1 Department of Neuroscience, New York State Psychiatric Institute, New York, USA, 2 Department of Pharmacology, Columbia University, New York, NY 10032, USA, 3 Department of Neuroscience, Mount Sinai School of Medicine, New York, NY 10029, USA and 4 Department of Psychiatry, Columbia University, New York, NY 10032, USA
Address correspondence to Claudia Schmauss, Department of Psychiatry/Neuroscience, Columbia University, New York State Psychiatric Institute, 1051 Riverside Drive, New York, NY 10032, USA. Email: schmauss{at}neuron.cpmc.columbia.edu.
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Abstract |
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Key Words: anterior cingulate cortex cognitive control dopamine immunocytochemistry infralimbic cortex prelimbic cortex
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Introduction |
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In the present study, we sought to determine whether D2 and D3 receptor inactivation similarly or differentially affect the performance in tasks requiring sustained attention and hence, optimum functioning of the mPFC. Birrell and Brown (2000) described a two-choice discrimination attention-set-shifting task for rodents that enables investigations on the ability of the animal to acquire, maintain and shift attentional sets as well as the ability to alter behavior under reversal conditions. Bilateral lesions of the mPFC or the posterior parietal cortex of rats have been shown to selectively impair the performance of the animal in the extradimensional shift phase of the test (Birrell and Brown, 2000
; Fox et al., 2003
), and a recent study suggests that mPFC DA plays an important role modulating the ability to shift attention between different stimulus dimensions (Turnbridge et al., 2004
). In the present study, we tested the performance of wildtype and D2 and D3 mutant mice in the attention-set-shifting test developed by Birrell and Brown (2000)
and performed a quantitative immunocytochemical study to compare the test-induced c-fos responses of neurons in the mPFC. In contrast to the similar consequences of D2 and D3 receptor inactivation for spatial working memory, results of the present studies revealed differential effects of D2 and D3 receptor inactivation on both the performance in the cognitive task that requires sustained attention and the extent of task-induced neuronal activation in the mPFC.
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Materials and Methods |
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All procedures involving the animals adhered to guidelines laid out in the Principles of Laboratory Animal Care of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committees of Columbia University and the New York State Psychiatric Institute. Adult male congenic C57BL/6 mice lacking D2 and D3 receptors (Jung et al., 1999) and their wildtype littermates were used in this study. Wildtype and homozygous mutants were derived from cross-breeding of heterozygous mutants. After weaning the animals between postnatal day (P) 21 and P28, male animals of the same genotype, but derived from different litters, were group-housed (2 or 3 animals per cage) with unlimited access to food and water. Prior to testing at P60P90, their food consumption was restricted so that their body weights were gradually reduced (over a period of 710 days) to 85% of their starting weight. The animals remained group-housed during the period of food restriction. Daily recordings of body weights during this period revealed that all animals housed in the same cage lost weight to the same (gradual) extent and fights over food were not observed. Thus, single housing of animals was not required during food restriction. Behavioral testing was conducted during the light phase of a 12 h light/dark cycle (lights on at 6.00 a.m.).
Attention Set-shifting Paradigm
These experiments were conducted using a test apparatus made of Plexiglas that resembled the housing cage (dimensions: 32 x 27 x 15 cm). One-third of the apparatus was separated from the remaining two-thirds by a sliding door and served as a holding box. Terracotta pots were used as digging bowls and their rims were scented with perfumed oil to produce a lasting odor. The bowls contained a small food pellet (Prolab Isopro RMH 3000; PMI Nutrition, Brendwood, MO) buried underneath different digging media. In each trial, two pots were placed adjacent to each other in the larger section of the test box.
Mice were first trained to dig in the (unscented) small bowls filled with woodchip cage bedding to retrieve a food reward deeply buried underneath the bedding. After this habituation period (which lasted 5 to 10 min), mice were subjected to an attention-set-shifting paradigm originally described by Birrell and Brown (2000). In this study, we used a modification of this test described by Fox et al. (2003)
. In this modification, the number of reversal phases is reduced from three to one. This ensured that all animals completed the entire five phases of the test, a situation not given when the test was extended to seven phases. Briefly, mice were first trained on two simple discriminations (SD) of either odor (patchouli/jasmine, mango/vanilla, tea rose/dewberry, fuzzy peach/woody sandlewood) or digging media of different textures (wood chips/alpha dry bedding, glass beads/Eppendorf tube lids, ribbon/yarn, shredded paper/shredded rubber) to a criterion of six consecutive correct trials. The order of the two SDs and relevant stimulus dimensions (odor or digging medium) were randomized across each genotype such that 50% of animals per genotype started the experiments with odor guiding the location of the food pellet and the remaining 50% of the animals started with digging medium as the relevant stimulus. No differences in stimulus dimension preference or the ability to ignore a particular stimulus dimension was observed between the three genotypes. After successful completion of the SD, mice performed the series of discriminations described by Birrell and Brown (2000)
and Fox et al. (2003)
. Hence, after an SD between two odors or two digging media was presented, a compound discrimination (CD) followed in which a new dimension was added to the stimuli presented in the initial SD. This new dimension, however, is not a reliable indicator of the food location. The next test required an intradimensional shift of attention (IDS). The IDS is another CD in which both relevant and irrelevant stimuli are changed, but the previously relevant stimulus dimension (odor or digging medium) remains the same. This IDS was then subjected to reversal rules, i.e. the previously negative stimulus became a positive one but the irrelevant stimulus dimension was still not predicting the reward location. Finally, in a task requiring an extradimensional shift of attention (EDS), the formerly irrelevant dimension became relevant and the originally guiding dimension lost its predictive value. In all tasks, animals had to reach a criterion of six consecutive correct trials. During the course of the experiments, each sensory stimulus was used only once. All animals performed the entire series of tasks (including the habituation) in a single test session. The number of trials to criterion and the average time between stimulus presentation and response selection was computed, and the performance of the three different genotypes was first compared by an overall one-way analysis of variance (ANOVA; threshold of significance,
= 0.05) and significant differences were resolved post hoc using the TukeyKramer multiple comparisons test.
Immunocytochemistry and Stereological Analysis
One hour following completion of either the CD or the final EDS component of the attention set-shifting task, mice were deeply anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (15 mg/kg) and perfused transcardially with 100 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) at room temperature. Brains were postfixed for 1 h (in the same fixative) and cryoprotected overnight (30% sucrose in 0.1 M PB).
The expression of Fos immunoreactivity was analyzed in a series of adjacent, coronal, 40-µm-thick Microtome sections that were collected every 200 µm from 6.5 to 1.5 mm rostral to bregma. One section of each pair was mounted directly onto gelatin-coated microscope slides, dehydrated, and counterstained with 0.25% thionin. The remaining free-floating sections were processed to detect Fos-immunoreactivity. Non-specific staining was reduced by first incubating sections for 30 min in 0.1 M PB containing 0.5% bovine serum albumin (BSA). Sections were then incubated overnight at 4°C with a rabbit polyclonal antibody directed against the Fos protein (Ab-5; 1:10 000; Oncogene Sciences, Boston, MA) dissolved in PB containing 0.1% BSA and 0.25% Triton X-100. After incubation with primary antibody, sections were incubated for 30 min with biotinylated goat anti-rabbit IgG (1:400 in PB containing 0.1% BSA; Vector Laboratories, Burlingame, CA), followed by a 30-min incubation in avidin-biotin-peroxidase complex (Vectastain Elite Kit; 1:100 in PB; Vector Laboratories). To visualize bound immunoperoxidase, sections were incubated for 6 min in 0.022% 3,3'-diaminobenzidine (DAB; Aldrich, Milwaukee, WI) and 0.003% hydrogen peroxide in PB. All sections were rinsed in PB and mounted onto gelatin-coated slides.
All tissues were processed within 3 days of perfusion. In some experiments, tissues of all three genotypes subjected to the same behavioral test paradigm (CD or EDS) were processed in parallel and in other experiments, tissues of a single genotype subjected to both testing paradigms were processed in parallel, and consistent results were obtained for sections processed at different times. The intensity of the DAB immunoreaction product ranged from a paler brown to a deep brown color in all genotypes and treatment groups.
To obtain a quantitative estimate of the numbers of mPFC neurons expressing Fos immunoreactivity in wildtype and D2 and D3 single mutants, a stereologic counting method was used. For this analysis, a Zeiss Axioplan 2 photomicroscope (Oberkochen, Germany) equipped with a Dage-MTI (Michigan City, IN) DC-330 CCD camera and Ludl motorized stage, interfaced with a Gateway Athlon computer and StereoInvestigator (MicroBrightField, Wiliston, VT) was used.
The stereologic analysis was conducted on brain sections obtained from 46 mice per genotype. For each case, the thionin-labeled series of sections was used to identify the boundaries between layers II/III and V/VI. Total numbers of Fos-immunolabeled neurons were determined in three subregions of the mPFC, the infralimbic (IL), prelimbic (PL) and the anterior cingulate (AC) cortices (Hof et al., 2000). In addition, Fos-positive nuclei were counted in one subregion of the somatosensory cortex (barrel fields, SSbf) to establish a reference for general brain activation so that the magnitude of solely task-induced neuronal activation in the mPFC could be determined.
Stereological measures were taken from three (IL) or six (PL, AC and SSbf) sections from the series, using an unbiased stereologic method, the optical fractionator (West et al., 1991). Optical disector frame and counting grid sizes of 45 and 75 µm2, respectively, were chosen to permit systematic-random sampling of >3 neurons within an 8 µm focusing range for each sampling field. All sampling parameters were set such that at least 200 neurons per region were sampled in the cases with lowest Fos expression. This yielded an average of 498 for superficial or deep layers of mPFC regions and 1171 for SSbf, and resulted in an intrasample coefficients of error (CE), calculated as described previously (Schmitz and Hof, 2000
), that were always <0.05. There were no significant differences in CE values across genotypes and testing conditions. All regions were sampled at high magnification in Koehler illumination conditions using a Zeiss 63x Plan-Apochromat objective. The volume of the different laminar domains of interest in each of three mPFC regions and the entire SSbf was estimated using the Cavalieri principle. Because there were no differences in regional volumes as well as the number of neurons and glia between the three genotypes studied here (Glickstein et al., 2002
), the extent of Fos-labeling was expressed as the number of Fos-immunoreactive neurons per 0.1 mm3 so that the net activation per region could be compared across genotypes and test conditions. Hence, we report here densities of Fos-labeled neurons that were calculated by dividing the mean number of Fos-labeled neurons of each group by the average volume of the corresponding region. The estimated total numbers of Fos-labeled neurons ranged from: 1.07 x 104 to 3.84 x 104 (AC II/III), 1.19 x 104 to 4.63 x 104 (AC V/VI), 4.6 x 103 to 1.64 x 104 (PL/IL II/III), 7.01 x 103 to 2.60 x 104 (PL/IL V/VI), and 3.16 x 104 to 5.13 x 104 (SSbf).
For statistical analysis of the stereological data, a one-way ANOVA (threshold of significance, = 0.05) was performed, and significance of differences was analyzed post hoc using the StudentNewmanKeuls multiple comparisons test.
For fluorescence microscopy, additional sections were incubated in 0.5% BSA in PB, followed by immersion in primary antibody solutions as described except that the antibody directed against the Fos protein was used at a higher dilution (1:15 000). Sections were washed in PB containing 0.25% Triton X-100 and incubated with a fluorescein isothiocyanate-conjugated goat anti-rabbit IgG secondary antibody (dilution 1:200; Vector Laboratories) for 1 h at room temperature, washed in PB containing 0.25% Triton X-100, mounted onto gelatin-coated slides, air-dried and processed for light-microscopic viewing. Portions of the mPFC at 1.7 mm rostral to bregma were photographed at 10x magnification using the Zeiss Axiocam camera attached to a Zeiss Axiophot 2 microscope. Images were processed using Open Lab (Improvision, Lexington, MA) and Adobe Photoshop (version 6.0) software. Adjustments to image brightness and contrast were made for clarity of publication, but the resultant images reflect the experimental data.
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Results |
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To examine the ability of D2 and D3 mutants and their wildtype littermates to acquire, maintain, and shift attentional sets as well as to test their ability to alter behavior under reversal conditions, we employed the attention-set-shifting test originally developed by Birrell and Brown (2000) and later modified by Fox et al. (2003)
(see Materials and Methods). Figure 1 (top) summarizes the number of trials to the criterion of six consecutive correct trials for SD, CD, IDS, IDSRev, and EDS components of this test. In the SD, wildtype animals reached this criterion after a mean of 8 trials and completed the CD and IDS with a mean of nine trials. The more demanding tasks, IDSRev and EDS, were completed after a mean of 10 trials.
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Different results were obtained with D3 mutants. Whereas their number of trials to criterion in the SD and CD did not differ from wildtype, they exhibited a slightly better performance in the more difficult IDS, IDSRev and EDS, and a post hoc TukeyKramer multiple comparisons test revealed that their performance in the IDSRev was significantly better compared with wildtype (P < 0.03).
Figure 1 (bottom) compares the average response latencies of wildtype and mutants in all five components of the task. This response latency reflects the time between releasing the animal from the holding box and the initiation of digging for the food pellet, and an overall analysis of variance (ANOVA) indicated significant differences between them [F(14,125) = 8.7, P < 0.0001]. Post hoc TukeyKramer multiple comparisons revealed that, although the response latencies of D2 mutants are shorter for the IDS, IDSRev and EDS phases of the test compared with wildtype and D3 mutants, none of these differences reach statistical significance. In contrast, the response latencies of D3 mutants are longer in all five phases of the test, and their response latencies measured for the SD, CD and EDS phases of the test are significantly longer (P < 0.05) compared with wildtype and D2 mutants (Fig. 1, bottom).
We have previously shown that D2 mutants have spatial working memory deficits that are due to decreased prefrontal cortical D1 receptor activity, and that a single dose of methamphetamine (METH) rescued both the decreased agonist-promoted D1 receptor function as well as the working memory deficits of these mutants in a long-term manner (Glickstein et al., 2002). As shown in Table 1, however, an identical pharmacological manipulation with METH did neither affect the (impaired) performance of D2 mutants in the CD phase of the test nor did it alter the performance of wildtype and D3 mutants. In fact, when mice were treated with a single dose of METH (5 mg/kg) 1 week prior to testing, their number of trials to criterion were either similar (D3 mutants) or non-significantly higher (wildtype and D2 mutants) compared with the corresponding non-treated genotypes [wildtype: F(1,25) = 1.52, P = 0.23; D2 mutants: F(1,25) = 0.56, P = 0.5; D3 mutants: F(1,26) = 0.15; P = 0.7]. Moreover, pretreatment with methamphetamine did not significantly affect the response latencies of either genotype [wildtype: F(1,16) = 0.39, P = 0.5; D2 mutants: F(1,24) = 0.42, P = 0.5; D3 mutants: F(1,20) = 0.74; P = 0.4; Table 1].
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Additional experiments also revealed that the response selection of both wildtype and D3 mutants is equally and strongly guided by the relevant stimulus (odor or medium), and hence not by a sense of smell derived from the food pellet: after successful completion of the CD, the food pellet was removed from the bowl that contained the relevant stimulus and animals were allowed to approach the bowls one more time. Despite the absence of the food pellet, 80% of the animals chose the correct (but non-baited) bowl [n = 10 (wildtype) and n = 13 (D3 mutants) (not shown)]. Finally, in the SD, 60 and 63% of wildtype and D3 mutants, respectively, corrected a bowl-selection error in the subsequent trial after a 2 min delay period (one-trial learning) and maintained correct responses in the following trials.
In summary, mice lacking D2 receptors exhibit specific deficits in the acquisition of rules that govern the task when two stimulus dimensions and four stimulus properties are presented together (CD). The performance of D3 mutants, however, is significantly increased in the task that requires flexibility to reversal conditions (IDSRev). Moreover, the increased response accuracy of D3 mutants occurs together with increased response latencies when compared with wildtype and D2 mutants.
Basal and Test-induced Expression of Fos Immunoreactivity in the PFC
The following experiments used immunocytochemistry to examine task-induced expression of neuronal Fos immunoreactivity. Under basal conditions, the expression of Fos-immunoreactivity of non-tested wildtype and D2 and D3 mutant mice fed ad libitum is extremely low. There are typically fewer than five Fos-immunolabeled nuclei in the cortex in a field of view of 10x magnification (Fig. 2). After completion of the attention-set-shifting test, however, robust expression of Fos immunoreactivity was detected in both superficial and deep layers of the mPFC, and in several other brain regions, including orbital, motor and somatosensory cortices, hippocampus, dorsal striatum, nucleus accumbens, thalamus, hypothalamus and cholinergic nuclei.
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Our analysis focused on the mPFC. The top panel of Figure 2 illustrates the anatomic topography and cytoarchitecture of the mouse mPFC revealed by thionin-counterstaining of a coronal section taken 5.5 mm rostral to the interaural line (Fig. 2A), and a corresponding DAB-stained section illustrating the expression of Fos immunoractivity in wildtype (Fig. 2B). The bottom panels of Figure 2 show the expression of Fos immunofluorescence at the same rostrocaudal level in coronal sections of wildtype. Compared with mice fed ad libitum (Fig. 2C), the numbers of neurons expressing Fos-immunofluorescence and the intensity of labeling is substantially enhanced in food-restricted mice (Fig. 2D). Therefore, in order to determine the true magnitude of task-specific induction of expression of the c-fos gene, we conducted a detailed analysis of the numbers of Fos-immunoreactive nuclei in food-restricted mice with and without behavioral testing.
We used the optical fractionator to obtain a quantitative estimate of the numbers of mPFC neurons expressing Fos-immunoreactivity, calculated the densities of Fos-labeled neurons for each region (see Materials and Methods), and subtracted the regional densities calculated for food-restricted, but non-tested animals from corresponding densities obtained from animals subjected to the behavioral test. The measures for PL and IL were combined to allow for the sampling of at least 200 Fos-positive neurons within all genotypes so that the sampling parameters used for the AC, SSbf and PL/IL were identical.
There were no significant differences between genotypes in the volumes of any of the regions measured. Microscopic inspection of thionin-stained sections revealed no differences in the cytoarchitecture of the regions analyzed. Moreover, as previously reported for the same lines of mice used in this study, there are no differences between wildtype and mutants in the numbers of neurons or glia in the mPFC (Glickstein et al., 2002).
The mean densities of Fos-immunoreactive neurons in food restricted, non-tested mice determined for two laminar territories of each of two regions of the mPFC and the SSbf are summarized in Table 3. It is evident that food-restriction alone elevates Fos immunoreactivity substantially. A statistical analysis of variance (ANOVA) between genotypes revealed that, although there is a significantly greater density of Fos-positive neurons in the superficial AC of food-restricted D3 mutants relative to wildtype [F(2,12) = 5.6; P < 0.05], both D2 and D3 mutant mice had significantly reduced densities of Fos-immunolabeled neurons in the SSbf compared with food-restricted wildtype [F(2,12) = 5.95; P < 0.05].
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In order to determine the extent to which sustained attention required during the entire attention-set-shifting task elicits region-specific neuronal activation, the expression of Fos immunoreactivity in the mPFC and the SSbfs of wildtype and mutants was examined 1h after completion of the last phase of the test, the EDS. Moreover, because of the specific deficits of D2 mutants during the CD-phase of the test, additional experiments compared the expression Fos-immunoreactivity 1 h after completion of the CD.
Figure 3 illustrates Fos-immunofluorescence detected in the mPFC of mice after CD (top panel) or EDS testing (bottom panel). Compared with non-tested (food-restricted) mice (Fig. 2D), the number of Fos-labeled neurons is increased in CD-tested wildtype (Fig. 3, top left) and D3 mutants (Fig. 3, top center), especially in superficial mPFC layers. In CD-tested D2 mutants, however, no apparent increase was detected (Fig. 3, top right). Similar results were obtained for EDS-tested mice (Fig. 3, bottom).
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A comparison of the sum of all densities (without subtracting baseline activation) calculated for the entire AC and PL/IL after CD testing revealed a differential extent of dorsal versus ventral PFC activation in wildtype and D3 mutants. In wildtype, the density of Fos-immunolabeled neurons in the AC exceeds that in the PL/IL by 17%. In D3 mutants, however, Fos-immunoreactive neurons are more densely packed in the PL/IL where they exceed AC activation by >50%.
Remarkably, in D2 mutants, CD-testing did not elevate c-fos expression above the already elevated baseline expression and the slightly decreased densities of Fos-labeled neurons that are evident in Figure 4A are not significantly different from corresponding baseline densities. In the AC, however, the densities of Fos-immunoreactive neurons of CD-tested D2 mutants were significantly lower when compared with wildtype [AC II/III, F(2,12) = 9.64, P < 0.01; AC V/VI, F(2,12) = 5.83, P < 0.05]. Despite the absence of CD-induced c-fos expression in the mPFC of D2 mutants, their densities of Fos-positive neurons in the SSbfs were increased relative to wildtype [F(2,12) = 4.03, P < 0.05].
The mean regional densities of Fos-immunoreactive neurons determined after EDS testing are plotted in Figure 4B. In EDS-tested wildtype, the expression of Fos immunoreactivity exceeded baseline and CD-test values in all regions examined. In EDS-tested D3 mutants, the extent of c-fos-induction in all regions of the mPFC was significantly increased when compared with wildtype [AC II/III, F(2,15) = 23.93, P < 0.001; AC V/VI, F(2,15) = 22.57, P < 0.01; PL/IL II/III, F(2,15) = 18.37, P < 0.001; and PL/IL V/VI, F(2,15) = 16.93, P < 0.001]. The induction of c-fos in the SSbfs surpassed baseline values determined for food-restricted, non-tested D3 mutants, but did not significantly differ from wildtype and CD-tested D3 mutants despite the large increase in mPFC Fos immunoreactivity. Similar to the differential relative activation of the AC and PL/IL detected after CD testing, wildtype animals still displayed a greater activation of neurons in the AC (>50%) compared with PL/IL regions. However, c-fos is expressed uniformly throughout the mPFC of D3 mutants.
In EDS-tested D2 mutants, the expression of mPFC Fos immunoreactivity does not rise above baseline values, and is substantially lower compared with EDS-tested wildtype. These differences are significant in the entire AC of D2 mutants [AC II/III, F(2,15) = 23.93, P < 0.05; AC V/VI, F(2,15) = 22.57, P < 0.05], but they do not reach statistical significance in either superficial or deeper layers of the PL/IL. Despite the lack of EDS-specific c-fos induction in the mPFC, the expression of Fos-immunoreactivity in the SSbfs of EDS-tested D2 mutants was increased by 18% from baseline but did not significantly differ from corresponding values obtained from wildtype.
In summary, whereas wildtype and D3 mutants exhibit test-specific activation of mPFC neurons, mPFC c-fos induction in mice lacking D2 receptors is not elevated above baseline. Furthermore, in EDS-tested D3 mutants, the expression of c-fos in the mPFC is significantly increased compared with EDS-tested wildtype.
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Discussion |
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Whereas the performance of D2 mutants is indistinguishable from wildtype in simple discrimination tests, they have significant deficits in the compound discrimination. More precisely, deficits in rule acquisition become evident only when the complete set of stimulus dimensions and stimulus properties are presented for the first time. Our data indicate that D2 mutants have no deficit in attending to a perceptual dimension per se. Once these mutants acquired the rules of the CD, they proceed through attention-set-shifting tasks, including rule reversals, in a manner indistinguishable from wildtype. Interestingly, similar deficits in the acquisition of rules have previously been found to be responsible for the impaired performance of schizophrenic subjects in related cognitive tasks (Posada and Franck, 2002; Faraone et al., 1999
). The present data therefore suggest that decreased D2-receptor activity contributes specifically to deficits in the acquisition of rules that govern cognitive tasks. They further suggest that chronic treatment with neuroleptic drugs that block D2 receptors would further worsen this deficit.
Our previous study has shown that D2 mutants have spatial working memory deficits that are due to decreased prefrontal cortical D1 receptor activity. Whereas a single dose of METH rescued both the decreased agonist-promoted D1 receptor function as well as the working memory deficits of these mutants in a long-term manner (Glickstein et al., 2002), the present study shows that an identical pharmacological manipulation with METH had no effect on the (impaired) performance of D2 mutants in the CD phase of the test, suggesting that the deficits of rule acquisition in D2 mutants are not due to their decreased D1-receptor activity in the mPFC. In this regard, it is of interest to note that a recent study demonstrated that D2 receptors selectively modulate a specific component of working memory circuitry that is not affected by D1 receptors (Wang et al., 2004
). Similarly, the present study suggests that D2 receptors, but not D1 receptors, play a direct role in the acquisition of complex rules that govern cognitive tasks. Whether this requires activation of D2 receptors located on pyramidal cells or GABAergic interneurons of the mPFC (or both) remains to be investigated.
The present study also shows that D2 mutants exhibit virtually no task-specific activation of neurons in the mPFC after CD- and EDS-testing. This, however, does not imply that the neuronal mPFC activity is low in these mutants. Rather, our data revealed that in D2 mutants, the activity of mPFC neurons during tasks requiring sustained attention does not exceed the already elevated neuronal activation of non-tested, but food-deprived animals. This is clearly different from results obtained from D3 mutants. In SD and CD phases of the test, these mutants perform in a manner indistinguishable from their wildtype littermates. In intra- and extradimensional set-shifting phases, their performance is slightly enhanced, and they perform significantly better than wildtype in tests governed by reversal rules. Moreover, CD- and EDS-tests led to mPFC c-fos responses in these mutants that were significantly higher compared with the already elevated baseline levels of food-deprived, non-tested mutants. Furthermore, in EDS-tested D3 mutants, the densities of mPFC neurons expressing the c-fos gene were significantly higher compared with EDS-tested wildtype. Altogether, these data indicate that the extent of test-induced activation of neurons in the mPFC correlates with the performance of wildtype and mutants in the cognitive task and that the differences between wildtype and mutants are test-phase specific, i.e. they are evident for CD- and IDSRev-performances of D2 and D3 mutants, respectively, but the performance of either mutants in the EDS is indistinguishable from wildtype. These findings are different from those reported for rats with bilateral lesions of the mPFC (Birrel and Brown, 2000). These rats showed deficits only in the EDS phase of the task. Several reasons could account for these differences. First, bilateral lesions of the PFC are far more robust manipulations than inactivation of single genes. Second, differences in the use of anatomic circuitries activated during set-shifting could account for the differences between the two rodent species. Third, the attention-set shifting task developed by Birrell and Brown (2000)
may be more sensitive in detecting deficits in set-shifting in rats compared with mice.
A striking finding of the present study is the difference of neuronal activation in the AC relative to PL/IL in wildtype and D3 mutants. Whereas in wildtype, AC activation exceeds corresponding ones found in PL/IL subregions, in D3 mutants the number of neurons expressing c-fos in the AC never exceeded PL/IL numbers. In fact, in CD-tested D3 mutants, AC activation was lower than PL/IL activation. The rodent mPFC, anatomically defined by its reciprocal connections to the mediodorsal thalamus as well as reciprocal corticocortical connections, has many functional properties that are also characteristic for the primate dorsolateral PFC (working memory, attention, attention shifts). However, the rodent mPFC also has features of other cortical regions of primates, most notably the anterior cingulate cortex (Uylings et al., 2003). In primates, one interpretation of neuronal activation in the AC during tasks requiring attention and response selection is that the AC monitors conflict between different action plans (performance monitoring; MacDonald et al., 2000
; Botvinick et al., 2001
) to signal greater cognitive control to the dorsolateral PFC. This decreases conflict and in subsequent test trials, with correct response selections, activation of the AC decreases while activation of the dorsolateral PFC is increased (Kerns et al., 2004
). It is thus tempting to speculate that the higher response accuracy of D3 mutants is, at least in part, due to their relatively higher activation in the PL/IL compared with AC and that such an activation pattern reflects less conflict, greater cognitive control, and hence lower error rates.
D3 mutants also exhibit prolonged response latencies in the behavioral task that cannot simply be explained by decreased motivation or increased anxiety of these mutants (see Table 1). The differences in response latencies between wildtype, D2 mutants and D3 mutants together with the differences between their response accuracies suggest that longer response latencies are necessary for heightened attention. Indeed, D3 mutants spent more time checking both bowls and correcting approaches to the incorrect bowl than wildtype or D2 mutants. Thus, one role of D3 receptors in attention may be to speed up response selection, perhaps at the expense of response accuracy. However, the mechanisms underlying the improvement in attention in D3 knockout mice remain to be investigated.
Finally, a general concern of studies on constitutive knockout mice is that results obtained with such mutants are due to the lack of expression of the targeted gene during development. The extent to which the specific cognitive phenotypes of D2 and D3 mutants are related to adaptive developmental processes remains to be demonstrated. In the absence of evidence for both structural alterations in the PFC of the mutants examined in this study and a pivotal role of D1 receptors (whose function is diminished in these mutants) in the deficits described here, it is likely that sustained pharmacological blockade of D2 and D3 receptors reproduces the results obtained with the mutants. At present, however, a selective pharmacological inactivation of these two receptors must still await the successful development of drugs that, over a wide range of concentrations, clearly discriminate between D2 and D3 receptors expressed in vivo.
In summary, inactivation of D2 and D3 receptors in knockout mice results in different modulations of the performance in cognitive tasks requiring sustained attention. The lack of D2 receptors impairs the acquisition of complex rules that govern the task, and the lack of D3 receptors enhances performance in set-shifting/reversal phases of the task. Hence, DA exerts different modulatory effects on mPFC activity during tasks requiring sustained attention that are dependent upon the type of D2-like receptor that is activated.
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Acknowledgments |
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