The present experiment tested the effects of P11–15 treatment with MA at different doses on later learning using two different kinds of tests of learning: the Morris and Cincinnati water mazes under two rearing conditions. The principal findings were that using this shorter exposure period, MA impairs both path integration learning in the CWM and spatial learning in the MWM, however, the effects in the CWM was seen at all doses whereas the effects in the MWM were seen only at the highest dose tested. The results also showed that even a modest environmental enrichment (adding a stainless steel enclosure as partial enrichment) significantly improved initial MWM performance. Interestingly, it had no significant effect on CWM performance. Moreover, the partial enrichment rearing did not interact with the effects of MA at any dose on either test of learning.
The two maze tests we used rely on different learning strategies. The Morris water maze is a well-established test of spatial learning and reference memory in the hidden platform version. Spatial navigation is mediated primarily by the hippocampus (Brandeis et al., 1989
; Burgess, 2002
; D'Hooge and De Deyn, 2001
; McNamara and Skelton, 1993
; Morris et al., 2003
), although other brain regions are also involved. We have previously shown that P11–20 treatment with MA results in MWM spatial learning and reference memory deficits (Skelton et al., 2007
; Vorhees et al., 1994a
; Vorhees et al., 1998
; Vorhees et al., 1999
; Vorhees et al., 2000
; Vorhees et al., 2007
; Williams et al., 2002
; Williams et al., 2003c
; Williams et al., 2004
) indicating that hippocampal-dependent learning and memory are affected by this drug following exposure during this specific developmental window. Spatial navigation depends on the availability of distal cues in order to use an allocentric strategy to find the hidden platform in the MWM efficiently across a series of trials (Morris, 1981
). It has been established that disrupting distal cues in the MWM, disrupts performance, e.g. (Maurer and Derivaz, 2000
). Hence distal cues are essential to this form of learning through the use of cues located outside the swimming tank. Neonatal MA exposure, especially when treatment occurs between P11–20, reliably disrupts this type of learning while leaving learning using proximal cues, in the cued platform version of the maze, intact.
Learning to find the hidden platform in the MWM requires secondary or subordinate skills in addition to allocentric ability. For example, the animal must not only learn to orient to distal cues, it must simultaneously learn that the platform is not located around the perimeter of the maze, nor in the center, but in the region in between. It must also learn that there is a platform and that they must climb upon and remain on the platform in order to escape the water and to ultimately be removed from the maze by the experimenter. Cain and colleagues (Cain et al., 1996
; Cain et al., 2006
; Cain and Saucier, 1996
; Hoh and Cain, 1997
; Saucier et al., 1996
; Saucier and Cain, 1995
) and Morris and colleagues (Bannerman et al., 1995
) have shown that pretraining animals for these subordinate skills prior to exposure to the typical conditions, eliminates some types of drug-induced spatial impairments, suggesting that sensorimotor factors sometimes play a role. In the case of neonatal MA treatment, we tested for just such effects and showed that MA induces both spatial and subordinate skill MWM learning impairments and these can be distinguished from one another (Williams et al., 2002
), such that pretraining eliminated acquisition deficits, but not those seen during reversal training. Since reversal training occurs after subordinate skills are learned, these deficits represent a reliable spatial impairment.
Together with our previous data, the learning effects observed in the present experiment are unlikely to be attributable to sensorimotor deficits for three reasons: (1) no differences were seen on straight channel swimming times. This test requires no learning after the first trial in which animals determine that they need only swim to the opposite end in order to find the escape ladder. Once this is determined, animals may be observed to swim from one end to the other as rapidly as possible. This provides an index of swimming ability and motivation to escape. The data showed no differences among the MA-treated groups compared to saline controls. (2) We assessed swimming speed in the MWM and found no differences among groups in speed on learning or memory trials. (3) If MA-treated animals had significant sensorimotor impairments, one would expect differences on all phases of MWM testing, but in fact differences were selective, with no differences on reversal or shifted-reduced probe trials and no effects below the high dose on learning trials even though these lower dose groups showed impaired learning in the CWM. This pattern argues against any generalized sensorimotor impairment-based explanation for the learning effects obtained.
In the present experiment we focused on a shorter MA exposure period than used previously. We did this based on a prior experiment in which we found that MA treatment on P11–15, but not on P16–20, induced MWM learning deficits (Williams et al., 2003a
). In that experiment, a single dose of MA was tested. We treated neonatal rats with 10 mg/kg x 4 doses/d of MA on P11–15. In the present experiment, we treated rats on these same days with doses of 10, 15, 20, or 25 mg/kg x 4 doses/d. Surprisingly, we found MWM maze deficits only in the high dose group (25 mg/kg), but not in any of the lower dose groups, including the dose at which we had found effects previously. However, there is a significant procedural difference between the two experiments. In an attempt to make the task more sensitive in our previous experiment, we tested the rats in the MWM using the small (5 × 5 cm) platform during acquisition. An examination of the learning curves in that experiment (Williams et al., 2002
), shows that while it succeeded in revealing group differences, the rate of learning was very slow, even among controls, compared to experiments that have used a 10 × 10 cm platform in the same 210 cm diameter tank. Because of the poor learning in that experiment, we decided not to use this procedure in future experiments, but rather to use the smaller platform only after the animals had prior experience (acquisition and/or reversal) with the larger 10 × 10 cm platform. This approach improved the learning rate, yet still succeeded in showing the value of using the small platform to uncover learning differences that were not apparent using only the larger platform, e.g. (Vorhees et al., 2000
; Williams et al., 2003c
). Accordingly, in the present experiment we used this multiphasic MWM test procedure to evaluate animals, i.e., using a larger platform during acquisition and reversal and the smaller platform during shifted platform trials (i.e., with platform in the quadrant adjacent to that used during reversal). We predicted a dose-dependent, stepwise impairment in MWM performance at progressively higher doses. Unexpectedly, only the MA-25 group showed clear deficits, leading us to conclude that the P11–15 exposure period, while sensitive, is not the maximally sensitive period for spatial learning deficits. Rather, the data suggest that a 10-day (P11–20) exposure likely has an additive effect that produces a more pronounced effect such that lower doses, such as 10 mg/kg x 4/d, or even 0.625 mg/kg x 4/d, will induce MWM deficits (Williams et al., 2004
). Alternatively it could be argued that the higher dose group was more comparable to the total drug exposure seen with a 10 day dosing regimen of 10 mg/kg/dose, although no effects in the MWM were observed with the 20 mg/kg/dose group in this experiment. That this requirement for increased days of exposure was not apparent in the experiment comparing P11–15 vs. P16–20 exposure periods we now interpret as being attributable to the use of the small platform during acquisition training in our previous experiment. We suggest that the small platform made the task sufficiently difficult that it may have obscured differences present in the P16–20 MA-treated group, or that the P11–15 exposure period is required for spatial learning deficits, but these deficits are more pronounced when MA is delivered over a longer period of time.
In three previous experiments we tested the effects of P11–20 MA treatment on learning in a 9-unit multiple T-maze (Cincinnati water maze), and each time found trends toward impairment that fell short of significance (Vorhees et al., 1994a
; Vorhees et al., 2000
; Williams et al., 2003c
). At that time, we tested animals under standard house lighting. Subsequently, we changed the procedure to one that eliminated the use of distal cues. We did this because we already knew from the MWM findings that MA-treated neonates had a spatial impairment and wondered if the trend in the CWM might be the result of animals using combined or redundant allocentric and egocentric strategies to solve the maze, thereby showing no significant effect. To test this hypothesis we eliminated access to distal cues by testing in complete darkness and used an infrared camera to score performance. Not surprisingly, under these conditions the task becomes very difficult and the rats learn at a much slower rate. However, given enough trials, they do master the maze and ultimately perform quite well (see ). As the present results show, this change in procedure revealed that MA-treated rats at all doses showed impaired performance. The effects were dramatic and affected all aspects of performance, i.e., the MA-treated animals had longer latencies to reach the escape ladder, made more errors by entering more dead-end cul-de-sacs, and returned to the start more frequently than saline controls.
Path integration is conserved in organisms ranging from ants (Wittlinger et al., 2006
) and rodents, to humans (Etienne and Jeffery, 2004
). It is a form of egocentric learning that relies upon self-movement cues to locate places in an environment based on direction and rate of movement, i.e., a trajectory or vector through space rather than distal cue navigation (Etienne and Jeffery, 2004
). Unlike spatial or allocentric (landmark-based) learning, path integration is dependent on movement cues (primarily internal) rather than objects located at a distance (Etienne, 1992
; Etienne and Jeffery, 2004
). The neural circuits underlying path integration in rats partially overlap with those of spatial navigation inasmuch as some place cells in the hippocampus are activated during path integration, however, path integration depends upon head-direction cells in the presubiculum and grid cells in the entorhinal cortex (Fuhs and Touretzky, 2006
; McNaughton et al., 2006
; Rondi-Reig et al., 2006
; Sargolini et al., 2006
; Whishaw et al., 1997
). Path integration is critically dependent on connections between head direction and grid cells, especially those in layers II and III of the medial entorhinal cortex (Sargolini et al., 2006
). Although it remains to be proven that entorhinal cortex lesions selectively impair CWM learning compared to MWM learning, the fact that rats are severely impaired in the CWM when visual cues are removed but are only marginally affected when they are present, argues in favor of the view that egocentric learning is more sensitive to the effects of neonatal MA-treatment after exposure on P11–15. As such, this represents a potentially useful finding about the long-term effects of developmental MA treatment.
We tested the effects of a partial environmental enrichment provided to half the litters during development and throughout the experiment compared to the other half of the litters that were reared and housed as adults in standard box cages in order to determine whether such partial enrichment alters the effects of neonatal MA treatment. Even in conditions which are more enriched than in the past (i.e., box cages vs. older wire bottom cages, housing animals in pairs vs. single housing, and not weaning litters until P28; all practices designed to improve laboratory housing conditions for rodents), adding a simple item such as a stainless steel enclosure to each cage produced significant main effects on initial spatial learning. In all treatment groups, the animals raised with enrichment performed significantly better on all measures of MWM performance than those raised in standard box cages. Importantly, however, from the perspective of interest in the effects of MA, drug treatment did not interact significantly with rearing partial enrichment hence, alleviating concern over whether this change in housing practice might alter the basic effects of MA on brain development and behavior. Interestingly, however, this same enrichment did not produce a main effect (or interaction) on performance in the CWM. This suggests that this task is not sensitive to the added effects of placing an enclosure into box cages throughout the animals’ lives and continuing when they are pair housed after P28. This is not to suggest that more elaborate enrichment if compared to single, wire-bottom, P21 weaned rats might not reveal enrichment effects on CWM performance, but rather the incremental effect of the enclosure did not result in a significant effect on this task.
The mechanism(s) underlying the developmental effects of MA on learning are not known. P11–20 MA treatment causes increased release of corticosterone (Williams et al., 2000
), showing a U-shaped response function in which corticosterone release is larger 30 and 105 min after MA treatment on P1 or P3 than on P5, 7, 9, 11, or 13, and was higher again on P15, 17 and 19 (Williams et al., 2006
). This resembles what is termed the stress hyporesponsive period (SHRP) (Sapolsky and Meaney, 1986
), which is described as beginning shortly after birth and extending to P14. When the drug is given longer, for 4 days with one additional dose on the fifth day, 30 min corticosterone release was no longer U-shaped but progressively increasing with age, and 105 min levels were lower on all days than after the dose measured at 30 min. Moreover, these corticosterone effects remain evident 18 h after the final dose (Schaefer et al., 2006
; Schaefer et al., 2007
). We are currently testing the role of MA-induced corticosterone release on later learning.
Rats treated with 10 mg/kg x 4/day of MA on P11–20 and assessed at P90, also show 10–15% reductions in neostriatal 3
H-spiperone binding to D2-like receptors with no changes in affinity. They also show 20% reductions in neostriatal PKA activity, and 15% reductions in neostriatal dopamine and DOPAC concentrations (Crawford et al., 2003
). Rats given MA on P11, P11–15, or P11–20 and examined 24 h later, show no differences in dopamine levels (Schaefer et al., 2006
; Schaefer et al., 2007
), indicating that the long-term changes in dopamine in adulthood are not caused by neonatal reductions during treatment. As with the effects on corticosterone, it is not yet know if any of these neurotransmitter, receptor, or enzymes mediate the learning effects and future studies will be require to determine the relationship among these changes.