Glutamate inhibits GABA excitatory activity in developing neurons.

In contrast to the mature brain, in which GABA is the major inhibitory neurotransmitter, in the developing brain GABA can be excitatory, leading to depolarization, increased cytoplasmic calcium, and action potentials. We find in developing hypothalamic neurons that glutamate can inhibit the excitatory actions of GABA, as revealed with fura-2 digital imaging and whole-cell recording in cultures and brain slices. Several mechanisms for the inhibitory role of glutamate were identified. Glutamate reduced the amplitude of the cytoplasmic calcium rise evoked by GABA, in part by activation of group II metabotropic glutamate receptors (mGluRs). Presynaptically, activation of the group III mGluRs caused a striking inhibition of GABA release in early stages of synapse formation. Similar inhibitory actions of the group III mGluR agonist L-AP4 on depolarizing GABA activity were found in developing hypothalamic, cortical, and spinal cord neurons in vitro, suggesting this may be a widespread mechanism of inhibition in neurons throughout the developing brain. Antagonists of group III mGluRs increased GABA activity, suggesting an ongoing spontaneous glutamate-mediated inhibition of excitatory GABA actions in developing neurons. Northern blots revealed that many mGluRs were expressed early in brain development, including times of synaptogenesis. Together these data suggest that in developing neurons glutamate can inhibit the excitatory actions of GABA at both presynaptic and postsynaptic sites, and this may be one set of mechanisms whereby the actions of two excitatory transmitters, GABA and glutamate, do not lead to runaway excitation in the developing brain. In addition to its independent excitatory role that has been the subject of much attention, our data suggest that glutamate may also play an inhibitory role in modulating the calcium-elevating actions of GABA that may affect neuronal migration, synapse formation, neurite outgrowth, and growth cone guidance during early brain development.

Early synaptogenesis in vitro: role of axon target distance.

In contrast to some previous reports suggesting a delay in synapse formation in vitro, we found that under ideal conditions, most hippocampal and hypothalamic rat neurons were synaptically coupled after 3 or 4 days in vitro. Synaptophysin immunocytochemistry revealed strongly stained presynaptic boutons by 3 days in vitro. Studies with time-lapse laser confocal imaging of FM1-43 revealed that axonal boutons were recycling their synaptic vesicles, an indication of synapse formation, as early as 3 days after plating. To test the hypothesis that neurite outgrowth was enhanced in high-density cultures, thereby increasing the probability of synapse formation, neurons were transfected with the jellyfish green fluorescent protein (GFP) gene. After 2 days in high-density cultures, green fluorescent neurites were about three times longer than in sister neurons plated in low-density cultures. Even in single dishes, GFP-transfected cells in contact with other neurons had neurites that were at least three times longer and grew faster than more isolated cells. Neurons grew longer neurites (+51%) when growing on surface membranes of heat-killed neurons than on polylysine, underlining the importance of plasma membrane contact. Calcium imaging with fura-2 and whole cell recording showed that both GABA and glutamate presynaptic release occurred after 3 or 4 days in vitro in high-density cultures but was absent in low-density cultures at this time. Together, these morphological, cytochemical, and physiological data suggest that the distance an axon must grow to find a postsynaptic partner plays a substantial role in the timing of synapse formation. Although other factors in vitro may also play a role, the distance to a postsynaptic target, which defines the interval during which an axon grows to its target, can probably account for much of the difference in timing of synapse formation previously reported in vitro. A short intercell distance may increase the concentration of limited amounts of trophic factors available to a nearby cell, and once contact is made, a neuronal membrane provides a superior substrate for neuritic elongation.

GABAB receptor-mediated inhibition of GABAA receptor calcium elevations in developing hypothalamic neurons.

In the CNS, gamma-aminobutyric acid (GABA) affects neuronal activity through both the ligand-gated GABAA receptor channel and the G protein-coupled GABAB receptor. In the mature nervous system, both receptor subtypes decrease neural excitability, whereas in most neurons during development, the GABAA receptor increases neural excitability and raises cytosolic Ca2+ levels. We used Ca2+ digital imaging to test the hypothesis that GABAA receptor-mediated Ca2+ rises were regulated by GABAB receptor activation. In young, embryonic day 18, hypothalamic neurons cultured for 5 +/- 2 days in vitro, we found that cytosolic Ca2+ rises triggered by synaptically activated GABAA receptors were dramatically depressed (>80%) in a dose-dependent manner by application of the GABAB receptor agonist baclofen (100 nM-100 microM). Coadministration of the GABAB receptor antagonist 2-hydroxy-saclofen or CGP 35348 reduced the inhibitory action of baclofen. Administration of the GABAB antagonist alone elicited a reproducible Ca2+ rise in >25% of all synaptically active neurons, suggesting that synaptic GABA release exerts a tonic inhibitory tone on GABAA receptor-mediated Ca2+ rises via GABAB receptor activation. In the presence of tetrodotoxin the GABAA receptor agonist muscimol elicited robust postsynaptic Ca2+ rises that were depressed by baclofen coadministration. Baclofen-mediated depression of muscimol-evoked Ca2+ rises were observed in both the cell bodies and neurites of hypothalamic neurons taken at embryonic day 15 and cultured for three days, suggesting that GABAB receptors are functionally active at an early stage of neuronal development. Ca2+ rises elicited by electrically induced synaptic release of GABA were largely inhibited (>86%) by baclofen. These results indicate that GABAB receptor activation depresses GABAA receptor-mediated Ca2+ rises by both reducing the synaptic release of GABA and decreasing the postsynaptic Ca2+ responsiveness. Collectively, these data suggest that GABAB receptors play an important inhibitory role regulating Ca2+ rises elicited by GABAA receptor activation. Changes in cytosolic Ca2+ during early neural development would, in turn, profoundly affect a wide array of physiological processes, such as gene expression, neurite outgrowth, transmitter release, and synaptogenesis.

Glutamate hyperexcitability and seizure-like activity throughout the brain and spinal cord upon relief from chronic glutamate receptor blockade in culture.

Cortical structures such as the hippocampus and cerebral cortex are considered to be particularly susceptible to seizure and epileptiform electrical activity and, as such, are the focus of intense investigation relative to hyperexcitability. To determine whether parallel glutamate-mediated hyperexcitability and seizure-like activity in the rat can be generated by neurons irrespective of their origin within the CNS, we maintained cells from the spinal cord,hippocampus, olfactory bulb, striatum, hypothalamus, and cortex in the long-term presence of glutamate receptor antagonists 2-amino-5-phosphonovalerate and 6-cyano-7-nitroquinoxaline-2-3-dione. After removal of chronic (three to 11 weeks) glutamate receptor block, whole-cell patch-clamp recordings from current-clamped neurons (n = 94) revealed an immediate increase in large excitatory postsynaptic potentials and a depolarization of 20-35 mV that was often sustained for recording periods lasting 5 min (54% of 66 neurons from all six areas). The intense activity was not seen in age-matched control neurons not subjected to chronic glutamate receptor block. Selective blockade of ionotropic glutamate receptors showed that the hyperexcitability was due to an enhanced response through both AMPA/kainate and N-methyl-D-aspartate receptors. Relief from chronic glutamate receptor block also increased inhibitory activity, as revealed by an increase in inhibitory postsynaptic currents while neurons were voltage-clamped at -25 mV. These inhibitory postsynaptic currents could be blocked with bicuculline, indicating that they were mediated by an enhanced GABA release. This enhanced GABA activity reduced, but did not eliminate, the glutamate-mediated hyperactivity, shown by an increase in both intracellular Ca2+ and excitatory electrical activity when bicuculline was added. When the glutamate receptor block was removed, cells (n > 1000) from all six regions showed exaggerated Ca2+ activity, characterized by abnormally high increases in intracellular Ca2+, rising from basal levels of 50-100 nM up to 150-1600 nM. Cd2+ eliminated the hyperexcitability by blocking Ca2+ channels, and reducing excitatory transmitter release and response. Fura-2 digital imaging revealed Ca2+ oscillations with periods ranging from 4 to 60 s. Ca2+ peaks in oscillations in oscillations were synchronized among most neurons recorded simultaneously. That synchronization was dependent on a mechanism involving voltage-dependent Na+ channels was demonstrated with experiments with tetrodotoxin that blocked Ca2+ rises and synchronous cellular behavior. Removal of the glutamate receptor antagonists resulted in the glutamate-mediated death of 44% of the cells after 23 days of chronic block and 82% cell death after 40 days of chronic block. Nimodipine substantially reduced cell death, indicating that one mechanism responsible for the enhanced cell death after relief from chronic glutamate receptor block was increased intracellular Ca2+ entry through L-type voltage-gated calcium channels. These data indicate that glutamate is released by neurons from all areas studied, including the spinal cord. Sufficient amounts of glutamate can be released from axon terminals from all areas to cause cell hippocampal and cortical neurons, but also by neurons from any of the brain regions tested after chronic deprivation of glutamate receptor stimulation during development. This hyperexcitability is mediated by glutamatergic mechanisms independent of the specific excitatory connections existing in vivo. The epileptiform activity of neurons from one region is indistinguishable from that of another in culture, underlining the importance of synaptic connections in vivo that define the responses characteristic of neurons from different brain regions.

Growth cone calcium elevation by GABA.

Cytoplasmic calcium plays a key role in neurite growth. In contrast to previous work suggesting that gamma aminobutyrate's (GABA) role in regulating growth cone calcium is primarily to antagonize the effects of glutamate, we report that GABA can act in an excitatory manner on developing hypothalamic neurites, independently raising calcium in growing neurites and their growth cones. Time-lapse digital video and confocal laser microscopy with the calcium-sensitive dyes fluo-3 and fura-2 were used to study the influence of GABA on neurite calcium levels. GABA (10 microM) evoked a calcium rise in both bicarbonate- and Hepes-based buffers. The calcium rise was greatly reduced after chloride transport was blocked. GABA raised calcium by stimulating the cell body, resulting in an increase in calcium throughout the neuronal cell body and dendritic arbor. GABA also acted locally, stimulating a neuritic calcium rise only in a single dendrite or growth cone. In some neurites and growth cones during early development, GABA generated a greater calcium rise than did glutamate. Bicuculline, a GABAA receptor antagonist, reduced calcium levels in neurites of young synaptically coupled neurons, indicating that ongoing synaptic release of GABA raised neuritic calcium. These data suggest that during early development, GABA may play a significant role in regulating process growth and modulating the formation of early connections in the hypothalamus. Our data support the hypothesis that GABA receptors are functionally active and may play a calcium regulating role similar to that of glutamate in neuronal development. This is particularly true in early development, as later in development GABA's role becomes more inhibitory, and glutamate plays the primary excitatory role.

Excitatory actions of GABA after neuronal trauma.

GABA is the dominant inhibitory neurotransmitter in the CNS. By opening Cl- channels, GABA generally hyperpolarizes the membrane potential, decreases neuronal activity, and reduces intracellular Ca2+ of mature neurons. In the present experiment, we show that after neuronal trauma, GABA, both synaptically released and exogenously applied, exerted a novel and opposite effect, depolarizing neurons and increasing intracellular Ca2+. Different types of trauma that were effective included neurite transection, replating, osmotic imbalance, and excess heat. The depolarizing actions of GABA after trauma increased Ca2+ levels up to fourfold in some neurons, occurred in more than half of the severely injured neurons, and was long lasting (>1 week). The mechanism for the reversed action of GABA appears to be a depolarized Cl- reversal potential that results in outward rather than inward movement of Cl-, as revealed by gramicidin-perforated whole-cell patch-clamp recording. The consequent depolarization and resultant activation of the nimodipine sensitive L- and conotoxin-sensitive N-type voltage-activated Ca2+ channel allows extracellular Ca2+ to enter the neuron. The long-lasting capacity to raise Ca2+ may give GABA a greater role during recovery from trauma in modulating gene expression, and directing and enhancing outgrowth of regenerating neurites. On the negative side, by its depolarizing actions, GABA could increase neuronal damage by raising cytosolic Ca2+ levels in injured cells. Furthermore, the excitatory actions of GABA after neuronal injury may contribute to maladaptive signal transmission in affected GABAergic brain circuits.

Adenosine pre- and postsynaptic modulation of glutamate-dependent calcium activity in hypothalamic neurons.

1. Within the hypothalamus, adenosine has been reported to influence temperature regulation, sleep homeostasis, and endocrine secretions. The effects of adenosine on hypothalamic neurons have not been studied at the cellular level. Adenosine (5 nM-30 microM) showed no influence on intracellular Ca2+ or electrical activity in the presence of glutamate receptor antagonists D-2-amino-5-phosphonovalerate and 6-cyano-7-nitroquinoxaline-2,3-dione; consequently, we examined the role of adenosine in modulating the activity of glutamate in cultured hypothalamic neurons (n > 1,700) with fura-2 Ca2+ digital imaging and whole cell patch-clamp electrophysiology in the absence of glutamate receptor block. 2. When glutamate receptors were not blocked, adenosine (1-30 microM) and the selective adenosine A1 receptor agonist N6-cyclopentyl adenosine (CPA; 5 nM-1 microM) caused a large reduction in intracellular Ca2+ and electrical activity, suggesting that glutamate neurotransmission was critical for an effect of adenosine to be detected. Neuronal Ca2+ levels were reversibly depressed by CPA (50 nM), with a maximum depression of 90%, and these effects were blocked by coadministration of the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). 3. Ca2+ levels in immature neurons before the time of synaptogenesis were not affected by adenosine. Adenosine A1 receptor activation suppressed glutamate-mediated Ca2+ activity in neurons in vitro 8 to 73 days. 4. Adenosine (1 or 10 microM) caused a hyperpolarization of membrane potential and a reduction of large postsynaptic potentials arising from endogenously released glutamate. The administration of low concentrations of CPA (5 nM) decreased the frequency of glutamate-mediated, neuronally synchronized Ca2+ transients and the frequency of postsynaptic potentials. 5. To compare the relative effects of adenosine on hypothalamic neurons with cells from other brain regions, we assayed the effects of CPA on glutamate-mediated Ca2+ in hippocampal and cortical cultures. CPA (50 nM) reversibly depressed glutamate-mediated Ca2+ rises in hypothalamic neurons by 35%, compared with 54% in hippocampal neurons and 46% in cortical neurons. 6. If it does play a functional role, adenosine should be released by hypothalamic cells. In some neurons the adenosine A1 receptor antagonists cyclopentyltheophylline or DPCPX caused an increase in intracellular Ca2+, suggesting that adenosine was secreted by hypothalamic cells, tonically depressing glutamate-enhanced neuronal Ca2+. 7. To determine whether adenosine could exert a postsynaptic effect, we coapplied it with glutamate agonists in the presence of tetrodotoxin. Within subpopulations of hypothalamic neurons, adenosine and CPA either inhibited (18% of total neurons) or potentiated (6% of total neurons) responses to glutamate, N-methyl-D-aspartate, and kainate by > or = 20%. 8. In contrast to the modest effects found in neurons, responses of hypothalamic astrocytes to the application of glutamate or the metabotropic glutamate receptor agonist (+/-)-trans-1-amino-1,3-cyclopentanedicarboxylic acid were strongly potentiated by adenosine (mean +225%) and CPA. 9. Together, these findings suggest that adenosine exerts a major presynaptic effect and a minor postsynaptic effect in the modulation of glutamate neurotransmission in the hypothalamus, where it can play a significant role in blocking a large part of the glutamate-induced Ca2+ rise. In the absence of glutamate transmission, adenosine has relatively little effect on either neuronal intracellular Ca2+ or electrical activity.

GABA neurotransmission in the hypothalamus: developmental reversal from Ca2+ elevating to depressing.

GABA is the primary inhibitory transmitter of the adult hypothalamus, synthesized by many neurons and found in 50% of the presynaptic boutons. GABA causes a decrease in Ca2+ in mature hypothalamic neurons in vitro by depressing cellular activity through opening Cl- channels. Despite the early expression of GABAA receptors in the embryonic hypothalamus (E15), the cellular function of GABA in the developing hypothalamus has received little attention. In the present study the role of GABA in modulating intracellular Ca2+ in developing hypothalamic neurons was studied with fura-2 digital imaging. GABA (0.5-500 microM) applied to embryonic hypothalamic neurons elicited a dramatic and rapid increase in intracellular Ca2+ This Ca2+ rise could be completely blocked by the GABAA antagonist bicuculline (20 microM) and persisted in the presence of tetrodotoxin (1 microM). The Ca2+ elevation induced by GABA was greater than that of equimolar concentrations of the excitatory transmitter glutamate in early development. The number of E15 neurons that responded to GABA with a Ca2+ rise increased during the first few days of culture, reaching 78% after 4 d in vitro. The Ca2+ rise was 87% blocked by cadmium (100 microM) and 85% blocked by nimodipine (1 microM), indicating that the mechanism of Ca2+ increase was primarily via L-type voltage operated Ca2+ channels. Addition of bicuculline to synaptically coupled cultures caused a significant decrease in Ca2+ 4-10 d after culturing, indicating hypothalamic neurons were secreting GABA at an early age of development, and that sufficient GABA was released to elicit an increase in Ca2+. This effect was seen even after blocking all glutamatergic activity with glutamate receptor antagonists. In contrast, GABA elicited no Ca2+ rise in older neurons (> 18 d in vitro), and the action of bicuculline reversed and caused a large increase in Ca2+ in spontaneously active neurons. Similar findings were obtained in cultures enriched in GABAergic neurons from the suprachiasmatic nucleus. To determine if the Ca2+ stimulating role of GABA on developing neurons was restricted to the hypothalamus and a few other regions, or whether it might exist throughout the brain, we examined the Ca2+ responses in cultured olfactory bulb, cortex, medulla, striatum, thalamus, hippocampus, and colliculus. The majority (75%) of developing neurons from each region showed a Ca2+ rise in response to GABA. Together these data suggest that GABA elevates Ca2+ in developing, but not mature, neurons from the hypothalamus and all other brain regions examined.(ABSTRACT TRUNCATED AT 400 WORDS)

Embryonic hypothalamic expression of functional glutamate receptors.

Glutamate can play a number of roles in the developing brain, including modulation of gene expression, cell motility, neurite growth and neuronal survival, all critical for the final organization and function of the mature brain. These functions are dependent on the early expression of glutamate receptors and on glutamate release in developing neurons. This subject has received little attention in the hypothalamus, despite glutamate's critical role as an excitatory transmitter in hypothalamic control of circadian rhythms, endocrine secretion, temperature regulation, and autonomic control. A total of 10,922 rat hypothalamic neurons were studied with digital Ca2+ imaging with the ratiometric dye fura-2 to examine their responses to glutamate receptor agonists and antagonists during embryonic development and maturation in vitro. Functional glutamate receptors were found very early in development (embryonic day 15-E15) with both Ca2+ imaging and with patch clamp recording. This is a time when the hypothalamus is beginning to undergo neurogenesis. Ca2+ responses from N-methyl-D-aspartate receptors developed later than those from non-N-methyl-D-aspartate ionotropic receptors that responded to kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate. The responses of immature E15 cells after one day in vitro were compared with more mature cells after six days in vitro to examine the response to repeated 3 min applications of 100 microM kainate (n = 108). Immature cells showed similar Ca2+ rises (+232nM Ca2+) with each kainate stimulation. In contrast, more mature cells showed an initial Ca2+ rise of 307 nM, with the second rise only to 147 nM above the initial baseline. Immature cells more quickly returned to their pre-kainate baseline than did older cells. The expression of metabotropic glutamate receptors was studied with the selective agonist trans-1-amino-cyclopentyl-1,3-dicarboxylic acid and with glutamate stimulation in the absence of extracellular Ca2+ and presence of 1 mM EGTA. After five days in vitro. E16 astrocytes showed a greater response than did neurons to conditions that would activate the metabotropic glutamate receptor. A dramatic increase in the percentage of cells that responded to N-methyl-D-aspartate was found after only a few days in culture. Only a small number of E15 cells studied on the day of culture (4% of 694 cells) showed a response to 100 microM N-methyl-D-aspartate. Thirty-eight percent of 120 E18 cells cultured for one day in vitro showed an N-methyl-D-aspartate response.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium hyperexcitability in neurons cultured with glutamate receptor blockade.

1. The effects of culturing hypothalamic neurons in glutamate receptor antagonists were studied with fura-2 Ca2+ digital imaging of groups of synaptically coupled neurons. Removal of D-2-amino-5-phosphonovalerate (AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) from cultures chronically blocked for periods of 14-188 days caused a dramatic increase in neuronal Ca2+ to abnormally high levels 5- to- 10 fold greater than the normal intracellular levels of 50-100 nM. In most cases AP5/CNQX removal initiated spontaneous synchronized Ca2+ oscillations. 2. Ca2+ rises and oscillations were blocked by the reintroduction of AP5/CNQX or by the addition of tetrodotoxin to block action potentials. These data indicate that hypothalamic neurons were the source of the excitatory transmitter that activated glutamate receptors and consequently led to the Ca2+ hyperexcitability. 3. The Ca2+ spike amplitude and frequency increased in response to the removal of Mg2+ from the perfusion solution to facilitate N-methyl-D-aspartate (NMDA) receptor responses. Picrotoxin, a GABAA-receptor blocker, also increased Ca2+ activity. 4. Blocking either NMDA (with AP5) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-type (with CNQX) glutamate receptors reduced the level of Ca2+, but blocking both types was necessary for chronically blocked neurons to return to their basal Ca2+ level. 5. The survival of a large percentage of chronically blocked neurons was dependent on the presence of glutamate receptor blockade. Removal of AP5/CNQX from the tissue culture medium induced an immediate increase in Ca2+ levels in the majority of chronically blocked neurons and, with prolonged withdrawal of AP5/CNQX (3 h), 50% of the neurons lost the immediate ability to regulate internal Ca2+ levels. Excitotoxic cell death was induced in 40% of the neurons within 40 h of the removal of AP5/CNQX from neurons chronically blocked for 30 days. The number of neurons that survived for 70 days doubled when cultures were maintained in AP5/CNQX. 6. Relative to control cultures of the same period in vitro, chronically blocked neurons showed an enhanced Ca2+ influx when stimulated with the glutamate receptor agonists kainate (+70%), NMDA (+62%), or glutamate (+34%) in the presence of tetrodoxin. When the data from control and chronically blocked cultures stimulated with glutamate receptor agonists were pooled, without exception all the smallest responses were found in the control neurons. Compared with controls, chronically blocked neurons showed an exaggerated response to glutamate in the presence of nimodipine, indicating that Ca2+ hyperexcitability was not due to changes in voltage activated L-type Ca2+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)