dose-dependent potentiation of the frequency of spontaneous postsynaptic currents (sPSCs; 160 %) in developing neurones during a period when GABA evoked inward (depolarizing) current, as determined with gramicidin-perforated patch recordings. The NT-3 effect was long lasting; continued enhancement was found > 30 min after NT-3 wash-out. NT-3 evoked a substantial 202 % increase in total GABA-mediated inward current, measured as the time-current integral. Action potential frequency was also increased by NT-3 (to 220 %). 3. The frequency of GABA-mediated miniature postsynaptic currents in developing neurones in the presence of tetrodotoxin was potentiated (to 140%) by NT-3 with no change in the mean amplitude, suggesting a presynaptic locus of the effect. 4. In striking contrast to immature neurones, when more mature neurones were studied, NT-3 did not enhance the frequency of GABA-mediated spontaneous postsynaptic currents (sPSCs), but instead evoked a slight (16%) decrease. The frequency of miniature post-synaptic currents was also slightly decreased (16%) by the NT-3, with no change in amplitude. These results were recorded during a later period of neuronal maturity when GABA would evoke outward (hyperpolarizing) currents. NT-3 had no effect on the mean amplitude of GABA-evoked postsynaptic currents in either developing or mature neurones. 5. Intracellular application of K252a, a non-selective tyrosine kinase inhibitor, did not block the NT-3 effect postsynaptically. In contrast, bath application of K252a prevented the enhancement of sPSCs by NT-3, consistent with NT-3 acting through presynaptic induction of tyrosine kinase. Decreasing extracellular calcium with BAPTA or inhibiting calcium channels with Cd2+ blocked the augmentation of sPSC frequency by NT-3, suggesting that an increase of calcium entry may be required for the facilitation of NT-3. 6. Together, our results suggest NT-3 enhances GABA release during the developmental period when GABA is depolarizing and calcium elevating, but not later when GABA is inhibitory, suggesting that one mechanism through which NT-3 may influence neuronal development is via presynaptic potentiation of GABA excitation.

Hypothalamic hypocretin (orexin): robust innervation of the spinal cord.

Hypocretin (orexin) is synthesized by neurons in the lateral hypothalamus and has been reported to increase food intake and regulate the neuroendocrine system. In the present paper, long descending axonal projections that contain hypocretin were found that innervate all levels of the spinal cord from cervical to sacral segments, as studied in mouse, rat, and human spinal cord and not previously described. High densities of axonal innervation are found in regions of the spinal cord related to modulation of sensation and pain, notably in the marginal zone (lamina 1). Innervation of the intermediolateral column and lamina 10 as well as strong innervation of the caudal region of the sacral cord suggest that hypocretin may participate in the regulation of both the sympathetic and parasympathetic parts of the autonomic nervous system. Double-labeling experiments in mice combining retrograde transport of diamidino yellow after spinal cord injections and immunocytochemistry support the concept that hypocretin-immunoreactive fibers in the cord originate from the neurons in the lateral hypothalamus. Digital-imaging physiological studies with fura-2 detected a rise in intracellular calcium in response to hypocretin in cultured rat spinal cord neurons, indicating that spinal cord neurons express hypocretin-responsive receptors. A greater number of cervical cord neurons responded to hypocretin than another hypothalamo-spinal neuropeptide, oxytocin. These data suggest that in addition to possible roles in feeding and endocrine regulation, the descending hypocretin fiber system may play a role in modulation of sensory input, particularly in regions of the cord related to pain perception and autonomic tone.

Synaptic interaction between hypocretin (orexin) and neuropeptide Y cells in the rodent and primate hypothalamus: a novel circuit implicated in metabolic and endocrine regulations.

Hypocretin (orexin) has recently been shown to increase feeding when injected into the brain. Using both rat and primate brains, we tested the hypothesis that a mechanism of hypocretin action might be related to synaptic regulation of the neuropeptide Y (NPY) system. Hypocretin-immunoreactive terminals originating from the lateral hypothalamus make direct synaptic contact with neurons of the arcuate nucleus that not only express NPY but also contain leptin receptors. In addition, hypocretin-containing neurons also express leptin receptor immunoreactivity. This suggests a potential mechanism of action for hypocretin in the central regulation of metabolic and endocrine processes. The excitatory actions of hypocretin could increase NPY release, resulting in enhanced feeding behavior and altered endocrine regulation, whereas leptin, released from adipose tissue as an indicator of fat stores, would have the opposite effect on the same neurons, leading to a decrease in NPY and NPY-mediated hypothalamic functions. On the other hand, the innervation of hypocretin cells by NPY boutons raises the possibility that NPY may exert an effect on hypothalamic functions, at least in part, via mediation or feedback action on these lateral hypothalamic cells. Our data indicate that a direct interaction between leptin, hypocretin, and NPY exists in the hypothalamus that may contribute to the central regulation of metabolic and endocrine processes in both rodents and primates.