246

:~,lolecular Brain Re.search, 16 (1992) 246-254 ,.v, 1992 Elsevier Science Publishers B.V. All rights reserved 0169-328x/92/$05.00

BRESM 70519

Ontogeny of GABAA/benzodiazepine receptor subunit mRNAs in the murine inferior olive: transient appearance of/3 3 subunit mRNA and [3H]muscimol binding sites Adrienne Frostholm, Darko Zdilar, Vera Luntz-Leybman, Venugopal Janapati and Andrej Rotter Department of Pharmacology and the Neuroscience Program, The Ohio State University, ('olumbus, 0 t f 43210 (USA)

(Accepted 21 July 1992)

Key words." GABAA; Receptor subunil: Development: Mouse: In situ hybridization

The GABA A/benzodiazepine receptor consists of at least four subunits, o~,/3, y and 6, each comprised of several variants. The developmental expression of the oq, /31 .~, Ye and 6 subunits was studied in the murine inferior olivary nucleus by in situ hybridization with antisense cRNA probes. The postnatal appearance and distribution of [~H]flunitrazepam and [~H]muscimol binding sites, ~ and /3 subunit-specific ligands respectively, were also studied autoradiographically. The/33 subunit was transiently expressed in each of the subnuclei of the inferior olive: The signal was strong at birth, increased throughout postnatal week I and rapidly declined thereafter to low adult levels. A similar pattern of labeling was observed with [3H]muscimol. Detectable levels of orI subunit mRNA hybridization signal and [3H]flunitrazepam binding sites were also present in the inferior olive at birth, decreasing thereafter. Ix~w to moderate levels of/31, /32, and y_, subunit mRNAs were present in olivary neurons throughout postnatal development, while 6 mRNAs were largely absent. It has been reported previously that. during the 2rid postnatal week, the ratio of climbing fiber terminals to Purkinje cells is reduced from 3 : 1. as observed in neonates, to the 1 : I relationship observed in the aduh cerebellar cortex. Our results raise the possibility that the subunit composition of the GABA A/benzodiazepine receptor in inferior oliva~ neurons undergoes changes during development, and that this process may be related to the elimination of multiple climbing fiber innervation of cerebellar Purkinje cells.

INTRODUCTION

Olivary n e u r o n s receive G A B A e r g i c i n p u t 1s,~,~,~3 a n d possess G A B A A / b e n z o d i a z e p i n e r e c e p t o r s 37'5'~. T h e

T h e i n f e r i o r olive is a m e d u l l a r y n u c l e u s w h i c h gives rise to, a n d is the p r i m a r y s o u r c e of, c l i m b i n g fibers i n n e r v a t i n g the P u r k i n j e cells of t h e c e r e b e l l a r cortex ~2.

GABA/benzodiazepine r e c e p t o r is a p r o t e i n which consists of at least f o u r s u b u n i t s , c~, /3, y a n d & each c o m p r i s e d of several v a r i a n t s 5'~('. A c o m m o n l y e n c o u n -

D u r i n g d e v e l o p m e n t of the r o d e n t c e r e b e l l u m , e a c h P u r k i n j e cell is initially c o n t a c t e d by t h r e e c l i m b i n g fibers 929-3°. T h i s m u l t i p l e i n n e r v a t i o n is e l i m i n a t e d d u r -

t e r e d s u b u n i t c o m b i n a t i o n of the r e c e p t o r is (it, /32/3 a n d y2 ~. T h e c~ s u b u n i t c o n t a i n s the b i n d i n g site for [ 3 H ] f l u n i t r a z e p a m , while the /32 a n d /3~ s u b u n i t s possess the b i n d i n g site for [ ~ H ] m u s c i m o l 7'4~. T h e y , sub-

ing early p o s t n a t a l d e v e l o p m e n t l e a d i n g to t h e o n e - t o o n e r e l a t i o n s h i p o b s e r v e d in the a d u l t c e r e b e l l u m '~'~°. Little is k n o w n a b o u t c h a n g e s in t h e sensitivity of i n f e r i o r olivary n e u r o n s d u r i n g t h e p e r i o d in which this m u l t i p l e c l i m b i n g fiber i n n e r v a t i o n of t a r g e t cells is e l i m i n a t e d . R e d u c t i o n in t a r g e t i n n e r v a t i o n m a y result in c h a n g e s in the m e m b r a n e p r o p e r t i e s o f the a f f e r e n t n e u r o n , which m a y well b e r e f l e c t e d in r e c e p t o r expression.

unit, which is p r e s e n t in a l t e r n a t i v e l y spliced long (L) a n d short (S) forms z2, is t h o u g h t to m e d i a t e c o o p e r a tive i n t e r a c t i o n s b e t w e e n the c~ a n d /3 s u b u n i t s 25'3'~. T h e role of the 6 s u b u n i t 4s is, as yet, u n k n o w n . In t h e p r e s e n t study, we have u s e d in situ h y b r i d i z a t i o n a n d r e c e p t o r a u t o r a d i o g r a p h y to e x a m i n e the p o s t n a t a l a p p e a r a n c e of [1~1, /31 3, 'Y2 a n d 6 s u b u n i t mRNAs and [3H]flunitrazepam and [3H]muscimol

Correspondence: Department of Pharmacology and the Neuroscience Program, The Ohio State University, 333 West 10th Ave., Columbus, OH 4321(I, USA. Fax: (11 (614) 292-9805.

247

binding sites in the murine inferior olive. While ligand binding studies indicate the presence of receptors generally located within dendritic fields, in situ hybridization with subunit-specific cRNA probes indicates the cell bodies to which the mRNAs are largely constrained. MATERIALS AND METHODS Preparation of mouse cDNAs

ill, f12, /33, 3'2 and 6 mouse specific probes were generated by polymerase chain reaction (PCR) as follows: poly(A) + R N A was isolated from mouse cerebellum using the Fast Track m R N A isolation kit (Invitrogen, San Diego). First strand c D N A and P C R reactions were conducted according to the Perkin Elmer Cetus Gen e A m p R N A P C R protocol (Perkin Elmer Cetus, Norwalk). 0.5 p~g of poly(A) + R N A was reverse transcribed at 42°C for 30 min in a reaction mixture containing 5 m M MgCl2, 50 m M KCI, 10 m M Tris (pH 8.3), 1 m M dNTPs each, 1 U / / z L of RNase inhibitor, 2.5 U / t z l cloned Moloney Murine Leukemia Virus Reverse transcriptase and 2.5 p.M random hexamer, in a final volume of 20 /.tl. T h e mixture was heat inactivated at 99°C for 5 min and prepared for PCR. P C R reactions were conducted as follows: 1 - 5 / z l of the reverse transcription reaction mixture was amplified for 30 cycles, with a denaturation step at 94°C for 1 min (first cycle for 3 min), an annealing step at 55°C (/31,/33, 8) or 60°C (f12, Y2 ) for 1 min, and an extension step at 72°C for 1 min (last cycle for 5 rain) in a total volume of 100/zl containing 2 m M MgClz, 50 m M KCI, 10 m M Tris (pH 8.3), 200 m M dNTPs each, 2.5 U AmpliTaq D N A polymerase and 0.2 IzM each of upstream and downstream primer. Primers, used for amplification of the above probes, spanned a region of the M 3 - M 4 fragment, the most variable portion of the G A B A A receptor subunits. The sequence of the primers was based on published sequences 22'24'39'4s'58, with HindIII or BamHI restriction sites and three additional nucleotides, as follows: 5 ' - A T A - A A G C T T - G G A G C G A G C A A A C A A G A C C A - 3 ' (upstream /3t primer); 5 '-ATA-GGATCC-TGGCGCTGTCGTATGAGTAC-3' (downstream /31 primer); 5 '-ATA-AAGCTT-GAGAAGATGCGCCTGGATGTC-3' (upstream /32 primer); 5 ' - A T A - G G A T C C - G C A C G T C T C C T C A G G C G A C T T - 3 ' (downstream /32 primer); 5 ' - A T A - A A G C T T - C T A G C A C C G A T G G A T G T T C A C - 3 ' (upstream /33 primer); 5'-ATA-GGATCC-TGCTTCTGTCTCCCATGTACC-3' (downstream /33 primer); 5'-ATA-AAGCTT-TCAGCAACCGGAAGCCAAGCA-3' (upstream 3'2 primer) 5'-ATA-GGATCC-ACTGGCACAGTCCTTGCCATC-3' (downstream 3'2 primer). 5'-ATA-AAGCTT-AGCCAAGGTCAAGGTCACGAAG-3' (upstream 6 primer) 5 '-ATA-GGATCC-GATGTCGATGGTGTCTGCATCG-3' (downstream 6 primer). Amplified fragments of 202b (/31), 266b (/32), 157b (/33) , 212b (3'2) and 272b (6) were digested with HindlII and BamHI restriction enzymes and ligated "in gel" in HindlIIBamHl treated, dephosphorylated pBluescript II S K [ + ] phagemid vector (Stratagene, La Jolla) as described elsewhere TM.The recombinant plasmid was purified, alkali denatured, and sequenced using Taq polymerase (Perkin Elmer Cetus), as described by the manufacturer. In vitro transcription In addition to the above mouse probes, rat cDNAs, coding for a full length a I and a 0.90 kb fragment of f13 subunit m R N A were obtained from Dr. Allan Tobin (UCLA, Los Angeles). All plasmids were linearized with HindIII and BamHI (/31, /32, /33, 3'2 and 6),

and SalI and PstI (rat a l , /33 ) (Boehringe'r, Indianapolis) for the subsequent production of antisense and sense R N A probes. The linearized templates were transcribed at 37°C for 60 min in a reaction mixture containing 40 m M Tris, 8 m M MgC12, 50 m M NaC1, 2 m M spermidine, 10 m M dithiothreitol (DTT), 0.5 units RNaseBlock II, 500 ~ M ATP, 500 p,M CTP, 500 p~M G T P (Stratagene, La Jolla), 500 ng of linearized template and 25 /~M [35S]UTP (Amersham, S.A. > 800 C i / m m o l ) ; the reaction was initiated by the addition of 25 units of T3 R N A polymerase (for antisense RNA), or 25 units of T7 R N A polymerase (for sense R N A ) (Stratagene, La Jolla), pH 8.0, to a final volume of 10 ~l. After digestion of template with 5 units of DNase (Stratagene, La Jolla) for 30 rain at 37°C, the riboprobe was purified over a Nensorb 20 column (NEN, Wilmington). The rat a 1 and 133 probes were then dried and subjected to limited alkaline hydrolysis (200 p.1 volume, containing 40 m M N a H C O 3 / 6 0 m M NaECO3, pH 10.2) for 65 min to generate probes with an average length of 100-150 nucleotides. Finally, all probes were precipitated by adding sequentially 6.6 pA 3 M sodium acetate, pH 6.0, 1.3 ~ l glacial acetic acid, 2 p,l (20 p,g) t R N A and 500 /~l EtOH. The mixture was then placed on dry ice for 30 min, centrifuged (12,000× g / 1 5 min), washed in ice-cold 70% E t O H and dried. Probes were stored at - 2 0 ° C in 100 m M DTF. Specific activity was 1 - 2 × 109 d p m / t z g . Each of the riboprobes was specifically designed to include regions complementary to the most variable portion of the corresponding m R N A , in order to maximize specificity of hybridization. The n u m b e r and size of the m R N A species recognized by the various probes was determined by Northern blot hybridization which revealed bands of approximately 4.3 kb (al), 11 kb (/31), 8.2 kb (/32), 6.1 kb (/33), 4.3 and 2.5 kb (Y2) and 1.8 and 2.5 kb (8).

In situ hybridization Two C 5 7 B L / 6 mice (Jackson Laboratories, Bar Harbor) were sacrificed daily between postnatal days (P) 1 and 12, every second day from P12 to P20, and every fourth day from P20 to P35 (day of birth = P1). Animals were decapitated after inhalation anesthesia (Metofane, Pitman-Moore Inc., N J). Brains were rapidly removed, frozen on dry ice and stored at - 7 0 ° C . Coronal sections, 20 /zm thick, were thaw-mounted onto 3 × s u b b e d (300 bloom gelatin & chrome alum) slides and stored at - 7 0 ° C . Prior to hybridization, sections were fixed for 30 min in 4% paraformaldehyde in 1 × PBS (pH 7.4), and washed ( 2 x 5 min) in PBS. Sections were then acetylated (0.25% acetic anhydride diluted in 0.1 M triethanolamine/0.9% NaC1, pH 8.0) for 10 rain at room temperature (R.T.), and dehydrated (1 rain each) through 70%, 80%, 90%, and 100% EtOH. Hybridization was conducted as previously described 17. After hybridization, R N A s e treated sections were washed at increasing stringency, as shown in Table I. Sections were then dehydrated (1

TABLE I

Stringency conditions for GABA A / B Z receptor subunit cRNA probes N u m b e r s in the first 6 columns refer to the concentration of SSC in washing buffer.

al

/31

/32

/33

72

6

mm

°C

2.000

2.000

2.000

2.000

2.000

2.000

1.000

1.000

1.000

1.000

1.000

1.000

0.500 0.250 . 0.250 . . .

0.500 0.250 . 0.250 . . .

0.500 0.250 . . 0.250 . . . . . .

0.500 0.250 0.125

0.500 0.500 -

0.125

0.500 -

0.500 0.250 0.125 0.063 0.031 0.031

10 10 10 60 10 60 10 10 10 10 60 10

70 * 70 * 70 * 70 * 70 * 70* 65 R.T. R.T. 65 65 65

.

. . .

* All washes for the 8 probe were conducted at 65°C.

248 min each) through 70~/~, 80%, 90% and 100% EtOH. All washing solutions, except RNase A solution, contained 10 mM 2mercaptoethanol to prevent non-specific binding of riboprobe. The non-specific hybridization signal was determined by exposing adjacent sections to sense RNA probes.

Ligand binding [3H]Muscimol binding. Sections were preincubated in phosphate buffered saline (PBS), pH 7.4, for 30 min at 4°C, followed by a 40 rain incubation in PBS containing 5 nM [3H]muscimol (9 Ci/mmol, NEN) at 4°C; sections were washed (2×30 s) in ice-cold buffer to remove unbound radioactivity. The slides were then dipped into ice cold double-distilled water to remove residual salts. Control slides for non-specific binding were labeled in the presence of 200 p,M GABA. [3H]Flunitrazepam binding. Sections were incubated in 170 mM Tris-HCl buffer, pH 7.4, containing 2 nM [3H]flunitrazepam (81 Ci/ retool, Amersham) for 40 rain at 4°C and washed twice for 5 min in ice-cold buffer alone to remove unbound radioactivity. The slides were then dipped into ice cold double-distilled water to remove residual salts. Control slides for non-specific binding were labeled in the presence of 1 /~M diazepam.

P1

P14

+

A utoradiography Autoradiograms were generated as follows: Acid-washed coverslips (25×77 ram, No. 0 Corning Glass Works, NY), previously coated with a uniform layer of photographic emulsion (llford ICS-D. Polysciences, Inc., Warrington, PA) were apposed to the slidemounted sections and clamped together with binder clips under minimum sodium safelight illumination. The assemblies were placed in lightproof boxes containing desiccant and exposed for 5 days at 4°C. Coverslips containing autoradiographs were developed in Kodak D-19 developer (diluted 1 : 1 with distilled water) for 3.5 rain at 20°C, fixed for 3.5 min in Kodak Rapid-Fix, and washed for 30 min. Coverslips were dehydrated in ascending alcohols and mounted onto microscope slides with Depex (Bio/medical Specialties, Santa Monica, CA). After the coverslips were developed, slides containing the sections were dipped in llford K5-D photographic emulsion at 42°C. dried overnight, exposed for 4 days (in situ hybridization), 21 days ([3H]flunitrazepam) and 60 days ([3H]muscimol) at 4°C, and developed as above. After development, emulsion dipped sections were counterstained with cresyl fast violet. Sections and autoradiograms were photographed (Kodak Panatomic-X or T-Max 100 film) under brightfield or darkfield illumination using a Nikon SMZ-10 stereomicroscope (low magnification), or a Nikon Optiphot compound microscope (high magnification).

)

P25 Fig. 1. Cresyl fast violet-stained sections of the brainstem of postnatal day (P) 1, 14 and 25 mice. io, inferior olive; dmcc, dorsomedial cell column: dc, dorsal cap; DAO, dorsal accessory olive: MAO. medial accessory olive; PO, principal olive. Magnification: P1, 10 ×: PI4, 7×: P25, 21)×

subunit mRNA. and

P r o b e s b a s e d o n the rat s e q u e n c e (a~

/33), a l t h o u g h

of greater

length,

also i n c l u d e d

p o r t i o n s c o d i n g for t h e i n t r a c e l l u l a r loops. N o r t h e r n blot s t u d i e s s h o w e d no c r o s s - h y b r i d i z a t i o n b e t w e e n t h c

RESULTS

c R N A p r o b e s , a n d t h e sizes o f t h e s u b u n i t m R N A s w e r e s i m i l a r to t h o s e r e p o r t e d p r e v i o u s l y 21'22'48'54'58'~'°, with t h e e x c e p t i o n o f t h e / 3 3 p r o b e w h e r e a 2.5 kb b a n d

Specificity of probes Based on the following considerations,

it m a y be

o b s e r v e d in t h e rat was a b s e n t in t h e m o u s e . B o t h rat

c o n c l u d e d t h a t t h e c R N A p r o b e s u s e d in t h e s e s t u d i e s

a n d m o u s e /33 p r o b e s g e n e r a t e d

w e r e s e l e c t i v e for t h e i r r e s p e c t i v e m e s s a g e s : T h e p r o b e s

graphic

b a s e d o n m o u s e s e q u e n c e s (/31,/32,/33, 72 a n d ~) w e r e c o n s t r u c t e d to b e c o m p l e m e n t a r y to t h e r e g i o n o f

s p e c i f i c [ 3 5 S ] o l i g o n u c l e o t i d e p r o b e s , also c o m p l e m e n -

mRNA

was s i m i l a r to t h a t o f t h e c R N A

ing t h e

c o d i n g for t h e p u t a t i v e i n t r a c e l l u l a r l o o p linkM3

and

M4 transmembrane

segments,

this

s e g m e n t b e i n g t h e m o s t v a r i a b l e r e g i o n in e a c h subunit. S e q u e n c i n g o f P C R g e n e r a t e d f r a g m e n t s r e v e a l e d a g r e a t e r t h a n 9 5 % i d e n t i t y w i t h o u r / 3 ~ , /32, /33, a n d p r o b e s to t h o s e p r e v i o u s l y r e p o r t e d for other s p e c i e s 45'48'5~. T h e s e q u e n c e for m o u s e 3'2 was i d e n t i c a l to t h a t o f Y2e p r e v i o u s l y published22;

however, the

p r o b e r e c o g n i z e s b o t h s h o r t a n d l o n g f o r m s o f t h e 72

distributions.

The

identical autoradio-

distribution

of

subunit-

tary to t h e r e g i o n c o d i n g for t h e i n t r a c e i l u l a r loops, probes (unpublished

observations).

] SH]Flunitrazepam and [ SH]muscimol binding sites T h c i n f e r i o r olivary n u c l e u s is l o c a t e d at t h e v e n t r a l surface of the medulla. The characteristic cytoarchitect u r e is largely f o r m e d by P1, a n d r e m a i n s s t a b l e into a d u l t h o o d (Fig. 1). M o d e r a t e

levels o f [ 3 H ] f l u n i t r a z e -

p a r e b i n d i n g sites w e r e o b s e r v e d t h r o u g h o u t t h e e n t i r e

249 rostrocaudal extent of the olivary nucleus at birth. By P7, only the medial accessory olive was of moderate density; labeling over the remaining subnuclei was low (Fig. 2B). Grain density over this region slowly decreased throughout development into adulthood (Fig. 2D). During postnatal weeks 1 and 2, [3H]muscimol binding sites were spread diffusely throughout the medullary region with the most intense labeling observed over each subnucleus of the inferior olive. Although high autoradiographic grain density was present over the olive at birth, labeling increased markedly up to P7-P9, after which a sudden reduction in grain density was observed (Fig. 3). By P14-16, labeling over the olive was low, and by P30 (Fig. 3), labeling was barely discernible over the olive and was similar to that of other medullary regions.

than the surrounding brainstem. These levels were maintained into adulthood. Unlike the other /3 subunits, the /33 m R N A hybridization signal in olivary neurons was considerably higher than that of surrounding medullary regions at birth (Fig. 5). As with [3H]muscimol binding sites, grain density increased during postnatal week 1 and reached a peak between P7 and 9. A rapid decrease in labeling was observed between P10 and 16, followed by a slower decline to low adult levels by P25-30 (Fig. 5). The variation in /33 subunit expression was uniformly distributed among all the subnuclei of the inferior olive. The hybridization signals of sense probes for the subunit mRNAs, and the non-specific binding control sections for each ligand, were of low grain density and were uniformly distributed throughout the control sections (data not shown).

In situ hybridization of cRNAs coding for GABA A / B Z receptor subunits

DISCUSSION

The distribution of a~, /3J-3, 3'2 and fi subunit mRNAs within the olivary nucleus was examined by in situ hybridization autoradiography. When compared to the surrounding medullary region, the a~ hybridization signal in olivary neurons was low throughout postnatal week 1 (Figs. 2A and 4); grain density further decreased during postnatal weeks 2-4 (Fig. 2C). In general, the in situ hybridization signals for the remaining subunit mRNAs, with the exception of /33, were also relatively low during postnatal week 1 (Fig. 4), with the inferior olive being labeled at the same level, or lower,

y-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter whose effects are mediated through chloride channels formed by the GABAA/benzodiazepine receptor multisubunit complex. Although a variety of subunit combinations have been shown to constitute a functional receptor 36'5°, a frequent composition in the central nervous system appears to be that of a~,/32/3 and 3'2 subunits~- The a~ subunit contains the binding site for [3H]flunitrazepam, the /32 and /33 subunits possess the binding site for [3H]muscimolT,

C Fig. 2. Autoradiographiclocalizationof tr I subunit mRNA (left panels) and [3H]flunitrazepambinding sites (right panels) in the inferior olive; emulsion-dipped sections and emulsion coated coverslips, respectively,photographed under dark field illumination.A,B: postnatal day (P) 7; C,D: postnatal day (P) 32; arrowheadsindicate the inferiorolive; smallarrow, medial accessoryolive. Magnification:A,B, 6 x ; C,D, 4 ×.

25{) and the Y2 subunit appears to mediate cooperative interactions between the o~ and /3 subunits 25'3~. The present results demonstrate that, in the inferior olive, discernible quantities of c~ subunit mRNAs and [~H]flunitrazepam binding sites are present at birth,

!i

i!i~

Fig. 3. Autoradiographic localization of [3H]muscimol binding sites in the inferior olivary nucleus of the developing mouse; emulsion coated coverslips, photographed under dark-field illumination. Postnatal day (P) 1, 7. I1, and 30. Arrows indicate the inferior olive. Magnification: 10 × .

after which they slowly decrease: /31, /32, Y2 subunit mRNAs remain stable throughout development, and the 3 subunit is largely absent. In contrast, /33 subunit mRNA and [3H]muscimol binding sites are transiently expressed within the inferior olive; high levels of grain density arc present at birth and increase during the first postnatal week, after which they are rapidly downregulated to low adult levels. Although the role, and precise subunit composition, of the GABAA/benzodiazepine receptor in olivocerebellar circuitry is presently unclear, immunocytochemical studies show a high concentration of glutamic acid decarboxylase (GAD), the GABA-synthesizing enzyme, to be present in the inferior olive of the adult mouse~( The source of this GABAergic innervation has been identified in the rat, where small, GAD-containing cells in the lateral and interpositus regions of the deep cerebellar nuclei have been shown to project to the inferior olive ~5. This suggests a possible mechanism by which climbing fibers, which originate in the olive and innervate the Purkinjc cells of the cerebellum, are modulated by cerebellar nuclear input. The observation that GABA A receptor /33 subunit mRNA and [3H]muscimol binding sites are transiently expressed in the inferior olive is by no means unique, since various receptors in both the peripheral and central nervous system of several species have been shown to undergo similar transient changes. For example, opiate receptor binding sites are present in the external germinal layer of the cerebellar cortex, a region consisting of actively proliferating cells; however, the progeny of these neuroblasts, the granule cells, do not express detectable levels of opiate receptors~C( Serotonin receptors (5-HTIA) are also transiently expressed in the cerebellum ~~, as are nerve growth factor receptors and their mRNA 53, Transient expression of neurotransmission related molecules has also been observed in olivary neurons: in the human inferior olive, neurotensin receptors are present only during early development; labeling is absent by the 4th postnatal month 26. The transient expression of both insulin growth factor (IGF-I) and its mRNA within the rat inferior olive 3 is almost identical to that of the murinc /33 subunit mRNA and [3H]muscimol binding sites. There are a number of studies which suggest that the properties of receptors are altered during development. In the immature rat cerebellum, exposure to glycine does not evoke a significant increase in glutamate binding to the N-methyl-D-aspartate receptor as it does in adult animals, suggesting that subunit composition may change during development 6. In addition, certain subunits of the peripheral nicotinic acetylcholine receptor are also transiently expressed: AI-

251 though the 3' subunit is present during early development, it is later replaced by the e subunit 3z'47. This subunit substitution, which causes dramatic changes in receptor properties, is possibly regulated by calcitonin gene related peptide (CGRP), which is present in, and released by, motoneuron terminals 34. The reduction of /33 subunit m R N A in the adult olivary nucleus does not appear to be accompanied by a loss of GABA-related enzymes ~9, which may indicate that some functional form of the receptor is still present on olivary neurons. Although /3 subunits appear to function almost interchangeably when reconstituted in Xenopus oocytes with a and 3' su bunits24"38,58, some form of /3 subunit does appear to be required for appropriate receptor function 5°. Recent in situ hybridization studies have shown that, although a2, a 4 and "/1 subunit mRNAs are present at high density in the adult rat inferior olive 23, all the known /3 variants, in both rats and mice, appear to be quite low. Thus, if the rapid downregulation of /3.~ mRNA indicates a change in subunit composition,

the replacement subunit does not appear to include any of the currently known /3 variants. Several cellular events may lead to the transient expression of receptor subunits. During early development, neurons bearing the receptor in question may be eliminated, or subunit expression may be shut off after initial acquisition. In the latter case, subunit downregulation may be genetically programmed within the cell expressing the receptor, or may possibly occur in response to modifications in cellular interactions, such as the formation of transient presynaptic or postsynaptic contacts. The observed postnatal downregulation of the/33 subunit m R N A and [3H]muscimol binding sites within the inferior olive may be related to one, or a combination, of these developmental events. It is unlikely to be related to presynaptic influences for the following reasons: firstly, GABAergic innervation of the olive is not, to our knowledge, transient, i.e. once established during development it persists into adulthood. Secondly, there are indications that, in some

@

@

@

@

Fig. 4. In situ hybridization signal of various GABA A / B Z receptor subunit probes in the developing inferior olivary nucleus at postnatal day 7; during this time, the/33 probe signal was at its peak, while oq,/31-2, Y2 and ~ signals were relatively low. All probes, with the exception of a 1, were based on mouse sequences. Emulsion-dipped sections, photographed under dark-field illumination. Magnification: 20×.

252 cases, CNS receptors appear before synapses are established ~"'17'42, thus resembling the development of receptors at the neuromuscular junction I~. A much stronger correlation exists between the transient expression of the /33 message and the formation of connections between the inferior olivary axons and Purkinje cells. During development of the olivocerebellar system, there is a change in the numerical relationship between climbing fibers and their targets, the Purkinje cells. From birth to P5-7, each Purkinje cell receives synaptic input from three olivary climbing fibers ~'1°'27'28'29'3°. These multiple contacts between climbing fibers and Purkinje cells regress to the adult pattern of a 1 : 1 relationship during the 2nd postnatal week, following a very similar time-course to the downregulation of /33 subunit and [3H]muscimol binding sites. The loss of multiple climbing fiber innervation of Purkinje cells is thought to be due mostly to the

regression of olivary a x o n collaterals 4, and has been attributed to competition with parallel fibers for a trophic factor within the Purkinje cell s. Since parallel fibers release glutamate onto Purkinje cell receptors, it is of interest that application of D,L-APV (a selective N M D A receptor antagonist) to the surface of the cerebellar vermis before the period of synapse elimination, prevents the regression of climbing fiber synapses4~( These results suggest that activation of N M D A glutamate receptors may well be prerequisite for the regression of functional synapses during development. There is a considerable body of evidence which suggests that a variety of neural properties are dependent upon interactions between the nerve cell and its target. These cellular properties include the cell's ability to receive afferent contacts 25~, the maintenance of normal m e m b r a n e properties 3~, conservation of normal dendritic arborization 52 and control of neurotransmit-

Fig. 5. Autoradiographic localization of /33 mRNAs in the developing inferior olivary nucleus. Probe was based on rat sequence. Emulsiondipped sections were photographed under dark-field illumination. Postnatal day (P) 1, 4.8, 11, 14 and 25. Magnification: 15×.

253 ter phenotype 46. In the hypoglossal nucleus, upon disconnection from the target, neurons undergo chromatolysis; there is in addition, a generalized increase in the synthesis and turnover of RNA and structural proteins 5s. In contrast, the synthesis of neurotransmission related molecules, and the enzymes responsible for their synthesis and degradation, decrease during this period. For example, levels of muscarinic acetylcholine receptors 4L43, choline acetyltransferase 57 and acetylcholinesterase~4"56 decline when hypoglossal neurons are disconnected from their peripheral targets. In addition to muscarinic receptors, glycine44 and NMDA (unpublished observations) receptor levels also have been shown to be dependent upon the integrity of the synapse between the hypoglossal nerve axon and the skeletal muscle of the tongue. If this connection is experimentally disrupted, the number of functional receptors on hypoglossal neurons is considerably reduced. When the nerve regenerates and reestablishes connections with the target muscle, normal levels of receptors are restored 41'43'44. Although the molecular basis for these changes is, as yet, unknown, they have been interpreted as being due to the influence of a retrogradely transported factor 35. It is conceivable, therefore, that the reduction in olivary axonal synapses with Purkinje cells may be related to the transient expression of /33 subunit m R N A and [3H]muscimol binding sites within olivary neurons. It remains to be determined whether this could be due to competition for atrophic factor. Acknowledgments. We are very grateful to Dr. Allan Tobin for providing us with the rat a I and /33 cDNA clones. This publication was made possible by grants fore the National Institute on Alcohol Abuse and Alcoholism and the National Institute of Neurological Disorders and Stroke. V.L. was supported by training Grant NS07291.

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