Brain research. Developmental brain research最新文献

Indomethacin-sensitive mechanisms involved in inducible heat shock protein 70 (iHSP 70) synthesis were investigated at 6 h after global cerebral ischemia in parietal cortex and hippocampus. In anesthetized piglets, increased intracranial pressure was used to produce 5 or 10 min of cerebral ischemia. Brain regions were sampled for immunoblot analysis, immunohistochemistry and morphology. Immunoblots revealed differential expression of iHSP 70 in untreated brains. Cerebellum contained substantial amounts of iHSP 70 while lower levels were present in parietal cortex and hippocampus. Detectable increases in iHSP 70 were observed at 2 h after ischemia in parietal cortex and hippocampus. Using immunoblot data, calculation of percent change from control at 6 h after ischemia revealed significant (p<0.05) increases in iHSP 70 of 111±39% (&xmacr;±sem) (n=6) in parietal cortex and 195±69% (n=8) in hippocampus. Increased iHSP 70 immunoreactivity occurred primarily in the granular/subgranular area of the dentate gyrus 6 h after ischemia. Histological staining revealed little cellular injury at 6 h after ischemia in the granular/subgranular region injury whereas the CA3 region, which lacked iHSP 70 staining, displayed modest cellular injury. Cellular injury was also observed in cortical layers II/III and VI. At 6 h after ischemia, indomethacin pretreatment (5 mg/kg, i.v.) attenuated the iHSP 70 increases in parietal cortex and hippocampus (7±30% and 89±30%, respectively n=5; p<0.05 compared to ischemia). Also, the increase in iHSP 70 immunoreactivity and appearance of cellular injury were not detected with indomethacin pretreatment. Thus, prior administration of indomethacin is associated with attenuation of ischemia-induced increases in iHSP 70 and cellular injury.

Phenotypes of septal neurons, dissociated from 19-day-old fetal rat brains and then cultured in a medium containing nerve growth factor for 4 weeks, were examined using gamma-aminobutyric acid (GABA), calbindin D-28k, parvalbumin and choline acetyltransferase immunohistochemistry, and acetylcholinesterase histochemistry. There were primarily four groups of neurons identified in this septal culture: the first group (12.7% of 212 neurons examined) displayed a cholinergic, but not GABAergic, phenotype and had an average diameter of 13.6 +/- 2.7 microm (mean +/- S.D.); the second group (31.6%) displayed both cholinergic and GABAergic phenotypes and had a diameter of 12.2 +/- 2.8 microm; the third group (31.0%) displayed only a GABAergic phenotype and had a diameter of 10.4 +/- 2.3 microm; and the fourth group (24.7%) displayed neither a GABAergic nor cholinergic phenotype and had a diameter of 10.4 +/- 2.1 microm. Neurons in the first two groups described were significantly larger than those in the second two groups; neurons in the third and fourth groups were the same size. Calbindin D-28k was expressed in some neurons of each group (31.3%, 18.8%, 9.6% and 15.7%, respectively). These results demonstrate that septal neurons have the ability to express a variety of phenotypes when grown in vitro. This culture will be a useful tool for studying mechanisms of phenotype expression in septal neurons.

Cultured explants obtained from the dentate gyrus of rat embryos (embryonic day 19-20) were used to investigate synapse formation and morphological differentiation of neuron types in the absence of extrinsic afferents. Synaptogenesis was studied by whole-cell recordings of postsynaptic currents and by ultrastructural analysis. Neurons were visualized using Lucifer Yellow filling or staining with DiI. In short-term (3-5 days) cultured explants postsynaptic currents were rarely evoked by extracellular stimulation and synapses were almost completely absent at the ultrastructural level. After 6-10 days in vitro, the incidence of evoking postsynaptic currents mediated by glutamate and GABA(A) receptors was strongly increased. At the ultrastructural level, the density of synapses increased more than 20-fold. These results demonstrate de novo formation of synapses in cultured embryonic dentate gyrus explants. Neuron types could be discriminated by their dendritic arborizations and by their electrophysiological properties. After 6-10 days in vitro, mossy-like cells exhibited 3-4 primary dendrites branching in a characteristic pattern and showed moderate spike-frequency adaptation. Application of serotonin (5-HT) to cultured explants elicited GABA(A)-receptor-mediated postsynaptic currents in mossy-like cells, indicating synaptic GABA release from local interneurons. Comparison to 5-HT evoked GABA release in mossy cells in age-matched, acute slices revealed only slight quantitative differences. In contrast to mossy cells, granule cells showing several primary dendrites originating at one cell pole were almost completely absent in cultured explants, suggesting an involvement of extrinsic afferents in the differentiation of granule cells.

Considering the novel functions for both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the developing nervous system (reviewed in Layer and Willbold, Prog. Histochem. Cytochem., 1995) a quantitative survey of the spatiotemporal developmental profiles of both AChE and BuChE activity in the neonatal rat brain would be extremely useful. To that end, we collected six brain regions at seven developmental time points, (postnatal day 1, 4, 7, 12, 17, 21, adult; n > or = 3) and measured AChE and BuChE activity using both biochemical and histological methods. These results indicated that the developmental pattern of AChE and BuChE activity varied with respect to brain region and age: (1) the ontogeny of either AChE or BuChE specific activity in one region was not necessarily indicative of the developmental pattern of the same cholinesterase in other regions; (2) the AChE developmental profile in a given region did not necessarily predict the BuChE developmental pattern for that same region. The data were also analyzed from a different perspective, i.e., the ratio of BuChE-AChE activity, in order to determine if BuChE activity preceded AChE activity during development as has been proposed for the chick nervous system (Layer, Proc. Natl. Acad. Sci. USA, 1983). Our analysis showed that, in general, the BuChE-AChE ratio decreased as the region matured, data which parallel the pattern of development of these esterases in the chick nervous system.

The postnatal development of immunoreactivity for the neuronal calcium-binding protein calretinin in the pineal gland of the Mongolian gerbil was investigated using immunostaining of serial semithin sections. Calretinin-positive pineal cells could readily be visualized from the day of birth (P0) onwards and coexpressed the intermediate filament (IF) protein vimentin. During the first half of the first postnatal week, many of the calretinin-/vimentin-positive cells were also immunopositive for synaptophysin and neuron-specific enolase (NSE) and thus corresponded to pinealocytes. However, the expression of calretinin in pinealocytes was only transitory and declined towards the end of the first postnatal week. Thereafter, calretinin immunoreactivity became restricted to vimentin-positive interstitial glial cells. Therefore, in the gerbil pineal gland, calretinin obviously is not required in mature pinealocytes but instead serves as yet unknown functions in interstitial cells. The unusual calretinin expression pattern adds to the notion that pineal interstitial cells differ from glial cells of other brain regions. This conclusion is also underlined by our present detection of the neuronal marker protein PGP 9.5 in interstitial cells during postnatal development.

Phenotypes of septal neurons, dissociated from 19-day-old fetal rat brains and then cultured in a medium containing nerve growth factor for 4 weeks, were examined using gamma-aminobutyric acid (GABA), calbindin D-28k, parvalbumin and choline acetyltransferase immunohistochemistry, and acetylcholinesterase histochemistry. There were primarily four groups of neurons identified in this septal culture: the first group (12.7% of 212 neurons examined) displayed a cholinergic, but not GABAergic, phenotype and had an average diameter of 13.6±2.7 μm (mean±S.D.); the second group (31.6%) displayed both cholinergic and GABAergic phenotypes and had a diameter of 12.2±2.8 μm; the third group (31.0%) displayed only a GABAergic phenotype and had a diameter of 10.4±2.3 μm; and the fourth group (24.7%) displayed neither a GABAergic nor cholinergic phenotype and had a diameter of 10.4±2.1 μm. Neurons in the first two groups described were significantly larger than those in the second two groups; neurons in the third and fourth groups were the same size. Calbindin D-28k was expressed in some neurons of each group (31.3%, 18.8%, 9.6% and 15.7%, respectively). These results demonstrate that septal neurons have the ability to express a variety of phenotypes when grown in vitro. This culture will be a useful tool for studying mechanisms of phenotype expression in septal neurons.

In adult rats, combined lesions of the striatum and globus pallidus (GP) cause transsynaptic cell death of neurons in the substantia nigra pars reticulata (SNr) which becomes apparent 1-2 weeks after the lesions. This delayed cell death of SNr neurons has been explained to be caused by over-excitation of SNr neurons which results from an imbalance between excitatory and inhibitory inputs due to two simultaneous events: acceleration of the excitatory input from the disinhibited subthalamic nucleus (STN) and deprivation of the inhibitory input from the striatum. To examine whether the transsynaptic neuronal death in SNr is caused by the same lesions in developing rats, we destroyed the striatum and GP in rats on postnatal days 10 (P10), P15, P20, P25, P30, P35 and P60 by injecting ibotenic acid. We found that cell death did not occur in SNr neurons in rats younger than P20 and that Fos expression induced in STN neurons after these striatopallidal lesions in P10 and P20 rats was lower than that in P30 or P60 rats. These findings suggest that excitation of STN neurons is not enough to cause cell death of SNr neurons in rats younger than P20. Immature functional connection between the cerebral cortex and STN in the early developing animals may contribute to the resistivity of SNr neurons to transsynaptic delayed cell death.

The postnatal development of immunoreactivity for the neuronal calcium-binding protein calretinin in the pineal gland of the Mongolian gerbil was investigated using immunostaining of serial semithin sections. Calretinin-positive pineal cells could readily be visualized from the day of birth (P0) onwards and coexpressed the intermediate filament (IF) protein vimentin. During the first half of the first postnatal week, many of the calretinin-/vimentin-positive cells were also immunopositive for synaptophysin and neuron-specific enolase (NSE) and thus corresponded to pinealocytes. However, the expression of calretinin in pinealocytes was only transitory and declined towards the end of the first postnatal week. Thereafter, calretinin immunoreactivity became restricted to vimentin-positive interstitial glial cells. Therefore, in the gerbil pineal gland, calretinin obviously is not required in mature pinealocytes but instead serves as yet unknown functions in interstitial cells. The unusual calretinin expression pattern adds to the notion that pineal interstitial cells differ from glial cells of other brain regions. This conclusion is also underlined by our present detection of the neuronal marker protein PGP 9.5 in interstitial cells during postnatal development.

In the mouse nasopalate papilla and in the trenches of the foliate and vallate papillae, taste buds accumulated primarily during the first 2 weeks after birth. Null mutation for brain-derived neurotrophic factor caused extensive death of embryonic taste neurons, with the secondary outcome that most taste buds failed to form. However not all taste neurons died; functional redundancy rescued a variable number. The primary research objective was to identify the likely site of the taste neuron rescue factor that substituted for BDNF. In this quest taste bud abundance served as a useful gauge of taste neuron abundance. The proportion of taste buds that developed was variable and uncorrelated among the nasopalate, vallate, and foliate gustatory papillae within each bdnf null mutant mouse. Thus, in spite of shared IXth nerve innervation, the vallate and foliate papillae independently varied in residual gustatory innervation. This variation rules against the rescue of gustatory neurons by system-wide factors or by factors acting on the IXth ganglion or nerve trunk. Therefore it is likely that surviving BDNF-deprived taste neurons were stochastically rescued by a redundant neurotrophic factor at the level of the local gustatory epithelium. These findings broaden the classic expectation that target tissue supplies only a single neurotrophic factor that can sustain sensory (taste) neurons.

Neurotransmitters influence a wide variety of developmental processes. We hypothesize that N-methyl-D-aspartate (NMDA) glutamate receptors influence proliferation of populations of forebrain neurons. As our model, we use a subclass of GABAergic striatal interneurons that express the calcium binding protein parvalbumin (PV). To separate proliferative and post-proliferative effects of NMDA receptor antagonists on PV neurons, we first determined the birth-date of rat striatum PV neurons at the coronal level selected for analysis. Dividing striatal progenitor cells were marked by intraperitoneal injections of 5'-bromodeoxyuridine (BrdU) given to timed pregnant rats at selected time points between embryonic days (E) 12-22. Double immunohistochochemistry for BrdU and PV was used in adult progeny to determine the time course of neurogenesis of striatal PV neurons. The results of the neurogenetic analysis were then used for rational timing of treatment with competitive (CGS 19755) and non-competitive (MK-801) NMDA receptor antagonists. In comparison to pair-fed and vehicle-injected controls, gestational rats given CGS-19755 and MK-801 during the proliferative phase (E15-E18) showed a marked reduction of striatal PV neuron cell density as adults. In contrast, animals given NMDA antagonists during the post-proliferative period (E18-E21) showed no significant reduction in PV neuron cell density compared to pair-fed controls. These results suggest that glutamate influences cell proliferation of a population of striatal neurons by an NMDA-mediated mechanism, providing evidence for a novel role for excitatory amino acids in early forebrain development.