Neil R. Krieger

3α-Hydroxysteroid Oxidoreductase in Rat Brain

Abstract: We describe a simple procedure for the microassay of 3α‐hydroxysteroid oxidoreductase in homogenates of rat brain. This enzyme converts dihydrotestosterone to 3α‐androstandiol. We have mapped the distribution of the enzymatic activity in 14 regions of the rat brain. The highest activities were observed in homogenates of olfactory bulb (51/nmol/mg protein/h) and olfactory tubercle (29 nmol/mg protein/h). Substantially lower values were seen in the other brain regions, including thalamus, caudate nucleus, frontal cortex, hippocampus, hypothalamus, and preoptic area (6–20 nmol/mg protein/ h).

Testosterone 5α‐Reductase in Rat Brain

Abstract: We describe a simple procedure for the microassay of testosterone 5α‐reductase in homogenates of rat brain. This enzyme converts testosterone to dihydrotestosterone. We have used this assay to characterize the enzymatic activity and to map its distribution. The apparent Km is 4.1 × 10−6 M and the Vmax is 85.6 pmol/mg protein/h. The pH optimum is broad and extends from pH 6.0 to 8.0. For the brain regions surveyed, testosterone 5α‐reductase activity varied over a 10‐fold range. The highest activities were observed in homogenates of the midbrain and pons (37–39 pmol/mg protein/h). The lowest were seen in homogenates of the thalamus, caudate nucleus, frontal cortex, hippocampus, hypothalamus, olfactory tubercle, and preoptic area (3–7 pmol/mg protein/h).

Testosterone 5α‐Reductase in Spinal Cord of Xenopus laevis

Abstract: Testosterone 5α‐reductase, the enzyme that converts testosterone to 5α‐dihydrotestosterone, is present in the spinal cord of Xenopus laevis. In adult males the enzymatic activity is optimal at pH 7.4 and 27°C; the apparent Km is 2.0 × 10−5m and the Vmax is 10.0 pmol/mg protein/h. Enzymatic activity was assayed in segments of the spinal cord in each of four groups: control untreated males, females, castrated males, and sexually active clasping males. Striking differences in both the amount of dihydrotestosterone produced with time and in the pattern of its distribution were seen in spinal cords of clasping males compared with those of the other groups. The differences are greatest in the basal medulla and rostral segments of the spinal cord. Neurons in these segments innervate the muscles primarily involved in clasping.

The olfactory bulb is rich in TRH immunoreactivity

We report that the rat olfactory bulb is rich in thyrotropin releasing hormone (TRH) immunoreactivity. TRH content was determined according to the radioimmunoassay method of Bassiri and Utiger. The concentration (mean +/- SEM., n = 10) of TRH in olfactory bulb (60 +/- 10 pg/mg wet weight) was 23% of the concentration in the hypothalamus, and was at least twice that of other brain regions examined. The 2 olfactory bulbs (mean wet weight 65 mg/2 bulbs) contained 3.9 +/- 0.3 ng TRH. The TRH immunoreactivity could be separated into high and low molecular weight forms. The low molecular weight form co-chromatographed with authentic TRH (mol. wt. 362) on gel filtration and thin layer adsorption chromatography and caused the release of thyrotropin from pituitary tissue incubated in vitro. Since the neuronal organization and functions of the olfactory bulb are well described, studies of the localization and metabolism of TRH in this region may help to clarify the role of this tripeptide in the central nervous system.

Localization of dopamine-sensitive adenylate cyclase within the rat olfactory tubercle.

There are three histological layers within the rat olfactory tubercle: plexiform, pyramidal, and polymorphic. We have assayed dopamine-sensitive adenylate cyclase in homogenates of frozen sections cut parallel to these layers. Consecutive sections (16 micrometer) were homogenized in groups of 6. Every seventh section was stained with toluidine blue to monitor the depth and orientation of the plane of section. I report the laminar distribution of dopamine-sensitive adenylate cyclase at two locations within the tubercle where the local topographies are quite different from one another. In the caudomedial tubercle where the parallel alignment of the neuronal layers is clearest, there is a marked gradient in the activity of dopamine-sensitive adenylate cyclase. Its distribution corresponds very closely to the distribution of pyramidal cell bodies and their dendrites in this region of the olfactory tubercle.

Pathways of TRH degradation in rat brain

Abstract We have investigated the TRH degradative enzymes in brain by examining the pattern of metabolites formed in vitro . The homogenate and three subcellular fractions of rat brain were separately incubated for 1, 5, and 15 minutes with 3H-TRH (pyro-glu-his-3H-proNH2) at 37°C. TRH and its metabolites were separated on silica gel thin-layer chromatography plates. The crude homogenate and subcellular fractions each produced a characteristic pattern of metabolism. The homogenate metabolized TRH to TRH-OH, proline, and prolineamide. With the P1 fraction, prolineamide and proline were the major metabolites. In both the homogenate and P1 fraction incubations, histidyl-prolineamide appeared as a minor component. The P2 fraction produced prolineamide and histidyl-prolineamide as the major metabolites while the cytosol metabolized TRH primarily to TRH-OH. Proline was formed during incubation with both cytosol and P2 fractions. The TRH deamidase is found in the soluble fraction of brain tissue homogenate while the pyroglutamate aminopeptidase and the prolineamide cleaving enzyme are associated with particulate fractions. Histidyl-prolineamide is further degraded in the homogenate and P1 fractions by a secondary metabolic pathway. Proline salvaging enzymes are present in all subcellular fractions of rat brain.

TRH concentration in rat olfactory bulb is undiminished by deafferentation

The effect of deafferentation of the rat olfactory bulb on bulbar TRH concentration was studied. TRH concentrations in the lesioned bulbs did not decline when compared to concentrations in sham-lesioned bulbs for the post-lesion intervals of 1 h through 14 days. Since TRH concentrations did not decline following deafferentation, TRH in the olfactory bulb does not derive from centrifugal neurons.

Glutamic acid decarboxylase in sea lamprey (Petromyzon marinus): characterization, localization, and developmental changes.

Abstract: We have carried out assays for glutamic acid decarboxylase (GAD) in homogenates of brain and spinal cord from larval and adult sea lamprey (Petromyzon marinus). The enzyme had similar characteristics in both stages. Optimal pH was 6.8; optimal temperature was 27–30° C; Km at 27°C was 5 mM. GAD activity was distributed uniformly along the length of the spinal cord. Specific activities for the larval cord and brain were 26 and 63 nm CO2/mg protein/h. respectively. The specific activities for the adult cord and brain were 29 and 236 nm CO2/mg protein/h, respectively. Thus, the activity of cord homogenates did not change significantly between larval and adult stages, but that of the brain increased about fourfold.

A method for stimulation of cyclic AMP levels in vivo by intracerebral injection in the rat olfactory tubercle

A method is described for stimulation of cAMP levels in brain by direct injection of dopamine (DA) and other neuroactive substances. Intracerebral microinjection was preceded by intraperitoneal injection of 3-isobutyl-1-methylxanthine (IBMX) to inhibit cyclic nucleotide phosphodiesterase. In vivo adenylate cyclase and phosphodiesterase activities were terminated by focused microwave radiation and the injected tissue assayed for protein and cAMP content. Increases in cAMP levels in response to injections of DA were both time- and dose-dependent. Animals receiving only vehicle or sham injections into the olfactory tubercle had basal cAMP levels of 5 pmol/mg protein. Up to five-fold increases above basal (25 pmol cAMP/mg protein) were observed for DA. With the injection of other neuroactive substances, values ranging from 160 pmol cAMP/mg protein for norepinephrine (NE), to 15 pmol cAMP/mg protein for gamma-amino butyric acid (GABA) were observed. The present study demonstrates that neuroactive substances can stimulate cAMP production in vivo when injected directly into brain tissue.

Localization of choline acetyltransferase in laminae of the rat olfactory tubercle.

Abstract: We report the distribution of choline acetyltransferase (ChAT) activity in the laminae of the rat olfactory tubercle. Within its posterior medial portion, the tubercle contains three parallel histological laminae that can be separated by cutting tangential sections from frozen tissue. ChAT was measured in homogenates of consecutive sections (16 μm) cut parallel to these laminae. The distribution of ChAT activity, as a function of tubercle depth, showed a broad peak centered at 500 μm from the ventral surface of the brain. Enzyme activity measured at this depth (85 pmol acetylcholine formed/μg protein/h) was 2 1/2 times greater than that measured in the outermost, plexiform, layer. Stereotaxic injections of kainic acid (1 μg in 1 μ1) made directly into the tubercle were used to eliminate intrinsic neurons. Three days after injection, histological examination revealed the almost total absence of neuronal cell bodies and the proliferation of glial cells. The greatest decreases in ChAT activity (50%) were seen at depths of 300–600 μm whereas no loss of activity occurred in the plexiform layer.