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Histochemical study of RNA content of neurones in the dorsal lateral geniculate nucleus during postnatal development
Mechanisms of Ageing and Development, 57 (1991) 275--282
275
Elsevier Scientific Publishers Ireland Ltd.
HISTOCHEMICAL STUDY OF RNA CONTENT OF NEURONES IN THE DORSAL LATERAL GENICULATE NUCLEUS DURING POSTNATAL DEVELOPMENT
A. VILLENA, V. REQUENA, F. DIAZ and I. PEREZ DE VARGAS Departamento de Morfologla Normal y Patol6gica, Facultad de Medicina, Universidad de M~laga (Spain) (Received August 25th, 1990)
SUMMARY
Nuclear and cytoplasmic dLGN neurons were investigated by cytophotometric measurements of RNA. This study has been carried out in rats from birth to adulthood. In order to quantify the RNA content a cytophotometer was used. Extinction mean values were obtained which indicated RNA concentrations per surface unit. The nuclear and cytoplasmic surface were calculated simultaneously and from the product of the mean extinction and the surface the RNA total content was calculated. Our results have suggested that the changes are age-related. From day I to day 21 the neuronal size and RNA content increase; this may somehow be involved with the differentiation process. Around post-natal day 21 neuronal maturation may begin, reaching its optimal phase around day 42, on which the RNA concentration per surface unit, surface neuronal content and RNA total content are stable.
Keywords: Gallocyanin; Cytophotometric; RNA content; Dorsal lateral geniculate nucleus INTRODUCTION
The dorsal lateral geniculate nucleus (dLGN) has been the centre of many studies that have shown various changes which it undergoes during post-natal development. In this respect, there have been many morphological and quantitative studies which have typified and evaluated various parameters in the dLGN neurones during the post-natal phases. These studies, carried out in various species, under both physiological [lm14] and experimental conditions [15--22] have been complemented with histochemical [23--27], biochemical [28--30], immunohistochemical [31--35] and Correspondence to: Dr. A. Villena, Departmento de Morfologia Normal y Patol6gica, Facultad de Medicine, Universitad de M~laga, Spain. 0047-6374/91/$03.50 Printed and Published in Ireland
© 1991 Elsevier Scientific Publishers Ireland Ltd.
276 electrophysiological studies [36--39] which have helped to establish the chronology and post-natal behaviour of this nucleus in the optic tract. In this study, a histochemical-quantitative analysis was carried out on the RNA content of the d L G N neurones, at a nuclear and cytoplasmic level, in order to study the possible changes during the post-natal development and its relation with the various stages of this development. MATERIALS AND METHODS Ninety Sprague--Dawley albino rats were divided into 9 groups according to their ages, i.e., 1, 7, 14, 21, 28, 35, 42, 60 and 90 post-natal days (pnd). They were then anesthetized and perfused with saline solution and 10070 formol. Their brains were then removed, embedded in paraffin and serially sectioned in the transversal plane at 10 tam. These sections were subsequently stained according to the Einarson gallocyanine technique [40] which is specific for nucleic acids. In order to quantify the R N A content a Leitz MPV2 cytophotometer with a 58118 nm interference filter was used. The mean extinction method (E) was used on 10 values obtained in the nucleus and 10 in the cytoplasm to carry out the measurements. The results were shown in arbitrary units (a.u.) which indicated R N A concentrations per surface unit. The nucleus and cytoplasm surface (A) were calculated simultaneously using drawings of their profiles traced by a camera lucida at a magnification of × 100 which were then measured with a Kontron M O P - A M . The RNA total content (T.C.), shown in arbitrary units, was found using the product of the mean extinction and the area (T.C. = E X A). Finally, an analysis of the variance (ANOVA at P < 0.05) was used to statistically examine the results of the above calculations. RESULTS A t a nuclear level
The values of the mean extinction oscillated between a m a x i m u m of 0.3538 a.u. on the day 1 of post-natal life and a minimum of 0.1133 a.u. (P < 0.05) on day 21 with intermediate values for the other ages studied. Having worked out the nuclear surface, shown in Table I and Fig. l, we went on to find the RNA content whose value ranged between a minimum of 3.9554 a.u. at 1 pnd and a m a x i m u m of 12.511 a.u. (P < 0.05) at 14 pnd. During the third post-natal week there was an important decrease in the RNA content, reaching a value of 7.5664 a.u. at 21 pnd (Table I, Fig. 1). A t a c y t o p l a s m i c level
The values of the mean extinction oscillated between a maximum of 0.3337 a.u. at 1 pnd and a minimum of 0.1154 a.u. (P < 0.05) at 90 pnd. At 21 pnd a high RNA
277
TABLE I EACH VALUE REPRESENTS THE MEAN ± S.E.M. OF EXTINCTION (E), SURFACE (A) AND RNA TOTAL CONTENT (T.C.) AT A NUCLEAR LEVEL DURING THE POST-NATAL DEVELOPMENT
Age in
Mean extinction
Surface
Total content
days
(-x (a.u.) ± S.E.M.)
(-x (~n 2) ± S.E.M.)
(~ (a.u.) ± S.E.M.)
1 7 14 21 28 35 42 60 90
0.3538 0.2684 0.2501 0.1133 0.1503 0.1567 0.2283 0.2268 0.1566
13.13 25.07 48.58 55.21 57.22 60.60 62.72 53.29 53.13
3.9554 9.2239 12.5110 7.5664 8.4393 8.9532 11.4073 10.4829 7.9906
± ± ± ± ± ± ± ± ±
0.0054 0.0100 0.0081 0.0038 0.0056 0.0062 0.0060 0.0064 0.0042
± ± ± ± ± ± ± ± ±
0.39 0.37 1.12 1.09 1.41 0.96 1.22 1.01 0.94
± ± ± ± ± ± ± ± ±
0.07 0.30 0.46 0.36 0.36 0.36 0.39 0.34 0.26
concentration was found per surface unit. Once the cytoplasmic surface was determined we went on to find the R N A content which had values oscillating between 1.8696 a.u. at 1 pnd and 12.4372 a.u. ( P < 0.05) at 21 pnd. From 21 to 28 pnd there is a significant decreases reaching a value of 6.9649 a.u. This also being true from the 42nd pnd onwards (Table II, Fig. 2).
i IS"
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i O 9g mg X u.m
"50 - 30 -10
'0.3 ~0.1
7
I~1 2"1 28
35 42
60
9() AGE (lli dol))
Fig. 1. Post-natal changes of mean extinction ( • • ), surface ( • (O O) at a nuclear level during the post-natal development.
•
) and RNA total content
278
TABLE II E A C H VALUE R E P R E S E N T S T H E M E A N ± S.E.M. OF E X T I N C T I O N (E), SURFACE (A) AND RNA TOTAL C O N T E N T (T.C.) AT A C Y T O P L A S M I C LEVEL D U R I N G TH E P O S T-N A TA L DEVELOPMENT
Age in days
Mean extinction f f (a.u.) ± S.E.M.)
Surface (x (#m9 ± S.E.M.)
Total content f f (a.u.) ± S.E.M.)
1 7 14 21 28 35 42 60 90
0.3377 0.2479 0.2465 0.2913 0.1721 0.1652 0.1703 0.1588 0.1154
8.75 18.09 34.28 43.00 42.97 41.78 47.56 42.28 41.71
1.8696 5.9101 8.9417 12.4372 6.9649 6.0347 7.6183 6.7372 5.2759
± ± ± ± ± ± ± ± ±
0.0060 0.0064 0.0083 0.0051 0.0038 0.0038 0.0042 0.0056 0.0041
± ± ± ± ± ± ± ± ±
0.18 0.78 1.55 1.77 1.73 1.75 1.70 1.45 1.59
± ± ± ± ± ± ± ± ±
0.06 0.22 0.42 0.51 0.32 0.33 0.29 0.32 0.19
A N
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AGE ( 1 i dags) • ), surface ( • Fig. 2. Post-natal changes of mean extinction ( • O) at a cytoplasmic level during the post-natal development. (o
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) and RNA total content
279 DISCUSSION The dLGN, which forms part of the optic tract as a relay centre, shows a grade of complexity, when referring to its layers and synaptic connections, which is proportional to the evolutive grade o f the species [41--43]. Its neurogenesis has been described by Stroer [44], Coggeshall [45] and McAllister and Das [46] in the rat and by Keyser [47] and ZiUes [48] in other species, showing that in the rat the process begins around the 12th pre-natal day [49]. During days 16--18 of this period the dLGN neurones do not form a well-defined isolated nucleus since they are joined to the neurones neighbouring nuclei [50]. The dLGN becomes well defined around prenatal days 20--21, showing both its dorsal and ventral areas [49]. After birth the neurones show a rapid growth with a parallel development to the ergastoplasm [44,45]. Since there are few histochemical references with respect to the post-natal period [23,24,27,51] our histochemical research began in order to show the neuronal growth, differentiation and maturation stages which characterize this period. To do this a histochemical-quantitative study was carried out on the variations in the RNA content in the neurones from birth to adulthood. The values obtained correspond to both the nuclear and cytoplasmic RNA since, a previous study [14] showed that there was no neuronal increase from at least the 7th post-natal day onwards and therefore, the DNA content must be constant. The studies at a nuclear level showed that during the first post-natal week there is a decrease in the RNA concentration per surface unit, yet this increases when there is a parallel increase of the nuclear area. For the second week the concentration is stable at the same time as both the area and content increases, reaching a maximum value at the end of the second week. These results show that during the first 2 weeks of post-natal development, there is intense activity at a nuclear level which is related to the transcription process and at the same time, the somatic area increases its surface area to three times its size [141. During the third week, both the RNA concentration and content decrease, a period in which an increase of RNA is found at a cytoplasmic level. The concentration per surface unit, the area and RNA content increase between pnd 21--42 which shows the existence of a moderate nuclear activity which prepares the neurones to start their specific activities. The values decrease from pnd 42 onwards in such a way that has been classed as insignificant. The studies carried out on the RNA at a cytoplasmic level show a similar behaviour to that in the nuclear level during the first 2 weeks. The RNA increase corresponds to the progressive development of the Nissl bodies observed by Raedler and Sievers [47]. Likewise this increase indicates that the neurones have begun their differentiation, a period characterized by specific protein synthesis. During the third week, contrary to what happens in the nucleus, there is an important increase in the RNA content which reaches its highest level on the pnd 21. This difference could be due to the fact that, in this stage, there is an RNA transfer from the nucleus to the
280
cytoplasm where they carry out their functions. These functions are closely linked to the neurotransmitter synthesis proved by McDonald et al. [51 ] and Kvale et al. [52] with the increase in number and development o f the synapsis [ 13] in the same way as the development o f organelles and dendrites as described by Parnavelas et al. [2] and Purpura and Schade [53]. During the fourth week the R N A content decreases until it becomes stable. From this point on it is considered that the neurones have reached the functional level, which finally ends in complete neuronal maturation. In summary, we can conclude that the evaluation o f our histochemical results and those from other studies have allowed us to establish three stages in the dLGN neurogenesis: the first or proliferation stage [44--46] which occurs during the pre-natal life and which is characterized by an increase in the number of neurones with the corresponding increase in the D N A content; the second of growth or differentiation stage o f the neurones which consists o f the first 3 weeks o f post-natal life, in which the area o f the nucleus and the total R N A content increase and the organeUes and dendritic tree develop; and the third or maturation stage o f the neurones which begins about pnd 21 and reaches its optimal phase around pnd 42, a date on which the concentration values, area and total R N A content are stable and which the neurophil growth ceases. REFERENCES l 2 3
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10 11 12
H. Elgeti, R. Elgeti and K. Fleischhauer, Post-natal growth of the dorsal lateral geniculate nucleus of the cat. Anat. EmbryoL, 149 (1976) 1--3. J.G. Parnavelas, R. Bradford, E.J. Mounty and A.R. Lieberman, Post-natal growth of neuronal perikarya in the dLGN of the rat. Neurosci. Lett., 5 (1977) 33--37. J.G. Parnavelas, E.J. Mounty, R. Bradford and A.R. Lieberman, The post-natal development of neurons in the dorsal lateral geniculate nucleus of the rat: a Golgi study. J. Comp. NeuroL, 171 (1977) 481--500. R.E. Kalil, Development of the dorsal lateral geniculate nucleus in the cat. J. Comp. Neurol., 182 (1978) 265--292. D.C. Linden, R.W. Guillery and J. Cucchiaro, The dorsal lateral geniculate nucleus of the normal ferret and its post-natal development. J. Comp. Neurol., 203 (1981) 189--211. J.K. Brunso-Bechtold and V.A. Casagrande, Early post-natal development of laminar characteristics in the dorsal lateral geniculate nucleus of the tree shrew. J. Neurosci., 2 (1982) 589--597. P.F. Hitchcock, T.L. Flickey and C.G. Dunkel, Genesis of morphologically identified neurons in the dorsal lateral geniculate nucleus of the cat. J. Comp. Neurol., 228 (1984) 200--209. M.J. Webster and M.H. Rowe, Morphology of identified relay cells and interneurones in the dorsal lateral geniculate nucleus of the rat. Exp. Brain Res., 56 (1984) 469--474. K. Brauer, J. Hamori and E. Winkelmann, On the ontogenetic development of the rat dLGN. 1. GCR-neurons at post-natal day 7. A Golgi electron microscopic study. Anat. Embryol., 172 (1985) 39--48. J. Satorre, J. Cano and F. Relnoso-Su~trez, Stability of the neuronal population of the dorsal lateral geniculate nucleus (LGNd) of aged rats. Brain Res., 339 (1985) 375--377. J. Satorre, J. Cano and F. Reinoso-Su~trez, Quantitative cellular changes during post-natal development of the rat dorsal lateral geniculate nucleus. Anat. EmbryoL, 174 (1986) 321--327. J.M. Fritschy and L.J. Garey, Postnatal development of dendrites of relay neurons in the lateral geniculate nucleus of the marmoset (Callithrixjacchus): a quantitative Golgi study. J. Comp. NeuroL, 268 (1988) 234--247.
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A.M. Sillito, J.A. Kemp and N. Berardi, The cholinergic influence on the function of the cat dorsal geniculate nucleus (dLGN). Brain Res., 280 (1983) 299--307. S. Gabriel, H.J. Gabriel, U. Zippel and H. Brandl, Regional distribution of fast and slow geniculocortical relay cells (GCR-cells) within the rat's dorsal lateral geniculate nucleus (LGND). Exp. Brain Res., 61 (1985) 210--213. Y. Kayama, Ascending, descending and local control of neuronal activity in the rat lateral geniculate nucleus. Vision Res., 25 (1985) 339--347. G.A. Marks, S.G. Speciale, K. Cobbey and H.P. Roffwarg, Serotonergic inhibition of the dorsal lateral geniculate nucleus. Brain Res., 418 (1987) 76--84. L. Einarson, On the theory of gallocianin-chromalum staining and its applications for quantitative estimation of basophilia. A selective staining of exquisite progressivity. Acta Pathol. Microbiol. Scand., 28 (1951) 82. R.W. Guillery and M. Colonnier, Synaptic patterns in the LGNd of the monkey. Z. Zellforsch. Mikrosk. Anat., 103 (1970) 90--108. J.K. Brunso-Bechtold and S.L. Vinsant, Cellular interrelationships during laminar segregation in the dorsal lateral geniculate nucleus. J. Neurosci., 8 (1988) 2693--2706. B.E. Reese, "Hidden lamination" in the dorsal lateral geniculate nucleus: the functional organization of this thalamic region in the rat. Brain Res., 472 (1988) 119--137. W.E.H. Stroer, Studies on the diencephalon. I. The embryology of the diencephalon of the rat. J. Comp. Neurol., 105 (1956) 1--24. R.E. Coggeshall, A study of diencephalic development in the albino rat. J. Comp. Neurol., 122 (1964) 241--270. J.P. McAllister and G. Das, Neurogenesis in the epithalamus, dorsal thalamus and ventral thalamus of the rat: an autoradiographic and cytological study. J. Comp. Neurol., 172 (1977) 647--686. A. Keyser, The development of the diencephalon of the Chinese hamster. Acta Anat., 83, Suppl. 59 (1972) pp. 1--181. K.J. Zilles, Ontogenesis of the visual system. Adv. Anat. EmbryoL CelIBiol., 54 (1978) 5--138. A. Raedler and J. Sievers, The development of the visual system of the albino rat. Adv. Anat., 50, Fasc. 3 (1975), Springer-Verlag, Berlin, Heidelberg, New York. R.D. Lund and M.J. Mustari, Development of the geniculocortical pathway in rats. J. Comp. Neurol., 173 (1977) 289--306. J.K. McDonald, S.G. Speciale and J.G. Parnavelas, The development of glutamic acid decarboxylase in the visual cortex and the dorsal lateral geniculate nucleus of the rat. Brain Res., 217 (1981) 364--367. 1. Kvale, V.M. Fosse and F. Fonnum, Development of neurotransmitter parmeters in lateral geniculate body, superior colliculus and visual cortex of the albino rat. Dev. Brain Res., 7 (1983) 137-- 145. D.P. Purpura and J.P. Schade, Progress in Brain Research. Growth and Maturation o f the Brain, Vol. 4, 1964, Elsevier, The Netherlands.
275
Elsevier Scientific Publishers Ireland Ltd.
HISTOCHEMICAL STUDY OF RNA CONTENT OF NEURONES IN THE DORSAL LATERAL GENICULATE NUCLEUS DURING POSTNATAL DEVELOPMENT
A. VILLENA, V. REQUENA, F. DIAZ and I. PEREZ DE VARGAS Departamento de Morfologla Normal y Patol6gica, Facultad de Medicina, Universidad de M~laga (Spain) (Received August 25th, 1990)
SUMMARY
Nuclear and cytoplasmic dLGN neurons were investigated by cytophotometric measurements of RNA. This study has been carried out in rats from birth to adulthood. In order to quantify the RNA content a cytophotometer was used. Extinction mean values were obtained which indicated RNA concentrations per surface unit. The nuclear and cytoplasmic surface were calculated simultaneously and from the product of the mean extinction and the surface the RNA total content was calculated. Our results have suggested that the changes are age-related. From day I to day 21 the neuronal size and RNA content increase; this may somehow be involved with the differentiation process. Around post-natal day 21 neuronal maturation may begin, reaching its optimal phase around day 42, on which the RNA concentration per surface unit, surface neuronal content and RNA total content are stable.
Keywords: Gallocyanin; Cytophotometric; RNA content; Dorsal lateral geniculate nucleus INTRODUCTION
The dorsal lateral geniculate nucleus (dLGN) has been the centre of many studies that have shown various changes which it undergoes during post-natal development. In this respect, there have been many morphological and quantitative studies which have typified and evaluated various parameters in the dLGN neurones during the post-natal phases. These studies, carried out in various species, under both physiological [lm14] and experimental conditions [15--22] have been complemented with histochemical [23--27], biochemical [28--30], immunohistochemical [31--35] and Correspondence to: Dr. A. Villena, Departmento de Morfologia Normal y Patol6gica, Facultad de Medicine, Universitad de M~laga, Spain. 0047-6374/91/$03.50 Printed and Published in Ireland
© 1991 Elsevier Scientific Publishers Ireland Ltd.
276 electrophysiological studies [36--39] which have helped to establish the chronology and post-natal behaviour of this nucleus in the optic tract. In this study, a histochemical-quantitative analysis was carried out on the RNA content of the d L G N neurones, at a nuclear and cytoplasmic level, in order to study the possible changes during the post-natal development and its relation with the various stages of this development. MATERIALS AND METHODS Ninety Sprague--Dawley albino rats were divided into 9 groups according to their ages, i.e., 1, 7, 14, 21, 28, 35, 42, 60 and 90 post-natal days (pnd). They were then anesthetized and perfused with saline solution and 10070 formol. Their brains were then removed, embedded in paraffin and serially sectioned in the transversal plane at 10 tam. These sections were subsequently stained according to the Einarson gallocyanine technique [40] which is specific for nucleic acids. In order to quantify the R N A content a Leitz MPV2 cytophotometer with a 58118 nm interference filter was used. The mean extinction method (E) was used on 10 values obtained in the nucleus and 10 in the cytoplasm to carry out the measurements. The results were shown in arbitrary units (a.u.) which indicated R N A concentrations per surface unit. The nucleus and cytoplasm surface (A) were calculated simultaneously using drawings of their profiles traced by a camera lucida at a magnification of × 100 which were then measured with a Kontron M O P - A M . The RNA total content (T.C.), shown in arbitrary units, was found using the product of the mean extinction and the area (T.C. = E X A). Finally, an analysis of the variance (ANOVA at P < 0.05) was used to statistically examine the results of the above calculations. RESULTS A t a nuclear level
The values of the mean extinction oscillated between a m a x i m u m of 0.3538 a.u. on the day 1 of post-natal life and a minimum of 0.1133 a.u. (P < 0.05) on day 21 with intermediate values for the other ages studied. Having worked out the nuclear surface, shown in Table I and Fig. l, we went on to find the RNA content whose value ranged between a minimum of 3.9554 a.u. at 1 pnd and a m a x i m u m of 12.511 a.u. (P < 0.05) at 14 pnd. During the third post-natal week there was an important decrease in the RNA content, reaching a value of 7.5664 a.u. at 21 pnd (Table I, Fig. 1). A t a c y t o p l a s m i c level
The values of the mean extinction oscillated between a maximum of 0.3337 a.u. at 1 pnd and a minimum of 0.1154 a.u. (P < 0.05) at 90 pnd. At 21 pnd a high RNA
277
TABLE I EACH VALUE REPRESENTS THE MEAN ± S.E.M. OF EXTINCTION (E), SURFACE (A) AND RNA TOTAL CONTENT (T.C.) AT A NUCLEAR LEVEL DURING THE POST-NATAL DEVELOPMENT
Age in
Mean extinction
Surface
Total content
days
(-x (a.u.) ± S.E.M.)
(-x (~n 2) ± S.E.M.)
(~ (a.u.) ± S.E.M.)
1 7 14 21 28 35 42 60 90
0.3538 0.2684 0.2501 0.1133 0.1503 0.1567 0.2283 0.2268 0.1566
13.13 25.07 48.58 55.21 57.22 60.60 62.72 53.29 53.13
3.9554 9.2239 12.5110 7.5664 8.4393 8.9532 11.4073 10.4829 7.9906
± ± ± ± ± ± ± ± ±
0.0054 0.0100 0.0081 0.0038 0.0056 0.0062 0.0060 0.0064 0.0042
± ± ± ± ± ± ± ± ±
0.39 0.37 1.12 1.09 1.41 0.96 1.22 1.01 0.94
± ± ± ± ± ± ± ± ±
0.07 0.30 0.46 0.36 0.36 0.36 0.39 0.34 0.26
concentration was found per surface unit. Once the cytoplasmic surface was determined we went on to find the R N A content which had values oscillating between 1.8696 a.u. at 1 pnd and 12.4372 a.u. ( P < 0.05) at 21 pnd. From 21 to 28 pnd there is a significant decreases reaching a value of 6.9649 a.u. This also being true from the 42nd pnd onwards (Table II, Fig. 2).
i IS"
m
i O 9g mg X u.m
"50 - 30 -10
'0.3 ~0.1
7
I~1 2"1 28
35 42
60
9() AGE (lli dol))
Fig. 1. Post-natal changes of mean extinction ( • • ), surface ( • (O O) at a nuclear level during the post-natal development.
•
) and RNA total content
278
TABLE II E A C H VALUE R E P R E S E N T S T H E M E A N ± S.E.M. OF E X T I N C T I O N (E), SURFACE (A) AND RNA TOTAL C O N T E N T (T.C.) AT A C Y T O P L A S M I C LEVEL D U R I N G TH E P O S T-N A TA L DEVELOPMENT
Age in days
Mean extinction f f (a.u.) ± S.E.M.)
Surface (x (#m9 ± S.E.M.)
Total content f f (a.u.) ± S.E.M.)
1 7 14 21 28 35 42 60 90
0.3377 0.2479 0.2465 0.2913 0.1721 0.1652 0.1703 0.1588 0.1154
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279 DISCUSSION The dLGN, which forms part of the optic tract as a relay centre, shows a grade of complexity, when referring to its layers and synaptic connections, which is proportional to the evolutive grade o f the species [41--43]. Its neurogenesis has been described by Stroer [44], Coggeshall [45] and McAllister and Das [46] in the rat and by Keyser [47] and ZiUes [48] in other species, showing that in the rat the process begins around the 12th pre-natal day [49]. During days 16--18 of this period the dLGN neurones do not form a well-defined isolated nucleus since they are joined to the neurones neighbouring nuclei [50]. The dLGN becomes well defined around prenatal days 20--21, showing both its dorsal and ventral areas [49]. After birth the neurones show a rapid growth with a parallel development to the ergastoplasm [44,45]. Since there are few histochemical references with respect to the post-natal period [23,24,27,51] our histochemical research began in order to show the neuronal growth, differentiation and maturation stages which characterize this period. To do this a histochemical-quantitative study was carried out on the variations in the RNA content in the neurones from birth to adulthood. The values obtained correspond to both the nuclear and cytoplasmic RNA since, a previous study [14] showed that there was no neuronal increase from at least the 7th post-natal day onwards and therefore, the DNA content must be constant. The studies at a nuclear level showed that during the first post-natal week there is a decrease in the RNA concentration per surface unit, yet this increases when there is a parallel increase of the nuclear area. For the second week the concentration is stable at the same time as both the area and content increases, reaching a maximum value at the end of the second week. These results show that during the first 2 weeks of post-natal development, there is intense activity at a nuclear level which is related to the transcription process and at the same time, the somatic area increases its surface area to three times its size [141. During the third week, both the RNA concentration and content decrease, a period in which an increase of RNA is found at a cytoplasmic level. The concentration per surface unit, the area and RNA content increase between pnd 21--42 which shows the existence of a moderate nuclear activity which prepares the neurones to start their specific activities. The values decrease from pnd 42 onwards in such a way that has been classed as insignificant. The studies carried out on the RNA at a cytoplasmic level show a similar behaviour to that in the nuclear level during the first 2 weeks. The RNA increase corresponds to the progressive development of the Nissl bodies observed by Raedler and Sievers [47]. Likewise this increase indicates that the neurones have begun their differentiation, a period characterized by specific protein synthesis. During the third week, contrary to what happens in the nucleus, there is an important increase in the RNA content which reaches its highest level on the pnd 21. This difference could be due to the fact that, in this stage, there is an RNA transfer from the nucleus to the
280
cytoplasm where they carry out their functions. These functions are closely linked to the neurotransmitter synthesis proved by McDonald et al. [51 ] and Kvale et al. [52] with the increase in number and development o f the synapsis [ 13] in the same way as the development o f organelles and dendrites as described by Parnavelas et al. [2] and Purpura and Schade [53]. During the fourth week the R N A content decreases until it becomes stable. From this point on it is considered that the neurones have reached the functional level, which finally ends in complete neuronal maturation. In summary, we can conclude that the evaluation o f our histochemical results and those from other studies have allowed us to establish three stages in the dLGN neurogenesis: the first or proliferation stage [44--46] which occurs during the pre-natal life and which is characterized by an increase in the number of neurones with the corresponding increase in the D N A content; the second of growth or differentiation stage o f the neurones which consists o f the first 3 weeks o f post-natal life, in which the area o f the nucleus and the total R N A content increase and the organeUes and dendritic tree develop; and the third or maturation stage o f the neurones which begins about pnd 21 and reaches its optimal phase around pnd 42, a date on which the concentration values, area and total R N A content are stable and which the neurophil growth ceases. REFERENCES l 2 3
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