Bekkers JM, Stevens CF. Arancio et al. (1995)found that long-lasting potentiation can be induced reliably at synapses between pairs of hippocampal neurons in tradition by high-frequency activation of the presynaptic neuron during brief removal of Mg2+ from your extracellular answer, which unblocks the NMDA receptor channels. This process is effective even when both cells are held under ruptured whole-cell voltage clamp, permitting injection of substances into either neuron. Further experiments using this method have suggested that, during induction of the potentiation, a retrograde messenger, NO, is definitely synthesized in the postsynaptic neuron by a Ca2+-dependent enzyme, NO-synthase, and diffuses to the presynaptic terminals, where it generates a long-lasting increase in transmitter launch (O’Dell et al., 1991; Arancio et al., 1996). NO appears to take action by stimulating soluble guanylyl cyclase leading to the production of cGMP, and injection of cGMP into the presynaptic (but not the postsynaptic) neuron generates activity-dependent long-lasting potentiation (Arancio et al., 1995). However, cGMP might take action through several pathways besides cGK, including 7ACC2 cyclic nucleotide-gated channels and phosphodiesterases (Lincoln and Cornwell, 1993), both of which are also present in hippocampal neurons (Repasko et al., 1993; Kingston et al., 1996; Bradley et al., 1997). We have therefore now investigated the possible presynaptic part of cGK during the induction of long-lasting potentiation in hippocampal cell tradition. MATERIALS AND METHODS Sprague Dawley rats (Hilltop Laboratory, Scottdale, PA) were housed and cared for in accordance with the guidelines of Columbia University or college. Dissociated cell ethnicities of hippocampal neurons from postnatal day time 1 animals were prepared as explained previously (O’Dell et al., 1991). The bath and electrode solutions were also as explained previously (Arancio et 7ACC2 al., 1995). We examined monosynaptic EPSCs between pairs of pyramidal-shaped neurons 7C21 d after plating. Both the presynaptic and postsynaptic neurons were managed under ruptured whole-cell voltage clamp throughout LIFR the experiments, and the holding currents and input resistances of both cells were checked for constancy. EPSCs were evoked once every 10 sec by applying a 10 msec positive 7ACC2 voltage step to the presynaptic neuron of adequate amplitude to elicit an inward current in that neuron. Currents were recorded with an Axopatch 1A and Axopatch 200B (Axon Devices, Foster City, CA) and filtered at 1 kHz. EPSC amplitudes were measured automatically between the peak and the mean during the interval just before the start of the EPSC using the pClamp system from Axon Devices. A peptide inhibitor of cGK (synthesized at Albert Einstein College of Medicine), a peptide comprising the same amino acids with their sequence scrambled (synthesized at Columbia University or college), or a peptide inhibitor of cAMP-dependent protein kinase (Upstate Biotechnology, Lake Placid, NY) were included in the presynaptic or postsynaptic electrode answer throughout the experiments. The isozyme of cGK type I (Promega, Madison, WI) was delivered to the tip of the electrode by a fast internal perfusion method that has been explained previously (Arancio et al., 1995). All other chemicals were from Sigma (St. Louis, MO) unless normally indicated. Data are demonstrated as mean SEM percent of the baseline EPSC amplitude. The data were analyzed by a two-way ANOVA (treatment and time) followed by planned pairwise comparisons of the treatments overall and at each time point. Hippocampi microdissected from male Sprague Dawley rats (150C200 gm, anesthetized with Nembutal) were homogenized in PBS comprising protease inhibitors (5 g/ml leupeptin, 1.5 mm benzamidine, 200 U/ml aprotinin, 2 g/ml pepstatin A, 10 g/ml PMSF, and 1 mm EDTA), and then the homogenates were mixed with SDS-PAGE quit solution. Cultured hippocampal cells were scraped collectively directly in quit answer. Samples were analyzed by 8% SDS-PAGE and Western blotting with ECL detection (Amersham Pharmacia Biotech, Piscataway, NJ) using cGK I antiserum diluted 1:3000 or affinity-purified cGK II antibody (Markert et al., 1995) diluted 1:1000. In independent experiments, the immunolabeling specificity of the cGK antibodies was shown by antibody preabsorption with recombinant cGK I or cGK II purified from Sf9 cells. Total RNA from cultured hippocampal neurons was isolated using Trizol Reagent from Existence Technologies (Grand Island, NY), and reverse transcription (RT) was performed using a kit from Life Systems with oligo-dT priming. For cDNA amplification, the primers utilized for human being cGK I were.