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A N van den Pol

Publications and source records attributed to A N van den Pol.

At least 19 recordsLinked to original sources
PubMed2003

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.

Animals↗
PubMed2001

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.

2-Amino-5-phosphonovalerate↗
PubMed2001

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.

Animals↗
PubMed2001

GABA, not glutamate, a primary transmitter driving action potentials in developing hypothalamic neurons.

Neuronal activity is critical for many aspects of brain development. It has often been assumed that the primary excitatory transmitter driving this activity is glutamate. In contrast, we report that during early development, synaptic release of GABA, the primary inhibitory neurotransmitter in the mature brain, is not only excitatory but in addition plays a more robust role than glutamate in generating spike activity in mouse hypothalamic neurons. Based on gramicidin perforated whole cell and extracellular recording, which leave intracellular Cl(-) unperturbed in brain slices and cultures, the GABA(A) receptor antagonist bicuculline induced a dramatic decrease in spike frequency (83% decrease) in developing neurons, three times greater than that generated by glutamate receptor antagonists 2-amino-5-phosphono-pentanoic acid and 6-cyano-7-nitroquinoxalene-2,3-dione. Thus a number of factors related to spike-dependent stabilization of neuronal connections, including Hebbian mechanisms, that are generally applied to glutamate transmission may also participate in stabilization of GABA circuits.

Action Potentials↗
PubMed2001

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.

2-Amino-5-phosphonovalerate↗
PubMed2000

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.

Animals↗
PubMed2000

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.

2-Amino-5-phosphonovalerate↗
PubMed1999

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.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1999

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.

Animals↗
PubMed1999

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.

Animals↗
PubMed1999

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.

2-Amino-5-phosphonovalerate↗
PubMed1999

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.

2-Amino-5-phosphonovalerate↗
PubMed1999

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.

Animals↗
PubMed1998

Selective neuronal expression of green fluorescent protein with cytomegalovirus promoter reveals entire neuronal arbor in transgenic mice.

In simple nervous systems, identified groups of neurons can be studied in depth. To allow the same advantage in the mammalian brain, we have generated green fluorescent protein (GFP) transgenic mice in which only a few types of neurons are strongly labeled with a fluorescent molecule, which the neurons synthesize internally, allowing the cells, their dendrites, filopodia, and axons to be identified in both living and fixed CNS, in slices and culture. The same neurons, with GFP expression controlled by part of the major immediate early promoter of human cytomegalovirus (CMV), show GFP beginning early in development, from one generation to the next, allowing cellular and physiological studies of axonal and dendritic growth, fate mapping, anatomical connections, and synapse formation in identified neurons. The human CMV promoter sequence we used was different from that used in previous work with other reporter genes and gave a dramatically different pattern of expression. Two transgenic lines with the same CMV promoter show similar anatomical patterns of expression in the present study. Strong GFP labeling was found in a subpopulation of mossy fibers that innervated parasagittal bands in the cerebellar cortex and olfactory axons that projected into the olfactory bulb, subsets of motoneurons and dorsal root ganglion cells, granule but not mitral cells of the olfactory bulb, and a group of neurons in the hypothalamic suprachiasmatic nucleus. A novel type of neuron was strongly labeled in the olfactory bulb external plexiform layer. In normal brains, CMV does not constitute a threat, but in the developing brain, CMV can cause debilitating neurodegeneration and death; studies using the CMV promoter aid in understanding the affinity of CMV that has been suggested for specific brain regions.

Animals↗
PubMed1998

Glutamate inhibits GABA excitatory activity in developing neurons.

In contrast to the mature brain, in which GABA is the major inhibitory neurotransmitter, in the developing brain GABA can be excitatory, leading to depolarization, increased cytoplasmic calcium, and action potentials. We find in developing hypothalamic neurons that glutamate can inhibit the excitatory actions of GABA, as revealed with fura-2 digital imaging and whole-cell recording in cultures and brain slices. Several mechanisms for the inhibitory role of glutamate were identified. Glutamate reduced the amplitude of the cytoplasmic calcium rise evoked by GABA, in part by activation of group II metabotropic glutamate receptors (mGluRs). Presynaptically, activation of the group III mGluRs caused a striking inhibition of GABA release in early stages of synapse formation. Similar inhibitory actions of the group III mGluR agonist L-AP4 on depolarizing GABA activity were found in developing hypothalamic, cortical, and spinal cord neurons in vitro, suggesting this may be a widespread mechanism of inhibition in neurons throughout the developing brain. Antagonists of group III mGluRs increased GABA activity, suggesting an ongoing spontaneous glutamate-mediated inhibition of excitatory GABA actions in developing neurons. Northern blots revealed that many mGluRs were expressed early in brain development, including times of synaptogenesis. Together these data suggest that in developing neurons glutamate can inhibit the excitatory actions of GABA at both presynaptic and postsynaptic sites, and this may be one set of mechanisms whereby the actions of two excitatory transmitters, GABA and glutamate, do not lead to runaway excitation in the developing brain. In addition to its independent excitatory role that has been the subject of much attention, our data suggest that glutamate may also play an inhibitory role in modulating the calcium-elevating actions of GABA that may affect neuronal migration, synapse formation, neurite outgrowth, and growth cone guidance during early brain development.

Animals↗
PubMed1998

Neurons containing hypocretin (orexin) project to multiple neuronal systems.

The novel neuropeptides called hypocretins (orexins) have recently been identified as being localized exclusively in cell bodies in a subregion of the tuberal part of the hypothalamus. The structure of the hypocretins, their accumulation in vesicles of axon terminals, and their excitatory effect on cultured hypothalamic neurons suggest that the hypocretins function in intercellular communication. To characterize these peptides further and to help understand what physiological functions they may serve, we undertook an immunohistochemical study to examine the distribution of preprohypocretin-immunoreactive neurons and fibers in the rat brain. Preprohypocretin-positive neurons were found in the perifornical nucleus and in the dorsal and lateral hypothalamic areas. These cells were distinct from those that express melanin-concentrating hormone. Although they represent a restricted group of cells, their projections were widely distributed in the brain. We observed labeled fibers throughout the hypothalamus. The densest extrahypothalamic projection was found in the locus coeruleus. Fibers were also seen in the septal nuclei, the bed nucleus of the stria terminalis, the paraventricular and reuniens nuclei of the thalamus, the zona incerta, the subthalamic nucleus, the central gray, the substantia nigra, the raphe nuclei, the parabrachial area, the medullary reticular formation, and the nucleus of the solitary tract. Less prominent projections were found in cortical regions, central and anterior amygdaloid nuclei, and the olfactory bulb. These results suggest that hypocretins are likely to have a role in physiological functions in addition to food intake such as regulation of blood pressure, the neuroendocrine system, body temperature, and the sleep-waking cycle.

Animals↗
PubMed1998

Early synaptogenesis in vitro: role of axon target distance.

In contrast to some previous reports suggesting a delay in synapse formation in vitro, we found that under ideal conditions, most hippocampal and hypothalamic rat neurons were synaptically coupled after 3 or 4 days in vitro. Synaptophysin immunocytochemistry revealed strongly stained presynaptic boutons by 3 days in vitro. Studies with time-lapse laser confocal imaging of FM1-43 revealed that axonal boutons were recycling their synaptic vesicles, an indication of synapse formation, as early as 3 days after plating. To test the hypothesis that neurite outgrowth was enhanced in high-density cultures, thereby increasing the probability of synapse formation, neurons were transfected with the jellyfish green fluorescent protein (GFP) gene. After 2 days in high-density cultures, green fluorescent neurites were about three times longer than in sister neurons plated in low-density cultures. Even in single dishes, GFP-transfected cells in contact with other neurons had neurites that were at least three times longer and grew faster than more isolated cells. Neurons grew longer neurites (+51%) when growing on surface membranes of heat-killed neurons than on polylysine, underlining the importance of plasma membrane contact. Calcium imaging with fura-2 and whole cell recording showed that both GABA and glutamate presynaptic release occurred after 3 or 4 days in vitro in high-density cultures but was absent in low-density cultures at this time. Together, these morphological, cytochemical, and physiological data suggest that the distance an axon must grow to find a postsynaptic partner plays a substantial role in the timing of synapse formation. Although other factors in vitro may also play a role, the distance to a postsynaptic target, which defines the interval during which an axon grows to its target, can probably account for much of the difference in timing of synapse formation previously reported in vitro. A short intercell distance may increase the concentration of limited amounts of trophic factors available to a nearby cell, and once contact is made, a neuronal membrane provides a superior substrate for neuritic elongation.

2-Amino-5-phosphonovalerate↗