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Endogenous opioid peptides modulate neural transmission in the hippocampus. Procnkephalin-derived peptides have been demonstrated to act at mu and delta opioid receptors to inhibit GABA release from inhibitory interneurons, resulting in increased excitability of hippocampal pyramidal cells and dentate gyrus granule cells. Prodynorphin-derived peptides primarily act at presynaptic kappa opioid receptors to inhibit excitatory amino acid release from perforant path and mossy fiber terminals. Opioid receptors reduce membrane excitability by modulating ion conductances, and in this way they may decrease voltage-dependent calcium influx and transmitter release. Synaptic plasticity in the hippocampus also is modulated by endogenous opioids. Enkephalins facilitate long-term potentiation, whereas dynorphins inhibit the induction of this type of neuroplasticity. Further, opioids may play important roles in hippocampal epilepsy. Recurrent seizures induce changes in the expression of opioid peptides and receptors. Also, enkephalins have proconvulsant effects in the epileptic hippocampus, whereas dynorphins may function as endogenous anticonvulsants.

The granule cell population response to perforant path stimulation decreased significantly within seconds following release of endogenous dynorphin from dentate granule cells. The depression was blocked by the opioid receptor antagonists naloxone and norbinaltorphimine, suggesting that the effect was mediated by dynorphin activation of kappa 1 type opioid receptors. Pharmacological application of dynorphin B in the molecular layer was effective at reducing excitatory synaptic transmission from the perforant path, but application in the hilus had no significant effect. These results suggest that endogenous dynorphin peptides may be released from a local source within the dentate molecular layer. By light microscopy, dynorphin-like immunoreactivity (dynorphin-LI) was primarily found in granule cell axons in the hilus and stratum lucidum with only a few scattered fibers evident in the molecular layer. At the extreme ventral pole of the hippocampus, a diffuse band of varicose processes was also seen in the molecular layer, but this band was not present in more dorsal sections similar to those used for the electrophysiological studies. Electron microscopic analysis of the molecular layer midway along the septotemporal axis revealed that dynorphin-LI was present in dense-core vesicles in both spiny dendrites and unmyelinated axons with the majority (74%) of the dynorphin-LI-containing dense-core vesicles found in dendrites. Neuronal processes containing dynorphin-LI were observed throughout the molecular layer. The results suggest that dynorphin release from granule cell processes in the molecular layer regulates excitatory inputs entering the hippocampus from cerebral cortex, thus potentially counteracting such excitation-induced phenomena as epileptogenesis or long-term potentiation.

Release of endogenous norepinephrine was detected in guinea pig hippocampal slices using a radioligand displacement assay. Focal electrical stimulation released endogenous norepinephrine and caused a calcium-dependent reduction in specific [3H]propranolol binding at beta-adrenergic receptors in the brain slice. The mu-opioid agonist PL017 decreased norepinephrine release, and the inhibition by PL017 could be blocked by the opioid antagonist naloxone. Endogenous opioid peptides concomitantly released by tissue stimulation also decreased norepinephrine release in a naloxone-sensitive manner. These results support the hypothesis that endogenous opioids can regulate excitability in the hippocampus by presynaptic modulation of norepinephrine release.

In the guinea pig hippocampus, kappa 1-opioid binding sites were primarily localized in the molecular layer of the dentate gyrus as shown by autoradiography using either the kappa 1-selective radioligand 3H-U69,593 or the nonselective radioligand 3H-diprenorphine in the presence of unlabeled mu- and delta-blocking ligands. In this region, the electrophysiological effects of kappa 1-receptor activation were identified using extracellular and intracellular recordings of dentate granule cell responses. The amplitude of the extracellularly recorded population spike was reduced by U69,593 with an EC50 of 26 nM; this effect was reversible and blocked by the opioid antagonist naloxone. The kappa 1-selective antagonist norbinaltorphimine also blocked the effect of U69,593 with an apparent equilibrium dissociation constant (Ki) of 0.26 nM determined by Schild analysis in the physiologic assay. This value agreed well with the Ki for norbinaltorphimine at kappa 1-binding sites measured by radioligand binding displacement (0.24 nM). These results indicate that the electrophysiologic response observed was likely mediated by kappa 1-receptors. As seen with U69,593, dynorphin B, an endogenous opioid peptide that is present in the dentate gyrus, also inhibited the population spike response. mu- and delta-selective opioid agonists had no effect on the amplitude of the maximally evoked response. Intracellular recordings of dentate granule cells showed no direct effects of U69,593 on the granule cells themselves. However, analysis of synaptic potentials revealed that U69,593 significantly reduced the amplitude of glutaminergic EPSPs evoked by afferent stimulation without affecting IPSP amplitudes. The specific effect of U69,593 application on granule cell EPSPs indicates that presynaptic kappa 1-receptor activation inhibits glutamate release from perforant path terminals in the molecular layer of the dentate gyrus. These results suggest that endogenous dynorphins present in the granule cells may act as feedback inhibitors of the major excitatory input to the dentate gyrus.

The stimulus parameters needed for the release of endogenous opioid peptides were investigated using an in vitro radioligand displacement assay in living guinea pig hippocampal slices. Electrical stimulation of the enkephalin-containing fibers in the perforant path caused the release of endogenous opioid peptides and the subsequent displacement of [3H]-[D-Ala2,N-methyl-Phe4,glyol5]enkephalin binding. High frequency trains of stimuli (10 Hz for 1 sec every 10 sec) were more effective than lower frequency stimulation (1 Hz continuous) at evoking opioid peptide release. Having identified an effective stimulation paradigm able to release endogenous opioids, the electrophysiological effects of endogenous opioids on CA3 pyramidal cells were measured in the guinea pig hippocampal slice preparation. Unlike exogenously applied opioids, stimulated release of endogenous opioid peptides from the perforant path did not significantly reduce inhibitory postsynaptic potential (IPSP) amplitudes recorded in CA3 pyramidal cells. However, perforant path stimulation in the presence of naloxone did cause a dramatic increase in IPSP amplitudes. CA3 pyramidal cells were not directly affected by perforant path stimulation. The naloxone-sensitive increase in IPSPs was delayed 3 min in onset and lasted for several minutes. In addition, the increase in the IPSPs was specifically blocked either by the beta adrenergic antagonist propranolol or by pretreating the animals with reserpine. These findings indicate that endogenous opioids regulate the effects of norepinephrine in the CA3 region of the guinea pig hippocampus. In addition, endogenously released norepinephrine appeared to act on GABAergic interneurons to increase the amplitude of the IPSP recorded in CA3 pyramidal cells.

The mu-opioid agonist, [N-MePhe3,D-Pro4]morphiceptin (PL017), significantly decreased the conductance changes measured during both the early and late inhibitory postsynaptic potentials (IPSP) in CA1 pyramidal cells. Although the conductance change during the early IPSP was much larger than that during the late IPSP, the relative decrease in conductance caused by 1 microM PL017 was similar for both. Chronic morphine treatment of rats prior to hippocampal slice preparation resulted in a loss of PL017 (1 microM) effects on both the early and late IPSPs. These results suggest that opioids have an equal ability to alter both early and late IPSPs in the CA1, that these effects are equally sensitive to chronic morphine, and that these measurements are a sensitive means of determining opioid tolerance in the hippocampus.

Physiological release of endogenous opioids in the rat hippocampus was detected by an in vitro radioligand displacement assay using [3H][D-Ala2,N-methyl-Phe4,glyol5]enkephalin ([3H]DAGO), a mu selective opioid agonist. In this assay, radioligand binding to opioid receptors in the in vitro hippocampal slice was reduced by competition with endogenous opioids released following tissue depolarization. Veratridine-induced opioid release caused displacement of [3H]DAGO that could be blocked by either tetrodotoxin addition or calcium removal from the incubation buffer. Maximal displacement of [3H]DAGO also required the presence of peptidase inhibitors in the incubation buffer. None of the buffer composition changes directly affected [3H]DAGO binding to rat brain membranes. Calcium-dependent displacement of [3H]DAGO binding from mu receptor sites elicited by focal electrical stimulation depended on the intensity and frequency of stimulation and positioning of the electrode in the slice. Maximal displacement of [3H]DAGO binding was observed following high intensity (150-300 microA), high frequency (10-50 Hz) stimulation of the perforant path, a major afferent fiber system to the hippocampus previously shown to contain proenkephalin-derived opioids. Low frequency stimulation (0.1-1 Hz) was ineffective. Stimulation of the mossy fibers (containing both dynorphins and enkephalins) also significantly reduced mu receptor binding, but to a lesser extent. Electrical stimulation of the hippocampal slice at sites not containing opioid peptides did not cause mu receptor displacement. These results demonstrate that under physiological conditions, the release of endogenous opioids from the major opioid containing pathways can be detected in a single hippocampal slice following high frequency stimulation.

The localization of opioid peptides in the rat hippocampal formation and the epileptogenic action of beta-endorphin and certain enkephalin analogues have led to speculations that opioids may play a role in limbic seizures. These immunochemical and electroencephalographic data are compatible with single-unit electrophysiological studies showing predominant excitations of hippocampal pyramidal neurons in CA1 and CA3 fields produced by iontophoresis of endorphins or enkephalins. These excitations are naloxone sensitive and appear to arise from a disinhibitory mechanism due to inhibition of inhibitory interneurons. Thus, intracellular recordings in in vitro preparations of hippocampus usually show opioid-induced reduction of inhibitory postsynaptic potentials. However, more recent studies suggest that a major opioid-containing pathway in the hippocampus, the mossy fiber projection from the dentate gyrus to CA3 pyramidal neurons, contains more pro-dynorphin-derived peptides than pro-enkephalin. Intracerebroventricular dynorphin does not induce epileptiform activity in the rat, and single-unit and field-potential studies show mixed effects on CA3 neuronal excitability, with more inhibitory responses than are seen with the enkephalins. Selective inactivation of mu opioid receptors reveals that dynorphin, which was previously shown to express specificity for kappa receptors, can act on delta receptors in CA1. Furthermore, a specific kappa agonist, U50,488H, has inhibitory actions when applied directly to CA3 neurons. These data suggest the presence of multiple opioid receptor types in the hippocampus. These multiple receptors may point to heterogeneous functions of the different families of opioid peptides in various regions of the hippocampus, and could explain the divergent effects reported for the various opioids and naloxone to promote or prevent paroxysmal activity.

The effects of dynorphin-A (Dyn) and [Leu5]-enkephalin (Enk) were compared in the vitro hippocampal slice preparation, using extracellular field potential and intracellular voltage recordings. In the CA1 region, Dyn, like Enk, consistently increased the size of the extracellularly recorded population spike (PS) evoked by stratum radiatum (StR) stimulation of the Schaffer collaterals. These responses were naloxone reversible. In contrast, in the CA3 region, Dyn both increased and decreased the PSs evoked by mossy fiber stimulation, whereas Enk slightly enhanced the PS. Intracellular recordings from CA3 pyramidal neurons (HPNs) revealed both excitatory and inhibitory actions of Dyn on spontaneous activity. Associated membrane potential changes were variable. In contrast, Enk had only weak effects on spontaneous activity and no effect on membrane potential. These data suggest regional differences in the effect of Dyn and Enk on hippocampal activity.

The contents and molecular forms of five different prodynorphin-derived opioid peptides were compared in extracts of rat hippocampus by radioimmunoassay after C18-HPLC resolution. Dynorphin (Dyn) A(1-17) immunoreactivity (ir) and Dyn B-ir were heterogeneous in form; Dyn A(1-8)-ir, alpha-neoendorphin (alpha neo)-ir and beta-neoendorphin (beta neo)-ir each eluted as single homogeneous peaks of immunoreactivity. The fraction of immunoreactivity having the same retention as the appropriate synthetic standard was used to estimate the actual hippocampal content of each peptide. Comparison of these values showed that the concentrations of Dyn B, alpha neo, and Dyn A(1-8) were nearly equal, whereas both Dyn A(1-17) and beta neo were 1/5th to 1/10th the value of the other three. Calcium-dependent K+-stimulated release of these prodynorphin-derived opioids from hippocampal slices was detected. The stimulated rates of release were highest for Dyn B-ir followed by alpha neo-ir, then beta neo-ir and Dyn A(1-8)-ir with Dyn A(1-17)-ir lowest. The relative rates of stimulated release were in agreement with the relative proportions of peptide present within the tissue. This evidence of the presence and release of these opioid peptides considerably strengthens the hypothesis that this family of endogenous opioids plays a neurotransmitter role in the hippocampus.

In the guinea pig ileum myenteric plexus--longitudinal muscle preparation, dynorphin-(1--13) and the prototypical kappa agonist ethylketocyclazocine had equally poor sensitivity to naloxone antagonism and showed selective cross protection in receptor inactivation experiments with the alkylating antagonist beta-chlornaltrexamine. In binding assays with membranes from guinea pig brain, ethylketocyclazocine and dynorphin-(1--13) amide were more potent in displacing tritium-labeled ethylketocyclazocine than in displacing typical mu and delta opioid receptor ligands. In the two preparations studied, the dynorphin receptor appears to be the same as the kappa opioid receptor.

Pharmacological depolarization by KCl or veratrine reduced [3H]diprenorphine binding to opioid receptors in the hippocampal slice in a transient, calcium-dependent, and peptide-sensitive manner. These results suggest that endogenous opioid peptides were released from synaptic terminals and competitively displaced [3H]diprenorphine binding to opioid receptors. [3H]diprenorphine binding was significantly reduced by calcium-dependent depolarization throughout the hippocampus as determined by subsequent receptor autoradiography and quantitative densitometry. Displacement of binding was evident at sites in the CA1 and CA3 regions, the dentate gyrus, and the subiculum. The most dramatic reduction was evident in stratum lacunosum moleculare of CA3. Correlating the sites of maximal [3H]diprenorphine displacement with the previously described distribution of the opioid peptides suggests that the perforant path fibers release enkephalins in stratum lacunosum moleculare of CA3 and stratum moleculare of the dentate gyrus, and that mossy fibers may release both dynorphins and enkephalins near stratum pyramidale of CA3 and stratum granulosum. The lack of complete overlap between the distribution of opioid terminals and the sites of displacement indicates that these peptides may diffuse a moderate distance to their sites of action. Radioligand displacement defines the sites of endogenous opioid binding, suggests the likely sources of peptide release, and thus predicts the sites of endogenous opioid action within the hippocampus.

Opioid receptors on guinea pig brain membranes were alkylated by the naltrexone analogue beta-chlornaltrexamine. Binding of the prototypical mu and kappa ligands, [3H]dihydromorphine and [3H]ethylketocyclazocine, was more readily affected by the reagent than was binding of the delta ligand, 3H-labeled [D-Ala2, D-Leu5]enkephalin. Treatment of membranes with beta-chlornaltrexamine in the presence of dynorphin resulted in significant protection of [3H]ethylketocyclazocine binding sites, without protection of [3H]dihydromorphine or 3H-labeled [D-Ala2, D-Leu5]enkephalin sites. Similarly, [D-Ala2, D-Leu5]enkephalin and sufentanil selectively protected binding sites for 3H-labeled [D-Ala2, D-Leu5]enkephalin and [3H]dihydromorphine, respectively. Scatchard analysis of [3H]ethylketocyclazocine binding to untreated membranes suggested two types of binding site with 40-fold difference in affinities. Membranes treated with beta-chlornaltrexamine in the presence of dynorphin retained about 40% of the high-affinity sites and lost the low-affinity sites. Selective protection of sites with high affinity for dynorphin and ethylketocyclazocine was confirmed in competition binding assays. These results strongly suggest that the three types of opioid receptor are not interconvertible and provide further evidence that the endogenous peptide dynorphin is a highly selective ligand of the kappa opioid receptor.

Dynorphin-A1-17 and dynorphin-B increased the evoked response of hippocampal CA1 pyramidal cells, as did other opioids tested. Treatment of the hippocampal slice with beta-funaltrexamine, a mu-receptor selective antagonist, blocked the effects of normorphine, dynorphin-A and dynorphin-B, but did not change the response to D-Ala2, D-Leu5-enkephalin. The low potency of kappa selective agonists and the antagonism by beta-funaltrexamine of the dynorphins' effect indicate that kappa-opioid receptors may not be involved in these observed responses. Our data suggest that both mu- and delta-receptors are functionally represented and provide evidence that the dynorphins or their derivatives may also be agonists at the mu-receptor.

The molecular forms of Dynorphin A immunoreactivity (Dyn A-IR) detected in hippocampus were resolved by reverse-phase high pressure liquid chromatography (C18-HPLC). Peptide co-eluting with synthetic Dyn A(1-17) represents 20% of the total Dyn A-IR. Dyn A-IR was released from hippocampal slices in vitro by a Ca++-dependent mechanism stimulated by superfusion with K+, kainate or veratrine. Released peptides were resolved by C18-HPLC and shown to contain Dyn A-IR co-eluting with synthetic Dyn A(1-17). These observations are consistent with the hypothesis that Dyn A(1-17) acts as a neurotransmitter at synapses in hippocampus.

The structural features responsible for the high potency and opiate receptor specificity of the opioid peptide dynorphin in the guinea pig ileum myenteric plexus were examined. Successive removal of COOH-terminal amino acids from dynorphin-(1--13) demonstrated important contributions of lysine-13, lysine-11, and arginine-7 to the potency. Removal of the NH2-terminal tyrosine abolished the biologic activity. Several other structural modifications were shown to affect potency: substitution of D-alanine for glycine-2 reduced the potencies of dynorphin-(1--13) amide, -(1--11), and -(1--10); and methyl esterification of the COOH terminus enhanced the potencies of dynorphin-(1--12), -(1--10), -(1--9), -(1--8), and -(1--7). Within the dynorphin sequence, lysine-11 and arginine-7 were found to be important for selectivity of interaction with the dynorphin receptor, which is distinguishable from the mu receptor in this tissue.

The opioid receptor antagonists, naloxone and beta-chlornaltrexamine, were used to determine whether activation of endogenous opioid peptide containing pathways produced pharmacologically reversible opioid actions. Extracellularly recorded responses of the hippocampal CA3 pyramidal cells were evoked by stimulation of the dynorphin-containing mossy fiber pathway. Neither naloxone nor beta-chlornaltrexamine pretreatment significantly changed the evoked response. However, both antagonists blocked the effect of applied dynorphin-A(1-17) on CA1 pyramidal cell evoked responses. Thus, our data demonstrate that if endogenous opioids are released from this pathway, the peptides cannot be responsible for the evoked response measured in hippocampal CA3 cellular field. With no direct evidence for endogenous opioid peptides acting through opioid receptors, the neurotransmitter role of dynorphins in rat hippocampus remains obscure.