Immunolocalization of aromatic L-amino acid decarboxylase, tyrosine hydroxylase, dopamine, and serotonin in the forebrain of Ambystoma mexicanum.

To improve basic knowledge about the neurochemical organization of the urodele brain, and to study discrepancies in the localization of monoaminergic markers, we immunohistochemically charted the distribution of four such markers (tyrosine hydroxylase, aromatic L-amino acid decarboxylase, dopamine, and serotonin) in the axolotl (Ambystoma mexicanum) forebrain. Catecholaminergic and serotoninergic systems were found in similar locations to those seen in other Urodela. As seen in other vertebrates, the localization of the different monoaminergic markers reveals some inconsistencies. Cells that are exclusively tyrosine hydroxylase-immunoreactive are observed in the olfactory bulb, anterior olfactory nucleus/nucleus accumbens region, the epichiasmatic portion of the preoptic nucleus, and in the pars intercalaris thalami, whereas cells that are only labelled by aromatic L-amino acid decarboxylase are seen in the anterior olfactory nucleus/nucleus accumbens region, the bed nuclei of the anterior commissure, the posterior portion of the preoptic nucleus, the ventral hypothalamus, and the pars intercalaris thalami. The presence of cells solely serotonin (5-HT)-immunoreactive is suggested for the nucleus infundibularis dorsalis. Conversely, there were no areas that appeared to be exclusively immunoreactive for dopamine. Double-labelling for aromatic L-amino acid decarboxylase/tyrosine hydroxylase and aromatic L-amino acid decarboxylase/serotonin, together with cell counting, confirmed the existence of neurons that express only one monoaminergic marker in amphibian, supporting the hypothesis that these cells are universally present in the central nervous system of vertebrates.

Isolation and characterization of ovine tyrosine hydroxylase mRNA.

Tyrosine hydroxylase (TH) cDNA has been characterized in rodents and primates, but only a few studies have been developed in ungulates, except in cows. Because sheep is a species used for many physiological studies, it was of interest to clone TH cDNA in this species. Ovine TH cDNA was purified from a library of sheep adrenal glands. The entire cDNA was 1,721 bp long. It presented a higher percentage of similarity with bovine TH cDNA (93%) than with rodent cDNAs (75%). The deduced amino acid sequence was 490 amino acids long and had 96% similarity with the bovine amino acid sequence. The entire cDNA and different fragments obtained with endonuclease restriction enzymes were cloned in plasmid pUC 18 and were labeled with 35S-dATP to detect TH mRNA by in situ hybridization. Strong labelings were observed on adrenal medulla and on noradrenergic and dopaminergic neurons in the sheep but also in the cow and pig. This labeling matched completely TH immunohistochemical staining obtained on the same sections with anti-TH antibodies. Ovine TH cDNA is a useful tool to study the variations of TH mRNA levels in sheep catecholaminergic neurons.

Progesterone receptor immunoreactivity in aromatic L-amino acid decarboxylase-containing neurons of the guinea pig hypothalamus and preoptic area.

A double-labeling immunofluorescence procedure was used to determine whether progesterone receptor (PR)-immunoreactive (IR) neurons in the preoptic area and hypothalamus of female guinea pigs also contained aromatic L-amino acid decarboxylase (AADC), an enzyme involved in the synthesis of both catecholamines and serotonin. Immunostaining was performed on cryostat sections prepared from ovariectomized guinea pigs primed by estradiol to induce PR. The nuclear presence of PR was visualized by a red fluorescence while the AADC-containing perikarya showed a yellow-green fluorescence. The topographic distribution of AADC-IR neurons was investigated by using a specific antiserum obtained by immunization of rabbits with a recombinant protein beta-galactosidase-AADC in the two regions known to contain the densest populations of estradiol-induced PR-IR cells: the preoptic area and the mediobasal hypothalamus. The localization of PR-IR and AADC-IR cell populations showed considerable overlap in these areas, mainly in the medial and periventricular preoptic nuclei and in the arcuate nucleus. A quantitative analysis of double-labeled cells estimated that about 15% to 23% of AADC-IR cells in the preoptic area and about 11% to 21% of AADC-IR cells in the arcuate nucleus possessed PR. This colocalization persisted throughout the rostrocaudal extent of these areas and represented 3% to 9% of the population of PR-IR cells. These findings provide neuroanatomical evidence that a subset of AADC neurons is directly regulated by progesterone. The exact physiological role of this enzyme in target cells for progesterone is not understood. AADC may be involved in functions other than that for the synthesis of the classical neurotransmitters.

Polynucleotide binding to macrophage scavenger receptors depends on the formation of base-quartet-stabilized four-stranded helices.

Macrophage scavenger receptors exhibit unusually broad, but circumscribed, polyanionic ligand-binding specificity. For example, the polyribonucleotides poly(I) and poly(G) are ligands but poly(A) and poly(C) are not. To further investigate the molecular basis of this polynucleotide-binding specificity, we tested the capacity of various oligodeoxyribonucleic acids to inhibit the scavenger receptor-mediated degradation of 125I-labeled acetylated low density lipoprotein by Chinese hamster ovary cells expressing the type I bovine scavenger receptor. A series of short oligodeoxyriboguanines (dGn, where 5 < or = n < or = 37) were effective inhibitors. The dG6, dG12, and dA5G37 members of this series were shown by circular dichroism and UV spectroscopy to be assembled into four-stranded helices stabilized by G-quartets. [32P]dA5G37 bound directly to scavenger receptors. Partial or complete denaturation of the quadruplex structures of these oligonucleotides by boiling destroyed their inhibitory activity. Receptor activity was also inhibited by d(T4G4)4, a telomere-like oligonucleotide which forms an intramolecular quadruplex. In addition, conversion of the four-stranded potassium salt of poly(I) to the single-stranded lithium salt dramatically reduced its inhibitory activity. Addition of KCl to the Li+ salt resulted in the reformation of poly(I)'s quadruplex structure and restoration of its inhibitory activity. A variety of single-stranded and double-stranded oligo- and polydeoxyribonucleotides (e.g. dA37, HaeIII restriction fragments of phi X174) exhibited very little or no inhibitory activity. Thus, a base-quartet-stabilized four-stranded helix appears to be a necessary structural determinant for polynucleotide binding to and inhibition of scavenger receptors. This conformational requirement accounts for the previously unexplained polyribonucleotide-binding specificity of scavenger receptors. The spatial distribution of the negatively charged phosphates in polynucleotide quadruplexes may form a charged surface which is complementary to the positively charged surface of the collagenous ligand-binding domain of the scavenger receptor.

Quantitative autoradiographic analysis of estradiol retention by cells in the preoptic area, hypothalamus and amygdala.

These experiments were done to compare quantitatively, on a cell-by-cell basis, estradiol retention by cells in the medial preoptic area, arcuate nucleus, ventrolateral subdivision of the ventromedial nucleus, and the caudal half of the medial nucleus of the amygdala. The steroid autoradiograms were prepared from 2 mu sections of brains from ovariectomized, adrenalectomized adult female rats that had been infused intravenously with [3H] estradiol (E2) in a regimen which kept circulating hormone concentration at or above proestrus levels for 3-4 h. Even in these brain regions, containing the most dense collections of E2-concentrating cells, a maximum of only 27-61% of the cells concentrated E2. Therefore, in these regions only a particular subset of the cells retain hormone; other cells in the region do not retain hormone. Frequency distribution histograms of the number of grains per cell versus the number of cells in each region showed a wide range in the amount of E2 retained per cell, and no modes among E2-retaining cells. The data followed a distribution markedly different from that predicted by a simple Poisson distribution, confirming that E2-retention does not result from a random, passive process such as diffusion. The overall quantitative characteristics of the frequency distribution histograms were similar across the four brain areas. Therefore, we propose that the different E2-sensitive functions of these brain areas must depend on differences in the neural connectivity or differences in hormone regulated peptide content of the areas.

The use of amphotericin B to detect inhibitors of cellular cholesterol biosynthesis.

Pores formed in the membranes of animal cells by complexes of sterols and the polyene antibiotic amphotericin B can efficiently kill the cells. Thus, in the absence of exogenous sources of cholesterol, inhibitors of enzymes in the cholesterol biosynthetic pathway render cells resistant to amphotericin B. Preincubation of Chinese hamster ovary cells with compactin or 25-hydroxycholesterol, inhibitors of the synthesis of the key intermediate mevalonate, protected cells from amphotericin B killing and this protection was reversed by the addition of exogenous mevalonate. The ability of compactin to confer amphotericin B resistance on normal cells was abolished when cells were provided exogenous cholesterol by the receptor-mediated endocytosis of low density lipoprotein. Low density lipoprotein receptor-defective Chinese hamster ovary cells were not subject to this low density lipoprotein-dependent amphotericin B killing. Exogenous mevalonate did not prevent 4,4,10 beta-trimethyl-trans-decal-3 beta-ol, an inhibitor of mevalonate conversion to sterols, from protecting cells from amphotericin B. A simple two-step protocol in which cells are preincubated (15-24 h) with potential inhibitors and then treated (3-6 h) with amphotericin B was devised to provide a sensitive method for detecting direct (e.g., competitive) and regulatory inhibitors of cholesterol biosynthesis. This protocol may prove useful in detecting potential antihypercholesterolemia drugs and is currently being used to isolate mutants in receptor-mediated endocytosis.

Autoradiographic identification of estradiol-concentrating cells in the spinal cord of the female rat.

The topography and number of estradiol (E)-concentrating cells in the lower lumbar and sacral segments of the spinal cord of the female rat have been examined by the steroid autoradiography method. A nuclear-saturating does of E was administered by intravenous infusion, which kept blood estrogen at or above proestrus levels for 3.5-4 h, much longer than usual for steroid receptor studies. The cord segments selected for examination are known to receive somatosensory information relevant for estrogen-dependent behavior, and to contain some of the motoneurons for epaxial muscles responsible for this behavior. Small numbers of E-concentrating cells were found in the dorsal portion of the gray matter of L4, L5, L6 and the sacral segments. These cells were found in lamina II, in the midline region which includes lamina X, and the medial portions of laminae III, IV and V when they cross in the midline. E-concentrating cells were also found in the lateral portions of laminae III, IV, and V, and in lamina VII. Virtually no E-concentrating cells were found in the ventral portion of the gray matter or in the white matter. The spinal cord had few E-concentrating cells compared to the hypothalamus.

An autoradiographic study of the efferent connections of the ventromedial nucleus of the hypothalamus.

Efferent projections from the ventromedial nucleus of the hypothalamus (VMN) were traced using tritiated amino acid autoradiography in albino rats. Ascending fibers passed through the anterior hypothalamus. Labelled fibers and terminal fields were seen in the preoptic area, bed nucleus of the stria terminalis, substantia innominata, the anterior amygdaloid area, diagonal bands of Broca and lateral septum. Fibers also projected laterally from VMN and entered the supraoptic commissures and zona incerta. These lateral projections were responsible for the fibers observed in the cerebral peduncle, the amygdala, the thalamus and the reticular formation. Fibers descending in a medial position projected through the posterior hypothalamus and then swept dorsally to terminate in the mesencephalic and pontine central grey. A projection from VMN into the median eminence was noted. The overall patterns of projection from different parts of VMN were similar; differences that existed were primarily in the relative strengths of the different projections. The efferent projections from VMN are extensive, well organized, and would appear capable of supporting significant physiological actions on extra-hypothalamic structures.