GABAB receptors are the G protein-coupled receptors for the main inhibitory neurotransmitter in the brain, gamma-aminobutyric acid (GABA). Molecular diversity in the GABAB system arises from the GABAB1a and GABAB1b subunit isoforms that solely differ in their ectodomains by a pair of sushi repeats that is unique to GABAB1a. Using a combined genetic, physiological, and morphological approach, we now demonstrate that GABAB1 isoforms localize to distinct synaptic sites and convey separate functions in vivo. At hippocampal CA3-to-CA1 synapses, GABAB1a assembles heteroreceptors inhibiting glutamate release, while predominantly GABAB1b mediates postsynaptic inhibition. Electron microscopy reveals a synaptic distribution of GABAB1 isoforms that agrees with the observed functional differences. Transfected CA3 neurons selectively express GABAB1a in distal axons, suggesting that the sushi repeats, a conserved protein interaction motif, specify heteroreceptor localization. The constitutive absence of GABAB1a but not GABAB1b results in impaired synaptic plasticity and hippocampus-dependent memory, emphasizing molecular differences in synaptic GABAB functions.

G-protein-coupled inwardly rectifying Kϩ channels (Kir3 channels) coupled to metabotropic GABA B receptors are essential for the control of neuronal excitation. To determine the distribution of Kir3 channels and their spatial relationship to GABA B 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 ϩ 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.

GABA B receptors are the G-protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the brain. GABA B receptors are abundant on dendritic spines, where they dampen postsynaptic excitability and inhibit Ca 2+ 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 GABA B -mediated inhibition. We found that NMDA application to cultured hippocampal neurons promotes dynamindependent endocytosis of GABA B receptors. NMDA-dependent internalization of GABA B receptors requires activation of Ca 2+ /Calmodulindependent protein kinase II (CaMKII), which associates with GABA B receptors in vivo and phosphorylates serine 867 (S867) in the intracellular C terminus of the GABA B1 subunit. Blockade of either CaMKII or phosphorylation of S867 renders GABA B receptors refractory to NMDA-mediated internalization. Time-lapse two-photon imaging of organotypic hippocampal slices reveals that activation of NMDA receptors removes GABA B receptors within minutes from the surface of dendritic spines and shafts. NMDA-dependent S867 phosphorylation and internalization is predominantly detectable with the GABA B1b 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 GABA B receptors to activate K + channels. Thus, our data support that NMDA receptor activity endocytoses postsynaptic GABA B receptors through CaMKIImediated phosphorylation of S867. This provides a means to spare NMDA receptors at individual glutamatergic synapses from reciprocal inhibition through GABA B receptors.γ-aminobutyric acid | spines | trafficking | synaptic plasticity | GABAB

We report on an optimized method for the in vitro culture of tissue cyst-forming Neospora caninum bradyzoites in Vero cells and the separation of viable parasites from host cells. Treatment of tachyzoite-infected Vero cell cultures with 17 M sodium nitroprusside for 8 days severely scaled down parasite proliferation, led to reduced expression of tachyzoite surface antigens, and induced the expression of the bradyzoite marker NcBAG1 and the cyst wall antigen recognized by the monoclonal antibody MAbCC2. Transmission electron microscopy demonstrated that intracellular parasites were located within parasitophorous vacuoles that were surrounded by a cyst wall-like structure, and the dense granule antigens NcGRA1, NcGRA2, and NcGRA7 were incorporated into the cyst wall. Adhesion-invasion assays employing purified tachyzoites and bradyzoites showed that tachyzoites adhered to, and invaded, Vero cells with higher efficiency than bradyzoites. However, removal of terminal sialic acid residues from either the host cell or the parasite surface increased the invasion of Vero cells by bradyzoites, but not tachyzoites.

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 Ն1 M, 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.