Characterization of opioid receptor types modulating acetylcholine release in septal regions of the rat brain.

Presynaptic opioid receptors of the delta- and mu-types have been shown to inhibit the release of acetylcholine (ACh) in the rat striatum and hippocampus, respectively, but it is unknown whether opioid receptors modulate the release of ACh also in the region of origin of the hippocampal cholinergic innervation, the septum. To answer this question, slices (350 microm) of the medial septal area and of the diagonal band of Broca, as well as (for comparison) of the hippocampus, were prepared from adult male Wistar rats. The slices were incubated with [3H]choline, superfused in the presence of hemicholinium-3 (10 microM) and stimulated twice (S1, S2) by electrical fields (360 pulses, 3 Hz, 2 ms, 60 mA); opioid receptor agonists were present during S2. The preferential mu-agonist [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO) inhibited the evoked ACh release by maximally about 40% in hippocampal slices and acted even more strongly in the medial septal area, or the diagonal band of Broca (about 60% or 75% maximal inhibition, respectively). These effects were reduced or abolished by the preferential mu-antagonist naloxone, which showed no effects when given alone. Using naloxone in the presence of a cocktail of peptidase inhibitors, no evidence for an endogenous tone of opioid peptides was found in the medial septal area, diagonal band of Broca or the hippocampus. Using the preferential delta-agonist [D-Pen2, D-Pen5]enkephalin (DPDPE) and the delta-antagonist naltrindole, a delta-opioid receptor inhibiting evoked ACh release was clearly detectable both in the medial septal area and the diagonal band of Broca, but not in the hippocampus, whereas the preferential kappa-agonist trans-3,4-dichloro-N-methyl-N-[2(1-pyrrolidinyl)cyclo-hexyl] benzeneacetamide (U50,488H) had only weak or no effects. In addition to the functional experiments, double in-situ hybridization studies were performed, in which cells containing mRNA for choline acetyltransferase (ChAT) were labeled by an antibody-linked enzymatic staining procedure, whereas mRNAs for mu- or delta-opioid receptors were detected with radioactive probes. These experiments revealed that in the septal region mainly mu-opioid receptors were expressed by neurons positive for ChAT mRNA, whereas in the rat striatum the expression of delta-opioid receptors prevailed in those neurons. We conclude that in the septal area of the rat brain, in contrast to the rat striatum and hippocampus, both presynaptic mu- and delta-opioid receptors modulate the evoked release of ACh. Whether presynaptic mu- and delta-opioid receptors occur on the same or on different septal cells or axon terminals remains to be clarified.

Stable expression of recombinant AMPA receptor subunits: binding affinities and effects of allosteric modulators.

Homomeric AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-type glutamate receptors (GluRs) were stably expressed in kidney cells from cDNAs encoding GluR1 flop, GluR2 flip, GluR2 flop, and GluR3 flop subunits. The recombinant receptors were of the expected size and showed functional properties in whole-cell recording as previously reported. [3H]AMPA binding to all subunits was increased to a similar extent by the chaotropic ion thiocyanate (SCN-). Significant differences were found in the Scatchard plots, however, which were linear and of high affinity for GluR1 and -3 receptors (K(D) values of 33 and 52 nM, respectively) but showed curvature for GluR2 receptors, indicating the presence of two components with distinct affinities. As with brain AMPA receptors, solubilization of GluR2 receptors reduced the number of lower-affinity sites and correspondingly increased the number of higher-affinity sites. The sulfhydryl reagent p-chloromercuriphenylsulfonic acid, which increases binding to brain receptors, produced only minor changes except in the case of GluR2 flip. These results indicate that GluR2, among the subunits examined here, most closely resembles the native AMPA receptors in brain membranes. [3H]AMPA binding was inhibited in a noncompetitive manner by two drugs that change the desensitization kinetics of the AMPA receptor. In agreement with physiological observations, the apparent affinity of cyclothiazide for GluR2 flip (EC50 = 7 microM) was higher than that for receptors made of flop subunits (49-130 microM). In contrast, BDP-37, a member of the benzamide family of drugs, exhibited a lower potency for GluR2 flip (58 microM) than for any of the flop isoforms (18-40 microM). These results predict that the action of centrally active AMPA-receptor modulators varies across brain regions depending on their flip/flop composition.

Distinct distributions of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits and a related 53,000 M(R) antigen (GR53) in brain tissue.

Polyclonal antibodies against specific carboxy-terminal sequences of known alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits (GluR-4) were used to screen regional homogenates and subcellular fractions from rat brain. Affinity purified anti-GluR1 (against amino acids 877-899), anti-GluR2/3 (850-862), and anti-GluR4a and anti-GluR4b (868-881) labeled distinct subunits with the expected molecular weight of approximately 105,000. These antigens were shown to have distinct distributions in the brain. While GluR2/3 epitopes had a distribution profile similar to that of the presynaptic marker synaptophysin, GluR1 was notable for its abundance in the hippocampus and its relatively low density in neocortical areas, and GluR4 was highly enriched in cerebellar tissue. An additional antigen (glutamate receptor-related, GR53) of lower molecular weight (50,000-59,000) was recognized in rat, human, frog, chick and goldfish brain samples by anti-GluR4a as well as by anti-GluR1 at, an antibody that specifically recognizes the extracellular aminoterminal domain of GluR1 (amino acids 163-188). Both antibodies also labeled antigens of approximately 105,000 mol. wt in brain tissue from all species tested. The approximately 53,000 mol. wt antigen was concentrated 10-20-fold in synaptic membranes vs homogenates across rat brain regions. Both the 105,000 and the 53,000 mol. wt proteins were also concentrated in postsynaptic densities, and neither of the two antigens were evident in seven non-brain tissue samples. These data indicate that AMPA receptors have regionally different subunit combinations and that some AMPA receptor composites include proteins other than the conventional 105,000 mol. wt GluR subunits.

Relative concentrations and seizure-induced changes in mRNAs encoding three AMPA receptor subunits in hippocampus and cortex.

In situ hybridization was used to determine 1) the relative concentrations of mRNAs encoding different subunits of the alpha-amino 3-hydroxy-5-methyl-4- isoxazolepropionate receptor family in select regions of rat forebrain and 2) whether limbic seizures alter the balances of the subunit mRNAs. GluR1 and GluR2 mRNA levels were about equal and were much greater than GluR3 mRNA levels in the principal neurons of each hippocampal subdivision. Probable interneurons in hippocampal molecular layers had much higher levels of GluR1 mRNA than of either GluR2 or GluR3 mRNA. Pyramidal cell layers in neo- and paleocortex had a balance of mRNAs that was significantly different from the balance in hippocampus: GluR1 mRNA and GluR3 mRNA levels were about equal and were substantially lower than those of GluR2 mRNA. Lesion-induced limbic seizures caused transient changes in mRNA levels that were differentiated with regard to subunit and brain region. All three mRNAs were decreased in the pyramidal layers of cortex, and changes in hippocampal pyramidal cells were smaller. Seizure-induced changes in granule cells of the dentate gyrus differed from all other regions examined: GluR1 mRNA was reduced to a greater degree than GluR2 mRNA, whereas GluR3 mRNA content was markedly increased. These data strongly suggest that the subunit composition of alpha-amino 3-hydroxy-5-methyl-4-isoxazolepropionate receptors differs significantly between areas of the cortical telencephalon. Furthermore, the data indicate that aberrant patterns of physiological activity differentially influence the expression of subunit mRNAs in a region-specific and/or cell-type-specific manner.