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Notes: Abstract The expression of somatostatin mRNA within the allocortex of the rat was examined by in situ hybridization with an alkaline phosphatase labeled probe. We sought to determine whether parcellation of the allocortex could be based upon the number and laminar location of the hybridized cells and to contrast the allocortical features with those of the isocortical areas. The cingulate region was characterized by intense, moderate, and faint cells, small to medium in size throughout the laminae. The retrosplenial region demonstrated a somewhat stratified appearance with an abundance of cells expressing somatostatin mRNA in the upper portion of the composite layer II–IV and also in the upper portion of layer VI. The insular region displayed more heterogeneity. The distribution of the cells hybridized for somatostatin mRNA formed distinctive configurations within the insular region (dorsal and ventral agranular insular areas) with no obvious generality. The perirhinal area resembled the ventral agranular insular area, and the cell distribution of the entorhinal and prepiriform areas displayed a common characteristic in that the primary axis of the perikarya of somatostatin mRNA expressing cells within the lower layers were oriented at almost every possible angle. The conclusion of the investigation is that in situ hybridization for somatostatin mRNA provides a means by which the areal boundaries within the allocortex may be drawn.

Notes: Abstract The expression of somatostatin mRNA within the neocortex of the rat was examined by in situ hybridization with an alkaline phosphatase-labeled probe. We sought to determine whether parcellation of the neocortex could be based upon the number and laminar location of the hybridized cells. Our investigation demonstrated that the boundaries of the neocortical areas can be determined by the distribution pattern of neurons expressing somatostatin mRNA. Few hybridized cells were located within layer IV, and this sparsity of cells within their wide granular layer marked the primary sensory areas. The occipital region was stratified, with insensely labeled cells in layers II/III and VI and faintly labeled cells in layer V. The parietal region carried a similar stratification, but more space between intensely labeled cells in layers III and V and between layers V and VI gave the region a three-tiered appearance. The temporal region displayed intensely labeled cells dispersed throughout layers III and VI and many in layer V as well as those faintly labeled without any breaks between the laminae. The distribution of the cells hybridized for somatostatin mRNA formed two configurations within the frontal region. It was difficult to identify any lamination in the first area, whereas the second area demonstrated a stratification reminiscent of the parietal region, but with only two tiers. The conclusion of the investigation is that in situ hybridization for somatostatin mRNA provides an exceptional means by which the areal boundaries within the neocortex may be drawn.

Notes: Abstract Increased levels of somatostatin (SS) and neuropeptide Y (NPY) have been demonstrated in the hippocampal formation after kindling. The increase might be specifically associated with kindling, or be an effect of repeated seizures per se. In order to separate these two components we studied the effects of repeated electroconvulsive shocks (ECS) on hippocampal SS-like and NPY-like immunoreactivity and SS mRNA and NPY mRNA in situ hybridization. ECS elicit seizures without having a demonstrable kindling effect. Rats were subjected to 10, 20, or 36 ECS (50 mA, 0.5 s), given as one shock per day, 5 days per week. One, 2 and 30 days after the last ECS, the rats were killed, together with sham-treated control rats, and processed for immunocytochemistry and non-radioactive in situ hybridization. There was a bilateral increase in SS-like and NPY-like immunoreactivity 1 and 2 days after the last ECS in the outer part of the dentate molecular layer. This is the terminal field of the hilar SS-containing and NPY-containing neurons, which displayed both increased immunoreactivity and hybridization signal of the cell bodies. There was also a bilateral de novo expression of NPY-like immunoreactivity in the mossy fiber system, but this was not accompanied by the appearance of a detectable NPY hybridization signal over the parent dentate granule cell bodies. The increase in SS-like immunoreactivity and hybridization signal was most pronounced in the rats that had received the largest number of ECS. This was not observed for the NPY-like immunoreactivity and hybridization signal, where the increase appeared similar after 10, 20 and 36 ECS. One month after the last ECS, both the SS-like and NPY-like immunoreactivity and the in situ hybridization signals had decreased towards normal levels. Since increased SS and NPY levels are also induced by repeated ECS, these changes are accordingly not specific to kindling-induced seizures. In a second experiment, the perforant path to the fascia dentata was transected 1 month prior to the ECS treatment. Removal of such major afferent input did not abolish the ECS-induced increase in hippocampal SS-like and NPY-like immunoreactivity, suggesting that the neuropeptide changes were not caused by afferent stimulation via the perfant path fibers, but rather may be an effect of direct electrical activation of the relevant cells.

Notes: Summary This study examines the effect of the immunosuppressive drug Cyclosporin A (CyA) on the survival and differentiation of solid grafts of fetal (E16–17) mouse hippocampi transplanted to the brain of adult rats. The CyA was given as daily subcutaneous injections of 20 mg/kg from the day before transplantation with reduction of the dose to 15 mg/kg after 14 days. Five weeks after transplantation neuron containing xenografts were recovered in 11 out of 17 CyA-treated recipients (65%). After 8 weeks 9 out of 21 grafts were found (43%). In the control groups, treated only with the vehicle olive oil, 8 out of 14 xenografts were recovered after 5 weeks survival (36%) and only 3 out of 17 after 8 weeks (18%). All xenografts were infiltrated with mononuclear lymphocytic-like cells, but the infiltration was least extensive and least dense in the CyA treated animals. An observed correlation between this cellular infiltration and the gliosis in the xenografts suggested that CyA also directly or indirectly influenced the glial reaction. Most surviving xenografts were located next to the lateral ventricles or the choroid fissure. They were organotypically organized with identifyable cell and neuropil layers, and their connectional organization was similar to rat and mouse allografts grafted to adult recipients. In the absence of major extrinsic afferents the intrinsic pathways observed with Timm staining had reorganized according to known principles for aberrant growth and collateral sprouting. Ingrowth of extrinsic host afferents was only demonstrated for AChE positive host fibers. We conclude that CyA treatment of adult rat recipients can increase the survival of intracerebral fetal mouse hippocampal xenografts and reduce the histological signs of rejection. Xenografting combined with CyA treatment thereby permits the use of a wider spectrum of donor neurons for studies of neuronal interaction and repair.

Notes: Abstract In order to establish an in vitro model of Huntington's disease, we prepared slice cultures of striatal tissue from newborn rats. The striatal cultures were grown for 12–39 days in the absence of any other brain tissue. The presence of specific cell markers was shown by immunocytochemistry, histochemistry and in situ hybridization with alkaline-phosphatase-labeled oligonucleotide probes. We focused on (1) the medium-sized, aspiny interneurons, which in vivo express the neuropeptides somatostatin and neuropeptide Y and the nitric oxide synthesizing enzyme nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, and which are spared in Huntington's disease and (2) the enkephalinergic, medium-sized projection neurons, which are particularly vulnerable in Huntington's disease. Similar basic morphologies of the presumed interneurons and double staining of NADPH-diaphorase positive and somatostatin immunoreactive neurons suggest that the two neuropeptides and NADPH-diaphorase are extensively colocalized in the cultures, as in vivo. In the newborn rats, included as controls, a patch-matrix distribution of the NADPH-diaphorase staining is described for the first time. In the striatal slices the distribution of the NADPH-diaphorase staining stayed uneven after 3–5 weeks in culture, with areas almost devoid of staining alternating with more heavily stained areas. This pattern may represent an intermediate stage between the patch-matrix distribution in the newborn and the homogeneous staining in the adult rat striatum. From quantitative estimates we found the same mutual rank order of the numbers of neuropeptide Y- and somatostatin-immunoreactive neurons and NADPH-diaphorase positive neurons in vivo and in vitro. Both in the slice cultures and in the brain, the number of enkephalin mRNA-containing neurons significantly exceeded that of neuropeptide Y- and somatostatin mRNA-containing neurons. This implies that the mutual distribution of presumed interneurons and projection neurons was preserved in the slice cultures. Comparison of cell numbers per unit volume showed that, in the cultures, the number of presumed interneurons, with the exception of NPY mRNA-containing neurons, significantly exceeded that in vivo. In contrast, the enkephalin mRNA-containing neurons, which in vivo are projection neurons, were significantly fewer in the cultures. The relative loss of projection neurons and preservation of interneurons in single slice cultures of striatal tissue apparently mimick some of the neurodegenerative changes of Huntington's disease. From the finding that the number of neuropeptide Y mRNA-containing neurons both in vitro and in vivo was significantly higher than the number of neuropeptide Y-immunoreactive neurons — unlike the number of somatostatin mRNA containing neurons — it is suggested that somatostatin and neuropeptide Y are differentially regulated at the level of translation. The peptides are also suggested to be differentially regulated at the level of transcription because the number of somatostatin mRNA-containing neurons increased significantly in the slice cultures compared with in vivo, while the number of neuropetide Y mRNA-containing neurons was unchanged.

Notes: Abstract Thalamic neuronal degeneration after neocortical lesions involve both anterograde and retrograde components. This study deals with the thalamic microglial response after neocortical aspiration lesions, using fluorogold fluorescent prelabeling, to identify retrogradely degenerating thalamocortical neurons, combined with histochemical or immunohistochemical staining of microglial cells. Adult male Wistar rats were injected with the retrograde fluorescent tracer fluorogold, in the right sensorimotor cortex (forepaw area) in order to retrogradely label thalamic neurons projecting to this area. After 1 week, the fluorogold injection site was removed by aspiration, axotomizing at the same time the thalamic projection neurons now retrogradely labeled with fluorogold. After 3, 7, 14, and 28 days the animals were killed and processed for nucleoside diphosphatase histochemistry or complement type 3 receptor immunohistochemistry and class I and II major histocompatibility complex immunohistochemistry using OX42, OX18, and OX6 antibodies. The histological analysis showed a prominent and progressive nucleoside diphosphatase-,OX42-, and OX6-positive microglial cell response in the ventrolateral, posterior, and ventrobasal thalamic nuclei with ongoing retrograde and anterograde neuronal degeneration. Initially the reactive microglia had a bushy morphology and were succeeded by ameboid microglia and microglial cluster cells as the reaction progressed. However, in the reticular thalamic nucleus, which suffered exclusively anterograde neuronal degeneration, a different picture was seen with only bushy microglia. The neurons undergoing retrograde degeneration in the ventrolateral, posterior, and ventrobasal thalamic nuclei were retrogradely labeled by the fluorogold tracer. Individual nucleoside diphosphatase-, OX42-, or OX6-positive microglial cells extended long cytoplasmic processes surrounding fluorogold-labeled neurons and had in some cases apparently phagocytized these. Several microglial cells were thus double-labeled with nucleoside diphosphatase or OX42 and fluorogold. In addition, small nucleoside diphosphatase-positive, fluorogold-labeled perivascular cells were observed in the neocortex near the fluorogold-injected and ablated neocortical areas and in the ipsilateral thalamus. This study demonstrates: (1) that the microglial response to thalamic degeneration after neocortical lesion is graded with a limited reaction to the well-known massive anterograde axonal degeneration and a more extended reaction to the axotomy-induced retrograde cell death; and (2) that also perivascular cells and possibly macrophages may contribute to this reaction, as seen by uptake of fluorogold from axotomized neurons in the degenerating thalamic nuclei.