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Neonatal depletion of cortical dopamine: Effects on dopamine turnover and motor behavior in juvenile and adult rats
Developmental Brain Research 156 (2005) 167 – 175 www.elsevier.com/locate/devbrainres
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Neonatal depletion of cortical dopamine: Effects on dopamine turnover and motor behavior in juvenile and adult rats P.J. Boyce, J.M. FinlayT,1 Department of Psychology, Western Washington University, 220 Miller Hall, MS 9089, Bellingham, WA 98225, USA Accepted 15 February 2005 Available online 7 April 2005
Abstract Abnormal development of mesoprefrontal dopamine (DA) neurons may contribute to the pathophysiology of schizophrenia. Consistent with this hypothesis, DA nerve terminal density is decreased in the cortex of schizophrenic subjects [M. Akil, J.N. Pierri, R.E. Whitehead, C.L. Edgar, C. Mohila, A.R. Sampson, and D.A. Lewis, Lamina-specific alterations in the dopamine innervation of the prefrontal cortex in schizophrenic subjects, Am. J. Psychiatry, 156 (1999) 1580–1589]. This abnormality may be present early in development, giving rise to dysfunction as an individual matures. The present studies examined the effects of early partial loss of medial prefrontal cortex (mPFC) DA on DA turnover and locomotor behavior in juvenile, pubertal, and adult rats (30, 45, and 60 days of age, respectively). Local infusions of 6hydroxydopamine on postnatal day (PN) 12–14 produced persistent decreases in basal tissue DA concentrations and increases in 3,4dihydroxyphenylacetic acid (DOPAC):DA ratios in the mPFC. In the nucleus accumbens of lesioned rats, basal DA concentrations were decreased and DOPAC:DA ratios were increased on PN30, but not PN45 or 60. Footshock (30 min at 0.6 mA) increased DOPAC and DOPAC:DA ratios in the mPFC of PN30 and 60 control rats. These effects were attenuated in age-matched rats previously sustaining ~50% loss of mPFC DA on PN12–14. Footshock did not affect DOPAC:DA ratios in the nucleus accumbens of control or lesioned rats. The lesion also failed to alter basal or stress-evoked motor activity. The present data suggest that a decreased density of mPFC DA nerve terminals occurring early in development results in persistent alterations in basal and stress-evoked activity of mesoprefrontal DA neurons, but not mesoaccumbens DA neurons. D 2005 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Neurotransmitter systems and channels Keywords: Footshock stress; 3,4-dihydroxyphenylacetic acid; Schizophrenia; Development; 6-hydroxydopamine; Mesoprefrontal dopamine
1. Introduction Abnormal function of dopamine (DA) neurons projecting to the prefrontal cortex (PFC) has long been thought to contribute to the expression of schizophrenia in some individuals (for review, see Ref. [10]). One hypothesis is that hypoactivity of the mesocortical DA innervation leads to hyperactivity of mesolimbic DA neurons and that, together, these pathophysiologies contribute to the negative T Corresponding author. Fax: +1 360 650 7305. E-mail address: [email protected] (J.M. Finlay). 1 This work was supported by USPHS Grant MH61616. 0165-3806/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2005.02.006
and positive symptoms of the illness, respectively [7,13,42]. Results of experimental studies support the view that mesocortical DA neurons regulate the activity of mesolimbic DA neurons. For example, partial loss of mesoprefrontal DA neurons disrupts DA turnover and release in the nucleus accumbens (NAS), electrophysiological activity of ventral tegmental area DA neurons, and behaviors thought to be modulated by mesoaccumbens DA neurons such as motor behavior [6,8,14,24,25,34]. It has been suggested that the primary pathological event(s) associated with schizophrenia occur during pre- or peri-natal development [2,31,33]. The functional consequences of such developmental disturbances are then expressed
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when the affected neural networks are fully mature, accounting for the appearance of clinical symptoms of schizophrenia in late adolescence or early adulthood. Although postmortem analyses have revealed a decreased density of fibers immunoreactive for tyrosine hydroxylase and DA transporter protein in the PFC of schizophrenic subjects [1], it is not known when this structural brain abnormality occurs. The effects of PFC DA depletions on the activity of mesolimbic DA neurons have been examined predominantly in rodents sustaining lesions in late adolescence/early adulthood. Furthermore, studies assessing the effects of depletions sustained early in development, used electrolytic lesions of the ventral tegmental area (VTA) or intraventricular administration of 6-hydroxydopamine (6-OHDA), resulting in damage to non-DA neurons and widespread loss of DA [9,17,19–21,28,30]. In the developing rat, DA fibers destined to innervate deep and superficial cortical layers reach the PFC by embryonic day 16 and postnatal day 2, respectively [3,4,18,38,39,41]. The DA projections to the rat PFC then continue to increase in density until adulthood. Thus, the DA innervation of the rat PFC, like that of primates [11,29,36,40], undergoes a protracted development from gestation to adulthood. Because rat neurodevelopment at birth resembles that seen in primates during the second trimester [26], the impact of lesions sustained in the early postnatal period may be relevant to hypotheses that adverse events occurring in utero contribute to the pathophysiology of schizophrenia [35]. The goal of the present studies was to examine the effects of partial depletion of PFC DA sustained on PN12–14, on DA turnover in the PFC and nucleus accumbens (NAS) of juvenile and adult rats. Motor activity was monitored as behavioral correlate of the activity of mesolimbic DA neurons [22]. Neurochemical activity and motor behavior were measured under baseline conditions and in response to a challenge stimulus, footshock stress. This stimulus was chosen because stress can precipitate some of the symptoms of schizophrenia [5].
2. Methods and materials 2.1. Animals Litters of Sprague–Dawley rats (Hilltop Lab Animals, Scottsdale, PA) consisted of a lactating dam and 12 male pups age PN8–10. Upon arrival, litters were housed in cages (30 cm2 13 cm) with cob bedding. Pups were weaned on PN21 and housed 4 per cage. On PN30, rats were tested and sacrificed or housed singly in hanging wire-mesh cages (24 cm 20 cm2) where they remained until testing and sacrifice on PN45 or 60. At all times, rats were housed in temperature-controlled rooms (20–22 8C) with the lights on from 8:00 am to 8:00 pm. Rat chow and water were available ad libitum. All treatments were performed during the light phase of the light/dark cycle. Procedures for the
treatment of rats were approved by the Institutional Animal care and Use Committee at Western Washington University using criteria established by the U.S. Animal Welfare Act and the National Institutes of Health Guide for the Case and Use of Laboratory Animals. 2.2. Neonatal 6-hydroxydopamine lesions of the rat mPFC On PN12–14, rats randomly assigned to the lesion condition were removed from the litter, pretreated with the norepinephrine (NE) uptake inhibitor desipramine hydrochloride (0.125 mg base/0.025 ml/10 g in 0.9% NaCl, subcutaneous; Sigma-Aldrich, St. Louis, MO), and housed in a plastic container (42 cm 25 cm 15 cm) lined with a heating pad (Vetko, Model V-21, Colorado Springs, CO). Thirty minutes later, rats were anesthetized with 4.5% isoflurane (Abbott Laboratories, Abbott Park, IL) in oxygen and placed in a stereotaxic instrument equipped with a neonatal rat adaptor and anesthesia mask (David Kopf Instruments, Models 900, 970, and 907, Tujunga, CA). Isofluorane (1–2%) was delivered continuously during surgery. A reusable heat packet (Harvard Apparatus, Holliston, MA) was placed under the anesthetized rat to provide warmth during surgery. A glass pipette (tip o.d. = 50–100 Am) was positioned in the medial PFC (mPFC) using stereotaxic coordinates: AP +2.1 mm and ML F0.3 mm from bregma, DV 2.4 mm from dura with the skull flat (stereotaxic coordinates were established in a pilot study using dye injections delivered via glass pipettes). The pipette was left in place for 5 min after which 1.0 Ag 6-OHDA base (Sigma-Aldrich or ICN Biomedicals, Irvine, CA) in 0.25 Al of vehicle (0.9% NaCl containing 0.03% ascorbic acid) was infused over 5 min using pressure ejection (World Precision Instruments, PV820 Pneumatic picopump, Sarasota, Fl). A stock solution of 6-OHDA, prepared fresh each day, was stored on ice in darkness. Immediately prior to being positioned in the left or right mPFC, the injection pipette was filled with sufficient toxin for a single infusion. Rats assigned to the PN45 testing group received a second infusion of 1.0 Ag 6-OHDA in 0.25 Al of vehicle at a site located DV 3.7 mm from dura. After each infusion, the pipette was left in place for 5 min to allow for dispersal of the toxin. The infusion procedure was repeated in the contralateral hemisphere, the pipette was then removed, and the scalp closed with sutures. Upon resuming normal motor activity, pups were returned to the litter where they remained until weaned on PN21. Control rats were unoperated, remaining with dam and littermates until weaning. Each rat was handled 2–3 times/week by an animal care technician. 2.3. Horizontal locomotor activity Horizontal locomotor activity was monitored in control and lesioned rats on PN30 and 60. Rats were placed in a
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cylindrical Plexiglas cage (41 28 cm in diameter) with a metal rod floor (16 metal rods each 5 mm in diameter and spaced 14 mm apart). The Plexiglas cage was located in one corner of a custom built motor-activity chamber (51 cm2) comprised of a single layer of photocells (20 cells per side) forming a horizontal grid. Photocells were 2.5 cm apart and 5 cm above the floor of the chamber. Photocell interruptions were recorded 3 times per second for 30 min, beginning immediately after the rat was placed in the cage. Control and lesioned rats assigned to the baseline condition remained undisturbed in the cage for 30 min. Rats assigned to the stress condition were exposed to 0.6 mA scrambled footshock delivered via a shock generator and scrambler (BRS-LVE, Models SG903 and SC922, Laurel, MD). Footshock was delivered for 2 s every 5 s for a total of 12 2-s shocks in 60 s. This series was repeated every 5 min for 30 min, resulting in a total of 6 shock cycles. 2.4. Tissue dissection and neurochemical analyses Rats used for analysis of motor activity on PN30 and 60 were removed from the motor-activity chamber and immediately decapitated. All other rats were decapitated immediately following removal from their cages in the colony room on PN30, 45, or 60. The brain of each rat was rapidly removed and frozen onto a microtome stage. Thick coronal sections were made at the level of the mPFC and NAS (2.9–4.3 and 1.0–2.4 mm anterior to bregma, respectively; [32]). The left and right mPFC and NAS were freehand dissected, as described in a previous publication [23]. Tissue samples were weighed, sonicated in 250 Al of 0.2 M hydrochloric acid, and centrifuged at 14,000 rpm for 25 min at 4 8C. The supernatants were filtered through 0.2-Am nylon filters by centrifugation at 10,000 rpm for 10 min at 4 8C. Supernatants were stored at 80 8C until analysis. Tissue DA, NE, and DOPAC concentrations were determined using high performance liquid chromatography (HPLC) with amperometric or coulometric detection. The HPLC systems consisted of a manual injector (Rheodyne, Model 9125, Rohnert Park, CA), an HPLC pump (Waters Co., Model 540, Milford, MA; ESA, Model 580, Chelmsfod, MA), and a pulse dampener (SSI, Model LP-21, State College, PA). Peak separation was accomplished using a reversed-phase C18 column (3.2 mm 150 mm, 3 um; ESA, Part #70-0636) and mobile phase (ESA, Part #71-1332). The mobile phase was circulated through the HPLC system at a flow rate of 0.4 ml/min. Amperometric detection was performed using a potentiostat (Waters, Model 460) and glassy carbon working electrode set at +870 mV relative to an Ag/AgCl reference electrode. Coulometric detection was performed using a potentiostat (ESA, Model 5100 A) that controlled a conditioning cell (ESA, Model 5020) maintained at +400 mV and an analytical cell (ESA, Model 5014) with
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the first and second electrodes maintained at 140 and +125 mV, respectively; analytes were quantified using the oxidation signal from the second analytical electrode. DA, NE, and DOPAC concentrations in the samples were determined by comparing oxidation peaks produced by samples to those produced by standards of known concentrations. The detection limit of the assay was ~0.5 pg/20 Al of sample for each analyte. Acquisition and analysis of chromatograms were performed via computer (Varian, Star Chromatography Workstation, Walnut Creek, CA and Rainin Instrument, Dynamax Macintegrator HPLC Method Manager, Rainin Instrument Co., Emeryville, CA). 2.5. Statistical analyses mPFC and NAS tissue from the left and right hemisphere of each rat was analyzed separately using HPLC with electrochemical detection. Data from the two hemispheres were then used to calculate average DA, NE, and DOPAC concentrations in the mPFC and NAS of individual subjects. Baseline and stress-evoked tissue DA and NE concentrations (ng/mg tissue) and DOPAC to DA ratios in the mPFC and NAS of control and lesioned rats were analyzed using independent samples t tests with layered Bonferroni corrections for multiple comparisons. The effects of lesions and stress on horizontal locomotor activity (photocell beam interruptions per 5 min) were analyzed using univariate repeated measures ANOVAs with degrees of freedom appropriate for the Geisser– Greenhouse F test followed by independent samples t tests with layered Bonferroni corrections. Results of all statistical analyses appear in the appropriate figure caption. In all cases, sample size indicates the number of rats per treatment condition.
3. Results 3.1. Effects of neonatal 6-OHDA lesions of the mPFC on basal catecholamine concentrations in the mPFC and NAS of juvenile and adult rats Local 6-OHDA infusions delivered on PN12–14 decreased baseline DA and, to a lesser extent, NE concentrations in the mPFC of rats sacrificed on PN30, 45, and 60 (Figs. 1A and B). DOPAC to DA ratios in the mPFC were increased in lesioned rats sacrificed on PN30 and 45, relative to age-matched control rats (Fig. 1C). Although lesioned rats sacrificed on PN60 exhibited a 25% increase in metabolite to DA ratios in the mPFC, this trend did not reach statistical significance. Infusions of 6-OHDA into the mPFC on PN12–14 also reduced DA concentrations and increased DOPAC to DA ratios in the NAS of rats sacrificed on PN30, but not PN45 and 60 (Figs. 2A and B).
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Fig. 1. Effects of 6-OHDA infused into the rat mPFC on PN12–14 on local tissue concentrations of DA (A) and NE (B) and DOPAC to DA ratios (C) in rats sacrifice on PN30, 45, and 60, under baseline conditions (n = 10–14/group). 6-OHDA lesions of the mPFC decreased local DA and, to a lesser extent, NE content in rats sacrifice on PN30, 45, and 60 [DA: t(21) = 6.0, t(25) = 10.05, and t(19) = 4.63, respectively; NE: t(21) = 4.21, t(26) = 4.08, t(20) = 3.45, respectively]. DOPAC to DA ratios in the mPFC were significantly increased in rats sacrificed on PN30 and 45, but not PN60 [t(21) = 2.59 and t(24) = 4.10, respectively]. Data are presented as group mean F SEM. *Significantly different from age-matched control rats (t tests with layered Bonferroni correction, P V 0.017–0.05).
3.2. Effects of neonatal 6-OHDA lesions of the mPFC on stress-evoked catecholamine concentrations in the mPFC and NAS of juvenile and adult rats Control rats and rats previously sustaining 6-OHDA lesions of the mPFC on PN12–14 were exposed to acute footshock (30 min at 0.6 mA) on PN30 or 60 and immediately sacrificed for analysis of tissue catecholamine concentrations in the mPFC and NAS. Data from stressexposed control and lesioned rats were compared to that from age-matched control and lesioned rats tested under baseline conditions, as reported above. Consistent with our findings in non-stressed lesioned rats, local 6-OHDA on PN12–14 reduced DA and, to a lesser extent, NE concentrations in the mPFC of rats subsequently exposed to stress and sacrificed on PN30 and 60 (DA: 0.03 F 0.002 and 0.04 F 0.004 ng/mg tissue, respectively; NE: 0.18 F 0.01 and 0.27 F 0.03 ng/mg tissue, respectively). Similarly, we again found that 6-OHDA infusions into the mPFC on PN12–14 decreased NAS DA content on PN30, but not PN60 (4.2 F 0.6 and 6.7 F 0.6 ng/mg tissue, respectively). Acute footshock increased DOPAC to DA ratios in the mPFC of control and lesioned rats sacrificed on PN30 and 60 (Figs. 3A and B). Absolute stress-evoked increases in metabolite to DA ratios were similar in lesioned and age-
matched control rats. However, this represented a smaller percent increase in turnover above baseline in lesioned than age-matched control rats (% increases above baseline are indicated on corresponding bars in Figs. 3A and B). Absolute stress-evoked increases in tissue DOPAC concentrations were also attenuated in lesioned rats tested on PN30 and 60, relative to age-matched control rats (% increases above baseline are indicated on corresponding bars in Figs. 4A and B). Footshock did not significantly affect DOPAC to DA ratios in the NAS of control and lesioned rats tested on PN30 and 60 (Figs. 5A and B). Although there was a trend for a stress-evoked increase in DA turnover in control rats sacrificed on PN30, this effect failed to reach criterion for significance using a layered Bonferroni correction (see Fig. 5 legend). 3.3. Effects of neonatal 6-OHDA lesions of the rat mPFC on locomotor activity in juvenile and adult rats Immediately prior to sacrifice for analysis of tissue catecholamine concentrations, baseline and footshockevoked horizontal locomotor activity was monitored in all control and lesioned rats tested on PN30. Due to equipment failure, motor activity data were acquired from only a subset of control and lesioned rats sacrificed on PN60 (n = 6–8/
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Fig. 2. Effects of 6-OHDA infused into the rat mPFC on PN12–14 on tissue DA concentrations (A) and DOPAC to DA ratios (B) in the NAS of rats sacrificed on PN30, 45, and 60, under baseline conditions (n = 10–14/group). 6-OHDA lesions of the mPFC decreased DA concentrations and increased DOPAC to DA ratios in the NAS of rats sacrificed on PN30 [t(19) = 2.80 and t(19) = 3.39, respectively]. On PN45 and 60, basal DA levels and DA turnover in the NAS of lesioned rats did not differ from control values. Data are presented as group mean F SEM. *Significantly different from age-matched control rats (t tests with layered Bonferroni correction, P V 0.017–0.05).
group of the total n = 11–12/group available for analysis of tissue catecholamines). Concentrations of DA and NE in the mPFC of lesioned rats used for analysis of locomotor behavior on PN60 were 54 F 5% and 73 F 2% of control values, confirming that lesions in this subset were representative of the larger group. Baseline horizontal motor activity on PN30 and 60 was similar in control rats and rats previously sustaining 6-OHDA lesions of the mPFC on PN12–14 (Figs. 6A and B). Exposure to acute footshock (30 min at 0.6 mA) on PN30 or 60 reduced locomotor activity of control and lesioned rats. The stress-evoked behavioral response of lesioned rats did not differ from that of age-matched control rats.
4. Discussion 4.1. Effects of 6-OHDA lesions of the rat mPFC on PN12–14 on tissue DA concentrations and turnover in the mPFC and NAS of juvenile and adult rats under baseline conditions In the present studies, 6-OHDA infusions into the rat mPFC during early postnatal development induced a persistent and relatively selective loss of local tissue DA concentrations. Specifically, in rats sustaining 6-OHDA lesions of the mPFC on PN12–14, local DA concentrations
were reduced by ~70% on PN45 and ~50% on PN30 and 60. The presence of larger lesions in rats sacrificed on PN45, than 30 and 60, was likely due to administration of a higher concentration of 6-OHDA (2.0 versus 1.0 Ag per hemisphere, respectively). Although an NE uptake inhibitor was administered prior to the 6-OHDA infusions, NE concentrations in the mPFC were decreased by ~20% in lesioned rats of all age groups. In addition, NAS DA concentrations were reduced by ~35% in rats tested on PN30, but not 45 and 60, suggesting that this loss was a transient consequence of 6-OHDA infusions into the mPFC. Previous investigators used electrolytic lesions of the VTA or intraventricular injections of 6-OHDA to damage mesoprefrontal DA projections, resulting in extensive damage to non-DA neurons and widespread loss of brain DA [9,15,17,19,20,21,28,30]. For example, electrolytic lesions of the VTA on PN1 result in a persistent decrease in DA concentrations in the NAS and limbic forebrain [9,19]. In the present study, the transient increase in DOPAC/DA ratio in the NAS on PN30 may be (1) a direct result of diffusion of 6-OHDA to the NAS and a concomitant loss of DA terminals in this region or (2) an indirect effect on mesoaccumbens DA cell bodies in the VTA, mediated by degeneration of neighboring mesoprefrontal DA cell bodies. Nonetheless, our finding that NAS DA concentrations return to normal levels by PN45 suggests either that VTA DA cells projecting to the NAS are largely
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possible given that NAS and limbic forebrain DA concentrations were also reduced under the conditions of these previous studies. 4.2. Effects of 6-OHDA lesions of the rat mPFC on PN12–14 on tissue DA concentrations and turnover in the mPFC and NAS of juvenile and adult rats under stress conditions
Fig. 3. Effects of footshock (30 min at 0.6 mA) on DOPAC to DA ratios in the mPFC of control rats and rats previously sustaining 6-OHDA lesions of the mPFC on PN12–14. Control and lesioned rats were tested on PN30 (A) and 60 (B) (n = 8–12/group). Footshock increased DOPAC to DA ratios in the mPFC of control and lesioned rats sacrificed on PN30 [t(21) = 5.62 and t(22) = 2.65, respectively] and PN60 [t(19) = 3.19 and t(16) = 2.25]. Absolute stress-evoked increases in DOPAC to DA ratios were similar in lesioned and control rats. However, because the lesion itself increased basal DA turnover, the effects of stress were attenuated in lesioned rats when the data were represented as a percent of baseline (% increase above baseline appears on the corresponding bar). Data are presented as group means F SEM. *Significantly different from age-matched control rats under baseline conditions (data from Fig. 1). **Significantly different from corresponding age-matched control and lesioned rats under baseline conditions (t tests with layered Bonferroni correction, P V 0.017–0.05).
spared following local infusions of 6-OHDA into the mPFC on PN12–14 or that damage is sufficiently limited that the remaining neurons can fully compensate. As a measure of DA turnover, we also examined the effects of our lesion on basal DOPAC to DA ratios in the mPFC and NAS. In rats sustaining 6-OHDA lesions of the mPFC on PN12–14, DOPAC to DA ratios in the mPFC were increased on PN30, 45, and 60. The latter findings are consistent with previous studies indicating that early partial loss of mPFC DA results in a persistent increase in local DA turnover. Specifically, rats sustaining electrolytic lesions of the VTA or intraventricular 6-OHDA infusions (PN1 and 3, respectively) exhibit increased DOPAC to DA ratios in the mPFC in adulthood ([9,19,30]; however, see also Ref. [28]). In the present study, lesioned rats exhibited increased DOPAC to DA ratios in the NAS on PN30, but not 45 or 60. Our finding appears to be consistent with a previous report indicating that electrolytic lesions of the VTA on PN1 do not affect DA turnover in the NAS or limbic forebrain of adult rats [9,19]. However, a direct comparison is not
Thirty minutes of footshock evoked similar absolute increases in DOPAC to DA ratios in the mPFC of control and lesioned rats tested on PN30 and 60 (rats were not tested under stress conditions on PN45). However, because baseline DA turnover was increased in the mPFC of lesioned rats, the effects of stress on DOPAC to DA ratios were attenuated by ~35% when the data were represented as a percent of baseline values. In addition, absolute stressinduced DOPAC concentrations were ~50% lower in lesioned rats tested on PN30 and 60 relative to age-matched controls. The latter findings are consistent with the observation that footshock-evoked increases in mPFC DOPAC concentrations are attenuated in adult rats previ-
Fig. 4. Effects of footshock (30 min at 0.6 mA) on DOPAC concentrations in the mPFC of control rats and rats previously sustaining 6-OHDA lesions of the mPFC on PN12–14. Control and lesioned rats were tested on PN30 (A) and 60 (B) (n = 8–12/group). Footshock increased DOPAC in the mPFC of control rats sacrificed on PN30 and 60 [t(21) = 3.27 and t(20) = 5.13, respectively]. In lesioned rats, stress-evoked increases in tissue DOPAC concentrations were significantly attenuated relative to agematched control rats tested on PN30 and 60 [t(22) = 3.14 and t(17) = 3.84, respectively]. Data are presented as group means F SEM (% increase above baseline appears on the corresponding bar). *Significantly different from age-matched control rats under stress conditions. **Significantly different from corresponding age-matched control rats under baseline conditions (t tests with layered Bonferroni correction, P V 0.0125–0.05).
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activate mesoaccumbens DA neurons [8,9,16]. However, it has also been reported that stimuli sub-threshold for activating mesoaccumbens DA neurons in control subjects increase DA turnover in the NAS of rats previously sustaining loss of mesoprefrontal DA during puberty or as adults [8,24]. In contrast, results of the present studies indicate that mPFC DA depletions sustained early in development do not alter basal or stress-evoked DA turnover in the NAS of adult rats. 4.3. Effects of neonatal 6-OHDA lesions of the rat mPFC on PN12–14 on locomotor activity in juvenile and adult rats under baseline and stress conditions In the present study, loss of ~50% of mPFC DA on PN12–14 did not significantly alter baseline or footshock-
Fig. 5. Effects of footshock (30 min at 0.6 mA) on DOPAC to DA ratios in the NAS of control rats and rats previously sustaining 6-OHDA lesions of the mPFC on PN12–14. Control and lesioned rats were tested on PN30 (A) and 60 (B) (n = 8–13/group). Although footshock increased metabolite to DA ratios in the NAS of control rats sacrificed on PN30 by ~40%, this increase failed to reach the level of significance required by the layered Bonferroni correction [t(21) = 2.32, P = 0.03]. In contrast, stress did not affect DA turnover in the NAS of lesioned rats tested on PN30 or control and lesioned rats tested on PN60. Data are presented as group means F SEM. *Significantly different from age-matched control rats under baseline conditions (data from Fig. 2).
ously sustaining electrolytic lesions of the VTA on PN1 [9]. Furthermore, our observation that the stress-evoked increases in DOPAC concentrations were attenuated in lesioned rats tested on PN30 and 60 indicates that this effect of the lesion does not change as a function of the age of the rat at the time of testing. It has been hypothesized that hypoactivity of mesoprefrontal DA neurons, in turn, leads to hyperactivity of mesoaccumbens DA neurons [7,12,42]. In support of this view, depletion of mPFC DA in pubertal and adult rats increases basal and/or stress-evoked activity of mesoaccumbens DA neurons [6,8,24,25,34,37]. As discussed above, results of the present study indicate that loss of mPFC DA on PN12–14 results in a transient increase in DA turnover in the NAS evident on PN30, but not 45 or 60. We also examined whether loss of mPFC DA sustained on PN12–14 enhances the responsiveness of mesoaccumbens DA neurons to stress later in development. Footshock failed to significantly affect DOPAC to DA ratios in the NAS of control or lesioned rats tested on PN30 and 60. Our data are consistent with studies indicating that lower intensity footshock selectively activates mesoprefrontal DA neurons in control rats, whereas higher intensities are required to
Fig. 6. Effects of 6-OHDA lesions of the mPFC sustained on PN12–14 on horizontal locomotor activity (number of photocell beam interruptions per 5 min) under baseline conditions and in response to footshock (30 min at 0.6 mA). Control and lesioned rats were tested on PN30 (A; n = 12–15/ group) and 60 (B; n = 6–8/group). Locomotor activity over the 30 min testing period varied as a function of treatment condition in rats tested on both PN30 and PN60 [PN30 group time: F(10, 166) = 5.33; PN60 group time: F(11, 92) = 8.21]. Footshock decreased locomotor activity in control and lesioned rats tested on PN30 relative to the respective nonshocked control and lesioned rats [ F(3, 32) = 10.91 and F(3, 78) = 3.90, respectively]. Similarly, stress decreased the activity of control and lesioned rats tested on PN60 [ F(3, 49) = 16.36 and F(3, 28) = 8.22, respectively]. Partial loss of mPFC DA sustained on PN12–14 did not significantly alter basal or stress-evoked locomotor activity in rats tested on PN30 or 60, relative to age-matched control rats. Data are presented as group mean F SEM. *Significantly different from corresponding lesioned and control rats under baseline conditions (t tests with layered Bonferroni correction, P V 0.008–0.05).
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evoked locomotor activity in rats tested on PN30 or 60. In contrast, results of a recent study indicate that 6-OHDA lesions of the mPFC sustained on PN11 resulted in ~20% loss of local tissue DA and increased baseline and amphetamine-evoked locomotor activity in adult rats [27]. It is unclear whether lesion-induced alterations in motor behavior in the latter study may have been due to effects of 6OHDA on subcortical DA neurons, since the integrity of those neurons does not appear to have been assessed. Studies examining the effects of early postnatal VTA electrolytic lesions have yielded inconsistent results. VTA electrolytic lesions sustained on PN4 decrease PFC DA by ~45% and increase locomotor activity on ~PN23, but not 60 [15]. In contrast, other investigators report that VTA electrolytic lesions inducing smaller loss of PFC DA (lesion size not quantified) increase locomotor activity on PN25, but not 35, whereas larger loss decreases motor activity at both ages [21]. Again, the non-specific effects of electrolytic lesions make it difficult to attribute changes in motor behavior specifically to loss of mesocortical DA neurons. To our knowledge, previous studies have not examined the effects of stress on motor activity in rats sustaining early partial loss of mPFC DA. Results of our studies indicate that relatively selective loss of PFC DA sustained early in development does not affect basal- or stress-evoked horizontal motor activity, suggesting that effects observed following less selective lesion methods may be due to effects on non-DA and/or subcortical DA neurons. In addition, it remains to be determined whether early postnatal depletion of mPFC DA consistently affects other, more subtle, aspects of motor behavior such as stress-induced immobilization. 4.4. Conclusions and clinical implications It has been hypothesized that developmental abnormalities in the mesoprefrontal DA innervation may contribute to the expression of schizophrenia as an individual matures. Results of the present study confirm that early partial loss of mPFC DA results in a persistent hypofunction of the remaining mesoprefrontal DA neurons. Once present, this dysfunction does not change as the organism matures. In contrast, effects of the lesion on mesoaccumbens DA neurons (which may be a direct effect of 6-OHDA or a secondary consequence of loss of mesoprefrontal DA neurons) are transient. Thus, it is possible that partial loss of mesoprefrontal DA nerve terminals occurring early in development cannot be compensated for, resulting in a persistent hypoactivity of the remaining nerve terminals.
Acknowledgments We thank Jeff Frost and Clint Burgess of WWU Scientific Technical Services for designing and constructing the locomotor activity monitors and data acquisition software.
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Research report
Neonatal depletion of cortical dopamine: Effects on dopamine turnover and motor behavior in juvenile and adult rats P.J. Boyce, J.M. FinlayT,1 Department of Psychology, Western Washington University, 220 Miller Hall, MS 9089, Bellingham, WA 98225, USA Accepted 15 February 2005 Available online 7 April 2005
Abstract Abnormal development of mesoprefrontal dopamine (DA) neurons may contribute to the pathophysiology of schizophrenia. Consistent with this hypothesis, DA nerve terminal density is decreased in the cortex of schizophrenic subjects [M. Akil, J.N. Pierri, R.E. Whitehead, C.L. Edgar, C. Mohila, A.R. Sampson, and D.A. Lewis, Lamina-specific alterations in the dopamine innervation of the prefrontal cortex in schizophrenic subjects, Am. J. Psychiatry, 156 (1999) 1580–1589]. This abnormality may be present early in development, giving rise to dysfunction as an individual matures. The present studies examined the effects of early partial loss of medial prefrontal cortex (mPFC) DA on DA turnover and locomotor behavior in juvenile, pubertal, and adult rats (30, 45, and 60 days of age, respectively). Local infusions of 6hydroxydopamine on postnatal day (PN) 12–14 produced persistent decreases in basal tissue DA concentrations and increases in 3,4dihydroxyphenylacetic acid (DOPAC):DA ratios in the mPFC. In the nucleus accumbens of lesioned rats, basal DA concentrations were decreased and DOPAC:DA ratios were increased on PN30, but not PN45 or 60. Footshock (30 min at 0.6 mA) increased DOPAC and DOPAC:DA ratios in the mPFC of PN30 and 60 control rats. These effects were attenuated in age-matched rats previously sustaining ~50% loss of mPFC DA on PN12–14. Footshock did not affect DOPAC:DA ratios in the nucleus accumbens of control or lesioned rats. The lesion also failed to alter basal or stress-evoked motor activity. The present data suggest that a decreased density of mPFC DA nerve terminals occurring early in development results in persistent alterations in basal and stress-evoked activity of mesoprefrontal DA neurons, but not mesoaccumbens DA neurons. D 2005 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Neurotransmitter systems and channels Keywords: Footshock stress; 3,4-dihydroxyphenylacetic acid; Schizophrenia; Development; 6-hydroxydopamine; Mesoprefrontal dopamine
1. Introduction Abnormal function of dopamine (DA) neurons projecting to the prefrontal cortex (PFC) has long been thought to contribute to the expression of schizophrenia in some individuals (for review, see Ref. [10]). One hypothesis is that hypoactivity of the mesocortical DA innervation leads to hyperactivity of mesolimbic DA neurons and that, together, these pathophysiologies contribute to the negative T Corresponding author. Fax: +1 360 650 7305. E-mail address: [email protected] (J.M. Finlay). 1 This work was supported by USPHS Grant MH61616. 0165-3806/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2005.02.006
and positive symptoms of the illness, respectively [7,13,42]. Results of experimental studies support the view that mesocortical DA neurons regulate the activity of mesolimbic DA neurons. For example, partial loss of mesoprefrontal DA neurons disrupts DA turnover and release in the nucleus accumbens (NAS), electrophysiological activity of ventral tegmental area DA neurons, and behaviors thought to be modulated by mesoaccumbens DA neurons such as motor behavior [6,8,14,24,25,34]. It has been suggested that the primary pathological event(s) associated with schizophrenia occur during pre- or peri-natal development [2,31,33]. The functional consequences of such developmental disturbances are then expressed
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when the affected neural networks are fully mature, accounting for the appearance of clinical symptoms of schizophrenia in late adolescence or early adulthood. Although postmortem analyses have revealed a decreased density of fibers immunoreactive for tyrosine hydroxylase and DA transporter protein in the PFC of schizophrenic subjects [1], it is not known when this structural brain abnormality occurs. The effects of PFC DA depletions on the activity of mesolimbic DA neurons have been examined predominantly in rodents sustaining lesions in late adolescence/early adulthood. Furthermore, studies assessing the effects of depletions sustained early in development, used electrolytic lesions of the ventral tegmental area (VTA) or intraventricular administration of 6-hydroxydopamine (6-OHDA), resulting in damage to non-DA neurons and widespread loss of DA [9,17,19–21,28,30]. In the developing rat, DA fibers destined to innervate deep and superficial cortical layers reach the PFC by embryonic day 16 and postnatal day 2, respectively [3,4,18,38,39,41]. The DA projections to the rat PFC then continue to increase in density until adulthood. Thus, the DA innervation of the rat PFC, like that of primates [11,29,36,40], undergoes a protracted development from gestation to adulthood. Because rat neurodevelopment at birth resembles that seen in primates during the second trimester [26], the impact of lesions sustained in the early postnatal period may be relevant to hypotheses that adverse events occurring in utero contribute to the pathophysiology of schizophrenia [35]. The goal of the present studies was to examine the effects of partial depletion of PFC DA sustained on PN12–14, on DA turnover in the PFC and nucleus accumbens (NAS) of juvenile and adult rats. Motor activity was monitored as behavioral correlate of the activity of mesolimbic DA neurons [22]. Neurochemical activity and motor behavior were measured under baseline conditions and in response to a challenge stimulus, footshock stress. This stimulus was chosen because stress can precipitate some of the symptoms of schizophrenia [5].
2. Methods and materials 2.1. Animals Litters of Sprague–Dawley rats (Hilltop Lab Animals, Scottsdale, PA) consisted of a lactating dam and 12 male pups age PN8–10. Upon arrival, litters were housed in cages (30 cm2 13 cm) with cob bedding. Pups were weaned on PN21 and housed 4 per cage. On PN30, rats were tested and sacrificed or housed singly in hanging wire-mesh cages (24 cm 20 cm2) where they remained until testing and sacrifice on PN45 or 60. At all times, rats were housed in temperature-controlled rooms (20–22 8C) with the lights on from 8:00 am to 8:00 pm. Rat chow and water were available ad libitum. All treatments were performed during the light phase of the light/dark cycle. Procedures for the
treatment of rats were approved by the Institutional Animal care and Use Committee at Western Washington University using criteria established by the U.S. Animal Welfare Act and the National Institutes of Health Guide for the Case and Use of Laboratory Animals. 2.2. Neonatal 6-hydroxydopamine lesions of the rat mPFC On PN12–14, rats randomly assigned to the lesion condition were removed from the litter, pretreated with the norepinephrine (NE) uptake inhibitor desipramine hydrochloride (0.125 mg base/0.025 ml/10 g in 0.9% NaCl, subcutaneous; Sigma-Aldrich, St. Louis, MO), and housed in a plastic container (42 cm 25 cm 15 cm) lined with a heating pad (Vetko, Model V-21, Colorado Springs, CO). Thirty minutes later, rats were anesthetized with 4.5% isoflurane (Abbott Laboratories, Abbott Park, IL) in oxygen and placed in a stereotaxic instrument equipped with a neonatal rat adaptor and anesthesia mask (David Kopf Instruments, Models 900, 970, and 907, Tujunga, CA). Isofluorane (1–2%) was delivered continuously during surgery. A reusable heat packet (Harvard Apparatus, Holliston, MA) was placed under the anesthetized rat to provide warmth during surgery. A glass pipette (tip o.d. = 50–100 Am) was positioned in the medial PFC (mPFC) using stereotaxic coordinates: AP +2.1 mm and ML F0.3 mm from bregma, DV 2.4 mm from dura with the skull flat (stereotaxic coordinates were established in a pilot study using dye injections delivered via glass pipettes). The pipette was left in place for 5 min after which 1.0 Ag 6-OHDA base (Sigma-Aldrich or ICN Biomedicals, Irvine, CA) in 0.25 Al of vehicle (0.9% NaCl containing 0.03% ascorbic acid) was infused over 5 min using pressure ejection (World Precision Instruments, PV820 Pneumatic picopump, Sarasota, Fl). A stock solution of 6-OHDA, prepared fresh each day, was stored on ice in darkness. Immediately prior to being positioned in the left or right mPFC, the injection pipette was filled with sufficient toxin for a single infusion. Rats assigned to the PN45 testing group received a second infusion of 1.0 Ag 6-OHDA in 0.25 Al of vehicle at a site located DV 3.7 mm from dura. After each infusion, the pipette was left in place for 5 min to allow for dispersal of the toxin. The infusion procedure was repeated in the contralateral hemisphere, the pipette was then removed, and the scalp closed with sutures. Upon resuming normal motor activity, pups were returned to the litter where they remained until weaned on PN21. Control rats were unoperated, remaining with dam and littermates until weaning. Each rat was handled 2–3 times/week by an animal care technician. 2.3. Horizontal locomotor activity Horizontal locomotor activity was monitored in control and lesioned rats on PN30 and 60. Rats were placed in a
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cylindrical Plexiglas cage (41 28 cm in diameter) with a metal rod floor (16 metal rods each 5 mm in diameter and spaced 14 mm apart). The Plexiglas cage was located in one corner of a custom built motor-activity chamber (51 cm2) comprised of a single layer of photocells (20 cells per side) forming a horizontal grid. Photocells were 2.5 cm apart and 5 cm above the floor of the chamber. Photocell interruptions were recorded 3 times per second for 30 min, beginning immediately after the rat was placed in the cage. Control and lesioned rats assigned to the baseline condition remained undisturbed in the cage for 30 min. Rats assigned to the stress condition were exposed to 0.6 mA scrambled footshock delivered via a shock generator and scrambler (BRS-LVE, Models SG903 and SC922, Laurel, MD). Footshock was delivered for 2 s every 5 s for a total of 12 2-s shocks in 60 s. This series was repeated every 5 min for 30 min, resulting in a total of 6 shock cycles. 2.4. Tissue dissection and neurochemical analyses Rats used for analysis of motor activity on PN30 and 60 were removed from the motor-activity chamber and immediately decapitated. All other rats were decapitated immediately following removal from their cages in the colony room on PN30, 45, or 60. The brain of each rat was rapidly removed and frozen onto a microtome stage. Thick coronal sections were made at the level of the mPFC and NAS (2.9–4.3 and 1.0–2.4 mm anterior to bregma, respectively; [32]). The left and right mPFC and NAS were freehand dissected, as described in a previous publication [23]. Tissue samples were weighed, sonicated in 250 Al of 0.2 M hydrochloric acid, and centrifuged at 14,000 rpm for 25 min at 4 8C. The supernatants were filtered through 0.2-Am nylon filters by centrifugation at 10,000 rpm for 10 min at 4 8C. Supernatants were stored at 80 8C until analysis. Tissue DA, NE, and DOPAC concentrations were determined using high performance liquid chromatography (HPLC) with amperometric or coulometric detection. The HPLC systems consisted of a manual injector (Rheodyne, Model 9125, Rohnert Park, CA), an HPLC pump (Waters Co., Model 540, Milford, MA; ESA, Model 580, Chelmsfod, MA), and a pulse dampener (SSI, Model LP-21, State College, PA). Peak separation was accomplished using a reversed-phase C18 column (3.2 mm 150 mm, 3 um; ESA, Part #70-0636) and mobile phase (ESA, Part #71-1332). The mobile phase was circulated through the HPLC system at a flow rate of 0.4 ml/min. Amperometric detection was performed using a potentiostat (Waters, Model 460) and glassy carbon working electrode set at +870 mV relative to an Ag/AgCl reference electrode. Coulometric detection was performed using a potentiostat (ESA, Model 5100 A) that controlled a conditioning cell (ESA, Model 5020) maintained at +400 mV and an analytical cell (ESA, Model 5014) with
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the first and second electrodes maintained at 140 and +125 mV, respectively; analytes were quantified using the oxidation signal from the second analytical electrode. DA, NE, and DOPAC concentrations in the samples were determined by comparing oxidation peaks produced by samples to those produced by standards of known concentrations. The detection limit of the assay was ~0.5 pg/20 Al of sample for each analyte. Acquisition and analysis of chromatograms were performed via computer (Varian, Star Chromatography Workstation, Walnut Creek, CA and Rainin Instrument, Dynamax Macintegrator HPLC Method Manager, Rainin Instrument Co., Emeryville, CA). 2.5. Statistical analyses mPFC and NAS tissue from the left and right hemisphere of each rat was analyzed separately using HPLC with electrochemical detection. Data from the two hemispheres were then used to calculate average DA, NE, and DOPAC concentrations in the mPFC and NAS of individual subjects. Baseline and stress-evoked tissue DA and NE concentrations (ng/mg tissue) and DOPAC to DA ratios in the mPFC and NAS of control and lesioned rats were analyzed using independent samples t tests with layered Bonferroni corrections for multiple comparisons. The effects of lesions and stress on horizontal locomotor activity (photocell beam interruptions per 5 min) were analyzed using univariate repeated measures ANOVAs with degrees of freedom appropriate for the Geisser– Greenhouse F test followed by independent samples t tests with layered Bonferroni corrections. Results of all statistical analyses appear in the appropriate figure caption. In all cases, sample size indicates the number of rats per treatment condition.
3. Results 3.1. Effects of neonatal 6-OHDA lesions of the mPFC on basal catecholamine concentrations in the mPFC and NAS of juvenile and adult rats Local 6-OHDA infusions delivered on PN12–14 decreased baseline DA and, to a lesser extent, NE concentrations in the mPFC of rats sacrificed on PN30, 45, and 60 (Figs. 1A and B). DOPAC to DA ratios in the mPFC were increased in lesioned rats sacrificed on PN30 and 45, relative to age-matched control rats (Fig. 1C). Although lesioned rats sacrificed on PN60 exhibited a 25% increase in metabolite to DA ratios in the mPFC, this trend did not reach statistical significance. Infusions of 6-OHDA into the mPFC on PN12–14 also reduced DA concentrations and increased DOPAC to DA ratios in the NAS of rats sacrificed on PN30, but not PN45 and 60 (Figs. 2A and B).
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Fig. 1. Effects of 6-OHDA infused into the rat mPFC on PN12–14 on local tissue concentrations of DA (A) and NE (B) and DOPAC to DA ratios (C) in rats sacrifice on PN30, 45, and 60, under baseline conditions (n = 10–14/group). 6-OHDA lesions of the mPFC decreased local DA and, to a lesser extent, NE content in rats sacrifice on PN30, 45, and 60 [DA: t(21) = 6.0, t(25) = 10.05, and t(19) = 4.63, respectively; NE: t(21) = 4.21, t(26) = 4.08, t(20) = 3.45, respectively]. DOPAC to DA ratios in the mPFC were significantly increased in rats sacrificed on PN30 and 45, but not PN60 [t(21) = 2.59 and t(24) = 4.10, respectively]. Data are presented as group mean F SEM. *Significantly different from age-matched control rats (t tests with layered Bonferroni correction, P V 0.017–0.05).
3.2. Effects of neonatal 6-OHDA lesions of the mPFC on stress-evoked catecholamine concentrations in the mPFC and NAS of juvenile and adult rats Control rats and rats previously sustaining 6-OHDA lesions of the mPFC on PN12–14 were exposed to acute footshock (30 min at 0.6 mA) on PN30 or 60 and immediately sacrificed for analysis of tissue catecholamine concentrations in the mPFC and NAS. Data from stressexposed control and lesioned rats were compared to that from age-matched control and lesioned rats tested under baseline conditions, as reported above. Consistent with our findings in non-stressed lesioned rats, local 6-OHDA on PN12–14 reduced DA and, to a lesser extent, NE concentrations in the mPFC of rats subsequently exposed to stress and sacrificed on PN30 and 60 (DA: 0.03 F 0.002 and 0.04 F 0.004 ng/mg tissue, respectively; NE: 0.18 F 0.01 and 0.27 F 0.03 ng/mg tissue, respectively). Similarly, we again found that 6-OHDA infusions into the mPFC on PN12–14 decreased NAS DA content on PN30, but not PN60 (4.2 F 0.6 and 6.7 F 0.6 ng/mg tissue, respectively). Acute footshock increased DOPAC to DA ratios in the mPFC of control and lesioned rats sacrificed on PN30 and 60 (Figs. 3A and B). Absolute stress-evoked increases in metabolite to DA ratios were similar in lesioned and age-
matched control rats. However, this represented a smaller percent increase in turnover above baseline in lesioned than age-matched control rats (% increases above baseline are indicated on corresponding bars in Figs. 3A and B). Absolute stress-evoked increases in tissue DOPAC concentrations were also attenuated in lesioned rats tested on PN30 and 60, relative to age-matched control rats (% increases above baseline are indicated on corresponding bars in Figs. 4A and B). Footshock did not significantly affect DOPAC to DA ratios in the NAS of control and lesioned rats tested on PN30 and 60 (Figs. 5A and B). Although there was a trend for a stress-evoked increase in DA turnover in control rats sacrificed on PN30, this effect failed to reach criterion for significance using a layered Bonferroni correction (see Fig. 5 legend). 3.3. Effects of neonatal 6-OHDA lesions of the rat mPFC on locomotor activity in juvenile and adult rats Immediately prior to sacrifice for analysis of tissue catecholamine concentrations, baseline and footshockevoked horizontal locomotor activity was monitored in all control and lesioned rats tested on PN30. Due to equipment failure, motor activity data were acquired from only a subset of control and lesioned rats sacrificed on PN60 (n = 6–8/
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Fig. 2. Effects of 6-OHDA infused into the rat mPFC on PN12–14 on tissue DA concentrations (A) and DOPAC to DA ratios (B) in the NAS of rats sacrificed on PN30, 45, and 60, under baseline conditions (n = 10–14/group). 6-OHDA lesions of the mPFC decreased DA concentrations and increased DOPAC to DA ratios in the NAS of rats sacrificed on PN30 [t(19) = 2.80 and t(19) = 3.39, respectively]. On PN45 and 60, basal DA levels and DA turnover in the NAS of lesioned rats did not differ from control values. Data are presented as group mean F SEM. *Significantly different from age-matched control rats (t tests with layered Bonferroni correction, P V 0.017–0.05).
group of the total n = 11–12/group available for analysis of tissue catecholamines). Concentrations of DA and NE in the mPFC of lesioned rats used for analysis of locomotor behavior on PN60 were 54 F 5% and 73 F 2% of control values, confirming that lesions in this subset were representative of the larger group. Baseline horizontal motor activity on PN30 and 60 was similar in control rats and rats previously sustaining 6-OHDA lesions of the mPFC on PN12–14 (Figs. 6A and B). Exposure to acute footshock (30 min at 0.6 mA) on PN30 or 60 reduced locomotor activity of control and lesioned rats. The stress-evoked behavioral response of lesioned rats did not differ from that of age-matched control rats.
4. Discussion 4.1. Effects of 6-OHDA lesions of the rat mPFC on PN12–14 on tissue DA concentrations and turnover in the mPFC and NAS of juvenile and adult rats under baseline conditions In the present studies, 6-OHDA infusions into the rat mPFC during early postnatal development induced a persistent and relatively selective loss of local tissue DA concentrations. Specifically, in rats sustaining 6-OHDA lesions of the mPFC on PN12–14, local DA concentrations
were reduced by ~70% on PN45 and ~50% on PN30 and 60. The presence of larger lesions in rats sacrificed on PN45, than 30 and 60, was likely due to administration of a higher concentration of 6-OHDA (2.0 versus 1.0 Ag per hemisphere, respectively). Although an NE uptake inhibitor was administered prior to the 6-OHDA infusions, NE concentrations in the mPFC were decreased by ~20% in lesioned rats of all age groups. In addition, NAS DA concentrations were reduced by ~35% in rats tested on PN30, but not 45 and 60, suggesting that this loss was a transient consequence of 6-OHDA infusions into the mPFC. Previous investigators used electrolytic lesions of the VTA or intraventricular injections of 6-OHDA to damage mesoprefrontal DA projections, resulting in extensive damage to non-DA neurons and widespread loss of brain DA [9,15,17,19,20,21,28,30]. For example, electrolytic lesions of the VTA on PN1 result in a persistent decrease in DA concentrations in the NAS and limbic forebrain [9,19]. In the present study, the transient increase in DOPAC/DA ratio in the NAS on PN30 may be (1) a direct result of diffusion of 6-OHDA to the NAS and a concomitant loss of DA terminals in this region or (2) an indirect effect on mesoaccumbens DA cell bodies in the VTA, mediated by degeneration of neighboring mesoprefrontal DA cell bodies. Nonetheless, our finding that NAS DA concentrations return to normal levels by PN45 suggests either that VTA DA cells projecting to the NAS are largely
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possible given that NAS and limbic forebrain DA concentrations were also reduced under the conditions of these previous studies. 4.2. Effects of 6-OHDA lesions of the rat mPFC on PN12–14 on tissue DA concentrations and turnover in the mPFC and NAS of juvenile and adult rats under stress conditions
Fig. 3. Effects of footshock (30 min at 0.6 mA) on DOPAC to DA ratios in the mPFC of control rats and rats previously sustaining 6-OHDA lesions of the mPFC on PN12–14. Control and lesioned rats were tested on PN30 (A) and 60 (B) (n = 8–12/group). Footshock increased DOPAC to DA ratios in the mPFC of control and lesioned rats sacrificed on PN30 [t(21) = 5.62 and t(22) = 2.65, respectively] and PN60 [t(19) = 3.19 and t(16) = 2.25]. Absolute stress-evoked increases in DOPAC to DA ratios were similar in lesioned and control rats. However, because the lesion itself increased basal DA turnover, the effects of stress were attenuated in lesioned rats when the data were represented as a percent of baseline (% increase above baseline appears on the corresponding bar). Data are presented as group means F SEM. *Significantly different from age-matched control rats under baseline conditions (data from Fig. 1). **Significantly different from corresponding age-matched control and lesioned rats under baseline conditions (t tests with layered Bonferroni correction, P V 0.017–0.05).
spared following local infusions of 6-OHDA into the mPFC on PN12–14 or that damage is sufficiently limited that the remaining neurons can fully compensate. As a measure of DA turnover, we also examined the effects of our lesion on basal DOPAC to DA ratios in the mPFC and NAS. In rats sustaining 6-OHDA lesions of the mPFC on PN12–14, DOPAC to DA ratios in the mPFC were increased on PN30, 45, and 60. The latter findings are consistent with previous studies indicating that early partial loss of mPFC DA results in a persistent increase in local DA turnover. Specifically, rats sustaining electrolytic lesions of the VTA or intraventricular 6-OHDA infusions (PN1 and 3, respectively) exhibit increased DOPAC to DA ratios in the mPFC in adulthood ([9,19,30]; however, see also Ref. [28]). In the present study, lesioned rats exhibited increased DOPAC to DA ratios in the NAS on PN30, but not 45 or 60. Our finding appears to be consistent with a previous report indicating that electrolytic lesions of the VTA on PN1 do not affect DA turnover in the NAS or limbic forebrain of adult rats [9,19]. However, a direct comparison is not
Thirty minutes of footshock evoked similar absolute increases in DOPAC to DA ratios in the mPFC of control and lesioned rats tested on PN30 and 60 (rats were not tested under stress conditions on PN45). However, because baseline DA turnover was increased in the mPFC of lesioned rats, the effects of stress on DOPAC to DA ratios were attenuated by ~35% when the data were represented as a percent of baseline values. In addition, absolute stressinduced DOPAC concentrations were ~50% lower in lesioned rats tested on PN30 and 60 relative to age-matched controls. The latter findings are consistent with the observation that footshock-evoked increases in mPFC DOPAC concentrations are attenuated in adult rats previ-
Fig. 4. Effects of footshock (30 min at 0.6 mA) on DOPAC concentrations in the mPFC of control rats and rats previously sustaining 6-OHDA lesions of the mPFC on PN12–14. Control and lesioned rats were tested on PN30 (A) and 60 (B) (n = 8–12/group). Footshock increased DOPAC in the mPFC of control rats sacrificed on PN30 and 60 [t(21) = 3.27 and t(20) = 5.13, respectively]. In lesioned rats, stress-evoked increases in tissue DOPAC concentrations were significantly attenuated relative to agematched control rats tested on PN30 and 60 [t(22) = 3.14 and t(17) = 3.84, respectively]. Data are presented as group means F SEM (% increase above baseline appears on the corresponding bar). *Significantly different from age-matched control rats under stress conditions. **Significantly different from corresponding age-matched control rats under baseline conditions (t tests with layered Bonferroni correction, P V 0.0125–0.05).
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activate mesoaccumbens DA neurons [8,9,16]. However, it has also been reported that stimuli sub-threshold for activating mesoaccumbens DA neurons in control subjects increase DA turnover in the NAS of rats previously sustaining loss of mesoprefrontal DA during puberty or as adults [8,24]. In contrast, results of the present studies indicate that mPFC DA depletions sustained early in development do not alter basal or stress-evoked DA turnover in the NAS of adult rats. 4.3. Effects of neonatal 6-OHDA lesions of the rat mPFC on PN12–14 on locomotor activity in juvenile and adult rats under baseline and stress conditions In the present study, loss of ~50% of mPFC DA on PN12–14 did not significantly alter baseline or footshock-
Fig. 5. Effects of footshock (30 min at 0.6 mA) on DOPAC to DA ratios in the NAS of control rats and rats previously sustaining 6-OHDA lesions of the mPFC on PN12–14. Control and lesioned rats were tested on PN30 (A) and 60 (B) (n = 8–13/group). Although footshock increased metabolite to DA ratios in the NAS of control rats sacrificed on PN30 by ~40%, this increase failed to reach the level of significance required by the layered Bonferroni correction [t(21) = 2.32, P = 0.03]. In contrast, stress did not affect DA turnover in the NAS of lesioned rats tested on PN30 or control and lesioned rats tested on PN60. Data are presented as group means F SEM. *Significantly different from age-matched control rats under baseline conditions (data from Fig. 2).
ously sustaining electrolytic lesions of the VTA on PN1 [9]. Furthermore, our observation that the stress-evoked increases in DOPAC concentrations were attenuated in lesioned rats tested on PN30 and 60 indicates that this effect of the lesion does not change as a function of the age of the rat at the time of testing. It has been hypothesized that hypoactivity of mesoprefrontal DA neurons, in turn, leads to hyperactivity of mesoaccumbens DA neurons [7,12,42]. In support of this view, depletion of mPFC DA in pubertal and adult rats increases basal and/or stress-evoked activity of mesoaccumbens DA neurons [6,8,24,25,34,37]. As discussed above, results of the present study indicate that loss of mPFC DA on PN12–14 results in a transient increase in DA turnover in the NAS evident on PN30, but not 45 or 60. We also examined whether loss of mPFC DA sustained on PN12–14 enhances the responsiveness of mesoaccumbens DA neurons to stress later in development. Footshock failed to significantly affect DOPAC to DA ratios in the NAS of control or lesioned rats tested on PN30 and 60. Our data are consistent with studies indicating that lower intensity footshock selectively activates mesoprefrontal DA neurons in control rats, whereas higher intensities are required to
Fig. 6. Effects of 6-OHDA lesions of the mPFC sustained on PN12–14 on horizontal locomotor activity (number of photocell beam interruptions per 5 min) under baseline conditions and in response to footshock (30 min at 0.6 mA). Control and lesioned rats were tested on PN30 (A; n = 12–15/ group) and 60 (B; n = 6–8/group). Locomotor activity over the 30 min testing period varied as a function of treatment condition in rats tested on both PN30 and PN60 [PN30 group time: F(10, 166) = 5.33; PN60 group time: F(11, 92) = 8.21]. Footshock decreased locomotor activity in control and lesioned rats tested on PN30 relative to the respective nonshocked control and lesioned rats [ F(3, 32) = 10.91 and F(3, 78) = 3.90, respectively]. Similarly, stress decreased the activity of control and lesioned rats tested on PN60 [ F(3, 49) = 16.36 and F(3, 28) = 8.22, respectively]. Partial loss of mPFC DA sustained on PN12–14 did not significantly alter basal or stress-evoked locomotor activity in rats tested on PN30 or 60, relative to age-matched control rats. Data are presented as group mean F SEM. *Significantly different from corresponding lesioned and control rats under baseline conditions (t tests with layered Bonferroni correction, P V 0.008–0.05).
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evoked locomotor activity in rats tested on PN30 or 60. In contrast, results of a recent study indicate that 6-OHDA lesions of the mPFC sustained on PN11 resulted in ~20% loss of local tissue DA and increased baseline and amphetamine-evoked locomotor activity in adult rats [27]. It is unclear whether lesion-induced alterations in motor behavior in the latter study may have been due to effects of 6OHDA on subcortical DA neurons, since the integrity of those neurons does not appear to have been assessed. Studies examining the effects of early postnatal VTA electrolytic lesions have yielded inconsistent results. VTA electrolytic lesions sustained on PN4 decrease PFC DA by ~45% and increase locomotor activity on ~PN23, but not 60 [15]. In contrast, other investigators report that VTA electrolytic lesions inducing smaller loss of PFC DA (lesion size not quantified) increase locomotor activity on PN25, but not 35, whereas larger loss decreases motor activity at both ages [21]. Again, the non-specific effects of electrolytic lesions make it difficult to attribute changes in motor behavior specifically to loss of mesocortical DA neurons. To our knowledge, previous studies have not examined the effects of stress on motor activity in rats sustaining early partial loss of mPFC DA. Results of our studies indicate that relatively selective loss of PFC DA sustained early in development does not affect basal- or stress-evoked horizontal motor activity, suggesting that effects observed following less selective lesion methods may be due to effects on non-DA and/or subcortical DA neurons. In addition, it remains to be determined whether early postnatal depletion of mPFC DA consistently affects other, more subtle, aspects of motor behavior such as stress-induced immobilization. 4.4. Conclusions and clinical implications It has been hypothesized that developmental abnormalities in the mesoprefrontal DA innervation may contribute to the expression of schizophrenia as an individual matures. Results of the present study confirm that early partial loss of mPFC DA results in a persistent hypofunction of the remaining mesoprefrontal DA neurons. Once present, this dysfunction does not change as the organism matures. In contrast, effects of the lesion on mesoaccumbens DA neurons (which may be a direct effect of 6-OHDA or a secondary consequence of loss of mesoprefrontal DA neurons) are transient. Thus, it is possible that partial loss of mesoprefrontal DA nerve terminals occurring early in development cannot be compensated for, resulting in a persistent hypoactivity of the remaining nerve terminals.
Acknowledgments We thank Jeff Frost and Clint Burgess of WWU Scientific Technical Services for designing and constructing the locomotor activity monitors and data acquisition software.
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