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ACTH 4–9 analog can retard spatial alternation learning in brain damaged and normal rats
BEHAVIORAL AND NEURAL BIOLOGY
52, 271-278 (1989)
BRIEF REPORTS ACTH 4-9 Analog Can Retard Spatial Alternation Learning in Brain Damaged and Normal Rats W. F. MCDANIEL, E. J. DAVALL, AND P. E. WALKER 1 Department of Pxychology, Georgia College, Milledgeville, Georgia 31061 Thirty adult male hooded rats (Long-Evans strain) were assigned randomly to one of three lesion groups (n = 10) and prepared with medial frontal, posterior parietal, or sham neocortical injuries. Following a recovery interval of 10-12 days, access to water was limited to 30 min per day and the rats were shaped to traverse a T-maze for a reward of sweetened water. After a pretraining criterion was attained, osmotic minipumps (Alzet 2002) were installed subcutaneously. The minipumps delivered chronically for the next 14 to 15 days either 0 or 1.2 /xg of ACTH 4-9 dissolved in bacteriostatic saline per day while the rats were trained on a reinforced spatial alternation task. Analysis of the number of errors made to a criterion of at least 80% correct alternations in two consecutive training sessions, or a ceiling of 62 errors (attained by two rats with parietal lesions), revealed that learning was impaired in the rats with parietal injuries. Contrary to our hypothesis, animals receiving ACTH 4-9 committed more errors than their counterparts receiving only saline. © 1989AcademicPress, Inc.
Administration of the ACTH-like peptides can facilitate neural and functional recovery following nervous system injury. For example, Bijlsma, Jennekens, Schotman, and Gispen (1983) have shown that ACTH 4-10, a fragment that fails to exert an endocrine effect upon the adrenal cortex, accelerates recovery of sensorimotor functions following sciatic nerve crush in rats. This functional amelioration may be due to the peptide's neurotrophic properties which result in increases in fiber density and regeneration rates in damaged axons (Girlanda et al., 1988). Postsurgical administration of ACTH fragments that fail to evoke an adrenal endocrine response can also improve some behavioral functions following central nervous system injury. Isaacson and Poplawsky (1983) reported that administering 1/zg of an analog of the ACTH 4-9 fragment This research was made possible by a Georgia College Faculty Research Grant awarded to W. F. McDaniel. We thank Mr. Tim Vacula for expert assistance with the photography. Address correspondence and reprint requests to W. F. McDaniet. 271 0163-1047/89 $3.00 Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
272
MCDANIEL, DAVALL, AND WALKER
for 4 consecutive days immediately following bilateral septal lesions reduced the amount of hyperemotionality and hastened the rate of declining hyperemotionality seen with repeated testing. A subsequent study with 1 and 10 ~g of the 4-10 fragment failed to mimic the previous beneficial results (Isaacson & Poplawsky, 1985). This is probably due to the greater metabolic stability (Verhoef & Witter, 1976), and hence potency (Fekete & DeWied, 1982), of the 4-9 analog. Veldhuis, Nyakas, and DeWied (1985) demonstrated that chronic administration (eight injections total) of either ACTH 4-9 (10 or 50 t~g sc, beginning the third day after surgery) or o~-MSH (same doses) attenuated or reversed a cognitive impairment associated with bilateral parafascicular thalamic nucleus injury. T-maze position reversal learning was significantly improved by the 10-~g dose of either neuropeptide, but still inferior to the sham-operated controls; however, 50-t~g doses resulted in acquisition rates that were parallel to those observed in normal rats. The peptides did not significantly influence the learning rates observed in noninjured rats, and motor dysfunctions that result from a parafascicular nucleus lesion were not altered. In another experiment, acute administration of the peptides 1 h prior to daily training sessions failed to improve learning in the brain-injured rats. The main purpose of this experiment was to test the possibility that chronic exposure to 1 t~g of ACTH 4-9 analog per day, simultaneous with training, might attenuate learning impairments in a cognitive task that are associated with neocortical injuries. Removal of the medial frontal (MF; Kolb, 1984) and posterior parietal neocortices (PP; McDaniel & Wall, 1988) results in a constellation of behavioral deficiencies in instrumental tasks with spatial demands. MF lesions have been observed to retard the learning of a reinforced spatial alternation strategy, but apparently the effects of PP lesions upon this task have not been assessed. Thirty adult male hooded rats bred in this laboratory were handled 5 min daily for 5 days prior to being assigned randomly to one of the three lesion groups. The animals had been entrained to a reversed dark-light schedule since birth (off, 8:00 AM; on, 9:00 PM), and they had been housed individually in standard stainless-steel cages since approximately 50 days of age. After being anesthetized with 55 mg/kg Nembutal, the scalp was shaved and scrubbed with a 10% (USP) Bovidone-iodine solution. All neurosurgery was performed with the animal mounted in a Kopf stereotaxic instrument and sterile instruments were used. Rats designated for sham operations received only a scalp incision. For lesion animals, the cranium overlying the MF and PP cortices was drilled with a 2.5mm trephine and enlarged with rongeurs to approximate the shape of the underlying cortical sector. The dura was incised, and the underlying neocortex was aspirated through a pipet using a dissecting microscope. The incision was closed with sterile wound clips and Mycitracin triple
ACTH 4 - 9 AND RECOVERY
273
antibiotic was applied to the incision. The rats were handled daily during a 10- to 12-day postoperative r e c o v e r y period. Access to water was restricted to a 30-rain period per day prior to shaping the rats to traverse a T-maze for a reward of water sweetened with table sugar (3.0%). The apparatus was constructed from 1-cm-thick plywood. The stem measured 15 cm wide by 30 cm long, the choice area 15 cm by 15 cm, and each goal arm measured 15 cm wide by 29 cm long. Initially the animals were allowed to explore the maze for 15 min and discover water in each goal arm. Then they were trained o v e r a sequence of days to traverse the maze for a reward of 5 s access to sweetened water which was delivered through standard water bottles available in each arm. When an animal selected a particular arm for 5 consecutive trials, the arm was blocked by a guillotine door on the next trial. Spatial alternation training began after a rat completed 10 runs for 5 days. Approximately 24 days elapsed between surgery and spatial alternation training. Upon reaching the pretraining criterion, all animals were anesthetized lightly with chloroform. Their backs were shaved posterior to the neck and scrubbed with iodine. A 1.5- to 2.0-cm lateral incision was made and an osmotic minipump (Alzet Model 2002) inserted subcutaneously. The incision was closed with sterile wound clips and Mycitracin antibiotic ointment was applied to the wound. The 200-/zl pump reservoir was filled with either bacteriostatic saline or A C T H 4-9 (Sigma Chemical Co.). The pump has a nominal life for constant drug delivery of 14 days, infuses its contents at a rate o f 0.5/~l/h, and exhausts its contents after 16 days. The dosage of 1.0 to 1.2 /zg of A C T H 4-9 per day was attained by dissolving 5 mg of the peptide in 60 ml bacteriostatic saline. One day after installing the osmotic pumps, training of the spatial alternation strategy began. One rat from each of the factorial groups was trained one session of 16 trials per day. All remaining animals were trained two sessions of 11 trials each per day, one in the morning and one in the late afternoon. This change in training strategies was necessary to maximize the training effort while A C T H 4 - 9 levels were maximal. Since the dependent variable was errors to criterion, and since equivalent number of animals from each group experienced the two training techniques, it was deemed appropriate to pool the behavioral data for purposes of statistical analysis. Training occurred 7 days per week until a criterion of two successive sessions of 80% correct alternations was attained or to a ceiling of 62 errors. The initial arm selection was determined randomly and forced by blocking access to the other arm. On subsequent trials, clear Plexiglas barriers blocked access to both water bottles. Selection of the correct arm led to removal of the barrier and 5 s access to water. U p o n making an error, that is entering an incorrect alley with all four feet, the barrier to water was not removed and the
274
MCDANIEL, DAVALL, AND WALKER
rat was returned immediately to the starting point for the next trial. On each succeeding trial, the animal was rewarded only for alternating it's choice by selecting the side opposite the last rewarded site. The intertrial interval was approximately 5 s. At the completion of training, the animals were sacrificed with an overdose of Nembutal and perfused through the heart first with 0.85% saline and then with 10% formalin. The brains were removed, soaked in formalin, and then soaked in a 30% sucrose10% formalin solution before being photographed from the dorsal perspective. Brains were blocked to include the lesion and thalamus and sliced through the coronal plane into 40-/xm sections. Every 10th section was mounted on a slide and stained with cresyl violet. An analysis of variance (ANOVA; two lesions x two drug conditions) was performed on the proportion of the neocortex visible from the dorsal view that was removed by the ablation. These determinations were estimated using the dot-grid method (Thomas & Peacock, 1965). The ANOVA confirmed that the PP injuries (M = 0.27, SD = 0.05) were larger than the MF injuries (M = 0.15, SD = 0.03), F(1, 16) = 43.91, p < .001. No other differences approached significance. Microscopic examination of the brains (see Fig. 1) showed that the MF lesions extended from the frontal pole to the genu of the corpus callosum, usually sparing the tissue along the medial walls and the anterior cingulate cortex. No retrograde necrosis was observed in the thalamus following this lesion. PP lesion included the tissue rostral to visual cortex and extended rostrally into area 3. The underlying corpus callosum was damaged in most preparations, and moderate to heavy gliosis was observed in the lateral, posterior, and ventral thalamic nuclei. One rat with a MF lesion stopped running when the alternation training began and despite much effort completed only a total of 75 trials. Histological evaluation revealed that this animal had the largest and the deepest MF lesion. On the basis of these observations, this animal's data were deleted from the study. An unweighted means ANOVA for a completely randomized factorial design was performed on the errors to criterion or ceiling (62 errors; obtained by two rats with PP lesions), and Fig. 2 summarizes these results. The difference between lesion groups was significant, F(2, 23) = 4.59, p = .02. Fisher's LSD test revealed that the rats with PP injuries committed more errors than the shamoperated rats. Rats with MF lesions were intermediate between these two extremes and not significantly different from either group. The ANOVA also revealed a significant main effect for the peptide condition, F(1, 23) = 5.77, p = .02. This result demonstrates, as is shown by Fig. 2, that chronic administration of ACTH 4-9 (totaling 1.0 /zg per day) impaired learning relative to saline. The nonsignificant interaction of lesion by dose, F(2, 23) = 0.27, p > > .05, demonstrates that this effect was consistent across brain lesion and sham-operated groups. Correlation
275
ACTH 4-9 AND RECOVERY MF
PP
5.6 0.0
4,0 1.6
IN
2.4
}~:i
30
FIG. 1. (A) Photographs from the dorsal perspective depicting location of the lesions in representative cases. (B) Photographs of three cornonal sections through the brain shown in A. Numerals indicate millimeter anterior or posterior ( - ) to bregma.
coefficients between the estimated proportion of neocortex removed and errors did not approach significance for either lesion group. The results of this study demonstrate that PP injuries retard the learning of a spatial alternation strategy. This observation was expected since injuries in the PP cortex have been associated with learing and retention deficits in a large number of instrumental tasks with spatial demands (McDaniel & Wall, 1988). Failure to detect a significant impairment in the rats with MF injuries was initially surprising, but may be due to sparing of the anterior cingulate cortex. A recent study by Passingham, Meyers, Rawlins, Lightfoot, and Fearn (1988) has shown that medial
276
MCDANIEL, DAVALL, AND WALKER
SPATIAL ALTERNATION ACQUISITION 60
50
z
_
40
I--
if: o t-
30
m
Y
o
~
20
10
SHAM
SALINE
FIG. 2. groups.
MEDIAL FRONTAL
POSTERIOR PARIETAL
BRAIN LESIONS ACTH 4 - 9
Mean and SEM errors to criterion or ceiling for rats in each of the six factorial
injuries within the rat's frontal cortex that spare the cortical tissue occupying the walls of the sagittal sulcus result in a learning impairment in a visual conditional motor task, but not a delayed spatial alternation task. The authors suggest that this portion of the medial frontal cortex is both homologous and analogous two primate premotor cortex. Also, two of our nine rats with MF lesions attained criterion after making only one or two errors. We have no explanation for this spontaneous alternation tendency because the lesions in these animals did not differ from those seen in the more deficient learners. These results show that administration of a low dose of ACTH 4-9 during training on the spatial alternation task can retard acquisition. In a review of the behavioral and neurochemical literature, van Wimersma Greidanus et al., (1983) concluded that ACTH-like substances facilitate memory retrieval by enhancing attentional processes. A similar conclusion was reached by Born, Fehm, and Voight (1986) concerning ACTHlike peptides and human behavior. The authors of both reviews have concluded that these peptides stimulate limbic structures and thereby facilitate general attentional mechanisms. However, this enhanced disinhibitory influence is not without costs; habituation and selective attention are impaired, making the animal or person readily distracted by
ACTH 4-9 AND RECOVERY
277
irrelevant stimuli. This alteration of attentional mechanisms could be responsible for the learning impairment associated with ACTH 4-9 seen here. The ACTH 4-9-treated rats may have been easily distracted by events that took place during the intertrial interval. Memories of the spatial choice and consequences of the previous trial may have been diminished by these potentially interfering events. This conclusion may not apply to higher levels of ACTH-like peptides, to paradigms demanding less use of spatial memory, or to studies assessing postsurgical retention. There is a study that supports the ACTH 4-9 results reported here. Rigter, Veldhuis, and de Kloet (1984) studied the influence of chronic exposure to ACTH 4-9 upon the number of hippocampal corticosterone receptors and learning of an 18-arm spatial task, analogous to the radial arm maze, in young adult (6 months) and old (26 months) rats. Exposure to the peptide increased hippocampal corticosterone receptors in the old rats to parallel the numbers observed in young rats, but failed to improve spatial learning in either age group. Not only did the old animals administered ACTH 4-9 remain significantly impaired in spatial learning, but although not reported by the authors chronic exposure to 0.5 /zg/0.5 /xl/h ACTH 4-9 (i.e., 12 /xg per day) retarded learning in the senescent rats relative to saline controls. Our analysis of their only behavioral measure that proved to be a sensitive index of an age-related spatial learning dysfunction (see p. 394, Table 1) revealed that the senescent rats exposed to ACTH 4-9 required more time to find the correct goal box than the old rats administered saline, t(16) = 3.19, p < .01. Young adult rats administered ACTH 4-9 were not significantly influenced by the peptide. It appears that ACTH-like fragments exert beneficial functional effects, possibly because of their neurotrophic properties, shortly after a neural injury. Delaying administration to coincide with new learning can produce a deleterious outcome. Efforts are underway to examine the hypotheses presented here. REFERENCES Bijlsma, W. A., Jennekens, F. G. I., Schotman, P., & Gispen, W. H. (1983). Stimulation by ACTH 4-10 of nerve fiber regeneration following sciatic nerve crush. Muscle and Nerve, 6, 102-110. Born, J., Fehm, H. L., & Voigt, K. H. (1986). ACTH and attention in humans: A review. Neuropsychobiology, 15, 165-186. Fekete, M., & DeWied, D. (1982). Potency and duration of action of the ACTH 4-9 analog (ORG 2766) as compared to ACTH 4-10 and (D-Phe7) ACTH 4-10 on active and passive avoidance behavior of rats. Pharmacology, Biochemistry & Behavior, 16, 387392. GManda, P., Muglia, U., Vita, G., Dattola, R., Santoro, M., Toscano, A., Venuto, C., Roberto, M. L., Baradello, A., Romano, M., & Messina, C. (1988). Effect of ACTH 4-10 on nerve fiber regeneration after sciatic nerve crush in rabbits: An electrophysiological and morphological study. Experimental Neurology, 99, 454-460.
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Isaacson, R. L., & Poplawsky, A. (1983). An ACTH 4-9 analog (ORG 2766) speeds recovery from septal hyperemotionality in the rat. Behavioral and Neural Biology, 39, 52-59. Isaacson, R. L., & Poplawsky, A. (1985). ACTH 4-10 produces a transient decrease in septal hyperemotionality. Behavioral and Neural Biology, 43, 109-113. Kolb, B. (1984). Functions of the frontal cortex of the rat: A comparative review. Brain Research Reviews, 8, 65-98. McDaniel, W. F., & Wall, T. T. (1988). Visuospatial functions in the rat following injuries to straite, peristriate, and parietal neocortical sites. Psychobiology, 16, 251-260. Passingham, R. E., Meyers, C., Rawlins, N., Lightfoot, V., & Fearm, S. (1988). Premotor cortex in the rat. Behavioral Neuroscience, 102, 101-109. Rigter, H., Veldhuis, H. D., & de Kloet, E. R. (1984). Spatial learning and the hippocampal corticosterone receptor system of old rats: Effect of the ACTH 4-9 analogue ORG 2766. Brain Research, 309, 393-398. Thomas, R. K., Jr., & Peacock, L. J. (1965). A method of measuring brain lesions. Psychonomic Science, 3, 184. Van Wimersma Greidanus, T. B., Bohus, B., Kovacs, G. L., Versteeg, D. H. G., Burbach, J. P. H., & DeWied, D. (1983). Sites of behavioral and neurochemical action of ACTHlike peptides and neurohypophyseal hormones. Neuroscience & Biobehavioral Reviews, 7, 453-463. Veldhuis, H. D., Nyakas, C., & DeWied, D. (1985). Neuropeptides and functional recovery after brain damage. In B. E. Will, P. Schmitt, & J. C. Dalrymple-Alford (Eds.), Brain plasticity, learning, and memory (pp. 473-480). New York: Plenum. Verhoef, J., & Witter, A. (1976). In vivo fate of a behaviorally active ACTH 4-9 analog in rats after systemic administration. Pharmacology, Biochemistry & Behavior, 4, 583590.
52, 271-278 (1989)
BRIEF REPORTS ACTH 4-9 Analog Can Retard Spatial Alternation Learning in Brain Damaged and Normal Rats W. F. MCDANIEL, E. J. DAVALL, AND P. E. WALKER 1 Department of Pxychology, Georgia College, Milledgeville, Georgia 31061 Thirty adult male hooded rats (Long-Evans strain) were assigned randomly to one of three lesion groups (n = 10) and prepared with medial frontal, posterior parietal, or sham neocortical injuries. Following a recovery interval of 10-12 days, access to water was limited to 30 min per day and the rats were shaped to traverse a T-maze for a reward of sweetened water. After a pretraining criterion was attained, osmotic minipumps (Alzet 2002) were installed subcutaneously. The minipumps delivered chronically for the next 14 to 15 days either 0 or 1.2 /xg of ACTH 4-9 dissolved in bacteriostatic saline per day while the rats were trained on a reinforced spatial alternation task. Analysis of the number of errors made to a criterion of at least 80% correct alternations in two consecutive training sessions, or a ceiling of 62 errors (attained by two rats with parietal lesions), revealed that learning was impaired in the rats with parietal injuries. Contrary to our hypothesis, animals receiving ACTH 4-9 committed more errors than their counterparts receiving only saline. © 1989AcademicPress, Inc.
Administration of the ACTH-like peptides can facilitate neural and functional recovery following nervous system injury. For example, Bijlsma, Jennekens, Schotman, and Gispen (1983) have shown that ACTH 4-10, a fragment that fails to exert an endocrine effect upon the adrenal cortex, accelerates recovery of sensorimotor functions following sciatic nerve crush in rats. This functional amelioration may be due to the peptide's neurotrophic properties which result in increases in fiber density and regeneration rates in damaged axons (Girlanda et al., 1988). Postsurgical administration of ACTH fragments that fail to evoke an adrenal endocrine response can also improve some behavioral functions following central nervous system injury. Isaacson and Poplawsky (1983) reported that administering 1/zg of an analog of the ACTH 4-9 fragment This research was made possible by a Georgia College Faculty Research Grant awarded to W. F. McDaniel. We thank Mr. Tim Vacula for expert assistance with the photography. Address correspondence and reprint requests to W. F. McDaniet. 271 0163-1047/89 $3.00 Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
272
MCDANIEL, DAVALL, AND WALKER
for 4 consecutive days immediately following bilateral septal lesions reduced the amount of hyperemotionality and hastened the rate of declining hyperemotionality seen with repeated testing. A subsequent study with 1 and 10 ~g of the 4-10 fragment failed to mimic the previous beneficial results (Isaacson & Poplawsky, 1985). This is probably due to the greater metabolic stability (Verhoef & Witter, 1976), and hence potency (Fekete & DeWied, 1982), of the 4-9 analog. Veldhuis, Nyakas, and DeWied (1985) demonstrated that chronic administration (eight injections total) of either ACTH 4-9 (10 or 50 t~g sc, beginning the third day after surgery) or o~-MSH (same doses) attenuated or reversed a cognitive impairment associated with bilateral parafascicular thalamic nucleus injury. T-maze position reversal learning was significantly improved by the 10-~g dose of either neuropeptide, but still inferior to the sham-operated controls; however, 50-t~g doses resulted in acquisition rates that were parallel to those observed in normal rats. The peptides did not significantly influence the learning rates observed in noninjured rats, and motor dysfunctions that result from a parafascicular nucleus lesion were not altered. In another experiment, acute administration of the peptides 1 h prior to daily training sessions failed to improve learning in the brain-injured rats. The main purpose of this experiment was to test the possibility that chronic exposure to 1 t~g of ACTH 4-9 analog per day, simultaneous with training, might attenuate learning impairments in a cognitive task that are associated with neocortical injuries. Removal of the medial frontal (MF; Kolb, 1984) and posterior parietal neocortices (PP; McDaniel & Wall, 1988) results in a constellation of behavioral deficiencies in instrumental tasks with spatial demands. MF lesions have been observed to retard the learning of a reinforced spatial alternation strategy, but apparently the effects of PP lesions upon this task have not been assessed. Thirty adult male hooded rats bred in this laboratory were handled 5 min daily for 5 days prior to being assigned randomly to one of the three lesion groups. The animals had been entrained to a reversed dark-light schedule since birth (off, 8:00 AM; on, 9:00 PM), and they had been housed individually in standard stainless-steel cages since approximately 50 days of age. After being anesthetized with 55 mg/kg Nembutal, the scalp was shaved and scrubbed with a 10% (USP) Bovidone-iodine solution. All neurosurgery was performed with the animal mounted in a Kopf stereotaxic instrument and sterile instruments were used. Rats designated for sham operations received only a scalp incision. For lesion animals, the cranium overlying the MF and PP cortices was drilled with a 2.5mm trephine and enlarged with rongeurs to approximate the shape of the underlying cortical sector. The dura was incised, and the underlying neocortex was aspirated through a pipet using a dissecting microscope. The incision was closed with sterile wound clips and Mycitracin triple
ACTH 4 - 9 AND RECOVERY
273
antibiotic was applied to the incision. The rats were handled daily during a 10- to 12-day postoperative r e c o v e r y period. Access to water was restricted to a 30-rain period per day prior to shaping the rats to traverse a T-maze for a reward of water sweetened with table sugar (3.0%). The apparatus was constructed from 1-cm-thick plywood. The stem measured 15 cm wide by 30 cm long, the choice area 15 cm by 15 cm, and each goal arm measured 15 cm wide by 29 cm long. Initially the animals were allowed to explore the maze for 15 min and discover water in each goal arm. Then they were trained o v e r a sequence of days to traverse the maze for a reward of 5 s access to sweetened water which was delivered through standard water bottles available in each arm. When an animal selected a particular arm for 5 consecutive trials, the arm was blocked by a guillotine door on the next trial. Spatial alternation training began after a rat completed 10 runs for 5 days. Approximately 24 days elapsed between surgery and spatial alternation training. Upon reaching the pretraining criterion, all animals were anesthetized lightly with chloroform. Their backs were shaved posterior to the neck and scrubbed with iodine. A 1.5- to 2.0-cm lateral incision was made and an osmotic minipump (Alzet Model 2002) inserted subcutaneously. The incision was closed with sterile wound clips and Mycitracin antibiotic ointment was applied to the wound. The 200-/zl pump reservoir was filled with either bacteriostatic saline or A C T H 4-9 (Sigma Chemical Co.). The pump has a nominal life for constant drug delivery of 14 days, infuses its contents at a rate o f 0.5/~l/h, and exhausts its contents after 16 days. The dosage of 1.0 to 1.2 /zg of A C T H 4-9 per day was attained by dissolving 5 mg of the peptide in 60 ml bacteriostatic saline. One day after installing the osmotic pumps, training of the spatial alternation strategy began. One rat from each of the factorial groups was trained one session of 16 trials per day. All remaining animals were trained two sessions of 11 trials each per day, one in the morning and one in the late afternoon. This change in training strategies was necessary to maximize the training effort while A C T H 4 - 9 levels were maximal. Since the dependent variable was errors to criterion, and since equivalent number of animals from each group experienced the two training techniques, it was deemed appropriate to pool the behavioral data for purposes of statistical analysis. Training occurred 7 days per week until a criterion of two successive sessions of 80% correct alternations was attained or to a ceiling of 62 errors. The initial arm selection was determined randomly and forced by blocking access to the other arm. On subsequent trials, clear Plexiglas barriers blocked access to both water bottles. Selection of the correct arm led to removal of the barrier and 5 s access to water. U p o n making an error, that is entering an incorrect alley with all four feet, the barrier to water was not removed and the
274
MCDANIEL, DAVALL, AND WALKER
rat was returned immediately to the starting point for the next trial. On each succeeding trial, the animal was rewarded only for alternating it's choice by selecting the side opposite the last rewarded site. The intertrial interval was approximately 5 s. At the completion of training, the animals were sacrificed with an overdose of Nembutal and perfused through the heart first with 0.85% saline and then with 10% formalin. The brains were removed, soaked in formalin, and then soaked in a 30% sucrose10% formalin solution before being photographed from the dorsal perspective. Brains were blocked to include the lesion and thalamus and sliced through the coronal plane into 40-/xm sections. Every 10th section was mounted on a slide and stained with cresyl violet. An analysis of variance (ANOVA; two lesions x two drug conditions) was performed on the proportion of the neocortex visible from the dorsal view that was removed by the ablation. These determinations were estimated using the dot-grid method (Thomas & Peacock, 1965). The ANOVA confirmed that the PP injuries (M = 0.27, SD = 0.05) were larger than the MF injuries (M = 0.15, SD = 0.03), F(1, 16) = 43.91, p < .001. No other differences approached significance. Microscopic examination of the brains (see Fig. 1) showed that the MF lesions extended from the frontal pole to the genu of the corpus callosum, usually sparing the tissue along the medial walls and the anterior cingulate cortex. No retrograde necrosis was observed in the thalamus following this lesion. PP lesion included the tissue rostral to visual cortex and extended rostrally into area 3. The underlying corpus callosum was damaged in most preparations, and moderate to heavy gliosis was observed in the lateral, posterior, and ventral thalamic nuclei. One rat with a MF lesion stopped running when the alternation training began and despite much effort completed only a total of 75 trials. Histological evaluation revealed that this animal had the largest and the deepest MF lesion. On the basis of these observations, this animal's data were deleted from the study. An unweighted means ANOVA for a completely randomized factorial design was performed on the errors to criterion or ceiling (62 errors; obtained by two rats with PP lesions), and Fig. 2 summarizes these results. The difference between lesion groups was significant, F(2, 23) = 4.59, p = .02. Fisher's LSD test revealed that the rats with PP injuries committed more errors than the shamoperated rats. Rats with MF lesions were intermediate between these two extremes and not significantly different from either group. The ANOVA also revealed a significant main effect for the peptide condition, F(1, 23) = 5.77, p = .02. This result demonstrates, as is shown by Fig. 2, that chronic administration of ACTH 4-9 (totaling 1.0 /zg per day) impaired learning relative to saline. The nonsignificant interaction of lesion by dose, F(2, 23) = 0.27, p > > .05, demonstrates that this effect was consistent across brain lesion and sham-operated groups. Correlation
275
ACTH 4-9 AND RECOVERY MF
PP
5.6 0.0
4,0 1.6
IN
2.4
}~:i
30
FIG. 1. (A) Photographs from the dorsal perspective depicting location of the lesions in representative cases. (B) Photographs of three cornonal sections through the brain shown in A. Numerals indicate millimeter anterior or posterior ( - ) to bregma.
coefficients between the estimated proportion of neocortex removed and errors did not approach significance for either lesion group. The results of this study demonstrate that PP injuries retard the learning of a spatial alternation strategy. This observation was expected since injuries in the PP cortex have been associated with learing and retention deficits in a large number of instrumental tasks with spatial demands (McDaniel & Wall, 1988). Failure to detect a significant impairment in the rats with MF injuries was initially surprising, but may be due to sparing of the anterior cingulate cortex. A recent study by Passingham, Meyers, Rawlins, Lightfoot, and Fearn (1988) has shown that medial
276
MCDANIEL, DAVALL, AND WALKER
SPATIAL ALTERNATION ACQUISITION 60
50
z
_
40
I--
if: o t-
30
m
Y
o
~
20
10
SHAM
SALINE
FIG. 2. groups.
MEDIAL FRONTAL
POSTERIOR PARIETAL
BRAIN LESIONS ACTH 4 - 9
Mean and SEM errors to criterion or ceiling for rats in each of the six factorial
injuries within the rat's frontal cortex that spare the cortical tissue occupying the walls of the sagittal sulcus result in a learning impairment in a visual conditional motor task, but not a delayed spatial alternation task. The authors suggest that this portion of the medial frontal cortex is both homologous and analogous two primate premotor cortex. Also, two of our nine rats with MF lesions attained criterion after making only one or two errors. We have no explanation for this spontaneous alternation tendency because the lesions in these animals did not differ from those seen in the more deficient learners. These results show that administration of a low dose of ACTH 4-9 during training on the spatial alternation task can retard acquisition. In a review of the behavioral and neurochemical literature, van Wimersma Greidanus et al., (1983) concluded that ACTH-like substances facilitate memory retrieval by enhancing attentional processes. A similar conclusion was reached by Born, Fehm, and Voight (1986) concerning ACTHlike peptides and human behavior. The authors of both reviews have concluded that these peptides stimulate limbic structures and thereby facilitate general attentional mechanisms. However, this enhanced disinhibitory influence is not without costs; habituation and selective attention are impaired, making the animal or person readily distracted by
ACTH 4-9 AND RECOVERY
277
irrelevant stimuli. This alteration of attentional mechanisms could be responsible for the learning impairment associated with ACTH 4-9 seen here. The ACTH 4-9-treated rats may have been easily distracted by events that took place during the intertrial interval. Memories of the spatial choice and consequences of the previous trial may have been diminished by these potentially interfering events. This conclusion may not apply to higher levels of ACTH-like peptides, to paradigms demanding less use of spatial memory, or to studies assessing postsurgical retention. There is a study that supports the ACTH 4-9 results reported here. Rigter, Veldhuis, and de Kloet (1984) studied the influence of chronic exposure to ACTH 4-9 upon the number of hippocampal corticosterone receptors and learning of an 18-arm spatial task, analogous to the radial arm maze, in young adult (6 months) and old (26 months) rats. Exposure to the peptide increased hippocampal corticosterone receptors in the old rats to parallel the numbers observed in young rats, but failed to improve spatial learning in either age group. Not only did the old animals administered ACTH 4-9 remain significantly impaired in spatial learning, but although not reported by the authors chronic exposure to 0.5 /zg/0.5 /xl/h ACTH 4-9 (i.e., 12 /xg per day) retarded learning in the senescent rats relative to saline controls. Our analysis of their only behavioral measure that proved to be a sensitive index of an age-related spatial learning dysfunction (see p. 394, Table 1) revealed that the senescent rats exposed to ACTH 4-9 required more time to find the correct goal box than the old rats administered saline, t(16) = 3.19, p < .01. Young adult rats administered ACTH 4-9 were not significantly influenced by the peptide. It appears that ACTH-like fragments exert beneficial functional effects, possibly because of their neurotrophic properties, shortly after a neural injury. Delaying administration to coincide with new learning can produce a deleterious outcome. Efforts are underway to examine the hypotheses presented here. REFERENCES Bijlsma, W. A., Jennekens, F. G. I., Schotman, P., & Gispen, W. H. (1983). Stimulation by ACTH 4-10 of nerve fiber regeneration following sciatic nerve crush. Muscle and Nerve, 6, 102-110. Born, J., Fehm, H. L., & Voigt, K. H. (1986). ACTH and attention in humans: A review. Neuropsychobiology, 15, 165-186. Fekete, M., & DeWied, D. (1982). Potency and duration of action of the ACTH 4-9 analog (ORG 2766) as compared to ACTH 4-10 and (D-Phe7) ACTH 4-10 on active and passive avoidance behavior of rats. Pharmacology, Biochemistry & Behavior, 16, 387392. GManda, P., Muglia, U., Vita, G., Dattola, R., Santoro, M., Toscano, A., Venuto, C., Roberto, M. L., Baradello, A., Romano, M., & Messina, C. (1988). Effect of ACTH 4-10 on nerve fiber regeneration after sciatic nerve crush in rabbits: An electrophysiological and morphological study. Experimental Neurology, 99, 454-460.
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