Electrophysiological study of mammalian neurons from ventral mesencephalon grown in primary dissociated cell culture

Mammalian neurons from ventral mesencephalon were grown in primary dissociated cell culture. While dopaminergic neurons were found in culture and used as a marker for this area of the nervous system, our study did not segregate the neurons in terms of their dopaminergic nature. Indeed all of the results were probably from nondopaminergic neurons. Action potentials dependent on sodium and potassium could be elicited from neurons placed in a balanced salt solution. Following blockage of sodium current by tetrodotoxin, and reduction of potassium current by tetraethylammonium and 3-aminopyridine, long-duration action potentials could be elicited by intracellular stimulation. These action potentials were most probably calcium-dependent since they could not be elicited in bathing solution containing manganese, a calcium-conductance blocker.     

Droperidol suppresses spontaneous electrical activity in neurons cultured from ventral midbrain. Implications for neuroleptanesthesia

Droperidol is used in anesthesia as an antiemetic and as a component in neuroleptanalgesia. Its mechanism of action is thought to involve dopamine receptor blockade in the brain. The electrophysiological consequences associated with this action however, have not been elucidated. In this study we demonstrate a dose-dependent electrophysiological response to droperidol in central nervous system (CNS) neurons that express dopamine receptors that is absent in CNS neurons that do not express dopamine receptors. Primary dissociated cell (PDC) cultures were prepared from embryonal tissue dissected from ventral mesencephalon (VM) and spinal cord (SC). VM neurons were used to investigate the electrophysiological action of droperidol, a dopamine receptor antagonist, since these cultures contain neurons having dopamine receptors on their surface. SC neurons were used as a control as they do not express dopamine receptors. Some dopamine receptors are on dopaminergic neurons and therefore are called autoreceptors, while others are on nondopaminergic neurons, such as GABA producing (GABAergic) neurons. All neurons in both PDC cultures were spontaneously active. The percentage of neurons which spontaneously generated action potentials was reduced in a dose-dependent manner by droperidol (1 nM-10 microM) only in PDC cultures of VM. Exposure to droperidol had no effect on neurons from PDC cultures of SC which lack dopamine receptors. Our results suggest that droperidol modulates the electrophysiological properties of VM neurons with dopamine receptors possibly through facilitation of inhibitory interneurons. The reduced activity of VM neurons might contribute to the antiemetic and/or anesthetic activity of droperidol, since the concentrations of droperidol used in this study are at clinically relevant concentrations.     

Neuronal maturation in mammalian cell culture is dependent on spontaneous electrical activity

Fetal mouse spinal cord (SC) and dorsal root ganglion (DRG) neurons undergo a process of maturation in cell culture lasting a month or more. We have investigated the role of electrical activity in this maturational process with the use of tetrodotoxin (TTX), the specific blocker of the voltage-sensitive sodium channel responsible for action potential generation. This agent completely eliminates the spikes and related synaptic activity which occur abundantly in untreated cultures. Such blockade of electrical activity in the cultures, when begun early (day 1 or day 8 in vitro), results in a 85-95% reduction in the number of large SC neurons, without affecting DRG neuron numbers. TTX treatment initiated when cultures are mature (day 70) has no significant effect on either DRG or SC neurons. Intermediate effects are obtained when treatment is initiated at day 35 in vitro. The activity of the nerve-specific enzyme choline acetyltransferase, is significantly decreased by early TTX treatment, while DNA and protein content of the cultures (primarily contributed by glial and fibroblastic cells) is not affected.     

Selective serotonin reuptake inhibitors reduce the spontaneous activity of dopaminergic neurons in the ventral tegmental area

Electrophysiological techniques were used to study the effects of paroxetine, sertraline, and fluvoxamine on the basal activity of dopaminergic neurons in the ventral tegmental area (VTA) of rats. Acute i.v. administrations of paroxetine (20–1280 μg/kg), sertraline (20–1280 μg/kg), and fluvoxamine (20–1280 μg/kg) caused a slight but significant reduction in the firing rate of the VTA dopaminergic cells studied. Paroxetine produced a maximal inhibitory effect of 10 ± 11% at the cumulative dose of 160 μg/kg. Sertraline induced a dose-related inhibition of VTA dopaminergic neurons, which reached its maximum (10 ± 7%) at the cumulative dose of 1280 μg/kg. The effect of fluvoxamine on the basal firing rate of VTA dopaminergic neurons was more pronounced as compared to that of paroxetine and sertraline, in that it produced a maximal inhibition of 17 ± 12% at the cumulative dose of 1280 μg/kg. Acute i.v. injections of paroxetine (20–1280 μg/kg), sertraline (20–1280 μg/kg), and fluvoxamine (20–5120 μg/kg) caused a dose-dependent decrease in the basal firing rate of serotonergic neurons in the dorsal raphé nucleus (DRN). Paroxetine and sertraline stopped the spontaneous firing of serotonergic neurons at the cumulative dose of 1280 μg/kg, whereas fluvoxamine reached the same effect only at the cumulative dose of 5120 μg/kg. Pretreatment with the 5-HT_1A receptor antagonist tertatolol (1 mg/kg, i.v.) reduced the inhibitory effects of paroxetine, fluvoxamine, and sertraline on the basal activity of serotonergic neurons in the DRN. Administration of tertatolol induced a 15-fold increase in the ED₅₀ for fluvoxamine. The antagonistic effect of tertatolol was much less evident in blocking the inhibitory action exerted by paroxetine and sertraline on the activity of serotonergic neurons. Pretreatment with tertatolol (1 mg/kg, i.v.) potentiated the inhibitory effect of fluvoxamine on the basal activity of VTA dopaminergic neurons. Tertatolol did not affect the inhibitory action exerted by paroxetine and sertraline on these neurons. It is concluded that inhibition of the basal firing rate of dopaminergic neurons in the VTA is a common characteristic of selective serotonin reuptake inhibitors (SSRIs). The effects of SSRIs on VTA dopaminergic cell activity might be relevant for their therapeutic action and may explain the origin of the reported cases of akathisia.     

Calcium-dependent action potentials in mouse spinal cord neurons in cell culture

Following blockade of membrane potassium conductance with tetraethylammonium ions or 3-aminopyridine, long-duration action potentials were recorded from mouse spinal cord neurons in primary dissociated cell culture. The action potentials were calcium-dependent since they: (1) were not blocked by the sodium-channel blocker tetrodotoxin, (2) could be recorded in sodium-free, calcium-containing medium (3) could not be evoked in sodium-containing, calcium-free medium, (4) were blocked by calcium channel blockers manganese and cobalt and (5) had overshot amplitudes that varied linearly with the log of the extracellular calcium concentration (slope of 27.5 mV/decade change in calcium concentration).     

Hydrogen ions have multiple effects on the excitability of cultured mammalian neurons

A neuronal culture system has been developed that has demonstrated to induce myelin formation by added oligodendrocytes. Networks of dissociated dorsal root ganglion neurons were prepared by suppressing non-neuronal cells (i.e. fibroblasts and Schwann cells) with a continuous 2 week exposure to 10^?5 M fluorodeoxyuridine in the culture medium. After drug withdrawal, neuroglial cells were introduced in optic nerve implants from 1–2 week-old rats. These added glial cells migrated extensively over the unensheathed neurities and central myelin was formed by 2 weeks after the implant addition.     

Platelet-activating factor antagonists reduce excitotoxic damage in cultured neurons from embryonic chick telencephalon and protect the rat hippocampus and neocortex from ischemic injury in vivo

The neuroprotective effects of the platelet-activating factor (PAF) antagonists BN 52020 and BN 52021 were determined in a temperature-controlled model of transient forebrain ischemia in the rat (occlusion of both common carotid arteries combined with lowering of the mean arterial blood pressure to 40 mm Hg for 10 min). After 7 days of recirculation, the ischemic neuronal damage was evaluated histologically within the hippocampus and neocortex. Combined pre- and post-treatment with the PAF antagonists (2 × 25 mg/kg, s.c.) significantly reduced the resulting neuronal damage of the CA1 and CA3 hippocampal subfields and of the occipital and parietal cerebral cortex. The two PAF antagonists were also tested for their neuroprotective activity in primary neuronal cultures isoalted from embryonic chick telencephalon. Since an excessive activation of excitatory amino acid receptors is discussed to be of importance for the ischemic brain damage, the cultured neurons were exposed to the excitatory amino acid L-glutamate (1 mM) for a period of 60 min. Twenty hours after the excitotoxic insult, BN 52020- and BN 52021- treated cultured (1–100 μM) showed both a better preserved morphology, as well as a dose-dependent increase in cell viability and protein content compared to the control cultures. Our results demonstrate that the PAF antagonists BN 52020 and BN-52021 have the capacity to protect brain tissue against ischemic neuronal damge independent of hypothermic effects and are also capable of reducing excitotoxic damage of telencephalic neurons from chick embryos cultured in the absence of glial or endothelial cells. We thus propose that PAF plays an important role in the pathophysiology of ischemic/excitotoxic neuronal injury via a direct action on neurons. © 1993 Wiley-Liss, Inc.     

Ketamine protects cultured neocortical neurons from hypoxic injury

The general anesthetic ketamine, which has recently been reported to block the excitation of cortical neurons by N-methyl-d-aspartate (NMDA), was found to markedly reduce neuronal loss in murine neocortical cell cultures exposed to a hypoxic atmosphere or to cyanide. These observations may be relevant to attempts to find pharmacological means of minimizing hypoxic brain damage in the clinical setting.     

Characterization of dopamine receptors on neurons grown in primary dissociated cell culture from ventral mesencephalon of mouse

Mammalian neurons from ventral mesencephalon were grown in primary dissociated cell culture. These cultures were examined for dopamine sensitive adenylate cyclase activity and specific ligand binding of [3H]spiroperidol and [3H]flupenthixol. No stimulation of adenylate cyclase activity by 10 microM dopamine was demonstrable in cell culture homogenates. [3H]Spiroperidol bound to cell culture homogenates with high affinity and was displaced by (+)-butaclamol but not by 5-hydroxytryptamine, suggesting that the [3H]spiroperidol was bound to dopamine receptors. While [3H]flupenthixol binding was also present, it could be displaced by spiroperidol indicating that the dopamine receptor was probably of the D2 subtype. Binding of spiroperidol was proportional to the amount of cell culture homogenate, and was saturable. Are these receptors autoreceptors? The toxin 1-methyl-4-phenylpyridine (MPP+) was used to destroy dopaminergic neurons; spiroperidol binding in these cultures was found to be increased, demonstrating that most of these D2 receptors are not autoreceptors.     

Localization of cholecystokinin-octapeptide-like-immunoreactivity in neurons cultured from rat cerebral cortex

Cholecystokinin-octapeptide-like-immunoreactivity (CCK-8-LI) is demonstrable by the indirect immunofluorescent technique in cerebral cortical neurons cultured from fetal rats. Prominent and widespread staining is seen in widely ramifying cellular processes throughout the cultures. Staining in neuronal cell bodies is enhanced by treatment of the cultures with colchicine prior to fixation and can be seen to extend from the perikaryal region into dendrites and axonal processes. While several morphological types of neurons contain CCK-8-LI, there is a relative preponderance of small and moderate-sized bipolar and, to a lesser extent, multipolar neurons. These findings are in concordance with the findings of several investigators in studies of brain sections. The further finding of frequent groupings of CCK-8 neurons is noted and discussed.     

Synchronization of circadian firing rhythms in cultured rat suprachiasmatic neurons

The circadian clock in mammals is located in the suprachiasmatic nucleus (SCN) which consists of multiple oscillating neurons. Integration of the cellular oscillations is essential for the generation of a single circadian period in the SCN. By using a multielectrode dish (MED), we measured circadian firing rhythms in individual SCN neurons for more than 2 weeks continuously, and examined the involvement of synaptic communication in the synchronization of circadian rhythms. Cross-correlation analysis of spontaneous action potentials revealed that a neuron pair was functionally connected by synapses when their circadian rhythms were synchronized. No correlation was found between the paired neurons whose circadian rhythms were not synchronized. Calcium (Ca2+)-dependent synaptic transmission in the cellular communication was indicated by dose-dependent lengthening of an intercellular spike interval and loss of spike correlation with a Ca2+ channel blocker. Approximately 60% of the SCN neurons in culture were immunoreactive to antibodies against gamma-aminobutyric acid (GABA) or glutamic acid decarboxylase (GAD). Spontaneous firing of all the neurons tested was either increased or decreased by bicuculline, the GABAA receptor antagonist. These findings indicate that synaptic communication plays a critical role in the synchronization of circadian rhythms in individual SCN neurons and the GABAergic transmission is involved in the synchronization mechanism.     

Effects of nerve growth factor on electrical membrane properties of cultured dorsal root ganglia neurons from normal and trisomy 21 human fetuses

Trisomy 21 (Down syndrome) results in abnormalities of electrical membrane properties of cultured human fetal dorsal root ganglion (DRG) neurons; namely, faster rates of depolarization and repolarization of the action potential, and a shortened spike duration. A possible role of nerve growth factor (NGF) in the expression of abnormal electrical membrane properties fetal human DRG neurons from trisomy 21 subjects was examined. DRG neurons obtained from normal and trisomy 21 abortuses of 16–20 weeks gestation were cultured in the presence or absence of 40 nM 7S NGF. After 1 week in culture, action potentials were recorded using the whole cell patch-clamp technique, in current clamp mode. At the testing membrane potential, normal (diploid) neurons grown without NGF showed reduced maximal rates of depolarization (−41.3%) and of repolarization (−31.4%), a decreased spike amplitude (−14.2%) and a prolonged action potential (+49.2%), when compared to normal cells cultured with NGF. Trisomy 21 neurons showed similar changes, but had a greater relative decrease in the rates of action potential depolarization and repolarization. These changes were evident at different membrane potentials. Normal and trisomic DRG neurons cultured without NGF showed differences in action potential parameters similar to those previously described using NGF-supplemented culture medium. These data indicate that NGF can regulate electrical membrane properties in cultured human fetal DRG neurons, but apparently is not responsible for the abnormalities observed in trisomy 21 neurons.     

Dopamine induces apoptosis in cultured rat striatal neurons; possible mechanism of D2-dopamine receptor neuron loss during aging

We examined D₂-dopamine receptor containing neurons in cultures of neonatal rat striatum for apoptosis following dopamine treatment. Exposure to cultures to micromolar concentrations of dopamine resulted in 60–70% killing of D₂-dopamine receptor neurons within 24 hr. We also utilized a double labeling procedure to determine that treatment with dopamine induced apoptosis in D₂-dopamine receptor containing neurons. These results suggest that loss of D₂-dopamine receptor containing neurons during aging could be due to an apoptotic effect of dopamine. J. Neurosci. Res. 47:393–399, 1997. © 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Neonatal mammalian spinal cord neurons and motoneurons in monolayer culture

Previously, embryonic tissues have been used to produce monolayer cultures containing mammalian spinal cord neurons (SCN) and motoneurons (MN) for studies of the pathophysiology of motoneuron diseases. We demonstrate here that viable SCN and MN were observed in dissociated cultures from neonatal rat and mouse. These SCN and MN produced neurites and expressed acetylcholinesterase, neuron-specific enolase and neurofilament protein. These results indicate that cultured postnatal SCN and MN are capable of survival, neurite extension, and phenotypic expression in culture.     

Early ischemia enhances action potential-dependent, spontaneous glutamatergic responses in CA1 neurons

Two types of quantal spontaneous neurotransmitter release are present in the nervous system, namely action potential (AP)-dependent release and AP-independent release. Previous studies have identified and characterized AP-independent release during hypoxia and ischemia. However, the relative contribution of AP-dependent spontaneous release to the overall glutamate released during transient ischemia has not been quantified. Furthermore, the neuronal activity that mediates such release has not been identified. Using acute brain slices, we show that AP-dependent release constitutes approximately one-third of the overall glutamate-mediated excitatory postsynaptic potentials/currents (EPSPs/EPSCs) measured onto hippocampal CA1 pyramidal neurons. However, during transient (2 mins) in vitro hypoxia–hypoglycemia, large-amplitude, AP-dependent spontaneous release is significantly enhanced and contributes to 74% of the overall glutamatergic responses. This increased AP-dependent release is due to hyper-excitability in the presynaptic CA3 neurons, which is mediated by the activity of NMDA receptors. Spontaneous glutamate release during ischemia can lead to excitotoxicity and perturbation of neural network functions.     

Fetal human cortical neurons grown in culture: morphological differentiation, biochemical correlates and development of electrical activity

Cultured fetal human cortical neurons derived from second trimester human fetal cortex were analyzed with regard to their morphological differentiation and expression of cell-specific markers. The culture method was adapted from standardized protocols originally developed for the isolation and culture of rodent oligodendrocytes and astrocytes. This technique takes advantage of the different adhesive properties and stratification of central nervous system cells in vitro. Under these culture conditions fetal human cortical neurons underwent morphological differentiation, expressed neuron-specific markers and voltage- and ligand-gated ion channels. Highly enriched cultures of microglia and astrocytes generated from the same starting material also expressed cell-type specific markers. These cultures serve as a valuable tool for the establishment of normative data and as experimental models for neurodevelopmental and neurodegenerative studies.     

Aromatase activity in cultured brain cells: difference between neurons and glia

At the level of the central nervous system (CNS) of several mammalian and non-mammalian species, estrogens may be intracellularly formed from circulating androgens through the action of the aromatase complex. Estrogenic steroids play a crucial role in organizing and directing certain behavioral and neuroendocrine responses both during the fetal/neonatal life and in adulthood. Biochemical and immunocytochemical studies have shown that the aromatase is particularly concentrated in CNS areas involved in the control of reproductive functions, such as the hypothalamus, the preoptic area and the limbic system; despite this large body of evidence, the exact cellular localization of this enzymatic complex within the different cell populations of the brain is still uncertain. In the experiments described here, the presence of the aromatase has been evaluated in the two main cellular components of the brain: the neurons and the glia. In these experiments, cultures of neurons obtained from the brains of 14-15-day-old rat embryos, mixed glial cells from 1-day-old rats and type 1 astrocytes derived from cultured glial cells, have been utilized. The aromatase has been also evaluated in oligodendrocytes isolated from adult male rat brain by density gradient ultracentrifugation. The aromatase activity has been assayed by an 'in vitro' radiometric method which quantifies the production of tritiated water from [1 beta-3H]-androstenedione as an index of estrogen formation. The validity of the method has been verified both on the placental microsomes and on rat hypothalamic tissue, in which the actual formation of estrogens has also been measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Attenuation by PACAP of glutamate-induced neurotoxicity in cultured retinal neurons

The effects of pituitary adenylate cyclase activating polypeptides (PACAPs: PACAP27, PACAP38) on glutamate-induced neurotoxicity were examined using cultured retinal neurons obtained from 3- to 5-day old Wistar rats. Cell viability was evaluated by double staining with fluorescein diacetate and propidium iodide. Effects of PACAPs on the increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in retinal neurons was investigated using the Ca(2+) image analyzing system with fura-2. The cAMP contents and the mitogen-activated protein (MAP) kinase activity in retinal cultures were measured by radioimmunoassay. Concomitant application of PACAPs (10 nM-1 microM) with glutamate (1 mM) for 10 min inhibited the delayed death of retinal neurons, which was observed 24 h after glutamate (1 mM) treatment in a dose-dependent manner. Protection by PACAPs (100 nM) against glutamate-induced neurotoxicity was antagonized by PACAP6-38 (1 microM), a PACAP antagonist, and H-89 (1 microM), a protein kinase A (PKA) inhibitor. However, PACAPs did not affect the glutamate-induced increase in [Ca(2+)](i), but PACAPs (1-100 nM) increased the cAMP levels in a dose-dependent manner. In addition, activation of MAP kinase by PACAP38 (1 microM) was inhibited by simultaneous application with H-89 (1 microM). These findings suggest that PACAPs attenuate glutamate-induced delayed neurotoxicity in cultured retinal neurons by activating MAP kinase through the activation of cAMP-stimulated PKA.