Changes in neurotransmitter actions in the aged rat hippocampus

Intracellular activity was recorded from pyramidal cells in region CA1 of young and aged rat hippocampal slices. There were no apparent differences between the two age groups in the passive or active membrane properties tested. These included resting membrane potential, input resistance, spike size and overshoot, after potentials, and EPSP's. There were marked differences between the two age groups in reactivity to chemical stimulation. The response to acetylcholine (ACh) in young rat hippocampal cells consisted of an initial hyperpolarization followed by a late, slow depolarization. These were accompanied by a decrease in reactivity of the recorded cell to afferent stimulation. In the old cells the initial hyperpolarization and late depolarization were markedly reduced, yet the decreased reactivity to afferent stimulation was maintained. Both young and old cells were depolarized by GABA to the same magnitude, yet the recovery was slower in the old cells. 5-HT evoked a smaller response in old compared to young cells. These data indicate that some specific physiological processes which are not paralleled by reported biochemical changes are affected by aging. The mechanisms underlying these changes are subject to further investigations.     

Thyroid hormone regulates neurotransmitter release in neonatal rat hippocampus

Thyroid hormone is essential for the normal maturation and function of the mammalian CNS. Thyroid hormone deficiency during a critical period of development profoundly affects cognitive functions such as learning and memory. However, the possible electrophysiological alterations that could underlie these learning deficits in hypothyroid animals remain largely unexplored. In this work, we have studied the possible effect of thyroid hormone on short-term synaptic plasticity, which is hypothesized to be a neural substrate of short-term memory. We compared short-term modification of the excitatory postsynaptic potential in hippocampal slices between control and hypothyroid rats. Electrophysiological studies reveal that paired-pulse facilitation is strongly altered in the hypothyroid rats. In addition, hypothyroid rats exhibit an increase in the Ca(2+)-dependent neurotransmitter release. These alterations are basically reversible when thyroid hormone is administered. In order to examine the possible molecular mechanisms underlying these synaptic changes, we compared the expression of synapsin I, synaptotagmin I, syntaxin, and alpha-Ca(2+)/calmodulin kinase II between control and hypothyroid hippocampus. Our results show that the levels of synapsin I and synaptotagmin I are increased in the hypothyroid rats, which suggests that the genes encoding these proteins are implicated in the action of thyroid hormone on neurotransmitter release.Taken together, the results from this study suggest that thyroid hormone may modulate the probability of neurotransmitter release.     

Effects of oxiracetam on neurotransmitter release from rat hippocampus slices and synaptosomes.

The effects of the nootropic drug oxiracetam on the K(+)-evoked overflow of [3H]D-aspartic acid ([3H]D-ASP), [3H]acetylcholine ([3H]ACh), [3H] gamma-aminobutyric acid ([3H]GABA), [3H]noradrenaline ([3H]NA) and [3H]5-hydroxytryptamine ([3H]5-HT) have been studied in superfused rat hippocampal slices. The overflow of [3H]D-ASP was enhanced by low concentrations of oxiracetam (0.01-1 microM) but not by high concentrations (10-100 microM) which showed some tendency to inhibit it. Similarly, low concentrations of oxiracetam increased, although less effectively, the depolarization-evoked overflow of [3H]ACh, whereas higher concentrations were without effect. At the concentrations active on [3H]D-ASP and [3H]ACh overflow oxiracetam did not affect that of [3H]GABA, [3H]NA or [3H]5-HT. The oxiracetam effects present in slices could not be observed in hippocampal synaptosomes. Thus oxiracetam may selectively increase the release of glutamate and acetylcholine in hippocampus by a mechanism which appears not to be sited in the releasing nerve terminals.     

海马内主要神经递质系统递质释放的异源受体介导作用

海马的主要神经递质包括谷氨酸 (Glu)能、γ-氨基丁酸(GABA)能、乙酰胆碱 (ACh)能、去甲肾上腺素 (NA)能和 5 -羟色胺 (5 - HT)能等系统。这些递质系统神经元的胞体、树突和 /或轴突上存在递质的受体 ,它们介导调节神经递质的释放。这些受体根据其所接受递质来源的不同分为自身受体(autoreceptor)、异源受体 (heteroreceptor)和同源受体 (ho-moreceptor)。自身受体是指接受自身神经元释放的递质的作用而发挥介导作用的受体 ;异源受体系指接受不同类型神经元释放的递质的作用而发挥介导作用的受体 ;同源受体系指接受其它同类型神经元释…     

海马内主要神经递质系统和递质释放的自身受体介导作用

海马在学习与记忆功能方面发挥着关键作用 ,因此 ,近2 0年来 ,在神经科学研究中它成为主要的对象之一 ,在此期间 ,积累了大量的神经化学资料。本文主要介绍在海马内发现的主要神经递质系统 ,包括谷氨酸 (Glu)能、(γ-氨基丁酸(GABA)能、乙酰胆碱 (ACh)能、去甲肾上腺素 (NA)能和 5 -羟色胺 (5 - HT)能系统 ,以及海马内各种结构上的自身受体在神经递质释放的突触前调制中的作用。1.　海马结构内主要的神经递质系统1.1　 Glu能系统Glu能通路和细胞在海马结构内形成三级突触环路 ,即由内嗅皮质来的主要 Glu能传入纤维与齿状回区的主细胞…     

Regional heterogeneity of glycoconjugate distribution in the glomerulus revealed by lectin-gold cytochemistry and SDS-PAGE.

The authors have used SDS-PAGE and lectin overlay analysis in parallel with lectin-gold cytochemistry to identify Helix pomatia lectin (HPL) binding glycoconjugates in rat kidney glomeruli. Previous work revealed HPL binding sites only beneath podocyte foot process bases, where they contact the glomerular basement membrane. It is shown here that after neuraminidase digestion of thin sections of glomeruli before incubation with HPL-gold complexes, the number of HPL binding sites is markedly increased. These new sites are mainly associated with the podocyte free surface (adjacent to the urinary space) and with capillary endothelial cells. By lectin overlays, this neuraminidase-dependent HPL binding was shown to be due to reaction of the lectin with desialylated podocalyxin. In contrast, HPL binding sites detected prior to neuraminidase digestion are associated with a novel glycoconjugate having a lower electrophoretic mobility than podocalyxin. Although any role for this glycoconjugate is at present speculative, it is strategically positioned at the site of interaction between foot process bases and the glomerular basement membrane. Its presence correlates with normal podocyte architecture, as shown by our previous studies on developmental and aminonucleoside nephrosis-associated changes in HPL binding to podocytes.     

Quantified distribution of the serotonin innervation in adult rat hippocampus

To quantify the serotonin innervation in adult rat hippocampus, serotonin axon terminals (varicosities) were uptake-labeled for light microscope radioautography in whole hemisphere slices incubated with 1 microM [3H]serotonin. The labeled varicosities were visualized as small aggregates of silver grains and counted with the aid of an image analysis system across all layers in representative sectors of subiculum, Ammon's horn (CA1, CA3-a, CA3-b) and dentate gyrus (medial blade, crest and lateral blade). Counts were obtained in six rats at three equidistant horizontal levels from the ventral two-thirds of the hippocampus. After double correction for duration of radioautographic exposure and section thickness, and measurement of the mean diameter of labeled varicosities in electron microscope radioautographs, the results were expressed in number of varicosities per mm3 of tissue. The overall density of hippocampal serotonin innervation was thus evaluated at 2.7 x 10(6) varicosities per mm3, and appeared significantly higher in subiculum (3.6 x 10(6)) and Ammon's horn (3.1 x 10(6)) than in dentate gyrus (2.2 x 10(6)). Subiculum and dentate gyrus-crest (2.0 x 10(6)) had the highest and lowest regional densities. There was a marked heterogeneity also in terms of laminar distribution. For example, the stratum moleculare of subiculum and CA1, and the stratum oriens of CA3 (5.2 x 10(6)) varicosities in CA3-a), showed much higher values than the pyramidal cell layer (0.7, 1.1 and 0.7 x 10(6) in CA1, CA3-a and CA3-b, respectively). Similarly, the granular layer of dentate gyrus had a much lower density (1.1 x 10(6)) than did the molecular (2.8 x 10(6)) and the polymorph layer (2.4 x 10(6)). From these data, it was possible to evaluate the mean endogenous amine content per hippocampal serotonin varicosity (0.05-0.07 fg), and the average number of serotonin varicosities per hippocampal neuron in both CA3 (130) and dentate gyrus (20-35). In the context of current data on the distribution of serotonin receptors and diverse actions of serotonin at the cellular level in hippocampus, such quantified information provides new insights on some basic properties of serotonin in this part of the brain.     

Regional distribution of cholinergic nerves in rat parietal pericardium

The innervation pattern of parietal pericardium was studied in normal as well as chemically sympathectomized rats using the cholinesterase histochemical method. The existence of important regional variations in the distribution of cholinergic nerves within various portions of parietal pericardium studied was observed. The atria appear more richly innervated than ventricles, while the innervation of atria is characterized by the existence of thin and thick cholinergic nerve fibers not organized in plexuses and of elbow-shaped acetylcholinesterase cholinergic nerve fibers. Small blood vessels and islands of adipocytes receive a cholinergic innervation as well. The chemical sympathectomy does not alter the pattern of stained cholinergic nerve fibers. A possible afferent significance of the atrial innervation is discussed.     

Regional differences in the distribution of capsaicin-sensitive target-identified adult rat dorsal root ganglion neurons.

A retrograde labelling technique combined with a cobalt uptake assay in cultured adult rat dorsal root ganglion (DRG) neurons were applied to study the distribution of capsaicin sensitivity in relation to different peripheral targets. This study shows that there are regional differences between skin, skeletal muscle and urinary bladder; 20-30% of skin afferents, 40% of muscle afferents and 60% of bladder afferents were found to be capsaicin-senditive. This may reflect differences in the proportion of chemosensitive afferents innervating different peripheral tissues.     

Autoradiographic analysis of [H]ryanodine binding sites in rat brain: Regional distribution and the effects of lesions on sites in the hippocampus

Quantitative and qualitative autoradiographic methods together with lesion approaches were used to determine the distribution of [3H]ryanodine binding sites in rat brain and the neuronal localization of these sites in the hippocampus. In normal animals, levels of [3H]ryanodine binding sites ranged from a low of about 1 fmol/mg tissue in subcortical structures to a high of 12-18 fmol/mg tissue in subregions of the hippocampus and the olfactory bulb. Relatively high densities of sites (5-9 fmol/mg tissue) were also seen in the olfactory tubercle, most areas of the cerebral cortex, accumbens nucleus, striatum, lateral septal nuclei, pontine nucleus, superior colliculus and granule cell layer of the cerebellum. Specific binding was undetectable in white matter. In experimental animals, intracerebral injections of kainic acid caused neuronal degeneration and a near total depletion of [3H]ryanodine binding sites in the dentate gyrus and in fields CA1, CA2 and CA3 of the hippocampus. Injections of kainic acid that left dentate granule cells largely intact while destroying all neurons in field CA3 had no effect on binding sites in the dentate gyrus. However, these lesions substantially reduced the density of binding in field CA3, leaving a narrow band of sites outlining the position of the degenerated CA3 pyramidal cells. Mechanical knife-cut lesions that severed the granule cell mossy fiber input to field CA3 reduced the density of binding sites in the CA3 region. The results indicate that [3H]ryanodine binding sites in brain are heterogeneously distributed and suggest that a proportion of these sites in the hippocampus may be contained in mossy fiber terminals where a presumptive calcium channel/ryanodine receptor complex may be involved in the regulation of calcium mobilization and/or neurotransmitter release.     

Regional differences in the expression of DNA topoisomerase IIbeta in the pyramidal neurons of the rat hippocampus

A detailed analysis of the differential expression of a nuclear enzyme, DNA topoisomerase (topo) IIbeta, was performed in the rat hippocampal pyramidal layer. Three-dimensional (3-D) reconstruction from serial sections immunostained with anti-topo IIbeta antibody showed that the immunoreactivity was apparently weak in the entire CA3 region. Almost all CA1 pyramidal cells showed similar immunoreactivity to that seen in the dentate granular cells, the subicular neurons, and the cerebral neocortical neurons. In addition, immunoblotting analysis in the adult dorsal hippocampus showed that the expression level of topo IIbeta in the CA3 was significantly lower than that in the CA1 region. The dissociation in the expression level between CA1 and CA3 occurred in postnatal days 4 (P4) through P6. The present finding suggests that the enzyme is possibly involved in activities of the hippocampal pyramidal neurons.     

Regional and laminar distributions of alpha 1-adrenoceptors and their subtypes in human and rat hippocampus.

The distributions of the alpha 1-adrenoceptor and its subtypes (alpha 1A and alpha 1B) in human and rat hippocampus are analysed by quantitative receptor autoradiography. alpha 1-Adrenoceptors are labelled by [3H]prazosin. The alpha 1A subtype is visualized by [3H]prazosin after irreversible blockade of alpha 1B adrenoceptors with chloroethylclonidine or directly by [3H]5-methyl-urapidil. The alpha 1B subtype is investigated by [3H]prazosin binding in the presence of the alpha 1A antagonist 5-methyl-urapidil. Considerable differences in the regional and laminar patterns of alpha 1-adrenoceptors are found between rat and human hippocampi. The rat hippocampus is characterized by a low overall density and a rather homogeneous regional and laminar distribution. This is in contrast to the human pattern, which shows a much higher overall level of alpha 1 receptor density and a restriction of alpha 1 receptors to the CA3 region of Ammon's horn and the dentate gyrus. Moreover, alpha 1A and alpha 1B receptors of the human hippocampus are differentially distributed with the alpha 1A subtype concentrated in the hilus and lucidum layer of CA3, and the alpha 1B subtype concentrated in the molecular layer of the dentate gyrus. Additionally, the distribution of alpha 1 receptors is compared with the distribution of 5-hydroxytryptamine 1A receptors. The subtype specific pattern is correlated with the distribution of glutamatergic systems in the human (but not in the rat) hippocampus. alpha 1A Receptor localization coincides with the target area of the mossy fibre system, and alpha 1B receptors are preferentially localized in the target area of the hippocampal associational fibres and partly of the perforant pathway. This result points to possible interactions between noradrenaline- and glutamate-mediated neurotransmission differentiated by topographically segregated alpha 1-adrenoceptor subtypes.     

Effects of the unilateral striatal lesion on neurotransmitter markers in the contralateral striatum and both substantia nigrae of the rat

We investigated quantitative changes in possible neurotransmitters and their biosynthetic enzymes in the contralateral striatum and both substantia nigrae following unilateral electrothermic lesions of the striatum in the rat. Two types of changes were observed: (1) the first ones were long-lasting (up to 56 post-operative days) effects and consisted in a decrease of GABA content and tyrosine hydroxylase activity in the ipsilateral substantia nigra due to the anterograde and retrograde degeneration of striatal efferent and afferent fibres, respectively, and in a marked increase of glutamate and GABA contents in the contralateral striatum resulting possibly from a modified activity of the collaterals of the glutamatergic corticostriatal fibres and a subsequent secondary increase of GABA. The latter interpretation was supported by the finding that the changes observed were abolished by an additional callosal transection; (2) the second series of changes were transient (only found at 3-7 post-operative days) effects represented by an increase in GABA content, a decrease of tyrosine hydroxylase activity, and a decrease of dopamine content, which mostly appeared in the contralateral substantia nigra. The decrease of dopamine markers may be a subsequent secondary effect of the increase of GABA in the substantia nigra. These results suggest that the contralateral increase of the amino acid transmitters in the striatum and the increase followed by decrease of transmitter markers in the contralateral substantia nigra could be a "plastic" or "compensatory" biochemical response to the unilateral striatal lesions.     

Regional distribution of phosphoinositide-specific phospholipase C activity in rat brain

The phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP₂) hydrolytic activities of phosphoinositide-specific phospholipase C (PLC) were measured in membrane and cytosol fractions from 7 discrete areas of the rat brain. Both the PI-PLC and PIP₂-PLC specific activities were found to differ significantly among the 7 discrete brain areas. In the membrane fraction, the PIP₂-PLC activity was higher than that of PI-PLC in each region, suggesting that the PLC in membranes prefers PIP₂ to PI as substrate. The PIP₂-PLC activities in the membrane were high in prefrontal cortex and cerebellum, but rather low in medulla oblongata and hypothalamus. The PI-PLC specific activity in the cytosol was significantly higher than that in the membrane of all brain areas examined. The PI-PLC specific activity in membranes is inversely proportional to its activity in the cytosol. In the cytosol fraction, the distribution pattern of PI-PLC specific activity resembled that of PIP₂-PLC. These results indicate that PLCs are differently distributed in various regions of rat brain, and suggest the regional differences in neuronal transduction.     

Cocaine-responsive gene expression changes in rat hippocampus.

Chronic cocaine use is known to elicit changes in the pattern of gene expression within the brain. The hippocampus plays a critical role in learning and memory and may also play a role in mediating behaviors associated with cocaine abuse. To profile the gene expression response of the hippocampus to chronic cocaine treatment, cDNA hybridization arrays were used to illuminate cocaine-regulated genes in rats treated non-contingently with a binge model of cocaine (45 mg/kg/day, i.p.) for 14 days. Validation of mRNA changes illuminated by hybridization array analysis was accomplished by measuring immunoreactive protein (via specific immunoblots). The induction of protein kinase Calpha, potassium channel 1.1, and metabotropic glutamate receptor 5 seen by hybridization arrays was confirmed at the level of protein. Immunoblot screening of previously described cocaine-responsive genes demonstrated increased levels of protein tyrosine kinase 2, beta-catenin, and protein kinase Cepsilon. While some of these changes exist in previously described cocaine-responsive models, others are novel to any model of cocaine use. The inductions of potassium channel 1.1, protein tyrosine kinase 2 and metabotropic glutamate receptor 5 are novel findings to hippocampal cocaine-responsive gene expression. These proteins have been shown to subserve learning and memory and/or long-term potentiation functions within the hippocampus. Additionally, these genes are known to interact with one another, forming a more complex pattern of gene expression changes.The findings suggest altered expression of genes with a number of different functions in the rat hippocampus after a 'binge' style of non-contingent cocaine administration. These changes in gene expression may play roles in neuronal plasticity and the behavioral phenomena associated with cocaine abuse.     

Immunomorphologic studies of mast cell heterogeneity, location, and distribution in the rat conjunctiva

Mast cells are crucial components of immediate and some delayed-type hypersensitivity reactions. They play a pivotal role in allergic conjunctivitis and other immunoinflammatory disorders of the ocular surface, yet little is known of their distribution and heterogeneity in the conjunctiva of potential animal models, such as the rat. In this study, mast cell types were investigated in histologic sections and corneal-conjunctival-lid whole mounts by using toluidine blue, alcian blue–safranin, and immunohistochemical staining methods (anti-rat mast cell proteinase [RMCP] antibodies). Quantitative analyses were performed on corneal-conjunctival-lid whole mounts by using the optical dissector procedure to obtain the density of mast cells per unit volume in different regions of the conjunctiva. Single and double immunohistochemical analyses revealed that the mast cells in the conjunctiva of the limbus, fornices, and lid margin were strongly RMCP I⁺, suggesting that they were of the connective tissue phenotype. Mast cells containing the mucosal mast cell proteinase RMCP II were not present in the normal conjunctiva. Histochemical analysis revealed that the maturity of the connective tissue mast cells, as assessed by the presence or absence of safranin (heparin)-positive granules in their cytoplasm varied in different regions. In the lid margin 60% to 78% of the mast cells were solely alcian blue–positive, whereas in the fornices 68% to 78% were safranin-positive. In the limbus the predominant type of mast cell was either safranin-positive or contained mixed granules. Mast cell densities were greatest close to the lid margin (10,000 to 12,000 cells/mm³), followed by the limbus (3400 to 4800 cells/mm³) and were rare in the remainder of the conjunctiva (500 to 1000 cells/mm³), with the exception of the region around the nictitating membrane. This study of rat conjunctival mast cells provides essential baseline data for future studies of the role of mast cells in models of allergic conjunctivitis. (J ALLERGY CLIN IMMUNOL 1996;97:1375-86.)