Beta amyloid is neurotoxic in hippocampal slice cultures.

We examined the neurotoxicity of the 40 amino acid fragment of beta amyloid peptide (A beta 1-40) in cultured hippocampal slices. When injected into area CA3, A beta 1-40 produced widespread neuronal damage. Injection of the reverse sequence peptide, A beta 40-1, or vehicle alone produced little damage. The distribution A beta 1-40 was highly correlated with the area of neuronal damage. Thioflavine S and electron microscopic analysis confirmed that injected A beta 1-40 formed 7-9 nm AD type amyloid fibrils in the cultures. A beta 1-40 also altered the number of GFAP immunoreactive astrocytes and ED-1 immunoreactive microglia/macrophages within and around the A beta 1-40 deposit. The observed neurotoxicity of A beta 1-40 in hippocampal slice cultures provides evidence that this peptide may be responsible for the neurodegeneration observed in AD.

Morphology of identified interneurons in the CA1 regions of guinea pig hippocampus.

Identified interneurons in the CA1 region of guinea pig hippocampus were examined using light and electron microscopic (EM) techniques. The HRP was intracellularly injected into cells, recorded in the in vitro slice preparation, which met physiological criteria for interneurons. These neurons were characterized at the light and electron microscopic level, and used as standards for identifying interneurons which had not been HRP-labeled. Pyramidal basket cell somata were found in stratum oriens and stratum pyramidale; they were easily identifiable by their convoluted nucleus, dense endoplasmic reticulum, and numerous organelles. Aspinous dendrites reached into stratum oriens, where they received profuse synaptic input (primarily asymmetric synapses). Many of the asymmetric synapses degenerated following commissural lesions, suggesting that much of the input to interneurons was from extrinsic afferents. Dendrites were characterized by their spindled appearance, especially at distal sites. They showed postsynaptic degenerative changes following commissural lesions. Interneuron axons were extremely fine, with regular enlargements or "beads" which made apparent synaptic contacts primarily on pyramidal cell somata. The axon of a single, HRP-injected interneuron made many apparent contacts on large numbers of pyramidal cells; axons arborized over distances of several hundred micra within stratum pyramidale. This study provides direct evidence that neurons, with an identified inhibitory interneuron function in hippocampus, can mediate feed-forward as well as feed-back (recurrent) inhibition. Interneuron output showed extreme divergence, with influence over large distances. The high density of intracellular organelles in these cells suggested high metabolic activity and demand, perhaps making these interneurons exceptionally vulnerable to trauma-induced damage.

Axon arbors and synaptic connections of hippocampal mossy cells in the rat in vivo.

The axon collateralization patterns and synaptic connections of intracellularly labeled and electrophysiologically identified mossy cells were studied in rat hippocampus. Light microscopic analysis of 11 biocytin-filled cells showed that mossy cell axon arbors extended through an average of 57% of the total septotemporal length of the hippocampus (summated two-dimensional length, not adjusted for tissue shrinkage). Axon collaterals were densest in distant lamellae rather than in lamellae near the soma. Most of the axon was concentrated in the inner one-third of the molecular layer, with the hilus containing an average of only 26% of total axon length and the granule cell layer containing an average of only 7%. Ultrastructural analysis was carried out on three additional intracellularly stained mossy cells, in which axon collaterals and synaptic targets were examined in serial sections of chosen axon segments. In the central and subgranular regions of the hilus, mossy cell axons established a low density of synaptic contacts onto dendritic shafts, neuronal somata, and occasional dendritic spines. Most hilar synapses were made relatively close to the mossy cell somata. At greater distances from the labeled mossy cell (1-2 mm along the septotemporal axis), the axon collaterals ramified predominantly within the inner molecular layer and made a high density of asymmetric synaptic contacts almost exclusively onto dendritic spines. Quantitative measurements indicated that more than 90% of mossy cell synaptic contacts in the ipsilateral hippocampus are onto spines of proximal dendrites of presumed granule cells. These results are consistent with a primary mossy cell role in an excitatory associational network with granule cells of the dentate gyrus.

Synaptic connections of dentate granule cells and hilar neurons: results of paired intracellular recordings and intracellular horseradish peroxidase injections.

Simultaneous intracellular recordings were made in the dentate gyrus of rat hippocampal slices, from pairs of the following cell types: granule cells, interneurons located in the granule cell layer, hilar interneurons, and spiny hilar "mossy cells". Granule cells were found to have strong excitatory effects on mossy cells and interneurons. Interneurons inhibited granule cells as well as other interneurons. No synaptic connections from mossy cells onto other cell types were found, within the confines of the slice, using intracellular recording methods. However, at the ultrastructural level, axon terminals of horseradish peroxidase-filled mossy cells were found making synaptic contacts in the hilus on dendrites of interneurons. These studies provide the first step towards determining the functional interactions of the various cell types in the fascia dentata.

Ultrastructure of acetylcholine receptor aggregates parallels mechanisms of aggregation.

BACKGROUND: Acetylcholine receptors become aggregated at the developing neuromuscular synapse shortly after contact by a motorneuron in one of the earliest manifestations of synaptic development. While a major physiological signal for receptor aggregation (agrin) is known, the mechanism(s) by which muscle cells respond to this and other stimuli have yet to be worked out in detail. The question of mechanism is addressed in the present study via a quantitative examination of ultrastructural receptor arrangement within aggregates. RESULTS: In receptor rich cell membranes resulting from stimulation by agrin or laminin, or in control membrane showing spontaneous receptor aggregation, receptors were found to be closer to neighboring receptors than would be expected at random. This indicates that aggregation proceeds heterogeneously: nanoaggregates, too small for detection in the light microscope, underlie developing microaggregates of receptors in all three cases. In contrast, the structural arrangement of receptors within nanoaggregates was found to depend on the aggregation stimulus. In laminin induced nanoaggregates receptors were found to be arranged in an unstructured manner, in contrast to the hexagonal array of about 10 nm spacing found for agrin induced nanoaggregates. Spontaneous aggregates displayed an intermediate amount of order, and this was found to be due to two distinct population of nanoaggregates. CONCLUSIONS: The observations support earlier studies indicating that mechanisms by which agrin and laminin-1 induced receptor aggregates form are distinct and, for the first time, relate mechanisms underlying spontaneous aggregate formation to aggregate structure.

Transgenic animal models for Alzheimer's disease.

The neuropathology of Alzheimer's disease is characterized by the deposition of abnormal protein aggregates. The main constituent of the deposition is beta-amyloid protein. A seminal role of this protein is supported by the discovery of point mutations in the gene of its precursor protein in certain forms of familial Alzheimer's disease. In vitro (cultured neuronal cells), overexpression of the precursor protein or a part of the precursor leads to degeneration of neurons, suggesting neurotoxicity of its derivatives. At this time, all of the reported transgenic mice bearing DNA construct for the precursor or a part of the precursor, however, have not developed convincing pathological changes similar to what is observed in patients with Alzheimer's disease. This interesting discrepancy between in vitro and in vivo suggests suppressors in vivo which ameliorate beta-amyloid precursor protein derivative-mediated neurotoxicity.

Electron microscopy of intracellularly labeled neurons in the hippocampal slice preparation.

We have assessed the properties of three intracellular markers, horseradish peroxidase, biocytin/Neurobiotin, and Lucifer Yellow, and have compared their usefulness as neuronal markers for light and electron microscopic visualization. Neurons in the acute slice preparation of rat hippocampus were filled with one of these markers, and the marker was converted to an optical and electron-dense reaction product. Dimethylsulfoxide (DMSO) greatly facilitated penetration of recognition reagents while preserving membrane integrity. The markers were compared with respect to injection parameters, mobility and recognition, stability and visibility, and ultrastructural clarity. Horseradish peroxidase (HRP)-labeled neurons, recognized histochemically with diaminobenzedine (DAB), were easily visualized by the density of the DAB reaction product; however, the electron density was often so great as to obscure ultrastructural details. Biocytin (BC)-/Neurobiotin (NB)-labeled neurons were recognized by avidin-HRP, followed by histochemical localization of HRP with DAB. The optically dense reaction product gave complete visualization of the soma and processes at the light microscopic level. The electron density was homogeneously distributed throughout the cell, so that ultrastructural features were easily identified. Lucifer Yellow (LY), a fluorescent marker, was converted to an optical and electron-dense reaction product via immunocytochemical staining with a rabbit anti-LY antibody, followed by goat anti-rabbit IgG-HRP and DAB histochemical localization. Similar to BC/NB, the reaction product was evenly dispersed, providing good light microscopic and ultrastructural clarity. Under our experimental conditions, BC/NB and LY were superior markers that could be used routinely to label neurons, and give excellent visualization not only at the light but also at the electron microscopic level.

Development of rabbit hippocampus: anatomy.

The postnatal development of the CA1 region of rabbit hippocampus was studied using a variety of light and electron microscopic (EM) techniques. Nissl and Golgi stains showed high cellular density, small cell soma area, and sparse dendritic branching in neurons of immature animals (less than 1 week old); dendritic spines were also relatively infrequent during this period. Cell branching and spine frequency reached near-adult levels by 3 weeks, with the major area of hippocampal expansion seen in the apical dendritic layer. EM examination of synapse patterns was made using osmicated and E-PTA-treated tissue. Both techniques showed that the vast majority of synapses in immature animals (less than 2 weeks old) occurred in the dendritic region and were of the asymmetric type. Axosomatic synapses became less rare by 2 weeks; they were usually of the symmetric synapse type. The pattern of synaptic contacts in immature hippocampus resembled the mature pattern by 3-4 weeks. These data suggest a relatively late development of inhibitory postsynaptic potentials in CA1 pyramidal cells.

Developmental and regional differences in the localization of Na,K-ATPase activity in the rabbit hippocampus.

Regional differences in Na,K-ATPase activity, and development of Na,K-ATPase activity were examined in rabbit hippocampus using a histochemical marker of enzyme activity. Stratum lucidum of CA3/CA2, corresponding to the mossy fiber terminal field, showed high Na,K-ATPase activity compared to stratum radiatum of CA1. A significant increase in Na,K-ATPase activity was found between 8 and 15 days postnatal. Tissues with limited Na,K-ATPase activity (immature hippocampus, the mature CA1 region) appear particularly prone to seizure-like abnormalities, perhaps reflecting an inability to regulate extracellular potassium.

Intracellular dyes mask immunoreactivity of hippocampal interneurons.

The results of several studies have suggested that local circuit neurons, or interneurons, of area CA1 of hippocampus use gamma-aminobutyric acid (GABA) as their neurotransmitter. However, when these cells were labelled by intracellular dye injection, and examined immunocytochemically with antisera raised against GABA, none of the interneurons were immunoreactive. Numerous non-injected interneurons in the same tissue section were clearly immunoreactive. These results suggest that intracellular dyes interfere with immunocytochemical staining of hippocampal interneurons.

Changes in gamma-aminobutyric acid and somatostatin in epileptic cortex associated with low-grade gliomas.

The role of specific neuronal populations in epileptic foci was studied by comparing epileptic and non-epileptic cortex removed from patients with low-grade gliomas. Epileptic and nearby (within 1 to 2 cm) non-epileptic temporal lobe neocortex was identified using electrocorticography. Cortical specimens taken from four patients identified as epileptic and nonepileptic were all void of tumor infiltration. Somatostatin- and gamma-aminobutyric acid (GABAergic)-immunoreactive neurons were identified and counted. Although there was no significant difference in the overall cell count, the authors found a significant decrease in both somatostatin- and GABAergic-immunoreactive neurons (74% and 51%, respectively) in the epileptic cortex compared to that in nonepileptic cortex from the same patient. It is suggested that these findings demonstrate changes in neuronal subpopulations that may account for the onset and propagation of epileptiform activity in patients with low-grade gliomas.

Effect of styrene oxide on rat brain glutathione.

Glutathione (GSH) plays a primary role in protecting cells from oxidative stress and in detoxifying foreign compounds. The functions and regulations of GSH in nervous tissue have not been thoroughly investigated. This study examines the effects of styrene oxide, a reactive metabolite of the neurotoxic solvent styrene, on GSH metabolism in six regions of the rat brain (cortex, cerebellum, medulla-pons, hippocampus, striatum and hypothalamus). Control levels of GSH in brain regions ranged from 1.6 mM in medulla-pons to 2.7 mM in striatum. Styrene oxide (100-400 mg/kg, ip) depleted GSH in a dose- and time-dependent manner in all brain regions studied. Histochemical studies indicated a predominantly glial distribution of GSH and confirmed the depletion of GSH by styrene oxide in brain. Studies with [8(-14)C] styrene oxide revealed no differences in the distribution of styrene oxide/metabolites among brain regions. gamma-Glutamylcysteine synthetase, the rate-limiting enzyme in GSH biosynthesis, was not affected by styrene oxide in any brain region, either in vitro or following in vivo administration. Glutathione S-transferase activity in different brain regions, measured using p-nitrostyrene oxide as a substrate, correlated quantitatively with GSH depletion by styrene oxide. Depletion of brain GSH by styrene oxide may contribute to oxidative injury to neuronal and glial cells and may be involved in styrene neurotoxicity.

Developmental changes in the ultrastructure of the Sauromatum guttatum (Araceae) mitochondria.

Electron microscopic studies show that the electron density of the mitochondria of the thermogenic appendix of Sauromatum guttatum inflorescences changes during development. Two to 3 days before heat-production, the mitochondria accumulate osmiophilic, electron-dense material between the inner and outer membranes. During heat-production and the release of volatiles compounds the osmiophilic material disappears from the inter membrane space. Deposits with the same electron density are also found in the endoplasmic reticulum. It is concluded that the mitochondria may also have the capacity to accumulate that material in the inter membrane space.

Structural organization of developing acetylcholine receptor aggregates.

We report the first quantitative ultrastructural analysis of newly formed acetylcholine receptor aggregates. Aggregates were induced in Xenopus muscle cell cultures with agrin, labeled with gold particles, and detected using high resolution scanning electron microscopy. Aggregates are readily discernible at the ultrastructural level within 2 h of stimulation by agrin. The size and density profiles of the developing aggregates show that receptors reach maximal density very quickly in small "nano-aggregates" and that the aggregation process is not limited by the diffusion rate of the receptor. Quantitative analysis of label locations indicates that the receptor distribution within aggregates is nonrandom. Instead, the newly aggregated receptors appear to be bound to a localized scaffold conforming to a hexagonal (close-packed) geometry with a spacing of approximately 9.9 nm.

Ultrastructural characterization and GAD co-localization of somatostatin-like immunoreactive neurons in CA1 of rabbit hippocampus.

Immunocytochemical techniques have been used to identify a striking interneuronal population which is immunoreactive for the peptide, somatostatin. The cell population, which is seen most densely in stratum oriens and at the oriens/alveus border of the CA1 region of rabbit hippocampus, was characterized in light and electron microscopic observations. The cells have dendrites which extend parallel to and into the alveus, with occasional processes ascending through stratum pyramidale toward the hippocampal fissure. The dendrites receive numerous synaptic contacts directly onto aspinous dendritic shafts. Axon collaterals ramify profusely within the pyramidale region, and among the proximal apical and basal pyramidal cell dendrites in areas of stratum radiatum and stratum oriens. Somatostatin-like immunoreactive terminals make synaptic contact, primarily of the symmetric type, with the somata and proximal dendrites of pyramidal neurons. Somatostatin-like neurons are found at approximately equal density in the hippocampus of immature (8 days postnatal) and mature (30 days postnatal) rabbit. Double-labelling techniques, to identify both somatostatin-like and glutamic acid decarboxylase (GAD) immunoreactive neurons, demonstrated that a large proportion of the somatostatin neurons were also GABAergic.

Electrophysiology and morphology of the developing hippocampus of fetal rabbits.

The pyramidal neurons of fetal rabbit hippocampus were studied using intracellular electrophysiological techniques in in vitro slice preparations. Correlative light and electron microscopic analyses were carried out on hippocampus during the 21st through the 29th day of fetal gestation. In intracellular experiments, neurons with all-or-none action potentials and near-adult level resting potentials were found even in the youngest preparations. Synaptic activity, however, was rare until about 24 days of gestation. CA1 neurons showed primarily excitatory synaptic potentials during fetal development, whereas CA3 neurons displayed both inhibitory and excitatory postsynaptic potentials at early stages. Anatomical studies suggested that pyramidal cell precursors were still dividing and migrating at 21 days; by 29 days, cellular migration was completed, and cellular intercommunication in the form of synapses was increasing. These experiments demonstrate that fetal central nervous system (CNS) material can be studied electrophysiologically without growing tissue in culture. Our results suggest that the newly differentiated hippocampal neurons have a limited repertoire of activities. Such data may provide a link between in vivo studies of postnatal CNS development and cell and tissue culture investigations of the properties of immature neurons.

Further studies of the glandular tissue of the Sauromatum guttatum (Araceae) appendix.

Electron microscopic studies showed that the trans-Golgi network (trans indicates the polarity of cisternae within the Golgi apparatus; it is opposite to the cis-face that is adjacent to the rough endoplasmic reticulum) was involved in the processing of the osmiophilic material present in the appendix of the inflorescence of Sauromatum guttatum. This material accumulated in the rough endoplasmic reticulum and in special pockets of the plasma membrane prior to heat production. Associations between the endoplasmic reticulum and trans-Golgi network were observed. The Golgi apparatus was composed of 5-6 dictyosomes on one side and one or two somewhat detached cisternae on the other side. Various nonosmiophilic Golgi-derived vesicles were observed: small ones covered with spike-like material, large ones with a smooth surface, and irregularly shaped ones. These electron-translucent vesicles seemed to accumulate in specific localities at the plasma membrane surface in the vicinity of the osmiophilic material; they were not found when the aroma was released. During heat production, the Golgi structures shrank and the activity of the trans-Golgi network seemed to be reduced. At the same time, coated pits were seen at the plasma membrane surface. In some cells, hypertrophic Golgi apparatuses were seen with only 2-3 dictyosomes that contained granulated material in their lumens. Finally, the osmiophilic material was also found in the plasmodesmata.

Electrophysiological actions of somatostatin (SRIF) in hippocampus: an in vitro study.

The electrophysiological actions of somatostatin (somatotropin release inhibiting factor; SRIF) were investigated in the in vitro hippocampal slice preparation. Intracellular recordings were obtained from pyramidal neurons in area CA1 in slices of hippocampus from guinea pigs and rabbits. Somatostatin, applied via micropressure ejection to CA1 pyramidal-cell somata, was primarily excitatory. The effects, however, were quite variable, with nearly all cells displaying pronounced tachyphylaxis. A majority of cells was depolarized by SRIF, but hyperpolarizations or biphasic depolarization/hyperpolarization responses were also recorded. Only minimal conductance changes were associated with the SRIF-induced voltage changes. Depletion of SRIF, by injection of the intact animal with cysteamine several hours before preparing slices, resulted in no obvious abnormalities in hippocampal slice electrophysiology. Our results obtained with application of exogenous SRIF are consistent with the concept that SRIF acts as an excitatory neurotransmitter/neuromodulator in hippocampus. However, our attempts to demonstrate endogenous SRIF action have thus far been unsuccessful.

Ultrastructure of stratum lacunosum-moleculare interneurons of hippocampal CA1 region.

Intracellular recordings were obtained from nonpyramidal neurons (interneurons) in stratum lacunosum-moleculare (L-M) of the CA1 region of guinea pig hippocampal slices. These interneurons had response characteristics that distinguish them from pyramidal cells and other interneuron types: the L-M neurons had relatively broad action potentials with large spike afterhyperpolarizations, high input resistance and little spike-firing adaptation, and low spontaneous activity. Lucifer Yellow (LY) and horseradish peroxidase (HRP) were injected intracellularly into physiologically identified L-M interneurons, and the cells were characterized morphologically using light and electron microscopy. L-M somata were fusiform-shaped (15 x 25 micron), had multiple processes, and were located at the border between stratum (str.) lacunosum-moleculare and str. radiatum. L-M dendrites coursed through str. lacunosum-moleculare and projected into str. radiatum. L-M axons made axodendritic synaptic contacts primarily in str. lacunosum-moleculare and str. radiatum, but also in str. moleculare of the dentate gyrus. These axodendritic synaptic contacts were made onto spiny dendritic processes (presumably pyramidal cell and granule cell dendrites) and onto aspinous dendrites (presumably interneuron dendrites), and appeared to be of the symmetric type (type 2), characteristic of inhibitory synapses. In separate groups of animals, selective lesions were made of afferents to the CA1 and dentate regions of hippocampus, and subsequent degeneration of contacts and L-M interneuron somata and dendrites was examined at the ultrastructural level. Fibers originating from contralateral and ipsilateral CA3 region, and from ipsilateral entorhinal cortex, were found to make synaptic contact onto presumed L-M interneurons. Degenerating terminals appeared to be of the asymmetric type (type 1), characteristic of excitatory synapses. These morphological data are consistent with electrophysiological results showing that L-M interneurons can mediate feedforward inhibition of CA1 pyramidal cells.

Development of dopamine-beta-hydroxylase-positive fiber innervation of the rat hippocampus.

Development of the noradrenergic fiber innervation of the rat hippocampus by the locus coeruleus was examined immunohistochemically in fixed tissue from animals aged 4 days through 55 days postnatal. The presence of tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) immunoreactive cells and fibers was evaluated in sections of hippocampus and locus coeruleus. Large, multipolar TH- and DBH-positive cells with long beaded fibers were visible within locus coeruleus at all ages; no immunopositive cell bodies were found in hippocampus. In hippocampal sections from mature animals (PN55), the highest density of DBH-stained fibers was found in stratum lucidum of CA3 and in the hilus and inner molecular layer of the dentate gyrus. Whereas similar patterns of fiber positivity were found at PN21 and PN10 (although with somewhat reduced density of immunopositive fibers), the pattern was quite different at PN4. Although fiber staining was relatively sparse at PN4, relative density of DBH fibers was highest in stratum radiatum of CA1 and subiculum. This change in staining pattern suggests that noradrenergic function in hippocampus may change as the rat matures. Double immunofluorescence techniques showed an overlap of DBH and TH positive fibers in all hippocampal regions at all ages. DBH immunostaining appeared to be somewhat more sensitive than the TH staining. These data made it impossible to confirm the presence of significant numbers of nonnoradrenergic, catecholamine-containing fibers in hippocampus.