Fetal Rat Cerebral Cortex Research Articles

Morphometric analysis of developing rat cerebral cortex following acute prenatal ethanol exposure

Morphologic alterations of fetal rat cerebral cortex were quantified by morphometric analysis following acute ethanol exposure on Gestational Day 14, a critical period of development of cerebral cortex. Pregnant rats were intubated with a total dose of 9 g/kg of ethanol on Gestational Day 14. Maternal blood ethanol levels ranged from 51 to 202 mg% during the period of ethanol exposure. Fetal brains were examined on Gestational Day 15, 24 h after the last dose of ethanol. The acute morphologic changes associated with ethanol exposure include enlargement of subventricular zone nuclei, cortical swelling, and dilation of pial blood vessels over the affected cortex. In some fetuses, cortical swelling was accompanied by the protrusion into the lateral ventricles of cytoplasmic blebs of ventricular zone cells. It is concluded that maternal ethanol exposure during a critical period of brain development produces measurable morphologic changes in fetal rat cerebral cortex within 24 h after ethanol exposure.

Developmentally expressed O-acetyl ganglioside GT3 in fetal rat cerebral cortex

Monoclonal antibody M6704, established against the chick neural tube, was shown to recognize a trisialosyl residue, NeuAcα2-8NeuAcα2-8NeuAcα2-3-R of C-series gangliosides. Using this antibody, the developmental changes of C-series gangliosides in fetal rat cerebral cortex have been examined. Two dimensional thin layer chromatography (TLC) enzyme-immunostaining analysis revealed that alkali treatment resulted in a great increase in GT3 that amounted to more than 85% of the total GT3 detected. The alkalilabile form was easily degraded to form GT3 by the action of the receptor-destroying enzyme of influenza C virus, sialate O-acetylesterase, indicating that the antigen was most probably 9- O-acetyl-NeuAc containing GT3. The ganglioside was highly enriched at the 14th gestation day, gradually decreased, and was not detected in adult rat cerebral cortex.

Direct extracellular electrical stimulation influences density dependent aggregation of fetal rat cerebrocortical neurons in vitro.

Cells isolated from fetal (E17) rat cerebral cortex form aggregates in a density-dependent manner. Direct current electrical stimulation influences the formation of neuronal aggregates by promoting neuronal survival at a calculated current density of 15 nA/cm2 (with an average calculated field strength of 1 microV/cm), and inhibiting neuronal survival at calculated current densities of both 1 nA/cm2 and 150 nA/cm2. Electrical stimulation had no observable effect on neuronal aggregation independent of its effect on cell survival.

Accelerated neural network formation in rat cerebral cortex cultures chronically disinhibited with picrotoxin

Our aim was to determine if chronic blockade of GABAergic inhibitory synaptic activity, monitored electrophysiologically at the neuronal level, would affect synapse formation and ultrastructure in dissociated fetal rat cerebral cortex cultures. This was achieved by adding picrotoxin to the serum-free growth medium in a dose that induced continuous epileptiform discharges throughout the culture period. Light and electron microscopic analysis suggested an accelerated synaptic network formation in the experimental cultures during the first 2 weeks in vitro. The elimination of excess synapses (mainly on spines), which normally takes place during the fourth week in vitro, occurred 1 week earlier in the presence of picrotoxin. Finally, the experimental cultures showed smaller spine synapses throughout the entire culture period. Because these effects were opposite those induced by chronic tetrodotoxin-blockade of spontaneous bioelectric activity in a previous study, the underlying causal factor could be the respective intensification and suppression of neuronal activity in the two experiments. An appropriate balance between excitatory and inhibitory synaptic drive seems therefore to be important for normal maturation of neocortical circuitry.

Enhanced coupling of neonatal muscarinic receptors in rat brain to phosphoinositide turnover.

The relationship between the density of the muscarinic receptor in developing rat cerebral cortex and its coupling to phosphoinositide turnover is examined. Tissue slices from rats of various ages were incubated with myo-[2-3H]inositol, and the effect of carbamoylcholine on the release of total inositol phosphates was determined. Binding of [3H]quinuclidinyl benzilate was determined in the same tissue. Although muscarinic receptor density in day-18 embryonic cortex was only 5% of that in the adult, the maximal response of stimulated phosphoinositide turnover to carbamoylcholine (1-10 mM) was at the adult level (i.e., three-fold increase). Comparison of the dependence of the turnover on carbamoylcholine concentration revealed that in neonates, the dose-response curve was shifted to the left, giving a half-maximal effect at concentrations approximately tenfold lower than that in the adult. In addition, the partial muscarinic agonists oxotremorine-2 and bethanechol were both more efficacious in young rats than in adults. The differences could not be accounted for either by alterations in agonist affinity for the receptor or by the presence of "spare" muscarinic receptors. These results indicate that muscarinic receptors in fetal and newborn rat cerebral cortex are more efficiently coupled to stimulation of phosphoinositide turnover than in the adult.

Tetrodotoxin enhances initial neurite outgrowth from fetal rat cerebral cortex cells in vitro

The effect of two different concentrations of the sodium channel blocker tetrodotoxin on neurite outgrowth from fetal rat cerebral cortex neurons was studied in vitro. A concentration of 10 −6M tetrodotoxin enhanced after two days in vitro: (1) the percentage of isolated cells and reaggregates which formed neurites, (2) the number of neurites per cell soma, and (3) neurite elongation and subsequent branching. The effects observed after treatment with 10 −7M tetrodotoxin were on the whole intermediate between control and 10 −6M tetrodotoxin.

Thyroid hormones reversibly suppress somatostatin secretion and immunoreactivity in cultured neocortical cells

Thyroid hormone effects on brain somatostatin-like immunoreactivity (SRIF-LI) were studied in an in vitro model system. Serum was removed from the nutrient culture medium of fetal day-18 rat cerebral cortex cells maintained in primary, long-term, dispersed monolayer culture. Chronic administration of either T 3 or T 4 in serum-free medium was associated with suppressed release of SRIF-LI into the culture medium (36–43 h accumulation), cell content of peptide and acute release in response to potassium-induced depolarization. Suppression was dose-dependent with an IC 50 of less than 1 nM for T 3. The most dramatic effects were observed for K +-induced release. Thirty-five to 50% suppression was typically observed with T 3 at a near maximum dose (3 nM). Reverse T 3 and diiodotyrosine were less potent and effective than T 3. TRIAC and diiodothyronine also possessed significant suppressive activity. T 3 suppression of release depended on duration of pretreatment. Administered for less than 16 h, T 3 failed to significantly suppress K +-induced release, but significant suppression was observed for pretreatment periods of 16 h or longer. Indirect fluorescent immunohistochemical examination revealed a reduction in the number of cells positively stained for SRIF-LI in T 3-treated dishes relative to controls. Upon removal of T 3 and subsequent recovery in serum supplemented medium for 24 h, T 3-treated and control cells exhibited similar levels of SRIF-LI release and cell content. T 3-treated and control cells incorporated [ 3H] leucine into trichloracetic acid precipitable counts to similar extents. Dexamethasone and several sex steroids failed to modify the effects of T 3 and did not independently influence SRIF-LI levels. Acute cycloheximide administration did not reverse T 3 effects. The data indicate that primary brain cell cultures may be useful models to examine direct peripheral hormone actions on nervous tissue. Thyroid hormones suppress SRIF-LI levels in a dose, time and structure-dependent manner, which appears to be reversible. The findings are consistent with a possible integration of peripheral hormone and brain peptide physiology.

Cholecystokinin-octapeptide enhances synaptic activity in neurons cultured from rat cerebral cortex

Cholecystokinin-octapeptide (CCK-8) was applied by local microperfusion techniques to neurons cultured from fetal rat cerebral cortex while membrane properties and synaptic activity were monitored with intracellular recording techniques. There were no consistent effects of CCK-8 on resting membrane potential, membrane input resistance, or excitability of the recorded neuron. Nevertheless, the application of CCK-8 was associated both with an increase in the frequency of ongoing synaptic activity and with an initiation of new synaptic activity not seen during control periods of recording prior to peptide application. CCK-8 was more potent evoking increased synaptic activity than its non-sulfated CCK-8 analogue. The existence of areas in proximity to the recorded neuron which were particularly sensitive to the effect of CCK-8 was noted. These results suggest that CCK-8 has a predominantly excitatory effect on cerebral cortical neurons. While a mechanism cannot be determined from the present data, a presynaptic site of action is suggested.

Towards an improved serum-free, chemically defined medium for long-term culturing of cerebral cortex tissue

The present study describes a series of experiments which have led to a substantially improved serum-free, chemically defined medium (CDM) for long-term culturing of reaggregated fetal rat cerebral cortex tissue. A reduction of the original medium concentrations of the hormones insuline, T3 and corticosterone, on the one hand, and an enrichment of the medium with the vitamins A, C and E, the unsaturated fatty acids linoleic and linolenic acid, and biotin, L-carnitine, D(+)-galactose, glutathione (reduced) and ethanolamine, on the other hand, formed the most important chemical adjustments of the medium. With the aid of this CDM (encoded R12), the light- and electron microscopic architecture of the tissue could be kept in a good condition (superior to that seen earlier in serum-supplemented medium) up to 23 days in vitro. From that time on, the neuronal network lying between the reaggregates degenerated for the largest part, while a portion of the large neurons (probably pyramidal cells) plus some of the neuronal network within the reaggregates degenerated too. This degeneration process continued during the following weeks, but the reaggregates nevertheless retained most of their mass, so that both small and large neuronal cell bodies (visible in transparent regions at the edge of the reaggregates) remained in good condition up to at least 103 DIV. Stout, thick nerve bundles interconnecting the reaggregates, also survived up to this point. Electron microscopic evaluation of such ‘aged’ reaggregates revealed degenerating as well as healthy regions. The latter had indeed retained healthy-looking pyramidal and non-pyramidal neurons, embedded within a dense neuropil which was often traversed by myelinated axons. The numerical synapse density in such selected, healthy tissue regions reached its maximum during the sixth week in vitro, followed by a rapid decrease and a stabilization at about half the peak values. The present culture system has opened the possibility for performing controlled quantitative studies on the relationship between structure and function of cerebral cortex tissues during development and aging, on its dependence on nutrients, hormones and drugs, and on special factors synthesized by the tissue and released into the nutrient medium.

Neuron generation in dissociated cell cultures from fetal rat cerebral cortex

Examination of dissociated cell cultures of rat fetal cortex revealed that mature cultures contained more neurons (identified by phase contrast morphology and tetanus toxin staining) than the number of cells initially adhering to the coverslips. Labeling with [ 3H]thymidine confirmed that neuronal precursors were undergoing mitosis throughout at least the first 10 days in vitro.

Dopamine-β-hydroxylase-like immunoreactivity in the fetal cerebral cortex of the rat: Noradrenergic ascending pathways and terminal fields

A topographical analysis of the noradrenergic innervation in the fetal rat cerebral cortex was carried out from embryonic day 15 (E15) until birth using antibodies raised against dopamine-β-hydroxylase (DBH). During late gestation DBH-like immunoreactive axons were coursing through the basal forebrain along three pathways: 1.(1) a medial component reached the medial cortex and then ran caudally along the anlage of the cingulum bundle;2.(2) a lateral component reached the frontal pole and curved ventro-dorsally in the primordium of the external capsule;3.(3) a few fibers were observed along the ventral amygdaloid bundle toward the amygdaloid complex and the surrounding cortex. No DBH positive fibers were observed in the main body of the internal capsule.The first noradrenergic axons were seen at E17 in the frontal pole, the lateral frontal cortex, and in the medial frontal cortex which also receives a dopaminergic input. The innervation then extended caudally, but the dorsal part of the cortex was reached after a 2-day delay when compared to the medial and lateral parts. The arrival of noradrenergic axons did not parallel the gradient of cortical neurogenesis; however, all cortical areas were innervated at birth. DBH positive fibers reached a given cortical region simultaneously through the marginal and intermediate zones and then invaded the cortical plate.

Nerve outgrowth, synaptogenesis and bloelectric activity in fetal rat cerebral cortex tissue cultured in serum-free, chemically defined medium

Dissociated and non-dissociated cerebral cortex of fetal rat was successfully cultured in a serum-free, chemically defined medium for at least 18 days without any preincubation in serum-supplemented medium. Neurite outgrowth, synapse formation and spontaneous bioelectric activity, in its qualitative and quantitative aspects, were essentially the same as was observed in medium containing 20% heat-inactivated horse serum.

A quantitative electron microscopic study on synapse formation in dissociated fetal rat cerebral cortex in vitro

Occipital cortex of 19-day-old fetal rats was dissociated and cultured in vitro for 2–5 weeks in horse serum supplemented Eagle's MEM. The outgrowing neurons rapidly formed a dense network which started to degenerate after 3 weeks in vitro. By means of electron microscopy the numerical development of 6 different categories of synapses was followed during the time in culture. This approach revealed a sigmoid growth curve emerging over the first 3 weeks for the category of axo-dendritic synapses (which comprised the bulk of all synapses counted). Thereafter a decline in number set in. Chronic exposure of these dissociated cerebral cortex cultures to 50 μg/ml xylocaine (a concentration adequate to block all measurable bioelectrical activity) did not prevent the formation of functional synaptic networks having normal synaptic ultrastructure. These results are in agreement with previous studies on fetal cerebral explants in culture 8,15. However, in some groups of our cultures, xylocaine led to a retardation in neurite outgrowth and in numerical synapse formation. Since these xylocaine effects were dose-related at a concentration double that required to silence the cultures, the growth retardation was probably not due to selective suppression of bioelectric activity, but rather to some general cytotoxic effect of the drug. In one of the xylocaine-treated groups (50 μg/ml) which showed an exceptionally rapid neuronal outgrowth by the use of horse serum obtained from a different source than in the other groups, no deficit was noted in the number of synapses formed. Extracellular recording in this latter drug-treated group of cultures during the third week revealed (after return to control medium) spontaneous isolated action potentials, burst patterns and slow waves which were indistinguishable from the bioelectric activity seen in the control cultures. It is concluded that bioelectric activity in dissociated cortex cultures is not a prerequisite for the formation of apparently normal, functional synaptic networks.

Quantitative Assessment of Radiation and Hypothermia-Hypercapnia Interaction Effects on the Formation of the Fetal Rat Cerebral Cortex

In the formation of the fetal rat cerebral cortex, undifferentiated cells proliferate in the primordial ependemyl layer on day 13 of gestation and begin to migrate through the intermediate zone on day 14 to form the cortical plate. The aims of this study were to evaluate whole-body gamma radiation (50, 100, 150, 200 R) and hypothermia-hypercapnia exposure on the 13th day of gestation on the formation of the cortical hemispheres on day 19 of gestation. Fetal brains were removed and subjected to qualitative and morphometric assessment of the various exposure effects on cellular and tissue derangements in the cerebral hemispheres and the cross-sectional area ( $\mu {\rm m}^{2}\times 10^{4}$) of the cortical plate. Dose-dependent cellular and tissue derangements were observed in the cortical wall and in the mean cross-sectional area of the cortical plate. Significantly lesser effects on the cerebral hemispheres and cortical plate were observed after two fractionated exposures, with an intervening 6-hr interva...