Dysbindin mutant mouse breeders on the DBA2J background were obtained from Roswell Park Cancer Institute (Conyers GA). Experimental mice were generated by heterozygote crosses, and genotypes were determined by polymerase chain reaction. The wt product [472 bp] was amplified with the following primers: TGAGCCATTAGGAGATAAGAGCA and AGCTCCACCTGCTGAACATT; the dys− product [274 bp] was amplified with the following primers: TCCTTGCTTCGTTCTCTGCT and CTTGCCAGCCTTCGTATTGT). The fragments were separated on a 3% agarose gel.
Group-housed male mice were used in the behavioral and electrophysiological studies. The mice in the behavioral experiments were 60–100 days of age during experimentation, while the mice used in the recording studies were 45–60 days of age. All experimental protocols were approved by the Chancellor’s Animal Research Committee at UCLA or the Medical University of South Carolina Institutional Animal Care and Use Committee.
Working memory testing
Mice were trained and tested using a delayed non-match-to-position test (Marrs et al. 2005; Aarde and Jentsch 2006) in small aluminum and Plexiglas operant conditioning chambers (Med-Associates Inc., St Albans VT), fitted with a horizontal array of five nose-poke apertures on one-side of the box and a 20-mg pellet delivery magazine on the opposite side. This delayed non-match-to-position task emphasizes retrospective encoding and maintenance of spatial information in a manner analogous to spatial delayed response tests used to measure working memory in rats, monkeys and humans.
Each trial in the delayed non-match to sample task consists of both a sample phase and a choice phase. The inter-trial interval and time-out periods were 5 s and 3 s, respectively. In the sample phase, one of the 5 nose-poke apertures was chosen at random and illuminated for up to 15 s. A response into the illuminated aperture (correct sample-phase response) caused the aperture light to extinguish and the magazine light to be illuminated. The magazine remained lit until an entry into the magazine is detected, after which the choice phase was initiated (see below). In the sample phase, the trial was aborted and a time-out ensues if the mouse makes a response in an unlit nose-poke aperture during stimulus presentation (incorrect sample-phase response) or failed to respond to the illuminated aperture during the 15 s presentation period (sample-phase omission). During the choice phase of the trial, two apertures were illuminated: the sample aperture as well as another, randomly selected aperture (the non-match location). Both of these apertures were illuminated for up to 15 s. A response in any aperture other than that of the non-match location resulted in a time-out, and an incorrect choice-phase response was recorded. Failure to make a response while the apertures were illuminated resulted in a time-out, and a choice-phase omission was recorded. Responses to the non-match location (correct response) triggered magazine illumination and pellet delivery.
During the first 30 days of training, there was no imposed delay, allowing the mice to acquire the task under relatively low memory load conditions. After this initial training, probe sessions were administered in which minimum delay periods were imposed between a correct sample-phase response and the first response into the magazine that initiated the choice phase. The imposed delays were either 0.5, 5 or 10 s and were interpolated in pseudorandom order and at equivalent frequencies across the session. The tasks ended after 75 trials are completed or 60 min passes, whichever comes first.
Incorrect choices and omissions during the sample phase and omissions during the choice phase were calculated and analyzed by one-way analyses of variance, with genotype as the factor. Accuracy of responding during the choice phase was analyzed by repeated measures ANOVA with genotype as the factor and delay length as the repeated measure.
Brain slices were prepared from male wt/wt, wt/dys− and dys−/ dys− mice. Subjects were anesthetized with the inhalant isoflurane (Abbott Laboratories). The brain was then removed and coronal slices were cut at 300 µm thickness in ice-cold high-sucrose solution containing (in mM): sucrose, 200; KCl, 1.9; Na2HPO4, 1.2; NaHCO3, 33; MgCl2, 6; CaCl2, 0.5; glucose, 10; ascorbic acid, 0.4. Slices were incubated at 33°C for at least 1 h before recordings; the incubation medium was an artificial cerebrospinal fluid solution containing (in mM): NaCl, 125; KCl, 2.5; NaH2PO4,1.25; NaHCO3, 25; MgCl, 4, CaCl, 1, d-glucose, 10; sucrose, 15; ascorbic acid, 0.4, continuously aerated with 5%CO2/95%O2. After incubation, slices were transferred to a submerged chamber and superfused with oxygenated artificial cerebrospinal fluid (in mM: 125 NaCl, 2.5 KCl, 25 NaHCO3, 2.0 CaCl2, 1.3 MgCl2, 10 glucose and 0.4 ascorbic acid) at room temperature. Recordings were made using a Multiclamp 700B amplifier (Axon Instruments, CA), connected to a computer running Windows XP and Axograph X® software and later analyzed off-line. All recordings were obtained from neurons in layers V or VI of the prelimbic or infralimbic cortex, identified using infrared-differential interference contrast optics and video-microscopy.
For current-clamp recordings, thick-walled borosilicate pipettes (3–7 MΩ tip resistance) were filled with (in mM): 125 K+-gluconate, 3 KCl, 2 MgCl2, 10 HEPES, 0.1 EGTA. A series of current steps (1000 ms duration, −100 to +300 pA, at 1 Hz) were injected to evoke spike firing at various steady-state membrane potentials. In between the steps, cells were held as close to −80 mV as possible via DC current injection (referred to as holding current in ). Measures of the intrinsic membrane excitability (rheobase current, holding current, number of evoked spikes, input resistance, action potential threshold, amplitude, and half-width) were compared across genotypes.
Membrane properties of prefrontal cortex neurons recorded in wild-type and heterozygous or homozygous dysbindin null mutant mice.
For voltage-clamp recordings, electrodes (3–7 MΩ resistance in situ) were filled with a solution containing (in mM): 135 CsCl, 10 HEPES, 2 MgCl2, 1 EGTA, 4 NaCl; 2 Na-ATP, 0.3 tris-GTP, 1 QX-314, 10 phosphocreatine; 285 mOsmols. All the voltage-clamp experiments were performed in the presence of 100 µM picrotoxin. Series resistances (10–20 MΩ) and input resistances were continually monitored throughout the experiment via a −1 mV (100 ms) hyperpolarizing pulse. Evoked EPSCs (eEPSCs) were elicited by applying low-intensity, square-wave pulses (50–150 µA; 100 µsec in duration) using a bipolar concentric electrode placed within 200 µm of the recording electrode. The eEPSC amplitude was defined as the mean amplitude during a 1–2 ms window at the peak of the EPSC minus the amplitude during a similar window immediately before the stimulus artifact. Pulses were administered every 30 seconds and peak eEPSC amplitude was measured. Stimulus intensity was gradually increased in order to construct an input-output curve from 10 µA up to 500 µA (in some cases), until maximum amplitude was reached, meaning that response amplitude remained consistent regardless of increasing stimulation intensity. The responses included in the analysis were limited to those measured at 75% of maximum amplitude. Paired pulse stimuli were delivered at a frequency of 0.3Hz, with an ISI of 50 msec.
Miniature EPSCs (mEPSCs) were obtained from 50–200 sweeps per cell; TTX (1 µM) was added to the recording buffer when mEPSCs were assessed. Amplitude and frequencies were calculated using MiniAnalysis software® with a detection threshold of 8 pA, events were then manually checked for accuracy.
Parametric analyses of variance (ANOVA) were used to examine main effects (e.g., genotype) and interactions with repeated measures, when appropriate. Significant main effects or interactions were followed up with post hoc tests. In some cases, paired t-tests were used to evaluate within subject, a priori hypothesized effects. All figures present data as mean ± SEM.