Autoradiographic analysis of second messenger and neurotransmitter system receptors in the gerbil hippocampus following transient forebrain ischemia.

Changes in second messenger and neurotransmitter system receptor ligand binding induced by transient forebrain ischemia were studied in the gerbil hippocampus. The animals were allowed variable periods of recovery ranging from 2 h to 7 days after 5-min bilateral carotid artery occlusion. The binding of second messenger systems ([3H]inositol 1,4,5-trisphosphate ([3H]IP3)to inositol 1,4,5-triphosphate, [3H]forskolin to adenylate cyclase and [3H]phorbol 12,13-dibutylate to protein kinase C) and neurotransmitter receptor systems ([3H]PN200-110 to L-type calcium channels. [3H]N6-cyclohexyl-adenosine to adenosine A1 and [3H]quinuclidinyl benzilate to muscarinic cholinergic receptor) were assayed using quantitative autoradiography. In the CA1 subfield, 2 h after ischemia, [3H]IP3, [3H]forskolin, and [3H]quinuclidinyl benzilate binding activities significantly decreased by 25, 17 and 13%, respectively, though no morphological abnormalities were obvious. Six hours after ischemia, the [3H]phorbol 12,13-dibutylate binding activity in the stratum oriens of the CA1 subfield increased by 15%. One day after ischemia, [3H]PN200-110 binding activity in this subfield decreased by 26%, and 7 days after ischemia, [3H]phorbol 12,13-dibutylate and [3H]N6-cyclohexyl-adenosine receptor binding activities decreased in this subfield. In particular, at 7 days after ischemia, [3H]IP3 binding activity in the CA1 subfield showed a complete decline. In the CA3 subfield, [3H]PN200-110 binding activity decreased 2 days after ischemia, and [3H]IP3 and [3H]N6-cyclohexyl-adenosine binding activities decreased 7 days after ischemia. In the dentate gyrus, the structure of which remained histologically intact after ischemic insult, [3H]IP3 and [3H]forskolin binding activities decreased 7 days after ischemia. In contrast, the [3H]phorbol 12,13-dibutylate binding activity increased in the molecular layer of the dentate gyrus 7 days after ischemia. These results indicate that marked alteration of intracellular signal transduction precedes neuronal damage in the hippocampal CA1 subfield and that the histologically intact CA3 and dentate gyrus also shows modulated neuronal transmission after ischemia.     

Autoradiographic evidence that NMDA receptor-coupled channels are located postsynaptically and not presynaptically in the perforant path-dentate granule cell system of the rat hippocampal formation

In vitro quantitative autoradiography with [3H]MK-801 was used to determine Kd and Bmax values for the NMDA receptor-coupled channel in subregions of the rat hippocampal formation. A single form of the channel with an apparent Kd in the 15-20 nM range was found for [3H]MK-801 binding in the presence of both 1 microM glutamate and 1 microM glycine. Specific binding was highest in the molecular layer of the dentate gyrus, followed by CA1 stratum radiatum and CA1 stratum oriens. Fewer binding sites were observed in the hilus of the dentate gyrus, cerebral cortex, CA1 stratum pyramidale, CA3 subregion (stratum oriens, stratum pyramidale, stratum radiatum), and thalamus. Selective destruction of dentate granule cells by colchicine microinjections reduced the amount of specific [3H]MK-801 binding by half in the molecular layer of the dentate, compared to intact tissue. [3H]MK-801 binding did not change in other hippocampal subregions as a consequence of colchicine injection. Electrolytic entorhinal cortical lesions produced no changes in regional MK-801 binding site density in any of the regions under study. To address the tissue shrinkage following entorhinal cortex lesions, detailed analysis of the binding site density per fixed (16 microns) length of granule cell dendrite, and of the aggregate density across the entire molecular layer revealed no change in the number of MK-801 binding sites per unit length of dendrite in the molecular layer of the dentate gyrus. These findings indicate that NMDA receptor-coupled channels are confined to a postsynaptic location in the perforant path-dentate granule cell system of the adult rat.     

Ischemia-induced neuronal cell loss is associated with loss of atypical angiotensin type-1 receptor expression in the gerbil hippocampal formation

The hippocampal formation of Mongolian gerbils expresses high amounts of atypical angiotensin II type-1 receptors. We studied the expression of these receptors by in situ hybridization using specific [³⁵S]-labeled riboprobes and by receptor autoradiography using [¹²⁵I]Sarcosine¹-angiotensin II. Angiotensin II receptor mRNA was found in the pyramidal cell layer of the CA1, CA2 and CA3 subfields, with the highest expression in the CA2 subfield, and in the granular cell layer of the dentate gyrus. Angiotensin II binding was detected in the stratum oriens and stratum radiatum of the CA1 and CA2 subfields, in the stratum oriens of the CA3 subfield, and in the molecular layer of the dentate gyrus. We then studied the effect of ischemia on hippocampal angiotensin II receptor expression, 1, 4 and 15 days after bilateral occlusion of the common carotid arteries for 5 min. No changes in angiotensin II receptor mRNA or binding were detected 1 day after ischemia. Delayed, progressive loss of angiotensin II mRNA and binding occurred 4 and 15 days after ischemia, in the CA1, CA2 and CA3 subfields. The decline was faster in the CA1 subfield, and paralleled the loss of neurons after ischemia. In the dentate gyrus, angiotensin II receptor mRNA and angiotensin II binding were not changed when compared to sham operated controls. The decrease of angiotensin II receptor expression may reflect the loss of angiotensin II receptor-producing neurons rather than a down-regulation of receptor expression.     

Calcium antagonist, adenosine A1, and muscarinic bindings in rat hippocampus after transient ischemia

The protective roles of Ca2+ channel blockers against ischemic hippocampal damage are still debated. We used autoradiography to study postischemic L-type Ca2+ channels (1,4-dihydropyridine Ca2+ channel blocker binding), adenosine A1 receptors, and muscarinic cholinergic receptors in the rat hippocampus using [3H]PN200-110 (PN), [3H]cyclohexyladenosine (CHA), and [3H]quinuclidinyl benzilate (QNB), respectively, in 49 rats subjected to 20 minutes of forebrain ischemia. The rats were decapitated after 1 (n = 7), 3 (n = 7), 6 (n = 8), 12 (n = 7), 24 (n = 6), 48 (n = 6), or 168 (n = 8) hours of recirculation; eight control rats were sham-operated but experienced no cerebral ischemia. Reduced receptor binding preceding the delayed death of CA1 pyramidal cells was first observed in the stratum oriens of the CA1 subfield. Significant reductions in [3H]PN, [3H]CHA, and [3H]QNB bindings of this stratum compared with control were noticed after 3 (35%, p less than 0.01), 12 (31%, p less than 0.01), and 1 (10%, p less than 0.05) hours of recirculation, respectively. By 168 hours after ischemia (when the populations of CA1 pyramidal cells were depleted) all strata in the CA1 subfield had lost most of their receptor sites, and [3H]PN, [3H]CHA, and [3H]QNB bindings in the stratum oriens were decreased to 23%, 30%, and 63% of control (p less than 0.01). Although [3H]PN binding in the CA3 subfield did not change significantly during 168 hours after ischemia, the histologically intact dentate gyrus exhibited a 31% loss of binding sites compared with control (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of GTP binding proteins in ischemic brain damage: autoradiographic and histophatological study

Cerebral ischemia produces perturbation of signal transduction systems in neurons. In order to estimate the contribution of guanine nucleotide-binding protein (G-protein) to hippocampal neuronal death, the effect of pertussis toxin (PTX) on the CA1 pyramidal cell damage after transient forebrain ischemia in rats was examined. PTX was administered 3 days before 20 min of transient forebrain ischemia. PTX injection into the CA1 failed subfield to alter the number of ischemic-damaged CA1 pyramidal cells. In contrast, ventricular PTX injection exacerbated CA1 pyramidal cell damage. We also studied postischemic alteration of GTP binding sites in the hippocampal formation using quantitative in vitro autoradiography. Autoradiographic imaging demonstrated predominant distribution of GTP binding sites in synaptic areas in the hippocampus. No significant change of GTP binding activity was observed in the hippocampus until 2 days after recirculation. Seven days after ischemia, when the CA1 pyramidal cells were depleted, the GTP binding sites of the strata oriens and radiatum in the CA1 subfield had reduced by 32% and 31%, respectively. In contrast, GTP binding in the CA3 subfield and the dentate gyrus remained unaltered throughout the reperfusion period. These results suggest that the amount of G-proteins as estimated by GTP binding remained unaltered in the hippocampus during the early recirculation period, when the CA1 pyramidal cells were morphologically intact, and that signal transduction pathways mediated by G_i and G_o do not play a major role in delayed death of the CA1 pyramidal cells.     

Sequential changes in muscarinic acetylcholine, adenosine A1 and calcium antagonist binding sites in the gerbil hippocampus following repeated brief ischemia.

We performed quantitative autoradiography to determine sequential alterations in the binding of muscarinic cholinergic and adenosine A1 receptors and of an L-type calcium channel blocker in the gerbil hippocampus following repeated brief ischemic insults. [3H]Quinuclidinyl benzilate (QNB). [3H]cyclohexyladenosine (CHA) and [3H]PN200-110 were used to label muscarinic and adenosine A1 receptors and L-type calcium channels, respectively. Changes at 1 h, 6 h, 1 day, 4 days and 1 month after three 2-min ischemic insults were compared with changes after single 2- or 6-min ischemia. Two-minute ischemia, which causes no histopathological neuronal damage, produced no persistent alterations in binding sites. We observed a transient and mild increase in binding activities, especially in [3H]CHA binding, at 1 h of recirculation. Following 6-min ischemia and three 2-min ischemic insults. [3H]QNB and [3H]PN200-110 binding decreased by more than 50% in the CA1 subfield by 1 month, but [3H]CHA binding decreased transiently by 20-30% at 4 days when delayed neuronal death of hippocampal CA1 pyramidal cells took place. Reductions in binding, especially in [3H]QNB binding, following three 2-min ischemic insults were greater and appeared earlier than those after 6-min ischemia. Furthermore, alterations extended to the CA3 subfield and the dentate gyrus following repeated insults. Thus, alterations in receptor binding after repeated ischemic insults were greater than those after equivalent single period of ischemia.     

Excitatory amino acid binding sites in the rat hippocampus after transient forebrain ischemia

The influence of transient forebrain ischemia on the temporal alteration of glutamate receptors in the hippocampal formation was analyzed by means of in vitro quantitative receptor autoradiography. We compared the binding of N-methyl-D-aspartate (NMDA) receptors using [3H]3-[+/-)2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), noncompetitive NMDA antagonist binding sites using [3H]N-(1-(2-thienyl)-cyclohexyl)-3,4-piperidine (TCP), and kainate (KA) receptors. In the CA1 subfield of the hippocampus, the number of NMDA receptors and noncompetitive NMDA antagonist binding sites remained constant during the early stage of recirculation when the CA1 pyramidal cells remained histologically intact. A significant reduction of these receptor densities was observed 7 days following ischemia, when NMDA receptors and noncompetitive NMDA antagonist binding sites lost 64 and 29% of their binding sites in the stratum radiatum of the CA1, respectively. The KA receptor density in the CA1 subfield decreased by 44% 7 days after ischemia. Marked loss of the above-mentioned receptors in the CA1 after selective depletion of the CA1 pyramidal cells indicated that NMDA receptors, noncompetitive NMDA antagonist binding sites, and KA receptors in the CA1 are predominantly localized on the CA1 pyramidal cells. NMDA receptor density in the CA3 gradually decreased during the recirculation period. The stratum moleculare of the dentate gyrus, whose structure was histologically intact after ischemic insult, also showed a reduction of NMDA receptors 7 days following ischemia. [3H]KA receptor density in the stratum lucidum of the CA3 and in the hilus also decreased during recirculation. These     

Alterations in [3H]kainate and N-methyl-D-aspartate-sensitive L-[3H]-glutamate binding in the rat hippocampal formation following fimbria-fornix lesions.

Following lesions of the fimbria-fornix, there is a time-dependent increase in interictal spikes and seizure susceptibility. This may result from sprouting of local excitatory and inhibitory circuits in response to the loss of subcortical and commissural innervation of the hippocampal formation. We used receptor autoradiography to examine the density of N-methyl-D-aspartate (NMDA)-sensitive L-[3H]glutamate and [3H]-kainate (KA) binding sites in the hippocampal formation at 5 days, 3 months, and 1 year following bilateral aspiration lesions of the fimbria-fornix. At 5 days post-lesion, the CA3 and CA1 strata radiatum and oriens displayed a decrease (20-42%, P less than 0.01) in NMDA-sensitive L-[3H]glutamate binding. The initial decrease was followed by a moderate recovery at later time points but was still evident at 1 year postlesion. This may reflect a lesion-induced turnover of synaptic complexes, down-regulation of postsynaptic receptors, or loss of presynaptic receptors. Five days following fimbria-fornix lesion there was also a decrease (13-15%, P less than 0.05) in [3H]KA binding in CA3 strata radiatum and pyramidale. However, at 3 months postlesion KA receptor density was elevated by 29-33% (P less than 0.01) in the outer molecular layer of the dentate gyrus with no significant change in binding to the inner molecular layer. By 1 year postlesion, the density of [3H]KA binding sites was not significantly different from that observed in control animals of the same age. The increase in KA receptor density in the outer molecular layer 3 months after fimbria-fornix lesion may reflect sprouting of the perforant path input or mossy fibers to this region and contribute to the increase in interictal spikes and seizures susceptibility.     

Alz-50 immunoreactive neuropil differentiates hippocampal complex subfields in Alzheimer's disease

The topographic distribution of Alz-50 containing profiles was determined within the hippocampal formation and anterior parahippocampal gyrus by using a monoclonal antibody directed against the A68 protein in normal and Alzheimer's diseased (AD) brains. Although there was a paucity of immunoreactive neuropil in the normal hippocampal complex, there were a few Alz-50 positive neurons that occupied the hippocampal subfield, CA2. In most AD cases, Alz-50 immunoreactive neuropil was prominent in the outer two-thirds of the molecular layer of the dentate gyrus, although a few cases exhibited staining in the inner third of the molecular layer. CA2 was characterized by an increased density of neuropil staining within stratum pyramidale. The neuropil in subfield CA1 was stained densely with Alz-50 in strata oriens, pyramidale, and at the border between strata lacunosum-moleculare and radiatum. Alz-50 immunostained neurites occupied primarily the lateral two-thirds of the subiculum proper, whereas only sparse staining was seen in the adjacent presubiculum. Alz-50 neuropil and neuronal staining displayed three distinct laminar patterns along the mediolateral extent of the entorhinal cortex, whereas the perirhinal cortex exhibited a bilaminar pattern of immunoreactivity involving heavy staining in layers 1-3 as compared to layer 5. In general, the density of Alz-50 neurite staining in the neuropil appeared inversely proportional to the distribution of Alz-50 immunoreactivity within dendritic and somal compartments. Interestingly, the patterns of Alz-50 staining observed in the hippocampal complex in AD coincides with patterns of well-characterized afferent fiber pathways to these regions, thus further supporting the suggestion that hippocampal subfield specific pathology effectively disconnects medial temporal structures from adjacent neocortex in AD.     

Postischemic neuroprotection in the ischemia-tolerant state gerbil hippocampus is associated with increased ligand binding to inhibitory GABA<Subscript>A</Subscript> receptors

Excitotoxic activation of glutamate receptors is thought to play a key role in delayed neuronal death (DND) of highly vulnerable hippocampal CA1 neurons after transient global ischemia. DND can be prevented by a short sublethal preconditioning (PC) stimulus. Recently, we demonstrated that ischemic PC, but not a single period of 5-min ischemia elicits a transient up-regulation of hippocampal [(3)H]muscimol binding to GABA(A) receptors. This indicates that activation of the GABAergic system may participate in the acquisition of neuroprotection. The present study was designed to test whether postischemic modulation of receptor binding also occurs in the ischemia-tolerant state, i.e., after a PC stimulus of 2.5-min ischemia and a subsequent normally lethal period of 5-min ischemia 4 days apart. Using receptor autoradiography, [(3)H]AMPA and [(3)H]muscimol binding to excitatory AMPA and inhibitory GABA(A) receptors was analyzed in hippocampal subfields CA1, CA3 and dentate gyrus at recirculation intervals of 30 min, 8, 24, 48, 96 h and 3 weeks. Postischemic hippocampal ligand binding to AMPA receptors remained unchanged at any time point investigated, but [(3)H]muscimol binding to GABA(A) receptors in CA1 neurons rendered tolerant to ischemia was up-regulated between 30 min and 48 h of recirculation. Our data suggest that a relative shift between excitatory and inhibitory neurotransmission may promote postischemic survival of CA1 neurons.     

Mapping second messenger systems in the rat hippocampus after transient forebrain ischemia: in vitro [3H]forskolin and [3H]inositol 1,4,5-trisphosphate binding

Autoradiographic imaging demonstrated predominant and reciprocal localization of forskolin and inositol 1,4,5-trisphosphate (IP3) binding sites in synaptic areas in the hippocampus. We produced selective damage to the CA1 pyramidal cells in the rat hippocampus by means of transient forebrain ischemia and analyzed the alteration of the intracellular signal transduction using quantitative autoradiography of these second messenger systems. The dendritic fields (stratum oriens, radiatum and lacunosummoleculare) in the CA1 showed 20% decrease in [3H]IP3 binding activity 3 h after ischemia, when no morphological abnormalities were obvious. Thereafter, grain density in these layers decreased and half of the binding sites were lost 2 days after ischemia. By contrast, the stratum pyramidale of the CA1 showed no significant change until 2 days after recirculation. Seven days after ischemia, when CA1 pyramidal cells were depleted, all layers in the CA1 subfield lost 85% of [3H]IP3 binding sites. In the CA3 subfield, only a small and transient alteration in the [3H]IP3 binding was noticed during recirculation. Postischemic reduction of [3H]forskolin binding sites was obvious in the CA1 only 1 h after ischemia followed by loss of 50% of binding activity 7 days after recirculation. These results suggest that forskolin and IP3 binding sites are predominantly distributed on the pyramidal cells in the CA1 subfield and that marked alteration of intracellular signal transduction precedes the delayed CA1 pyramidal cell death.     

Regional and selective effects of oestradiol and progesterone on NMDA and AMPA receptors in the rat brain

We investigated the effect of 10 months ovariectomy and a correction therapy, 2 weeks before the rats were killed, of oestradiol, progesterone or their combination on NMDA and AMPA receptor binding in the hippocampus, dentate gyrus, striatum, nucleus accumbens and frontal cortex of the rat brain as well as on amino acid levels in frontal cortex. NMDA and AMPA binding densities were assayed by autoradiography using, respectively, L-[3H]glutamate and [3H]AMPA; amino acid concentrations were measured by high performance liquid chromatograhy (HPLC) coupled with UV detection. Ovariectomy was without effect on NMDA and AMPA binding density in all brain regions assayed except in the hippocampal CA1 region and dentate gyrus where it decreased NMDA binding density compared to intact rats values. Oestradiol restored and increased NMDA binding density in the CA1 subfield and the dentate gyrus of ovariectomized rats but, by contrast, it decreased binding density in the striatum and in the frontal cortex while having no effect in the CA2/3 subfield of the hippocampus and in the nucleus accumbens. Oestradiol was without effect on AMPA binding density in the hippocampus and the dentate gyrus but it reduced AMPA binding density in the striatum, the frontal cortex and the nucleus accumbens. Progesterone, and oestradiol combined with progesterone, decreased NMDA but not AMPA binding density in the frontal cortex of ovariectomized rats, and they were without effect on these receptors in the other brain regions assayed. Amino acid concentrations in the frontal cortex were unchanged after ovariectomy or steroid treatments. The effect of oestradiol in the hippocampus confirmed in the present study and our novel findings in the frontal cortex, striatum and nucleus accumbens may have functional significance for schizophrenia and neurodegenerative diseases.     

Postnatal development of the hippocampal formation: a stereological study in macaque monkeys

We performed a stereological analysis of neuron number, neuronal soma size, and volume of individual regions and layers of the macaque monkey hippocampal formation during early postnatal development. We found a protracted period of neuron addition in the dentate gyrus throughout the first postnatal year and a concomitant late maturation of the granule cell population and individual dentate gyrus layers that extended beyond the first year of life. Although the development of CA3 generally paralleled that of the dentate gyrus, the distal portion of CA3, which receives direct entorhinal cortex projections, matured earlier than the proximal portion of CA3. CA1 matured earlier than the dentate gyrus and CA3. Interestingly, CA1 stratum lacunosum-moleculare, in which direct entorhinal cortex projections terminate, matured earlier than CA1 strata oriens, pyramidale, and radiatum, in which the CA3 projections terminate. The subiculum developed earlier than the dentate gyrus, CA3, and CA1, but not CA2. However, similarly to CA1, the molecular layer of the subiculum, in which the entorhinal cortex projections terminate, was overall more mature in the first postnatal year compared with the stratum pyramidale in which most of the CA1 projections terminate. Unlike other hippocampal fields, volumetric measurements suggested regressive events in the structural maturation of presubicular neurons and circuits. Finally, areal and neuron soma size measurements revealed an early maturation of the parasubiculum. We discuss the functional implications of the differential development of distinct hippocampal circuits for the emergence and maturation of different types of "hippocampus-dependent" memory processes, including spatial and episodic memories.     

Postischemic alteration of muscarinic acetylcholine, adenosine A1 and calcium antagonist binding sites in selectively vulnerable areas: an autoradiographic study of gerbil brain.

We performed receptor autoradiography to determine sequential alterations in the binding of muscarinic cholinergic and adenosine A1 receptors and of a voltage dependent L-type calcium channel blocker 1 h-1 month after transient cerebral ischemia in the gerbil brain. [3H]Quinuclidinyl benzilate (QNB), [3H]cyclohexyladenosine (CHA) and [3H]PN200-110 were used to label muscarinic and adenosine A1 receptors and L-type calcium channels, respectively. Transient ischemia was induced for 10 min. [3H]QNB and [3H]CHA binding showed no significant alteration in selectively vulnerable areas at an early stage (1-24 h) of recirculation. However, the dentate molecular layer which was resistant to ischemia revealed a significant decrease in the [3H]CHA binding sites 24 h after ischemia. Thereafter, the [3H]QNB and [3H]CHA binding showed significant reduction in most of selectively vulnerable areas. Marked reduction was especially found in the dorsolateral part of striatum and the hippocampal CA1 sector which was the most vulnerable to ischemia. In contrast, [3H]PN200-110 binding showed a transient elevation in the hippocampal CA1 sector, the dentate molecular layer and the thalamus 1 h of recirculation. However, the striatum and neocortex revealed no alteration in the [3H]PN200-110 binding. Thereafter, the reduction in the [3H]PN200-110 binding was seen only in the dorsolateral part of the striatum and the hippocampal CA1 sector. The results suggest that transient cerebral ischemia can cause the alterations in the binding of muscarinic cholinergic and adenosine A1 receptors and of L-type calcium channel blocker in most of selectively vulnerable areas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of [3H]inositoltrisphosphate binding in kainic acid-lesioned and postischemic rat hippocampus

A quantitative autoradiographic study was made on the binding of the phosphatidylinositol system ligand [3H]inositol(1,4,5)-trisphosphate (IP3) to forebrain sections from rats decapitated various times after 10 min of forebrain ischemia. To investigate the effect of a deafferentation of the hippocampal CA1, kainic acid-induced CA3-lesioned rats with or without 10 min of cerebral ischemia, were also included. The highest binding was found in the hippocampal CA1. Ten min of cerebral ischemia did not change the binding significantly. Between 5 min and 1 h of recirculation there was a 25-35% binding decline in all regions. In the CA1, where the pyramidal cells became necrotic 6 days after ischemia, there was a further decline to 16% of control. In the cortex, where there is no necrosis in this model, binding did not return to control values until day 14. Four days after a selective CA3 lesion with kainic acid, there was a significant 25% decline in the cortex, dentate gyrus and CA1, whereas in the necrotic CA3 binding declined to 54% of control. Ten min of ischemia did not alter this binding significantly. This decrease in calcium mobilizing intracellular receptors after ischemia and seizures could be due to increased membrane degradation, or to a more specific down-regulation following increased intracellular concentration of calcium and IP3.     

Mu- and delta-opioid receptor binding peaks and kappa-homogeneity in the molecular layers of rat hippocampal formation

The sublaminar binding profiles of (D-Ala-NMe-Phe-Gly-ol)-enkephalin (DAGO), (2-D-penicillamine), 5-D-penicillamine)-enkephalin (DPDPE), and dynorphin A(1-8) (DYN) were studied in the CA1 subfield and dentate gyrus of rat hippocampal formation. Binding was assayed on cryomicrotome sections using coverslip autoradiographic and single grain counting techniques. DAGO, an agonist for mu-sites, had peak binding in the stratum pyramidale with a secondary peak in the distal part of the stratum radiatum. Binding of DAGO in the dentate gyrus was homogeneous. DPDPE, a delta-site agonist, also had peak binding in the stratum pyramidale, but there was no secondary peak in the molecular layer of the hippocampus. In the dentate gyrus, DPDPE binding was highest in the inner one-third of the molecular layer immediately adjacent to the granular cell layer. The endogenous opioid DYN had a laminar binding profile that mimicked that of DAGO. However, when tritiated DYN was coincubated with unlabeled DAGO and DPDPE, as much as 90% of DYN binding was blocked and remaining binding was homogenous though a small peak remained in the stratum pyramidale. The secondary peak of DAGO binding in the stratum radiatum corresponds to an area previously determined to contain processes immunoreactive for enkephalin and gamma aminobutyric acid. This correspondence suggests that opioid compounds may mediate disinhibition of the distal dendrites of hippocampal pyramidal neurons. In addition, DYN binding patterns indicate that its action in rat hippocampus is likely by both mu- and delta-receptors.     

Release of endogenous opioid peptides displaces [3H]diprenorphine binding in rat hippocampal slices

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.     

Autoradiographic localization of opioid and spirodecanone receptors in the gerbil hippocampus as compared with the rat hippocampus

Opioid ([3H]naloxone) and spirodecanone ([3H]spiperone) binding sites in the hippocampus were visualized in the Mongolian gerbil and in the rat using in vitro autoradiography. In the hippocampus, marked differences were noted in the stratum (sr.) pyramidale of the CA1 subfield where opioid and spirodecanone (assayed in the presence of mianserin and sulpiride) binding activities were very low in gerbils, but high in rats. Gerbils exhibited a high concentration of [3H]naloxone binding sites in the sr. pyramidale of the CA3 subfield, as observed in the rat. In addition, the gerbil has a very high opioid receptor density in the hilar region and in the sr. moleculare of the dentate gyrus. The cellular localization of opioid and spirodecanone receptor sites was studied in the rat hippocampus using selective neuronal damage to CA1 and CA3 neurons by means of ischemia and kainic acid treatment, respectively. The results suggest that the gerbil differs from the rat with respect to the characteristic pyramidal cells (spirodecanone binding site) and interneurons (opioid receptor) in the CA1 subfield of the hippocampus. Distinct localization of opioid and spirodecanone receptors in the gerbil provides a good model with which to investigate the electrophysiological and biochemical roles of opioid peptides and butyrophenone spirodecanone drugs.     

Effects of ischemia on regional ligand binding to adrenoceptors in the rat brain.

Changes in ligand binding to adrenoceptors ([3H]prazosin to alpha 1-receptors, [3H]idazoxan to alpha 2-receptors and [125I]cyanopindolol to beta-receptors) following transient cerebral ischemia were investigated using autoradiographic methods. The binding was quantified in brain sections from control rats, rats subjected to 15 min of 2-vessel occlusion ischemia, and rats with recirculation times of 1 h, 1 week or 4 weeks after ischemia. No significant change in alpha 1-receptor binding was observed during and immediately following ischemia, but a decrease was noted in the vulnerable hippocampal CA1 region following 1 week's survival. In the parietal cortex, the ligand binding to alpha 1-receptors increased at 4 weeks. A reduced [3H]idazoxan binding was observed 1 h after ischemia in the temporal cortex and amygdala. No change in ligand binding to beta-receptors was seen in the early phase postischemia, but a marked increase had occurred in the hippocampal CA1 region at 1 and 4 weeks after ischemia (+163% and +142%, respectively), presumably due to accumulation of macrophages expressing beta-receptors. The early postischemic changes in receptor binding may represent downregulation of the adrenoceptors by processes activated during ischemia, while neuronal degeneration, compensatory mechanisms in surviving neurons and proliferation of non-neuronal cells may account for the subsequent changes.