Effects of traumatic brain injury in rats on binding to forebrain opiate receptor subtypes

Sprague-Dawley rats were subjected to a moderate level (2.2 atm) of traumatic brain injury (TBI) using fluid percussion. Injured animals were allowed to survive posttrauma for periods of 5 min, 3 h, and 24 h. The effect of TBI on binding to forebrain opiate receptors was assessed using quantitative receptor autoradiography, and compared to a sham control group. Binding of [³H]DAGO to mu receptors in neocortex and the CA1 pyramidal layer of the hippocampus was significantly decreased in the 24-h group (p<0.05). [³H]Bremazocine binding to kappa receptors was unchanged at 5 min and 24 h, but showed large decreases 3 h after TBI in the CA1 pyramidal layer (65%,p<0.05) and dentate gyrus (43%,p<0.05). Levels of delta binding (measured with [³H]DSLET) and lambda binding (measured with [³H]naloxone) were unaffected by TBI. These data support previous suggestions of a role for endogenous opioids in TBI, and provide further evidence that mu and kappa opioid receptor subtypes in neocortex and hippocampus may have different functions in TBI.     

Evidence for the presynaptic localization of opiate binding sites on striatal efferent fibers

Using quantitative receptor autoradiography, [3H]D-Ala-D-Leu-enkephalin (DADL) and [3H]naloxone binding were studied in rat striatum and striatal projection areas (globus pallidus (GP) and substantia nigra pars reticulata (SNr] after unilateral striatal kainic acid lesions. [3H]DADL and [3H]naloxone binding were each examined by two methods. Initially, [3H]DADL binding was performed in 50 mM Tris-HCl (pH 7.4), 30 mM NaCl, 3 mM manganese acetate and 2 microM GTP; [3H]naloxone binding was carried out in 50 mM Tris-HCl (pH 7.4) and 100 mM NaCl. Subsequent studies were carried out in 150 mM Tris-HCl (pH 7.4) and either [3H]DADL plus 500 nM morphiceptin (to block [3H]DADL binding to mu receptors) or [3H]naloxone plus 10 nM delta receptor peptide (to block [3H]naloxone binding to delta receptors). At one and eight weeks in the lesioned striatum, [3H]DADL binding was reduced by 70% and 82%, respectively, when compared to the control side. [3H]Naloxone binding was reduced by 35% and 20%. In GP and SNr, [3H]DADL binding was reduced by 31% and 41%, respectively, at one week and 27% and 26% at eight weeks. [3H]Naloxone binding was reduced 19% in GP at eight weeks. A parsimonious explanation of these results is that opiate binding sites are located on presynaptic terminals of striatal efferent fibers to globus pallidus and substantia nigra pars reticulata as well as on local striatal axon collaterals. Since opiate peptides have recently been found to coexist with GABA in some striatal neurons, opiate peptides may play a role in striatal function by controlling GABA release from striatal efferent fibers. It is possible that pallidal and nigral opiate binding could be utilized as a marker for striatal terminals.     

Interaction of dynorphin with kappa opioid receptors in bovine adrenal medulla

Dynorphin (Dyn) and various prototypic kappa opioid ligands were tested for their ability to bind to opioid receptors in a membrane preparation of bovine adrenal medulla and to modulate the release of catecholamines (CA) from isolated adrenal chromaffin cells. Saturation binding studies with [3H]-ethylketocyclazocine ([3H]-EKC) were performed at 37 degrees C for 30 min in the presence of [D-Ala2,Me-Phe4,Gly-ol5]-enkephalin (DAGO) and [D-Ser2,Thr6]-Leu-enkephalin (DSLET), two specific ligands for crossreacting mu and delta opioid receptors, respectively. Scatchard plot analysis of the data revealed the presence of two receptor sites: a high affinity binding site (kappa) with a KD of 0.66 nM and a Bmax of 12 pmoles/g protein and a low affinity binding site (kappa 2) with a KD of 11.1 nM and a Bmax of 56 pmoles/g protein. The presence of kappa opioid receptors in the membrane preparation was also supported by competition studies. U-50, 488H and Dyn-(1-13), two selective kappa opioid ligands, were potent inhibitors of [3H]-EKC binding with Ki (high affinity binding sites) of 2.5 and 2.3 nM, respectively. Among the various ligands tested for each class of opioid receptors (mu, delta, kappa), U-50, 488H and Dyn-(1-13) were the most potent inhibitors of the acetylcholine-evoked CA secretions from isolated adrenal chromaffin cells with IC50 of 0.31 and 1.14 microM, respectively. The inhibitory effect of U-50, 488H was significantly antagonized by diprenorphine and MR-2266, two opioid antagonists with a high affinity for the kappa opioid receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological and anatomical evidence of selective mu, delta, and kappa opioid receptor binding in rat brain

While the distribution of opioid receptors can be differentiated in the rat central nervous system, their precise localization has remained controversial, due, in part, to the previous lack of selective ligands and insensitive assaying conditions. The present study analyzed this issue further by examining the receptor selectivity of [3H]DAGO (Tyr-D-Ala-Gly-MePhe-Gly-ol), [3H]DPDPE (2-D-penicillamine-5-D-penicillamine-enkephalin), [3H]DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr) and [3H](-)bremazocine, and their suitability in autoradiographically labelling selective subpopulations of opioid receptors in rat brain. The results from saturation, competition, and autoradiographic experiments indicated that the three opioid receptor subtypes can be differentiated in the rat brain and that [3H]DAGO and [3H]DPDPE selectively labelled mu and delta binding sites, respectively. In contrast, [3H]DSLET was found to be relatively non-selective, and labelled both mu and delta sites. [3H]Bremazocine was similarly non-selective in the absence of mu and delta ligands and labelled all three opioid receptor subtypes. However, in the presence of 100 nM DAGO and DPDPE, concentrations sufficient to saturate the mu and delta sites, [3H]bremazocine did label kappa sites selectively. The high affinity [3H]bremazocine binding sites showed a unique distribution with relatively dense kappa labelling in the hypothalamus and median eminence, areas with extremely low mu and delta binding. These results point to the selectivity, under appropriate conditions, of [3H]DAGO, [3H]DPDPE and [3H]bremazocine and provide evidence for the differential distribution of mu, delta, and kappa opioid receptors in rat brain.     

Characterization of kappa opiate receptors in rat spinal cord-dorsal root ganglion cocultures and their regulation by chronic opiate treatment

We have investigated the expression and regulation of kappa opiate receptors in rat spinal cord-dorsal root ganglion primary cocultures. The density of opiate receptors increased markedly during the differentiation of the cultures; after 10 days in vitro the number of [3H]diprenorphine binding sites reached 244 +/- 47 fmol/mg protein. Most of the binding sites were of the kappa type, representing about 65-80% of total opiate receptors, while mu sites were expressed at a lower density (ca. 20% of total opiate sites). Following this period of development, the number of kappa and mu receptors did not change significantly. No detectable delta sites were observed at any time of culture (up to 4 weeks in vitro). Chronic opiate agonist treatment (24 h) of the cultured cells with either 10 microM U50488 (a selective kappa agonist), or 1 microM etorphine (a nonselective opiate agonist), did not change the number of kappa receptors and their binding affinity to [3H]diprenorphine. On the other hand, 50% of the mu receptor sites down-regulated following 24 h treatment with 1 microM etorphine. Chronic antagonist exposure (5 days) with 10 microM naloxone, markedly up-regulated the mu receptors (261% of control), whereas kappa sites exhibited a much weaker upregulation (164% of control). These data demonstrate that kappa opiate receptors are expressed at high concentration in spinal cord-dorsal root ganglion cocultures and that contrary to mu sites, kappa receptor density is less susceptible to modulation by chronic opiate treatment. The results also suggest that postreceptor components are important in regulating the kappa receptor function following prolonged opiate exposure.     

Opiate-induced seizures: a study of mu and delta specific mechanisms

Two groups of experiments were conducted to determine if morphine- and enkephalin-induced seizures are specifically mediated by the mu and delta receptor, respectively. In the first experiments, designed to assess the ontogeny of mu- or delta-seizures, rats from 6 h to 85 days of age received implanted cortical and depth electrodes as well as an indwelling cannula in the lateral ventricle. Various amounts of the mu-receptor agonists, morphine and morphiceptin, and the delta agonists, D-Ala2-D-Leu5-enkephalin (DADL) and Tyr-D-Ser-Gly-Phe-Leu-Thr (DSLET), were then administered intracerebroventricularly (icv) with continuous EEG monitoring. The second experiments entailed use of the nonspecific opiate antagonist, naloxone, as well as the specific delta antagonist, ICI 154,129, against seizures induced by icv-administered morphine, morphiceptin, DADL, or DSLET. Both morphine and morphiceptin produced electrical seizure activity in rats as young as 5 days after birth. The drugs produced similar seizure activity in terms of electrical morphology, observed behavior, ontogeny, threshold dose, and reversibility with small doses of naloxone. In the pharmacologic experiments, icv naloxone blocked all opiate-induced seizures. ICI 154,129 blocked DSLET seizure, had little effect on enkephalin or DADL seizures, and no effect on morphine or morphiceptin seizures. These data indicate that DSLET seizures are delta-specific but that all other opiate-induced seizures studied may involve multiple opiate receptor-mediated mechanisms.     

Naltrexone-induced opiate receptor supersensitivity

Chronic administration of the long-lived narcotic antagonist naltrexone resulted in a marked increase in brain opiate receptors. Similar changes in receptor density were observed for binding of the putative mu agonist [3H]dihydromorphine, the mu antagonist [3H]naloxone, the putative delta ligand [3H]D-Ala2,D-Leu5-enkephalin and [3H]etorphine. In addition, the sensitivity of agonist binding to guanyl nucleotide inhibition increased significantly. In contrast, no such changes in opiate binding were observed following acute administration of naltrexone. The increase in opiate receptor number following chronic naltrexone was highest in the mesolimbic and frontal cortex areas, and lowest in the dorsal hippocampus and periaqueductal gray. These results indicate a degree of plasticity in the opiate receptor system that may correlate with specific functional pathways.     

Opiate binding sites in the chick, rabbit and goldfish retina

The characteristics of opiate binding sites in the retina of the chick, rabbit and goldfish have been investigated. In the newly hatched chick retina, 131 fmol/mg of binding sites for [D-Ala2-D-Leu5]-[3H]enkephalin are present; competition studies with the delta selective peptide [D-Thr-Leu5]-enkephalin (DTLET) and the mu selective peptide morphiceptin show that all of the [D-Ala2-D-Leu5]-[3H]-enkephalin binding sites are of the delta subtype. Dihydro[3H]morphine binds poorly to the chick retina; 13.2 fmol/mg of this binding is displaceable by morphiceptin and corresponds to mu binding sites. Benzomorphan sites are defined as sites occupied by [3H]diprenorphine which is displaceable by low concentrations of ethylketocyclozacine but not by high concentrations of D-Ala2-D-Leu5-enkephalin and morphiceptin. At least 88 fmol/mg of benzomorphan sites are present in the chick retina. [3H]diprenorphine binding to the rabbit and fish retina was measured. The rabbit retina bound 60 fmol/mg, and the fish retina 42 fmol/mg of [3H]diprenorphine. These findings are discussed in the light of the studies on the localization and physiological effects of enkephalin in the retina.     

Opioid peptide receptor studies. 12. Buprenorphine is a potent and selective mu/kappa antagonist in the [35S]-GTP-gamma-S functional binding assay.

We utilized the [(35)S]-GTP-gamma-S functional binding assay to determine the selectivity of opioid receptor agonists in guinea pig caudate membranes. The study focused on two opioid agonists used for treating opioid-dependent patients: methadone and buprenorphine. Selective antagonists were used to generate agonist-selective conditions: TIPP + nor-BNI to measure mu receptors, CTAP + nor-BNI to measure gamma receptors and TIPP + CTAP to measure kappa receptors. The assay was first validated with opioid agonists of known subtype specificity (DAMGO for mu, SNC80 for delta, and U69, 593 for kappa receptors). Methadone-stimulated [(35)S]-GTP-gamma-S binding was mu-specific and less potent and efficacious than etorphine (K(d) = 1,537 nM vs. K(d) = 7.8 nM). Buprenorphine failed to stimulate [(35)S]-GTP-gamma-S binding but inhibited agonist-stimulated [(35)S]-GTP-gamma-S binding. The antagonist-K(i) values (nM) of buprenorphine at mu, delta, and kappa receptors were 0.088 nM, 1.15 nM, and 0.072 nM, respectively. The antagonist-K(i) values (nM) of naloxone at mu, delta, and kappa receptors were 1.39 nM, 25.0 nM, and 11.4 nM, respectively. Autoradiographic studies showed that buprenorphine failed to stimulate [(35)S]-GTP-gamma-S binding in caudate-level rat brain sections but blocked DAMGO-stimulated [(35)S]-GTP-gamma-S binding. In cells expressing the cloned rat mu receptor, buprenorphine was a partial agonist and potent mu antagonist. Administration of buprenorphine to rats produced a long-lasting (>24 h) decrease in mu and kappa2 receptor binding and attenuated mu-stimulated [(35)S]-GTP-gamma-S binding. Viewed collectively, these data indicate that, in this assay system, buprenorphine is a potent mu and gamma receptor antagonist. The clinical implications remain to be elucidated. Synapse 34:83-94, 1999. Published 1999 Wiley-Liss, Inc.     

Autoradiographic visualization of adenosine A1 receptors in the gerbil hippocampus: changes in the receptor density after transient ischemia

N6-cyclohexyl-[3H]adenosine [( 3H]CHA) was used for the in vitro visualization of the hippocampal adenosine A1 receptors in the gerbil. The strata radiatum and oriens of the hippocampus showed particularly high binding activity. Depletion of pyramidal cells and consequent severe decrease in [3H]CHA binding activity in the CA1 subfield were observed after transient ischemic insult. These results suggest that most adenosine receptors in the CA1 region are localized in association with pyramidal cells.     

The distribution of multiple opiate receptors in bovine brain

The distribution of mu and delta opiate receptors in bovine brain has been investigated using the selective radioligands [3H]morphine and D-[3H]Ala2, D-Leu5-enkephalin. Their distributions were found to vary independently through different brain areas with up to a 10-fold difference between the ratio of mu to delta binding sites for the substantia nigra and the dentate gyrus of the hippocampus.     

Characterization of dermorphin binding to membranes of rat brain and heart

Binding of dermorphin to the two major opioid receptor types, mu and delta, in rat brain membranes was examined by displacement of [3H] [D-Ala2, MePhe4, Gly-(ol)5]enkephalin (DAGO) and [3H]-[D-Ala2,D-Leu5]-enkephalin (DADLE) binding. Affinity of dermorphin binding to mu sites, Kd = 1.24 nM, was almost 3 times greater than that of DAGO, Kd = 3.35 nM. In contrast, the Kd value of dermorphin binding to delta sites was 78 nM only, as compared to Kd = 2.27 nM for DADLE. Dermorphin was ineffective in displacing [3H]ethylketocyclazocine (EKC) binding to kappa receptors after prior blocking of [3H]EKC binding to mu and delta sites. Studies of dermorphin binding to mu sites revealed that the potency of dermorphin increased in the presence of Na+ (+31%) but decreased in the presence of Mn2+ (-81%) or Gpp(NH)p (-44%). Displacement of bound [3H]diprenorphine (DPN) by dermorphin from atrial membranes of the rat heart, left side, was detectable, suggesting the presence of mu sites in this section of the heart.     

Autoradiographic study of irreversible binding of [3H]beta-funaltrexamine to opioid receptors in the rat forebrain: comparison with mu and delta receptor distribution.

beta-Funaltrexamine (beta-FNA) is an irreversible mu antagonist and a reversible kappa agonist in in vivo and in vitro tests. However, whether it produces irreversible delta antagonism is controversial. In binding studies, it is clear that beta-FNA does not bind irreversibly (it does reversibly) to kappa receptors. Yet there is no consensus as to whether beta-FNA binds irreversibly to mu and/or delta receptors. In this study, irreversible binding of [3H]beta-FNA to opioid receptors was examined in rat forebrain sections in the presence of 200 mM NaCl and its distribution compared with those of mu and delta opioid receptors, labeled by [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin ([3H]DAMGO) and [3H][D-Pen2,D-Pen5]enkephalin ([3H]DPDPE), respectively. Irreversible binding of [3H]beta-FNA was determined as the binding that remained following 5 washes at room temp. for 1, 5, 20, 20, and 20 min each. Non-specific binding was defined by including 10 microM naloxone, beta-chlornaltrexamine (beta-CNA), or beta-FNA in the incubation mixture. At 37 degrees C, specific irreversible binding of [3H]beta-FNA to opioid receptors reached a plateau at 10 nM in 60 min, and constituted 50-70% of total irreversible binding. Series of 4 sections of similar anatomical levels were labeled with [3H]DAMGO, [3H]beta-FNA, [3H]beta-FNA + 10 microM naloxone, beta-CNA, or beta-FNA, and [3H]DPDPE, resp., and exposed to [3H]-Ultrofilm. The distribution of [3H]beta-FNA (5 nM) irreversible labeling is very similar to that of [3H]DAMGO, i.e. patches and subcallosal streaks in caudate-putamen, patches in nucleus accumbens, dense labeling in thalamus, and more binding in the rostral than caudal striatum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential postnatal development of mu and delta opiate receptors

We found a differential postnatal development of mu and delta opiate receptors. Mu receptors labelled with low concentrations of [3H]naloxone appeared to develop earlier than did delta receptors labelled with [3H]D-Ala2-D-Leu5-enkephalin (0.5 nM). Competition binding studies also revealed a delayed appearance of delta receptors (day 12 postnatal).     

Dog cerebral cortex contains μ-, δ- and κ-opioid receptors at different densities: apparent lack of evidence for subtypes of the κ-receptor using selective radioligands

The biochemical and pharmacological properties of mu (mu), kappa (kappa) and delta (delta) opioid receptors were ascertained in dog cerebral cortex homogenates. The selective peptides, [3H]D-Pen2-D-Pen5enkephalin [( 3H]DPDPE) and [3H]D-Ala2-MePhe4-Glyol5-enkephalin [3H]Glyol; [3H]DAMGO), bound to delta- and mu-opioid receptors with high affinity (dissociation constants, Kd values = 4.7 and 1.6 nM) but to different densities of binding sites (Bmax values of 49.2 and 6.6 fmol/mg protein, respectively) in washed homogenates of dog cerebral cortex. In contrast, the non-peptides, [3H]U69593 [( 3H]U69) and [3H]etorphine [( 3H]ET), labeled a high concentration of kappa-opioid receptors (respective Bmax values of 67.2 and 76.6 fmol/mg protein) of high affinity (respective Kds of 1.4 and 0.47 nM) in the same tissue homogenates. Thus, the relative rank order of opioid receptor densities was: kappa greater than delta much greater than mu. The selective labeling of the kappa-receptors with two different drugs [( 3H]U69 and [3H]ET) failed to reveal the possible existence of multiple kappa-sites based on the relative Bmax values of the two radioligands. This conclusion was further supported by the similarity of the pharmacological specificity of both [3H]U69 and [3H]ET binding, where all the opioids tested produced 100% inhibition of these labels and where the rank order of potency of opioids at inhibiting the binding of these probes was: U50488 greater than U69593 greater than dynorphin-(1-8) greater than naloxone much greater than morphine much greater than Glyol (DAMGO) greater than DPDPE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of multiple opiate receptors in opioid kindling

D-Tyr-Ser-Gly-Phe-Leu-Thr (DSLET), beta-endorphin, morphiceptin and morphine were microinjected at 48-h intervals into the amygdala or hippocampus of awake rats in an attempt to identify the opiate receptor types involved in opioid kindling. DSLET, beta-endorphin, morphiceptin and morphine were injected into the lateral ventricle to assess the possibility of kindling seizures by this route. The delta-receptor agonist DSLET effectively kindled convulsions when microinjected into amygdala or ventral hippocampus. The convulsions were suppressed or strongly attenuated by ICI 174,864, a specific antagonist of the delta-receptor, microinjected into the same brain site, but were not affected by ICI 174,864 administered peripherally. When microinjected into amygdala or hippocampus, beta-endorphin and morphiceptin also kindled convulsions, which were antagonized by naloxone but not by ICI 174,864. Morphine evoked EEG epileptiform activity but did not kindle convulsions from limbic brain sites. DSLET occasionally evoked epileptiform spiking and submaximal convulsions when injected into ventricle, and morphiceptin evoked epileptiform spiking only, but tolerance to these effects occurred after repetition of the injections. Thus, convulsions can be kindled by activation of either mu-, delta- or epsilon-receptors when opioids are injected directly into limbic tissue. However, the ability of these compounds to kindle seizures is markedly reduced when they are administered into ventricle. The striking differences between the present results and previous results obtained by peripheral or intraventricular administration of opioid peptides suggest that the route of administration, among other variables, is a crucial factor in assessing the epileptogenic properties of opioid peptides.     

Preserved neurotransmitter receptor binding following ischemia in preconditioned gerbil brain

Preconditioning the brain with sublethal ischemia induces tolerance to subsequent ischemic insult. Using [3H]quinuclidinyl benzilate (QNB), [3H]MK 801, [3H]cyclohexyladenosine, [3H]muscimol, and [3H]PN200-110, we investigated the alterations in neurotransmitter receptor and calcium channel binding in the gerbil hippocampus following ischemia with or without preconditioning. Two-minute forebrain ischemia, which produced no neuronal damage, resulted in no alterations in binding except for a slight reduction in [3H]QNB binding in the CA1 subfield. Three-minute ischemia destroyed the majority of CA1 pyramidal cells and caused, in CA1, reductions in binding of all ligands used. Preconditioning with 2-min ischemia followed by 4 days of reperfusion protected against CA1 neuronal damage and prevented the reductions in binding although [3H]QNB and [3H]PN200-110 binding transiently decreased in the early reperfusion period, suggesting down-regulation. Thus, preconditioning protects against damage to the neurotransmission system as well as histopathological neuronal death.     

Changes in NPY-mediated modulation of hippocampal [3H]D-aspartate outflow in the kindling model of epilepsy

The anticonvulsant effect of NPY may depend on Y(2) and/or Y(5) receptor-mediated inhibition of glutamate release in critical areas, such as the hippocampus. However, Y(2) and Y(5) receptor levels have been reported to increase and decrease, respectively, in the epileptic hippocampus, implicating that the profile of NPY effects may change accordingly. The aim of this study was to evaluate the differential effects of NPY on glutamate release in the normal and in the epileptic hippocampus. Thus, we pharmacologically characterized the effects of NPY on the release of [(3)H]D-aspartate, a valid marker of endogenous glutamate, from synaptosomes prepared from the whole hippocampus and from the three hippocampal subregions (dentate gyrus and CA1 and CA3 subfields) of control and kindled rats, killed 1 week after the last stimulus-evoked seizure. In the whole hippocampus, NPY does not significantly affect stimulus-evoked [(3)H]D-aspartate overflow. In synaptosomes prepared from control rats, NPY significantly inhibited 15 mM K(+)-evoked [(3)H]D-aspartate overflow only in the CA1 subfield (approx. -30%). Both Y(2) and Y(5) receptor antagonists (respectively, 1 microM BIIE0246 and 1 microM CGP71683A) prevented this effect, suggesting the involvement of both receptor types. In contrast, in synaptosomes prepared from kindled rats NPY significantly inhibited 15 mM K(+)-evoked [(3)H]D-aspartate overflow in the CA1 subfield and in the dentate gyrus (approx. -30%). Only the Y(2) (not the Y(5)) antagonist prevented these effects. These data indicate a critical role for the Y(2) receptor in the inhibitory control of glutamate release in the kindled hippocampus and, thus, suggest that the anticonvulsant effect of NPY in the epileptic brain is most likely Y(2), but not Y(5), receptor-mediated.     

Administration of kainic acid and colchicine alters mu and lambda opiate binding in rat hippocampus

Quantitative in vitro autoradiography was used to assess the effects of kainic acid (KA) and colchicine (COL) on mu and lambda opiate binding in the rat hippocampus. Rats were treated with either systemic KA, a neurotoxin that damages CA3 pyramidal cells and causes seizures and wet-dog shakes, or intrahippocampal COL to destroy dentate granule cells and their mossy fibers, or both toxins. Moderate levels of mu binding were detected in the pyramidal layer and in the stratum lacunosum-moleculare; binding was greater in the ventral hippocampus. Levels of mu binding were markedly increased in all regions 48 h after treatment with KA. Two weeks after COL treatment, there was a modest decrease in mu binding; COL plus KA gave results similar to COL alone. Dense lambda binding was present over the mossy fibers in the stratum lucidum, but was absent over the pyramidal layer. In contrast to mu binding, lambda binding was greater in the dorsal hippocampus. KA alone had little effect on lambda binding, whereas COL alone caused large decreases. KA plus COL caused even larger decreases in lambda binding, to as much as 85% below control. These results demonstrate that mu and lambda binding are localized to different parts of the hippocampus, respond differently to neurotoxin lesions, and likely serve different roles in this brain region. The number of mu sites is responsive to the release of enkephalin; these receptors appear to be linked to opiate-induced hippocampal seizure activity, especially wet-dog shakes. Lambda sites may serve as autoreceptors on mossy fibers.     

An in vitro autoradiographic analysis of mu and delta opioid binding in the hippocampal formation of kindled rats

Recent studies have shown that opioid peptide levels are altered in hippocampal formation of kindled animals. We therefore studied the distributions of mu and delta opioid binding sites in hippocampal formation of kindled and control rats using quantitative in vitro autoradiography. Animals received daily stimulations of the amygdala until they experienced 3 class 5 seizures. Paired control animals underwent implantation of electrodes but were not stimulated. Mu binding sites were labeled with 125I-FK-33824. Twenty-four hours after the last kindled seizure, mu binding was decreased by 32% in stratum pyramidale of CA1 and stratum radiatum of CA2 and by 17-27% throughout most of the rest of CA1, CA2, and CA3. Few, if any, differences were seen between kindled and control animals at 7 or 28 days after the last kindled seizure. Delta binding sites were labeled with 125I-[D-Ala2,D-Leu5]enkephalin in the presence of the morphiceptin analog PL-032. Twenty-four hours after the last kindled seizure, delta binding was decreased only in stratum moleculare of the dentate gyrus. Seven days after the last kindled seizure, delta binding was decreased by 11-17% throughout CA1, CA3, and the dentate gyrus. At 28 days after the last seizure, however, no differences were found between kindled and control animals. Since the decreases in mu and delta opioid binding are transient, they are unlikely to be the molecular basis of the permanent kindling phenomenon. Rather, these changes in opioid binding may represent responses to repeated seizures.