Autoradiographic analysis on second-messenger systems and local cerebral blood flow in ischemic gerbil brain

Alterations of the second-messenger systems, adenylate cyclase (AC) and protein kinase C (PKC), and local cerebral blood flow (lCBF) were evaluated during experimental cerebral ischemia in gerbils employing a quantitative autoradiographic method, which permitted these three parameters to be measured in the same brain. Ischemia was induced by occlusion of the right common carotid artery for 6 h. Animals attaining more than 5 in their ischemic scores were utilized for further experiments. At the end of ischemia, lCBF was measured by the [14C]iodoantipyrine method. The AC and PKC activities were estimated by the autoradiographic technique developed in our laboratory using [3H]forskolin (FK) and [3H]phorbol-12,13-dibutyrate (PDBu), respectively. The lCBF fell below 10 ml/100 g/min in most cerebral regions on the ligated side. The greatest reduction in FK binding was noted in the olfactory tubercle, caudate-putamen, and globus pallidus, followed by the hippocampus and cerebral cortices. The FK binding tended to be low at lCBF less than 20 ml/100 g/min in the cerebral cortices. However, the PDBu binding was relatively well preserved in each cerebral structure, and no significant correlation between lCBF and PDBu binding was noted in the cerebral cortices. The AC system may thus be vulnerable to ischemic insult over extensive brain regions, while the PKC system may be relatively resistant to ischemia.     

Activation of protein kinase C by phorbol dibutyrate modulates GABAA receptor binding in rat brain slices

Effects of protein kinase C (PKC) activation on the function of the GABA/benzodiazepine receptor-chloride complex were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam and [³⁵S]TBPS in rat brain slices. The density of [³H]muscimol binding was highest in cerebellar granular layers and high in both the frontal cortex and thalamus, but binding levels in the hippocampus were low. After activation of PKC by 100 nM phorbol-12,13-dibutyrate (PDBu), [³H]muscimol binding was decreased in the frontal cortex, striatum and thalamus, but binding levels were not changed in the hippocampus or cerebellum. The density of [³H]flunitrazepam binding was high in the cortex, hippocampus and molecular layers of cerebellum but was low in thalamus. PDBu increased the [³H]flunitrazepam binding only in the striatum and in part of the cortex and thalamus after activation of PKC. After activation of PKC by PDBu, [³⁵S]TBPS binding was increased in most areas, but binding levels were not changed in the brainstem or cerebellum. The receptor binding was markedly decreased in almost all areas by the addition of 2.5 mM Mg²⁺. Elevated [³⁵S]TBPS binding produced by PDBu was significantly inhibited by the addition of Mg²⁺. These results suggest that the activation of PKC potentiates benzodiazepine and TBPS binding, but decreases muscimol binding in a region-specific manner in the rat brain.     

Alteration of protein kinase C activity in the postischemic rat brain areas using in vitro [3H]phorbol 12,13-dibutyrate autoradiography

Summary Chronological changes of protein kinase C (PKC) activity were measured using in vitro [³H]phorbol 12,13-dibutyrate (PDBu) autoradiography to investigate the postischemic alteration of this second messenger system in the rat brain. Transient ischemia was induced by the occlusion of the middle cerebral artery (MCA) for 90 min and such occlusion followed by various recirculation periods of up to 4 weeks. After 90 min of ischemia followed by 3 hours of recirculation, [³H]PDBu binding sites were found to be significantly decreased in the cerebral cortex and lateral segment of the caudate putamen, both supplied by the occluded MCA; thereafter, the binding sites decreased progressively in those ischemic foci. On the contrary, there was no alteration on day 1, but 3 days after ischemic insult, a significant decrease of [³H]PDBu binding sites was first detected in the ipsilateral thalamus and the substantia nigra, which both areas had not been directly affected by the original ischemic insult. This postischemic delayed phenomenon observed in the thalamus and the substantia nigra developed concurrently with⁴⁵Ca accumulation, which was detected there in our previous study. These results suggest that alteration of second messenger (PKC) pathways may be involved not only in the ischemic foci, but also in neuronal degeneration of the exo-focal remote areas in relation to the disruption of intracellular calcium homeostasis which plays a key role in the pathogenesis of postischemic neuronal damage and that marked alteration of intracellular signal transduction may precede the neuronal damage in the exo-focal postischemic brain areas.     

Activation of protein kinase C by phorbol dibutyrate modulates GABAA receptor binding in rat brain slices

Effects of protein kinase C (PKC) activation on the function of the GABA/benzodiazepine receptor-chloride complex were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam and [³⁵S]TBPS in rat brain slices. The density of [³H]muscimol binding was highest in cerebellar granular layers and high in both the frontal cortex and thalamus, but binding levels in the hippocampus were low. After activation of PKC by 100 nM phorbol-12,13-dibutyrate (PDBu), [³H]muscimol binding was decreased in the frontal cortex, striatum and thalamus, but binding levels were not changed in the hippocampus or cerebellum. The density of [³H]flunitrazepam binding was high in the cortex, hippocampus and molecular layers of cerebellum but was low in thalamus. PDBu increased the [³H]flunitrazepam binding only in the striatum and in part of the cortex and thalamus after activation of PKC. After activation of PKC by PDBu, [³⁵S]TBPS binding was increased in most areas, but binding levels were not changed in the brainstem or cerebellum. The receptor binding was markedly decreased in almost all areas by the addition of 2.5 mM Mg²⁺. Elevated [³⁵S]TBPS binding produced by PDBu was significantly inhibited by the addition of Mg²⁺. These results suggest that the activation of PKC potentiates benzodiazepine and TBPS binding, but decreases muscimol binding in a region-specific manner in the rat brain.     

Distribution of protein kinase C in the brain of Apteronotus leptorhynchus as revealed by phorbol ester binding.

An antibody to the mammalian protein kinase C alpha (PKCalpha) subunit and brain dissection was used for immunoblot analysis of this protein in various brain regions of Apteronotus leptorhynchus. Western blots revealed that the antibody labeled a band of the expected molecular mass (approximately 80 kDa) for this enzyme in mammalian cortex and electric fish brain, suggesting that this protein is also found in gymnotiform brain. The 80-kDa band was enriched in fish forebrain and cerebellum compared with hypothalamus and brainstem areas. [3H]Phorbol 12,13-dibutyrate ([3H]PDBu) binding was used as a marker for the distribution of protein kinase C (PKC). [3H]PDBu binding was nearly completely displaced by excess cold PDBu; specific [3H]PDBu binding sites were heterogenously distributed with high densities in some gray matter regions and negligible densities in fiber tracts. A very high density of [3H]PDBu binding sites were found in the dorsal forebrain with far lower densities in most ventral forebrain nuclei. Low binding densities were observed in preoptic and hypothalamic areas with the exception of the nucleus diffusus and nucleus tuberis anterior. The thalamus and midbrain also had only low levels of binding. The cerebellar molecular layer had dense binding, in contrast to the granule cell layer where binding was negligible. In the electrosensory lateral line lobe (ELL), there was moderate binding in the dorsal molecular layer, which contains cerebellar parallel fibers; the other layers of the ELL had far lower binding densities.     

Protein kinase C in focal ischemic rat brain: dual autoradiographic analysis of [C]iodoantipyrine (IAP) and [H]phorbol-12,13-dibutyrate (PDBu)

Protein kinase C (PKC) activity was measured in rat brain with 2 h of middle cerebral artery (MCA) and common carotid artery (CCA) occlusion, using dual autoradiography of [¹⁴C]iodoantipyrine (IAP) and [³H]phorbol-12,13-dibutyrate (PDBu). In the ischemic brain, it required more than 120 min of incubation to obtain a plateau in PDBu binding. In contrast, the binding of PDBu in non-ischemic brain reached a plateau with incubation for 60 min. This delay of PDBu binding in the ischemic brain suggests that the affinity of this ligand is reduced due to a change in structure of the cell membrane caused by ischemia. PDBu binding in the ischemic brain increased significantly compared to the non-ischemic brain. This finding provides further evidence that excessive activation of PKC in the ischemic brain may play an important role in ischemic neuronal damage. ©1997 Elsevier Science B.V. All rights reserved.     

Stress persistently increases NMDA receptor-mediated binding of [H]PDBu (a marker for protein kinase C) in the amygdala, and re-exposure to the stressful context reactivates the increase

The long-term consequences of acute stress on [³H]phorbol 12,13-dibutyrate ([³H]PDBu) binding, a marker for protein kinase C (PKC) activity, were investigated. In the first experiment, exposure to acute restraint and intermittent tail-shock increased [³H]PDBu binding in the amygdala but not in the hippocampus or cerebral cortex. The increase was persistent, lasting at least 24 h after stressor cessation. In the second experiment, it was determined that the stress-induced increase in binding in the amygdala was dependent on NMDA receptor activation; rats injected with a competitive NMDA receptor antagonist prior to the stressor did not exhibit the increased binding in the amygdala 24 h later. In the third experiment, re-exposure to the stressful context 96 h after stressor cessation reactivated the stress-induced increase the binding of [³H]PDBu in the amygdala. Re-exposure to the context also increased binding in the thalamus and area CA1 of the hippocampus. [³H]PDBu binds preferentially to PKC in the membrane and, therefore, these results suggest that stress induces the translocation of PKC from its resting compartments in the cytosol to the membrane. Its dependence on NMDA receptor activation implicates isoforms of PKC that are sensitive to intracellular calcium, such as PKCγ. The results further suggest that a ‘psychological' manipulation, viz. context re-exposure, can reactivate the persistent increase in [³H]PDBu binding in the amygdala. © 1997 Elsevier Science B.V. All rights reserved.     

Autoradiographic analysis of second-messenger systems in the gerbil hippocampus following repeated brief ischemic insults.

Using [3H]inositol 1,4,5-triphosphate (IP3), [3H]phorbol 12,13-dibutyrate (PDBu) and [3H]forskolin, we performed quantitative autoradiography to determine sequential alterations in second-messenger systems in the gerbil hippocampus following repeated brief ischemic insults. Changes following three 2-min ischemic insults were compared with those following single 2- or 6-min ischemia. [3H]IP3 binding was extremely sensitive to ischemic insult, and more than 80% of the binding sites were lost after destruction of CA1 pyramidal cells following 6-min ischemia and three 2-min ischemic insults. Furthermore, a 30% reduction was observed after 2-min ischemia which leads to no neuronal loss. [3H]PDBu binding in the CA1 subfield decreased by 1 day after three 2-min ischemic insults and by 4 days after 6-min ischemia, and 40-50% reductions were observed at 1 month. In contrast, [3H]forskolin binding was relatively preserved. [3H]PDBu and [3H]forskolin binding transiently increased early in the reperfusion period. We also observed a difference in the pattern and severity of alterations between repeated ischemic insults and single ischemia.     

Hypothermia attenuates the activation of protein kinase C in focal ischemic rat brain: dual autoradiographic study of [H]phorbol 12,13-dibutyrate and iodo[C]antipyrine

Using phorbol 12,13-dibutyrate (PDBu) autoradiography, we investigated the effect of hypothermia on protein kinase C (PKC) activation in rat brain 2 h after focal ischemia. In normothermia, a significant increase of PDBu binding was observed in ischemic brain. Hypothermia suppressed the increase of PDBu binding in degree and extent. These observations suggest that intraischemic hypothermia attenuates the activation of PKC, and this may in part be participate in the protective effect of hypothermia.     

[H]Phorbol ester binding sites and neuronal plasticity in the hippocampus following entorhinal cortex lesions

Entorhinal cortex lesioning (ECL) produces a loss of more than 80% of the synapses in the outer molecular layer of the hippocampus. However, the loss of synapses is transient. Beginning a few days after denervation, new synapses are formed, virtually replacing the lost inputs within 2 months. Synaptic remodelling induced by ECL is associated with specific modifications of neurotransmitters, hormones and growth factors. Particularly, protein kinase C (PKC) plays important functional roles in receptor-mediated transmembrane signal transduction. PKC is also involved in various aspects of synaptic plasticity, such as cellular growth and differentiation. To investigate further the potential roles of PKC in synaptic plasticity observed in the ECL model, [³H]phorbol 12,13-dibutyrate ([³H]PDBu) binding, a putative marker of PKC, was examined at different times post-lesion. [³H]PDBu binding sites transiently decreased bilaterally at 2 and 8 days post-lesion (20%) in different laminae and sub-fields of the rostral hippocampus but returned to control values at 14 and 30 days post-lesion. In caudal portion of the hippocampus, [³H]PDBu binding was also decreased at 2 days post-lesion but only on the contralateral side. Interestingly, [³H]PDBu binding sites in the cortex increased by up to 30% in the contralateral side while no significant change was observed in the ipsilateral side at any time post-lesion. It is known that PKC can be regulated by different systems following alterations of neuronal and glial activity. We suggest that these could be involved in the response of PKC and [³H]PDBu binding sites following ECL. Moreover, PKC seemed to be modified in different brain areas in addition to the hippocampal formation in this model. This can be associated to a rather general reorganization observed following losses of neuronal inputs from the entorhinal cortex and the subsequent reinnervation process.     

Postreceptor modulation of cAMP accumulation in rat brain particulate fraction after ischemia— Involvement of protein kinase C

The brain cyclic AMP generation was studied in rats subjected to 15 min of cardiac arrest. We have used a particulate, synaptoneurosomal fraction to demonstrate the effect of ischemia in vivo on the responsiveness of adenylate cyclase (AC) system. It has been shown that, although there is a slight decrease in AC activity after ischemia, the in vitro fractions produce more cAMP in response to a variety of stimuli, suggesting an indirect, nonadenylate cyclase activation mechanism. For elucidation of this mechanism we have probed phorbol-12,13-dibutyrate (PDBu) as a direct PKC activator, forskolin to activate the catalytic subunit of AC, and cholera toxin (CT) for stabilizing the active, GTP-bound form of stimulatory guanine nucleotide binding protein (G_s). All these postreceptor AC modulators as well as the receptor activators such as adenosine and α₁-adrenergic agonists markedly enhanced cAMP production in the rat brain particulate fraction, although the postischemic hyperactive response to these stimuli was still present. However, when AC was stimulated by the combination of CT and PDBu, cAMP responses were identical in both control and postischemic fractions. The data, taken together, support the hypothesis that ischemia increases cAMP accumulation by facilitating the postreceptor AC activation through a PKC-involving pathway and by promoting the stronger coupling of membrane AC receptors with G-protein. Protein kinase C (PKC) activity during cerebral ischemia was also investigated. In contradistinction to our expectation PKC decreased significantly in the ischemic brain to 85% of the control activity in the cytosol and 72% in the membranes. However, in the incubated postischemic brain particulate fraction a relative increase in the membranebound form of the enzyme, from 30% for control to 53% for ischemia, was observed. This may suggest that ischemia-induced membrane changes could promote the enzyme translocation/activation during recovery, resulting in the sensitization of cAMP producing system.     

Regional changes in [H]inositol 1,4,5-triphosphate binding in the gerbil brain following repeated sublethal ischemia

We investigated the regional changes in [³H]inositol 1,4,5-triphosphate (IP₃) binding in the brain following ischemia using in vitro autoradiography. Three 2-min ischemic insults at 1-hr intervals and a 6-min period of ischemia were induced in gerbils and they were killed after 1, 4, and 28 days. Normal animals had high [³H]IP₃ binding in the CA1 subfield of the hippocampus and the striatum. The binding in the CA1 decreased strikingly after both 6-min ischemia and three 2-min ischemic insults. The [³H]IP₃ binding also decreased in the lateral striatum after three 2-min ischemic insults but not after 6 min of ischemia. Histological observations confirmed neuronal damage to these areas of reduced binding. By contrast, we found a marked increase in [³H]IP₃ binding in the ventral thalamus 28 days after three 2-min ischemic insults. Histological observations with Nissl staining revealed an accumulation of fine granular deposits there. Thus, repeated ischemic insults produced more extensive neuronal damage and changes in [³H]IP₃ binding than a single equivalent period of ischemia. The increased [³H]IP₃ binding in the thalamus coincidentally with an accumulation of Nissl-positive granules at the chronic stage after repeated ischemia is of considerable interest.     

Cerebral blood flow and metabolism in soman-induced convulsions

Regional cerebral blood flow (CBF) and regional cerebral glucose utilization (CGU) were studied by quantitative autoradiographic techniques in rats. Animals were treated either with a toxic dose of soman, an irreversible organophosphorus cholinesterase inhibitor, that produced convulsions or with saline as controls. An increased arterial blood pressure (mean increase = 41% of control) always preceded onset of convulsions. Convulsive activity was associated with an increase of plasma glucose concentration and marked increases over controls of CGU [average of all regions: control = 75 +/- 5 mumol.100 g-1.min-1, n = regions/animals (304/8); seizures = 451 +/- 20 mumol.100 g-1.min-1, n = 190/5] and CBF [average of all regions: control = 135 +/- 6 ml.100 g-1.min-1, n = 190/5; seizures = 619 +/- 29 ml.100 g-1.min-1, n = 190/5). Regional distribution of these effects revealed a greater proportional increase of CBF over CGU in cingulate, motor, and occipital cortex and caudate-putamen. In contrast, a lower proportional increase of CBF over CGU in CA3 region of hippocampus, dentate gyrus, medial thalamus, and substantia nigra was observed, implying the existence of a relative ischemia in these brain areas. These findings may be relevant to the pathogenesis of brain lesions associated with soman-induced convulsions.     

The effect of 4β-phorbol-12,13-dibutyrate and staurosporine on the extracellular glutamate levels during ischemia in the rat striatum

Hypothermia diminishes the ischemia-induced protein kinase C (PKC) translocation and inhibition, and also reduces transmitter release during ischemia. To study the role of PKC in the mechanism of glutamate release during ishcemia, we measured extracellular glutamate levels in the striatum with the microdialysis technique, in the presence and absence in the dialysate of the PKC activator 4β-phorbol-12,13-dibutyrate (PDBu) and the protein kinase inhibitor staurosporine. We confirm that hypothermia attenuates the elevation of extracellular levels of glutamate in the striatum during ischemia. In the presence of PDBu, the glutamate levels in the dialysate increased from 0.3 μmol/L to an end ischemic level of 4.8 μmol/L during hypothermic ischemia (33°C). These levels were significantly higher than in hypothermic ischemia (33°C) without added PDBu. Staurosporine significantly mitigated the glutamate levels during normothermic ischemia. Our data suggest that PKC is involved in the temperature-dependent elevations of extracellular glutamate levels in the striatum during ischemia, and we propose that compounds preventing PKC activation may mimic the hypothermic protective action against ischemic brain damage.     

Autoradiographic analysis of [3H]desmethylimipramine binding in the human brain postmortem

In vitro quantitative autoradiography of high-affinity [3H]desmethylimipramine (DMI) binding sites was performed on 24 human brains postmortem. Highest densities of binding sites were found in portions of the amygdala, the granular layer of the dentate gyrus, the pyramidal layer of CA4. Next, in descending order were other portions of the hippocampus and amygdala, the head of the caudate nucleus, putamen, hypothalamus, insular cortex, prefrontal cortex, parietal, frontal and temporal cortex, anterior, medial and posterolateral nuclei of the thalamus. Lowest densities, hardly above background, were found in the two divisions of the globus pallidus, the centromedian, ventrolateral and posteroventral lateral nuclei of the thalamus, substantia nigra, red nucleus and white matter. Specific high-affinity [3H]DMI binding was not affected by age, sex and suicide in any of the regions studied. It was negatively correlated with postmortem delay in several regions.     

Age-related changes of sodium-dependentd-[3H]aspartate and [3H]FK506 binding in rat brain

Summary We investigated age-related changes in excitatory amino acid transport sites and FK506 binding protein (FKBP) in 3-week-, and 6-, 12-, 18- and 24-month-old Fischer 344 rat brains using receptor autoradiography. Sodium-dependentd-[³H]aspartate and [³H]FK506 were used to label excitatory amino acid transport sites and immunophilin (FKBP), respectively. In immature rats (3-week-old), sodium-dependentd-[³H]aspartate binding was lower in the frontal cortex, parietal cortex, striatum, nucleus accumbens, whole hippocampus, thalamus and cerebellum as compared to adult animals (6-month-old), whereas [³H]FK506 binding was significantly lower only in the hippocampus, thalamus and cerebellum. [³H]FK506 binding exhibited no significant change in the brain regions examined during aging. However, sodium-dependentd-[³H]aspartate binding showed a conspicuous reduction in the substantia nigra in 18-month-old rats. Thereafter, a significant reduction in sodium-dependentd-[³H]aspartate binding was found in the thalamus, substantia nigra and cerebellum in 24-month-old rats. Other regions also showed about 10–25% reduction in sodium-dependentd-[³H]aspartate binding. The results indicate that excitatory amino acid transport sites are more susceptible to aging process than immunophilin. Further, our findings demonstrate the conspicuous differences in the developmental pattern between excitatory amino acid transport sites and immunophilin in immature rat brain.     

Induction of tolerance to ischemia: Alterations in second-messenger systems in the gerbil hippocampus

Preconditioning the brain with sublethal ischemia protects against neuronal damage following subsequent ischemic insult. Using [3H]inositol 1,4,5-triphosphate (IP3), [3H]phorbol 12,13-dibutyrate (PDBu), [3H]cyclic adenosine monophosphate (cAMP) and [3H]rolipram, we performed quantitative autoradiography to determine postischemic alterations in second-messenger systems in the gerbil hippocampus following preconditioning the brain with sublethal ischemia. At 7 days of reperfusion, no alterations were observed in brains subjected to 2 min of forebrain ischemia which produced no neuronal damage. However, 3-min ischemia caused a 75% reduction in [3H]IP3 binding (p < 0.01 vs. control) and 15-25% reductions in [3H]forskolin (p < 0.01 vs. control), [3H]cAMP (p < 0.05 vs. control), and [3H]rolipram (p < 0.01 vs. control) binding in the CA1 subfield coincident with histopathological CA1 pyramidal cell destruction, but no significant alterations in [3H]PDBu binding. Preconditioning the brain with 2 min of ischemia followed by 4 days of reperfusion prevented both histopathological cell death and the reductions in binding following subsequent 3 min of ischemia. Interestingly, [3H]IP3 and [3H]rolipram binding in CA1 showed a transient reduction, by 30% and 20% (both p < 0.01 vs. control), respectively, in the early reperfusion period. This downregulation of the IP3 system may play a role in the protection against cell death.     

Differential visualization of dopamine and norepinephrine uptake sites in rat brain using [3H]mazindol autoradiography

Mazindol is a potent inhibitor of neuronal dopamine (DA) and norepinephrine (NE) uptake. DA and NE uptake sites in rat brain have been differentially visualized using [3H]mazindol autoradiography. At appropriate concentrations, desipramine (DMI) selectively inhibits [3H]mazindol binding to NE uptake sites without significantly affecting binding to DA uptake sites. The localization of DMI-insensitive specific [3H] mazindol binding, reflecting DA uptake sites, is densest in the caudate-putamen, the nucleus accumbens, the olfactory tubercle, the subthalamic nucleus, the ventral tegmental area, the substantia nigra (SN) pars compacta, and the anterior olfactory nuclei. In contrast, the localization of DMI-sensitive specific [3H]mazindol binding, representing NE uptake sites, is densest in the locus coeruleus, the nucleus of the solitary tract, the bed nucleus of the stria terminalis, the paraventricular and periventricular nuclei of the hypothalamus, and the anteroventral thalamus. The distribution of DMI-insensitive specific [3H]mazindol binding closely parallels that of dopaminergic terminal and somatodendritic regions, while the distribution of DMI-sensitive specific [3H]mazindol binding correlates well with the regional localization of noradrenergic terminals and cell bodies. Injection of 6-hydroxydopamine, ibotenic acid, or colchicine into the SN decreases [3H]mazindol binding to DA uptake sites in the ipsilateral caudate-putamen by 85%. In contrast, ibotenic acid lesions of the caudate-putamen do not reduce [3H]mazindol binding to either the ipsilateral or contralateral caudate-putamen. Thus, the DA uptake sites in the caudate-putamen are located on the presynaptic terminals of dopaminergic axons originating from the SN.     

Quantitative autoradiographic analysis of muscarinic cholinergic and adenosine A1 binding sites after transient forebrain ischemia in the gerbil

The influence of transient forebrain ischemia on adenosine A₁ and muscarinic cholinergic receptors in the gerbil brain 1–27 days after recirculation was studied. The topographical distribution and the alteration in the adenosine A₁ and muscarinic receptor sites were analyzed by means of quantitative receptor autoradiography using [³H]cyclohexyladenosine ([³H]CHA) and [³H]quinuclidinyl benzilate ([³H]QNB), respectively. In most regions examined, the temporal profiles of the alteration of the receptor density were in accordance with the histopathological findings. [³H]CHA binding activity decreased suddenly after neuronal damage, while [³H]QNB grain density showed a gradual decrease in the dorsolateral caudate-putamen and in the CA₁ subfield of the hippocampus. In the caudate-putamen, [³H]CHA and [³H]QNB binding activity in the dorsal aspect was markedly reduced 1–27 days after ischemia. [³H]CHA binding activity in the ventromedial region of the caudate-putamen also decreased 1–3 days after ischemia, though neuronal damage was restricted to the dorsolateral aspect. Neuronal death in CA₁ was preceded by the decrease in [³H]QNB binding activity in the stratum radiatum 1 and 2 days after ischemia. Marked decrease in [³H]QNB and [³H]CHA binding activity was noted in the CA₁ subfield 3–27 days after recirculation. Three to 27 days after ischemia, the A₁ binding activities in the CA₃ subfield of the hippocampus and int he dentate gyrus were reduced despite the normal appearance of these areas throughout the reperfusion period. Muscarinic binding sites in the CA₃ subfield were also reduced 27 days after ischemia. Despite minimal neuronal damage in the lateral septal nucleus and in the substantia nigra, the A₁ binding activity in these regions was reduced by 70% and 50%, respectively. These results provide further evidence that the muscarinic receptors in the dorsolateral region of the caudate-putamen are localized postsynaptically on small and medium-sized neurons and that those in the CA₁ subfield of the hippocampus are localized on the CA₁ pyramidal cells.     

Alterations of 45Ca accumulation and [3H]inositol 1,4,5-trisphosphate binding using autoradiography in the exo-focal postischemic brain areas of the rat.

We studied the alterations of calcium accumulation and intracellular signal transduction using autoradiography of the second messenger system in order to clarify the mechanisms of the delayed neuronal changes in the remote areas of rat brain after transient focal ischemia. Chronological changes of 45Ca accumulation and [3H]inositol 1,4,5-trisphosphate (IP3) binding sites were determined after 90 min of right middle cerebral artery (MCA) occlusion and after such occlusion followed by different periods of recirculation. After the ischemic insult, 45Ca accumulation extended to the lateral segment of the caudate putamen and to the cerebral cortex, both supplied by the occluded MCA. One day after the ischemia, [3H]IP3 binding sites decreased significantly compared with the control values in these ischemic areas. Moreover, 3 days after the ischemia, 45Ca accumulation was first detected in the ipsilateral thalamus and the substantia nigra, which lay outside the ischemic areas. In the substantia nigra, a significant decrease of [3H]IP3 binding sites and concurrent 45Ca accumulation were observed. In the thalamus, however, there was not alteration until 1 week after the ischemia, and then [3H]IP3 binding sites increased significantly 2 weeks (P less than 0.05) and 4 weeks (P less than 0.01) after the ischemia. Based on the present study, we speculate that different mechanisms associated with signal transduction systems may be responsible for exo-focal postischemic delayed neuronal changes in the thalamus and the substantia nigra. The increase of [3H]IP3 binding sites of the thalamus in the chronic stage may be new evidence of plasticity related to neurotransmission.