Appearance of parvalbumin-specific immunoreactivity in the cerebral cortex and hippocampus of the developing rat and gerbil brain

Summary Developmental changes in the distribution of parvalbumin-specific immunoreactivity in the brain, in particular in the cerebral cortex and hippocampus, were followed immunohistochemically in two different species, the rat and the Mongolian gerbil (Meriones unguiculatus) using an antibody raised against for rat parvalbumin. The gerbil is known to develop its auditory and visual capacity later than rat. In both the rat and gerbil, parvalbumin-specific immunoreactivity appeared after birth in both the cerebral cortex and hippocampus. The timing of the development of expression of parvalbumin varied among different parts of the cerebral cortex. The parietal cortex showed evidence of the earliest expression of parvalbumin whilst the occipital and temporal cortices expressed parvalbumin at a later stage of a development. This feature was common to both the rat and gerbil but occurred at a relatively later stage in the gerbil. The profile of the distribution of parvalbumin in the brain of the developing and adult gerbil was similar to that of the rat, but there were some differences. The frequency of bead-like structures on the dendrites of the parvalbumin-positive cells in the CA1 region of the hippocampus was markedly lower in the gerbil; instead, straight non-beaded fibers which ran vertically into the pyramidal layer were stained. Parvalbumin-positive fibers were also found in the cerebral cortex of the gerbil.     

Developmental Expression of the Glycine Transporters GLYT1 and GLYT2 in Mouse Brain

Abstract: Using immunocytochemical localization, the distribution of the glycine transporters GLYT1 and GLYT2 in the developing mouse brain was studied. GLYT1 and GLYT2 immunoreactivity begins during the period of fiber outgrow and synaptogenesis. GLYT2 is first expressed in spinal and spinothalamic white matter and is followed by the expression of synaptophysin. In the postnatal stages, GLYT2 staining in the white matter disappears, and a punctuated pattern in the gray matter emerges. In contrast, in the fetal brain GLYT1 immunoreactivity coincides with gray matter neuropil and processes of radial glia. GLYT1 is distributed over a much wider area of the brain than GLYT2. However, the distribution of these two GLYTs implies that GLYT1 and GLYT2 operate in concert within the area where both are present. At the day 12 embryo stage, GLYT1 antibodies stain the liver, and later they also react with the pancreas and the gastroduodenal junction. No other organs exhibit significant GLYT1 immunoreactivity. We additionally observed the presence of GLYT1 in rat fetal cerebral cortex and hippocampus, which was not detected in fetal mouse brain. Moreover, GLYT1 immunoreactivity was found in the mouse floor plate and the ventral commissure but was not present in the same regions in rats. These findings suggest possible differences in the expression of GLYT1 between these two species.     

Comparative studies on the GABA-transaminase immunoreactivity in rat and gerbil brains.

Gamma-aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the central nervous system (CNS). Degradation of GABA in the CNS is catalyzed by the action of GABA transaminase (GABA-T). However, the neuroanatomical characteristics of GABA-T in the gerbil, which is a useful experimental animal in neuroscience, are still unknown. Therefore, we performed a comparative analysis of the distribution of GABA-T in rat and gerbil brains using immunohistochemistry. GABA-T immunoreactive neurons were observed in the regions which contained GABAergic neurons of both animals: corpus striatum; substantia nigra, pars reticulata; septal nucleus; and accumbens nucleus. GABA-T + neurons were restricted to layers III and V in the rat. Unlike the rat GABA-T + neurons were observed in layers II, III, and V of the gerbil cerebral cortex. These results suggest that the expression of GABA-T in the gerbil brain may be similar to that in the rat brain, except in the cerebral cortex.     

Distribution and Developmental Regulation of Metabotropic Glutamate Receptor 7a in Rat Brain

Abstract: To determine the regional and cellular distribution of the metabotropic glutamate receptor mGluR7a, we used rabbit anti-peptide polyclonal-targeted antibodies against the C-terminal domain of mGluR7a. Here we report that immunocytochemistry at the light-microscopic level revealed that mGluR7a is widely distributed throughout the adult rat brain, with a high level of expression in sensory areas, such as piriform cortex, superior colliculus, and dorsal cochlear nucleus. In most brain structures, mGluR7a immunoreactivity is characterized by staining of puncta and fibers. However, in some regions, including the locus ceruleus, cerebellum, and thalamic nuclei, both cell bodies and fibers are immunopositive. The changes in levels of mGluR7a during development were investigated with immunoblotting and immunocytochemical analysis. Immunoblot analysis revealed that the levels of mGluR7a are differentially regulated across brain regions during postnatal development. In cortical regions (hippocampus, neocortex, and olfactory cortex), mGluR7a levels were highest at postnatal day 7 (P7) and P14, then declined in older rats. In contrast, mGluR7a levels were highest at P7 in pons/medulla and cerebellum and decreased markedly between P7 and P14. In these regions, mGluR7a immunoreactivity was at similar low levels at P14 and P21 and in adults. Immunocytochemical analysis revealed that staining for mGluR7a was exceptionally high in fiber tracts in P7 animals relative to adults. Furthermore, the pattern of mGluR7a immunoreactivity in certain brain structures, including cerebellum, piriform cortex, and hippocampus, was significantly different in P7 and adult animals. In summary, these data suggest that mGluR7a is widely distributed throughout the rat brain and that this receptor undergoes a dynamic, regionally specific regulation during postnatal development.     

Distribution of neurotensin-like immunoreactivity in the central nervous system of the Formosan monkey

The distribution of neurotensin-like immunoreactivity was investigated in the central nervous system of the Formosan monkey employing immunohistochemical techniques. Neurotensin-containing cells were found to be widely distributed in the forebrain. The principal densities of neurotensin-like neuronal perikarya were located in the limbic system, the basal ganglion and the cerebral cortex; particularly in the amygdala, the septum, the neostriatum, the claustrum and the insula. The stria terminalis and the preoptic area were also rich in immunostained neurotensin-like neurons. A large number of immunoreactive fibers were observed from the cerebral cortex to the spinal cord in locations such as the median eminence, the arcuate nucleus, the hippocampus, the central gray and the dorsal horn of the spinal cord. We analyzed in detail the distribution of neurotensin-like immunoreactivity in the brain of the Formosan monkey, and compared these results with those obtained in the brain of the rat, Japanese monkey and human. Some possible implications regarding differences in location of this peptide are also briefly discussed.     

Parvalbumin in Human Brain

Parvalbumin was isolated from human cerebral cortex and biceps and triceps muscles by HPLC. The immunological properties of the human protein and the mobility in two-dimensional polyacrylamide gels were similar to that of parvalbumin isolated from the muscles of rat, mouse, rabbit, and chicken. The tryptic peptide maps of the human parvalbumin, however, differed considerably from all other parvalbumins, indicating a distinct primary structure. The immunolabeled cells in the hippocampus of the human brain were of different sizes and forms; they occurred in all subfields and probably represent interneurons.     

Altered PLCβ-1 expression in the gerbil hippocampal complex following spontaneous seizure

Although the phospholipase C (PLC)β-1 isoform is associated with spontaneous seizure and distinctively expressed in the telencephalon, the distribution of PLCβ-1 expression in the epileptic gerbil hippocampus remains controversial. Therefore, we determined whether PLCβ-1 is associated with spontaneous seizure in an animal model of genetic epilepsy. In the present study, PLCβ-1 immunoreactivity was down-regulated in seizure-sensitive (SS) gerbils more than in seizure-resistant (SR) gerbils. The expression of PLCβ-1 within calretinin (CR)- positive neurons was rarely detected within the dentate hilar region of SS gerbils. PLCβ-1 immunoreactivity in the hippocampus was significantly elevated as compared to that in pre-seizure SS gerbil 3 h post-ictal. These findings suggest that alterations in PLCβ-1 immunoreactivity in the SS gerbil hippocampus may be closely related to the epileptic state of the gerbil brain and transiently elevated PLCβ-1 protein levels following seizure episodes. Such alterations may be compensatory responses in the SS gerbil hippocampus.     

Increased expression of glucose transporter 3 in gerbil brains following magnesium sulfate treatment and focal cerebral ischemic injury

Glucose is the primary energy substrate for neurons. Glucose transporter 3 (Glut3) localizes at the neuronal cellular membrane, which transports glucose from the extracelluar space into neurons. Ischemia results in an increased energy demand that is associated with profound changes in brain energy metabolism. Magnesium sulfate (MgSO₄) ameliorates ischemia‐induced neuronal death in the rat and gerbil model. We investigated the effects of MgSO₄ administration on the expression of Glut3 in cortex and hippocampus of gerbils during ischemia. The focal cerebral ischemia was produced by unilateral occlusion of the right common carotid artery and right middle cerebral artery. Following ischemia, Glut3 expression increased significantly versus non‐ischemic (contra‐lateral) cortex and hippocampus. MgSO₄ treatment significantly increased the level of Glut3 expression in the non‐ischemic and ischemic cortex and hippocampus. We found that the MgSO₄‐induced increase in Glut3 expression was not reversed by administration of U0126, a MEK kinase inhibitor. These results suggest that other factors may function to modulate the MgSO₄‐induced Glut3 response. In all, our data showed that MgSO₄ increases the expression of Glut3 in the cortex and hippocampus of gerbil brains both in non‐ischemia and ischemia status. However, the MEK signaling pathway might not be involved in MgSO₄‐induced Glut3 expression following focal ischemia. Copyright © 2010 John Wiley & Sons, Ltd.     

Proteomics in neurodegenerative diseases

The effects of amyloid-beta (Aβ) protein on the expression of m1, m2 subunits of mAChR and on α7nAChR were analyzed in the cerebral cortex and in the hippocampus of rats following injections of Aβ (1–40) (BACHEM, 2 μg in 1 μL of PBS) into the left retroesplenial cortex (RSg) and injections of 1 μL of PBS into the right RSg. Sections were immunoreacted for the localization of α7, m1, m2, GABA, somatostatin and parvalbumin. Injections of Aβ resulted in loss of neurones expressing α7- and m1-like immunoreactivity (IR) in frontal, RSg cortices, hippocampus and subicular complex. A decrease of α7, m1- and m2-like-IR fibers and structures-like terminals was also seen in hippocampus, subicular and cerebral cortex. α7nAChR and m1, m2 subuntis of mAChRs were most commonly identified on GABAergic interneurones. These results point to an effect of Aβ on the synthesis of α7nAChR and mAChRs and suggest an important role of cholinoceptive interneurones in the dysfunction of hippocampus and cerebral cortex seen in AD.     

Alteration in NCX-3 immunoreactivity within the gerbil hippocampus following spontaneous seizures

Although NCX-3 is highly expressed in the brain, the distribution of NCX-3 in the epileptic hippocampus is still controversial. Therefore, to assess the distribution and pattern of NCX-3 expression in epileptic hippocampus, we performed a comparative analysis of NCX-3 immunoreactivities in the hippocampus of seizure-resistant (SR) and seizure-sensitive (SS) gerbils. In SR gerbils, NCX-3 immunoreactivity was higher than pre-seizure SS gerbils, particularly in the pavalbumin (PV)-positive interneurons. Three h post-ictal, NCX-3 immunoreactivity in the SS gerbil hippocampus was markedly elevated to the level of SR gerbils. Six h post-ictal, the expression of NCX-3 was reduced to the level of pre-seizure SS gerbils. Therefore, the results of the present study suggest that down-regulation of NCX-3 expression in the SS gerbil hippocampus may be involved in the hyperexcitability of SS gerbils due to an imbalance of intracellular Na(+)/Ca(2+) homeostasis and Ca(2+) concentration.     

Localization of plasminogen in mouse hippocampus,cerebral cortex,and hypothalamus

Although the tissue plasminogen activator/plasminogen system contributes to numerous brain functions, such as learning, memory, and anxiety behavior, little attention has as yet been given to the localization of plasminogen in the brain. We have investigated the localization of plasminogen in the adult mouse brain by using immunohistochemistry. In the hippocampus, plasminogen immunoreactivity was seen in the pyramidal cell layer as numerous punctate structures in neuronal somata. An electron-microscopic study further demonstrated that the plasminogen-immunoreactive punctate structures represented secretory vesicles and/or vesicle clusters. In the cerebral cortex, plasminogen immunoreactivity was evident in the somata of the layer II/III and V neurons. A quantitative analysis revealed that parvalbumin (PV)-positive neurons had more plasminogen-immunoreactive puncta compared with those of PV-negative neurons in the hippocampus and cerebral cortex. Plasminogen immunoreactivity was present throughout the hypothalamus, being particularly prominent in the neuronal somata of the organum vasculosum laminae terminalis, ventromedial preoptic nucleus, supraoptic nucleus, subfornical organ, medial part of the paraventricular nucleus (PVN), posterior part of the PVN, and arcuate hypothalamic nucleus. Thus, plasminogen is highly expressed in specific populations of hippocampal, cortical, and hypothalamic neurons, and plasminogen-containing vesicles are mainly observed at neuronal somata.     

Differential Expression of c-fos mRNA and Fos Protein in the Rat Brain After Restraint Stress or Pentylenetetrazol-Induced Seizures

1. c-fos mRNA expression and Fos protein expression were investigated by in situ hybridization and immunohistochemistry after 30 min of forced restraint stress or pentylenetetrazol (PTZ; 64 mg/kg, i.p.)-induced seizures.2. Forced restraint stress and PTZ-induced seizures generated c-fos mRNA expression of distinct intensities, but in similar brain regions, including the hippocampus, the amygdala, the piriform cortex, the paraventricular hypothalamic nucleus, the habenula, and parts of the cerebral cortex.3. The distribution of Fos-like immunoreactivity induced by stress or seizures only partially overlap. No Fos-like expression was found in the hippocampus or the habenula after restraint stress. Nevertheless, both areas presented Fos-like expression after PTZ-induced seizures.4. Our results support the suggestion that immediate early gene expression in vivo may exhibit both region- and stimulus-specific expression.     

Increased Expression of Apolipoprotein D Following Experimental Traumatic Brain Injury

Increasing evidence suggests that apolipoprotein D (apoD) could play a major role in mediating neuronal degeneration and regeneration in the CNS and the PNS. To investigate further the temporal pattern of apoD expression after experimental traumatic brain injury in the rat, male Sprague-Dawley rats were subjected to unilateral cortical impact injury. The animals were killed and examined for apoD mRNA and protein expression and for immunohistological analysis at intervals from 15 min to 14 days after injury. Increased apoD mRNA and protein levels were seen in the cortex and hippocampus ipsilateral to the injury site from 48 h to 14 days after the trauma. Immunohistological investigation demonstrated a differential pattern of apoD expression in the cortex and hippocampus, respectively: Increased apoD immunoreactivity in glial cells was detected from 2 to 3 days after the injury in cortex and hippocampus. In contrast, increased expression of apoD was seen in cortical and hippocampal neurons at later time points following impact injury. Concurrent histopathological examination using hematoxylin and eosin demonstrated dark, shrunken neurons in the cortex ipsilateral to the injury site. In contrast, no evidence of cell death was observed in the hippocampus ipsilateral to the injury site up to 14 days after the trauma. No evidence of increased apoD mRNA or protein expression or neuronal pathology by hematoxylin and eosin staining was detected in the contralateral cortex and hippocampus. Our results reveal induction of apoD expression in the cortex and hippocampus following traumatic brain injury in the rat. Our data also suggest that increased apoD expression may play an important role in cortical neuronal degeneration after brain injury in vivo. However, increased expression of apoD in the hippocampus may not necessarily be indicative of neuronal death.     

Developmental and Regional Studies of the Metabolism of Inositol 1,4,5-Trisphosphate in Rat Brain

Coupling of CNS receptors to phosphoinositide turnover has previously been found to vary with both age and brain region. To determine whether the metabolism of the second messenger inositol 1,4,5-trisphosphate also displays such variations, activities of inositol 1,4,5-trisphosphate 5'-phosphatase and 3'-kinase were measured in developing rat cerebral cortex and adult rat brain regions. The 5'-phosphatase activity was relatively high at birth (approximately 50% of adult values) and increased to adult levels by 2 weeks postnatal. In contrast, the 3'-kinase activity was low at birth and reached approximately 50% of adult levels by 2 weeks postnatal. In the adult rat, activities of the 3'-kinase were comparable in the cerebral cortex, hippocampus, and cerebellum, whereas much lower activities were found in hypothalamus and pons/medulla. The 5'-phosphatase activities were similar in cerebral cortex, hippocampus, hypothalamus, and pons/medulla, whereas 5- to 10-fold higher activity was present in the cerebellum. The cerebellum is estimated to contain 50-60% of the total inositol 1,4,5-trisphosphate 5'-phosphatase activity present in whole adult rat brain. The localization of the enriched 5'-phosphatase activity within the cerebellum was examined. Application of a histochemical lead-trapping technique for phosphatase indicated a concentration of inositol 1,4,5-trisphosphate 5'-phosphatase activity in the cerebellar molecular layer. Further support for this conclusion was obtained from studies of Purkinje cell-deficient mutant mice, in which a marked decrement of cerebellar 5'-phosphatase was observed. These results suggest that the metabolic fate of inositol 1,4,5-trisphosphate depends on both brain region and stage of development.     

Differential Effect of Cerebral Ischemia on Monoamine Content of Discrete Brain Regions of the Mongolian Gerbil (Meriones unguiculatus)

The effect of bilateral cerebral ischemia on noradrenaline, dopamine, and serotonin concentrations in six brain regions (i.e., the cerebral cortex, striatum, hippocampus, midbrain-diencephalon, cerebellum, and pons-medulla oblongata) was examined in the gerbil stroke model. The relative changes in regional cerebral blood flow after bilateral common carotid occlusion were also assessed using the radioactive microsphere technique. At 1 h after bilateral carotid occlusion, a significant decrease of monoamine concentration was observed in the cerebral cortex, striatum, hippocampus, and midbrain-diencephalon whereas no significant change was detected in the cerebellum and pons-medulla oblongata. The fall in NA content was most prominent in the cerebral cortex and hippocampus and percentage reductions of dopamine and serotonin were greatest in the striatum and cerebral cortex, respectively. These results suggest that the monoamine neurons in various brain regions might have different vulnerabilities to ischemic insult and show no evidence of transtentorial diaschisis.     

The Somatostatin 2A Receptor Is Enriched in Migrating Neurons during Rat and Human Brain Development and Stimulates Migration and Axonal Outgrowth

The neuropeptide somatostatin has been suggested to play an important role during neuronal development in addition to its established modulatory impact on neuroendocrine, motor and cognitive functions in adults. Although six somatostatin G protein-coupled receptors have been discovered, little is known about their distribution and function in the developing mammalian brain. In this study, we have first characterized the developmental expression of the somatostatin receptor sst2A, the subtype found most prominently in the adult rat and human nervous system. In the rat, the sst2A receptor expression appears as early as E12 and is restricted to post-mitotic neuronal populations leaving the ventricular zone. From E12 on, migrating neuronal populations immunopositive for the receptor were observed in numerous developing regions including the cerebral cortex, hippocampus and ganglionic eminences. Intense but transient immunoreactive signals were detected in the deep part of the external granular layer of the cerebellum, the rostral migratory stream and in tyrosine hydroxylase- and serotonin- positive neurons and axons. Activation of the sst2A receptor in vitro in rat cerebellar microexplants and primary hippocampal neurons revealed stimulatory effects on neuronal migration and axonal growth, respectively. In the human cortex, receptor immunoreactivity was located in the preplate at early development stages (8 gestational weeks) and was enriched to the outer part of the germinal zone at later stages. In the cerebellum, the deep part of the external granular layer was strongly immunoreactive at 19 gestational weeks, similar to the finding in rodents. In addition, migrating granule cells in the internal granular layer were also receptor-positive. Together, theses results strongly suggest that the somatostatin sst2A receptor participates in the development and maturation of specific neuronal populations during rat and human brain ontogenesis.