Appearance of parvalbumin-specific immunoreactivity in the cerebral cortex and hippocampus of the developing rat and gerbil brain

Developmental changes in the distribution of parvalbumin-specific immunoreactivity in the brain, in particular in the cerebral cortex and hippocampus, were followed immunohistochemically in two different species, the rat and the Mongolian gerbil (Meriones unguiculatus) using an antibody raised against for rat parvalbumin. The gerbil is known to develop its auditory and visual capacity later than rat. In both the rat and gerbil, parvalbumin-specific immunoreactivity appeared after birth in both the cerebral cortex and hippocampus. The timing of the development of expression of parvalbumin varied among different parts of the cerebral cortex. The parietal cortex showed evidence of the earliest expression of parvalbumin whilst the occipital and temporal cortices expressed parvalbumin at a later stage of a development. This feature was common to both the rat and gerbil but occurred at a relatively later stage in the gerbil. The profile of the distribution of parvalbumin in the brain of the developing and adult gerbil was similar to that of the rat, but there were some differences. The frequency of bead-like structures on the dendrites of the parvalbumin-positive cells in the CA1 region of the hippocampus was markedly lower in the gerbil; instead, straight non-beaded fibers which ran vertically into the pyramidal layer were stained. Parvalbumin-positive fibers were also found in the cerebral cortex of the gerbil.     

Appearance of parvalbumin-specific immunoreactivity in the cerebral cortex and hippocampus of the developing rat and gerbil brain

Summary Developmental changes in the distribution of parvalbumin-specific immunoreactivity in the brain, in particular in the cerebral cortex and hippocampus, were followed immunohistochemically in two different species, the rat and the Mongolian gerbil (Meriones unguiculatus) using an antibody raised against for rat parvalbumin. The gerbil is known to develop its auditory and visual capacity later than rat. In both the rat and gerbil, parvalbumin-specific immunoreactivity appeared after birth in both the cerebral cortex and hippocampus. The timing of the development of expression of parvalbumin varied among different parts of the cerebral cortex. The parietal cortex showed evidence of the earliest expression of parvalbumin whilst the occipital and temporal cortices expressed parvalbumin at a later stage of a development. This feature was common to both the rat and gerbil but occurred at a relatively later stage in the gerbil. The profile of the distribution of parvalbumin in the brain of the developing and adult gerbil was similar to that of the rat, but there were some differences. The frequency of bead-like structures on the dendrites of the parvalbumin-positive cells in the CA1 region of the hippocampus was markedly lower in the gerbil; instead, straight non-beaded fibers which ran vertically into the pyramidal layer were stained. Parvalbumin-positive fibers were also found in the cerebral cortex of the gerbil.     

The relationship between cellular nucleic acids in the developing rat cerebral cortex

1. In the rat cerebral cortex net DNA synthesis ceases when the animal has reached about 25g. body weight (18 days of age). There is then little further change in the DNA content per cortex. 2. Nuclear and transfer RNA follow a similar pattern to DNA. 3. Microsomal and ribosomal RNA content increases up to 25g. body weight but then declines. The decrease in ribosomal and microsomal RNA content is associated with a change in RNA base composition. 4. Incorporation of [(14)C]orotic acid into nuclear RNA proceeds at a similar rate in 4-day-old and adult animals. However, there is a lag period of about 60min. in the young animals during which incorporation into the ribosome fractions proceeds slowly. In the adult animals the lag period is not seen.     

Splice variants of the OB receptor gene are differentially expressed in brain and peripheral tissues of mice

A high affinity receptor for OB protein was recently cloned from the choroid plexus of mice. At least six alternatively spliced forms of the OB receptor (OB-R) gene have been described, all of which encode proteins containing the OB-R extracellular domain. One splice variant encodes a receptor with a long intracellular domain, OB-RL, that has been implicated in OB-R signaling. Here, we have used in situ hybridization to examine the localization of OB-R splice variants in brain and peripheral tissues of adult and newborn mice. Using a probe hybridizing with all known splice variants, we confirmed that OB-R mRNA was widely distributed in the adult tissues. In the CNS, choroid plexus was the major site of expression. We now demonstrate that OB-R mRNA is expressed in peripheral tissues; primarily associated with connective tissues. In addition, OB-R mRNA was detected at higher levels in peripheral tissues of newborn mice than in adult mice. With a probe specific for OB-RL, we confirmed that high mRNA expression was detected in hypothalamic nuclei, while low levels were observed in choroid plexus. We now report that in peripheral tissues of adult mice, OB-RL mRNA expression was either very low or undetectable. In newborn mice, the pattern of OB-RL message expression in the CNS was similar to that of adult mice, while bone was the site of highest OB-RL message expression in the peripheral tissue. These data suggest different biological roles for OB-R splice variants encoding the short and long forms of OB-R. The localization of OB-RL to hypothalamic nuclei supports the idea that OB-RL is the brain receptor that mediates OB protein signaling and actions. In addition, the expression of OB-R message in newborn mice also suggests a biological role of OB-R during development in mice.     

Characterizing exons 11 and 1 promoters of the mu opioid receptor (Oprm) gene in transgenic mice

Background  

The complexity of the mouse mu opioid receptor (Oprm) gene was demonstrated by the identification of multiple alternatively spliced variants and promoters. Our previous studies have identified a novel promoter, exon 11 (E11) promoter, in the mouse Oprm gene. The E11 promoter is located ~10 kb upstream of the exon 1 (E1) promoter. The E11 promoter controls the expression of nine splice variants in the mouse Oprm gene. Distinguished from the TATA-less E1 promoter, the E11 promoter resembles a typical TATA-containing eukaryote class II promoter. The aim of this study is to further characterize the E11 and E1 promoters in vivo using a transgenic mouse model.     

The interaction of nivazol with the glucocorticoid receptor from rat and rhesus monkey target tissues

Steroid hormone receptor competition techniques were used to evaluate the glucocorticoid receptor binding properties of nivazol and its 11 beta-hydroxy derivative, Win 44577 in rat and monkey target tissues. These agents competitively inhibited the binding of 3H-dexamethasone to the glucocorticoid receptor from the liver and anterior pituitary from both rat and monkey with relative binding affinities of Win 44577 greater than dexamethasone greater than nivazol greater than cortisol in all cases. However, nivazol and Win 44577 had approximately twice the affinity for the anterior pituitary glucocorticoid receptor from both species. Neither compound demonstrated any significant binding to rat estrogen, progestin or androgen receptors. These results are consistent with a glucocorticoid receptor mediated mechanism of action for nivazol and Win 44577; however, the difference in the endocrine profile of nivazol in the rhesus monkey versus the rat does not appear to be due to a species selectivity in the affinity of nivazol for the glucocorticoid receptor from central or peripheral target tissue.     

Portacaval anastomosis causes selective alterations of peripheral-type benzodiazepine receptor expression in rat brain and peripheral tissues.

There is a growing body of evidence to suggest that peripheral-type benzodiazepine receptors (PTBRs) and their endogenous ligands are implicated in the pathogenesis of end-organ failure in chronic liver disease. Portal-systemic encephalopathy, a major neuropsychiatric complication associated with chronic liver disease, results in activation of brain PTBR and probably in peripheral organs. In order to address these issues, PTBR mRNA was measured using semi-quantitative RT-PCR in extracts of cerebral cortex, kidney and testis of rats four weeks after end-to-side portacaval anastomosis and sham-operation (controls). Densities of PTBR sites were measured concomitantly by in vitro receptor binding using the selective PTBR ligand [3H]PK11195. Portacaval shunting resulted in a 2 to 3-fold increase in expression of PTBR in brain and kidney and a 37% reduction in expression in testis. Densities of [3H]PK11195 sites changed in parallel with the alterations of gene expression. These findings suggest that selective alterations of PTBR expression are implicated in the pathogenesis of peripheral tissue hypertrophy (kidney) and/or atrophy (testis) which accompanies portal-systemic shunting in chronic liver failure. In brain, activation of PTBR could result in an increase in the production of neurosteroids with potent inhibitory action in the CNS, which could contribute to the pathogenesis of portal-systemic encephalopathy.     

Functional analysis of multiple promoters of the rat insulin-like factor II gene

We have characterized the multiple promoters of the rat insulin-like growth factor II (rIGFII) gene by in vivo transient expression assay using a series of deletion mutant templates. Among the four promoters (P1, P2, P3 and P6), two (P2 and P3) showed relatively strong promoter activities compared with the other two. One of the four promoters, P2, was further characterized by gel band-shift and footprinting analysis using HeLa cell nuclear extract, showing two retarded bands and at least one protected sequence stretch. The results indicated that P2 has a very simple structure like P3, and consists of no more than 141 base-pairs (bp) including a TATA box and two GC core hexanucleotides. Promoter strength shown by in vivo transient expression in different cell types failed to explain the differential employment of P2 and P3 in these cells, suggesting the involvement of other regulatory mechanisms that might operate only in the native state.     

Protein synthesis by membrane-bound and free ribosomes of the developing rat cerebral cortex

1. Rates of RNA and protein synthesis were measured in rat cerebral-cortex slices, and compared with amino acid incorporation into protein by membrane-bound and free ribosomes from the same tissue, in the first 3 weeks of life. 2. A rapid age-dependent decline in the incorporation of labelled precursors into both RNA and protein was observed, which was more marked for amino acid incorporation into protein. 3. Although membrane-bound ribosomes comprise only a small fraction of total ribosomes, they were more active in incorporating amino acids into protein than were free ribosomes, especially immediately after birth. The decline in activity with age was more marked in the membrane-bound fraction than in free ribosomes. This loss of activity was largely independent of alterations in soluble factors or endogenous mRNA content and appeared to involve some alteration of the function of the ribosome itself, with relatively small alterations in the ratio of membrane-bound to free ribosomes. 4. Thyroidectomy, performed soon after birth, had no effect on the incorporation of radioactive precursors into RNA or protein by either slices or the cell-free preparations during the first 3-4 weeks of life.     

Asymmetric distribution and down-regulation of the muscarinic acetylcholine receptor in rat cerebral cortex

The distribution and down-regulation of the muscarinic acetylcholine receptor (mAChR) were studied in dissociated cells from right (RCC) and left (LCC) cerebral cortex. For this purpose [³H]quinuclidinyl benzilate (QNB) and [³H]pirenzepine (Pz), two muscarinic antagonists, were used. The mAChR binding sites detected with [³H]QNB were asymmetrically distributed between the two hemispheres, the majority being found in the RCC. Asymmetry was also evident in the distribution of the mAChR subtypes (M₁ and M₂) detected with [³H]Pz. Under basal conditions the RCC had roughly 50% more M₁ subtype than the LCC. The pharmacological and kinetic parameters were similar for both antagonists in RCC and LCC, indicating that the observed lateralization was due to a different density of the receptor rather than to different kinetics of binding of the two radioligands. After sustained stimulation with the agonist carbamoylcholine, the receptor sites detected with [³H]Pz, i.e. the M₁ subtype of mAChR, decreased at a higher rate in the RCC (44%) than in the LCC (25% of controls), demonstrating that the down-regulation process is more active in the right than in the left cortex, and thus implying that there is better coupling between the stimulated mAChR and its effector system in the former.     

Glutamate as a precursor of GABA in rat brain and peripheral tissues

Summary The formation of GABA from L-glutamate was investigated in homogenates of rat brain, liver, and kidney, using highly purified [¹⁴C]-L-glutamic acid as substrate and a thin-layer chromatographic separation of products. In agreement with other workers, liberation of [¹⁴C]-CO₂ was found to be stoichiometric with GABA formation in brain homogenates, but not in liver or kidney extracts. Subcellular fractionation and dialysis experiments suggested that most of the GABA synthesis in these peripheral tissues, unlike brain, does not occur via a direct decarboxylation of glutamate and requires one or more cofactors other than pyridoxal phosphate. NAD stimulated GABA formation in dialyzed extracts, and inhibition of GABA-transaminase, bothin vitro andin vivo, caused marked inhibition of GABA formation from glutamate in peripheral extracts. Although a very low GAD activity in liver and kidney cannot be excluded, these experiments suggest a major pathway from glutamate to GABA in these homogenates which includes (1) conversion of glutamate to -ketoglutarate by glutamate dehydrogenase or transaminases, (2) conversion of -ketoglutarate to succinic semialdehyde, and (3) formation of GABA from succinic semialdehyde and glutamate by GABA-transaminase.