Clusters of GABA<sub>A</sub> receptors on cultured hippocampal cells correlate only partially with functional synapses

Kannenberg, Kai; Sieghart, Werner; Réuter, H.

doi:10.1046/j.1460-9568.1999.00533.x

Cited by 47 publications

(34 citation statements)

References 52 publications

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“…Consistent with previous reports (20,25,26,39,53), our immunocytochemical data clearly demonstrate that, in addition to synaptic receptors, also extrasynaptic ones are arranged in clusters and that they become uniformly distributed throughout the cell surface after nocodazole treatment. In agreement with other groups (39, 54 -56), we have found that extrasynaptic receptors in the hippocampus include the ␦ and ␥ 2 subunits.…”

Section: Discussionsupporting

confidence: 92%

Clustering of Extrasynaptic GABAA Receptors Modulates Tonic Inhibition in Cultured Hippocampal Neurons

Petrini

Marchionni

Zacchi

et al. 2004

Journal of Biological Chemistry

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Tonic inhibition plays a crucial role in regulating neuronal excitability because it sets the threshold for action potential generation and integrates excitatory signals. Tonic currents are known to be largely mediated by extrasynaptic ␥-aminobutyric acid type A (GABA A ) receptors that are persistently activated by submicromolar concentrations of ambient GABA. We recently reported that, in cultured hippocampal neurons, the clustering of synaptic GABA A receptors significantly affects synaptic transmission (Petrini, E. M., Zacchi, P., Barberis, A., Mozrzymas, J. W., and Cherubini, E. (2003) J. Biol. Chem. 278, 16271-16279). In this work, we demonstrated that the clustering of extrasynaptic GABA A receptors modulated tonic inhibition. Depolymerization of the cytoskeleton with nocodazole promoted the disassembly of extrasynaptic clusters of ␦ and ␥ 2 subunitcontaining GABA A receptors. This effect was associated with a reduction in the amplitude of tonic currents and diminished shunting inhibition. Moreover, diffuse GABA A receptors were less sensitive to the GAT-1 inhibitor NO-711 and to flurazepam. Quantitative analysis of GABA-evoked currents after prolonged exposure to submicromolar concentrations of GABA and model simulations suggest that clustering affects the gating properties of extrasynaptic GABA A receptors. In particular, a larger occupancy of the singly and doubly bound desensitized states can account for the modulation of tonic inhibition recorded after nocodazole treatment. Moreover, comparison of tonic currents recorded during spontaneous activity and those elicited by exogenously applied low agonist concentrations allows estimation of the concentration of ambient GABA. In conclusion, receptor clustering appears to be an additional regulating factor for tonic inhibition.Similar to many other neurotransmitter receptors, ␥-aminobutyric acid type A (GABA A ) 1 receptors are localized at synaptic and extrasynaptic levels. Whereas synaptic GABA A receptors are involved in phasic inhibition (1), extrasynaptic ones are responsible for tonic inhibition (2-9). Tonic inhibition is due to persistent inhibitory conductance that contributes to "signal integration" in the brain since it sets the threshold for action potential generation (10, 11) and shunts excitatory synaptic inputs (2,(12)(13)(14)(15). This conductance is maintained by "ambient" GABA, which represents the amount of neurotransmitter present in the extracellular space. Ambient GABA originates from spillover of the neurotransmitter released at neighboring synapses (3, 5, 11), from astrocytes (16, 17), or from non-vesicular release (18,19). Tonic inhibition has been well characterized in the cerebellum, where ␣ 6 subunit-containing receptors act as high affinity sensors for GABA (4,11,20,21). Persistent GABA conductance has also been identified in other brain regions, including the hippocampus (6,8,9,22,23). However, in this structure, the subunit composition of the receptors involved has not been fully elucidated. In the past years, immunocytochemi...

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Section: Discussionsupporting

confidence: 92%

Clustering of Extrasynaptic GABAA Receptors Modulates Tonic Inhibition in Cultured Hippocampal Neurons

Petrini

Marchionni

Zacchi

et al. 2004

Journal of Biological Chemistry

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“…S1I) and 65.3 ± 5.2% were apposed to FM4-64 labeled presynaptic terminals (Fig. S1J, in agreement with previous reports for the proportion of native GABA A R clusters found opposite active FM dye-labeled presynaptic terminals) (16,17). However, only 9.1 ± 1.3% were found apposed to excitatory synapses labeled with homer1 (Fig.…”

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confidence: 91%

NMDA receptors regulate GABA _A receptor lateral mobility and clustering at inhibitory synapses through serine 327 on the γ2 subunit

Muir¹,

Arancibia-Cárcamo²,

MacAskill³

et al. 2010

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

134

218

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Modification of the number of GABA A receptors (GABA A Rs) clustered at inhibitory synapses can regulate inhibitory synapse strength with important implications for information processing and nervous system plasticity and pathology. Currently, however, the mechanisms that regulate the number of GABA A Rs at synapses remain poorly understood. By imaging superecliptic pHluorin tagged GABA A R subunits we show that synaptic GABA A R clusters are normally stable, but that increased neuronal activity upon glutamate receptor (GluR) activation results in their rapid and reversible dispersal. This dispersal correlates with increases in the mobility of single GABA A Rs within the clusters as determined using singleparticle tracking of GABA A Rs labeled with quantum dots. GluRdependent dispersal of GABA A R clusters requires Ca 2+ influx via NMDA receptors (NMDARs) and activation of the phosphatase calcineurin. Moreover, the dispersal of GABA A R clusters and increased mobility of individual GABA A Rs are dependent on serine 327 within the intracellular loop of the GABA A R γ2 subunit. Thus, NMDAR signaling, via calcineurin and a key GABA A R phosphorylation site, controls the stability of synaptic GABA A Rs, with important implications for activitydependent control of synaptic inhibition and neuronal plasticity.ion channels | plasticity | trafficking | diffusion | calcineurin S ynaptic inhibition plays a critical role in regulating neuronal excitability and information processing in the brain. The number of GABA A receptors (GABA A Rs) in the surface membrane and at synaptic sites is an important determinant of inhibitory synapse strength (1), but the mechanisms that rapidly control synaptic GABA A R number and stability remain poorly understood. Activation of Ca 2+ -permeable ionotropic glutamate receptors (GluRs) during plasticity and in pathology can result in down-modulation of inhibitory synapse strength and GABA A R function (2-5) but the molecular and cellular mechanisms underlying GluR-dependent changes in the strength of GABAergic inhibition remain unclear.A major mechanism for modulating GABA A R activity is the direct phosphorylation of residues within the intracellular loops of GABA A R subunits, which can regulate synaptic inhibition, GABA A R channel kinetics, and trafficking (6-9). The rapid movement of neurotransmitter receptors (including GABA A Rs) (10-12) into and out of synapses has also recently emerged as an important mechanism for regulating synaptic strength (13). However, whether GABA A R phosphorylation can directly regulate the synaptic stability of GABA A Rs and their lateral diffusion and movement into and out of synapses is unknown.Here, by live cell imaging of surface GABA A R clusters with pHsensitive superecliptic pHluorin (SEP) and single GABA A Rs with quantum dots (QDs), we investigate the mechanisms that regulate activity-dependent control of the lateral diffusion, clustering, and stability of GABA A Rs at inhibitory synapses. We find that Ca 2+ entry through NMDA receptors (NMDARs) l...

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“…GABA A receptors can also cluster at glutamatergic terminals in mass hippocampal cultures that contain many more synapses than autapses (Kannenberg et al, 1999;Christie et al, 2002). It is unclear whether presynaptically silent GABA synapses also occur in vivo, although synapses that colocalize postsynaptic receptors for two fast neurotransmitters have been reported in slice preparations (Jonas et al, 1998;Jo and Schlichter, 1999;Walker et al, 2001).…”

Section: Discussionmentioning

confidence: 99%

Presynaptically Silent GABA Synapses in Hippocampus

Bekkers

2005

J. Neurosci.

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Mammalian central synapses commonly specialize in one fast neurotransmitter, matching the content of their presynaptic vesicles with the appropriate receptors in their postsynaptic membrane. Here, I show that hippocampal cultures contain autaptic glutamatergic synapses that contravene this rule: in addition to postsynaptic glutamate receptors, they also express clusters of functional postsynaptic GABA A receptors yet lack presynaptic GABA. Hence, these synapses are presynaptically silent with respect to GABA. They can be unsilenced by loading GABA into presynaptic vesicles by endocytosis, after which a postload IPSC appears. This IPSC is similar to native IPSCs recorded from GABAergic interneurons in the same cultures. Thus, these "mistargeted" GABA A receptors, which apparently lack a signal that confers synaptic specificity, function almost normally. After GABA loading, glutamatergic miniature postsynaptic currents acquire a slow tail that is mediated by GABA A receptors, showing that synaptic vesicles can accommodate both the usual concentration of native glutamate and a saturating concentration of loaded GABA. After brief Ca 2ϩ -dependent exocytosis, endocytosis of GABA can proceed in low-Ca 2ϩ external solution. The amplitude of the postload IPSC declines exponentially with repetitive stimulation as the endocytosed GABA passes through the presynaptic vesicle cycle and is depleted. Hence, by using GABA as an exogenous but physiological tracer, the properties of these presynaptically silent synapses can provide novel insights into the content and cycling of vesicles in presynaptic terminals.

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Clusters of GABA_A receptors on cultured hippocampal cells correlate only partially with functional synapses

Cited by 47 publications

References 52 publications

Clustering of Extrasynaptic GABAA Receptors Modulates Tonic Inhibition in Cultured Hippocampal Neurons

Clustering of Extrasynaptic GABAA Receptors Modulates Tonic Inhibition in Cultured Hippocampal Neurons

NMDA receptors regulate GABA _A receptor lateral mobility and clustering at inhibitory synapses through serine 327 on the γ2 subunit

Presynaptically Silent GABA Synapses in Hippocampus

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Clusters of GABAA receptors on cultured hippocampal cells correlate only partially with functional synapses

Cited by 47 publications

References 52 publications

Clustering of Extrasynaptic GABAA Receptors Modulates Tonic Inhibition in Cultured Hippocampal Neurons

Clustering of Extrasynaptic GABAA Receptors Modulates Tonic Inhibition in Cultured Hippocampal Neurons

NMDA receptors regulate GABA A receptor lateral mobility and clustering at inhibitory synapses through serine 327 on the γ2 subunit

Presynaptically Silent GABA Synapses in Hippocampus

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Clusters of GABA_A receptors on cultured hippocampal cells correlate only partially with functional synapses

NMDA receptors regulate GABA _A receptor lateral mobility and clustering at inhibitory synapses through serine 327 on the γ2 subunit