Age-dependent changes in second messenger and rolipram receptor systems in the gerbil brain

Summary Age-related alterations in binding sites of major second messengers and a selective adenosine 3,5-cyclic monophosphate (cyclic-AMP) phospho-diesterase (PDE) in the gerbil brain were analysed by receptor autoradiography. [³H]Phorbol 12,13-dibutyrate (PDBu), [³H]inositol 1,4,5-trisphosphate (IP₃), [³H]forskolin, [³H]cyclic-AMP, and [³H]rolipram were used to label protein kinase C (PKC), IP₃ receptor, adenylate cyclase, cyclic-AMP dependent protein kinase (PKA), and Ca²⁺/calmodulin-mdependent cyclic-AMP PDE, respectively. In middle-aged gerbils (16 months old), [³H]PDBu binding was significantly reduced in the hippocampal CA 1 sector, thalamus, substantia nigra, and cerebellum, compared with young animals (1 month old). [³H]IP₃ binding revealed significant elevations in the nucleus accumbens, hippocampal CA 1 sector, dentate gyrus, and a significant reduction in cerebellum of middle-aged gerbils. [³H]Forskolin binding in middle-aged animals was significantly increased in the nucleus accumbens and hilus of dentate gyrus, but was diminished in the substantia nigra and cerebellum. On the other hand, in middle-aged animals, [³H]cyclic-AMP binding revealed a significant elevation only in the hippocampal CA3 sector, whereas [³H] rolipram binding showed a significant reduction in the thalamus and cerebellum. Thus, the age-related alteration in these binding sites showed different patterns among various brain regions in middle-aged gerbils indicating that the binding sites of PKC, IP₃, and adenylate cyclase are more markedly affected by aging than those of PKA and cyclicAMP PDE and that the hippocampus and cerebellum are more susceptible to these aging processes than other brain regions. The findings suggest that in-tracellular signal transduction is affected at an early stage of senescence and this may lead to neurological deficits.     

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.     

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.     

Age-related changes in bindings of second messengers in the rat brain

Age-related alterations in bindings of major second messengers in the brain were studied in 3-week- and 6-, 12-, 18- and 24-month-old Fisher 344 rats using receptor autoradiography. [³H]Phorbol 12,13-dibutyrate (PDBu) and [3H]forskolin were used to label protein kinase C (PKC) and adenylate cyclase, respectively. In immature rats (3-week-old), [³H]PDBu binding showed a significant decrease only in the cerebellum as compared to adult rats (6-month-old), whereas [³H]forskolin binding exhibited a significant reduction in the neocortex, nucleus accumbens, thalamus and substantia nigra. In aged rats, [³H]PDBu binding showed no significant change in all brain areas. In contrast, [³H]forskolin binding showed a conspicuous reduction in various brain areas in 18-month-old rats as compared to adult animals. The age-related reduction was especially observed in the cerebral cortex, hippocampal CA3 pyramidal cell layer, dentate gyrus, thalamus and molecular layer of cerebellum of 24-month-old rats. The results indicate that adenylate cyclase system in the rat brain is more susceptible to aging processes than phosphoinositide cycle system. Furthermore, our data demonstrate that the change in the adenylate cyclase system is more pronounced than that in the phosphoinositide cycle system in immature rat brain. These findings suggest that the adenylate cyclase system is primarily affected in aging processes and this may lead to age-related neurological deficits.     

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.     

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 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.     

Autoradiographic mapping of a selective cyclic adenosine monophosphate phosphodiesterase in rat brain with the antidepressant [3H]rolipram

Rolipram is a clinically effective antidepressant with selective cAMP phosphodiesterase (PDE) inhibiting properties. (+/-)-[3H]Rolipram binds with high affinity (Kd = 2.52 +/- 0.47 nM) to sections of rat brain (Hill number = 0.90 +/- 0.05). Binding is stereospecific. Association of (+/-) [3H]rolipram to sections is rapid (47% of specific binding in the first minute, kobs = 0.52 min-1). Dissociation of (+/-)-[3H]rolipram exhibits non first order kinetics (3 component model; t1/2 = 2.5 min, 50 min and 6 h, respectively). A number of PDE inhibitors reduce (+/-)-[3H]rolipram binding to the level of nonspecific binding ((-)-rolipram, IC50 = 0.9 nM; (+/-)-rolipram, IC50 = 1.5 nM; Ro 20-1724, IC50 = 11 nM; ICI 63.197, IC50 = 35 nM; medazepam, IC50 = 240 nM; diazepam, IC50 = 1200 nM; IBMX, IC50 = 3800 nM). In vitro autoradiography reveals high binding site densities in the cerebellum, olfactory bulb, lateral septal nucleus, frontal cortex, subiculum and CA1 of hippocampus. Most of the labeled structures are part of the limbic system. In vivo autoradiography of (+/-)-[3H]rolipram binding shows much more nonspecific binding than in vitro, nevertheless the distribution pattern of (+/-)-[3H]rolipram binding sites is similar. A comparison of the distribution pattern of (+/-)-[3H]rolipram binding sites with that of an antidepressant (monoamine oxidase inhibitor, monoamine uptake inhibitor) reveals no overlap. Limited, though significant correlations exist with the distribution of beta 1-adrenergic, adenosine1 and glutamate/quisqualate receptors as well as protein kinase C, but not with beta 2-adrenergic receptors and forskolin binding sites.     

Withdrawal from butorphanol dependence alters binding of [3H]phorbol dibutyrate to protein kinase C, but not of [3H]forskolin to adenylate cyclase

The time course of autoradiographic binding of major second messengers in the rat brain was studied at 2, 7, and 24 h after withdrawal from butorphanol infusion. [3H]Forskolin and [3H]phorbol 12,13-dibutyrate (PDBu) were used to label adenylate cyclase and protein kinase C (PKC), respectively. Rats were rendered dependent by intracerebroventricular infusion of butorphanol (26 nmol microliter-1 h-1) via osmotic minipumps for 3 days. Withdrawal was initiated by abrupt cessation of the butorphanol infusion. The levels of [3H]forskolin binding were not changed at any time or in any brain area, except for an increase following 7 h of withdrawal in the brainstem only. The levels of [3H]PDBu binding were significantly increased (13-47%) in multiple areas of the rat brain following 7 h of withdrawal from butorphanol infusion. These findings suggest that the phosphoinositide cycle system is more susceptible to alteration during butorphanol dependence than is the adenylate cyclase system in the rat brain.     

Autoradiographic analysis of second-messenger systems in the gerbil brain

Quantitative in-vitro autoradiographic study was performed to localize two prominent second-messenger systems (the adenylate cyclase and phosphoinositide systems) in the normal gerbil brain. [3H] Forskolin and [3H] phorbol 12, 13-dibutyrate (PDBu) were used to identify the regional distribution of adenylate cyclase and protein kinase C, respectively. The localization of the forskolin binding was not uniform, being particularly concentrated in the striatum, the accumbens nucleus, the olfactory tubercle, the substantia nigra, the CA3 region of the hippocampus and the molecular layer of the cerebellum. On the other hand, the PDBu binding was rather uniform, although the superficial layer of the cerebral neocortices, the strata oriens of the CA1 region of the hippocampus and the molecular layer of the cerebellum showed relatively dense binding. Quantitative autoradiography of the second-messenger systems in the brain is expected to provide important information concerning the role of neurotransmitters in the pathophysiology of various conditions.     

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.     

Changes in [H]PAF binding and PAF concentrations in gerbil brain after bilateral common carotid artery occlusion: a quantitative autoradiographic study

This study investigated the distribution of platelet activating factor (PAF) binding sites in the brain and their involvement in global ischemia in a model of bilateral common carotid occlusion in the gerbil. In sagittal sections of gerbil brain, labeling with [³H]PAF was mainly located in the cortex, hippocampus and cerebellum. The corpus striatum, the superior and inferior colliculi showed lower binding, while the thalamus was only weakly labeled. Scatchard analysis of the data obtained from displacement curves with unlabeled PAF revealed the presence of one or two populations of binding sites with different affinity constant values depending on the brain structures. When the gerbils were submitted to 10 min ischemia, similar autoradiography with [³H]PAF demonstrated a dramatic reduction of labeling in all brain structures, particularly in the hippocampus. Immunoreactive endogenous PAF concentrations in brain tissues showed a marked increase in ischemic animals: (8977.3±1113 pg/g wet weight) as compared to sham-operated control: (997.7±77 pg/g wet weight). Endogenous PAF levels returned to basal values following 30 min reperfusion. These results indicate that PAF may be involved in the early stages of brain ischemia in the gerbil and suggest that endogenous PAF produced during ischemia may contribute to the down-regulation of [³H]PAF binding sites in the brain.     

Sequential alteration of [H]rolipram binding in gerbil brain after transient cerebral ischemia

The time course of rolipram (Ca²⁺/calmodulin independent cyclic adenosine monophosphate inhibitor) binding sites changes following gerbil transient forebrain ischemia was determined using receptor autoradiography. Gerbils subjected to 10-min ischemia revealed a significant reduction in rolipram binding in most selectively vulnerable regions early in the recirculation (1–5 h). Marked reduction in the rolipram binding was seen in the selectively vulnerable areas 48 h or 7 days after ischemia. Thereafter, the rolipram binding in the hippocampal CA1 and CA3 sectors, which were most vulnerable to ischemia, was severely reduced up to 1 month after recirculation. In contrast, the reduction of the rolipram binding activity in other regions recovered to sham-operated level or showed a slight recovery. Interestingly, the dentate gyrus, which was resistant to ischemia, also exhibited a significant reduction of the rolipram binding activity up to I month after ischemia. Eight months after ischemia, the hippocampal CA 1 and CA3 sectors showed severe shrinkage and marked reduction in the rolipram binding. Other regions exhibited no significant reduction in the rolipram binding except for a slight reduction in the thalamus. These results demonstrate that transient cerebral ischemia causes severe reduction in rolipram binding sites in selectively vulnerable areas, and this reduction precedes the neuronal cell loss. These findings may reflect the alteration of an intracellular phosphodiesterase activity after ischemia.     

Withdrawal from butorphanol dependence alters binding of [H]phorbol dibutyrate to protein kinase C, but not of [H]forskolin to adenylate cyclase

The time course of autoradiographic binding of major second messengers in the rat brain was studied at 2, 7, and 24 h after withdrawal from butorphanol infusion. [³H]Forskolin and [³H]phorbol 12,13-dibutyrate (PDBu) were used to label adenylate cyclase and protein kinase C (PKC), respectively. Rats were rendered dependent by intracerebroventricular infusion of butorphanol (26 nmol μl⁻¹ h⁻¹) via osmotic minipumps for 3 days. Withdrawal was initiated by abrupt cessation of the butorphanol infusion. The levels of [³H]forskolin binding were not changed at any time or in any brain area, except for an increase following 7 h of withdrawal in the brainstem only. The levels of [³H]PDBu binding were significantly increased (13–47%) in multiple areas of the rat brain following 7 h of withdrawal from butorphanol infusion. These findings suggest that the phosphoinositide cycle system is more susceptible to alteration during butorphanol dependence than is the adenylate cyclase system in the rat brain.     

Heterogeneous localization of protein kinase C in rat brain: autoradiographic analysis of phorbol ester receptor binding

Protein kinase C is a calcium- and phospholipid-stimulated enzyme present in high concentration in the brain. Phorbol esters are potent tumor promoters that bind to specific receptors with high affinity. Several lines of evidence indicate that the phorbol ester receptor is identical to protein kinase C. To determine the distribution of protein kinase C, we have localized phorbol ester receptors in the rat brain by autoradiography, using [3H]phorbol 12,13-dibutyrate ([3H]PDBu) and have performed a variety of lesions to assess the nature of the cellular elements possessing the binding sites. The [3H]PDBu binding sites in the rat brain are discretely localized and primarily associated with neurons. Evidence is presented for localization to intrinsic neurons of the cortex and hippocampus, terminals of the striatonigral projection, a projection to the molecular layer of the dentate gyrus, and to dendrites of Purkinje cells.     

Effects of naftidrofuryl oxalate, a 5HT2 antagonist, on neurotransmission and transduction systems in the gerbil hippocampus

We investigated the effects of age and naftidrofuryl oxalate (Naftidrofuryl), a 5-HT₂ antagonist, on neurotransmission and transduction systems in the gerbil hippocampus using quantitative autoradiography. [³H]Quinuclidinyl benzilate (QNB), [³H]cyclohexyl-adenosine (CHA), [³H]MK-801, and [³H]muscimol were used to label muscarinic acetylcholine, adenosine A1, N-methyl-d-aspartate (NMDA), and γ-aminobutyric acid-A (GABA_A) receptors, respectively. [³H]PN200-110 labeled L-type Ca²⁺ channels. [³H]Forskolin, [³H]cyclic adenosine monophosphate (cAMP), [³H]phorbol 12,13-dibutyrate (PDBu), and [³H]inositol 1,4,5-triphosphate (IP₃) were used to label adenylate cyclase, cAMP-dependent protein kinase, protein kinase C (PKC), and IP₃ receptors, respectively. Approximately 20% reductions in [³H]QNB, [³H]forskolin, and [³H]PDBu binding were observed in the hippocampus of 9-month-old gerbils in comparison with 5-week-old gerbils. Treatment with Naftidrofuryl (10 mg/kg, i.p., once a day for 7 days) ameliorated these reductions. No changes were found in [³H]CHA, [³H]MK-801, [³H]muscimol, [³H]PN200-110, [³H]cAMP, and [³H]IP₃ binding. The results suggest that Naftidrofuryl may have beneficial effects on the age-related alterations in signal transmission and transduction systems in the brain. Because the acetylcholine system, adenylate cyclase, and PKC are considered to be involved in learning and memory processes, the result may have clinical implications.     

Effects of chronic treatment with a cyclic AMP-selective phosphodiesterase inhibitor, rolipram, on excitatory amino acid neurotransmission systems in young and aged rat brains

Summary Rolipram selectively inhibits cyclic AMP-specific phosphodiesterase, and leads to an increase in cyclic AMP levels in the brain. In this study, we investigated the effects of chronic rolipram treatment on excitatory and inhibitory amino acid neurotransmission systems in young and aged Wistar rat brains. We used in vitro autoradiography with [³H]MK-801, [³H]glycine, D-[³H]aspartate, and [³H]muscimol to label N-methyl-D-aspartate (NMDA) receptors, glycine modulatory sites, glutamate transport sites, and -aminobutyric acid-A (GABA) receptors, respectively. Rolipram (0.01 or 0.1 mg/kg, per os) or its vehicle (distilled water) was administered once a day for 4 weeks. The highest binding of [³H]MK-801, [³H]glycine, and d-[³H]aspartate was seen in the hippocampus in vehicle-treated rats. No significant differences in these binding activities were seen between young and aged rat brains. [³H]Muscimol binding was the highest in the cerebellum, and decreased in many brain regions in aged rats. The chronic rolipram treatment resulted in (1) an increase in [³H]MK-801 binding in the dentate gyrus in both young and aged rats, (2) remarkable reductions in D-[³H]aspartate binding in many regions of both young and aged rats, and (3) no or minimal changes in [³H]glycine and [³H]muscimol binding. These results suggest that the chronic rolipram treatment modifies the excitatory amino acid neurotransmission system.     

Evaluation of CA1 damage using single-voxel 1H-MRS and un-biased stereology: Can non-invasive measures of N-acetyl-asparate following global ischemia be used as a reliable measure of neuronal damage?

Global brain ischemia provoked by transient occlusion of the carotid arteries (2VO) in gerbils results in a severe loss of neurons in the hippocampal CA1 region. We measured the concentration of the neuron specific N-acetyl-aspartate, [NAA], in the gerbil dorsal hippocampus by proton MR spectroscopy (1H-MRS) in situ, and HPLC, 4 days after global ischemia. The [NAA] was correlated with graded hippocampus damage scoring and stereologically determined neuronal density. A basal hippocampal [NAA] of 8.37+/-0.10 and 9.81+/-0.44 mmol/l were found from HPLC and 1H-MRS, respectively. HPLC measurements of [NAA] obtained from hippocampus 4 days after 2VO showed a 20% reduction in the [NAA] following 4 min of ischemia (P<0.001). 1H-MRS measurements on gerbils subjected to 4 or 8 min of ischemia showed a similar 24% decline in the [NAA] (P<0.05). Thus, there was correlation between the HPLC and 1H-MRS determined NAA decline. There was also a significant correlation between 1H-MRS [NAA] and the corresponding reduction in CA1 neuronal density (P<0.004). In summary our findings show that single voxel 1H-MRS can be used as a supplement to histological evaluation of neuronal injury in studies after global brain ischemia. Accordingly, volume selective spectroscopy has a potential for assessment of neuroprotective therapeutic compounds/strategies with respect to neuronal rescue for delayed ischemic brain damage.     

Alteration of second-messenger ligand binding following 2-hr hemispheric ischemia in the gerbil brain.

The alterations of second-messenger ligand binding and cerebral blood flow (CBF) were evaluated in the gerbil brain after 2-h unilateral common carotid artery occlusion. [3H]Forskolin (FK) and [3H]phorbol-12,13-dibutyrate (PDBu) were used as specific ligands for adenylate cyclase (AC) and protein kinase C (PKC) activity estimation, respectively. CBF was determined at the end of the experiment by the [14C]iodoantipyrine method. A quantitative autoradiographic method permitted simultaneous measurement of the three parameters in the same brain. The levels in the caudate-putamen, globus pallidus, and hippocampus were analyzed. The animals were divided into three groups: Group 1 with severe ischemia (CBF in the lateral nuclei of the thalamus (CBFt) less than 50 ml/100 g/min), Group 2 with mild ischemia (CBFt greater than or equal to 50 ml/100 g/min), and the Sham Group. The PDBu binding revealed a statistically significant increase in the caudate-putamen, lateral nuclei of the thalamus and hippocampus (CA1 and CA3 regions and dentate gyrus) on the ischemic side in Group 1 as compared to that in Group 2 and the Sham Group. In contrast, the FK binding did not show any significant changes in any of the regions. These data and our previous findings for 6-h ischemia suggest that (1) PKC translocation to the cell membrane may occur at the early ischemic phase in particular regions including the caudate-putamen, lateral nuclei of the thalamus and hippocampus, with the translocated PKC gradually diminishing during the subsequent ischemic period; and (2) the suppression of the AC system observed in 6-h ischemia may not appear in the early ischemic phase.     

[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.