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ABSTRACT: In mammals, olfactory bulb granule cells (GCs) are generated throughout life in the subventricular zone. GABAergic inputs onto newborn neurons likely regulate their maturation, but the details of this process remain still elusive. Here, we investigated the differentiation, synaptic integration, and survival of adult-born GCs when their afferent GABAergic inputs are challenged by conditional gene targeting. Migrating GC precursors were targeted with Cre-eGFP-expressing lentiviral vectors in mice with a floxed gene encoding the GABA(A) receptor α2-subunit (i.e., Gabra2). Ablation of the α2-subunit did not affect GC survival but dramatically delayed their maturation. We found a reduction in postsynaptic α2-subunit and gephyrin clusters accompanied by a decrease in the frequency and amplitude of GABAergic postsynaptic currents beginning ∼14 d post-injection (dpi). In addition, mutant cells exhibited altered dendritic branching and spine density. Spine loss appeared with mislocation of glutamatergic synapses on dendritic shafts and a reduction of spontaneous glutamatergic postsynaptic currents, underscoring the relevance of afferent GABAergic transmission for a proper synaptic integration of newborn GCs. To test the role of GABAergic signaling during much early stages of GC maturation, we used a genetic strategy to selectively inactivate Gabra2 in precursor cells of the subventricular zone. In these mice, labeling of newborn GCs with eGFP lentiviruses revealed similar morphological alterations as seen on delayed Gabra2 inactivation in migrating neuroblasts, with reduced dendritic branching and spine density at 7 dpi. Collectively, these results emphasize the critical role of GABAergic synaptic signaling for structural maturation of adult-born GCs and formation of glutamatergic synapses.
Journal of Neuroscience 06/2012; 32(26):9103-15. · 7.11 Impact Factor
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ABSTRACT: Knowledge of the functional organization of the GABAergic system, the main inhibitory neurotransmitter system, in the CNS has increased remarkably in recent years. In particular, substantial progress has been made in elucidating the molecular mechanisms underlying the formation and plasticity of GABAergic synapses. Evidence available ascribes a key role to the cytoplasmic protein gephyrin to form a postsynaptic scaffold anchoring GABA(A) receptors along with other transmembrane proteins and signaling molecules in the postsynaptic density. However, the mechanisms of gephyrin scaffolding remain elusive, notably because gephyrin can auto-aggregate spontaneously and lacks PDZ protein interaction domains found in a majority of scaffolding proteins. In addition, the structural diversity of GABA(A) receptors, which are pentameric channels encoded by a large family of subunits, has been largely overlooked in these studies. Finally, the role of the dystrophin-glycoprotein complex, present in a subset of GABAergic synapses in cortical structures, remains ill-defined. In this review, we discuss recent results derived mainly from the analysis of mutant mice lacking a specific GABA(A) receptor subtype or a core protein of the GABAergic postsynaptic density (neuroligin-2, collybistin), highlighting the molecular diversity of GABAergic synapses and its relevance for brain plasticity and function. In addition, we discuss the contribution of the dystrophin-glycoprotein complex to the molecular and functional heterogeneity of GABAergic synapses.
Cellular and Molecular Life Sciences CMLS 02/2012; 69(15):2485-99. · 6.57 Impact Factor
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ABSTRACT: The ventral tegmental area (VTA) is widely implicated in drug addiction and other psychiatric disorders. This brain region is densely populated by dopaminergic (DA) neurons and also contains a sparse population of γ-aminobutyric acid (GABA)ergic cells that regulate the activity of the principal neurons. Therefore, an in-depth knowledge of the organization of VTA GABAergic circuits and of the plasticity induced by drug consumption is essential for understanding the mechanisms by which drugs induce stable changes in brain reward circuits. Using immunohistochemistry, we provide a detailed description of the localization of major GABA(A) and GABA(B) receptor subunits in the rat VTA. We show that DA and GABAergic cells express both GABA(A) and GABA(B) receptors. However VTA neurons differ considerably in the expression of GABA(A) receptor subunits, as the α1 subunit is associated predominantly with non-DA cells, whereas the α3 subunit is present at low levels in both types of VTA neurons. Using an unbiased stereological method, we then demonstrate that α1-positive elements represent only a fraction of non-DA neurons and that the ratio of DA and non-DA cells is quite variable throughout the rostro-caudal extent of the VTA. Interestingly, DA and non-DA cells receive a similar density of perisomatic synapses, whereas axo-dendritic synapses are significantly more abundant in non-DA cells, indicating that local interneurons receive prominent GABAergic inhibition. These findings reveal a differential expression of GABA receptor subtypes in the two major categories of VTA neurons and provide an anatomical basis for interpreting the plasticity of inhibitory circuits induced by drug exposure.
PLoS ONE 01/2012; 7(10):e46250. · 4.09 Impact Factor
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ABSTRACT: Pyramidal cells express various GABA(A) receptor (GABA(A)R) subtypes, possibly to match inputs from functionally distinct interneurons targeting specific subcellular domains. Postsynaptic anchoring of GABA(A)Rs is ensured by a complex interplay between the scaffolding protein gephyrin, neuroligin-2 and collybistin. Direct interactions between these proteins and GABA(A)R subunits might contribute to synapse-specific distribution of GABA(A)R subtypes. In addition, the dystrophin-glycoprotein complex, mainly localized at perisomatic synapses, regulates GABA(A)R postsynaptic clustering at these sites. Here, we investigated how the functional and molecular organization of GABAergic synapses in CA1 pyramidal neurons is altered in mice lacking the GABA(A)R α2 subunit (α2-KO). We report a marked, layer-specific loss of postsynaptic gephyrin and neuroligin-2 clusters, without changes in GABAergic presynaptic terminals. Whole-cell voltage-clamp recordings in slices from α2-KO mice show a 40% decrease in GABAergic mIPSC frequency, with unchanged amplitude and kinetics. Applying low/high concentrations of zolpidem to discriminate between α1- and α2/α3-GABA(A)Rs demonstrates that residual mIPSCs in α2-KO mice are mediated by α1-GABA(A)Rs. Immunofluorescence analysis reveals maintenance of α1-GABA(A)R and neuroligin-2 clusters, but not gephyrin clusters, in perisomatic synapses of mutant mice, along with a complete loss of these three markers on the axon initial segment. This striking subcellular difference correlates with the preservation of dystrophin clusters, colocalized with neuroligin-2 and α1-GABA(A)Rs on pyramidal cell bodies of mutant mice. Dystrophin was not detected on the axon initial segment in either genotype. Collectively, these findings reveal synapse-specific anchoring of GABA(A)Rs at postsynaptic sites and suggest that the dystrophin-glycoprotein complex contributes to stabilize α1-GABA(A)R and neuroligin-2, but not gephyrin, in perisomatic postsynaptic densities.
The Journal of Physiology 08/2011; 589(Pt 20):4959-80. · 4.72 Impact Factor
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Rosa C Paolicelli,
Giulia Bolasco,
Francesca Pagani,
Laura Maggi,
Maria Scianni, Patrizia Panzanelli,
Maurizio Giustetto,
Tiago Alves Ferreira,
Eva Guiducci,
Laura Dumas,
Davide Ragozzino,
Cornelius T Gross
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ABSTRACT: Microglia are highly motile phagocytic cells that infiltrate and take up residence in the developing brain, where they are thought to provide a surveillance and scavenging function. However, although microglia have been shown to engulf and clear damaged cellular debris after brain insult, it remains less clear what role microglia play in the uninjured brain. Here, we show that microglia actively engulf synaptic material and play a major role in synaptic pruning during postnatal development in mice. These findings link microglia surveillance to synaptic maturation and suggest that deficits in microglia function may contribute to synaptic abnormalities seen in some neurodevelopmental disorders.
Science 07/2011; 333(6048):1456-8. · 31.20 Impact Factor
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ABSTRACT: New olfactory bulb granule cells (GCs) are GABAergic interneurons continuously arising from neuronal progenitors and integrating into preexisting bulbar circuits. They receive both GABAergic and glutamatergic synaptic inputs from olfactory bulb intrinsic neurons and centrifugal afferents. Here, we investigated the spatiotemporal dynamic of newborn GC synaptogenesis in adult mouse olfactory bulb. First, we established that GABAergic synapses onto mature GC dendrites contain the GABA(A) receptor alpha2 subunit along with the postsynaptic scaffolding protein gephyrin. Next, we characterized morphologically and electrophysiologically the development of GABAergic and glutamatergic inputs onto newborn GCs labeled with eGFP (enhanced green fluorescent protein) using lentiviral vectors. Already when reaching the GC layer (GCL), at 3 d post-vector injection (dpi), newborn GCs exhibited tiny voltage-dependent sodium currents and received functional GABAergic and glutamatergic synapses, recognized immunohistochemically by apposition of specific presynaptic and postsynaptic markers. Thereafter, GABAergic and glutamatergic synaptic contacts increased differentially in the GCL, and at 7 dpi, PSD-95 clusters outnumbered gephyrin clusters. Thus, the weight of GABAergic input was predominant at early stages of GC maturation, but not later. Newborn GC dendrites first reached the external plexiform layer at 4 dpi, where they received functional GABAergic contacts at 5 dpi. Reciprocal synapses initially were formed on GC dendritic shafts, where they might contribute to spine formation. Their presence was confirmed ultrastructurally at 7 dpi. Together, our findings unravel rapid synaptic integration of newborn GCs in adult mouse olfactory bulb, with GABAergic and glutamatergic influences being established proximally before formation of output synapses by apical GC dendrites onto mitral/tufted cells.
Journal of Neuroscience 12/2009; 29(48):15039-52. · 7.11 Impact Factor
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ABSTRACT: In the olfactory bulb, mitral and tufted cells receive GABAergic inhibition at dendrodendritic synapses with granule cells. Recent studies have revealed a remarkable variability in the subunit composition of GABAa receptors in dendrodendritic microcircuits, with differential expression patterns of the α1 and α3 subunits in different subtypes of mitral and tufted cells. In particular, all mitral cells express the α1 subunit, whereas GABAaα3 is restricted to a subgroup of mitral cells, as well as to several subtypes of tufted cells. To assess the functional relevance of this heterogeneity, we investigated a mouse strain carrying a genetic deletion of the α1 subunit. Elimination of GABAaα1 was partially compensated for in mitral cells by receptors containing the α3 subunit, substantially decreasing the frequency of spontaneous inhibitory postsynaptic currents, as well as prolonging their decay time. Evoked inhibition between granule and mitral cells was slower to rise and decay and had smaller amplitude in α1 mutants. Remarkably, these changes in synaptic inhibition were accompanied by a significant reduction in the frequency of field oscillations. Therefore, the subunit composition of GABAa receptors strongly influences rhythmic activities in the olfactory bulb network. Together, these data indicate that dendrodendritic circuits in the external plexiform layer segregate into parallel pathways involving distinct GABAa receptors that are expressed by different subtypes of mitral and tufted cells.
Annals of the New York Academy of Sciences 08/2009; 1170(1):259 - 263. · 3.15 Impact Factor
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ABSTRACT: In the olfactory bulb, mitral and tufted cells receive GABAergic inhibition at dendrodendritic synapses with granule cells. Recent studies have revealed a remarkable variability in the subunit composition of GABA(a) receptors in dendrodendritic microcircuits, with differential expression patterns of the alpha1 and alpha3 subunits in different subtypes of mitral and tufted cells. In particular, all mitral cells express the alpha1 subunit, whereas GABA(a)alpha3 is restricted to a subgroup of mitral cells, as well as to several subtypes of tufted cells. To assess the functional relevance of this heterogeneity, we investigated a mouse strain carrying a genetic deletion of the alpha1 subunit. Elimination of GABA(a)alpha1 was partially compensated for in mitral cells by receptors containing the alpha3 subunit, substantially decreasing the frequency of spontaneous inhibitory postsynaptic currents, as well as prolonging their decay time. Evoked inhibition between granule and mitral cells was slower to rise and decay and had smaller amplitude in alpha1 mutants. Remarkably, these changes in synaptic inhibition were accompanied by a significant reduction in the frequency of field oscillations. Therefore, the subunit composition of GABA(a) receptors strongly influences rhythmic activities in the olfactory bulb network. Together, these data indicate that dendrodendritic circuits in the external plexiform layer segregate into parallel pathways involving distinct GABA(a) receptors that are expressed by different subtypes of mitral and tufted cells.
Annals of the New York Academy of Sciences 08/2009; 1170:259-63. · 3.15 Impact Factor
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ABSTRACT: Fluctuations in the brain concentrations of neurosteroids are accompanied by changes in the expression of GABA(A) receptor subunits in the cerebral cortex and hippocampus. Here, we investigated the expression of the postsynaptic molecule gephyrin in the cerebral cortex and hippocampus of pregnant rats, as well as in rats treated chronically with contraceptive drugs. The amounts of gephyrin mRNA and protein did not change during pregnancy and after delivery, as well as in rats treated with ethynylestradiol (EE) and levonorgestrel (LNG) for 4 weeks. Similarly, using immunofluorescence and laser scanning confocal microscopy, we did not detect significant changes in the number and size of gephyrin-immunopositive clusters, which likely represent inhibitory postsynaptic sites. These findings indicate that the expression of gephyrin and the density of cortical inhibitory synapses are not influenced by endogenous neurosteroids.
Brain Research 10/2007; 1169:1-8. · 2.73 Impact Factor
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Pietro Pilo Boyl,
Alessia Di Nardo,
Christophe Mulle,
Marco Sassoè-Pognetto, Patrizia Panzanelli,
Andrea Mele,
Matthias Kneussel,
Vivian Costantini,
Emerald Perlas,
Marzia Massimi,
Hugo Vara,
Maurizio Giustetto,
Walter Witke
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ABSTRACT: Profilins are actin binding proteins essential for regulating cytoskeletal dynamics, however, their function in the mammalian nervous system is unknown. Here, we provide evidence that in mouse brain profilin1 and profilin2 have distinct roles in regulating synaptic actin polymerization with profilin2 preferring a WAVE-complex-mediated pathway. Mice lacking profilin2 show a block in synaptic actin polymerization in response to depolarization, which is accompanied by increased synaptic excitability of glutamatergic neurons due to higher vesicle exocytosis. These alterations in neurotransmitter release correlate with a hyperactivation of the striatum and enhanced novelty-seeking behavior in profilin2 mutant mice. Our results highlight a novel, profilin2-dependent pathway, regulating synaptic physiology, neuronal excitability, and complex behavior.
The EMBO Journal 07/2007; 26(12):2991-3002. · 9.20 Impact Factor
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ABSTRACT: In the olfactory bulb (OB), odorants induce oscillations in the gamma range (20-80 Hz) that play an important role in the processing of sensory information. Synaptic transmission between dendrites is a major contributor to this processing. Glutamate released from mitral cell dendrites excites the dendrites of granule cells, which in turn mediate GABAergic inhibition back onto mitral cells. Although this reciprocal synapse is thought to be a key element supporting oscillatory activity, the mechanisms by which dendrodendritic inhibition induces and maintains gamma oscillations remain unknown. Here, we assessed the role of the dendrodendritic inhibition, using mice lacking the GABA(A) receptor alpha1-subunit, which is specifically expressed in mitral cells but not in granule cells. The spontaneous inhibitory postsynaptic current frequency in these mutants was low and was consistent with the reduction of GABA(A) receptor clusters detected by immunohistochemistry. The remaining GABA(A) receptors in mitral cells contained the alpha3-subunit and supported slower decaying currents of unchanged amplitude. Overall, inhibitory-mediated interactions between mitral cells were smaller and slower in mutant than in WT mice, although the strength of sensory afferent inputs remained unchanged. Consequently, both experimental and theoretical approaches revealed slower gamma oscillations in the OB network of mutant mice. We conclude, therefore, that fast oscillations in the OB circuit are strongly constrained by the precise location, subunit composition and kinetics of GABA(A) receptors expressed in mitral cells.
Proceedings of the National Academy of Sciences 05/2007; 104(17):7259-64. · 9.68 Impact Factor
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ABSTRACT: Vesicular glutamate transporters (VGLUTs) mediate the packaging of the excitatory neurotransmitter glutamate into synaptic vesicles. Three VGLUT subtypes have so far been identified, with distinct expression patterns in the adult brain. Here, we investigated the spatial distribution of the three VGLUTs in the rat olfactory bulb, a brain region containing a variety of glutamate synapses, both axodendritic and dendrodendritic. Using multilabelling confocal microscopy and electron microscopic immunocytochemistry, we showed that each VGLUT isoform has a highly selective localization in olfactory bulb synapses. VGLUT1 is present at dendrodendritic synapses established by the output neurones (mitral and tufted cells) with bulbar interneurones in the glomerular layer and external plexiform layer, as well as in axonal synapses of the granule cell layer. By contrast, VGLUT2 is strongly expressed in axon terminals of olfactory sensory neurones, which establish synapses with second-order neurones in the glomerular neuropil. VGLUT2 is also found in the outer part of the external plexiform layer and in the granule cell layer but colocalizes only partially with VGLUT1. Finally, we showed that VGLUT3 is exclusively located in the glomerular neuropil, where it colocalizes extensively with the vesicular inhibitory amino acid transporter vesicular GABA transporter, suggesting that it is associated with a subset of inhibitory synapses. Together, these observations extend previous findings on VGLUT distribution in the forebrain, and suggest that each VGLUT subtype has a specific function in the distinct features of axodendritic and dendrodendritic synapses that characterize the olfactory bulb circuit.
European Journal of Neuroscience 03/2007; 25(5):1373-83. · 3.63 Impact Factor
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ABSTRACT: Elucidating the molecular organization of synapses is essential for understanding brain function and plasticity. Immunofluorescence, combined with various fluorescent probes, is a sensitive and versatile method for morphological studies. However, analysis of synaptic proteins in situ is limited by epitope-masking after tissue fixation. Furthermore, postsynaptic proteins (such as ionotropic receptors and scaffolding proteins) often require weaker fixation for optimal detection than most intracellular markers, thereby hindering simultaneous visualization of these molecules. We present three protocols, which are alternatives to perfusion fixation, to overcome these restrictions. Brief tissue fixation shortly after interruption of vital functions preserves morphology and antigenicity. Combined with specific neuronal markers, selective detection of -aminobutyric acid A (GABAA) receptors and the scaffolding protein gephyrin in relation to identified inhibitory presynaptic terminals in the rodent brain is feasible by confocal laser scanning microscopy. The most sophisticated of these protocols can be associated with electrophysiology for correlative studies of synapse structure and function. These protocols require 2–3 consecutive days for completion.
Nature Protocols 11/2006; 1(4):1887-1897. · 9.92 Impact Factor
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Loredana Serpe,
Marilena Guido,
Roberto Canaparo,
Elisabetta Muntoni,
Roberta Cavalli, Patrizia Panzanelli,
Carlo Della Pepal,
Alessandro Bargoni,
Alessandro Mauro,
Maria Rosa Gasco,
Mario Eandi,
Gian Paolo Zara
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ABSTRACT: The structure of both carrier and anticancer drug affects the intracellular fate of a transported drug. The study investigated in vitro intracellular accumulation and cytotoxic activity of doxorubicin-loaded solid lipid nanoparticles (SLN), doxorubicin in pegylated liposomes (Caelyx) and free doxorubicin. Intracellular doxorubicin levels and cytotoxic activity were determined by high performance liquid chromatography with fluorescence detection, and by the trypan blue dye exclusion assay, respectively. Doxorubicin-loaded SLN inhibited cell growth more strongly than either free or liposomal doxorubicin, in human colorectal adenocarcinoma, HT-29, retinoblastoma Y79, and glioblastoma U373 cell lines. The IC50 values for doxorubicin-loaded SLN were significantly lower after 24 h exposure than those for free doxorubicin in all cell lines; after 48 h exposure they were lower than those for liposomal doxorubicin in HT-29 and Y79 cells. The enhanced cytotoxic activity of doxorubicin-loaded SLN was associated with increased drug incorporation in cells: intracellular doxorubicin levels were significantly enhanced after exposure to drug-loaded SLN versus either free or liposomal drug. Rate of intracellular accumulation and cytotoxic activity also differed among different cell lines; in particular, cells of epithelial origin were found to be more sensitive to doxorubicin-loaded SLN. In conclusion, the greater sensitivity of HT-29, Y79, and U373 cells to doxorubicin-loaded SLN than to the other drug formulations may be due to the capability of the delivery system to enhance drug action, through a marked uptake and accumulation of SLN within the cell.
Journal of Nanoscience and Nanotechnology 08/2006; 6(9-10):3062-9. · 1.56 Impact Factor
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ABSTRACT: 1. Adhesion of polymorphonuclear cells (PMNs) to vascular endothelial cells (EC) is a critical step in recruitment and infiltration of leukocytes into tissues during inflammation. High doses of butyric acid have been shown to ameliorate inflammation in inflammatory bowel diseases (IBD). Cholesteryl-butyrate solid lipid nanoparticles (chol-but SLN) as prodrug are a possible delivery system for butyric acid. 2. Sodium butyrate or chol-but SLN were coincubated with human PMNs and human umbilical vein EC (HUVEC); adhesion was quantified by computerized microimaging fluorescence analysis. Both chol-but SLN and sodium butyrate displayed antiadhesive effects on FMLP- and IL-1beta-stimulated cells in a concentration-response curve (10(-8)-10(-5) M), but chol-but SLN were in all cases more active. Moreover, chol-but SLN inhibited FMLP-induced adhesion of PMNs to FCS-coated plastic wells, thus showing a direct effect on PMNs, while sodium butyrate had little effect. Confocal microscopy showed that fluorescent SLN entered PMNs and HUVEC after 10 min incubation. Chol-but SLN acted either on activated PMN or HUVEC. 3. Chol-but SLN inhibited O2-* production and myeloperoxidase release by PMNs evoked by FMLP, in a dose-dependent, but not time-dependent, manner and were more active than sodium butyrate. 4. In conclusion, in all tests chol-but SLN were more active than sodium butyrate. Thus, chol-but SLN might be a valid alternative to sodium butyrate in the anti-inflammatory therapy of ulcerative colitis, avoiding complications related to the administration of sodium butyrate.
British Journal of Pharmacology 08/2006; 148(5):648-56. · 4.41 Impact Factor
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ABSTRACT: Synapse formation and maintenance require extensive transsynaptic interactions involving multiple signal transduction pathways. In the cerebellum, Purkinje cells (PCs) receive GABAergic, axo-dendritic synapses from stellate cells and axo-somatic synapses from basket cells, both with GABAA receptors containing the alpha1 subunit. Here, we investigated the effects of a targeted deletion of the alpha1 subunit gene on GABAergic synaptogenesis in PCs, using electrophysiology and immunoelectron microscopy. Whole-cell patch-clamp recordings in acute slices revealed that PCs from alpha1(0/0) mice lack spontaneous and evoked IPSCs, demonstrating that assembly of functional GABAA receptors requires the alpha1 subunit. Ultrastructurally, stellate cell synapses on PC dendrites were reduced by 75%, whereas basket cell synapses on the soma were not affected, despite the lack of GABAA-mediated synaptic transmission. Most strikingly, GABAergic terminals were retained in the molecular layer of adult alpha1(0/0) mice and formed heterologous synapses with PC spines characterized by a well differentiated asymmetric postsynaptic density. These synapses lacked presynaptic glutamatergic markers and postsynaptic AMPA-type glutamate receptors but contained delta2-glutamate receptors. During postnatal development, initial steps of GABAergic synapse formation were qualitatively normal, and heterologous synapses appeared in parallel with maturation of dendritic spines. These results suggest that synapse formation in the cerebellum is governed by neurotransmitter-independent mechanisms. However, in the absence of GABAA-mediated transmission, GABAergic terminals in the molecular layer apparently become responsive to synaptogenic signals from PC spines and form stable heterologous synapses. In contrast, maintenance of axo-somatic GABAergic synapses does not depend on functional GABAA receptors, suggesting differential regulation in distinct subcellular compartments.
Journal of Neuroscience 04/2006; 26(12):3245-55. · 7.11 Impact Factor
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Annals of the New York Academy of Sciences 02/2006; 868(1):693 - 696. · 3.15 Impact Factor
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ABSTRACT: Elucidating the molecular organization of synapses is essential for understanding brain function and plasticity. Immunofluorescence, combined with various fluorescent probes, is a sensitive and versatile method for morphological studies. However, analysis of synaptic proteins in situ is limited by epitope-masking after tissue fixation. Furthermore, postsynaptic proteins (such as ionotropic receptors and scaffolding proteins) often require weaker fixation for optimal detection than most intracellular markers, thereby hindering simultaneous visualization of these molecules. We present three protocols, which are alternatives to perfusion fixation, to overcome these restrictions. Brief tissue fixation shortly after interruption of vital functions preserves morphology and antigenicity. Combined with specific neuronal markers, selective detection of gamma-aminobutyric acid A (GABA(A)) receptors and the scaffolding protein gephyrin in relation to identified inhibitory presynaptic terminals in the rodent brain is feasible by confocal laser scanning microscopy. The most sophisticated of these protocols can be associated with electrophysiology for correlative studies of synapse structure and function. These protocols require 2-3 consecutive days for completion.
Nature Protocol 02/2006; 1(4):1887-97. · 8.36 Impact Factor
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ABSTRACT: GABAA receptors form heteromeric GABA-gated chloride channels assembled from a large family of subunit genes. In cerebellum, distinct GABAA receptor subtypes, differing in subunit composition, are segregated between cell types and synaptic circuits. The cerebellum therefore represents a useful system to investigate the significance of GABAA receptor heterogeneity. For instance, studies of mice carrying targeted deletion of major GABAA receptor subunit genes revealed the role of alpha subunit variants for receptor assembly, synaptic targeting, and functional properties. In addition, these studies unraveled mandatory association between certain subunits and demonstrated distinct pharmacology of receptors mediating phasic and tonic inhibition. Although some of these mutants have a profound loss of GABAA receptors, they exhibit only minor impairment of motor function, suggesting activation of compensatory mechanisms to preserve inhibitory networks in the cerebellum. These adaptations include an altered balance between phasic and tonic inhibition, activation of voltage-independent K+ conductances, and upregulation of GABAA receptors in interneurons that are not affected directly by the mutation. Deletion of the alpha1 subunit gene leads to complete loss of GABAA receptors in Purkinje cells. A striking alteration occurs in these mice, whereby presynaptic GABAergic terminals are preserved in the molecular layer but make heterologous synapses with spines, characterized by a glutamatergic-like postsynaptic density. During development of alpha1(0/0) mice, GABAergic synapses are initially formed but are replaced upon spine maturation. These findings suggest that functional GABAA receptors are required for long-term maintenance of GABAergic synapses in Purkinje cells.
The Cerebellum 02/2006; 5(4):275-85. · 3.21 Impact Factor
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ABSTRACT: Mitral and tufted cells of the olfactory bulb receive strong gamma-aminobutyric acid (GABA)-ergic input and express GABA(A) receptors containing the alpha1 or alpha3 subunit. The distribution of these subunits was investigated in rats via multiple immunofluorescence and confocal microscopy, by using gephyrin as a marker of GABAergic synapses. A prominent immunoreactivity was detected throughout the external plexiform layer (EPL) and glomerular layer (GL). However, although staining for the alpha1 subunit was uniform throughout the EPL, that of the alpha3 subunit was most intense in the outer one-third of this layer. All mitral cells were positive for the alpha1 subunit. In contrast, the alpha3 subunit was restricted to a subpopulation of mitral cells, many of which also expressed calretinin. Likewise, external tufted cells could be subdivided into distinct groups, either singly labeled for the alpha1 or alpha3 subunit or doubly labeled. At the subcellular level, staining for the alpha1 and alpha3 subunits was punctate, forming clusters partially colocalized with gephyrin. However, many alpha1- and alpha3-positive clusters lacked gephyrin, suggesting the existence of either nonsynaptic GABA(A) receptor clusters or synaptic receptors not associated with gephyrin. Quantitative analysis of colocalization among the three markers in the inner EPL, outer EPL, and GL revealed considerable heterogeneity, suggestive of a differential organization of GABA(A) receptor subtypes in the apical and basal dendrites of mitral and tufted cells. Together these results reveal a complex subunit organization of GABA(A) receptors in the olfactory bulb and suggest that mitral and tufted cells participate in different synaptic circuits controlled by distinct GABA(A) receptor subtypes.
The Journal of Comparative Neurology 04/2005; 484(1):121-31. · 3.81 Impact Factor
