Immunofluorescence in brain sections: simultaneous detection of presynaptic and postsynaptic proteins in identified neurons

Gasser, Edith M. Schneider; Straub, Carolin J.; Panzanelli, Patrizia; Weinmann, Oliver; Sassoè‐Pognetto, Marco; Fritschy, Jean‐Marc

doi:10.1038/nprot.2006.265

Cited by 128 publications

(137 citation statements)

References 38 publications

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“…Double and triple immunofluorescence was performed on cryostat sections as described in ref. 48. See SI Materials and Methods for antibody specification and characterization.…”

Section: Methodsmentioning

confidence: 99%

Synapse formation and clustering of neuroligin-2 in the absence of GABA _A receptors

Patrizi

Scelfo

Viltono

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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114

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GABAergic synapses are crucial for brain function, but the mechanisms underlying inhibitory synaptogenesis are unclear. Here, we show that postnatal Purkinje cells (PCs) of GABAA␣1 knockout (KO) mice express transiently the ␣3 subunit, leading to the assembly of functional GABAA receptors and initial normal formation of inhibitory synapses, that are retained until adulthood. Subsequently, down-regulation of the ␣3 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 GABAA receptors and the scaffold molecule gephyrin, but is absent from mismatched synapses, despite innervation by GABAergic axons. Our data indicate that GABAA 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.gephyrin ͉ Purkinje cell ͉ synaptogenesis T he identification of the factors that are critical for the assembly, maturation, and stabilization of synaptic connections remains a central puzzle in developmental neuroscience. Studies on cultured neurons have been invaluable for uncovering the molecular mechanisms of synaptogenesis (1, 2). Unfortunately, such studies can be influenced by varying experimental conditions, and their results often have not been replicated by in vivo analyses of gene knockout (KO) mice (3). As an alternative approach, gene deletion techniques allow investigating the in vivo function of genes that are believed to be critical for synapse formation (4-8). However, the premature death of newborn mutant mice has often prevented long-term analyses of synapse maturation and function. In the present study, we took advantage of a KO model that survives up to adulthood without a major phenotype, despite extensive loss of GABA-mediated function in specific populations of neurons.Specifically, we investigated the in vivo effects of a selective silencing of GABAergic transmission on the formation and longterm stability of GABAergic synapses, which provide the major inhibitory control over neuronal activity in the brain (9). By analogy to glutamatergic synapses, assembly of GABAergic synapses is believed to involve selective trans-synaptic interactions between adhesion molecules and cytosolic interactions with scaffolding proteins (2). Neuroligin-2 (NL2) is a postsynaptic adhesion molecule that localizes at GABAergic synapses and triggers synapse formation by interacting with presynaptic neurexins (10, 11). NL2 belongs to a family of related proteins also comprising NL1, NL3, and NL4 (12). Importantly, NL1 is targeted to glutamatergic synapses (13,14), suggesting that different NLs may play an important role in specifying distinct types of synapse and in determining a balance between n...

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“…Double and triple immunofluorescence was performed on cryostat sections as described in ref. 48. See SI Materials and Methods for antibody specification and characterization.…”

Section: Methodsmentioning

confidence: 99%

Synapse formation and clustering of neuroligin-2 in the absence of GABA _A receptors

Patrizi

Scelfo

Viltono

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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114

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“…Immunohistochemistry of frozen coronal sections (20 μm) of the adult mouse brain was performed as described by Schneider Gasser et al (33) by using the primary antibodies against ephrinA5 (Abcam), EphA4, EphA5 (for both, Abcam or Santa Cruz), pErk1/2 (Santa Cruz), GAD67, TH (Millipore), gephyrin (Synaptic Systems), and GFAP (Sigma Aldrich). The secondary antibodies used were Alexa488-or Alexa594-conjugated goat IgG (Molecular Probes), and specific immunoreactivity was confirmed by performing immunohistochemical analysis without addition of the primary antibody.…”

Section: Methodsmentioning

confidence: 99%

Pathway-specific engagement of ephrinA5-EphA4/EphA5 system of the substantia nigra pars reticulata in cocaine-induced responses

Kimura

Hikida

Yawata

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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The nucleus accumbens (NAc) serves as a key neural substrate that controls acute and adaptive behavioral responses to cocaine administration. In this circuit, inputs from the NAc are transmitted through two parallel pathways, named the direct and indirect pathways, and converge at the substantia nigra pars reticulata (SNr). Our previous study using reversible neurotransmission blocking (RNB) of each pathway revealed that the dual stimulation of the SNr by both pathways is necessary for the acute response, but that the direct pathway predominantly controls the adaptive response to repeated cocaine administration. This study aimed at exploring the pathway-specific mechanism of cocaine actions at the convergent SNr. We examined a genome-wide expression profile of the SNr of three types of experimental mice: the direct pathway-blocked D-RNB mice, the indirect pathway-blocked I-RNB mice, and wild-type mice. We identified the up-regulation of ephrinA5, EphA4, and EphA5 specific to D-RNB mice during both acute and adaptive responses to cocaine administration. The activation by EphA4 and EphA5 in the SNr of wild-type mice by use of the immunoadhesin technique suppressed the adaptive response to repeated cocaine administration. Furthermore, cocaine exposure stimulated the phosphorylation of Erk1/2 in ephrinA5-expressing SNr cells in a direct pathway-dependent manner. The results have demonstrated that the ephrinA5-EphA4/EphA5 system plays an important role in the direct pathway-dependent regulation of the SNr in both acute and adaptive cocaine responses and would provide valuable therapeutic targets of cocaine addiction.basal ganglia | drug addiction | Eph-ephrin signaling | gene regulation | transmission blocking T he basal ganglia are the key neural substrates that control motor balance and reward-based and aversive learning (1, 2). Dysfunction of the basal ganglia leads to devastating neurological disorders, such as Parkinson disease and drug addiction (3-5). The projection neurons in the striatum and the nucleus accumbens (NAc), the ventral part of the striatum, are GABAcontaining medium-sized spiny neurons, which are divided into two subpopulations: striatonigral neurons in the direct pathway and striatopallidal neurons in the indirect pathway (1, 3, 6). The inputs of these two pathways converge at the substantia nigra pars reticulata (SNr) and control the dynamic balance of the basal ganglia-thalamocortical circuitry (1, 7). Cocaine and other psychostimulants massively increase dopamine levels in the NAc and the striatum and induce abnormal behavioral responses both acutely and chronically (8). We previously developed a genemanipulating technique that allows separate and reversible neurotransmission blocking (RNB) of the direct pathway (D-RNB mice) and the indirect pathway (I-RNB mice) in vivo (9). The use of this technique revealed the distinct regulatory function of the two pathways in acute and chronic responses to cocaine exposure (9). Blockade of the direct pathway abrogates the acute response and then markedl...

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“…Brains were postfixed overnight, dehydrated in 20% sucrose in 0.1 M PBS overnight, cut in 30 m sections, and stored in cryoprotective solution. For analysis of gephyrin distribution (Schneider Gasser et al, 2006), 10 rats (LPS, n ϭ 6; vehicle, n ϭ 4) were anesthetized and decapitated. Brains were dissected and placed in ice-cold aCSF, cut in transverse 300-m-thick sections, placed in gassed aCSF for 20 min and in PFA for 10 min, rinsed in PBS, dehydrated in 30% sucrose in 0.1 M PBS, cut in 14 m sections, and stored at Ϫ20°C for at least 1 h.…”

Section: Methodsmentioning

confidence: 99%

Inflammation Regulates Functional Integration of Neurons Born in Adult Brain

Jakubs¹,

Bonde²,

Iosif³

et al. 2008

J. Neurosci.

132

117

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Inflammation influences several steps of adult neurogenesis, but whether it regulates the functional integration of the new neurons is unknown. Here, we explored, using confocal microscopy and whole-cell patch-clamp recordings, whether a chronic inflammatory environment affects the morphological and electrophysiological properties of new dentate gyrus granule cells, labeled with a retroviral vector encoding green fluorescent protein. Rats were exposed to intrahippocampal injection of lipopolysaccharide, which gave rise to longlasting microglia activation. Inflammation caused no changes in intrinsic membrane properties, location, dendritic arborization, or spine density and morphology of the new cells. Excitatory synaptic drive increased to the same extent in new and mature cells in the inflammatory environment, suggesting increased network activity in hippocampal neural circuitries of lipopolysaccharide-treated animals. In contrast, inhibitory synaptic drive was more enhanced by inflammation in the new cells. Also, larger clusters of the postsynaptic GABA A receptor scaffolding protein gephyrin were found on dendrites of new cells born in the inflammatory environment. We demonstrate for the first time that inflammation influences the functional integration of adult-born hippocampal neurons. Our data indicate a high degree of synaptic plasticity of the new neurons in the inflammatory environment, which enables them to respond to the increase in excitatory input with a compensatory upregulation of activity and efficacy at their afferent inhibitory synapses.

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Immunofluorescence in brain sections: simultaneous detection of presynaptic and postsynaptic proteins in identified neurons

Cited by 128 publications

References 38 publications

Synapse formation and clustering of neuroligin-2 in the absence of GABA _A receptors

Synapse formation and clustering of neuroligin-2 in the absence of GABA _A receptors

Pathway-specific engagement of ephrinA5-EphA4/EphA5 system of the substantia nigra pars reticulata in cocaine-induced responses

Inflammation Regulates Functional Integration of Neurons Born in Adult Brain

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Immunofluorescence in brain sections: simultaneous detection of presynaptic and postsynaptic proteins in identified neurons

Cited by 128 publications

References 38 publications

Synapse formation and clustering of neuroligin-2 in the absence of GABA A receptors

Synapse formation and clustering of neuroligin-2 in the absence of GABA A receptors

Pathway-specific engagement of ephrinA5-EphA4/EphA5 system of the substantia nigra pars reticulata in cocaine-induced responses

Inflammation Regulates Functional Integration of Neurons Born in Adult Brain

Contact Info

Product

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Synapse formation and clustering of neuroligin-2 in the absence of GABA _A receptors

Synapse formation and clustering of neuroligin-2 in the absence of GABA _A receptors