GABAergic hub neurons orchestrate synchrony in developing hippocampal networks.

Brain function operates through the coordinated activation of neuronal assemblies. Graph theory predicts that scale-free topologies, which include "hubs" (superconnected nodes), are an effective design to orchestrate synchronization. Whether hubs are present in neuronal assemblies and coordinate network activity remains unknown. Using network dynamics imaging, online reconstruction of functional connectivity, and targeted whole-cell recordings in rats and mice, we found that developing hippocampal networks follow a scale-free topology, and we demonstrated the existence of functional hubs. Perturbation of a single hub influenced the entire network dynamics. Morphophysiological analysis revealed that hub cells are a subpopulation of gamma-aminobutyric acid-releasing (GABAergic) interneurons possessing widespread axonal arborizations. These findings establish a central role for GABAergic interneurons in shaping developing networks and help provide a conceptual framework for studying neuronal synchrony.

Differential properties of dentate gyrus and CA1 neural precursors.

In the present article we investigated the properties of CA1 and dentate gyrus cell precursors in adult rodents both in vivo and in vitro. Cell proliferation in situ was investigated by rating the number of cells incorporating BrdU after kainate-induced seizures. CA1 precursors displayed a greater proliferation capacity than dentate gyrus precursors. The majority of BrdU-labeled cells in CA1 expressed Nestin and Mash-1, two markers of neural precursors. BrdU-positive cells in the dentate gyrus expressed Nestin, but only a few expressed Mash-1. In animals pretreated with the antimitotic azacytidine, the capacity of kainate to enhance the proliferation was higher in CA1 than in the dentate gyrus. Differences in intrinsic progenitor cell activity could underlie these different expansion capacities. Thus, we compared the renewal- expansion and multipotency of dentate gyrus and CA1 precursors isolated in vitro. We found that the dissected CA1 region, including the periventricular zone, is enriched in neurosphere-forming cells (presumed stem cells), which respond to either EGF or FGF-2. Dentate gyrus contains fewer neurosphere-forming cells and none that respond to FGF-2 alone. Neurospheres generated from CA1 were multipotent and produced neurons, astrocytes, and oligodendrocytes, while dentate gyrus neurospheres mostly produced glial cells. The analysis of the effects of EGF on organotypic cultures of hippocampal slices depicted similar features: BrdU and Nestin immunoreactivities increased after EGF treatment in CA1 but not in the dentate gyrus. These results suggest that CA1 precursors are more stem-cell-like than granule cell precursors, which may represent a more restricted precursor cell.

The establishment of GABAergic and glutamatergic synapses on CA1 pyramidal neurons is sequential and correlates with the development of the apical dendrite.

We have performed a morphofunctional analysis of CA1 pyramidal neurons at birth to examine the sequence of formation of GABAergic and glutamatergic postsynaptic currents (PSCs) and to determine their relation to the dendritic arborization of pyramidal neurons. We report that at birth pyramidal neurons are heterogeneous. Three stages of development can be identified: (1) the majority of the neurons (80%) have small somata, an anlage of apical dendrite, and neither spontaneous nor evoked PSCs; (2) 10% of the neurons have a small apical dendrite restricted to the stratum radiatum and PSCs mediated only by GABA(A) receptors; and (3) 10% of the neurons have an apical dendrite that reaches the stratum lacunosum moleculare and PSCs mediated both by GABA(A) and glutamate receptors. These three groups of pyramidal neurons can be differentiated by their capacitance (C(m) = 17.9 +/- 0.8; 30.2 +/- 1.6; 43.2 +/- 3.0 pF, respectively). At birth, the synaptic markers synapsin-1 and synaptophysin labeling are present in dendritic layers but not in the stratum pyramidale, suggesting that GABAergic peridendritic synapses are established before perisomatic ones. The present observations demonstrate that GABAergic and glutamatergic synapses are established sequentially with GABAergic synapses being established first most likely on the apical dendrites of the principal neurons. We propose that different sets of conditions are required for the establishment of functional GABA and glutamate synapses, the latter necessitating more developed neurons that have apical dendrites that reach the lacunosum moleculare region.

Abnormal connections in the malformed cortex of rats with prenatal treatment with methylazoxymethanol may support hyperexcitability.

Prenatal treatment with methylazoxymethanol (MAM) in rats generates animals with a diffuse cortical malformation associated with hyperexcitability. These alterations are reminiscent of the cortical malformations associated with epilepsy in children. We hypothesised that one of the mechanisms supporting hyperexcitability in MAM rats could be the presence of abnormal cortical connections in the malformed cortex. Using a variety of anatomical techniques, we provide evidences for three types of such abnormal connections: (i) tangential bundles of corticocortical fibres in and below the neocortical molecular layer; (ii) partial deafferentation of neocortical heterotopias by afferent cortical fibres whatever their location; (iii) exuberant innervation of hippocampal CA3 pyramidal cells by mossy fibres that form ectopic mossy boutons on their basal dendrites. We conclude that these abnormal intrinsic cortical connections may support the propagation of paroxymal activity in the neocortex of MAM-treated rats.

Neocortex in the hippocampus: an anatomical and functional study of CA1 heterotopias after prenatal treatment with methylazoxymethanol in rats.

Migration disorders cause neurons to differentiate in an abnormal heterotopic position. Although significant insights have been gained into the etiology of these disorders, very little is known about the anatomy of heterotopias. We have studied heterotopic masses arising in the hippocampal CA1 region after prenatal treatment with methylazoxymethanol (MAM) in rats. Heterotopic cells were phenotypically similar to neocortical supragranular neurons and exhibited the same temporal profile of migration and neurogenesis. However, they did not express molecules characteristic of CA1 neurons such as the limbic-associated membrane protein. Horseradish peroxidase injections in heterotopia demonstrated labeled fibers not only in the neocortex and white matter but also in the CA1 stratum radiatum and stratum lacunosum. To study the pathophysiological consequences of this connectivity, we compared the effects of neocortical and limbic seizures on the expression of Fos protein and on cell death in MAM animals. After metrazol-induced seizures, Fos-positive cells were present in CA1 heterotopias, the only hippocampal region to be activated with the neocortex. By contrast, kainic acid-induced seizures caused a prominent delayed cell death in limbic regions and in CA1 heterotopias. Together, these results suggest that neocortical heterotopias in the CA1 region are integrated in both the hippocampal and neocortical circuitry.

Transient increase of tenascin-C in immature hippocampus: astroglial and neuronal expression.

In the present report we describe the anatomical localization of cells expressing tenascin-C, an extracellular matrix glycoprotein, in the hippocampal complex of developing rats. We report a development-dependent down regulation of both tenascin-C protein and mRNA. The highest levels of expression of tenascin-C was observed in rat pups from embryonic day 18 to postnatal day 7. Double labelling experiments performed with a tenascin-C antibody or tenascin-C probes combined with specific markers of astrocytes (GFAP) or neurons (MAP2 and Tau) allowed us to demonstrate that tenascin-C is expressed by both immature astrocytes and neurons in immature hippocampus. The temporal and topographic distribution of cells expressing tenascin-C (in the hilus and the stratum oriens of CA3) correlate with the localization and period of migration and maturation of post-mitotic cells. In view of these data we discuss the hypothesis that tenascin-C, as a mediator of neuron-glia interactions, may contribute to the development of hippocampal cells.

The HIV-1 envelope protein gp120 induces neuronal apoptosis in hippocampal slices.

The HIV-1 envelope protein gp120 produces neuronal cell damage in primary cultures of a variety of cell types including hippocampal and retinal ganglion cell neurons. The properties of primary cell cultures are, however, often markedly different from those of cells living in their normal environment. We now report that gp120 induces widespread chromatin condensation and lesions in pyramidal granular neurones and in interneurones of rat hippocampal organotypic slice cultures. This damage is clearly of an apoptotic (programmed cell death) type. The use of an in vitro organized structure will enable the molecular and cellular mechanism of action of gp120 to be examined in conditions which are particularly suitable and relevant to the in vivo situation.

Distribution of caldesmon and of the acidic isoform of calponin in cultured cerebellar neurons and in different regions of the rat brain: an immunofluorescence and confocal microscopy study.

Caldesmon and calponin are two F-actin-binding and calcium-calmodulin-dependent proteins. In smooth muscle and nonmuscle cells both proteins are localized on actin filaments. Using one- or two-dimensional gel electrophoresis followed by the Western blot technique, and by immunofluorescence studies, we have given evidence that calponin is also present in rat and pig brain. In the present study, for the first time, we demonstrate caldesmon- and calponin-specific immunoreactivities in cerebellar cultured neurons. In the rat central nervous system these antibodies mainly stain neuronal cell bodies and dendrites. By confocal analysis we observed that calponin and caldesmon are located in the actomyosin domain although the total actin and myosin were not saturated. In many cases it is clear that these two proteins are adjacent rather than superimposed in the same domain of the cell. These results are compatible with the functional role of caldesmon and calponin in the regulation of the actomyosin activity as described by others and suggest that they are part of the contractile apparatus of neural cells.

Gliosis and axonal sprouting in the hippocampus of epileptic rats are associated with an increase of tenascin-C immunoreactivity.

Temporal lobe epilepsy is associated with neuronal death, gliosis and sprouting of mossy fibres in the hippocampus of human and rats. In the present study we show that immunoreactivity for tenascin-C (an extracellular matrix glycoprotein) increase in the hippocampus of epileptic rats. However, this increase was only observed in the cases displaying neuronal cell loss and glial reaction (i.e. after kainate treatment but not after kindling). Tenascin-C increase was particularly striking at Ammon's horn, where the antibody labelled both reactive astrocytes (confirmed by double-labelling experiments) and axonal plasma membranes. In the molecular layer tenascin-C immunoreactivity remained unchanged in both kindled or kainate treated rats. It is interesting that increased tenascin-C immunoreactivity was observed within zones in which axonal regeneration did not occur (the CA3 area in kainate-treated animals) whereas zones in which reactive synaptogenesis occurred (such as the CA3 area of kindled rats or the molecular layer of both kindled and kainate-treated rats) were devoid of tenascin-C immunoreactivity. We infer from these results that tenascin-C impedes the terminal sprouting of mossy fibres in CA3 of kainate-treated rats.

Proliferative astrocytes may express fibronectin-like protein in the hippocampus of epileptic rats.

Kainic acid treatment, a model of temporal lobe epilepsy, induces in CA3-CA4 fields of hippocampal complex a neuronal degeneration associated with glial hypertrophy and proliferation. After treatment with kainate, fibronectin (an extracellular matrix protein) immunoreactivity increases in CA3-CA4. Fibronectin antibodies stain proliferative cells (simultaneously labelled by [3H]thymidin) of astrocytic type (double-immunostained by GFAP antibodies). This result constitutes the first direct demonstration of astroglial fibronectin expression in vivo. In the molecular layer of kainate-treated rats there is an axon-terminal degeneration of association-fibers. This is associated with a transient hypertrophy of resident astrocytes but not with any glial proliferation. Reactive astrocytes do not express (or faintly) fibronectin immunoreactivity in this layer. Since fibronectin is involved in astroglial proliferation in vitro, the present observations suggest that astrocytes contribute in vivo to the astroglial proliferation by an autocrin mechanism.

NCAM immunoreactivity on mossy fibers and reactive astrocytes in the hippocampus of epileptic rats.

Sprouting and synaptogenesis of mossy fibers develop in adult hippocampus after epilepsy. In control conditions, mossy fibers constitute the main afferent pathway to the Ammon's horn, where they mainly innervate CA3 pyramidal cells, but after treatment with the convulsant agent, kainate, mossy fibers also innervate granule cell dendrites generating recurrent excitatory circuits which may contribute to the maintenance of the epileptic condition. In the present study we show an enhanced immunoreactivity to neural cell adhesion molecules (NCAMs), a family of membrane glycoproteins involved in axonal growth. NCAM immunoreactivity is enriched on cytoplasmic membranes of axon shafts that are likely to be mossy fiber collaterals. NCAM immunoreactivity was also observed on the cytoplasmic membranes of reactive astrocytes, at the axon-glial contacts. Our results therefore suggest that there is an interaction of newly developed mossy fibers with other fibers and glial cells. This interaction may be mediated by NCAMs. Taking into account the trophic properties of NCAMs we suggest that they regulate the sprouting, growing and synaptogenesis of mossy fibers in epileptic conditions.

Epilepsy induced collateral sprouting of hippocampal mossy fibers: does it induce the development of ectopic synapses with granule cell dendrites?

In the present study, using Golgi and electron microscopy techniques, experimentally induced epilepsy (kindling and kainate treatment) elicited collateral sprouting of mossy fibers in rat hippocampus. Collateral branches invade the hilus, cross the granule cell layer, and distribute throughout the inner third of the molecular layer. These newly developed collaterals may acquire the typical features of mossy fibers including giant fiber varicosities (mousses), although the mean surface of these mousses was thinner in these collaterals than in terminal branches. Granule cell dendrites may develop giant thorny excrescences, suggesting that the targets of these collaterals are granule cells. Giant synaptic boutons appear in the inner third of molecular layer of epileptic rats. These boutons acquire the morphological features of mossy fiber boutons and made multiple synaptic contacts with dendritic spines. The analysis of the profile types suggests that some of the newly developed collateral mossy fibers made hypotrophic synaptic contacts.

Cycloheximide Reduces the Effects of Anoxic Insult In Vivo and In Vitro.

In vivo and in vitro techniques were utilized to examine the influence of a protein synthesis blocker, cycloheximide (CHX), on the damaging effects of anoxia in the rat. CHX administered 1 h before transient (30 min) forebrain ischaemia increased the survival of animals, decreased body weight loss and reduced the occurrence of delayed degeneration in the CA1 pyramidal region. The same dose of CHX injected 1 h after ischaemia induced status epilepticus, a decrease in survival rate, and did not reduce weight loss or CA1 damage in any of the surviving rats. Electrophysiological techniques were then used to determine the effects of various periods of anoxia and aglycaemia (AA) on CA1 field excitatory postsynaptic potentials (EPSPs) in hippocampal slices incubated in the presence or absence of CHX. In CHX-treated slices, recuperation of EPSP amplitude (45 +/- 16%) was significantly greater than in control slices (9 +/- 9%) following an AA episode of 3 min 45 s. No difference was seen in the percent recuperation of EPSPs in the control and CHX-treated slices after shorter or longer episodes of AA. From these studies, it appears that CHX protects against the damaging effect of ischaemia in vivo or AA in vitro.