Postnatal ontogenesis of neurons containing GABAAα1 subunit mRNA in the rat forebrain

Molecular Brain Research, 16 (1992) 193-203

193

© 1992 Elsevier Science Publishers B.V. All rights reserved 0169-328x/92/$05.00 BRESM 70508

Postnatal o n t o g e n e s i s of n e u r o n s containing G A B A A a 1 subunit m R N A in the rat forebrain Jian-Hua

Zhang, Makoto

Sato, Toshiyuki Araki and Masaya Tohyama

Department of Anatomy and Neuroscience, Osaka Unicersity Medical School, Osaka (Japan)

(Accepted 23 June 1992)

Key words: Gene expression; aj Subunit; GABAA receptor; Brain; Rat

The expression of G A B A A receptor crI subunit mRNA in the postnatal rat forebrain was examined by in situ hybridization histochemistry. In most regions, including the isocortex, olfactory bulb, amygdala, septum, nucleus of the diagonal band, bed nucleus of the stria terminalis, basal ganglia, thalamus, and hypothalamus, the expression of a I subunit mRNA was low at birth but showed a dramatic increase during the early postnatal period. Adult levels of expression were reached at around the second or third week of life in these regions. However, in the caudate-putamen, and the nucleus accumbens, the expression of this suhunit was only transient.

INTRODUCTION The y-aminobutyric acid A receptor (GABAA-R) is one of the main targets of GABA, which is a major inhibitory neurotransmitter in the vertebrate brain 37. Molecular cloning studies have shown that the G A B A A - R is composed of many subunits and subunit variants, such as al_ 6, /31-4, /3~, ~/i-3, ~, and p6.11,18,21,22,35,39,41,44,46 48. The in vitro co-expression of these subunit m R N A s has shown that many G A B A A - R subtypes, each with different pharmacological properties, can be formed by complicated combinations of these different subunits 34'43. For example, three G A B A A - R subtypes were generated when one of the three a subunit m R N A s (Of1, a2, and a 3) was co-expressed with /31 and /32 m R N A in transfected human embryonic kidney cells (i.e., a I +/31 + Y2, a2 +/31 + Y2, and of3 +/31 q'- y34). In addition, using oligonucleotide probes for the a I and /3 subunit m R N A s (/31, /32, and /33), respectively, we have found that each subunit m R N A shows region-specific expression in the rat brain 4'5'17'2°:5'49-51, suggesting the existence of distinct G A B A A - R subtypes in rats.

Recently, it has been shown that in addition to functioning as an inhibitory neurotransmitter, G A B A can also act as a trophic factor in the immature brain 15"19'24-26'28'29'42, with this trophic effect being mediated by the G A B A A - R 26. Therefore, G A B A may be involved in variou.s functions in the brain and this might be reflected by the heterogeneity of GABAA-R. Our recent study indicated that the /3 subunit expressed in the thalamus changed from /33 (neonatal) to /32 (adult) during postnatal ontogenesis 52. In addition, although the three/3 subunit m R N A s (/31, /32, and /33) are expressed in most regions of the adult rat forebrain, including the isocortex, hippocampal formation, septum, amygdala, and hypothalamus, only /33 m R N A is highly expressed in these regions during the early postnatal period 51. Therefore, it seems that the composition of the G A B A A - R subtypes mediating the trophic effect of G A B A during brain development may differ from that mediating its inhibitory effect in adults. Recently, developmental expression of a I subunit m R N A was examined in the rat brain by Northern blotting 14"24 or in situ hybridization histochemistry using film autoradiography 12:3. These studies revealed

Correspondence: M. Tohyama, Department of Anatomy and Neuroscience, Osaka University Medical School, 2-2 Yamadaoka Suita-shi, Osaka 565, Japan. Fax: (81) 6-875-7221. * Present address: Department of Anatomy, The Fourth Military Medical School, Shaanxi, Xi'an, P.R. China.

194

P--1

GI ~

~

P--7

.~'~---~-A O B

P-14

G! ~

o,

AOB

.t~ r/-;. M

i

~

AOE

"

( .'~..,e,

t.

)':11

y

<.,:,.~:.;.. . ~ : ~ ~:¢:~% ; : • ;. , ". t" .'~:,

¢~

\i~ ~i~.:i

csv - - ~ n

~u ,cb

VDB~:'/''t;::'~

DEn

D

,.

I "

=

!e'.'.k~/ i"

,_/~{~~!.

'/':t''

O,

" ~ ; ' ~ A c bj,", ~ L : "~

F I ~::-'.:*-~'.'.'".'-.

L-:-W ~ ' ~ : - " . ~ . % CA~ _ ,.~ .•. : . .~'-'."=-'..:.::'~'~: ~ ~ ~ ::-.,.-.:.~

~:." ~"~).:.~.:,.'J', ,,.'.;~ 1~ L # CAID(~ _ ~ ~ eL ~ ~ ' ~ ~ i

ADq~.~S A V ~ ~ ,

~," DLG ~ :~ ..k~ "k\

\ ":"..'.:?~; CPU~/ ~i:i~/

BST - - ~ / ~ . : ~ - ~

/';i":.~

__

p-r "~ ... pc~

LH ' - ~ . . ~/~. { ~ ' ACo

..~'FIL

TU

"):.; ~:/)

r'

°°

:;'~::~,, >.....;:-"

° ,"

•," .;: . . ! . p::. .. ",'~ • VL ~ . L po.:,-:-,~.;"." "" ~:,.-i ~ / I " ' " . ' . " : ~ " ~ ' ~ ~ c~

I

..v:-,.

H

~ " plr BMA

.

% ~ : ~ ,L" - ~ : . : ' . :~.\\ ~";"~"-~'~ " • ~"~" ".;~II /. "" " \" " :;:11

,~&.:.:'~::<. oo;~)~,

,

V M H ~ ~ MePD

.

7,/-~:"

MCPO

PIr

I

ft~. ::. .'.:.,.; :":.~, PrS L

Po\

~ V P L

VPM~

CL~

ZI-- ='~!i"iii'~l // ~ ' . , ~ STh LH-- - - " f J ~ 9 ; , . ~ , V M H --

~"/:'~

;~

o ~

M ~ ~ ~ , VLG V

MD-~

.

..~

~v..:.:, • --

i ~ ' I ~ VLG

~

" "'"" ~ A

Plr

LP

:'~ ~ * ~ ' ; ~ / ' Zl "......... ".":i ~... ',; E ~ ~ ";'"!)'J"v:'~

A 1

"~LLaDL V I ~ I B

VMH

• ~-- : . Y . ' : . ~ . ~ . .

/~'i.~.'~ ~,~.,.'~ . .~'" -, ; : ' , . "" _-,'x.... .'. t

.

3 L"DL

i ' " ) . ~ : ' / . , ' ""

'

H~'";"

' ; . . ;

:L:P"/~ i''~'~''~:: "-2"~':-.~.\

~'~::_.. ~ :~.~1. ' I ~ '= .. = V,_.~.L. . ...~. "~ ;"-¢;~."t.~ ~.';~ ." ~ : ~t ' : ~~ : VM ~ : " ~ ' ~ ~ ' . " ~ Zl I':':" :.~'~'~;~.;'~"/ -;'.:.

C=--

~

~

L

a

D

- ..

,

. _

LU "_-77"-DLG VPM

L

~"" BLA

AC(

BMA

K

TC ~ ~ M e P V ~ ' ~ PLCo

~'BLP ~ Pit BLV

L

-

Fig. I. Diagrams showing the location of n e u r o n s containing G A B A A receptor cr~ subunit m R N A in the rat forcbrain on postnatal day I (left columnL day 7 (middle column), and day 14 (right column). Each dot represents 3 - 5 positive neurons. Sections are shown in the frontal phme from rostral (upper) to caudal (lower). Bar ~ I ram. The nomenclature used in the diagrams was based upon the atlas of Paxinos and Watson 25.

195 that a~ mRNA markedly increases between postnatal days 7 and 38. However, the overall developmental pattern of neurons expressing a~ subunit mRNA of GABA A receptor is still obscure. In the present study, we examined the postnatal expression of a~ subunit mRNA in the rat forebrain.

Oligonucleotide probe The oligonucleotide probe employed in the present study was the same as that previously described 17. This probe (48 mer) was complementary to bases 851-898 of the bovine GABAA-R a t subunit cDNA 39. Using terminal deoxynucleotidyl transferase, the probe was labeled by adding [a-35S]dATP [1000-1500 C i / m m o l (37-55.5 T B q / m m o l ) , NEN] to its 3' end to form an [a-35S]dA tail u. The specific activity of the labeled probe was about 1.0-1.8x10 y dpm//xg.

MATERIALS

In situ hybridization The procedure for in situ hybridization was essentially the same as that described previously 51. In brief, after being warmed to room temperature (about 1 h), the slide-mounted sections were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) (all steps at room temperature unless otherwise indicated). Then these sections were rinsed for 1 h in 4 x SSC (pH 7 . 2 ) ( I x SSC contained 0.15 M sodium chloride and 0.015 M sodium citrate) and immersed in 4 x SSC containing 1x Denhardt's solution [ I x Denhardt's solution contained 0.02% bovine serum albumin (fraction V, Sigma), 0.02% Ficoll 400 (Pharmacia), and 0.02% polyvinylpyrrolidone K-30 (Nakarai Tesque Inc.)]. After being dehydrated through an ethanol series and defatted in chloroform, the sections were incubated for 48 h at 38°C in a buffer [4× SSC, 50% deionized formamide, 1% N-lauroylsarcosine sodium salt, 0.12 M phosphate buffer (pH 7.2),

AND METHODS

Animals and tissue preparation Male Wistar rat were used at postnatal days 1, 3, 5, 7, 10, 14, 21, and 35, and as adults (rats weighing 200-250 g). Each group contained at least two rats. Since the rats generally gave birth late at night and we always decapitated them in late morning, we assigned the day of birth as postnatal day 1. All animals were decapitated under sodium pentobarbital (50 m g / m l ) anesthesia (50 mg/kg, i.p.). Their forebrains were removed quickly and were immediately frozen on powdered dry ice. Serial sections of 10/zm (postnatal days 1, 3, 5, and 7) or 15 um (postnatal days 10, 14, 21, 35, and adult) in thickness were cut on a cryostat and thaw-mounted onto gelatin-coated slides. The slides were then stored at - 8 0 ° C for about 1 month until use.

Fig. 2. Dark-field photomicrographs showing labeled neurons in the isocortex ( A - C ) and the cingulate cortex (Cg) (D, E) at different postnatal ages. A: postnatal day 1. B: postnatal day 7. C: postnatal day 14. D: postnatal day 5. E: postnatal day 14. CP, cortical plate; Mz, marginal zone; SP, subplate; l - V f , layers I-VI. Bar in C = 100/~m and the scale is common to A and B, while bar in E = 100/zm and the scale is common to D.

196 1x Denhardt's solution, 2.5~'~ tRNA, and 10% dextran sulfate] containing the [o~-3sS]dATP-labeled at probe. Then, the sections were rinsed 3-4 times in 1 × SSC (pH 7.2) at 55°C (20 rain each), coated with Ilford K-5 emulsion diluted 1 : 1 with water, and exposed for 4-6 weeks at 4°C. After being developed in D-19 (Kodak) developer, fixed with photographic fixer, and washed with tap water, the sections were counterstained with thionin solution to allow morphological identification.

background density 3~, because sections from younger rat brains were 33% thinner than those taken from older rats. RESULTS

]socort~L~On postnatal

SpecificiO' and contro& The probe employed in this experiment was complementary to bases 851-898 of bovine a I mRNA39, and showed 90% complementary to the corresponding portion of rat a t mRNA TM. The specificity of the probe was determined in several ways. First, Northern blot analysis showed that our probe hybridized with the GABA A receptor ¢~i subunit mRNA in the rat brain 17. Secondly, no appreciable hybridization signals were detected in a competition experiment, in which the sections were prehybridized with an excess amount of t.nlabeled probe '7. Thirdly, some sections were pretreated with a buffer containing 10 mM Tris-HCl (pH 7.5), 1 mM ethylenediaminetetraacetic acid, and pancreatic RNase A (10/zm/ml, Sigma). Again, no hybridization signals were seen in these sections.

day

1, w e a k l y l a b e l e d n e u r o n s

were

s c a t t e r e d in t h e c o r t i c a l p l a t e (Figs. 1D, G , J a n d 2 A ) . By p o s t n a t a l d a y 7, t h e p o s i t i v e c e l l s c o u l d b e d i v i d e d i n t o t w o g r o u p s . O n e g r o u p w a s s i t u a t e d in t h e c o r t i c a l plate and formed

a densely packed

zone of positive

cells. T h e o t h e r g r o u p w a s f o r m e d by s c a t t e r e d l a b e l e d c e l l s lying in t h e z o n e b e n e a t h

the cortical plate which

b e l o n g t o t h e d e v e l o p i n g l a y e r s V a n d V I (Figs. 1E a n d 2B). O n

postnatal

day

14, a n u m b e r

of moderately

l a b e l e d n e u r o n s w e r e s e e n in l a y e r s 11 t o V I (Figs. I F , I, L a n d 2C). T h e d i s t r i b u t i o n a n d l a b e l i n g i n t e n s i t y o f t h e n e u r o n s w a s s i m i l a r at t h i s s t a g e t o t h o s e f o u n d in

Nomenclature and charting The terminology used to describe the rat forebrain was based upon that used in the atlas of Paxinos and Waston 3~. Using a camera lucida and brightfield illumination, selected sections of the rat forebrain at postnatal days 1, 7, and 14 were depicted. The positive neurons in the same sections were charted under darkfield illumination and confirmed under high-power magnification. To identify the neurons expressing a I subunit mRNA, the grain densities of the neurons in some brain areas and the background densities of the sections were determined, as reported elsewhere 31. Neurons were only considered to be positive if the density of the silver grains on the perikaryon was three times (for the animals on postnatal days 1, 3, 5 and 7) and two times (for the animals on postnatal days 10, 14, 21, 35 and adult) greater than the

adult

rats.

layers II-IV

Labeled

neurons

were

more

common

in

t h a n in l a y e r s V a n d V I (Fig. 2C).

Allocortex Weakly labeled neurons

were detected

throughout

t h e c o r t i c a l p l a t e in t h e a l l o c o r t e x o n p o s t n a t a l d a y 1, including the anterior

cingulate

cortex,

retrosplenial

c o r t e x , i n s u l a r c o r t e x a n d p e r i r h i n a l c o r t e x (Fig. 1D, G , J). T h e r e a f t e r ,

p o s i t i v e cells i n c r e a s e d in n u m b e r

and

i n t e n s i t y . Fig. 2 D s h o w s t h e p o s i t i v e c e l l s i n t h e c i n g u -

Fig. 3. Dark-field photomicrographs showing the labeling of the main olfactory bulb (MOB) (A-C) and the piriform cortex (Pir) (D-F) at different postnatal ages. A: postnatal day 1. B: postnatal day 3. C: postnatal day 14. D: postnatal day 1. E, F: postnatal day 14. EPL, external plexiform layer: GCL, glomerular layer; MCL, mitral cell layer; I-III, layers I-III of the Pir. Bar in F = 2(10 /.tm and the scale is common to A-E.

197 late cortex on postnatal day 5 when layers I I / I I I can be clearly identified. Moderately labeled positive cells were concentrated in these layers (Fig. 2D, cf. Fig. 1E, H). The areas beneath the developing layers II and III contained positive cells with moderate to weak labeling. However, it was difficult to distinguish layers V and VI at this stage. Fig. 2E (cf. Fig. IF, I) shows the neurons expressing a~ subunit m R N A in the cingulate cortex on postnatal day 14. At this stage, each cortical layer can be clearly distinguished. Positive cells had increased in number and intensity to reach the adult levels by this time. Neurons with strong labeling were packed densely in layers II and III and were also abundant in layer V. In addition, positive cells were also seen in layer VI, but they were much less numerous than in layer V.

Olfactory bulb and related regions In the main olfactory bulb, most of the mitral cells already showed the moderate expression of a~ subunit mRNA on postnatal day I (Figs. 1A and 3A). However, hybridization signals in the other regions were still at the background level (Figs. 1A and 3A). Thereafter,

labeling of the mitral cells became progressively stronger and reached a plateau by postnatal day 14 (Figs. IC and 3A-C). On postnatal day 3, scattered weakly positive neurons appeared in the border region between the glomerular layer and the external plexiform layer (positive periglomerular neurons). An increase in the labeling of these neurons was obvious between postnatal day 7 and 10 (Fig. 1B, cf. Fig. 3B, C), and labeling reached the adult level by day 14 when a large number of strongly labeled neurons were found in this region (Figs. 1C and 3C). Other layers lacked any labeled cells during all developmental stages examined. In the accessory olfactory bulb, no labeled neurons were seen on postnatal day 1 (Fig. IA). By postnatal day 3, moderately labeled neurons were identified in the external plexiform layer-mitral cell layer complex. They increased markedly in number during the subsequent days and reached a plateau on postnatal day 14 (Fig. 1A-C). Labeling of the piriform cortex was first identified on postnatal day I (Figs. 1D, G and 3D). At this stage, there was weak positive labeling of many cells in layers

t*

Fig. 4. Dark-field photomicrographs showing the changes of labeling in the hippocampal formation ( A - C ) and the subicular complex (Sub) at different postnatal (P) ages. A: postnatal day 3. B: postnatal day 14. C: postnatal day 21. D: postnatal day 1. E: postnatal day 7. Bar in C = 200 p.m and the scale is common to A and B and white bar in E = 100 # m and the scale is common to D.

198 il where the neurons were densely packed. In layer II1, a few weakly labeled neurons were seen, and no labeled cells were detected in layer I at this stage. Thereafter, the labeling intensity and number of positive neurons in layer II increased markedly and reached the adult stage by postnatal day 14 (Figs. IF, I and 3E, F). In addition, the intensity of labeling also increased progressively with advancing age in layer III (Fig. 3 D F). The adult state was reached by postnatal day 14 (Fig. 3F).

Hippocampal formation In adult rats, most of the pyramidal cells in A m m o n ' s horn and the granule cells in the dentate gyrus were labeled moderately. No labeled cells were seen in the other layers. The distribution of the labeled cells was similar throughout ontogenesis (Fig. 1G-L). However, the ontogenesis of m R N A expression differed between the various subregions of the hippocampus. On postnatal day 1, weak expression of a~ subunit m R N A was already detected in regions CAI and CA2 but not in CA3 (Fig. 1G, J). Fig. 4A shows the neurons expressing al subunit m R N A on postnatal day 3. At this stage, the m R N A was moderately expressed by most of the pyramidal

cells in regions CA1 and CA2, whereas expression of this subunit in CA3 and by the granule cells of the dentate gyrus was very low or absent. From this time onwards, the increase in the labeling of the pyramidal cells of CA3 and the granule cells of the dentate gyrus was much more marked than that seen in CA I and CA2 (Fig. 1H, K). Therefore, most of the pyramidal cells and granule cells throughout the hippocampus showed moderate to strong expression of this subunit by postnatal day 14 (Fig. 4B). Thereafter, the expression of c~j subunit m R N A decreased slightly and reached a plateau on postnatal day 21 (Fig. 4C). A dense band of moderately positive neurons was already present on postnatal day I in the zone of densely packed neurons in the subiculum (Figs. 1J and 4D), and in layers I1-VI of the entorhinal cortex. The number of positive cells and their labeling intensity increased slightly thereafter to reach a plateau on postnatal day 7 (Figs. I K and 4E).

Amygdala Labeled neurons in the medial, basolateral, and basomedial amygdaloid nuclei were already present on postnatal day 1 (Fig. IG, J), while those in other subnuclei were detected on postnatal day 3. At this

Fig. 5. Darkfield photomicrographs showing the developmental changes of labeling in the globus pallidus (GP) (A-C) and the magnocellular preoptic nucleus (D, E). A: postnatal day 3. B: postnatal day 7. C: postnatal day 14. D: postnatal day 5. E: postnatal day 14. Bar in E - 200/,~m and the scale is common to A-D.

199 stage, the labeled neurons were few and their labeling was weak. Subsequently, they increased in number and labeling intensity to reach the adult level on postnatal day 21.

Septal region and bed nucleus of the stria terminalis (BST) Labeled neurons in the lateral septal area and BST first appeared on postnatal day 3, although the number of labeled cells was few and their labeling was weak. Thereafter, they increased slightly in number and intensity until postnatal day 14 (Fig. lI). At this stage, scattered weakly labeled cells were seen. From postnatal day 14, no significant changes of the labeling of these neurons were detected. No labeled neurons were found in the medial septal region throughout ontogenesis.

Tenia tecta In the adult animals, most of the neurons in the tenia tecta were strongly labeled. Labeling of these neurons was weak on postnatal day I, but there was marked increase in both the number and intensity of labeled cells until postnatal day 14 when a plateau was reached.

Diagonal band of Broca On postnatal day 3, scattered weakly labeled neurons were first observed in both the ventral and horizontal limbs of the diagonal band. Subsequently, the labeled cells increased in both number and intensity (Figs. 1E, H). The increase was particularly marked during postnatal days 7-14 and labeling reached the adult state on day 14 (Fig. 1F, I). At this stage, many neurons with moderate and moderate-to-strong labeling were seen in the vertical and horizontal limbs, respectively.

Magnocellular preoptic nucleus Neurons in the magnocellular preoptic nucleus were first labeled on postnatal day 3 (Fig. 5D). At this stage, there were few labeled neurons and the labeling intensity was weak. However, the labeled cells then increased rapidly in number to reach the adult level on postnatal day 14 (Fig. lI and 5E). Many strongly positive neurons were detected in the nucleus at this stage.

Basal ganglia No positive cells were seen in the basal ganglion including the globus pallidus, caudate-putamen, ventral pallidum, olfactory tubercle and nucleus accumbens on postnatal day 1 (Fig. 1D, G, J). The time of initial appearance of the positive cells differed between the

nuclei of the basal ganglia. Positive cells first appeared on postnatal day 3 in the globus pallidus, the ventral pallidum, and the neuropil between the ventral pallidum and the olfactory tubercle. They were sparse and their labeling was faint. Thereafter, the labeled cells increased in number to reach a plateau on postnatal day 14 (Figs. 1E, F, H, I). At this stage, the globus pallidus and ventral pallidum contained numerous strongly labeled neurons, while the neuropil between the ventral pallidum and the olfactory tubercle contained widely disseminated moderately labeled neurons. Positive cells in the caudate-putamen and the nucleus accumbens were first detected on postnatal days 3 and 5, respectively. The initial labeling was very weak and there were few labeled neurons. More labeled cells were seen in these two regions on postnatal day 7 (Fig. 1E, H). However, the hybridization signals in these regions decreased subsequently, and few or no labeled cells were seen on postnatal day 35 and at the adult stage. No positive cells were detected in the olfactory tubercle throughout ontogenesis.

Thalamus and habenular complex (epithalamus) Positive cells first appeared in the thalamus on postnatal day 5 in the parafascicular nucleus. On postnatal day 7, positive cells were first identified in all the other nuclei of the thalamus, except the reticular thalamic nucleus (Fig. 1K). From that time on, the labeled cells increased markedly in number and intensity to reach the adult state on postnatal day 14 (Fig. 1L). No labeled neurons were seen in the reticular thalamic nucleus at any stage. In the habenular complex, positive cells were first identified on postnatal day 3 in the medial habenular nucleus and on postnatal day 7 in the lateral habenular nucleus, respectively (Fig. 1K). Thereafter, although the labeled cells increased in number and intensity to reach a plateau on postnatal day 14, the increase was not so conspicuous as that seen in the thalamus. Therefore, on postnatal day 14, the habenular complex contained scattered weakly labeled neurons (Fig. 1L).

Hypothalamus In agreement with the findings of a previous study 49, the hypothalamus generally had few labeled neurons. Some weakly labeled neurons were scattered through the medial and lateral preoptic areas, the anterior hypothalamic nucleus, the paraventricular nucleus, the dorsomedial hypothalamic nucleus, the ventromedial hypothalamic nucleus, the posterior hypothalamic nucleus, and the lateral hypothalamus on postnatal day 21 and in adult animals. Faintly labeled neurons were first detected in these regions on postnatal day 7 (Figs.

200 I H, K) and subseqfiently there was a slight increase in the number and intensity of the labeled cells until day 21. Subthalamic area and substantia innominata Labeled cells were first detected on postnatal day 3 in the zona incerta and the subthalamic nuclei. On the other hand, the positive cells in the substantia innominata appeared on postnatal day 5. From that time on, they increased in number and intensity to reach the adult level on postnatal day 14, when many strongly to moderately labeled neurons were seen in these areas (Fig. 1L). DISCUSSION Pattern of postnatal expression of c~I subunit rnRNA and its possible significance Using Northern blot analysis, Garrett et al. H and MacLennan et al. 24 examined the ontogenesis of a~ subunit m R N A in the rat brain. Their studies revealed that a~ m R N A levels increased postnatally and reached the adult level by 3 weeks of age. Recently, G a m b a r a n a et al. 12'13 examined the expression of a t subunit m R N A during ontogenesis by in situ hybridization histochemistry. They confirmed the results by Northen blot analysis and further showed that m R N A levels of this subunit in the cerebral cortex, olfactory bulb, inferior colliculus and hippocampus increased during the second and third postnatal weeks, while an increase of the m R N A levels of this subunit in the cerebellum was seen during subsequent developmental stages. However, these studies did not permit analysis at the cellular level. In contrast, our study revealed the precise developmental pattern of the a~ subunit in the rat forebrain at the cellular level. The general profile of a t subunit elucidated in this study was as follows, although there were minor discrepancies among the various brain regions. In most regions of the rat forebrain, the expression of a t subnit m R N A was very low or absent at birth, but showed a dramatic increase subsequently to reach a plateau by postnatal day 14 or 21. However, the caudate-putamen, nucleus accumbens, and hippocampal formation were exceptions. Expression of aj subunit m R N A in the caudate-putamen and nucleus accumbens was only transient, while in the hippocampal formation, its expression decreased from postnatal day 14 to reach a plateau by day 21. It has been shown that the a subunit carries the benzodiazepine binding sites 4~' and the localization of these binding sites 3z'3~ roughly coincides with that of the neurons containing the a~ subunit in the adult

brain ~7'4'~.However, mismatches occurred in some brain regions such as the ventral pallidum, reticular thalamic nucleus and hypothalamic nuclei. In these areas, benzodiazepine binding sites are rich but neurons expressing a~ subunit m R N A are not seen. Since receptor proteins are mainly located on the dendrites, it is likely that neurons expressing the o~ subunit exist near these regions and that their processes invade these regions. Benzodiazepine binding sites have been detected in the brain at a very early prenatal stage s'4°. In contrast, bindings sites for [3H]muscimol or [3H]GABA were very low at birth, but then increased markedly to adult levels in the early postnatal period 2'42. Therel~bre, the postnatal pattern of expression of a~ subunit m R N A was very similar to that of G A B A binding sites in the brain, indicating that the aj subunit plays an important role in forming the G A B A or ligand recognition site in the developing brain, or that the a~ subunit itself is the ligand recognition site. Using immunocytochemistry with antiserum against G A B A or a 50 kDa benzodiazepine-binding subunit of the GABAA-R and in situ hybridization histochemistry with probe against the GABAA-R a~ subunit, Meinecke and Rakic ~° investigated the relationship between the expression of G A B A and GABAA-R in a wellcharacterized cerebellar circuit formed by granule cells and their synapses with Golgi II neurons in the rat cerebellum at ages from birth to 21 days. They demonstrated that the granule cells expressed GABAA-R a~ subunit m R N A only after they had completed migration and their dendrites had become involved in specific synapse circuits, including those with GABAergic afferents. Therefore, it seems that expression of the GABAA-R might be induced by G A B A itself. This hypothesis is confirmed by the report that G A B A or its agonists, muscimol and 4,5,6,7-tetrahydroisoxazolo[54-c]pyridin-3-ol, can induce expression of the low-affinity GABAA-R, but only in thc early stages of developmerit 7'~5"2¢'. In the present study, we found that at birth the expression of a~ subunit m R N A was very low or absent in most of the forebrain regions. However, at this time G A B A concentration in the whole brain and in most regions, such as the parietal cortex, cerebellum, corpus striatum, hypothalamus, and midbrainthalamus, was approximately 50% of the adult level ~¢~'~¢'. In addition, during the early postnatal period when synapses were being established extensively in the brain and the concentration of G A B A increased rapidly 13"W¢', the level of ¢~1 subunit m R N A was low and the a~ subunit was just started to be expressed in the rat forebrain. Therefore, it seems that in rats the expression of a~ subunit m R N A may be stimulated by G A B A itself (sec also ref. 13).

201

Comparison of the developmental expression of a I and /3 subunit mRNAs In previous studies, we have shown that a 1- and /32 subunit mRNA-containing neurons are often found at the same sites in the rat forebrain 49'5~. These areas are the isocortex, olfactory bulb, hippocampal formation, amygdala, septal region, nucleus of the diagonal band of Broca, bed nucleus of the stria terminalis, subthalamus, thalamus, ventral pallidum, globus pallidus and magnocellular preoptic nucleus. Therefore, it is likely that the al and /32 subunits are co-expressed by single neurons in these regions. Indeed, we have found neurons containing both a~ and /32 subunit mRNA on serial sections of the magnocellular preoptic nucleus 54. A comparison of the development of /32 subunit mRNA53 with that of al subunit mRNA in these regions revealed two patterns. In one case, the development of a~ and/32 subunit mRNA was similar, while in the other case their developmental patterns differed. The isocortex, olfactory bulb, hippocampal formation, amygdala, septal region, nucleus of the diagonal band of Broca, bed nucleus of the stria terminalis, subthalamus, and thalamus all belong to the first category. In the regions belonging to the first category, close functional linking between the c~ and /32 subunits many occur both for GABA a receptor formation and GABAergic transmission. In the latter group of cells, strong labeling for the 132 subuit was already detected at birth 53, while no signals for the cq subunit were detected at the same time. However, by postnatal day 14 these two subunit mRNAs showed the same level of expression. These results suggest that in the ventral pallidum, the globus pallidus, and the magnocellular preoptic nucleus, the GABAA-R subtypes present in the neonatal stage might differ from those found in adult rats. Acknowledgements. This work was supported by the Ministry of Education, Science, and Culture of Japan, by the Japan Brain Foundation and by Senri Life Science Center.

ABBREVIATIONS

Acb ACo AD AHP AM AMPO AOB AOE AV BL

accumbens nucleus anterior cortical amygdaloid nucleus anterodorsal thalamic nucleus anterior hypothalamic area, posterior part anteromedial thalamic nucleus anterior medial preoptic nucleus accessory olfactory bulb anterior olfactory nucleus, external part anteroventral thalamic nucleus basolateral amygdaioid nucleus

BLA BLP BLV BMA BMP BST CA1-3 Ce CeL CeM CL C1 CM CPu DEn DG DLG EP EPL G GL GP GrA Hb HDB ICj ICjM IGL La LaDL LaVL LD LH LP LPO LSD LSI LSV MCPO MD MeAD MePD MePV Mi MPA MS PC Pir PLCo PMCo Po

basolateral amygdaloid nucleus, anterior part basolateral amygdaloid nucleus, posterior part basolateral amygdaloid nucleus, ventral part basomedial amygdaloid nucleus, anterior part basomedial amygdaloid nucleus, posterior part bed nucleus of the stria terminalis fields CA1-3 of Ammon's horn central amygdaloid nucleus central amygdaloid nuclerus, lateral division central amygdaloid nucleus, medial division centrolateral thalamic nucleus claustrum central medial thalamic nucleus caudate-putamen (striatum) dorsal endopiriform nucleus dentate gyrus dorsal lateral geniculate nucleus entopeduncular nucleus external plexiform layer of the olfactory bulb gelatinosus thalamic nucleus glomerular layer of the olfactory bulb globus pallidus granular cell layer of the accessory olfactory bulb habenula nucleus of the horizontal limb of the diagonal band islands of Calleja islands of Calleja, major island internal granular layer of the olfactory bulb lateral amygdaloid nucleus lateral amygdaloid nucleus, dorsolateral part lateral amygdaloid nucleus, ventrolateral part laterodorsal thalamic nucleus lateral hypothalamic area lateral posterior thalamic nucleus lateral preoptic area lateral septal nucleus lateral septal nucleus, intermediate part lateral septal nucleus, ventral part magnocellular preoptic nucleus mediodorsal thalamic nucleus medial amygda[oid nucleus, anterodorsal part medial amygdaloid nucleus, posterodorsal part medial amygdaloid nucleus, posteroventral part mitral cell layer of the olfactory bulb medial preoptic area medial septal nucleus paracentral thalamic nucleus piriform cortex posterolateral cortical amygdaloid nucleus posteromedial cortical amygdaloid nucleus posterior thalamic nuclear group

202 PrS PT Re Rt STh TC Tu

VDB VL VLG VM VMH VO VP VPL VPM ZI

presubiculum paratenial thalamic nucleus reuniens thalamic nucleus reticular thalamic nucleus subthalamic nucleus tuber cinereum olfactory tubercle nucleus of the vertical limb of the diagonal band ventrolateral thalamic nucleus ventrolateral geniculate nucleus ventromedial thalamic nucleus ventromedial hypothalamic nucleus ventral orbital cortex ventral pallidus ventral posterolateral thalamic nucleus ventral posteromedial thalamic nucleus zona incerta

REFERENCES 1 Aghajanian, G.K. and Bloom, F.E., The formation of synaptic junctions in developing rat brain: a quantitative electron microscopic study, Brain Res., 6 (1967) 716-727. 2 Aldinio, C., Balzano, M.A. and Toffano, G., Ontogenic developmerit of G A B A recognition sites in different brain areas, Pharmacol. Res. Commun., 12 (1980) 495-500. 3 Altman, J. and Das, G.D., Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats, J. (~)mp. Neurol., 124 (19651 319-336. 4 Araki, T. and Tohyama, M., Region-specific expression of GABA~x receptor ~.~ and ~4 subunits m R N A s in the rat brain, Mol. Brain Res., 12 (1992) 293 314. 5 Araki, T., Sato, M., Kiyama, H., Manabe, Y. and Tohyama, M., Localization of GABAA-receptor y2-subunit mRNA-containing neurons in the rat central nervous system, Neuroscience, 47 (1992) 45-61. 6 Bateson, A., Lasham, A and Darlison, M.G., y-Aminobutyric acids receptor heterogeneity is increased by alternative splicing of a novel /3-subunit gene transcript, J. Neurochem, 56 (19911) 1437-14411. 7 Belhage, B., Hansen, G.H., Schousboe, A. and Meier, E., G A B A agonist promoted formation of low affinity G A B A receptors on cerebellar granule cells restricted to early development, hit..l. Det'. Neurosci., 6 (1988) 125-128. 8 Braestrup, C. and Nielsen, M., Ontogenetic development of benzodiazepine receptors in the brain, Brain Res., 147 (1978) 170 173. 9 Collins, M.L. and Hunsaker, W.R., Improved hybridization assays employing tailed oligonucleotide probes: a direct comparison with 5'-end-labeled oligonucleotide probes and nick-translated plasmid probes, Anal Biol., 151 (1985) 211-224. 111 Coyle, J.T. and Enna, S.J., Neurochemical aspects of the ontogenesis of GABAergic neurons in the rat brain, Brain Res., 111 119761 119-133. 11 Cutting, G.R., I,u, L., O'Hara, B.F., Kasch, L.M., MontroseRafizadeh, C., Donovan, D.M., Shimada, S., Antonarakis, S.E., Guggino, W., Uhl, G.R. and Kazazian, H.H., Cloning of the y-aminobutyric acid (GABA) Pl eDNA: a G A B A receptor subunit highly expressed in the retina, Proc. NatL Acad. Sci. USA, 88 ( 1991 ) 2673-2677. 12 Gambarana, C., Beattie, C.E., Rodriguez, Z.R. and Siegel, R.E., Region-specific expression of messenger R N A s encoding G A B A A receptor subunits in the developing rat brain, Neuroscience, 45 ( 1991 ) 423-432.

13 Gambarana, C., Pittman, R. and Siegel, R.E., Developmental expression of the G A B A A receptor ~rt subunit m R N A in the rat brain, J. Neurobiol., 21 (1990) 1169 1179. 14 Garrett, K.M., Duman, R.S., Saito, N., Blume, A.J., Vitek, M.P. and Tallman, J.F., Isolation of a e D N A clone for the alpha subunit of the h u m a n G A B A - A receptor, Biochem. Biophys. Res. Commun., 156 (1988) 1(139-1(145. 15 Hansen, G., Belhage, B., Schousboe, A. and Meier, E., Temporal development of G A B A agonist induced alterations in ultrastructure and G A B A receptor expression in cultured cerebellar granule cells, hit. J. Dec. Neurosci., 5 (1987) 263-269. 16 Hedner, T., lversen, K. and Lundborg, P., Central G A B A mechanisms during postnatal development in the rat: neurochemical characteristics, J. Neural Transm., 59 (1984) 105-118. 17 Hironaka, T., Morita, Y., Hagihira, S., Tateno, E., Kita, H. and Tohyama, M., Localization of GABAA-receptor ~el subunit mRNA-containing neurons in the lower brainstem of the rat, Mol. Brain Res., 7 (1990) 335-345. 18 Khrestchatisky, M., Maclennan, A.J., Chiang, M-Y. Xu, W., Jackson, M.B., Brecha, N., Sternini, C., Olsen, R.W. and Tobin, A.J.. A novel a subunit in rat brain G A B A A receptors. Neuron, 3 11989) 745-753. 19 Lauder, J.M., Hart, V.K.M., Henderson, P., Verdoorn, T. and Towle, A.C., Prenatal ontogeny of the GABAergic system in the rat brain: an immunocytochemical study, Neuroscience, 19 (19861 465 -493. 20 Laurie, D.J., Seeburg, PAl. and Wisden, W., The distribution of 13 G A B A A receptor subunit m R N A s in the rat brain, lI. Olfactory bulb and cerebellum, Z Neurosci., 12 (1992) 1063-1076. 21 Levitan, E.S., Schofield. P.R., Burt, D.R., Rhee, L.M., Wisden, W., Kohler, M., N. Fujita, Rodriguez, H.F., Stephenson, A., Darlison, M.G., Barnard, E.A. and Seeburg, P.H., Structural and functional basis for GABA,x receptor heterogeneity, Nature, 335 11988) 76 79. 22 Luddens, H., Pritchett, D.B., Kohler, M., Fillisch, 1., Keinanen, K., Monyer, H., Sprengel, R. and Seeburg, P.H., Cerebellar G A B A A receptor selective for a behavioral alcohol antagonist, Nature, 346 (1990) 648-651. 23 Madtes, P. Jr. and Redburn, D.A. G A B A as a trophic factor during development, Lift" Sci., 33 (1983) 979-984. 24 MacLennan, A.J.. Brecha, N., Khrestchatisky, M., Sternini, ('_ Tillakaratne, N.J.K., Chiang, M.-Y., Anderson, K., Lai, M. and Tobin, A.J., Independent cellular and ontogenetic expression of m R N A s encoding three (~ polypeptides of the rat GABA/x receptor, Neuroscience, 43 (1991) 369-380. 25 Mattson, M.P., Kater, S.B., Excitatory and inhibitory neurotrans mitters in the generation and degeneration of hippocampal neuroarchitecture, Brain Res., 478 11989) 337 348. 26 Meier, E., Drejer, J. and Schousboe, A.. G A B A induces function ally active low-affinity G A B A receptors on cultured cerebellar granule cells, J. Neurochem., 43 (1984) 1737 1744. 27 Meier, E., Hansen, G . H and Schousboe, A., The trophic effect ot G A B A on cerebellar granule cells is mediated by GABA-receptors, Int. J. Dec. NeuroscL, 3 (1985) 4111-407. 28 Meier, E. and Jorgensen, O.S., y-Aminobutyric acid affects the developmental expresskm of neuro-associated proteins in cerebellar granule cell cultures, ,L Neurochem., 46 (1986) 1256-1262. 29 Meier, E., Jorgensen, O.S. and Schousboe, A., Effect of repeated treatment with a 7-aminobutyric acid receptor agonist on postnatal neuronal development in r~tts, J. Neurochem., 49 (1987) 14621470. 30 Meinecke. D.L. and Rakic, P., Developmental expression of G A B A and subunits of the G A B A A receptor complex in an inhibitory synaptic circuit in the rat cerebellum, Det'. Brain Res., 55 (19911) 73 86. 31 Noguchi, K., Morita, Y., Kiyama, H., ()no, K. and Tohyama, M., A noxious stimulus induces the preprotachykinin-A gene expression in the rat dorsal root ganglion: a quantitative study using in situ hybridization histochemistry, Mol. Brain Res., 4 (1988) 31-35. 32 Olsen, R.W., McCabe, R.T. and Wamsley, J.K., G A B A A receptor subtypes: autoradiographic comparison of GABA. benzodi-

203

33 34

35

36

37

38

39

40

41

42

43

azepine, and convulsant binding sites in the rat central nervous system, J. Chem. Neuroanat., 3 (1990) 59-76. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1986. Pritchett, D.B., Luddens, D.B. and Seeburg, P.H., Type 1 and Type II GABAA-benzodiazepine receptors produced in transfected cells, Science, 245 (1989) 1389-1392. Pritchett, D.B., Sontheimer, H., Shivers, B.D., Ymer, S., Kettenmann, H., Schofield, P.R. and Seeburg, P.H., Importance of a novel GABA A receptor subunit for benzodiazepine pharmacology, Nature, 338 (1989) 582-585. Richards, G., Mohler, H. and Haefely, W., Mapping benzodiazepine receptors in the CNS by the radiohistochemistry and immunohistochemistry. In P. Panula, H. Paivarinta and S. Soinila (Eds.), Neurology and Neurobiology, Vol. 16, Neurohistochemistry: Modern Methods and Applications, Alan R. Liss, New York, 1986, pp. 629-677. Roberts, E., GABA: the road to neurotransmitter status. In R.W. Olsen and J.C. Venter (Eds.), Receptor Biochemistry and Methodology, Vol. 5, Alan R. Liss, New York, 1986, pp. 1-39. Rothe, T., Middleton-Price, H. and Bigl, V., The ontogeny of GABA receptors and glutamic acid decarboxylase in regions of the rat brain: effect of prenatal exposure of diazepam, Neuropharmacology, 27 (1988) 661-667. Schofield, P.R., Darlison, M.G., Fujita, N., Burt, D.R., Stephenson, F.A., Rodriguez, H., Rhee, L.M., Ramachandran, J., Reale, V., Glencorse, T.A., Seeburg, P.H. and Barnard, E.A., Sequence and functional expression of the GABA A receptor shows a ligand-gated receptor superfamily, Nature, 328 (1987) 221-227. Schlumpf, M., Richards, J.G., Lichtensteiger, W. and Mohler, H., An autoradiographic study of the prenatal development of benzodiazepine-binding sites in rat brain, J. Neurosci., 3 (1983) 14781487. Shivers, B.D., Killisch, 1., Sprengel, R., Sontheimer, H., Kohler, M., Schofield, P.R. and Seeburg, P.H., Two novel GABA A receptor subunits exist in distinct neuronal subpopulations, Neuron, 3 (1989) 327-337. van Eden, C.G., Mrzljak, L., Voorn, P. and Uylings, H.B.M., Prenatal development of GABA-ergic neurons in the neocortex of the rat, J. Comp. Neurol., 289 (1989) 213-227. Verdoorn, T.A., Draguhn, A., Ymer, S., Seeburg, P.H. and Sakmann, B., Functional properties of recombinat rat GABA A receptors depend upon subunit composition, Neurons, 4 (1990) 919-928.

44 Whiting, P., Mckernan, R.M. and Iversen, L.L., Another mechanism for creating diversity in y-aminobutyrate type A receptors: RNA splicing directs expression of two forms of Y2 subunit, one of which contains a protein kinase C phosphorylation site, Proc. Natl. Acad. USA, 87 (1990) 9966-9970. 45 Wisden, W., Laurie, D.J., Monyer, H. and Seeburg, P.H., The distribution of 13 GABA A receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon, J. Neurosci., 12 (1992) 1040-1062. 46 Ymer, S., Draguhn, A., Kohler, M., Schofield, P.R. and Seeburg, H., Sequence and expression of a novel GABA A receptor a subunit, FEBS Lett., 258 (1989) 119-122. 47 Ymer, S., Draguhn, A., Wisden W., Werner, P., Keinanen, K., Schofield, P.R., Spreng¢l, R., Pritchett, D.B. and Seeburg, P.H., Structural and functional characterization of the yl subunit of GABA A/benzodiazepine receptors, EMBO J., 9 (1990) 32613267. 48 Ymer, S., Schofield, P.R., Draguhn, A., Werner, P., Kohler, M. and Seeburg, P.H., GABA A receptor fl subunit heterogeneity: functional expression of cloned cDNAs, EMBO J., 8 (1989) 1665 - 1670. 49 Zhang, J.-H., Araki, T., Sato, M. and Tohyama, M., Distribution of GABAA-receptor a 1 subunit gene expression in the rat forebrain, Mol. Brain Res., 11 (1991) 239-247. 50 Zhang, J.-H., Sato, M. and Tohyama, M., The differential expression patterns of the mRNAs encoding fl subunits (ill, f12 and f13) of GABA A receptor in the olfactory bulb and its related areas in the rat brain, Neurosci. Lett., 119 (1990) 257-260. 51 Zhang, J.-H., Sato, M. and Tohyama. M., The region-specific expression of the mRNAs encoding /3 subunits (/3t, /32 and /33) of GABA n receptor in the rat brain, J. Comp. Neurol., 304 (1991) 1-21.

52 Zhang, J.-H., Sato, M. and Tohyama, M., Different postnatal ontogenic profiles of neurons containing /3 (/31, /32 and /33) subunit mRNAs of GABA A receptor in the rat thalamus, Det'. Brain Res., 58 (1991) 289-292. 53 Zhang, J.-H., Sato, M. and Tohyama, M., Different postnatal development profiles of neurons containing distinct GABA A receptor/3 subunit mRNAs (/31,/32, and /33) in the rat forebrain, J. Comp. Neurol., 308 (1991) 586-613. 54 Zhang, J.-H., Sato, M. and Tohyama, M., Co-expression of the a~ and/32 subunit genes of the GABA A receptor in the magnocellular preoptic nucleus, Mol. Brain Res., 15 (1992) 174-178.