F. Nürnberger

Opsin-immunoreactive outer segments in the pineal and parapineal organs of the lamprey (Lampetra fluviatilis), the eel (Anguilla anguilla), and the rainbow trout (Salmo gairdneri)

SummaryThe pineal complex of Lampetra fluviatilis, Anguilla anguilla and Salmo gairdneri was studied by means of the indirect immunohistochemical antiopsin reaction.Opsin-immunoreactive material was demonstrated in the outer segments of the photoreceptor cells in the pineal organ of all three species investigated. In the lamprey, the opsin-positive outer segments were located in the lumen of the pineal vesicle and atrium. In the two teleost species, the immunoreactive outer segments were observed in abundance in the pineal end-vesicle and stalk. These structures were found to accumulate in the prominent initial portion of the pineal stalk of the eel. In the rainbow trout, immunoreactive outer segments occurred in the wide orifice of the pineal recess at the roof of the third ventricle.In addition, outer segments of photoreceptor cells of the parapineal organ (“parapinealocytes”) displayed opsin immunoreactivity. In the lamprey, opsin immunoreactivity was restricted to the central portion of the ventral parapineal retina, while the parapinealocytes in the lateral portions did not bind the antibody. In the two teleosts, immunoreactive outer segments displayed a scattered pattern.These immunocytochemical results provide direct evidence that the photosensitivity of the pineal demonstrated electrophysiologically in lampreys and teleosts (cf. Dodt 1973) is based on an opsin-containing photopigment. The presence of opsin in cells of the parapineal organ strengthens the view that also this organ may be capable of direct light perception. In the lamprey, the exclusive opsin immunoreactivity of a circumscribed group of parapineal cells suggests the existence of two types of parapinealocytes. The significance of opsin-containing photoreceptor outer segments occurring in the most proximal portion of the teleost pineal stalk is discussed, especially with regard to the interpretation of results obtained from pinealectomy experiments.

The efferent connections of the lateral septal nucleus in the guinea pig: projections to the diencephalon and brainstem.

SummaryThe anterograde Phaseolus vulgaris-leucoagglutinin (PHA-L) tracing technique was used to determine the distribution of efferent fibers originating in the lateral septal nucleus of the guinea pig. For complementary detection of the chemical identity of the target neurons, double-labeling immunocytochemistry was performed with antibodies to PHA-L and to vasopressin, oxytocin, vasoactive intestinal polypeptide, serotonin or dopamine β-hydroxylase, respectively. The hypothalamus received the majority of the PHA-L-stained septofugal fibers. Here, a specific topography was observed. (1) The medial and lateral preoptic area, (2) the anterior, lateral, dorsal, posterior hypothalamic and retrochiasmatic area, (3) the supraoptic, paraventricular, suprachiasmatic, dorsomedial, caudal ventromedial and arcuate nuclei, and (4) the tuberomammillary, medial and lateral supramammillary, dorsal and ventral premammillary nuclei always contained PHA-L-labeled fibers. The rostral portion of the ventromedial nucleus and the medial and lateral mammillary nucleus only occasionally showed weak terminal labeling. In other diencephalic areas, termination of PHA-L-labeled fibers was observed in the epithalamus and the nuclei of the midline region of the thalamus. In the mesencephalon, terminal varicosities occurred in the ventral tegmental area, interfascicular and interpeduncular nucleus, and periaqueductal gray. In addition, the dorsal and medial raphe nuclei of the metencephalon, together with the locus coeruleus and the dorsal tegmental nucleus, received lateral septal efferents.

Pattern of afferents to the lateral septum in the guinea pig

SummaryThe septal region represents an important telencephalic center integrating neuronal activity of cortical areas with autonomous processes. To support the functional analysis of this brain area in the guinea pig, the afferent connections to the lateral septal nucleus were investigated by the use of iontophoretically applied horseradish peroxidase (HRP). Retrogradely labeled perikarya were located in telencephalic, diencephalic, mesencephalic and metencephalic sites. The subnuclei of the lateral septum (pars dorsalis, intermedia, ventralis, posterior) receive afferents from the (i) medial septal nucleus, diagonal band of Broca (pars horizontalis and pars ventralis), and the principal nucleus of the stria terminalis, the hippocampus, and amygdala (nucleus medialis); (ii) the medial habenular nucleus, and the para- (peri-) ventricular, parataenial and reuniens nuclei of the thalamus; the anterior, lateral and posterior hypothalamic areas in particular, the medial and lateral preoptic, suprachiasmatic, periventricular, paraventricular, arcuate, premammillary, and supramammillary nuclei; (iii) the periaquaeductal grey, ventral tegmental area, nucleus interfascicularis, nucleus reticularis linearis, central linear nucleus, interpeduncular nucleus; (iv) dorsal and medial raphe complex, and locus coeruleus. Each subnucleus of the lateral septum displays an individual, differing pattern of afferents from the above-described regions. Based on a double-labeling method, the vasopressinergic and serotonergic afferents to the lateral septum were found to originate in the nucleus paraventricularis hypothalami and the raphe nuclei, respectively.

Oxytocin-and vasopressin-immunoreactive nerve fibers in the pineal gland of the hedgehog, Erinaceus europaeus L.

SummaryOxytocin-and vasopressin-immunoreactive nerve fibers, apparently originating from a dorsal subunit of the paraventricular nucleus, were demonstrated in the pineal gland of the hedgehog. The majority of these fibers (pinealopetal projections) is intimately related to the capillaries of the pineal organ, whereas only a few elements are scattered throughout the pineal parenchyma. The number of peptidergic elements observed in the central portion of the pineal organ exceeds that of fibers located at the periphery. In relation to the functional state of the animals, the amount of immunoreactive material in these pinealopetal nerve fibers exhibits conspicuous variations. In hibernating hedgehogs (group 1), these nerve fibers were considerably richer in oxytocin than in non-hibernating or arousing winter animals (group 2 and 3). In contrast, only weak immunoreactivity for vasopressin was found in intrapineal nerve fibers of hibernating hedgehogs (group 1), whereas the fibers of arousing or non-hibernating hedgehogs (group 2 and 3) contained slightly larger amounts of vasopressin.In the pineal organ of animals sacrificed during the summer period (group 4), no immunoreactivity for both neuropeptides was found.The functional significance of the connection between the hypothalamic paraventricular nucleus and the pineal organ is discussed with special reference to the vascular terminals of the pinealopetal peptidergic nerve fibers.

The efferent connections of the lateral septal nucleus in the guinea pig: intrinsic connectivity of the septum and projections to other telencephalic areas.

SummaryThe distribution of efferent fibers originating in the lateral septal nucleus was investigated in guinea pigs by means of anterograde tracing with Phaseolus vulgaris-leucoagglutinin (PHA-L). Special emphasis was placed on the intraseptal fiber systems. The fibers originating from the different subnuclei of the lateral septal nucleus formed massive horizontal connections in the rostrocaudal axis. Projections to the contralateral, congruent subnuclei were also detected. In the medial septum/diagonal band of Broca complex the largest number of PHA-L-stained fibers was found after application of the tracer into the dorsal subnucleus of the lateral septal nucleus; the density of the efferent fibers decreased progressively after injection into the intermediate or ventral subnuclei. In all cases the diagonal band contained a much higher number of efferent fibers from the lateral septal nucleus than from the medial septal nucleus. In the medial septal nucleus, terminal labeling was generally sparse. Other telencephalic areas (organum vasculosum of the lamina terminalis, nucleus accumbens, bed nucleus of the stria terminalis, amygdala, hippocampal complex, and other cortical areas) contained varying numbers of labeled projections. In double-labeling experiments, a close spatial relationship between PHA-L-stained fibers and vasoactive intestinal polypeptide-immunoreactive perikarya was observed in several of these target areas.

Effects of illumination and enucleation on substance-P-immunoreactive structures in subcortical visual centers of golden hamster and Wistar rat.

The undecapeptide substance P is found in different entities of the visual system that control eye movement and synchronize endogenous rhythms with the light cycle (i.e., superior colliculus, suprachiasmatic nucleus, intergeniculate leaflet). Immunocytochemical methods were used to compare the reactivity to substance P in the brain of five groups of golden hamsters and two groups of Wistar rats: (1) untreated hamsters kept under 14L:10D and sacrificed at noon; (2) identically maintained animals sacrificed at midnight; (3) enucleated animals kept under control conditions; (4) hamsters kept under constant darkness; (5) hamsters kept under the same conditions as the controls, but intraventricularly injected with colchicine. The results obtained in golden hamsters of groups (1) and (3) were compared with findings in Wistar rats treated accordingly [groups (6) and (7)]. Substance P-immunoreactive perikarya were found in the suprachiasmatic nucleus and superior colliculus of hamsters and Wistar rats. Substance P-immunoreactive nerve fibers were abundant in the hypothalamic area ventral to the paraventricular nucleus, in the intergeniculate leaflet, in some thalamic nuclei, and in the superior colliculus. Immunoreactivity to substance P in the suprachiasmatic nucleus and intergeniculate leaflet did not vary among the experimental groups. However, a conspicuous decrease in reactivity to substance P was observed in the superficial layers of the superior colliculus of enucleated hamsters and rats, compared with all other groups. These results indicate that substance P immunoreactivity in the superior colliculus, but not that in the suprachiasmatic nucleus or intergeniculate leaflet, depends on the integrity of the retinal projection.

Effects of hibernation on somatostatin-like immunoreactivity in the brain of the ground squirrel (Spermophilus richardsonii) and European hedgehog (Erinaceus europaeus)

SummaryThe localization of the somatostatin system in the brains of Richardsons ground squirrels (Spermophilus richardsonii) and European hedgehogs (Erinaceus europaeus) was described by use of immunocytochemical methods. In addition, (i) chemically differing types of somatostatin and (ii) different activity phases of the somatostatin system during the hibernation cycle were investigated in the ground squirrel by means of high pressure liquid chromatography (HPLC) and radioimmunoassay (RIA).In both species, the hypothalamic component of the somatostatin system (periventricular nuclei, fiber projections to the median eminence) is more prominent than the widespread extrahypothalamic representation of the system displaying mainly scattered perikarya and nerve fibers. The reactivity pattern of the somatostatin system varied among hibernating, aroused, and non-hibernating animals; moreover, the interspecific differences were pronounced. The activity of the hypothalamic somatostatin system in the hibernating ground squirrel appeared to be suppressed when compared to non-hibernating controls, whereas in the hibernating hedgehog this system showed signs of increased activity in comparison to non-hibernating controls. In contrast, in the present material the extrahypothalamic components of the somatostatin system did not exhibit significant changes in their activity.

Hibernation-related changes in the immunoreactivity of neuropeptide systems in the suprachiasmatic nucleus of the ground squirrel, Spermophilus richardsonii

SummaryThe pattern of distribution and reactivity of the neuropeptides vasopressin (AVP), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), substance P (SP), and thyrotropin-releasing hormone (TRH) were studied in the suprachiasmatic nucleus (NSC) of 20 Richardsons ground squirrels (and 7 European hedgehogs) of both sexes during hibernation and euthermia. The total area of immunostained structures revealed by application of the individual immunocytochemical techniques was measured by means of computer-aided image analysis. In both species, elements of all peptide systems examined were related to particular subdivisions of the NSC. The pattern of immunoreactivity was strongly correlated with the physiological stage of hibernation or euthermia both in ground squirrels and hedgehogs. The immunoreactivities to AVP and SP increased in area during hibernation (AVP: 25%; SP: 25%), whereas the respective area immunoreactive to NPY and VIP decreased (NPY: 45%; VIP: 100%) in comparison to nonhibernating controls. The TRH-immunoreactive nerve fibers were rare and rather scattered; thus, the quantitative procedure was not applicable for this immunoreaction.

The serotonin-immunoreactive system of the suprachiasmatic nucleus in the hibernating ground squirrel, Spermophilus richardsonii *

SummaryIn the suprachiasmatic nucleus (NSC) of hibernating and non-hibernating ground squirrels, the distribution of serotonin-immunoreactive (5HT-IR) fibers was studied by the use of the peroxidase-antiperoxidase technique. The cytology of perikarya giving rise to these suprachiasmatic 5HT-IR fibers was investigated in the anterior raphe nuclei. Differences in the immunoreactivity of suprachiasmatic fibers between hibernating and non-hibernating ground squirrels were determined by digital image analysis. The cellular activity was determined densitometrically after RNA-staining in anterior raphe neurons and suprachiasmatic perikarya. Abundant 5HT-IR fibers were observed in the medial and ventromedial portions of the NSC. Frequently, the fibers were found in close contact with perikarya of suprachiasmatic neurons. The central portion of the nucleus and the surrounding hypothalamic areas contained only a few scattered 5HT-IR fibers. Inside the raphe nuclei, 5HT-IR fibers and perikarya formed a dense network. In hibernating ground squirrels, the immunoreactivity to serotonin was approximately 45% higher than in non-hibernating controls. This difference is in accordance with signs of higher neuronal activity (40% higher RNA-content, 20% larger cell nuclei) in 5HT-IR perikarya of the raphe nucleus and the persisting activity of the NSC during hibernation; the activity of other brain regions dropped conspicuously in torpid animals.

SOMATOSTATIN-IMMUNOREACTIVE CELLS IN THE GASTROINTESTINAL TRACT OF THE FROG RANA ESCULENTA

The morphology and topographic distribution of somatostatin-immunoreactive cells in the stomach and small intestine of the frog Rana esculenta were studied at the light-microscopic level by the use of the peroxidase-antiperoxidase method. Scattered immunostained cells occurred in all regions of the gastrointestinal tract investigated. In the small intestine, the number of these cells decreased gradually in the oral to anal direction, i.e. from the pyloric (antral) stomach to the entrance into the colon. Most of the immunostained cells possessed thick, short cytoplasmic processes, which did not display a preferential spatial orientation. Other somatostatin-immunoreactive cells, which were exclusively located in the small intestine, gave rise to a single long extension oriented toward the lumen. In both stomach and small intestine, a complete penetration of the epithelial surface by these processes of somatostatin-immunoreactive cells was observed only occasionally. The morphological features of the somatostatin-immunostained cells speak in favor of endocrine, paracrine, and possibly also intraluminal secretory functions of the enteroendocrine somatostatin system in frogs.