Neonatal monoaminergic depletion in mice (Mus musculus) improves performance of a novel odor discrimination task

Acknowledgement: This research was supported by National Science Foundation Grant IBN94585101, by the Whitehall Foundation, and through Morgan State University. We thank U. Berger and K. Frick for critically reviewing the manuscript, D. Smith for help in designing the odor discrimination protocol, E. Caliguri for aid in neurochemical analyses, and L. Baldwin for statistical advice. Portions of the research reported in this article have appeared previously in abstract form (Libbey, Berger-Sweeney, & Hohmann, 1996).

The organization of the mature cerebral cortex is influenced by several subcortical afferent systems. In rodents, noradrenergic, serotonergic, dopaminergic, and cholinergic fibers innervate the cortex just prior to birth, the most dynamic period of cortical differentiation and synapse formation. A wealth of studies suggest that these afferents regulate cortical maturation and establish neural circuitry that is important for cognition (Berger-Sweeney & Hohmann, 1997). Previously, we have shown that electrolytic lesions to the nucleus basalis/basal forebrain (nBM/BF) region on Postnatal Day 1 (PND1) interrupt fibers projecting to the cortex and produce long-lasting alterations in cortical morphology and behavior (Bachman, Berger-Sweeney, Coyle, & Hohmann, 1994; Hohmann & Ebner, 1985). The cortical abnormalities correlate with depletion of cholinergic innervation of the cortex, suggesting that acetylcholine is critical for normal cortical development and behavior. Electrolytic lesions, however, are not specific; in addition to cholinergic neurons, the lesions destroy noncholinergic neurons in the nBM as well as monoaminergic medial forebrain bundle fibers (MFB) en route to the cortex and hippocampus (Parent, Descarries, & Beaudet, 1981). Therefore, reduced monoaminergic innervation of the cortex may contribute to the cortical and behavioral abnormalities seen after electrolytic lesions. Understanding the effects of the monoamines on cortical development and cognition is particularly interesting because of their reported involvement in attention deficit hyperactivity disorder (ADHD; Arnsten, Steere, & Hunt, 1996; Shekim, Dekirmenjian, Chapel, Javaid, & Davis, 1979).

Norepinephrine (NE) fibers from the locus coeruleus and serotonin (5HT) fibers from the raphe nuclei arrive at the cortical anlage around Embryonic Day 17 and penetrate all cortical layers within the 1st week of life (Lidov & Molliver, 1982; Wallace & Lauder, 1983). In vitro studies strongly suggest that 5HT and NE act as morphological differentiation signals in the developing brain (Lauder, 1990). Few in vivo studies, however, have shown that amine depletion at birth affects cortical morphology and behavior. Ventricular NE lesions at birth have subtle behavioral effects, some of which are apparent only when combined with other manipulations (Kolb & Sutherland, 1992; Saari & Pappas, 1978). Neonatal 5HT depletions delay thalamocortical ingrowth into the cortex (Blue, Erzurumlu, & Jhaveri, 1991) but produce behavioral deficits only on pharmacological challenge (Breese, Vogel, & Mueller, 1978).

Indirect evidence suggests that alterations in 5HT around the time of birth may have different effects on the two sexes: (a) Maternal stress that alters cortical 5HT receptors in the offspring reportedly feminizes male rats' cerebral cortex such that a characteristic female laterality pattern is displayed (Alonso, Castellano, & Rodriguez, 1991; Fleming, Anderson, Rhees, Kinghorn, & Bakaitis, 1986; Stewart & Kolb, 1988) and (b) neonatal ventral tegmental lesions in rats that deplete cortical dopamine and 5HT affect cortical morphology and behavior differently in females and males (Kalsbeek et al., 1987; Kalsbeek, De, Bruin, Feenstra, Matthijssen, & Uylings, 1988; Kalsbeek, DeBruin, Matthijssen, & Uylings, 1989; Kalsbeek, Matthijssen, & Uylings, 1989). Furthermore, there are sex differences in spatial navigation performance after neonatal electrolytic nBM/BF lesions (which affect cholinergic, serotonergic, and noradrenergic markers in the cortex) in BALB/c mice (Arters, Hohmann, Mills, Olaghere, & Berger-Sweeney, in press). We therefore hypothesized that neonatal monoaminergic lesions would have different effects on behavior and cortical neurochemistry in the two sexes.

The present study examined the developmental role of monoaminergic projections on cognitive development and neurochemistry. The BALB/cByJ mice used in our previous studies are slow to learn spatial navigation tasks because of their limited visual capabilities (Bachman et al., 1994). We therefore developed two tasks that do not depend primarily on visual cues to assess cognitive performance: a simple odor discrimination (SOD) task, adapted for mice fromBunsey and Eichenbaum (1995), and a novel odor delayed nonmatch-to-sample (DNMS) task. The SOD task requires the mouse to form an association between a scent and a reward and demands intact sensory processing, attention, motivation, and learning. The DNMS task additionally requires the mouse to retain this scent–reward association across a delay. The mouse must then attend to a novel odor and restrain the previously reinforced response. Because each response in DNMS is trial dependent, and odors are reused but with novel pairing, it is considered a working memory task. It is interesting to note that few working memory tasks are available for use in mice. Some working memory tasks that can be performed readily by rats, such as delayed nonmatch-to-place (Markowska, Price, & Koliatsos, 1996), are not readily transferable to mice (J. Berger-Sweeney, unpublished, observations, & September, 1997). To assess the mouse's performance on simple odor discrimination (SOD) and delayed nonmatch-to-sample (DNMS), we evaluated choice accuracy, rate of errors, and latency. Choice accuracy is traditionally used to assess precision in conditional discrimination performance (e.g., Gaffan, 1974), whereas errors provide a measure of the perseverance with which the mouse performs the task. Latency to retrieve the reward is more sensitive to locomotor activity, goal-directed attention, and motivation.

Groups of female and male mice received bilateral 5,7 dihydroxytryptamine (DHT) lesions to the nBM/MFB region on PND 1 to deplete hippocampally and cortically projecting NE and 5HT fibers. Mice grew to adulthood and were tested on a battery of tasks: SOD and DNMS, passive avoidance (PA), and locomotor activity. Other mice were killed for neurochemical analyses.

BALB/cByJ mice (Mus musculus) were bred at the Wellesley College colony and placed randomly into the following groups: unoperated controls, sham-operated mice, and bilateral DHT-lesioned mice. All mice were maintained on a 12-hr light–dark cycle (lights on at 7 a.m.), and behavioral testing was conducted during the light cycle. Food and water were available ad libitum during activity and PA testing. During SOD and DNMS, mice were deprived of food 4–5 hr before testing.

Fifty-one mice were used for behavioral testing: 21 unoperated controls (10 females and 11 males), 16 ascorbic acid sham-lesioned mice (10 females and 6 males), and 14 bilateral DHT-lesioned mice (9 females and 5 males). These mice underwent SOD, DNMS, PA, and general motor activity measurements. Thirty mice were used for neurochemical analyses: 10 unoperated controls, 10 bilateral ascorbic acid sham-lesioned mice, and 10 bilateral DHT-lesioned mice. Equal numbers of females and males were used. The ascorbic acid sham-operated mice were not significantly different from unoperated controls on any of the behavioral tasks or in neurochemical measures. The data from these two groups, therefore, were combined, and they are referred to as controls.

Pups were removed from their mothers 12 to 24 hr after birth. Mice were anesthetized by hypothermia (placed on ice for 5 min until movement ceased) and were injected in the nBM/MFB (ventromedial globus pallidus) region with the monoaminergic toxin 5,7-DHT (Sigma Chemical, St Louis, MO; 0.5 μl of a 5 μg/μl solution of 5,7-DHT containing 0.1 mg/ml ascorbic acid) or ascorbic acid (sham-operated controls). The lesion coordinates, as previously reported, were 1 mm anterior to bregma, 1.5 mm lateral to bregma, and lowered 3.5 mm, 4.0 mm, and 4.5 mm ventral from the dura (Hohmann et al., 1988). Mice were injected with toxin (DHT-lesioned) or ascorbic acid (sham-lesioned) into first the right and then the left nBM/MFB region. Unoperated controls underwent hypothermia anesthesia only. After surgery, the pups were placed on a heating pad to speed recovery. After recovery, pups were returned to their mothers, whose noses were swabbed with vanilla extract to prevent cannibalization of the pups.

Odor discrimination

Shaping and testing for SOD and DNMS tasks were conducted in standard (30 × 19 cm) clear mouse cages (Allentown Caging, Allentown, PA) with bedding. Either one or two bait cups were located at one end of the rectangular cage about 3 cm apart (seeFigure 1). All cups were filled with sterilized sand and attached to the bottom of the cage with Velcro. Various powdered spices and flavorings were mixed with the sterilized sand for the odor presentations. The concentrations of the spices are listed as mg/5 g of sand: allspice (10 mg), clove (10 mg), cinnamon (15 mg), chili powder (20 mg), cumin (15 mg), curry (20 mg), ginger (10 mg), garlic (20 mg), mace (10 mg), marjoram (20 mg), mustard (20 mg), nutmeg (10 mg), onion (15 mg), paprika (20 mg), thyme (20 mg), turmeric (15 mg), and unsweetened Kool-Aid flavors (15 mg). Cinnamon, curry, and turmeric were Spice Island brand spices; chili powder, garlic, and mustard were Spice Rack brand spices; all others were Durkee brand spices.

Passive avoidance

The test enclosure (52.5 × 52.5 × 30 cm) had an automatic gate that separated a light (white walls) and a dark (black walls) chamber. The grid floor of the black chamber was electrified to give a 0.2-mA footshock for 3 s. The data were collected with GEMINI software (Gemini Active Avoidance System, San Diego Instruments, San Diego, CA).

Motor activity

The Cage Rack activity system (San Diego Instruments, San Diego, CA) consisted of a clear acrylic cage (43 × 22 cm) surrounded by a bracket containing three photobeam projectors. The beams were oriented to transect the short axis of the cage and were located 2.5 cm from the cage floor. The photobeam breaks were recorded with PAS software (Photobeam Activity System, San Diego Instruments San Diego CA).

Testing began when the mice reached adulthood, 8–12 weeks after the surgery. Before testing began, all mice were handled by the experimenter for 1 week.

Odor discrimination

Mice were deprived of food 4–5 hr prior to odor discrimination shaping and testing procedures. Baited cups contained 1–3 small pieces of chocolate (≈ 15 mg); unbaited cups did not contain chocolate. Mice were placed in a holding cage for 30 s between trials.

Shaping

On Day 1, the mouse was presented with a baited cup in which one piece of chocolate was visible, one was semivisible, and another was buried. The mouse was allowed a maximum of 1 hr to retrieve all three of the chocolate pieces. If the mouse did not begin to dig within 15 min, another piece of chocolate was placed on top of the sand, and the mouse was given an additional 5 min. On Day 2, the mouse was presented with two unscented cups, one baited and one unbaited. In the baited cup, one piece of chocolate was visible to the mouse, and one was buried. Each mouse underwent four trials with a maximum trial length of 15 min. If the mouse did not begin digging for the buried piece of chocolate within 10 min, then an additional piece of chocolate was placed on top. The position of the baited cup (left side vs. right side) was randomized. On Day 3, the mouse was introduced to two scents, cinnamon and curry. Each mouse was assigned a baited scent of either cinnamon or curry, and this association was maintained throughout the SOD task. Assignments were counterbalanced across groups.

Simple odor discrimination

The location of the baited cup was varied so that it was never in the same location for more than two trials. A dig was defined by displacement of sand in the cup by the paws or snout; sniffing or standing on the cup was not counted as a dig. For each trial, a mouse was presented with two cups (one cinnamon, one curry) and given a maximum of 5 min to retrieve the buried chocolate; if the mouse had not begun digging within 5 min, a piece of chocolate was dropped on the top of the correct cup, and the mouse was given an additional 2 min to find the chocolate. Choice accuracy (percentage correct on the first choice), number of errors (total number of digs in the unbaited cup), and latency to retrieve the chocolate were recorded. Each mouse was given four trials per day for 5 consecutive days. On the day after SOD testing, two probe trials were conducted to ensure that the mouse was using odor cues to guide the choice. For the probe trials, each mouse was presented with two scented cups, both unbaited. The first cup choice was recorded. Once the correct cup was chosen, a piece of chocolate was placed on the sand, and the mouse was allowed to retrieve it. For each test, two experimenters performed the task. After initial training, observations between the two experimenters were identical.

Delayed nonmatch-to-sample

This task began 1 day after completion of the SOD. Each trial began with the presentation of one baited cup (sample presentation) filled with sand mixed with a scent. The mouse was allowed to retrieve the chocolate from this cup and was then removed from the test cage for a 30-s delay. At the end of the delay, the mouse was placed in the choice cage and presented with two cups (choice presentation). One cup (match) was identical to the sample but was unbaited. The other cup (nonmatch) contained a novel scent and was baited. The mouse was allowed to dig until the buried chocolate was found in the nonmatch cup. For each choice presentation, the cup in which the mouse dug first (choice accuracy), the latency to retrieve the chocolate piece, and the errors (digs in the unrewarded match cup) were recorded. Scents were reused across the trials but were never paired with the same scent as before. Mice were given three trials per day for 4 consecutive days.

Passive avoidance

This protocol has been previously reported (Berger-Sweeney, Arnold, Gabeau, & Mills, 1995). Each mouse performed two acquisition trials, separated by 1 hr, and followed by a 24-hr retention period. In the first acquisition, the mouse was placed in the light chamber of the PA apparatus and given 30 s to adapt to the environment. After 30 s, the light chamber was illuminated, and the door to the dark chamber was opened. The mouse had 60 s to cross into the black chamber. Mice that did not enter the dark chamber within 60 s were dropped from testing and excluded from the analysis. If the mouse entered the dark chamber, the door closed, and the mouse received a footshock of 0.2 mA for 3 s. The mouse was then removed immediately from the chamber and placed in its home cage. The latency to cross was recorded. An hour later, the acquisition was repeated. A retention trial was conducted 24 hr after the first acquisition. As in the acquisition trials, the mouse was given 30 s to adapt to the environment. After the adaptation period, the light was turned on in the light chamber and the door was opened. Mice were given a maximum of 180 s to enter the dark chamber. If the mouse crossed over into the dark chamber, the door was closed but the mouse did not receive a footshock. The latency to enter the dark chamber was recorded for data analysis. To examine footshock sensitivity, control and lesioned mice were placed individually into the light chamber of the PA apparatus. Each mouse then received increasing levels of footshock (0.05–0.2 mA for 3 s), and responses were observed. Freezing, running, jumping, and vocalizations at the different shock levels were recorded.

Motor activity

Animals were placed in individual activity cages at 4:30 p.m., and activity, as assessed by photobeam breaks, was recorded and expressed as beam break totals per hour.

At 12–17 weeks of age, groups of lesioned and control mice were killed by cervical dislocation after carbon dioxide anesthesia (Berger-Sweeney, Berger, Sharma, & Paul, 1994). Both the right and left hemispheres of the frontoparietal cortex and hippocampus were dissected. Tissues were immediately frozen on dry ice and then stored at −70° C until analysis. The two hemispheres were combined and analyzed by high performance liquid chromatography. The tissue was sonicated in a 1:10 dilution of extraction solution (50 mM perchloric acid). The samples were then centrifuged at 14,000 G for 15 min. Supernatants were then recentrifuged in 0.2-μm filter Eppendorf tubes at 12,000 G for an additional 15 min. Twenty-μl samples were injected by autosampler onto a reverse phase C-18 column and analyzed by electrochemical detection (Bioanalytical Systems, Inc.). The mobile phase consisted of 0.61 ml concentrated phosphoric acid, 10 mg ethylenediamine tetra acetic acid (EDTA; Sigma Chemical, St. Louis, MO), 300 mg octyl sulfate, 9.60 mg sodium phosphate, and 100 ml acetonitrile brought to a final volume of 1L and a pH of 3.1. An internal standard of10−6 M 3,4-dihydroxybenzylamine was used to monitor sample degradation. Data were collected and analyzed with Gilson software (712 HPLC System Controller, Gilson Medical Electronics Middleton WI).

Behavioral and neurochemical data were analyzed by a two-factor, repeated measures analysis of variance (ANOVA). Lesion status and sex were examined as the main effects, and the tasks (SOD and DNMS), acquisition trials (PA), or hours (activity) were examined as the repeated measures. PA retention trials, neurochemical, and probe data were analyzed in two-factor ANOVAs with lesion status and sex as main effects.T tests were used to compare pairwise means between groups. Significance level was set atp < .05 for all analyses.

The measure of errors was not recorded during the SOD of the first group of mice tested. Therefore, there were 6 mice (3 female control, 3 male control) that were excluded from the SOD analysis. Errors were recorded during the DNMS portion of testing for those 6 mice and are included in the DNMS analysis. In addition, 1 mouse did not cross over to the black chamber during PA and was therefore dropped from the analysis.

Simple odor discrimination

Choice accuracy increased significantly across the 5 days of testing for all groups,F(4, 164) = 6.7,p = .001 (seeFigure 2 A). All groups were performing at almost 70% accuracy on the 1st day of testing and were performing with more than 90% accuracy by the 5th day of testing. There were no significant differences overall between the lesioned and control groups or between the sexes nor were there any significant interactions.

Errors for all groups decreased significantly across the 5 days of testing,F(4, 164) = 8.7,p = .001 (seeFigure 2 B). Mean errors ranged from 0.9–0.3 per day on the 1st day of testing and decreased to 0.1 per day by the 5th day of testing. There were no significant differences between the control and lesioned groups or between the sexes, nor were there any significant interactions on this measure.

Latency to retrieve the chocolate also decreased significantly across the 5 days of testing for all groups,F(4, 164) = 11.9,p = .0001 (seeFigure 2 C). Generally, latencies decreased by at least 50% across the 5 days of discrimination testing for all groups. There were no main effects of either lesion or sex. There was, however, a significant interaction of Lesion × Sessions,F(4, 164) = 3.0,p = .02.T tests revealed that the interaction occurred because the control mice were slower at finding the chocolate than the lesioned mice on the 1st day of testing (p = .036), which resulted in a steeper overall latency decrease in the control mice as compared with the lesioned mice.

On the probe trials, all groups performed with 82–100% accuracy, which suggests that all mice were using odor cues to guide SOD performance. There were no significant differences in probe accuracy between the lesioned groups or the sexes.

Delayed nonmatch-to-sample

Overall, the lesioned mice performed the DNMS with higher choice accuracy than did control mice,F(1, 47) = 5.6, p = .02 (seeFigure 3 A). Performance did not vary significantly by sex or across the days of testing, and there were no significant interactions.

The lesioned mice also made significantly fewer errors than did control mice,F(1, 47) = 5.8,p = .02 (seeFigure 3 B). There was also a nearly significant Lesion × Sessions interaction,F(3, 141) = 2.6, p = .056; control mice showed a steady decrease in errors over the 4 days, whereas the lesioned mice made few errors throughout testing. There was no significant main effect of sex or session.

During the choice presentation, control mice had significantly longer latencies to find the piece of chocolate than did DHT-lesioned mice,F(1, 47) = 7.8,p = .007, on the DNMS task (seeFigure 3 C). There were no overall significant effects of sex or days of testing nor were there any significant interactions.

Passive avoidance

Control and lesioned mice of both sexes acquired the PA task similarly; there were no significant differences in acquisition among the groups. The latency to enter the dark chamber was significantly higher on the second acquisition trial for all groups,F(1, 46) = 7.3,p = .009. At the 24-hr retention trial, however, a significant main effect of lesion was apparent. The control mice had significantly longer latencies to enter the dark compartment (better performance) than did the DHT-lesioned mice,F(1, 46) = 26.6,p = .01 (seeFigure 4). There was also a significant main effect of sex,F(1, 46) = 4.7,p = .04; females had longer latencies on the 24-hr retention trial.T tests revealed that the sex difference was mainly due to the poor performance of the lesioned males. Control males were not significantly different than control females, which had near maximum latencies on the retention trial (p = .1276). There were no differences among groups in sensitivity to varying shock intensities, which ranged from 0.05–0.2 mA. All mice exhibited freezing at 0.05 mA, running and jumping at 0.1 mA, and vocalization in response to shock between 0.1 and 0.2 mA.

Motor activity

Locomotor activity in control and lesioned mice was similar; there were no significant differences between the groups. In all groups, activity decreased significantly over the 12 hr of testing,F(11, 517) = 50.6,p = .0001 (seeFigure 5). There was a significant main effect of sex,F(1, 47) = 18.2,p = .0001; females were significantly less active overall than males. There was a highly significant interaction between sex and hour of testing,F(11, 517) = 6.4,p = .0001.T tests showed that males were significantly more active on all but the final 2 hr of activity monitoring (allp s < .03). There was also a significant Lesion Status × Hour of Testing interaction,F(11, 517) = 2.3,p = .009; DHT-lesioned mice were hypoactive as compared with controls in the 1st and 11th hours (p s < .04).

Bilateral neonatal DHT lesions resulted in significant decreases in NE and 5HT levels in both the frontoparietal cortex and hippocampus in lesioned mice relative to controls,F(1, 16) ranged from 22.1 to 74.5, allp s < .01 (seeTable 1). The NE decreases ranged from 23% to 28% in the hippocampus and cortex. The 5HT decreases were more dramatic, ranging from 40% to 45% in the cortex and from 50% to 54% in the hippocampus. There were no significant sex differences in NE or 5HT levels in either the hippocampus or frontoparietal cortex.

The results of this study show that DHT lesions on PND 1 interrupt monoaminergic projections to the cortex and hippocampus and have long-lasting effects on behavior and neurochemistry. Neonatal DHT-lesioned mice performed SOD with similar accuracy, perseverance, and speed as compared with control mice. In contrast, the lesioned mice performed DNMS faster, more accurately, and with less perseverance than controls. This lesion-induced improvement in performance is not attributable to increased locomotor activity or different accuracy on probe (unrewarded) trials. The improved performance in the lesioned mice relative to controls could result from improved mnemonic or attentional abilities or differences in digging strategies. In addition to persistent behavioral alterations, the neonatal DHT lesions lead to significant depletions in cortical and hippocampal NE and 5HT in the adult mice. This study provides one of the first demonstrations of the long-lasting effects of such lesions and provides insights into the developmental role of monoaminergic input to the cortex and hippocampus.

Choice accuracy and errors on SOD were similar for the control and lesioned mice. All mice performed SOD well (more than 70% accuracy) on the 1st day and improved to more than 90% accuracy by the 5th day of the task. Moreover, errors (total number of incorrect digs) decreased over the 5 days of SOD, which suggests that the mice did not continue to persevere in the incorrect cup as testing continued. There were no overall differences between control and lesioned mice on latencies on SOD trials; however, the rate of latency changes over the 5 days did differ between the groups. Control and lesioned mice also had a similar sensitivity to odor cues (J. Berger-Sweeney, unpublished, observations, & May, 1998), performed similarly during unrewarded probe trials, and appeared to have a similar motivation to associate an odor cue with a reward, as shaping was similar in the two groups. The control and lesioned mice appeared to be able to learn a “win–stay” strategy with similar speed and accuracy. In contrast, control and lesioned mice differed significantly in accuracy and perseverance in the performance on the DNMS task; lesioned mice of both sexes significantly outperformed (better choice accuracy and fewer errors) the controls. In addition, the DHT-lesioned mice performed DNMS faster than their control littermates of both sexes. General locomotor activity, however, did not differ between control and lesioned groups, which makes this an unlikely explanation for their performance differences on DNMS. As such, the lesioned mice appeared to adopt the “win–shift” strategy more readily than controls.

The DHT-lesioned mice were significantly impaired in 24-hr retention of PA, although they acquired the PA task similarly to controls and responded to shock stimuli similarly to controls. What could account for the improved performance of the lesioned mice on DNMS and their impaired performance on PA retention trials? One possibility is a response inhibition deficit in the DHT-lesioned mice. Response inhibition refers to interrelated processes that permit a delay in the decision to respond, allow for distraction by competing events, and inhibit a response that is immediately reinforced (Barkley, 1997). The DHT-lesioned mice may have been unable to inhibit the directly reinforced odor response, leading to faster responses; they may have also been unable to inhibit entering the dark chamber in the PA paradigm, leading to shorter latencies. In addition, if short-term working memory in rodents decays over a matter of seconds, as it does in primates (see, e.g., Zola-Morgan & Squire, 1986), then impaired inhibition and faster responding in the DHT-lesioned mice could actually lead to improved choice accuracy of a working memory task, such as the improvements seen in DNMS in the current study. Behavioral inhibition has been used to describe children with ADHD (Barkley, 1997), and likely arises from neural networks in the prefrontal cortex (Fuster, 1995) and perhaps the hippocampus (Olton & Werz, 1978). Because neonatal DHT lesions affect both cortical and hippocampal catecholamine levels, our studies can be considered indirect support for cortical and hippocampal involvement in behavioral inhibition.

Consistent with the results of the current study, performance improvements have been reported after serotonergic lesions in adulthood. In one study, rats (Rattus norvegicus) were required to switch from pressing one lever to pressing another lever at a specific time for reinforcement (Ho et al., 1995). Rats with DHT lesions to the medial septal nucleus (which provides the primary cholinergic projection to the hippocampus) can time the switch of levers more accurately than control rats. Similar to the mice in the current study, DHT-lesioned rats may have been unable to inhibit responses, thus making the transition faster than that of control animals. In another study, adult rats injected with the serotonergic neurotoxin p-chloroamphetamine (PCA) made fewer Stone maze errors than did controls (Altman, Ogren, Berman, & Normile, 1989).

The noradrenergic and serotonergic systems have also been associated with contextual learning and attention. PCA-treated adult rats, for example, respond differently to contextual cues than control rats (Altman et al., 1989). NE has been associated with selective attention to context in adult rats (Sara, 1985), attention in adult humans (Posner & Petersen, 1990), and olfactory learning in infant rats (Sullivan, Wilson, & Leon, 1989). When these data are considered, it is possible that in the present study, the lesioned mice performed the DNMS tasks more accurately than controls because they were less distracted by contextual cues in the odor-testing apparatus and were better able to focus on the task at hand.

Another possible explanation for the improved performance of the lesioned mice on DNMS is an indirect cholinergic-induced improvement in working memory. Serotonergic lesions can reportedly increase acetylcholine turnover in the cortex (Steckler & Sahgal, 1995), which suggests that 5HT may modulate acetylcholine release and enhance mnemonic processes through the cholinergic system (Decker & McGaugh, 1989). Further neurochemical studies should shed light on this potential cholinergic–serotonergic interaction.

In addition to altering behavior, the 5,7-DHT lesions affected cortical and hippocampal neurochemistry, which suggests that MFB fibers projecting to these structures were affected. Our previous results after 5,7-DHT lesions (which used the same lesion protocol) showed that 4 days postlesion, NE and 5HT levels in the cortex were 53% and 75%, respectively, relative to controls (Hohmann, Brooks, & Coyle, 1988). In the present study, there were still significant reductions in NE (26% in males and females combined) and 5HT (43% in males and females combined) levels in the cortex 3 months postlesion, at the time of behavioral testing. In addition, we noted significant reductions in hippocampal NE (25% in males and females combined) and 5HT (52% in males and females combined). Whereas the behavioral deficits noted in the present study could result from chronic monoaminergic depletion beginning at birth or acute depletion at the time of behavioral testing, we believe that the former is a likely explanation of our data. Neonatal 5,7-DHT lesions produce long-lasting alterations in cortical thickness and connectivity that suggest a lesion-induced reorganization of cortical circuits (Hohmann et al., 1997) that could lead to long-lasting alterations in cognitive behaviors such as those seen in the present study.

Sex differences in performance have been reported on a wide variety of behavioral tasks (Beatty, 1979; Berger-Sweeney et al., 1995). Generally, males make fewer errors and need less time to reach criterion. In the present study, sex differences existed in locomotor activity; females were hypoactive relative to males. We have noted previously that female BALB/c mice, unlike many other strains of rats and mice, are hypoactive relative to males (Arters et al., in press). In the present study, although control females were less active than control males, SOD and DNMS errors and choice accuracy were similar. These data strongly suggest that both sexes perform SOD and DNMS with similar precision and accuracy despite differences in motor propensities. Moreover, no sex differences were noted in any of the neurochemical measures in the control or lesioned mice. In total, the data suggest that sex differences are not a key component in this lesion model.

Researchers have shown previously that electrolytic lesions to the nBM on PND1 cause significant deficits in spatial learning and PA retention and alter cortical morphology in adult mice (Bachman et al., 1994). These deficits were attributed to the cholinergic system because of the high percentage of cholinergic neurons in the region and because monoaminergic lesions alone did not produce the same cortical abnormalities (Hohmann et al., 1988). The present study suggests, however, that some of the behavioral deficits after electrolytic lesions may be due to the loss of monoaminergic as well as cholinergic fibers. Interactions between the monoaminergic and cholinergic systems appear to be critical for successful performance of many learning and memory tasks (Decker & McGaugh, 1989).

The present study provides convincing evidence that serotonergic and noradrenergic fibers play an important role in the normal development of cognitive behaviors. Further studies will be necessary to determine whether 5HT, NE, or both are responsible for the observed performance alterations. Developmental monoaminergic lesions likely lead to alterations in the cortex and hippocampus that result in deficits in response inhibition and alterations in integration of contextual cues. Interestingly, poor behavioral inhibition is considered to be a central deficiency in ADHD (Barkley, 1997). Children with ADHD are plagued by inappropriate impulsiveness as well as difficulty in withholding responses and resisting distractions by competing events. It has also been suggested that children with ADHD abnormally focus cortical attention systems on external stimuli (Pliszka, McCracken, & Maas, 1996). These behavioral findings, combined with evidence of altered monoaminergic neurochemistry in children with ADHD (e.g., Hanna, Ornitz, & Hariharan, 1996; Mefford & Potter, 1989), suggest that some of the symptoms of the disorder are due to derailed monoaminergic innervation of cortical or hippocampal structures (or both) at birth.

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Submitted: December 12, 1997 Revised: May 6, 1998 Accepted: June 16, 1998