Facilitated sexual behavior reversed and serotonin restored by raphe nuclei transplanted into denervated hypothalamus.

Fetal raphe cells transplanted into the hypothalamus reversed facilitation of feminine sexual behavior in rats with brain lesions induced by 5,7-dihydroxytryptamine. Immunocytochemical and chemical analyses of serotonin indicate that reinnervation of the ventromedial nucleus of the hypothalamus by the transplants is associated with behavioral recovery. The findings suggest that transplanted fetal tissue can exert functional regulation over an innate, complex, hormone-dependent behavior in adult rats.

Postnatal development of the serotonin innervation of the hippocampus and dentate gyrus following raphe implants.

Serotoninergic (5-HT) neurons derived from the embryonic raphe nuclear area (brainstem, embryonic days (E 16-18) were implanted into the entorhinal cortex of 6-day-old (P6) neonatal rat recipients which had received a fimbria lesion and entorhinal cortex ablation on P3. The hippocampus, dentate gyrus, and the raphe implant area were examined with 5-HT immunohistochemistry 7, 14, 21, 30, and 60 days after implantation. The pattern of 5-HT reinnervation was compared to that of normal and lesioned animals, and to previous studies in which rats received septal or striatal implants. In the hippocampus adjacent to the implant 5-HT-immunoreactive fibers were first observed by 7 days postimplantation and increased in density and in their septotemporal and dorsoventral extent with increasing time postimplantation. Moderately dense fiber networks were diffusely distributed in the hippocampus and dentate gyrus at 30 and 60 days postimplant. Little, if any, indication of lamination was present. Retrogradely labeled neurons (the majority of which contained 5-HT immunoreactivity) were observed in the raphe implant following injections of Fast Blue into the hippocampal formation. A few retrogradely labeled cells did not contain 5-HT, methionine-enkephalin (ME), or substance P (SP) immunoreactivity, although ME- and SP- immunoreactive neurons were observed in the implants. The lamination patterns and the increased density of 5-HT-immunoreactive fibers following a raphe implant into the entorhinal cortex clearly differ from the normal 5-HT pattern and from the patterns of lamination following a striatal or septal implant.

Interactions between grafted serotonin neurons and adult host rat hippocampus.

We have followed the development of physiological and functional properties of serotonin-containing raphe neurons grafted into an adult host hippocampus as a model system for graft-host interactions. These raphe cells have no clear identifying properties on the day of grafting: they develop them while growing in the host. Raphe neurons, recorded 1 month after grafting, possess adult normal physiological properties. These include high input resistance, slow membrane time constant, lack of inward rectification, a transient outward rectification, broad spikes having a Ca2+ component, lack of accommodation, and a large afterhyperpolarization. The graft is first innervated by host fibers and later projects to the host tissue. When stimulated, postsynaptic hyperpolarized responses are recorded in hippocampal neurons. In the freely moving rat, raphe grafts can restore sleep-wakefulness variations in an evoked population response of the hippocampus to afferent stimulation, which is eliminated by depletion of serotonin. These studies illustrate that grafted serotonin neurons develop functional relations with a host brain.

Functional activity of raphe neurons transplanted to the hippocampus and caudate-putamen. An immunohistochemical and neurochemical analysis in adult and aged rats.

Adult (3-month-old) and aged (28-month-old) rats that had been pretreated with 5,7-DHT in both lateral ventricles received grafts of cell suspensions taken from the RR or MR regions taken from the embryonic stages E12-21. These cell suspensions were implanted unilaterally into the rostral part of the hippocampus or the caudate-putamen for immunohistochemical and neurochemical studies. MR and RR cell suspensions have the potency to regenerate serotonergic fibers in the previously denervated adult and aged hippocampus and caudate-putamen. The RR cell suspension, however, also showed outgrowth of other transmitter-specific neuronal systems, specifically noradrenaline and substance P. To evaluate the functional activity of the serotonergic reinnervation, we have combined immunohistochemistry and neurotransmitter release studies on adjacent hippocampus slices of adult rats. Results showed that after a survival time of 10 weeks, the serotonergic innervation of the hippocampus was greatly restored and, moreover, that the K+-induced Ca2+-dependent release of 5-HT amounted to about 80% of normal values. There appeared to be a striking similarity between the immunohistochemical and neurochemical data regarding the increase in the number of newly formed serotonergic fibers, the increase of the release of radiolabeled 5-HT, and the extent of the outgrowth in the hippocampus.

Stimulation of serotonergic neuronal maturation after fetal mesencephalic raphe transplantation into the 5,7-DHT-lesioned hippocampus of the adult rat.

Neurotoxin lesioning of 5-HT fibers selectively induced the homotypic collateral sprouting of spared 5-HT fibers in the hippocampus. We have used this model to investigate the possibility that the neurotoxin-primed hippocampus will enhance the development of transplanted fetal serotonergic neurons in the brain. The neurotoxin 5,7-DHT, when microinjected into the FF, produced a specific and partial depletion of 5-HT in the hippocampus of adult rats. The ability of the 5,7-DHT-primed hippocampus to selectively support the neurochemical maturation of fetal serotonergic cells was tested by assaying the transplanted fetal raphe or LC 1 month after neuronal transplantation. The neurochemical maturation of fetal 5-HT and NE neurons was dramatically different when they were transplanted in the 5,7-DHT-FF-lesioned hippocampus as compared to the normal hippocampus. The transplanted 5-HT neurons had 480% more SHAU of [3H]5-HT and had a 250% greater content of 5-HT in the partially denervated hippocampus than in the normal hippocampus after 1 month. Furthermore, extracts obtained from lesioned hippocampus enhanced the 5-HT content of 5-HT neurons transplanted in the normal hippocampus, to a level similar to that seen in neurons transplanted in the lesioned hippocampus. In contrast, the implanted NE neurons of fetal LC had a lower NE level in the 5-HT partially denervated hippocampus than in normal hippocampus after 1 month in the host site. The growth of the NE transplants was not facilitated by the vacant postsynaptic space produced by the 5,7-DHT lesion. These results suggest that the 5-HT denervation triggered a trophic signal selectively enhancing the development of the 5-HT neurons but not the NE neurons. Our results are consistent with previous studies showing homotypic collateral sprouting in 5,7-DHT-primed hippocampus.

Survival of adult 5-HT neurons following intraocular transplantation.

The present study was undertaken to provide an improved environment in which to examine the capacity of more mature CNS tissue to survive following transplantation. Tissue containing serotonin (5-HT) neurons from nucleus raphe dorsalis of 4- and 8-week-old rats was transplanted to the anterior chamber of the eye. Baseline conditions were improved by minimizing the time of the grafting procedure and enhancing the nutrition and oxygenation of the grafting medium. Additional treatment of the grafts during the 4 weeks of in oculo development included either: (1) intermittent hyperbaric oxygen (HBO), (2) continuous hyperoxia, or (3) control. In vivo measurement revealed that all grafts decreased significantly in size, a majority of which still demonstrated a small degree of vascularization. Microscopically, a significant percentage of the grafts demonstrated 5-HT-immunoreactive (5-HT-ir) fiber outgrowth into the host irides, although 5-HT-ir cell bodies could not always be discerned. In terms of percentage of grafts with surviving 5-HT-ir fibers, the best results were seen with the grafts treated with continuous hyperoxia (3 out of 4), as compared to HBO-treated grafts (4/18) and the control group (3/24). For both the HBO-treated and control groups, slightly better results were seen with 4-week-old vs 8-week-old donor tissue. The density and the surface area covered by the 5-HT-ir fibers was not correlated with either treatment or donor age. Thus, while continuous hyperoxia or HBO treatment may have a positive effect, the enhanced baseline conditions appear to provide an environment in which to demonstrate that 5-HT neurons from 4- and 8-week-old rats possess the capacity to survive transplantation.

Fetal raphe neurons grafted into the hippocampus develop normal adult physiological properties.

Embryonic midbrain raphe was grafted into serotonin-deficient adult rat hippocampus. Serotonin-containing neurons in the graft survive for at least 6 months after grafting. Grafted neurons develop physiological properties, not present on the day of grafting, identical to those of normal adult serotonin-containing neurons. These include (a) high input resistance and slow membrane time constant, (b) lack of inward rectification in response to hyperpolarizing current pulses and (c) a potent, 4-aminopyridine-sensitive transient outward rectification. The grafted neurons innervate the host tissue with axons that have a slow conduction velocity and refractoriness. It is suggested that grafted CNS neurons may possess normal physiological properties.

Reinnervation of serotonin fibers in the denervated rat subcommissural organ by fetal raphe transplants. An immunohistochemical study.

The ability of axonal outgrowth of serotonin neurons in the implanted brain tissue of subcommissural organ (SCO) was immunohistochemically studied. The serotonin neuron system of the experimental rats was completely destroyed by the intraventricular injection of 5,6-dihydroxytryptamine. The raphe region of normal fetal rats was implanted into the caudal part of the third ventricle of the neurotoxic drug pretreated rats. The host brain was examined 3 months after transplantation. The numerous serotonin fibers were distributed in the SCO and periventricular region of the third ventricle of the host brain. The outgrowing serotonin fibers from the raphe transplant seemed to innervate the SCO with the target specificity.

Transplanted raphe and hippocampal fetal neurons do not displace afferent inputs to the dorsal hippocampus from serotonergic neurons in the median raphe nucleus of the rat.

To determine if fetal transplants can substitute for or suppress intrinsic serotonergic (5-HT) innervation, we studied the relationship between transplanted and the endogenous raphe neurons projecting to the hippocampus. Fetal raphe transplants produced a 5-HT hyperinnervation of dorsal hippocampus in adult rats. Yet, transplants of fetal raphe tissue did not affect the number of median raphe nucleus (MRN) neurons, approximately 300, which retrogradely transported HRP from the hippocampus. This provides evidence that transplanted 5-HT neurons can co-exist with intrinsic 5-HT nerve terminals in the target area for at least one month. In the second part of this study, fetal hippocampal tissue was transplanted into the host hippocampus. Intrinsic 5-HT immunoreactive fibers innervated the transplanted fetal tissue. Nevertheless, the number of MRN neurons innervating the host tissue as revealed by HRP retrograde transport remained unchanged. Changes in the innervation pattern and 5-HT level in the dorsal hippocampus occur following transplantation of fetal tissue. These changes are discussed and suggest that both the target tissue and afferent neurons readjust to accommodate extrinsic transplanted tissue.

Immunocytochemical localization of serotonin in the rat dentate gyrus following raphe transplants.

The ultrastructural features of the serotoninergic innervation of the rat dentate gyrus in normal adults and in animals receiving raphe nuclear area transplants was investigated using an antibody to serotonin (5-HT). Neonatal rats received a lesion of the fimbria-fornix and entorhinal cortex. Three days later, a portion of embryonic (E-16-18) raphe nuclear area was transplanted to the entorhinal cavity and the animals were allowed to survive for 60 days. Animals were processed for the immunocytochemical localization of 5-HT using the peroxidase-antiperoxidase method. Light microscopic observation showed that 5-HT-containing fibers from transplanted raphe neurons densely innervated the hilar and molecular zones of the dentate gyrus. Electron microscopic analysis showed that 5-HT immunoreactivity was contained only in axons and axon varicosities. There were no differences in the ultrastructural characteristics of axons and axon terminals between normal animals and those which had received raphe transplants. A mixture of both conventional synaptic junctions and non-synaptic axonal swellings were found in both groups.

Serotonin neurons grafted to the adult rat hippocampus. I. Time course of growth as studied by immunohistochemistry and biochemistry.

The maturation and growth of fetal serotonergic raphe neurons have been studied immunohistochemically and biochemically between 1 week and 5 months after grafting to the hippocampal formation in 5,7-dihydroxytryptamine-pretreated adult rats. The average number of surviving neurons in each group was 1800, which is equivalent to approximately 20% of the potential number of serotonin neurons contained in the grafted cell suspension. The fetal raphe cells, which were taken from 12-14-day-old embryos, had developed strong serotonin immunoreactivity at 1 week after transplantation, and the number of serotonin cells present at 1 week was similar to that found at later time points. Fiber outgrowth was demonstrable already at 1 week but the serotonin-positive fibers were restricted to the areas close to the graft. Single fibers, however, could be traced for distances of up to 500-800 microns into the host hippocampus and dentate gyrus. At later time points, the graft-derived serotonin-immunoreactive fiber network extended to cover the entire hippocampal formation. At the longest postoperative time point, 7 weeks and 5 months, some of the animals exhibited extensive hyperinnervation patterns throughout the dorsal parts of the hippocampus and the dentate gyrus. Consistent with these immunohistochemical observations, supranormal serotonin levels developed with time after transplantation in the grafted hippocampi from an average of 5% of normal at 1 week, to 28% of normal at 3 weeks, 146% of normal at 7 weeks, and 216% of normal at 5 months. Although the recovery of 5-hydroxyindoleacetic acid (5-HIAA) paralleled that of serotonin (5-HT), the increase in the metabolite concentrations was less than that of the amine, indicating a change in the turnover or metabolism of serotonin in the grafted neurons over time. Thus, the 5-HIAA:5-HT ratio was higher than normal at 3 weeks post-grafting (when the host hippocampus was only partially reinnervated); it was similar to normal at 7 weeks, and it tended to be lower than normal in the hyperinnervated specimens at 5 months' survival. A regression analysis revealed a significant inverse correlation between the hippocampal 5-HT concentration and the 5-HIAA:5-HT ratio in the graft-reinnervated hippocampal formation. In conclusion, the grafted serotonergic raphe neurons, in contrast to other types of aminergic neurons, exhibited a prominent tendency to form extensive hyperiinnervation patterns in the previously dennervated host target.(ABSTRACT TRUNCATED AT 400 WORDS)

Raphe cells grafted into the hippocampus can ameliorate spatial memory deficits in rats with combined serotonergic/cholinergic deficiencies.

The ability of embryonic raphe cells grafted into the hippocampus to restore spatial learning ability was tested in rats with combined serotonergic/cholinergic deficits. Embryonic raphe cells (E14) were transplanted into the hippocampus of serotonin-depleted rats. Two to 3 months after transplantation, control, lesioned and grafted rats were tested in a spatial memory task (a water maze) with and without the addition of atropine. All 3 groups could negotiate the water maze equally well, in non-drug conditions. The injection of atropine caused a severe disruption of performance only in the serotonin depleted rats. The presence of an active serotonergic graft was examined in the intact rat hippocampus using the serotonin releasing drug fenfluramine (FFA). A pronounced depression of hippocampal EEG was observed in control and grafted but not in lesioned rats 15 min after the injection of FFA. These results suggest the involvement of serotonin in cognitive functions in the rat. Furthermore, it is suggested that an interaction between serotonergic and cholinergic neurotransmission occurs in the hippocampus.

In vivo voltammetric measurement of extracellular 5-hydroxyindoleacetic acid in the denervated striatum after transplantation of mesencephalic raphe neurons.

In vivo differential pulse voltammetry with carbon fiber electrodes was used to monitor extracellular 5-hydroxyindoleacetic acid (5-HIAA) levels in a serotonergic denervated target area, the striatum, before and after transplantation of mesencephalic raphé nuclei into the lateral ventricle in the rat. The amplitude of the electrochemical signal recorded in the striatum on the transplanted side was found to be comparable to that measured in control striatum whereas the signal measured from the contralateral nontransplanted striatum was negligible. These findings suggest that mesencephalic raphé nuclei transplants establish connections with the host tissue.

Adult age destruction versus neonatal age destruction of the serotonin system: two models to investigate the survival of serotonin neurons transplanted in adult rats.

Mesencephalic raphe nuclei were transplanted via the cisterna magna in the IVth ventricle of adult rats having received either a neonatal or an adult age destruction of the serotonin system. In neonatally treated rats both serotonin (5-HT) and glial fibrillary acidic protein (GFA) immunocytochemistry showed an absence of survival of 5-HT neurons in the transplants. However, many 5-HT immunoreactive cell bodies and processes were detected in 10-month-old transplants in adult treated animals. These results suggest that recognition processes between the target tissue and the implant may be a prerequisite for the survival of grafted neurons.

The viability of transplants of mesencephalic raphe nuclei in the IVth ventricle of the adult rat.

Mesencephalic raphe nuclei were transplanted via the cisterna magna near the locus coeruleus. The transplants were observed at different time intervals ranging from 24 h to 4 months. Microscopic observations revealed that from 3 weeks to 4 months the transplant developed with a well-defined neural organization. Radioautographic results showed that some of the neurons in the implant at 3 weeks captured and retained [3H] serotonin. These results indicate that the intact IVth ventricle seems to be a likely culturing floor for mesencephalic raphe nuclei.

Serotonin turnover in raphe neurons transplanted into rat hippocampus.

Injections of the neurotoxin, 5,7-dihydroxytryptamine, into rostral raphe nuclei in rats reduced serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in hippocampus to 35-50% of normal levels but only reduced 5-HT synthesis and catabolism by 20-35%. The small reduction in 5-HT metabolism as compared to depletion of 5-HT suggests that 5-HT turnover was increased in nerve terminals that survived the neurotoxin lesion. Transplantation of rat fetal raphe cells into the 5-HT-denervated hippocampus restored 5-HT and 5-HIAA levels and 5-HT synthesis to 125-150% of normal. This demonstrates that transplantation of fetal raphe tissue can restore metabolism to normal levels in the 5-HT denervated hippocampus.

Formation of a glial scar following microinjection of fetal neurons into the hippocampus or midbrain of the adult rat: an immunocytochemical study.

Raphe tissue (minced or dissociated) from fetal rat was microinjected into adult hippocampus or midbrain, and the response of astrocytes observed by immunocytochemical staining for the astrocyte specific marker, glial fibrillary acidic protein (GFA). Astrocytes were observed to form a border around the transplanted cells. This border was visible as early as 7 days post-transplant and was still present up to 6 months later. The transplanted serotonergic cells, identified by immunocytochemical staining with a primary antibody to serotonin, appeared at the edge of the border but not beyond. This border may be responsible for preventing the complete integration of the transplanted cells with the adult host hippocampal and midbrain tissue.

Fetal serotoninergic transplants in the fourth ventricle of adult rodents reverse sleep disturbances induced by neonatal administration of 5,7-dihydroxytryptamine.

Rats received a neonatal (at 4 days of age) intracisternal injection of 5,7-dihydroxytryptamine which eliminates serotonin (5-HT) throughout adulthood. Sleep recordings, performed on these rats at adult age, demonstrated significant decreases in paradoxical sleep. Dissociated fetal 5-HT cell suspensions transplanted in the IVth ventricle of these rats restored paradoxical sleep. However, fetal neuronal noradrenergic or cholinergic transplants did not restore paradoxical sleep. These results suggest that paradoxical sleep is directly or indirectly mediated through serotoninergic mechanisms.

Intraspinal transplants of serotonergic neurons in the adult rat.

Adult male Sprague-Dawley rats were made paraplegic by a complete transection of the spinal cord at lower thoracic level. One week later they were transplanted, below the level of the section, with a cell suspension prepared from the raphe region of 14-day embryos. After survival periods of 10 days to 1 year, the animals were sacrificed and the spinal cord processed for the immunocytochemical detection of 5-HT. Axons from grafted cells grew extensively into the grey matter of the host, and established axosomatic and axodendritic synapses in the anterior horn and intermediolateral column, similar to those of the intact animal. In addition, a group of transplanted animals was tested for sexual reflexes which are under the control of serotonin. It was found that ejaculation reflexes, which are absent in paraplegic rats, are restored in transplanted animals.

Raphe dorsalis-spinal cord cografts in oculo: electrophysiological evidence for an excitatory serotonergic innervation of transplanted spinal neurons.

Intraocular replicas of descending serotonergic bulbospinal pathways were constructed by means of sequential intraocular grafting of nucleus raphe dorsalis and spinal cord. Using extracellular recordings we have studied the functional connections between such double grafts. Superfusion of single spinal cord grafts with serotonin causes an increase in spontaneous activity. This excitation is reversibly blocked by the specific 5-hydroxytryptamine (5-HT) antagonist metergoline. Stimulation of the raphe part of nucleus raphe dorsalis-spinal cord double grafts causes a long-lasting excitation of the spinal neurons similar to that seen in single spinal cord grafts given serotonin. The electrically induced excitation could also be reversibly blocked with metergoline. It is concluded that serotonin-containing nerves from grafts of nucleus raphe dorsalis are not only morphologically organotypic, but also form functional contacts with neurons in cografted spinal cord. The results further support an excitatory or modulatory role of the descending spinal serotonergic pathways and demonstrate that functional contacts can be established between isolated CNS grafts when 5-HT fibers invade immature or mature spinal cord tissue.