Cellular and subcellular localization of gamma-aminobutyric acidB receptors in the rat olfactory bulb.

Olfactory nerve axons terminate in rounded regions of the olfactory bulb, termed glomeruli, where they make excitatory synapses with the dendrites of second-order neurons. Neurotransmission from the olfactory nerve to the postsynaptic targets is negatively regulated by gamma-aminobutyric acid (GABA), and there is evidence that inhibition of sensory input is mediated, at least in part, by GABA(B) receptors. Using an antiserum that recognizes two GABA(B) receptor splice variants (GBR1a and GBR1b), we show here that GABA(B) receptors are located on the axon terminals of the olfactory nerve, where they are concentrated at sites of axodendritic apposition. Taken with previous data, these results indicate that GABA(B) receptors act presynaptically to regulate the release of glutamate from olfactory nerve terminals.

Immunocytochemical localization of glutamate and gamma-aminobutyric acid in the accessory olfactory bulb of the rat.

The synaptic organization of the accessory olfactory bulb (AOB) was studied in the rat with antibodies against the excitatory neurotransmitter glutamate (Glu) and the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). To a large extent, the immunoreactivity patterns produced by the two antibodies were complementary. Glu-like immunoreactivity (-LI) was observed in the glomerular neuropil, in the mitral cells, and in large neurons located in the periglomerular region. Immunogold electron microscopy revealed particularly high levels of Glu-LI in the axon terminals of vomeronasal neurons. GABA-LI was present in granule and periglomerular cells and in their processes. The dendritic spines of granule cells, which were presynaptic to mitral cells, were strongly labelled by the antiserum against GABA. Labelling of serial semithin sections showed that the GABA-positive and Glu-positive neurons of the periglomerular region are generally distinct, and colocalization of Glu and GABA occurred only in a few cells. These results are consistent with electrophysiological studies indicating that the synaptic organization of the AOB is similar to that of the main olfactory bulb. In both systems, Glu is the neurotransmitter used by primary afferents and output neurons, whereas GABA is involved in the circuits underlying lateral and feed-back inhibition.

Localization of the clustering protein gephyrin at GABAergic synapses in the main olfactory bulb of the rat.

The tubulin-binding protein gephyrin is essential for the formation of postsynaptic glycine-receptor clusters in cultured spinal neurons. In addition, there is increasing evidence that gephyrin can also be present at nonglycinergic synapses. Here we analyzed immunocytochemically the subcellular localization of gephyrin in the main olfactory bulb of the rat and compared its distribution with that of gamma-aminobutyric acid (GABA) and of two major GABA(A)-receptor subunits. Gephyrin was selectively localized to the postsynaptic side of symmetric synaptic junctions, where the presynaptic terminals contained GABA. Moreover, gephyrin colocalized extensively with the alpha1 and gamma2 subunits of the GABA(A) receptor. In contrast, gephyrin was not detected at presumed glutamatergic synapses. These results indicate that gephyrin is not uniquely associated with glycine receptors, but can also be found at distinct GABAergic synapses. Thus, they raise the possibility that gephyrin is involved in anchoring certain GABA(A)-receptor subtypes in the postsynaptic membrane.

Co-localization of carnosine and glutamate in photoreceptors and bipolar cells of the frog retina.

Immunocytochemical methods were used to visualize carnosine (beta-alanyl-L-histidine)-like immunoreactivity (-LI) in the frog retina and to compare its localization with that of glutamate. Carnosine-LI was conspicuous in photoreceptors and bipolar cells. The axon terminals of labelled bipolar cells formed five bands in the inner plexiform layer. A few presumed amacrine and ganglion cells, as well as Müller cell endfeet, were also labelled. Post-embedding immunocytochemistry revealed particularly high levels of glutamate-LI in the synaptic axon terminals of bipolar cells, with a mean gold particle density 5 x higher than that of amacrine cells. Photoreceptor terminals were also labelled, but with a labelling intensity about half that of bipolar cells. Labelling of serial semithin sections showed co-localization of carnosine and glutamate in photoreceptors and bipolar cells. These findings are consistent with the notion that glutamate is the neurotransmitter of neuronal elements that transfer information vertically through the retina. We propose that carnosine may modulate GABA and/or glutamate receptors by virtue of its ability to chelate Zn2+ and other ions.

Glutamate receptors in the olfactory bulb synaptic circuitry: heterogeneity and synaptic localization of N-methyl-D-aspartate receptor subunit 1 and AMPA receptor subunit 1.

In this study, we analysed the molecular heterogeneity and synaptic localization of the N-methyl-D-aspartate receptor subunit 1 and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit 1 in the olfactory bulb glomerular synaptic circuitry. Semiquantitative reverse transcriptase polymerase chain reaction showed that approximately 40% of the N-methyl-D-aspartate receptor subunit 1 messenger RNA splice variants contain the N1 exon, which conveys specific functional properties on the channel. In other forebrain and hindbrain regions that we examined, the ratio of the N1-containing (receptor subunit 1(1XX)) to N1-lacking (receptor subunit 1(0XX)) N-methyl-D-aspartate receptor subunit 1 messenger RNAs varied considerably. The cellular and subcellular distribution of N-methyl-D-aspartate receptor subunit 1 and AMPA receptor subunit 1 was investigated with antibodies generated against the C-terminal domain of the individual subunits [Petralia R. S. et al. (1994) J. Neurosci. 14, 667 696; Wenthold R. J. et al. (1992) J. biol Chem. 267, 501 507]. Both N-methyl-D-aspartate receptor subunit 1 and AMPA receptor subunit 1 were localized to the postsynaptic density of asymmetric synapses established by olfactory receptor neuron terminals with the dendrites of mitral and tufted cells. Not all of these synapses, however, were labelled. These results are consistent with the notion that glutamate is the neurotransmitter at the olfactory nerve to mitral and tufted cell synapses, and suggest a high heterogeneity in the expression of the postsynaptic glutamate receptors.

Developmental study of ultrastructural and biochemical changes in isolated chick brain microvessels.

The morphology of the endothelial junctions, the expression of the alkaline phosphatase (ALKP) and gamma-glutamyltranspeptidase (GGT) activities, and the transport systems for neutral amino acids (NAA) and for d-glucose were studied in parallel in isolated microvessels from the brains of 9-, 12-, and 21-day chick embryos and 30-day chickens using freeze-fracturing and biochemical techniques. In the 9-day embryos, the endothelium junctional plasma membranes show alignments of discrete intramembrane particles (Imps) on the replica P-faces. In the 12-day embryos, the junctional membranes show short fibrils of loosely interconnected fused Imps. In the 21-day embryos, the junction is characterized by simple networks, and in the 30-day chickens, by very dense meshworks of continuous, fibrillary strands. In early embryonic life, the ALKP activity is expressed at high levels, more markedly in the microvessels than in the whole brain. ALKP progressively declines in later embryos and even more in adulthood, when the activity is fivefold higher in the microvessels than in the whole brain. No GGT activity is expressed in either embryonic or adult microvessels. The transport systems for NAA and for d-glucose are most active in 12-day embryonic microvessels, and progressively less expressed by the 21st day and in the adult. The results taken as a whole suggest a close relationship between the morphological and the metabolic maturation of the endothelial barrier of cerebral vessels.

Reperfusion damage to the bile canaliculi in transplanted human liver.

In 19 patients who have undergone orthotopic liver transplantation (OLT), the trend and degree of cholestasis was statistically monitored in terms of plasma levels of L-gamma-glutamyl transferase (GGT) and total bilirubin. In addition, the ultrastructure of the bile canaliculus was examined during the entire OLT procedure, i.e., during explantation, cold ischemia, and after 60 to 90 minutes of organ reperfusion. Cholestasis was evident from the second day after surgery, with a peak after approximately 10 to 16 days. Defined, small changes in the functional state of actin filaments were noted in the bile canalicular area after prolonged ischemia. But the morphological status of the bile canaliculi changed dramatically after reperfusion. In fact, the mean area and perimeter of the canaliculi had increased significantly, and there was a marked loss in the number of bile microvilli per unit of canalicular area. The bile canaliculus appears to be one of the liver structures most susceptible to ischemia-reperfusion damage. A series of biochemical changes occurring during ischemia and after reoxygenation of the transplanted liver, especially, would provide a reason for the observed early morphological damage of the bile canaliculus, which, in turn, would explain the cholestasis of these patients in the first posttransplantation period.

Glutamate and carnosine in the vestibular system of the frog.

We demonstrate that both glutamate-like and carnosine-like immunoreactivities are present in hair cells and in fibers of the vestibular organ of the frog inner ear. Comparison of the two immunoreactivity patterns indicates that glutamate and carnosine might be colocalized in some hair cells. The presence of glutamate-like immunoreactivity in hair cells is consistent with biochemical and pharmacological data indicating glutamate as the excitatory neurotransmitter in these sensory receptors. There is also evidence that carnosine might have a neuromodulatory function.

Orthogonal arrays of particles (OAPs) in perivascular astrocytes and tight junctions in endothelial cells. A comparative study in developing and adult brain microvessels.

The plasmamembranes of the astrocyte processes, which envelop the capillaries of the adult brain, contributing to the blood-brain barrier constitution, are characterized by peculiar aggregates of intramembrane particles (IMPs) packed in orthogonal arrays (orthogonal arrays of particles, OAPs). With the aim of investigating the maturation sequence of the cerebral microvasculature, the IMPs distribution has been analysed in the plasmamembranes of both perivascular astrocytes and endothelial cells of fractured microvessels, in 16-, 20-, 21-day chick embryo and 10-day chicken optic tecta. The IMPs distribution undergoes remarkable changes from late embryonic to early postnatal life in the astrocytes and endothelial cells as well. In the astrocyte plasmamembranes, short chains of individual particles and linear units of packed ones precede the appearance of complete OAPs; in the endothelium junctional plasmamembranes, fibrils of fused particles precede the formation of fibrillary networks which express the tight junction setting up. The parallel formation of the astrocytic OAPs and the endothelial tight junctions further supports the suggestion that mutual relationships establish between perivascular glia and endothelium in the cerebral microvasculature differentiation during the blood-brain barrier development.

Presynaptic co-localization of carnosine and glutamate in olfactory neurones.

Olfaction plays a dominant role in modulating behaviour in most vertebrate species and the olfactory bulb is considered a model system for characterizing principles of neural computation. Nevertheless, although the physiology and neurochemistry of the olfactory circuits have been widely studied, the neurotransmitter released by olfactory receptor neurones remains unknown. We now describe the ultrastructural localization of the dipeptide carnosine and the excitatory amino acid glutamate in the glomerular layer of the mouse olfactory bulb. We demonstrate that both carnosine-like and glutamate-like immunoreactivities are selectively co-localized in the olfactory neurone boutons. These observations, taken with the recent findings of glutamate-receptor subunit expression in rodent olfactory bulb, argue compellingly for a role of glutamate in olfactory neurotransmission and suggest a modulatory effect of carnosine.

Expression of carnosine-like immunoreactivity during retinal development in the clawed frog (Xenopus laevis).

The development of neurons immunoreactive to carnosine (beta-alanyl-L-histidine) was studied in the retina of Xenopus laevis during the premetamorphic period. Carnosine-like immunoreactivity was detected in photoreceptors from stage 39/40 (according to Nieuwkoop and Faber [Normal Tables of Xenopus laevis (Daudin), Elsevier, Amsterdam, 1956]) and in bipolar cells and their processes in the inner plexiform layer from stage 44/45. At all the developmental stages studied, neuroepithelial cells at the ciliary margin were completely unstained, suggesting that carnosine is only present in postmitotic retinal neurons. This study demonstrates a correlation between the times of appearance of carnosine-like immunoreactivity during retinal development and the onset of visual function.

Carnosine-like immunoreactivity is associated with synaptic vesicles in photoreceptors of the frog retina.

The distribution of carnosine-like immunoreactivity in photoreceptors of the frog retina was studied by post-embedding electron microscope immunocytochemistry. Different fixation and embedding procedures were applied and the best results were achieved on sections from tissue embedded in the hydrophilic resin LR White. All photoreceptor types present in the frog retina (red and green rods, single and double cones) were intensely reactive for the carnosine antiserum. The immunoreactivity was particularly prominent in the synaptic terminal, nucleus and myoid, but decreased in the ellipsoid and in the outer segment. Müller glial cells and horizontal and bipolar cell processes in the outer plexiform layer were completely unstained. At the level of the photoreceptor terminals, the presence of gold particles within the synaptic vesicles could be demonstrated. This study is the first to provide the anatomical evidence for the presence of carnosine in the synaptic vesicles of a nervous cell. The present data may be relevant to the understanding of the biological functions of carnosine (and related dipeptides) and offer an exploitable system in which to verify its proposed role as a neurotransmitter or neuromodulator in sensory systems.

Comparative study of glial fibrillary acidic protein (GFAP)-like immunoreactivity in the retina of some representative vertebrates.

A polyclonal glial fibrillary acidic protein (GFAP) antiserum was used to study the distribution of GFAP-like immunoreactivity in the retina of adult vertebrates (teleosts, amphibians, reptiles, birds and mammals). GFAP-positive Müller cells were demonstrated in all the species studied, although with different degrees and patterns of immunoreactivity. In nonmammalian vertebrates, Müller cells were the only immunoreactive retinal elements. The staining was located throughout the retina of the species examined, with the exception of the rabbit, which exhibited regional variability in the expression of GFAP. The data indicate that GFAP expression in retinal Müller cells is a common feature of a wide variety of adult vertebrate species.

Tight endothelial junctions in the developing microvasculature: a thin section and freeze-fracture study in the chick embryo optic tectum.

The development of the interendothelial tight junctions was studied in the microvessels of the otpic tectum of chick embryos, at the 14th-16th and 18th-20th incubation day (i.d.), and in post-hatching chickens, using thin sections and freeze-fracture techniques. At the 14th-16th i.d., the junctional plasmamembranes of the endothelial cells are simply apposed or fused for brief tracts showing a pentalaminar or trilaminar configuration. In the replicas the P-faces of the fractured junctional membranes are either lacking in intramembrane particles (IMPs) and characterized by finger-like depressions, or provided with discrete IMPs aligned in rows. At the 18th-20th i.d., the thin peripheral expansions of the endothelial cells are superimposed and welded by continuous pentalaminar junctions. Their fracture P-faces display junctional strands formed by parallel fibrils of fused IMPs, with or without interconnections. In the 10-day-old chickens the junctions consist of highly complex networks of fibrils. The results have made it possible to recognize precise relationships between the features of the developing endothelial junctions in the ultrathin sections and, respectively, in the replicas. Moreover, the observations suggest that tight junction formation occurs progressively in the cerebral microvessels by processes of alignment and fusion of the IMPs, which conclude with the arrangement of fibrils in networks.

[Presence of calretinin in neurons of the human intestine]. / Presenza di calretinina nei neuroni dell'intestino umano.

Calcium-binding proteins are present in different neuron populations in the Central Nervous System. As concerns the Enteric Nervous System, only a few studies have been performed. In the present work we investigated immunohistochemically the localization of Calretinin in neurons of the human intestinal wall. Our results showed the presence of stained cell bodies and fibers with antibodies against Calretinin in the Auerbach plexus. Since most of the enteric neurons are characterized by a slow phase after hyperpolarization caused by Ca2+ dependent K+ channels, the role of Calretinin could be to modulate this particular electrophysiological behaviour.

A freeze-fracture study of human bronchial epithelium in normal, bronchitic and asthmatic subjects.

Tight junctions (TJ) play a major role in maintaining the integrity of epithelia. Damage of conducting airway surface epithelium is commonly observed in asthma, and recent data suggest that epithelial cells modulate airway smooth muscle tone by the production of relaxant factor(s). To evaluate the ultrastructure of tight junctions (TJ) in human bronchial epithelium of normal and diseased lung, biopsy samples were obtained by fiberoptic bronchoscopy in three normal healthy subjects, four asthmatic patients with bronchial hyperreactivity to methacholine and one heavy smoker with chronic bronchitis. Specimens were examined by electron microscopy, using both thin sections and freeze-fracture techniques. In normal subjects two types of TJ were identified, based on 27 type I, 23 type II junctional areas. Epithelium from the patient with chronic bronchitis showed extensive mucous metaplasia and only junctions of the second type. Extensive epithelial damage was detected in asthmatic subjects, so that a few TJ could be found. Varying degrees of TJ abnormalities were observed and the possible role of such ultrastructural derangements was discussed.

Morphology of Purkinje cell axon terminals in intracerebellar nuclei following inferior olive lesion.

We have examined the ultrastructural changes of axons and synaptic boutons in the intracerebellar nuclei of the rat at 3 days to one year after inferior olive lesion performed by means of electrocoagulation or 3-acetylpyridine injection. A large number of preterminal segments and axons terminals undergoes remarkable ultrastructural changes after total or subtotal olivary lesion. Large membrane bound vacuoles and clusters of small synaptic vesicles characterize a good number of these terminals at 3 days up to one month after the lesion. Tightly packed tubules and cisternae of smooth endoplasmic reticulum appear during the first week in an increasing number of axon terminals. Boutons with large whorled bodies formed by smooth membranes increase in number during the second half of the first month and further increase in density until the sixth month. They are still present in large amounts at one year. Immunoreactivity for 3',5'-guanosine-phosphate-dependent protein kinase, which is specific for Purkinje neurons, can be detected in the axons and synaptic terminals displaying the ultrastructural changes described above. These results are discussed in relation to a possible trophic action of the climbing fibers on the Purkinje cells. We suggest that, at least in part, these alterations may be the consequence of the intense Purkinje cell hyperactivity which is present for up to one month from inferior olive lesion.

Subsurface cisternae in retinal double cones.

Double cones of tench and goldfish retina are characterized by extensive subsurface cisternae underlying the plasma membranes at the appositional area between the principal and accessory cone. Such a membrane system is absent in double cones of turtle and salamander retina. Measurements on both transverse and longitudinal sections gave a total appositional area of about 75 square microns, the subsurface cisterna in each element of the double cone being around 8-10% smaller due to multiple fenestrations at the level of the paraboloid. No gap junctions joining the inner segments of tench and goldfish double cones were detected, while gap junctions could be observed at the level of the ellipsoid and paraboloid of turtle double cones. The possible role of the subsurface cisternae in functional interactions between double cone elements is discussed.

Presence of intra-mucosal smooth muscle cells in normal human and rat colon.

Intramucosal smooth muscle cells surrounding the crypts and originating from the muscularis mucosae were observed in normal human and rat colon. Immunohistochemical techniques, using anti-desmin and anti-actin antibodies, along with ultrastructural procedures were employed to investigate the nature and distribution of these cells. Desmin-positive and actin-rich smooth muscle cells sprouting from the muscularis mucosae into the lamina propria and surrounding the crypts were observed both in rat and human colon. The intramucosal smooth muscle cells may play an important role in some pathophysiological conditions.