Odor regulates the expression of the mitogen-activated protein kinase phosphatase gene hVH-5 in bilateral entorhinal cortex-lesioned rats

Since it is known that several immediate early genes are induced by olfactory stimuli, we determined whether an olfactory stimulus also induces the expression of the mitogen-activated protein kinase (MAPK) phosphatase gene hVH-5 (homologue of vaccinia virus H1 phosphatase gene, clone 5), a member of a novel class of immediate early genes encoding dual-specificity protein phosphatases. The expression was studied by in situ hybridization in different brain structures involved in odor processing, in control and bilateral entorhinal cortex (EC) lesioned rats. EC-lesion did not significantly affect hVH-5 gene expression in the glomerular cell layer of the olfactory bulb (OB), while odor stimulation induced it in both control and EC-lesioned groups. In contrast, odor-induced expression of hVH-5 gene in mitral/granular cell layers was only evident after lesion of the EC. Similar results were obtained in the piriform cortex (PCx), a structure intimately connected to the mitral cell layer. In the CA1 hippocampal subfield, odor stimulation induced hVH-5 gene expression in both control and EC-lesioned animals, the increase being potentiated in lesioned rats. CA3 and dentate gyrus exhibited a similar pattern of gene expression, the odor stimulating gene expression in both control and lesioned groups. The amygdala (Am) displayed no significant change. It appears that through the induction of a MAPK phosphatase, the EC controls MAPK activities differently after odor stimulation in OB, PCx and hippocampus (Hip). The results illustrate the notion that odor representation in the brain requires plastic modifications at both anatomical and functional levels.     

Lesion of the lateral entorhinal cortex amplifies odor-induced expression of c-fos, junB, and zif 268 mRNA in rat brain

Paradoxical facilitation of olfactory learning following entorhinal cortex (EC) lesion has been described, which may result from widespread functional alterations taking place within the olfactory system. To test this hypothesis, expression of the immediate early genes c-fos, junB, and zif 268 was studied in response to an olfactory stimulation in several brain areas in control and in EC-lesioned rats. Olfactory stimulation in control rats induced the expression of the three genes in the granular/mitral and glomerular layers of the olfactory bulb, as well as c-fos and junB expression in the piriform cortex. However EC lesion was devoid of effects in nonstimulated animals; it significantly amplified the odor-induced expression of the three genes in these areas, as well as in the amygdala, hippocampus, and parietal-temporal cortices. The data suggest that EC lesion modifies the neural processing of odor by suppressing an inhibitory influence on brain areas connected to this cortex.     

Increased proportion of high-affinity dopamine D2 receptors in rats with excitotoxic damage of the entorhinal cortex, an animal model of schizophrenia

Excitotoxic lesions of the left entorhinal cortex (EC) cause dopamine supersensitivity. In order to determine if these lesions selectively alter the high-affinity state of dopamine D2 receptors (D2(High)), these high-affinity states were measured by competition between dopamine and [3H]domperidone in striata from lesioned rats and sham-operated animals. The proportion of D2(High) sites was significantly elevated by 200% in the EC-lesioned rats while that of the D1(High) sites, measured by dopamine/[3H]SCH23390 competition, was unaltered. These results provide a biochemical basis for behavioral supersensitivity in rats with EC lesions.     

Systemic injection of kainic acid: gliosis in olfactory and limbic brain regions quantified with [3H]PK 11195 binding autoradiography

Neurodegenerative diseases may result from excessive stimulation of excitatory amino acid receptors by endogenous ligands. Because neuronal degeneration is associated with glial proliferation and hypertrophy, the degenerative changes throughout rat brain following the systemic administration of kainic acid (12 mg/kg) were mapped with quantitative autoradiography of [3H]PK 11195. This radioligand binds to a mitochondrial benzodiazepine binding site (MBBS) on microglia and astrocytes. Analysis of eight horizontal and four coronal brain levels revealed up to 16-fold increases in [3H]PK 11195 binding from 1 to 5 weeks but not 1 day after kainate injection. Increases in [3H]PK 11195 binding were predominantly in ventral limbic brain regions and olfactory projections to neocortical areas, with the olfactory cortex greater than subiculum/CA1 greater than anterior olfactory nucleus, medial thalamic nucleus, and piriform cortex greater than cingulate cortex and rostral hippocampus greater than dentate gyrus, septum, and amygdala greater than entorhinal cortex and temporal cortex. Little or no enhancement of [3H]PK 11195 binding was observed in numerous regions including the caudate-putamen, substantia nigra, nucleus accumbens, olfactory tubercle, cerebellum, thalamic nuclei, choroid plexus, medulla, parietal or occipital cortex, or pons. A 2-fold greater extent of neurodegeneration was obtained in ventral portions of the olfactory bulb, entorhinal cortex, temporal cortex, and dentate gyrus compared with the dorsal portions of these structures. The pattern of increase in [3H]PK 11195 binding closely matched the patterns of neuronal degeneration reported following parenteral kainate injection. These findings strengthen the notion that quantitative autoradiography of [3H]PK 11195 is a valuable tool to quantify the extent of neuronal degeneration. Furthermore, the quantitative changes in [3H]PK 11195 binding in different limbic structures parallel their relative variation in neuropathology observed in Alzheimer's disease but not Huntington's chorea. These findings are in agreement with the idea that excessive stimulation of excitatory amino receptors may contribute to the etiology of Alzheimer's disease.     

[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.     

Development of phorbol ester (protein kinase C) binding sites in cat visual cortex

Tritiated phorbol-12,13-dibutyrate [( 3H]PDBu), a phorbol ester, was utilized to autoradiographically localize protein kinase C (PKC) in the cat visual cortex. Thin, slide-mounted sections of adult cat brain were used to characterize binding of [3H]PDBu. This was found to be saturable, reversible, and more readily displaced by phorbol ester than by synthetic diacylglycerols. Binding sites displayed a tissue concentration of 20 pmol/mg protein, and a dissociation constant of 8.0 nM. [3H]PDBu was slow to associate with its receptor, requiring 9.5 h to reach equilibrium. Autoradiograph revealed that PKC is heterogeneously distributed in the cat brain, and displays a laminar-specific pattern in the visual cortex. This laminar distribution undergoes marked changes during the first two months of postnatal life. In the visual cortex of neonatal kittens, [3H]PDBu binding is confined to layers I and V. Layer III acquires high levels of binding by postnatal day 15, layer II by 28 days, and layer VI becomes labelled by 40 days of age. Adult animals exhibit high levels of binding in all laminae except layer IV. Age-dependent changes in PKC's laminar distribution do not seem to be correlated with specific anatomical, neurochemical, or behavioural events during development. PKC appears to be associated with cell bodies or processes intrinsic to the visual cortex, and is probably not located on the terminals of cortical afferents.     

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.     

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.     

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.     

Phorbol ester binding sites in human brain: Characterization,regional distribution,age-correlation,and alterations in Parkinson’s disease

We have characterized and localized phorbol ester binding sites in human autopsied brains, using [³H]phorbol 12,13-dibutyrate ([³H]PDBu). When the tissue was homogenized in the absence of Ca²⁺ chelator (10 mM EGTA/2 mM EDTA), Scatchard analysis of the specific [³H]PDBu bindings to both particulate and soluble fractions yielded a single class of high-affinity binding site (K _d = 7.1 and 7.4 nM:B _max = 45.4 and 3.1 pmol/mg protein, respectively). The particulate fraction retained the majority of [³H]PDBu binding (98% of total binding activity), while the soluble fraction was almost devoid of binding activity (2%). In the presence of Ca²⁺ chelator, more of the activity was found in the soluble fraction (30%). The binding of [³H]PDBu was potently inhibited by active phorbol esters and related diterpenes withK _i of nanomolar concentration but not by inactive ones. Diolein (OAG), a synthetic diacylglycerol, and polymixin B, an inhibitor of protein kinase C (PKC), inhibited the binding moderately (K _i = 5.8 and 1.3 μM, respectively). H-7, an inhibitor of PKC and cyclic nucleotides-dependent kinase, did not compete with [³H]PDBu for the binding sites (K _i > 100,000 nM). The regional distribution of specific [³H]PDBu binding in the human brain was rather uneven and resembled that of [³H]PDBu autoradiograms and PKC-like immunoreactivities in the rat brain. The binding capacities were generally in the order: rhinencephalon > basal ganglia > cerebral cortex > diencephalon > cerebellum > mesencephalon. Age-related loss of binding sites was observed in the prefrontal cortex of the subjects 33–81 years of age. In Parkinson’s disease, the phorbol ester binding showed a significant reduction in the substantia nigra, caudate putamen, and pallidum, whereas it was unchanged in the prefrontal cortex and caudate nucleus of schizophrenics, when compared with the relevant controls. This work was supported by a Grant-in-Aid for Special Project Research of Selected Intractable Neurological Disorders from the Ministry of Education, Science and Culture, Japan.     

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.     

Odor-induced fast waves in the dentate gyrus depend on a pathway through posterior cerebral cortex: effects of limbic lesions and trimethyltin

Previous research has shown that the odor of a variety of organic solvents and of components of the anal scent gland excretions of various predators will elicit a burst of fast waves of about 20 Hz in the olfactory bulb, pyriform cortex, and dentate gyrus of the rat. The present experiments show that large lesions of the caudal cerebral cortex, involving particularly the entorhinal and subicular cortices and the angular bundle, abolish the olfactory fast wave response of the dentate gyrus, but not the similar response of the olfactory bulb. In confirmation of previous work, such a lesion also abolishes an average evoked response elicited in the dentate gyrus by electrical stimulation of the olfactory bulb. Systemic treatment with the neurotoxin trimethyltin abolished the olfactory fast wave response in the olfactory bulb and both the olfactory fast wave and the olfactory evoked potential in the dentate gyrus. Large lesions of the amygdala or the septal nuclei did not eliminate either the dentate olfactory evoked potential or the odor-induced dentate fast wave response. However, the septal lesion reduced the amplitude of both spontaneous and odor-induced dentate fast wave activity. It is suggested that olfactory stimuli elicit 20 Hz dentate fast waves via a pathway from the olfactory bulb through the entorhinal cortex and, further, that cholinergic interneurons in the dentate gyrus may be essential to the dentate fast wave response.     

抑郁症患者血小板蛋白激酶C水平的变化

目的：探讨抑郁症患者血小板细胞浆和细胞膜蛋白激酶C(PKC)的变化及其意义。方法：采用[^3H]12，13-二丁酰佛波醇酯(PDBu)结合法，测定23例抑郁症患者和10名正常对照者的血小板细胞浆和细胞膜PKC水平。结果：与正常对照组比较，抑郁症组血小板胞膜PKC水平显著降低，血小板胞浆PKC水平差异无显著性。结论：蛋白激酶C的改变在抑郁症发病机制中可能起一定作用。     

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.     

Postnatal development of [3H]yohimbine binding in the olfactory bulb of male and female rats

A postnatal increase in [3H]yohimbine binding sites was observed in the rat olfactory bulb. The maximal increase in binding site density was between postnatal days 1 and 14 and corresponded to that of peak interneuron (granule and periglomerular) production and maturation, suggesting a possible interneuron location for the alpha 2-adrenoceptors in the rat olfactory bulb. No differences in [3H]yohimbine binding in the olfactory bulb attributable to sex or stage of the estrous cycle were observed.     

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.     

One-trial olfactory learning enhances olfactory bulb responses to an appetitive conditioned odor in 7-day-old rats

The expression of a conditioned odor preference and focal uptake of [14C]2-deoxyglucose (2-DG) within the olfactory bulb was assessed in neonatal rat pups that had undergone a single olfactory classical conditioning trial. At 6 days of age, rat pups were simultaneously exposed for 10 min to an odor (peppermint) and to a reinforcing tactile stimulation similar to that received from the dam. Three control groups received only the odor, only the stimulation, or neither of these stimuli. The next day, pups were either assessed for differential olfactory bulb activity using the 2-DG technique or tested for their olfactory preference behavior. Only pups that received simultaneous odor and tactile stimulation exhibited an attraction to the conditioned odor in the two-odor choice test. Furthermore, such pups had greater focal 2-DG uptake in the olfactory bulb glomeruli that were responsive to the odor than pups in all other groups. Thus, the olfactory bulb responds differentially to an odor which has acquired attractive value.     

The role of olfactory bulb norepinephrine in early olfactory learning.

Wistar rat pups were implanted with bilateral olfactory bulb cannulas on postnatal day 5 (PN5). On PN6, pups were trained in an olfactory classical conditioning task with peppermint odor as the CS and tactile stimulation/stroking as the UCS. Pups were randomly assigned to either PAIRED, BACKWARD or ODOR-only conditions. Half the pups in each group received intrabulbar infusions of 100 microM propranolol and half received intrabulbar infusions of saline during the training session. Propranolol infusions blocked acquisition of the learned odor preference expressed by PAIRED saline-infused pups. Diffusion of the infusate was checked in additional pups by infusing [3H]NE and performing LSC analysis. Infusate concentration did not significantly differ between the anterior and posterior halves of the bulb, but were sharply lower in the olfactory peduncle and more posterior areas. The results suggest that olfactory bulb NE is critical for early olfactory learning.     

A quantitative autoradiographic study of [3H]cAMP binding to cytosolic and particulate protein kinase A in post-mortem brain staged for Alzheimer's disease neurofibrillary changes and amyloid deposits

The cAMP-dependent protein kinase (PKA) has been implicated in the Alzheimer's disease pathology of abnormal tau phosphorylation leading to neurofibrillary tangle (NFT) formation, as well as in amyloid precursor protein alpha-secretase processing. In the present study, we determined whether [3H]cAMP binding to cytosolic and particulate PKA showed any relationship to the extent of Alzheimer's disease pathology at post-mortem. Autoradiographic [3H]cAMP binding to cytosolic and particulate PKA was measured in sections of entorhinal cortex/hippocampal formation from 23 cases that had been staged for Alzheimer's disease-related neurofibrillary changes and amyloid deposits according to Braak and Braak [H. Braak, E. Braak, Neuropathological staging of Alzheimer's-related changes, Acta Neuropathol. 82 (1991) 239-259]. [3H]cAMP binding to cytosolic PKA showed statistically significant reductions in the entorhinal cortex (P<0.01, ANOVA) with respect to neurofibrillary changes. Post-hoc analysis with Fisher's PLSD test showed significant reductions of [3H]cAMP binding to cytosolic PKA at the isocortical stages (V and VI), compared to the non-pathological (O) (by 55%, P<0.01), transentorhinal (I and II) (by 58%, P<0.001) and limbic (III and IV) (by 45%, P<0.05) stages. A significant reduction (by 25%, P<0.05) was also seen in the transentorhinal compared to the limbic stages. [3H]cAMP binding to cytosolic PKA showed no significant alterations with respect to neurofibrillary changes in either the subiculum, CA1-CA4 subfields of the hippocampus or the dentate gyrus. [3H]cAMP binding to cytosolic PKA also showed significant declines in the entorhinal cortex (P<0.01) and subiculum (P<0.05) with respect to staging for amyloid deposits. Post-hoc analysis with Fisher's PLSD test showed significant reductions of [3H]cAMP binding to cytosolic PKA in the entorhinal cortex at amyloid stage C compared to stages O (by 41%, P<0.01) and A (by 38%, P<0.01). In the subiculum, there were significant reductions of [3H]cAMP binding at stages C (by 41%, P<0.01) and B (by 40%, P<0.05), respectively, compared to stage O. [3H]cAMP binding to particulate PKA did not show significant relationships to staging for either neurofibrillary changes or amyloid deposits in either the entorhinal cortex or any of the hippocampal subregions. These findings suggest that whereas [3H]cAMP binding to cytosolic PKA in the entorhinal cortex is reduced with progression of neurofibrillary and amyloid pathology, other hippocampal regions show a preservation of cytosolic and particulate PKA even in late stage pathologies.     

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.