Gomathy Gopinath

Role of medial preoptic area beta adrenoceptors in the regulation of sleep-wakefulness

The role of the medial preoptic area (mPOA) beta adrenergic receptors in the regulation of sleep-wakefulness (S-W) was investigated in this study. S-W was assessed on the basis of polygraphic recording of EEG, EMG and EOG in free moving rats. Intracerebral microinjection of beta agonist, isoproterenol, into the mPOA produced arousal. The study was also conducted on another set of rats in which noradrenergic (NE) innervation to the mPOA was destroyed by injecting 6-hydroxydopamine into the ventral noradrenergic bundle, in the brain stem. Local application of isoproterenol, into the mPOA, in these animals, did not produce any significant change in S-W. Thus, the increase in awake period obtained on isoproterenol administration was the result of its action on the presynaptic NE terminals. Possible involvement of other responses in the isoproterenol induced increase in wakefulness, is discussed.

Developing substantia nigra in human: a qualitative study

Midbrains from 43 fresh human embryos and fetuses at 8-22 weeks of gestation were processed for routine histology, Golgi staining, tyrosine hydroxylase (TH) immunolabelling and retrograde tracing with the fluorescent dye DiI. Cells were immature and densely packed between 8 and 10 weeks. By 13 weeks cells could be identified as neurons and glia. Neurons matured gradually and achieved adult characteristics by 20-22 weeks. Neurons in the paramedian regions of the tegmentum, raphae region and substantia nigra were positive for TH from 13 weeks onwards, the earliest age group used for this technique. The presence of TH-positive neurons in the paramedian part of the tegmentum until 18 weeks and radial glial fibers extending from the aqueductal lining to the ventral part until 20 weeks were suggestive of migration of neurons to the ventral mesencephalon region. DiI labelling of the neurons and fibers of ipsilateral nigra from the caudate as early as 10 weeks demonstrated early nigrostriatal connections. The mature nature of the neurons appeared only by 13 weeks by this method. The present study shows that the nigral neurons in the human migrate and mature until mid-gestation. The nigrostriatal connection at 10 weeks suggests a trophic influence of nigra on the proliferating and maturing neurons of the striatum.

Ultrastructural observations in experimental hydrocephalus in the rabbit

Changes in the ependyma and periventricular brain tissues of the lateral, 3rd and 4th ventricles and the cervical spinal canal were studied electron-microscopically in young rabbits on the 9th day after injecting kaolin into the cisterna magna. The ependyma of the lateral ventricle overlying the white matter was notably stretched causing increased egress of CSF and disorganisation of the normal architecture of the white matter and capillaries. The neurons and glial cells close to the white matter showed edematous changes. The changes in the ependymal lining and the underlying grey matter were less severe in the dorsal part of the 3rd and the 4th ventricle. The ventral part of the 3rd ventricle was the least affected. The height and the arrangement of the ependymal cells, the surrounding grey matter with narrow interstitial spaces and the absorbing tanycytes seemed to be factors which were responsible for the minimal changes in these regions. The changes appeared to be reversible if the CSF pressure was relieved at this stage. The spinal canal remained unaffected in the majority of our hydrocephalic animals, which could probably be attributed to the type of animal and the degree of hydrocephalus.

Ageing changes in the transplants of fetal substantia nigra grafted to striatum of adult rat

Fetal nigral neurons from 16 and 17 gestational days were transplanted into the intact striatum of adult rat. On different post-transplantation days (30-360 days), the structural and immunohistochemical details of the transplants were studied. The grafted neurons matured and showed phenotypical characteristics comparable to that of normal nigral neurons in adult rats until 180 days. Tyrosine hydroxylase-positive neurons were seen not only in the transplant but also in the adjacent host striatum. Tyrosine hydroxylase-positive fibres were also seen extending for a short distance into the host striatum. A large number of synapses in the transplants were of asymmetric type, containing clear round vesicles. These synapses resembled the few intrinsic type present in the normal substantia nigra. On the other hand, the predominant type 2 synapses with pleomorphic vesicles in the normal nigra were infrequently encountered in the transplants. On the 300th day, the cytoplasm of a few of the neurons showed ageing changes in the form of clear spaces, paucity of organelles especially rough endoplasmic reticulum, membrane-bound vacuoles and increase in the lipofuscin population. In addition, localized thickening of the soma and the dendrites were seen in relation to randomly distributed neurons. By 360 days, more than one quarter (26%) of the total neurons showed these changes indicating ageing. The number per unit volume of normal neurons decreased significantly when compared to the transplants on 60 and 90 days. In the substantia nigra of age-matched control, except for an increase in the lysosomal population, other ageing changes were not detectable. The neurons of intact substantia nigra of the host rat, chronologically 4-8 months older than the transplanted neurons, also appeared normal but for lipofuscin granules. The present study provides morphological evidence for rapid ageing of neurons in the long term nigral transplants. These observations raise fresh doubts regarding permanent survival of grafted neurons in the host brain. Studies so far conducted are after prior nigral lesions. Trophic factors following lesions of the host tissue may have influenced the long term survival of the transplanted neurons. On the other hand, such changes may have been missed since no detailed morphological investigations of the long term transplants have been done so far.

Fetal Nigral Transplants in the Lateral Ventricle of Adult Rats

The substantia nigra from rats of 16 and 17 gestational days grafted into the lateral ventricle of adult rats grew into isolated or partially integrated transplants in 80% of the rats. The morphological details of the transplants were studied at intervals of 30-360 days. The neurons grew, differentiated and were comparable to age-matched controls until 150 days. Thereafter, the neurons showed progressive aging changes like dendritic thickening, clear spaces, membrane-bound vacuoles and increase in lysosomes in the cytoplasm. The numerical density per unit volume (Nv) of normal neurons significantly decreased with simultaneous increase in Nv of glial cells by 360 days. These changes were slower to develop in the partially integrated transplants: it is concluded that target tissue interaction is necessary for prolonged survival of the grafted tissue.

Cell surface molecules (NCAM and LI) in intrastriatal transplants of embryonic mesencephalon in rats

Cell surface molecules, NCAM and L1, reported to have a role in synaptogenesis, growth and fasciculation of the neurites in the brain, were traced in the embryonic nigral transplants in the host striatum of adult rats. Substantia nigra of five, 15 and 25 postnatal days were also examined for the same molecules. Tyrosine hydroxylase label was used as a marker to localize the nigral neurons and glial fibrillary acidic protein to detect if glial scar present. In the control as well as transplants large neurons had expressed tyrosine hydroxylase. By 15th postnatal day tyrosine hydroxylase neurons appeared mature and were scattered, suggesting a well-formed neuropil. NCAM and L1 reaction was seen as a peripheral rim in most of the cells on the fifth postnatal day. The reaction was mainly in relation to the large cells and more extensive on the 15th day. Thereafter on the 25th day, activity was negligible. Large neurons demonstrated strong reactivity for NCAM and L1 during early post-transplantation days. After 30 days only smaller cells were reactive, many of which could be identified as neurons. Strong reaction for these molecules was present only until 60 days, though faint reaction could be detected even on the 90th day. These observations indicate that the growth promoting molecules, the type seen in the neonatal period, can be detected normally only until the neurons mature. Prolonged expression of these molecules by the grafted neurons indicate delay in the maturation of these cells due to absence of adequate target sites for synaptic connections. Some of the smaller cells expressing these molecules after 30 days of transplantation could be astroglia, either proliferating or reactive.

Fetal nigral grafts in the anterior eye chamber of adult rats: A long-term morphological study

Substantia nigral grafts of 16 and 17 gestation days showed phenotypic characteristics in the anterior eye chamber of adult rats until the third month after transplantation. Thereafter by the sixth month a number of neurons showed somal and dendritic thickening, reduced population of endoplasmic reticulum, increase in lysosomes, and clear areas devoid of organelles, indicating age changes. These changes were progressive and affected more neurons by the end of 1 year, the longest period studied. The observations suggest that the maturation of nigral neurons is independent of specific afferent input, whereas target influence is necessary for the continued maintenance of the mature neurons. All the synapses observed in the transplant were of the asymmetric variety, reminiscent of the few intrinsic synapses of the intact nigra. This suggested establishment of mutual connectivity among the transplanted neurons in the absence of a target and the type of synapse formed may have been influenced by the local environment. Large glial processes, very prominant during the 4- to 6-month period became less significant afterward but continued to be present until the end of the period studied. Though there was no morphological evidence of lymphocytic infiltration, this might suggest an immunologic reaction.

Effect of internal irradiation on the maturing Purkinje cells in the rat: a Golgi study

Continuous irradiation in utero is reported to produce mental retardation and gross abnormalities of the brain in the human. A few experimental studies conducted so far also report gross brain defects in animals exposed to continuous irradiation in utero. Despite the increasing use of nuclear energy for power and radioisotopes in medicine, there is hardly any literature available on the effect of continuous irradiation on the structural details of the developing brain. After intraperitoneal injections of different doses of 131I (8, 18 and 32 microCi) and 32P (10 microCi) in new-born rats on the 6th postnatal day, cerebella stained by Golgi techniques were cut sagittally and the sections were examined on the 10th, 15th and 21st postnatal days. In the animals injected with 18 and 32 microCi of 131I and 10 microCi of 32P a large number of Purkinje cells showed morphological alterations not seen in the control groups or in the groups injected with 8 microCi of 131I. The changes observed included persistence of the perisomatic processes beyond the 10th postnatal day, multiple primary dendrites, angulation of the primary dendrites, long segments of primary dendrites without branches and significantly reduced dendritic volume. The number of affected cells was less on the 21st postnatal day. The effective radiation dose estimated in these groups ranged from 15 to 26 rad. Since the rats irradiated with 6 rad had not shown such changes it is believed that there is a threshold dose of radiation beyond which only changes are perceptible at neuronal level.

Adrenal Medullary Autografts in Anterior Eye Chamber, Lateral Ventricle and Striatum of Adult Rats: A Long Term Study

The neurobiological basis for the short-term recovery in Parkinsons patients and experimental animals grafted with adrenal medulla is not yet clear. Structural details of the grafted chromaffin cells are also not available. In the present study, autografts of adrenal medulla in the anterior eye chamber, lateral ventricle and striatum of adult rats were studied for 360 days. Though a large number of cells degenerated, a few healthy chromaffin cells survived up to 360 days in the anterior eye chamber. In the ventricular and striatal regions, cells degenerated more rapidly, and a few surviving cells were seen only up to 120 and 150 days, respectively. Degeneration of the cells was evident from the alteration of the cytoplasmic granules, appearance of vacuoles and lysosomes, rapid decline in the number of TH and DBH positive cells and diffusion of enzymes in the intercellular region. Only lymphocytes and connective tissue cells were seen in the ventricle after 120 days, while outlines of a few chromaffin cells and background fluorescence were still evident in the striatum up to 150 days. In some of the intrastriatal transplants, morphologically identifiable Schwann cells were present and, in one transplant, there was evidence of myelination of axons by Schwann cells. These axons were obviously derived from the adjacent host tissue. From the findings it has been concluded that autografts of adrenal medulla survive for only a Limited period of time on transplantation to the central nervous system and anterior eye chamber. Survival seemed to be better in the anterior eye chamber than at the sites preferentially chosen for treating Parkinsons patients or experimental animals.

Fetal Dopaminergic Neurons Transplanted to the Normal Striatum of Neonatal or Adult Rats and to the Denervated Striatum of Adult Rats

Fetal ventral mesencephalon from the 15th gestational day was grafted into the striatum of neonatal and adult rats. In one group of adult rats, fetal nigra was transplanted into normal striatum. In a second group, the tissue was transplanted at sites where dopaminergic fibers were denervated with 6-hydroxydopamine. The behavior of the dopaminergic neurons and glial reactions were studied by staining with cresyl violet to localize the transplants and by immunolabeling tyrosine hydroxylase (TH) and glial fibrillary acidic protein. In normal adults, the transplants were small. At the edge of the transplants, TH-positive neurons were packed into clusters, and an interface without any significant crossover of TH-positive fibers was present. Glial reaction was minimal in and around the transplant. In the denervated striatum, transplants were generally larger than those in normal striatum and surrounded by a glial scar. TH-positive neurons were both closely packed and loosely arranged at the periphery of the transplants. Processes could be clearly defined and could be traced to the adjacent host striatum through the TH-free denervated area. In neonates, the transplants were large and at times extended beyond the striatum. Most TH-positive neurons were arranged linearly along the periphery of the transplant. Cell bodies were widely separated and a well-developed neuropil was present. Fibers from the transplant mingled freely with the host striatum without any interface. In all three transplant groups, tracing the TH-positive neurites was easy because they were thicker and coarser than other elements. No apparent glial reaction occurred in the neonates. Thus, the growth and maturation of dopaminergic neurons seemed to vary in different environments. The most conducive environment appears to be neonatal brain in which growth factors are readily available.