Enhanced nucleus accumbens dopamine and plasma corticosterone stress responses in adult rats with neonatal excitotoxic lesions to the medial prefrontal cortex

Neuroscience Vol. 96, No. 4, pp. 687–695, 2000 687 Copyright q 2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00

Neonatal cortical lesions and subcortical dopamine

Pergamon PII: S0306-4522(00)00002-6 www.elsevier.com/locate/neuroscience

ENHANCED NUCLEUS ACCUMBENS DOPAMINE AND PLASMA CORTICOSTERONE STRESS RESPONSES IN ADULT RATS WITH NEONATAL EXCITOTOXIC LESIONS TO THE MEDIAL PREFRONTAL CORTEX W. G. BRAKE,* G. FLORES,† D. FRANCIS,* M. J. MEANEY,* L. K. SRIVASTAVA* and A. GRATTON*‡ *Douglas Hospital Research Centre, Department of Psychiatry, McGill University, 6875 LaSalle Boulevard, Montre´al, Canada †Instituto de Fisiologia, Universidad Autonoma de Puebla, 14 SUR 6301, 72570 Puebla, Puebla, Mexico

Abstract—The medial prefrontal cortex modulates the nucleus accumbens dopamine response to stress and has been implicated in feedback regulation of hypothalamic–pituitary–adrenal axis activation by stress. Here we report on the effects of bilateral neonatal (postnatal day 7) ibotenate-induced lesions to the medial prefrontal cortex on nucleus accumbens dopamine and neuroendocrine function in adult rats. Voltammetry was used to monitor the dopamine response to each of five, once-daily exposures to tail-pinch stress whereas alterations in neuroendocrine function were determined from the plasma corticosterone response to a single 20-min episode of restraint stress. Potential lesion-induced deficits in sensory-motor gating were assessed by measuring prepulse inhibition of the acoustic startle response before and after repeated stress. Our data show that each daily stress episode elicited larger and longer-lasting dopamine increases in prefrontal cortex-lesioned animals than in sham-lesioned controls. Furthermore, greater stress-induced elevations in plasma corticosterone were seen in lesioned animals than in their sham-lesioned counterparts. However, while repeated stress potentiated startle responses in animals of both groups, there was no effect of lesion on the amplitude or on prepulse inhibition of the startle response. Together, these findings indicate that neonatal prefrontal cortex damage can lead to changes in mesolimbic dopamine and neuroendocrine function during adulthood. They also add to a growing body of experimental and clinical evidence implicating abnormal prefrontal cortex neuronal development in the pathophysiology of schizophrenia and other disorders linked to central dopamine dysfunction. q 2000 IBRO. Published by Elsevier Science Ltd. Key words: voltammetry, glucocorticoids, HPA axis, ibotenic acid, prepulse inhibition, schizophrenia.

The ventral tegmental area (VTA) dopamine (DA) projection to medial prefrontal cortex (PFC) is strongly activated by stress. 1,25,79 Glucocorticoid receptors are found in relatively high numbers in PFC 42,61,62,74 and increasing evidence points to a functional interaction between central DA, the PFC and the hypothalamic–pituitary–adrenal (HPA) axis. 5,6,17,26,32,44,73,74 The infralimbic and prelimbic cortices, in particular, can influence neuroendocrine and autonomic function via any one of several direct and indirect projections to diencephalic, brainstem and spinal control centers. 2,45,66,83 The VTA also contains glucocorticoid receptors as well as DA neurons that innervate nucleus accumbens (NAcc) where stress will activate DA transmission. 1,27 We have previously reported that adult rats that had been subjected to a transient anoxic episode during Cesarean birth will sensitize more readily to the effects of repeated stress on NAcc DA release and amphetamine-induced locomotor activity. 10,11 Furthermore, these animals were found to display a lateralized blunting of the PFC DA stress response. 39 This finding raises the possibility that the NAcc DA stress response is enhanced as an indirect consequence of perinatal anoxic damage to PFC. Such a possibility would be consistent with evidence indicating that DA-sensitive neurons in PFC can

indirectly modulate activation of NAcc DA transmission by a variety of stimuli including stress. 16,23,28,63 Taken together, these findings could have important implications for research on major psychiatric disorders, notably schizophrenia which has been linked to a functional hyperactivity of mesolimbic DA neurons and is thought to involve in some cases a disruption of neuronal development in the PFC. 75,76,84 Adult animals that have sustained neonatal lesions are increasingly recognized as useful models to study neurodevelopmental deficits that are postulated to underlie major mental disorders. Evidence indicates that neonatal lesions of the PFC alter cortical structure and connectivity differently than do PFC lesions in adult animals. 21,52–55 Yet, there is a relative paucity of information concerning the effects of neonatal PFC lesions on mesolimbic DA. Furthermore, conflicting results have emerged from the few available studies. In one study, Flores et al. 34 reported evidence that adult animals with neonatal ibotenate-induced damage to PFC display an increased expression of ventral striatal DA D2 receptors and an enhanced sensitivity to the locomotor stimulant effect of amphetamine. In a more recent study, however, Lipska et al. 60 did not observe any such DA receptor changes following similar lesions and also reported that these animals were subsensitive to the behavioral effects amphetamine. Thus, it is presently unclear whether neonatal excitotoxic damage to PFC would lead to an enhancement of NAcc DA responsivity analogous to that resulting from perinatal anoxia. Therefore, one objective of the present study was to investigate how repeated stress-induced activation of NAcc DA transmission is altered in adult rats that had received PFC lesions as neonates. We also measured in these animals prepulse inhibition (PPI) of acoustic startle prior to and

‡To whom correspondence should be addressed. Tel.: 1 1-514-762-3048 ext. 23937; fax: 1 1-514-762-3034. E-mail address: [email protected] (A. Gratton). Abbreviations: AA, ascorbic acid; ACTH, adrenocorticotropin hormone; CORT, corticosterone; DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; EAA, excitatory amino acid; EDTA, ethylenediaminetetraacetate; HPA, hypothalamic–pituitary–adrenal axis; NAcc, nucleus accumbens; PFC, prefrontal cortex; PND, postnatal day; PPI, prepulse inhibition; VTA, ventral tegmental area. 687

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following repeated stress. Prepulse inhibition, a measure of sensorimotor gating, is attenuated by treatments that stimulate mesolimbic DA transmission (for review, see Ref. 81). It has been shown also to be diminished in schizophrenic patients 7–9 and in adult animals with DA-depleting lesions to PFC. 13 Finally, given evidence that PFC is involved in modulating the neuroendocrine response to stress, we also examined the effects of neonatal PFC damage on stress-induced increases in plasma corticosterone (CORT) levels. EXPERIMENTAL PROCEDURES

Neonatal prefrontal cortex lesions All procedures were carried out in accordance to the guidelines established by the Canadian Council on Animal Care. Sprague– Dawley pups (Charles River Canada; St Constant, Quebec, Canada) were used in this study. Upon birth, pups were culled to litters of six to eight males and on postnatal day 7 (PND 7; body weight ˆ 15–17 g) were randomly assigned to either a PFC-lesioned or sham-lesioned group. Anesthesia was induced by placing the pups on wet ice for 10–15 min. The animals were then placed in a stereotaxic apparatus adapted for neonatal surgery. A 30-gauge stainless steel cannula was lowered into the left and right PFC (2.5 mm anterior to bregma, ^0.4 mm lateral to midline, 2.2 mm below dura) and 0.3 ml of either an ibotenic acid solution (RBI, 10 mg/ml; lesion group) or vehicle (0.9% saline; sham-lesion group) was infused into each site over 2 min. The cannula was left in place for 5 min following each infusion to allow the excitotoxin to disperse away from the cannula tip. A code consisting of a small amount of indelible ink injected into one of the paws was used to later identify lesioned and sham-lesioned animals. Following surgery, pups were placed under a heat lamp and gently stimulated manually to help initiate post-operative recovery. Pups were later returned to their respective dams (each litter contained equal numbers of lesioned and sham-lesioned pups) where they remained until weaning at PND 21 at which time animals were housed in groups of two to three per cage. Acoustic startle and prepulse inhibition PPI of the acoustic startle response was measured using a commercially available system (SR-LAB, San Diego Instruments, San Diego, U.S.A.) that comprised two sound-attenuating chambers each equipped with a cylindrical Plexiglas animal enclosure (length ˆ 16 cm; inner diameter ˆ 8.2 cm). Ventilation was provided by a small electric fan which also generated a 70 dB background noise. Tone pulses were presented by a speaker positioned 24 cm directly above the animal enclosure. A piezoelectric accelerometer afixed to the animal enclosure frame was used to detect and transduce motion resulting from the animals’ response. Tone pulse parameters were controlled by a microcomputer using a commercial software package (SR-LAB) and interface assembly which also digitized (0-4095), rectified and recorded stabilimeter readings. Animals were tested twice for acoustic startle and PPI, once at four months-of-age when they were naı¨ve to experimental conditions and again two to three weeks later, on the day following the last of five once-daily exposures to tail-pinch stress. The same procedure was followed for both test sessions. Animals were placed in the Plexiglas enclosure and allowed to acclimatize to the environment for 5 min before being tested during 37 discrete trials. On the first two trials, the magnitude of the startle response to a 50 ms duration 120 dB tone was measured. On the subsequent 35 trials, the startle tone was either presented alone or 100 ms after presentation of a 30 ms duration prepulse. Prepulse intensity ranged from 3 to 15 dB above background noise (73–85 dB) and was varied randomly between trials in 3 dB steps. Measures were taken at each of the five prepulse intensities on five trials; animals were randomly presented with the startle tone alone during the other 10 trials. The same stimulus condition was never presented on more than two consecutive trials. The interval between each trial was programmed to a variable time schedule with an average duration of 15 s (range 5 to 30 s). A measure of startle response amplitude was derived from the mean of 100 digitized data points collected from stimulus onset at a rate of 1 kHz. Prepulse effectiveness in suppressing the startle response was expressed as a percentage based on the mean amplitude of responses to the startle tone alone (n ˆ 12)

relative to those recorded under the five prepulse conditions (n ˆ 5/ condition). In vivo electrochemistry Surgery. Within three days following the first test for acoustic startle and PPI, rats were injected with atropine sulfate (0.1 mg/kg, s.c.), anesthetized with sodium pentobarbital (60 mg/kg, i.p.), and implanted with a voltammetric recording electrode aimed at the NAcc. The flat skull coordinates were: 1.6 mm anterior to bregma, 1.5 mm lateral to the midline and 7.5 mm below dura. 69 A Ag/AgCl reference electrode and a stainless-steel ground wire were implanted in the contralateral and ipsilateral parietal cortex, respectively. The entire assembly was secured with dental cement anchored to four stainless-steel screws threaded into the cranium. Voltammetric probes. Voltammetric electrodes each comprised three 30-mm diameter carbon fibers (Avco Speciality Materials, Lowell, MA, U.S.A.), that extended 50–100 mm beyond the sealed tip of a pulled glass capillary. The exposed fiber bundle was repeatedly dipped in a 5% solution of Nafion (Aldrich, Milwaukee, WI, U.S.A.) and dried at 1208C for 5 min. This treatment has been shown to promote the exchange of cations such as DA while impeding that of interfering anionic species notably ascorbic acid (AA) and the primary metabolite of DA, 3,4-dihydroxyphenylacetic acid (DOPAC). 12,15,20,36 Electrodes were calibrated immediately prior to implantation to determine their sensitivity to DA and their selectivity for DA against AA. Calibrations were performed in 0.1 M phosphate-buffered saline (pH 7.4) containing 250 mM AA. Only electrodes with a DA-to-AA selectivity greater than 1000:1 and a linear response (r . 0.997) to increasing concentrations of DA were used. Chronoamperometric recordings. Recordings were performed using a computer-controlled, chronoamperometric instrument (Medical Systems, Greenvale, NY, U.S.A.). An oxidative potential of 10.55 V (with respect to the reference electrode) was applied to the electrode for 100 ms at a rate of 5 Hz. The amplitude of the resulting oxidation current was digitized and integrated over the last 80 ms of each pulse. Every 10 digitized current measures were automatically summed and converted into equivalent values of DA concentration using the in vitro calibration factor. Values were displayed on a video monitor at 2-s intervals. The reduction current generated when the potential was stepped down to 0.0 V for 100 ms was digitized and summed in the same manner. With Nafion-coated electrodes and a sampling rate of 5 Hz, the magnitude of the reduction current flow produced by an increase in DA concentration is typically 60–80% of the corresponding increase in oxidation current (red:ox ˆ 0.6–0.8). Whereas the oxidation of AA is virtually irreversible (red:ox ˆ 0), that of DOPAC is almost entirely reversible (red:ox ˆ 0.9–1.0); the reduction-to-oxidation ratios for norepinephrine and serotonin are 0.4– 0.5 and 0.1–0.3, respectively. Extensive discussions concerning the interpretation of chronoamperometric data have been published previously. 11,40,68 Electrochemical recordings began three days after surgery; animals were allowed to acclimatize to the testing environment during the intervening days. Immediately before a recording session, the in vitro calibration factor for the animal’s electrode—the slope of the function relating increases in oxidation current to increases in DA concentration—was entered in the data acquisition software. This allowed on-line conversion of an increase in oxidation current to a value equivalent to the mM change in DA concentration that was required to produce an equal signal increase in vitro. The animals were placed in a recording chamber and connected to the chronoamperometric instrument via a shielded cable and a low impedance multi-channel commutator (Airflyte, Bayonne, NJ, U.S.A.). Electrical interference was minimized by connecting a preamplifier configured as a current-to-voltage converter (gain ˆ 1 × 10 8) directly into the animal’s head assembly. Baseline electrochemical signals were recorded for approximately 180 min on the first test day. On each of the five subsequent days, animals received 15 min of tail-pinch stress after obtaining 30 min of a stable baseline recordings. Tail-pinch consisted in placing a wooden clothespin on the rat’s tail approximately 1.5 cm from its base. Changes in electrochemical signal were recorded throughout the stress period and for 80 min after removing the clothespin.

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Fig. 1. Photomicrographs ( × 6.3) of Thionin-stained coronal section of the PFC taken at the injection site of neonatally (A) sham-lesioned and (B) ibotenatelesioned; black arrows point to region of cell loss and open arrow points to area of irregularly shaped and aligned cells. (C) Schematic diagram depicting minimum (black) and maximum (hatched) extent of damage seen in PFC-lesioned animals; shaded rectangle corresponds to area of PFC shown in photomicrographs.

Electrochemical data format and analysis. Because of the inherent differences in sensitivity between Nafion-coated electrodes, in vivo changes in oxidation current recorded with different electrodes (in different animals) cannot be assumed to be equivalent. Thus, valid comparisons are possible only if the sensitivity of each electrode is calibrated against a standard and the electrochemical data are expressed as standard equivalent values. In the present study, DA was used as the standard to calibrate electrode sensitivity. Accordingly, in vivo changes in oxidation current are expressed as mM equivalent values of DA concentration. Averaged data are presented as changes in electrochemical signal (mM DA equivalent) relative to the signal level immediately prior to onset of tail-pinch stress (Time 0). Since the record at Time 0 was the reference point for changes in electrochemical signal that followed, it was given a value of 0. A value of 0 mM, therefore, is not meant to correspond to the absolute concentration of extracellular DA. Rather, the data reflect relative changes in the DA signal elicited by stress. Statistical comparisons were based on group differences in the amplitude of electrochemical signal increases taken at 5-min intervals from onset of tail-pinch stress. Amplitude is defined here as the electrochemical signal level relative to that seen at Time 0. Corticosterone radioimmunoassay The day following the second PPI test session, animals from both PFC-lesioned and sham-lesioned groups were tested for stress-induced changes in plasma CORT levels. Sampling began at 10:00 h (2 h following lights on) when basal CORT levels are lowest (e.g., 26). Blood samples ( < 200 ml) were taken from the tail vein into tubes containing EDTA and trasylol (48C) which were then centrifuged and stored at 2208C until assayed. A pre-stress sample was taken immediately upon removing the animals from their home cages thus

providing a reliable estimate of basal CORT levels. Animals were placed in Plexiglas restrainers (16 × 8 cm diameter) for 20 min and samples were collected at 0 (pre-stress/basal), 10 and 20 min into the restraint session. After being released from the restrainers, animals were allowed to recover in a quiet room where post-stress blood samples were taken 20, 60 and 120 min later. Plasma CORT was measured using the radioimmunoassay of Krey et al. 77 with a highly specific antiserum-B3-163-(Endocrine Sciences, Tarzana, CA, U.S.A.) and [ 3H] CORT as a tracer (101.0 mCi/mmol; New England Nuclear, Boston, MA, U.S.A.). The detection threshold of the assay is 1 mg/dl. The antiserum cross-reacts slightly with desoxycorticosterone (approx. 4%) but not with cortisol (,1%). The intra- and inter-assay coefficients of variation are 9.2% and 10.3%, respectively. Histology All animals included in the study were analysed for PFC damage and NAcc electrochemical probe placement. Animals were deeply anesthetized with sodium pentobarbital (75 mg/kg, i.p.) and transcardially perfused with 0.9% saline followed by a 10% formalin solution. The brains were stored in 10% formalin and subsequently cryo-protected in a 30% sucrose-saline solution for 48 h prior to being sliced in 40-mm coronal sections. Sections were stained with Thionin to confirm electrode and lesion placements. RESULTS

Neonatal excitotoxic lesions to prefrontal cortex Histological analysis revealed bilateral damage to the PFC of all animals lesioned as neonates. Although there was some variability in the location and extent of the lesions, none of the animals were excluded from the study as a result of this analysis (Fig. 1C). As can be seen in Fig. 1B, the lesion site was characterized by neuronal loss, atrophy, slight cavitation, and an apparent alteration in morphological development of PFC; no such evidence of damage to PFC was observed in sham-lesioned animals (Fig. 1A). Stress-induced changes in dopamine signals

Fig. 2. Photomicrograph ( × 1) of tissue damage produced by a typical electrode placement in the NAcc (left); arrow indicates point of deepest electrode penetration. Asterisks in opposite hemisphere represent the distribution of voltammetric electrode placements.

Only data from animals with histologically confirmed electrode placements in NAcc were used in this study (Fig. 2). Figure 3 presents the mean changes in DA signal recorded in NAcc of PFC-lesioned (n ˆ 6) and sham-lesioned (n ˆ 5) during each of five once-daily episodes of tail-pinch stress. A three-way repeated measures analysis of variance (ANOVA) revealed that stress elicited significantly greater

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Fig. 3. Mean (^S.E.M.) DA signal increases recorded in NAcc of neonatally PFC-lesioned and sham-lesioned animals in response to each of five consecutive once-daily exposures to tail-pinch stress. Stress-induced signal increases of PFC-lesioned animals were significantly greater than those of sham-lesioned controls (P , 0.0001). Length of horizontal bar corresponds to duration of tail-pinch stress.

DA signal increases in PFC-lesioned animals than in their sham-lesioned counterparts (F1,560 ˆ 32.570, P , 0.0001). However, there was no effect of test day nor were there any significant interactions; that is, stress-induced NAcc DA signal increases within the PFC-lesioned and sham-lesioned groups of animals did not differ across test days. The mean reduction-to-oxidation ratios (^S.E.M.) were 0.679 ^ 0.027 and 0.686 ^ 0.034 for the lesioned and sham-lesioned groups of animals, respectively. Ratios in the 0.6 to 0.8 range are typically seen when DA is the predominant electroactive species contributing to an increase in oxidation current. Plasma corticosterone levels Stress-induced changes in plasma CORT levels in the PFClesioned (n ˆ 8) and sham-lesioned (n ˆ 8) groups of animals are shown in Fig. 4. There was no significant difference in basal (pre-stress) CORT values between the two lesion

groups. In both groups of animals, restraint for 20 min resulted in significant increases in plasma CORT levels that peaked between 20 and 60 min after stress. An ANOVA of the entire sampling period using area under the curve integration revealed higher CORT levels in PFC-lesioned animals when compared to sham-lesioned controls (F1,13 ˆ 5.734, P ˆ 0.0324). The mean (^S.E.M.) integrated values were 18.8 ^ 2.37 and 12.0 ^ 1.62 mg/dl/min for the PFC-lesioned and sham-lesioned groups of animals, respectively. Prepulse inhibition of acoustic startle Figure 5A shows the mean startle response amplitude of PFC-lesioned (n ˆ 8) and sham-lesioned (n ˆ 8) rats recorded prior to (pre-stress) and following (post-stress) repeated once daily exposure to tail-pinch stress. A two-way ANOVA revealed that the amplitude of the startle response was greater in both groups of animals following repeated stress

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Fig. 4. Mean (^S.E.M.) plasma CORT levels of sham- and PFC-lesioned animals measured just prior to (220 min) during and after 20 min of restraint stress; length of horizontal black bar corresponds to duration of restraint. Integrated area under the curves values differed significantly (P , 0.05) between the two groups.

(F1,16 ˆ 7.983, P ˆ 0.012); however, there was no significant effect of lesion nor of a lesion by stress interaction. The effects of lesion, repeated stress and prepulse intensity on the startle response were tested for significance using a three-way repeated measures ANOVA. (Fig. 5B). The analysis revealed a significant effect of stress but none of lesion as well as a significant interaction between prepulse intensity and stress (F4,128 ˆ 7.378, P , 0.0001); further analysis revealed that repeated stress attenuated prepulse inhibition at 6 and 9 dB above background (P , 0.05; Scheffe´ F-test). No other significant effects or interactions were uncovered.

DISCUSSION

Dopamine stress response One of the main findings of this study is that the NAcc DA response to stress is potentiated in adult animals with neonatal excitotoxic damage to PFC. This effect is probably due to a disruption in the development of the PFC circuitry involved in regulating DA transmission in NAcc and other subcortical regions. Stress stimulates DA transmission in PFC and converging evidence suggests that the NAcc DA stress response is modulated by a DA-sensitive mechanism in PFC. 16,23,28,63 Stress will also increase levels of excitatory amino acids (EAA) in PFC 3,64,65 and the DA stress response there appears to be regulated by glutamate. 46 There is evidence implicating also PFC EAA-containing projection neurons some of which are known to terminate in VTA and NAcc. 4,29,38,47,77,78 The PFC also contains GABA interneurons and these too appear to be part of the cortical circuitry regulating stress-induced activation of NAcc DA transmission. 30,41,67 Together, these findings point to a number of potential mechanisms by which disruption of PFC development, as a consequence of neonatal excitotoxic damage, might lead to NAcc DA hyperfunction. Developmental neuropathology is increasingly thought to be an etiological factor in schizophrenia (for review, see

Fig. 5. (A) Mean (^S.E.M.) amplitude of acoustic startle response measured in neonatally PFC- and sham-lesioned animals before (Pre-stress) and after (Post-stress) five once-daily exposures to tail-pinch stress. (B) Mean (^S.E.M.) per cent inhibition of acoustic startle response produced at different prepulse tone intensities (dB, decibels) in neonatally PFC- and sham-lesioned animals before (pre) and after (post) repeated exposure to stress. In both panels, asterisks denote significant differences at P , 0.05.

Ref. 85). In particular, it has been suggested that some of the cardinal symptoms of this illness reflect a neurodevelopmental deficit of PFC function. 84 We have previously reported evidence indicating that the acute NAcc DA response to repeated stress is enhanced in adult animals born by Cesarean section and particularly so when delivery is accompanied by an anoxic episode. 11 This finding has added to increasing evidence suggesting that perinatal complications may contribute to the pathophysiology of psychiatric disorders, in particular those that involve central DA dysfunction such as schizophrenia. Presently, we can only speculate on the mechanisms that might mediate the long-term effects of perinatal complications on meso-NAcc DA function. However, based on the findings reported here it is tempting to hypothesize that perinatal damage to PFC contributed, at least in part, to the development of NAcc DA hyperfunction. Such a possibility would be consistent with the finding that the PFC DA response to stress is attenuated in adult animals that sustained perinatal complications 39 and with several lines of evidence suggesting that stress-induced DA release in NAcc is dampened by the concurrent activation of PFC DA transmission. 16,24,28 Still, the effects of neonatal PFC damage on the NAcc DA stress response did differ from those of perinatal anoxia in that augmented DA stress responses were observed on the first and

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each subsequent test day. Thus, there was no progressive dayto-day enhancement that often characterizes the development of sensitized DA reponses to stress. It should be pointed out, however, that sensitized Nacc DA responses are usually seen with repeated intermittent exposure to more potent stressors than tail-pinch. Indeed, under conditions very similar to those of the present study, the acute NAcc DA stress response in intact, untreated animals was found to sensitize with repeated restraint 27 but not tail-pinch stress. 11,27 Thus, the fact that NAcc DA transmission in sham-lesioned animals did not sensitize to tail-pinch stress is, at least, consistent with our own previous findings. Be that as it may, the fact that augmented DA responses were seen in lesioned animals on the very first test day suggests a fundamental difference between the effects of neonatal excitotoxic PFC damage and those resulting from perinatal anoxia. The potentiated DA stress response seen in the present study is generally consistent with the data of Flores et al. 34 showing that adult rats with comparable neonatal excitotoxic lesions to PFC are spontaneously hyperactive and also more sensitive to the locomotor stimulant action of amphetamine. The same study also revealed higher DA D2 receptor densities and mRNA levels in the NAcc of animals with neonatal PFC lesions. Interestingly, these receptor changes were seen in the shell but not the core region of NAcc. The significance of this finding in the context of the present study is difficult to assess since electrochemical data on the NAcc DA stress response were obtained from the core region. The present data do not preclude the possibility, however, that changes in NAcc shell DA D2 receptor densities and in Nacc core DA release both contributed to the greater locomotor response to amphetamine observed by Flores et al. 34 It is also noteworthy that Flores et al. 34 did not observe comparable neonatal PFC lesion-induced changes when animals were tested before they reached puberty (PND 35). Postpubertal emergence of altered DA function has been observed also in animals with neonatal excitotoxic lesions to ventral hippocampus. 33,58,59 This feature is particularly relevant to research on schizophrenia given that symptoms of the disease typically start to emerge during or shortly after adolescence and early adulthood. The main conclusion of the present study is in contrast to that suggested recently by Lipska et al. 60 In that study, adult animals with neonatal (PND 7) ibotenate-induced lesions to PFC were found to be subsensitive to the locomotor-stimulant effects of amphetamine and dizocilpine maleate (MK-801). These authors also reported greater apomorphine-induced stereotypy in neonatally lesioned animals, suggesting a facilitation of DA transmission postsynaptically. Yet unlike Flores et al., 34 they failed to observe any change in NAcc D2 receptor mRNA levels. The discrepencies between the findings of Lipska et al. 60 and those reported here and by Flores et al. 34 are not easily explained. In adult animals at least, the location of the lesion within the PFC appears to be a critical factor (e.g., Ref. 71). Evidence of regional specialization within PFC is supported by data from tract-tracing studies showing that inputs to NAcc core originate dorsally in the cingulate and prelimbic cortices whereas the NAcc shell is innervated primarily by neurons located more ventrally in the infralimbic cortex. 4,77 However, attributing the discrepencies between the present findings and those reported by Lipska et al. 60 to a difference in lesion placement is somewhat tenuous, given that the lesion procedure used in the two studies (including

stereotaxic coordinates) are identical. The possibility appears even more remote when considering the fact that these lesions were performed in seven-day-old pups. Given the relative size and proximity of PFC subregions in neonates and the volume of excitotoxin injected (0.3 ml/side), it seems improbable that sufficiently localized damage could be achieved as to allow the apparent dissociation suggested by the study of Lipska et al. 60 The timing of neonatal PFC lesions along with any one of several early environmental factors (e.g., housing and rearing conditions, amount of handling, mother pup interactions, etc.) may ultimately prove to be more important than lesion placement alone. It is known, for instance, that animals with early (PND 1–4) neonatal lesions are more severely affected on behavioral measures of cognitive performance than similarly lesioned adult animals. Yet cognitive function is relatively spared in animals lesioned at a slightly older age (PND 7– 10). 21,35,48,53 It should be noted that not all behaviors are spared following early cortical damage; little or no sparing of sensorimotor and maze learning has been reported in PND 7 frontal cortical-lesioned rats. 49 In contrast to these previous studies, the present data suggest that little recovery had occurred and that the effects of “late” neonatal PFC damage on the NAcc DA stress response persist into adulthood. In this respect, at least, the effects of neonatal PFC lesions reported here are more in line with those observed in non-human primates. 37 As suggested previously, recovery from neonatal PFC damage depends upon the integrity of catecholamine inputs to PFC as well as enrichment of the environment 51,52 and may reflect changes in dendritic branching or spine density or both. 50,56 Neuroendocrine response to stress The present study indicates that neonatal damage to PFC also leads to greater stress-induced elevations in plasma CORT levels. Converging lines of evidence suggest that this greater neuroendocrine responsivity may have contributed indirectly to the enhanced NAcc DA stress response seen in PFC-lesioned animals. Glucocorticoid receptors are found on VTA DA neurons 42 and also in PFC 61,62,65 where glucocorticoids are thought to exert negative feedback regulation over the HPA axis. 26,32,44 Evidence from a variety of sources also indicate that DA transmission and the DAmediated behavioral effects of stimulant drugs are influenced by corticotropin-releasing factor and circulating CORT levels. 14,17,19,22,70,72 Furthermore, CORT acting at Type II glucocorticoid receptors was recently reported to facilitate EAA-induced activation of VTA DA cell firing. 18 That neonatal PFC lesions resulted in an enhancement of the plasma CORT stress response is in general agreement with the results of Diorio et al. 26 who reported enhanced plasma CORT and adrenocorticotropin hormone (ACTH) responses to restraint, but not ether stress in adult animals with thermolytic PFC lesions. The same authors found that CORT implants into PFC decreased the ACTH response to restraint, but not ether stress suggesting that the PFC is a stress-specific target site for HPA negative feedback. More recently, the plasma CORT response to restraint stress was found to be positively correlated with stress-induced DA release in PFC DA, suggesting that neuroendocrine and cortical DA stress responses are linked positively. 79 Thus, the present findings add to a growing body of evidence that

Neonatal cortical lesions and subcortical dopamine

implicates the PFC in mediation of HPA negative feedback regulation. Prepulse inhibition of acoustic startle Previous studies have reported that adult excitotoxic lesions to PFC of adult rats have little effect on PPI. 57,82 The present results indicate that this is true also of neonatal PFC lesions. Comparable acoustic startle responses were observed in PFC-lesioned and sham-lesioned rats. Interestingly, however, the amplitude of this response was significantly augmented in both groups of animals following repeated daily stress. Similarly, while pre-pulse effectiveness (at 6 and 9 dB) in inhibiting the acoustic startle response was noticeably lower in lesioned animals, it was significantly diminished in both groups of animals by repeated daily stress. Thus, on measures of both acoustic startle and PPI, it appears that a history of repeated daily stress was more important than neonatal PFC damage in determining the animals’ response. These findings are unexpected in light of the other findings reported here and of evidence from other sources showing that PPI is disrupted under conditions of increased NAcc DA transmission. 31,80 The fact that repeated stress produced in PFC-lesioned and sham-lesioned animals comparable increases in acoustic startle responses is difficult to reconcile with the fact that the NAcc DA stress response in the two groups of animals differed markedly. That PPI was disrupted after animals had been repeated stressed might be expected given that stress has the potential of sensitizing NAcc DA transmission and DA-mediated behaviors to the effects other stimuli, including presumably, a startling tone. This conclusion is based on the assumption that acoustic startle is associated with increased NAcc DA. Recent evidence suggests, however, that this assumption may not be well founded. 43 Still, if the disruptive effect of repeated stress on

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PPI was indeed a consequence of enhanced NAcc DA responsivity, then this effect should have been greatest among PFClesioned animals given that the NAcc DA stress response of these animals was greater than that of sham controls. Clearly this was not the case. Excitoxic PFC lesions during adulthood were previously reported to have little effect on PPI unless animals were pretreated with a low dose of apomorphine. 82 This would suggest, as do the present data, that excitotoxic damage to PFC is, alone, insufficient to disrupt sensorimotor gating function as measured by PPI. It would appear rather that a minimal level of activation of subcortical DA transmission is required for the expression of the disruptive effects of such lesions. CONCLUSIONS

Neonatal excitotoxic damage to PFC resulted in an enhancement of the NAcc DA and plasma CORT responses to stress. Under the conditions of the present study, sensorimotor gating as measured by PPI was found to be disrupted by prior repeated exposure to stress but not by neonatal PFC lesions. The present data indicate that the PFC plays a modulatory role in stress responsivity and provide experimental evidence consistent with the idea that developmental pathology of the frontal cortex contributes to the etiology of schizophrenia and other disorders involving increased subcortical DA activity. Acknowledgements—This study was made possible by grants from the Medical Research Council of Canada (MRCC), the National Alliance for Research on Schizophrenia and Depression (NARSAD) and les Fonds de la recherche en sante´ du Que´bec (FRSQ) to A.G. and a Natural Sciences and Engineering Research Council of Canada (NSERCC) Scholarship to W.G.B. Funding was provided also by MRCC grants to L.K.S. and M.J.M. Shakti Sharma’s assistance with the corticosterone radioimmunoassay is gratefully acknowledged.

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