The targeting construct for the striatum-specific Cre mice was made with a genomic sequence containing exons 17-19 of the RGS9 gene from clones that were generously provided by the laboratory of Eric J. Nestler (1). Between the translational stop codon and transcriptional termination sequence within exon 19, a cassette was placed that contained an internal ribosome entry site (IRES), a coding region for a nuclear-localized Cre protein, and an SV-40 poly(A) sequence. This was then followed by an FRT-flanked PGK-neo-poly(A) cassette. The NMDAR1-loxP targeting construct contained loxP sequences flanking exons 9 and 10 of the NMDAR1 gene and was described previously (2). A PGK-neo cassette without a polyA sequence was inserted after the second loxP sequence in intron 10.

Stem cell transfections with the targeting vectors, Southern blot analyses using external probes, and generation of chimeras and germline-transmitted RGS9-cre knockin mice and NMDAR1-loxP heterozygous mice were performed as described previously (3). The PGK-neo cassette in the RGS9-cre mice was removed by crossing them with FLP mice (stock no. 003946; The Jackson Laboratory). Both lines were mated to obtain regional knockout (RGS9-cre+/- NMDAR1-loxP-/-) and control (RGS9-cre+/- NMDAR1-loxP+/- or RGS9-cre+/+ NMDAR1-loxP-/-) mice. Animals were genotyped using NMDAR1 primers and cre primers described elsewhere (4). These mice had a mixed genetic background of C57BL/6, 129/Sv, and BALB/c. Mice were housed in a facility that conforms to National Institutes of Health guidelines, and the animal protocols described in this paper were approved by the Division of Animal Resources at the University of Illinois.

To confirm the location of Cre expression of the RGS9-cre mice, germline-transmitted RGS9-cre mice were mated with ROSA26 reporter mice (5), purchased from The Jackson Laboratory (strain no. 3309). Progenies were genotyped using the ROSA primers described in the original report on these mice (5). Mice carrying RGS9-cre and the ROSA2 reporter genes at postnatal day (P) 8-P90 were stained for b-galactosidase activity as described previously (2). Briefly, animals were perfused with ice-cold 100 mM PBS, followed by 4% paraformaldehyde in 100 mM PBS. The brains were removed and postfixed for 15 min. Brains were washed three times with 100 mM phosphate buffer, pH 7.3, 2 mM MgCl₂, 0.01% sodium deoxycholate, and 0.02% Nonidet P-40 and cryoprotected in 30% sucrose overnight. Brains were sectioned (50 mm), postfixed in 0.2% paraformaldehyde for 10 min, washed with PBS, and stained overnight at 37°C with X-gal staining solution [1 mg/ml X-gal, 4 mM K₄Fe(CN)₆, 4 mM K₃Fe(CN)₆, and 2 mM MgCl₂ in 100 mM phosphate buffer, pH 7.5].

hybridization was performed using the protocol from Bessert and Skoff (6) with few modifications. The RNA probe template of NMDAR1 (7) was labeled using a digoxigenin RNA labeling kit with T3 polymerase (Roche Applied Science) according to the manufacturer's protocol. Animals were perfused with 0.9% NaCl solution followed by 4% paraformaldehyde in 100 mM PBS, pH 7.4. Brains were soaked in paraformaldehyde overnight and transferred to and soaked for 16-18 h in 30% sucrose in 100 mM PBS. Sections were made on a sliding microtome. Brain sections were washed with 100 mM PBS and treated with 0.3% Triton X-100 in 100 mM PBS followed by proteinase K (0.001% proteinase K in 50 mM Tris•HCl and 5 mM EDTA). Sections were fixed with 4% paraformaldehyde in 100 mM PBS. Endogenous alkaline phosphatase was eliminated with a 200-mM HCl wash of the sections. Sections were then treated with 0.25% acetic anhydride in Tris•HCl and 0.9% NaCl. Four prehybridization buffers were used that contained 4´ SSC, 50% formamide, 0.25 mg/ml each of salmon sperm DNA and yeast tRNA, 0.1 mM DTT, 5´ Denhardt's solution, and 0.05, 0.1, 0.15, or 0.2 g/ml dextran sulfate. To prevent tissue sections from curling, they were first incubated in prehybridization buffer with the lowest concentrations of dextran sulfate and subsequently incubated in buffer with increasing amounts of dextran sulfate for 15 min each at 55°C. Sections were then hybridized in buffer containing the highest concentration of dextran sulfate with labeled probes for 18 h at 50°C. They were then washed in 5´ SSC and then in 4´ SSC with 50% formamide at 60°C. Sections were treated with 1 mg/ml RNaseA in 2´ SSC and washed with 1´ SSC with10 mM DTT and subsequently with 0.5´ SSC with 10 mM DTT. Sections were blocked with 100 mM Tris•HCl, pH 7.6, and 1´ Denhardt's solution. Detection of digoxigenin-labeled nucleic acids was performed using a digoxigenin nucleic acid detection kit (Roche Applied Science) according to the manufacturer's instructions.

Patch pipettes were filled with an internal solution containing 120 mM cesium methane sulfonate, 5 mM NaCl, 10 mM tetraethylammonium chloride, 10 mM Hepes, 4 mM lidocaine N-ethyl bromide, 1.1 mM EGTA, 4 mM Mg-ATP, and 0.3 mM Na-GTP, pH-adjusted to 7.2 with CsOH and osmolarity set to 298 mOsm with sucrose. For current-clamp recordings, pipettes were filled with an internal solution containing 150 mM K-gluconate, 2 mM MgCl₂, 1.1 mM EGTA, 10 mM Hepes, 3 mM Na-ATP, and 0.2 mM Na-GTP, pH-adjusted to 7.2 with KOH and osmolarity set to 315 mOsm with sucrose.

Stimulation was delivered by a Master-8 stimulator and optical stimulus isolation unit (A.M.P.I., Jerusalem, Israel) through a bipolar twisted tungsten wire that was placed in the white matter above the striatum. Stimulus pulse duration ranged from 0.04 to 0.09 ms, and stimulation intensity ranged from 0.4 to 1 mA. With pipette resistance ranging from 2.5 to 6 MW, recordings were made with the aid of differential interference contrast-enhanced visual guidance. Recordings were only accepted if the series resistance, which was not compensated, remained below 20 MW and stable throughout. Synaptic currents and membrane potentials were recorded with an Axopatch 1D amplifier (Axon Instruments, Foster City, CA) filtered at 5 kHz and digitized at 10 kHz. The results were analyzed using peak detection software in pCLAMP8 (Molecular Devices, Union City, CA).

To measure AMPAR- and NMDAR-mediated synaptic current, neurons were voltage-clamped at +40 mV, and the NMDAR antagonist dl-2-amino-5-phosphonopentanoic acid (APV, 50 mM) was applied to the bath after a stable baseline had been established. The EPSCs recorded after 5 min of APV application are used as a measure of AMPAR-mediated currents, and the amplitudes of the NMDAR-mediated currents are obtained by subtracting the AMPAR-mediated response from the baseline response.

Pipettes with resistances ranging from 2 to 4 MW were pulled from borosilicate glass capillaries on a Flaming-Brown micropipette puller (Sutter Instruments, Novato, CA) and filled with 1 M NaCl. Stimulation, given at 0.05 Hz, was delivered as described above. The recording electrode was placed in the dorsomedial striatum, as described previously (8). Stimulation intensity was adjusted to yield an evoked population spike amplitude approximately one-half the amplitude of the maximal evoked response. Stimulus pulse duration ranged from 0.02 to 0.05 ms, and stimulation intensity ranged from 0.5 to 0.9 mA. Data were filtered (highpass, 0.1 Hz; lowpass 3 kHz) and then amplified and digitized using an Axoclamp 1D amplifier and Digidata 1322 interface (Axon Instruments/Molecular Devices, Sunnyvale, CA). To induce LTP, four trains of 100-Hz stimulation were delivered 10 s apart at an intensity that evoked the maximal population spike. The induction protocol for striatal LTD is the same as that for striatal LTP; the only difference is that LTD is recorded from the dorsolateral, or sensorimotor, striatum.

Parasagittal slices containing the ventral striatum (≈350 mm) were prepared from 1.5- to 3-month-old CT and KO mice. Slices were maintained in an incubation chamber with artificial cerebrospinal fluid (aCSF) buffer for at least 1 h. aCSF was made of 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 26 mM NaHCO_3, 1.3 mM MgSO₄, 2.4 mM CaCl₂, and mM 0.05 picrotoxin (Sigma-Aldrich, St. Louis, MO) and equilibrated with 95% O₂/5% CO₂ (9). An individual slice was then transferred to a submerge-recording chamber and continuously perfused with aCSF at a rate of ≈2 ml/min at room temperature (≈24°C) (10). Extracellular field potentials were recorded in the NAc core region close to the anterior commissure with a glass electrode filled with 1 M NaCl. A bipolar tungsten stimulating electrode was placed within the NAc »200 mm away from the recording electrode. Field potentials were evoked by applying currents (100-ms duration) at 30-s intervals (11). To induce LTD, a 10-Hz stimulation was applied for 10 min (12). Recordings were performed with Axopatch-1D. Signals were filtered at 2 kHz and sampled at 10 kHz. Data were collected and analyzed with pCLAMP9 (Axon Instruments). The values for LTD were taken »20 min after 10-Hz stimulation. DL-APV (Sigma-Aldrich) was used at a concentration of 75 mM.

Corticostriatal slices, prepared as described above for dorsal striatum field potential recording, were incubated for 20 min in normal aCSF in the presence of 0.6 mg/ml pronase (Calbiochem/EMD Bioscience, Darmstadt, Germany) at 37°C. Slices were removed to modified aCSF containing 124 mM NaCl, 4.5 mM KCl, 0.5 mM CaCl₂, 4 mM MgCl₂, 26 mM NaHCO₃, 1.2 mM NaH₂PO₄, and 10 mM d-glucose for trituration. The cortex and white matter were then removed from the slices, leaving only dorsal striatum. Striatal tissue was then triturated through a series of pipette tips of decreasing size in a 35-mm diameter culture dish and allowed to settle to the dish bottom for at least 10 min before recording.

Whole-cell patch-clamp recordings were performed on the stage of an inverted Nikon (Melville, NY) TE200 microscope at room temperature. Cells were constantly superfused with an external solution containing 150 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl₂, 0.01 mM glycine, 10 mM Hepes, 4 mM QX-314, and 10 mM d-glucose, pH-adjusted to 7.4 with NaOH and osmolarity adjusted to &asymp;340 mOsM with sucrose. Recordings were made from neurons with medium-sized somata (4-10 pF whole-cell capacitance) under direct visualization using patch pipettes filled with 100 mM N-methyl-d-glucamine, 100 mM CH₃SO₃H, 40 mM CsF, 1 MgCl₂, 10 mM Hepes, and 5 mM EGTA, pH-adjusted with CsOH to 7.4, osmolarity 290-300 mOsM. Pipette resistance was 2-4 MW, and series resistance was <10 MW for all recordings. When possible, neurons were lifted free of the bottom of the culture dish after initiation of whole-cell recording, to facilitate solution exchange around the neuron. All recordings were made at a holding potential of -50 mV. Data were acquired by using a multiclamp 700-A amplifier and Digidata 1322 interface (Axon Instruments). Currents were filtered at 2 kHz and digitized at >5 kHz.

Receptor agonists and other drugs were applied to neurons by rapid solution application using a Warner Perfusion Fast-Step system (Warner Instruments, Hamden, CT) and two separate drug delivery barrels. Solution exchange time is »50 ms in whole-cell mode with this system. In experiments examining maximal AMPAR-mediated current, cyclothiazide (100 mM) was applied for at least 30 s before application of AMPA to fully block desensitization.

Peak current amplitude activated by agonists was measured using cursor-based protocols in Clampex version 8.0 (Axon Instruments) as the mean of three consecutive responses. Whole-cell capacitance was then estimated from transients generated by a 10-mV hyperpolarizing voltage step from a -50-mV holding potential. Current density was calculated as peak current amplitude divided by whole-cell capacitance.

1. Rahman Z, Gold SJ, Potenz MN, Cowan CW, Ni YG, He W, Wensel TG, Nestler EJ (1999) J Neurosci 19:2016-2026.

2. Jin XL, Guo H, Mao C, Atkins N, Wang H, Avasthi PP, Tu YT, Li Y (2000) Biochem Biophys Res Commun 270:978-982.

3. Dang M, Yokoi F, McNaught KS, Jengelley TA, Jackson T, Li J, Li Y (2005) Exp Neurol 196:452-463.

4. Campos VE, Du M, Li Y (2004) Biochem Biophys Res Commun 320:606-614.

5. Soriano P (1999) Nat Genet 21:70-71.

6. Bessert DA, Skoff RP (1999) J Histochem Cytochem 47:693-702.

7. Li Y, Erzurumlu RS, Chen S, Jhaveri S, Tonegawa S (1994) Cell 76:427-437.

8. Partridge JG, Tang K, Lovinger DM (2000) J Neurophysiol 84:1422-1429.

9. Wang Y, Aghajanian GK (1990) J Neurosci 10:3335-3343.

10. Wang Y, Xu R, Sasaoka T, Tonegawa S, Kung MP, Sankoorikal EB (2000) J Neurosci 20:8305-8314.

11. Stevens CF, Tonegawa S, Wang Y (1994) Curr Biol 4:687-693.

12. Hoffman AF, Oz M, Caulder T, Lupica CR (2003) J Neurosci 23:4815-4820.