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.

Growth cone localization of neural cell adhesion molecule on central nervous system neurons in vitro.

Ultrastructural analysis of colloidal gold immunocytochemical staining and immunofluorescence microscopy has been used to study the presence of neural cell adhesion molecule (NCAM) on the surface of neuronal growth cones. The studies were carried out with cultures of rat hypothalamic and ventral mesencephalic cells, using morphology and expression of tyrosine hydroxylase, neurofilaments, and glial fibrillary acidic protein as differential markers for neurons and glia. NCAM was found on all plasmalemmal surfaces of neurons including perikarya and neurites. The density of NCAM varied for different neurons growing in the same culture dish, and neurons had at least 25 times more colloidal gold particles on their plasmalemmal membranes than astroglia. Of particular interest in the present study was a strong labeling for NCAM on all parts of neuritic growth cones, including the lamellar and filopodial processes that extend from the tip of the axon. The density of NCAM was similar on different filopodia of the same growth cone. Therefore, in situations where homophilic (NCAM-NCAM) binding might contribute to axon pathfinding, a choice in direction is more likely to reflect differences in the NCAM content of the environment, rather than the distribution of NCAM within a growth cone. On the other hand, the variation in NCAM levels between single neurons in culture was significant and could provide a basis for selective responses of growing neurites.

Silver-intensified gold and peroxidase as dual ultrastructural immunolabels for pre- and postsynaptic neurotransmitters.

An ultrastructural immunostaining method that uses silver-intensified gold was combined with another procedure that uses biotin peroxidase conjugates to allow simultaneous identification of two neurotransmitter-related antigens in the central nervous system. Tyrosine hydroxylase-immunoreactive neurons labeled with silver-intensified gold could be differentiated at both light and electron microscopic levels from glutamate decarboxylase-immunoreactive neurons labeled with peroxidase. Cross reactivity of the second group of immunoreagents with the first group was reduced by the heavy metal silver shell formed around the colloidal gold immunoglobulin complex. With this dual pre-embedding method, peroxidase-stained axons containing the inhibitory neurotransmitter gamma-aminobutyric acid were found to synapse directly on silver-stained dopamine neurons in the rat dorsomedial hypothalamus. This approach can be used in combination with a post-embedding immunocytochemical colloidal gold procedure, allowing ultrastructural identification of three neurotransmitter-related antigens in the same tissue section.

Glutamate, the dominant excitatory transmitter in neuroendocrine regulation.

Glutamate has been found to play an unexpectedly important role in neuroendocrine regulation in the hypothalamus, as revealed in converging experiments with ultrastructural immunocytochemistry, optical physiology with a calcium-sensitive dye, and intracellular electrical recording. There were large amounts of glutamate in boutons making synaptic contact with neuroendocrine neurons in the arcuate, paraventricular, and supraoptic nuclei. Almost all medial hypothalamic neurons responded to glutamate and to the glutamate agonists quisqualate and kainate with a consistent increase in intracellular calcium. In all magnocellular and parvocellular neurons of the paraventricular and arcuate nuclei tested, the non-NMDA (non-N-methyl-D-aspartate) glutamate antagonist CNQX (cyano-2,3-dihydroxy-7-nitroquinoxaline) reduced electrically stimulated and spontaneous excitatory postsynaptic potentials, suggesting that the endogenous neurotransmitter is an excitatory amino acid acting primarily on non-NMDA receptors. These results indicate that glutamate plays a major, widespread role in the control of neuroendocrine neurons.

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.

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.

Glutamate and GABA presence and action in the suprachiasmatic nucleus.

Glutamate and gamma-aminobutyrate (GABA) are two neurotransmitters that appear to play an important role in the hypothalamic suprachiasmatic nucleus (SCN) and in the adjacent areas of the medial hypothalamus. Converging evidence based on ultrastructural immunocytochemistry, molecular biology, calcium imaging, and electrophysiology suggests not only that GABA and glutamate are used for inhibitory and excitatory activity, respectively, in this region of the rat brain, but that these two fast-acting amino acids may account for the majority of neurotransmission there.

Metabotropic glutamate receptor mGluR1 distribution and ultrastructural localization in hypothalamus.

The metabotropic glutamate receptor mGluR1 is a G-protein-coupled glutamate receptor whose activation induces phosphotidylinositol hydrolysis and increases diacylglycerol and cytoplasmic calcium. By using affinity-purified antisera against a partial amino acid sequence of mGluR1 alpha, deduced from the nucleotide sequence of the cloned gene, the heterogeneous expression of this glutamate receptor was studied immunocytochemically with light and electron microscopy in the rat hypothalamus. Immunoreactivity was restricted to cell bodies and dendrites throughout many regions of the adult hypothalamus, including the preoptic area, anterior hypothalamus, suprachiasmatic nucleus, dorsomedial hypothalamus, and periventricular region. Strong immunolabeling was found in the lateral hypothalamus where immunoreactivity could be detected as early as embryonic day 18. Intense immunoreactivity was also found in the medial mammillary nuclei. In contrast to the strong labeling in many other regions, the neuroendocrine neurons of the arcuate, supraoptic, and paraventricular nuclei showed relatively little staining in adults. With light microscopy, immunoperoxidase labeling was found distributed in patches on the cytoplasmic side of the plasma membrane of immunoreactive neurons. When the same tissue was examined ultrastructurally, the patches were not restricted to synaptic specializations but were also found distributed on perikaryal and dendritic membranes sometimes associated with synapses and sometimes not. Some immunoreactive membranes showed no immunolabeling at the synaptic junction. When the tissue was strongly stained, labeling could be found in the cytoplasm of immunoreactive cells. No immunostaining was found on axons or presynaptic boutons. Together with other evidence showing a widespread expression of many different subtypes of both ionotropic and metabotropic receptors, these data support the hypothesis that glutamate may regulate hypothalamic cellular activity with a number of physiologically different mechanisms, and these mechanisms include second-messenger systems activated by G proteins.

The magnocellular and parvocellular paraventricular nucleus of rat: intrinsic organization.

The magnocellular and paravocellular regions of the rat hypothalamic paraventricular nucleus (PVN) were examined in several hundred brains. Converging qualitative and quantitative anatomical methods, including Golgi impregnations, Nissl stains, silver stains, and immunocytochemistry were used to study the intrinsic organization of the PVN with light, scanning, and transmission electron microscopy. A computer-assisted quantitative analysis of dendritic branching patterns was used to examine total dendritic length, center of mass, orientation of dendritic tree, and several other parameters of dendritic organization and revealed statistically significant differences between cells in the lateral and posterolateral magnocellular and medial parvocellular areas of PVN. Electron microscopy, Golgi impregnation, and neurophysin immunohistochemistry showed that dendrites of posterolateral cells were generally oriented perpendicular to the third ventricle; dendrites of cells in the lateral PVN usually projected medially from the perikaryon. Cells in the medial zone of PVN had dendritic trees which often paralleled the third ventricle. Large numbers of axons entered and left PVN ventrally near the midline and laterally in the area of the posterolateral PVN; axons generally were oriented parallel to the mean major axis of dendritic trees in these areas. Ultrastructural examination of serial thin sections showed a peculiar astroglia multiple lamellar isolation of axodendritic synaptic contacts. Intrinsic axons commonly arose from parvocellular but not from magnocellular neurons and contacted dendrites of both medial parvocellular and more lateral magnocellular neurons. Synapses were found on shafts and spines of dendrites, on perikarya and somatic appendages, and invaginated into the soma. Both dendrites axons with large neurosecretory vesicles and immunostained with neurophysin antiserum were found postsynaptic to other axons. Presynaptic neurosecretory axons were not found within the PVN. A semiquantitative analysis of catecholamine axons identified with the glyoxylic acid method and fibers immunoreactive with ACTH and Substance P antisera indicated that the parvocellular region of PVN received ggreater innervation than the lateral magnocellular area; similarly, a reater density of stained fibers was found in the medial parvocellular PVN region with Golgi impregnations and silver stains. With a stereological analysis of 1-micrometer plastic sections, the parvocellular area had a significantly greater neuropil to cell volume ration, with cells accounting for 48 +/- 9% in the lateral magnocellular zone, but only for 26 +/- 7% in the parvocellular area. A quantitative analysis of vasculature from thin sections showed that the PVN had 3.3 times more blood vessels, and 3.6 times more lumen perimeter than a control area ventrolateral to PVN; an interesting finding here was that the medial parvocellular PVN had a high degree of vascularity, not significantly different from the lateral magnocellular zone...

Presynaptic metabotropic glutamate receptors in adult and developing neurons: autoexcitation in the olfactory bulb.

The mitral cell of the olfactory bulb is the primary relay neuron that transmits information from the olfactory receptors to the rest of the brain. This excitatory neuron releases glutamate from presynaptic dendrites and axon terminals. All rat mitral cells studied showed strong, selective, and widespread metabotropic glutamate receptor mGluR1 alpha immunoreactivity on the presynaptic membrane of dendrites, often at the synaptic vesicle release site, when examined with light and electron microscopy. The finding of glutamate receptors on mitral cell secondary dendrites supports the conclusion that not all dendritic membrane with glutamate receptors necessarily have gray type I asymmetrical synaptic specializations. In contrast, the metabotropic glutamate receptor mGluR5 was not found in mitral cells but was expressed by granule cells and astrocytes around mitral dendrites. Both mGluR1 alpha and mGluR5 were expressed early in development, with strong immunostaining present by postnatal day 1. MGluR1 alpha staining at birth mirrored the adult staining pattern. MGluR5 staining at birth showed different patterns of immunostaining than that found in the adult, particularly in the external plexiform layer. In vitro olfactory bulb neurons and their dendrites from embryonic day (E) 18 olfactory bulbs responded to t-ACPD and quisqualate, selective and nonselective metabotropic glutamate receptor agonists, and to several ionotropic glutamate agonists with increases in intracellular Ca2+ as studied with fura-2 digital imaging. These data indicate that the receptors were functionally active at an early stage of development. Application of the glutamate receptor blockers d-2-amino-5-phosphonovalerate (AP5) and 6-cyano-7-nitroquinoxaline (CNQX) to E17 olfactory bulb neurons after only 4 days in vitro resulted in a dramatic decrease in Ca2+ levels in 70% of 128 cells tested, suggesting that embryonic neurons after a short time in vitro can actively secrete glutamate. The presence of glutamate receptors on the long mitral cell dendrite suggests that it would be able to respond to release of its own excitatory transmitter, probably at an early stage of development. In the probable absence of other excitatory input to the secondary mitral dendrites, it would be the only excitatory "input." This autoexcitatory response would be modulated by release of GABA from olfactory interneurons occurring milliseconds after glutamate release induced by olfactory nerve activation. This novel type of neuronal microcircuitry would potentially amplify signal transmission and current spread along the long mitral dendrites and could play an important role in lateral inhibition of olfactory neurons.

GABA immunoreactivity in hypothalamic neurons and growth cones in early development in vitro before synapse formation.

GABA (gamma-aminobutyrate) is the most prevalent inhibitory transmitter in the mature hypothalamus. In contrast, in the developing hypothalamus, GABA may exert depolarizing actions leading to neuronal excitation. To determine whether GABA is present in hypothalamic neurons early in development, and whether there is a preferential expression in axonal growth cones, immunogold and peroxidase studies were used with light and whole mount transmission electron microscopy. At embryonic day 15, a stage of development at the beginning of hypothalamic neurogenesis, histological sections showed GABA immunoreactivity in fibers and weakly stained perikarya. Hypothalamic neurons (13%) cultured at embryonic day 15 were immunoreactive after 1 day in vitro. The percentage of neurons stained, and the intensity of staining increased during the next few days to 39% at 4 days in vitro. Neuritic growth cones, including lamellipodia and long filopodia, showed strong immunoreactivity before synaptogenesis. By using neuronal whole mounts studied with transmission electron microscopy and GABA silver-enhanced immunogold staining, a quantitative comparison of growth cones after a day and a half in culture revealed that the growth cone of the longest process, the putative axon, had a greater level of immunogold labeling than that of the shorter processes, the putative dendrites. This finding is one of the earliest biochemical differences between putative axons and dendrites. Astrocytes in the same cultures showed no immunolabeling. These results indicate that GABA is present very early in the development of hypothalamic neurons and is in a position to be released.

NILE/L1 and NCAM-polysialic acid expression on growing axons of isolated neurons.

The neuron adhesion molecules NILE/L1 and NCAM may be involved in axonal guidance and cell recognition. To investigate all exposed membrane domains of single neurons, something which has not previously been done for any adhesion molecule, we used digitally processed scanning electron microscopy with a high-energy backscatter electron detector. This allowed a quantitative analysis of immunogold staining densities on all surfaces of isolated rat hippocampal neurons in culture to study NILE/L1 and NCAM expression independent of potentially inductive innervation. During early stages of neuritic extension, all growth cones showed similar NILE/L1 expression, but as soon as a single process extended farther than the others (by 20 hours), this putative axon and its growth cone generally showed a stronger level of NILE/L1 immunogold labeling than the other neurites. This is the earliest evidence of plasma membrane differentiation between axons and dendrites. With further neuritic growth, the relative NILE/L1 expression on axons and their growth cones continued to increase. In contrast to some earlier reports, NILE/L1 was expressed on axonal growth cones growing on both polylysine-coated glass and astrocyte substrates. Strong immunostaining for NCAM-related polysialic acid (PSA) was found on axonal growth cones and filopodia, suggesting that the homophilic adhesive action of NCAM may be reduced during axonal growth. PSA showed greater labeling on distal axons than on other areas of the neuron, indicating a variable NCAM-mediated adhesion on different regions of the same cell. Neither NILE/L1, NCAM, nor PSA appeared to show regional differences in axons fasciculating or defasciculating on themselves. A strong intercellular heterogeneity of NILE/L1, NCAM, and PSA expression levels on neurons in the same culture dish was found, suggesting that subsets of cells from the hippocampus may express biologically relevant differences in adhesion molecules compared to neighboring neurons. In light of the growing body of evidence pointing to the multifaceted array of homophilic and heterophilic binding interactions that NILE/L1 and NCAM may exhibit, and the functional importance of molecular densities, the quantitative data here support the hypothesis that sufficient cellular and subcellular heterogeneity exists for these molecules to be involved in some aspects of axonal guidance.

Paraventriculospinal tract as a model for axon injury: spinal cord.

The response of immunocytochemically identified hypothalamic axons innervating the rat spinal cord was examined at varying times after cord hemisection in a model of axonal injury using the paraventriculospinal projection. The purpose was to determine whether these long descending peptidergic axons would show signs of regrowth after injury. From 1 to 180 days after hemisection, horizontal sections of the spinal cord were stained with peroxidase immunocytochemistry. Antiserum against neurophysin was used to identify axons projecting from the hypothalamic paraventricular nucleus to the spinal cord. The paraventricular nucleus innervates all rostrocaudal segments of the cord, yet the projection is not massive, allowing the trauma response of individual axons to be studied. Immediately caudal to a T4 hemisection, axons began decreasing in number by 2 days after surgery. Ten days postoperatively, only a few axons could be found caudal to the cut; these remaining axons arose from the contralateral cord. A substantial increase in the number of stained axons was found rostral to the hemisection 3-12 weeks after surgery. In that an increase in axon number could be due to both increasing staining efficacy and sprouting, the orientation of axons in control and hemisected rats was studied. Three millimeters rostral to the hemisection, axons had a greater variance in orientation and were more likely to project medially out of the dorsolateral white matter compared with the contralateral control side. Rostral to the hemisection, a statistically significant two- to fourfold increase in the number of branches per axons was found in comparison to the contralateral control side. Axons were found in the dorsal white matter 4 months after surgery; in controls, immunostained axons were not found here. At all intervals after surgery, structures suggestive of growth were found, including terminal growth cones and lateral filopodia and lamellipodia extending from axons whose distal ends had been severed by hemisection. Similar structures were not found in control spinal cord. Together, these data suggest that after cord hemisection, axons from the paraventricular nucleus sprout rostral to the injury.

Synaptic relationships between neurons containing vasopressin, gastrin-releasing peptide, vasoactive intestinal polypeptide, and glutamate decarboxylase immunoreactivity in the suprachiasmatic nucleus: dual ultrastructural immunocytochemistry with gold-substituted silver peroxidase.

In order to examine the morphological substrates for neuronal connections between cells of the hypothalamic suprachiasmatic nucleus (SCN) that contain immunoreactivity for different neurotransmitters, a double ultrastructural immunocytochemical analysis was used. For double immunostaining, the first neuroactive substance antigen was labeled with gold-substituted silver-intensified peroxidase (GSSP), which results in a granular gold deposit of high electron and light opacity. The second antigen was labeled with peroxidase and a diaminobenzidine chromagen. The GSSP reaction product greatly increased the visibility of immunoreactive structures, with both light and electron microscopy. Intensification with the GSSP method worked at all depths of thick tissue sections as determined with analysis of immunostained sections cut perpendicular to their flat surface, and with analysis of thick 80-micron sections of brain tissue into which horseradish peroxidase (HRP) has been microinjected. On a nitrocellulose dot-blot comparison of different substrates for HRP, the GSSP intensification compared favorably with tetramethylbenzidine, but unlike tetramethylbenzidine, the GSSP was stable in a wide range of buffers. In addition to diaminobenzidine, the GSSP reaction was used to intensify and stabilize both the Hanker-Yates reagent and tetramethylbenzidine on the nitrocellulose model system. Through the use of the GSSP reaction, five new synaptic relationships in the suprachiasmatic nucleus were revealed. By increasing the sensitivity of the peroxidase method by silver-gold intensification, immunoreaction product could be found in dendrites at a greater distance from the perikaryon than in nonintensified material. Because of this greater sensitivity, the neuroactive substance contained in the cell of origin of a dendrite could sometimes be identified. Boutons immunoreactive for vasopressin-associated neurophysin were found to make synaptic contact with postsynaptic dendrites that also contained vasopressin-neurophysin immunoreactivity. Similarly, boutons containing gastrin-releasing peptide immunoreactivity made synaptic contact with cells also exhibiting gastrin-releasing peptide immunoreactivity. Neurons stained with GSSP reaction product could be easily discriminated from those containing only HRP-precipitated diaminobenzidine, allowing the simultaneous use of these two markers in the same 30-micron tissue section and subsequently in ultrathin sections for electron microscopy.(ABSTRACT TRUNCATED AT 400 WORDS)

Converging GABA- and glutamate-immunoreactive axons make synaptic contact with identified hypothalamic neurosecretory neurons.

To study the neurochemical identity of axons in synaptic contact with identified hypothalamic neurosecretory neurons in rats, we combined retrograde axonal transport of a marker molecule with postembedding immunogold staining for amino acid neurotransmitters. After intravenous injections of horseradish peroxidase, neurosecretory neurons with axons in the median eminence or neurohypophysis transported the peroxidase retrogradely back to the cell body of origin. Serial ultrathin sections from the paraventricular and arcuate nuclei were immunostained with glutamate or GABA antisera. Peroxidase-labeled neurons and their dendrites received synaptic contact from colloidal gold-labeled axons immunoreactive for GABA or for glutamate. Axons which were highly immunoreactive for GABA and other axons immunoreactive for glutamate but not for GABA consistently made converging synaptic contact with the same peroxidase-labeled cell. Some of the peroxidase-labeled neurons from the arcuate nucleus which were postsynaptic to both GABA and glutamate axons were themselves identified as being GABA immunoreactive. Serial ultrathin sections revealed that multiple presynaptic axons immunoreactive for glutamate or GABA made repeated contacts with single neurons. These results suggest a widespread convergence of the major inhibitory and excitatory amino acid transmitter on the neurons which control both the anterior and posterior pituitary hormones.

Growth cone calcium elevation by GABA.

Cytoplasmic calcium plays a key role in neurite growth. In contrast to previous work suggesting that gamma aminobutyrate's (GABA) role in regulating growth cone calcium is primarily to antagonize the effects of glutamate, we report that GABA can act in an excitatory manner on developing hypothalamic neurites, independently raising calcium in growing neurites and their growth cones. Time-lapse digital video and confocal laser microscopy with the calcium-sensitive dyes fluo-3 and fura-2 were used to study the influence of GABA on neurite calcium levels. GABA (10 microM) evoked a calcium rise in both bicarbonate- and Hepes-based buffers. The calcium rise was greatly reduced after chloride transport was blocked. GABA raised calcium by stimulating the cell body, resulting in an increase in calcium throughout the neuronal cell body and dendritic arbor. GABA also acted locally, stimulating a neuritic calcium rise only in a single dendrite or growth cone. In some neurites and growth cones during early development, GABA generated a greater calcium rise than did glutamate. Bicuculline, a GABAA receptor antagonist, reduced calcium levels in neurites of young synaptically coupled neurons, indicating that ongoing synaptic release of GABA raised neuritic calcium. These data suggest that during early development, GABA may play a significant role in regulating process growth and modulating the formation of early connections in the hypothalamus. Our data support the hypothesis that GABA receptors are functionally active and may play a calcium regulating role similar to that of glutamate in neuronal development. This is particularly true in early development, as later in development GABA's role becomes more inhibitory, and glutamate plays the primary excitatory role.

Trauma-induced Golgi-like staining of neurons: a new approach to neuronal organization and response to injury.

A new esterification-silver approach to the directed staining of the dendritic trees of traumatized neurons is described. Stained neurons compare favorably to those labeled with silver chromate Golgi impregnations in the visualization of dendritic arbors. Cells in all parts of the brain, including the hypothalamus, hippocampus, cerebral cortex, cerebellum, striatum, spinal cord, thalamus, and olfactory bulb, can be detected after focal trauma to that region. Selection of neurons to be stained is made by increasing their affinity for silver with many different types of directed neuronal trauma, including micropipette wounds, pressure, surgical incision, chemical cytotoxicity, and impact trauma. Trauma to one area of the brain results in dendritic arbors staining only in the injured area; other areas of the brain are free of dendritic staining. Injury can be produced either in vivo or in vitro. In vitro injury to neurons allows a high degree of localization and facilitates the analysis of neuronal response to trauma in the absence of complicating factors such as blood flow and secondary injury. The selective affinity for silver staining in this approach is increased very rapidly, allowing detection of traumatized cells fixed minutes after injury. Brains of all mammals used appear to stain similarly, including the rat, mouse, pig, and human. Axons, although labeled less frequently than dendritic arbors, are induced to stain just as rapidly as dendrites. The ability to visualize a large part of the dendritic tree after trauma allows the segregation of neuronal subtypes on the basis of their differential response to injury. Subpopulations of cells in the same area of the brain appear to respond differently to trauma. Differential response of neurons to trauma can easily be detected. For instance, slight variations in trauma can be used to label selectively the major subpopulations of neurons in the hippocampus, including pyramidal cells, interneurons near the pyramidal cell layer, or granule cells. Similarly, neurons of the hypothalamic paraventricular and arcuate nuclei respond to compression trauma much more dramatically than other cell types in the same region of the hypothalamus. Hypothalamic neurons were studied extensively, particularly in regions that are difficult to routinely stain with Golgi impregnations, including the arcuate, paraventricular, supraoptic, and suprachiasmatic nuclei. Trauma to these areas was made in vitro after removal of the brain from the skull, allowing easy access to the ventral surface of the brain.(ABSTRACT TRUNCATED AT 400 WORDS)

GABA: a dominant neurotransmitter in the hypothalamus.

To study the organization and distribution of the inhibitory amino acid neurotransmitter GABA in the medial hypothalamus, we used a postembedding immunocytochemical approach with colloidal gold. Quantitative analysis showed that half (49%) of all synapsing boutons studied were immunoreactive for GABA, based on immunogold staining of the suprachiasmatic, arcuate, supraoptic, and paraventricular nuclei. This was corroborated with pre-embedding peroxidase immunostaining with antisera against glutamate decarboxylase, the GABA synthetic enzyme. These data suggest that GABA is the numerically dominant neurotransmitter in the hypothalamus, and emphasize the importance of inhibitory circuits in the hypothalamus. Serial ultrathin sections were used to reconstruct GABA immunoreactive boutons and axons in three dimensions. With this type of analysis we found less morphological heterogeneity between GABA immunoreactive boutons than with single ultrathin sections. Single sections sometimes showed boutons containing only small clear vesicles, and other with both clear vesicles and small dense core vesicles. However, with serial sections through individual boutons, dense core vesicles were consistently found at the periphery of the pre-synaptic GABA immunoreactive boutons, suggesting probable co-localization of GABA with unidentified peptides in most if not all boutons throughout the hypothalamus. A positive correlation was found between the density of small clear vesicles and the intensity of immunostaining with colloidal gold particles. GABA immunoreactive axons generally made symmetrical type synaptic specializations, although a small percentage made strongly asymmetrical synaptic specializations. Vesicles in GABA immunoreactive boutons were slightly smaller than those in non-reactive boutons. Synaptic efficacy is related to the position of the synapse on the post-synaptic neuron. While the majority of GABA immunoreactive axons made synaptic contact with dendrites, the distribution of GABA immunoreactive synapses on somata and dendrites was the same as would be expected from a random distribution of all boutons. No preferential innervation of cell bodies by GABA immunoreactive terminals was found. Serial ultrathin sections showed that a GABA immunoreactive axon would sometimes make repeated synaptic contacts with a single postsynaptic neuron, indicating a high degree of direct control by the presynaptic GABAergic cell. Other immunoreactive axons made synaptic contact with a number of adjacent dendrites and cells, suggesting a role for GABA in synchronizing the activity of hypothalamic neurons. Based on the density of immunogold particles per unit area, varying concentrations of immunoreactive GABA were found in different presynaptic boutons in the hypothalamus.

Metabotropic glutamate receptor mGluR5 subcellular distribution and developmental expression in hypothalamus.

The metabotropic glutamate receptor mGluR5 is a G-protein coupled receptor that plays a key role in release of Ca2+ from internal stores via inositol triphosphate mobilization. Western and Northern blot analyses revealed a greatly enhanced expression of mGluR5 in rats during early stages of hypothalamic development compared with the adult. This enhanced developmental expression provides an explanation for the dramatic physiological response of developing neurons to metabotropic glutamate receptor activation and supports the argument that metabotropic glutamate receptors may play an important role in hypothalamic development. During development, expression of the mGluR5 gene was reduced, not only in the hypothalamus but also in other regions of the brain. A differential decrease in mGluR5 protein was found in different brain regions with Western blot analysis. The hypothalamus showed a sixfold decrease in mGluR5 with development, whereas the cortex showed only a threefold decrease. Immunocytochemistry with an affinity-purified antibody against a peptide deduced from the cloned mGluR5 gene revealed selective expression in some regions in the adult hypothalamus. In the adult and developing (postnatal day 10) brain, immunoreactive neurons were found in the suprachiasmatic nucleus, preoptic area, lateral hypothalamus, and mammillary region, areas where the related metabotropic glutamate receptor mGluR1 is also found. In contrast, the ventromedial nucleus, an area critically involved in the regulation of food intake and metabolic balances, showed strong mGluR5 immunoreactivity but no mGluR1 immunoreactivity. Little or no mGluR5 staining was found in the neurosecretory neurons of the paraventricular, supraoptic, and arcuate nuclei. Ultrastructurally, mGluR5 was associated with the cytoplasmic face of the plasmalemma on hypothalamic dendrites, dendritic spines, and neuronal perikarya in the adult. The strongest immunoreactivity was found in patches on the membrane, sometimes associated with the postsynaptic side of synapses and sometimes associated with nonsynaptic dendritic or perikaryal membrane. Intense immunostaining was found on some astrocyte processes surrounding synaptic complexes containing asymmetrical synapses. These astrocytes would be in an ideal position to receive excitatory signals from glutamatergic axons. Unlike the punctate appearance of immunolabeling on neuronal membranes, astrocytes showed continuous staining along the plasma membrane.

VGF expression in the brain.

VGF is a neuronal polypeptide first identified as a cDNA clone in a gene library from nerve growth factor-stimulated PC12 cells. In the present paper, the expression of VGF is examined for the first time throughout the adult rat central nervous system with immunocytochemistry and Northern blot analysis. VGF RNA was found in all brain regions studied, including hypothalamus, hippocampus, cerebellum, olfactory bulb, and cortex. In contrast to the relatively strong immunostaining of hypothalamic neurons, the level of VGF RNA expression in the hypothalamus was relatively low in comparison with other brain regions. With the aid of antisera raised against bacterially produced recombinant proteins containing parts of the VGF sequence, immunoreactive neurons were detected throughout the brain, including regions of the olfactory tubercle, caudate-putamen, thalamus, cortex, amygdala, hypothalamus, midbrain, and hippocampus. VGF-immunoreactive neurons did not contain detectable amounts of nerve growth factor receptor; other neurons that showed nerve growth factor receptor immunoreactivity expressed no VGF immunoreactivity. The lack of colocalization of VGF and nerve growth factor receptor suggests that, unlike expression in PC12 cells, VGF expression in neurons from the central nervous system does not require nerve growth factor stimulation. Within the hippocampus, the location of VGF-immunoreactive cells was suggestive of inhibitory interneurons. VGF-immunoreactive axons and terminals were found throughout the brain. These observations extend our earlier work on VGF expression in the hypothalamus to other regions of the brain and support the conclusion that although VGF expression is only detected in subsets of neurons in each brain region, these subsets are widely distributed throughout the central nervous system.