Alida Amadeo

Increased Ethanol Resistance and Consumption in Eps8 Knockout Mice Correlates with Altered Actin Dynamics

Dynamic modulation of the actin cytoskeleton is critical for synaptic plasticity, abnormalities of which are thought to contribute to mental illness and addiction. Here we report that mice lacking Eps8, a regulator of actin dynamics, are resistant to some acute intoxicating effects of ethanol and show increased ethanol consumption. In the brain, the N-methyl-D-aspartate (NMDA) receptor is a major target of ethanol. We show that Eps8 is localized to postsynaptic structures and is part of the NMDA receptor complex. Moreover, in Eps8 null mice, NMDA receptor currents and their sensitivity to inhibition by ethanol are abnormal. In addition, Eps8 null neurons are resistant to the actin-remodeling activities of NMDA and ethanol. We propose that proper regulation of the actin cytoskeleton is a key determinant of cellular and behavioral responses to ethanol.

Heterogeneous expression of SNAP‐25 in rat and human brain

Synaptosomal associated protein of 25 kDa (SNAP‐25) is a SNARE component of the exocytotic apparatus involved in the release of neurotransmitter. We used multiple‐labeling immunofluorescence, confocal microscopy, and ultrastructural immunocytochemistry to examine the expression of SNAP‐25 in excitatory and inhibitory terminals from different rat and human brain areas. Glutamatergic and GABAergic terminals were identified by staining for the vesicular glutamate transporter (vGLUT1), glutamic acid decarboxylase (GAD67), or the vesicular GABA transporter (vGAT). In all examined areas GABAergic terminals did not display detectable levels of SNAP‐25, whereas glutamatergic terminals expressed the protein to a variable extent. Codistribution analysis revealed a high colocalization between pixels detecting SNAP‐25 labeling and pixels detecting vGLUT1 immunoreactivity. On the contrary, a low degree of pixel colocalization, comparable to that between two unrelated antigens, was detected between SNAP‐25 and vGAT, thus suggesting a random overlap of immunofluorescence signals. Our immunofluorescence evidence was supported by ultrastructural data, which clearly confirmed that SNAP‐25 was undetectable in GABAergic terminals identified by both their typical morphology and specific staining for GABA. Interestingly, our ultrastructural results confirmed that a subset of glutamatergic synapses do not contain detectable levels of SNAP‐25. The present study extends our previous findings obtained in rodent hippocampus and provides evidence that SNAP‐25 expression is highly variable between different axon terminals both in rat and human brain. The heterogeneous distribution of SNAP‐25 may have important implications not only in relation to the function of the protein as a SNARE but also in the control of network excitability. J. Comp. Neurol. 506:373–386, 2008.

GABAA-receptor immunoreactivity in the rat dorsal thalamus: An ultrastructural investigation

The ultrastructural localization of GABAA-receptor (GABAA-R) immunoreactivity (ir) in representative nuclei of the rat dorsal thalamus was investigated using the monoclonal antibody 62-3G1 to the beta 2 and beta 3 subunits of the GABAA-R [8]. The pattern of distribution and the subcellular localization of ir were similar in all the thalamic nuclei examined, with the exception of the reticular nucleus that was unlabeled. The reaction product was present along somatic and dendritic plasma membranes of thalamic neurons and on their intracellular membranes. No labelling was observed in glial cells. The ir was present in areas of plasma membranes related and non related to terminals containing flat vesicles, and also on invaginated plasma membranes suggesting a recycling process of the receptor complex. The distribution and mismatches between GABA neurotransmitter and its receptor localization are discussed.

Developmental and neurochemical features of cholinergic neurons in the murine cerebral cortex

BackgroundThe existence and role of intrinsic cholinergic cells in the cerebral cortex is controversial, because of their variable localization and morphology in different mammalian species. We have applied choline acetyltransferase (ChAT) immunocytochemistry to study the distribution of cholinergic neurons in the murine cerebral cortex, in the adult and during postnatal development. For more precise neurochemical identification of these neurons, the possible colocalization of ChAT with different markers of cortical neuronal populations has been analyzed by confocal microscopy. This method was also used to verify the relationship between cholinergic cells and cortical microvessels.ResultsChAT positive cells appeared at the end of the first postnatal week. Their density dramatically increased at the beginning of the second postnatal week, during which it remained higher than in perinatal and adult stages. In the adult neocortex, cholinergic neurons were particularly expressed in the somatosensory area, although their density was also significant in visual and auditory areas. ChAT positive cells tended to be scarce in other regions. They were mainly localized in the supragranular layers and displayed a fusiform/bipolar morphology.The colocalization of ChAT with pyramidal neuron markers was negligible. On the other hand, more than half of the cholinergic neurons contained calretinin, but none of them expressed parvalbumin or calbindin. However, only a fraction of the ChAT positive cells during development and very few in adulthood turned out to be GABAergic, as judged from expression of GABA and its biosynthetic enzymes GAD67/65. Consistently, ChAT showed no localization with interneurons expressing green fluorescent protein under control of the GAD67 promoter in the adult neocortex. Finally, the cortical cholinergic cells often showed close association with the microvessel walls, as identified with the gliovascular marker aquaporin 4, supporting previous hypotheses on the role of cholinergic cells in modulating the cortical microcirculation.ConclusionOur results show that the development of the intracortical cholinergic system accompanies the cortical rearrangements during the second postnatal week, a crucial stage for the establishment of cortical cytoarchitecture and for synaptogenesis. Although intrinsic ChAT positive cells usually expressed calretinin, they displayed a variable GABAergic phenotype depending on marker and on cortical developmental stage.

The role of nicotinic acetylcholine receptors in autosomal dominant nocturnal frontal lobe epilepsy

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a focal epilepsy with attacks typically arising in the frontal lobe during non-rapid eye movement (NREM) sleep. It is characterized by clusters of complex and stereotyped hypermotor seizures, frequently accompanied by sudden arousals. Cognitive and psychiatric symptoms may be also observed. Approximately 12% of the ADNFLE families carry mutations on genes coding for subunits of the heteromeric neuronal nicotinic receptors (nAChRs). This is consistent with the widespread expression of these receptors, particularly the α4β2* subtype, in the neocortex and thalamus. However, understanding how mutant nAChRs lead to partial frontal epilepsy is far from being straightforward because of the complexity of the cholinergic regulation in both developing and mature brains. The relation with the sleep-waking cycle must be also explained. We discuss some possible pathogenetic mechanisms in the light of recent advances about the nAChR role in prefrontal regions as well as the studies carried out in murine models of ADNFLE. Functional evidence points to alterations in prefrontal GABA release, and the synaptic unbalance probably arises during the cortical circuit maturation. Although most of the available functional evidence concerns mutations on nAChR subunit genes, other genes have been recently implicated in the disease, such as KCNT1 (coding for a Na+-dependent K+ channel), DEPD5 (Disheveled, Egl-10 and Pleckstrin Domain-containing protein 5), and CRH (Corticotropin-Releasing Hormone). Overall, the uncertainties about both the etiology and the pathogenesis of ADNFLE point to the current gaps in our knowledge the regulation of neuronal networks in the cerebral cortex.

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.

Orchestration of “Presto” and “Largo” Synchrony in Up-Down Activity of Cortical Networks

It has been demonstrated using single-cell and multiunit electrophysiology in layer III entorhinal cortex and disinhibited hippocampal CA3 slices that the balancing of the up-down activity is characterized by both GABAA and GABAB mechanisms. Here we report novel results obtained using multi-electrode array (60 electrodes) simultaneous recordings from reverberating postnatal neocortical networks containing 19.2 ± 1.4% GABAergic neurons, typical of intact tissue. We observed that in each spontaneous active-state the total number of spikes in identified clusters of excitatory and inhibitory neurons is almost equal, thus suggesting a balanced average activity. Interestingly, in the active-state, the early phase is sustained by only 10% of the total spikes and the firing rate follows a sigmoidal regenerative mode up to peak at 35 ms with the number of excitatory spikes greater than inhibitory, therefore indicating an early unbalance. Concentration-response pharmacology of up- and down-state lifetimes in clusters of excitatory (n = 1067) and inhibitory (n = 305) cells suggests that, besides the GABAA and GABAB mechanisms, others such as GAT-1-mediated uptake, Ih, INaP and IM ion channel activity, robustly govern both up- and down-activity. Some drugs resulted to affect up- and/or down-states with different IC50s, providing evidence that various mechanisms are involved. These results should reinforce not only the role of synchrony in CNS networks, but also the recognized analogies between the Hodgkin–Huxley action potential and the population bursts as basic mechanisms for originating membrane excitability and CNS network synchronization, respectively.

Organization of radial and non‐radial glia in the developing rat thalamus

The organization of glia and its relationship with migrating neurons were studied in the rat developing thalamus with immunocytochemistry by using light, confocal, and electron microscopy. Carbocyanine labeling in cultured slice of the embryonic diencephalon was also used. At embryonic day (E) 14, vimentin immunoreactivity was observed in radial fascicles spanning the neuroepithelium and extending from the ventricular zone to the lateral surface of the diencephalic vesicle. Vimentin‐immunopositive fibers orthogonal to the radial ones were also detected at subsequent developmental stages. At E16, radial and non‐radial processes were clearly associated with migrating neurons identified by the neuronal markers calretinin and γ‐aminobutyric acid. Non‐radial glial fibers were no longer evident by E19. Radial fibers were gradually replaced by immature astrocytes at the end of embryonic development. In the perinatal period, vimentin immunoreactivity labeled immature astrocytes and then gradually decreased; vimentin‐immunopositive cells were only found in the internal capsule by the second postnatal week. Glial fibrillary acidic protein immunoreactivity appeared at birth in astrocytes of the internal capsule, but was not evident in most of the adult thalamic nuclei. Confocal and immunoelectron microscopy allowed direct examination of the relationships between neurons and glial processes in the embryonic thalamus, showing the coupling of neuronal membranes with both radial and non‐radial glia during migration. Peculiar ultrastructural features of radial glia processes were observed. The occurrence of non‐radial migration was confirmed by carbocyanine‐labeled neuroblasts in E15 cultured slices. The data provide evidence that migrating thalamic cells follow both radial and non‐radial glial pathways toward their destination. J. Comp. Neurol. 428:527–542, 2000.

Hypocretin (Orexin) Regulates Glutamate Input to Fast-Spiking Interneurons in Layer V of the Fr2 Region of the Murine Prefrontal Cortex

We studied the effect of hypocretin 1 (orexin A) in the frontal area 2 (Fr2) of the murine neocortex, implicated in the motivation-dependent goal-directed tasks. In layer V, hypocretin stimulated the spontaneous excitatory postsynaptic currents (EPSCs) on fast-spiking (FS) interneurons. The effect was accompanied by increased frequency of miniature EPSCs, indicating that hypocretin can target the glutamatergic terminals. Moreover, hypocretin stimulated the spontaneous inhibitory postsynaptic currents (IPSCs) on pyramidal neurons, with no effect on miniature IPSCs. This action was prevented by blocking 1) the ionotropic glutamatergic receptors; 2) the hypocretin receptor type 1 (HCRTR-1), with SB-334867. Finally, hypocretin increased the firing frequency in FS cells, and the effect was blocked when the ionotropic glutamate transmission was inhibited. Immunolocalization confirmed that HCRTR-1 is highly expressed in Fr2, particularly in layer V–VI. Conspicuous labeling was observed in pyramidal neuron somata and in VGLUT1+ glutamatergic terminals, but not in VGLUT2+ fibers (mainly thalamocortical afferents). The expression of HCRTR-1 in GABAergic structures was scarce. We conclude that 1) hypocretin regulates glutamate release in Fr2; 2) the effect presents a presynaptic component; 3) the peptide control of FS cells is indirect, and probably mediated by the regulation of glutamatergic input onto these cells.

Parvalbumin and GABA in the developing somatosensory thalamus of the rat: an immunocytochemical ultrastructural correlation.

The calcium binding protein parvalbumin (PV) is widely distributed in the mammalian nervous system and its relationship with GABAergic neurons differs within thalamic nuclei and animal species. In the rat somatosensory thalamus PV immunoreactive (ir) neurons were found only in the GABAergic reticular thalamic nucleus (RT), while a dense PVir neuropil is present in the ventrobasal complex (VB). In this study the distribution and relationship of PV and GABA were investigated in RT and VB during postnatal development at electron microscopic level. The pre-embedding immunoperoxidase detection of PV was combined with the post-embedding immunogold localization of GABA. In RT, at all developmental ages, neuronal cell bodies, dendrites and rare axonal terminals were both PVir and GABAir. In VB during the first postnatal week several small vesicle-containing profiles were double-labelled and some of them were identifiable as synaptic terminals. From postnatal day 7 (P7) to P9 the medial part of VB was more intensely PVir than the lateral one and some differences in the sequence of maturation of PVir terminals were noted between these two VB subdivisions. Single-labelled PVir profiles were first observed at P8, whereas single-labelled PVir terminals appeared at P12 and at P15 they became more frequent and larger, showing the typical morphology of ascending afferents described in adult VB. These results demonstrate the late expression of PV and acquisition of adult morphology in ascending terminals of rat VB during postnatal development in comparison with the innervation arising from the GABAergic RT.