Brain Research, 590 (1992) 95-108 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00

95

BRES 18047

Regional distribution of cells expressing glycine receptor a 2 subunit m R N A in the rat brain Kohji Sato

a,

Hiroshi Kiyama b and Masaya Tohyama

b

a Department of Neuroanatomy, Biomedical Research Center and t, Department of Anatomy and Neuroscience, Osaka Unicersity Medical School, Osaka (Japan) (Accepted 7 April 1992)

Key words: Glycine receptor; a~ Subunit; Cellular localization; In situ hybridization histochemistry; Rat brain

The a 2 subunit of the glycine receptor is expressed transiently in the rat brain during early development suggesting that this subunit may be replaced by the a I subunit in the adult brain. The expression of glycine receptor a 2 subunit mRNA was investigated in the 7-day-old rat brain by in situ hybridization histochemisfry using oligonucleotide probes specific for this subunit. Neurons expressing a 2 subunit mRNA were found to be widely and abundantly distributed throughout brain. We compared the distribution of neurons expressing a 2 subunit mRNA with that of neurons expressing a t or/3 subunit mRNA. In the lower brainstem, the location of the neurons expressing a 2 subunit mRNA was very similar to that of the neurons with t~t or/3 subunit mRNA. Neurons expressing/3 subunit mRNA were widespread and numerous in the forebrain, where neurons with otI subunit mRNA were uncommon. The locations of the neurons labeled by the a 2 probe were very similar to those of the cells labeled by the ~ probe. These findings suggest that the t~2 subunit is not only expressed by immature neurons containing the t~I subunit, but is also common to w~est immature neurons having the glycine receptor. However, it should be noted that several neurons contained /3 and/or al subunit mRNA but lacked a : subunit mRNA, suggesting that the glycine receptor is heterogeneous in its composition during brain development.

INTRODUCTION Giycine is a major inhibitory neurotransmitte.r in the central nervous system 4'5. The glycine receptor (GIy-R) is thought to be composed of two subunits (the as and /3 subunits) and a 93 kDa protein which is associated with the cytoplasmic domain of the Gly-R core 2'3. The primary structures of the a t and/3 subunits have been determined by eDNA sequencing 7'8. Additional t~ variants have be~'n ,:loned recently 9'tt-tS, and designated as the ~2, a3, and a4 subunits. The a 3 and a4 variants are expressed in a few regions of the adult brain but the level of expression of these subunits is very low t2'tS. On the other hand, the mRNA for the a 2 subunit variant is highly expressed in the prenatal and early postnatal periods ~5. Thereafter, the expression of this subunit mRNA decreases to become very low or undetectable in the adult brain ~5. It is thought that the a2 subunit is the ligand-binding subunit of the neonatal Gly-R isoform 9't°'t3, and that the role of this subunit

may be taken over by the a~ subunit during postnatal development ~'H,. If this were so, the location of the neurons expressing a 2 subunit mRNA at a very young age would coincide with that of the neurons containing at subunit mRNA in the adult brain. We have previously determined the overall distribution of neurons containing at and /3 subunit mRNA in the adult rat brain 6'is. In the present study, to explore the above possibility, we investigated the overall distribution of neurons containing a 2 subunit mRNA in the postnatal day 7 rat brain, and compared the localization of ot2 subunit mRNA expression with that of mRNA for the a I or/3 subunits. MATERIALS AND METHODS

Animals and tissue preparation Five 7-day-old male Wistar rats were decapitated under sodium pentobarbital anesthesia (50 mg/kg, i p.). The fresh brains were quickly removed and immediately frozen on powdered dry ice. Serial sections (15-20 /zm thick) were cut on a cryostat, thaw-mounted

Correspondence; K. Sato, Department of Neuroanatomy, Biomedical Research Center, Osaka University Medical School, 2-2 Yamadaoka, Suita-shi, Osaka-fu, 565 Japan. Fax: (81) (06) 875 7359.

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98 onto gelatin-coated slides, and stored at -80°C until use. Microscopic examination revealed no appreciable changes in the hybridization signals with storage.

In situ hybridization The procedure used for in situ hybridization was essentially the same as that described previously t6. After being warmed to room temperature (all steps were performed at room temperature unless otherwise indicated), the slide-mounted sections were fixed for 5 rain in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2), and then rinsed 3 times for 5 rain each in 4×SSC (pH 7.2) (1 xSSC contained 0.15 M sodium chloride and 0.015 M sodium citrate). Sections were then dehydrated through a graded ethanol series (70%-!00%), defatted with chloroform for 5 rain, and immersed twice in 100% ethanol for 5 rain each time before being subjected to hybridization. Hybridization was performed by incubating the sections with buffer (4xSSC, 50% deionized formamide, 0.12 M phosphate buffer [pH 7.2], Denhardt's solution, 2.5% tRNA, and 10% dextran sulfate) containing two kinds of [a-dsS]dATP [1,006-I,500 Ci/mmol (37-55.5 TBq/mmol), NEN]-Iabeled probes (3-6×10 ~ dpm/ml, 0.3 ml/slide) for 24 h at 41°C. ~ e r hybridization, the sections were rinsed in I×SSC (pH 7.2) for 10 rain, followed by rinsing three times in l xSSC at 55°C for 20 rain each time. The sections were then dehydrated through a graded ethanol series (70-100%) and coated with llford K-5 emulsion diluted 1:1 with water. Sections were then exposed at 4°C for 3 weeks in a tightly sealed dark box. After being developed in D-19 developer (Kodak), fixed with photographic fixer, and washed with tap water, the sections were counterstained with thionin solution to allow morphological identification. OligonucleotJde probes Two a., oligonucleotide probes (48mer) were synthesized with an Applied Biosystems DNA synthesizer and then purified using a Hitachi high-performance liquid chromatograph (ODS column chromatography). These probes were complementary io bases 1,555-1,602 and 1.714-1,761 of the rat Gly.R a,* subunit mRNA La. (These segments are located between the putative membrane spanning regions 3 and 4 of the Gly-R a~subunit cDNA, and these segments are also common in other t~, variants', a : A anti a,B~.) A computer.assisted homology search (Gen Bank R 68.0, June 1991, National Institutes of Health, USA) showed that each of the Gly-R a.,subunit probes had less than 61% homology with any sequences contained in the gene bank, The probes were labeled at the 3' end using [a-'~sS]dATP [1,(~0-1,500 Ci/mmol (37-55.5 TBq/mmol), NEN] to a specific activity of approximately 1.0-2.0x 10~ dpm/#g, Control studies We carried out control competition experiments using a 100-fold excess of unlabeled probe together with the labeled probe, and other controls involving RNAase A pretreatment (20 pg/ml)just before hybridization. These experiments showed no positive signals. Nomenclature The terminology used is according to the atlas of Paxinos and Watson ~7.

RESULTS Identification of neurons expressing the GIy-R a 2 subunit mRNA was based upon the criteria used in our previous study 26. Positive neurons were divided into 3 categories by a visual comparison of the extent of silver grain accumulation. The neurons labeled in the 7-dayold rat brain are summarized in Fig. 1.

L Telencephalon Olfactory bulb and related areas No labeled neurons were seen in the main olfactory bulb and the accessory olfactory bulb. However, a number of moderately labeled neurons were seen in each of the anterior olfactory subnuclei (external, medial, lateral, dorsal, ventral, and posterior) (Figs, 1A, B and 2A). Moderately labeled neurons were densely packed in layer II of the piriform cortex and were also found in layer III, while few or no labeled neurons were detected in layer I (Figs. 1B-I and 2B). Cerebral cortex Isocortex. A number of labeled neurons were detected i,1 layers II to VI. However, the distribution pattern and the labeling intensity varied between the different cortical layers. Most of the labeled neurons were concentrated in layer V, and the deep zone of layer VI, while the other layers contained only scattered labeled neurons (Figs. 1B-M and 2D). The intensity of the labeling in layer V, and the deep zone of layer VI was moderate, while the labeling in the other layers was only weak. Both pyramidal and non-pyramidal cells were labeled. Allocortex. All the subregions of the allocortex, such as the anterior cingulate cortex, and the retrosplenial cortex, contained unevenly distributed moderately to weakly labeled neurons. Labeled neurons were particularly prominent in layer V, and the deep zone of layer VI (Fig. 1C-M). Hippocampal formation Moderately labeled neurons were densely packed in the medial and lateral entorhinal areas, parasubiculum, and subiculum, while the presubiculum lacked any positive neurons (Figs. 1K-M and 2E). In Ammon's horn, moderately labeled neurons were densely packed in pyramidal cell layers of CA1, CA2, and CAd, while the labeling of cells in the lateral part of CA3 (adjacent to CA2) was quite weak (Figs. 1F-M and 2C). Some weakly labeled neurons were also found in the stratum radiatum and stratum oriens. In the dentate gyrus, most of the granule cells were labeled moderately (Figs. 1G-M and 2C). Some weakly labeled neurons were also found in the polymorphic layer. Endopiriform cortex and claustrum A number of strongly labeled neurons were found in the endopiriform cortex (Figs. 1C-L and 2G), while the claustrum contained moderately labeled neurons (Fig. 1C-E).

99

Septum and nuclei of the diagonal band of Broca

Amygdala and bed nucleus of the stria terminalis

Strongly labeled neurons were assembled compactly in the tenia tecta (Fig. 2A). The medial and lateral septal areas contained a number of moderately labeled neurons (Fig. 1C, D). Numerous moderately labeled neurons were seen in both the vertical and horizontal limbs of the diagonal band of Broca (Fig. 1C).

The cortical amygdaloid subnucleus and amygdalohippocampal area contained many strongly labeled neurons (Figs. 1 E - L and 2G), while the medial subnuclei were moderately labeled (Figs. 1 F - H and 2F). The other subnuclei apart from the basolateral nucleus contained only weakly labeled neurons (Figs. 1F-I and

Fig. 2. Dark-field photomicrographs showing expression of Gly-R a 2 subunit mRNA in the telencephalon. A, B: note strong expression in the

tenia tecta, posterior part of anterior olfactory nucleus and piriform cortex. C: hippocampus. The arrowhead shows weak labeling of pyramidal cells in the lateral part of CA3. D: cortex. Note the moderately labeled neurons in the layer V (arrow heads), and the deep zone of layer VI (the arrow). E: subiculum. The subiculum shows moderate expression, while the presubiculum lacks expression (arrowheads). F~ G: amygdala. Note strong expression in dorsal endopiriform cortex, amygdalohippocampalarea and posteromedial cortical amygdaloid nucleus. Bar = 500/zm.

l~d) 2F, G). The bed nucleus of the stria terminal is contained a number of moderately labeled neurons (Fig. ID).

Basal gangli,: Some large neurons which were labeled modcrately were observed in the ventral and lateral parts of the caudate putamen, while scattered neurons with weak

labeling were seen in the other parts (Figs. 1 C - H and 2F). The globus pallidus contained moderately labeled neurons (Figs. 1D-F). Many of the moderately labeled neurons were in the cell layer corresponding to layer II of the olfactory tubercle (Fig. 1C, D). The entopeduncular nucleus lacked any labeled neurons (Fig. 1G, H), while neurons in the nucleus accumbens were moderately labeled (Fig. 1C).

Fig. 3. Dark-field photomicrographs showing expression of GIy-R a z subunit mRNA in the diencephalon. A-D: expression in the thalamus. Expression is absent in the reticular thalamic nucleus (arrowheads in A and B), and in the medial and lateral habenular nuclei (arrows in B). The ventral lateral geniculate body also shows weak expression (arrowheads in C). E and F: expression in the hypothalamus. Note strong labeling in the ventrolateral part of tlle ventral medial hypothalamic nucleus (the broken line in F). The dorsomedial hypothalamic nucleus lacks expression (arrowheads in F.) Bar = 500/zm.

101

II. Diencephalon Thalamus, subthalamus, and habenular complex The anterior thalamic nuclei (dorsal, ventral and medial subnuclei) contained a large number of moderately labeled cells. Among these subnuclei, the anterior ventral nucleus had several strongly labeled cells that

tended to be localized in its ventrolateral part (Figs. 1E, F and 3A). The medial geniculate body and the dorsal lateral geniculate nucleus contained strongly labeled neurons, while the ventral lateral geniculate nucleus was only labeled weakly (Figs. l I - K and 3C, D). No labeled cells were seen in the reticular nucleus (Figs. 1 E - H and 2A,B), and the other thalamic nuclei

Fig. 4. Dark-field photomicrographs showing expression of Gly-R a z subunit mRNA in the lower brain. A: expression at the level of the red nucleus. Note lack of expression in the oculomotor nucleus (arrowheads), interpeduncular nucleus (the arrow) and weak expression in the red nucleus. B, C: strong expressio, is observed in the pontine nucleus and dorsal raphe nucleus. D and E: expression at the level of the motor trigeminal nucleus. Note strong expression in the parabrachial nuclei, locus coeruleus and motor trigeminal nucleus The trigeminal mesencephalic nucleus lacks expression (the arrowhead in D). F: expression at the level of the hypoglossai nucleus. Note weak expression in the spinal trigeminal nucleus. G: expression in the facial nucleus. H: expression in the spinal cord. Strongly labeled neurons are seen in the ventral horn. Bar = 1 mm (A); 500 #m (B-H).

102 contained moderately to weakly labeled neurons (Figs. 1E-L and 2A-D). The zona incerta contained weakly labeled neurons, but no labeling could be identified in the habenular nuclei or the subthalamus (Figs. IG-I and 2B). Preoptic area and hypothalamus The medial and lateral preoptic areas contained many moderately labeled neurons, with the number of positive cells being greater in the medial area (Fig. ID). In the hypothalamus, moderately to weakly labeled neurons were found in the anterior, lateral, ventromedial, and ventral premammiilary nuclei (Figs. IE-I and 3E, F). The dorsomedial hypothalamic nucleus and the dorsal premammillary nucleus both lacked labeled neurons (Figs. IH, I and 3F). In the ventromedial nucleus, positive cells were localized in the dorsomedial and ventrolateral subnuclei, whereas the central subnuclei contained fewer labeled cells (Figs. IG, H and 3F). The area surrounding the most caudal part of the 3rd ventricle contained numerous strongly labeled cells, while moderately labeled neurons were found in the arcuate nucleus (Figs. 1H-J and 3F). The paraventricular hypothalamic nucleus contained a small number of weakly labeled neurons (Fig. IF). The mammillary nucleus complex was devoid of labeled cells, except for the supramammillary nucleus which contained some moderately labeled neurons (Fig. IJ, K). The periventricular zone of the preoptic area, the suprachiasmatic nucleus, and the supraoptic nucleus also lacked labeled neurons (Figs. ID-H and 3E). ill. Midbra#t A number of moderately labeled neurons were observed in the ventral tegmental area and the substantia nigra (both the pars compacta and the pars reticulata), while 'he labeling of the red nucleus was weak (Figs. IJ-L and 4A). A number of moderately labeled neurons were distributed throughout the midbrain gray matter (Figs. I L-N and 4A). No characteristic labeling pattern of any of the subnuclei of the midbrain gray matter could be detected. The nucleus of Darkschewitsch contained moderately labeled neurons, and the interstitial nucleus of Cajal contained weakly labeled neurons (Fig. IK, L). A number of moderately to weakly labeled neurons were seen in the pretectal nucleus (Figs. IJ-L and 3D). In the superior colliculus, the superficial and intermediate gray layers contained several moderately labeled neurons, and the other layers contained scattered weakly labeled neurons. (Fig. IL-M). The parabigeminal nucleus was labeled moderately (Fig. IM).

IV. Pons and Medulla Visceromotor system The dorsal motor nucleus of the vagus contained moderately labeled neurons (Figs. 1T and 4F), which were especially concentrated in the medial part. The Edinger-Westphal nucleus had no labeled neurons (Fig. 1L). General somatomotor and brachiomotor systems Strongly labeled neurons were concentrated in the trigeminal motor nucleus (Fig. 10, P and 4D, E), facial nucleus (Fig. 1R and 4G), ambiguus nucleus (Fig. 1S, T), and hypoglossal nucleus (Figs. 1T and 4F), while no labeled neurons were found in the oculomotor nucleus (Figs. IM and 4A). In the hypoglossal nucleus, strongly labeled cells were concentrated in the ventral and lateral parts. Viscerosensory system Scattered neurons labeled with a moderate intensity were seen in the nucleus of the solitary tract (Figs. IS, T and 4F). General somatosensory system The principal and spinal trigeminal nuclei contained scattered weakly labeled neurons (Figs. I O - T and 4D, F), while no labeled neurons were tbund in the mescncephalic trigcminal nucleus (Figs. IO, P and 4D). The gracile and cuneate nuclei and the external cuneate nucleus all contained moderately labeled neurons (Fig. IS, T). Special somatosensoty system Auditory system. The dorsal cochlear nucleus contained many moderately labeled neurons, while the ventral cochlear nucleus lacked positive neurons (Fig. 1P-R) No labeled neurons were found in the trapezoid nucleus (Fig. IN-Q), while the superior olivary complex was labeled moderately (Fig. IO-Q). Moderately labeled neurons were dispersed throughout the inferior colliculus (Fig. IN, O), and a group of moderately labeled neurons was seen in the lateral lemniscus (Fig. IN). Vestibular system Moderately labeled neurons were found in the lateral vestibular nucleus, while the medial and spinal vestibular nuclei contained weakly labeled neurons (Fig. 1R, S). The superior vestibular nuclei lacked any positive neurons (Fig. IQ, R).

103 Reticular formation and raphe nuclei Most of the reticular formation contained moderately labeled neurons (Figs. 1M-T and 4A-G), but the lateral reticular nucleus was devoid of labeled cells (Fig. 1T). Many strongly labeled neurons were found in the dorsal and median raphe nuclei (Figs. 1N and 4C). The other raphe nuclei, such as the raphe pontis, raphe magnus, raphe pallidus, and raphe obscurus, contained moderately labeled neurons (Fig. 1N-T). Pontine tegmental area The dorsal tegmental nucleus contained weakly labeled neurons (Figs. 10, P and 4E), while the ventral tegmental nucleus of Gudden lacked any positive neurons (Fig. IN). Many neurons in the locus coeruleus and the median and lateral parabrachial nuclei were labeled strongly (Figs. 1N-P and 4D, E), while Barrington's nucleus contained moderately labeled neurons (Fig. 1P). Other lower brainstem areas A number of strongly labeled neurons were see~, in the pontine nucleus (Figs. 1M and 4A, B). The inferior olivary complex also contained strongly to moderately labeled neurons, with more intense labeling tending to occur in the lateral component (Fig. IS, T). The prepositus hypoglossal nucleus contained moderately labeled neurons (Figs. IR, S), but the interpedur:cular nucleus had no labeled neurons (Figs. IL, M and 4A). E Cerebellum

The medial and interposed cerebellar nuclei contained several weakly labeled neurons, while the lateral cerebellar nucleus lacked positive neurons (Fig. I R). The cerebellar cortex had no labeled neurons. VI. Spinal cord

In the spinal cord, numerous motoneurons in lamina IX were strongly labeled, and some strong labeling was also found in lamina VIII. In addition, many moderately labeled neurons were found in laminae II-VII (Figs. 1N and 4H). DISCUSSION I. Location of neurons containing Gly-R a 2 subunit mRNA, compared with neurons containing a t or /3 subunit mRNA The present study demonstrated the overall distribution of neurons containing GIy-R a 2 subunit mRNA in the brain of the postnatal day 7 rat. Positive neurons

were widely and abundantly distributed throughout the brain in animals of this age. We have previously reported on the cellular localization of mRNAs for the a~ subunit (the putative glycine and strychnine binding site) and the/3 subunit (considered to be a homologous polypeptide constituting the GIy-R) of the GIy-R in the adult rat brain 6'18. Neurons containing a~ subunit mRNA are concentrated in the lower brainstem and the forebrain is devoid of a~ subunit mRNA, except in a few regions. On the other hand,/3 subunit mRNA is expressed strongly and widely throughout the entire brain, with such neurons being abundant in both the lower brainstem and in the forebrain. Although it is almost difficult to compare the distributions among a~, a2 and /3 subunit mRNAs in the adult brain because of the transient appearance of a 2 subunit mRNA during development ~'~5, it would be of significance to at least compare the distribution of a 2 subunit mRNA in 7-day-old with other subunits in the adult for the understanding of Gly-R functions in both immature and mature animals. A comparison of the distribution of neurons containing a 2 subunit mRNA in the 7-day-old rat brain elucidated in this study with that of the neurons containing a~ or/3 subunit mRNA in the adult rat brain showed that the neurons expressing mRNA for the a 2 subunit almost coincided with that of neurons contain!ng mRNA of/3 subunit. The locations of the neurons expressing mRNAs for the a~, a 2, and /3 subunits are summarized in Table II. Positive neurons could be divided into 4 types (Table I). Neurons belonging to Type l contained all 3 subunit mRNAs, although the levels of expression of each one varied. Neurons belonging to Types II or Ill contained mRNAs for two of the 3 subunits, i.e. a 2 and /3 subunit mRNAs (Type II) or a~ and/3 subunit mRNAs (Type lIl), respectively. Neurons containing only /3 subunit mRNA were designated Type IV. We found no other combinations in this study, but further investigation of mRNA coexpression is still needed. Besides,

TABLE ! Classification of the combinations of expression of Gly-R subunit mRNAs Expression was investigatedin the adult rat (a I,/3) and the 7-day-old rat (a 2) central nervous system: + = detected; - = not detected. Type

mRNA

at

a2

I

+

+

+

II III IV

-

+

+

+ -

-

+ +

104 since the s t u d i e s 6as

present

study

revealed

the

together

with

distributions

our of

transcripts,

there remains a possibility of difference between transcript and protein

levels. T h e n ,

II. Possible replacement o f the el 2 subunit by other Gly-R subunits during brain development

previous the

M a n y o f t~'c l o w e r b r a i n s t e m a r e a s c o n t a i n e d T y p e I

the protein level

neurons, and many of the forebrain regions contained

should further investigated.

TABLE II

Distribution and relative abundance of Gb'-R subunit mRNAs in different regions of the adult (a i, .8) and 7-day-old rat (or2) central nervous system Area Olfactory bulb Accessory olfactory bulb Anterior olfactory nucleus Piriform cortex Cerebral cortex lsocortex Allocortex Hippocampal formation Subiculum Presubiculum Dentate gyrus CAi CA2 CA3 Endopiriform cortex Claustrum Septum Tenia tecta Medial septal nucleus Lateral septal nucleus Diagonal hand Amygdala (\~rtical nucleus Medial nucleus Basolateral nucleus Others Bed nucleus of the stria terminals Basal ganglia Olobus pallidus Entopeduncular nucleus

C'audate putamen Olfactory tubercle Accumbens nucleus Thalamus Reticular nucleus Parafascicular nucleus Cuntromedial nucleus Anterodorsal nucleus Others Geniculate nuclei Medial nucleus Dorsolateral nucleus Ventrolateral nucleus Habenular complex Subthalamic nucleus Zona incerta Preoptic area Hypothalamus Paraventricular area Supramamillary nucleus Mamillary nucleus Arcuate nucleus Periventricular zone Anterior hypothalamic nucleus Lateral hypothalamus Others

mRNA

Type

al

a2

.8

n.d. n.d. n.d. n.d.

n.d. n.d. ++ ++

+++ +++ +++ +++

IV IV Ii |I

n.d. n.d.

++ ++

+++ +++

I! II

++ ++ ++ ++ ++ ++ ++

.4-++ +++ + + +++ +++ +++ +++ +++

II IV It II II II II I!

++ ++ ++ ++

+++ + + + ++

I! 11 I! I

++ ++ n.d. + + +

+ + + + + +

!! I! IV !! I!

++ n.d, + ++ + +

++ + + + ++ +

II IV !! ii I!

n.d. + + + n,d. n.d.

n,d. ++

+ + + +++

IV !

++ ++ ++

++ +++ ++

I ll I!

n.d, n.d. n.d. n.d. n.d. + + n,d.

++ ++ +

+ + + + + q' + +++ ++

II II II IV IV I

+ +

II

+ + + + + + + ++ ++ +

II It IV II IV I I II

n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n,d.

n.d. n,d, + n,d, n.d. n.d. n.d.

n,d. n.d. n,d,

n.d. n.d. n.d.

n.d, n.d, n,d,

n,d, n,d, + + + +

n,d,

n.d.

n,d. n,u, + ++

+ + + n.d. + + n.d. + ++ + +

105 T y p e II c e l l s . T i l e n u c l e i w i t h o t h e r w e r e f e w in n u m b e r , and

the

Neurons

trapezoid

types of neurons

e.g., t h e v e n t r a l c o c h l e a r n u c l e u s , nuclei

contained

Type

Ill

cells.

and

amygdaloid

nucleus, reticular thalamic nu-

cleus, subthalamic

nucleus, mammillary

nuclei, trigemi-

nal mesencephalic

nucleus, ventral nucleus of Gudden,

nucleus,

as w e l l as t h e P u r k i n j e

cells and the granule cells of the cerebellum The

if2

subunit

ligand-binding

has

subunit

f o r m L'~'I3. T r a n s c r i p t i o n the

prenatal

and

been of

the

considered neonatal

of the a 2 subunit

early

postnatal

Substantia nigra Pars compacta Pars reticulata Red nucleus Central gray matter Nucleus of Darkschewtisch Visceromo~or system Dorsal motor nucleus of the vagus Edinger-Westphal nucleus General motor system Trigeminal motor nucleus Facial nucleus Ambiguus nucleus Hypoglossal nucleus Oculomotor nucleus Viscerosensory system Nucleus of the solitary tract General somatosensory system Trigeminal mesencephalic nucleus Trigeminal spinal nucleus Trigeminal principal nucleus Gracile nucleus Cuneate nucleus External cuneate nucleus Auditory system Dorsal cochlear nucleus Ventral cochlear nucleus Trapezoid nuclei Superior olivary complex Inferior colliculus Lateral lemniscus Vestibular system Medial nucleus Lateral nucleus Spinal nucleus Superior nucleus Visual system Pretectal nucleus Superior colliculus Parabigeminal nucleus Reticular formation Reticular tegmentai nucleus Lateral reticular nucleus Others Raphe nuclei Dorsal raphe nucleus Median raphe nucleus Others Pontine tegmental area Dorsal nucleus of Gudden Ventral nucleus of Gudden Locus coeruleus Parabrachial nucleus Barrington's nucleus

mRNA

Type

al

a2

n.d. + + + + + n.d. n.d.

+ + + + +

+ +

+ + + + +

+ +

+ + n.d.

+ +

+ + + + +

+ + + +

+ + + +

+ n.d. + + + + + + + + + +

+ + + + + + + + +

/3 + +

+ + + + + + + + n.d.

+ + + +

+ + + + +

+ + + + +

I1 I I II II I III

+ + + + +

+ + + + +

I I I ! ill

+ +

+

1

n.d. + + + + + + + +

+ + + + + +

+ + + + + + +

IV I 1 ! I I

+ + + + + +

+ + + + +

! Ill I!! i 1 !

+ + + + +

I i ! It!

+ + + + + +

+ + + +

+ +

+ + n.d. n.d. + + + + + +

+ + + +

+ + + +

+ + + + n.d.

+ + + +

n.d. + n.d.

+ + + + + +

+ + + + + + +

II 1 ll

+ + + + + + + + +

+ + n.d + +

+ + + + + + + +

I llI I

n.d. n.d. n.d.

+ + + + + + + +

+ + +

lI II II

+ + + n.d. n.d. + + + +

+ n.d. + + + + + + + +

+ + + + +

+ + + +

+ + + + +

to

be

Gly-R appears

s t a g e s mS, a n d

TABLE II (continued).

Area

belonged

to Type IV.

in t h e o l f a c t o r y b u l b , a c c e s s o r y o l f a c t o r y b u l b ,

basolateral

interpeduncular

! IV II I I

the isoat de-

!06 TABLE !1 (continued). Area

Other lower brainstem areas Pontine nucleus Nucleus prepositus hypoglossi lnterpeduncular nucleus Inferior olivary complex Cerebellum Cerebellar nuclei Medial nucleus Interposed nucleus Lateral nucleus Purkinje cell layer Granular cell layer Spinal cord ViII-IX iI-Vll

mRNA al

a2

/3

Type

n.d. +++ n.d. +

+++ ++ n.d. +++

+ + + +

++ ++ + +

11 I IV I

++ ++ + n.d. n.d.

+ + n.d. n.d. n.d.

+ + + + +

+ + + +

I ! II! IV IV

+++ ++

+++ ++

+++ ++

+ + + +

I I

Relative expression was estimated by visual comparison of exposed emulsion-coated slides, n.d. = not detected; + = low; + + = moderate; + + + = high; see discussion for details of the type classification (Types I-IV).

creases markedly after that time, with little expression of this subunit being identified in the adult brain. Therefore, it has been suggested that the o~2 subunit is replaced by the a~ subunit during brain d e v e l o p m e n t m. in fact, we found a high level of a 2 subunit m R N A expression in the lower brainstem of the postnatal day 7 rat, a site where a~ subunit m R N A is strongly expressed in adult animals, in addition, the distribution of the neurons labeled by the a,. probe was very similar to that oi tllose labeled by the ot a probe in these regions. These findings give some morphological support to the hypothesis m e n t i o n e d above. However, it should bc noted that a.,.. subunit m R N A is expressed strongly and widely not only in the lower brainstem but also in many forebrain areas that generally lack a~ subunit m R N A . As previously reported 7.ts, it seems likely that the /3 subunit pairs with some unknown Gly-R subunit to form the Gly-R in most brain areas. Therefore, it may be suggested that the a , subunit in " these areas is replaced by the u n k n o w n subunits with advancing age. For example, in the pyramidal cells of A m m o n ' s horn, the a 3 subunit is expressed at a very low level ~-~, and it is possible that the a , subunit is replaced by the a 3 subunit in this region during development. In Type IV neurons, only the/3 subunit m R N A is expressed. In these neurons, it seems likely that during m a t u r a t i o n the unidentified n e o n a t a l Gly-R isoform is replaced by the unknown subunit which pairs with t h e / 3 subunit, The present study demonstrated that, except in a few brain regions, the o~2 subunit is expressed abundantly in most of the neurons containing t h e / 3 subunit at a very young age These findings show that the ~2 subunit is a c o m m o n neonatal subunit of the Gly-R

expressed t h r o u g h o u t the brain, suggesting that it has a role in the m a t u r a t i o n of the entire glycinergic transmission system. Acknowledgements. This work was supported in part by the Ministry of Education, Science and Culture of Japan.

ABBREVIATIONS 3 ~ 7 7n Sn 10 12 12n ac Ach

AD AH AHi AM Arab AOB AOE AOL AOP APir APT Arc AV AVDM AVVL Bar BIC bic BL BM BST CAI-3 Ce CG cIc

oculomotor nucleus abducens nucleus facial llUCleUS htcial nerve or its root vcstibulocochlear nerve dorsal motor nucleus of vagus hypoglossal nucleus root of hypoglossal nerve anterior commissure accumbens nucleus anterodorsal thalamic nucleus

anterior hypothalamic area amygdalohippocampal area anteromedial thalamic nucleus ambiguus nucleus accessory olfactory bulb anterior olfactory nucleus, external part anterior olfactory nucleus, lateral part anterior olfactory nucleus, posterior part amygdalopiriform transition area anterior pretectal nucleus arcuate hypothalamic nucleus anteroventral thalamic nucleus anteroventral thalamic nucleus, dorsomedial part anteroventral thalamic nucleus, ventrolateral part Barrington's nucleus nucleus of the brachium of the inferior coiliculus brachium of the inferior colliculus basolateral amygdaloid nucleus basomedial amygdaloid nucleus bed nucleus of the stria terminalis fields CAI-3 of Ammon's horn central amygdaloid nucleus central gray central nucleus of the inferior colliculus

107 CL CI CLi CM CnF Co cp CPu Cu DC DEn DO DH Dk DLG DLL DM DpG DPO DR DTg ECu En EP EPi EW f fi fr G g7 Ol GP Gr OrA Hb HDB IAM IC ic

lcj icp IOr IMLF lag Int lnW IO IP IPI La La| LC LD LDT LH LHb LM LOT LP LPB LRt LS LSO LVe LVPO m5 mcp MCPO MD MdD MdV

centrolateral thalamic nucleus claustrum caudal linear nucleus of the raphe central medial thalamic nucleus cuneiform nucleus cortical amygdaloid nucleus cerebral peduncle caudate putamen cuneate nucleus dorsal cochlear nucleus dorsal endopiriform nucleus dentate gyrus dorsal horn of the spinal cord nucleus of Darkschewitsch dorsal lateral geniculate nucleus dorsal nucleus of the lateral lemniscus dorsomedial hypothaiamic nucleus deep gray layer of the superior colliculus dorsal periolivary region dorsal raphe nucleus dorsal tegmental nucleus external cuneate nucleus endopiriform nucleus entopeduncular nucleus external plexiform layer of the olfactory bulb Edinger-Westphal nucleus fornix fimbria of the hippocampus fasciculus retroflexus gelatinosus thalamic nucleus genu of the facial nerve glomerular layer of the olfactory bulb globus pallidus gracile nucleus granular cell layer of the accessory olfactory bulb habenular nuclei nucleus of the horizontal limb of the diagonal band interanteromedial thalamic nucleus inferior colliculus internal capsule islands of Calleja inferior cerebellar peduncle internal grattular layer of the olfactory bulb interstitial nucleus of the medial longitudinal fasciculus intermediate gray layer of the superior colliculus interposed cerebellar nucleus intermediate white layer of the superior colliculus inferior olive interpeduncular nucleus internal plexiform layer of the olfactory bulb lateral amygdaloid nucleus lateral cerebellar nucleus locus coeruleus laterodorsal thalamic nucleus iaterodorsal tegmental nucleus lateral hypothalamic area lateral habenular nucleus lateral mammillary nucleus nucleus of the lateral olfactory tract lateral posterior thalamic nucleus lateral parabrachial nucleus lateral reticular nucleus lateral septal nucleus lateral superior olive lateral vestibular nucleus lateroventral periolivary nucleus motor root of the trigeminal nerve middle cerebellar peduncle magnocellular preoptic nucleus mediodorsal thalamic nucleus medullary reticular nucleus, dorsal part medullary reticular nucleus, ventral part

ME Me . me5 Med mfb MG MHb Mi ML ml mlf MM MnPO MnR Mo5 MP mp MPA MPB MPT MS MSO mt MVe MVPO ON Op OPT opt OT ox PaV PB PBL PBM PC pc Pe PF PH Pir PMCo PMD PMn PMV Pn PnC PnO Po PP PPT Pr5 PrH PrS PT PV PVA PY R

Re Rh RMg ROb RPa RPO Rt RtTg S s5 SC Sch scp

median eminence medial amygdaloid nucleus mesencephalic trigeminal tract medial cerebellar nucleus medial forebrain bundle medial geniculate nucleus medial habenular nucleus mitral cell layer of the olfactory bulb medial mammillary nucleus, lateral part medial lemniscus medial longitudinal fasciculus medial mammillary nucleus, medial part median preoptic nucleus median raphe nucleus motor trigeminal nucleus medial mammiilary nucleus, posterior part mammillary peduncle medial preoptic area medial parabrachial nucleus medial pretectal nucleus medial septal nucleus medial superior olive mammillothalamic tract medial vestibular nucleus medioventral periolivary nucleus olfactory nerve layer optic nerve layer of the superior colliculus olivary pretectal nucleus optic tract nucleus of the optic tract optic chiasm paraventricular hypothalamic nucleus parabrachial nucleus lateral parabrachial nucleus medial parabrachial nucleus paracentral thalamic nucleus posterior commissure periventricular hypolhalamic nucleus parafascicular thalamic nucleus posterior hypothalamic area piriform cortex posteromedial cortical amygdaloid nucleus premammillary nucleus, dorsal part paramedian reticular nucleus premammillary nucleus, ventral part pontine nuclei pontine reticular nucleus, caudal part pontine reticular nucleus, oral part posterior thalamic nuclear group peripeduncular nucleus posterior pretectal nucleus principal sensory trigeminal nucleus prepositus hypoglossai nucleus presubiculum paratenial thalamic nucleus paraventricular thalamic nucleus paraventricular thalamic nucleus, anterior part pyramidal tract red nucleus reuniens thalamic nucleus rhomboid thalamic nucleus raphe magnus nucleus raphe obscurus nucleus raphe pallidus nucleus rostal periolivary region reticular thalamic nucleus reticulotegmentai nucleus of the pons subiculum sensory root of the trigemninal nerve superior colliculus suprachiasmatic nucleus superior cerebellar peduncle

108 SFi SHi S! sm

SNC SNR SO Sol sol Sp5 sp5 SPO SpVe st SuG SuM SuVe Tu "f'I" Tz tz unc

VC VDB VII VL VLG VLL VM VMH VN VP VPL VPM VTA VTg ZI

septofimbria nucleus septohippocampal nucleus substantia innominata stria medullaris of the thalamus substantia nigra, compact part substantia nigra, reticular part supraoptic nucleus nucleus of the solitary tract solitary tract spinal trigeminal nucleus spinal trigeminal tract superior paraolivary nucleus spinal vestibular nucleus stria terminalis superficial gray layer of the superior colliculus supramammillary nucleus superior vestibular nucleus olfactory tubercle tenia tecta nucleus of the trapezoid body trapezoid body uncinate fasciculus ventral cochlear nucleus nucleus of the vertical limb of the diagonal band ventral horn of the spinal cord ventrolateral thalamic nucleus ventral lateral geniculate nucleus ventral nucleus of the lateral lemniscus ventromedial thalamic nucleus ventromedial hypothalamic nucleus vomeronasal nerve layer ventral pallidum ventral posterolateral thalamic nucleus ventral posteromedial thalamic nucleus ventral tegmental area ventral tegmental nucleus zona incerta

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