Takeshi Hashitani

Long-term survival of grafted cells, dopamine synthesis/release, synaptic connections, and functional recovery after transplantation of fetal nigral cells in rats with unilateral 6-OHDA lesions in the nigrostriatal dopamine pathway

In animal models of hemi-Parkinsons disease, survival of grafted nigral cells, their synaptic connections, dopamine (DA) synthesis/release, and recovery from motor disturbances were investigated, and these were compared among 3 groups of animals raised for 3 months, 1 year and 2 years after the transplantation. Fetal nigral DAergic cell suspensions were transplanted in the ipsilateral caudate nucleus of rats with unilateral 6-OHDA lesions in the nigrostriatal DA pathway. Motor disturbances, assessed by methamphetamine-induced rotation, recovered partly in the 2nd week, significantly in the 4th week after the grafting, and remained stable thereafter. Many tyrosine hydroxylase (TH)-positive cells were detected along the grafting tracks. The number of TH-positive cells was similar in the 3 groups of animals. These TH-positive cells made synaptic connections in the host caudate. By in vivo microdialysis measurement, extracellular DA, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) around the grafted sites recovered to 30-100% of those of controls. No significant differences were observed in the concentration of DA, DOPAC and HVA among 3 groups of animals. They also responded to methamphetamine loading though the magnitudes were smaller. Using a TH cDNA probe, TH-positive cells were found to express TH mRNA in in situ hybridization-autoradiographic analysis. Data indicate that grafted fetal DAergic cells survive, synthesize and release DA, make synaptic connections in the host brain and ameliorate motor disturbances for over 2 years. There were no differences in these parameters among the 3 groups of animals, and no untoward side effects were observed even at 2 years after the grafting. Thus it was confirmed that the grafting of neuronal cells into the brain is a promising approach to restore disturbed function.

Pathophysiological process after transient ischemia of the middle cerebral artery in the rat

For the understanding of pathophysiology of the cerebral ischemia, we made a transient intraluminal occlusion of the middle cerebral artery in the rat and investigated the appearance of collapsed dark neurons and the extravasation of serum proteins using argyrophil III method and immunohistochemistry. In the acute stage (minutes to 3 days), dark neurons appeared in the lateral half of the ipsilateral striatum and adjacent cortex which formed the ischemic core of this model. Dark neurons also appeared in the ipsilateral reticular thalamic nucleus, hippocampus, and amygdala. The extravasation of serum proteins, albumin, leucocyte common antigen, immunoglobulin G, complement factor C3, as well as heat shock protein 70, was observed not only in the ischemic but sometimes also in the contralateral hemisphere. Among these, the expression of IgG and C3 was most prominent in the ischemic core. In the chronic stage (1 to 3 months), the ischemic core changed into the porencephaly, and the ventrobasal nucleus of the thalamus got also involved in the necrosis. A strong microgliosis was observed in the substantia nigra pars reticulata. Data suggest, that among many mechanisms that contribute to ischemic neuronal death, the activation of immune response, due to the damage of blood-brain barrier and the extravasation of serum proteins could promote the ischemic cell death in the brain.

Dopamine has inhibitory and accelerating effects on ischemia-induced neuronal cell damage in the rat striatum

Dopaminergic (DAergic) influence on ischemic neuronal cell damage in the dorsolateral striatum was studied. Intact and 6-hydroxydopamine (6-OHDA) lesioned rats, with and without pretreatment by D1 and D2 DA antagonists, were subjected to 20 min forebrain ischemia. Extracellular DA and glutamate (Glu) were measured using microdialysis technique. Histological examination was performed on the dorsolateral striatum and the hippocampal CA1 area 24 h after ischemia. DA increased 400-500 times the control level during ischemia among the groups except the 6-OHDA lesioned group. No significant changes were observed in the concentration of 3,4-dihydroxyphenylacetic acid (DOPAC), but a transient decrease was seen in homovanillic acid (HVA). Due to ischemia, Glu increased up to about 5 times the control level among the groups. Neuronal damage in the dorsolateral striatum was slightly attenuated by 6-OHDA lesion. Treatment by spiperone (D2 antagonist, 7 micrograms/kg IP) alone attenuated the damage strongly. Treatment by SCH23390 (D1 antagonist, 2.5 mg/kg IP) alone or both D1 and D2 antagonists had no effects. Data suggest that excessive Glu and DA are involved in neuronal cell damage. DA might enhance the damage via D2 but inhibit via D1 receptor.

Improvement of passive avoidance task after grafting of fetal striatal cell suspensions in ischemic striatum in the rat

Behavioral recovery and cell survival/growth after grafting of fetal striatal cell suspensions in the ischemic striatum of rats were investigated. Ischemia was induced by one hour intraluminal occlusion of the right middle cerebral artery under halothane anesthesia. During the ischemia rats usually manifested signs of hemiparesis and sometimes rotations. Behavioral function was measured by a passive avoidance task and radial arm maze test at 1-2 weeks and 6-7 weeks after ischemia. The size of the ischemic lesions depended on each animal, but the ischemic animals showed deficits in both passive avoidance task and radial maze test. Two weeks after ischemia, fetal striatal cells, marked with DiI, were transplanted into the ischemic striatum. The transplantation improved the ischemia-induced deficit in the passive avoidance task but not in radial maze test. Although there were variations in the size of the grafts, many DiI-positive cells with dendritic outgrowth were detected under fluorescent microscopy. Immunohistochemical study revealed that many choline acetyltransferase (ChAT) positive cells and GABA-positive cells survived in the grafts. However, striosome-matrix compartments were not evident inside the grafts. Thus, partial recoveries in both cytoarchitectural and behavioral aspects were obtained by striatal cell grafts, suggesting that neural transplantation could be a useful approach in reconstructing ischemic brain function.

Reconstruction of GABAergic transmission and behavior by striatal cell grafts in rats with ischemic infarcts in the middle cerebral artery.

Fetal striatal cell suspensions were grafted stereotaxically into the infarcted striatum of rats, and reconstruction of striatopallidal GABA transmission and behavior were investigated. Occlusion of the middle cerebral artery (MCA) for one hour induced ischemic infarcts mainly in the lateral striatum, as detected by magnetic resonance imaging (MRI) and histology. Ischemic rats had deficits in the performance of a passive avoidance task, both acquisition and retention, but no changes in general circadian actograms. In these animals pallidal GABA, detected by microdialysis, decreased to about half of control levels. There were suggestions of an improvement in passive avoidance performance in the grafted animals. Pallidal GABA concentrations recovered almost to control levels, and were increased by infusions of the GABA uptake blocker nipecotic acid. These data indicate that neural transplantation is a promising approach to improve the deficits in chemical transmission and behavior following ischemic infarcts in rat striatum.

tGS ganglioside induces peculiar morphological features in grafted dopaminergic cells and promotes motor recovery in rats with unilateral lesions in the nigrostriatal dopamine pathway

A cell suspension of substantia nigra from fetal rats was introduced into the ipsilateral caudate nucleus of rats with unilateral lesions in the nigrostriatal dopamine pathway, and effects of bovine total ganglioside (tGS) and monosialoganglioside (GM1) treatment on the morphological features of the transplanted cells and recovery from motor imbalance (rotation induced by methamphetamine) were investigated. Gangliosides (30 mg/kg) were administered intraperitoneally once a day for 2 weeks after transplantation to test animals while control animals received saline alone. tGS animals showed definite motor recovery in the 2nd week (P less than 0.05) while control and GM1 animals exhibited slight recovery only. At 6 weeks after transplantation, motor imbalance disappeared in all 3 groups. Tyrosine hydroxylase (TH) immunocytochemical staining revealed that in the 2nd week TH-positive cells in tGS animals had more primary dendrites and more large neurites (meganeurites) than did controls. TH-positive cells of all 3 groups often had spiny processes at that time. In the 20th week, TH-positive cells became more multigonal and had wider dendritic fields in all groups, and had less meganeurites and spines. Motor recovery of each animal was dependent on the number of TH-positive cells and no significant difference was observed in the number of TH-positive cells among the three groups. tGS treatment for 2 weeks without grafting induced immunohistologically no axonal sprouting in the substantia nigra, medial forebrain bundle, accumbens and caudate nucleus when the chemical lesions were complete. Data suggest that tGS induces hypertrophy but not hyperplasia of the transplanted nigral cells, and increases the morphological plasticity. This might be the basis for promotion of recovery in motor function after transplantation.

Dopamine-denervation enhances the trophic activity in striatum: evaluation by morphological and electrophysiological development in PC12D cells.

To evaluate the possibility that dopamine (DA) denervation enhances the trophic activity in striatum, normal or DA-depleted striatal tissue extract (N- or L-extract, respectively) was obtained, and their trophic effects on PC12D cells were investigated from the viewpoints of differentiation using morphological and electrophysiological analyses. Treatment with N- or L-extract induced neurite outgrowth in a concentration-dependent manner, and induced the enlargement of cell size. These effects were stronger in L-extract than in N-extract. Cation currents were investigated in whole cell patch-clamp mode. Development of cation current started with delayed-rectifier type K+ current (IK) and transient type K+ current (IA), followed by Ca2+ current (ICa) and tetrodotoxin-sensitive Na+ current (INa). INa was expressed more frequently in L-extract treated cells than N-extract treated cells at D7-9. The larger IK amplitude in L-extract treatment at D7-9 seemed to be related to the expression of INa. Development of IA was similar at any stage for both treatments. ICa development started at D3-5 after treatments, and the amplitude and current density were similar in both treatments. ICa was strongly blocked by omega-conotoxin GVIA (3 microM), indicating that N-type channels were mainly expressed after treatments. The data suggests that L-extract has stronger effects to hasten the differentiation of PC12D cells than N-extract by promoting the neurite outgrowth, cell enlargement and expression of voltage-dependent cation channels, especially INa and IK.

Dynamic regulation of striatal dopaminergic grafts during locomotor activity

The present experiment was designed to estimate the neurochemical activity of dopaminergic grafts in hemiparkinsonian model rats during locomotion and to examine the functional importance of dynamic regulation of the grafted neurons in the host brain. Rats were trained to run on a straight treadmill at various speeds (300, 660, 1200, 1800 cm/min), and extracellular dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were measured by in vivo microdialysis during and after running. Grafted rats were divided into two groups depending on their running ability and data were compared with those of normal and lesioned controls. Although the tonic level of extracellular DA in grafted rats recovered to 70% of control, levels of DOPAC and HVA remained 15-20% of controls. A small number of grafted rats showed full recovery in treadmill running tasks. In these animals, the percentage increase in DOPAC and HVA showed similar time courses and magnitudes as those in normal rats. Most grafted rats showed partial recovery in locomotor ability. The percentage increase in DOPAC and HVA in these animals remained at a lower level than that in normal rats, though the tonic levels of DA, DOPAC and HVA were not lower than those of fully recovered rats. Data suggest that grafted DAergic cells in functionally well recovered rats were dynamically regulated in the host brain in an actual behavior and that well-controlled release of DA might be involved in the recovery of complex motor behavior, such as high speed locomotion.

Phenotypic plasticity of grafted catecholaminergic cells in the dopamine-depleted caudate nucleus in the rat

Catecholamine-producing cells were grafted in the caudate nucleus of model rats with hemi-Parkinsons disease--animals with unilateral 6-hydroxydopamine (6-OHDA) lesions in the nigrostriatal dopaminergic pathway. Survival of the grafted cells, dopamine synthesis/release, and recovery from motor imbalance were investigated. Motor imbalance (methamphetamine rotations) recovered in more than 90% of animals by grafting of nigral dopaminergic (DAergic) cells, in 40-60% of animals by grafting of locus coeruleus noradrenergic (LC-NAergic) cells, and in 0-30% of animals by grafting of adrenal medullary cells. Many tyrosine hydroxylase (TH)-positive cells survived in the host caudate after grafting of DAergic cells. A moderate number of TH-positive but dopamine-beta-hydroxylase (DBH)-negative cells survived after grafting of NAergic cells. A few TH-positive neuron-like cells survived after grafting of adrenal medullary cells. In vivo microdialysis revealed that extracellular DA recovered up to 50-80% of that of control level, and dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) up to 25-50% of those of controls in animals that showed motor recovery after grafting. Using a TH cDNA probe, TH-positive cells were found to express TH mRNA in an in-situ hybridization-autoradiographic study. Animals that received 6-OHDA lesion only or animals that received grafting but had no surviving cells showed neither behavioral nor chemical recovery. The data suggest that grafted catecholaminergic cells survive, make phenotypic plasticity, synthesize/release dopamine, and ameliorate motor function.

Descending influences from nucleus raphe magnus on fusimotor neurone activity in rats

Abstract 1. 1.Single fusimotor fibres were isolated in the ventral roots of lumbosacral segments of urethane-anaesthetized rats, and effects of electrical stimulation of the nucleus raphe magnus (NRM) on their spontaneous activity were investigated. The experiments were carried out in rats whose bilateral preoptic and anterior hypothalamic regions (PO/AH) were electrolytically destroyed to eliminate the influences of these regions to fusimotor activity. 2. 2.Of 44 fusimotor fibres studied, 38 (86%) were found to be affected by NRM stimulation. The effects of NRM stimulation were classified according to their response pattern: primary depression (D-type, n = 24), facilitation followed by depression (F-D-type, n = 5) and primary facilitation (F-type, n = 8). The most predominant effect of NRM stimulation upon fusimotor activity was characterized by a strong depression followed by a complete cessation of firing lasting either for a short period or for more than 30 min (D- and F-D-type). 3. 3.In three fusimotor fibres studied in the different preparations, it was observed that a NRM-evoked depression response was blocked by an intraperitoneal administration of a serotonin antagonist, p -chlorophenylalamine ( p -CPA) (10 mg/kg). 4. 4.The results indicate that the NRM exerts descending inhibitory or facilitatory influences on fusimotor neurones, and suggest that cold shivering is controlled by modulating fusimotor neurone activity via the serotonergic raphe-signal pathways.