Synapse formation and clustering of neuroligin-2 in the absence of GABAA receptors.

GABAergic synapses are crucial for brain function, but the mechanisms underlying inhibitory synaptogenesis are unclear. Here, we show that postnatal Purkinje cells (PCs) of GABA(A)alpha1 knockout (KO) mice express transiently the alpha3 subunit, leading to the assembly of functional GABA(A) receptors and initial normal formation of inhibitory synapses, that are retained until adulthood. Subsequently, down-regulation of the alpha3 subunit causes a complete loss of GABAergic postsynaptic currents, resulting in a decreased rate of inhibitory synaptogenesis and formation of mismatched synapses between GABAergic axons and PC spines. Notably, the postsynaptic adhesion molecule neuroligin-2 (NL2) is correctly targeted to inhibitory synapses lacking GABA(A) receptors and the scaffold molecule gephyrin, but is absent from mismatched synapses, despite innervation by GABAergic axons. Our data indicate that GABA(A) receptors are dispensable for synapse formation and maintenance and for targeting NL2 to inhibitory synapses. However, GABAergic signaling appears to be crucial for activity-dependent regulation of synapse density during neuronal maturation.

Expression and distribution of CB1 cannabinoid receptors in the central nervous system of the African cichlid fish Pelvicachromis pulcher.

Neuroanatomical investigation of the cannabinoid system in a lower vertebrate group such as teleost fishes might improve our understanding of the physiological role of such a signaling system. In the present study, the expression of a CB1 cannabinoid receptor has been demonstrated in the CNS of a teleost fish, the cichlid Pelvicachromis pulcher. Moreover, CB1-like immunoreactivity has been analyzed by using a purified antibody against the CB1 receptor amino-terminus. Immunostained neurons and varicosities were found through the telencephalon as well as in the preoptic area and lateral infundibular lobes of the hypothalamus. Stained cells were observed in the pituitary gland. Several cell bodies and nerve terminals containing an intense CB1-like immunoreactivity were found in the pretectal central nucleus and posterior tuberculum, both lying in a transitional region between diencephalon and mesencephalon. In the brainstem, the CB1 immunopositivity was more restricted than in the prosencephalon, with the exception of some large, intensely immunopositive nerve cells within the dorsal mesencephalic tegmentum, possibly motor neurons of the third cranial nerve. In the cerebellum, among a majority of immunonegative granule cells, a subset of them was immunostained. Some positive Purkinje cells were also observed. In the spinal cord, ventral gray matter, several alpha-motoneurons were stained. Similarities to and discrepancies from the CB1 receptor distributions in other vertebrate CNS are discussed, paying particular attention to the abundant CB1 immunoreactivity observed in the area encompassing the pretectum and glomerular nucleus, which is characterized by a peculiar differentiation in bony fishes.

Selective localization of collybistin at a subset of inhibitory synapses in brain circuits.

Collybistin is a brain-specific guanine nucleotide exchange factor (GEF) that is crucial for the postsynaptic accumulation of gephyrin and γ-aminobutyric acid A receptors (GABA(A) Rs) at a specific subset of inhibitory synapses. Our understanding of the in vivo function of collybistin has been hampered by lack of information about the synaptic localization of this protein in brain circuits. Here we describe the subcellular localization of endogenous collybistin by using antibodies raised against distinct molecular domains that should recognize the majority of endogenous collybistin isoforms. We show that collybistin co-clusters with gephyrin and GABA(A) Rs in synaptic puncta and is recruited to postsynaptic specializations early during synapse development. Notably, collybistin is present in only a subset of gephyrin-positive synapses, with variable co-localization values in different brain regions. Moreover, collybistin co-localizes with GABA(A) Rs containing the α1, α2, or α3 subunits, arguing against a selective association with specific GABA(A) R subtypes. Surprisingly, we found that collybistin is expressed only transiently in Purkinje cells, suggesting that in these cerebellar neurons collybistin plays a selective role during the initial assembly of postsynaptic specializations. These data reveal a remarkable heterogeneity in the organization of GABAergic synapses and provide an anatomical basis for interpreting the variable effects caused by disruption of the collybistin gene in human X-linked intellectual disability and mouse knockout models.

Quantitative organization of GABAergic synapses in the molecular layer of the mouse cerebellar cortex.

In the cerebellar cortex, interneurons of the molecular layer (stellate and basket cells) provide GABAergic input to Purkinje cells, as well as to each other and possibly to other interneurons. GABAergic inhibition in the molecular layer has mainly been investigated at the interneuron to Purkinje cell synapse. In this study, we used complementary subtractive strategies to quantitatively assess the ratio of GABAergic synapses on Purkinje cell dendrites versus those on interneurons. We generated a mouse model in which the GABAA receptor alpha1 subunit (GABAARalpha1) was selectively removed from Purkinje cells using the Cre/loxP system. Deletion of the alpha1 subunit resulted in a complete loss of GABAAR aggregates from Purkinje cells, allowing us to determine the density of GABAAR clusters in interneurons. In a complementary approach, we determined the density of GABA synapses impinging on Purkinje cells using alpha-dystroglycan as a specific marker of inhibitory postsynaptic sites. Combining these inverse approaches, we found that synapses received by interneurons represent approximately 40% of all GABAergic synapses in the molecular layer. Notably, this proportion was stable during postnatal development, indicating synchronized synaptogenesis. Based on the pure quantity of GABAergic synapses onto interneurons, we propose that mutual inhibition must play an important, yet largely neglected, computational role in the cerebellar cortex.

Microvesicles derived from adult human bone marrow and tissue specific mesenchymal stem cells shuttle selected pattern of miRNAs.

BACKGROUND: Cell-derived microvesicles (MVs) have been described as a new mechanism of cell-to-cell communication. MVs after internalization within target cells may deliver genetic information. Human bone marrow derived mesenchymal stem cells (MSCs) and liver resident stem cells (HLSCs) were shown to release MVs shuttling functional mRNAs. The aim of the present study was to evaluate whether MVs derived from MSCs and HLSCs contained selected micro-RNAs (miRNAs). METHODOLOGY/PRINCIPAL FINDINGS: MVs were isolated from MSCs and HLSCs. The presence in MVs of selected ribonucleoproteins involved in the traffic and stabilization of RNA was evaluated. We observed that MVs contained TIA, TIAR and HuR multifunctional proteins expressed in nuclei and stress granules, Stau1 and 2 implicated in the transport and stability of mRNA and Ago2 involved in miRNA transport and processing. RNA extracted from MVs and cells of origin was profiled for 365 known human mature miRNAs by real time PCR. Hierarchical clustering and similarity analysis of miRNAs showed 41 co-expressed miRNAs in MVs and cells. Some miRNAs were accumulated within MVs and absent in the cells after MV release; others were retained within the cells and not secreted in MVs. Gene ontology analysis of predicted and validated targets showed that the high expressed miRNAs in cells and MVs could be involved in multi-organ development, cell survival and differentiation. Few selected miRNAs shuttled by MVs were also associated with the immune system regulation. The highly expressed miRNAs in MVs were transferred to target cells after MV incorporation. CONCLUSIONS: This study demonstrated that MVs contained ribonucleoproteins involved in the intracellular traffic of RNA and selected pattern of miRNAs, suggesting a dynamic regulation of RNA compartmentalization in MVs. The observation that MV-highly expressed miRNAs were transferred to target cells, rises the possibility that the biological effect of stem cells may, at least in part, depend on MV-shuttled miRNAs. Data generated from this study, stimulate further functional investigations on the predicted target genes and pathways involved in the biological effect of human adult stem cells.

Synaptic inhibition of Purkinje cells mediates consolidation of vestibulo-cerebellar motor learning.

Although feedforward inhibition onto Purkinje cells was first documented 40 years ago, we understand little of how inhibitory interneurons contribute to cerebellar function in behaving animals. Using a mouse line (PC-Deltagamma2) in which GABA(A) receptor-mediated synaptic inhibition is selectively removed from Purkinje cells, we examined how feedforward inhibition from molecular layer interneurons regulates adaptation of the vestibulo-ocular reflex. Although impairment of baseline motor performance was relatively mild, the ability to adapt the phase of the vestibulo-ocular reflex and to consolidate gain adaptations was strongly compromised. Purkinje cells showed abnormal patterns of simple spikes, both during and in the absence of evoked compensatory eye movements. On the basis of modeling our experimental data, we propose that feedforward inhibition, by controlling the fine-scale patterns of Purkinje cell activity, enables the induction of plasticity in neurons of the cerebellar and vestibular nuclei.

Synaptogenesis in the cerebellar cortex: differential regulation of gephyrin and GABAA receptors at somatic and dendritic synapses of Purkinje cells.

In rodent cerebellar cortex, synaptogenesis occurs entirely postnatally, allowing study of the mechanisms of synapse formation in vivo. Here we monitored the clustering of GABA(A) receptors and the scaffolding protein gephyrin at GABAergic postsynaptic sites during rat cerebellar development. We found that GABA(A) receptors and gephyrin co-aggregate at nascent synapses in the molecular and Purkinje cell layers with a similar time course. With few exceptions, gephyrin and GABA(A) receptor subunits clustered selectively in front of presynaptic boutons expressing the vesicular inhibitory amino acid transporter VIAAT and no ectopic localization of these molecules was observed. Surprisingly, gephyrin clusters outlining the cell body of Purkinje cells were transient, and disappeared rapidly at the end of the second postnatal week. The loss of gephyrin from perisomatic synapses was coincident with a significant reduction in the size of GABA(A) receptor clusters. Furthermore, these changes were accompanied by a developmental decrease in the size of synaptic appositions, as documented by electron microscopy. These findings suggest that gephyrin takes part in the initial assembly of postsynaptic specializations and reveal an unsuspected heterogeneity in the molecular organization of the postsynaptic apparatus at somatic and dendritic synapses of mature Purkinje cells.