Stereological quantification of extraocular muscles in humans / Cuantificación estereológica de músculos extraoculares en humanos

SUMMARY: The aim of this study is to quantify muscular and connective tissue volumes of extraocular muscles (EOM) in humans with no ophthalmological disease using stereology. EOM from five cadaveric non-strabismic humans were obtained. The number of muscle fibers in 5,000 µm2 and volume density (Vv) of muscle and collagen were measured using stereology. Comparisons between antagonist EOM were conducted using Wilcoxon signed rank test for paired samples. A secondary analysis examining differences between pairs of EOM was also conducted. Bilateral tests were performed, and significance was set at 0.05. The horizontal rectus muscles (medial and lateral rectus) had the highest Vv of muscle and the lowest Vv of collagen. The inferior rectus muscle tended to have a fewer number of fibers per 5,000 µm2 than the rest of the EOM. However, these differences did not reach statistical significance. This is the first published study describing the normal histology of human EOM using stereology. Our investigation, through the quantification of the proportion of muscle and collagen tissue, as well as the number of muscle fibers in 5,000 µ2, establishes normal stereological parameters for EOM of humans without ophthalmological disease.

RESUMEN: El objetivo de este estudio es cuantificar el volumen de tejido muscular y conectivo de los músculos extraoculares en humanos sin enfermedad oftalmológica conocida utilizando estereología. Los músculos extraoculares fueron obtenidos de cinco cadáveres humanos sin estrabismo. El número de fibras musculares en 5.000 µm2 y la densidad de volumen (Vv) de músculo y colágeno fueron medidas usando estereología. Las comparaciones entre músculos extraoculares antagonistas se realizaron a través de la prueba de los rangos con signo de Wilcoxon para muestras pareadas. Un análisis secundario examinando diferencias entre pares de músculos extraoculares también fue llevado a cabo. Se realizaron pruebas bilaterales y la significancia fue fijada en 0,05. Los músculos rectos horizontales (recto medial y lateral) tuvieron el mayor Vv de músculo y el menor Vv de colágeno. El músculo recto inferior tuvo la tendencia a poseer menos número de fibras por 5.000 µm2 que el resto de los músculos extraoculares. No obstante, estas diferencias no llegaron a ser estadísticamente significativas. Este es el primer estudio publicado describiendo la histología normal de los músculos extraoculares usando estereología. Nuestra investigación, a través de la cuantificación de la proporción de tejido de músculo y colágeno, así como el número de fibras musculares en µm2, establece parámetros estereológicos normales para músculos extraoculares en humanos sin enfermedad oftalmológica.

Late acute humoral rejection in low-risk renal transplant recipients induced with an interleukin-2 receptor antagonist and maintained with standard therapy: preliminary communication.

Low-risk renal transplant recipients treated with standard immunosuppressive therapy including interleukin-2 receptor (IL-2R) antagonist show a low incidence of early rejection episodes but few reports have examined the incidence and severity of late rejection processes. This study evaluated retrospectively cellular and antibody-mediated rejection (AMR) among 42 recipients selected because they showed low panel-reactive-antibodies, short cold ischemia time, no delayed graft function, and therapy including basiliximab (Simulect) induction. The mean observation time was 6.6 years. Sixty-seven percent of donors were deceased. Ten-year patient and death-censored graft survivals were 81% and 78%, respectively. Seven patients lost their kidneys due to nonimmunologic events. The seven recipients who experienced cellular rejection episodes during the first posttransplant year had them reversed with steroids. Five patients displayed late acute AMR causing functional deterioration in four cases including 1 graft loss. De novo sensitization occurred in 48% of recipients including patients without clinical rejection. In conclusion, long-term follow-up of kidney transplant recipients selected by a low immunologic risk showed a persistent risk of de novo sensitization evolving to acute AMR in 11% of cases. Although immunologic events were related to late immunosuppressive reduction, most graft losses were due to nonimmunologic factors.

Prenatal ozone exposure abolishes stress activation of Fos and tyrosine hydroxylase in the nucleus tractus solitarius of adult rat.

Ozone (O3) is widely distributed in the environment, with high levels of air pollution. However, very few studies have documented the effects on postnatal development of O3 during pregnancy. The long-term effects of prenatal O3 exposure in rats (0.5 ppm 12 h/day from embryonic day E5 to E20) were evaluated in the adult nucleus tractus solitarius (NTS) regulating respiratory control. Neuronal response was assessed by Fos protein immunolabeling (Fos-IR), and catecholaminergic neuron involvement by tyrosine hydroxylase (TH) labeling (TH-IR). Adult offspring were analyzed at baseline and following immobilization stress (one hour, plus two hours' recovery); immunolabeling was observed by confocal microscopy. Prenatal O3 increased the baseline TH gray level per cell (p < 0.001). In contrast, the number of Fos-IR cells, Fos-IR/TH-IR colabeled cells and proportion of TH double-labeled with Fos remained unchanged. After stress, the TH gray level (p < 0.001), number of Fos-IR cells (p < 0.001) and of colabeled Fos-IR/TH-IR cells (p < 0.05) and percentage of colabeled Fos-IR/TH-IR neurons against TH-IR cells (p < 0.05) increased in the control group. In prenatal-O3 rats, immobilization stress abolished these increases and reduced the TH gray level (p < 0.05), indicating that prenatal O3 led to loss of adult NTS reactivity to stress. We conclude that long-lasting sequelae were detected in the offspring beyond the prenatal O3 exposure. Prenatal O3 left a print on the NTS, revealed by stress. Disruption of neuronal plasticity to new challenge might be suggested.

Glial vascular endothelial growth factor overexpression in rat brainstem under tolerable hypoxia: evidence for a central chemosensitivity.

The ventilatory response to hypoxia is mediated by peripheral inputs arising from the arterial chemoreceptors. In their absence, hypoxic adaptation can be achieved, possibly as a result of central cellular reorganization. To study this reorganization, we used chemodenervated rats to investigate the expression and localization of vascular endothelial growth factor (VEGF) in the brainstem. VEGF is a target gene of hypoxia-inducible factor (HIF) that is responsible for the morphofunctional remodeling induced by hypoxia. Intact and chemodenervated rats were subjected to normoxia or hypoxia for 6 hr (10% O(2) in N(2)). VEGF protein was quantified in micropunches of brainstem tissue. Only chemodenervated animals showed an increased VEGF expression in response to hypoxia, whereas, in normoxia, VEGF expression was not modified by chemodenervation. The same hypoxic condition was repeated for 8 days before immunocytochemical staining with anti-VEGF; antiglial fibrillary acidic protein (GFAP), a marker of astrocytes; and anti-rat endothelial cell antigen-1 (anti-RECA-1) that recognizes endothelial cells. Confocal analysis showed a cellular colocalization of GFAP and VEGF, indicating that VEGF was overexpressed predominantly in astrocytes. Increased RECA-1 immunolabeling indicated an enhanced angiogenesis in chemodenervated rats subjected to hypoxia. These results indicate that glial cells and the vascular network contribute to the brainstem remodeling. The peripheral chemodenervation reveals a central O(2) chemosensitivity involving a cascade of gene expression triggered by hypoxia, which in intact animals may act synergically with peripheral chemosensory inputs.

8-oxoguanine DNA glycosylase, but not Kin17 protein, is translocated and differentially regulated by estrogens in rat brain cells.

8-oxoguanine DNA glycosylase and Kin17 are proteins widely distributed and phylogenetically conserved in the CNS. 8-oxoguanine DNA glycosylase is a DNA repair enzyme that excises 7,8-dihydro-8-oxoguanine present in DNA damaged by oxidative stress. Kin17 protein is involved in DNA repair and illegitimate recombination in eukaryotic cells. The present study evaluates the effect of ovarian hormones on the expression of both proteins in the magnocellular paraventricular nucleus of the hypothalamus and the bed nucleus of the stria terminalis in female and male rat brains. In the paraventricular nucleus, ovariectomy induced a significant decrease in the number of 8-oxoguanine DNA glycosylase-positive nuclei as well as in their relative fluorescent intensity as compared with ovariectomized-estradiol treated and proestrous groups. Confocal microscopy observation demonstrated that oxoguanine DNA glycosylase protein is located in the Hoechst-dyed nuclei and cytoplasm in male and ovariectomized rats. Surprisingly, following estradiol administration to ovariectomized and proestrous rats, the 8-oxoguanine DNA glycosylase immunolabeling was observed in the nucleolus, the cytoplasm and the dendrites of cells, while Kin17 protein was always localized in the cell nuclei. In the bed nucleus of the stria terminalis, the number of 8-oxoguanine DNA glycosylase-positive nuclei during proestrous was significantly lower than the number obtained in males and ovariectomized rats and similar to the number of ovariectomized-estradiol-treated groups. In contrast to these observations, no significant differences were observed in the expression of kin17 protein. Our results suggest that estrogens differentially regulate the expression of 8-oxoguanine DNA glycosylase, but not that of Kin17 protein, in specific regions of the rat brain and that estradiol can translocate the 8-oxoguanine DNA glycosylase protein within nuclei and to other subcellular compartments.

Tyramine-immunoreactive neuronal structures in the rat brain: abundance in the median eminence of the mediobasal hypothalamus.

Immunoreactivity to p-tyramine, one of the natural trace amines, was studied in the rat brain by an anti-p-tyramine antibody. Immunoreactivity to this amine is very weak in the nigrostriatal dopaminergic neurons and terminals, and weak in the locus coeruleus noradrenergic ones. It was intensified in these structures after monoamine oxidase inhibition. On the other hand, this amine was highly concentrated in the median eminence of the mediobasal hypothalamus, in which its physiological function on prolactin release has been demonstrated.

Expression of Kin17 and 8-OxoG DNA glycosylase in cells of rodent and quail central nervous system.

Kin17 and 8-Oxoguanine DNA glycosylase (Ogg1) are proteins, respectively, involved in illegitimate recombination and DNA repair in eukaryotic cells. To characterize the expression of these proteins in cell types of rodent and avian brains, we combined immunocytochemistry for either Kin17 or Ogg1 proteins with glial fibrillary acidic protein (GFAP, an astrocyte marker) immunodetection on the same tissue section. Both Kin17 and Ogg1 proteins were localized in cell nuclei and were extensively distributed in neuronal populations of quail and rodent brains. However, GFAP-immunoreactive cells were never labeled by Kin17 protein. This was observed in nerve fiber tracts, in the cerebral cortex, the hippocampal formation, the hypothalamic region, and the periventricular regions of the brain of both species studied. These results were confirmed by combining in situ hybridization of kin17 mRNA and GFAP immunodetection. On the contrary, GFAP-immunoreactive cells were often labeled by the Ogg1 protein in brain structures such as fiber tracts, the cortical surface, the cerebellum, and the ependymal surface of both quail and mouse brains. Our results suggest that the expression of the Kin17 protein (observed in neurons) and that of the Ogg1 protein (observed in neurons and glial cells) is conserved in brain phylogeny.

Serotonergic retinopetal projections from the dorsal raphe nucleus in the mouse demonstrated by combined [(3)H] 5-HT retrograde tracing and immunolabeling of endogenous 5-HT.

The present study demonstrated a direct serotonergic retinopetal projection in the mouse stemming from the lateral portion of the dorsal raphe nucleus bilaterally. A double-labeling technique was employed combining: (1) radioautography and retrograde axonal tracing following intraocular injection of [(3)H] 5-HT and (2) immunocytochemical identification of endogenous 5-HT. Radiolabeled neurons were only observed within the dorsal raphe nucleus and were always double-labeled with the 5-HT antibody. The radiolabeling appeared to be specific resulting from the retrograde transport of a radioactive 5-HT derivative product following uptake of the neurotransmitter by intraretinal terminals.

Detection of 8-oxoG DNA glycosylase activity and OGG1 transcripts in the rat CNS.

The oxoguanine DNA glycosylase (Ogg1) is a DNA repair enzyme that excises 7,8-dihydro-8-oxoguanine present in DNA damaged by oxidative stress. We have investigated the expression of the OGG1 gene in different regions of the rat CNS. Biochemical studies on brain homogenates of adult rats have shown that Ogg1 nicking activity is present at relatively similar levels in the cerebral cortex, the hypothalamus, the pons and the cerebellum. Following in situ hybridization with radiolabeled OGG1 cDNA or specific antisense oligonucleotides, OGG1 transcripts showed a widespread but heterogeneous distribution pattern among distinct brain regions of adult rats: high levels of this transcript were detected in the CA1-CA3 layers and the gyrus dentate of the hippocampal formation, the piriform cortex, the supraoptic nuclei, the olivary complex as well as in the pyramidal cells of layer V of the cortex and the Purkinje cells of the cerebellum. In peripheral organs such as the lungs, the stomach and the spleen, OGG1 transcript is however expressed in specific subpopulations of cells. Using a semi-quantitative reverse transcription - polymerase chain reaction assay on total mRNA from the frontal cortex, OGG1 mRNA was determined to be expressed with relatively the same levels in 1-day-old and 7-day-old rats as well as in adult rats. These results provide evidence for the widespread expression of the OGG1 gene in developing and adult brains.

Medullary noradrenergic neurons projecting to the bed nucleus of the stria terminalis express mRNA for the NMDA-NR1 receptor.

The bed nucleus of the stria terminalis pars ventralis (vBNST) receives dense noradrenergic terminals and contains the highest concentration of noradrenaline (NA) in the brain. We used autoradiography following retrograde axonal transport of [(3)H]-NA to identify selectively whether noradrenergic neurons innervating the vBNST originate in the medulla oblongata and/or the locus coeruleus. In combination with this technique, non-isotopic in situ hybridization for the NMDA-NR1 receptor subunit mRNA was used to examine, on the same brain sections, its expression in noradrenergic neurons that innervate the vBNST. The results showed that 60 +/- 6% and 35 +/- 7% of the total number of radiolabeled cells detected after injection of [(3)H]-NA in the vBNST were located in brainstems A1 and A2 noradrenergic cell groups, respectively. In addition, 18.5 +/- 4.2% of radiolabeled cells in A1 and 15.7 +/- 5% in A2 also expressed the mRNA for the NMDA-NR1 receptor subunit. In contrast, only 4 +/- 3% of the radiolabeled cells were present in the locus coeruleus, and none of these cells was positive to NMDA-NR1 receptor subunit mRNA. The present results provide evidence that BNST noradrenergic fibers and terminals originate predominantly from A1 and A2 noradrenergic cell groups, and that a significant number of these noradrenergic neurons also express the mRNA for the NMDA-NR1 receptor subunit. The observation that brainstem noradrenergic neurons innervating the vBNST express NMDA receptor mRNA gives anatomical support to the regulation of NA release by NMDA presynaptic receptors.

Kin protein expression: laminar specificity during rat cerebral cortex development.

Kin is a mammalian nuclear protein involved in DNA recombination-repair and the regulation of gene expression. The present study explored the expression of the Kin nuclear protein during postnatal development of the rat cerebral cortex, using immunocytochemistry with anti-RecA antibodies. The immunostaining of the Kin protein preferentially occurs within layers IV-V and VIb of the cortex in early postnatal developing brain, whereas in the adult rat this expression is observed unequivocally in all cortical layers. 35S-isotopic in situ hybridization for Kin-17 mRNA confirmed this Kin protein expression pattern and demonstrated its transcription in cortical neurons. This gradual age-related expression during development may have functional implications in the maturation processes of the cortex.

Raphe serotonergic neurons projecting to the olfactory bulb contain galanin or somatostatin but not neurotensin.

Retrograde axonal transport with [3H]5-HT has been developed as a specific tracing technique to identify serotonergic projections. This method, in combination with immunocytochemistry, offers considerable advantage of specificity and sensitivity to study pathways of multitransmitter-containing neurons. In this work, we studied the presence of galanin, somatostatin, and neurotensin in serotonergic neurons of dorsal and median raphe, which project to the olfactory bulbs. After [3H]5-HT injections into the rat olfactory bulbs, double galanin-immunoreactive and [3H]5-HT radiolabelled cells were located in the dorsal, lateral, and ventral region of dorsal raphe, but they were never seen in the median raphe. In the dorsal raphe, galanin-radiolabelled neurons represented 28% of the total number of radiolabelled cells. Double somatostatin-immunoreactive and radiolabelled neurons were located in the dorsal and median raphe. In the dorsal raphe, double somatostatin-radiolabelled neurons represented only 11% of the radiolabelled cells and they were mainly located ventral to the aqueduct. In the median raphe, 15% of radiolabelled cells were also immunopositive for somatostatin. In contrast, neurotensin-immunoreactive cells in the dorsal and median raphe were distributed among [3H]5-HT radiolabelled neurons but they were never radiolabelled. Our results show subpopulations of serotonergic raphe-olfactory bulb projection neurons containing either galanin or somatostatin, but not neurotensin.

Expression of Kin, a nuclear protein binding to curved DNA, in mammal and avian brains.

Kin is a nuclear protein which presents cross-immunoreactivity with the bacterial RecA protein and which efficiently binds to curved DNA. This genomic interaction could be implied in DNA repair and illegitimate recombination in eukaryotic cells. Using immunocytochemistry with anti-RecA antibodies, we report the ubiquitous presence of the Kin protein in the CNS of mice and quails. However, some brain structures such as the hippocampal area, the locus coeruleus and Purkinje cells are preferentially immunolabelled and show some homologies between the two species. In conclusion, the expression of the Kin protein is preserved in the phylogeny of the brain of higher vertebrates.

Preferential expression of kin, a nuclear protein binding to curved DNA, in the neurons of the adult rat.

The KIN17 gene product has been identified by cross immunoreactivity with anti-RecA antibodies and by DNA recombination techniques, and is probably part of the DNA recombination-repair machinery. Following Western blotting and immunocytochemistry using anti-RecA antibodies, and in situ hybridization with specific KIN17 cDNA probes, we here report the detection of high levels of KIN protein and KIN17 mRNA in the CNS of adult rats. The RecA cross-reacting protein has an apparent molecular weight of 41 kDa and is located in the nucleus of brain cells. Both the KIN17 transcript and the protein were found to be widespread, but they were present in different proportions, depending on the type of brain cells. High levels of KIN protein were seen in neurons of the motor nuclei of the brainstem, the locus coeruleus, hippocampal formation, entorhinal cortex, Purkinje cells, pyramidal cells of the cortex and mitral cells. In contrast, using a combination of KIN17 mRNA in situ hybridization and GFAP immunocytochemistry (a marker of glial cells) showed that the KIN17 messenger is preferentially transcribed in neurons, the post-mitotic and long lived brain cells. We postulate that KIN17 play a role in the illegitimate recombination of DNA sequences and/or the repair of alterations of the genome in neurons.

NMDA-NR1 receptor subunit mRNA expression in rat brain after 6-OH-dopamine induced lesions: a non-isotopic in situ hybridization study.

Antisense digoxigenin-labeled deoxyoligonucleotides probes and non-isotopic in situ hybridization (HIS) techniques have been used to explore the NMDA-NR1 receptor subunit mRNA distribution in different brain areas of rats which had their dopaminergic nigrostriatal pathway previously lesioned with intracerebral administration of 6-OH-dopamine (6-OH-DA). Intense and significant hybridization signals for NR1 mRNA were found in dentate gyrus and regions CA1-CA2-CA3 of the hippocampus, in layers II-III and V-VI of the cerebral cortex, and in the cerebellum of sham-treated rats. Basal ganglia structures such as the striatum exhibited few NR1 mRNA hybridization signals as compared to the hippocampus and cerebral cortex. In contrast, both zona compacta and reticulata of substantia nigra (SN) showed a reduced number of cells with nevertheless intense NR1 mRNA HIS signals. The NR1 mRNA distribution in the brain was affected in a brain regional selective manner by 6-OH-DA induced lesions of DA neuronal systems. A striking increase in NR1 mRNA HIS signals was observed in both striata after unilateral lesioning with 6-OH-DA. Instead, in SN compacta but not in reticulata, a moderate but significant bilateral reduction of NR1 mRNA was observed after unilateral 6-OH-DA injection. No significant changes in NR1 mRNA were detected in cerebral cortex and other brain regions after 6-OH-DA treatment. These studies, and others reported in the literature, support the view that extensive lesions of nigrostriatal DA-containing neurons in the brain may trigger compensatory or adaptative responses in basal ganglia structures such as striatum and substantia nigra which involve glutamateric neurons and the genic expression of NMDA receptors.

The mouse Kin-17 gene codes for a new protein involved in DNA transactions and is akin to the bacterial RecA protein.

We have sought to characterize the molecular basis of the sensitivity to ionising radiation and to identify the genes involved in the cellular response of mammalian cells to such radiation. Using the Escherichia coli model, we tested the hypothesis that functional domains of RecA protein are represented in proteins of mammalian cells. We review here the results obtained in the detection of nuclear proteins of mammalian cells that are recognized by anti-RecA antibodies. We have called them kin proteins. Kin proteins likely play a role in DNA metabolism. We summarize the cloning of the mouse Kin-17 cDNA and our work on the identification and preliminary characterisation of the biochemical properties of mouse kin17 protein, a new nuclear protein able to recognize bent DNA and suspected to be involved in illegitimate recombination. We briefly describe our latest experiments on the molecular characterisation of the mouse Kin-17 gene. Finally, we discuss the properties of kin17 protein and the possible participation of kin17 protein in DNA transactions like transcription or recombination.

High expression of noradrenaline, choline acetyltransferase and glial fibrillary acidic protein in the epileptic focus consecutive to GABA withdrawal. An immunocytochemical study.

Interruption of a chronic GABA infusion into the rat somatosensory cortex induces the appearance of focal epileptic manifestations, known as the 'GABA withdrawal syndrome' (GWS). The aim of the present study was to determine, by immunocytochemistry, if neurotransmitters other than GABA are involved in GWS, namely: noradrenaline (NA), serotonin, choline acetyltransferase (CAT), cholecystokinin, neuropeptide Y, somatostatin and glial fibrillary acid protein (GFAP). Immunocytochemical data were compared in three animal groups: GABA-, saline- and L-aspartate (L-Asp)-infused rats. Only GABA-infused rats presented epileptic manifestations after interruption of the infusion. Saline- and L-Asp-infused rats served as controls. Observations were limited to the region surrounding the cortical infusion site. GABA-infused rats showed in the zone of the epileptic focus a number of cell bodies strongly immunoreactive to NA antibodies much larger than control rats. In addition, NA-immunoreactive fibers formed a dense plexus and some of them were observed around perikarya. In saline- and L-Asp-infused rats, the NA-immunolabelled fibers were sparse and NA immunolabelling was rarely observed in cell bodies. These results contrast to those obtained for the serotonergic system, where no significant difference was observed among the three groups of rats. CAT immunolabelling was observed in cell bodies, but not in nerve terminals in rats of the three groups. The number of CAT-immunoreactive cell bodies was much greater in GABA-infused rats than in the control animals. GFAP immunolabelling showed an important number of astrocytes throughout the cortex of the GABA-infused hemisphere, whereas, astrocytic reaction was limited to the infusion site in controls. Immunocytochemical data concerning peptides revealed cortical neuronal elements labelled similarly in the three groups of rats. Noradrenergic, cholinergic and glial modifications observed mainly in GABA-infused rats can result from lesion and from a specific action of GABA in chronic infusion. These modifications may contribute to the epileptogenesis of GWS, as recently demonstrated by electrophysiological recordings that show a modulating action of NA on firing activity of neurons involved in the epileptic focus.

Some noradrenergic neurons of locus ceruleus-olfactory pathways contain neuropeptide-Y.

We report a specific tracing technique for studying projections of noradrenergic neurons that contain other transmitters. Autoradiography after retrograde axonal transport of tritiated-noradrenaline ([3H]NA) was combined with immunocytochemical detection of endogenous NA or neuropeptide Y (NPY). The specificity of [3H]NA retrograde labeling was dependent on the concentration of [3H]NA injected at the terminal region. At 2 x 10(-3) M, the specificity of [3H]NA retrograde labeling was confirmed by immunodetection of endogenous NA in radiolabeled cell bodies of the locus ceruleus. Combination of autoradiography of [3H]NA retrograde labeling with immunodetection of NPY demonstrated that a some scattered locus ceruleus noradrenergic neurons (about 26%) projecting to the olfactory bulb do contain NPY.

Distribution of substance P-like immunoreactivity in the chameleon brain.

The distribution of substance P-like immunoreactivity in the chameleon brain and spinal cord was studied with immunohistochemical methods using polyclonal antibodies against substance P. In the telencephalon, immunoreactive cell bodies and fibers were located primarily in the striatum and in the globus pallidus. In addition, few substance P-like fibers were observed in the cortical areas, in the septum, and in the amygdala. In the diencephalon, a high density of immunostained neurons and fibers were seen in the periventricular and ventrolateral hypothalamus. Another group of cell bodies was located in the optic tectum and particularly in the stratum griseum central. A large number of immunoreactive fibers were also detected in the thalamic nuclei and in the median eminence. In the mesencephalon, few immunoreactive neurons were observed in the ventral tegmental area, in the substantia nigra, and in the nucleus reticularis isthmi. These latter nuclei, the periventricular area, the posterior commissure, the nucleus lentiformis mesencephali, the oculomotor nucleus, and the raphe nuclei contained a dense plexus of substance P immunoreactive fibers. No immunoreactive cell bodies were observed in raphe nuclei. In the spinal cord, no substance P-like immunoreactive neurons were observed, but a large number of substance P immunostained fibers were seen in the dorsal and lateral part of the dorsal horn and surrounding the dorsal parts of the central canal. The results of the present study are discussed with respect to those obtained in other species of reptiles, the main differences concerning the lateral septum, the habenula, the area of the paraventricular organ, and the raphe nuclei.

Identification of a Kin nuclear protein immunologically related to RecA protein in the rat CNS.

A polypeptide, called Kin, has been identified in cells of the central nervous system (CNS) by using antibodies raised against RecA protein of E. coli and by in situ hybridization with identified cDNA. RecA protein is a recombination enzyme associated with DNA repair. The RecA cross-reacting polypeptide was immunocytochemically demonstrated in the nuclei of various cells of adult rats. Following Western blot analysis using anti-RecA antibodies, the Kin protein showed a band with an apparent molecular weight of 41 kDa. Double labelling experiments, using in situ hybridization of Kin-17 mRNA with serotonin immunocytochemistry, demonstrated a cytoplasmic distribution of radiolabelling indicating the translation of this messenger RNA in serotonergic neurons. These data indicate the presence of a Kin nuclear protein in the CNS and suggest that neurons may possess some DNA-repair pathways analogous to those described in bacteria.