Combined mesencephalic and hypothalamic transplants reverse lesion-induced sexual behavior deficits in the male rat.

Studies of sexual behavior in rodent animal models have provided evidence about the relevant role played by the medial preoptic area of the anterior hypothalamus and the central tegmental field within the mesencephalon in the control of this behavior. Bilateral lesions of the anterior hypothalamus or central tegmental field as well as combined unilateral lesions of both these regions result in sexual behavior deficits. Studies using fetal hypothalamic transplants have been shown to reverse sexual behavior deficits induced either by lesions or aging. However, no previous study has evaluated the effect of combined homotopic transplants into both the anterior hypothalamus and the mesencephalon. In the present study male Wistar animals received two electrolytic lesions, one aimed at the ipsilateral medial preoptic area of the anterior hypothalamus and the other at the contralateral central tegmental field. Following these lesions, unilateral homotopic fetal hypothalamic and mesencephalic transplants were placed into the lesioned areas. Sexual behavior recovered gradually and by weeks 14-15 after transplantation, above 90% of animals with bilateral transplants showed mounts, intromissions, and ejaculations. Only animals with viable transplants located within both lesioned areas showed recovery. These results indicate that the behavioral deficits induced by combined unilateral lesions of hypothalamic and mesencephalic regions can be reversed by homotopic fetal transplants and that this recovery could be the result of the restoration of a behavioral relevant circuit between transplants and host brain nuclei separated by as much as 5 mm, which makes this an excellent model to study mechanisms underlying behavioral recovery after transplantation.

GAP-43 mediates retinal axon interaction with lateral diencephalon cells during optic tract formation.

GAP-43 is an abundant intracellular growth cone protein that can serve as a PKC substrate and regulate calmodulin availability. In mice with targeted disruption of the GAP-43 gene, retinal ganglion cell (RGC) axons fail to progress normally from the optic chiasm into the optic tracts. The underlying cause is unknown but, in principle, can result from either the disruption of guidance mechanisms that mediate axon exit from the midline chiasm region or defects in growth cone signaling required for entry into the lateral diencephalic wall to form the optic tracts. Results here show that, compared to wild-type RGC axons, GAP-43-deficient axons exhibit reduced growth in the presence of lateral diencephalon cell membranes. Reduced growth is not observed when GAP-43-deficient axons are cultured with optic chiasm, cortical, or dorsal midbrain cells. Lateral diencephalon cell conditioned medium inhibits growth of both wild-type and GAP-43-deficient axons to a similar extent and does not affect GAP-43-deficient axons more so. Removal or transplant replacement of the lateral diencephalon optic tract entry zone in GAP-43-deficient embryo preparations results in robust RGC axon exit from the chiasm. Together these data show that RGC axon exit from the midline region does not require GAP-43 function. Instead, GAP-43 appears to mediate RGC axon interaction with guidance cues in the lateral diencephalic wall, suggesting possible involvement of PKC and calmodulin signaling during optic tract formation.

Effect of hyperbaric oxygen therapy on fetal spinal grafts: an experimental study.

In neural transplantation, lack of oxygen supply to the graft in acute stage is an important problem. This study was undertaken to evaluate the effects of hyperbaric oxygen (HBO2) on fetal spinal grafts. Spinal cord tissues obtained from 16-day fetal rats were transplanted into the spinal cord of adult Wistar rats (n = 30). After transplantation, they were randomly divided into two groups (n = 15 for each group); Group 1 received transplant alone, group 2 received transplant and HBO2 therapy consisting of 100% oxygen at 2.5 atm abs for 90 min twice a day for 7 days. Seven days after their surgery, all animals were killed for histologic examination. Degenerative changes, parenchymal integrity, host spinal cord edema, and vascularization of the graft were scored on a 3-point scale. Scores of these parameters were statistically analyzed. The graft survival rates in groups 1 and 2 were 54.5% and 71.4%, respectively. Animals treated with HBO2 showed statistically less spinal cord edema than the untreated groups (P < 0.05). Parenchymal integrity was also significantly better in this group (P < 0.05). The results indicate that edema reduction effect of HBO2 prevents the displacement of graft from the gap and contributes to the integration between the graft and host.

Ablative surgery and deep brain stimulation for Parkinson's disease.

Surgical options for Parkinson's disease (PD) are rapidly expanding and include ablative procedures, deep brain stimulation, and cell transplantation. The target nuclei for ablative surgery and deep brain stimulation are the motor thalamus, the globus pallidus, and the subthalamic nucleus. Multiple factors have led to the resurgence of interest in the surgical treatment of PD: 1) recognition that long-term medical therapy for PD is often unsatisfactory, with patients eventually suffering from drug-induced dyskinesias, motor fluctuations, and variable responses to medication; 2) greater understanding of the pathophysiology of PD, providing a better scientific rationale for some previously developed procedures and suggesting new targets; and 3) use of improved techniques, such as computed tomography- and magnetic resonance imaging-guided stereotaxy and single-unit microelectrode recording, making surgical intervention in the basal ganglia more precise. We review the present status of ablative surgery and deep brain stimulation for PD, including theoretical aspects, surgical techniques, and clinical results.

Restoration of circadian behavior by anterior hypothalamic grafts containing the suprachiasmatic nucleus: graft/host interconnections.

Destruction of the hypothalamic suprachiasmatic nucleus (SCN) disrupts circadian behavior. Transplanting SCN tissue from fetal donors into SCN-lesioned recipients can restore circadian behavior to the arrhythmic hosts. In the transplantation model employing fetal hamster donors and SCN-lesioned hamsters as hosts, the period of the restored circadian behavior is hamster-typical. However, when fetal rat anterior hypothalamic tissue containing the SCN is implanted into SCN-lesioned rats, the period of the restored circadian rhythm is only rarely typical of that of the intact rat. The use of an anterior hypothalamic heterograft model provides new approaches to donor specificity of restored circadian behavior and with the aid of species-specific markers, provides a means for assessing connectivity between the graft and the host. Using an antibody that stains rat and mouse neuronal tissue but not hamster neurons, it has been demonstrated that rat and mouse anterior hypothalamic heterografts containing the SCN send numerous processes into the host (hamster) neuropil surrounding the graft, consistent with graft efferents reported in other hypothalamic transplantation models in which graft and host tissue can be differentiated (i.e., Brattleboro rat and hypogonadal mouse). Moreover, SCN neurons within anterior hypothalamic grafts send an appropriately restricted set of efferent projections to the host brain which may participate in the functional recovery of circadian locomotor activity.

Region-specific migration of embryonic glia grafted to the neonatal brain.

Cell fate determination and region-specific migration among neurons from the developing brain have been widely studied. Because similar attributes have been mostly unexplored in reference to glia, the present study has characterized the migratory responses of glia from diverse regions of the embryonic mouse brain after their transplantation to the brains of early postnatal (still developing) rats. Through the use of the mouse-specific, glial-specific marker M2, immunocytochemical processing of host tissues three to four weeks after transplantation revealed notable difference in the migratory patterns of phylogenetically diverse populations of glia. While glia from the ventral mesencephalon, cerebral cortex, and cerebellar neuroepithelium all showed a similar affinity for the nigropallidal tract after grafting to the internal capsule, only ventral mesencephalon-derived glia showed restricted migration toward and into the substantia nigra after transplantation to the thalamus or pontine tegmentum. These results suggest the presence of a highly favourable substrate for glial migration along developing fibre tracts, but, more importantly, indicates the potential for certain glia to respond to particular (region-specific) distal cues within the developing brain.

Fetal grafts containing suprachiasmatic nuclei restore the diurnal rhythm of CRH and POMC mRNA in aging rats.

We assessed whether fetal tissue containing the suprachiasmatic nuclei (SCN) can restore age-related changes in the diurnal rhythm of hypothalamic corticotropin-releasing hormone (CRH) and anterior pituitary proopiomelanocortin (POMC) mRNA. Young, middle-aged, and middle-aged SCN-transplanted rats were killed at seven times of day. In young rats, CRH mRNA exhibited a diurnal rhythm in the dorsomedial paraventricular nuclei but not in other subdivisions of the nuclei. No rhythm was detected in aging rats. SCN transplants restored a rhythm in CRH mRNA, but the timing was not precisely the same as in young animals. POMC mRNA exhibited a daily rhythm in young rats. Aging abolished the rhythm and decreased the average mRNA level; fetal transplants restored the rhythm, but the amplitude remained attenuated. These data are the first demonstration that fetal tissue can restore the diurnal rhythm of a neuroendocrine axis that is driven by the SCN. We conclude that the neuroendocrine substrate from the aging host remains capable of responding to diurnal cues to express diurnal rhythmicity in CRH/POMC mRNA when fetal SCN transplants confer the appropriate signals.

Motor function analysis of myelin mutant mice using a rotarod.

We have examined motor control in normal and shiverer mutant mice using the rotarod assay, a forced motor activity which tests for balance and co-ordination. Shiverer mice carry a deletion of the myelin basic protein (MBP) gene, resulting in CNS dysmyelination and characteristic motor dysfunction. Homozygous mutant mice had a significant increase in cumulative falls from the rotarod relative to heterozygous mice. Non-acclimated animals of both genotypes showed progressive improvement in performance when tested on successive days. The rotarod test also discriminated shiverer mutants from animals that received gene therapy intervention. Shiverer animals carrying an MBP transgene showed gene-dosage-dependent improvements in motor function, and mutants which received thalamic transplants of wild type oligodendrocyte precursor cells showed improvement relative to sham operated and non-transplanted controls. Thus the rotarod is a sensitive measure of motor function in hypomyelinated mice, and may be useful for assessing the results of experimental manipulations including transgenic gene therapy and cell transplantation.

Morphological maturation of thalamic neurons as studied in fetal neural transplants.

This study attempts to determine whether fetal thalamic neuroblasts from rat embryos (embryonic age 15 days) labeled with horseradish peroxidase (HRP) can differentiate into their normal dendritic phenotype when transplanted as a cell suspension into a lesioned site in the adult somatosensory thalamus. The HRP labeling provided a Golgi-like staining of numerous neurons up to 12-14 days after transplantation. There were three main results. 1) As early as 2 days after transplantation three morphologic cell types were observed: Two were bipolar and the third multipolar. These cellular profiles are characteristic of adult ventroposterolateral, reticular, and ventroposteromedial neurons and suggest that transplanted neurons can take shape in the absence of specific arrangements of afferent fibers. 2) The initial stage of dendritic growth was characterized by numerous growing specializations and consisted of a rapid, arborizing growth that appeared to proceed at an accelerated rate relative to normal development. During the later stage, which was characterized by the great reduction of growing specializations, dendritic remodeling resulted in a simpler morphology, and the transplanted neurons did not achieve an adult morphology. 3) Putative axons exhibiting growth cones were present in impressive densities in the transplants, and a number of them grew into the neuron-depleted host thalamus. A very small number of axons grew into host gray matter outside the lesioned area, indicating that neurodegenerative areas provide a better substrate for neurite outgrowth than intact tissue. In rare instances axons were visible in the internal capsule, indicating that the biochemical inhibition provided by mature myelin and oligodendrocytes may not be an absolute obstacle to axonal growth.

Transmitter release from transplants of fetal ventral mesencephalon or locus coeruleus in the rat frontal cortex and nucleus accumbens: effects of pharmacological and behaviorally activating stimuli.

The present study was performed in order to establish whether dopamine (DA) release from behaviorally functional intracerebral DA transplants is dependent on changes in neuronal impulse flow, and is under control of the host brain. Rats were subjected to combined intraventricular and ventral tegmental injections of 6-hydroxydopamine (6-OHDA) in order to obtain a severe bilateral lesion of the ascending mesocorticolimbic DA projections. Cell suspension grafts of fetal ventral mesencephalic neurons were thereafter implanted into the medial frontal cortex (MFC) and the nucleus accumbens (NAc). Since the neurotoxin injections removed also the ascending noradrenergic systems, fetal locus coeruleus neurons were added to the graft suspension in one group of animals. Age-matched lesion-only and normal animals served as controls. The lesion-induced alterations in spontaneous, amphetamine- and apomorphine-induced locomotor activity and in a skilled paw reaching task were evaluated before transplantation, and at 3 and 6 months post-grafting. Microdialysis probes were finally implanted in the MFC and NAc in order to monitor extracellular DA and noradrenaline (NA) levels (i) during administration of pharmacological agents which augment or depress catecholamine release in the intact brain; (ii) during exposure of the rats to stressful manipulations (handling and immobilization) or appetitive stimuli (eating) known to enhance cortical and limbic DA or NA release in intact animals. The lesion-induced reduction in amphetamine-induced locomotor activity was reversed in all grafted animals, which also showed a higher than normal spontaneous overnight activity. Daytime spontaneous locomotor activity (which was reduced in the lesion-only rats) as well as apomorphine-induced hyperactivity was reversed by the grafts of DA neurons only. By contrast, the lesion-induced impairment in skilled forelimb use was not alleviated by the grafts. The grafted DA neurons restored normal steady-state DA overflow in the NAc, whereas they enhanced cortical DA overflow to significantly higher than normal levels. Restoration of both cortical and striatal NA overflow was observed in the group that received mixed DA and NA grafts, whereas animals that received DA grafts only did not differ from the lesioned controls. The changes in extracellular DA and NA levels measured in the grafted MFC and NAc under potassium depolarization (100 mM KCl), inhibition of terminal catecholamine reuptake (10 microM nomifensine), and sodium channel blockade (1 microM TTX) indicated that graft-derived DA or NA release had normal neuronal properties, and was dependent on an intact axonal impulse flow.(ABSTRACT TRUNCATED AT 400 WORDS)

Grafting fetal suprachiasmatic nuclei in the hypothalamus of old hamsters restores responsiveness of the circadian clock to a phase shifting stimulus.

In the present study, 18-25-month-old hamsters free-running in constant dim light were injected, both before and after receiving fetal grafts containing either cerebellar tissue or the suprachiasmatic nuclei (SCN), with a dose of triazolam given at a time known to reliably phase shift the rhythm of locomotor activity in young hamsters. SCN-grafted animals, but not control animals implanted with fetal cerebellar tissue, showed a significantly greater response to the phase shifting effects of triazolam, demonstrating that at least some age-related changes in the circadian system can be reversed by neuronal transplantation. These results raise the possibility that neuropharmacological interventions that can simulate the effects of fetal SCN grafts might be useful in the treatment of age-related disorders in circadian function.

Effects of intraventricular locus coeruleus transplants on seizure severity in genetically epilepsy-prone rats following depletion of brain norepinephrine.

Audiogenic seizures (AGS) in genetically epilepsy-prone rats (GEPR) of the moderate-seizure substrain (GEPR-3s) were investigated to determine whether norepinephrine (NE) depletion induced by 6-hydroxydopamine (6-OHDA) microinfusion into the locus coeruleus (LC) could alter the efficacy of intraventricular NE tissue grafts in promoting reductions in seizure severity in AGS. GEPR-3s were stereotaxically infused with 6-OHDA (4 micrograms/side/rat), or vehicle into the region of the LC. Following 6-OHDA treatment all animals were subjected to 3 AGS tests. GEPR-3s seizure severities were increased in 39.5% of the animals after microinfusion of 6-OHDA into the region of the LC. Following the third AGS test, each rat was stereotaxically implanted with 17 gestational day rat fetal tissue obtained from the dorsal pons and containing the primordia of the LC or with tissue obtained from the neocortex or were sham-grafted. Subsequent to grafting, rats were subjected to 3 additional AGS tests. 53% (10/19) of 6-OHDA treated GEPRs showed a significant reduction in seizure severity following transplantation of fetal LC tissue. In contrast, only 20% (1/5) of GEPRs infused with saline rather than 6-OHDA showed a reduction of seizure severity following fetal LC transplantation. NE content in the cortex and pons/medulla was decreased by 78% and 46% respectively following 6-OHDA microinfusion into the LC. Prominent grafts with numerous TH positive neurons and neurites were present within the third ventricle of grafted animals, while cortex grafts contained no TH immunostained structures. These findings suggest that the efficacy of fetal LC tissue to promote reductions in seizure severity in GEPRs is increased following depletion of central NE by microinfusion of 6-OHDA.

Migration pathways, differentiation and survival of macroglial cells from a xenograft implanted into the thalamus of newborn mice.

Embryonic rabbit corpus callosum transplants were grafted into thalamus of newborn shiverer mice in order to compare the fates of oligodendroglial and astroglial cells derived from the transplants. Our model allowed the identification of the two populations of macroglial cells. The thalamus was chosen as site of implantation because of its situation at a crossroad of numerous neuronal fascicles. Previous studies, where the dorsal striatum was used as site of implantation, had shown that corpus callosum was one of the favorite routes of migration for both populations of macroglial cells. In the present study special attention was given to the comparison of the migration pathways and areas of settlement of implanted astroglia and oligodendroglia. The internal capsule, the medial lemniscus, the crus cerebri and the thalamic radiations were used by both populations of transplant derived macroglial cells for their migrations through the host parenchyma. They integrated into the host tissue on these routes or further away in areas such as the putamen, the mesencephalon or the colliculi. Signs of degeneration of the implanted astroglia were often observed after 1 month post-implantation.

Hypothalamic but not cortical grafts induce recovery of sexual behavior and connectivity in medial preoptic area-lesioned rats.

We have previously shown that hypothalamic fetal brain grafts induced recovery of sexual behavior in medial preoptic area (MPOA)-lesioned male rats. In the present series of experiments, male rats with completely abolished sexual behavior by MPOA lesions received either hypothalamic or frontal cortical fetal grafts. The animals that received hypothalamic grafts showed a gradual recovery of sexual behavior. In contrast, those animals who received cortical grafts did not recover sexual behavior during the 15 weeks after the graft. In addition, to evaluate the connectivity of the grafted tissue with the host brain, a retrograde tracer, fluorogold, was injected in the dorsal tegmental area. Fluorogold-labeled cells were found in the hypothalamic, but not in the cortical grafts. These results suggest that specificity of the grafted tissue and connectivity between brain grafts and host tissue are necessary for the recovery of male sexual behavior in MPOA-lesioned rats.

Basal forebrain grafts in the rat neocortex restore in vivo acetylcholine release and respond to behavioural activation.

Acetylcholine release in the frontal cortex of awake rats after acute or chronic lesions of the nucleus basalis magnocellularis and grafting of cholinergic-rich basal forebrain tissue was studied by in vivo microdialysis. Three to four weeks and five months after a unilateral quisqualic acid lesion of the nucleus basalis, and five months after lesion and cortical implantation of a basal forebrain cell suspension, acetylcholine release was characterized during a range of pharmacological and behavioural manipulations. Neostigmine (5 microM) was added to the perfusion fluid in order to inhibit the degradation of acetylcholine. The extracellular levels of acetylcholine in normal animals increased three- to four-fold when KCl (100 mM) was added to the perfusion medium and was reduced by 80% after addition of tetrodotoxin (1 microM). The nucleus basalis lesion resulted in a 60% reduction in baseline acetylcholine levels compared to normal and the response to KCl-evoked depolarization was significantly reduced. There were no differences between the acute and chronic lesion groups during any of the manipulations performed. Rats with grafts showed baseline levels of acetylcholine about 70% higher than normal, and responded to both KCl (two-fold increased acetylcholine release) and tetrodotoxin (85% reduced levels). All groups showed lower acetylcholine levels during halothane anaesthesia (on average 70-85% reduction). Sensory stimulation by handling resulted in a two-fold increase in acetylcholine release in normal animals, whereas the absolute responses in the lesioned controls were significantly weaker. Rats with grafts increased their acetylcholine release after handling to an extent not different to normal or lesioned controls. Immobilization stress induced an almost two-fold increase in cortical acetylcholine levels in normal rats, whereas the effect in the lesion-only groups was very weak. The grafts responded to the immobilization with an enhanced acetylcholine overflow that was significantly higher than in lesioned controls. The results showed that the reduction in frontocortical acetylcholine release induced by excitotoxic lesions of the nucleus basalis did not recover spontaneously over several months. Intracortical cholinergic-rich grafts obtained from the fetal basal forebrain provided a source of acetylcholine release with firing-dependent properties which could be modulated by behaviourally stressful stimuli. The ability of the grafts to respond to behavioural manipulation strongly suggests that the host brain can functionally influence graft neuronal activity during ongoing behaviour. Host control of graft activity may play a role in the recovery of the lesion-induced deficits seen with these types of grafts.

Time course of fiber outgrowth from fetal anterior hypothalamic heterografts.

Fetal anterior hypothalamic (AH) heterografts can restore circadian rhythmicity to animals rendered arrhythmic following ablation of the suprachiasmatic nucleus (SCN). Behavioral restoration of circadian activity typically begins between two and six weeks post-implantation. The time course of fiber outgrowth from fetal AH heterografts was examined to determine whether neuronal outgrowth from the implants precedes the typically observed effects of such implants upon circadian behavior. Fetal mouse or rat AH tissue containing the SCN was implanted into the third ventricle of SCN-lesioned hamsters. Using species-specific monoclonal antibodies generated against mouse or rat neuronal elements, fiber outgrowth into the host hypothalamus was examined at 2, 4, 7, 14, 30 and 45 days after implantation. Fibers were observed to have emerged from the implant at the earliest time point examined. Four days after surgery, individual fibers had extended up to 0.6 mm into the host neuropil. By 14 days post-implantation, outgrowth from the implant had formed a dense fiber plexus in the host hypothalamus. This observation demonstrates that neuronal integration of the implant with the host brain begins within 48 hours of implantation, and is extensively established well before a restoration of rhythmicity is typically observed. Thus, on the basis of the time course of fiber outgrowth, it is clear that neuronal contact between graft and host may mediate the observed restoration of circadian rhythmicity.

2-Deoxyglucose autoradiographic evidence that fetal substantia nigra grafts exert contralateral effects in 6-hydroxydopamine-lesioned animals who recover motor asymmetries.

Fetal substantia nigra grafts decrease turning behavior induced by dopaminergic agonists in rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal system. The purpose of this study was to determine, through the [14C]2-Deoxyglucose autoradiographic method, whether substantia nigra grafts have an effect on efferent striatal nuclei and other related structures. The results showed an unexpected enhancement of metabolic activity in the substantia nigra pars reticulata and striatum on the side contralateral to the lesion and a bilateral enhancement in the thalamus and frontoparietal cortex, in rats bearing fetal substantia nigra grafts. The results are discussed in terms of the possibility that recovery of turning behavior may in part be caused by an increased activity of the striatum contralateral to the grafted side.

Treadmill running test for evaluating locomotor activity after 6-OHDA lesions and dopaminergic cell grafts in the rat.

A treadmill apparatus was used for estimating the recovery of locomotor activity after dopaminergic grafts to rats with unilateral 6-OHDA lesions in the nigrostriatal DAergic pathway. Naive control rats, 6-OHDA lesioned rats, and grafted rats were trained to run on the uphill treadmill for 20 min per day for 7 successive days. Weak electrostimulation (ES), less than 1 mA, was used to force running, i.e., if rats did not run they received ES from the grids fixed behind the treadmill belt. The numbers of ES that the rats received were counted because they reflected the frequency with which the rats failed to meet the treadmill speed. Control rats received 5-10 ES/10 min on the first day but only 0-1 ES after the second day. Lesioned rats and grafted rats received over 100 and 50-100 ES/10 min, respectively, during the first 3 days. Thereafter, lesioned rats still received frequent ES (80-100/10 min), whereas grafted rats received less (20-30/10 min). Results indicate that motor ability in grafted rats may not be ameliorated completely despite the recovery from drug-induced rotations. This treadmill running test was found to be a useful method for quantitative evaluation of motor ability.

Effects of neural transplantation on seizures in the immature genetically epilepsy-prone rat.

To study the hypothesis that neural transplantations can alter seizure susceptibility in a genetic animal model of epilepsy, 93 pubescent genetically epilepsy-prone rats with stage 9 seizures received either bilateral inferior colliculi (N = 21) or lateral ventricle (N = 42) transplants or sham transplants (N = 30). The grafts consisted of embryonic locus ceruleus, neocortical, or cerebellar tissue. Starting 2 days after the transplantation the rats were subjected to audiogenic stimulations every other day for 61 days. Latency to the running and tonic phase, seizure severity score, and duration of the tonic and clonic phase were compared in the neural transplant and sham-operated controls. Rats that received transplants had a longer latency to the tonic phase and a shorter duration of the clonic phase than the controls. At age 110 days the rats had electrodes implanted bilaterally into the angular bundle and were kindled. No difference in kindling rate was found between the rats that received neural grafts and the sham-operated controls. Cerebrospinal fluid concentration of norepinephrine was not altered by the transplants. This study demonstrates that the anticonvulsant effects of neural transplants, using the genetically epilepsy-prone model of epilepsy, are mild.