GABA transporter subtype 1 (GAT1) knock-out (KO) mice display normal reproduction and life span but have reduced body weight (female, Ϫ10%; male, Ϫ20%) and higher body temperature fluctuations in the 0.2-1.5/h frequency range. Mouse GAT1 (mGAT1) KO mice exhibit motor disorders, including gait abnormality, constant 25-32 Hz tremor, which is aggravated by flunitrazepam, reduced rotarod performance, and reduced locomotor activity in the home cage. Open-field tests show delayed exploratory activity, reduced rearing, and reduced visits to the central area, with no change in the total distance traveled. The mGAT1 KO mice display no difference in acoustic startle response but exhibit a deficiency in prepulse inhibition. These open-field and prepulse inhibition results suggest that the mGAT1 KO mice display mild anxiety or nervousness. The compromised GABA uptake in mGAT1 KO mice results in an increased GABA A receptor-mediated tonic conductance in both cerebellar granule and Purkinje cells. The reduced rate of GABA clearance from the synaptic cleft is probably responsible for the slower decay of spontaneous IPSCs in cerebellar granule cells. There is little or no compensatory change in other proteins or structures related to GABA transmission in the mGAT1 KO mice, including GAT1-independent GABA uptake, number of GABAergic interneurons, and GABA A -, vesicular GABA transporter-, GAD65-, and GAT3-immunoreactive structures in cerebellum or hippocampus. Therefore, the excessive extracellular GABA present in mGAT1 KO mice results in behaviors that partially phenocopy the clinical side effects of tiagabine, suggesting that these side effects are inherent to a therapeutic strategy that targets the widely expressed GAT1 transporter system.

GABA transporter subtype 1 (GAT1) molecules were counted near GABAergic synapses, to a resolution of approximately 0.5 microm. Fusions between GAT1 and green fluorescent protein (GFP) were tested in heterologous expression systems, and a construct was selected that shows function, expression level, and trafficking similar to that of wild-type (WT) GAT1. A strain of knock-in mice was constructed that expresses this mGAT1-GFP fusion in place of the WT GAT1 gene. The pattern of fluorescence in brain slices agreed with previous immunocytochemical observations. [3H]GABA uptake, synaptic electrophysiology, and subcellular localization of the mGAT1-GFP construct were also compared with WT mice. Quantitative fluorescence microscopy was used to measure the density of mGAT1-GFP at presynaptic structures in CNS preparations from the knock-in mice. Fluorescence measurements were calibrated with transparent beads and gels that have known GFP densities. Surface biotinylation defined the fraction of transporters on the surface versus those in the nearby cytoplasm. The data show that the presynaptic boutons of GABAergic interneurons in cerebellum and hippocampus have a membrane density of 800-1300 GAT1 molecules per square micrometer, and the axons that connect boutons have a linear density of 640 GAT1 molecules per micrometer. A cerebellar basket cell bouton, a pinceau surrounding a Purkinje cell axon, and a cortical chandelier cell cartridge carry 9000, 7.8 million, and 430,000 GAT1 molecules, respectively; 61-63% of these molecules are on the surface membrane. In cultures from hippocampus, the set of fluorescent cells equals the set of GABAergic interneurons. Knock-in mice carrying GFP fusions of membrane proteins provide quantitative data required for understanding the details of synaptic transmission in living neurons.

A common inflammatome signature, as well as disease-specific expression patterns, was identified from 11 different rodent inflammatory disease models. Causal regulatory networks and the drivers of the inflammatome signature were uncovered and validated.

ATP-gated P2X2 receptors are widely expressed in neurons, but the cellular effects of receptor activation are unclear. We engineered functional green fluorescent protein (GFP)-tagged P2X 2 receptors and expressed them in embryonic hippocampal neurons, and report an approach to determining functional and total receptor pool sizes in living cells. ATP application to dendrites caused receptor redistribution and the formation of varicose hot spots of higher P2X2-GFP receptor density. Redistribution in dendrites was accompanied by an activation-dependent enhancement of the ATP-evoked current. Substate-specific mutant T18A P2X2-GFP receptors showed no redistribution or activation-dependent enhancement of the ATP-evoked current. Thus fluorescent P2X 2-GFP receptors function normally, can be quantified, and reveal the dynamics of P2X 2 receptor distribution on the seconds time scale.ion channel ͉ ATP ͉ filopodia C ationic P2X receptors mediate the ''fast'' milliseconds time scale actions of ATP in the nervous system (1, 2). The identity of most natively expressed P2X receptors is unclear, but many neurons express P2X 2 mRNA, P2X 2 proteins, and functional P2X 2 -like receptors (2). For example, ATP mediates synaptic transmission in a portion of CA1 neurons (3), and postnatal hippocampal neurons express P2X receptors, which include P2X 2 subunits (3-7). Moreover, cytosolic ATP concentration is 1-5 mM, and ATP released during tissue damage activates neuronal P2X receptors in the periphery (1). ATP released as a synaptic transmitter and during ischemia of brain neurons may contribute to pathophysiology, but there are no available data on the cellular consequences of P2X 2 receptor activation or on the dynamic aspects of P2X 2 receptor distribution in brain neurons.This study used P2X 2 receptors tagged with green fluorescent protein (GFP) in a quantitative method to study receptors expressed with recombinant Sindbis virus in embryonic hippocampal neurons. We report (i) the properties of functional GFP-tagged P2X 2 receptors, (ii) an optical and electrophysiological approach to measuring receptor numbers in living cells, and (iii) the cellular effects of P2X receptor activation. Materials and MethodsMolecular Biology. By PCR the P2X 2 stop codon was removed and the FLAG (f) epitope was inserted in frame with the P2X 2 cDNA cDNA (9). In the same PCR we inserted an XhoI site in the DNA. We generated GFP37 (10) with a XhoI site before the start codon and subcloned it into P2X 2 -f between the XhoI site 3Ј of the FLAG epitope and HindIII in the pcDNA3 polylinker to yield P2X 2 -GFP. The P2X 2 -f-GFP fragment was inserted into pSinRep5 between the StuI and ApaI sites, and infective Sindbis particles were generated with the use of the Sindbis Expression System (http:͞͞www.invitrogen.com͞). Site-directed mutagenesis was performed on the cDNAs with the use of synthetic oligonucleotides to generate K69A and T18A mutants (Quick Change; Stratagene).Electrophysiology and Imaging. All cell preparations, twoelectrode voltage-clamp record...