histochemical study of rna content of neurones in the dorsal lateral

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"
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
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
..
,s.
A
•
It
v
~.,
W
O
m
m
4It
=-
I-,
m
tO
m
I-X
161
m
5-
io
s,-
$0
I0
,0.3
8
;
;
'4
21
zo
ss
4~
.0.1
,'.
+b
AGE ( 1 i dags)
• ), surface ( •
Fig. 2. Post-natal changes of mean extinction ( •
O) at a cytoplasmic level during the post-natal development.
(o
•
) 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.
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