Distribution and morphology of the catecholaminergic neural elements in the human hypothalamus.

Previous studies have demonstrated that catecholaminergic, tyrosine hydroxylase (TH)-immunoreactive (IR) perikarya and fibers are widely distributed in the human hypothalamus. Since TH is the key and rate-limiting enzyme for catecholaminergic synthesis, these IR neurons may represent dopaminergic, noradrenergic or adrenergic neural elements. However, the distribution and morphology of these neurotransmitter systems in the human hypothalamus is not entirely known. Since the different catecholaminergic systems can be detected by identifying the neurons containing the specific key enzymes of catecholaminergic synthesis, in the present study we mapped the catecholaminergic elements in the human hypothalamus using immunohistochemistry against the catecholaminergic enzymes, TH, dopamine beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT). Only a few, PNMT-IR, adrenergic neuronal elements were found mainly in the infundibulum and the periventricular zone. DBH-IR structures were more widely distributed in the human hypothalamus occupying chiefly the infundibulum/infundibular nucleus, periventricular area, supraoptic and paraventricular nuclei. Dopaminergic elements were detected by utilizing double label immunohistochemistry. First, the DBH-IR elements were visualized; then the TH-IR structures, that lack DBH, were detected with a different chromogen. In our study, we conclude that all of the catecholaminergic perikarya and the majority of the catecholaminergic fibers represent dopaminergic neurons in the human hypothalamus. Due to the extremely small number of PNMT-IR, adrenergic structures in the human hypothalamus, the DBH-IR fibers represent almost exclusively noradrenergic neuronal processes. These findings suggest that the juxtapositions between the TH-IR and numerous peptidergic systems revealed by previous reports indicate mostly dopaminergic synapses.

Tight regulation from a single tet-off rAAV vector as demonstrated by flow cytometry and quantitative, real-time PCR.

Vectors suitable for delivery of therapeutic genes to the CNS for chronic neurodegenerative diseases will require regulatable transgene expression. In this study, three self-regulating rAAV vectors encoding humanized green fluorescent protein (hGFP) were made using the tetracycline (tet)-off system. Elements were cloned in different orientations relative to each other and to the AAV internal terminal repeat (ITRs). The advantage of this vector system is that all infected cells will carry both the 'therapeutic' gene and the tet-regulator. To compare the efficiency of the vectors, 293T cells infected by each vector were grown in the presence or absence of the tet-analog doxycycline (dox). Cells were analyzed by flow cytometry for hGFP protein expression, and quantitative RT-PCR (QRT-PCR) for levels of hGFP mRNA and the tet-activator (tTA) mRNA. In the presence of dox, cells infected with one of the vectors, rAAVS3, showed less than 2% total fluorescent intensity and mRNA copy number than cells grown without dox. The other two vectors were significantly more leaky. Levels of tTA mRNA were not affected by dox. The S3 vector also displayed tight regulation in HeLa and HT1080 cells. To assess regulation in the brain, the S3 vector was injected into rat striatum and rats maintained on regular or dox-supplemented water. At 1 month after vector injection, numerous positive cells were observed in rats maintained on regular water whereas only rare positive cells with very low levels of fluorescence were observed in rats maintained on water containing dox. The QRT-PCR analysis showed that dox inhibited expression of hGFP mRNA in brain by greater than 99%. These results demonstrate that exceedingly tight regulation of transgene expression is possible using the tet-off system in the context of a self-regulating rAAV vector and that the specific orientation of two promoters relative to each other and to the ITRs is important. Regulatable vectors based on this design are ideal for therapeutic gene delivery to the CNS.

Postmortem stability of mRNA for glucocorticoid and mineralocorticoid receptor in rodent brain.

The relative postmortem stability of the mRNA's for glucocorticoid (GR) and mineralocorticoid (MR) receptor in rodent brain was determined using semi-quantitative in situ hybridization (ISH). Rats were killed by CO2 asphyxiation and their brains removed immediately (0 h) or following 12 h or 24 h delays. Specific hybridization of GR and MR anti-sense [35S]RNA-probe to tissue mRNA encoding these receptors was detected using film and emulsion autoradiography. The most intense labeling for GR mRNA was in the dentate gyrus followed by the CA1 hippocampal region. Lower, but still detectable signal, was apparent over CA3-CA4 pyramidal cell regions. MR mRNA was detected throughout the CA1-4 pyramidal cell fields of the hippocampus and the granular cells of the dentate gyrus. Film images demonstrated that even in the 24 h postmortem delay group intense specific signal was present in sections hybridized with both anti-sense GR and MR probes, although there was some diminution in signal intensity in cortical areas at this later postmortem delay. These initial experiments with rat brain demonstrate that the mRNA's for both GR and MR, as detected with ISH, are stable for up to 24 h following death.

Association of glucocorticoid receptor immunoreactivity with cell membrane and transport vesicles in hippocampal and hypothalamic neurons of the rat.

The aim of the present study was to reveal at the ultrastructural level cytoplasmic loci that display glucocorticoid receptor (GR) immunoreactivity in pyramidal neurons of the CA1 sector of the hippocampus and in cells of the medial parvicellular subnucleus of the hypothalamic paraventricular nucleus (PVN). Adrenalectomized male rats were injected intraperitoneally with corticosterone (CS) (1 mg/100 g bw) and sacrificed within 4 hr. Vibratome sections of the perfusion-fixed forebrains were processed for immunocytochemical detection of type 2 GR by means of the BuGr, anti-rat liver GR monoclonal antibody and silver-gold-intensified diaminobenzidine chromogen. The corticosterone administration gradually shifted the GR immunoreactivity (IR) from the cytoplasm to the nucleus. Samples taken 20-40 min after the steroid treatment demonstrated pyramidal cells expressing GR IR in both the cytoplasmic and nuclear pools. Although the chromatin-associated appearance of GR in the nucleus was identifiable at the light microscopic level, the nature of immunoreactive intracytoplasmic loci was not. Ultrastructural analysis of the cytoplasm indicated that fine silver-gold grains marking GR-immunoreactive sites associated with the plasma membrane and coated and regular vesicles. Noted occasionally beneath the plasma membrane of the cell bodies and dendrites, the vesicles also appeared at deeper locations in dendritic processes and around the cell nuclei. These results suggest that glucocorticoid receptors participate in signal transduction at the level of the cell membrane, as well as at the level of the genome in the cell nucleus.

Glucocorticoid receptor-immunoreactivity in C1, C2, and C3 adrenergic neurons that project to the hypothalamus or to the spinal cord in the rat.

A combined retrograde transport-double immunohistochemical staining method was used to determine the extent to which rat liver glucocorticoid receptor-immunoreactivity (GR-ir) is contained within phenylethanolamine-N-methyltransferase (PNMT)-ir neurons that project to the paraventricular nucleus of the hypothalamus (PVH) or the spinal cord. The results confirmed that cells in the C1, C2, and C3 adrenergic cell groups each contribute to the adrenergic innervation of the PVH, and indicated that the great majority of retrogradely labeled neurons in each group (80% overall) also express GR-ir. Following injections in the upper thoracic segments of the spinal cord, the bulk of adrenergic neurons that were retrogradely labeled were found in the C1 cell group, though 31% of the total number PNMT-ir cells that could be retrogradely labeled following spinal injections were localized in the C2 and C3 regions. Of these spinally projecting PNMT-ir neurons, 62% displayed GR-ir. The results suggest all three medullary adrenergic cell groups contribute projections to the spinal cord and/or the PVH, and that the capacity to express the GR phenotype is a common, though perhaps not universal, attribute of PNMT-ir neurons. No pronounced differences in the expression GR-ir were observed in adrenergic neurons as a function of their location or efferent projections. Brainstem adrenergic neurons may play a role in integrating neuronal and hormonal controls of adrenal function via ascending and descending projections.

Distribution of the mineralocorticoid and the glucocorticoid receptor mRNAs in the rat hippocampus.

The cellular localization of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) gene expression in the rat hippocampus was studied by in situ hybridization using 35S-labeled RNA-probes, complementary to either 513 bases of the rat brain mineralocorticoid receptor (MR)-mRNA or 500 bases of the rat liver glucocorticoid receptor (GR)-mRNA. Neurons in CA1, CA2, and the dentate gyrus expressed both receptor genes at high levels. The MR-mRNA was demonstrated in all pyramidal cell fields (CA1-4) of the hippocampal formation and the granular neurons of the dentate gyrus. In contrast, GR-mRNA was mainly restricted to CA1 and CA2 pyramidal cell fields and the dentate gyrus. This pattern of hybridization was found to agree with the cellular distribution of the two types of corticosteroid receptors detected previously in the hippocampus by autoradiography of the radio-labeled receptors and by immunocytochemistry of the receptor protein. These observations suggest that the corticosteroid receptors described previously as type 1 and type 2 are encoded by MR- and GR-mRNA, respectively. Although both the MR and GR genes are co-expressed in some hippocampal neurons, the unique patterns of distribution of the two receptor mRNAs in the hippocampal formation suggest that the genes for these receptors are differentially regulated. Moreover, the microanatomy of MR and GR expression provides insight into molecular mechanisms underlying the characteristic action of various steroids on behaviors involved in stress and circadian regulation.