GABAB receptor, localization and regulation

GABAB receptors are G-protein coupled receptors for gamma-amino butyric acid, the main inhibitory neurotransmitter in the brain. Functional GABAB receptors are obligate heterodimers composed of GABAB1 and GABAB2 subunits. The GABAB1 subunit exists in two isoforms, GABAB1a and GABAB1b, that can be differentiated by a pair of sushi domains exclusively located on the ectodomain of GABAB1a. As a consequence, two distinct receptor subtypes, GABAB(1a,2) and GABAB(1b,2), are present in the brain. Depending on their subcellular localization, GABAB receptors exert distinct regulatory effects on synaptic transmission. Presynaptically, GABAB receptors inhibit Ca2+ influx by closing voltage-gated Ca2+-channels therefore regulating neurotransmitter release. Postsynaptically, GABAB receptors activate inwardly rectifying Kir3-type K+-channels leading to hyperpolarisation of the postsynaptic membrane. Recently, it has become clear that GABAB(1a,2) and GABAB(1b,2) receptors convey individual functions, which are, at least in part, related to their distinct subcellular distribution. The aim of this thesis was to gain further insight into the function of GABAB receptors by characterizing their localization at the ultrastructural level in respect to effector channels and subtype composition. Moreover, it was of interest to study the dynamic regulation of GABAB receptors in response to synaptic activity. In the first part of this thesis, the localization of GABAB receptors and Kir3-type effector channels was investigated in the CA1 region of the hippocampus. It could be demonstrated that postsynaptic GABAB receptors colocalize with the Kir3.2 subunit of K+-channels in dendritic spines, but not in dendritic shafts of CA1 pyramidal cells (chapter 6.1.; Kulik et al., 2006). The differential distribution of GABAB1 subunit isoforms at the mossy fiber-CA3 pyramidal neuron synapse was investigated in the second part of this work. Due to the lack of isoform specific antibodies, mice selectively expressing GABAB1a or GABAB1b were used. It could be shown that mainly the GABAB1a subunit isoform contributes to the composition of presynaptic GABAB receptors whereas GABAB1b is the predominant GABAB1 subunit isoform on the postsynaptic side. Electrophysiological recordings were used to assess the contribution of the two different GABAB1 subunit isoforms to functional pre- and postsynaptic receptors in response to pharmacological as well as physiological GABAB receptor activation. The findings illustrate that the spatial segregation of GABAB1 subunit isoforms at mossy fiber terminals is sufficient to produce a strictly subtype–specific response (chapter 6.2.; Guetg et al., 2009). In the third part of this work, a new mouse model containing a GABAB1-eGFP transgene, allowing the visualization of GABAB receptors, was generated. Crossing the GABAB1-eGFP transgene into the GABAB1 deficient background allowed the study of GABAB receptors tagged with a fluorescent protein under expression of endogenous promoter elements. Therefore these mice provide a useful tool to visualize the spatio-temporal distribution of GABAB receptors in vivo and in vitro (chapter 6.3.; Casanova et al., 2009). The dynamic regulation of surface GABAB receptors induced by glutamate was investigated in primary hippocampal neurons and the results are presented in the last part of this thesis. Activation of NMDA receptors resulted in a decrease of surface GABAB receptor levels. This decrease involved Ca2+-dependent activation of CaMKII. A CaMKII phosphorylation site within the cytoplasmic domain of the GABAB1 subunit was identified. Evidence that phosphorylation of this site is essential for the observed effect of NMDA receptor activation on GABAB surface receptors is presented in this thesis. In conclusion, it could be demonstrated that GABAB receptors are dynamically regulated and interact with other receptors and kinases. The results obtained, implicate that activity-dependent regulation of GABAB receptors is potentially involved in the modulation of synaptic strength (chapter 6.4.)

Stochastic models and dynamic measures for the characterization of bistable circuits

During the last few years, a great deal of interest has risen concerning the applications of stochastic methods to several biochemical and biological phenomena. Phenomena like gene expression, cellular memory, bet-hedging strategy in bacterial growth and many others, cannot be described by continuous stochastic models due to their intrinsic discreteness and randomness. In this thesis I have used the Chemical Master Equation (CME) technique to modelize some feedback cycles and analyzing their properties, including experimental data. In the first part of this work, the effect of stochastic stability is discussed on a toy model of the genetic switch that triggers the cellular division, which malfunctioning is known to be one of the hallmarks of cancer. The second system I have worked on is the so-called futile cycle, a closed cycle of two enzymatic reactions that adds and removes a chemical compound, called phosphate group, to a specific substrate. I have thus investigated how adding noise to the enzyme (that is usually in the order of few hundred molecules) modifies the probability of observing a specific number of phosphorylated substrate molecules, and confirmed theoretical predictions with numerical simulations. In the third part the results of the study of a chain of multiple phosphorylation-dephosphorylation cycles will be presented. We will discuss an approximation method for the exact solution in the bidimensional case and the relationship that this method has with the thermodynamic properties of the system, which is an open system far from equilibrium.In the last section the agreement between the theoretical prediction of the total protein quantity in a mouse cells population and the observed quantity will be shown, measured via fluorescence microscopy

The GABAB1a isoform mediates heterosynaptic depression at hippocampal mossy fiber synapses

GABA(B) receptor subtypes are based on the subunit isoforms GABA(B1a) and GABA(B1b), which associate with GABA(B2) subunits to form pharmacologically indistinguishable GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Studies with mice selectively expressing GABA(B1a) or GABA(B1b) subunits revealed that GABA(B(1a,2)) receptors are more abundant than GABA(B(1b,2)) receptors at glutamatergic terminals. Accordingly, it was found that GABA(B(1a,2)) receptors are more efficient than GABA(B(1b,2)) receptors in inhibiting glutamate release when maximally activated by exogenous application of the agonist baclofen. Here, we used a combination of genetic, ultrastructural and electrophysiological approaches to analyze to what extent GABA(B(1a,2)) and GABA(B(1b,2)) receptors inhibit glutamate release in response to physiological activation. We first show that at hippocampal mossy fiber (MF)-CA3 pyramidal neuron synapses more GABA(B1a) than GABA(B1b) protein is present at presynaptic sites, consistent with the findings at other glutamatergic synapses. In the presence of baclofen at concentrations <or=1 microm, both GABA(B(1a,2)) and GABA(B(1b,2)) receptors contribute to presynaptic inhibition of glutamate release. However, at lower concentrations of baclofen, selectively GABA(B(1a,2)) receptors contribute to presynaptic inhibition. Remarkably, exclusively GABA(B(1a,2)) receptors inhibit glutamate release in response to synaptically released GABA. Specifically, we demonstrate that selectively GABA(B(1a,2)) receptors mediate heterosynaptic depression of MF transmission, a physiological phenomenon involving transsynaptic inhibition of glutamate release via presynaptic GABA(B) receptors. Our data demonstrate that the difference in GABA(B1a) and GABA(B1b) protein levels at MF terminals is sufficient to produce a strictly GABA(B1a)-specific effect under physiological conditions. This consolidates that the differential subcellular localization of the GABA(B1a) and GABA(B1b) proteins is of regulatory relevance

A mouse model for visualization of GABA(B) receptors

GABA(B) receptors are the G-protein-coupled receptors for the neurotransmitter gamma-aminobutyric acid (GABA). Receptor subtypes are based on the subunit isoforms GABA(B1a) and GABA(B1b), which combine with GABA(B2) subunits to form heteromeric receptors. Here, we used a modified bacterial artificial chromosome (BAC) containing the GABA(B1) gene to generate transgenic mice expressing GABA(B1a) and GABA(B1b) subunits fused to the enhanced green fluorescence protein (eGFP). We demonstrate that the GABA(B1)-eGFP fusion proteins reproduce the cellular expression patterns of endogenous GABA(B1) proteins in the brain and in peripheral tissue. Crossing the GABA(B1)-eGFP BAC transgene into the GABA(B1) (-/-) background restores pre and postsynaptic GABA(B) functions, showing that the GABA(B1)-eGFP fusion proteins substitute for the lack of endogenous GABA(B1) proteins. Finally, we demonstrate that the GABA(B1)-eGFP fusion proteins replicate the temporal expression patterns of native GABA(B) receptors in cultured neurons. These transgenic mice therefore provide a validated tool for direct visualization of native GABA(B) receptors

Compartment-dependent colocalization of Kir3.2-containing K+ channels and GABA(B) receptors in hippocampal pyramidal cells

G-protein-coupled inwardly rectifying K+ channels (Kir3 channels) coupled to metabotropic GABAB receptors are essential for the control of neuronal excitation. To determine the distribution of Kir3 channels and their spatial relationship to GABAB receptors on hippocampal pyramidal cells, we used a high-resolution immunocytochemical approach. Immunoreactivity for the Kir3.2 subunit was most abundant postsynaptically and localized to the extrasynaptic plasma membrane of dendritic shafts and spines of principal cells. Quantitative analysis of immunogold particles for Kir3.2 revealed an enrichment of the protein around putative glutamatergic synapses on dendritic spines, similar to that of GABA(B1). Consistent with this observation, a high degree of coclustering of Kir3.2 and GABA(B1) was revealed around excitatory synapses by the highly sensitive SDS-digested freeze-fracture replica immunolabeling. In contrast, in dendritic shafts receptors and channels were found to be mainly segregated. These results suggest that Kir3.2-containing K+ channels on dendritic spines preferentially mediate the effect of GABA, whereas channels on dendritic shafts are likely to be activated by other neurotransmitters as well. Thus, Kir3 channels, localized to different subcellular compartments of hippocampal principal cells, appear to be differentially involved in synaptic integration in pyramidal cell dendrites

NMDA receptor-dependent GABAB receptor internalization via CaMKII phosphorylation of serine 867 in GABAB1

GABAB receptors are the G-protein–coupled receptors for GABA, the main inhibitory neurotransmitter in the brain. GABAB receptors are abundant on dendritic spines, where they dampen postsynaptic excitability and inhibit Ca2+ influx through NMDA receptors when activated by spillover of GABA from neighboring GABAergic terminals. Here, we show that an excitatory signaling cascade enables spines to counteract this GABAB-mediated inhibition. We found that NMDA application to cultured hippocampal neurons promotes dynamin-dependent endocytosis of GABAB receptors. NMDA-dependent internalization of GABAB receptors requires activation of Ca2+/Calmodulin-dependent protein kinase II (CaMKII), which associates with GABAB receptors in vivo and phosphorylates serine 867 (S867) in the intracellular C terminus of the GABAB1 subunit. Blockade of either CaMKII or phosphorylation of S867 renders GABAB receptors refractory to NMDA-mediated internalization. Time-lapse two-photon imaging of organotypic hippocampal slices reveals that activation of NMDA receptors removes GABAB receptors within minutes from the surface of dendritic spines and shafts. NMDA-dependent S867 phosphorylation and internalization is predominantly detectable with the GABAB1b subunit isoform, which is the isoform that clusters with inhibitory effector K+ channels in the spines. Consistent with this, NMDA receptor activation in neurons impairs the ability of GABAB receptors to activate K+ channels. Thus, our data support that NMDA receptor activity endocytoses postsynaptic GABAB receptors through CaMKII-mediated phosphorylation of S867. This provides a means to spare NMDA receptors at individual glutamatergic synapses from reciprocal inhibition through GABAB receptors