Ontogeny and distribution of GABAA receptors in rat brainstem and rostral brain regions

Neuroscience Vol. 49, No. 4, pp. 973-989, 1992 Printed in Great Britain

0306-4522/92 $5.00 + 0.00

Pergamon Press Ltd 0 1992IBRO

ONTOGENY AND DISTRIBUTION OF GABA, RECEPTORS IN RAT BRAINSTEM AND ROSTRAL BRAIN REGIONS Y. XIA

and G. G. HADDAD*

Department of Pediatrics, Section of Respiratory Medicine (Laboratory of Respiratory Neurobiology), Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, U.S.A. Abstract-Previous studies from our laboratory and others have shown that there are major age-related differences in brainstem neuronal function. Since GABA, receptors are major targets for GABA-mediated inhibitory modulation and play a key role in regulating cardiorespiratory function, especially during 0, deprivation, we examined differences in GABA, receptor density and distribution during postnatal development. Using quantitative receptor autoradiography, the present study was performed to examine the postnatal expression of GABA, receptors in the rat brainstem and rostra1 brain areas at five ages, i.e. postnatal day 1 (Pl), P5, PlO, P21 and Pl20. Ten-micrometer brain sections at different brain levels were labelled with [3H]muscimol in Triscitrate buffer. We found that (i) GABA, receptors appeared very early in almost all the brainstem as well as rostra1 areas; (ii) at PI, the brainstem had a higher GABA, receptor binding density than rostra1 areas and its density peaked at PS or PlO; and (iii) receptor densities of the cerebellum and rostra1 brain areas such as cortex, thalamus and dentate gyrus increased with age, especially between PlO and P21, but most other subcortical areas like caudate-putamen and hippocampal CA1 area did not increase remarkably after birth. We conclude that: (i) GABA, receptors exist in most brain areas at birth; (ii) there are several patterns of postnatal development of GABA, receptors in the CNS with dramatic differences between the brainstem and cortex; (iii) brainstem functions rely more on GABA, receptors in early postnatal life than at more mature stages. We speculate that GABA, receptors develop earlier in phylogenetically older structures (such as brainstem) than in newer brain regions (such as cortex).

It is now well established that GABA is a key inhibitory neurotransmitter in the mammalian CNS and that it plays an important role in a wide variety of physiologic activities and pathologic alterations,2~“~‘3~39~45~So~s0a~S7 The molecular mechanisms underlying the action of many centrally acting agents (including sedative/hypnotic, anxiolytic, anticonvulsant and muscle relaxant) are directly or indirectly related to the GABAergic system. For example, enhancement of GABA activity has been considered a major component of benzodiazepine action.45 So far, two classes of GABA receptors have been well characterized, i.e. GABA, and GABAg.6*33s39The major receptor type for the inhibitory action of the neurotransmitter GABA is GABA,.“s~‘*~ It is a heteroligomeric protein composed of several distinct polypeptides which constitute a supramolecular complex with a benzodiazepine binding site and a chloride channel.‘8,“.45,55,6L,65 Activation of GABA, receptors causes an increase in Cl - flux across neuronal membranes.7s42This subtype is sensitive to muscimol and antagonized by bicuculline.7*‘2~42 A large body of neurophysiological and neurochemical evidence suggests that GABA is involved in cardiorespiratory control during both normoxia and O2 deprivation.‘6~‘7~19*24*2*~30*42@@ Centrally applied

GABA or muscimol, a GABA, receptor agonist, causes respiratory depression, hypotension and bradycardia. m-70Previous studies have also shown that GABA accumulates in the CNS during hypoxia and may be one of the mechanisms underlying hypoxic tolerance.3.M.42a Previous data from our laboratory have shown that the cardiorespiratory response to hypoxia in the newborn is different from that in the adult.27 Also, it has been shown that newborn mammals survive O2 deprivation longer than the adult, and that the survival period depends on postnatal age and species maturity at birth. 20.3LBecause of the importance of brainstem neurons in maintaining cardiorespiratory homeostasis and other autonomic functions, we have recently studied the electrophysiologic responsiveness of brainstem neurons to hypoxia/anoxia and found that the responsiveness is blunted in the newborn but not in the adult rat.” The age-related functional differences may be attributed to differences in membrane properties between neonatal and adult CNS, including the presence and functional activity of neuronal GABA receptors. However, little is known about the ontogeny and distribution of GABA, receptors in brainstem nuclei although some data are available on the development of GABA GABAergic neur_ receptors, 1,9,14,15,*1,23,40,4~48.52~Y,59.62

*To whom correspondence should be addressed. P, postnatal day.

ons14*22,35,36 or functional maturation of GABAergic inhibition.3*sw Therefore, the purpose of the present

Abbreviations:

913

974

Y.

XlA

aMi

study is to examine the ontogeny and distribution of GABA, receptors in the brainstem and to compare the results with those from rostra] brain areas in rats using in vitro quantitative autoradiographic techniques. Since muscimol has been widely used as a GABA, receptor ligand,4,1’.4’ we used [‘H]muscimol to label brain tissues. We specifically sought to explore the following questions: (i) What

c;

G.

HADDAD

are the differences in GABA, receptor distribution and density in the brainstem at birth as compared with those in the adult? (ii) How do GABA, distribution and density change in the brainstem after birth? (iii) Is there any difference in postnatal development of GABA, receptors between the brainstem and phylogenetically newer structures like the neocortex?

l-5 7

lobules l-5 of cerebellum facial nerve

MRD

AL AP AV BS CA1 CA3 CB cG

ansifonn lobules area postrema anteroventral thalamic nucleus brainstem field CA1 of Ammon’s horn field CA3 of Ammon’s horn cerebellum cerebellar granular layer central gray central nucleus of the inferior cotliculus, dorsomedial part cerebeilar molecular layer caudate putamen frontal cortex cuneate nucleus dorsal cochear nucleus dentate gyms dorsal lateral geniculate nucleus dorsomedial hypothalamic nucleus dorsal parabrachial nucleus dorsal raphe nucleus dorsal tegmental nucleus entorhinal cortex fornix facial nucleus fasciculus retroflexus frontoparietal cortex, motor area frontop~etal cortex, somatosensory area globus pallidus gracile nucleus habenular nucleus hypoglossal nucleus interanteromedial thalamic nucleus inferior colliculus inferior olive lateral amygdaloid nucleus laterodorsal thalamic nwleus lateral hypothalamic area lateral posterior thalamic nucleus lateral reticular nucleus mammillary nucIeus medial geniculate nucleus medial mammillary nucleus, posterior part

MRv

%DM CM CPU Crx Cu DCo DG DLG DM DPB DR DTg Ent f Fa or FA fr

FrPaM FrPaSS GP z HYP IAM IC IO La or LA LD LH LP LRt or LRT M MG MP

mt MVE NTS NTSu NTSR PCg PCRT PFL PMCo PMN PnC PO PO PR PrH or PaH RMg or RMc ROb or ROB RPn or RPN RSpl SGe or SGE SLVE SNR so spsc Sp5I or SF51 Str SuG VL VMH VPB VPL VPM 21

medullary reticular nucleus, dorsal part medullary reticular nucleus, ventral part mammillothalamic tract medial vestibular nucleus nucleus of the solitory tract nucleus of the solitory tract, medial part nucleus of the solitory tract, rostra1 part posterior cingulate cortex parvocellular reticular nucleus para~o~uIus posterom~i~ cortical amygdaloid nucleus paramedial reticular nucleus pontine reticular nucleus, caudal part posterior thalamic nuclear group primary olfactory nucleus pontine reticular nucleus prepositus hypoglossal nucleus raphe magnus nucleus raphe obscurus nucleus raphe pontis nucleus retrosplenial cortex suprageniculate nucleus of the pons superior and Iateral vestibular nucleus substantia nigra, reticular part superior olive nucleus of the spinal tract of the trigeminal nerve, caudal part nucleus of the spinal tract of the trigeminal nerve, interpolar part striate cortex superficial grey layer of superior colliculus ventrolateral thalamic nucleus ventromedial hypothalamic nucleus ventral parabrachial nucleus ventroposter~or thalamic nucleus, lateral part ven~~sterior thalamic nucleus, medial part zona incerta

Fig. 1. Serial autoradiographic images in a saturation study. Note that GABA, receptor binding was saturable. The sections were obtained from an adult rat brain and incubated in 50 mM Tris-citrate buffer (PH 7.0) with increasing concentration (&250nM) of [3H]muscimol. The film was exposed to the incubated brain sections for two weeks. (a) 0 nM; (b) 1 nM; (c) 12.5 nM; (d) 25 nM; (e) 50 nM; (f) 100 nM; (g) 200 nM; (h) 250 nM. Scale bar = 2 mm.

Ontogeny of GABA, receptors in rat brain

Fig. 1

975

I’. XIA and G G. HADDAD

976 Table

I. Mean Kd values of GABA,

in developing

PlO

P5

PI MK, n S.E.

receptors

rat brain P120

P21

High

Low

High

Low

High

Low

High

Low

High

Low

3.3 12 0.1

48.2 12 5.5

3.4 16 0.3

41.8 16 2.7

2.2 15 0.4

41.1 15 7.5

3.5 15 0.3

59.4 15 3.9

3.1 I2 0.5

46.7 12 9.3

Values are expressed in nM. MK,,, mean K,, values; n. number

EXPERIMENTAL

of nuclei measured

PROCEDURES

Mareriuls [‘H]muscimol (30.3 Ci/mmol) was purchased from New England Nuclear. GABA was purchased from Sigma Chemical Co. (St Louis, MO). All other compounds were purchased from Aldrich (Milwaukee, WI) or Sigma Chemical Co. Animals Sprague-Dawley rats (Camm Research, Wayre, NJ) at postnatal day 1 (Pl), P5, PlO, P21 and P120 (adult) were used. Handling of rat pups was similar to that in our previous work. ” In brief, several rat pups of the same litter at Pl were shipped to our institution and three pups were immediately used. The rest of the same litter were used at P5. The PIO, P21 and adult rats were obtained from different litters. For the autoradiographic study of ontogeny and distribution, we had three rats at each age.

Tissue preparation Rats from each age were decapitated under inhalational anesthesia (methoxyflurane) and their brains were rapidly removed and divided into several blocks in cold Ringer’s solution. The blocks were frozen in isopentane pre-cooled in a dry-ice/methanol bath, and stored in -70°C freezer. Pt. PIO, P21 and adult brains were obtained on the same day and P5, four days later. Using a Hacker-Bright cryostat, IO-pm-thick coronal sections were cut sequentially at -20°C. The sections were thaw-mounted onto pre-cleaned and gelatin-coated microscope slides and stored in wellsealed slide boxes at -70°C. Autoradiography The methods were similar to those of our previous work.7’.72 In brief, all tissue sections from the same group were performed under the exact same conditions including buffer, ligand concentration, temperature and time. Three sets of incubations were performed, each containing sections

a

d

e

Fig. 2. Distribution of GABA, receptors in the adult rat brainstem. Note that the GABA, receptor density was low throughout the brainstem except for some nuclei in the dorsal area such as dorsal tegmental nucleus and the nucleus of the solitary tract. The brain sections were labelled with 50 nM [3H]muscimol in 50 mM Trisscitrate buffer (pH 7.0) and the films were exposed for four weeks. Scale bar = 2 mm. From a to f. rostra1 to caudal level.

Ontogeny of GABA, from one brain at each age. The section-mounted slides were gradually brought up to room temperature and dried in cool air for 10 min immediately prior to binding procedure. (1) Pre-incubation: to deplete the tissue of endogenous ligands, the thawed and dried sections were pre-incubated for 30 min at 25°C in 50 mM Tris-citrate buffer (pH 7.0), and then were blown dry with a stream of cool air at room temperature. (2) Receptor binding: the sections were incubated for 45 min at 4°C with 50nM [‘H]muscimol in 50 mM Trisscitrate

‘eceptors in rat brain

917

buffer (pH 7.0). Nonspecific binding was determined from adjacent sections in parallel incubations in the presence of 100 p M unlabeled GABA. (3) Rinsing: to reduce nonspecific binding, incubated slides were rinsed in seven different jars (2 s each) containing 500 ml of cold 50 mM Tris-citrate buffer (pH 7.0) and dipped in one jar containing 500 ml of cold distilled water. (4) Exposure and development: the rinsed slides were dried rapidly under a stream of cold and dry air and stored in a desiccated, and pressure-reduced jar

Fig. 3. Distribution of GABA, receptors in the adult rat rostra1 brain. Note that GABA, receptor distribution was very heterogeneous and the density was extremely high in the rostra1 brain regions as compared with those in the brainstem (see Fig. 2). The experimental conditions were the same as those for Fig. 2. Scale bar = 2 mm. From a to f, rostra1 to caudal level.

Y. Xrn and G. G. HADDAD

97x

at 4°C for three days. Subsequently, slides were placed in exposure cassettes, along with sH-polymer and ‘H-tissue7’.” standards, all of which were bought (polymer, Amersham) or made (tissue) at the same time, covered with a tritiumsensitive film (-‘H-LJltrofilm, LKB) and stored at 4°C. After four weeks, the films were developed in Kodak D- 19 at 19°C and fixed.

Table

2. Regional

distribution

Saturation

stuiy

The experiments were carried out at the level of 3.8-5.8 mm posterior to the bregma according to Paxinos and Watson’s atlas.4q Brain structures including the cortex, hippocampus, thalamus, hypothalamus and midbrain were present on these sections. The same incubation conditions

of muscimol

binding

Area Cortex Frontoparietal cortex, motor area layers 1 & 2 layers 3 & 4 layers 5 & 6 Frontoparietal cortex, somatosensory area layers 1 & 2 layers 3 & 4 layers 5 & 6 Striate cortex layers 1 & 2 layers 3 & 4 layers 5 & 6 Anterior cingulate cortex Posterior cingulate cortex Temporal cortex, auditory area Primary olfactory cortex Retrosplenial cortex Entorhinal cortex Hippocampus Dentate gyms Field CA1 of Ammon’s horn Field CA2 of Ammon’s horn Field CA3 of Ammon’s horn Field CA4 of Ammon’s horn Basal ganglia Caudate-putamen Globus pallidus Ventral pallidus Amygdala Basolateral amygdaloid nucleus Basomedial amygdaloid nucleus Lateral amygdaloid nucleus Central amygdaloid nucleus Medial amygdaloid nucleus Amygdalohippocampal area Posteromedial cortical amygdaloid nucleus Thalamus Central medial thalamic nucleus Gelatinosus nucleus of thalamus Laterodorsal thalamic nucleus Lateral posterior thalamic nucleus Mediodorsal thalamic nucleus Habenular nucleus Posterior thalamic nuclear group Ventroposterior thalamic nucleus, medial part Ventroposterior thalamic nucleus, lateral part Medial geniculate nucleus, dorsal part Medial geniculate nucleus, ventral part Zona incerta Hypothalamus Ventromedial hypothalamus nucleus Lateral hypothalamic area Posterior hypothalamic nucleus Mamrnilary nucleus Cerebellum Granular layer Molecular layer

sites in adult Binding (fmol/mg

rat brain

density protein)

2690 &- 101 3677 + 115 2970 & 267 2630 f 178 3010 + 165 2551 + 30 2324 + 60 3335 f 37 2814 f 196 2898 + 38 2297 f 46 2791 f 124 2391 k 47 2706 f 175 2925+41 2965 _C 120 2157 + 32 1813 f 87 1336 f 64 1598+58 1666+49 1505 rf: 76 2467 & 50 2055 + 103 1993 f 49 2927 + 166 2073 f 96 2133 f 32 2343 f 87 2610+45 2920 & 139 5491 + 248 3560 + 204 3079 & 278 3497 If 148 946+76 3093 f 137 3480 * 164 2098 f 72 2082 f 217 3408 f 180 1081 + 63 168Ok 11 1266 & 126 1488 k 22 1088 5 23 9280 + 674 3354 * 103 continued

Gntogeny of GABA,, receptors in rat brain

979

Table 2. (continued) Binding density (fmol/mg protein)

Area Midbrain Superior colliculus superior gray layer intermediate gray layer Inferior colliculus dorsahnedial part of the central nucleus ventrolateral part of the central nucleus Central gray Anterior pretectal area Linear nucleus of the raphe Red nucleus Interpeduncular nucleus Substantia nigra Dorsal raphe nucleus Pons Dorsal tegmental nucleus Laterodorsal tegmental nucleus Pontine reticular nucleus Dorsal raphe nucleus Raphe pontis nucleus Raphe pallidus nucleus Dorsal parabrachial nucleus Ventral parabrachial nucleus Kitlliker-Fuse nucleus Facial nucleus Superior vestibular nucleus Lateral vestibular nucleus Suprageniculate nucleus of the pons Medulla Superior vestibular nucleus Lateral vestibular nucleus Dorsal cochlear nucleus Prepositus hypoglossal nucleus Sninal vestibular nucleus Raphe obscurus nucleus Lateral reticular nucleus Paramedian reticular nucleus Medulla reticular nucleus dorsal part ventral part Nucleus of spinal tract of the trigeminal nerve oral part interpolar part caudal part Nucleus of the solitary tract rostra1 part caudal part Hypoglossal nucleus Inferior olive Cuneate nucleus Gracile nucleus Area postrema

261.5f 87 2040 + 178 2062 * 21 1410&20 1826 f 113 1100*31 5901t63 852 rf 23 1162 f 29 2202 f 100 1339 & 57 2490 + 229 1990 & 137 529 f 77 1198f48 488 rf: 80 327j,ll 1279 zf 35 1483 f 20 1109&22 576 f 33 364t28 287 + 10 862 f 56 451 f 19 375 If: 12 1014Jr28 1002 * 18 868 * 10 338 f 24 649& 16 418 & 15 817f26 620 rf: 20 415 + 18 530 f 42 724 j, 18 1000*30 1737 + 39 844_+30 847 & 37 728 & 19 762 rt: 16 1140+ 15

Values are exnressed in mean f S.E. from nine corresponding animals (three sections each rat). were used except for different concentrations (0, 1, 12.5,25, 50, 100,200 and 250 nM) of [‘H]muscimol. The films were exposed to incubated brain sections for two weeks. Arudysis

After developing each film, all brain sections were stained with Cresyl Violet for histological verification of anatomical structures which were identi&d by reference to the rat brain atlas.47+49 The autoradiographic images from various brain levels were quantified by a computerized imaging system (Recognition Technology Inc.). Sururrzrion unu!ysi.r. In our conditions, the optical densities varied from 0 to 0.46 for all images and this was far

sections of three

from saturation level. Since the optical density was linear with radioactivity within the range of O-O.5 (r = 0.99), we used optical density values as index of specific binding density after subtracting nonspecific binding and fitm background. In each animal, we measured Eve brain areas, i.e. cortex, hippocampus, thalamus, hypothalamus and midbrain and at least two nuclei from each area. Each value obtained from a given nucleus in a given animal was the average of six to eight measurements. O~rug~y arid struts. Optical density values in a brain region were converted to binding site density when compared with standards. The calibration values produced by 3H-standards were reproducible with &2%.

9x0

Y. X.4 and G G. HADDAD

Three corresponding sections in each ammal were quantitated for a given brain nucleus. The density values, in fmol/mg protein, were expressed as means i S.E. of averaged values from nine corresponding sections of three

animals at each age. RESULTS

Binding characteristics sections

sf‘ [3H]muscimol sites in brain

[3H]muscimol binding sites were saturable in either newborn or adult rats. Serial autoradiographic images of an adult rat brain incubated in different concentrations of [3H]muscimol (l-250 nM) are shown in Fig. 1. In most nuclei measured, Scatchard plots were curvilinear and suggested two binding sites, i.e. high- and low-affinity binding sites. Depending on the nucleus observed, high-affinity binding sites occupied 25-50% of total binding sites. Only a very few nuclei such as the dorsal hypothalamic area at Pl presented a linear pattern. Table 1 shows that Kd values of high- and low-affinity sites do not change in any major way as a function of age. In addition, little variability was obtained when comparing Kc, values of most areas measured. For example, the Kc, values of the high-affinity sites for the striate cortex, retrosplenial cortex, entorhinal cortex and hippocampus at Pl were (in nM) 2.6, 3.4, 2.1 and 1.9, respectively, while those in the adult were 2.8, 2.3, 2.3 and 2.1, respectively. Based on our saturation results, the concentration (50 nM) used in our autoradiographic study (below) roughly reflected 100% occupancy of the high-affinity binding sites and about 50% for the low-affinity binding sites in rats at all ages. GABA,

receptor distribution

in the adult rat CNS

GABA, receptor distribution in the brainstem (Figs 2, 4E, 7b) was very different from that in other brain regions (Figs 3, 45, 40, 7b). The quantification of GABA, receptor density from various brain nuclei is shown in Table 2. As seen in the table and figures, GABA, receptors were detected in almost all areas of gray matter. The density, however, varied from area to area with large differences between brainstem and other areas. Lower receptor density in brainstem than in rostra1 areas. The brainstem contained low GABA, receptor density (below 1000 fmol/mg protein), especially at

the levels of the caudal pons and rostra1 ntcdulla. Most brainstem nuclei had a density within the range of 300-800 fmol/mg protein with the lowest density in the raphe and vestibular nuclei. In some cardiorespiratory-related nuclei such as the nucleus of the solitary tract and the dorsal and ventral parabrachial nuclei, the receptor density was higher ( x 2). The highest density in the brainstem, i.e. around 2000 fmol/mg protein, was seen in the dorsal tepmental area of the pons (Fig. 2a, b). In sharp contrast to the brainstem, other brain regions including ‘the cerebellum and more rostra1 areas contained high GABA, receptor density. Most rostra1 brain regions contained 2300-3500 fmol/mg protein, with the highest densities seen in the cerebellum and cortex. In the granular layer of the cerebeilum, the density was as high as 9000 fmol/mg protein. More homogeneous distribution in brainstem than in rostra1 ureas and cerebellum. The brainstem had a more uniform distribution of GABA, receptor than other brain regions, as shown in Fig. 2. Although a few nuclei of the dorsal brainstem contained a moderate density, there were no major differences in the density between brainstem nuclei at most regions ot the pons and medulla. This was different from that in other brain areas (Figs 3, 45, 40, 7b). Very heterogeneous distribution was seen in the cerebellum and rostra1 brain areas such as the cortex. For example, layer 3 of the frontal cortex had a distinctly higher density than layer 6. In agreement with other studies, the whole cerebellum contained a high density of GABA,, however, the granular layer had clearly a different labeling density. D@erent receptor density within same nucleus. In some brainstem nuclei, GABA, receptors had different densities in different parts of the same nucleus although the distribution was more homogeneous in the brainstem than that in rostra1 brain regions. For example, the density in the caudal part of the nucleus of the solitary tract was 70% higher than that in its rostra1 part. Also, there was a 75% higher density in the caudal than rostra1 part of the nucleus of the trigeminal nerve spinal tract. Such differences were also observed at the same brainstem level. At the level of the area postrema, for example, GABA, receptor density was more than 30% higher in the dorsal than ventral part of the medullary reticular nucleus. The 3ifferences between parts of the same nucleus were

Fig. 4. Development of GABA, receptors in the rat brain. Note that: (i) the receptor density in the medulla was much higher in the newborn (A, B, C) than in the more mature (D) or the adult (E); (ii) the density in the cerebellum was low in the newborn, especially at Pl, (F), and increased with age; the density was several-fold higher in the adult (J) than at Pl; (iii) the central cerebellar structures (especially lobules l-3) developed GABA, receptors earlier than the outer structures like the ansiform lobules and paraflocculus of the cerebellum (F-H); and (iv) GABA, receptor distribution in the rostra1 brain was much more homogeneous and the density was relatively lower at PI (K) than in the adult (0). All brain sections were labeled with 50 nM [3H]muscimol in 50 mM Trisxitrate buffer (PH 7.0) and exposed to the films for four weeks. Medulla: A-E (Pl, P5, PIO, P21 and P120, respectively). Cerebellum: F-J (PI, P5, PlO, P21 and P120, respectively). Rostra1 brain: K-O, (PI, P5, PlO, P21 and P120, respectively). Scale bar in A = 2 mm for A-E; scale bar in F = 2 mm for F-J; scale bar in K = 2 mm for K--O. (pmol/mgP represents pmol/mg protein.)

Y. XIA and G G. HADDAV

982

also seen in the rostra1 brain. For instance, Fig. 3 shows that GABA, receptor densities were different between the rostra1 and caudal parts of caudateputamen. Ontogeny system

of GABA,

receptor in rat central nervous

GABA, receptor autoradiograms at three different brain levels in the developing rat are shown in Fig. 4

PR

2600

and comparative quantitation from representative nuclei of the brainstem and rostra1 brain areas is displayed in Figs 5 and 6. These images and data clearly indicate that GABA, receptors in the brainstem develop differently from those in other brain regions. Higher density in the newborn brainstem than in the adult. At PI, the brainstem contained moderate to

high densities of GABA, receptors depending on the

2500

SO

26co

2am

2an

1800

16CO

Km

too3

5io

SW

0

0 PI

Pa

PI0

Pi21

Pm3

st.vE

Pi

m

PlO

Ppt

2503

PlM

MVE

Pt

1wO

1EUJ

1603

lox

lam

1003

0

Pa

Iv0

P21

2sm

RPN

P6

PlO

Fzl

lx0

PI20

RH6

Pl

zoao

2cu3

16Ul

ImJ

tan

loo0

1COU

1003

0

0 m

PI0

P21

Ps

Pi0

mi

Pi20

m

2030

mm

zco3

1500

1500

1600

ICC0

lau

1CUJ

PlO

P?t

P120

Pi0

P21

Pwo

605

0 Ps

P6

MRV

ml0

Pt

PWO

ROB

Pi

2x0

0

Pm

0 Pt

P120

6al

PlO

aa,

0 Pl

t%

2scn

zam

tnx

Plza

0 Pt

Fwa

Pm

mo

0 P1

PI0

s6E

2oco

500

P6

2Kn

Moo

em

FA

0 Pi

P6

Pto

Fig. S(a)

PIi

PI20

Pi

w

P10

P21

P120

Ontogeny of GABA, receptors in rat brain 2sm

PMN

2soa

2ceo

Moo

UUl

1tiXl

lae

lccc

om

983

LRT

em

0

0

i-1

F8

PW

Pa

Pi20

DCo

zsocl

Pi

PO

PI0

Fzi

s!mJ

Iwo

laa

IaD

looo

cc0

an

0

Pl

RI

PlO

PZI

PI20

PI

6%

Pi0

F-21

PI20

PI

Fs

PlO

P21

Pi20

PRH

zsca

2coo

Plzm

0 P1

PJ

PW

Pzi

Pml

NTSR

Pi

P6

Pi0

F21

PI20

HYP

2500

!xoo

leoo

lcoo

600

0 L

0

cu

uco

Pi

Pa

Pi0

Pm

Pl20

AP

!mo

2om

lax,

zcuo 1030 1COl

fm 0 Pi

Pa

PM

at

PI20

0 PI

w

Pi0

Fst

Pi20

Pt

Fs

Pi0

P!a

Pa0

Fig. S(b) Fig. 5. Multiple patterns of GABA, receptor ontogeny in various brainstem nuclei. Note that GABA, receptors reached peak levels at or before PI0 and decreased substantially after PlO. See text. The numbers of y-axis indicate the binding density (fmol/mg protein). The values are expressed in mean f S.E. from nine corresponding sections of three rats (three sections for each rat).

nucleus (Fig. 5). In general, most areas had higher density at Pl than in adulthood and the density increased and peaked at P5 or PlO. It is noteworthy that nuclei which contained low or very low GABA, receptor density in adulthood presented with high or very high receptor density in the new-

born. For example, the inferior dive showed an eight-fold higher density at Pl than in the adult (Figs 4, 7). Almost all brainstem nuclei had much higher GABA, receptor density in the newborn than in more mature animals (P21 or the adult, as shown in Figs 4, 5).

984

Y. XIA and G. G. HADDAD

More heterogeneous distribution in newborn brainstem than other regions. In contrast to a rather homogeneous distribution in the adult brainstem, there was a heterogeneous distribution found in the newborn, especially at P1 and P5 (Fig. 4A, B). For example, there was more than a 10-fold difference in the density in the medulla between the highest and the 5O0O

CTX

~oo

4OOO

~00

1000

50OO

PO

4000

ml]

-

lo00

0

0 PI

r~coo

lowest levels at P1, The opposite is true for the rostral brain regions and cerebellum, i.e. G A B A A receptors distribution of these regions was more homogeneous in early life than in the adult (Fig. 4). Multiple developing patterns in the brainstem. The postnatal development of G A B A A receptors varied (Fig. 5). Most brainstem nuclei developed according

P5

PlO

F'2I

PI20

nnN PI

5OOO

DG

P6

PIO

1>21

2O3O

1000

0 P120

5O00

CAI

4OOO

4OOO

30<30

3O0O

3000

2000

2OOO

looo

1000

0 PI

5COO

P5

PIO

P21

P120

P1

C:~O0

PHCo

UmijJ P6

PIO

P21

J|mn| P1

4000

0

CPu

4OOO

0

P120

PIO

P21

PI20

LA

mlJl 1 P1

LD

P6

P5

PIO

P21

PIO

P21

P12C'

VPH

4OOO

OOO(3

B~30

20O0

'2000 1000

IOO0 0

0

I:'1

~o3o

P5

PIO

P21

P120

r~oo

DH

0 P!

4E~O

400o

8OOO

8o0o

P5

PIO

P2f

P120

VMH

lOOO

0

o PI

P5

PlO

I~t

P120

P1

~-

P5

J P120

ZI

8000

2000

1000

.i

|

P1

P5

PIO

1:'21

P120

mmmj m P1

P5

PrO

P21

Fig. 6. Developing patterns of GABA A receptors in rostral brain areas. Note that the density increased with age and peaked at or after P21. The adult levels were close to peak levels in most areas. The numbers of y-axis indicate the binding density (fmol/mg protein). The values are expressed in mean + S.E. from nine corresponding sections of three animals (three sections for each rat).

P120

Ontogeny of GABA, receptors in rat brain

to one of the following three patterns. (1) Nuclei, like the parvocellular reticular nucleus, the nucleus of the solitary tract and medial vestibular nucleus, increased in density after birth and reached peak levels at P5 (Pig. 4B). The density then decreased slowly from PS to PI0 and decreased rapidly after PIO. (2) Other nuclei, such as the hypoglossal nucleus, prepositus hypoglossal nucleus, dorsal cocblear nucleus and cuneate nucleus, continued to increase in density after P5 until they peaked at PlO, and then decreased. (3) Still other nuclei, such as the lateral reticular nucleus, changed little in density from PI to PlO, but decreased in a major way from PlO to P21. Only the inferior olive showed a peak density at Pl and decreased thereafter postnatally. Difirences in GABA, receptor ontogeny between bru~ste~ and other brain areas. The cortex, thalamus and hip~~rnp~ increased in GABAA receptor density with age and the density was much higher at P21 or in the adult than in the newborn (Fig. 6). Most of the subcortical structures like the hypothalamus changed little in receptor density during maturation. In general, the cerebellum had a much lower density than the brainstem and rostra1 brain areas in the newborn (Fig. 4F) but increased in density remarkably with postnatal age, reaching the highest density in the adult rat brain (Fig. 45). It is interesting to note that some structures of the cerebellum like the central areas (e.g. lobules l-4), relatively old structures, had a much higher density than the ansifo~ lobules and paraflocculus in early postnatal life (Fig. 4F-H). The dramatic differences in GABA, receptor distribution between the brainstem and cerebellum at different postnatal stages are shown clearly in Fig. 7. DISCI_ISSION

The GABA, receptor is the major target for the inhibitory neurotransmitter GABA in the CNS. This receptor belongs to the l&and-gated ion channel family of receptors, the activation of which leads to the opening of chloride channels and neuronal inhibition.2~4s.ss.56A large body of literature suggests now that GABA, receptors play an important role in modulating a number of physiological functions, and many agents of clinical significance have been targeted to GABA,-mediated synaptic inhibition. 34*45 Because of its importance, the GABA, receptor has been well studied and recently little is known cloned 5,10~1R.25~r),*1.37,441.51.58.M).63.67 However about the postnatal development and expression of GABA, receptors in the developing mammalian CNS, especially in the brainstem. Our present study is the first one to investigate the postnatal ontogeny of GABA, receptor in the rat brainstem nuclei and a compa~son with that in rostra1 brain regions. Our data demonstrate that GABA, receptor densities change markedly from area to area and with age postnatally. Although receptor binding affinity may alter the binding density, we believe that the changes

Fig. 7. Comparison between medulla and cerebellum in GABA, receptar distribution at two different ages. Note that the GABA, receptor density is much lower in the cerebellum than in tbe brainstem in the newborn and the opposite is true in the adult. The brain sections were incubated at the same time in 50 mM Tris-citrate bnffer (pH 7.0) containing 50nM [3H)muscimol and were exposed to the same film for four weeks. Scale bar = 2 mm. (a) P5; (b) P-120.

in density observed are mainly due to differences in GABA, receptor number. This idea is supported by previous work showing that GABA receptor affinity does not change significantly with age or within brain areas8.‘5,48,59 and by our saturation experiments. For example, the ;U, values of the striate cortex at PI was 2.2-2.9 nM and 44.2-49.8 nM, respectively, for highand low-a~nity binding sites, whereas those at P21 were somewhat higher (around 3.3 and 66nM, respectively). But GABA, receptor density more than doubled in this same region at P21 as compared with that at PI. Another example is that although the cortex and hippocampus had similar Iri, values for [-‘H]muscimol binding, they had different binding densities. Thus, the differences in GABA, receptor density between brain areas or between ages are mostly related to differences in receptor number. This is also in agreement with our observations regarding other receptors, including those of opioid7’ and sulfonylurea?* receptors. We have made several important observations in this work: (i) the GABA, receptor appears very early in almost all brain areas; (ii) the brainstem has a higher binding density than in rostra1 areas at birth; (iii) the newborn brainstem has a much higher

Y. XIA and <;. G. HAWAII

986

GABA, receptor density than the adult brainstem and peaks in receptor density at P5 or PlO; and (iv) cerebellum and rostra1 brain areas such as the cortex, thalamus and dentate gyrus increase in GABA, receptor density with age, especially between PI0 and P21, but most other subcortical areas like caudateputamen, hippocampal CA1 area and thalamus do not increase remarkably after birth. One of the most interesting findings in the present study is the difference in the developmental profile of GABA, receptors between the brdinstem and other brain areas such as the cortex, thalamus and cerebellum. Several differences in GABA, receptor development existed between brainstem and other brain areas. First, brainstem nuclei had generally a higher receptor density than those in other brain areas in the newborn, especially at Pl. The average density in the medulla (Fig. 4A), for example, was more than 100% higher than that in the cerebellum (Fig. 4F). This was completely different from the distributional pattern in the adult (Figs 4E, J). Second, the distribution of GABAA receptor was much more heterogeneous in the brainstem than in other regions in the newborn. In sharp contrast to the several-fold differences in density between newborn brainstem nuclei, differences in receptor density among major rostra1 brain areas were less than 100%. Third, the density changed remarkably more in the brainstem than in the other areas from Pl to PlO. For instance, the nucleus of the solitary tract increased more than 50% in density from Pl to P5, whereas the primary olfactory cortex, thalamus and hypothalamus increased by only around 5% during the same period. Fourth, brainstem nuclei reached peak density earlier postnatally than rostra1 brain regions. Most brainstem nuclei peaked at or before PlO, but almost all other brain areas reached peak at P21 or subsequently (Figs 4-6). Finally, it is noteworthy that GABA, receptor density in brainstem nuclei decreased markedly after peak and the density was much lower in the adult than in the newborn, while most of other brain areas decreased little to reach adult levels after their peak (Fig. 6). Since GABA, receptors play an important role in a wide variety of CNS functions, the observed differences may have an important physiological significance. GABA, receptors mature after birth in a parallel fashion to cerebellum and rostra1 brain function. The large increase in GABA, receptor density after PlO in the cortex, cerebellum and thalamus must account for the need of GABAergic inhibitory modulation with maturation postnatally. In fact, a recent electrophysiologic study documented that in immature rat neocortical neurons, Cl flux mediated at GABAergic receptors is relatively ineffective.” This functional observation

is consistent with our results which demonstrate quantitatively lower GABA, receptor density in the immature than in the adult rat cortex (Figs 4, 6). Our results would suggest that the newborn brdinstem relies more on GABA, receptors than the adult brainstem. Indeed, GABA, receptor density in the brainstem is higher at Pl than in adulthood and peak level is reached by P5 or PlO in almost all brdinstem areas. This is different from other receptors that WC have previously studied. For example, we have shown that opioid mu and delta receptors,” and sulfonylurea receptors” mature in most brainstem areas after PlO or by adulthood. Generally, these receptors reached peak levels after PlO or even P2 1. It seems, therefore, that GABA, receptors develop earlier than other receptors in the brainstem. The reason(s) for the difference between GABA and other receptors is not known. However, several laboratories have demonstrated that brainstem GABA,\ receptors mediate important inhibitory mechanisms for regulating cardiovascular and respiratory ac70 and it is possible that these tivity 16.17.19.24.28.32.42,66.68 mechanisms may be more important in early life than during adulthood. Except for some phylogenetically newer structures (e.g. neocortex), most newborn brain areas have an equal or higher density of GABA, receptors as compared with that in the adult, suggesting that GABA, receptors develop very early in the CNS. Actually, GABA* receptors can be detected in almost all brain areas at Pl. In addition, recent studies have demonstrated that in the brain of reptiles (e.g. turtle). there is a rich distribution of GABA, receptor binding (unpublished observation from our laboratory) and that GABA, receptors are functional even in embryonic neurons.57 We speculate that GABA,, receptors may be phylogeneticatly old receptors and appear in lower vertebrates and in early prenatal life of mammals. Evidence to date suggests that excitatory amino acids are main factors in hypoxia/ischemic brain injury, while inhibitory amino acids and activation of GABA receptors can protect against neuronal damage. Thus, high GABA, receptor density in newborn brainstem may mediate important mechanisms during hypoxia. Very high GABA, receptor density in the newborn brainstem may help maintain important autonomic functions such as cardiorespiratory activity during hypoxia. This may account, to some degree. for the differences in hypoxia responsiveness between newborn and adult. work was supported by Grants HD 15736 and HL 39924 from the National Institute of Health

Acknowledgemenrs-This

REFERENCES 1.

Aldino C., Balzano M. and Toffano G. (1980) Ontogenetic development of GABA recognition sites in different areas.

Pharmac.

Res. Commun. 12, 495-500.

brain

Ontogeny of GABA, receptors in rat brain

987

2. Alger B. E. (1985) GABA and glycine: postsynaptic actions. In Neurotransmitter Actions in the Vertebrate Nervous System (eds Rogawski M. A. and Barker J. L.), pp. 33-69. Plenum Press, New York. 3. Baxter C. F. (1976) Some recent advances in studies of GABA metabolism and compartmentation. In GAB.4 in Neroour SystemFwrction (eds Robert E., Chase T. N. and Towers D. B.), pp. 61-87. Raven press, New York. 4. Beales M., Lorden J. F., Walz E. and Oltmans G. (1990) Quantitative autoradiography reveals selective changes in cerebellar GABA receptors of the rat mutant dystonic. J. Neurosci IO, 1874-1885. 5. Beaumont K., Chifton W. S., Yamamura H. I. and Enna S. J. (1978) Muscimol binding in rat brain: association with synaptic GABA receptors. Bra& Res. 148, 153-162. 6. Bowery N. G., Hudson A. L. and price G. W. (1987) GABA, and GABAs receptor site dist~bution in the rat central nervous system. N~rosc~~~ 20, 365-383. 7. Bowery N. G., Frice G. W., Hudson A. L., Hill D. R., Wilkin G. P. and Turnbull M. J. (1984) GABA receptor multiplicity: visualization of different receptor types in the mammalian CNS. Neurophurmaco~ogy 23, 219-231. 8. Brooksbank B. W., Atkinson D. J. and Bal&zs R. (1981) Biochemical development of the human brain. II. Some parameters of the GABA-ergic system. Deaf Neurosci. 4, 188-200. 9. Carpenter M. K., Parker I. and Miledi R. (1988) Expression of GABA and glycine receptors by messenger RNAs from the developing rat cerebral cortex. Proc. R. Sot. Land. (Biol.) 234, 159-170. 10. Caspary D. M., Raza A., Lawhorn-Armour B. A., Fippin J. and Americ S. P. (1990) Immunocytochemical and neurochemical evidence for age-related loss of GABA in the inferior colliculus: imolications for neural oresbvcusis. _ . J. Neurosci. 10, 2363-2372. 11. Chen J. C. and Chesler M. (1990) A bicarbonate-dependent increase in extracellular pH mediated by GABA, receptors in turtle cerebellum. Neurosci. Lett, 116, 130-135. 12. Chesnut T. J. and Swann J. W. (1989) L&inhibitory actions of the GABA, agonist muscimol in immature hippocampus. Brain Res. SBZ, 365-374. 13. Chronwag B. and Wolff J. R. (1980) Prenatal and postnatal d~elopment of GABA-adulating cells in the occipital neocortex of rat. J. camp. Neurol. 190, 18%2OP. 14. Cobas A., Fairen A., Alvarez-Bolado G. and Sanchez M. P. (1991) prenatal development of tb intrinsic neurons of the rat neocortex: a comparative study of tbe distribution of GABA-immunoreactive ceils and the GABAA receptor. Neuroscience 40, 375-397.

15. Coyle J. T. and Enna S. J. (1976) Neurochemical aspects of the ontogenesis of GABAergic neurons in the rat brain. Brain Res. 111, 119-133. 16. DeFeudis F. V. (1982) GABA-an inhibitory neurotransmitter that is involved in cardiovascular control. Pharmac. Res. Commun. 14, 567-575. 17. Denovit-Saubie M. and Champagnat J. (1975) The effect of some depressing amino acids on bulbar respiratory and non-respiratory neurons. Brain Res. 97, 356361. 18. Dir&ding R., Myers S. and Nicholas R. A. (1990) Molecular biology of mammalian amino acid receptors. Fe&r Am. Sac. exp. Biol. J. 4, 26362645.

19. Elliot K. A. and Hobbiger F. (1959) Gamma aminobutyric acid: circulatory and respiratory effects in different species; re-investigation of the anti-strychnine action in mice. J. Physiol., Land. 146, 70-84. 20. Fazekas J. F., Alexander F. A. D. and Himwich H. E. (1941) Tolerance of the newborn to anoxia. Am. J. Physiof. 134, 28 i-287. 21. Fiszer-de-Plazas S., Rores V. and Rios H. (1986) GABA receptors in the developing chick visual system: influence of light and darkness. A&. biochem. Psychopharmac. 42, 25-38. 22. Fosse V. M., Heggelund P. and Fonnum F. (1989) Postnatal development of glutamatergic, GABAergic, and cholonergic neurotransmitter phenotypes in the visual cortex, lateral geniculate nucleus, Fulvinar, and superior colliculus in cats. J. Neurosci. 9, 426-435. 23. Frostholm A. and Rotter A. (1987) The ontogeny of [3H]muscimol binding sites in the C57BL/6J mouse cerebellum. Brain Res. 465, 157-166. 24. Gillis R. A., DiMicco J. A., Williford D. J., Hamilton B. L. and Gale K. (1980) Importance of CNS GABAergic mechanisms in the regulation of cardiovascular function. Brain Res. BUN. 5, Suppl. 2, 303-315. 25. Guastelia J., Nelson N., Nelson H., Czyzvk L., Kevnan S., Miedel M. C., Davidson N.. Lester H. A. and Kanner B. I. (1990) Cloning and expression of a rat brain GABA transporter. Science 249, 1303-i306. 26. Haddad G. G. and Donnelly D. F. (1990) Or deprivation induces a major depolarization in brainstem neurons in the adult but not in the neonatal rat. J. Physiol., Lot&. 429,411-428. 27. Haddad G. G. and Mellins R. B. (1985) Hypoxia and respiratory control in early Iife. A. Rev. PhysioZ. 46, 629-643. 28. Hebebrand J., Fried1 W., Reichelt R., Schmitz E., Moller P., hopping P. (1988) The shark GABA-~~i~pine receptor: further evidence for a not so late phyio~netic appearance of the benzodiazepine receptor. Brain Rex 446, 251-261. 29. Hendry S. H. C., Fuch J., deBlas A. L. and Jones E. G. (1990) Distribution and plasticity of immunocytochemically localiied GABA, receptors in adult monkey visual cortex. J. Neurosci. 10, 2438-2450. 30. HitzigB. M., Kneussl M. P., Shih V., Brandstetter R. D. and Kazemi H. (1985) Brain amino acid concentrations during diving and acid-based stress in turtles. J. appl. Physiol. 58, 1751-1754. 31. Kabat H. (1940) The greater resistance of very young animals to arrest of the brain circulation. Am. J. Physiol. 130, 588-599.

32. Khrestchatisky M., MacLennan A. J., Chiang M., Xu W., Jackson M. B., Brecha N., Sternini C., Olsen R. W. and Tobin A. J. (1989) A novel a subunit in rat brain GABA. receotors. Neuron 3. 745-753. 33. Kim K. H. aud Takeuchi H. (1990) Pharmacological cbara&risti& of two ditferedt types of inhibitory GABA receptors on Achatina fulica neurons. Eur. J. Pharmac. 182, 49-62. 34. Kirkness E. F. (1989) Steroid modulation reveals further complexity of GABA,, receptors. Trends pharmac. Sci. 10, 6-7. 35. Kuriyama K., Tomono S., Kishi M., Mukainaka T. and Ohkuma S. (1987) Development of Emma-~inobuty~c acid (GABA)ergic neurons in cerebral cortical neurons in primary culture. Brain Res, O&7-21. 36. Lauder J. M., Han V. K. M., Henderson P., Verdoom T. and Towle A. C. (1986) prenatal ontogeny of the GABAergic system in the rat brain: an immunocytochemicaf study. Neuroscience 19, 465-493.

E. S.. Schofield P. R., Burt D. R., Rhee L. M., Wisden W., Kohler M., Fujita N., Rodriguez H. F.. Stephenon A. and Darlison M. G., Barnard E. A. and Seeburg P. H. (1988) Structural and functional basis for GABA ~rcceptcv heterogeneity. Nurure 335, 76-79. 38. Luhmann H. J. and Prince D. A. (1991) POStned maturation of the GABAergic system in rat neocortex J. Neurophysiol. 65, 247-263. 39. Lliddens H., Pritchett D. B.. KGhler Killisch I., Keinlnen K., Monyer H., Sprengel R. and Seeburg P. l-1. (1990) Cerebellar GABA, receptor selective for a behavioural alcohol antagonist. Nature 346, 648.,651. 40. Madtes P. Jr and Bashir-Elahi R. (1986) GABA receptor binding site “induction” in rabbit retina after nipecotic acid treatment: changes during postnatal development. ~e~r~c~e~, Res. 11, 55-61. 41. Malherbe P., Sigei E., Baur R., Persohn E., Richards J. G. and Mohler H. (1990) Functional characteristics and sites of gene expression of the a,, B,, @soform of the rat GABA, receptor. J. Neurosci. f0, 233iS2337. 42. Nakagawa T.. Wakamori M.. Shirasaki T., Nskaya T. and Akaike N. (1991) y-Aminobutyric acid-induced response in acutely isolated nucleus solitarii neurons of the rat. Am. J. Physiol. 260, (CeN P&iol. 29), C745-C749. 42a. Nilsson G. E. and Lutz P. L. (1991) Release of inhibitorv neurotransmitters in resDonse to anoxia in turtle brain. Am. J. Physiol. 261, (Regulatory In’tegtkive camp. Physiol. 36), R32-R37. 43. Olpe H. R.. Steinmann M. W., Hall R. G., Brugger F. and Pozza M. F. (1988) GABA, and GABA, receptors in locus coeruleus: effects of blockers. Eur. J. Phurmuc. 149, 183-185. 44. Olsen R. W. and Tobin A. J. (1990) Molecular biology of GABA, receptors. Fedn Am. Sot. exp. Biol. J. 4, 1469-1480. 45. Olsen R. W. and Venter J. C. (1986) Benzodiazepine/GABA receptors and chloride channels: structural functional properties. In Receptor Binchenktry and Methodology (eds Venter J. C. and Harrison I.. C.). Vol. 5, pp. I 351. Alan R. Liss. New York. 46. Palacios J. M., Niehoff D. L. and Kuhar M. J. {I979) Ontogeny of GABA and benzodiazepine receptors: effects of Triton X-100, bromide and muscimol. Brnin Res. 179, 390-395. 47. Palkovits M. and Brownstein M. J. (1988) Maps and Guide to ~~crodisseetio~ of’the Rut Bruin. Elsevier, New York. 48. Pate1 A. J.. Smith R. M., Kingsbury A., Hunt A. and Balazs R. (1980) Effects of thyroid state on brain development: muscarinic acetylchoiine and GABA receptors. Bruin Res. 198, 389402. 49. Paxinos G. and Watson C. (1982) The Rat Brain in Stereotaxic Coordinates, Academic Press, New York. 50. Peng Y. Y. and Frank E. (1989) Activation of GABA, receptors causes presynaptic and postsynaptic inhibition at synapses between muscle spindle afferents and motoneurons in the spinal cord of bullfrogs. J. Neurosci. 9, 1516-1522. 50a. Pennv J. B. Jr. Pan H. S., Young A. B.. Frev K. A. and Dauth G. W. (1981)I_Ouantitative autoradioeranhv _ . _ of [“H]m&cimol binding in rat brain.-.Science 214.<1036-1038. 51. Polenzani L., Woodward R. M. and Miledi R. (1991) Expression of mammalian y-aminobutyric acid receptors with distinct pharmacology in Xenopus oocytes. Prot. nam. Acad. Sci. U.S.A. 88, 43184322. 52. Rios H.. Flores V. and Fiszer-de-Plazas S. (1987) Effects of light- and dark-rearing on the postnatal development GABA receptor sites in the chick optic lobe. Inr. J. devil Neurosci. 5, 319-325. 53. Rothe T.. Middleton-Price H. and Big1 V. (1988) The ontogeny of GABA receptors and glutamic acid decarboxylase in regions of the rat brain. Neuropharmacoiugy 27, 661.-667. 54. Schiiebs R. and Rothe T. (1988) Development of GABA, receptors in the central visual structures of rat brain. Effect of visual pattern deprivation. Gen. P~.~.~i~/.Biophys. 7, 281-291. 55. Schofield P. R., Da&son M. G.. Fujita N., Burt D. R., Stephenson F. A., Rodriguez H., Rhee L. M., Rama~handran J., Reale V., Glencorse T. A., Seeburg P. H. and Barnard E. A. (1987) Sequence and functional expression of the GABA-A receptor shows a &and-gated receptor superfamily. Nature 328, 221-227. 56. Schofield P. R., Shivers B. D. and Seeburg P. H. (1991) The role of the receptor subtype diversity in the CNS. Trends 3’7. Levitan

Neurosci. 13, 8-I

1.

57. Shen J. M., Huguenard J. R. and Kriegstein A. R. (1988) Development of GABA responsiveness in embryonic turtle cortical neurons. Neurosci. Left. 89, 335-341. 58. Sigel E., Baur R., Trube G., Mohler H. and Malherbe P. (1990) The effect of subunit composition of rat brain GABA, receptors on channel function. Neuron 5, 703-711. 59. Skerritt J. H. and Johnston G. A. R. (1982) Postnatal development of GABA binding sites and their endogeneous inhibitors in rat brain. Deul Neurosci. 5, 189--197. 60. Somogyi P., Takagi H., Richards J. G. and Mohler H. (1989) Subcellular localization of benzodiazepine/CABA, receptors in the cerebellum of rat, cat, and monkey using monoclonal antibodies. J. Neurosci. 9, 2197-2209. 61. Stephenson F. A. (1988) Understanding the GABAA receptor: a chemically gated ion channel. J. B&hem. 249, 21-32. 62. Tehrani M. H. and Barnes E. M. Jr (1986) Ontogeny of the GABA receptor complex in chick brain: studies In z%:o and in vitro. ~r~j~ Rex 390, 91-98. 63. Telang S., Fuller G., Wiggins R. and Enna S. J. (1984) Early undernutrition and r3HJgamma-aminobutyric acid binding in rat brain. J. Neurochem. 43, 640645. 64. Tirelli E. (1989) Functional maturation of the GABAergic inhibition on dopamine-mediated behaviours during the neonatal period in the mouse. Behav. Brain Res. 33, 83-95. 65. Unnerstall J. R., Kuhar M. J., Niehoff D. L. and Palacios J. M. (1981) Benzodiazepine receptors are coupled to a subpopulation of y-aminobutyric acid (GABA) receptors: evidence from a quantitative autoradiographic study. J. Pharmuc. exp. Ther. 218, 797-804.

66. Williford D. J., Hamilton B. L., Dias Souza J., Williams T. P., DiMicco J. A. and Gillis R. A. (1980) Central nervous system mechanism involving GABA influence arterial pressure and heart rate in the cat. Circulation Res. 47, 80-88. 67. Wisden W., Morris B. J., Darlison M. G., Hunt S. P. and Barnard E. A. (1989) Localization of GABA, receptor alpha-subunit mRNAs in relation to receptor subtypes. M&c. Bruin Res. 5, 305-310. 68. Wood J. D. (1967) A possible role for gamma-aminobutyric acid in the homeostatic control of brain metabolism under conditions of hypoxia. Expl Bruin Res. 4, 81.-84. 69. Yamada K. A., Hamosh R. and Gillis R. A. (1981) Respiratory depression produe& by activation of GABA receptors in hindbrain of cat. J. uppI. Pkysioi. 51, 1278-1286. 70. Yamada K. A., Norman W. P., Hamosh P. and Gillis R. A. (1982) Medullary ventral surface GABA receptors affect respiratory and cardiorespiratory function. Brain Res. 248, 71-78.

Ontogeny of GABA, receptors in rat brain

989

71. Xia Y. and Haddad G. G. (1991) Ontogeny and distribution of opioid receptors in the rat brainstem. Brain Rex 549, 181-193. 72. Xia Y. and Haddad G. G. (1991) Major differences in sulfonylurea receptors between rat (newborn, adult) and turtle. J. camp. Neurol. 314, 278-289. (Accepted 11 February 1992)