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To investigate dopamine (DA) levels as well as DA metabolism by which the striatal DAergic grafts may bring the functional recovery to hemiparkinsonian model rats, a microdialysis study was performed in the striatum, and an autoradiographic analysis for DA transporter was made. In hemiparkinsonian model rats, the concentrations of DA, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in striatal perfusates, decreased considerably (less than 5%, of control levels). In grafted rats that showed motor recovery, the concentration of DA recovered to almost control level, and DOPAC and HVA to about 20% of controls' suggesting that the rate of DA metabolism is low. L-DOPA loading to grafted rats induced a big release of DOPAC and HVA, thus the DOPAC/DA ratio was close to that of the controls'. Methamphetamine loading increased the concentration of DA but did not change the level of DOPAC and HVA. Haloperidol loading increased DA, DOPAC and HVA. [3H]mazindol binding that reflects the activity of the DA transporter decreased considerably in hemiparkinsonian model rats, but it reappeared more or less in grafted rats. Data indicated that in grafted striatum, the extracellular DA level is almost normal level while the rate of DA metabolism is low. By L-DOPA loading, the grafts show the capacity to synthesize, release and metabolize DA and then the DOPAC/DA ratio is normalized. Responses to methamphetamine and haloperidol, as well as the results of the autoradiographic study suggest that the grafts are under a good feedback regulation of DA metabolism.

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.

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 bloodbrain barrier and the extravasation of serum proteins could promote the ischemic cell death in the brain.

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.

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.

Circadian rhythms of running wheel activity under the ambient temperatures of 25, 16, and 30°C were examined in spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). Temperature coefficie

The effects of 6-hydroxydopamine lesions of the ventral tegmental area and following intra-amygdala or intra-accumbens dopaminergic (DAergic) grafts on methamphetamine (MAP)-induced locomotor activity were investigated in rats. Intra-accumbens DAergic grafts from rat embryos restored the locomotor hyperactivity response to MAP 5 weeks after grafting, while intra-amygdala grafts did not restore responses by 10 weeks after grafting. Biochemical measurements of extracellular DAergic activity in the amygdala (AMY) by in vivo microdialysis after grafting showed no significant change in the basal levels of dopamine (DA) and partial restoration of metabolite levels. MAP induced an increase of DA efflux and a decrease in dihydroxyphenylacetic acid without a significant change in homovanillic acid, which is the same pattern of response seen in control animals. These biochemical changes are similar to those seen previously after intra-accumbens grafts. The results show that restoration of DAergic activity in the AMY in the presence of DAergic denervation of accumbens does not have an effect on MAP-induced locomotion.

1. Catecholaminergic cells were grafted in the caudate nucleus of hemi-Parkinsonian model rats, and the following problems were discussed. 2. Phenotypic plasticity and development of catecholamine-producing cells. 3. Mechanism of motor recovery. 4. Trophic factor. 5. Regulation of immunological responses. 6. Long-term survival and security.

In animal models of hemi-Parkinson's 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 event 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.

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.

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.

6-Hydroxydopamine (6-OHDA) lesions in the ventral tegmental area (VTA) in rats prevented the hyperactivity response to methamphetamine in an open field. Transplantation of mesencephalic dopaminergic cells, obtained from rat embryos, into the nucleus accumbens (NAC) of 6-OHDA-lesioned animals restored the hyperactivity 4 weeks after grafting. By microdialysis of the NAC in freely moving rats no significant differences in baseline concentration of dopamine (DA) among the 3 groups (control, lesioned, grafted) were observed. However, after methamphetamine administration, DA increased significantly during the first 80 min in control animals, during the first 40 min in grafted animals, but did not increase in lesioned animals. On the other hand baseline concentrations of dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) decreased to one sixth to one third of those of controls in 6-OHDA-lesioned animals, and they did not respond to methamphetamine. After grafting, however, DOPAC and HVA restored to control levels and responded to methamphetamine with decreases as was observed in control animals. Data suggest that grafts not only restore the ability to release DA but also improve DA metabolism in the NAC. This might be a reason for recovery of locomotor activity.

The circadian rhythms of running-wheel activity and body temperature in a strain of spontaneously hypertensive rats (Aoki) and a strain of normotensive rats (Wistar Kyoto) were analysed. When the rats

Extracts from dopamine (DA)-depleted striatal tissue (lesion extract) and from intact striatal tissue (intact extract) were prepared, and trophic activities in these extracts were evaluated using survival and neurite extension of DAergic neurons as indices. Levels of brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), glial cell-line derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) in extracts were measured using enzyme-linked immunosorbent assay (ELISA). The lesion extract exhibited a stronger trophic activity on survival and neurite extension of DAergic neurons than intact extract. In lesion extract, bFGF was slightly and GDNF was significantly increased, while BDNF and NT-3 were the same level in each extract. The peak increase of bFGF and GDNF was during 2 to 3 weeks after DA depletion. Trophic activity of extract was strongly attenuated after immunoprecipitation of GDNF and partly attenuated after immunoprecipitation of bFGF. In parallel immunohistological study, no significant variations were found for striatal microtubule-associated protein-2 (MAP-2)- nor OX-41-immunoreactive cells, while the number of strongly labeled glial fibrillary acidic protein (GFAP)-immunoreactive cells were increased in DA-depleted striatum, suggesting reactive gliosis. Data suggest that bFGF is a minor, while GDNF is a major component of trophic activity for DAergic neurons in DA-depleted striatum, and increased bFGF and GDNF levels may be mediated partly by reactive gliosis.

Trophic actions of alpha-sialyl cholesterol (SC) and its sialidase-tolerant derivative, alpha-(3 beta-hydroxysialyl) cholesterol (SCt), were carried out on the development of midbrain neurons both in vitro and in vivo transplantation studies. Low to moderate concentrations of SC (0.01 to 0.05 micrograms/ml) facilitated neurite extension but had no effects on cell survival of primary cultured midbrain neurons. However, high concentration of SC (0.1 micrograms/ml) disturbed both neurite genesis and cell survival. SCt had a similar effect on midbrain neurons. At higher concentrations, SC and SCt induced concentration-dependent morphological changes in astrocytes from flat to fibrous. The effect on astrocytes was stronger in SCt than SC. At highest concentration tested (20 micrograms/ml), the proliferation of astrocytes was completely blocked, cells became detached and finally died. This effect of SC and SCt was partially blocked by simultaneous application of aFGF. Following dopaminergic cell grafting in vivo, SC and SCt had biphasic effects: a low dose (0.2 mg/kg, SC) enhanced motor recovery at 4 and 6 weeks after transplantation, while the highest dose (20 mg/kg, SC) disturbed motor recovery at all periods tested. These effects on motor recovery were paralleled by an effect on neurite genesis as studied by tyrosine hydroxylase immunostaining. Thus, at low concentrations, SC and SCt are neurotrophic agents that stimulate the development and differentiation of dopaminergic neurons.

Publisher Summary This chapter examines the phenotypic plasticity of locus coeruleus (LC) noradrenergic neurons after transplantation into the dopamine-depleted caudate in the rat. The locus coeruleus noradrenergic (NAergic) cells in the caudate nucleus of rats is grafted with unilateral lesions in the nigrostriatal dopamine (DA) pathway and is investigated for cell survival, transmitter phenotypic plasticity, DA levels, and motor recovery. The chapter examines the effect of grafting NAergic cells in these lesioned animals. If these cells are effective in reversing motor deficits, they would provide an alternative and more abundant source of donor tissue, because NAergic cells are found in the peripheral nervous system, such as sympathetic ganglia, and are rather easy to obtain for autografting in Parkinsonian patients. The grafting of LC NAergic cells into the DA-depleted caudate in the rat is quite effective in improving motor disturbances if grafts are performed soon after DA-depletion. LC NAergic cells are obtained from late stage embryos, thus they had already migrated to the proper site in the dorsal midbrain and sent NAergic projections into the rostra1 brain. The plasticity of phenotypic expression of LC NAergic cells indicates a mutual interrelationship between grafted embryonic cells and the microenvironment of the hosts. It also indicates that LC NAergic neurons in late stage embryos still retain some plasticity in phenotypic expression. Thus, young NAergic cells could be one further source of tissue for grafting to ameliorate Parkinsonian motor deficiencies.

Catecholamine-producing cells were grafted in the caudate nucleus of model rats with hemi-Parkinson's 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-β-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.

Grafting of fetal nigral dopaminergic (DAergic) cells, in DA depleted caudate has been reported to produce the recovery of motor disturbances in animal models of hemi-parkinson’s disease1. In the clinical field, graftings of adrenal medullary tissue into patients with Parkinson’s disease have been performed in about two hundred cases2. However, the results so far are not always satisfactory. It because the basic problems remain to be solved. In the present study, to make clear how long the grafted cells survive, function, and ameliorate motor performance, we grafted nigral DAergic cells in model rats of hemi-parkinson’s disease and investigated the survival of the grafted cells, DA synthesis/release, receptor activity and motor recovery for more than 2 years. Some results have been reported elsewhere3.

An impairment of dopaminergic (DAergic) transmission in the mesocorticolimbic system results in deficiencies such as hypoexploration, loss of amphetamine-induced hyperactivity, supersensitive locomotor responses to apomorphine, and other sensorimotor deficiencies. DAergic cell transplantation into the nucleus accumbens (NAC) is reported1,2 to alleviate these deficiencies. In the present study, methamphetamine-induced locomotor activity in an open-field was investigated in order to determine the effects of 6-hydroxydopamine (6-OHDA) lesions in the ventral tegmental area (VTA) and intra-accumbens DAergic grafts. Using an in vivo microdialysis-HPLC (high-performance liquid chromatography) detection system, we aimed to clarify the extent to which a deficiency in locomotor activity and its subsequent recovery are related to dopamine (DA) metabolism in the NAC and the degree to which DA is released. Our present study shows that DAergic grafting restores metabolism and release of DA in the NAC.

1. 1.|Changes in fusimotor activity were observed during local cooling or warming of the midbrain reticular formation (MRF) of urethan-anaesthetized rats, whose bilateral preoptic and anterior hypothalamus were electrolytically lesioned. 2. 2.|At the normal MRF temperature, 23 (36%) of the 64 fusimotor fibres isolated were found to exhibit periodic fluctuations in spontaneous discharge whereas the nest sustained spontaneous discharge. 3. 3.|Of 38 fusimotor fibres examined, 27(71%) were affected by changes in local temperature of the MRF. In most (93%) of these fibres, the discharge frequency was increased by cooling of the MRF below normal body temperature (37.5°C) and decreased by heating of the MRF above this temperature. In the temperature reactive fusimotor fibres, temperature dependencies of discharge activity were obtained; −1.5 ± 0.1 imp./s °C ( 37.5°C). 4. 4.|Results indicate that the fusimotor activity is enhanced in the hypothermic range and suppressed in the hyperthermic range, and suggest that the descending signals derived from the thermo-sensitive structures in the MRF control shivering threshold at the spinal level.

1. The relationship between thermal salivation (TS) and thermoregulation was studied in anesthetized rats. 2. Of the 6 anesthetics used, ketamine-anesthetized rats secreted the largest amount of saliva. Salivation, however, was thermal and not induced by ketamine itself. 3. Ketamine-anesthetized rats readily secreted saliva at core temperatures less than 40 degrees C but TS was remarkably enhanced by hyperthermia of 40-42.5 degrees C. 4. The equilibrium phase in the triphasic heat response of core temperature was a consequence of equilibrium between heat gain and heat loss by salivation.

In a male patient suffering from congenital ectodermal dysplasia with sweating only on the plantar and palmar surface, we found that the anhidrotic eccrine sweat glands became able to produce sweat after repeated local application of acetylcholine. We ultrastructurally examined the sweat glands in skin biopsies before and after acetylcholine treatment. The secretory portion of the sweat glands of the right plantar region was characterized by well-developed basal infoldings and intercellular interdigitations, and also by dilated intercellular spaces and canaliculi. In the flexor region of the right brachium (anhidrotic area), the secretory portion showed hypoplastic features, especially denoted by the presence of few locally distributed basal infoldings and intercellular canaliculi. The secretory cells seemed to be hypofunctional with the nuclei containing much heterochromatin. After treatment, in the flexor region of the left brachium and the right infraclavicular region, where sweating was induced, the secretory portion appeared to be considerably activated, though hypoplastic structures were unchanged: the secretory cell nuclei contained dispersed chromatin, the Golgi apparatus in the dark cells was well developed, and both in the clear cells and the myoepithelial cells micropinocytotic vesicle-like structures increased in number. In ductular cells, mitochondria also increased in number.