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The asymmetry of the habenular nuclei of female and male frogs in spring and in winter
Brain Research, 517 (1990) 251-255
251
Elsevier BRES 15496
The asymmetry of the habenular nuclei of female and male frogs in spring and in winter M. Kemali, V. Guglielmotti and L. Fiorino lstituto di Cibernetica del CNR, Naples (Italy)
(Accepted 27 October 1989) Key words: Habenula; Male; Female; Frog; Morphometry
Male and female frog brains stained according to the Nissl method and cut transversely, 15 ~m thick, at the level of the habenular nuclei, were investigated in spring and winter. Right and left habenular nuclei were examined. The volume and the standard deviations were calculated in each portion of the habenular nuclei investigated. The frog habenula consists of a single cell group: one on the right and two on the left side of the brain which differ, among themselves, both in the volumes of the neuropil and of the cellular ring. Functional corollaries of this striking asymmetry are still unknown. However, female and male frogs' habenular nuclei are longer and larger in spring - - when frogs are sexually active - - than in winter. We propose that structural brain asymmetries may be sex linked and may be induced by steroid hormonal effect in the central nervous system. INTRODUCTION A s y m m e t r y is a fundamental feature of neuroanatomy 1°. In explaining right-left differences in cerebral activities, Berlucchi! suggested that their morphological substrate is represented by microanatomical asymmetries (different extension or different neural density) of bilateral brain structures. The use of quantitative m o r p h o m e t r y has revealed morphological asymmetries between the two halves of the brain which otherwise would have passed unnoticed and, even more remarkable, has disclosed sexual dimorphism in several brain structures 12. Such a link between gender and brain has been explicitly proposed by Geschwind and Behan 4, who suggested that testosteronemediated neurohumoral interaction in intrauterine life may induce sex-dependent differences in certain neuronal assemblies which result in different cerebral organization of the brain of adult males and females. Lateralization of brain functions may thus have different peculiarities depending on whether it occurs in males or females. In human psychopathology the lateralized disorganization of hemispheric functions, which may underlie certain mental disorders, has indeed been reported to have different characteristics in the two sexes 3. Sex-dependent structural asymmetries in the brain of vertebrates are thus of wide interest. See, for instance, the sexually dimorphic nucleus of the amphibian amygdala and its seasonal variability 11.
Recently, a sex-dependent asymmetry of the medial habenular nucleus of the chicken has been morphometrically established 5. In a previous paper 2 we reported that the habenular complex of low vertebrates is strikingly asymmetric, the left one being more lobated than its fight counterpart. The presence of what was considered a habenular 'extra nucleus' on the left side of the brain only, having histological features different from the remaining cell population, was related to the changes occurring during phylogenesis between the habenulae and other epithalamic structures, viz. the pineal extracranial complex 6. That study 2 was conducted, however, only from a qualitative point of view, disregarding the sex of the animal and the season of the year during which the observations were made. In the study here reported we examined male and female frogs in two seasons, in order to see the effect of the circulating endogenous hormones by comparing the habenular nuclei of the two sexes; in addition, we compared, always from the qualitative point of view, the right habenula and the lateral portion of the left habenula in order to see if an asymmetry might also exist between these two portions. MATERIALS AND METHODS Forty frogs of the species Rana esculenta were used. They were divided in two groups (20 males and 20 females). The observations were carried out in spring and in winter when the frogs are sexually active and sexually quiescent, respectively. The animals of both groups were anaesthetized in a water solution
Correspondence: M. Kemali, Istituto di Cibernetica, 80072 Arco Felice, Naples, Italy.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B,V. (Biomedical Division)
252 TABLE I Arithmetical media (in microns) of the length of each portion of the frog habenular nuclei in the two seasons and in the two sexes
Note that in spring frogs, which are sexually active, the habenula is longer in the female (underlined) than it is in the other cases. Right habenula
Left habenula Medial portion
Lateral portion
Spring 10 Females 10 Males
447 396
408 363
409 365
Winter 10 Females 10 Males
395 390
339 328
355 324
animals, belonging to both sexes and taken in both seasonal conditions, were drawn on a Visopan (Reichert) microscope and observed afterwards in a Zeiss light microscope. Then the area occupied by the cells and that occupied by the neuropii of the right and left habenula was calculated on a computer equipped to calculate the area of irregular figures. The area multiplied for 15 (the thickness of the section) gave the value of the volume of the section. A statistical analysis (arithmetical media, standard deviation) was carried out for the external (cellular) and the internal (neuropil) compartment as reported on Table II. A schematic tridimensional model of the frog habenulae was constructed from 2.5-mm-thick wax plates, each conforming to a serial section of the right or left habenula and drawn, on a projecting microscope, at a magnification of x220.
RESULTS
(1:3000) of tricaine methanesulphonate (MS 222, Sandoz). Then the frogs were perfused first with saline (NaCI 0.75%) in order to wash away the blood circulating in the vessels, and then with phosphatebuffered formaline (pH 7.4). Brains were then removed immediately from the cranium and fixed with the same fixative used for perfusion (formaline) for at least 5 days. The brains were embedded in paraffin and sectioned at the level of the habenular nuclei in serial frontal sections of 15/~m thickness. Care was taken to orient the length of the brain strictly parallel to the length of the animal in order to have the sections perfectly oriented in the transverse direction. The slides were stained according to the Nissl method. The length of each portion of the left and of the right habenula was determined in a light microscope, multiplied for 15/zm in order to obtain the actual length of the habenular nuclei. The results are reported on Table I. The boundaries of the various portions of the habenulae of all
In males and females, the h a b e n u l a is formed by an undivided, single cell group on the right, and a bipartite cell mass on the left side of the brain. The left habenula is composed of a medial and a lateral part. The medial portion of the left h a b e n u l a consists of two parts which, extending caudally, eventually fuse, giving origin to a massive nucleus which is larger than the right habenula. In the side opposite to the ventricle, the left h a b e n u l a gives origin to a small lateral nucleus which ends caudally to the fused medial nucleus of the left h a b e n u l a and continues as far caudally as the right h a b e n u l a (Fig. 1). We have followed the shapes of the h a b e n u l a r nuclei of male and female frogs, in the two seasons of the year, in a Zeiss microscope, and have counted the sections in which the various portions of the h a b e n u l a e appeared.
TABLE II Arithmetical media of the volume of the right and the left habenular nuclei (in I~m ) of l Ofemale and 10 male frogs in spring and in winter
Standard deviation (S.D.) is shown. Med. Port., medial portion; Lat. Port., lateral portion. Right habenula
Left habenula S.D.
Med. Port.
S.D.
Lat. Port.
S.D.
Spring female Total volume Cellular volume Neuropil volume
7,122,137 3,838,611 3,283,525
366,447 308,043 281,692
6,683,760 3,643,097 3,040,662
659,232 715,977 540,432
4,821,025 2,848,234 1,972,791
671,076 435,520 260,260
Spring male Total volume Cellular volume Neuropil volume
7,240,757 4,013,648 3,227,108
578,250 251,693 367,777
6,426,265 3,549,214 2,877,051
719,222 425,193 352,151
4,491,094 2,562,090 1,892,014
415,774 252,026 198,969
Winter female Total volume Cellular volume Neuropil volume
6,183,600 3,376,577 2,807,022
405,174 191,299 226,534
5,418,548 2,838,265 2,580,282
731,356 376,487 408,934
3,953,948 2,236,448 1,717,500
443,064 262,343 220,819
Winter male Total volume Cellular volume Neuropil volume
5,822,505 3,276,367 2,546,137
140,685 104,424 87,135
5,526,247 2,989,612 2,536,635
704,904 477,715 386,178
3,993,937 2,368,687 1,625,250
412,506 246,699 186,128
253
Fig. 1. right ot third ventricle; CP, choroidal piexus; DH, dorsal"habenula; VH, ventral habenula~
Table I lists the lenght of the various portions of the habenulae as calculated from these numbers. It is evident that the habenular nuclei are longer in the females and, to a lesser degree in males, in spring than in winter. In order to see exactly the beginning of the lateral nucleus of the left habenula, with reference also to the right habenula, we have made a tridimensional model reconstructing the habenular nuclei in wax sheets. Photographs of the model appear in Fig 2. In Table II we have reported the values of the volume of the inner portion (neuropil) and the outer portion (cellular) of the habenular nuclei. The data have been used for a statistical analysis and the standard deviations have been reported. DISCUSSION The present study has convincingly demonstrated a pronounced asymmetry in the size and composition of the frog's habenular complex. The largest nucleus is the medial subnucleus of the left habenula (adjacent to the third ventricle), while the smallest one is that disposed on the left habenula, laterally to the huge medial subnu-
the III,
cleus. In addition, the cellular area is bigger than the neuropil area both in the two subnuclei of the left and in the right habenular nucleus. Furthermore, from the results it is clear that in spring, especially in females, but to a lesser degree also in males, the habenulae are longer (see Table I) and larger (see Table II) than in winter. Since in spring the frogs are sexually active, it is reasonable to think that the hormones responsible for reproduction have an effect of some sort on the size of the habenular nuclei, especially on the medial portion of the left habenula, the one close to the third ventricle. It is of interest to recall that a neuroendocrine function of the ependymal part of the habenulae lining the third ventricle was suggested a. The injection of horseradish peroxidase in the habenular nuclei 7 showed the connections of these nuclei, but shed no light on the asymmetry of this zone, which thus far remains unexplained 7. Having examined frogs of both sexes, in spring and in winter, we propose that structural brain asymmetries may be sex-linked and may be induced by steroid effects occurring during development in the central nervous system (cf. ref. 9).
254
Fig. 2. a: model reconstruction on wax of the habenular nuclei, done from serial sections of Nissl-stained material. The reconstruction is seen from the top of the epithalamus. On the left side, the left habenula; on the right side, the right habenula. The distance of the III ventricle as well as the habenulae, are of the real size. The left habenula is formed by a lateral (L) and a medial (M) portion. The lateral portion is smaller than the medial portion. This is better visible in b, which is a ventral view of the wax model of the left habenula, seen from the bottom of the epithalamus. A cut was done in order to see the internal of the habenulae. The arrows (a and b) mark the place where the cut was done in order to obtain c and d. From these we can see that the right habenula (d) is formed by a single nucleus. The left habenula (c) is formed by a medial (M) and a lateral (L) portion. This asymmetry is the same for females and males, in winter and in spring. The wax reconstruction permits also to evaluate the shapes of the different portions of the habenular nuclei and their mutual relationship, x220. R, rostral; C, caudal, b: ventral view of the wax-reconstructed model of the left habenula seen from the bottom of the epithalamus. We can see, from this side, the small extension of the lateral (L) portion of the habenular nuclei, in comparison with the medial (M). ×220. c,d: cut of the wax-reconstructed model of the habenular nuclei at the level of the arrows in a and b. Note the difference between the left (c) and the right (d) internal habenular nuclei, x220. M, medial portion; L, lateral portion; V, ventral, D, dorsal.
255 REFERENCES 1 Berlucchi, G., Basi nervose del comportamento: alcuni sviluppi recenti, Fed. Med., XXXVI (1983) 213-220. 2 Braitenberg, V. and Kemali, M., Exception to bilateral symmetry in lower vertebrates, J. Comp. Neurol., 138 (1970) 137-146. 3 Flor-Henry, P., Functional hemispheric asymmetry and psychopathology, lntegr. Psychiatry, 1 (1983) 46-52. 4 Geshwind, N. and Behan, P., Left-handedness: association with immune disease, migrain, and developmental learning disorder, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 5097-5100. 5 Gurusijnge, C.J. and Ehrlich, D., Sex-dependent structural asymmetry of the medial habenular nucleus of the chicken brain, Cell Tissue Res., 240 (1985) 149-152. 6 Kemali, M., The non-retinal photoreceptive system of the frog. An hypothesis for epithalamic asymmetries, Z. Mikrosk.Anatom. Forsch., 103 (1989) 353-358. 7 Kemali, M., Guglielmotti, V. and Gioffrr, D., Neuroanatomical identification of the frog habenular connections using Horserad-
ish Peroxidase (HRP), Exp. Brain Res., 38 (1980) 341-347. 8 Kumar, K. and Anand-Kumar, T.C., The habenular ependyma: a neuroendocrine component of the epithalamus in the Rhesus monkey. In W.E. Stumpf and L.D. Grant (Eds.), Anatomical Neuroendocrinology, Karger, Basel 1975, pp. 49-52. 9 Merrell, J.I. and Pfaff, D.W., A neuroendocrine approach to brain function: localization of sex steroid concentrating cells in vertebrate brain, Am. Zool., 18 (1978) 447-460. 10 Sherman, G.F., Galabuda, A.M. and Geshwind, N., Neuroanatomical asymmetries in non-human species, Trends Neurosci., 5 (1982) 429-431. 11 Takami, S. and Urano, A., The volume of the toad medial amygdala - - anterior preoptic complex is sexually dimorphic and seasonally variable, Neurosci. Lett., 44 (1984) 253-258. 12 Van Eden C.G., Uylings, H.B.M. and Van Pelt, J., Sexdifference and left-right asymmetries in the prefrontal cortex during postnatal development in the rat, Dev. Brain Res., 12 (1984) 146-153.
251
Elsevier BRES 15496
The asymmetry of the habenular nuclei of female and male frogs in spring and in winter M. Kemali, V. Guglielmotti and L. Fiorino lstituto di Cibernetica del CNR, Naples (Italy)
(Accepted 27 October 1989) Key words: Habenula; Male; Female; Frog; Morphometry
Male and female frog brains stained according to the Nissl method and cut transversely, 15 ~m thick, at the level of the habenular nuclei, were investigated in spring and winter. Right and left habenular nuclei were examined. The volume and the standard deviations were calculated in each portion of the habenular nuclei investigated. The frog habenula consists of a single cell group: one on the right and two on the left side of the brain which differ, among themselves, both in the volumes of the neuropil and of the cellular ring. Functional corollaries of this striking asymmetry are still unknown. However, female and male frogs' habenular nuclei are longer and larger in spring - - when frogs are sexually active - - than in winter. We propose that structural brain asymmetries may be sex linked and may be induced by steroid hormonal effect in the central nervous system. INTRODUCTION A s y m m e t r y is a fundamental feature of neuroanatomy 1°. In explaining right-left differences in cerebral activities, Berlucchi! suggested that their morphological substrate is represented by microanatomical asymmetries (different extension or different neural density) of bilateral brain structures. The use of quantitative m o r p h o m e t r y has revealed morphological asymmetries between the two halves of the brain which otherwise would have passed unnoticed and, even more remarkable, has disclosed sexual dimorphism in several brain structures 12. Such a link between gender and brain has been explicitly proposed by Geschwind and Behan 4, who suggested that testosteronemediated neurohumoral interaction in intrauterine life may induce sex-dependent differences in certain neuronal assemblies which result in different cerebral organization of the brain of adult males and females. Lateralization of brain functions may thus have different peculiarities depending on whether it occurs in males or females. In human psychopathology the lateralized disorganization of hemispheric functions, which may underlie certain mental disorders, has indeed been reported to have different characteristics in the two sexes 3. Sex-dependent structural asymmetries in the brain of vertebrates are thus of wide interest. See, for instance, the sexually dimorphic nucleus of the amphibian amygdala and its seasonal variability 11.
Recently, a sex-dependent asymmetry of the medial habenular nucleus of the chicken has been morphometrically established 5. In a previous paper 2 we reported that the habenular complex of low vertebrates is strikingly asymmetric, the left one being more lobated than its fight counterpart. The presence of what was considered a habenular 'extra nucleus' on the left side of the brain only, having histological features different from the remaining cell population, was related to the changes occurring during phylogenesis between the habenulae and other epithalamic structures, viz. the pineal extracranial complex 6. That study 2 was conducted, however, only from a qualitative point of view, disregarding the sex of the animal and the season of the year during which the observations were made. In the study here reported we examined male and female frogs in two seasons, in order to see the effect of the circulating endogenous hormones by comparing the habenular nuclei of the two sexes; in addition, we compared, always from the qualitative point of view, the right habenula and the lateral portion of the left habenula in order to see if an asymmetry might also exist between these two portions. MATERIALS AND METHODS Forty frogs of the species Rana esculenta were used. They were divided in two groups (20 males and 20 females). The observations were carried out in spring and in winter when the frogs are sexually active and sexually quiescent, respectively. The animals of both groups were anaesthetized in a water solution
Correspondence: M. Kemali, Istituto di Cibernetica, 80072 Arco Felice, Naples, Italy.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B,V. (Biomedical Division)
252 TABLE I Arithmetical media (in microns) of the length of each portion of the frog habenular nuclei in the two seasons and in the two sexes
Note that in spring frogs, which are sexually active, the habenula is longer in the female (underlined) than it is in the other cases. Right habenula
Left habenula Medial portion
Lateral portion
Spring 10 Females 10 Males
447 396
408 363
409 365
Winter 10 Females 10 Males
395 390
339 328
355 324
animals, belonging to both sexes and taken in both seasonal conditions, were drawn on a Visopan (Reichert) microscope and observed afterwards in a Zeiss light microscope. Then the area occupied by the cells and that occupied by the neuropii of the right and left habenula was calculated on a computer equipped to calculate the area of irregular figures. The area multiplied for 15 (the thickness of the section) gave the value of the volume of the section. A statistical analysis (arithmetical media, standard deviation) was carried out for the external (cellular) and the internal (neuropil) compartment as reported on Table II. A schematic tridimensional model of the frog habenulae was constructed from 2.5-mm-thick wax plates, each conforming to a serial section of the right or left habenula and drawn, on a projecting microscope, at a magnification of x220.
RESULTS
(1:3000) of tricaine methanesulphonate (MS 222, Sandoz). Then the frogs were perfused first with saline (NaCI 0.75%) in order to wash away the blood circulating in the vessels, and then with phosphatebuffered formaline (pH 7.4). Brains were then removed immediately from the cranium and fixed with the same fixative used for perfusion (formaline) for at least 5 days. The brains were embedded in paraffin and sectioned at the level of the habenular nuclei in serial frontal sections of 15/~m thickness. Care was taken to orient the length of the brain strictly parallel to the length of the animal in order to have the sections perfectly oriented in the transverse direction. The slides were stained according to the Nissl method. The length of each portion of the left and of the right habenula was determined in a light microscope, multiplied for 15/zm in order to obtain the actual length of the habenular nuclei. The results are reported on Table I. The boundaries of the various portions of the habenulae of all
In males and females, the h a b e n u l a is formed by an undivided, single cell group on the right, and a bipartite cell mass on the left side of the brain. The left habenula is composed of a medial and a lateral part. The medial portion of the left h a b e n u l a consists of two parts which, extending caudally, eventually fuse, giving origin to a massive nucleus which is larger than the right habenula. In the side opposite to the ventricle, the left h a b e n u l a gives origin to a small lateral nucleus which ends caudally to the fused medial nucleus of the left h a b e n u l a and continues as far caudally as the right h a b e n u l a (Fig. 1). We have followed the shapes of the h a b e n u l a r nuclei of male and female frogs, in the two seasons of the year, in a Zeiss microscope, and have counted the sections in which the various portions of the h a b e n u l a e appeared.
TABLE II Arithmetical media of the volume of the right and the left habenular nuclei (in I~m ) of l Ofemale and 10 male frogs in spring and in winter
Standard deviation (S.D.) is shown. Med. Port., medial portion; Lat. Port., lateral portion. Right habenula
Left habenula S.D.
Med. Port.
S.D.
Lat. Port.
S.D.
Spring female Total volume Cellular volume Neuropil volume
7,122,137 3,838,611 3,283,525
366,447 308,043 281,692
6,683,760 3,643,097 3,040,662
659,232 715,977 540,432
4,821,025 2,848,234 1,972,791
671,076 435,520 260,260
Spring male Total volume Cellular volume Neuropil volume
7,240,757 4,013,648 3,227,108
578,250 251,693 367,777
6,426,265 3,549,214 2,877,051
719,222 425,193 352,151
4,491,094 2,562,090 1,892,014
415,774 252,026 198,969
Winter female Total volume Cellular volume Neuropil volume
6,183,600 3,376,577 2,807,022
405,174 191,299 226,534
5,418,548 2,838,265 2,580,282
731,356 376,487 408,934
3,953,948 2,236,448 1,717,500
443,064 262,343 220,819
Winter male Total volume Cellular volume Neuropil volume
5,822,505 3,276,367 2,546,137
140,685 104,424 87,135
5,526,247 2,989,612 2,536,635
704,904 477,715 386,178
3,993,937 2,368,687 1,625,250
412,506 246,699 186,128
253
Fig. 1. right ot third ventricle; CP, choroidal piexus; DH, dorsal"habenula; VH, ventral habenula~
Table I lists the lenght of the various portions of the habenulae as calculated from these numbers. It is evident that the habenular nuclei are longer in the females and, to a lesser degree in males, in spring than in winter. In order to see exactly the beginning of the lateral nucleus of the left habenula, with reference also to the right habenula, we have made a tridimensional model reconstructing the habenular nuclei in wax sheets. Photographs of the model appear in Fig 2. In Table II we have reported the values of the volume of the inner portion (neuropil) and the outer portion (cellular) of the habenular nuclei. The data have been used for a statistical analysis and the standard deviations have been reported. DISCUSSION The present study has convincingly demonstrated a pronounced asymmetry in the size and composition of the frog's habenular complex. The largest nucleus is the medial subnucleus of the left habenula (adjacent to the third ventricle), while the smallest one is that disposed on the left habenula, laterally to the huge medial subnu-
the III,
cleus. In addition, the cellular area is bigger than the neuropil area both in the two subnuclei of the left and in the right habenular nucleus. Furthermore, from the results it is clear that in spring, especially in females, but to a lesser degree also in males, the habenulae are longer (see Table I) and larger (see Table II) than in winter. Since in spring the frogs are sexually active, it is reasonable to think that the hormones responsible for reproduction have an effect of some sort on the size of the habenular nuclei, especially on the medial portion of the left habenula, the one close to the third ventricle. It is of interest to recall that a neuroendocrine function of the ependymal part of the habenulae lining the third ventricle was suggested a. The injection of horseradish peroxidase in the habenular nuclei 7 showed the connections of these nuclei, but shed no light on the asymmetry of this zone, which thus far remains unexplained 7. Having examined frogs of both sexes, in spring and in winter, we propose that structural brain asymmetries may be sex-linked and may be induced by steroid effects occurring during development in the central nervous system (cf. ref. 9).
254
Fig. 2. a: model reconstruction on wax of the habenular nuclei, done from serial sections of Nissl-stained material. The reconstruction is seen from the top of the epithalamus. On the left side, the left habenula; on the right side, the right habenula. The distance of the III ventricle as well as the habenulae, are of the real size. The left habenula is formed by a lateral (L) and a medial (M) portion. The lateral portion is smaller than the medial portion. This is better visible in b, which is a ventral view of the wax model of the left habenula, seen from the bottom of the epithalamus. A cut was done in order to see the internal of the habenulae. The arrows (a and b) mark the place where the cut was done in order to obtain c and d. From these we can see that the right habenula (d) is formed by a single nucleus. The left habenula (c) is formed by a medial (M) and a lateral (L) portion. This asymmetry is the same for females and males, in winter and in spring. The wax reconstruction permits also to evaluate the shapes of the different portions of the habenular nuclei and their mutual relationship, x220. R, rostral; C, caudal, b: ventral view of the wax-reconstructed model of the left habenula seen from the bottom of the epithalamus. We can see, from this side, the small extension of the lateral (L) portion of the habenular nuclei, in comparison with the medial (M). ×220. c,d: cut of the wax-reconstructed model of the habenular nuclei at the level of the arrows in a and b. Note the difference between the left (c) and the right (d) internal habenular nuclei, x220. M, medial portion; L, lateral portion; V, ventral, D, dorsal.
255 REFERENCES 1 Berlucchi, G., Basi nervose del comportamento: alcuni sviluppi recenti, Fed. Med., XXXVI (1983) 213-220. 2 Braitenberg, V. and Kemali, M., Exception to bilateral symmetry in lower vertebrates, J. Comp. Neurol., 138 (1970) 137-146. 3 Flor-Henry, P., Functional hemispheric asymmetry and psychopathology, lntegr. Psychiatry, 1 (1983) 46-52. 4 Geshwind, N. and Behan, P., Left-handedness: association with immune disease, migrain, and developmental learning disorder, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 5097-5100. 5 Gurusijnge, C.J. and Ehrlich, D., Sex-dependent structural asymmetry of the medial habenular nucleus of the chicken brain, Cell Tissue Res., 240 (1985) 149-152. 6 Kemali, M., The non-retinal photoreceptive system of the frog. An hypothesis for epithalamic asymmetries, Z. Mikrosk.Anatom. Forsch., 103 (1989) 353-358. 7 Kemali, M., Guglielmotti, V. and Gioffrr, D., Neuroanatomical identification of the frog habenular connections using Horserad-
ish Peroxidase (HRP), Exp. Brain Res., 38 (1980) 341-347. 8 Kumar, K. and Anand-Kumar, T.C., The habenular ependyma: a neuroendocrine component of the epithalamus in the Rhesus monkey. In W.E. Stumpf and L.D. Grant (Eds.), Anatomical Neuroendocrinology, Karger, Basel 1975, pp. 49-52. 9 Merrell, J.I. and Pfaff, D.W., A neuroendocrine approach to brain function: localization of sex steroid concentrating cells in vertebrate brain, Am. Zool., 18 (1978) 447-460. 10 Sherman, G.F., Galabuda, A.M. and Geshwind, N., Neuroanatomical asymmetries in non-human species, Trends Neurosci., 5 (1982) 429-431. 11 Takami, S. and Urano, A., The volume of the toad medial amygdala - - anterior preoptic complex is sexually dimorphic and seasonally variable, Neurosci. Lett., 44 (1984) 253-258. 12 Van Eden C.G., Uylings, H.B.M. and Van Pelt, J., Sexdifference and left-right asymmetries in the prefrontal cortex during postnatal development in the rat, Dev. Brain Res., 12 (1984) 146-153.