We have developed "pure" neuronal cultures (< 1% astrocytes) from mouse neocortex to study the effect of glial cells on the response of neurons to injury. Cortical neurons were found to require glial-conditioned medium to survive. Immature neurons, 2-4 d in vitro, deprived of glial-conditioned medium, underwent apoptosis over 48 hr, as suggested by condensed nuclear morphology, DNA fragmentation, and protection by inhibition of macromolecular synthesis. Apoptosis induced by trophic factor deprivation has been described for other neuronal populations, such as superior cervical ganglion and dorsal root ganglion cells. Cortical neurons in pure culture provide another neuronal population for the study of apoptosis induced by trophic factor deprivation. We then studied the interaction of neurons and glia under excitotoxic conditions. Experiments on mature cultures showed that pure neuronal cultures were at least 10-fold more sensitive to acute glutamate exposure than were neuronal-glial ("mixed") cocultures. The difference in sensitivity between pure neurons and mixed cultures was reduced when mixed cultures were treated with the glutamate uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid (trans-PDC). In 24 hr exposure to N-methyl-D-aspartate (NMDA), or oxygen, glucose deprivation, pure neurons were more sensitive than mixed cultures; trans-PDC again increased the sensitivity of mixed cultures to nearly that of pure neuronal cultures. In contrast, mixed and pure neuronal cultures exposed to NMDA for 10 min, or to kainate for 24 hr, had similar injury dose-response curves, suggesting that glial glutamate uptake is a less important protective mechanism in these excitotoxic injuries. Surprisingly, pure neurons were less sensitive than mixed cultures to (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) toxicity at concentrations up to 100 microM. This does not reflect astrocyte toxicity, as AMPA at concentrations to 1 mM did not injure astrocyte cultures. Glial cultures showed increased levels of glutamate in the extracellular medium in response to exposure to AMPA, but not NMDA or kainate. However, pure neuronal and mixed cultures exposed to the same concentration of AMPA did not have elevated levels of glutamate in the media. We found that glia were generally neuroprotective under excitotoxic conditions, likely through their ability to clear extracellular glutamate. However, the presence of glia exacerbated AMPA neurotoxicity.

Our results confirm earlier findings that clinically relevant concentrations of volatile anesthetics depress glutamate-mediated synaptic transmission. The observed increases in synaptic facilitation support recent findings from biochemical and electrophysiologic studies indicating presynaptic sites of action contribute to anesthetic-induced depression of excitatory transmission. This anesthetic-induced reduction in glutamate release would contribute to the central nervous system depression associated with anesthesia by adding to postsynaptic depressant actions on glutamate receptors.

1 Prostanoid receptor-mediated sensitization, or excitation, of sensory nerve ®bres contributes to the generation of hyperalgesia. To characterize the prostanoid receptors present on sensory neurones, biochemical assays were performed on primary cultures of adult rat dorsal root ganglia (DRG) and the F-11 (embryonic rat DRG6neuroblastoma hybrid) cell line. 2 In DRG cultures, the IP receptor agonists, cicaprost and carbaprostacyclin (cPGI 2 ) stimulated cyclic AMP accumulation. Prostaglandin E 2 (PGE 2 ) also increased cyclic AMP levels, but to a lesser extent, while carbocyclic thromboxane A 2 (cTxA 2 ), PGD 2 and PGF 2a had negligible e ects. The rank order of agonist potency was cicaprost 4PGE 2 =BMY45778=cPGI 2 =PGI 2 . In the F-11 cells, the rank order of agonist potency for the stimulation of cyclic AMP accumulation was: cicaprost4iloprost=cPGI 2 =P-GI 2 =BMY457784PGE 2 =cTXA 2 . In DRG cultures, cicaprost induced signi®cantly more accumulation of inositol phosphates than PGE 2 . 3 To examine the e ects of prostanoids on C-®bre activity, extracellular recordings of d.c. potentials from the rat isolated vagus nerve were made with the`grease-gap' technique. PGI 2 (0.1 nM ± 10 mM) produced the largest depolarizations of the nerve. The rank order of agonist potency was: PGI 2 =cPGI 2 =PGE 1 4cTXA 2 4PGE 2 =PGD 2 =TXB 2 4PGF 2a . 4 Prior depolarization of nerves with either forskolin (10 mM) or phorbol dibutyrate (1 mM) alone signi®cantly reduced the response to PGI 2 (10 mM), while simultaneous application of both forskolin and phorbol dibutyrate attenuated PGI 2 responses almost completely. 5 Putative EP 1 and/or TP receptor-selective antagonists had no e ect on the responses to PGI 2 , cPGI 2 or PGE 2 in the three preparations studied. 6 Collectively, these data are consistent with a positive coupling of IP receptors to both adenylyl cyclase and phospholipase C in sensory neurones. These ®ndings suggest that IP receptors play a major role in the sensitization of rat sensory neurones.

Inhibitors of the voltage-gated sodium channel Na V 1.7 are being investigated as pain therapeutics due to compelling human genetics. We previously identified Na V 1.7-inhibitory peptides GpTx-1 and JzTx-V from tarantula venom screens. Potency and selectivity were modulated through attribute-based positional scans of native residues via chemical synthesis. Herein, we report JzTx-V lead optimization to identify a pharmacodynamically active peptide variant. Molecular docking of peptide ensembles from NMR into a homology model-derived Na V 1.7 structure supported prioritization of key residues clustered on a hydrophobic face of the disulfide-rich folded peptide for derivatization. Replacing Trp24 with 5-Br-Trp24 identified lead peptides with activity in electrophysiology assays in engineered and neuronal cells. 5-Br-Trp24 containing peptide AM-6120 was characterized in X-ray crystallography and pharmacokinetic studies and blocked histamine-induced pruritis in mice after subcutaneous administration, demonstrating systemic Na V 1.7-dependent pharmacodynamics. Our data suggests a need for high target coverage based on plasma exposure for impacting in vivo end points with selectivity-optimized peptidic Na V 1.7 inhibitors.

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