Fiorenzo Conti

Biological activity of chitosan: ultrastructural study

Reparative processes are reconstructive phenomena in which cellular elements and extracellular matrix glycoproteins interact to build the injured tissue. Biomaterials can be used to improve or stimulate reconstruction. In the present experimental investigation, tissue repair induced by chitosan, an 86.8% deacetylated poly(GlcNAc), was monitored by morphological analysis. To evaluate its biological role, chitosan was positioned in contact with dura mater or was used as a dura mater substitute. This polysaccharide, having structural characteristics similar to glycosaminoglycans, seems to mimic their functional behaviour. The inductive and stimulatory activity of chitosan on connective tissue-rebuilding is clearly demonstrated, and it is suggested that chitosan could be considered a primer on which a normal tissue architecture is organized.

Generating diversity at GAB Aergic synapses

Abstract GABA-mediated transmission is characterized by high variability of synaptic responses. Major contributors to this variability are: presynaptic factors, including release probability and number of release sites; factors that determine synaptic GABA transients in the cleft, including diffusion and the actions of GABA transporters; and postsynaptic factors, including GABA A receptors subtypes, their location and number, their modulation by endogenous and exogenous factors, and their interactions with postsynaptic-anchoring proteins.

Characterization of antisera to glutamate and aspartate.

Antisera were raised in rabbits against glutamate (Glu) and aspartate (Asp) conjugated to the invertebrate carrier protein hemocyanin (HC) with glutaraldehyde (GA). The antisera were characterized by testing their immunocytochemical staining properties on sections cut at the level of the ventral cochlear nucleus (VCN) from fixed brains of normal rats after absorption with conjugates of compounds structurally similar and biologically relevant to Glu and Asp. Optimal staining with Glu antiserum was obtained at a dilution of 1:10,000 and was completely blocked by 303 micrograms/ml of the Glu-HC conjugate. No crossreactivity with any of 11 compounds tested was observed. Optimal staining with the Asp antiserum was obtained at 1:8000 dilution and was completely blocked by 225 micrograms/ml of the Asp-HC conjugate. Of 10 compounds tested for crossreactivity, only L-asparagine demonstrated a measurable (about 10%) crossreactivity with the Asp antiserum. The specificity of the two antisera was also tested by immunoblot analysis against 11 compounds conjugated to HC with GA. Listed in order of staining intensity, from greatest to least, conjugates that reacted with the Glu antiserum were Glu greater than Gly-Glu greater than Asp-Glu = Asp greater than N-carbamyl (NC)-Glu greater than Asn = Gln = GABA. Conjugates that reacted with the Asp antiserum, in order of decreasing staining intensity, were Asp greater than Glu-Asp = Asn greater than Gly-Asp greater than Glu. No other compounds tested for crossreactivity reacted with the two antisera in the immunoblot analysis. Glu-like immunoreactivity in rat dorsal root ganglia and somatosensory cortex, and the comparative distribution of Glu- and Asp-like immunoreactivities in the latter tissue, are presented as examples of staining patterns obtained with the two antisera.

Expression of NR1 and NR2A/B subunits of the NMDA receptor in cortical astrocytes.

Ionotropic glutamate (Glu) receptors of the N‐methyl‐D‐aspartate type (NMDA) play a fundamental role in many cortical functions. Native NMDA receptors are composed of a heteromeric assembly of different subunits belonging to two classes: NMDAR1 (NR1) and NMDAR2 (NR2). To date, NMDA receptors are believed to be expressed only in neurons, although electrophysiological and in situ hybridization studies have suggested that this class of Glu receptors might be also expressed by some astrocytes. In this study, we have investigated in the cerebral cortex of adult rats the presence of astrocytes expressing NR1 and NR2A/B subunits by immunocytochemistry with specific antibodies, and we show that some distal astrocytic processes, but only rarely astrocytic cell bodies, contain immunoreaction product indicative of NR1 and NR2A/B expression. These findings suggest that at least part of the role NMDA has in cortical functions might depend on the activation of astrocytic NMDA receptors; the subcellular localization of NR1 and NR2A/B subunits in distal processes suggests that NMDA receptors contribute to monitoring Glu levels in the extracellular space.

Neuronal and glial localization of GAT-1, a high-affinity gamma-aminobutyric acid plasma membrane transporter, in human cerebral cortex: with a note on its distribution in monkey cortex

High‐affinity γ‐aminobutyric (GABA) plasma membrane transporters (GATs) influence the action of GABA, the main inhibitory neurotransmitter in the human cerebral cortex. In this study, the cellular expression of GAT‐1, the main cortical GABA transporter, was investigated in the human cerebral cortex by using immunocytochemistry with affinity‐purified polyclonal antibodies directed to the C‐terminus of rat GAT‐1.

Increased expression of the astrocytic glutamate transporter GLT-1 in the prefrontal cortex of schizophrenics.

To verify whether altered glial glutamate uptake contributes to the reduced efficacy of glutamatergic transmission reported in the prefrontal cortex of schizophrenics, we studied the expression of GLT‐1, the transporter responsible for most glutamate transport, in autoptic samples of prefrontal cortex using real time quantitative RT‐PCR, immunocytochemistry, and functional assays. GLT‐1 mRNA levels in medication‐free patients were 2.5‐fold higher than in controls, whereas they were normal or reduced in patients treated with antipsychotics. We also observed a 4‐fold increase in L‐[3H]‐Glu uptake in Xenopus oocytes injected with mRNA from the prefrontal cortex of a medication‐free schizophrenic and a 2‐fold increase in GLT‐1 protein in the same cortical area of another medication‐free patient. Results suggest that GLT‐1 mRNA, protein and function are increased in prefrontal cortex of schizophrenics.

The expression of glutamate transporter GLT-1 in the rat cerebral cortex is down-regulated by the antipsychotic drug clozapine

We show here that clozapine, a beneficial antipsychotic, down-regulates the expression of the glutamate transporter GLT-1 in the rat cerebral cortex, thereby reducing glutamate transport and raising extracellular glutamate levels. Clozapine treatment (25–35 mg kg−1 day−1 orally) reduced GLT-1 immunoreactivity in several brain regions after 3 weeks; this effect was most prominent after 9 weeks and most evident in the frontal cortex. GLT-1 protein levels were reduced in the cerebral cortex of treated rats compared with controls and were more severely affected in the anterior (71.9 ± 4.5%) than in the posterior (53.2 ± 15.4%) cortex. L-[3H]-glutamate uptake in Xenopus laevis oocytes injected with mRNA extracted from the anterior cerebral cortex of rats treated for 9 weeks was remarkably reduced (to 30.6 ± 8.6%) as compared to controls. In addition, electrophysiological recordings from oocytes following application of glutamate revealed a strong reduction in glutamate uptake currents (46.3 ± 10.2%) as compared to controls. Finally, clozapine treatment led to increases in both the mean basal (8.1 ± 0.7 μM) and the KCl-evoked (28.7 ± 7.7 μM) output of glutamate that were 3.1 and 3.5, respectively, higher than in control rats. These findings indicate that clozapine may potentiate glutamatergic synaptic transmission by regulating glutamate transport.

Neuronal, glial, and epithelial localization of γ-aminobutyric acid transporter 2, a high-affinity γ-aminobutyric acid plasma membrane transporter, in the cerebral cortex and neighboring structures

Neuronal and glial high‐affinity Na+/Cl −‐dependent plasma membrane γ‐aminobutyric acid (GABA) transporters (GATs) contribute to regulating neuronal function. We investigated in the cerebral cortex and neighboring regions of adult rats the distribution and cellular localization of the GABA transporter GAT‐2 by immunocytochemistry with affinity‐purified polyclonal antibodies that react monospecifically with a protein of 82 kDa. Conventional and confocal laser‐scanning light microscopic studies revealed intense GAT‐2 immunoreactivity (ir) in the leptomeninges, choroid plexus, and ependyma. Weak GAT‐2 immunoreactivity also was observed in the cortical parenchyma, where it was localized to puncta of different sizes scattered throughout the radial extension of the neocortex and to few cell bodies. In sections double‐labeled with GAT‐2 and glial fibrillary acidic protein (GFAP) antibodies, some GAT‐2‐positive profiles also were GFAP positive. Ultrastructural studies showed GAT‐2 immunoreactivity mostly in patches of varying sizes scattered in the cytoplasm of neuronal and nonneuronal elements: GAT‐2‐positive neuronal elements included perikarya, dendrites, and axon terminals forming both symmetric and asymmetric synapses; nonneuronal elements expressing GAT‐2 were cells forming the pia and arachnoid mater; astrocytic processes, including glia limitans and perivascular end feet; ependymal cells; and epithelial cells of the choroid plexuses. The widespread cellular expression of GAT‐2 suggests that it may have several functional roles in the overall regulation of GABA levels in the brain. J. Comp. Neurol. 409:482–494, 1999.

GABAergic signaling at mossy fiber synapses in neonatal rat hippocampus

In the adult rat hippocampus, granule cell mossy fibers (MFs) form excitatory glutamatergic synapses with CA3 principal cells and local inhibitory interneurons. However, evidence has been provided that, in young animals and after seizures, the same fibers can release in addition to glutamate GABA. Here we show that, during the first postnatal week, stimulation of granule cells in the dentate gyrus gave rise to monosynaptic GABAA-mediated responses in principal cells and in interneurons. These synapses were indeed made by MFs because they exhibited strong paired-pulse facilitation, high sensitivity to the metabotropic glutamate receptor agonist l-AP-4, and short-term frequency-dependent facilitation. MF responses were potentiated by blocking the plasma membrane GABA transporter GAT-1 with NO-711 or by allosterically modulating GABAA receptors with flurazepam. Chemical stimulation of granule cell dendrites with glutamate induced barrages of GABAA-mediated postsynaptic currents into target neurons. Furthermore, immunocytochemical experiments demonstrated colocalization of vesicular GABA transporter with vesicular glutamate transporter-1 and zinc transporter 3, suggesting that GABA can be taken up and stored in synaptic vesicles of MF terminals. Additional fibers releasing both glutamate and GABA into principal cells and interneurons were recruited by increasing the strength of stimulation. Both the GABAergic and the glutamatergic component of synaptic currents occurred with the same latency and were reversibly abolished by l-AP-4, indicating that they originated from the MFs. GABAergic signaling may play a crucial role in tuning hippocampal network during postnatal development. Low-threshold GABA-releasing fibers may undergo elimination, and this may occur when GABA shifts from the depolarizing to the hyperpolarizing direction.

Presynaptic NMDA receptors in the neocortex are both auto- and heteroreceptors.

We used electron microscopic immunocytochemistry with antibodies against NR1 and NR2A and B subunits to study the distribution of N-methyl-D-aspartate (NMDA) receptors in presynaptic axon terminals in the rat cerebral cortex. In all sections examined, NR1 and NR2A/B immunoreactivities were observed in axon terminals: NR1- and NR2A/B-positive axon terminals made both symmetrical and asymmetrical synapses on unlabelled dendritic profiles. Combined pre- and postembedding studies showed that all NR1 and NR2A/B-positive axon terminals making symmetrical synapses were gamma-aminobutyric acid (GABA)-positive. These observations show that both auto- and hetero- NMDA receptors do exist in the cerebral cortex, and indicate that part of the effects of NMDA receptor activation might be determined by modulating glutamate and GABA release.