Presynaptic kainate receptors regulate spinal sensory transmission.

Small diameter dorsal root ganglion (DRG) neurons, which include cells that transmit nociceptive information into the spinal cord, are known to express functional kainate receptors. It is well established that exposure to kainate will depolarize C-fiber afferents arising from these cells. Although the role of kainate receptors on sensory afferents is unknown, it has been hypothesized that presynaptic kainate receptors may regulate glutamate release in the spinal cord. Here we show that kainate, applied at low micromolar concentrations in the presence of the AMPA-selective antagonist (RS)-4-(4-aminophenyl)-1, 2-dihydro-1-methyl-2-propyl-carbamoyl-6,7-methylenedioxyphthalazine++ +, suppressed spontaneous NMDA receptor-mediated EPSCs in cultures of spinal dorsal horn neurons. In addition, kainate suppressed EPSCs in dorsal horn neurons evoked by stimulation of synaptically coupled DRG cells in DRG-dorsal horn neuron cocultures. Interestingly, although the glutamate receptor subunit 5-selective kainate receptor agonist (RS)-2-alpha-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA) (2 micrometer) was able to suppress DRG-dorsal horn synaptic transmission to a similar extent as kainate (10 micrometer), it had no effect on excitatory transmission between dorsal horn neurons. Agonist applications revealed a striking difference between kainate receptors expressed by DRG and dorsal horn neurons. Whereas DRG cell kainate receptors were sensitive to both kainate and ATPA, most dorsal horn neurons responded only to kainate. Finally, in recordings from dorsal horn neurons in spinal slices, kainate and ATPA were able to suppress NMDA and AMPA receptor-mediated EPSCs evoked by dorsal root fiber stimulation. Together, these data suggest that kainate receptor agonists, acting at a presynaptic locus, can reduce glutamate release from primary afferent sensory synapses.

Glutamate receptor-mediated calcium entry in neurons derived from P19 embryonal carcinoma cells.

We have examined the control of calcium elevation by glutamate in neurons derived from the mouse P19 embryonal carcinoma cell line. Following transient exposure to retinoic acid, P19 cells differentiate into neurons that express both NMDA and non-NMDA glutamate receptor subtypes. Fluorescence videomicroscopy using the indicator fura-2 revealed concentration-dependent elevation in cytosolic calcium levels with exposure to NMDA or kainate. Replacement of extracellular sodium with N-methylglucamine significantly reduced the action of kainate. Exposure to high K+ medium also elicited an elevation of cytosolic calcium in P19 cells, which was partially inhibited by the calcium channel antagonist nimodipine. These experiments suggest that the elevation in calcium produced by kainate involves the activation of voltage-gated calcium channels as a consequence of membrane depolarization, in contrast to direct calcium entry through NMDA receptor channels. Whole-cell recordings revealed that P19 NMDA receptors were highly permeable to calcium (PCa/PNa = 5.6 +/- 0.2). In most cells, channels gated by kainate displayed low permeability to calcium; the median permeability ratio, PCa/PNa, was 0.053 (range 0.045 to 0.132). Activation of peak currents by NMDA, glycine, and kainate was half-maximal at 24 microM, 240 nM, and 81 microM, respectively. In addition, cadmium-sensitive currents through voltage-gated calcium channels were recorded in P19 cells bathed in barium/TEA chloride. Staining with antibodies directed against AMPA receptor subunits revealed wide-spread immunoreactivity for anti-GluR-B/C and anti-GluR-B/D. About half of the P19 cells were stained with antibodies selective for GluR-D but there was little or no immunoreactivity for the GluR-A subunit.

Synapse formation and establishment of neuronal polarity by P19 embryonic carcinoma cells and embryonic stem cells.

A number of different cell lines that exhibit a partial neuronal phenotype have been identified, but in many cases the full extent of their neuronal differentiation has not been directly addressed by functional studies. We have used electrophysiology and immunofluorescence to examine the formation of synapses and the development of neuronal polarity by murine embryonic stem (ES) cells and the mouse P19 embryonic carcinoma cell line. Within 2-3 weeks after induction by retinoic acid, subsets of P19 and ES cells formed excitatory synapses, mediated by glutamate receptors, or inhibitory synapses, mediated by receptors for GABA or glycine. In ES-cell cultures, both NMDA and non-NMDA receptors contributed to the excitatory postsynaptic response. Staining with antibodies to growth-associated protein-43 and microtubule-associated protein-2 revealed segregation of immunoreactivity into separate axonal and somato-dendritic compartments, respectively. Consistent with our physiological evidence for synapse formation, intense punctate staining was observed with antibodies to the synaptic vesicle proteins synapsin, SV2, and synaptophysin. These results demonstrate the in vitro acquisition by pluri-potent cell lines of neuronal polarity and functional synaptic transmission that is characteristic of CNS neurons.

Glutamate receptor-mediated currents and toxicity in embryonal carcinoma cells.

While primary neuronal cell cultures have been used to investigate excitotoxicity, development of cell lines exhibiting glutamate receptor-mediated death is desirable. P19 mouse embryonal carcinoma cells, exposed to retinoic acid and plated onto a layer of cultured mouse cortical glial cells, differentiated into neuron-like elements immunoreactive for neurofilaments and neuron-specific enolase. Whole-cell recordings revealed inward currents in response to extracellular application of either NMDA or kainate. The NMDA-induced currents exhibited a voltage-dependent blockade by magnesium, required glycine for maximal activation, and were blocked by the NMDA antagonist dizocilpine. Kainate-induced currents were blocked by the AMPA/kainate receptor antagonist CNQX. Exposure to 500 microM NMDA for 24 h destroyed most P19 cells (EC50 approximately 70 microM); death was prevented by dizocilpine or D-APV. Exposure to 500 microM kainate also resulted in widespread death reduced by CNQX. Thus differentiated P19 cells exhibited both excitatory amino acid responses and vulnerability to excitotoxicity, characteristic of CNS neurons. These cells may provide a genetically open system useful for studying glutamate receptor-mediated phenomena at a molecular level.

Primary culture of identified neurons from the visual cortex of postnatal rats.

We have examined the properties of neurons from the visual cortex of postnatal Long Evans rats in dissociated cell culture. Visual cortex from rat pups 1-15 d old was subjected to enzymatic and mechanical dissociation to yield a suspension of single cells. Neurons plated onto collagen or a feeder layer of astrocytes rapidly extended processes and survived for 4-10 weeks. Antisera to glutamic acid decarboxylase, choline acetyltransferase, and vasoactive intestinal polypeptide stained 22 +/- 2, 2.3 +/- 0.3, and 2.4 +/- 0.2% of all neurons, respectively, suggesting that different neuronal classes survived roughly in proportion to their number in vivo. In order to study a particular identified class of cortical neurons, we prelabeled cells in vivo by retrograde transport of a fluorescent tracer. Neurons in layer V of visual cortex that project to the superior colliculus were labeled after injecting fluorescent latex microspheres into the colliculus. Retrogradely labeled neurons were readily identified immediately after dissociation and throughout the period in vitro. After 2 weeks in culture, labeled cells exhibited many ultrastructural features characteristic of pyramidal neurons in vivo. Intracellular recording techniques were used to evaluate the response properties of labeled layer V neurons, as well as other, unlabeled neurons, to excitatory amino acid agonists and antagonists. Glutamate and aspartate--as well as the synthetic agonists N-methyl-D-aspartate (NMDA), kainate, and quisqualate--excited every cortical neuron tested. The antagonist 2-amino-5-phosphonovaleric acid had no effect on responses to quisqualate and kainate but completely blocked depolarizations due to NMDA and aspartate and reduced depolarizations elicited by low concentrations of glutamate. Kynurenic acid, piperidine dicarboxylic acid, and gamma-D-glutamylglycine antagonized responses to all 5 of the agonists. These results provide evidence that corticocollicular neurons in culture express both NMDA-type and non-NMDA receptors for excitatory amino acids.