Physiology& Behavior,Vol. 22, pp. 7-9. PergamonPress and Brain ResearchPubl., 1979. Printed in the U.S.A.
Effects of the Albino Gene on Self-Stimulation Behavior in the Lateral Hypothalamus in the Mouse PIERRE CAZALA AND JEAN-LOUIS GUENET
Laboratoire de Psychophysiologie, institut de Biologie Animale, Universit~ de Bordeaux !, Avenue des Facult~s 33405 Talence Cedex France and lnstitut Pasteur, D~partement de Biologie Mol~culaire, 25 Rue du Docteur Roux, 75015 Paris Cedex France ( R e c e i v e d 30 M a r c h 1978) CAZALA, P. AND J. L. GUENET. Effects of the albino gene on se(f-stimulation behavior in the lateral hypothalamus in the mouse. PHYSIOL. BEHAV. 22(1) 7-9, 1979.-- Intraeranial self-stimulation behavior (ICSS) in the lateral hypothalamus has been studied in two coisogenic mice groups differing at the albino locus. Heterozygous mice (+/c) had significant lower ICSS thresholds and higher performances than homozygous mice (c/c). Both groups however showed identical sensitivity for the triggering of convulsions suspending ICSS behavior. These results suggest that the albino mutation correlates with some changes in the normal function of the central nervous system. Self-stimulation
Lateral hypothalamus
Albino mutation
AS A RESULT of the large number of investigations which have been carried out concerning the gene which causes albinism, it is now possible to distinguish between albino and nonalbino mice from their anatomical, physiological, biochemical and behavioral features (for review see [6]). Among the behavioral pleiotropic effects which seem to be controlled by the c locus, it has been demonstrated that strains of albino mice in a water maze obtain lower learning scores than strains of pigmented mice [16]. A similar observation has also been made for an albino mutant of the C57BL/6 strain [5]. It has also been reported that the acquisition of a visual discrimination [10], as well as avoidance behavior [7,10] is slower in these albino mutants than it is in pigmented littermatcs. During the course of work which has been carried out to estimate the degree of complexity in the genetic control of intracranial self-stimulation behavior (ICSS) in the mouse, some observations were made which suggested that the albino gene could be involved in this particular behavioral pattern. From studies which involved genetic crosses between the related strains BALB/c and DBA/2, we have been able to establish (1) that the albino strain BALB/c had the lowest ICSS thresholds, and the highest level of performances [3], (2) the F1 generation had a very similar ICSS pattern as the DBA/2 parental strain; also, we have studied ICSS in the backcross progeny between the FI generation and the "recessive" strain BALB/c [2]. We have observed that among the offspring of this cross which self-stimulated vigorously like BALB/c, the majority (9 out of 11) were unpigmented. The aim of the present study is to try to determine what
Coisogenic mice
are the eventual effects of albinism on the various ICSS parameters. METHOD
Animals and Surgery Twenty-nine male mice 10 to 12 weeks old were used in this study. These animals were fwst generation hybrids resulting from a cross between BALB/c J hornozyguus albino females (c/c) and C57BL6/Jc males beterozyguus for albinism (+/c). The resulting FI generation consisted of two groups of coisogenic mice, one of which was albino (c/c), and the other was hcterozygous for c (+/c). The two groups showed an identical body weight: +/c mean=26.7 g (SEM +__0.8); c/c mean=27.3 g (SEM *-- 0.9). Twenty-three mice (13 c/c and 10 +/c) were anesthetised by an intraperitoneal injection of sodium thiopental (80 mg/kg). A bipolar electrode of two twisted platinum wires of 0.09 mm dia. was implanted under stereotaxic control into each mouse. The electrode was implanted into the ventral portion of the lateral hypothalamus (LH) which is the most sensitive area for the triggering of convulsions during ICSS behavior [1]. The stereotaxic coordinates used were previously determined in 3 mice from each group. +/c: antero-posterior distance refering to interaural line +2.00 ram; lateral distance refering to sagittal line - 1.00 ram; depth from the surface of the skull +5.70 nun. c/c: antero-posteriority + 1.90 ram; laterality _ 1.0 ram; depth +5.60 ram.
C o p y r i g h t © 1979 Brain R e s e a r c h Publications Inc.mO031-9384/79/OlO007-03502.00/O
8
CAZALA AND GUENET
Procedure
4000.
Each ICSS cage contained two identical levers which could be partially or completely separated by a removable partition's device [31. On the first day, no electrical stimulation was administered, in order to study the '*operant activity" (i.e., the mean baseline bar pressing). Then, with an experimental procedure previously described [3] the ICSS threshold for each mouse was determined, by comparing the number of presses on the two levers when one was connected to the stimulator, and the other indicated the basal activity which was used as reference. The connections of the 2 levers were changed every 20 rain. The intensity of stimulation was increased by 5 t~A each day until it has reached 30 ~A. The cage was then separated into two parts, and the intensity of stimulation was increased by 10/zA each day from 40 ~A to 100/.cA in order to study ICSS performances on one lever, as well as the possible triggering of convulsions. Each ICSS session lasted 40 min. Each central stimulation lasted 0.2 see (100 Hz, sinewave). At the end of the experiments the animals were killed. Their brains were kept in a 10% Formalin solution. Frontal frozen sections were made (40 /zm) and stained with 1%0 thionin solution.
T
T
I-
¢-
~"
3000
T_ r... -J''"
!/
,,,o..o --o
ri
#
0 1,1
m
:E
2000
Z
'0
emme "~/C
N=9
o - - o e/c
N.n
RESULTS
Operant Activity In the absence of electrical stimulation, the number of presses on the two levers was slightly higher in the group of albino mice: mean=151 (SEM _ 18) than in the group of pigmented mice: mean= 120 (SEM ± 19). This difference, however, was not statistically significant (Student ttest= 1.06; 0.30>p>0.20).
so
6o
eo
,oo
INTENSITY (~JA) FIG. 1. Self-stimulation performances, as a function of stimulation intensity, in mice homozygote (c/c) or heterozygote (+/c) for the albino gene. Abscissae: s t i m u l ~ intensities (/~A). Ordinate: mean number of presses in 40 min+standard error.
ICSS Behavior Among the 23 implanted mice, 21 (12 c/c and 9 +/c) showed ICSS behavior. ICSS thresholds were lower in the +/c groups: mean value 16 ~A, median value 15 ~.A (interquartile ranges 10-20/~A), than in the eli group: mean value 21/,~A, median value 20/,rA (interquartile ranses 15-25/~A). The difference observed between the two groups was stat/sticaily significant (Mann Whitney U test: U 12/9=80.6; 0.05>p>0.02). At all current intensities the mean leSS performances of the +/c group was superior to that of the c/c mice (Fig. I). This difference was confirmed by a trend analysis [151: F(1,19) =6.81; 0.02>p >0.01.
Con vulsions Convulsions were found to occur more frequently in albino mice (5/12) during ICSS (initiated at a mean threshold value of 98/~A), as opposed to I/9 of the pigmented mice (initiated at a threshold value of 100 /~A). However the difference in the frequency of convulsions between the tw'x) groups was not significant (X ~= 1.09, 0.30>p>0.20). All the convulsions observed were of minor type, composed solely of a clonic phase.
Histological Control Histological control revealed that in -all the animals that exhibited ICSS behavior the electrode had been correctly
implanted in the ventral ~ of the L H, in a frontal plane cormspondip41 to the third p o ~ r i o r part o f the v ¢ ~ medialis nucleus of the hylPothalamus. Scatter was id~tical among c/c and +/c mice. In the two mice (I +/c and 1 c/c) which did not self-stimulate the electrode was beneath the brain. DISCUSSION
The two groups of mice studied have been shown to exhibit very different ICSS responses. Since these two coisogenic groups differ only at the albino locus, the different behavioral pattern that we have observed can only be correlated to the pleiotropic effect of this mutation. These results have prompted us to make the following comments: First, it has been frequently stated that the poorer i~rformances observed in the albino mice after training was due to a reduced motor activity since the absence of reticular pigments in these animals results in the photophobic behavior patterns [ 13]. Several authors have also observed that these behavioral differences between albino and pigmented mice can be abolished if the intensity of light is reduced [4, 8, 14]. Our study shows that in spite of a different behavioral pattern concerning the ICSS phenomenon, the mice of the two coisogenic groups display a similar operant activity (un-
S E L F - S T I M U L A T I O N IN A L B I N O MICE
9
der normal natural day light). The lower ICSS performances of the albino IFoup is thus unrelated to the locomotor activity and to the photophobia. It is likely to consider that the behavioral differences which we measured in the present study correspond to changes occurring in the functioning o f the central nervous system associated with the albino mutation. On the other hand, in a series of previous studies [2,3], we have observed lower ICSS thresholds and higher performances in nonpigmented animals. The present study however appears conflicting as we have observed higher thresholds and lower performances. Two hypotheses could explain these results: (1) Either an "inhibitive" influence o f the albino mutation also exists in the animals of the BALB/c inbred strain and in some animals of the backcross progenies B A L B / c × F I but remains unde-
tected because of an intense ICSS behavior determined by other genomic components. (2) Or, this "inhibitive" influence does not occur in the course of our study because of the effects of the genetic background on the mutant gene [ I 1]. Various data obtained by some authors for other pleiotropic effects of the albino locus on behavioral traits [9, 10, 12] seem to agree with the latter hypothesis.
ACKNOWLEDGEMENTS We wish to thank Mrs. A. M. Perret, J. Ducout and A. Zielinski for their technical assistance. This investigation was supported by the C.N.R.S. (ERA No. 416).
REFERENCES 1. Cazala, P., Y. Cazals and B. Cardo. Hypothalamic selfstimulation in three inbred strains of mice. Brain Res. 81: 159167, 1974. 2. Cazala, P. and J. L. Guenet. The role of genetic factors in the determination of self-stimulation hehaviour in the mouse: back-cross analysis. Behav. Proc. 1: 93-96, 1976. 3. Cazala, P., J. L. Gnenet et B. Cardo. D6terminisme g~n~tique des m~canismes contr61ant le comportement d'autostimulation chaz la souris: analyse prt,qiminaire. C.r. hebd. S~anc. Acad. Sci., Paris 2'/8: 2657-2660, 1974. 4. DeFries, J. C., J. P. Hegman and M. W. Weir. Open-field hehavior in mice: Evidence for a major gene effect mediated by the visual system. Science 154: 1577-1579, 1966. 5. Fuller, J. L. Effects of the albinos Bene upon behavior in mice. Anim. Behav. 15: 467--470, 1967. 6. Henry, K. R. and M. M. Haythorn. Albinism and auditory function in the laboratory mouse. I. Effects of single 8ene substitutions on auditory physiology, andiogenic seizures and developmontal processes. Behav. Genet. $: 137-149, 1975. 7. Henry, K. R. and K. Schlesinger. Effect of the albino and dilute loci on mouse behavior. J. comp. physiol. Psycho/. 84: 430--435, 1973.
8. McCiearn, G. E. Strain differences in activity of mice: influence of illumination. J. comp. physiol. Psychol. 53: 142-143, 1960. 9. Owen, K., D. D. Thiessen and G. Lindzey. Acrophobic and photophobic responses associated with the albino locus in mice. Behav. Genet. !:249-255, 1971. 10. Rhoades, R. W. and K. R. Henry. Effects of single albino gene substitutions on the performance of mice in a compound avoidance-discrimination task. Physiol. Behav. 19: 87-92, 1977. 11. Rouhertoux, P. et M. Carlier. G~nOtiqae et Comportement. edited by Masson. Paris: 1976, 227 p. 12. Tyler, P. A. Coat color differences and runway learning in mice. Behav. Genet. !1: 149--155, 1970. 13. Wahlsten, D. Genetic experiments with animal learning: a critical review. Behav. Biol. 7: 148--182, 1972. 14. Wahlsten, D. Contribution of the gene albinism (c) and retinal degeneration (rd) to a strain-by-training procedure interaction in avoidance learning. Behav. Genet. 3: 303--316, 1973. 15. Wirier, B. J. Statistical Principles hi Experimental De.~#,m. International student, edited by Kogakusha. New York: McGraw Hill, 1971, 907 pp. 16. Winston, H. D. and G. Lindzey. Albinism and water escape performance in the mouse. Science 144: 189-191, 1964.
Effects of the Albino Gene on Self-Stimulation Behavior in the Lateral Hypothalamus in the Mouse PIERRE CAZALA AND JEAN-LOUIS GUENET
Laboratoire de Psychophysiologie, institut de Biologie Animale, Universit~ de Bordeaux !, Avenue des Facult~s 33405 Talence Cedex France and lnstitut Pasteur, D~partement de Biologie Mol~culaire, 25 Rue du Docteur Roux, 75015 Paris Cedex France ( R e c e i v e d 30 M a r c h 1978) CAZALA, P. AND J. L. GUENET. Effects of the albino gene on se(f-stimulation behavior in the lateral hypothalamus in the mouse. PHYSIOL. BEHAV. 22(1) 7-9, 1979.-- Intraeranial self-stimulation behavior (ICSS) in the lateral hypothalamus has been studied in two coisogenic mice groups differing at the albino locus. Heterozygous mice (+/c) had significant lower ICSS thresholds and higher performances than homozygous mice (c/c). Both groups however showed identical sensitivity for the triggering of convulsions suspending ICSS behavior. These results suggest that the albino mutation correlates with some changes in the normal function of the central nervous system. Self-stimulation
Lateral hypothalamus
Albino mutation
AS A RESULT of the large number of investigations which have been carried out concerning the gene which causes albinism, it is now possible to distinguish between albino and nonalbino mice from their anatomical, physiological, biochemical and behavioral features (for review see [6]). Among the behavioral pleiotropic effects which seem to be controlled by the c locus, it has been demonstrated that strains of albino mice in a water maze obtain lower learning scores than strains of pigmented mice [16]. A similar observation has also been made for an albino mutant of the C57BL/6 strain [5]. It has also been reported that the acquisition of a visual discrimination [10], as well as avoidance behavior [7,10] is slower in these albino mutants than it is in pigmented littermatcs. During the course of work which has been carried out to estimate the degree of complexity in the genetic control of intracranial self-stimulation behavior (ICSS) in the mouse, some observations were made which suggested that the albino gene could be involved in this particular behavioral pattern. From studies which involved genetic crosses between the related strains BALB/c and DBA/2, we have been able to establish (1) that the albino strain BALB/c had the lowest ICSS thresholds, and the highest level of performances [3], (2) the F1 generation had a very similar ICSS pattern as the DBA/2 parental strain; also, we have studied ICSS in the backcross progeny between the FI generation and the "recessive" strain BALB/c [2]. We have observed that among the offspring of this cross which self-stimulated vigorously like BALB/c, the majority (9 out of 11) were unpigmented. The aim of the present study is to try to determine what
Coisogenic mice
are the eventual effects of albinism on the various ICSS parameters. METHOD
Animals and Surgery Twenty-nine male mice 10 to 12 weeks old were used in this study. These animals were fwst generation hybrids resulting from a cross between BALB/c J hornozyguus albino females (c/c) and C57BL6/Jc males beterozyguus for albinism (+/c). The resulting FI generation consisted of two groups of coisogenic mice, one of which was albino (c/c), and the other was hcterozygous for c (+/c). The two groups showed an identical body weight: +/c mean=26.7 g (SEM +__0.8); c/c mean=27.3 g (SEM *-- 0.9). Twenty-three mice (13 c/c and 10 +/c) were anesthetised by an intraperitoneal injection of sodium thiopental (80 mg/kg). A bipolar electrode of two twisted platinum wires of 0.09 mm dia. was implanted under stereotaxic control into each mouse. The electrode was implanted into the ventral portion of the lateral hypothalamus (LH) which is the most sensitive area for the triggering of convulsions during ICSS behavior [1]. The stereotaxic coordinates used were previously determined in 3 mice from each group. +/c: antero-posterior distance refering to interaural line +2.00 ram; lateral distance refering to sagittal line - 1.00 ram; depth from the surface of the skull +5.70 nun. c/c: antero-posteriority + 1.90 ram; laterality _ 1.0 ram; depth +5.60 ram.
C o p y r i g h t © 1979 Brain R e s e a r c h Publications Inc.mO031-9384/79/OlO007-03502.00/O
8
CAZALA AND GUENET
Procedure
4000.
Each ICSS cage contained two identical levers which could be partially or completely separated by a removable partition's device [31. On the first day, no electrical stimulation was administered, in order to study the '*operant activity" (i.e., the mean baseline bar pressing). Then, with an experimental procedure previously described [3] the ICSS threshold for each mouse was determined, by comparing the number of presses on the two levers when one was connected to the stimulator, and the other indicated the basal activity which was used as reference. The connections of the 2 levers were changed every 20 rain. The intensity of stimulation was increased by 5 t~A each day until it has reached 30 ~A. The cage was then separated into two parts, and the intensity of stimulation was increased by 10/zA each day from 40 ~A to 100/.cA in order to study ICSS performances on one lever, as well as the possible triggering of convulsions. Each ICSS session lasted 40 min. Each central stimulation lasted 0.2 see (100 Hz, sinewave). At the end of the experiments the animals were killed. Their brains were kept in a 10% Formalin solution. Frontal frozen sections were made (40 /zm) and stained with 1%0 thionin solution.
T
T
I-
¢-
~"
3000
T_ r... -J''"
!/
,,,o..o --o
ri
#
0 1,1
m
:E
2000
Z
'0
emme "~/C
N=9
o - - o e/c
N.n
RESULTS
Operant Activity In the absence of electrical stimulation, the number of presses on the two levers was slightly higher in the group of albino mice: mean=151 (SEM _ 18) than in the group of pigmented mice: mean= 120 (SEM ± 19). This difference, however, was not statistically significant (Student ttest= 1.06; 0.30>p>0.20).
so
6o
eo
,oo
INTENSITY (~JA) FIG. 1. Self-stimulation performances, as a function of stimulation intensity, in mice homozygote (c/c) or heterozygote (+/c) for the albino gene. Abscissae: s t i m u l ~ intensities (/~A). Ordinate: mean number of presses in 40 min+standard error.
ICSS Behavior Among the 23 implanted mice, 21 (12 c/c and 9 +/c) showed ICSS behavior. ICSS thresholds were lower in the +/c groups: mean value 16 ~A, median value 15 ~.A (interquartile ranges 10-20/~A), than in the eli group: mean value 21/,~A, median value 20/,rA (interquartile ranses 15-25/~A). The difference observed between the two groups was stat/sticaily significant (Mann Whitney U test: U 12/9=80.6; 0.05>p>0.02). At all current intensities the mean leSS performances of the +/c group was superior to that of the c/c mice (Fig. I). This difference was confirmed by a trend analysis [151: F(1,19) =6.81; 0.02>p >0.01.
Con vulsions Convulsions were found to occur more frequently in albino mice (5/12) during ICSS (initiated at a mean threshold value of 98/~A), as opposed to I/9 of the pigmented mice (initiated at a threshold value of 100 /~A). However the difference in the frequency of convulsions between the tw'x) groups was not significant (X ~= 1.09, 0.30>p>0.20). All the convulsions observed were of minor type, composed solely of a clonic phase.
Histological Control Histological control revealed that in -all the animals that exhibited ICSS behavior the electrode had been correctly
implanted in the ventral ~ of the L H, in a frontal plane cormspondip41 to the third p o ~ r i o r part o f the v ¢ ~ medialis nucleus of the hylPothalamus. Scatter was id~tical among c/c and +/c mice. In the two mice (I +/c and 1 c/c) which did not self-stimulate the electrode was beneath the brain. DISCUSSION
The two groups of mice studied have been shown to exhibit very different ICSS responses. Since these two coisogenic groups differ only at the albino locus, the different behavioral pattern that we have observed can only be correlated to the pleiotropic effect of this mutation. These results have prompted us to make the following comments: First, it has been frequently stated that the poorer i~rformances observed in the albino mice after training was due to a reduced motor activity since the absence of reticular pigments in these animals results in the photophobic behavior patterns [ 13]. Several authors have also observed that these behavioral differences between albino and pigmented mice can be abolished if the intensity of light is reduced [4, 8, 14]. Our study shows that in spite of a different behavioral pattern concerning the ICSS phenomenon, the mice of the two coisogenic groups display a similar operant activity (un-
S E L F - S T I M U L A T I O N IN A L B I N O MICE
9
der normal natural day light). The lower ICSS performances of the albino IFoup is thus unrelated to the locomotor activity and to the photophobia. It is likely to consider that the behavioral differences which we measured in the present study correspond to changes occurring in the functioning o f the central nervous system associated with the albino mutation. On the other hand, in a series of previous studies [2,3], we have observed lower ICSS thresholds and higher performances in nonpigmented animals. The present study however appears conflicting as we have observed higher thresholds and lower performances. Two hypotheses could explain these results: (1) Either an "inhibitive" influence o f the albino mutation also exists in the animals of the BALB/c inbred strain and in some animals of the backcross progenies B A L B / c × F I but remains unde-
tected because of an intense ICSS behavior determined by other genomic components. (2) Or, this "inhibitive" influence does not occur in the course of our study because of the effects of the genetic background on the mutant gene [ I 1]. Various data obtained by some authors for other pleiotropic effects of the albino locus on behavioral traits [9, 10, 12] seem to agree with the latter hypothesis.
ACKNOWLEDGEMENTS We wish to thank Mrs. A. M. Perret, J. Ducout and A. Zielinski for their technical assistance. This investigation was supported by the C.N.R.S. (ERA No. 416).
REFERENCES 1. Cazala, P., Y. Cazals and B. Cardo. Hypothalamic selfstimulation in three inbred strains of mice. Brain Res. 81: 159167, 1974. 2. Cazala, P. and J. L. Guenet. The role of genetic factors in the determination of self-stimulation hehaviour in the mouse: back-cross analysis. Behav. Proc. 1: 93-96, 1976. 3. Cazala, P., J. L. Gnenet et B. Cardo. D6terminisme g~n~tique des m~canismes contr61ant le comportement d'autostimulation chaz la souris: analyse prt,qiminaire. C.r. hebd. S~anc. Acad. Sci., Paris 2'/8: 2657-2660, 1974. 4. DeFries, J. C., J. P. Hegman and M. W. Weir. Open-field hehavior in mice: Evidence for a major gene effect mediated by the visual system. Science 154: 1577-1579, 1966. 5. Fuller, J. L. Effects of the albinos Bene upon behavior in mice. Anim. Behav. 15: 467--470, 1967. 6. Henry, K. R. and M. M. Haythorn. Albinism and auditory function in the laboratory mouse. I. Effects of single 8ene substitutions on auditory physiology, andiogenic seizures and developmontal processes. Behav. Genet. $: 137-149, 1975. 7. Henry, K. R. and K. Schlesinger. Effect of the albino and dilute loci on mouse behavior. J. comp. physiol. Psycho/. 84: 430--435, 1973.
8. McCiearn, G. E. Strain differences in activity of mice: influence of illumination. J. comp. physiol. Psychol. 53: 142-143, 1960. 9. Owen, K., D. D. Thiessen and G. Lindzey. Acrophobic and photophobic responses associated with the albino locus in mice. Behav. Genet. !:249-255, 1971. 10. Rhoades, R. W. and K. R. Henry. Effects of single albino gene substitutions on the performance of mice in a compound avoidance-discrimination task. Physiol. Behav. 19: 87-92, 1977. 11. Rouhertoux, P. et M. Carlier. G~nOtiqae et Comportement. edited by Masson. Paris: 1976, 227 p. 12. Tyler, P. A. Coat color differences and runway learning in mice. Behav. Genet. !1: 149--155, 1970. 13. Wahlsten, D. Genetic experiments with animal learning: a critical review. Behav. Biol. 7: 148--182, 1972. 14. Wahlsten, D. Contribution of the gene albinism (c) and retinal degeneration (rd) to a strain-by-training procedure interaction in avoidance learning. Behav. Genet. 3: 303--316, 1973. 15. Wirier, B. J. Statistical Principles hi Experimental De.~#,m. International student, edited by Kogakusha. New York: McGraw Hill, 1971, 907 pp. 16. Winston, H. D. and G. Lindzey. Albinism and water escape performance in the mouse. Science 144: 189-191, 1964.
