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Filtrates of a water extract of commercially available garden thyme (Thymus vulgaris L.) were used for studying its possible bactericidal effect on Brachyspira hyodysenteriae, the causative agent of swine dysentery, by agar-diffusion technique. Five of the six studied Brachyspira strains have proven to be sensitive and one moderately sensitive in the in vitro tests. It was concluded that water extract of garden thyme possesses inhibitory effects against B. hyodysenteriae. In vivo experiments are needed to check the validity of this conclusion.

The CFTR High Expresser (CHE) cells express eightfold higher levels of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl−channel compared with neighboring enterocytes and were first identified by our laboratory (Ameen et al., Gastroenterology 108: 1016, 1995). We used double-label immunofluorescence microscopy to further study these enigmatic epithelial cells in rat intestine in vivo or ex vivo. CHE cells were found in duodenum, most frequent in proximal jejunum, and absent in ileum and colon. CFTR abundance increased in CHE cells along the crypt-villus axis. The basolateral Na+K+Cl−cotransporter NKCC1, a key transporter involved in Cl−secretion, was detected at similar levels in CHE cells and neighboring enterocytes at steady state. Microvilli appeared shorter in CHE cells, with low levels of Myosin 1a, a villus enterocyte-specific motor that retains sucrase/isomaltase in the brush-border membrane (BBM). CHE cells lacked alkaline phosphatase and absorptive villus enterocyte BBM proteins, including Na+H+exchanger NHE3, Cl−/HCO3−exchanger SLC26A6 (putative anion exchanger 1), and sucrase/isomaltase. High levels of the vacuolar-ATPase proton pump were observed in the apical domain of CHE cells. Levels of the NHE regulatory factor NHERF1, Na-K-ATPase, and Syntaxin 3 were similar to that of neighboring enterocytes. cAMP or acetylcholine stimulation robustly increased apical CFTR and basolateral NKCC1 disproportionately in CHE cells relative to neighboring enterocytes. These data strongly argue for a specialized role of CHE cells in Cl−-mediated “high-volume” fluid secretion on the villi of the proximal small intestine.

Changes in intestinal luminal pH affect mucosal ion transport. The aim of this study was to compare how luminal pH and specific second messengers modulate the membrane traffic of four major ion transporters (CFTR, NHE3, NKCC1, and NBCe1) in rat small intestine. Ligated duodenal, jejunal, and ileal segments were infused with acidic or alkaline saline, 8-Br-cAMP, or the calcium agonist carbachol in vivo for 20 min. Compared with untreated intestine, lumen pH was reduced after cAMP or carbachol and increased following HCO3−-saline. Following HCl-saline, lumen pH was restored to control pH levels. All four secretory stimuli resulted in brush-border membrane (BBM) recruitment of CFTR in crypts and villi. In villus enterocytes, CFTR recruitment was coincident with internalization of BBM NHE3 and basolateral membrane recruitment of the bicarbonate transporter NBCe1. Both cAMP and carbachol recruited NKCC1 to the basolateral membrane of enterocytes, while luminal acid or HCO3−retained NKCC1 in intracellular vesicles. Luminal acid resulted in robust recruitment of CFTR and NBCe1 to their respective enterocyte membrane domains in the upper third of the villi; luminal HCO3−induced similar membrane changes lower in the villi. These findings indicate that each stimulus promotes a specific transporter trafficking response along the crypt-villus axis. This is the first demonstration that physiologically relevant secretory stimuli exert their actions in villus enterocytes by membrane recruitment of CFTR and NBCe1 in tandem with NHE3 internalization.

SUMMARY Effects of 2 dietary levels (0.6 and 1.0 ppm) of T-2 toxin, and the possible protective capacity of a mycotoxin-inactivating feed additive (Detoxa Plus) were investigated in growing White Pekin ducklings in a 49-d trial comprising 6 treatment groups of 10 ducks/group. The experimental design consisted of 1 negative and 1 positive control and 4 test groups, as follows: group 1, negative control (no T-2 toxin and no feed additive added to the feeds); group 2, positive control (no T-2 toxin added, but the feeds were supplemented with feed additive at 2 kg/t); group 3, feeds were complemented with 0.6 ppm of purified T-2 toxin (no feed additive added); group 4, feeds were complemented with 0.6 ppm of T-2 toxin and 2 kg/t of the feed additive was provided; group 5, feeds were complemented with 1.0 ppm of T-2 toxin (no feed additive added); group 6, feeds were complemented with 1.0 ppm of T-2 toxin and 2 kg/t of the feed additive was provided. From wk 4 until the end of the trial, the daily BW gain of group 3 ducks was inferior to that of control ducks, and the differences in means were statistically significant (P ≤ 0.05) at wk 4 and 7. The final BW of this group was also significantly lower than that of the control (P ≤ 0.001). The BW gain of ducks in group 5 was also depressed by the toxin treatment. The adverse effect of 0.6 ppm of T-2 toxin was fully counteracted by the feed additive in terms of daily BW gain, cumulative daily BW gain, and BW at exsanguination. The T-2 toxin at both treatment levels depressed the blastogenic response of lymphocytes to nonspecific mitogens (concanavalin A and phytohaemagglutinin), which was counteracted by the feed additive at the lower dietary concentration of T-2 toxin. No such effect was observed with 1.0 ppm of T-2 toxin. Metabolic blood parameters and hematological data showed no consistent treatment effects. We conclude that this feed additive is able to counteract the adverse effects of T-2 toxin at dietary concentrations that might be encountered under field conditions.

We examined the cell-specific subcellular expression patterns for sodium- and potassium-coupled chloride (NaK2Cl) cotransporter 1 (NKCC1), Na+bicarbonate cotransporter (NBCe1), cystic fibrosis transmembrane conductance regulator (CFTR), and Na+/H+exchanger 3 (NHE3) to understand the functional plasticity and synchronization of ion transport functions along the crypt-villus axis and its relevance to intestinal disease. In the unstimulated intestine, all small intestinal villus enterocytes coexpressed apical CFTR and NHE3, basolateral NBCe1, and mostly intracellular NKCC1. All (crypt and villus) goblet cells strongly expressed basolateral NKCC1 (at approximately three-fold higher levels than villus enterocytes), but no CFTR, NBCe1, or NHE3. Lower crypt cells coexpressed apical CFTR and basolateral NKCC1, but no NHE3 or NBCe1 (except NBCe1-expressing proximal colonic crypts). CFTR, NBCe1, and NKCC1 colocalized with markers of early and recycling endosomes, implicating endocytic recycling in cell-specific anion transport. Brunner's glands of the proximal duodenum coexpressed high levels of apical/subapical CFTR and basolateral NKCC1, but very low levels of NBCe1, consistent with secretion of Cl−-enriched fluid into the crypt. The cholinergic agonist carbachol rapidly (within 10 min) reduced cell volume along the entire crypt/villus axis and promoted NHE3 internalization into early endosomes. In contrast, carbachol induced membrane recruitment of NKCC1 and CFTR in all crypt and villus enterocytes, NKCC1 in all goblet cells, and NBCe1 in all villus enterocytes. These observations support regulated vesicle traffic in Cl−secretion by goblet cells and Cl−and HCO3−secretion by villus enterocytes during the transient phase of cholinergic stimulation. Overall, the carbachol-induced membrane trafficking profile of the four ion transporters supports functional plasticity of the small intestinal villus epithelium that enables it to conduct both absorptive and secretory functions.

The actin motor myosin VI regulates endocytosis of cystic fibrosis transmembrane conductance regulator (CFTR) in the intestine, but the endocytic adaptor linking CFTR to myosin VI is unknown. Dab2 (Disabled 2) is the binding partner for myosin VI, clathrin, and alpha-AP-2 and directs endocytosis of low density lipoprotein receptor family members by recognizing a phosphotyrosine-binding domain. However, CFTR does not possess a phosphotyrosine-binding domain. We examined whether alpha-AP-2 and/or Dab2 were binding partners for CFTR and the role of myosin VI in localizing endocytic adaptors in the intestine. CFTR co-localized with alpha-AP-2, Dab2, and myosin VI and was identified in a complex with all three endocytic proteins in the intestine. Apical CFTR was increased in the intestines of Dab-2 KO mice, suggesting its involvement in regulating surface CFTR. Glutathione S-transferase pulldown assays revealed binding of CFTR to alpha-AP-2 (but not Dab2) in the intestine, whereas Dab-2 interacted with alpha-AP-2. siRNA silencing of alpha-AP-2 in cells significantly reduced CFTR endocytosis, further supporting alpha-AP-2 as the direct binding partner for CFTR. alpha-AP-2 and Dab2 localized to the terminal web regions of enterocytes, but Dab2 accumulated in this location in Snell's Waltzer myosin VI((sv/sv)) intestine. Ultrastructural examination revealed that the accumulation of Dab2 correlated with prominent involution and the loss of normal positioning of the intermicrovillar membranes that resulted in expansion of the terminal web region in myosin VI((sv/sv)) enterocytes. The findings support alpha-AP-2 in directing myosin VI-dependent endocytosis of CFTR and a requirement for myosin VI in membrane invagination and coated pit formation in enterocytes.

We previously demonstrated greater HPA axis activation in adult men compared to adult women following low-dose administration of the anticholinesterase inhibitor, physostigmine (PHYSO). Because blood sampling was done infrequently following PHYSO, the rise times of AVP, ACTH1-39, and cortisol could not be determined. In the present study, we determined the sequence of hormone increases by frequent blood sampling following PHYSO. Twelve adult women and 12 adult men underwent three test sessions 5-7 days apart: PHYSO, saline control, and repeat PHYSO. As in the earlier study, PHYSO produced no side effects in half the subjects and mild side effects in the other half, with no significant female-male differences. None of the hormone responses was significantly correlated with the presence or absence of side effects. In both women and men, the AVP increase preceded the ACTH1-39 increase, which in turn preceded the cortisol increase. The AVP and ACTH AUCs were significantly positively correlated in both women and men, supporting AVP as an acute stimulus to ACTH secretion. Also as in the earlier study, the AVP response to PHYSO was more than twice as great in men as in women, but the difference was not statistically significant. We therefore analyzed the results of both studies combined (N=26 women and 26 men). The men had a significantly greater AVP response and a trend toward a greater ACTH1-39 response compared to the women. These findings further support the concept of sexual diergism (functional sex difference) in the influence of CNS cholinergic systems on HPA hormone secretion.

Multiple doses of the dietary supplement l -ephedrine can cause severe hyperthermia and modest dopamine depletions in the rat brain. Since d -amphetamine treatment can result in neurodegeneration, the potential of l -ephedrine to produce similar types of degeneration was investigated. Adult male rats, some implanted in the caudate/putamen (CPu) for microdialysis, were given four doses of 25 mg/kg l -ephedrine or 5 mg/kg d -amphetamine (2 h between doses) at an ambient temperature of 23 °C. l -ephedrine-induced degeneration in the forebrain was dependent on the degree of hyperthermia. Layer IV of the parietal cortex was the most sensitive to l -ephedrine treatment with peak body temperatures of at most 40.0 °C necessary to produce degeneration. Extensive neurodegeneration in the parietal cortex after l -ephedrine treatment was as pronounced as that previously described for d -amphetamine treatment and also occurred in the intralaminar, ventromedial and ventrolateral thalamic nuclei in rats with severe hyperthermia (peak body temperatures>41.0 °C). The neurodegeneration induced by l -ephedrine may have resulted in part from excitotoxic mechanisms involving the indirect pathways of the basal ganglia and related areas. No differences were observed between microdialysis and non-implanted rats with respect to degree of tyrosine hydroxylase (TH) loss in the CPu after either d -amphetamine or l -ephedrine treatment. However, neurodegeneration resulting from d -amphetamine and l -ephedrine was reduced in the microdialysis animals in the hemisphere ipsilateral to the probe, which raises concerns when using the technique of in vivo microdialysis to evaluate neurodegeneration. The results of this study, in conjunction with human clinical evaluation of ephedrine neurotoxicity, indicate that regionally specific damage may occur in the cortex of some humans exposed to ephedrine in the absence of stroke or hemorrhage.

Estrogen receptors (ER) play a significant role in the development of some regions of the mammalian brain. Recently, ER-beta (ERβ) mRNA and protein were shown to be expressed in the rat cerebellum. In the present study, the ontogeny of ERβ protein expression was examined in the rat cerebellum during postnatal development. Western blot analysis indicated that a single ERβ-like immunoreactive species of ∼55 kDa was present in protein lysates prepared from the cerebella of female and male Sprague-Dawley rat pups. Immunocytochemical analysis of cerebellar sections from the midline vermis revealed that during development, the expression of ERβ varied with age and cell-type, but not sex. In the developing cerebellum, highest levels of ERβ-immunoreactivity (IR) were detected in neurons during neurite growth, and in some glia during migration. Throughout the first postnatal week, ERβ-IR was localized to differentiating granule cells in the external germinal layer and to migrating glia. Differentiating granule cells expressed detectable levels of ERβ throughout development. In Purkinje cells, ERβ-IR was first detected on postnatal day 6 (P6), with peak intensities of immunostaining coinciding with the initiation of axonal and dendritic growth that occurs between P7 and P8. Expression of ERβ-IR remained high during maturation of Purkinje cell dendrites, and then decreased to a lower level maintained in the adult. From the third postnatal week, ERβ-IR was also detected in the later developing Golgi, stellate, and basket neurons. These results suggest that ERβ may play a role in growth-related mechanisms during differentiation of cerebellar neurons and glia. J. Comp. Neurol. 430:396–409, 2001. © 2001 Wiley-Liss, Inc.

An emerging concept of cortical network organization is that distinct segments of the pyramidal neuron tree are controlled by functionally diverse inhibitory microcircuits. We compared the expression of two serotonin receptor subtypes, the G-protein-coupled 5-hydroxytryptamine2A receptors and the ion-channel gating 5-HT3 receptors, in cortical neuron types, which control these microcircuits. Here we show, using light and electron microscopic immunocytochemical techniques, that 5-HT2A receptors are segregated from 5-HT3 receptors in the macaque cerebral cortex. 5-HT2A receptor immunolabel was found in pyramidal cells and also in GABAergic interneurons known to specialize in the perisomatic inhibition of pyramidal cells: large and medium-size parvalbumin- and calbindin-containing interneurons. In contrast, 5-HT3 label was only present in small GABA-, substance P receptor-, and calbindin-containing neurons and in medium-size calretinin-containing neurons: interneurons known to preferentially target the dendrites of pyramidal cells. This cellular segregation indicates a serotonin-receptor-specific segmentation of the GABAergic inhibitory actions along the pyramidal neuron tree.

The present study provides a complete quantitative three-dimensional analysis of neurons in primate prefrontal cortex targeted by catecholaminergic axons. Individual pyramidal and nonpyramidal cells in fixed slices were filled with Lucifer yellow (LY) and recovered with anti-LY antibody combined with anti-tyrosine hydroxylase (TH) antisera to reveal catecholaminergic axons. The total number of TH contacts and TH apposition density (THAD) was obtained for pyramidal and nonpyramidal cells in different layers. Four TH contacts (two on spines and two on shafts) were selected for correlated electron microscopic examination and serially sectioned; all four were confirmed as membrane appositions. Quantitative analysis revealed 90 TH contacts per pyramidal neuron in layer III, with a density of 0.8 per 100 μm of dendritic length (i.e., averaging one contact per basal dendrite). Remarkably, pyramids of layers III, V, and VI had the same THAD values, with a highly regular distribution of TH terminals on their spiny dendritic trees. In contrast, TH contacts on nonpyramidal neurons in layer III were half as dense and, moreover, were distributed irregularly and showed large variation from cell to cell. Neurons in layers II and superficial III had the highest THAD, as compared with deeper layers (1.4 vs 0.7 per 100 μm of dendritic length for pyramids; 0.53 vs 0.4 for interneurons). The highly organized TH innervation of pyramidal neurons, with at least one contact on virtually every dendrite, indicates that catecholaminergic, presumably dopaminergic, terminals are placed strategically along the entire dendritic tree to modulate most, if not all, of the excitatory input of a neuron. At the same time, the sparsity of contacts per dendrite may explain cortical vulnerability in diseases involving dopamine.

It has been shown that sexual dimorphic morphology of certain hypothalamic and limbic areas underlie gender-specific sexual behavior and neuroendocrine mechanisms. The key role played by locally formed estrogen in these developmental events has been revealed during a critical perinatal period. In this study, we aimed to document the presence of estrogen-synthetase (aromatase)-immunoreactive elements in the involved limbic system and hypothalamus of the developing rat brain. On postnatal day 5, animals of both sexes were perfusion-fixed, and sections from the forebrain and hypothalamus were immunolabelled for aromatase using an antiserum that was generated against a 20 amino acid sequence of placental aromatase. Aromatase-immunoreactivity was present in neuronal perikarya and axonal processes in the following limbic structures: the central and medial nuclei of the amygdala, stria terminalis, bed nucleus of the stria terminalis (BNST), lateral septum, medial septum, diagonal band of Broca, lateral habenula and all areas of the limbic (cingulate) cortex. In the hypothalamus, the most robust labelling was observed in the medial preoptic area, periventricular regions, ventromedial and arcuate nuclei. The most striking feature of the immunostaining with this antiserum was its intracellular distribution. In contrast to the heavy perikaryal labelling that can be observed with most of the currently available aromatase antisera, in the present experiments, immunoperoxidase was predominantly localized to axons and axon terminals. All the regions with fiber staining corresponded to the projection fields of neuron populations that have previously been found to express perikaryal aromatase. Our results confirm the presence of aromatase-immunoreactivity in developing limbic and hypothalamic areas. The massive expression of aromatase in axonal processes raises the possibility that estrogen formed locally by aromatase may not only regulate the growth, pathfinding and target recognition of its host neuronal processes, but may also exert paracrine actions on structures in close proximity, including the target cells.

Reorganization of the cerebellar glomerulus, the main synaptic complex within the granule cell layer, was investigated using quantitative morphological techniques. All afferents to the cerebellar cortex, including mossy-fibers, were surgically destroyed by undercutting the cerebellar vermis. Fifteen days after the operation, which resulted in the removal of the main excitatory afferent to the glomerulus, a significant reorganization of the whole synaptic complex was observed, whereas the structural integrity of the glomerulus was remarkably well preserved. This was indicated by the observation that the number of granule cell dendrites (approximately 50 per glomerulus), as well as the number of dendritic digits (approximately 210 per glomerulus) bearing most of the approximately 230 synaptic junctions per glomerulus, did not change significantly after mossy-fiber degeneration. The total number of synapses in the reorganized glomerulus did not change either, despite the disappearance of two-thirds of (excitatory) synaptic junctions caused by mossy-fiber degeneration. In the reorganized glomeruli, however, the inhibitory, GABA-containing Golgi axonal varicosities became the dominant synaptic type-about four-fifths (approximately 200) of all synapses within the glomerulus-whereas the dendritic synapses between the granule cells represented only one-fifth of all synaptic junctions. The quantitative data of the reorganized cerebellar glomerulus demonstrate both a remarkable constancy and a plasticity of the excitatory granule cells and inhibitory Golgi neurons building up this synaptic complex. Constancy (the preservation of certain specific structural features) is represented by an eventually unchanged number of dendrites and synaptic junctions within the deafferented glomerulus. Such constancy was made possible, however, by the morphogenetic plasticity of both nerve-cell types to produce new, dendro-dendritic and axo-dendritic synapses to compensate for the loss of mossy-fiber synapses.

The Brunner's glands of the proximal duodenum exert barrier functions through secretion of glycoproteins and antimicrobial peptides. However, ion transporter localization, function, and regulation in the glands are less clear. Mapping the subcellular distribution of transporters is an important step toward elucidating trafficking mechanisms of fluid transport in the gland. The present study examined 1) changes in the distribution of intestinal anion transporters and the aquaporin 5 (AQP5) water channel in rat Brunner's glands following second messenger activation and 2) anion transporter distribution in Brunner's glands from healthy and disease-affected human tissues. Cystic fibrosis transmembrane conductance regulator (CFTR), AQP5, sodium-potassium-coupled chloride cotransporter 1 (NKCC1), sodium-bicarbonate cotransporter (NBCe1), and the proton pump vacuolar ATPase (V-ATPase) were localized to distinct membrane domains and in endosomes at steady state. Carbachol and cAMP redistributed CFTR to the apical membrane. cAMP-dependent recruitment of CFTR to the apical membrane was accompanied by recruitment of AQP5 that was reversed by a PKA inhibitor. cAMP also induced apical trafficking of V-ATPase and redistribution of NKCC1 and NBCe1 to the basolateral membranes. The steady-state distribution of AQP5, CFTR, NBCe1, NKCC1, and V-ATPase in human Brunner's glands from healthy controls, cystic fibrosis, and celiac disease resembled that of rat; however, the distribution profiles were markedly attenuated in the disease-affected duodenum. These data support functional transport of chloride, bicarbonate, water, and protons by second messenger-regulated traffic in mammalian Brunner's glands under physiological and pathophysiological conditions.

The substance P receptor (SPR) gene is expressed at high levels in basal ganglia, but the paucity of information about localization of the encoded receptor protein has limited our understanding of this peptide's involvement in cellular and subcellular mechanisms in this region. Morphological evidence in the rodent striatum indicates that SPRs are expressed in postsynaptic neuronal elements, while pharmacological studies suggest the existence of presynaptic SPRs in this structure. We have examined the issue of subcellular distribution of this receptor protein in rat and primate neostriatal tissue, employing an antiserum raised against SPR. Electron microscopic analysis revealed that SPR immunoreactivity is present in presynaptic and postsynaptic neuronal elements in both species. In agreement with earlier studies, SPR immunoreactivity was found predominantly in perikarya and dendrites of a small subset of striatal neurons, the large and medium-sized aspiny interneurons. In addition, a small but significant proportion of the immunoreaction product was localized in presynaptic profiles, both in axons and axon terminals. The majority of SPR immunoreactive boutons formed asymmetric synapses with dendrites and dendritic spines. The association of SPRs with asymmetric synapses provides a morphological substrate for peptidergic modulation of excitatory neurotransmission of extrastriatal origin. A minor proportion of immunolabeled axons established symmetric synaptic junctions with unlabeled dendrites. The presence of SPRs in these synapses suggests a presynaptic peptidergic modulation of intrinsic striatal transmitter systems. The observations in this study also indicate that SPR mediates a complex combination of postsynaptic and presynaptic effects on acetylcholine release in the mammalian striatum. © 1996 Wiley-Liss, Inc.

The striatal distribution of the substance P receptor (SPR) protein was examined in relation to its ligand, the neuro-peptide SP, as well as to the neurochemical and compartmental composition of the neostriatum in rhesus monkeys (Macaca mulatta) in immunohistochemical experiments. About 2% of striatal neurons, displaying varicose, virtually spine-free dendrites characteristic of large and medium-sized aspiny interneurons, expressed SPR immunoreactivity. SPR/choline acetyltransferase, SPR/somatostatin, SPR/GABA, SPR/calbindin D28k, and SPR/parvalbumin double immunolabeling experiments demonstrated that SPR-positive cells are either cholinergic or somatostatinergic. Comparison of SP and SPR immunoreactivities in double-labeled and adjacent single-labeled sections revealed compartment-specific match and mismatch between the densities of the peptide and receptor. A matching high density of SP fibers and SPR cells and dendrites was only observed in the rim of the striosome compartments. To our knowledge, this is the first evidence for an anatomical border comprised of dendritic processes that separate striatal compartments. We have termed these zones “striocapsules,” because they encircle and encapsulate striosomal cell islands. In the striatal matrix, an abundance of SPR-labeled profiles was complemented with light SP staining. By contrast, in the core of the striosomes, SPR labeling was sparse and SP staining intense. SP-positive axon-like puncta frequently contacted SPR-positive dendrites in all striatal compartments. The SP receptor/ligand match indicates a sharp increase in the efficacy of SP action in the striocapsules, and suggests that the influence of SP might be heightened in this striatal subcompartment. © 1996 Wiley-Liss, Inc.

Intraneuronal production of estradiol from testosterone has been shown to play a pivotal role in gender-specific brain development of most vertebrates, and to participate in numerous functions of the adult central nervous system. Previous biochemical and morphological approaches demonstrated that estrogen synthetase (aromatase) is present in specific limbic and hypothalamic structures. On the other hand, less attention has been paid to revealing its subcellular distribution. The possibility of aromatase presence in axonal processes has been indicated by recent biochemical and morphological observations suggesting new insights for the role of aromatase in neural functions. The objective of the present study was to provide morphological evidence for the subcellular location of aromatase in neurons of different vertebrate species including Japanese quail, rat, monkey, and human. Immunocytochemistry using a purified polyclonal antiserum against human placental aromatase localized immunoreactivity to hypothalamic and limbic cell groups in all of these species. Light and electron microscopic examination of vibratome sections revealed the presence of aromatase immunoreactivity throughout the neuronal perikarya, including dendrites and axonal processes. In each species there were numerous boutons which contained labeled small clear synaptic vesicles. Many of these axon terminals formed synapses with immuno-negative and immuno-positive dendrites and perikarya. This study furnishes the first immunolocalization of aromatase in the brains of two primate species, humans and monkeys. The provision of further evidence for estrogen synthesis in axons and axon terminals may help resolve apparent differences between the measurement of aromatase activity and the lack of aromatase-immunopositive cell bodies in previous studies. The present findings may be coupled with recent evidence regarding the molecular biology and the diversity of functional properties of P450 aromatase to indicate previously unexpected effects of brain aromatase at the synaptic level.

In two sets of experiments eight groups of seven-week-old pigs weighing about 9 kg were fed for three weeks a prestarter that contained 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 10.0 or 15.0 mg/kg of highly purified T-2 toxin. The feed of the two control groups was free from T-2 toxin. Average daily intakes of toxin by the pigs were 0.38, 0.81, 1.24, 1.43, 0.93, 0.81, 0.99 and 2.5 mg, respectively. The weight gains, the feed intakes, the extent of feed refusal, the parameters of energy and protein metabolism and the serum concentrations of calcium, inorganic phosphorus and magnesium were affected to different extents by the different doses of T-2 toxin, but the data indicated that feed consumption was reduced and the activity of aspartate aminotransferase was increased by the smallest amount of T-2 toxin tested.

Lubiprostone is a chloride channel activator in clinical use for the treatment of chronic constipation, but the mechanisms of action of the drug are poorly understood. The aim of this study was to determine whether lubiprostone exerts secretory effects in the intestine by membrane trafficking of ion transporters and associated machinery.Immunolabeling and quantitative fluorescence intensity were used to examine lubiprostone-induced trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR), sodium/potassium-coupled chloride co-transporter 1 (NKCC1), electrogenic sodium/bicarbonate co-transporter 1 (NBCe1), down-regulated in adenoma (DRA), putative anion transporter 1 (PAT1), sodium/proton exchanger 3 (NHE3), Ca(2+) activated chloride channel 2 (ClC-2) serotonin and its transporter SERT, E prostanoid receptors EP4 and EP1, sodium/potassium ATPase (Na-K-ATPase) and protein kinase A (PKA). The effects of lubiprostone on mucus exocytosis in rat intestine and human rectosigmoid explants were also examined.Lubiprostone induced contraction of villi and proximal colonic plicae and membrane trafficking of transporters that was more pronounced in villus/surface cells compared to the crypt. Membrane trafficking was determined by: (1) increased membrane labeling for CFTR, PAT1, NKCC1, and NBCe1 and decreased membrane labeling for NHE3, DRA and ClC-2; (2) increased serotonin, SERT, EP4, EP1 and PKA labeling in enterochromaffin cells; (3) increased SERT, EP4, EP1, PKA and Na-K-ATPase in enterocytes; and (4) increased mucus exocytosis in goblet cells.These data suggest that lubiprostone can target serotonergic, EP4/PKA and EP1 signaling in surface/villus regions; stimulate membrane trafficking of CFTR/NBCe1/NKCC1 in villus epithelia and PAT1/NBCe1/NKCC1 in colonic surface epithelia; suppress NHE3/DRA trafficking and fluid absorption; and enhance mucus-mobilization and mucosal contractility.

The aromatase enzyme (estrogen synthetase) catalyzes the conversion of testosterone to estrogen in peripheral and central nervous tissue. Light and electron microscopic immunocytochemistry was used to study the localization of this enzyme in the septal area of adult male and female albino rats. Aromatase-immunoreactivity was found restricted to neuronal somata and dendritic arbors, and no sex differences were detected in its distribution or intensity. Most aromatase-immunoreactive neurons formed two oblique bands in the lateral and the medial zones of the lateral septum; in addition, labeled cells were present in the septohippocampal nucleus and the laterodorsal portion of the bed nucleus of the stria terminalis. Electron microscopy revealed that the majority of aromatase-positive neurons in the lateral septum exhibit somatic spines, a characteristic marker of a neuron population that is known to contribute to local and extraseptal projections. The presence of aromatase in lateral septal somatospiny neurons suggests that estrogen formed by these neurons may be critically involved in the septal control of steroid-dependent behaviors.

The aromatase (estrogen synthetase) enzyme catalyzes the conversion of androgens to estrogens in peripheral tissues, as well as in the brain. Our study aimed at comparing the brain distribution of aromatase-immunoreactive neurons in male and female, normal and gonadectomized rats. Light microscopic immunostaining was employed using a purified polyclonal antiserum raised against human placental aromatase. Two anatomically separate aromatase-immunoreactive neuronal systems were detected in the rat brain: A “limbic telencephalic” aromatase system was composed by a large population of labeled neurons in the lateral septal area, and by a continuous “ring” of neurons of the laterodorsal division of the bed nucleus of stria terminalis, central amygdaloid nucleus, stria terminalis, and the substantia inominata-ventral pallidum-fundus striati region. The other, “hypothalamic” aromatase system consisted of neurons scattered in a dorsolateral hypothalamic area including the paraventricular, lateral and dorsomedial hypothalamic nuclei, the subincertal nucleus as well as the zona incerta. In addition, a few axon-like processes (unresponsive to gonadectomy) were present in the preoptic-anterior hypothalamic complex, the ventral striatum, and midline thalamic regions. No sexual dimorphism was observed in the distribution or intensity of aromatase-immunostaining. However, 3 days, 2, 3, 8, 16, or 32 weeks after gonadectomy, aromatase-immunoreactive neurons disappeared from the hypothalamus, whereas they were still present in the limbic areas of both sexes. The results indicate the existence of two distinct estrogen-producing neuron systems in the rat brain: (1) a “limbic ring” of aromatase-labeled neurons of the lateral septum-bed nucleus-amygdala complex unresponsive to gonadectomy; and (2) a sex hormone-sensitive “hypothalamic” aromatase neuron system.

Electron microscopic immunocytochemistry was combined with acute anterograde axon degeneration, following transection of the fimbria-fornix, to describe the innervation of somatospiny neurons by vasopressin-immunoreactive and degenerated hippocamposeptal axon terminals in the rat lateral septal area. Vasopressin-immunopositive boutons characterized by symmetric synaptic membrane specializations, and the degenerated hippocamposeptal axon terminals which form asymmetric synaptic contacts, frequently terminate on the same dendritic and somatic profiles, and particularly on the somata of somatospiny neurons. Although hippocamposeptal fibers predominantly form axospinous synapses in the lateral septal area, they terminate mainly on the dendritic shafts and soma of the vasopressin-receptive neurons. Of 720 vasopressin-immunoreactive terminals in the mediolateral part of the lateral septal area, 80% form synaptic contacts with dendritic shafts: 50% on small (distal) dendritic profiles and 30% on large (proximal) dendrites. Synaptic contacts between vasopressin-immunoreactive terminals and dendritic spines were not observed. The remaining 20% of immunoreactive boutons formed axosomatic synaptic contacts with a total of 58 neurons; 31% of these neurons exhibited somatic spines in the plane of the section analysed. Previous studies have demonstrated that in the lateral septal area vasopressin modulates the action of the excitatory amino acid-containing hippocamposeptal fibers, and also plays a role in the maintenance of long term potentiation evoked by fimbria-fornix stimulation. The convergent vasopressinergic and hippocampal input onto the same somatospiny neurons of the lateral septal area suggests that these neurons are targets of these physiological actions.

Gene expression profiles in several brain regions of adult male rats were evaluated following a d -amphetamine (AMPH) exposure paradigm previously established to produce AMPH neurotoxicity. Escalating doses of AMPH (5–30 mg/kg) were given over the course of 16 h per day in an 18 °C environment for 2 days. This paradigm produces neurotoxicity but eliminates or minimizes the hyperthermia and seizure activity that might influence gene expression in a manner unrelated to the neurotoxic effects of AMPH. The expression of 1185 genes was monitored in the striatum, parietal cortex, piriform cortex and posteriolateral cortical amygdaloid nucleus (PLCo) using cDNA array technology, and potentially significant changes were verified by RT–PCR. Gene expression was determined at time points after AMPH when neurodegeneration was beginning to appear (16 h) or maximal (64 h). Expression was also determined 14 days after AMPH to find long-term changes in gene expression that might be biomarkers of a neurotoxic event. In the parietal cortex there was a two-fold increase in neuropeptide Y precursor protein mRNA whereas nerve growth factor-induced receptor protein I-A and I-B mRNA decreased 50% at 16 h after the end of AMPH exposure. Although these changes in expression were not observed in the PLCo, insulin-like growth factor binding protein 1 mRNA was increased two-fold in the PLCo at 16 and 64 h after AMPH. Changes in gene expression in the cortical regions were all between 1.2- and 1.5-fold 14 days after AMPH but some of these changes, such as annexin V increases, may be relevant to neurotoxicity. Gene expression was not affected by more than 1.5-fold at the time points in the striatum, although 65% dopamine depletions occurred, but the plasma membrane-associated dopamine transporter and dopamine D2 receptor were decreased about 40% in the substantia nigra at 64 h and 14 days post-AMPH. Thus, the 2-day AMPH treatment produced a few changes in gene expression in the two-fold range at time points 16 h or more after exposure but the majority of expression changes were less than 1.5-fold of control. Nonetheless, some of these lesser fold-changes appeared to be relevant to the neurotoxic process.

The dopamine-releasing and depleting substance amphetamine (AMPH) can make cortical neurons susceptible to damage, and the prevention of hyperthermia, seizures and stroke is thought to block these effects. Here we report a 2-day AMPH treatment paradigm which affected only interneurons in three cortical regions with average or below-average dopamine input. AMPH (six escalating doses/day ranging from 5 to 30 mg/kg for 2 days) was given at 17-18 degrees C ambient temperature (T) to adult male rats. During the 2-day AMPH treatment, peak body T stayed below 38.9 degrees C in 40% of the AMPH treated rats. In 60% of the rats, deliberate cooling suppressed (

The cellular and subcellular localization of muscarinic receptor proteins m1 and m2 was examined in the neostriatum of macaque monkeys by using light and electron microscopic immunocytochemical techniques. Double-labeling immunocytochemistry revealed m1 receptors in calbindin-D28k–positive medium spiny projection neurons. Muscarinic m1 labeling was dramatically more intense in the striatal matrix compartment in juvenile monkeys but more intense in striosomes in the adult caudate, suggesting that m1 expression undergoes a developmental age-dependent change. Ultrastructurally, m1 receptors were predominantly localized in asymmetric synapse-forming spines, indicating that these spines receive extrastriatal excitatory afferents. The association of m1-positive spines with lesion-induced degenerating prefronto-striatal axon terminals demonstrated that these afferents originate in part from the prefrontal cortex. The synaptic localization of m1 in these spines indicates a role of m1 in the modulation of excitatory neurotransmission. To a lesser extent, m1 was present in symmetric synapses, where it may also modulate inhibitory neurotransmission originating from local striatal neurons or the substantia nigra. Conversely, m2/choline acetyltransferase (ChAT) double labeling revealed that m2-positive neurons corresponded to large aspiny cholinergic interneurons and ultrastructurally, that the majority of m2 labeled axons formed symmetric synapses. The remarkable segregation of the m1 and m2 receptor proteins to projection and local circuit neurons suggests a functional segregation of m1 and m2 mediated cholinergic actions in the striatum: m1 receptors modulate extrinsic glutamatergic and monoaminergic afferents and intrinsic GABAergic afferents onto projection neurons, whereas m2 receptors regulate acetylcholine release from axons of cholinergic interneurons. J. Comp. Neurol. 434:445–460, 2001. © 2001 Wiley-Liss, Inc.

In the period between December 5, 1991 and September 17, 1998, 760 maize, 367 wheat, 119 soybean, 222 barley, 85 bran, 32 triticale, 60 oat, 14 rye and 22 sunflower samples were investigated for the presence and concentration of seven fusariotoxins (T-2 toxin, zearalenone, deoxynivalenol, nivalenol, diacetoxyscirpenol, HT-2 toxin, fusarenone-X) and OTA. The comparison of analytical data with those of the relevant literature revealed that although the incidence rate and/ or concentration of Fusarium mycotoxins and OTA in Hungarian-grown cereals is occasionally considerable, the position of the country is not worse than the average of countries. Our findings indicate that soybean tends to be good substrate for trichothecene-producing fungi and the rate of contamination is regarded as substantial. The commodities were assorted into one of three quality categories. The proportion of objectionable samples was only 3.0, 2.2, 2.3 and 1.7% in maize, wheat, barley and soybean samples, respectively. However, this low rate of objection might still he a source of great economic loss. The proportion of objectionablc samples was much higher in the case of bran, oat and triticale (7.1, 6.7, and 6.3 %, respectively). The results of the present investigation indicate a need for regular screening for mycotoxins of importance and individual appraisal of each commodity from the point of their use in animal feeds.

To identify the cortical sites where 5-hydroxytryptamine 2A (5-HT 2A ) serotonin receptors respond to the action of hallucinogens and atypical antipsychotic drugs, we have examined the cellular and subcellular distribution of these receptors in the cerebral cortex of macaque monkeys (with a focus on prefrontal areas) by using light and electron microscopic immunocytochemical techniques. 5-HT 2A receptor immunoreactivity was detected in all cortical layers, among which layers II and III and layers V and VI were intensely stained, and layer IV was weakly labeled. The majority of the receptor-labeled cells were pyramidal neurons and the most intense immunolabeling was consistently confined to their parallelly aligned proximal apical dendrites that formed two intensely stained bands above and below layer IV. In double-label experiments, 5-HT 2A label was found in calbindin D28k-positive, nonphosphorylated-neurofilament-positive, and immuno-negative pyramidal cells, suggesting that probably all pyramidal cells express 5-HT 2A receptors. 5-HT 2A label was also found in large- and medium-size interneurons, some of which were immuno-positive for calbindin. 5-HT 2A receptor label was also associated with axon terminals. These findings reconcile the data on the receptor’s cortical physiology and localization by ( i ) establishing that 5-HT 2A receptors are located postsynaptically and presynaptically, ( ii ) demonstrating that pyramidal neurons constitute the major 5-HT 2A -receptor-expressing cells in the cortex, and ( iii ) supporting the view that the apical dendritic field proximal to the pyramidal cell soma is the “hot spot” for 5-HT 2A -receptor-mediated physiological actions relevant to normal and “psychotic” functional states of the cerebral cortex.

In spite of accumulating evidence on the potent neuromodulatory, neuroprotective, trophic and memory-enhancing effects of the neuropeptide substance P (SP) in the cerebral cortex, the excitatory or inhibitory nature of the cortical SP innervation remains unclear and the postsynaptic targets of SP fibers are not defined. To obtain further insight into these issues, we have examined SP-containing axons and their postsynaptic targets in the prefrontal cortex of adult monkeys with single- and double label immunocytochemistry combined with light and correlated electron microscopy. SP fibers in the primate prefrontal cortex, unlike those in the rat cortex, preferentially innervate cortical layers I, II and upper layer III. Our results demonstrate for the first time that all SP-immunoreactive boutons in all cortical layers contain GABA. Of the entire sample of SP boutons, 53% synapse on dendritic shafts, 39% on dendritic spines and 8% on cell bodies. Another new finding is that synapse-forming SP boutons, in addition to their known innervation of pyramidal cells, form pericellular baskets around interneurons in layers II and upper III, a subpopulation of which contains calbindin D28k. Finally, the study also revealed that SP boutons frequently participate in 'synaptic triads' with spines which receive another (asymmetric, putatively excitatory amino acid-utilizing) synapse. Our findings indicate that SP/GABA axons in the primate prefrontal cortex modulate excitatory amino acid-mediated neurotransmission and control feed-forward disinhibitory GABAergic circuits in supragranular cortical layers.

The electrophysiological observations that substance P administration to the lateral septal area elicits both excitatory and inhibitory responses, together with earlier reports on the multiple sources of substance P innervation of the septum, implies that these axons with distinct origins have different functions. This prompted us to examine the origin and neurochemical character of substance P afferents to the lateral septal area. Chronic surgical isolation of the septum from its ventral afferents and retrograde tracer experiments using wheat germ agglutinin-conjugated horseradish peroxidase, both followed by an immunostaining for substance P, were employed to elucidate the origin of these axon terminals. In order to assess the possible co-existence of substance P with other neurotransmitter substances in the parent cells of the septopetal projections, co-localization studies for substance P and choline acetyltransferase, as well as substance P and GABA, were performed. The comparative distribution of substance P fibers and septal calbindin-containing neurons was also investigated using correlated light and electron microscopic double immunostaining. The results are summarized as follows: (i) the substance P innervation of the lateral septal area derives from several hypothalamic nuclei (including the lateral and lateroanterior hypothalamic area, tuber cinereum and ventromedial hypothalamic nucleus) and tegmental nuclei (the majority of fibers from the laterodorsal and a few from the pedunculopontine tegmental nucleus), as well as intrinsic septal cells; (ii) the septopetal substance P fibers of tegmental origin are cholinergic; intraseptal substance P neurons located in the dorsolateral part of the lateral septum also contain GABA, while substance P neurons seen on the border between the medial and lateral septal area and septopetal hypothalamic substance P cells do not contain GABA or acetylcholine; (iii) substance P fibers from pericellular baskets around calbindin-containing lateral septal neurons with a high degree of selectivity; (iv) approximately 90% of the entire calbindin cell population are postsynaptic targets of substance P axons; (v) their terminals contact the soma and the dendrites of these cells, among them the somatospiny neurons; and (vi) the extrinsic substance P boutons establish asymmetric, while the intrinsic substance P axon terminals form symmetric membrane specializations. Because neurons in the lateral septal area receive hippocampal input and project massively to hypothalamic areas, the different types of substance P input on these neurons can modify the information flow arriving from the hippocampus to diencephalic brain structures at the level of the lateral septal area.

Four groups of seven-week-old pigs weighing about 9 kg were fed for three weeks a prestarter that contained 0.5, 1.0, 2.0 or 3.0 mg/kg of highly purified T-2 toxin. The average daily intakes of toxin by the pigs were 0.38, 0.81, 1.24 and 1.43 mg, respectively. The experimental and control pigs were immunised with 5 ml aluminum hydroxide gel-absorbed purified horse globulin on the first and fourth days of the treatment period. Blood samples were withdrawn on days 7, 14 and 21 and used for the determination of the titre of anti-horse globulin antibody, for an in vitro lymphocyte proliferation test, using purified horse globulin, phytohaemagglutinin and concanavalin-A and for determinations of the immune complex, the cytotoxic reaction and the phagocytic activity and phagocytic index of circulating granulocytes. The samples taken on day 21 were also used to determine the erythrocyte count, the mean cell volume of the erythrocytes, the haematocrit, the blood haemoglobin concentration, the leucocyte count and the proportion of T lymphocytes. At the end of the experiment samples were taken from the thymus, spleen and mesenteric lymph nodes for histological examination. The diets that contained 2 and 3 mg T-2 toxin/kg caused a significant decrease in the red blood cell count, the mean corpuscular volume and the haemoglobin concentration. A significant decrease in the leucocyte count and the proportion of T lymphocytes was observed in all the treatment groups. There were also dose-dependent, significant decreases in antibody formation and in the blastogenic transformation of lymphocytes, and mild to moderate reactive processes were observed histologically in the lymphoid organs.

This study deals with the synaptology, morphologically identified postsynaptic targets, and origin of somatostatin (SOM) fibers in the rat lateral septal area (LSA) with special reference to those forming pericellular baskets. Septal vibratome sections were immunostained for SOM-14 in 3 experimental groups: control animals, rats subjected to a chronic transection of the ascending afferents to the septum, and animals with acute fimbria-fornix lesion. Light microscopy revealed that the SOM-immunoreactive fibers form pericellular baskets predominantly in the intermediate and ventral parts of the caudal half of the LSA. Electron microscopic analysis showed that the somatospiny neurons are postsynaptic targets of these pericellular baskets. Eight days after a unilateral cut placed at the ventral border of the septum, virtually all SOM-immunoreactive axon terminals disappeared from the ipsilateral intermediate and ventral LSA, and they were substantially reduced in the dorsal LSA. However, in these rats SOM-positive neurons could be observed in the LSA on the lesioned, but not on the contralateral side. Furthermore, on the lesion side of the anterior periventricular hypothalamus an increase was detected both in the number and the intensity of immunostaining of SOM-positive neurons. Thirty-six h following a unilateral transection of the fimbria-fornix, the SOM-immunoreactive axon terminals in the LSA remained intact; only immunonegative degenerated hippocamposeptal boutons were detected forming synaptic contacts with somatospiny neurons. Axosomatic synapses of SOM-positive boutons regularly appeared at the neck of somatic spines which were postsynaptic to degenerated hippocamposeptal fibers. The results indicate that the septal SOM fibers are of multiple origin. Those forming pericellular baskets in the LSA originate in ventral extraseptal, probably periventricular hypothalamic areas. SOM fibers scattered in the dorsal LSA are most likely processes of local SOM neurons. The accumulation of immunoreactive SOM in some cells of the undercut septum is a sign of axonal lesion, indicating that these neurons project outside the septum. The SOM innervation of somatospiny neurons which also receive hippocampal input and have been reported to contain γ-aminobutyric acid (GABA) may be a morphological substrate of the SOM-related disinhibition in the LSA.

This study deals with the neurochemical characterization of the rat lateral septal area (LSA) somatospiny neurons and their innervation by hippocamposeptal, catecholaminergic, and GABAergic fibers. Electron microscopic single and double immunostaining methods were used to label catecholaminergic fibers and GABAergic cells and boutons. Axon terminals originating in the hippocampus were labeled by acute anterograde axon degeneration induced by fimbria-fornix transection 36 hours before sacrifice. Three types of experiments were performed. The convergent catecholaminergic and hippocamposeptal innervation of LSA somatospiny neurons was studied by combining immunostaining for tyrosine hydroxylase (TH) with fimbria-fornix transection. GABAergic neurons and their hippocamposeptal afferents were identified and characterized in colchicine pretreated animals immunostained for glutamic acid decarboxylase (GAD) combined with fimbria-fornix transection. The third experiment aimed at simultaneously visualizing the relationships between catecholaminergic boutons, hippocamposeptal excitatory amino acid containing axon terminals and GABAergic profiles by double immunostaining for TH (the PAP technique) and GAD (the immunogold method) combined with fimbria-fornix transection. The results are summarized as follows: 1) The same LSA somatospiny neurons receive synaptic inputs from the hippocampus and TH immunoreactive fibers which form pericellular baskets around these cells. 2) LSA somatospiny neurons are GABAergic and are postsynaptic targets of GABAergic boutons with unknown origin and hippocamposeptal axon terminals. 3) The double immunostaining experiment, finally, provided direct evidence that the same GABAergic somatospiny neurons are postsynaptic targets of both catecholaminergic and hippocamposeptal afferents. The synaptic interconnections described in this study provide anatomical basis for a better understanding of the action of catecholamines, excitatory amino acids, and GABA on the activity of LSA neurons.

The mediolateral part of the lateral septal area (LSA) is a common target of hippocamposeptal afferents, neuropeptide containing, catecholaminergic, cholinergic, and GABAergic pericellular baskets of different origins. This specific innervation pattern as well as electrophysiological data concerning this area suggest a convergent input from different sources to particular LSA neuron populations. Light and electron microscopy combined with Golgi impregnation and acute anterograde degeneration techniques following transection of the fimbria-fornix were employed to determine whether LSA neurons with hippocampal input have any characteristic and distinctive morphological signs. About 20% of all Golgi impregnated LSA neurons were found to have somatic spines. All of these somatospiny neurons are synaptic targets of hippocamposeptal fibers. The degenerated hippocamposeptal boutons establish asymmetric synaptic contacts on their soma, somatic and dendritic spines, and on dendritic shafts. Somatospiny neurons located in the most medial and dorsal parts of the LSA seem to project toward the medial septum while all of the others appear to send descending fibers to ventral areas. Somatospiny neuron axons occasionally give out recurrent collaterals. Quantitative analysis on the spatial distribution of the somatospiny neurons revealed that practically all of them are encountered in the mediolateral division of the LSA. This area includes the lateral part of the intermediolateral septal nucleus and adjacent lateral portions of the dorsolateral and the ventrolateral septal nuclei.

Computer-assisted stereological and quantitative morphological approaches were used to analyse cerebellar glomeruli of the “simple type” in serial ultrathin sections. It was found that, of the total volume (110–200 μm3) of the glomeruli studied, 53% was occupied by granule cell dendrites, 34% by mossy terminal and 13% by Golgi axons. None of the four analysed glomeruli contained Golgi cell dendrites. The mossy terminals that were studied received, on the average, 53 granule cell dendrites. All of the dendrites originated from different granule cells and all made synaptic contacts with mossy terminal. However only about 60% of granule cell dendrites made synapses with Golgi axons. The surface of the mossy terminals occupied by synaptic junctions, was found to be 5.4–5.5%. Each granule cell dendrite emitted 3–5 terminal protrusions (“dendritic digits”). Each digit receives one or more synaptic contact from either the mossy terminal (67% of all digits), or from Golgi axon varicosities (25%). Only about 8% of all digits were contacted synaptically by both types of axonal terminals. All of the dendritic digits that were observed made synaptic connections. Each digit was, on the average, connected by symmetric attachment plaques to 4 neighbouring digits. Three-dimensional reconstructions of mossy terminal and some of contacting granule cell dendrites demonstrated that the dendrites curved around the central mossy terminal and were much longer than expected from earlier Golgi-impregnation studies. In addition to mossy terminals and Golgi axons, an axon terminal of small calibre that contained large, empty, spheroid vesicles were occasionally observed. These terminals, which are most likely the axonal varicosities of ascending parallel fibers, made synaptic contacts exclusively with granule cell dendrites at the periphery of the glomeruli. The results demonstrate that, in the rat cerebellum, there is a high degree of convergence of granule cells at a glomernius (53 to 1); and that there is a rich inhibitory input to about 60% of all granule cell dendrites. It is also shown that the main postsynaptic targets, for both mossy and Golgi axons, are the dendritic digits. The presence of synaptic contacts between parallel-fiber-like varicosities and granule cell dendrites may be an additional source of excitation within the glomerulus.

Summary Mucopolysaccharides (MPS) have been studied in the vascular wall, various other tissues, and the serum of rabbits and cocks fed a cholesterol diet. Histological and histochemical investigations indicate that changes in the mucoid materials precede the intimal deposition of lipids. Sulphate ( 35 S) uptake of the MPS of aorta and cartilage was significantly increased in cholesterol-fed animals; that of liver and kidney MPS was unchanged. The concentrations of serum protein-bound hexose, hexosamine and sialic acid increased significantly after cholesterol feeding. It is suggested that an intimal lipid film, developed in hypercholesterolaemia, disturbs the metabolic processes of intimal MPS by creating a local oxygen deficiency. The consequently changed composition of the MPS may lead secondarily to the deposition of lipids and other substances.

Plasma fibronectin seems to be a component of mixed polyclonal cryoglobulins1,6. According to our previous studies, mixed cryoglobulins, consisting of monoclonal and polyclonal immunoglobulins contain fibronectin (unpublished results). In vivo activation of the complement system was demonstrated in all of our patients with essential mixed cryoglobulinemia. Moreover, in vitro studies showed that isolated cryoglobulins activated the complement system via the C1q. The purpose of this study was to analyze the binding of purified human fibronectin to potential components of the cryoglobulins, to monoclonal immunoglobulins and to C1q, in vitro. This was performed by binding of I125 labelled fibronectin to human C1q, to human C1 complex, to human C1q bound to immune complexes and to human myeloma IgG subclasse.

Cell surface receptors for fibronectin /FN/ were detected by immunofluorescence technique on isolated normal human neutrophils /PMN/.FN inhibited in vitro the chemiluminesoence response /CLR/ of PMN-s stimulated by opsonized zymosan /OZ/, zymosan /Z/ and fMet-Leu-Phe /fMLP/ synthetic peptid in a dose dependent manner. In the same concentration range, FN increased the phagocytosis of OZ particles by PMN-s.FN in a Boyden technique assay increased the chemotactic activity of PMN-s stimulated by Z activated serum /ZAS/ or fMLP.Binding of FN to their receptors on PMN-s can modify the production of oxygen radicals during the course of postphagocytic metabolic events and the response of cells to various chemotactic factors.

THE acid mucopolysaccharides (MPS) as macro-anions form complexes with proteins and thus they change the isoelectric point of the latter. Based on this property some MPS's are able to inhibit or activate many enzyme systems1.