Fetal Rat Hippocampal Neurons Research Articles

Propofol protects cultured rat hippocampal neurons against N-methyl-D-aspartate receptor-mediated glutamate toxicity.

The effect of propofol on the toxicity induced by glutamate (GLU), N-methyl-D-aspartate (NMDA), kainate (KA), and amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) was investigated on cultured fetal rat hippocampal neurons. The degree of neuronal injury was quantified by measuring the release of the neuron-specific enolase (NSE) into the culture media. The toxicity induced by brief exposure to GLU (10(-4) M) or to NMDA (10(-4) M) was significantly reduced by propofol, whereas that elicited by KA, AMPA (10(-4) M), or long GLU exposure was unaffected. In conclusion, high concentrations of propofol significantly attenuate NMDA receptor-mediated glutamate neurotoxicity in vitro. Further studies are needed to confirm this beneficial effect in vivo and to evaluate propofol as a neuroprotective anesthetic agent in pathologies involving glutamate release and NMDA-mediated toxicity.

Interleukin-6 protects cultured rat hippocampal neurons against glutamate-induced cell death

We examined the effect of interleukin-6 (human recombinant) on glutamate-induced neuronal death of cultured 20-day fetal rat hippocampal neurons. After 7 days in culture, the hippocampal neurons were markedly degenerated by the addition of l-glutamate and also N-methy- d-aspartate. The neuronal death was prevented by the addition of MK801, a potent N-methyl- d-aspartate antagonist. Interleukin-6 at the concentration of 50 ng/ml has a significant preventive effect on the glutamate-induced neuronal death. Basic fibroblast growth factor at the concentration of 100 ng/ml gave also significant protective effect on hippocampal neurons, but nerve growth factor was ineffective in preventing the toxicity. It has been postulated that glutamate plays an important role in the pathogenesis of neuronal death such as ischemia and the various neurological diseases. Interleukin-6 might have somewhat physiological or pathological role in these events.

N-methyl-D-aspartate induces a rapid, reversible, and calcium-dependent intracellular acidosis in cultured fetal rat hippocampal neurons

The ability of NMDA to alter intracellular pH (pHi) was studied in fetal rat hippocampal neurons and glia using the pH-sensitive fluorescent indicator 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Brief exposure (60 sec) of hippocampal neurons to NMDA (2.5-250 microM) results in a rapid, and in most cells reversible, reduction in pHi, with full recovery to baseline pHi values taking several minutes following removal of NMDA. In contrast, little or no change in pHi was observed in glial cells exposed to these same concentrations of NMDA. The NMDA-induced acidification of neurons was concentration and time dependent, with an EC50 of 39 microM and Emax (delta pH) of -0.53. More prolonged exposure to NMDA (> or = 10 min) resulted in a more prolonged reduction in pHi values over the ensuing 20 min observation period. The intracellular acidification resulting from NMDA exposure of hippocampal neurons was blocked by the NMDA receptor antagonist 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP). Moreover, removal of extracellular Ca2+ eliminated both the selective NMDA-induced elevation in [Ca2+]i and the reduction in pHi, indicating that Ca2+ influx may be required for the decrease in pHi induced by NMDA receptor activation. Finally, the NMDA-induced reduction in pHi was not significantly attenuated when extracellular [H+] was decreased by increasing extracellular pH to 8.0. The latter suggests that an intracellular source of H+ is responsible for the NMDA-induced reduction in neuronal pHi. The reduction in neuronal pHi induced by NMDA receptor activation may mediate some of the physiological and (or) pathophysiological actions of glutamate.

Chapter 2 Expression of Exogenous Proteins in Mammalian Cells with the Semliki Forest Virus Vector

Publisher Summary Expression of cloned sequences using carrier vectors is one of the central techniques in cellular and molecular biology. This chapter describes the preparation of recombinant Semliki Forest Viruses (SFVs), their usability in different cell types, and shows examples of expression of exogenous proteins in baby hamster kidney (BHK) cells and in primary cultures of fetal rat hippocampal neurons. The recombinant SFV vector with the inserted sequence encoding an exogenous protein and the helper plasmid are both linearized with Spe I ; runoff transcription is performed using SP6 RNA polymerase. The in vitro transcripts are introduced into BHK cells by electroporation. Transfection of the recombinant vector RNA alone results in transient expression of the exogenous protein, driven by the SFV RNA polymerase encoded in the same RNA molecule. If the vector and the helper RNAs are co-introduced into the cells, the SFV structural proteins encoded by the helper will form virions that package the recombinant vector RNA and can be used for nonreproductive infection of other cells.

Cl −/HCO 3− exchange function differs in adult and fetal rat hippocampal neurons

We studied Cl −/HCO 3 − exchange function in acutely dissociated single hippocampal neurons from adult and fetal day 18 rat using a fluorescent intracellular pH (pH i) indicator dye. The presence of Cl −/HCO 3 − exchange activity was assayed by observing the elevation in pH i upon acute reversal of the Cl − gradient. Resting intracellular pH in acutely dissociated neurons of both adult and fetal tissue was significantly higher than that of cultured fetal hippocampal neurons (day 10–12 in culture). Acute removal of extracellular Cl − caused a rapid and reversible increase in pH i by 0.25 pH units in adult neurons but had virtually no effect in similarly dissociated fetal neurons. Cl −/HCO 3 − exchange activity was also undetectable in fetal cultured hippocampal neurons. The mRNA for the anion exchanger AE3 is expressed abundantly in adult rodent neurons. AE3 is a potential candidate molecule for the observed Cl −/HCO 3 − exchange activity. In situ hybridization was used to monitor expression of the AE3 gene in these two age groups. We found that both adult and fetal neurons express AE3 mRNA. These results indicate that AE3 may not function as a Cl −/HCO 3 − exchanger in fetal neurons, in contrast to its possible role in the adult brain.

Pregnenolone sulfate augments NMDA receptor mediated increases in intracellular Ca2+ in cultured rat hippocampal neurons

The ability of the neuroactive steroid pregnenolone sulfate to alter N-methyl-d-aspartate (NMDA) receptor-mediated elevations in intracellular Ca2+ ([Ca2+]i) was studied in cultured fetal rat hippocampal neurons using microspectrofluorimetry and the Ca2+ sensitive indicator fura-2. Pregnenolone sulfate (5–250 μM) caused a concentration-dependent and reversible potentiation of the rise (up to ∼ 800%) in [Ca2+]i induced by NMDA. In contrast, the steroid failed to alter basal (unstimulated) [Ca2+]i or to modify the rise in [Ca2+]i that occurs when hippocampal neurons are depolarized by high K+ in the presence of the NMDA receptor antagonist CPP. These data suggest that the previously reported excitatory properties of pregnenolone sulfate may be due, in part, to an augmentation of the action of glutamic acid at the NMDA receptor.

A chloride channel in excised patches from cultured rat hippocampal neurons

A non-calcium-dependent chloride channel of conductance 62 pS, found in isolated inside-out patches of cultured fetal rat hippocampal neurons, is described. The channel does not require the presence of any neurotransmitter to be active and probably plays a part in maintaining the normal resting membrane potential.

Glucocorticoid toxicity in the hippocampus: in vitro demonstration

Glucocorticoids (GCs) disrupt the energy metabolism of neurons of the hippocampus, and thus leave them more vulnerable to a variety of damaging metabolic insults. In this manner, GCs appear to influence the rate of hippocampal neuron loss during aging in the rat, as well as the severity of hippocampal damage following hypoxia-ischemia or seizure. These GC actions could be secondary to their multitudinous peripheral actions. The present report, however, suggests that GCs directly endanger hippocampal neurons. Glucocorticoid-induced sensitization of neurons to damaging toxins was demonstrated in vitro. The viability of primary cultures of dispersed fetal rat hippocampal neurons was assessed following exposure to a variety of neurotoxins. Prior incubation of the cultures with the rodent-typical GC, corticosterone, significantly decreased neuronal viability in the face of the toxins. Such compounds included the glutaminergic excitotoxin kainic acid, the antimetabolite 3-acetylpyridine and the superoxide radical generator paraquat. As little as 10 −9 M corticosterone could potentiate damage, a concentration equivalent to low basal values in vivo. Higher concentrations of corticosterone could potentiate damage even further; these corticosterone concentrations were not themselves damaging. Administration of glucose increased neuronal viability in the face of the GC/toxin combination, without increasing viability following toxin alone. This suggests that a critical feature of the action of GCs on neurons might be the inhibition of glucose utilization (which is a hallmark of peripheral GC action).

Differential effects of nerve-growth factor and glioma-conditioned medium on neurons cultured from various regions of fetal rat central nervous system

When nerve-growth factor (NGF) was added to 17-day fetal rat central nervous system (CNS) septal neurons in culture using a defined medium, choline acetyltransferase (ChAT) activity was greatly induced. However, glioma-conditioned medium (GCM), which is expected to contain cholinergic neurotrophic factor(s) different from NGF, did not affect the ChAT activity of the cultured septal neurons. On the contrary, ChAT activity of 19-day fetal rat hippocampal neurons in culture was increased by the addition of GCM but not by NGF. These phenomena were confirmed in septal and hippocampal neuronal cultures obtained from the same embryonic day, i.e., 18-day fetal rat. The NGF-mediated increase in ChAT activity of cultured septal neurons was culture-time dependent (2.3-fold increase after 3 and 3.5-fold increase after 6 days in culture) and NGF-dose dependent (the ED 50 value was 0.8 ng/ml). The effect of NGF was completely abolished by the addition of specific anti-NGF antibodies. The differential effects of NGF and GCM on several other cultured cholinergic neurons from 17-day fetal rat spinal cord, striatum, brainstem and amygdala were measured. NGF tended to increase ChAT activities of cultured striatal and amygdala neurons but not cultured spinal cord and brainstem neurons. GCM increased ChAT activities in the latter two cultures, while having no effect on the former cultures. Although the extent of increase of NGF-mediated ChAT activities of cultured striatal and amygdala neurons were low, NGF-mediated increase of the striatal ChAT activity showed the pattern resembling that of cultured septal neurons as to time-course, dose-dependency and anti-NGF antibody sensitivity. NGF did not affect the tyrosine hydroxylase activity in the cultured brainstem catecholaminergic neurons.

Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension.

Basic fibroblast growth factor (FGF) has been found to increase neuronal survival and neurite extension in a highly purified population of fetal rat hippocampal neurons under well-defined serum-free cell culture conditions. In the presence of FGF, neuronal survival after 7 days in culture on a simple plastic substrate is increased 4-fold, to 54% of the initial population. Survival is increased 2-fold to 40% on polyornithine-laminin. When FGF was bound to plastic or heparin substrates, neurite outgrowth was significantly increased to lengths comparable to those seen with laminin; however, FGF produced no further increase in neurite outgrowth on laminin. Half-maximal survival was observed at FGF concentrations of about 15 pg/ml (1 pM); half-maximal process outgrowth occurred at about 375 pg/ml (20 pM). The responsive cells were identified as neurons by their labeling with tetanus toxin and by antibodies to neurofilaments and to the neuron-specific enolase. Astrocytes, identified by the presence of glial fibrillary acidic protein, constituted about 10% of cells present at 1 week both in the presence and in the absence of FGF. These results strongly suggest that, in addition to its known mitogenic effects on nonneuronal cells, FGF possesses neurotrophic activity for hippocampal neurons.