Quantification of c-Fos+ cells and overlap of c-Fos and tyrosine hydroxylase immunoreactivities in the ventral tegmental area and substantia nigra of chronic saline- and PCP-treated mice.

<p>(A) Representative full-focus images showing c-Fos and TH immunoreactivities in the VTA and SNC of chronic saline- and PCP-treated mice. Scale bar, 100 μm. (B and C) Quantification of c-Fos+ cells in the dSNC and mSNC of the SNC (B) and mVTA, lVTA and IF of the VTA of chronic saline- (0 min: n = 4, 90 min: n = 5) and PCP-treated mice (0 min: n = 4, 90 min: n = 5) (C). *<i>p</i> < 0.05, **<i>p</i> < 0.01 vs 0 min. Graphs show mean + SEM.</p

Working memory impairment, which is not delay-dependent in chronic PCP-treated mice.

<p>(A) Experimental schedules for the discrete paired-trial variable-delay task in T-maze and the drug treatments. (B) Schematic illustration of the forced alternation training sessions. (C) Significant decreases in latency to eat food pellets were observed over days during the forced alternation training with no group differences. (D) Schematic illustration of the discrete paired-trial delayed alternation training sessions. Before drug treatment, no group differences in days to reach criterion (E) and correct responses (F) were observed. (G) Schematic illustration of the variable delay test in T-maze. (H) Percent of correct responses by chronic saline- (n = 11 mice) and PCP-treated mice (n = 12 mice) for all variable delays (5, 15, and 30 sec). Note that the x-axis starts at 50%, the expected chance response accuracy. (I) No group differences in latency to reach the pellet cup during the variable delay test were observed. **<i>p</i> < 0.01, ^#<i>p</i> < 0.05; NS, no significance. Line graphs show mean ± standard error of the mean (SEM). Bar graphs show mean ± SEM.</p

Quantification of c-Fos+ cells in the thalamus of chronic saline and PCP-treated mice.

<p>Quantification of c-Fos+ cells in the PVT, MD and RE of the thalamus of chronic saline- (0 min: n = 4, 90 min: n = 5) and PCP-treated mice (0 min: n = 4, 90 min: n = 5). ***<i>p</i> < 0.001 vs 0 min. Graphs show mean + SEM.</p

Abnormal neural activation patterns underlying working memory impairment in chronic phencyclidine-treated mice

<div><p>Working memory impairment is a hallmark feature of schizophrenia and is thought be caused by dysfunctions in the prefrontal cortex (PFC) and associated brain regions. However, the neural circuit anomalies underlying this impairment are poorly understood. The aim of this study is to assess working memory performance in the chronic phencyclidine (PCP) mouse model of schizophrenia, and to identify the neural substrates of working memory. To address this issue, we conducted the following experiments for mice after withdrawal from chronic administration (14 days) of either saline or PCP (10 mg/kg): (1) a discrete paired-trial variable-delay task in T-maze to assess working memory, and (2) brain-wide c-Fos mapping to identify activated brain regions relevant to this task performance either 90 min or 0 min after the completion of the task, with each time point examined under working memory effort and basal conditions. Correct responses in the test phase of the task were significantly reduced across delays (5, 15, and 30 s) in chronic PCP-treated mice compared with chronic saline-treated controls, suggesting delay-independent impairments in working memory in the PCP group. In layer 2–3 of the prelimbic cortex, the number of working memory effort-elicited c-Fos+ cells was significantly higher in the chronic PCP group than in the chronic saline group. The main effect of working memory effort relative to basal conditions was to induce significantly increased c-Fos+ cells in the other layers of prelimbic cortex and the anterior cingulate and infralimbic cortex regardless of the different chronic regimens. Conversely, this working memory effort had a negative effect (fewer c-Fos+ cells) in the ventral hippocampus. These results shed light on some putative neural networks relevant to working memory impairments in mice chronically treated with PCP, and emphasize the importance of the layer 2–3 of the prelimbic cortex of the PFC.</p></div

Quantification of c-Fos+ cells in the frontal cortex of chronic saline and PCP-treated mice.

<p>(A) Schematic illustration of brain sampling schedule for brain-wide c-Fos mapping after working memory task. The timing of sampling for c-Fos immunohistochemistry is indicated with the red dashed lines. (B) Representative c-Fos staining (red) in the PL (B, top). Representative double staining for a layer 5 (and 6) marker, Ctip2 (magenta), and a layer 6 marker, Foxp2 (green), in sections adjacent to those used for c-Fos immunostaining in the PL (B, bottom). Scale bar, 100 μm. (C) Fluorescence microscopy images of c-Fos staining (red) in the frontal cortex of chronic saline- or PCP-treated mice. Scale bars, 100 μm. (D) Quantification of the total number of c-Fos+ cells in the ACC, PL, and IL of chronic saline- (0 min: n = 4, 90 min: n = 5) and PCP-treated mice (0 min: n = 4, 90 min: n = 5). (E–G) Quantitative laminar-specific c-Fos mapping in the ACC (E), PL (F) and IL (G). *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001 vs 0 min, ^##<i>p</i> < 0.01 vs saline (90 min). Graphs show mean + SEM.</p

Quantification of c-Fos+ cells in the hippocampus of chronic saline- and PCP-treated mice.

<p>(A and B) Quantification of c-Fos+ cells in the DG, CA1, and CA3 of the dHPC (A) and vCA1/subiculum of the vHPC (B) of chronic saline- (0 min: n = 4, 90 min: n = 5) and PCP-treated mice (0 min: n = 4, 90 min: n = 5). **<i>p</i> < 0.01 vs 0 min, ^#<i>p</i> < 0.05 vs saline. Graphs show mean + SEM.</p

Methamphetamine increases locomotion and dopamine transporter activity in dopamine d5 receptor-deficient mice.

Dopamine regulates the psychomotor stimulant activities of amphetamine-like substances in the brain. The effects of dopamine are mediated through five known dopamine receptor subtypes in mammals. The functional relevance of D5 dopamine receptors in the central nervous system is not well understood. To determine the functional relevance of D5 dopamine receptors, we created D5 dopamine receptor-deficient mice and then used these mice to assess the roles of D5 dopamine receptors in the behavioral response to methamphetamine. Interestingly, D5 dopamine receptor-deficient mice displayed increased ambulation in response to methamphetamine. Furthermore, dopamine transporter threonine phosphorylation levels, which regulate amphetamine-induced dopamine release, were elevated in D5 dopamine receptor-deficient mice. The increase in methamphetamine-induced locomotor activity was eliminated by pretreatment with the dopamine transporter blocker GBR12909. Taken together, these results suggest that dopamine transporter activity and threonine phosphorylation levels are regulated by D5 dopamine receptors

DAT phosphorylation in D5R-KO mice.

<p>DAT proteins were immunoprecipitated from whole brain lysates and were then immunoblotted with anti-phosphothreonine and anti-DAT antibodies. (a) Upper: Representative Western blot of phospho-threonine signals of immunoprecipitated DAT proteins. Lower: Total DAT protein levels of the same samples. WT, D5R-KO, and DAT-KO genotypes are indicated. (b) The intensities of the phosphothreonine bands were normalized to the intensities of the total DAT protein bands to quantify the phosphothreonine levels. WT mice, black bar; D5R-KO mice, gray bar. Threonine phosphorylation levels were significantly increased in D5R-KO mouse brains relative to WT mouse brains. Paired t-test: <i>t</i> = −2.59 and <i>p</i><0.05. * <i>p</i><0.05.</p

DAT levels in D5R-KO mice.

<p>(a) Dialysate samples were collected at a sampling rate of 2 µl/min for 20 min during a baseline period of 60 min and then for an experimental period of 120 min following the METH challenge (2.5 mg/kg; arrow). The interaction between blocker pretreatment and challenge was F_(5,120) = 6.66 and <i>p</i><0.0001. The simple main effects of blocker pretreatment at 40 and 60 min were F_(1,144) = 13.50 and 8.08, and <i>p</i><0.001 and <i>p</i><0.001, respectively. WT, filled circles (<i>n</i> = 7); D5R-KO, open circles (<i>n</i> = 7). DA, dopamine. The arrowhead indicates the time point of saline injection. (b) Dialysate samples were collected for 20 min during a baseline period of 60 min. The animals were then injected with GBR12909 (5 mg/kg) and samples were collected for 20 min during a pretreatment period of 80 min. The animals were then challenged with METH (2.5 mg/kg) and samples were collected throughout the 120 min experimental period. The arrowhead indicates the GBR12909 pretreatment and the arrow indicates the METH challenge. (c) Representative microdialysis probe placements. Dashed lines denote the boundaries of the NA and the anterior commissure (ac). (d) Schematic representations of probe placements in the experimental groups for microdialysis of the NA at two different rostrocaudal levels (1.1 mm and 0.8 mm rostral from the bregma). The short lines indicate the probe tracks. Seven WT and seven D5R-KO probe track cases are overlaid on representative sections.</p