Cortex Of Neonatal Rats Research Articles

Protein tyrosine phosphatases expressed in the developing rat brain

Previous studies of the developing nervous system have shown that cell-cell and cell-matrix interactions are involved in a variety of processes such as the proliferation, migration, and differentiation of neurons. While many cell-surface molecules have been identified, the signal transduction mechanisms through which they modify cellular responses are poorly understood. Recent studies have described a new and large family of enzymes, protein tyrosine phosphatases (PTPases), that may play a key role in transduction of cell surface events. Opposing the actions of protein tyrosine kinases (PTKs), PTPases can determine the state of tyrosine phosphorylation of a protein and regulate its function. Within the family of PTPases, two subgroups have been characterized: low-molecular-weight cytoplasmic (nonreceptor) PTPases and high-molecular-weight transmembrane (receptor) PTPases. Many receptor PTPases have fibronectin type III and/or Ig-like domains in their extracellular domains, suggesting that they have dual functions: cell adhesion and signal transduction. Such molecules may play a role in cellular recognition events that mediate the accurate assembly of the nervous system. Using polymerase chain reaction with degenerate primers and a neonatal rat cortex cDNA library, we have identified a number of putative PTPase domains expressed in brain. Three are characterized here. These three sequences are most abundantly expressed in the developing cortex and so are named cortex-enriched protein tyrosine phosphatases (CPTPs) 1, 2, and 3. CPTP1 and CPTP3 show sequence homology to receptor PTPases and detect multiple high-molecular-weight mRNAs that are expressed preferentially in the developing CNS. Analysis of a longer cDNA indicates that CPTP1 and CPTP3 are the first and second phosphatase domains of a single receptor PTPase. CPTP2 identifies a single, smaller mRNA species with sequence homology to nonreceptor PTPases. Within the CNS, mRNAs detected by all three CPTPs are expressed at highest levels during prenatal and early postnatal days and are downregulated in the adult. In situ hybridization demonstrates that the CPTPs are expressed by progenitor cells and developing neurons. The spatial and temporal regulation of CPTPs suggests that they may play a role in neuronal development.

Cytokines Regulate L‐Arginine‐Dependent Cyclic GMP Production in Rat Glial Cells

We have previously demonstrated that primary astrocyte cultures from neonatal rat cortex and C6 glioma cells express a calcium-independent nitric oxide synthase (NOS) on induction with bacterial endotoxin (lipopolysaccharide, LPS). One hypothesis regarding the mechanism of the LPS induction is that it causes release of cytokines from these cells which then induce the enzyme directly. Such cytokine induction of NOS has been demonstrated in many extraneural cell types. L-Arginine-dependent increases in cyclic GMP correlate with smaller increases in accumulation of nitrite, the major oxidation product of nitric oxide, and hence can serve as a more sensitive measure of nitric oxide production. Here we provide evidence that interferon-gamma (IFN-gamma), interleukin (IL)-1 beta and tumour necrosis factor-alpha induce L-arginine-dependent cyclic GMP synthesis in C6 cells and that a combination of IFN-gamma and IL-1 beta induce L-arginine-dependent cyclic GMP synthesis in astrocyte cultures, indicating that these cytokines induce NOS. In both cell types the induction by cytokines was less sensitive to inhibition by dexamethasone, IL-10 and IL-4 than was induction by LPS. These data suggest that cytokines can also induce a NOS in glial cells and that the mechanism of this induction may be more direct than that of LPS, since it is less sensitive to modulation by immunosuppressors. Due to the close associations of astrocytes with neurons and microvasculature, cytokine-induced NOS could have potentially important pathophysiological effects in the central nervous system.

Contact‐spacing among astrocytes is independent of neighbouring structures: In vivo and in vitro evidence

We have examined the morphology of astrocytes and the arrays they form in two situations, in retinas from which ganglion cells and blood vessels have been caused to degenerate, and in vitro. These observations were made to test whether the regularity of the spacing of astrocytes within normal central nervous tissue results from interaction among astrocytes, or from interaction between astrocytes and other elements of that tissue. Both in the partially degenerated cat retina, and in cultures of astrocytes from neonatal rat cortex, astrocytes make and maintain contact with neighbouring astrocytes, yet space their somas apart, giving regularity to the arrays. These results support the hypothesis that the regularity observed in arrays of astrocytes in intact tissue results from an interaction among astrocytes, independent of neighbouring structures, and lead us to suggest that the cell-cell interactions involved in contact spacing serve to distribute astrocytes through the central nervous system, and may, in other tissues, underlie the formation of epithelia.

Expression of the cholecystokinin gene in organotypic slice cultures of immature rat somatosensory cortex

The preprocholecystokinin gene is expressed in a subpopulation of cortical interneurons containing γ-aminobutyric acid. Slices of neonatal rat cortex were cultivated for 12 ± 2 days and examined for the presence and distribution of these neurons by in situ hybridization and immunocytochemistry. Like in situ, two layers of preprocholecystokinin-mRNA-expressing cells were present. Immunopositive fibers formed a dense network and established symmetric contacts on dendritic shafts and spines. It is concluded that cholecystokinin-expressing interneurons survive in cultured slices of rat cerebral cortex. These organotypic cultures may be useful to study the cellular interactions which regulate neuronal cholecystokinin expression.

CNTF promotes the survival of neonatal rat corticospinal neurons in vitro.

Corticospinal neurons were identified in cell cultures of neonatal rat cortex by immunostaining of cholera toxin B subunit (CTB), retrogradely transported from the cervical part of the spinal cord. The CTB-immunoreactive neurons were larger than the neurons in the overall (unstained) neuronal population and represented a small fraction of it (average of 0.3%) after 6 hours in vitro. The number of both total and CTB-labeled neurons declined progressively with time in culture. The neuronal death was, however, markedly faster in the CTB-labeled neuronal population than in the overall neuronal population. Ciliary neurotrophic factor (CNTF) promoted the survival of CTB-positive corticospinal neurons in a dose-dependent manner; with CNTF, the death rate of the CTB-labeled neurons became identical to that of the overall population.

Early development of the SI cortical barrel field representation in neonatal rats follows a lateral-to-medial gradient: an electrophysiological study.

Development of the barrel field in layer IV of SI cortex of neonatal rats was studied in vivo using electrophysiological recording techniques. This study was designed to determine (a) the earliest time SI cortex is responsive to peripheral mechanical and/or electrical stimulation and (b) whether the development of the SI cortical barrel field map of the body surface follows a differential pattern of development similar to the pattern previously demonstrated using peanut agglutinin (PNA) binding (McCandlish et al. 1989). Carbon fiber microelectrodes were used to record evoked responses from within the depth of the cortex in neonatal rats between postnatal day 1. (PND-1), defined as the day of birth, and PND-14. Evoked responses were first recorded approximately 12 h after birth. These responses in the youngest animals were of low amplitude, monophasic waveshape, and long latency, with long interstimulus intervals necessary to drive the cortex. Increases in amplitude and complexity of waveshape and decreases in latency were observed over subsequent postnatal days. The earliest responses recorded on middle PND-1 were evoked by stimulation of the face and/or mystacial vibrissae. The next responses were evoked approximately 24 h after birth (late PND-1) by stimulation of the forelimb. The last responses were evoked approximately 36 h after birth (middle PND-2), by stimulation of the hindlimb. The physiological map of the representation of the body surface follows a developmental gradient similar to the gradient observed using PNA histochemistry; however, the lectin-generated morphological map lagged approximately 48 h behind the physiological map.(ABSTRACT TRUNCATED AT 250 WORDS)

Glial cell-specific mechanisms of TGF-β1 induction by IL-1 in cerebral cortex

Transforming growth factor beta-1 (TGF-β1) immunoreactive product (IRP) has recently been detected in autopsied brains of individuals who died with central nervous system diseases and/or fever but not in normal individuals or in normal rodent brain. However, the mechanism(s) of induction of TGF-β1 in brain and the identity of cells expressing TGF-β1 need to be understood before a role, if any, for this potent pleiotropic cytokine in neuropathogenesis can be discerned. Towards this end we determined that IL-1 stimulated the production of TGF-β1 IRP in cells and TGF-β1 activity in culture fluids of all glial cells, astrocytes, microglial cells, and oligodendrocytes, derived from neonatal rat cortex and grown in cell type-enriched cultures. TGF-β1 production in vitro varied with the cell type and isoform of IL-1. Oligodendrocytes produced the most and astrocytes the least amount of TGF-β1. IL-1α stimulated TGF-β1 production in all glial cell types, whereas IL-1β did not. In vivo, TGF-β1 IRP was detected in human tissues from cerebral frontal cortex and subcortical white matter only when interleukin-1 (IL-1) was elevated in the same tissues. Moreover, the amount of detectable TGF-β1 was positively correlated with the amount of detectable IL-1 (rho = 0.605; P = 0.003), as determined by morphometry. Double-labelling of cells for their phenotypic markers and expression of TGF-β1 indicated that all glial cells, but not neurons, expressed TGF-β1. IL-1α and IL-1β IRPs were also detected in all three glial cell types, most frequently in astrocytes and least frequently in microglial cells. The cells containing both cytokine IRPs were also detected. These results indicate that TGF-β1 may be induced by IL-1 in all glial cells of the frontal cortex, by both autocrine and paracrine mechanisms.

EFA Commentary: A collaborative approach to epilepsy education in the school

Despite advances in antimicrobial therapy and advanced critical care neonatal bacterial meningitis has a mortality rate of over 10% and induces neurological sequelae in 20–50% of cases. Escherichia coli K1 (E. coli K1) is the most common gram-negative organism causing neonatal meningitis and is the second most common cause behind group B streptococcus. We previously reported that an E. coli K1 experimental meningitis infection in neonatal rats resulted in habituation and aversive memory impairment and a significant increase in cytokine levels in adulthood. In this present study, we investigated the oxidative stress profile including malondialdehyde (MDA) levels, carbonyl protein formation, myeloperoxidase activity (MPO) activity, superoxide dismutase (SOD) activity and catalase (CAT) activity 6, 12, 24, 48, 72 and 96 h after E. coli K1 experimental meningitis infection. In addition, sulfhydryl groups, nitrite and nitrate levels and activity of the mitochondrial respiratory chain enzymes were also measured in the frontal cortex and hippocampus of neonatal rats. The results from this study demonstrated a significant increase in MDA, protein carbonyls and MPO activity and a simultaneous decrease in SOD activity in the hippocampus of the neonatal meningitis survivors but the same was not observed in frontal cortex. In addition, we also observed a significant increase in complex IV activity in the hippocampus and frontal cortex of meningitis survivor rats. Thus, the results from this study reaffirmed the possible role of oxidative stress, nitric oxide and its related compounds in the complex pathophysiology of E. coli K1-induced bacterial meningitis.

Neuronal domains in developing neocortex.

The mammalian neocortex consists of a mosaic of columnar units whose development is poorly understood. Optical recordings of brain slices labeled with the fluorescent calcium indicator fura-2 revealed that the neonatal rat cortex was partitioned into distinct domains of spontaneously coactive neurons. In tangential slices, these domains were 50 to 120 micrometers in diameter; in coronal slices they spanned several cortical layers and resembled columns found in the adult cortex. In developing somatosensory cortex, domains were smaller than, and distinct from, the barrels, which represent sensory input from a single vibrissa. The neurons within each domain were coupled by gap junctions. Thus, nonsynaptic communication during cortical development defines discrete multicellular patterns that could presage adult functional architecture.

Astrocyte heterogeneity: endogenous amino acid levels and release evoked by non-N-methyl-D-aspartate receptor agonists and by potassium-induced swelling in type-1 and type-2 astrocytes.

The aim of the present study was to determine whether endogenous amino acids are released from type-1 and type-2 astrocytes following non-N-methyl-D-aspartate (NMDA) receptor activation and whether such release is related to cell swelling. Amino acid levels and release were measured by HPLC in secondary cultures from neonatal rat cortex, highly enriched in type-1 or type-2 astrocytes. The following observations were made. (a) The endogenous level of several amino acids (glutamate, alanine, glutamine, asparagine, taurine, serine, and threonine) was substantially higher in type-1 than in type-2 astrocytes. (b) The spontaneous release of glutamine and taurine was higher in type-1 than in type-2 astrocytes; that of other amino acids was similar. (c) Exposure of type-2 astrocyte cultures to 50 microM kainate or quisqualate doubled the release of glutamate and caused a lower, but significant increase in that of aspartate, glycine, taurine, alanine, serine (only in the case of kainate), and glutamine (only in the case of quisqualate). These effects were reversed by the antagonist CNQX. (d) Exposure of type-1 astrocyte cultures to 50-200 microM kainate or 50 microM quisqualate did not affect endogenous amino acid release, even after treating the cultures with dibutyryl cyclic AMP. (e) Exposure of type-1 or type-2 astrocyte cultures to 50 mM KCl (replacing an equimolar concentration of NaCl) enhanced the release of taurine greater than glutamate greater than aspartate. The effect was somewhat more pronounced in type-2 than in type-1 astrocytes. Veratridine (50 microM) did not cause any increase in amino acid release. (f) The release of amino acids induced by high [K+] appeared to be related to cell swelling, in both type-1 and type-2 astrocytes. Swelling and K(+)-induced release were somewhat higher in type-2 than in type-1 astrocytes. In contrast, neither kainate nor quisqualate caused any appreciable increase in cell volume. It is concluded that non-NMDA receptor agonists stimulate the release of several endogenous amino acids (some of which are neuroactive) from type-2 but not from type-1 astrocytes. The effect does not seem to be related to cell swelling, which causes a different release profile in both type-1 and type-2 astrocytes. The absence of kainate- and quisqualate-evoked release in type-1 astrocytes suggests that the density of non-NMDA receptors in this cell type is very low.

Receptor-coupled uptake of valproate in rat astroglial primary cultures

Various receptor ligands were investigated for their effects on the uptake of the antiepileptic drug valproic acid (VPA) in primary astroglial cultures from cerebral cortex of neonatal rats. After stimulation with the α 1-adrenoceptor agonist phenylephrine, 5-hydroxytryptamine (5-HT) or the glutamate receptor agonists glutamate, quisqualate and kainate, the V max and K m values for the drug transport increased. On the contrary, after exposure to the α 2-adrenoceptor agonist clonidine, V max and K m decreased. The effects were reversed in comparison to the control level in the presence of selective receptor antagonists. The data indicate a specific coupling between receptors and the uptake system for VPA. Furthermore, the results may have significant implications, as they suggest that receptors on astrocytes can be involved in the local regulation of drug transport in brain.

Beta-adrenoceptors are down-regulated by corticosterone in the brain cortex of neonatal rats via noradrenaline

Beta-adrenoceptors are down-regulated by corticosterone in the brain cortex of neonatal rats via noradrenaline

Brain and heart sodium channel subtype mRNA expression in rat cerebral cortex.

The expression of mRNAs coding for the alpha subunit of rat brain and rat heart sodium channels has been studied in adult and neonatal rat cerebral cortex using the reverse transcription-polymerase chain reaction (RT-PCR). Rat brain sodium channel subtype I, II, IIA, and III sequences were simultaneously amplified in the same PCR using a single oligonucleotide primer pair matched to all four subtype sequences. Identification of each subtype-specific product was inferred from the appearance of unique fragments when the product was digested with specific restriction enzymes. By using this RT-PCR method, products arising from mRNAs for all four brain sodium channel subtypes were identified in RNA extracted from adult rat cerebral cortex. The predominant component was type IIA with lesser levels of types I, II, and III. In contrast, the type II and IIA sequences were the predominant RT-PCR products in neonatal rat cortex, with slightly lower levels of type III and undetectable levels of type I. Thus, from neonate to adult, type II mRNA levels decrease relative to type IIA levels. Using a similar approach, we detected mRNA coding for the rat heart sodium channel in neonatal and adult rat cerebral cortex and in adult rat heart. These results reveal that mRNAs coding for the heart sodium channel and all four previously sequenced rat brain sodium channel subtypes are expressed in cerebral cortex and that type II and IIA channels may be differentially regulated during development.

Investigations of origins of serotonergic projection to developing rat visual cortex: A combined retrograde tracing and immunohistochemical study

The present study investigated whether the raphe neurons which give rise to the transient serotonergic fibers in the visual cortex of neonatal rats persist or disappear as the rats mature. Three experiments were performed employing the WGA-apoHRP-Au retrograde transport technique in conjunction with 5-HT or WGA-HRP immunohistochemical staining. WGA-apoHRP-Au was injected into the primary visual cortex of all rats 9 days postnatally. In the first experiment, the animals were examined after 2 days: retrogradely labeled cells were observed in the dorsal raphe nucleus (DR), the median raphe nucleus (MR), and in the B9 and B6 cell groups; the majority (82.5%) of the cells was serotonergic. In the second experiment, the examinations took place following a survival time of 8 weeks: virtually all of the original raphe-visual cortical serotonergic neurons were found to the present. In the third experiment, also performed after 8 weeks relabeling the raphe-visual cortical neurons by WGA-HRP, it was found that 37.2% of the raphe neurons which had projected to the neonatal visual cortex no longer possessed such projections.

Postnatal development of D 1 dopamine receptors in the medial prefrontal cortex, striatum and nucleus accumbens of normal and neonatal 6-hydroxydopamine treated rats: a quantitative autoradiographic analysis

The postnatal development of dopamine (DA) D 1 receptors in the medial prefrontal cortex (mPFC), striatum (STR) and nucleus accumbens (NAC) of control and perinatally 6-hydroxydopamine (6-OHDA) lesioned rats was examined using quantitative autoradiography of 3H-SCH 23390 binding. D 1 receptors are present at one week and increase only slightly to a stable level by 2 weeks in the STR and NAC. Their ontogeny is not altered by intracisternal injection of 6-OHDA 5 days after birth. A biphasic pattern of appearance of D 1 receptors was found in the mPFC. D 1 receptors are present in the mPFC at 1 week, increase 3-fold by 2–3 weeks, and then decline at 4 and 6 weeks. 6-OHDA lesions do not significantly alter this pattern. At all postnatal ages, D 1 receptor binding in the mPFC exhibits a laminar distribution with increased receptor density in deep cortical layers (V, VI) compared to more superficial cortical layers (I, II). Both superficial and deep layers of D 1 receptors in the mPFC show similar biphasic postnatal developmental patterns. DA turnover rates are consistently about 10-fold higher in frontal pole compared to remainder of forebrain at all postnatal ages. Early 6-OHDA lesions increase DA turnover in forebrain, but lead to a persistent reduction in DA turnover in frontal pole by 2 weeks of age.

Ammonia-induced astrocyte swelling in primary culture

The effect of ammonia on water space of astrocytes in culture was determined as a means of studying the neurotoxicity of ammonia in fulminant hepatic failure (FHF). Treatment of primary astrocyte cultures obtained from neonatal rat cortices with 10 mM NH4Cl for 4 days resulted in a 29% increase in astrocytic water space, as measured by an isotopic method utilizing 3-O-methyl-[3H]-glucose. This effect was time- and dose-dependent. The ammonia-induced swelling was reversible as the water space in cultures treated with 10 mH NH4Cl for 3 days, and then returned to normal culture media for 1 day, was similar to control cultures. These findings suggest that elevated levels of ammonia lead to astrocyte swelling and may contribute to the brain edema in FHF.

Repair and reconstruction of the cortical plate following closed cryogenic injury to the neonatal rat cerebrum

A cryogenic lesion was induced in the parietal cortex of neonatal rats at postnatal day 2, and the chronological sequence of cellular events during repair and reconstruction of the cortical plate examined. Serial sections of cerebra obtained at varying intervals ranging from 1 to 60 days postinjury were studied by light and electron microscopy and by immunocytochemistry for fibronectin, laminin, type IV collagen, vimentin and glial fibrillary acidic protein. In addition, localization of heavily labeled neurons (generated on embryonic day 20) in the cerebral cortical plate was examined by [3H]thymidine radioautography. Repair of a well-defined coagulative lesion was accomplished with little or no mesenchymal cell proliferation in either the necrotic zone or the leptomeninges. Eventually, fusion of the adjacent cortical plates took place with the formation of a microsulcus. Migration of neurons continued to take place along the outer margins of the lesion, and postmigratory neurons accumulated within the upper cortical layers. Around the microsulcus, heavily labeled neurons aligned themselves with layers II-III of the adjacent normal cortical plate. Irregular clusters of neurons closely abutting the leptomeningeal surface were frequently noted when repair took place without an intervening molecular layer and/or a well-defined pial-glial barrier. Supplementing intrinsic information inherent in migrating neurons, local environmental signals provided by the radial glia, glia limitans, basal lamina and pial-glial barrier appear to influence the polarity and final positioning of postmigratory neurons within the cortical plate. The necrotic zone within the deeper layers of the cortex eventually healed with a cell-sparse gliotic layer. The end result was a histological pattern that, in many respects, resembled that of human micropolygyria.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione levels in olfactory and non-olfactory neural structures of rats

Olfactory receptor neurons are a CNS entry point for a wide variety of airborne substances. Therefore, it is probable that detoxification mechanisms are present in these neurons to neutralize such agents. Glutathione (GSH) is an essential component of several detoxification schemes, and this study we examined the distribution and levels of GSH in the olfactory epithelium, olfactory bulb, cortex, hippocampus and cerebellum in neonatal, weanling, adult and aged rats. We report that GSH is primarily localized to the olfactory receptor neurons and their axons within the olfactory epithelium. It is also localized within the glomerular neuropil and granule cells of the olfactory bulb. Levels of GSH in the olfactory epithelium and hippocampus do not change as a function of age, although GSH levels decrease in several brain regions, including the olfactory bulb, cerebellum and cortex.

Changing interactions between astrocytes and neurons during CNS maturation

The environments of the developing brain and injured adult brain differ in their abilities to support axonal growth. To determine if astrocytes contribute to this difference, neurons were plated onto astrocytes cultured from the neonatal rat cortex and from the injured adult brain. Two patterns of neurite growth were observed in these two astrocyte culture systems. Neurons contacting the neonatal astrocytes had neurites that were twice as long as those contacting the injured adult astrocytes. Furthermore, in cultures with neonatal astrocytes, neurites faithfully followed the astrocytic processes, maximizing their contact, while in cultures of injured adult astrocytes, the neurites had a tendency to cross the processes orthogonally, minimizing their interaction with the astrocytes. When neurons were grown suspended over either neonatal or injured adult astrocytes, no difference in neurite length or the pattern of neurite growth was observed, indicating that neurite growth was not differentially affected by soluble factors released from the two populations of astrocytes. The addition of fetal calf serum, which is known to contain protease inhibitors, did not alter neurite growth when compared to serum-free medium, suggesting that a substantial difference in protease activity does not account for the variations in neurite length observed. Based on these results, it appears that the molecular components of the external surface of injured adult astrocytes do not support neurite growth to the same extent as those found on neonatal astrocytes. The differing abilities of these two populations of cultured astrocytes to support neurite growth in culture may reflect a change in the functional role of these cells that occurs during the development of the central nervous system.

Effects of variations in ionic concentrations on high affinity uptake of l-glutamate in non-glutamatergic neurons and non-neuronal cells cultured from neonatal rat cortex

High affinity uptake of l-glutamate in the CNS is known to be strongly dependent on Na +. However, the stoichiometry and the mechanism of the relationship between Na + and l-glutamate at the putative transport site (i.e. whether one or two Na + are co-transported with one molecule of l-glutamate and in which order they bind to the site) have been the subject of debate because the results of quantitative studies on Na +-dependence of l-glutamate uptake have varied from one experimental model to another. This communication discusses ionic requirements of l-glutamate uptake in two culture systems derived from neonatal rat cortex—one containing no neurons and the other including, in addition to glial cells, GABAergic neurons. The present results are consistent with a model in which one molecule of l-glutamate is co-transported with either one or two Na + and no fixed order is required for the binding of Na + and l-glutamate to the transport site. Also, in contrast to other studies, l-glutamate uptake was found to be significantly reduced in the absence of Ca 2+ and was not affected by depolarizing concentrations of K +.