[Vasopressinergic neurons in rats during ontogenesis: reaction to salt-loading and its modulation by noradrenergic afferents].

Salt-loading in adult mammals stimulates vasopressin secretion by vasopressinergic neurons of the supraoptic nucleus that is under control by a number of hormones and neurotransmitters including noradrenalin. This study was aimed to determine at what period of ontogenesis the vasopressinergic neurons begin to respond to salt-loading and when the noradrenergic control of this process is switched on. Rats on the 21st embryonic day (E), the 3rd postnatal day (P) and P13 were salt-loaded, sometimes under simultaneous treatment with prasozin, an inhibitor of al -adrenoreceptors. Thereafter, the hypothalamic nuclei of the animals were processed for immunocytochemistry and in situ hybridization. Salt-loading provoked increased synthesis of vasopressin mRNA and, most probably, vasopressin itself in rats in all studied age groups. Under salt-loading, the intraneuronal content of vasopressin increased significantly at E21 and P3, whereas it did not change at P13. No change in the intracellular contents of vasopressin mRNA and vasopressin was observed in foetuses following salt-loading and treatment with prasozin though the same treatment provoked an increase of both parameters at P3. These data show that noradrenalin provides an inhibitory control of vasopressin expression at least since P3. Thus, vasopressinergic neurons begin to respond to salt-loading at the since P3. Thus, life by the increased expression of vasopressin that is postnatally under the inhibitory control by noradrenalin.

Vasopressin and galanin expression in the hypothalamus of two African rodents, Taterillus gracilis and Steatomys caurinus, subjected to water-restriction.

The expression of arginine-vasopressin (AVP) and galanin (GAL) was studied by immunohistochemistry and in situ hybridization in the hypothalamus of two species of African rodents. In the wild, these animals experience successive arid and wet seasons that alternately stimulate their antidiuretic and diuretic systems. In this study, animals were subjected to both standardized laboratory conditions and to eight days of water-restriction. Under both sets of conditions, AVP and GAL were detected in the supraoptic nucleus (SON), paraventricular nucleus (PVN), and median eminence (ME). AVP and GAL responses to water-restriction differed in the two species, as did behavioral adaptations to the hot-dry season. In Taterillus gracilis, AVP- and GAL-LI (like immunoreactivity) peptide and mRNA levels increased in the SON. AVP-LI peptide and mRNA levels increased in the PVN, whereas only AVP-LI peptide levels increased in the ME. Pituitary gland AVP pools were unchanged by water deprivation, whereas urinary AVP levels and osmolality increased. The AVP response is typical of that of desert rodents, favoring survival under conditions of water-restriction. In Steatomys caurinus, which estivates, AVP and GAL-LI peptide levels decreased in the hypothalamus, as they did in the laboratory rat. In the SON, AVP, and GAL mRNA levels increased, whereas, in the PVN, only AVP mRNA levels increased. Pituitary gland AVP levels decreased, whereas urinary AVP levels and osmolality increased. In both species, the changes in the amount of GAL-LI peptide appeared to be closely linked to changes in AVP levels, suggesting that this peptide is involved in the osmoregulatory response to water-restriction.

Influence of serotonin on the development and migration of gonadotropin-releasing hormone neurones in rat foetuses.

This study used a pharmacological approach to evaluate the consequences of the metabolic perturbations of neurotransmitters on brain development. Pregnant rats received p-chlorophenylalanine (pCPA), an inhibitor of serotonin (5-hydroxytryptamine, 5-HT) synthesis, or saline (control) from the 11th day of gestation once or daily up to the 15th, 17th and 20th day, followed by processing of the forebrain and/or nasal cranium of foetal males and females for high-performance liquid chromatography of monoamines, radioimmunoassay of gonadotropin-releasing hormone (GnRH) and quantitative and semiquantitative immunocytochemistry for GnRH. The pCPA treatment resulted in a 50-70% depletion of 5-HT in the nasal crania and forebrains at any studied age. Radioimmunoassay showed no change in GnRH content in 5-HT deficient foetuses at E16 compared to controls, being higher in both cases in the rostral forebrain than in the hypothalamus. In controls at E21, the GnRH content in the hypothalamus exceeded that in the rostral forebrain, whereas in the 5-HT deficient group the opposite was found. These data suggest that 5-HT provided a stimulating effect on GnRH neurone migration, and this was confirmed by quantification of GnRH-immunoreactive neurones in the forebrain along the trajectory of their migration. At E18 and E21, the fractions of GnRH neurones in the rostral part of the trajectory in pCPA-treated foetuses were greater than those in control foetuses but the opposite was true for the caudal part of the trajectory. Moreover, 5-HT appeared to control the proliferation of the precursor cells of GnRH neurones and their differentiation, as derived from the observations of the increased number of GnRH neurones in the forebrain of foetuses of both sexes, as well as the region-specific decreased neuronal size and content of GnRH in 5-HT-deficient females. Thus, 5-HT appears to contribute to the regulation of the origin, differentiation and migration of GnRH neurones.

Axonal projections from the hypothalamus to the median eminence in rats during ontogenesis: DiI tracing study.

This study has determined the ontogenetic schedule of the arrival of the axons from the hypothalamus and the diagonal band in the median eminence in rats by using the fluorescent lipophilic carbocyanine dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) as a retrograde tracer. After fixation of the brain, the crystals of the dye were implanted in the median eminence on the 13th, 14th, 15th, 16th, 17th, 20th embryonic days, and on the 2nd postnatal day. This was followed by fluorescent staining of the neuronal cell bodies in the hypothalamus. According to our data, the axons of rare hypothalamic neurons first reached the primordium of the median eminence on the 14th embryonic day. For two subsequent days, the number of neurons projecting the axons to the median eminence appeared to increase considerably. They were widely distributed through the hypothalamus and in the ventromedial region of the more rostral forebrain. Till the 20th embryonic day, the majority of the fluorescent neurons were concentrated mainly in the paraventricular nucleus (dorsal and medial parts) and the arcuate nucleus, and to a lesser extent in the medial preoptic nucleus, the supraoptic nucleus, the diagonal band, and the retrochiasmatic nucleus. In neonates, DiI-labelled neurons appeared additionally in the accessory dorsolateral nucleus, medial preoptic area lateral to the diagonal band, anterior hypothalamic area, and in the anterior periventricular nucleus. Thus, the axons of differentiating neurons arrive in the median eminence from the 14th embryonic day till the neonatal period, providing the pathway for the neurohormone transfer to the hypophysial portal circulation.

Effects of centrally administered galanin (1-16) on galanin expression in the rat hypothalamus.

In the rat hypothalamic magnocellular neurons, galanin coexists with vasopressin and might be involved in hydro-osmotic regulation. In the present study, we investigated the ability of galanin to also regulate the osmotically stimulated expression of galanin itself in hypothalamic magnocellular neurons. Ten minutes after galanin injection, galanin mRNA rate decreased in salt-loaded rats whereas the level of galanin immunoreactivity increased. Both effects were suppressed by the injection of a galanin antagonist together with galanin. Moreover, electron microscope studies demonstrated synaptic contacts between galanin-containing fibers and magnocellular neurons. Galanin may exert inhibitory roles in the regulation of magnocellular neurons. However, galanin and vasopressin expression displayed differences upon galanin injection. Possible mechanisms underlying these discrepancies are further discussed.

Projections from the hypothalamus to the posterior lobe in rats during ontogenesis: 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate tracing study.

The objective of this study was to determine the schedule of the arrival of the axons from the hypothalamus to the posterior lobe of the pituitary (PL) in rats during ontogenesis by using the fluorescent lipophilic carbocyanine dye 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI) as a retrograde tracer. After preliminary fixation of the brain, DiI crystals were implanted in the PL on embryonic day 15 (E15), E16, E17, and E19 as well as on postnatal day 2 (P2) and P9. This was followed by a DiI retrograde diffusion along the plasma membrane and subsequent staining of hypothalamic neuronal cell bodies. The supraoptic nucleus (SO) contained an accumulation of fluorescent cells that extended toward the diamond-like swelling of the third ventricle as early as E15. These data suggest that the magnocellular neurons of the SO send their axons to the PL at the very beginning of differentiation, perhaps even before reaching their final position. The initial axons of the neurons of the paraventricular nucleus proper (PV) appeared to reach the PL significantly later, at E17. In addition to the SO and the PV, accessory magnocellular nuclei contributed to the innervation of the PL in perinatal rats. The neurons of the retrochiasmatic accessory nucleus first sent their axons to the PL on E16-E17. Axons that originated from other accessory hypothalamic nuclei reached the PL after birth, suggesting a delay in their involvement in the regulation of visceral functions compared with other magnocellular nuclei. Thus, the axons of magnocellular neurons reach the PL unexpectedly early in embryogenesis, raising the possibility of the functional significance of vasopressin and oxytocin as fetal neurohormones.

[The formation and regulation of the lactotrophic function of the hypophysis in the prenatal period of rat development. II. Dopamine inhibitory control of prolactin secretion]. / Stanovlenie i reguliatsiia laktotrofnoi funktsii gipofiza v prenatal'nom periode razvitiia krys. II. Dofaminovyi ingibitornyi kontrol' sekretsii prolaktina.

Using the technique of radioimmunoassay, we studied the secretion of prolactin and its control by dopaminergic system in 22-day-old rat fetuses under normal conditions and after pharmacological inhibition of dopamine receptors. In order to elucidate the origin of prolactin and dopamine participating in this process, we used decapitation and encephalectomy of fetuses in utero. Decapitation of fetuses did not result in any changes of baseline prolactin secretion into blood in males and insignificantly decreased it in females as compared with nonoperated controls. We conclude that prolactin detected in blood plasma of nonoperated fetuses does not originate in the pituitary, and any prolactin synthesized in the pituitary is not secreted into blood. Inhibition of dopamine receptors in decapitated fetuses did not result in any changes of prolactin level in blood. This provided evidence that in nonoperated fetuses, it is pituitary prolactin which is secreted in response to haloperidol, while the secretion of nonpituitary prolactin is not controlled by dopamine. Encephalectomy increased prolactin level in plasma and resulted in a drastic decrease of its level in the pituitary. The block of dopamine receptors did not affect the level of prolactin in blood plasma or pituitary of encephalectomized fetuses. We conclude that the inhibitory dopaminergic control of prolactin secretion by the pituitary during the prenatal period is accomplished just as in adult animals by dopaminergic neurons of hypothalamus.

Expression of galanin in hypothalamic magnocellular neurones of lactating rats: co-existence with vasopressin and oxytocin.

Lactation is a physiological condition known to upregulate the expression of the hypothalamic neurohormones, oxytocin and vasopressin, in the rat supraoptic and paraventricular nuclei. Other neuropeptides such as galanin are co-localized in the same magnocellular neurones and their expression has been demonstrated to be regulated by different experimental and physiological conditions. In the present study, we investigated the possible changes in galanin expression during lactation, using in situ hybridization and immunohistochemistry separately or in combination. Galanin messenger RNA concentrations decreased on day 3 of lactation in both the supraoptic and paraventricular nuclei and remained low on day 7 of lactation, but no differences were observed between control and 14-day lactating rats. In parallel, immunopositive cell bodies were almost undetectable on day 7 of lactation and immunoreactivity remained weak after 14 days of lactation, whereas galanin immunoreactive profiles in the supraoptic nucleus were more numerous than in the control group. Moreover, the subcellular distribution of immunostaining changed on day 14 of lactation. Galanin immunoreactivity was confined around the nucleus in the control females, but it became weaker and more homogenously distributed throughout the cytoplasm in the lactating rats. Electron microscopy using a pre-embedding technique confirmed that galanin immunoreactivity was no longer restricted to the Golgi complex, but was apparent throughout in the cytoplasm. Multiple labellings showed galanin and galanin messenger RNA to be co-localized with oxytocin messenger RNA in neurones of the dorsomedial part of the supraoptic nucleus during lactation. Some of those doubly labelled cells also expressed vasopressin messenger RNA in the same conditions as revealed by a triple-labelling procedure. As these co-localizations have not been observed in female control rats, lactation provided an example of a physiological condition inducing oxytocin and galanin co-synthesis in a subpopulation of magnocellular neurones. In conclusion, we have demonstrated plasticity of galanin expression during lactation in the hypothalamic magnocellular neurones. This plasticity could be caused by changes in galanin expression or in galanin processing in magnocellular neurones.

Birthdates of the tyrosine hydroxylase immunoreactive neurons in the hypothalamus of male and female rats.

This study determined the birthdates of the tyrosine hydroxylase-(TH) immunoreactive (IR) neurons in the zona incerta (ZI), periventricular nucleus (PeVN) and arcuate nucleus (AN) of male and female rats. 'Long-survival' [3H]thymidine autoradiography combined with TH immunocytochemistry, the first enzyme of catecholamine synthesis, was used. In males, TH-IR neurons originate in the ZI between embryonic days (E) 12 and 13, while in the PeVN and AN this process is prolonged until E16. The majority of TH-IR neurons became postmitotic at E12 in the ZI, between E12 and E14 in the PeVN and at E15 in the AN. The birthdate of TH-IR neurons was sexually dimorphic with (a) generation of the majority of TH-IR neurons in the ZI in males proceeding that in females, (b) generation of TH-IR neurons in the AN of males delayed as compared to females, and (c) average daily fractions of the newborn TH-IR neurons in each hypothalamic region of females exceeding that seen in males. This sexual dimorphism was observed prior to E16, i.e. before the onset of sex difference in androgen levels, implying a hormone-independent mechanism, determined at the genetic level.

Pharmacological model of catecholamine depletion in the hypothalamus of fetal and neonatal rats and its application.

1. The present study aimed to develop a pharmacological model of catecholamine (CA) depletion in the hypothalamus during the period of its morphofunctional development, i.e. in fetal and neonatal rats of both sexes. 2. In the first series of experiments, pregnant females and, hence, fetuses were systemically treated daily from the embryonic day (E) 13 to E20 with the inhibitor of the CA synthesis alpha-methyl-m-tyrosine. The CA concentrations were subsequently measured in the fetal hypothalamus at E21 by high performance liquid chromatography with electrochemical detection (HPLC-ED). In the second series of experiments, neonatal rats were injected with neurotoxin, 6-hydroxydopamine and/or alpha-methyl-m-tyrosine daily from the 2nd postnatal day (P2) to P10. 3. The HPLC-ED assay of hypothalamic catecholamines (CA's) at E21 and P11 showed that both in fetuses and neonates, alpha-methyl-m-tyrosine caused more than 50% depletion of hypothalamic noradrenaline and adrenaline, while the dopamine (DA) level remained unchanged. The combined treatment of neonatal rats with alpha-methyl-m-tyrosine and 6-hydroxydopamine resulted additionally in a 25% decreased level of DA. 4. The influence of CA deficiency on the developing hypothalamic CA system was further evaluated by measuring [3H]DA uptake by nervous tissue in vitro. 5. The CA deficiency caused a 50% drop of [3H]DA uptake by the hypothalamic tissue in treated fetuses suggesting a stimulating effect of CA's on the early development of the CA system. In pharmacologically treated neonatal rats [3H]DA uptake remained at the control level showing no influence of the CA deficiency on the developing CA system after birth. 6. The usefulness of the proposed pharmacological model for studying of CA influence on differentiating hypothalamic target neurons is discussed.

Development of the hypothalamic vasopressin system and nephrons in Meriones shawi during ontogenesis.

This study has evaluated the development of the hypothalamic vasopressin system and nephrons of the kidney in desert rodents, Meriones shawi, which effectively retain water by excretion of highly concentrated urine. The vasopressin system was studied immunocytochemically at the 18th fetal day, at the 2nd, 13th, 27th postnatal days and in adulthood. The kidneys were investigated at the 2nd, 13th postnatal days and in adulthood using microdissection technique. Occasional vasopressin-immunoreactive neurons were observed as early as the 18th fetal day, only in the paraventricular nucleus. From the 2nd postnatal day onwards, vasopressin neurons increased progressively in number, being mainly concentrated in the supraoptic and paraventricular nuclei, as well as in the ventral retrochiasmatic region. Transient neuronal populations were also observed at the 13th postnatal day in the lateral preoptic area and anterior hypothalamic nucleus. Apart from the neurons, the glandular cells of the tuberal lobe showed immunostaining from the 18th fetal day, the first age studied, until the 13th postnatal day. The fibers of differentiating vasopressin neurons grew towards the circumventricular/neurohemal organs, terminating in the organum vasculosum of the lamina terminalis and the lateral ventricles as early as the 18th fetal day, as well as the third ventricle, the posterior lobe and the external zone of the median eminence between the 2nd and 13th postnatal days. The kidney in 2-day-old Meriones comprised nephrons at different stages of development from an S-shaped body to well-differentiated nephrons. At the 13th postnatal day, as in adulthood, the nephrons were well differentiated and characterized by long, thin loops descending to different levels of papilla. Thus, according to our morphological data the hypothalamic vasopressin neurons and nephrons in the kidney of Meriones reach the definitive state by the end of the 2nd postnatal week.

Short-term effects of centrally administered galanin on the hyperosmotically stimulated expression of vasopressin in the rat hypothalamus. An in situ hybridization and immunohistochemistry study.

Galanin (GAL) is a 29-amino acid peptide that is present in the hypothalamic magnocellular neurons of the rat supraoptic nucleus (SON) and paraventricular nucleus (PVN). Since previous studies revealed a possible role of GAL in the hydro-osmotic regulation, we have investigated the effects of GAL on the vasopressin (AVP) mRNA content in the hypothalamic magnocellular neurons. We demonstrated by in situ hybridization (ISH) and immunohistochemistry that 100 pmol of GAL or GAL fragment (1-16) injected into the lateral ventricle of anesthetized dehydrated male rats induced a rapid (10 min time interval) decrease of AVP mRNA and AVP content in the magnocellular cell bodies of SON and PVN. These effects were quantified on an autoradiographic film and were also obvious at the cellular level by using ISH with a radiolabeled or digoxigenin-labeled oligonucleotide probe. Oxytocin mRNA content is not altered by the same injection either in dehydrated male or lactating female rats. Under the same conditions of lactation, AVP mRNA content is not modified and the i.c.v. injection of GAL has no effect. GAL antagonist (M15) injection suppressed the GAL-induced decrease of AVP mRNA in the dehydrated rats and increased AVP mRNA level in control rats. The efficacy of M15 in antagonizing GAL effects on AVP mRNA suggests the involvement of GAL receptors in the regulation of the vasopressinergic cell bodies. Moreover, endogenous GAL seems to depress the AVP mRNA content of SON and PVN in vivo. The possible origin of endogenous GAL and the mechanisms by which GAL can regulate the AVP mRNA content are also discussed.

Expression of a human insulin transgene in cholinergic neurons of the mouse medial habenula.

We explored the possibility that an insulin gene deleted in its 5'-flanking region is expressed in adult mouse brain. We used three independent lines of mice carrying a human insulin transgene which included the insulin gene transcription unit flanked by 168 base pairs upstream and 5.5 kb downstream. Using a reverse transcription-polymerase chain reaction assay, human insulin mRNAs were detected in whole brain extracts. In all three lines, human insulin mRNAs were localized by in situ hybridization in a single cerebral site, the medial habenula. With a monoclonal antibody specific for human C-peptide and human proinsulin, labelling was restricted to a subset of habenular cholinergic neurons, with rare immunostained fibers. No labelling was observed in the projection fibers of the retroflexus fasciculus or in their axon terminals in the interpeduncular nucleus. Electron microscope studies suggested that the transgene expressing cells. These findings demonstrate that the human insulin transgene tested here includes a habenula specific promoter which could be useful for physiological and molecular studies on the habenula.

Combination of non-radioactive and radioactive in situ hybridization with immunohistochemistry: a new method allowing the simultaneous detection of two mRNAs and one antigen in the same brain tissue section.

We describe here a simple method for combining non-radioactive and radioactive in situ hybridization and immunohistochemistry on the same brain tissue section. This approach was first developed on the well-characterized hypothalamo-neurohypophyseal system, facilitating the optimization of the triple-labeling procedure and the verification of labeling specificity. We report the simultaneous detection of vasopressin (VP) mRNA with a digoxigenin-labeled oligonucleotide, oxytocin (OT) mRNA with a 35S-labeled oligonucleotide, and OT peptide in the same 12-microns cryostat section. This was performed on floating sections as follows: first, the two probes were hybridized simultaneously; second, the peptide was detected with an immunoperoxidase-DAB procedure; third, the digoxigenin-labeled probe was detected with an alkaline phosphatase-NBT/BCIP technique; and finally, the 35S-labeled probe was detected by histological autoradiography. We also demonstrate that this approach is suitable for the simultaneous detection of tyrosine hydroxylase and two less abundant mRNAs, vasoactive intestinal peptide and vasopressin mRNAs, in the suprachiasmatic nucleus. The combination of the three techniques did not significantly diminish their specificity or sensitivity. In conclusion, this new method, permitting the simultaneous detection of three different products of gene expression in the same section, could be useful for further analysis of the phenotypic organization and its plasticity in endocrine or neural tissues.

[Demonstration by in situ hybridization and immunohistochemistry of human insulin gene expression cells in medial habenula of transgenic mice]. / Mise en évidence par hybridation in situ et immunohistochimie de cellules exprimant le gène humain de l'insuline dans l'habenula médiane de souris transgéniques.

In transgenic mice carrying a human insulin transgene, in situ hybridization and immunohistochemistry detected a cerebral site of expression of the human insulin gene. The gene construct that was introduced into the mouse genome included 168 base pairs (bp) upstream from the transcription unit of the human insulin gene. The human gene transcript and the corresponding proteins were detected in a cell subset of the medial habenula. This was found in 20 mice from three independent transgenic lines, indicating that expression was not dependent upon the insertion site of the transgene. When the DNA fragment upstream from the transcription unit was either larger (258 bp) or smaller (58 bp), this extrapancreatic expression of the human insulin transgene was not found. This result suggests that a sequence between nucleotides -58 and -168 might be responsible for an ectopic expression of the human insulin gene, specifically in habenular cells.

Hypothalamic galanin-immunoreactive neurons projecting to the posterior lobe of the rat pituitary: a combined retrograde tracing and immunohistochemical study.

UNLABELLED: To identify the galanin-immunoreactive neurons projecting to the posterior lobe of the pituitary in the rat hypothalamus, a retrograde tracer (complex of wheat germ agglutinin-enzymatically inactive horseradish peroxidase-colloidal gold) was injected into the posterior lobe of the pituitary. Sections of the hypothalamus were treated with a combination of silver enhancement of retrogradely transported tracer and immunohistochemistry of galanin. Of the total number of hypothalamic cells doubly labeled with retrograde tracing and galanin-immunostaining, 56-60% were found in the supraoptic nucleus, 18-23% in the retrochiasmatic nucleus, 8-10% in the lateral magnocellular portion of the paraventricular nucleus. The ratio of (number of doubly labeled cells/number of galanin-immunoreactive cells) in each of the above regions was similar to the ratio of (number of retrogradely labeled cells/number of Nissl-stained cells) in the supraoptic nucleus. Of all retrogradely labeled cells in the hypothalamus, 51-56% also contained galaninlike immunoreactivity. IN CONCLUSION: (1) galanin-immunoreactive fibers in the posterior lobe of the pituitary originate mainly in the supraoptic nucleus, retrochiasmatic nucleus, and lateral magnocellular portion of the paraventricular nucleus, (2) most of galanin-immunoreactive cells in these regions project to the posterior lobe of the pituitary, and (3) about half the neurons constituting the hypothalamo-neurohypophyseal system contain galaninlike immunoreactivity.

Ultrastructural localization of oxytocin mRNA in the rat hypothalamus by in situ hybridization using a synthetic oligonucleotide.

In situ hybridization (ISH) was used to study at the electron microscope level, the subcellular localization of oxytocin (OT) mRNA in the rat hypothalamic magnocellular neurons. Rat brains were fixed with paraformaldehyde and glutaraldehyde and vibratome slices were incubated with a 25-base synthetic oligonucleotide complementary to OT mRNA and labelled at the 3'-end with [3H]dCTP. Hybridized slices were embedded in Epon after post-fixation with osmium tetroxide and cut into ultrathin sections that were processed for ultrastructural radioautography. OT mRNA was observed in magnocellular neurons of supra-optic and paraventricular nuclei in the vibratome sections. On ultrathin sections, the cytological preservation appeared to be satisfactory. Except for a few silver grains over the nucleus, sometimes close to its membrane, most grains were localized over the cytoplasm of some magnocellular neurons, where they frequently overlapped the endoplasmic reticulum. To decrease exposure time, ISH was also performed with OT probes labelled with a long tritiated tail. In this case, clusters of silver grains occurred over the cell nuclei not only in magnocellular neurons but also in non-secretory neurons and even in glial cells. However, an excess of poly C added to the hybridization buffer strongly decreased this non-cytoplasmic labelling. In conclusion, the results obtained with the short-tailed oligonucleotides demonstrate that these synthetic oligonucleotides have possible applications for the ultrastructural localization of mRNAs and constitute a powerful tool for the dynamic study of cellular mRNA processing in several physiological and experimental conditions.

Origin of the met-enkephalinergic innervation of the lateral septum in the rat.

The location of the cells giving rise to the methionine-enkephalin (Met-Enk)-ergic innervation of the lateral septal nucleus has been investigated in the rat by combining immunohistochemistry and retrograde axonal tracing. Small volumes (0.06 microliter) of apo-horseradish peroxidase (Apo-HRP) conjugated to wheat-germ agglutinin (WGA) and coupled with colloidal gold particles (WGA-ApoHRP-gold) were injected into the lateral septum. The retrogradely labeled cell bodies were visualized by silver intensification of the gold particles on Vibratome sections that were subsequently processed for immunohistochemistry for Met-Enk. Cells labeled with WGA-ApoHRP-gold were observed in the septal area, throughout the hypothalamus (mainly in the perifornical and lateral nuclei) and in the mesencephalon. The localization of Met-Enk-immunoreactive cells was as previously described. With the exception of a few septal cells close to the injection site, doubly labeled cells were found only in the perifornical nucleus of the hypothalamus. Almost all perifornical magnocellular cells were doubly labeled ipsilateral to the injection site, whereas on the opposite side, only about 25% of the Met-Enk-immunoreactive cells contained WGA-ApoHRP-gold. Other brain regions containing retrogradely labeled or Met-Enk-immunoreactive cells (particularly the raphe nuclei) did not show double-labeled neurons. This study demonstrates, using a new and sensitive technique for specific neurochemical tracing of tracts, that the origin of the Met-Enk-ergic innervation of the rat lateral septal nuclei lies in the magnocellular perifornical nuclei of the hypothalamus. The precise involvement of this pathway in limbic functions remains to be determined.

Development of intracerebral dopaminergic grafts: a combined immunohistochemical and autoradiographic study of its time course and environmental influences.

The aim of the study was to obtain a description of some aspects of the development of intracerebral dopaminergic grafts, namely, the time course of the glial reaction and its relation to cell division on one hand, and the development of graft-originated innervation and its dependence on adequate matching of the implanted neurons and target site on the other hand. Cell suspensions obtained from the mesencephalon or hypothalamus of embryonic day (ED) 14 rat embryos were implanted into the striatum or lateral hypothalamus of adult rats following the destruction of the nigrostriatal system of the hosts. Animals were sacrificed at different postimplantation times, and the development of the graft was followed by immunohistochemistry by using antisera directed against tyrosine hydroxylase (TH) or glial fibrillary acidic protein (GFA). Furthermore, the existence of cell division at various times following implantation was examined by performing autoradiography on immunostained sections after prior intraventricular administration of 3H-thymidine to the host. The first stage of the development of intracerebral grafts was characterized by the existence of intense cell division within the grafted tissue, lasting about 2 weeks, and also in the host tissue surrounding the graft, lasting only about 6 days. The cell division in the host tissue was paralleled by the existence of a strong glial reaction which, however, did not extend into the graft itself. Glial reaction in the host tissue gradually decreased at later times and disappeared by 4 weeks postimplantation without leaving behind a noticeable glial scar. The graft itself was, however, transiently filled with a population of reactive astroglial cells between 3 and 6 weeks postimplantation. Within grafts of mesencephalic tissue located in the striatum TH-positive neurons were distributed evenly at short times postimplantation (2-6 days). At later time a compartmentation could be observed, with TH-positive neurons being aligned along the graft-host interface or clustered within the graft itself. Innervation of the host tissue by TH-positive fibers increased between 1 and 6 weeks postimplantation. On the other hand, no compartmentation and reinnervation of surrounding host tissue was observed for intrahypothalamic grafts of mesencephalic tissue or intrastriatal grafts of hypothalamic tissue. This last observation indicates that adequate matching of implanted neurons and target tissue plays an important role in the development of intracerebral dopaminergic grafts.