Overexpression of nerve growth factor in skin selectively affects the survival and functional properties of nociceptors.

Mice that overexpress nerve growth factor (NGF-OE) in the skin have double the normal number of cutaneous sensory neurons, have increased innervation of the skin and spinal cord, and are hyperalgesic. Here, we have asked whether the increased cutaneous NGF level results in a selective survival of only certain functional types of neurons and whether it changes the properties of cutaneous neurons. Using electron microscopy, we show that the number of both myelinated and unmyelinated nociceptors increases substantially in NGF-OE mice by a factor of 3.3 and 1.5, respectively. Using extracellular recordings from single units, we demonstrate that large myelinated (Abeta) fibers are unchanged in prevalence and receptive properties. In contrast, among thin myelinated (Adelta) fibers, the percentage of nociceptors increased from a normal 65 to 97%, consistent with a selective survival of nociceptors during embryogenesis. These afferents showed a twofold increase in their mechanical responsiveness, but their heat responsiveness remained normal. Among unmyelinated (C) fibers, there was a profound increase in the percentage of heat responsive neurons from a normal 42 to 96%. This change cannot be accounted for by a selective survival of heat-sensitive neurons. Unmyelinated nociceptors increased fourfold in their thermal responsiveness but decreased in mechanical responsiveness. Therefore, target-derived NGF selectively rescues nociceptors during the period of programmed cell death with different efficacy for thin myelinated or unmyelinated fibers. NGF also affects the response to noxious heat or mechanical stimuli in each group differently, implying specific regulations of transduction processes rather than general changes of excitability.

Estrogen-receptive neurons in the anteroventral periventricular nucleus are synaptic targets of the suprachiasmatic nucleus and peri-suprachiasmatic region.

The anteroventral periventricular nucleus (AVPv) in the rat preoptic area is a key site underlying control of the steroid dependent preovulatory gonadotropin surge. Estrogen and progesterone receptor-containing neurons in the preoptic/hypothalamic continuum, particularly those in the AVPv, are believed to transduce steroidal signals and, in turn convey this information to the LHRH system, which lacks steroid receptors. In addition to the influence of the gonadal steroids, the precise timing of the preovulatory gonadotropin surge is believed to be regulated by the hypothalamic suprachiasmatic nucleus (SCN). The SCN and peri-SCN neurons send efferent projections rostrally to the anterior preoptic area suggesting that circadian signals are communicated synaptically to steroid-responsive neurons in the AVPv. To test this hypothesis, ultrastructural double label immunocytochemistry was conducted to determine whether SCN efferents contact estrogen receptor-immunoreactive neurons in the AVPv. Brain sections with SCN injections of phaseolus vulgaris leucoagglutinin (PHA-L) were immunostained for estrogen receptors and PHA-L. Light and electron microscopic data show that the anterior preoptic area received robust PHA-L-immunoreactive efferents from SCN neurons and immediately adjacent subparaventricular zone. In particular, the AVPv contained abundant labeled fibers and terminal boutons. Ultrastructurally, SCN- and subparaventricular zone-derived terminals synaptically contacted the perikaryon of many estrogen receptor-immunoreactive neurons in the AVPv. The perikarya of unlabeled neurons were also contacted, but the majority of the labeled contacts were observed upon neuronal processes. These results demonstrate that estrogen responsive AVPv neurons are regulated by SCN efferents. Furthermore, the present data provide strong support to the idea of collective control of pituitary gonadotropin release by steroid sensitive and circadian signal neural pathways.

Effects of elevated intracellular calcium levels on the cytoskeleton and tau in cultured human cortical neurons.

Considerable evidence suggests that altered neuronal calcium homeostasis plays a role in the neuronal degeneration that occurs in an array of neurological disorders. A reduction in microtubules, the accumulation of 8-15 nm straight filaments, and altered antigenicity toward antibodies to the microtubule-associated protein tau and ubiquitin, as well as granulovacuolar degeneration, are observed in many human neurodegenerative disorders. Progress toward understanding how and why human neurons degenerate has been hindered by the inability to examine living human neurons under controlled conditions. We used cultured human fetal cerebral cortical neurons to examine ultrastructural and antigenic changes resulting from elevations in intracellular calcium levels. Elevation of intracellular calcium by exposure to a calcium ionophore or a reduced level of extracellular Na+ for periods of hours to days caused a loss of microtubules, an increase in 8-15 nm straight filaments, and increased immunostaining with Alz-50 and 5E2 (tau antibodies) and ubiquitin antibodies. Granulovacuolar degeneration was also observed. Antigenic changes in tau were sensitive to phosphatases, and the electrophoretic mobility of tau was altered in cells exposed to calcium ionophore, indicating that tau was excessively phosphorylated as the result of elevated intracellular calcium levels. Colchicine also caused an accumulation of straight filaments and altered tau immunoreactivity, suggesting that a disruption of microtubules secondary to altered calcium homeostasis may be a key event leading to altered tau disposition and neuronal degeneration. These data demonstrate that aberrant rises in intraneuronal calcium levels can result in changes in the neuronal cytoskeleton similar to those seen in neurodegenerative disorders, and suggest that this experimental system will be useful in furthering our understanding of the cellular and molecular mechanisms of human neurological disorders.

Colocalization of galanin and prolactin within secretory granules of anterior pituitary cells in estrogen-treated Fischer 344 rats.

Galanin is localized within specific cell types of the rat anterior pituitary gland (AP). Immunocytochemical studies at the light microscope level have shown that lactotrophs, somatotrophs, and thyrotrophs contain galanin in the intact female rat, whereas lactotrophs in the male AP do not. We recently reported that galanin and PRL release from estrogen-treated male and female pituitary cells in culture are coregulated by dopamine, TRH, and somatostatin. This suggested that galanin is stored within secretory granules, conceivably with PRL. Using postembedding immunocytochemistry at the ultrastructural level, the objectives of this study were to: 1) determine the subcellular location of galanin in the AP; 2) elucidate if galanin and PRL are colocalized within the same secretory granules; and 3) compare the cellular localization of galanin in the male and female AP. Male and ovariectomized female (OVEX) Fischer 344 rats were implanted with estradiol-containing or empty Silastic capsules for 2 weeks. Postembedding immunogold labeling was performed using rabbit (for galanin) and guinea pig (for PRL) generated antisera. Two different sizes of colloidal gold spheres were used to localize the hormones in the same tissue section. Galanin was primarily localized in secretory granules of adenohypophyseal cells. Based upon immunocytochemical results and morphological criteria, galanin was contained in somatotrophs but not lactotrophs in the male and OVEX AP. The AP of estrogen-treated rats contained more specific immunogold labeling for galanin than untreated rats. The increased immunoreactivity for galanin was notably associated with lactotrophs. After exposure to estrogen, galanin and PRL were colocalized within the same secretory granules of the male and OVEX pituitary cells. We conclude: 1) galanin is localized within secretory granules of the rat AP; 2) galanin and PRL are colocalized within secretory granules of the male and OVEX AP after estrogen treatment; and 3) galanin is localized in similar cell types in the male and OVEX AP, before and after estrogen treatment. These data provide a morphological basis for the coregulation of galanin and PRL secretion by hypothalamic factors.

Monoamine synaptic structure and localization in the central nervous system.

The monoamines dopamine, noradrenaline, adrenaline, and serotonin as well as the diamine histamine have a widespread distribution in the central nervous system within synaptic terminals and nonsynaptic varicosities. In certain regions of the central nervous system the monoamines are contained in varicosities that have no synaptic specialization associated with them, suggesting a possible neuromodulatory role for some of the monoamines. The majority of monoamine labelled structures are synaptic terminals which are characterized by the presence of small, clear vesicles (40-60 nm) and large, granular vesicles (70-120 nm) within the terminal. A third population of vesicles--small, granular vesicles--which are visible only after histochemical staining, are probably the equivalent of the small, clear vesicles present after either autoradiographic or immunohistochemical labelling. Most monoamine containing terminals contact dendrites and dendritic spines and, less frequently, neuronal somata and other axons. Both asymmetrical and symmetrical membrane specializations are associated with monoaminergic terminals; however, asymmetrical contacts are the most frequent type found. These ultrastructural results indicate that monoamine containing terminals and varicosities in general share many common morphological features, but still have diverse functions.