Biomedical subjects

K Obrietan

Publications and source records attributed to K Obrietan.

22 records · Page 2Linked to original sources

PubMed1995

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)

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PubMed1995

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)

2-Amino-5-phosphonovalerate↗

PubMed1995

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.

Adenosine↗

PubMed1995

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)

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