Bcl-2: prolonging life in a transgenic mouse model of familial amyotrophic lateral sclerosis.

Mutations in the gene encoding copper/zinc superoxide dismutase enzyme produce an animal model of familial amyotrophic lateral sclerosis (FALS), a fatal disorder characterized by paralysis. Overexpression of the proto-oncogene bcl-2 delayed onset of motor neuron disease and prolonged survival in transgenic mice expressing the FALS-linked mutation in which glycine is substituted by alanine at position 93. It did not, however, alter the duration of the disease. Overexpression of bcl-2 also attenuated the magnitude of spinal cord motor neuron degeneration in the FALS-transgenic mice.

Overexpression of BCL-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia.

Naturally occurring cell death (NOCD) is a prominent feature of the developing nervous system. During this process, neurons express bcl-2, a major regulator of cell death whose expression may determine whether a neuron dies or survives. To gain insight into the possible role of bcl-2 during NOCD in vivo, we generated lines of transgenic mice in which neurons overexpress the human BCL-2 protein under the control of the neuron-specific enolase (NSE) or phosphoglycerate kinase (PGK) promoters. BCL-2 overexpression reduced neuronal loss during the NOCD period, which led to hypertrophy of the nervous system. For instance, the facial nucleus and the ganglion cell layer of the retina had, respectively, 40% and 50% more neurons than normal. Consistent with this finding, more axons than normal were found in the facial and optic nerves. We also tested whether neurons overexpressing BCL-2 were more resistant to permanent ischemia induced by middle cerebral artery occlusion; in transgenic mice, the volume of the brain infarction was reduced by 50% as compared with wild-type mice. These animals represent an invaluable tool for studying the effects of increased neuronal numbers on brain function as well as the mechanisms that control the survival of neurons during development and adulthood.

Delaying caspase activation by Bcl-2: A clue to disease retardation in a transgenic mouse model of amyotrophic lateral sclerosis.

Molecular mechanisms of apoptosis may participate in motor neuron degeneration produced by mutant copper/zinc superoxide dismutase (mSOD1), the only proven cause of amyotrophic lateral sclerosis (ALS). Consistent with this, herein we show that the spinal cord of transgenic mSOD1 mice is the site of the sequential activation of caspase-1 and caspase-3. Activated caspase-3 and its produced beta-actin cleavage fragments are found in apoptotic neurons in the anterior horn of the spinal cord of affected transgenic mSOD1 mice; although such neurons are few, their scarcity should not undermine the potential importance of apoptosis in the overall mSOD1-related neurodegeneration. Overexpression of the anti-apoptotic protein Bcl-2 attenuates neurodegeneration and delays activation of the caspases and fragmentation of beta-actin. These data demonstrate that caspase activation occurs in this mouse model of ALS during neurodegeneration. Our study also suggests that modulation of caspase activity may provide protective benefit in the treatment of ALS, a view that is consistent with our recent demonstration of caspase inhibition extending the survival of transgenic mSOD1 mice.

Vasopressin in the brain of the golden hamster: the distribution of vasopressin binding sites and of immunoreactivity to the vasopressin-related glycopeptide.

Using in vitro light microscopic autoradiography and immunocytochemistry, the distribution of vasopressin binding sites and that of the vasopressin-related glycopeptide are described in the brain of golden hamster (Mesocricetus auratus). Vasopressin binding sites and immunoreactive axons were observed in the suprachiasmatic nucleus, in the anterior hypothalamus/median preoptic area, in the medial preoptic nucleus, in the bed nucleus of the stria terminalis, in the habenular complex, in the thalamic paraventricular nucleus, and in the nucleus of the solitary tract. In addition we observed binding sites in regions where no immunoreactivity could be evidenced: the lateral septal nucleus, the central amygdaloid nucleus, the subiculum, the dentate gyrus, the anterodorsal and anteroventral thalamic nuclei, the superior colliculus, the vestibular nuclei, and in the prepositus hypoglossal nucleus. In the golden hamster, exogenous vasopressin excites single neurones located in the suprachiasmatic nucleus and induces flank-marking behavior when microinjected into the preoptic area. Our results provide a morphological basis for similar effects exerted by endogenous vasopressin. A comparison of the present data with those previously described in the rat reveals marked species differences in the brain distribution of vasopressin and of its binding sites.

Vasopressin binding in the cerebral cortex of the Mongolian gerbil is reduced by transient cerebral ischemia.

In Mongolian gerbils, the content of vasopressin in the cerebral cortex, the striatum, and the hypothalamus is increased after induction of acute cerebral ischemia. We used an iodinated vasopressin analogue and light microscopic autoradiography to study the distribution of vasopressin V1 receptors in the brain of adult male gerbils and to evaluate the effects of a transient bilateral cerebral ischemia (6 minutes) on the density of this receptor population. The animals were killed immediately or 10, 30, or 100 hours after transient bilateral occlusion of the common carotid arteries. In control animals, specific [125I]-VPA binding sites were present in various structures of the brain (olfactory bulb, anterior olfactory nucleus, lateral septum, bed nucleus of the stria terminalis, median preoptic area, ventral pallidum, substantia innominata, amygdala, thalamus, hypothalamic mammillary nuclei, superior colliculus, subiculum, central gray, nucleus of the solitary tract, hypoglossal nucleus). The strongest labeling was detected in the cerebral cortex, layers 5-6. After 30-100 hours of survival time following ischemia there was a marked decrease in [125I]-VPA binding site density in these cerebral cortex layers. To a lesser degree, a decrease was also detected in the lateral septal nucleus. In contrast, labeling in other noncortical structures remained unchanged. All animals with 100 hours recovery showed a loss of cells in hippocampus (CA1 layer) and striatum. In addition, ischemia induced concomitant and proliferative changes in cortical and hippocampal astrocytes assessed by glial fibrillary acid protein immunoreactivity. These observations indicate a role for vasopressin in the cerebral cortex either on neurons or on glial cells and the modulation of vasopressin receptor expression by transient cerebral ischemia.

Postnatal distribution of cpp32/caspase 3 mRNA in the mouse central nervous system: an in situ hybridization study.

Apoptotic cell death is a major feature of the developing nervous system and of certain neurodegenerative diseases. Various gene effectors and repressors of this type of cell death have been identified. Among them, bcl-xl and bax, which encode for antiapoptotic and proapoptotic proteins, respectively, play major roles during development. The gene cpp32 encodes for the caspase 3 cysteine protease and is a critical mediator of cell death during embryonic development in the mammalian brain. To gain insight into the possible implications of these cell death genes during the postnatal development, we investigated the expression of bax, bcl-xl, and cpp32 mRNAs by in situ hybridization in the mouse brain from birth to adulthood. Whereas bax and bcl-xl mRNAs were expressed widely in neonates and adult mice, our results showed that cpp32 mRNA levels were decreased strongly from 12 postnatal days. From 1 postnatal day to 12 postnatal days, cpp32 mRNA was expressed ubiquitously in all brain nuclei, including areas where neurogenesis occurred. A positive correlation between areas displaying high levels of mRNA and apoptotic nuclei also was shown. In the adult, cpp32 mRNA was restricted to the piriform and entorhinal cortices, the neocortex, and to areas where neurogenesis is observed (e.g., olfactory bulb and dentate gyrus). The same pattern of expression was observed in adult mice over-expressing the antiapoptotic protein Bcl-2. These results demonstrate that the expression of cpp32 mRNA is highly regulated during the mouse postnatal period, leading to a specific distribution in the adult central nervous system. Moreover, the prevention of cell death by Bcl-2 likely is not linked to the regulation of caspase mRNA levels.

Vasopressin in the brain of a desert hibernator, the jerboa (Jaculus orientalis): presence of sexual dimorphism and seasonal variation.

The distribution of vasopressin innervation in the brain of the jerboa (Jaculus orientalis) was investigated, with special attention to sex differences and seasonal variations. Vasopressin perikarya were observed in the paraventricular and supraoptic nuclei, the suprachiasmatic nucleus, the periventricular nucleus, the medial preoptic area, the bed nucleus of the stria terminalis, and the medial amygdaloid nucleus. In addition, vasopressin cell bodies were observed in the ventral retrochiasmatic area. After treatment with colchicine, vasopressin perikarya were also observed around the organum vasculosum laminae terminalis, in the medial diagonal band of Broca, and in the dorsal medial preoptic nucleus. Vasopressin fibers were also found to be more widespread in the jerboa brain than in other rodents. Fibers were observed in the medial diagonal band of Broca, the stria medullaris, the tuber cinerum, the area postrema, the medial vestibular nucleus, and the dorsal motor nucleus of the vagus. Sexual dimorphism and seasonal variation in vasopressin immunoreactivity were observed in areas that not only showed a testosterone-dependent vasopressin innervation in other rodents but also in the paratenial and mediodorsal thalamic nuclei, the tuber cinerum, the supramammillary complex, the zona incerta, the interpeduncular complex, and the dorsal and medial raphe nuclei. A denser vasopressin innervation was observed in spring/summer (sexual active period) than in autumn. Numerous brain structures contained vasopressin receptors (cerebral cortex, hypothalamus, substantia nigra, dentate gyrus, thalamic nuclei, superior colliculus, dorsal cochlear nucleus, and cerebellum); no sex- or season-related differences were observed. These data indicate a high level of vasopressin in the jerboa brain, which may reflect an adaptation to its harsh bioclimatic environment.

Distribution of the C-terminal glycopeptide of the vasopressin prohormone in rat brain: an immunocytochemical study.

The distribution of the C-terminal glycopeptide of the vasopressin prohormone was mapped in rat brain by an immunocytochemical method using antibodies to the sheep glycopeptide. The antibodies did not react with vasopressin, oxytocin or their related neurophysins. Stained neural perikarya were observed in the hypothalamus (suprachiasmatic, paraventricular, and supraoptic nuclei) and in the bed nucleus of the stria terminalis. Fibres were detected in the hypothalamus and in extrahypothalamic regions (the frontal cortex, the lateral septum, the bed nucleus of the stria terminalis, the medial nuclei of the thalamus, the lateral habenula, the amygdala, the mesencephalic central gray, the raphe nucleus of the solitary tract and the cervical spinal cord). The distribution of glycopeptide immunoreactive cells was generally similar in young rats (8 weeks old) to the distribution in older rats (13 weeks old) except in the bed nucleus of the stria terminalis where stained neurons were relatively sparse or absent in the younger animals. Similarly, in the young rats the density of fibres containing the glycopeptide was reduced in territories innervated by the bed nucleus. In both young and old rats the neuronal distribution of the glycopeptide was identical to the distribution of vasopressin, which suggests that the glycopeptide and vasopressin are co-transported from the sites of biosynthesis to the sites of release.

Time course of axotomy-induced apoptotic cell death in facial motoneurons of neonatal wild type and bcl-2 transgenic mice.

In neonatal animals, axotomy of facial motoneurons induces cell death. Using the TUNEL technique, which labelled apoptotic DNA breaks in situ, the kinetics of motoneuron death were studied. Lesion of the right facial nerve were performed on two-day-old mice. Then, animals were perfused 8, 12, 16, 20, 24, 28, 32, 48, 72 and 120 h after the lesion. Our results provide direct evidence that, following an axotomy, facial motoneurons die through an apoptotic process. We showed that apoptotic neurons can be detected as early as 16 h after the lesion. Facial motoneurons die within 120 h, with a peak observed 28 h after the lesion. The kinetics of appearance of apoptotic cells were correlated with the loss of Cresyl Violet-stained motoneurons. Furthermore, labelled cells were observed in the contralateral side of the lesion, suggesting that spontaneous apoptotic cell death occurs during the postnatal period. The same study was performed on transgenic mice overexpressing the proto-oncogene bcl-2, a gene repressor of cell death. In these mice, no TUNEL-labelled cells were detected on the lesioned and unlesioned sides. In vivo, Bcl-2 may protect motoneurons from apoptotic death following axotomy and during naturally occurring cell death. These results suggest that these two types of cell death may occur via the same mechanism.

In vivo study of motoneuron death induced by nerve injury in mice deficient in the caspase 1/ interleukin-1 beta-converting enzyme.

The apoptotic cell death program is orchestrated by members of the caspase family. Among these caspases, several in vitro and in vivo reports indicate that the interleukin-1 beta-converting enzyme (or caspase 1) may be involved in neurodegenerative processes. In view of these findings, and in order to characterize the role of the interleukin-1beta-converting enzyme in mediating or modulating cell death processes in vivo, we have investigated the effects of its deletion on motoneuron survival after a facial nerve transection in newborn and adult interleukin-1 beta-converting enzyme knock-out mice. During the postnatal period of development, when facial motoneurons are highly vulnerable to axotomy, we did not observe any significant effect of the interleukin-1 beta-converting enzyme-deletion on the percentage of cell death in the lesioned nuclei. In addition, the spontaneous cell death characteristic of the postnatal period was not altered in knock-out mice. In contrast, in adult knock-out mice, a significant reduction (16%) in the number of surviving facial motoneurons was observed six weeks after axotomy. We therefore conclude that the interleukin-1 beta-converting enzyme does not appear to be critical for cell death during the postnatal period but may favor motoneuron survival during adulthood. Given the key role of caspase 3 in neuronal apoptosis during embryonic development of the central nervous system, we also investigated the role of this caspase in cell death following axotomy. Combined immunofluorescence revealed that, at least during the postnatal period, axotomized motoneurons that have apoptotic nuclear morphologies were immunopositive for the active form of caspase 3. Double-stained cells could be also observed on the unlesioned side. These results strongly suggest that caspase 3 may be involved in both the postnatal spontaneous- and axotomy-induced facial motoneuron death processes. Similar results were obtained in interleukin-1 beta-converting enzyme-deficient and wild-type mice, indicating that the interleukin-1 beta-converting enzyme may not be required for caspase 3 activation.

Nicotinic acetylcholine receptors in neonatal motoneurons are regulated by axotomy: an electrophysiological and immunohistochemical study in human bcl-2 transgenic mice.

Motoneuron axotomy was exploited as a model system for studying functional and morphological changes caused in motoneuron cell bodies by peripheral axon injury. Rodent facial motoneurons express functional nicotinic acetylcholine receptors. We have determined the effect of neonatal unilateral facial nerve transection on these receptors by using electrophysiological and immunohistochemical techniques. To avoid rapid apoptotic cell death of axotomized motoneurons, the study was done in mice overexpressing the human bcl-2 transgene. Intact motoneurons responded to acetylcholine by generating a rapidly rising inward current, which was insensitive to methyllycaconitine, a selective antagonist of alpha7-containing nicotinic receptors, but was suppressed by dihydro-beta-erythroidine, a broad-spectrum antagonist. This indicates that mouse facial motoneurons possess nicotinic receptors which are probably devoid of the alpha7 subunit. In striking contrast, axotomized motoneurons displayed little or no sensitivity to acetylcholine. Axotomy did not affect the sensitivity of facial motoneurons to the selective glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxaxolepropionic acid. Immunohistochemical studies revealed that the alpha4 nicotinic receptor subunit was present in intact motoneurons but was undetectable in axotomized motoneurons. By contrast, the beta2 subunit was comparable in intact and axotomized motoneurons. alpha3 immunoreactivity was undetectable, both in intact and in axotomized motoneurons.Thus, mouse facial nicotinic receptors are possibly of the alpha4beta2 type and axotomy interferes negatively with the expression of the alpha4 subunit. By down-regulating nicotinic receptors, peripheral nerve injury may facilitate motoneuron degeneration. Alternatively, nicotinic receptor downregulation and motoneuron degeneration may be independent consequences of peripheral axotomy.

Somatosensory systems and the milk-ejection reflex in the rat. I. Lesions of the mesencephalic lateral tegmentum disrupt the reflex and damage mesencephalic somatosensory connections.

Bilateral electrolytic lesions and unilateral tracer injections were performed in lactating rats in order to study the participation of the mesencephalic lateral tegmentum in the milk-ejection reflex. The release of oxytocin was detected as a rise in intramammary pressure during each milk ejection. In animals with lesions, the lateral part of the deep grey layers of the superior colliculus, the intercollicular area and the rostromedial portion of the external nucleus of the inferior colliculus were destroyed. The mesencephalic lateral tegmentum of animals in which the milk-ejection reflex was blocked sustained a larger damage than in rats where the frequency of the milk-ejection response was only slowed down. Solutions of True Blue, horseradish peroxidase or horseradish peroxidase coupled to wheat germ agglutinin were injected in the mesencephalic lateral tegmentum of rats with and without lesions. Retrogradely labelled cells were found in several nuclei of the somatosensory pathways: the principal sensory and spinal parts of the trigeminal complex, the cuneate and gracile nuclei, the lateral cervical nucleus and the nucleus proprius of the spinal cord. Labelled cells were also found in the ventral nucleus of the lateral lemniscus, the ventral parabrachial nucleus, the gigantocellular reticular nucleus, the lateral nucleus of the substantia nigra, the prerubral nucleus of the thalamus, the hypothalamic ventromedial nucleus, the zona incerta and in the anterior and lateral hypothalamic areas. Labelled fibres and "terminal-like" labelling were found in the anterior pretectal area, in the thalamic parafascicular nucleus, in the posterior nucleus and the ventroposterior complex, in the zona incerta and in the fields of Forel, but none were observed in the supraoptic or paraventricular nuclei. Injections made in the area of the lateral cervical nucleus and in the cuneate and gracile nuclei labelled fibres and "terminal-like" fields in the external nucleus of the inferior colliculus, the intercollicular area, the deep grey layers of the superior colliculus and in the mesencephalic lateral tegmentum. After injections in the posterior nucleus and ventroposterior complex of the thalamus, retrogradely labelled cells were found in the lateral tegmentum, the intercollicular area and the external nucleus of the inferior colliculus. These results indicate that bilateral lesioning of the mesencephalic lateral tegmentum, which disrupts the milk-ejection response, could damage somatosensory projections originating from the dorsal horn of the spinal cord, the lateral cervical nucleus, the dorsal column nuclei and the sensory and spinal trigeminal nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)

Somatosensory systems and the milk-ejection reflex in the rat. II. The effects of lesions in the ventroposterior thalamic complex, dorsal columns and lateral cervical nucleus-dorsolateral funiculus.

Bilateral electrolytic lesions of some somatosensory structures in the thalamus and spinal cord were made in order to assess their participation in the afferent limb of the milk-ejection reflex in the rat. Lesions involving the lateral cervical nucleus and a part of the dorsolateral funiculus region blocked the milk-ejection reflex, whereas animals with lesions in the dorsal column of the spinal cord or in the ventroposterior complex of the thalamus displayed milk-ejection reflexes similar to those of control animals. Unilateral injections of horseradish peroxidase coupled to wheat germ agglutinin were made after lesioning the lateral cervical nucleus and part of the dorsolateral funiculus. Anterograde labelling was seen mainly contralateral to the injection site: in the external nucleus of the inferior colliculus, the intercollicular zone, the brachium of the inferior colliculus, the lateral reticular nucleus of the thalamus and in the thalamic ventroposterior complex. Sparse projections ipsilateral to the injection site were also observed. These results, combined with our previous observations, suggest that the projection of the lateral cervical nucleus on the mesencephalon is part of the pathway which conveys the sensory information from the suckling stimulus.

Extra-hypothalamic afferent inputs to the supraoptic nucleus area of the rat as determined by retrograde and anterograde tracing techniques.

To detect neuronal cell bodies whose axon projects to the hypothalamic supraoptic nucleus, small volumes (10-50 nl) of 30% horseradish peroxidase or 2% fast blue solutions were pressure-injected into the area of one supraoptic nucleus of rats. Both dorsal and ventral approaches to the nucleus were used. In animals where the injection site extended beyond the limits of the supraoptic nucleus, retrogradely labelled cell bodies were found in many areas of the brain, mainly in the septum, the nucleus of the diagonal band of Broca and ventral subiculum in the limbic system; the dorsal raphe nucleus, the locus coeruleus, the nucleus of the dorsal tegmentum, the dorsal parabrachial nucleus, the nucleus of the solitary tract and the catecholaminergic A1 region in the brain stem; in the subfornical organ and the organum vasculosum of the lamina terminalis, as well as in the median preoptic nucleus. In contrast, when the site of injection was apparently restricted to the supraoptic nucleus, labelling was only clearcut in the two circumventricular organs, the median preoptic nucleus, the nucleus of the solitary tract and the A1 region. Injections of wheat germ agglutinin coupled with horseradish peroxidase (60-80 nl of a 2.5% solution) made in the septum and in the ventral subiculum anterogradely labelled fibers coursing in an area immediately adjacent to the supraoptic nucleus but not within it. In contrast, labelling within the nucleus was found following anterograde transport of tracer deposited in the A1 region and in an area that includes the nucleus of the solitary tract. Neurones located in the perinuclear area were densely labelled by small injections into the supraoptic nucleus; they may represent a relay station for some afferent inputs to the supraoptic nucleus. These results suggest that the supraoptic nucleus is influenced by the same brain areas which project to its companion within the magnocellular system, the paraventricular nucleus.

Expression of substance P and of a Ca2+-activated Cl- current in quail sensory trigeminal neurons.

A chloride current activated by an increase in intracellular calcium concentration is not present in all neurons of the trigeminal ganglion. It is not known whether the trigeminal neurons expressing calcium-activated chloride current belong to a defined class of neurons or whether they could belong to any class of sensory neurons. An answer to this question would be of importance because the physiological role of calcium-activated chloride current in neurons has not yet been completely established, nonetheless it is clear that this current, when activated, would act to modulate neuronal excitability. The goal of this study was to determine whether there was a difference in the expression of calcium-activated chloride current between neurons with and without substance P. The rationale was that the use of this morphological marker, which is present in a substantial fraction of embryonic trigeminal neurons, may give a first estimate of a possible inhomogeneity in the expression of calcium-activated chloride current among different classes of sensory neurons. The study was done on freshly dissociated neurons in order to minimize the influence of the culture conditions on the expression of the current or of substance P. By recording from large samples of neurons in cultures either enriched or depleted in substance P-containing neurons, we found that neurons with substance P expressed calcium-activated chloride current three times less frequently than neurons without substance P. This observation was confirmed by performing the immunocytochemical labelling for substance P immediately after the electrophysiological assessment of the presence or absence of calcium-activated chloride current. This result indicates that calcium-activated chloride current may not be randomly distributed in neurons of a sensory ganglion. It raises the possibility that neurons belonging to certain sensory modalities may need calcium-activated chloride current for their physiological functioning.

Early postnatal Müller cell death leads to retinal but not optic nerve degeneration in NSE-Hu-Bcl-2 transgenic mice.

Topographically localized over-expression of the human Bcl-2 protein in retinal glial Müller cells of a transgenic mice (line 71) leads to early postnatal apoptotic Müller cell death and retinal degeneration. Morphological, immunohistological and confocal laser microscopic examination of transgenic and wild-type retinas were achieved on paraffin retinal sections, postnatally. Apoptosis occurs two to three days earlier in the internal nuclear layer of transgenic retinae, than in wild-type littermates. In parallel there was a progressive disappearance of transgenic Hu-Bcl-2 over-expression, as well as of the Müller cell markers, cellular retinaldehyde-binding protein and glutamine synthetase. This phenomenon led to retinal dysplasia, photoreceptor apoptosis and then retinal degeneration and proliferation of the retinal pigment epithelium. The optic nerve, however, remains intact. Two complementary observations confirm the pro-apoptotic action of Bcl-2 over-expression in Müller cells: (i) in the peri-papillary and peripheral regions where the transgene Bcl-2 is not expressed, cellular retinaldehyde-binding protein or glutamine synthetase immunostaining persist and Müller glia do not die; and (ii) the retina conserves a normal organisation in these two regions in spite of total retinal degeneration elsewhere. We conclude that retinal dysplasia and degeneration are linked to primary Müller cell disruption. Besides its generally accepted anti-apoptotic function, over-expression of Bcl-2 also exerts a pro-apoptotic action, at least in immature Müller glia. One may suppose that Bcl-2 translocation resulting in its over-expression in retinal Müller cells could be a putative mechanism for early retinal degeneration.

cpp32 messenger RNA neosynthesis is induced by fatal axotomy and is not regulated by athanatal Bcl-2 over-expression.

In vivo, neuronal over-expression of the anti-apoptotic protein Bcl-2 prevents axotomy-induced motoneuron death and prolongs life in a mouse model of familial amyotrophic lateral sclerosis. The mechanism of these protective effects is still unknown. We have examined, in situ, the influence of Bcl-2 over-expression on the messenger RNA level of two pro-apoptotic, bax and cpp32, and one anti-apoptotic, bcl-xl, regulators of neuronal death. In neonates wild-type mice, cpp32 mRNA was increased in axotomized, dying motoneurons. No changes in bax and bcl-xl messenger RNAs expression were detected. A similar course was observed in protected axotomized neonate motoneurons of transgenic mice over-expressing Bcl-2. In adult wild-type mice no motoneuron death was detected one week after axotomy: bax and cpp32 messenger RNAs were increased and bcl-xl messenger RNA was decreased. Four weeks after the lesion, 60% of the lesioned facial motoneurons had disappeared. In the remaining motoneurons only cpp32 messenger RNA expression was superior to control level. In Bcl-2 transgenic mice, no axotomy-induced facial motoneurons death was detected but the course of the neosynthesis of cell death genes messenger RNAs was similar to wild-type mice. Bax, Bcl-x and CPP32 immunoreactivity were increased in facial motoneurons after axotomy. Thus, fatal axotomy induces cell death genes bax and cpp32 messenger RNAs neosynthesis which is not prevented by athanatal Bcl-2 over-expression. This suggests that the protective effect of Bcl-2 results from interactions with Bax and CPP32 at the post-translation level without repercussion at the messenger RNA level. Axotomy induces cell death messenger RNA neosynthesis potentially harmful at long-term despite Bcl-2 over-expression.

Autoradiographical localization of oxytocin-binding sites in the guinea-pig ovary at different stages of the oestrous cycle.

The discovery that oxytocin is synthesized and stored in corpora lutea of ruminants has fostered a renewed interest in the possible roles of oxytocin in ovarian function. In the present study we describe the distribution of binding sites for oxytocin in the guinea-pig ovary. Sections were reacted with a radioiodinated oxytocin antagonist (125I-labelled OTA) to yield autoradiograms on film. Specific binding sites for oxytocin were defined as those which bound 0.05 nmol 125I-labelled OTA/l and where 1 mumol non-radioactive oxytocin/l displaced the radioactivity. 125I-Labelled OTA consistently labelled the ovarian stroma and the theca interna, but not the corpora lutea, the granulosa cells or the theca externa. The amount of 125I-labelled OTA bound to ovarian stroma and theca interna was high in animals killed during dioestrus, and low during and shortly after oestrus. These data suggest that the binding sites are regulated by steroid hormone levels and that in the guinea-pig ovary oxytocin could exert a role in follicular steroidogenesis, maturation or ovulation rather than in luteal function. Oxytocin-binding sites were also shown in the uterus but their numbers varied only slightly during the cycle.

An autoradiographical study of binding sites for vasopressin located on corticotrophs in rat and sheep pituitary glands.

Arginine vasopressin (AVP) carried by hypophysial portal blood acts in the pituitary gland, in synergy with corticotrophin-releasing factor (CRF), to induce ACTH secretion. The relative importance of AVP and CRF in this secretory response depends upon the nature and intensity of stressful stimuli, and perhaps also on the species. The aim of the present work was to study, in isolated rat and sheep pituitary glands, the topography of binding sites for AVP and to establish whether they are indeed associated with corticotrophs. To this end, we performed contact autoradiography on tritium-sensitive film using 1.5-2.0 nmol [3H]AVP/l as ligand. ACTH immunoreactivity was detected on sections immediately adjacent to those used for autoradiography. In both species, specific binding sites for AVP were only present in the anterior lobe; the intermediate lobe was not labelled and the neural lobe showed non-specific labelling. Autoradiograms from experiments using [3H]AVP in competition with different synthetic structural analogues showed that, in rat and sheep anterior pituitary glands, receptors differ from the V1 and V2 subtypes. Specific [3H]AVP-binding sites formed an irregular, patchy pattern throughout the anterior lobe. In both species, this pattern was strikingly similar to that formed by cell clusters showing ACTH immunoreactivity, indicating that the AVP-binding sites are associated with the corticotrophs. Immunoreactive cells in the intermediate lobe had no [3H]AVP-binding sites. [3H]AVP binding was more intense in the sheep than in the rat anterior lobe, suggesting that AVP may be particularly important for ACTH secretion in the sheep.

Vasopressin-binding sites in the pig pituitary gland: competition by novel vasopressin antagonists suggests the existence of an unusual receptor subtype in the anterior lobe.

Arginine vasopressin (AVP) acts in the pituitary gland, in synergy with corticotrophin-releasing factor, to induce ACTH release in response to stressful stimuli. Pituitary AVP receptors in the rat are coupled to phospholipase C, as are the so-called V1-type AVP receptors. The present study examined [3H]AVP binding in membranes prepared from the anterior lobe of the pituitary gland of the pig. [3H]AVP, alone or in competition with analogues, bound to sites in the pig anterior lobe which are pharmacologically similar to those described previously by others in the rat pituitary gland. For comparison, the same competition studies were performed on membrane preparations from the rat liver which contain the classic V1-type AVP receptor. Pituitary and liver AVP-binding sites were dissimilar; both cyclic and linear V1 antagonists had, in general, a much lower affinity for pituitary AVP-binding sites than for those in the liver. Thus, Phaa-D-Tyr(Et)-Phe-Gln-Asn-Lys-Pro-Arg-NH2 (Phaa = phenylacetyl) has a 2500-fold greater affinity for the latter (negative logarithm of inhibition constant (pKi) = 9.64) than for the former (pKi = 6.22). One linear antagonist, Pa-D-Tyr-Phe-Val-Asn-Arg-Pro-Arg-Arg-NH2 (Pa = propionyl) had about equal affinities for liver and pituitary membranes (pKi = 6.39 and 6.53 respectively). Another compound, Phaa-D-Tyr-Phe-Val-Asn-Arg-Pro-Arg-Arg-NH2 had the highest affinity found to date for binding to AVP sites in the pituitary (pKi = 7.43). These findings suggest some ideas for the design of more potent and/or selective AVP analogues acting in the pituitary gland.