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Evidence of non-maternally mediated acceleration of eye-opening in ‘enriched’ artificially reared rat pups
Developmental Brain Research, 56 (1990) 141-143
141
Elsevier BRESD 60379
Evidence of non-maternally mediated acceleration of eye-opening in 'enriched' artificially reared rat pups J.L. Smart 1, A.C. McMahon 1, R.F. Massey 1, G.-N.K. Akbar 2 and M.A. Warren 2 IDepartment of Child Health, University of Manchester, Manchester (U. K.) and eDepartment of Biomedical Science, University of Sheffield, Sheffield ( U. K. )
(Accepted 19 June 1990) Key words: Eye-opening; Environmental enrichment; Environmental impoverishment; Stimulation in infancy; Artificial rearing; Rat
Rat pups were artificially reared, without their mothers, from postnatal day 5. They were exposed thereafter to impoverished or enriched environmental conditions. The enrichment included gentling, social interaction and homing experience. Enriched rats showed accelerated eye-opening compared with their impoverished littermates (P < 0.02). This is the first demonstration of stimulation-induced acceleration of development which cannot have been maternally mediated. There has been much investigation of the effects of stimulation in infancy on many aspects of development in rodents. Over two decades, mid 1950s to mid 1970s, a plethora of papers appeared on the effects of stimulating infant rats and mice in various ways: handling, gentling, exposure to cold and administration of electric shock, amongst others 7'8. The effects were usually interpreted as being beneficial, and included the acceleration of development in terms of the appearance of developmental milestones, most notably eye-opening 3'6. From the earliest days of early handling research there has been active investigation of possible mechanisms. Unfortunately we are little wiser today, except that the possibilities appear to be resolved into four categories: direct sensory stimulation, stress, cold exposure, and maternally mediated stimulation 8. The last category, maternal mediation, relates to the possibility that the mothers may respond differently towards handled/stimulated pups and that this difference in care may produce the subsequent effects. Maternal mediation has been a possible confounding factor in all previous studies of early stimulation. The technique for artificially rearing infant rats without their mothers, whereby they are fitted with a gastric cannula a few days postnatally and fed a milk or milk-substitute by infusion 4'9, affords the opportunity to study the effects of additional stimulation in a situation in which there can be no maternal mediation. The present paper reports the effects of stimulation on the timing of eye-opening. Morphometric findings from the brains of the same animals are presented elsewhere ~.
Rat pups of a black and white hooded Lister stock were fitted with a gastric cannula on postnatal day 5 (day 0 --- day of birth) by the method of Hall 4 and were subsequently artificially reared without their mothers on a rat milk substitute (RMS) given by intermittent gastric infusion (15 min infusion every hour). The RMS was prepared by ultrafiltering skimmed cows' milk and adding fat and vitamins to the resultant fluid. One volume of pasteurised skimmed cows' milk was ultrafiltered to produce 0.67 volumes of permeate (discarded) and 0.33 volumes of retentate, to which was added a mixture of oils, fat soluble vitamins and water soluble vitamins, to give the following calculated composition (g/l): total protein 93, lactose 43, fat 120, which is similar to that of rats' milk 2. The RMS was prepared and freeze-dried at the Hannah Research Institute (Ayr, Scotland) then reconstituted with sterile distilled water in Manchester. The volumes of milk infused per pup were increased from 2.7 ml on day 5-6 to 9.3 ml on day 19-20, in daily increments of 0.44 ml. Two male pups from each of 23 litters were cannulated and ascribed to either the impoverished condition (IC) or the enriched condition (EC). The IC was the usual artificial rearing condition9: pups were housed singly in polystyrene tubs, 46 mm deep and 82 mm base diameter, with wood shavings as bedding. These tubs were placed into identical tubs counter-weighted with a brass bolt through the base to prevent capsizing, and floated in a water bath at 42 °C. IC pups were stimulated morning and evening to urinate and defaecate by stroking the anogenital region with a small paint brush. They were
Correspondence: J.L. Smart, Department of Child Health, The Medical School, Oxford Road, Manchester, M13 9PT, U.K.
0165-3806/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
142 TABLE I Mean age (days + S.D.) at which both eyes were first seen to be open in artificially reared rats raised in improverished (1C) or enriched conditions (EC)
Numbers of rats in parentheses.
Litter-mate pairs Allrats
weight at 3-day intervals: k'1,34 = 0.900). Body weight did not differ between the IC and EC groups at the start of the experiment on day 5 (mean + S.D.: IC 12.1 ± 0.9, EC 12.1 + 0.6g, t37 = 0.008) o r o n day 13, the modal day of eye opening (IC 22.9 _+ 1.5, EC 23.3 ± 1.2 g; t37 0.866). Fourteen out of the total of 39 pups were seen to have one eye open before the other. In all 14 the left eye was the first to open, thus replicating our previous finding for artificially-reared rats 1°. The 14 were distributed exactly equally between the two treatment groups. It is necessary to examine the original evidence for the phenomenon of accelerated eye-opening resulting from additional stimulation in infancy, in order to put the present finding in context. In fact, the original, muchquoted paper is principally about 'emotionality' at weaning in rat pups whose cages had been moved about or shaken for two min a day from birth, and contains only informal observations on eye-opening6. There does not appear to have been any regular inspection of the rats for developmental milestones and no mean values are presented. It is merely remarked that the eyes opened at 12-14 days in the stimulated groups compared to about 16 days in the non-stimulated controls. The most substantial evidence is that of Barnett and Burn 3, who found eye-opening to be significantly accelerated by 0.6 to 0.7 of a day in mice which had been ear-marked for identification on day 6, irrespective of other treatment. Such mice, with holes punched in their ears, were observed to receive more parental attention. Barnett and Burn note the parallel between the findings of accelerated development and altered maternal care and suggest a causal association. Our present findings do not rule out the possible existence of this mechanism, but they do demonstrate that accelerated development can occur without maternal mediation. In mother-reared animals there may be separate and additive effects of additional stimulation acting directly on the pup and indirectly through the mother. It has been proposed that temperature may be an intervening variable in early stimulation studies; that is, that the effects of treatment on rodent pups, which have poorly developed thermoregulatory systems, may be mediated through changes in body temperature 8. Usually it is suggested that the treatment results, either directly or indirectly, in temporary cooling. The possibility cannot be ruled out that the effect on eye opening reported here may have been mediated in this way, since body temperature was not recorded. Continuous recording would have been necessary to provide conclusive evidence. Three aspects of the experimental design impinge on this question. (i) The tubs containing the pups of both artificially reared groups were equally distributed between 3 water :=
IC
EC
t
P
13.22+0.60(16) 13.30+0.66(20)
12.84+0.40(16) 12.89+0.39(19)
2.8181 <0.02 2.3482 <0.05
1 Paired comparisonst-test (dr 15). 2 t-test for unrelated samples (df 37). also weighed after the morning stimulation. The EC pups received the above treatment plus various 'enriching' experiences. The EC comprised two additions to the tub environment and, from day 6, twice daily 'gentling' and social experience, incorporating a 'homing' element from day 10. As well as the usual wood shavings a piece of synthetic fur (semicircle, 7.5 cm in diameter) was placed in each EC tub on day 5 and a piece of balsa wood (1.5 x 1 × 1 cm) on day 10. The fur, which lay free in the tub, could be moved by the pup and remained in the tub for the duration of artificial rearing. The balsa was replaced when it became gnawed. Twice a day, between feeds, EC pups were disconnected from the milk delivery system and placed in groups of six for 35 min in a plastic box (59 x 34 x 20 cm high) which was kept warm over a water bath at 42 °C. The floor of the box was of three different textures, plastic covered with wood shavings, synthetic fur and polystyrene; and the walls at one end were lined with synthetic fur. At the start of each enrichment session the pups were placed on the warmest surface (plastic) at the fur-walled end of the box. They were removed from the box one at a time, stimulated to urinate for 1 rain, 'gentled' (stroked with the fingers and thumb over much of the body surface) for 2 rain and then returned to the warm end of the box. From day 10, after gentling they were placed at the end of the box furthest from their peers, and they learned to 'home' to them. All of the pups displayed successful homing from day 10. The whole set of procedures, from disconnection to reconnection, took about 40 min. From day 11, pups were inspected for eye-opening twice daily at about 09.00 and 21.00 h. Eye-opening was defined as the slightest break in the membrane sealing the lids of both eyes. Additional stimulation (EC) resulted in significantly accelerated eye-opening by about 0.4 of a day, whether comparison was confined to the 16 complete litter-mate pairs or was extended to include all IC and EC rats (Table I). Growth in body weight between days 5 and 14 was unaffected by treatment (two-way ANOVA in body-
143 baths and hence there could not have been differences b e t w e e n the groups in m e a n water bath t e m p e r a t u r e . (ii) F u r t h e r m o r e , within the tubs, pups had some o p p o r t u n i t y for t h e r m o r e g u l a t i o n by moving the w o o d shavings ( + fur) to lie in contact with the floor of the tub or on the shavings ( + fur). (iii) The most likely occasions for different thermal experience were during the twice-daily enrichment sessions. The E C rats may have experienced lower a m b i e n t t e m p e r a t u r e s during the few minutes required at the beginning and end of the sessions for disconnection and reconnection to the p u m p and carrying them in their tubs b e t w e e n adjacent rooms, and during the 3-min p e r i o d in which the e x p e r i m e n t e r held the pups for anogenital stimulation and gentling.
The magnitude of the effect on e y e - o p e n i n g in the present investigation (0.4 day) is in line with that r e p o r t e d by Barnett and Burn 3 (0.6-0.7 day). In both studies the t r e a t m e n t was started on day 6, and hence it is possible that stimulation from an earlier age might have had a greater effect. In support of this, it is known that the effects of e p i d e r m a l growth factor on e y e - o p e n i n g are greater the earlier the t r e a t m e n t is c o m m e n c e d 5.
1 Akbar, G.-N.K., McMahon, A.C., Massey, R.F., Warren, M.A. and Smart, J,L., The effect of preweaning environmental diversity on the neuronal connectivity of artificially reared rats, Neurosci. Lett., Suppl. 38 (1990) S134. 2 Auestad, N., Korsak, R.A., Bergstrom, J.D. and Edmond, J., Milk-substitutes comparable to rat's milk; their preparation, composition and impact on development and metabolism in the artificially reared rat, Br. J. Nutr., 61 (1989) 495-518. 3 Barnett, S.A. and Burn, J., Early stimulation and maternal behaviour, Nature, 213 (1967) 150-152. 4 Hall, W.G., Weaning and growth of artificially reared rats, Science, 190 (1975) 1313-1315. 5 Hoath, S.B., Treatment of the neonatal rat with epidermal growth factor. Differences in time and organ response, Pediat.
Res., 20 (1986) 468-472. 6 Levine, S., The effects of differential infantile stimulation on emotionality at weaning, Can. J. Psychol., 13 (1959) 243-247. 7 Newton, G. and Levine, S., Early Experience and Behavior, Thomas, Springfield, IL, 1968, 785 pp. 8 Russell, P.A., 'Infantile stimulation' in rodents: a consideration of possible mechanisms, Psychol. Bull., 75 (1971) 192-202. 9 Smart, J.L., Stephens, D.N. and Katz, H.B., Growth and development of rats artificially reared on a high or a low plane of nutrition, Br. J. Nutr., 49 (1983) 497-506. 10 Smart, J.L., Tonkiss, J. and Massey, R.F., A phenomenon: left-biassed asymmetrical eye-opening in artificially reared rat pups, Dev. Brain Res., 28 (1986) 134-136.
We are grateful to the Medical Research Council (U.K.) and the National Fund for Research into Crippling Diseases (U.K.) for financial support. We also wish to thank Dr. Douglas Dalgleish (Hannah Research Institute, Ayr, Scotland) for kindly providing the ultrafiltered milk and Dr. Patricia Wainwright (University of Waterloo, Canada) for lively discussion and practical assistance in the early stages of the project.
141
Elsevier BRESD 60379
Evidence of non-maternally mediated acceleration of eye-opening in 'enriched' artificially reared rat pups J.L. Smart 1, A.C. McMahon 1, R.F. Massey 1, G.-N.K. Akbar 2 and M.A. Warren 2 IDepartment of Child Health, University of Manchester, Manchester (U. K.) and eDepartment of Biomedical Science, University of Sheffield, Sheffield ( U. K. )
(Accepted 19 June 1990) Key words: Eye-opening; Environmental enrichment; Environmental impoverishment; Stimulation in infancy; Artificial rearing; Rat
Rat pups were artificially reared, without their mothers, from postnatal day 5. They were exposed thereafter to impoverished or enriched environmental conditions. The enrichment included gentling, social interaction and homing experience. Enriched rats showed accelerated eye-opening compared with their impoverished littermates (P < 0.02). This is the first demonstration of stimulation-induced acceleration of development which cannot have been maternally mediated. There has been much investigation of the effects of stimulation in infancy on many aspects of development in rodents. Over two decades, mid 1950s to mid 1970s, a plethora of papers appeared on the effects of stimulating infant rats and mice in various ways: handling, gentling, exposure to cold and administration of electric shock, amongst others 7'8. The effects were usually interpreted as being beneficial, and included the acceleration of development in terms of the appearance of developmental milestones, most notably eye-opening 3'6. From the earliest days of early handling research there has been active investigation of possible mechanisms. Unfortunately we are little wiser today, except that the possibilities appear to be resolved into four categories: direct sensory stimulation, stress, cold exposure, and maternally mediated stimulation 8. The last category, maternal mediation, relates to the possibility that the mothers may respond differently towards handled/stimulated pups and that this difference in care may produce the subsequent effects. Maternal mediation has been a possible confounding factor in all previous studies of early stimulation. The technique for artificially rearing infant rats without their mothers, whereby they are fitted with a gastric cannula a few days postnatally and fed a milk or milk-substitute by infusion 4'9, affords the opportunity to study the effects of additional stimulation in a situation in which there can be no maternal mediation. The present paper reports the effects of stimulation on the timing of eye-opening. Morphometric findings from the brains of the same animals are presented elsewhere ~.
Rat pups of a black and white hooded Lister stock were fitted with a gastric cannula on postnatal day 5 (day 0 --- day of birth) by the method of Hall 4 and were subsequently artificially reared without their mothers on a rat milk substitute (RMS) given by intermittent gastric infusion (15 min infusion every hour). The RMS was prepared by ultrafiltering skimmed cows' milk and adding fat and vitamins to the resultant fluid. One volume of pasteurised skimmed cows' milk was ultrafiltered to produce 0.67 volumes of permeate (discarded) and 0.33 volumes of retentate, to which was added a mixture of oils, fat soluble vitamins and water soluble vitamins, to give the following calculated composition (g/l): total protein 93, lactose 43, fat 120, which is similar to that of rats' milk 2. The RMS was prepared and freeze-dried at the Hannah Research Institute (Ayr, Scotland) then reconstituted with sterile distilled water in Manchester. The volumes of milk infused per pup were increased from 2.7 ml on day 5-6 to 9.3 ml on day 19-20, in daily increments of 0.44 ml. Two male pups from each of 23 litters were cannulated and ascribed to either the impoverished condition (IC) or the enriched condition (EC). The IC was the usual artificial rearing condition9: pups were housed singly in polystyrene tubs, 46 mm deep and 82 mm base diameter, with wood shavings as bedding. These tubs were placed into identical tubs counter-weighted with a brass bolt through the base to prevent capsizing, and floated in a water bath at 42 °C. IC pups were stimulated morning and evening to urinate and defaecate by stroking the anogenital region with a small paint brush. They were
Correspondence: J.L. Smart, Department of Child Health, The Medical School, Oxford Road, Manchester, M13 9PT, U.K.
0165-3806/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
142 TABLE I Mean age (days + S.D.) at which both eyes were first seen to be open in artificially reared rats raised in improverished (1C) or enriched conditions (EC)
Numbers of rats in parentheses.
Litter-mate pairs Allrats
weight at 3-day intervals: k'1,34 = 0.900). Body weight did not differ between the IC and EC groups at the start of the experiment on day 5 (mean + S.D.: IC 12.1 ± 0.9, EC 12.1 + 0.6g, t37 = 0.008) o r o n day 13, the modal day of eye opening (IC 22.9 _+ 1.5, EC 23.3 ± 1.2 g; t37 0.866). Fourteen out of the total of 39 pups were seen to have one eye open before the other. In all 14 the left eye was the first to open, thus replicating our previous finding for artificially-reared rats 1°. The 14 were distributed exactly equally between the two treatment groups. It is necessary to examine the original evidence for the phenomenon of accelerated eye-opening resulting from additional stimulation in infancy, in order to put the present finding in context. In fact, the original, muchquoted paper is principally about 'emotionality' at weaning in rat pups whose cages had been moved about or shaken for two min a day from birth, and contains only informal observations on eye-opening6. There does not appear to have been any regular inspection of the rats for developmental milestones and no mean values are presented. It is merely remarked that the eyes opened at 12-14 days in the stimulated groups compared to about 16 days in the non-stimulated controls. The most substantial evidence is that of Barnett and Burn 3, who found eye-opening to be significantly accelerated by 0.6 to 0.7 of a day in mice which had been ear-marked for identification on day 6, irrespective of other treatment. Such mice, with holes punched in their ears, were observed to receive more parental attention. Barnett and Burn note the parallel between the findings of accelerated development and altered maternal care and suggest a causal association. Our present findings do not rule out the possible existence of this mechanism, but they do demonstrate that accelerated development can occur without maternal mediation. In mother-reared animals there may be separate and additive effects of additional stimulation acting directly on the pup and indirectly through the mother. It has been proposed that temperature may be an intervening variable in early stimulation studies; that is, that the effects of treatment on rodent pups, which have poorly developed thermoregulatory systems, may be mediated through changes in body temperature 8. Usually it is suggested that the treatment results, either directly or indirectly, in temporary cooling. The possibility cannot be ruled out that the effect on eye opening reported here may have been mediated in this way, since body temperature was not recorded. Continuous recording would have been necessary to provide conclusive evidence. Three aspects of the experimental design impinge on this question. (i) The tubs containing the pups of both artificially reared groups were equally distributed between 3 water :=
IC
EC
t
P
13.22+0.60(16) 13.30+0.66(20)
12.84+0.40(16) 12.89+0.39(19)
2.8181 <0.02 2.3482 <0.05
1 Paired comparisonst-test (dr 15). 2 t-test for unrelated samples (df 37). also weighed after the morning stimulation. The EC pups received the above treatment plus various 'enriching' experiences. The EC comprised two additions to the tub environment and, from day 6, twice daily 'gentling' and social experience, incorporating a 'homing' element from day 10. As well as the usual wood shavings a piece of synthetic fur (semicircle, 7.5 cm in diameter) was placed in each EC tub on day 5 and a piece of balsa wood (1.5 x 1 × 1 cm) on day 10. The fur, which lay free in the tub, could be moved by the pup and remained in the tub for the duration of artificial rearing. The balsa was replaced when it became gnawed. Twice a day, between feeds, EC pups were disconnected from the milk delivery system and placed in groups of six for 35 min in a plastic box (59 x 34 x 20 cm high) which was kept warm over a water bath at 42 °C. The floor of the box was of three different textures, plastic covered with wood shavings, synthetic fur and polystyrene; and the walls at one end were lined with synthetic fur. At the start of each enrichment session the pups were placed on the warmest surface (plastic) at the fur-walled end of the box. They were removed from the box one at a time, stimulated to urinate for 1 rain, 'gentled' (stroked with the fingers and thumb over much of the body surface) for 2 rain and then returned to the warm end of the box. From day 10, after gentling they were placed at the end of the box furthest from their peers, and they learned to 'home' to them. All of the pups displayed successful homing from day 10. The whole set of procedures, from disconnection to reconnection, took about 40 min. From day 11, pups were inspected for eye-opening twice daily at about 09.00 and 21.00 h. Eye-opening was defined as the slightest break in the membrane sealing the lids of both eyes. Additional stimulation (EC) resulted in significantly accelerated eye-opening by about 0.4 of a day, whether comparison was confined to the 16 complete litter-mate pairs or was extended to include all IC and EC rats (Table I). Growth in body weight between days 5 and 14 was unaffected by treatment (two-way ANOVA in body-
143 baths and hence there could not have been differences b e t w e e n the groups in m e a n water bath t e m p e r a t u r e . (ii) F u r t h e r m o r e , within the tubs, pups had some o p p o r t u n i t y for t h e r m o r e g u l a t i o n by moving the w o o d shavings ( + fur) to lie in contact with the floor of the tub or on the shavings ( + fur). (iii) The most likely occasions for different thermal experience were during the twice-daily enrichment sessions. The E C rats may have experienced lower a m b i e n t t e m p e r a t u r e s during the few minutes required at the beginning and end of the sessions for disconnection and reconnection to the p u m p and carrying them in their tubs b e t w e e n adjacent rooms, and during the 3-min p e r i o d in which the e x p e r i m e n t e r held the pups for anogenital stimulation and gentling.
The magnitude of the effect on e y e - o p e n i n g in the present investigation (0.4 day) is in line with that r e p o r t e d by Barnett and Burn 3 (0.6-0.7 day). In both studies the t r e a t m e n t was started on day 6, and hence it is possible that stimulation from an earlier age might have had a greater effect. In support of this, it is known that the effects of e p i d e r m a l growth factor on e y e - o p e n i n g are greater the earlier the t r e a t m e n t is c o m m e n c e d 5.
1 Akbar, G.-N.K., McMahon, A.C., Massey, R.F., Warren, M.A. and Smart, J,L., The effect of preweaning environmental diversity on the neuronal connectivity of artificially reared rats, Neurosci. Lett., Suppl. 38 (1990) S134. 2 Auestad, N., Korsak, R.A., Bergstrom, J.D. and Edmond, J., Milk-substitutes comparable to rat's milk; their preparation, composition and impact on development and metabolism in the artificially reared rat, Br. J. Nutr., 61 (1989) 495-518. 3 Barnett, S.A. and Burn, J., Early stimulation and maternal behaviour, Nature, 213 (1967) 150-152. 4 Hall, W.G., Weaning and growth of artificially reared rats, Science, 190 (1975) 1313-1315. 5 Hoath, S.B., Treatment of the neonatal rat with epidermal growth factor. Differences in time and organ response, Pediat.
Res., 20 (1986) 468-472. 6 Levine, S., The effects of differential infantile stimulation on emotionality at weaning, Can. J. Psychol., 13 (1959) 243-247. 7 Newton, G. and Levine, S., Early Experience and Behavior, Thomas, Springfield, IL, 1968, 785 pp. 8 Russell, P.A., 'Infantile stimulation' in rodents: a consideration of possible mechanisms, Psychol. Bull., 75 (1971) 192-202. 9 Smart, J.L., Stephens, D.N. and Katz, H.B., Growth and development of rats artificially reared on a high or a low plane of nutrition, Br. J. Nutr., 49 (1983) 497-506. 10 Smart, J.L., Tonkiss, J. and Massey, R.F., A phenomenon: left-biassed asymmetrical eye-opening in artificially reared rat pups, Dev. Brain Res., 28 (1986) 134-136.
We are grateful to the Medical Research Council (U.K.) and the National Fund for Research into Crippling Diseases (U.K.) for financial support. We also wish to thank Dr. Douglas Dalgleish (Hannah Research Institute, Ayr, Scotland) for kindly providing the ultrafiltered milk and Dr. Patricia Wainwright (University of Waterloo, Canada) for lively discussion and practical assistance in the early stages of the project.