POSTNATAL DEVELOPMENT OF GABAERGIC NEURONS IN RAT CEREBELLUM

Цель - оценка динамики формирования ГАМК-ергических нейронов в коре и ядрах развивающегося мозжечка белой крысы в постнатальном онтогенезе с помощью иммуногистохимического выявления глутаматдекарбоксилазы (ГДК). Материал и методы. Исследование выполнено на 16 беспородных белых крысах на 2-, 7-, 15-е сутки (ранний постнатальный период) и 45-е сутки после рождения (пубертатный период). Для иммуногистохимического выявления ГАМК-ергических нейронов применяли первичные моноклональные мышиные антитела GAD-67 к ГДК, являющейся ферментом синтеза γ-аминомасляной кислоты. Иммунореактивность ГДК изучали на парафиновых срезах околочервячной части мозжечка. Результаты. Установлено, что в цитоплазме тел всех типов ГАМК-ергических нейронов коры мозжечка ГДК выявлялась уже на 2-е сутки после рождения. В цитоплазме тел грушевидных нейронов (клеток Пуркинье, КП) она достигала максимума на 7-е сутки, а в их дендритах - на 15-е сутки, позднее снова уменьшалась в больших звездчатых нейронах (клетках Гольджи, КГ) - на 45-е сутки. В аксонах звездчатых и корзинчатых нейронов ГДК-иммунореактивность к 45-м суткам становилась значительно выше, они формировали сплетения (корзинки) вокруг тел КП. ГАМК-ергические синапсы выявлялись в коре мозжечка на 7-е сутки и достигали максимального развития на 45-е сутки. В цитоплазме тел некоторых нейронов шаровидного и пробковидного ядер мозжечка у двухсуточных крысят выявлялась умеренная ГДК-иммунореактивность. На 7-е сутки между нейронами обнаруживались ГДК-иммунопозитивные волокна и аксодендритические синапсы, а на телах нейронов - аксосоматические синапсы, число которых возрастало к 45-м суткам. Выводы. Иммуногистохимическое исследование ГДК позволило выявить чёткую динамику постнатальных структурных преобразований тел, отростков и синаптических окончаний ГАМК-ергических нейронов в мозжечке крысы. Objective - to evaluate the dynamics of the formation of GABAergic neurons in the cortex and nuclei of the developing cerebellum in albino rat postnatal ontogenesis by immunohistochemical detection of glutamate decarboxylase (GAD). Method. The study was performed on 16 outbred albino rats on the 2, 7, 15 day (early postnatal period) and 45 day after birth (puberty). For immunohistochemical detection of GABAergic neurons, primary monoclonal mouse antibodies GAD-67 raised against GAD, an enzyme for the synthesis of gamma-aminobutyric acid, were used. GAD immunoreactivity was studied on paraffin sections of the paravermal zone of the cerebellum. Results. In the cytoplasm of the cell bodies of all types of GABAergic neurons in the cerebellar cortex, GAD was detected as early as on the second postnatal day. In the cytoplasm of the cell bodies of Purkinje cells (PC) it reached the maximum on the 7 day, and in their dendrites - on 15 day, and then decreased; in Golgi cells, maximal GAD staining intensity was detected on 45 day. By the 45 day, in the axons of stellate and basket cells, GAD immunoreactivity became significantly higher; they formed plexuses (baskets) around the PC bodies. GABAergic synapses were detected in the cerebellar cortex on the 7 day and reached their maximal development on the 45 day after birth. Moderate GAD immunoreactivity was detected in the cytoplasm of the cell bodies of neurons in the globose and emboliform nuclei of two-day-old rats. On the 7th day, GAD immunopositive fibers and axodendritic synapses between the neurons, and axosomatic synapses were detected, the number of which increased by the 45 day. Conclusions. GAD immunohistochemical study demonstrates a distinct dynamics of postnatal structural transformations of bodies, processes and synaptic terminals of GABAergic neurons in rat cerebellum.

Ultrastructural Dendritic Changes Underlying Diaschisis After Capsular Infarct

Diaschisis has been described as functional depression distant to the lesion. A variety of neuroscientific approaches have been used to investigate the mechanisms underlying diaschisis. However, few studies have examined the pathological changes in diaschisis at ultrastructural level. Here, we used a rat model of capsular infarct that consistently produces diaschisis in ipsilesional and contralesional motor and sensory cortices. To verify the occurrence of diaschisis and monitor time-dependent changes in diaschisis, we performed longitudinal 2-deoxy-2-[18F]-fluoro-d-glucose microPET (FDG-microPET) study. We also used light and electron microscopy to identify the microscopic and ultrastructural changes at the diaschisis site at 7, 14, and 21 days after capsular infarct modeling (CIM). FDG-microPET showed the occurrence of diaschisis after CIM. Light microscopic examinations revealed no significant histopathological changes at the diaschisis site except a mild degree of reactive astrogliosis. However, electron microscopy revealed swollen, hydropic degeneration of axial dendrites and axodendritic synapses, although the neuronal soma (including nuclear chromatin and cytoplasmic organelles) and myelinated axons were relatively well preserved up to 21 days after injury. Furthermore, number of axodendritic synapses was significantly decreased after CIM. These data indicate that a circumscribed subcortical white-matter lesion produces ultrastructural pathological changes related to the pathogenesis of diaschisis.

The Effects of Maternal Deprivation on the Hippocampal Structure in Adult Rats

Objectives:To examine the ultrastructural effects of maternal deprivation during developmental periods of limbi-chypothalamo-pituitary-adrenal system on hippocampal dendritic structures in adult rats.Methods:The experiments were carried out with male and female wistar rats in our department. The rats were mated and, after birth, the pups were divided into four groups. The first group (control group) pups remained undisturbed with their dam until postweaning day 22. Maternal deprived groups were separated from their dams for 24 hours at postnatal day 4, 9 and 18. The subjects were provided with food and water ad libitum until 3-months-of-age. At the third month, the rats were transcardially perfused, samples were taken from CA1 and CA3 regions of the hippocampus. Tissues were prepared for electron microscopy.Results:When the data were analyzed, there were no differences between male and female rats in both ultrastructure and semiquantitative analysis of axodendritic synapses. The ultrastructure of Group 1 was seen as normal while in the second Group some neurons nuclear envelope made deep invagination into the nucleus. Additionally, axodendritic synapses were found normal. In Group 3, micrographs and axodendritic synapses were showed normal structure. However, in Group 4 in some neurons invaginations were seen similar to Group 2. Axodendritic synapses were found to be normal.Conclusion:These experiments establish that MD in rats produces slight ultrastructural changes and decreases the number of synapses in CA1 and CA3 subregions of the hippocampus.

Neuronal Vacuolation and Spinocerebellar Degeneration in Young Rottweiler Dogs

With the recent epizootic of bovine spongiform encephalopathy in Europe, the differential diagnosis of neuronal vacuolation and spongiform change in other species has become critically important. Four Rottweiler puppies of both sexes, presented at 3-8 months of age, had clinical signs of generalized weakness and ataxia that started at 6 weeks of age. In all pups, neurologic examination detected an ataxia and tetraparesis, most severe in the pelvic limbs, and slowed proprioceptive placing reactions. Subsequently, there was rapid progressive neurologic deterioration, with severe placing deficits, knuckling, severe ataxia, and quadraparesis by 8 months of age. At necropsy, no gross lesions were observed. Microscopic lesions were restricted to the nervous system. The major lesion in all dogs was an intracytoplasmic neuronal vacuolation that was most prominent in the cerebellar roof nuclei and in nuclei of the extrapyramidal system. Similar vacuolation was found in neurons in both dorsal nerve root ganglia, myenteric plexus, and other ganglia of the autonomic nervous system. The single or multiple empty vacuoles were between 1 and 45 μm in diameter. A mild spongiform change was seen in the adjacent neuropil. Purkinje cell vacuolation and degeneration with segmental cell loss was seen in the oldest dog. In ventromedial and dorsolateral areas of the spinal cord white matter, there was mild bilaterally symmetrical axonal degeneration. Immunoblotting and immunocytochemical staining of the brain for protease-resistant scrapie prion protein was negative. All forms of vacuoles were negative for immunohistochemical staining with a variety of lectins. Ultrastructurally, the vacuoles were bound by a single membrane and contained granular material and sometimes membranous profiles. There was mild distension of the cytocavitary network but no unequivocal connection with the vacuoles was found. Axosomatic and axodendritic synapses in affected neurons were intact both ultrastructurally and with synaptophysin immunostaining. The clinicopathologic findings were different from those seen in the other neurologic diseases of Rottweilers. The age of the dogs, distribution and type of the lesions, ultrastructural findings, and negative immunoblotting most likely rule out the possibility of a scrapie agent-associated spongiform encephalopathy. However, the etiology of this new disease was not determined.

Regional Differences of Serotonin-Mediated Synaptic Plasticity in the Chicken Spinal Cord With Development and Aging

Previous studies in our laboratory /3,17/ have demonstrated that serotonin (5-HT) appears to have a trophic-like effect in enhancing synapse formation and maintenance in both the developing and the adult central nervous system. In the present study, we focused on age-related changes in the density of the axosomatic and axodendritic synapses and the number of 5-HT-positive fibers in the chicken spinal cord, with special reference to differences between the ventral (laminae VII and IX) and the dorsal (lamina I) horn. At 1 week posthatching (PIW), a transient overproduction of synapses and 5-HT-immunoreactive fibers occurred in lamina IX; all parameters had returned to their initial levels by 1 month post-hatching (PIM). The density of synapses further decreased by about 40% between P6M and P2Y (2 years posthatching). Although the magnitude of the transient increase in lamina VII was less than that in lamina IX, the changing pattern of the synapses and the 5-HT-positive fibers was similar in both regions. In the ventral horn, thin 5-HT-positive fibers were most prominent at P1W and then decreased with development; thin 5-HT-positive fibers were still found at P6M but had almost disappeared by P2Y. By contrast, at P2Y the density of the synapses and the 5-HT-positive fibers in the dorsal horn was even higher than that of younger animals.Reduction of 5-HT levels in P2Y-old chickens by p-chlorophenylalanine (pCPA) administration decreased the synaptic density in lamina I but not in lamina IX. The results of this study demonstrate that 5-HT-mediated synaptic plasticity is markedly different in the ventral and dorsal horns of the aged chicken. In the ventral horn, synaptic plasticity reached a maximum at about P1W, remained stable in the young-adult period, and then finally disappeared in the aged chicken. Conversely, the results suggest that in the dorsal horn, 5-HT fibers continue to mediate the trophic influence on synaptic plasticity even in the old chicken.

Ultrastructural visualization and neurochemical characterization of spinal projections of primary sensory afferents from the nipple: combined use of transganglionic transport of HRP-WGA and glutamate immunocytochemistry.

We used transganglionic transport of the neuronal tracer horseradish peroxidase coupled to wheat germ agglutinin (HRP-WGA) and post-embedding immunogold staining to determine the spinal projections and neurochemical identity of sensory afferents originating from a discrete cutaneous area. After SC injection of tracer into the nipple of lactating rats and reaction with tetramethylbenzidine stabilized with diaminobenzidene (TMB-DAB) or DAB and cobalt (TMB-DAB-Co), we found labeled terminals in the internal part of the first two layers of the dorsal horn where they formed axodendritic synapses and, at times, central elements of glomeruli, synaptic complexes believed to be involved in the integration of sensory messages. Immunogold staining of ultra-thin sections of tissue reacted with TMB-DAB-Co revealed that many mammary afferents contained glutamate as putative neurotransmitter. This combined approach thus offers the possibility of marking a limited set of primary afferents, after capture of tracer by sensory receptors of restricted peripheral areas, to visualize their projections at the spinal level and to determine their neurochemical nature with electron microscopy.