Antipsychotic drugs reverse the disruption in prefrontal cortex function produced by NMDA receptor blockade with phencyclidine

Edited by Ranulfo Romo, National Autonomous University of Mexico, Mexico City, Mexico, and approved July 27, 2007
September 11, 2007
104 (37) 14843-14848

Abstract

NMDA receptor (NMDA-R) antagonists are extensively used as schizophrenia models because of their ability to evoke positive and negative symptoms as well as cognitive deficits similar to those of the illness. Cognitive deficits in schizophrenia are associated with prefrontal cortex (PFC) abnormalities. These deficits are of particular interest because an early improvement in cognitive performance predicts a better long-term clinical outcome. Here, we examined the effect of the noncompetitive NMDA-R antagonist phencyclidine (PCP) on PFC function to understand the cellular and network elements involved in its schizomimetic actions. PCP induces a marked disruption of the activity of the PFC in the rat, increasing and decreasing the activity of 45% and 33% of the pyramidal neurons recorded, respectively (22% of the neurons were unaffected). Concurrently, PCP markedly reduced cortical synchrony in the delta frequency range (0.3–4 Hz) as assessed by recording local field potentials. The subsequent administration of the antipsychotic drugs haloperidol and clozapine reversed PCP effects on pyramidal cell firing and cortical synchronization. PCP increased c-fos expression in PFC pyramidal neurons, an effect prevented by the administration of clozapine. PCP also enhanced c-fos expression in the centromedial and mediodorsal (but not reticular) nuclei of the thalamus, suggesting the participation of enhanced thalamocortical excitatory inputs. These results shed light on the involvement of PFC in the schizomimetic action of NMDA-R antagonists and show that antipsychotic drugs may partly exert their therapeutic effect by normalizing a disrupted PFC activity, an effect that may add to subcortical dopamine receptor blockade.
Schizophrenia is associated with alterations in several brain areas, including the thalamus, the hippocampus, the amygdala, and the prefrontal cortex (PFC), which are thought to underlie the deficits in working memory and executive functions exhibited by schizophrenic patients (13). Autopsy and neuroimaging studies have revealed the existence of a reduced PFC volume, reduced layer thickness, tight packing of pyramidal neurons and reduced neuropil in the brains of schizophrenic patients (1, 2, 4, 5). Moreover, alterations in key neurotransmitters such as glutamate, GABA, and dopamine have been reported in PFC (3, 5, 6).
Noncompetitive N-methyl-d-aspartate (NMDA) receptor (NMDA-R) antagonists such as the dissociative anesthetics ketamine and phencyclidine (PCP), have been extensively used as pharmacological models of schizophrenia because of their ability to evoke positive and negative symptoms of schizophrenia as well as the characteristic cognitive deficits of the illness in humans (3, 7). Neuroimaging studies suggest that these effects are associated with an increased PFC activity (8). On the other hand, NMDA-R antagonists evoke a behavioral syndrome in experimental animals characterized by hyperlocomotion, stereotypes and disruption in prepulse inhibition of the startle response which is totally or partly antagonized by antipsychotic drugs (9, 10). However, the cellular elements and brain networks involved in these actions are still poorly known. Of great interest is the knowledge of the neurobiological basis of the cognitive deficits evoked by NMDA-R antagonists, because these alterations are central to the pathology of schizophrenia and the improvement in cognitive performance may predict better therapeutic outcome (11).
The aim of the present study was to examine the effects of PCP on PFC function and the possible reversal of these effects by antipsychotic drugs, by using in vivo measures of cellular (single unit extracellular recordings) and population activity [local field potentials (LFPs)]. Additionally, we examined the effect of PCP on the expression of c-fos, a marker of neuronal activity, in PFC and afferent areas.

Results

Effects of PCP on the Activity of mPFC Pyramidal Neurons.

The effect of PCP was examined in 80 rats (one neuron per rat). The duration of the action potential was 0.97 ± 0.05 ms (up phase; n = 80). All recorded units were pyramidal neurons in layers V-VI (mainly in prelimbic PFC), as assessed by antidromic activation from midbrain (46 from dorsal raphe, 34 from ventral tegmental area; see Materials and Methods). Baseline firing rate was 2.2 ± 0.3 spikes/s (n = 80). Saline injections did not alter the firing rate of pyramidal neurons. PCP administration (0.25 mg/kg i.v.) evoked three types of responses. Forty-five percent of the neurons had their firing rate enhanced (to 286% of baseline), another 33% were inhibited (to 43% of baseline), and the rest (22%) were unaffected by PCP (Fig. 1). A detailed account of the effects of PCP on firing rate and burst firing is given in supporting information (SI) Table 1. Fig. 1 shows representative examples of neurons excited and inhibited by PCP as well as mean effects on firing rate.
Fig. 1.
PCP affects markedly the firing rate of pyramidal neurons in PFC. (A and B) Two representative examples of pyramidal neurons whose discharge was increased and decreased, respectively, by PCP administration (0.25 mg/kg i.v.; vertical arrows). The two traces below integrated firing rate histograms in each panel show the burst episodes in 1-min intervals during basal conditions (b) and after PCP administration (p) (shown in the abscissa of integrated firing rate histograms). In these horizontal traces, each vertical line corresponds to one burst episode. (C) Bar histogram showing the effect of PCP on the firing rate of pyramidal neurons according to the type of response. EXC, neurons excited by PCP (n = 36); INH, neurons inhibited by PCP (n = 26); NE, neurons unaffected by PCP (n = 18). *, P < 0.05 vs. baseline.
The effect of PCP administration (0.25 mg/kg i.v.) on LFPs was examined in 20 rats. In some of them, single units and LFPs were simultaneously recorded and the signal split by using the appropriate filters. PCP markedly reduced the cortical synchrony in the range 0.3–4 Hz (delta frequency). Saline injections did not alter LFPs. Overall, PCP reduced the power spectrum from 0.26 ± 0.04 to 0.10 ± 0.02 μV2/Hz (P < 0.001, Student's t test; n = 20). The reduction in the LFP amplitude was observed in all cases, irrespectively of whether the recorded unit was excited or inhibited by PCP. Fig. 2 shows examples of the effect of PCP on LFPs.
Fig. 2.
PCP induces a loss of synchronization of pyramidal spikes with the active phase of local field potentials in PFC. (A and B) Synchronization of the discharge of pyramidal neurons with the active phase of 0.3- to 4-Hz oscillations in mPFC in basal conditions. Note the temporal coincidence of spikes with active phases of the recorded LFPs (downward infections in the figure). (C and D) This temporal association was lost after PCP administration. (Scale bars: abscissa, 1 s; ordinate, 1 mV.)
PCP induced a temporal disorganization of the discharge of pyramidal neurons. In baseline conditions, pyramidal discharge occurred only during the active phase of the LFP [corresponding to depolarized or “up” states recorded intracellularly (12)] (Fig. 2). The percentage of spikes discharged during active (“up”) phases in baseline conditions was 90 ± 3%. PCP reduced this value to 59 ± 11% (P < 0.02, paired Student's t test; n = 9) (note that maximal effect size is to 50%, i.e., random discharge).

Antipsychotic Drug Reversal of PCP Effects.

Clozapine (CLZ, 1 mg/kg i.v.) or haloperidol (HAL, 0.1 mg/kg i.v.) had no significant effect on the spontaneous activity of mPFC pyramidal neurons, from 1.0 ± 0.3 to 0.7 ± 0.2 spike/s (CLZ; n = 9) and from 4.2 ± 1.8 to 3.6 ± 1.7 spikes/s (HAL; n = 5). However, CLZ and HAL reversed the PCP-induced increase in pyramidal cell firing (Fig. 3). Reversal was attempted in 16 neurons excited by 0.25 mg/kg i.v. PCP (9 with CLZ, 7 with HAL). CLZ (1 mg/kg, except 2 units at 2 mg/kg) reversed PCP effect (F2,16 = 13.09; P < 0.001). Likewise, HAL (0.1 mg/kg except in 2 units at 0.2 mg/kg) reversed PCP effect (F2,12 = 45.33, P < 0.0001) (Fig. 3C). Similarly, both antipsychotic drugs significantly reversed the increase in burst activity induced by PCP (baseline = 85 ± 28, PCP = 341 ± 138, PCP + CLZ = 113 ± 30, F2,14 = 5.55, P < 0.02; baseline = 50 ± 18, PCP = 338 ± 112, PCP + HAL = 77 ± 21, F2,12 = 6.03, P < 0.02; data in spikes fired in bursts/2 min). PCP-induced inhibitions were not reversed by antipsychotics (n = 5 with CLZ, n = 3 with HAL).
Fig. 3.
Reversal by antipsychotic drugs of the effects of PCP on PFC activity. (A1 and B1) Examples of two pyramidal neurons in mPFC showing an increase in the discharge rate after PCP administration (0.25 mg/kg i.v.; first arrow), which was reversed by the subsequent administration of clozapine (CLZ, 1 mg/kg i.v.) (A1) or haloperidol (HAL, 0.1 + 0.1 mg/kg i.v.) (B1). (A2 and B2) The corresponding LFP recordings (10 s each) obtained in basal conditions and after PCP and PCP plus antipsychotic treatments (10-s periods are shown in the abscissa of A1 and B1). Note the marked suppression of oscillations induced by PCP administration. (A3 and B3) The power spectra of the above recordings (1 min each, around the 10-s above 10-s periods). (C and D) Bar histograms showing the effects of PCP and reversal by CLZ and HAL on cell firing (C) and local field potentials (D). *, P < 0.05 vs. baseline; #, P < 0.05 vs. PCP alone.
CLZ and HAL also significantly reversed the loss in cortical synchrony in the 0.3- to 4-Hz range induced by PCP. A total of 17 rats were used in these experiments. The examples in Fig. 3 show concurrent effects on the firing rate of recorded unit and on LFP. One way ANOVA revealed a significant effect of treatment (F2,20 = 12.97, P < 0.0002 for PCP + CLZ, n = 11 and F2,10 = 25.02, P < 0.0001 for PCP + HAL, n = 6) (see also SI Fig. 5).

Induction of c-fos in Pyramidal Neurons by PCP.

PCP (10 mg/kg i.p.) administration increased the expression of c-fos in various fields of the PFC, but mainly its medial part (layers III–VI of the cingulate, prelimbic and infralimbic cortices) containing the area where extracellular recordings were made (Fig. 4). PCP also increased c-fos expression in the piriform cortex and in a narrow band of cells in the boundary between layers III and V, extending over dorsal a lateral aspects of the PFC (Fig. 4).
Fig. 4.
Effects of PCP and CLZ on c-fos expression in PFC. (A) Macroscopic dark-field images from emulsion-dipped coronal sections at the level of PFC (AP ≈+3.2 mm) from control and treated rats showing the localization of cells expressing c-fos mRNA. Note the PCP-induced expression of c-fos in various areas of the PFC, notably in the prelimbic area, where most of the extracellular recordings were made. CLZ antagonized the increase in c-fos expression induced by PCP. (B) High magnification photomicrographs showing the detection in mPFC (prelimbic area) of c-fos mRNA by using 33P-labeled oligonucleotides (silver grains) in pyramidal cells, visualized by hybridization with Dig-labeled oligonucleotides complementary to vGluT1 mRNA (dark precipitates). Note the increase in the number of c-fos positive cells and the density of silver grains per cell induced by PCP as well as the CLZ-induced antagonism of this effect. Red arrows mark some double-labeled cells, as follows: SAL + SAL (B1); SAL + PCP (B2); CLZ + SAL (B3); CLZ + PCP (B4). (C) Enlargement of the areas marked in B. (D) High magnification photomicrographs showing the expression of c-fos mRNA (silver grains) in GABAergic cells of the prelimbic PFC, visualized by GAD mRNA (dark precipitate). Note the increase in the number of c-fos positive cells not expressing GAD mRNA in the SAL + PCP group (C2) (blue arrows) and the increase in GAD mRNA positive cells in the CLZ + PCP group (C4). (E) Bar graph showing drug-induced changes in c-fos expression in the prelimbic mPFC. Bars show mean ± SEM of three rats per group and correspond to the optical density measured in films exposed to coronal sections of PFC (see Materials and Methods). (F) Bar graphs showing drug effects on the percentage of pyramidal [vGlut1-positive (Left)] and GABAergic neurons [GAD-positive (Right)] expressing c-fos mRNA after each treatment. *, P < 0.01 vs. SAL + SAL; #, P < 0.01 vs. SAL + PCP. (G) Bar graph showing drug effects on the individual cell expression of c-fos (number of silver grains per cell) in pyramidal [vGluT1-positive (Left)] and GABAergic neurons [GAD-positive (Right)]. *, P < 0.01 vs. SAL + SAL; #, P < 0.01 vs. SAL + PCP. (Scale bars: 20 μm.)
CLZ (5 mg/kg i.p., 30 min before PCP) did not affect c-fos expression but prevented the increase induced by PCP, as assessed by measuring optical density in films exposed to the hybridized sections (F3,11 = 7.10, P < 0.0001, one-way ANOVA; prelimbic area) (Fig. 4E). The same pattern of change was obtained in the infralimbic area and the piriform cortex (data not shown).
Double in situ hybridization experiments revealed that the enhanced c-fos signal seen in films corresponds to an increase in the number of pyramidal (vGluT1-positive) neurons expressing c-fos (Fig. 4 B, C, and F) and also to a parallel increase in cellular expression (Fig. 4 C and G). PCP increased 2.8-fold the proportion of c-fos positive pyramidal neurons (Fig. 4F) and 2.4-fold the individual c-fos expression, as measured by counting the number of silver grains in positive cells (Fig. 4G; compare also Fig. 4 B1/C1 and B2/C2). CLZ pretreatment prevented the increase in the number of c-fos-positive, vGluT1-positive cells (F3,11 = 37.52, P < 0.0001; see group differences in Fig. 4F) and the increase in individual cellular expression in the same neurons (F3,214 = 19.32, P < 0.00001; see group differences in Fig. 4G; silver grains were counted in a total of 218 vGluT1-positive cells).
CLZ alone did not modify c-fos expression in GABAergic interneurons but markedly enhanced it in rats treated also with PCP (Fig. 4 D–F). Given the occurrence of different subpopulations of local GABAergic neurons in different cortical layers and their differential involvement in schizophrenia (5), we analyzed separately drug effects in superficial (I, II/III) and deep layers (V, VI). There was no effect of treatment in superficial layers, yet one-way ANOVA revealed a significant effect of treatment on c-fos expression in deep layers (F3,11 = 9.4, P < 0.01) which was due to the increase in the number of c-fos-positive GABA cells in the CLZ + PCP group (Fig. 4F). A similar change was noted in the individual c-fos expression in GABAergic neurons, as judged by the number of silver grains per cell (F3,79 = 8.81, P < 0.00001; Fig. 4G; silver grains were counted in a total of 83 GAD-positive cells).

Expression of c-fos in Afferent Areas to PFC.

To identify afferent areas to PFC potentially involved in the increased PFC activity induced by PCP (see discussion below) we examined the effect of PCP on c-fos expression in the ventral hippocampus and the thalamus [mediodorsal (MD), centromedial (CM), and reticular nuclei(Rt)]. PCP induced a very small increase in the number of c-fos positive cells in the CA1/subiculum whereas it moderately increased c-fos positive cells in the adjacent entorhinal cortex. Double in situ hybridization (vGluT1 and c-fos mRNAs) revealed that c-fos expression took place exclusively in pyramidal cells (SI Fig. 6).
Unlike in hippocampus, PCP induced a dramatic increase in the number of c-fos-positive cells in the CM and MD nuclei as well as in adjacent midline and intralaminar nuclei. Optical density measures in films exposed to these sections indicated a significant effect of PCP on c-fos expression (from 0.133 ± 0.003 in SAL + SAL rats to 0.223 ± 0.019 in SAL + PCP rats n = 3 in both groups; P < 0.01). Double in situ hybridization revealed that c-fos was expressed by vGlut1-positive thalamic relay neurons in the CM and MD nuclei. However, PCP did not enhance c-fos expression in GABA neurons of the Rt nucleus (SI Fig. 7).

Discussion

The present findings indicate that the psychotomimetic actions of PCP are associated with a profound disruption of the PFC function. PCP affected 78% of the recorded pyramidal neurons (45% excited, 33% inhibited), and markedly reduced the cortical synchrony in the delta frequency range (0.3–4 Hz). Because the recorded units project to midbrain, it is unknown whether PCP may equally affect pyramidal neurons projecting to other areas (e.g., nucleus accumbens). These effects were reversed by the subsequent administration of a classical (HAL) and atypical (CLZ) antipsychotics, indicating a cellular convergence of the effects of both drugs in PFC, irrespectively of their different pharmacological properties. Further, PCP markedly increased the expression of c-fos in a substantial proportion of pyramidal neurons (50%) but not in GABA interneurons, an effect also prevented by CLZ administration. Two other studies examined the effect of PCP (13) and MK-801 (14) on the discharge rate of putative pyramidal PFC neurons in vivo. This study demonstrates that (i) PCP deeply affects cortical synchrony, and (ii) cellular and population effects of PCP in PFC are reversed by antipsychotic drugs. Further, c-fos data strongly support the involvement of thalamocortical inputs as mediators of PCP effects in PFC.
Delta waves reflect synchronized activity changes of cortical neuronal networks and are an emergent property of thalamocortical circuits dependent on the presence and interplay of specific currents [hyperpolarization-activated cation current (Ih) and transient low-threshold Ca2+ current (IT)] in thalamocortical neurons (12). Here, we show that PCP evokes a profound loss of the efficiency of cortical information processing, dramatically changing the activity patterns of PFC pyramidal neurons and suppressing delta rhythm. The loss of synchrony induced by PCP and the resulting random discharge of pyramidal neurons is likely to have profound effects on PFC-dependent functions. It may also affect subcortical regions as a result of the top-down processing of information in cortico-limbic circuits. Interestingly, PCP reduced the frequency and duration of depolarized (“up”) sates of nucleus accumbens neurons recorded intracellularly, which depends on hippocampal input, and affected their response to PFC stimulation (15).
Overall, these observations indicate that PCP disrupts the activity of cortico–limbic networks, compromising the functional connectivity between brain areas altered in schizophrenia (1, 2, 5). Yet it is unknown to what extent PCP-evoked changes in the PFC of anesthetized rats are related to the alterations in oscillations seen in the PFC of schizophrenic patients and the associated cognitive deficits (16, 17). Despite this limitation, it is interesting to note that two other agents modeling schizophrenia in rodents induce changes similar to those of PCP. Thus, the hallucinogen DOI (5-HT2A receptor agonist) alters the activity pattern of PFC pyramidal neurons similarly to PCP (18) and suppresses cortical delta rhythm (P.C., M. V. Puig, L. Díaz-Mataix, and F.A., unpublished observations). These common effects suggest a link between the altered PFC activity and the hallucinations induced by both agents. Also, the mitotoxic agent methylazoxymethanol, used as a neurodevelopmental model of schizophrenia, evoked a similar reduction of cortical synchrony in the delta range (19).
The reduction in cell firing evoked by PCP in some neurons is consistent with the excitatory role of cortical NMDA-R on pyramidal neurons and their blockade by PCP (13, 20). On the other hand, the increase in firing rate and c-fos expression in most PCP-sensitive neurons agrees with the increased PFC glutamate output induced by noncompetitive NMDA-R blockade (21, 22). Excitatory PCP effects on pyramidal neurons may be caused by disinhibition of PFC GABA interneurons, as suggested for MK-801 (14). They may be also caused by NMDA-R blockade of GABAergic neurons outside the PFC, and further disinhibition of excitatory neurons that project to PFC (3, 23, 24). The hippocampus has been claimed to mediate this effect via the CA1-PFC pathway (24). However, the present results do not support this view because PCP had a very minor effect on the number of glutamatergic cells expressing c-fos in CA1/subiculum. In contrast, PCP markedly increased the number of c-fos-positive cells in the CM and MD thalamic nuclei (vGluT1-positive), which densely project to PFC, but not in GABAergic cells of the reticular nucleus (Rt) which provides inhibitory feed-back to relay neurons in the rest of thalamic nuclei, including CM and MD.
Thus, PCP markedly affects reciprocal excitatory thalamocortical circuits. PCP may disinhibit corticothalamic PFC neurons by acting on PFC GABA interneurons. Likewise, it may increase thalamocortical excitatory inputs by blocking NMDA-R in Rt and/or afferent inhibitory areas to the thalamus (e.g., ventral pallidum). Both possibilities require further testing, yet the latter hypothesis is consistent with previous observations indicating that the removal of inhibitory GABAA tone in MD/CM nuclei increases c-fos expression and pyramidal discharge rate in PFC (18, 25) as observed here with PCP. The involvement of thalamocortical inputs is also suggested by the increased c-fos expression produced by PCP in deep layer III/superficial layer V of PFC and layer IV of parietal cortex (N.S., P.C., G.M., and F.A., unpublished observations), which are target of inputs from MD onto PFC pyramidal neurons in the rat (26). Additionally, given the increase in vGluT1-positive cells expressing c-fos in the entorhinal cortex and its direct connectivity with PFC (27), this pathway cannot be excluded.
Another relevant observation is that CLZ and HAL normalized the PCP-induced disruption of cortical function at cellular and population levels. Both drugs are equally effective to reverse the effect of NMDA-R blockade in some experimental models [e.g., increase in glutamate output (21, 22) yet differences have been noted in behavioral models (3, 10)]. CLZ and HAL possibly reverse PCP effects by antagonizing, at the doses used, 5-HT2A and dopamine D2 receptors, respectively. These actions prevented neurochemical and behavioral effects of NMDA-R antagonists (3, 28, 29) yet discrepant results have also been reported (30). The cellular basis for these effects is still poorly understood. 5-HT2A receptor blockade may attenuate glutamatergic transmission in mPFC (31). On the other hand ventral tegmental area stimulation excited fast spiking interneurons and concurrently inhibited PFC pyramidal neurons (32). Thus, HAL may attenuate PCP-induced pyramidal excitation by means of activation of dopamine D1 receptors secondary to an autoreceptor-mediated increase of PFC dopamine release.
Finally, the CLZ-induced prevention of PCP effects on c-fos expression in pyramidal PFC neurons agrees with the above electrophysiological observations, despite the different administration routes used. Interestingly, CLZ alone did not alter c-fos expression in GABAergic neurons yet increased it in animals treated with PCP, suggesting an state-dependent action. This effect occurred in deeper cortical layers, perhaps in large parvalbumin-positive GABAergic interneurons, which are densely expressed in layer V (33) and have been implicated in the pathophysiology of schizophrenia (5). The increased activity of GABAergic cells induced by CLZ + PCP may be related to the normalized pyramidal discharge through enhanced local inhibitory inputs.

Materials and Methods

Animals and Treatments.

Male albino Wistar rats weighing 250–320 g were used (Iffa Credo, Lyon, France). Animal care followed European Union regulations (Official Journal of the European Communities L358/1, December 18, 1986) and was approved by the Institutional Animal Care and Use Committee. Stereotaxic coordinates (in millimeters) were taken from bregma and dura mater (34). PCP and CLZ were from RBI (Natick, MA) and HAL (intramuscular preparation) was from Laboratorios Esteve (Barcelona, Spain).
In c-fos experiments, four groups of rats were administered i.p. with saline plus saline, saline plus PCP (10 mg/kg), CLZ (5 mg/kg) plus saline, and CLZ (5 mg/kg) plus PCP (10 mg/kg), respectively. Time between injections was 30 min, and rats were killed by anesthetic overdose and decapitated 1 h after the second injection. The brains were rapidly removed, frozen on dry ice, and stored at −20°C.

Electrophysiology: Single Unit and LFP Recordings.

Rats were anesthetized (chloral hydrate, 400 mg/kg, i.p. followed by 50–70 mg/kg per h using a perfusion pump) and positioned in a David Kopf stereotaxic frame (David Kopk, Tujunga, CA). Body temperature was maintained at 37°C with a heating pad. Pyramidal neurons were recorded extracellularly with glass micropipettes as described (18, 35). Briefly, single-unit extracellular recordings were amplified with a Neurodata IR283 (Cygnus Technology, Delaware Water Gap, PA), postamplified, and filtered with a Cibertec (Madrid, Spain) amplifier and computed on-line by using a DAT 1401plus interface system Spike2 software (Cambridge Electronic Design, Cambridge, U.K.). Descents in the mPFC were carried out at anteroposterior +3.2 to +3.4, lateral −0.5 to −1.0, dorsoventral −1.9 to −4.8 below brain surface. All recorded units were identified as pyramidal neurons by antidromic activation from dorsal raphe or ventral tegmental area and collision extinction with spontaneously occurring spikes as described (18). After stable baseline activity for 5 min, PCP was injected, followed by CLZ or HAL in reversal experiments. Only one neuron per rat was recorded. In some cases, recording electrodes were filled with Pontamine sky blue to verify the recording site. Brain sections were stained with neutral red, according to standard procedures.

In Situ Hybridization Histochemistry.

The oligonucleotide probes used were as follows: c-fos complementary to bases 131–178 (GenBank accession no. NM 022197); vesicular glutamate transporter vGluT1 (a glutamatergic cell marker) complementary to bases 127–172 and 1756–1800 (GenBank accession no. U07609); two oligonucleotides for each isoform glutamic acid decarboxylase (GAD65 and GAD67 to label GABAergic cells) complementary to bases 159–213 and 514–558 (GenBank accession no. NM_012563) and bp 191–235 and 1600–1653 (GenBank accession no. NM_017007). Probes were synthesized on a 380 Applied Biosystems DNA synthesizer (Applied Biosystems, Foster City, CA). Labeling of the probes, tissue sectioning and in situ hybridization procedures were carried out as described (35).

Data Analysis.

Changes in the firing rate or the proportion of burst firing in pyramidal neurons were assessed by using ANOVA or paired Student's t test, as appropriate. Values were quantified during the last 2 min of the 5-min baseline recording and during 2 min after PCP or antipsychotic administration (omitting the first minute after injection). Drug response was defined as a ± 30% change in firing rate from baseline. Bust analysis was carried out (Spike 2 software) by using the method of Laviolette et al. (36). Briefly, a burst episode was defined as the occurrence of two or more spikes with an interspike interval of <45 ms.
Power spectra were constructed by using Fast Fourier Transformations (FFT) of 1-min signal intervals (band-pass filter of 0.1–100 Hz) corresponding to baseline, PCP and PCP + antipsychotics with a resolution of 0.29 Hz (FFT size of 8192). Data are given as AUC of the power spectrum between 0.3–4 Hz. The temporal coincidence of discharged spikes with the active phase (up) of delta oscillations recorded in PFC was examined (1-min periods) with a custom made script for the Spike 2 software. Briefly, the mean LFP voltage value was calculated for each 1-min period and the number of spikes occurring below and above this mean value were computed.
Photomicrographs were obtained with a Nikon Eclipse E1000 microscope (Nikon, Tokyo, Japan). Cell counting was performed manually at the microscope. Only cellular profiles showing great abundance of both transcripts were considered to coexpress both mRNAs. A semiquantitative measure of the optical densities was conducted for in situ hybridization studies with the AISR computerized image analysis system (Imaging Research Inc., St. Catharines, ON, Canada). For each rat, individual values of optical densities and cell counts in prelimbic area of PFC were calculated as the mean of two adjacent sections of three rats per treatment group. The number of silver grains (representative of the level of expression of c-fos) in each c-fos-positive cell was counted manually in the same sections at a higher magnification in adjacent microscopic fields within the prelimbic area. Statistical analysis was performed by using a statistical software package (GraphPad Prism 4, GraphPad Software Inc, San Diego, CA). P < 0.05 was considered statistically significant.

Abbreviations

CLZ
clozapine
CM
centromedial nucleus
GAD
glutamate decarboxylase
HAL
haloperidol
LFP
local field potential
MD
mediodorsal nucleus
PCP
phencyclidine
mPFC
medial prefrontal cortex
vGluT1
vesicular glutamate transporter 1
Rt
reticular nuclei.

Acknowledgments

We thank Judith Ballart for skillful technical assistance and Ranulfo Romo and Kuei Y. Tseng for critical reading and helpful suggestions on the manuscript. This work was supported by Grant SAF 2004-05525 from the Ministry of Health. Support from Generalitat de Catalunya Grant 2005SGR00758 and Spanish Ministry of Health, Instituto de Salud Carlos III, RETICS Grant RD06/0011(REM-TAP Network) is also acknowledged. L.K. and N.S. are recipients of predoctoral fellowships from the Ministry of Science and Education.

Supporting Information

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References

1
PJ Harrison Brain 122, 593–624 (1999).
2
DA Lewis, JA Lieberman Neuron 28, 325–334 (2000).
3
JH Krystal, DC D'Souza, D Mathalon, E Perry, A Belger, R Hoffman Psychopharmacology (Berlin) 169, 215–233 (2003).
4
LD Selemon, PS Goldman-Rakic Biol Psychiatry 45, 17–25 (1999).
5
DA Lewis, T Hashimoto, DW Volk Nat Rev Neurosci 6, 312–324 (2005).
6
DR Weinberger, MF Egan, A Bertolino, JH Callicott, VS Mattay, BK Lipska, KF Berman, TE Goldberg Biol Psychiatry 50, 825–844 (2001).
7
DC Javitt, SR Zukin Am J Psychiatry 148, 1301–1308 (1991).
8
A Breier, AK Malhotra, DA Pinals, NI Weisenfeld, D Pickar Am J Psychiatry 154, 805–811 (1997).
9
M Carlsson, A Carlsson J Neural Transm 75, 221–226 (1989).
10
MA Geyer, K Krebs-Thomson, DL Braff, NR Swerdlow Psychopharmacology (Berlin) 156, 117–154 (2001).
11
B Elvevag, TE Goldberg Crit Rev Neurobiol 14, 1–21 (2000).
12
M Steriade, DA McCormick, TJ Sejnowski Science 262, 679–685 (1993).
13
Y Suzuki, E Jodo, S Takeuchi, S Niwa, Y Kayama Neuroscience 114, 769–779 (2002).
14
ME Jackson, H Honayoun, B Moghaddam Proc Natl Acad Sci USA 101, 8467–8472 (2004).
15
P O'Donnel, AA Grace Neuroscience 87, 823–830 (1998).
16
G Winterer, DR Weinberger Trends Neurosci 27, 683–690 (2004).
17
PJ Uhlhaas, W Singer Neuron 52, 155–168 (2006).
18
MV Puig, P Celada, L Diaz-Mataix, F Artigas Cereb Cortex 13, 870–882 (2003).
19
Y Goto, AA Grace Biol Psychiatry 60, 1259–1267 (2006).
20
WX Shi, XX Zhang J Pharmacol Exp Ther 305, 680–687 (2003).
21
B Adams, B Moghaddam J Neurosci 18, 5545–5554 (1998).
22
X Lopez-Gil, Z Babot, M Amargos-Bosch, C Sunol, F Artigas, A Adell Neuropsychopharmacology, in press. (2007).
23
G Tsai, JT Coyle Annu Rev Pharmacol Toxicol 42, 165–179 (2002).
24
E Jodo, Y Suzuki, T Katayama, KY Hoshino, S Takeuchi, S Niwa, Y Kayama Cereb Cortex 15, 663–669 (2005).
25
M Bubser, JM de Brabander, W Timmerman, MG Feenstra, EB Erdtsieck-Ernste, A Rinkens, JF van Uum, BH Westerink Synapse 30, 156–165 (1998).
26
M Kuroda, J Yokofujita, K Murakami Prog Neurobiol 54, 417–458 (1998).
27
R Insausti, MT Herrero, MP Witter Hippocampus 7, 146–183 (1997).
28
P Martin, N Waters, S Waters, A Carlsson, M Carlsson Eur J Pharmacol 335, 107–116 (1997).
29
I Ceglia, M Carli, M Baviera, G Renoldi, E Calcagno, RW Invernizzi J Neurochem 91, 189–199 (2004).
30
BW Adams, B Moghaddam Biol Psychiatry 50, 750–757 (2001).
31
GK Aghajanian, GJ Marek Brain Res 825, 161–171 (1999).
32
KY Tseng, N Mallet, KL Toreson, C Le Moine, F Gonon, P O'Donnell Synapse 59, 412–417 (2006).
33
AC Grobin, JA Lieberman, L Morrow J Neurosci 23, 1832–1839 (2003).
34
G Paxinos, C Watson The Rat Brain in Stereotaxic Coordinates (Academic, Sydney, 1998).
35
M Amargós-Bosch, A Bortolozzi, MV Puig, J Serrats, A Adell, P Celada, M Toth, G Mengod, F Artigas Cereb Cortex 14, 281–299 (2004).
36
SR Laviolette, WJ Lipski, AA Grace J Neurosci 25, 6066–6075 (2005).

Information & Authors

Information

Published in

Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 104 | No. 37
September 11, 2007
PubMed: 17785415

Classifications

Submission history

Received: May 23, 2007
Published online: September 11, 2007
Published in issue: September 11, 2007

Keywords

  1. neuronal oscillations
  2. NMDA receptor antagonists
  3. schizophrenia
  4. delta frequency
  5. thalamus

Acknowledgments

We thank Judith Ballart for skillful technical assistance and Ranulfo Romo and Kuei Y. Tseng for critical reading and helpful suggestions on the manuscript. This work was supported by Grant SAF 2004-05525 from the Ministry of Health. Support from Generalitat de Catalunya Grant 2005SGR00758 and Spanish Ministry of Health, Instituto de Salud Carlos III, RETICS Grant RD06/0011(REM-TAP Network) is also acknowledged. L.K. and N.S. are recipients of predoctoral fellowships from the Ministry of Science and Education.

Notes

This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0704848104/DC1.

Authors

Affiliations

Lucila Kargieman
Department of Neurochemistry and Neuropharmacology, Institut d'Investigacions Biomèdiques de Barcelona (CSIC), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain
Noemí Santana
Department of Neurochemistry and Neuropharmacology, Institut d'Investigacions Biomèdiques de Barcelona (CSIC), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain
Guadalupe Mengod
Department of Neurochemistry and Neuropharmacology, Institut d'Investigacions Biomèdiques de Barcelona (CSIC), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain
Department of Neurochemistry and Neuropharmacology, Institut d'Investigacions Biomèdiques de Barcelona (CSIC), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain
Francesc Artigas [email protected]
Department of Neurochemistry and Neuropharmacology, Institut d'Investigacions Biomèdiques de Barcelona (CSIC), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain

Notes

*To whom correspondence may be addressed. E-mail: [email protected] or [email protected]
Author contributions: P.C. and F.A. contributed equally to this work; G.M., P.C., and F.A. designed research; L.K. and N.S. performed research; L.K., N.S., G.M., P.C., and F.A. analyzed data; and P.C. and F.A. wrote the paper.

Competing Interests

The authors declare no conflict of interest.

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