An van den Pol

Neurotransmitters of the hypothalamic suprachiasmatic nucleus: immunocytochemical analysis of 25 neuronal antigens.

An immunocytochemical analysis with 33 antisera was undertaken to investigate the localization of 25 different neurotransmitter-related antigens in the hypothalamic suprachiasmatic nucleus in the rat. To obtain estimates of relative densities of immunoreactive axons a stereological approach was used involving counting of intersections of immunoreactive axons with a superimposed semi-circle test grid. All neurotransmitter-related antigens found in perikarya within the suprachiasmatic nucleus, including those stained with antisera against bombesin, gastrin-releasing peptide, neurophysin, vasopressin, somatostatin, gamma-aminobutyrate, glutamate decarboxylase and vasoactive intestinal polypeptide were also found in axons within the nucleus. A greater number of these immunoreactive axons was found within the nucleus than in the adjacent anterior hypothalamus. The size of all immunoreactive axons in the suprachiasmatic nucleus was consistently small; immunoreactive axons were found ramifying widely in the nucleus, often ending with terminal boutons near perikarya immunoreactive for the same antigen. All neurotransmitter-related substances found in perikarya of the suprachiasmatic nucleus were also found in axons crossing over the midline to innervate the contralateral nucleus, providing an anatomical substrate for a high degree of communication between the paired nuclei. Axons immunoreactive for other putative transmitters including serotonin arising outside the nucleus were also found in high densities within the nucleus and crossing over the midline between the nuclei. Immunoreactivity for some transmitters was found in axons of similar densities within and outside the nucleus, including antisera against tyrosine hydroxylase; a small number of dopamine beta-hydroxylase and a few phenylethanolamine N-methyltransferase-immunoreactive axons were found in the SCN, suggesting that dopamine, norepinephrine and epinephrine may occur in a limited number of axons in the nucleus. Small numbers of axons immunoreactive with antisera raised against cholecystokinin, prolactin, substance P, thyrotropin-releasing hormone and choline acetyltransferase were found within the suprachiasmatic nucleus. Axons immunoreactive for luteinizing hormone-releasing hormone, adrenocorticotropic hormone, alpha-melanocyte-stimulating hormone and neurotensin were rarely found within the suprachiasmatic nucleus; axons immunoreactive for luteinizing hormone-releasing hormone, adrenocorticotropic hormone, cholecystokinin and tyrosine hydroxylase were found in both horizontal and coronal sections in the area between the left and right suprachiasmatic nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)

Excitatory actions of GABA in developing rat hypothalamic neurones.

1. Gramicidin‐perforated patch clamp recording was employed to study GABA‐mediated responses in rat hypothalamic neurones (n = 102) with an intracellular Cl‐ concentration unaltered by the pipette solution. 2. In young cultures after 1‐7 days in vitro (DIV), GABA induced depolarizing membrane potentials (+16.5 +/‐ 1.3 mV) that often surpassed the threshold for the firing of action potentials (‐42 +/‐ 1 mV) and resulted in an increase in neuronal activity. The depolarizing responses to GABA in young cultures were dose dependent. The concentration of GABA necessary to evoke the half‐maximal depolarization (EC50) was 2.8 microM. In contrast, GABA induced hyperpolarizing membrane potentials (‐12.0 +/‐ 1.4 mV) and a decrease in neuronal activity in older neurones (20‐33 DIV). Both the depolarization and the hyperpolarization induced by GABA were blocked by bicuculline, indicating a mediation by GABAA receptors. 3. The reversal potentials of the GABA‐evoked currents were between ‐40 to ‐50 mV during the first week of culture, and shifted to below ‐70 mV after 3 weeks of culture. In parallel, neurones that were dissociated from older animals (postnatal day 5) had a more negative reversal potential for the GABA‐evoked currents than cells from younger animals (embryonic day 15), suggesting that the negative shift of the reversal potential occurs both in vitro and in vivo. Our data suggest that the mechanism for GABA‐induced depolarization is the depolarized Cl‐ reversal potential found in young but not older neurones. 4. Consistent with the depolarizing response to exogenous application of GABA, some spontaneous depolarizing postsynaptic potentials in young cultures were insensitive to AP5‐CNQX, but were eliminated by bicuculline, indicating that synaptically released GABA mediated excitatory synaptic transmission in early development. 5. By combining a rapid computer‐controlled delivery of GABA with subthreshold positive current injections into recorded neurones, we found in young cultures that the GABA‐evoked depolarization could directly trigger action potentials, facilitate some depolarizing input to fire action potentials, and shunt other depolarizing input. Whether the GABA‐induced depolarization is excitatory or inhibitory would be determined by the reversal potential of the GABA‐evoked current, and the temporal relationship between GABA‐evoked depolarizations and other excitatory events. 6. We conclude that the reversal potential of the GABA‐evoked current shifts negatively from depolarizing to hyperpolarizing in developing hypothalamus. Consequently, GABA neurotransmission may serve both excitatory and inhibitory roles during early development.

Tyrosine hydroxylase-immunoreactive neurons of the hypothalamus: a light and electron microscopic study.

The localization and morphology of neurons, processes, and neuronal groups in the rat hypothalamus containing tyrosine hydroxylase-like immunoreactivity were studied using an antiserum to bovine tyrosine hydroxylase. This antiserum was thoroughly characterized by precipitation of enzyme activity, immunoblotting, and precipitation of cell-free translation products; a single molecular weight band was recognized by the antiserum. Absorption of the antiserum with purified tyrosine hydroxylase abolished immunocytochemical staining, while addition of bovine dopamine beta-hydroxylase had no effect on immunostaining. Immunoreactive cells were found throughout the hypothalamus. Significant numbers of cells were found in the arcuate, periventricular, dorsomedial hypothalamus/zona incerta, posterior hypothalamic regions (A11-A14), and paraventricular nucleus, as previously described, and in addition, in the preoptic area, adjacent to the anterior commissure, medial and lateral to the suprachiasmatic nucleus, dorsal to and in the supraoptic nucleus, at the lateral borders of the ventromedial nucleus, and in the dorsal and ventral lateral hypothalamus. None of the immunoreactive cell groups are totally separated from adjacent cell groups. Dendritic overlap occurs between any two adjacent groups. From cell counts of 30 micron coronal sections, we estimate the hypothalamus has about 12,000 cells based on raw counts, or 8000 immunoreactive cells after correction for possible split cells. Mean soma size varied considerably from one immunoreactive group to another. Cells in the caudal part of the dorsomedial hypothalamus/zona incerta region were the largest, with a mean diameter of 25 micron, while cells in the anterior commissural and posterior hypothalamic group were among the smallest, with mean diameters of 10 micron. The largest immunoreactive cells in the hypothalamus had volumes in excess of ten times greater than the smallest immunoreactive cells. Tyrosine hydroxylase immunoreactivity was found in dendrites in every region of the hypothalamus, sometimes extending hundreds of micrometers from the perikaryon of origin. Although adjacent cell groups were not distinctly separated, the dendritic arbors of the different cell groups differed greatly. Dendritic and somatic appendages were found on some cells, particularly in the paraventricular nucleus. Immunoreactive dendritic arbors were particularly large in cells seen on horizontal sections through the caudal dorsomedial hypothalamic group and through the anterior hypothalamus. Only slight dendritic trees were observed in the rostral dorsomedial hypothalamus/zona incerta region, and in the pericommissural group.(ABSTRACT TRUNCATED AT 400 WORDS)

Cellular communication in the circadian clock, the suprachiasmatic nucleus

The hypothalamic suprachiasmatic nucleus functions as the circadian clock in the mammalian brain. Communication between the cells of the suprachiasmatic nucleus is likely to be responsible for the generation and accuracy of this biological clock. Communication between many cells of the brain is mediated by action potentials that pass down the axon and cause release of neurotransmitters at the neuronal synaptic junction. Additional mechanisms of cellular communication appear to operate in the suprachiasmatic nucleus. Several lines of evidence point to multiple modes of cellular communication: these include the continuing operation of the clock after Na(+)-mediated action potentials have been blocked, the orchestrated metabolic rhythms of suprachiasmatic nucleus cells prior to synaptogenesis, the entrainment of fetal to maternal rhythms, and the rapid recovery of function after suprachiasmatic nucleus transplants into arrhythmic rodents. Possible alternative means of intercellular communication in the suprachiasmatic nucleus are examined, including calcium spikes in presynaptic dendrites, ephaptic interaction, paracrine communication, glial mediation, and gap junctions. This paper identifies and examines some of the unanswered questions related to intercellular communication of suprachiasmatic nucleus cells.

Gamma-aminobutyrate, gastrin releasing peptide, serotonin, somatostatin, and vasopressin: Ultrastructural immunocytochemical localization in presynaptic axons in the suprachiasmatic nucleus

Abstract An ultrastructural immunocytochemical study was undertaken to identify neuroactive substances contained in presynaptic boutons in the hypothalamic suprachiasmatic nucleus. Axonal boutons containing immunoreactive γ-aminobutyrate, glutamate decarboxylase, neurophysin/vasopressin, gastrin releasing peptide/bombesin, somatostatin and serotonin were localized within the hypothalamic suprachiasmatic nucleus with pre-embedding peroxidase immunostaining. Synaptic contacts were found between boutons containing each of these substances and postsynaptic structures. While some variation in synaptic morphology existed, most of the immunoreactive contacts were of the symmetrical type. Previous work has indicated that neuroactive peptides may be found in highest concentrations in dense-core vesicles, to examine the subcellular localization of the amino acid inhibitory transmitter γ-aminobutyrate, ultrastructural immunocytochemistry with pre-embedding peroxidase was compared with post-embedding immunocytochemistry with colloidal gold. Ultracryothin sections were also used for ultrastructural localization of γ-aminobutyrate and glutamate decarboxylase immunoreactivity. Both γ-aminobutyrate and glutamate decarboxylase immunoreactivity were found throughout the cytoplasm of immunoreactive boutons when pre-embedding peroxidase was used; with post-embedding colloidal gold immunostaining, label was found over areas containing small clear vesicles, and over mitochondria of immunoreactive axons. At the dilutions used in this study, strongly immunoreactive γ-aminobutyrate dendrites, boutons forming asymmetrical synapses, and cell bodies were not found. Differences between pre-embedding and post-embedding immunostaining may be due to antigen and label diffusion caused by mild fixation and membrane damage necessary for antisera penetration during pre-embedding immunostaining. These results suggest that γ-aminobutyrate, gastrin releasing peptide, somatostatin and vasopressin are contained in axons making contact with neurons of the suprachiasmatic nucleus, and may function as neurotransmitters here. Since all of these substances can also be localized in perikarya within the suprachiasmatic nucleus, there is a strong possibility that at least some of the axons containing immunoreactivity for each of these substances may be involved in local circuit interactions between neurons within the suprachiasmatic nucleus.

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.

Glutamate hyperexcitability and seizure-like activity throughout the brain and spinal cord upon relief from chronic glutamate receptor blockade in culture

Cortical structures such as the hippocampus and cerebral cortex are considered to be particularly susceptible to seizure and epileptiform electrical activity and, as such, are the focus of intense investigation relative to hyperexcitability. To determine whether parallel glutamate-mediated hyperexcitability and seizure-like activity in the rat can be generated by neurons irrespective of their origin within the CNS, we maintained cells from the spinal cord,hippocampus, olfactory bulb, striatum, hypothalamus, and cortex in the long-term presence of glutamate receptor antagonists 2-amino-5-phosphonovalerate and 6-cyano-7-nitroquinoxaline-2-3-dione. After removal of chronic (three to 11 weeks) glutamate receptor block, whole-cell patch-clamp recordings from current-clamped neurons (n = 94) revealed an immediate increase in large excitatory postsynaptic potentials and a depolarization of 20-35 mV that was often sustained for recording periods lasting 5 min (54% of 66 neurons from all six areas). The intense activity was not seen in age-matched control neurons not subjected to chronic glutamate receptor block. Selective blockade of ionotropic glutamate receptors showed that the hyperexcitability was due to an enhanced response through both AMPA/kainate and N-methyl-D-aspartate receptors. Relief from chronic glutamate receptor block also increased inhibitory activity, as revealed by an increase in inhibitory postsynaptic currents while neurons were voltage-clamped at -25 mV. These inhibitory postsynaptic currents could be blocked with bicuculline, indicating that they were mediated by an enhanced GABA release. This enhanced GABA activity reduced, but did not eliminate, the glutamate-mediated hyperactivity, shown by an increase in both intracellular Ca2+ and excitatory electrical activity when bicuculline was added. When the glutamate receptor block was removed, cells (n > 1000) from all six regions showed exaggerated Ca2+ activity, characterized by abnormally high increases in intracellular Ca2+, rising from basal levels of 50-100 nM up to 150-1600 nM. Cd2+ eliminated the hyperexcitability by blocking Ca2+ channels, and reducing excitatory transmitter release and response. Fura-2 digital imaging revealed Ca2+ oscillations with periods ranging from 4 to 60 s. Ca2+ peaks in oscillations in oscillations were synchronized among most neurons recorded simultaneously. That synchronization was dependent on a mechanism involving voltage-dependent Na+ channels was demonstrated with experiments with tetrodotoxin that blocked Ca2+ rises and synchronous cellular behavior. Removal of the glutamate receptor antagonists resulted in the glutamate-mediated death of 44% of the cells after 23 days of chronic block and 82% cell death after 40 days of chronic block. Nimodipine substantially reduced cell death, indicating that one mechanism responsible for the enhanced cell death after relief from chronic glutamate receptor block was increased intracellular Ca2+ entry through L-type voltage-gated calcium channels. These data indicate that glutamate is released by neurons from all areas studied, including the spinal cord. Sufficient amounts of glutamate can be released from axon terminals from all areas to cause cell hippocampal and cortical neurons, but also by neurons from any of the brain regions tested after chronic deprivation of glutamate receptor stimulation during development. This hyperexcitability is mediated by glutamatergic mechanisms independent of the specific excitatory connections existing in vivo. The epileptiform activity of neurons from one region is indistinguishable from that of another in culture, underlining the importance of synaptic connections in vivo that define the responses characteristic of neurons from different brain regions.

Zika virus targeting in the developing brain

Zika virus (ZIKV), a positive-sense RNA flavivirus, has attracted considerable attention recently for its potential to cause serious neurological problems, including microcephaly, cortical thinning, and blindness during early development. Recent findings suggest that ZIKV infection of the brain can occur not only during very early stages of development, but also in later fetal/early neonatal stages of maturation. Surprisingly, after peripheral inoculation of immunocompetent mice on the day of birth, the first cells targeted throughout the brain were isolated astrocytes. At later stages, more neurons showed ZIKV immunoreactivity, in part potentially due to ZIKV release from infected astrocytes. In all developing mice studied, we detected infection of retinal neurons; in many mice, this was also associated with infection of the lateral geniculate, suprachiasmatic nuclei, and superior colliculus, suggesting a commonality for the virus to infect cells of the visual system. Interestingly, in mature mice lacking a Type 1 interferon response (IFNR−/−), after inoculation of the eye, the initial majority of infected cells in the visual system were glial cells along the optic tract. ZIKV microinjection into the somatosensory cortex on one side of the normal mouse brain resulted in mirror infection restricted to the contralateral somatosensory cortex without any infection of midline brain regions, indicating the virus can move by axonal transport to synaptically coupled brain loci. These data support the view that ZIKV shows considerable complexity in targeting the CNS and may target different cells at different stages of brain development. SIGNIFICANCE STATEMENT Zika virus (ZIKV) can cause substantial damage to the developing human brain. Here we examine a developmental mouse model of ZIKV infection in the newborn mouse in which the brain is developmentally similar to a second-trimester human fetus. After peripheral inoculation, the virus entered the CNS in all mice tested and initially targeted astrocytes throughout the brain. Infections of the retina were detected in all mice, and infection of CNS visual system nuclei in the brain was common. We find that ZIKV can be transported axonally, thereby enhancing virus spread within the brain. These data suggest that ZIKV infects multiple cell types within the brain and that astrocyte infection may play a more important role in initial infection than previously appreciated.

Embryonic hypothalamic expression of functional glutamate receptors

Glutamate can play a number of roles in the developing brain, including modulation of gene expression, cell motility, neurite growth and neuronal survival, all critical for the final organization and function of the mature brain. These functions are dependent on the early expression of glutamate receptors and on glutamate release in developing neurons. This subject has received little attention in the hypothalamus, despite glutamates critical role as an excitatory transmitter in hypothalamic control of circadian rhythms, endocrine secretion, temperature regulation, and autonomic control. A total of 10,922 rat hypothalamic neurons were studied with digital Ca2+ imaging with the ratiometric dye fura-2 to examine their responses to glutamate receptor agonists and antagonists during embryonic development and maturation in vitro. Functional glutamate receptors were found very early in development (embryonic day 15-E15) with both Ca2+ imaging and with patch clamp recording. This is a time when the hypothalamus is beginning to undergo neurogenesis. Ca2+ responses from N-methyl-D-aspartate receptors developed later than those from non-N-methyl-D-aspartate ionotropic receptors that responded to kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate. The responses of immature E15 cells after one day in vitro were compared with more mature cells after six days in vitro to examine the response to repeated 3 min applications of 100 microM kainate (n = 108). Immature cells showed similar Ca2+ rises (+232nM Ca2+) with each kainate stimulation. In contrast, more mature cells showed an initial Ca2+ rise of 307 nM, with the second rise only to 147 nM above the initial baseline. Immature cells more quickly returned to their pre-kainate baseline than did older cells. The expression of metabotropic glutamate receptors was studied with the selective agonist trans-1-amino-cyclopentyl-1,3-dicarboxylic acid and with glutamate stimulation in the absence of extracellular Ca2+ and presence of 1 mM EGTA. After five days in vitro. E16 astrocytes showed a greater response than did neurons to conditions that would activate the metabotropic glutamate receptor. A dramatic increase in the percentage of cells that responded to N-methyl-D-aspartate was found after only a few days in culture. Only a small number of E15 cells studied on the day of culture (4% of 694 cells) showed a response to 100 microM N-methyl-D-aspartate. Thirty-eight percent of 120 E18 cells cultured for one day in vitro showed an N-methyl-D-aspartate response.(ABSTRACT TRUNCATED AT 400 WORDS)