S. De Biasi

GABAergic Neurons in Mammalian Thalamus: A Marker of Thalamic Complexity?

The present study evaluated the occurrence, distribution, and number of GABAergic neurons in the thalamus of different mammalian species (bat, mouse, rat, guinea pig, rabbit, cat, monkey, humans), by means of light microscopical immunoenzymatic localization of GABA or of its biosynthetic enzyme glutamic acid decarboxylase and by ultrastructural immunogold detection of GABA. Our data demonstrated that: 1) GABAergic local circuit neurons were detected in the thalamic visual domain in all the species analyzed, whereas in other thalamic nuclei their presence and number varied among species; 2) the number of GABAergic local circuit neurons progressively increased in the dorsal thalamus of species with more complex behavior; 3) the presence of local circuit neurons conferred a similar intrinsic organization to the dorsal thalamic nuclei, characterized by complex synaptic arrangements; 4) in the reticular thalamic nucleus, whose neurons were GABA-immunoreactive in all the examined species, the cellular density decreased from the bat to humans. These findings strongly suggest that thalamic GABAergic local circuit neurons are not directly related to the ability to perform specific sensorimotor tasks, but they are likely to reflect an increasing complexity of the local information processing that occurs at thalamic level.

Immunoreactivity for the GABA transporter-1 and GABA transporter-3 is restricted to astrocytes in the rat thalamus. A light and electron-microscopic immunolocalization

GABA plasma membrane transporters mediate GABA uptake into presynaptic terminals and surrounding glial processes and thus play a key role in shaping the time course and spatial extent of GABAs action. In the present study we have investigated the cellular and subcellular localization of two GABA transporters (1 and 3) in the rat thalamus using affinity-purified polyclonal antibodies. GABA transporter-1 and -3 immunoreactivity, detected with immunoperoxidase and immunofluorescence methods, is present throughout the thalamus in small punctate structures scattered in the neuropil among unlabelled neuronal perikarya. Labelling for GABA transporter-3 is always more intense than that for GABA transporter-1. Astrocytic processes, identified by their immunoreactivity for glial fibrillary acidic protein, express both GABA transporters. Ultrastructural investigations confirm that GABA transporter-1 and -3 labelling is restricted to astrocytes. Labelled astrocytes are adjacent to terminals making either symmetric or asymmetric synaptic contacts, and are close to neuronal profiles that do not form synaptic contacts in the plane of the section. In double-labelled thin sections some GABA transporter-1- or -3-positive astrocytic processes, detected with immunoperoxidase labelling, surround GABA-positive terminals, detected with antibodies to GABA and immunogold labelling. These findings demonstrate that in rat thalamus the GABA uptake system mediated by GABA transporter-1 and -3 is localized exclusively to astrocytes near the synapses and in the neuropil, and absent from GABAergic terminals. Astrocytes play therefore an important role in mediating GABA transmission in the thalamus, compared to cortical regions.

Aspartate-like immunoreactivity in primary afferent neurons

There is now good evidence that amino acids act as neurotransmitters in primary afferent neurons of dorsal root ganglia. Glutamate is the primary candidate for such a role, and there are reasons to believe that release of glutamate may be accompanied by the release of other neuroactive substances. Using immunocytochemical techniques, we have tested the hypothesis that some dorsal root ganglion neurons contain elevated levels of aspartate as well as glutamate. Antisera raised against conjugates of aspartate or glutamate were used for this purpose. Blocking experiments confirmed that these antibodies were specific to their antigens in cryostat sections of dorsal root ganglia. Aspartate immunoreactivity was found in approximately 30% of neurons in cervical dorsal root ganglia. The relation between cell size and staining intensity for aspartate was examined using quantitative video microscopy; the great majority of cells immunopositive for aspartate were small (15-30 microns in diameter); about 85% of these cells stained for aspartate, although staining intensities varied over a wide range. By reacting consecutive sections with anti-aspartate and anti-glutamate it was shown that elevated levels of aspartate were found in the same cells which contained elevated levels of glutamate. By measuring the staining intensity of individual cells for both aspartate and glutamate, it was also shown that there was a positive correlation between staining intensity and, presumably, concentration of the two amino acids. The presence of high levels of aspartate in terminals located in the superficial laminae of the dorsal horn was verified by pre- and post-embedding immunocytochemistry with the electron microscope. Aspartate was demonstrated in scalloped terminals, including dark scalloped terminals believed to be associated with unmyelinated fibers of nociceptors. This evidence supports the hypothesis that aspartate as well as glutamate is present in the cell bodies and terminals of nociceptive primary afferents, and may be released by the terminals of these afferents to activate neurons in the superficial laminae of the dorsal horn.

Erythropoietin-mediated preservation of the white matter in rat spinal cord injury.

We investigated the effect of a single administration of recombinant human erythropoietin (rhEPO) on the preservation of the ventral white matter of rats at 4 weeks after contusive spinal cord injury (SCI), a time at which functional recovery is significantly improved in comparison to the controls [Gorio A, Necati Gokmen N, Erbayraktar S, Yilmaz O, Madaschi L, Cichetti C, Di Giulio AM, Enver Vardar E, Cerami A, Brines M (2002) Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma. Proc Natl Acad Sci U S A 99:9450-9455; Gorio A, Madaschi L, Di Stefano B, Carelli S, Di Giulio AM, De Biasi S, Coleman T, Cerami A, Brines M (2005) Methylprednisolone neutralizes the beneficial effects of erythropoietin in experimental spinal cord injury. Proc Natl Acad Sci U S A 102:16379-16384]. Specifically, we examined, by morphological and cytochemical methods combined with light, confocal and electron microscopy, i) myelin preservation, ii) activation of adult oligodendrocyte progenitors (OPCs) identified for the expression of NG2 transmembrane proteoglycan, iii) changes in the amount of the chondroitin sulfate proteoglycans neurocan, versican and phosphacan and of their glycosaminoglycan component labeled with Wisteria floribunda lectin, and iv) ventral horn density of the serotonergic plexus as a marker of descending motor control axons. Injured rats received either saline or a single dose of rhEPO within 30 min after SCI. The results showed that the significant improvement of functional outcome observed in rhEPO-treated rats was associated with a better preservation of myelin in the ventral white matter. Moreover, the significant increase of both the number of NG2-positive OPCs and the labeling for Nogo-A, a marker of differentiated oligodendrocytes, suggested that rhEPO treatment could result in the generation of new myelinating oligodendrocytes. Sparing of fiber tracts in the ventral white matter was confirmed by the increased density of the serotonergic plexus around motor neurons. As for chondroitin sulfate proteoglycans, only phosphacan, increased in saline-treated rats, returned to normal levels in rhEPO group, probably reflecting a better maintenance of glial-axolemmal relationships along nerve fibers. In conclusion, this investigation expands previous studies supporting the pleiotropic neuroprotective effect of rhEPO on secondary degenerative response and its therapeutic potential for the treatment of SCI and confirms that the preservation of the ventral white matter, which contains descending motor pathways, may be critical for limiting functional deficit.

Chronic erythropoietin-mediated effects on the expression of astrocyte markers in a rat model of contusive spinal cord injury.

Using a standardized rat model of contusive spinal cord injury (SCI; [Gorio A, Gokmen N, Erbayraktar S, Yilmaz O, Madaschi L, Cichetti C, Di Giulio AM, Vardar E, Cerami A, Brines M (2002) Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma. Proc Natl Acad Sci U S A 99:9450-9455]), we previously showed that the administration of recombinant human erythropoietin (rhEPO) improves both tissue sparing and locomotory outcome. In the present study, to better understand rhEPO-mediated effects on chronic astrocyte response to SCI in rat, we have used immunocytochemical methods combined with confocal and electron microscopy to investigate, 1 month after injury, the effects of a single rhEPO administration on the expression of a) aquaporin 4 (AQP4), the main astrocytic water channel implicated in edema development and resolution, and two molecules (dystrophin and syntrophin) involved in its membrane anchoring; b) glial fibrillary acidic protein (GFAP) and vimentin as markers of astrogliosis; c) chondroitin sulfate proteoglycans of the extracellular matrix which are upregulated after SCI and can inhibit axonal regeneration and influence neuronal and glial properties. Our results show that rhEPO administration after SCI modifies astrocytic response to injury by increasing AQP4 immunoreactivity in the spinal cord, but not in the brain, without apparent modifications of dystrophin and syntrophin distribution. Attenuation of astrogliosis, demonstrated by the semiquantitative analysis of GFAP labeling, was associated with a reduction of phosphacan/RPTP zeta/beta, whereas the levels of lecticans remained unchanged. Finally, the relative volume of a microvessel fraction was significantly increased, indicating a pro-angiogenetic or a vasodilatory effect of rhEPO. These changes were consistently associated with remarkable reduction of lesion size and with improvement in tissue preservation and locomotor recovery, confirming previous observations and underscoring the potentiality of rhEPO for the therapeutic management of SCI.

Expression of GABA transporters, GAT-1 and GAT-3, in the cerebral cortex and thalamus of the rat during postnatal development

The cellular and subcellular localization of two GABA transporters, GAT-1 and GAT-3, was investigated using immunocytochemical methods in the rat cerebral cortex and thalamus during postnatal development. The distribution of the transporters is compared with that of the neuronal marker GABA, and with that of vimentin and of glial fibrillary acidic protein, which identify immature and mature astrocytes, respectively. Our observations show that the two transporters are already expressed at birth in both brain areas with the same cellular localization as in adult rats, as GAT-1 is present in growth cones and terminals only in the cortex, whereas both transporters are expressed in astrocytes in the cortex and thalamus. The distribution of GAT-1 and GAT-3 undergoes postnatal changes reflecting in general the neurogenetic events of the neocortex and thalamus and, more specifically, the maturation of GABAergic innervation. The adult-like pattern of expression is achieved in the third postnatal week in the cortex and in the second postnatal week in the thalamus. The early expression of GAT-1 in GABAergic terminals confirms previous studies showing the existence of neuronal mechanisms of GABA uptake from the embryonic stages. As for the glial localization, the precocious existence of two astrocytic GABA transporters suggests that they operate through different functional mechanisms from birth, whereas their exclusively glial expression in the thalamus indicates that the astroglia plays a major role in the transport, recycling and metabolism of thalamic GABA.

GABAergic interneurons in the somatosensory thalamus of the guinea-pig: A light and ultrastructural immunocytochemical investigation

This work was performed to confirm previous data reporting the presence of GABAergic interneurons in the ventrobasal complex of guinea-pig, and to investigate the intrinsic organization of this nucleus compared to that of thalamic nuclei lacking interneurons. Immunocytochemical experiments were performed on the thalamus of adult guinea-pigs perfused with mixed aldehydes using an anti-GABA serum. At light microscopy, the immunoreaction on floating Vibratome sections showed that GABAergic neurons are present only in the reticular and lateral geniculate nuclei and in the ventrobasal complex. Quantitative evaluation of their number indicated that they are 20 and 15% of the total neuronal population in lateral geniculate nucleus and ventrobasal complex, respectively, while they are less than 1% in ventrolateral nucleus. At the ultrastructural level, the postembedding immunogold procedure showed the presence, in the ventrobasal complex, of GABA-labeled profiles involved in complex synaptic arrangements similar to those found in carnivores and primates. Conversely, GABA-labeled terminals in thalamic nuclei devoid of interneurons formed exclusively axo-dendritic or axo-somatic contacts, like in rats and mice. The present data suggest that GABAergic neurons in the ventrobasal complex of guinea-pigs give rise to functionally important rearrangements of its intrinsic synaptic organization and that they represent the morphological basis for an intrinsic modulatory mechanism that is absent in other thalamic nuclei lacking inhibitory interneurons. The phylogenetic implications of these findings are also discussed in comparison to other animal species.

Distinct expression pattern of microtubule-associated protein/microtubule affinity-regulating kinase 4 in differentiated neurons

Protein kinases of the microtubule affinity-regulating kinase (MARK) family were originally discovered because of their ability to phosphorylate tau protein and related microtubule-associated proteins (MAPs), and their role in the establishment of cell polarity in different contexts. Recent papers have indicated that microtubule affinity-regulating kinase 4 (MARK4) is a gene that is finely regulated at transcriptional level and expressed in two spliced isoforms called MARK4L and MARK4S. We here describe the characterization of the mouse orthologue of the human MARK4 gene. Interestingly, MARK4S is predominantly expressed in the brain, whereas MARK4L shows lower transcript levels in this organ. Using MARK4 antibodies specific for each isoform, we found that both isoforms have an identical expression pattern in the mouse CNS, and are present in a number of neuronal populations. We also found that human microtubule affinity-regulating kinase 4S (hMARK4S), whose expression is not detectable in human neural progenitor cells (HNPCs) and NTera2 (NT2) cells, is up-regulated in both cell systems from the very early stages of neuronal differentiation. This indicates that neuronal commitment is marked by MARK4S up-regulation. In conclusion, this study provides the first direct evidence suggesting that MARK4 is a neuron-specific marker in the CNS, and the up-regulation of MARK4S during neuronal differentiation suggests that it plays a specialized role in neurons.

Postnatal development of gaba-immunoreactive terminals in the reticular and ventrobasal nuclei of the rat thalamus : A light and electron microscopic study

The postnatal development of inhibitory GABAergic circuits in the thalamic reticular and ventrobasal nuclei was studied in rats ranging from the day of birth to the end of the third postnatal week by means of a postembedding immunogold staining procedure to visualize GABA. In the reticular nucleus, GABA labeling was present from birth in cell bodies, dendrites, growth cones and a few synaptic terminals, whereas in the ventrobasal nucleus it was exclusively in axonal processes identifiable as growth cones, vesicle-rich profiles and synaptic terminals. In both nuclei, GABA-labeled synaptic terminals were, however, very scarce and immature in neonatal animals and they became numerous and morphologically mature only after the end of the second postnatal week. These findings suggest that inhibitory synaptic responses in the somatosensory thalamus are not yet fully mature throughout the first two postnatal weeks and support the hypothesis that GABA may initially play trophic roles. The relatively late maturation of the thalamic GABAergic system may have important functional consequences, as the reticulothalamic circuits are responsible for the generation of spindle wave oscillations whose cellular mechanisms are also involved in the generation of spike-and-wave (absence) seizures in humans and in animal models.

Ultrastructural features of the isolated guinea-pig brain maintained in vitro by arterial perfusion

The morphological features of cerebral tissue in the isolated guinea-pig brain maintained in vitro by arterial perfusion are described. Light and electron microscopic analysis of the thalamus, the somatosensory cortex and the limbic cortices (hippocampus, piriform and entorhinal cortices) was performed after different periods of incubation in vitro (1, 7 and 12 h), in parallel with an electrophysiological study. The morphological analysis showed that neuronal elements retained their normal appearance at both cellular and subcellular level in the examined brain regions up to an incubation period of 12 h. Immunoreactivity for GABA was also preserved for up to 12 h of in vitro perfusion. Vasogenic edema and perivascular extracellular swelling appeared after 7 h, together with signs of progressive astrocytic deterioration. These findings show that normal electrophysiological recordings correlate with good anatomical preservation of the isolated guinea-pig brain preparation after prolonged times of arterial in vitro perfusion.