Neonatal Prefrontal Inactivation Results in Reversed Dopaminergic

Department of Molecular Animal Physiology, Radboud University Nijmegen, Donders Institute for Brain, Cognition and Behaviour, 6500 HB, Nijmegen, The N...
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Neonatal Prefrontal Inactivation Results in Reversed Dopaminergic Responses in the Shell Subregion of the Nucleus Accumbens to NMDA Antagonists Tiphaine Pouvreau,†,§ Emmanuelle Tagliabue,†,§ Yusuf Usun,† Séverine Eybrard,† Francisca Meyer,‡ and Alain Louilot*,† †

INSERM U 1114, Faculty of Medicine, FMTS, University of Strasbourg, Strasbourg 67085, France Department of Molecular Animal Physiology, Radboud University Nijmegen, Donders Institute for Brain, Cognition and Behaviour, 6500 HB, Nijmegen, The Netherlands

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ABSTRACT: Striatal dopaminergic dysregulation in schizophrenia could result from a prefronto-striatal dysconnectivity, of neurodevelopmental origin, involving Nmethyl-D-aspartate (NMDA) receptors. The dorsomedian shell part of the nucleus accumbens is a striatal subregion of particular interest inasmuch as it has been described as the common target region for antipsychotics. Moreover, NMDA receptors located on the dopaminergic endings have been reported in the shell. The present study examines in adult rats the effects of early functional inactivation of the left prefrontal cortex on behavioral and dopaminergic responses in the dorsomedian shell part of the nucleus accumbens following administration of two noncompetitive NMDA receptor antagonists, ketamine, and dizocilpine (MK-801). The results showed that postnatal blockade of the prefrontal cortex led to increased locomotor activity as well as increased extracellular dopamine levels in the dorsomedian shell following administration of both noncompetitive NMDA receptor antagonists, and, more markedly, after treatment with the more specific one, MK-801, whereas decreased dopaminergic levels were observed in respective controls. These data suggest a link between NMDA receptor dysfunctioning and dopamine dysregulation at the level of the dorsomedian shell part of the nucleus accumbens. They may help to understand the pathophysiology of schizophrenia in a neurodevelopmental perspective. KEYWORDS: Ketamine, MK-801, schizophrenia, animal modeling, TTX, in vivo voltammetry

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administration of subanesthetic doses of noncompetitive NMDA receptor antagonists (e.g., ketamine) led to symptoms reminiscent of schizophrenia in normal volunteers17,18 and enhanced such symptoms in patients.19,20 A better understanding of the pathophysiology of schizophrenia appears crucial given that commonly used treatments with antipsychotics have limited actions.21 Owing to the nature of schizophrenia, however, and from ethical point of view, clinical studies with untreated patients are difficult to conduct, such that animal modeling of the pathophysiology of the disease is of particular significance. The present work involving adult rats sets out to study the consequences of neonatal functional inactivation of the left PFC for the behavioral and dopaminergic responses in the dorsomedian shell part of the nucleus accumbens following administration of two noncompetitive NMDA receptor antagonists, ketamine and dizocilpine (MK-801). Functional inactivation of the PFC was achieved at postnatal day 8 (PND8) by local microinjection of tetrodotoxin (TTX), a potent and selective voltage-gated sodium channel blocker,22 in the left anteromedial PFC. PND8 is an important time point for brain development in rats, during

chizophrenia is a severe mental illness emerging in young adults. With a global lifetime prevalence of 0.3−0.7% (DSM-5, 2013),1 it is the most common psychotic disorder. However, the underlying pathophysiological mechanisms are not yet fully understood. According to recent proposals, schizophrenia would result from a functional disconnection between different integrative cerebral regions,2 stemming, at least in some cases, from neurodevelopmental failures.3,4 Among these regions, the prefrontal cortex (PFC) stands out as one structure particularly involved,5 namely, in the left hemisphere.6−8 Regarding the pathophysiology of schizophrenia, the existence of a striatal dopaminergic dysfunctioning is widely accepted.9−11 This dysfunctioning could be the consequence of a prefronto-striatal dysregulation involving N-methyl-D-aspartate (NMDA) receptors.10,11 In this respect, the dorsomedian shell part of the nucleus accumbens12 is a striatal subregion of particular interest insofar as it has been reported to be the common target structure for antipsychotics.13−15 Moreover, NMDA receptors located presynaptically on the dopaminergic terminals have been described in the shell part of the nucleus accumbens.16 Glutamatergic dysfunctions involving NMDA receptors may be important in the pathophysiology of schizophrenia and have received considerable attention in recent years.10,11 Pharmacological studies reported that © XXXX American Chemical Society

Received: March 18, 2016 Accepted: May 4, 2016

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ACS Chemical Neuroscience which electrical neuronal activity appears essential for developing fibers to establish appropriate connections in the brain’s target structures.23,24 It equates to the end of the second trimester of gestation in humans,25 reportedly a period of particular vulnerability for developing schizophrenia.3 Locomotor activity, a behavioral index generally considered as sensitive to dysregulated dopamine and glutamate manipulations,26 was assessed before and after ketamine and MK-801 were administered. Locomotor activity and extracellular dopamine levels in the dorsomedian shell part of the nucleus accumbens were measured simultaneously using in vivo voltammetry in freely moving grown-up rats.

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RESULTS Histology. No anatomical modifications, damage, or gliosis were detected with qualitative analysis of the brains of animals microinjected in the PFC at PND8 with either PBS (Figure 1A)

Figure 2. Ketamine-induced locomotor activity of adult animals microinjected at postnatal day 8 with PBS (upper graph) or TTX (lower graph). Adult animals received a subcutaneous (s.c.) injection (arrow) of NaCl 0.9% (white columns) or different ketamine doses (gray columns). Results are expressed as means ± SEM of the number of crossings in each 10 min period. n represents the number of animals per group. Statistical analysis was performed using factorial ANOVA.

microinjection (PBS or TTX) on locomotor activity was observed. More precisely, the general ANOVA for the 60 min postinjection period revealed a statistically significant doseeffect (NaCl 0.9%, ketamine 5 mg/kg, ketamine 10 mg/kg or ketamine 20 mg/kg) (F[3,69] = 26.29 p < 0.000001) and a significant effect of the microinjection (PBS/TTX) (F[1,69] = 8.59 p < 0.005), but no significant dose × microinjection interaction (F[3,69] = 1.81 ns). MK801-Induced Locomotor Activity. Fifty-six animals were used for the MK-801-induced locomotor activity study. Locomotor activity measured during the 60 min following MK-801 injection was found to be dose-dependent. A statistical effect of neonatal microinjection (PBS or TTX) on locomotor activity was observed (Figure 3). More precisely, the general ANOVA for the 60 min postinjection period showed a statistically significant dose-effect (NaCl 0.9%, MK-801 0.1 mg/kg or MK-801 0.2 mg/kg) (F[2,50] = 11.76 p < 0.0001) and a significant effect of the microinjection (PBS/TTX) (F[1,50] = 5.44 p < 0.05), but no significant dose × microinjection interaction (F[2,50] = 2.29 ns). Dopaminergic Changes Recorded in the Dorsomedian Shell. Only animals with working electrode recording sites clearly situated in the left dorsomedian shell (Figure 1, right) were taken into consideration for the voltammetric analysis. Ketamine-Induced Dopaminergic Changes in the Dorsomedian Shell. Fifty-two animals were included in the study of ketamine-induced dopaminergic changes. Variations in extracellular dopamine levels induced by ketamine were significantly dependent on the neonatal microinjection (PBS or TTX).

Figure 1. Typical microinjection sites in the left anteromedian prefrontal cortex (left sections) and typical recording sites in the left dorsomedian shell (right sections). Brain sections were performed in adult rats microinjected at postnatal day 8 either with PBS (A) or tetrodotoxin (TTX) (B). The postnatal microinjection sites in the left prefrontal cortex (arrows) were localized in adult animals by means of the vital dye Evans Blue included in the PBS and TTX solutions. Sections were stained with Neutral Red. The voltammetric recording sites in the dorsomedian shell (arrows) were identified by electrocoagulation carried out once the experiment was completed. Tissue sections were stained with Thionin Blue. Scale bar = 1 mm. Cx, cortex; CC, corpus callosum; Nacc, nucleus accumbens; ST, striatum.

or TTX (Figure 1B). Only animals with microinjection sites clearly located in the prelimbic/infralimbic part of the left PFC27 (Figure 1, left) were included in the locomotor activity analysis; 20 animals over a total of 153 were not taken into account for misplacement of the injection site. Locomotor Activity. Ketamine-Induced Locomotor Activity. Seventy-seven animals were used to study the ketamine-induced locomotor activity. No differences between PBS animals and TTX animals microinjected in the PFC were observed during the first 30 min following placement in the cage (data not shown). Locomotor activity measured during the 60 min following ketamine injection was found to be dosedependent (Figure 2). A significant effect of neonatal B

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injection effect (PBS/TTX) (F[1,33] = 29.70 p < 0.00001) and a significant dose × microinjection interaction (F[2,33] = 14.52 p < 0.00005), but no significant dose-effect (NaCl 0.9%, MK801 0.1 mg/kg or MK-801 0.2 mg/kg) (F[2,33] = 1.06 ns). For the 60 min postinjection period, the contrast analysis of ANOVA was performed to test the hypothesis that dopaminergic changes induced by the two MK-801 doses were significantly dependent on the neonatal microinjection (PBS/TTX). Significant microinjection effects (PBS/TTX) were found for the MK-801 0.2 mg/kg s.c. dose (F[1,33] = 56.93 p < 0.000001), but not for the MK-801 0.1 mg/kg s.c. dose (F[1,33] = 1.13 ns).

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DISCUSSION The present study concerned with animal modeling for schizophrenia used adult rats subjected to postnatal disconnection of the left PFC to investigate the impact of two noncompetitive NMDA receptor antagonists, ketamine and dizocilpine (MK-801), on the behavioral and dopaminergic responses recorded in the dorsomedian shell part of the nucleus accumbens, a striatal subregion described as a common target for antipsychotics.13−15 Our results showed that early functional TTX inactivation of the PFC led to increased dopaminergic levels in the dorsomedian shell after the pharmacological challenge with the two NMDA antagonists whereas decreased dopaminergic levels were observed in respective controls. Dopaminergic variations were more marked after treatment with MK-801, the more specific of the two NMDA antagonists. Locomotor responses measured in parallel to dopaminergic responses were found to be specifically increased for each NMDA-antagonist, and more markedly so in the animals neonatally inactivated with TTX. As regards locomotor activity, for the animals in the PBS and TTX groups, administration of the two noncompetitive NMDA receptor antagonists (ketamine, MK-801) induced drug-specific and dose-dependent increases in locomotor responses which for both antagonists were more elevated for the animals in the TTX groups. The present results are consistent with the pharmacokinetic profiles described for both NMDA antagonists which show that ketamine has a faster action onset and considerably shorter half-life of elimination,28 as well as a lesser affinity for NMDA receptors than that of MK-801.29,30 Concerning the animals in the PBS groups, the results obtained are in keeping with those of several studies showing that ketamine administered in the range of 2.5 to 50 mg/kg induced a dose-dependent increase in locomotor activity characterized by a return toward basal values after 20 to 50 min postinjection depending on the dose,31−33 whereas the increase in locomotor activity was still elevated 60 min after the injection of MK-801 in the range of 0.05 to 0.2 mg/kg.32,34,35 As regards the animals in the TTX groups, the present data obtained following administration of ketamine are in accordance with those reported after neonatal inactivation of the PFC, showing locomotor hyperactivity in grown-up rats that is more elevated for the TTX than the PBS animals.33 To the best of our knowledge, the impact of the postnatal functional blockade of the PFC by TTX on the locomotor hyperactivity induced by MK-801 has never been investigated before and, thus, cannot be directly compared to other studies. As regards the extracellular dopaminergic variations in the dorsomedian shell, the results we obtained in freely moving animals also appear to be original. Concerning the animals in the PBS groups, and based on the literature, it is nevertheless

Figure 3. MK-801-induced locomotor activity of adult animals microinjected at postnatal day 8 with PBS (upper graph) or TTX (lower graph). Adult animals received a subcutaneous (s.c.) injection (arrow) of NaCl 0.9% (white columns) or one of the two MK-801 doses (gray columns). Results are expressed as means ± SEM of the number of crossings in each 10 min period. n represents the number of animals per group. Statistical analysis was performed using factorial ANOVA.

Dopaminergic responses were dependent on the dose of ketamine, differently for the PBS and TTX groups (Figure 4). Moreover, for the 60 min postinjection period, variations in the dopamine signal differed significantly between the PBS and TTX groups for the two highest s.c. doses of ketamine (10 mg/ kg; 20 mg/kg). More precisely, the general ANOVA for the 60 min postinjection period highlighted a significant neonatal microinjection effect (PBS/TTX) (F[1,44] = 11.47 p < 0.005) and a significant microinjection × dose interaction (F[3,44] = 2.94 p < 0.05), but no significant dose-effect (NaCl 0.9%, ketamine 5 mg/kg, ketamine 10 mg/kg or ketamine 20 mg/kg) (F[3,49] = 2.11 ns). To determine whether dopaminergic changes induced by the different ketamine doses were statistically different depending on the neonatal microinjection (PBS/TTX), a contrast analysis of ANOVA was performed for the 60 min postinjection period. Significant microinjection effects (PBS/TTX) were shown for the 20 mg/kg dose (F[1,44] = 10.77 p < 0.005) and the 10 mg/kg dose (F[1,44] = 4.41 p < 0.05), but not for the 5 mg/kg dose (F[1,44] = 3.64 ns). However, a trend was observed (p = 0.063) for this lowest dose. MK-801-Induced Dopaminergic Changes in the Dorsomedian Shell. Thirty-nine animals were used to study MK-801induced dopaminergic changes in dorsomedian shell. Dopaminergic variations induced by MK-801 were statistically dependent on the neonatal microinjection (PBS or TTX), and dopaminergic responses were dependent on the MK-801 dose in a different way for the PBS and TTX groups (Figure 5). Variations in the dopamine signal for the PBS and TTX groups were statistically different for the highest dose of MK-801, 0.2 mg/kg s.c.. More precisely, the general ANOVA for the 60 min postinjection period revealed a significant neonatal microC

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Figure 4. Ketamine-induced dopaminergic variations in the left dorsomedian shell of adult rats microinjected at postnatal day 8 (PND8) in the anteromedian prefrontal cortex (PFC) with PBS (left graphs) or TTX (right graphs). Dopaminergic levels in the left nucleus accumbens following subcutaneous (s.c.) injection of NaCl (0.9%) (A) or ketamine ((B) 5 mg/kg, (C) 10 mg/kg, and (D) 20 mg/kg) were recorded every min using differential normal pulse voltammetry and computer-assisted numerical analysis in freely moving animals. Only mean values and SEM corresponding to each two voltammograms are presented. Where no SEM is given, the size is less than the radius of the symbol. The injection time is indicated by the arrow. n is the number of animals per group. Statistical analysis was performed using factorial ANOVA.

possible to propose, first, that the dopaminergic variations we observed are the result of interactions with excitatory glutamatergic pathways acting on the dopaminergic terminals. Indeed, NMDA receptors located presynaptically on the dopaminergic endings have been described in the shell part of the nucleus accumbens.16 Furthermore, it has been shown with rat striatal synaptosomes and tissue slices that stimulation of

presynaptic NMDA receptors induced an increase in dopamine release.36 Thus, conversely, a direct antagonist action of ketamine or MK-801 on the NMDA receptors may account for the present results, which are consistent with the rapid action onset and short half-life of elimination of ketamine compared to MK-801.28 However, in addition to direct excitatory glutamatergic pathways, the regulation of dopamine D

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Figure 5. MK-801-induced dopaminergic variations in the left dorsomedian shell of adult rats microinjected at postnatal day 8 (PND8) in the anteromedian prefrontal cortex (PFC) with PBS (left graphs) or TTX (right graphs). Dopaminergic levels in the left nucleus accumbens following subcutaneous (s.c.) injection of NaCl (0.9%) (A) or MK-801 (0.1 mg/kg and 0.2 mg/kg, respectively B and C) were recorded every min using differential normal pulse voltammetry and computer-assisted numerical analysis in freely moving animals. Only mean values and SEM corresponding to each two voltammograms are presented. Where no SEM is given, the size is less than the radius of the symbol. The time of injection is indicated by the arrow. n is the number of animals per group. Statistical analysis was performed using factorial ANOVA.

release in the dorsomedian shell part by NMDA receptors could involve more complex mechanisms, such as indirect inhibitory GABAergic pathways. Indeed, it has been shown by means of in vivo microdialysis that striatal infusion of NMDA induced an increase in extracellular GABA levels,37 whereas through presynaptic action on dopaminergic endings38 the stimulation or blockade of the GABAergic transmission in accumbal synaptosomes entailed a decrease or increase of dopamine release, respectively. Thus, the most parsimonious hypothesis is that NMDA receptors located on intrinsic GABAergic interneurons help regulate dopamine release in the dorsomedian shell. Blockade of these receptors by NMDA antagonists could lead to increased dopamine levels. To sum up, for the PBS groups, the dopaminergic variations obtained in the dorsomedian shell part of the nucleus accumbens after administration of ketamine or MK-801 could result mainly from the addition of excitatory effects dependent on NMDA receptors situated on the dopaminergic nerve terminals and indirect inhibitory effects involving NMDA receptors located

on GABAergic interneurons. This dual action may explain the delayed increases observed in the PBS animals with the two lowest doses of ketamine (5 mg/kg s.c.; 10 mg/kg s.c.) as well as the lack of any decrease observed with the lowest dose of MK-801 (0.1 mg/kg s.c.). In other respects, the prolonged decreases in extracellular dopamine levels observed in the present study with the highest doses of ketamine (20 mg/kg s.c.) and MK-801 (0.2 mg/kg s.c.) suggest that for the PBS groups the resultant of the two influences is in favor of the putative direct excitatory pathway involving presynaptic NMDA receptors. Concerning the animals in the TTX groups, the dopaminergic variations in the dorsomedian shell part of the nucleus accumbens differ from those observed in the PBS groups for both NMDA antagonists. Changes in TTX animals are characterized by dopamine signal increases, which are statistically distinct from the variations obtained in PBS animals with the highest doses of ketamine (10 mg/kg and 20 mg/kg s.c.) and MK-801 (0.2 mg/kg s.c.). Moreover, a more marked E

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light/dark cycle. All of the experimental procedures were conducted in accordance with the European Community guidelines for the care and use of experimental animals (Council Directive 86/609/EEC) and authorized by the French Ministry of Agriculture (authorization 67244 to Alain Louilot). All of the experimental protocol has been approved by the Regional Committee of Ethics regarding Animal Experimentation of Strasbourg (CREMEAS-CEEA35) with license number (AL/42/49/02/13). Furthermore, every effort was made to minimize the suffering and number of animals used. Surgery. Neonatal Functional Inactivation of the PFC. Neonatal functional blockade of the left anteromedial PFC was carried out at PND8. For each litter, half the pups, randomly chosen, received a local microinjection of PBS (control group) and the other half a local microinjection of TTX (experimental group). Surgery was performed under gas anesthesia using isoflurane (Isovet, CSP, Cournon-d’Auvergne, France) as previously described.33,48 More precisely, the microinjection cannula was implanted into the left anteromedial PFC at coordinates 1.5 mm anterior to bregma (AP), 0.4 mm lateral to the midline (L) and 3.9 mm below the cortical surface (H). TTX and PBS were injected in a volume of 0.3 μL over a period of 2 min 15 s. As previously discussed in detail,33,48 the infused TTX amount in the PFC corresponds to about 10 ng (100 μM × 0.3 μL) and is unlikely to spread efficiently over more than 0.67 mm in radius, with TTX effects lasting 4−48 h. Stereotaxic Implantation of the Microsystem in Adult Rats. Stereotaxic implantation of the microsystem was performed at PND70 in male rats (400 ± 25 g) microinjected at PND8 in the left anteromedial PFC with either PBS or TTX. Following chloral hydrate anesthesia (400 mg/kg i.p.), the animals were placed in a stereotaxic frame (Unimécanique, Epinay/Seine, France) (incisor bar set at 3.3 mm below the interaural line) and implanted in the left dorsomedian shell part of the nucleus accumbens at coordinates 10.6 mm anterior to the interaural line (AP), 0.8 mm lateral to the midline (L) and 6.2 mm below the cortical surface (H)49 with a specially designed microsystem (Unimécanique, Epinay/Seine, France),50 which allows behavioral and dopaminergic responses to be monitored in parallel in freely moving adult rats. After surgery, a postoperative recovery period of at least 7 days was observed before the start of pharmacological experiments including voltammetric recordings and behavioral testing. Pharmacological Experiments. Control rats received a subcutaneous (s.c.) injection of saline (NaCl 0.9%) at 0.5 mL/kg. Experimental animals received 0.5 mL/kg s.c. of either ketamine (Imalgene, Merial, Lyon, France) or MK-801 (Sigma, Saint-QuentinFallavier, France). Ketamine (5 mg/kg, 10 mg/kg and 20 mg/kg) and MK-801 (0.1 mg/kg and 0.2 mg/kg) were dissolved in saline. Ketamine and MK-801 solutions were prepared extemporaneously. A total of 77 animals and of 56 animals were used for the ketamine study and for the MK-801 study, respectively. Implanted adult male rats were placed in the experimental cage (24 cm wide x 27 cm long x 44 cm high) and habituated to it for about 1 h before receiving at random the s.c. injection of saline, ketamine (one of the three doses) or MK-801 (one of the two doses). The animals were then kept in the experimental cage for 1 h, during which their locomotor activity was measured. The behavior of the animals was recorded using an infrared camera (ref 51.8050, CA-H34C, Selectronic, Lille, France) placed in the ceiling of the cage and connected to a video monitor and video tape recorder. The floor of the cage was divided virtually into four equal quadrants so that locomotor activity could be assessed by directly observing each animal via the video recording and counting the number of times it crossed from one quadrant to another in each 10 min period.33,48 The procedures used for the voltammetric detection of dopamine were those reported in previous papers.33,48,51 Selective detection of extracellular dopamine levels in vivo was achieved using differential normal pulse voltammetry with electrochemically pretreated carbon fiber microelectrodes (diameter 12 μm, length 500 μm, ref AGT 8000, Serofim, Gennevilliers, France) combined with computerized mathematical analysis of the electrochemical signal. Variations in the dopaminergic signal, recorded min by min in the shell part of the nucleus accumbens, are expressed as percentages of the mean values

effect is observed with MK-801 than with ketamine, consistent with the higher affinity of the former compound for the NMDA receptors.29,30 The present data could be interpreted taking into account the elements and dual regulating pathways hypothesis presented above (see PBS animals). Indeed, a possible suggestion is that in TTX animals, the resultant of the pathways favors the indirect inhibitory pathways involving GABAergic interneurons (see above). According to this suggestion, ketamine and MK-801 acting on the NMDA receptors located on these interneurons would enhance extracellular dopamine levels in the dorsomedian shell. The differences between PBS and TTX groups in the effects of the noncompetitive NMDA receptor antagonists could result from premature changes induced by TTX occurring during cerebral development, and leading to a defect in the prefronto-striatal connectivity involving NMDA receptors. Anatomical data have shown that the shell part of the nucleus accumbens is the point where mesencephalic dopaminergic projections39 and glutamatergic projections originating from the PFC40,41 converge. Importantly, NMDA receptors are tetrameric molecules formed by two NR1 type subunits, essential for the constitution of a functional ionic channel, and two NR2 type subunits42 whose maturation and expression depend on neuronal electrical activity during postnatal development.43 In this context, it should be noted that NMDA receptors expressing the NR2B subunit are present in the nucleus accumbens from birth,44 and that at striatal level, GABAergic interneurons and medium spiny neurons possess NMDA receptors expressing the NR2A and NR2B subunits.45 Moreover, electrical activity is essential for establishing adequate connections of the cortical fibers, still in maturation at the eighth postnatal day, in target structures.23,24,46 Thus, blockade of neuronal activity by TTX at a crucial moment of brain development could cause NMDA receptors regulating the dopaminergic transmission to function abnormally. Interestingly, one of the recent hypotheses about the etiology and pathophysiology of schizophrenia suggests the expression and regulation of the NMDA receptors are disrupted, involving changes in cellular functioning as well as traffic of the receptors.47 In conclusion, the present results may provide a link between dysfunctioning of NMDA receptors and dopamine dysregulation at the level of the shell part of the nucleus accumbens, a striatal subregion described as the common target for antipsychotics. Furthermore, our study suggests our experimental approach may contribute to a better understanding of the pathophysiology of schizophrenia.

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METHODS

Animals and Experimental Design. Male Sprague−Dawley rats born to mothers from the R. Janvier breeding center (Le Genest-SaintIsle, France) were used in the present experiments. During gestation, females were housed in individual plexiglas cages on a 12 h/12 h light/ dark cycle (lights on at 7 am). Throughout the experiment, all the animals were kept at 22 ± 2 °C and had access to water and food ad libitum. The day of birth was defined as postnatal day 0 (PND0). At PND8, pups at random received a microinjection of either PBS (control group) or TTX (experimental group) in the PFC. At PND56, the male rats were individually housed in plexiglas cages on a 12 h/12 h reversed light/dark cycle (lights off at 11 am) before being implanted at PND70 with a microsystem specially designed so that behavioral and dopaminergic responses can be monitored in parallel in freely moving adult rats. Following surgery, animals were given at least 7 days to recover. At PND77, pharmacological experiments, including voltammetric recordings, were carried out during the dark phase of the F

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ACS Chemical Neuroscience corresponding to the average amplitude of the last 10 peaks of dopamine obtained during the control period preceding the s.c. injection. Statistics. Statistical analyses were carried out using a multifactorial analysis of variance (ANOVA) with repeated measurements on the time factor. Only between-subject ANOVAs were presented. Betweensubject variables were neonatal microinjection with two levels (PBS or TTX) and ketamine doses with four levels (NaCl 0.9%, ketamine 5 mg/kg, ketamine 10 mg/kg, or ketamine 20 mg/kg) or MK-801 doses with three levels (NaCl 0.9%, MK-801 0.1 mg/kg or MK-801 0.2 mg/ kg), while dependent variables were the number of crossings for the behavioral study and variations in the dopaminergic signal in the dorsomedian shell part of the nucleus accumbens for the voltammetric study. Post hoc contrast analyses of the ANOVA were used to test specific hypotheses.52 Statistical significance was set at p < 0.05 for all the analyses. Histology. At the end of the experiment, the animals were euthanized by lethal injection (240 mg/kg i.p.) of sodium pentobarbital (Doléthal) and intracardially perfused with NaCl 0.9% followed by a 4% paraformaldehyde solution. After removal from the skull, their brains were kept at 4 °C for at least 24 h. The postnatal microinjection sites in the left PFC were identified by means of the vital dye Evans Blue included in the PBS and TTX solutions microinjected at PDN8 and Neutral Red staining of the brain sections,33,48,53−56 whereas the voltammetric recording sites in the left shell part of the nucleus accumbens were visualized using electrocoagulation (see ref 53 for details) and Thionin Blue staining of the sections. The atlas of Paxinos and Watson49 was used as a reference.

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AUTHOR INFORMATION

Corresponding Author

*Mailing address: INSERM U 1114, Faculty of Medicine, University of Strasbourg, 11 rue Humann, Strasbourg 67085, France. E-mail: [email protected]. Author Contributions §

T.P. and E.T. contributed equally to this study.

Author Contributions

T.P., E.T., Y.U., S.E., and A.L. performed the experiments.T.P., E.T., and A.L. analyzed the data. F.M. and A.L. designed the experiments, prepared the figures, and wrote the manuscript. Funding

This research was supported by Electricité de France (EDF) (A.L.). INSERM (U1114SE13MA; U1114SE14MA), University of Strasbourg (RDGGPJ1301M; RDGGPJ1401M) to A.L. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Yasmine Makhloufi for her helpful assistance.

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DOI: 10.1021/acschemneuro.6b00087 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX