Behavioral Neuroscience 1992, Vol. 106, No. 6, 924-932
Medial Prefrontal Cortex Lesions and Spatial Delayed Alternation in the Developing Rat: Recovery or Sparing? John H. Freeman, Jr.
Mark E. Stanton
University of North Carolina at Chapel Hill
U.S. Environmental Protection Agency and University of North Carolina at Chapel Hill
In Experiment 1, Long-Evans rat pups received medial prefrontal cortex (PFC) aspirations or sham surgery on Postnatal Day 10 (PND10) and were then trained on PND23 to perform one of two T-maze tasks: discrete-trials delayed alternation (DA) or simple position discrimination. Early PFC damage produced a selective failure to learn the DA task. In Experiment 2, pups given the same lesion or sham surgery were trained on DA on PND19, PND27, or PND33. In relation to sham-operated controls, pups with PFC damage were impaired on PND19, somewhat impaired on PND27, and entirely unimpaired when tested on PND33. In Experiment 3, pups given larger lesions of the frontal cortex on PND10 were impaired on DA when tested on PND23 but not when tested on PND33. These findings indicate that early PFC lesions result in a memory deficit around the time of weaning, which then recovers over the next 10-14 days of development. Moreover, the early deficit is selective for a late developing cognitive process (or processes) that is involved in acquisition of DA.
Spatial delayed alternation may prove to be a useful rodent model for studying the neural basis of cognitive development (Freeman & Stanton, 1991; Green & Stanton, 1989; Stanton, 1992). Delayed alternation develops around the time of weaning in the rat (Green & Stanton, 1989) and can be contrasted with simple position discrimination (PD) learning, which appears much earlier (Green & Stanton, 1989; Kenny & Blass, 1977). In addition, early damage to the septohippocampal pathway disrupts the ontogeny of delayed alternation but does not disrupt position discrimination (Freeman & Stanton, 1991). These findings, together with those from recent studies of memory development in monkeys (Bachevalier, 1990; Bachevalier & Mishkin, 1984) and humans (Overman, 1990), suggest that there are (at least) two memory systems: one that develops early and another that develops later ("limbic" and "nonlimbic," respectively; see Bachevalier & Mishkin, 1984; Nadel & Zola-Morgan, 1984). The purpose of the present study was to examine the possible role of another late-developing brain region in the ontogeny of delayed alternation: the prefrontal cortex (PFC).
This cortical region is involved in delayed alternation in adult animals (for reviews see Foster, 1989; Kolb, 1984) and, like the hippocampal formation, shows postnatal maturation in the rat that continues into the weanling period of development. In fact, the medial PFC reaches its maximum volume on Postnatal Day 24 (PND24; Van Eden & Uylings, 1985), shortly after the age at which rat pups begin to acquire delayed alternation (Green & Stanton, 1989). In the present study, we examined the effects of early (PND10) lesions of the PFC on the ontogeny of delayed alternation in the rat. Studies of the effects of early PFC damage on learning and memory have been performed in both rodents (e.g., Kolb & Nonneman, 1978) and monkeys (e.g., Goldman-Rakic, Isseroff, Schwartz, & Bugbee, 1983). In the rodent, these studies have used a design in which lesions are performed early in infancy and memory testing occurs in adulthood. This design has established the basic principles of recovery or sparing after early cortical damage (for a review see Kolb & Whishaw, 1989). However, an obvious drawback of this design is the failure to test learning at earlier stages of development. In the context of the current literature, we feel that this is an important avenue to pursue for several reasons. First, it is difficult to differentiate between sparing and recovery of function in late-developing behaviors when animals that are given early lesions are tested as adults but not as infants. Early lesions could result in either no deficit or an early deficit that later recovers. The former case we will refer to as sparing, and the later case we will refer to as recovery (see also Kolb & Nonneman, 1978). The second reason is that any process of recovery that occurs after early brain damage may depend on a critical period of neural development. Van Eden and Uylings (1985) noted that there is a period of plasticity in the rodent PFC that occurs roughly from PND6 to PND30. It may be that recovery of function depends on some form of anatomical compensation that can only occur during this period of plasticity. If this is the case, then most of the recovery
Disclaimer: This article has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. We thank Craig Barry, Mary Coussons, Julia Davis, Beth Gregg, Chris Murchison, Bryant Murphy, Clark Spencer, and Dimitrios Tsoumbos for assistance in various phases of this research. We also thank Baker Bailey, Paul Killough, and Allen Lee for the construction, instrumentation, and software programming of the automated T-maze apparatus. Finally, we thank Bryan Kolb, Art Nonneman, and Rob Sutherland for their constructive criticism of an earlier draft of this article. Correspondence concerning this article should be addressed to Mark E. Stanton, Health Effects Research Laboratory (MD-74B), U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711.
924
PREFRONTAL CORTEX AND ONTOGENY OF MEMORY previously seen in animals that were tested as adults would have occurred during early development. The third reason is that there may be species differences in the effects of early PFC damage on subsequent memory development (Nonneman, Corwin, Sahley, & Vicedomini, 1984). Studies of such damage in nonhuman primates have shown that early lesions result in sparing of function in infants that later becomes a deficit in the adult (Goldman-Rakic et al., 1983). On the other hand, Diamond and Goldman-Rakic (1986) found that infant monkeys given dorsolateral PFC lesions were impaired on AB ("A not B") and delayed response (DR) when tested in early infancy. Thus, on the basis of available data, it is unclear whether infant rats would show sparing or an impairment of memory after early PFC lesions. The present study consisted of three experiments on the effects of early PFC lesions on the development of delayed alternation in the infant rat. In the first experiment, we determined the effects of medial PFC aspiration lesions, performed on PND10, on acquisition of discrete-trials delayed alternation or PD on PND23. We found that these infant lesions produced a selective impairment of delayed alternation. In the second experiment, we reexamined the effects of these medial PFC lesions on delayed alternation learning in rats that were 19, 27, or 33 days of age. There was again an impairment of performance at the earliest age that was no longer evident by PND33. In the third experiment, we asked whether larger lesions of the frontal cortex (FC) would prevent this apparent early recovery of function. Rat pups that received such damage on PND10 showed impaired delayed alternation learning on PND23 but not on PND33.
Experiment 1 Method Subjects. Subjects were 59 Long-Evans rat pups (29 males and 30 females; Charles River Labs., Raleigh, NC) that were derived from 12 litters. Surgery was performed when pups were 10 days old (PND10), and initial experimental procedures (deprivation) began on PND21. On PND21, pups that received PFC lesions weighed a mean of 50.0 g (range = 41.1-57.9 g), those that received control lesions (CTL) weighed a mean of 52.1 g (range = 45.3-59.5 g), and pups in the sham-operated group weighed a mean of 54.3 g (range = 46.2-64.9 g). Littermates were assigned to the different surgery conditions. Between surgery and testing, pups were housed with their dams (8 pups/litter) in 45 x 24 x 21 cm cages. The housing facility was maintained on a 12:12-hr light-dark cycle, with lights on at 7:00 a.m. The day of birth was designated as PNDO. Litters were culled to 8 pups (4 males and 4 females) on PND3. Surgery. The surgical procedure was similar to that used by Freeman and Stanton (1991). On PND10, pups were removed from their home cage and placed in a heated incubator. They were then removed from the incubator and anesthetized by inhalation of CO2Each pup's head was then antiseptically cleaned with a gauze pad that had been soaked with 100% ethanol, and a midline incision was made. A scalpel blade was used to remove a flap of skull (~3 x 3 mm) overlying the medial PFC. The medial PFC anterior to the caudate nucleus was removed by aspiration. The wound was packed with Gelfoam (Upjohn Co., Kalamazoo, MI), and the skull flap was returned to its appropriate position. The incision was sutured and bathed in a local anesthetic (0.5% Kainair [proparacaine] solution, Pharmphair, Inc., Hauppauge, NY), and pups were returned to the
925
incubator. Sham-operated animals received identical treatment except that the skull flap was not removed and no cortical tissue was aspirated. The CTL group consisted of rats that received lesions of the PFC that did not encroach on the medial area. Pups were then allowed 1 hr to recover before being returned to their dam. Two subjects died after surgery (6% of lesioned animals). Histology. Within 2 days after training (typically the next morning), 5 subjects per squad (4 lesion and 1 sham) were anesthetized with a lethal dose of Nembutal (sodium pentobarbital) and perfused intracardially with a saline flush, which was followed by a 10% phosphatebuffered formalin solution. Schematic drawings of the dorsal surface of the whole brain were made before brains were sliced. Brains were then embedded in paraffin, cut coronally in 40-u.m sections, and stained with cresyl violet. Lesions were characterized using a standard stereotaxic atlas (Paxinos & Watson, 1982). Apparatus. The apparatus was the same one used by Freeman & Stanton (1991), which can be consulted for a detailed description. Briefly, two identical T mazes were used to train subjects. They were enclosed Plexiglas mazes, scaled to the size of preweanling rats, with a start box and goal arms of equal dimensions and a choice point that connected the start box with the two goal arms. All Plexiglas walls were covered on their external surface with opaque brown paper. Computercontrolled guillotine doors separated the start box from the choice point and the choice point from the goal arms. A small metal cup was attached to the end of each goal arm. A photoelectric beam was directed across each maze arm 2 cm from the feeding cup. On rewarded trials, breaking the beam resulted in an infusion of light cream into the cup via computer-controlled syringe pumps. Between trials, animals were placed in intertrial interval (ITI) compartments, which were made of clear Plexiglas. Design and procedure. The design and procedure were the same as those used by Freeman and Stanton (1991, Experiment 1), which can be consulted for a detailed description. Briefly, all subjects received five 12-trial blocks of training on PND23. Pups were trained on one of two tasks: discrete-trials delayed alternation or PD. The two tasks involved somewhat different experimental designs. In the delayed alternation design, animals in both surgery conditions were assigned to either a contingently rewarded group (shift) or to a noncontingently rewarded control group (shift-noncontingent, SNC). This latter group was included to assess the effects of brain damage (or age) on baseline alternation rates (spontaneous alternation). Rewarding animals in the SNC group regardless of choice, but scoring their behavior as if an alternation contingency were in effect, provides a measure of spontaneous alternation (see Green & Stanton, 1989). This yielded a 2 (sham vs. PFC) x 2 (shift vs. SNC) x 5 (trial block) design. CTL pups (added in a follow-up experiment) were trained only on the shift task. This lesion group was compared with the others in a separate 3 (sham vs. CTL vs. PFC) x 5 (trial block) design. The PD design simply compared the performance of pups in both surgery groups, which yielded a 2 (sham vs. PFC) x 5 (trial block) design. For animals trained in the shift and SNC conditions, trials consisted of a pair of runs that were separated by a delay of 2 s. The first part of the trial was a forced run, in which the animal was presented with one of the maze arms and rewarded for entering it. The second part of the trial was a choice run, in which the animal was presented with a choice of the two arms. Animals in the shift group were rewarded only for entering the maze arm that they did not enter on the forced run of that trial. Animals in the SNC group were rewarded regardless of which arm they entered on the choice run. PD training consisted of only choice run trials, with one arm being correct on all trials. Half of the animals were rewarded for entering the left arm, whereas the other half were rewarded for entering the right arm. Pups were randomly assigned to these conditions as a control for maze arm preferences. Deprivation of food, water, and maternal and social contact was
926
JOHN H. FREEMAN, JR. AND MARK E. STANTON
Results and Discussion 100
O SHAM •
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PFC
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Medial Prefrontal Cortex Lesions and Spatial Delayed Alternation in the Developing Rat: Recovery or Sparing? John H. Freeman, Jr.
Mark E. Stanton
University of North Carolina at Chapel Hill
U.S. Environmental Protection Agency and University of North Carolina at Chapel Hill
In Experiment 1, Long-Evans rat pups received medial prefrontal cortex (PFC) aspirations or sham surgery on Postnatal Day 10 (PND10) and were then trained on PND23 to perform one of two T-maze tasks: discrete-trials delayed alternation (DA) or simple position discrimination. Early PFC damage produced a selective failure to learn the DA task. In Experiment 2, pups given the same lesion or sham surgery were trained on DA on PND19, PND27, or PND33. In relation to sham-operated controls, pups with PFC damage were impaired on PND19, somewhat impaired on PND27, and entirely unimpaired when tested on PND33. In Experiment 3, pups given larger lesions of the frontal cortex on PND10 were impaired on DA when tested on PND23 but not when tested on PND33. These findings indicate that early PFC lesions result in a memory deficit around the time of weaning, which then recovers over the next 10-14 days of development. Moreover, the early deficit is selective for a late developing cognitive process (or processes) that is involved in acquisition of DA.
Spatial delayed alternation may prove to be a useful rodent model for studying the neural basis of cognitive development (Freeman & Stanton, 1991; Green & Stanton, 1989; Stanton, 1992). Delayed alternation develops around the time of weaning in the rat (Green & Stanton, 1989) and can be contrasted with simple position discrimination (PD) learning, which appears much earlier (Green & Stanton, 1989; Kenny & Blass, 1977). In addition, early damage to the septohippocampal pathway disrupts the ontogeny of delayed alternation but does not disrupt position discrimination (Freeman & Stanton, 1991). These findings, together with those from recent studies of memory development in monkeys (Bachevalier, 1990; Bachevalier & Mishkin, 1984) and humans (Overman, 1990), suggest that there are (at least) two memory systems: one that develops early and another that develops later ("limbic" and "nonlimbic," respectively; see Bachevalier & Mishkin, 1984; Nadel & Zola-Morgan, 1984). The purpose of the present study was to examine the possible role of another late-developing brain region in the ontogeny of delayed alternation: the prefrontal cortex (PFC).
This cortical region is involved in delayed alternation in adult animals (for reviews see Foster, 1989; Kolb, 1984) and, like the hippocampal formation, shows postnatal maturation in the rat that continues into the weanling period of development. In fact, the medial PFC reaches its maximum volume on Postnatal Day 24 (PND24; Van Eden & Uylings, 1985), shortly after the age at which rat pups begin to acquire delayed alternation (Green & Stanton, 1989). In the present study, we examined the effects of early (PND10) lesions of the PFC on the ontogeny of delayed alternation in the rat. Studies of the effects of early PFC damage on learning and memory have been performed in both rodents (e.g., Kolb & Nonneman, 1978) and monkeys (e.g., Goldman-Rakic, Isseroff, Schwartz, & Bugbee, 1983). In the rodent, these studies have used a design in which lesions are performed early in infancy and memory testing occurs in adulthood. This design has established the basic principles of recovery or sparing after early cortical damage (for a review see Kolb & Whishaw, 1989). However, an obvious drawback of this design is the failure to test learning at earlier stages of development. In the context of the current literature, we feel that this is an important avenue to pursue for several reasons. First, it is difficult to differentiate between sparing and recovery of function in late-developing behaviors when animals that are given early lesions are tested as adults but not as infants. Early lesions could result in either no deficit or an early deficit that later recovers. The former case we will refer to as sparing, and the later case we will refer to as recovery (see also Kolb & Nonneman, 1978). The second reason is that any process of recovery that occurs after early brain damage may depend on a critical period of neural development. Van Eden and Uylings (1985) noted that there is a period of plasticity in the rodent PFC that occurs roughly from PND6 to PND30. It may be that recovery of function depends on some form of anatomical compensation that can only occur during this period of plasticity. If this is the case, then most of the recovery
Disclaimer: This article has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. We thank Craig Barry, Mary Coussons, Julia Davis, Beth Gregg, Chris Murchison, Bryant Murphy, Clark Spencer, and Dimitrios Tsoumbos for assistance in various phases of this research. We also thank Baker Bailey, Paul Killough, and Allen Lee for the construction, instrumentation, and software programming of the automated T-maze apparatus. Finally, we thank Bryan Kolb, Art Nonneman, and Rob Sutherland for their constructive criticism of an earlier draft of this article. Correspondence concerning this article should be addressed to Mark E. Stanton, Health Effects Research Laboratory (MD-74B), U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711.
924
PREFRONTAL CORTEX AND ONTOGENY OF MEMORY previously seen in animals that were tested as adults would have occurred during early development. The third reason is that there may be species differences in the effects of early PFC damage on subsequent memory development (Nonneman, Corwin, Sahley, & Vicedomini, 1984). Studies of such damage in nonhuman primates have shown that early lesions result in sparing of function in infants that later becomes a deficit in the adult (Goldman-Rakic et al., 1983). On the other hand, Diamond and Goldman-Rakic (1986) found that infant monkeys given dorsolateral PFC lesions were impaired on AB ("A not B") and delayed response (DR) when tested in early infancy. Thus, on the basis of available data, it is unclear whether infant rats would show sparing or an impairment of memory after early PFC lesions. The present study consisted of three experiments on the effects of early PFC lesions on the development of delayed alternation in the infant rat. In the first experiment, we determined the effects of medial PFC aspiration lesions, performed on PND10, on acquisition of discrete-trials delayed alternation or PD on PND23. We found that these infant lesions produced a selective impairment of delayed alternation. In the second experiment, we reexamined the effects of these medial PFC lesions on delayed alternation learning in rats that were 19, 27, or 33 days of age. There was again an impairment of performance at the earliest age that was no longer evident by PND33. In the third experiment, we asked whether larger lesions of the frontal cortex (FC) would prevent this apparent early recovery of function. Rat pups that received such damage on PND10 showed impaired delayed alternation learning on PND23 but not on PND33.
Experiment 1 Method Subjects. Subjects were 59 Long-Evans rat pups (29 males and 30 females; Charles River Labs., Raleigh, NC) that were derived from 12 litters. Surgery was performed when pups were 10 days old (PND10), and initial experimental procedures (deprivation) began on PND21. On PND21, pups that received PFC lesions weighed a mean of 50.0 g (range = 41.1-57.9 g), those that received control lesions (CTL) weighed a mean of 52.1 g (range = 45.3-59.5 g), and pups in the sham-operated group weighed a mean of 54.3 g (range = 46.2-64.9 g). Littermates were assigned to the different surgery conditions. Between surgery and testing, pups were housed with their dams (8 pups/litter) in 45 x 24 x 21 cm cages. The housing facility was maintained on a 12:12-hr light-dark cycle, with lights on at 7:00 a.m. The day of birth was designated as PNDO. Litters were culled to 8 pups (4 males and 4 females) on PND3. Surgery. The surgical procedure was similar to that used by Freeman and Stanton (1991). On PND10, pups were removed from their home cage and placed in a heated incubator. They were then removed from the incubator and anesthetized by inhalation of CO2Each pup's head was then antiseptically cleaned with a gauze pad that had been soaked with 100% ethanol, and a midline incision was made. A scalpel blade was used to remove a flap of skull (~3 x 3 mm) overlying the medial PFC. The medial PFC anterior to the caudate nucleus was removed by aspiration. The wound was packed with Gelfoam (Upjohn Co., Kalamazoo, MI), and the skull flap was returned to its appropriate position. The incision was sutured and bathed in a local anesthetic (0.5% Kainair [proparacaine] solution, Pharmphair, Inc., Hauppauge, NY), and pups were returned to the
925
incubator. Sham-operated animals received identical treatment except that the skull flap was not removed and no cortical tissue was aspirated. The CTL group consisted of rats that received lesions of the PFC that did not encroach on the medial area. Pups were then allowed 1 hr to recover before being returned to their dam. Two subjects died after surgery (6% of lesioned animals). Histology. Within 2 days after training (typically the next morning), 5 subjects per squad (4 lesion and 1 sham) were anesthetized with a lethal dose of Nembutal (sodium pentobarbital) and perfused intracardially with a saline flush, which was followed by a 10% phosphatebuffered formalin solution. Schematic drawings of the dorsal surface of the whole brain were made before brains were sliced. Brains were then embedded in paraffin, cut coronally in 40-u.m sections, and stained with cresyl violet. Lesions were characterized using a standard stereotaxic atlas (Paxinos & Watson, 1982). Apparatus. The apparatus was the same one used by Freeman & Stanton (1991), which can be consulted for a detailed description. Briefly, two identical T mazes were used to train subjects. They were enclosed Plexiglas mazes, scaled to the size of preweanling rats, with a start box and goal arms of equal dimensions and a choice point that connected the start box with the two goal arms. All Plexiglas walls were covered on their external surface with opaque brown paper. Computercontrolled guillotine doors separated the start box from the choice point and the choice point from the goal arms. A small metal cup was attached to the end of each goal arm. A photoelectric beam was directed across each maze arm 2 cm from the feeding cup. On rewarded trials, breaking the beam resulted in an infusion of light cream into the cup via computer-controlled syringe pumps. Between trials, animals were placed in intertrial interval (ITI) compartments, which were made of clear Plexiglas. Design and procedure. The design and procedure were the same as those used by Freeman and Stanton (1991, Experiment 1), which can be consulted for a detailed description. Briefly, all subjects received five 12-trial blocks of training on PND23. Pups were trained on one of two tasks: discrete-trials delayed alternation or PD. The two tasks involved somewhat different experimental designs. In the delayed alternation design, animals in both surgery conditions were assigned to either a contingently rewarded group (shift) or to a noncontingently rewarded control group (shift-noncontingent, SNC). This latter group was included to assess the effects of brain damage (or age) on baseline alternation rates (spontaneous alternation). Rewarding animals in the SNC group regardless of choice, but scoring their behavior as if an alternation contingency were in effect, provides a measure of spontaneous alternation (see Green & Stanton, 1989). This yielded a 2 (sham vs. PFC) x 2 (shift vs. SNC) x 5 (trial block) design. CTL pups (added in a follow-up experiment) were trained only on the shift task. This lesion group was compared with the others in a separate 3 (sham vs. CTL vs. PFC) x 5 (trial block) design. The PD design simply compared the performance of pups in both surgery groups, which yielded a 2 (sham vs. PFC) x 5 (trial block) design. For animals trained in the shift and SNC conditions, trials consisted of a pair of runs that were separated by a delay of 2 s. The first part of the trial was a forced run, in which the animal was presented with one of the maze arms and rewarded for entering it. The second part of the trial was a choice run, in which the animal was presented with a choice of the two arms. Animals in the shift group were rewarded only for entering the maze arm that they did not enter on the forced run of that trial. Animals in the SNC group were rewarded regardless of which arm they entered on the choice run. PD training consisted of only choice run trials, with one arm being correct on all trials. Half of the animals were rewarded for entering the left arm, whereas the other half were rewarded for entering the right arm. Pups were randomly assigned to these conditions as a control for maze arm preferences. Deprivation of food, water, and maternal and social contact was
926
JOHN H. FREEMAN, JR. AND MARK E. STANTON
Results and Discussion 100
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