Developmental changes of glutamate receptors in the rat cerebral cortex and hippocampus

We studied the immunohistochemial localization of the glutamate receptors (GluR-1, -2, and -3,) in the developing rat cerebral cortex and hippocampus using antibodies to GluR1 and to an epitope common to GluR2 and GluR3 (GluR2/3) subunits. In the cerebral cortex, GluR1 immunoreactivity appeared in the neurons from postnatal day (PND) 0, increased with maturation, was highest at PND?10, decreased until PND 30, and thereafter remained at the same level as on PND?0. GluR2/3 immunoreactivity appeared earlier in scattered neurons on embryonal day (ED) 18, increased with maturation and reached a peak between PND?10 and PND?15, after which the immunoreactivity gradually decreased and reached a plateau at PND?30. For both GluR1 and GluR2/3, some of the pyramidal neurons showed intense staining. In the pyramidal layers of the hippocampus, GluR1 and GluR2/3 immunoreactivity was found in all the pyramidal neurons of the CA1–4 area from ED?20. In the dentate gyrus of the hippocampus, GluR1 and GluR2/3 immunoreactivity was found in the neurons of the granule cells after PND?0. Immunoreactivity in the neurons of the subiculum was found after PND?5 and that of the polymorphic cell layers was found after PND?15–20. Our results indicate that the development of glutamate receptor subunits in the rat cerebral cortex and hippocampus is expressed in different spatial patterns and distinct temporal patterns throughout development and is scheduled during the early postnatal period, when synaptic plasticity or synaptic connection occurs in these regions.     

Developmental changes of glutamate receptors in the rat cerebral cortex and hippocampus

 We studied the immunohistochemial localization of the glutamate receptors (GluR-1, -2, and -3,) in the developing rat cerebral cortex and hippocampus using antibodies to GluR1 and to an epitope common to GluR2 and GluR3 (GluR2/3) subunits. In the cerebral cortex, GluR1 immunoreactivity appeared in the neurons from postnatal day (PND) 0, increased with maturation, was highest at PND 10, decreased until PND 30, and thereafter remained at the same level as on PND 0. GluR2/3 immunoreactivity appeared earlier in scattered neurons on embryonal day (ED) 18, increased with maturation and reached a peak between PND 10 and PND 15, after which the immunoreactivity gradually decreased and reached a plateau at PND 30. For both GluR1 and GluR2/3, some of the pyramidal neurons showed intense staining. In the pyramidal layers of the hippocampus, GluR1 and GluR2/3 immunoreactivity was found in all the pyramidal neurons of the CA1–4 area from ED 20. In the dentate gyrus of the hippocampus, GluR1 and GluR2/3 immunoreactivity was found in the neurons of the granule cells after PND 0. Immunoreactivity in the neurons of the subiculum was found after PND 5 and that of the polymorphic cell layers was found after PND 15–20. Our results indicate that the development of glutamate receptor subunits in the rat cerebral cortex and hippocampus is expressed in different spatial patterns and distinct temporal patterns throughout development and is scheduled during the early postnatal period, when synaptic plasticity or synaptic connection occurs in these regions. Accepted: 13 June 1996     

大鼠海马γ-氨基丁酸-A受体在生后发育中的表达

目的:研究大鼠生后发育过程中海马组织γ-氨基丁酸-A(GABA-A)受体的表达规律。方法:用免疫组织化学和PCR技术,检测不同年龄大鼠海马组织GABA-A受体及编码该受体的mRNA表达,并用图像分析方法进行定量研究。结果:大鼠海马组织在生后3 d已经出现GABA-A受体免疫反应,以后逐渐增强,到生后30 d达到最高值,海马各区GABA-A受体免疫反应强度没有显著的差别;编码GABA-A受体的mRNA表达也有类似的增龄性增多,但其表达高峰值提前到14 d。结论:生后大鼠海马GABA-A受体表达在一定时期内呈增龄性表达增强的趋势。     

Developmental expression of neural Wiskott-Aldrich syndrome protein (N-WASP) and WASP family verprolin-homologous protein (WAVE)-related proteins in postnatal rat cerebral cortex and hippocampus

The actin cytoskeleton plays a critical role in the cellular morphological changes. Its organization is essential for neurite extension and synaptogenesis under the processes of neuronal development. Recently, neural Wiskott-Aldrich syndrome protein (N-WASP) and WASP family verprolin-homologous protein (WAVE) have been identified as key molecules, which specifically participate in regulation of actin cytoskeleton through small GTPases. The functions of these factors have been investigated using cultured cells; however, in vivo developmental changes in these factors are not fully understood. In this study, we examined the expression levels and distributions of N-WASP, WAVE and their related proteins in the rat cerebral cortex and hippocampus during postnatal development. Protein levels of these factors were progressively increased during development, and actin was accumulated in membranous fractions. Immunoreactivities for these factors were widely but differentially observed in entire brain. In the developing brain, N-WASP and WAVE seemed to exist in the synapse-rich areas, such as stratum radiatum of hippocampal CA1 subfield. A similar tendency in the distributions of these factors was observed in the mature brain. Taken together, N-WASP, WAVE and their related proteins may participate in normal brain development and synaptic plasticity by regulating the actin cytoskeleton.     

Effect of melatonin on d-amphetamine-induced neuroglial alterations in postnatal rat hippocampus and prefrontal cortex

Most studies about cognitive functions in children with benign childhood epilepsy with centrotemporal spikes (BCECTS) have been conducted with alphabetic writing background subjects, however deficits observed might therefore potentially differ in a Chinese language environment. This study was designed to evaluate the intelligence quotient (IQ) profiles, especially the language abilities, in Chinese children with BCECTS and to investigate whether there is a relationship between clinical factors and disorders of cognitive functions. There are selective cognitive deficits in Chinese children with BCECTS, although the Full Scale Intelligence Quotient is within the normal range. There was a correlation between spike wave index (SWI) and language deficits in children with BCECTS, but the deficits are not dependent on age of onset, disease course, seizure frequency, spike location or seizure type. It is important that children with typical BCECTS undergo regular clinical investigations about language performance in order to start necessary interventions as early as possible.     

Region-specific modulation of electrically induced synchronous oscillations in the rat hippocampus and cerebral cortex

Strong tetanization induces synchronous membrane potential oscillations (seizure-like afterdischarge) in mature pyramidal cells of the hippocampal CA1 region. To investigate whether local networks in other brain regions can generate such an afterdischarge independently, we studied the inducibility of afterdischarge in individual 'isolated slices' of the rat hippocampal CA1 and CA3 regions, dentate gyrus (DG), entorhinal cortex (EC), and temporal cortex (TC) using intracellular and extracellular recordings. The strong tetanization constantly induced afterdischarges in the CA1 and CA3 pyramidal cells as well as in the EC and TC superficial principal cells. However, parameters of the afterdischarges, such as the frequency and duration of afterdischarges, varied among the regions. A mixture of N-methyl-D-aspartate (NMDA) and non-NMDA receptor antagonists or a GABA(A) receptor antagonist completely blocked the afterdischarges. Local GABA application during the afterdischarge elicited depolarization, rather than hyperpolarization. Moreover, reversal potentials of the afterdischarge were around -40 mV. In contrast, the tetanization resulted in occasional afterdischarge-like activities in DG slices, which were blocked by the non-NMDA or GABA(A) receptor antagonist. These findings suggest that the afterdischarges mediated through the excitatory GABAergic and glutamatergic transmissions might be common to, but be modulated differently by individual local networks in the hippocampus and cortex.     

Gerbil Angiotensin II AT1 receptors are highly expressed in the hippocampus and cerebral cortex during postnatal development

Increasing evidence suggests that Angiotensin II, classically known from its many effects regulating salt and water homeostasis, is also involved in brain development and cognitive functions through activation of AT₁ Angiotensin II receptors. The recently cloned gerbil AT₁ receptor is expressed in brain areas controlling hydro-mineral homeostasis, and particularly highly expressed in limbic areas such as the hippocampal formation. We quantified the gerbil AT₁ receptor messenger RNA expression and receptor binding by quantitative in situ hybridization and receptor autoradiography, respectively, in the hippocampal formation and cerebral cortex of gerbils during postnatal development. The receptor messenger RNA and binding were present from birth and showed a gradual and sustained increase through postnatal maturation in the CA1 and CA2 regions of the hippocampus and in the dentate gyrus. Conversely, in the CA3 region, no binding was detected while receptor messenger RNA peaked at 15 days after birth and disappeared in the adult. The highest receptor messenger RNA expression and binding were found in the septomedial portions of the CA1 region and at septal levels of the CA2 region.We detected the highest receptor messenger RNA expression at postnatal day one in the frontolateral pole of the cerebral hemispheres. In these areas, and in the frontoparietal and insular cortex, receptor messenger RNA dramatically decreased during postnatal life. Similarly, we found receptor messenger RNA expression in the cingulate, retrosplenial, perirhinal and infralimbic cortex with higher values during the first two weeks of development and decreased expression in the adult. However, receptor binding in the cerebral cortex, did not decrease during postnatal life.The differential profile of receptor messenger RNA expression and binding in the gerbil cortex and hippocampus during postnatal maturation suggest a role for AT₁ receptors in the development and function of the corticohippocampal system.     

Gerbil angiotensin II AT1 receptors are highly expressed in the hippocampus and cerebral cortex during postnatal development

Increasing evidence suggests that Angiotensin II, classically known from its many effects regulating salt and water homeostasis, is also involved in brain development and cognitive functions through activation of AT1 Angiotensin II receptors. The recently cloned gerbil AT1 receptor is expressed in brain areas controlling hydro-mineral homeostasis, and particularly highly expressed in limbic areas such as the hippocampal formation. We quantified the gerbil AT1 receptor messenger RNA expression and receptor binding by quantitative in situ hybridization and receptor autoradiography, respectively, in the hippocampal formation and cerebral cortex of gerbils during postnatal development. The receptor messenger RNA and binding were present from birth and showed a gradual and sustained increase through postnatal maturation in the CA1 and CA2 regions of the hippocampus and in the dentate gyrus. Conversely, in the CA3 region, no binding was detected while receptor messenger RNA peaked at 15 days after birth and disappeared in the adult. The highest receptor messenger RNA expression and binding were found in the septomedial portions of the CA1 region and at septal levels of the CA2 region. We detected the highest receptor messenger RNA expression at postnatal day one in the frontolateral pole of the cerebral hemispheres. In these areas, and in the frontoparietal and insular cortex, receptor messenger RNA dramatically decreased during postnatal life. Similarly, we found receptor messenger RNA expression in the cingulate, retrosplenial, perirhinal and infralimbic cortex with higher values during the first two weeks of development and decreased expression in the adult. However, receptor binding in the cerebral cortex, did not decrease during postnatal life. The differential profile of receptor messenger RNA expression and binding in the gerbil cortex and hippocampus during postnatal maturation suggest a role for AT1 receptors in the development and function of the corticohippocampal system.     

Proteomic analysis of rat cerebral cortex, hippocampus and striatum after exposure to morphine

Although a series of proteins in the brain have been shown to be qualitatively or quantitatively dysregulated following morphine administration, a systematic proteomic study has not been carried out so far. We therefore aimed to show the effect of morphine on protein levels in the rat brain. For this purpose rats were given a morphine base in subcutaneously placed pellets and subsequently the cerebral cortex, hippocampus and striatum were taken for proteomic studies after three days. Extracted proteins were run on two-dimensional gel electrophoresis, scanned and quantified by specific software. Proteins with significantly different levels were analysed by mass spectrometry (MALDI-TOF-TOF). Twenty-six proteins were found to be differentially expressed and were unambiguously identified. Dysregulated proteins were from several protein pathways and cascades including signaling, metabolic, protein handling, antioxidant and miscellaneous classes. These findings represent an initial approach to the generation of a 'morphinome' and may form the basis for further protein chemical studies as a valuable analytical tool. Moreover, the study reveals morphine-regulated proteins in different brain areas and indicates the pathways involved following morphine administration in the rat, the main species for pharmacological studies in the field.     

Late postnatal changes in rat somatosensory cortex

Summary Temporal and spatial developmental relationships between AChE and GABA-T reactivity in the sensory-motor cortex of rat were evaluated histochemically. Special attention was given to the barrels in layer IV of SmI that stain intensely for both enzymes. In the first and second postnatal weeks very low levels of diffuse GABA-T reactivity are seen in cortex, although cells and neuropil in the neostriatum are already clearly positive neonatally. There is little change until 16–18 days postnatal (dpn), when a steady increase in overall cortical reactivity for GABA-T has begun. In layer IV of SmI cortex, relatively intense foci of GABA-T staining begin to appear then, that overlap the barrel centers. GABA-T reactive non-pyramidal neurons at the periphery of these stained foci have distinctly stained processes that may enter the barrel centers. The intensity of GABA-T staining increases until 28 dpn when adult levels were reached. In contrast, AChE staining in cortex begins to appear at 2–3 dpn with prominent barrel staining seen by 6 dpn. When adult AChE reactivity levels are being achieved throughout all regions of neocortex, between 16–19 dpn, the barrel centers progressively lose demonstrable AChE-staining of their fiber plexus. Hence, the onset of GABA-T staining in the barrels during the third week postnatally coincides with the initiation of the progressive reduction in intensity of AChE staining in the same cortical zones. These observations on GABA-T correlate well with biochemical information on the time course of the maturation of the cortical GABAergic system. We should, therefore, consider the possibility that GABA-T staining may reflect the development of the GABAergic system in neocortex cerebri. The temporal linkage between AChE and GABA-T staining, raises the question of a possible developmental interaction between the non-cholinergic AChE-rich thalamic inputs to somatosensory cortex and the maturation of other more tardily developing components of the barrels in SmI. If the temporal relationship is only coincidental then the data still suggest that an intricate series of sequentially timed events characterize the ontogenesis of neocortical circuitry with relatively major developmental events occurring late in the postnatal period.     

Patterns of vascular sprouting in the postnatal development of the cerebral cortex of the rat

Light microscopic histochemistry for alkaline phosphatase was employed in a study of the development of vascular sprouting, with respect to time and distribution, in the rat cerebral cortex. Sprouts were counted in the full thickness of the cerebral cortex at each day from birth to 21 days of age. Several distinct bursts of sprouting activity were observed at specific times and levels of cortex. From birth to 4 days of age, sprouting was intense in the superficial third of the cortex. At 7 to 8 days, a burst of sprouting was found which was greatest in the middle third. Additional bursts of sprouting appeared at 10 and 14 days. Developing vessels with characteristics of arteries, capillaries, or sprouts were alkaline-phosphatase positive, while veins were not. It is concluded that alkaline phosphatase is a useful marker for identification of both mature and immature vasculature, as it reveals patent and nonpatent vessels, and the sprouts which are precursors of the mature vascular bed. New vessels developing in the cortex arise mainly from blind sprouts of capillaries, evidently in response to the metabolic demands imposed by the maturational process. At birth, the majority of intracortical vessels are capillaries. By 10 days of age, most perforating vessels from the surface have taken on arterial or venous characteristics. The findings are discussed in connection with morphological and biochemical differentiation and the pattern of vascularization in the mature cerebral cortex.     

Visualization of agonist-stimulated inositol phospholipid turnover in individual neurons of the rat cerebral cortex and hippocampus

A novel autoradiographic method to identify individual neurons responding to neurotransmitter stimulation with increased phosphoinositide turnover is described. When phosphoinositide-coupled receptors are activated, phosphatidylinositol 4, 5-bisphosphate is hydrolysed by phospholipase C generating the two second messengers, inositol 1, 4, 5-trisphosphate and diacylglycerol. During prolonged receptor stimulation, both second messengers are actively recycled to maintain the effective intracellular levels of agonist-sensitive phosphoinositides. Lithium ions inhibit this recycling pathway by blocking the recovery of free inositol from inositol 1, 4, 5-trisphosphate thus leading to the accumulation of phosphatidyl cytidine monophosphate, a membrane bound molecule which is the activated precursor of the synthesis of phosphoinositides. Therefore, addition of excess myo-inositol reverts the effects of lithium inhibition. Thus, taking advantage of this fact and using [³H]cytidine as precursor, phosphatidyl [³H]cytidine monophosphate accumulation was induced in rat neocortical and hippocampal slices after muscarinic or metabotropic glutamate receptor stimulation. The labelled slices were then fixed, dehydrated and embedded in Durcupan resin. Semithin sections (1 μm thick) were cut and exposed to autoradiographic emulsion for several weeks. Biochemical analysis of the incorporation of [³H]cytidine into the chloroform extracted (containing lipids) and the alkali-solubilized (containing nucleic acids and proteins) fractions were carried out in parallel with morphological studies. The stimulation of both receptor types induced labelling of neurons in neocortex and hippocampus. In labelled cells silver grains were characteristically observed over the cytoplasm surrounding the nucleus and main dendritic processes. The anatomical location and distribution of labelled cells as well as the levels of response obtained in both brain regions studied, was found to be receptor specific. Inclusion of 30 mM myo-inositol in the incubation media reversed completely both the accumulation of phosphatidyl [³H]cytidine monophosphate and the labelling of cells, thus demonstrating that the label detected autoradiographically corresponds to phosphatidyl [³H]cytidine monophosphate.

It is concluded that the method is sensitive and specific, allowing identification of individual neurons in both neocortical and hippocampal slices and after stimulation of both muscarinic and metabotropic glutamate receptor subtypes. The method may open a new means to study the phosphoinositide second messenger signalling pathway and the cells in which it takes place.     

Arginine vasopressin receptors in pig cerebral microvessels, cerebral cortex and hippocampus

We tested for the presence of arginine vasopressin (AVP) receptors in pig cerebral microvessels, cerebral cortex and hippocampus by specific binding methods with [3H]AVP as the ligand. The specific binding of [3H]AVP to all preparations was saturable and Scatchard analysis indicated a single class of high affinity binding sites (dissociation constant of 1-2 nM). Maximal binding capacity in cerebral microvessels was about 60% that of the cerebral cortex; and there were no apparent differences in the maximal binding capacity between cerebral cortex and hippocampus. These findings suggest the existence of AVP receptor sites in cerebral microvessels and support the hypothesis that AVP has a role in the control of the brain microcirculation.     

Developmental mechanics of the primate cerebral cortex

The idea that the brain is shaped through the interplay of predetermined ontogenetic factors and mechanisms of self-organization has a long tradition in biology, going back to the late-nineteenth century. Here we illustrate the substantial impact of mechanical forces on the development, morphology, and functioning of the primate cerebral cortex. Based on the analysis of quantitative structural data for prefrontal cortices of the adult rhesus monkey, we demonstrate that (1) the characteristic shape of cortical convolutions can be explained by the global minimization of axonal tension in corticocortical projections; (2) mechanical forces resulting from cortical folding have a significant impact on the relative and absolute thickness of cortical layers in gyri and sulci; (3) folding forces may affect the cellular migration during cortical development, resulting in a significantly larger number of neurons in gyral compared to non-gyral regions; and (4) mechanically induced variations of morphology at the cellular level may result in different modes of neuronal functioning in gyri and sulci. These results underscore the significant contribution of mechanical forces during the self-organization of the primate cerebral cortex. Taking such factors into account within a framework of developmental mechanics can lead to a better understanding of how genetic specification, the layout of connections, brain shape as well as brain function are linked in normal and pathologically transformed brains. Experiments involving animals were conducted according to the NIH guide for the Care and Use of Laboratory animals (NIH pub. 86–23, revised 1996), and experimental protocols were approved by the IACUC at Boston University School of Medicine., Harvard Medical School., and New England Primate Research Center     

Benzodiazepine receptors in rat cerebral cortex and hippocampus undergo rapid and reversible changes after acute stress

Rats were submitted to forced swimming and were killed 15 min after initiation of the stress and at 1 h, 1 day and 4 days thereafter. Immediately after the stress there was a decrease of 30% in the density of [³H]flunitrazepam binding sites in the cerebral cortex and of 27% in the hippocampal formation, with no changes in all the other brain areas studied. These changes in the number of benzodiazepine receptors were also corroborated by the binding of [³H]ethyl-β-carboline carboxylate. For both ligands there were no changes in affinity. These effects were selective for the benzodiazepine receptors and no changes in α₁, α₂ and β adrenoceptors and in dopaminergic receptors were observed. One hour after the stress, the number of benzodiazepine receptors had recovered in the cerebral cortex (8% above the control) and had increased greatly in the hippocampal formation ( + 53%). One day after the stress, the [³H]flunitrazepam binding in the cerebral cortex reached the normal level but it was still slightly elevated ( + 16%) in the hippocampus.These results are discussed in relation to some contradictory findings in the literature and to the fact that the hippocampal formation is related to neural mechanisms underlying behavior and neuroendocrine regulation.