Bruce E. Maley

Immunohistochemistry of γ-aminobutyric acid in the cat nucleus tractus solitarius

Abstract Using a new antibody directed solely against the γ-aminobutyric acid (GABA) molecule, distribution of GABA was studied in the nucleus tractus solitarii of the cat. Both immunoreactive puncta and cell bodies had a homogenous distribution within the nucleus. The one exception was in the parvocellular subdivision where very little immunoreactive puncta, but numerous immunoreactive cell bodies, were found. Results of this investigation provide immunohistochemical evidence of GABAs localization in an autonomic nucleus involved in cardiovascular regulation.

Progenitor proliferation in the adult hippocampus and substantia nigra induced by glial cell line-derived neurotrophic factor.

Neurogenesis is an ongoing process in the hippocampus and olfactory bulb of adult mammals, regulated in part by trophic factors. While glial cell line-derived neurotrophic factor (GDNF) is being directly delivered into the nigrostriatal system of the brain for the treatment of Parkinsons disease in clinical trials, little is known about its effects on cell genesis in the brain. Here, we investigated the effects of GDNF on progenitor cell proliferation and differentiation in two GDNF-responsive areas, the hippocampus and substantia nigra. GDNF (18 microg/day) was infused in the striatum of 2-month-old Sprague-Dawley rats for 28 days. New cells were identified by the nuclear incorporation of 5-bromo-2-deoxyuridine (BrdU) and analyzed by light and electron microscopic immunostaining and quantitative morphometric techniques. GDNF significantly increased cell proliferation in the hippocampus by 78% and in the substantia nigra by 52%. There was no evidence of neurogenesis in the substantia nigra, with new cells displaying glial features and none of the 1549 BrdU-positive cells co-labeled for the dopamine neuronal marker tyrosine hydroxylase (TH). Rather, GDNF upregulated TH in existing neurons, consistent with the restorative actions of this tropic factor. The hippocampus is a site that supports adult neurogenesis and new cells generated here were closely associated with granule cells in the dentate gyrus. Some were double labeled for the neuronal marker NeuN; others had features of astrocytes, the principal source of new adult neurons in the hippocampus. The effects of GDNF on the hippocampus are potentially important in memory and learning processes.

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.

Immunohistochemical demonstration of serotonin neurons in autonomic regions of the rat spinal cord

The immunohistochemical distribution of serotonin neurons in normal and transected spinal cords of rats was examined. Intraspinal serotonin neurons were immunostained as far rostral and caudal as T3 and Co1, respectively. All serotonin neurons were located in lamina VII and X, and most were located in spinal autonomic areas. Both bipolar and multipolar neurons were observed with many of the neurons oriented longitudinally to the long axis of the cord. Spinal neurons immunostained for serotonin were visible with and without L-tryptophan and monoamine oxidase inhibitor pretreatment.

Estrous cycle-associated axosomatic synaptic plasticity upon estrogen receptive neurons in the rat preoptic area.

This study examined the hypothesis that synaptic relationships change on a daily basis in the anterior preoptic area of the intact, cycling female rat. Specifically, the anteroventral periventricular nucleus (AVPv) was chosen for analysis due to its abundant estrogen- and progesterone-receptive neurons and its critical role in the control of gonadotropin secretion. Ultrastructural analysis revealed that in the 24 h between proestrus and estrus, there was a 39% increase in axosomatic synapses upon AVPv neurons. In the subsequent 24 h to metestrus, the number of synapses decreased by 22%. Additional data showed that ovariectomy resulted in more axosomatic synapses in the AVPv relative to proestrus (46%) and metestrus (35%). Another component of the study investigated synaptic relationships on estrogen receptor-immunoreactive (ER-ir) and non-ER-ir neurons across the estrous cycle. ER-ir neurons received significantly more synaptic input at proestrus and estrus than did non-ER-ir neurons. At metestrus and following ovariectomy, no significant differences were present. The present study indicates that estrous cycle-associated synaptic plasticity occurs in the AVPv and lends further support to the critical role of this nucleus in regulation of gonadotropin secretion. Moreover, it provides the first evidence that ER- and non-ER-ir neurons are differentially innervated.

Ornithine Decarboxylase Activity and Immunohistochemical Location in Postischemic Brain

Ornithine decarboxylase, rate-limiting in polyamine formation, has been found to be necessary for the development of vasogenic edema after cryogenic cerebral injury and is postulated to be of importance in late ischemic brain edema formation. Ornithine decarboxylase activity and accompanying edema was studied after transient cerebral ischemia in Mongolian gerbils. Bilateral carotid artery occlusion was utilized to produce dense forebrain ischemia. After 4 h of reperfusion a significant elevation in ornithine decarboxylase activity was present (72.5 ± 24.7 vs 8.5 ± 2 pmoles/mg protein/h, p < 0.05). Immunohistochemical localization of ornithine decarboxylase indicated its presence in cortical neurons of ischemic gerbils. This was typically located in the perinuclear cytoplasm and extended into proximal dendrites. Nonischemic animals did not contain ornithine decarboxylase immunoreactivity. These studies show the presence and location of ornithine decarboxylase in cerebral tissue sUbjected to transient ischemia. The increase in this marker of polyamine activity paralleled previous studies in this model of cerebral edema formation and reperfusion deficit in blood flow and evoked potential, suggesting that ornithine decarboxylase is a marker for and may be associated with those late metabolic events leading to progressive functional deterioration after incomplete cerebral ischemia.

Immunohistochemistry of choline acetyltransferase in the guinea pig brain

Choline acetyltransferase (ChAT) was localized immunohistochemically within the brain of the guinea pig using a monoclonal antibody. ChAT was found in the cytoplasm of cell bodies and primary dendrites of neurons located in striatum, basal forebrain, cranial nerve motor nuclei and scattered cells in the pons. The greatest numbers of immunoreactive neurons were located in the diagonal band of Broca, medial septum and striatum. Distinct immunoreactive fibers were not visible using this antibody, although a diffuse immunostaining was present in the same nuclear regions as well as in the nerve roots of cranial nerve nuclei and the interpeduncular nuclei. Results of the present study agree closely with other previous reports of acetylcholine distributions.

Immunocytochemical localization of γ-aminobutyric acid in the rat hippocampal formation

gamma-Aminobutyric acid (GABA) was detected immunocytochemically in the rat hippocampal formation utilizing a new antiserum made against GABA conjugated to bovine serum albumin. GABA immunoreactivity was found to be concentrated in pericellular networks of fibers around both dentate granule and hippocampal pyramidal neurons. Ultrastructural analysis indicated that GABA-immunoreactive (IR) terminals most often contained small clear vesicles and made symmetrical contacts with somata and dendritic shafts of hippocampal neurons. In addition, numerous GABA-IR neurons resembled basket interneurons that have been described in previous reports.

Ultrastructural evidence for serotonin-immunoreactive terminals contacting phrenic motoneurons in the cat

The innervation of the phrenic motor nucleus in the cat by serotonin-containing neurons has been studied using retrograde tracing combined with immunohistochemistry at the electron microscope level. It was found that phrenic motoneuron cell bodies and dendrites are contacted by serotonin-immunoreactive synaptic terminals. This finding suggests that the activity of phrenic motoneurons is directly affected by serotonergic neurons.

Enkephalin-immunoreactive neurons in the nucleus tractus solitarius project to the parabrachial nucleus of the cat

Enkephalin immunoreactive neurons within the nucleus tractus solitarius (NTS) were found to project to the parabrachial nucleus of the cat with the use of a combination of immunohistochemistry and retrograde transport of horseradish peroxidase. Double labelled neurons were located in the medial, parvocellular and commissural subdivisions of the NTS and were present predominantly ipsilateral to the injection site within the parabrachial nucleus. Only a few double labelled neurons were found in the contralateral NTS. The presence of neurons containing enkephalin immunoreactivity suggests that the role of enkephalin in the regulation of autonomic functions may be, in part, by circuits between the NTS and the parabrachial nucleus.