Quantitative analysis of transient GABA expression in embryonic and early postnatal rat spinal cord neurons

265

De~,elopmental Brain Research, 72 (1993) 265-276 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-3806/93/$06.00

BRESD 51607

Quantitative analysis of transient GABA expression in embryonic and early postnatal rat spinal cord neurons Anne E. Schaffner, Toby Behar, Suzan Nadi and Jeffery L. Barker Laborato~' o["Neurophysiologg', National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 (USA) (Accepted 10 November 1992)

Key words." GABA; Glutamic acid decarboxylase; Immunocytochemistry; Rat; Embryo; Spinal cord; Gradient

GABA expression was investigated using biochemical analysis of spinal cord homogenates and immunocytochemical analysis of cells acutely dissociated from the embryonic and postnatal rat spinal cord. 7-Aminobutyric acid (GABA) was detected by both methods as early as embryonic day 13 (El3). At El3, the percentage of neurons that were GABA + was 0.5%. This value increased during embryogenesis, peaked during the first two postnatal weeks to just over 50%, and declined to approximately 20% by the third postnatal week emphasizing the transient nature of GABA expression. At El7 there was a pronounced, positive ventro-dorsal and rostro-caudal gradient of GABA ~ cells that persisted until just before birth. At this time the gradients reversed in cervical and lumbosacral regions indicating that GABA immunoreactivity in discrete anatomical regions is also a transient phenomenon. During the embryonic period GABA immunoreactivity was diffusely distributed throughout cell bodies and proximal processes. At E21, both GABA and synaptophysin were present in the same cells. However the two antigens did not co-localize point for point. By postnatal day 21 GABA immunoreactivity appeared in puncta that co-localized entirely with puncta of synaptophysin immunoreactivity. The sizable percentage of neurons that transiently express GABA during development, and the fact that it can be detected prior to the synaptic form of glutamic acid decarboxylase (GADe,5), suggest that the amino acid may play a significant role during differentiation before it functions as an inhibitory neurotransmitter.

INTRODUCTION 7-Aminobutyric acid ( G A B A ) is considered to be the principal inhibitory neurotransmitter in the brain and, along with glycine, performs the same function in the spinal cord 1s,48,6°. G A B A circuits differentiate early as compared to other neurotransmitter systems. Lauder et al. 26 have described the immunohistochemical appearance of a GABAergic fiber network as early as E l 3 in the rat brain and GABAergic cell bodies at E l 4 in the cortex. The fibers were thought to originate from cell bodies in the brainstem and spinal cord. Fuji et al. t4 described glutamic acid decarboxylase (GAD)-immunoreactive fibers in the rat spinal cord at E l 6 in the dorsal, lateral and ventral funiculi. The amino acid has been shown to induce neurite outgrowth in cultured cerebellar neurons 15 and induce the expression of G A B A receptors in cultured rat cerebellar granule cells 39 and neonatal rabbit retinal cells in vivo 32. G A B A also affects the maturation of synapses in cultured

neuroblastoma cells 61. G A B A has been localized to glands and organs outside the nervous system as well 1°'53. The presence of the amino acid in nonneuronal as well as neuronal tissues, its effects on differentiation and its early embryonic expression have led to recent speculation that it may function as a t r o p h i c factor 6.25.37,40.52. Responses to G A B A have been detected in virtually all neurons of the central nervous system 4s. Functional receptors have been recorded on ventricular zone cells of E l 6 cortex zv. Studies in this laboratory have demonstrated the presence of G A B A a receptors on the majority of embryonic neurons derived from the developing rat spinal c o r d 34 and the hippocampus 13. The widespread distribution of responses to the amino acid also suggest that G A B A may play an important physiological role during embryonic development and differentiation. The myriad roles associated with G A B A and G A B A receptors prompted our investigation of the emergence

Correspondence: A.E. Schaffner, NIH, Building 36, Room 2C02, Bethesda, MD 20892, USA. Fax: (1) (301) 4(12-1565.

266

of the GABAergic phenotype in cells acutely dissociated from the developing rat spinal cord. Our results indicate that a large and transient population of GABAergic neurons is present during late embryogenesis and the early postnatal period. This population appears before, and continues into the period of spinal cord synaptogenesis 3s and the appearance of immunocytochemically detectable GAD6s (the synaptic form of GAD) 2'3't~'t2. These results are consistent with a developmental role for the amino acid.

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MATERIALS AND METHODS T h e following ages of Sprague-Dawley rat spinal cords were used in the study: embryonic day 13 (El3), El5, El7, E l 9 and E21 and postnatal day 5 (PN5), PN7, PNI0, PN14 and PN21. Embryonic age was determined by the appearance of a vaginal plug (day 1) and m e a s u r e m e n t of c r o w n - r u m p length. Birth generally occurred between E21 and E22. Fig. I illustrates the size and general physical characteristics of embryos used in the present study. T h e linear relationship between embryonic age and c r o w n - r u m p length is shown in Fig. 2. Our m e a s u r e m e n t s were identical to those previously published 17. Preparation

of acutely dissociated

cells

D a m s were euthanized with CO 2 and embryos delivered by cesarian. Pups older than 1 week were euthanized with CO2 before decapitation. Newborns were cooled for several minutes at 4°C and quickly decapitated. Spinal cords from littermates were removed, cleaned of meninges, pooled, and incubated with gentle rocking at 37°C for 45 min in Earle's Balanced Salt Solution (EBSS) containing 20 U / m l papain (Worthington Biochemical Corporation, Freehold, NJ), 0.005% D N a s e (Boehringer M a n n h e i m , Indianapolis, IN), 0.5

Embryonic Age (days) Fig. 2. There is a linear relationship between embryonic age and c r o w n - r u m p length (CRL). There is a significant, linear relationship between age and C R L from E l l through E22. Remarkably, the slope of the relationship doubled beginning at El7, C R L increased twice as fast as it did over E l 1-16.

m M E D T A , and 1 m M L-Cysteine2°. Cords were triturated through a 10ml pipet, spun at 300× g for 5 rain, and resuspended in EBSS containing l m g / m l bovine serum albumin (BSA; Sigma, St. Louis, MO.) and l m g / m l ovomucoid trypsin inhibitor (Sigma). The cell suspension was layered over 5 ml of EBSS containing 10 m g / m l each of BSA and ovomucoid and centrifuged at 80 x g for 7 min. Cells from PN10, 14 and 21 were run on an additional gradient to separate them from myelin 2. Twenty ml of a cell suspension in PBS was centrifuged through 10 ml of Lymphocyte Separation M e d i u m (LSM, Organon Technica) at 450× g for 15 min. Cells were recovered directly beneath the myelin layer which accumulated at the interface. All cells were resuspended in PBS with 17 m M glucose, and 1 x 106

Fig. 1. Rat embryos at different gestational ages exhibit a characteristic curvature and other distinct anatomical features. T h e insert shows how the c r o w n - r u m p length is measured, that is, from the vertex to the r u m p without distorting the natural curvature of the embryo.

267 cells were seeded onto poly-D-lysine-coated (53,000 mol.wt., 5 / z g / m l ) (Sigma), 35 m m N U N C culture dishes. Cells were allowed to adhere onto the dishes at 37°C for 1 h, then were processed for analysis by indirect immunofluorescence as described below.

Preparation of dissociated cells for topographical studies El5, El7, El9, E21 and PN5 pups were used for quantitative analysis of G A B A expression in different regions of the spinal cord. Spinal cords were dissected and separated into cervical, thoracic, and lumbosacral segments which were then divided into dorsal and ventral halves. Thus, each cord was divided into six regional segments: cervical dorsal, cervical ventral etc. Similar regional segments from littermates were pooled, and the segments were dissociated with papain and seeded onto 35 mm dishes as described above.

Incorporation of BrdU In some studies, E l 3 cells were analyzed for bromodeoxyuridine (BrdU) incorporation. Following enzyme dissociation, the cells were resuspended in Minimum Essential M e d i u m (GIBCO, G r a n d Island, NY.) supplemented with 5% Serum-Plus (JRH Biosciences, Lenexa, KS). The cells were plated and incubated 4 h at 37°C in the presence of 50 t~g/ml BrdU (Becton Dickinson, San Jose, CA), then processed for immunolabeling as described below.

Colchicine Twenty-day-old rat pups were initially anesthetized with chloral hydrate at 4(10 mg per kg body weight and maintained u n d e r metofane. Animals were placed in a stereotaxic device and colchicine (15 # g in 10 ~zl PBS) was injected into the fourth ventricle. Animals were allowed to recover and were held for 18 h prior to sacrifice. Spinal cords were removed and cells processed as described above.

Antibodies Two a n t i - G A B A antisera were used in the study: rabbit antiG A B A (Incstar, Stillwater MN) and guinea pig a n t i - G A B A (Eugene Tech Inc., Allendale, NJ). Two anti-GAD antisera were used in our studies. The antiserum designated as K-2 was purchased from Chemicon (Temecula, CA). It was prepared by immunizing rabbits with a G A D fusion protein shown to have G A D enzymatic activity. It has been shown to recognize a 67,000 Da form of G A D on W e s t e r n blots of feline brain, and has been used to localize G A D + cells by immunocytochemistry ~2. K-2 recognizes the protein encoded by the GAD67 gene. The antiserum designated as 1440 was the generous gift of I. Kopin (NINDS, Bethesda, MD). It was prepared by immunizing sheep with an antigen-antibody complex composed of rat G A D bound to immunoprecipitating anti-GAD antiserum 5°. This antibody has been used extensively to localize G A D + cells by immunocytochemistry, to identify G A D proteins on western blots, and to isolate G A D c D N A clones that encode cat and rat proteins from bacterial expression libraries. 1440 has been shown to recognize both 59-60,000 and 62-63,000 Da proteins on western blots of adult rat brain 2~'22,z3,27,2s'35'36"-s°and preferentially recognizes the product of the GADs5 gene. An IgM monoclonal anti-neurofilament (NF) antibody was the generous gift of Dr. C. Gibbs (NINDS, Bethesda, MD). T e t a n u s toxin and monoclonal anti-tetanus toxin antibody were the generous gifts of Dr. William Habig (FDA, Bethesda, MD). Monoclonal anti-galactocerebroside (GalC) was the generous gift of Dr. B. Ranscht (Cancer Research Institute, La Jolla, CA). A2B5 ascites was prepared from hybridoma cells purchased from American Type Culture Collection (Rockville, MD). Monoclonal anti-GFAP antibody was purchased from Sigma. Anti-BrdU antiserum was purchased from Becton Dickinson. Anti-synaptophysin was purchased from Boehringer M a n n h e i m Corporation.

Fixation and immunofluorescence A d h e r e n t cells were fixed in 4% paraformaldehyde (PF) in 0.1 M Sorenson's phosphate buffer, pH 7.2 and washed in PBS. Primary antisera were diluted in PBS with 0.25% Triton X-100. Exceptions to this fixation procedure and the use of a permeabilizing agent are

mentioned below for specific antisera. It should be noted that the guinea pig a n t i - G A B A from Eugene Tech was prepared using G A B A conjugated with glutaraldehyde to a carrier protein. However, the manufacturer has determined that immunocytochemical detection of G A B A does not require the presence of glutaraldehyde in the fixative and specifically r e c o m m e n d s 4% PF. We made extensive use of double-labeling immunofluorescence. Affinity purified secondary antibodies used in the study were rhodamine-conjugated (RITC) donkey anti-rabbit lgG, fluoresceine-conjugated (FITC) donkey anti-sheep IgG, FITC-goat anti-mouse IgM, RITC-goat anti-guinea pig IgG, FITC-rat anti-mouse IgG, and RITC-rat anti-mouse IgG, all purchased from Jackson Immunological Research (West Grove, PA). Incubations with secondary antibodies were carried out at room temperature. PBS with 0.25% Triton X-100 was used as the antibody diluent unless otherwise noted. GABA and K-2 (GAD671. Cells were fixed 30 min, washed 3 times, and incubated 48 h at 4°C in a mixture of guinea pig anti-GABA (1:5001 and rabbit K-2 (1:50). Cells were washed 3 times and incubated I h in R I T C goat anti-guinea pig lgG and FITC-donkey anti-rabbit IgG (1:401, then rinsed 5 times. GABA and 1440 (GAD65). Cells were fixed 30 min in 4% PF with 0.1% glutaraldehyde, washed 3 times, and incubated 48 h at 4°C in ~t mixture of rabbit anti-GABA (1:2,500) and sheep 1440 (1:700). The cells were washed 3 times, incubated 1 h in RITC-donkey anti-rabbit IgG and FITC-donkey anti-sheep IgG (1:40), and rinsed 5 times. GABA and neurofilament. Cells were fixed 30 min in 4% PF with 0.1% glutaraldehyde, and incubated overnight at 4°C in a mixture of rabbit a n t i - G A B A (1:2,500) and mouse anti-NF (tissue culture supernatant at 1:8). Cells were rinsed 3 times, then incubated 1 h in RITC-donkey anti-rabbit IgG and FITC-goat anti-mouse IgM (1:40), followed by 5 washes. GABA and tetanus toxin. A mouse monoclonal antibody (No. 1812126) and toxin were premixed for 1 h at room temperature at a final concentration of 1:2000 and 4 / x g / m l respectively. The mixture was applied to live, adherent cells for 30 rain at room temperature. Cultures were rinsed and incubated for 30 rain at room temperature in FITC-rat anti-mouse IgG (1:40). All incubations were carried out using PBS as diluent. Cells were then fixed in 4 ~ PF and processed for G A B A immunocytochemistry using guinea pig anti-GABA as described above. GABA and synaptophysin, Cells were fixed 30 min in 4% PF, incubated 10 min in 0.25% Triton X-100 in PBS, then incubated overnight at 4°C in a mixture of guinea pig anti-GABA and mouse anti-synaptophysin antisera (1:300 and 1:2, respectively). Following three washes in PBS, cells were incubated 1 h at room temperature in a mixture of FITC-goat anti-guinea pig IgG (1:40) and RlTC-rat anti-mouse IgG (1:40)without Triton. K-2 (GAD67) or G A B A and BrdU. Cells were fixed 10 rain in 2% PF, rinsed, and incubated in the following solutions followed by 2 - 3 washes in PBS: 10 rain at - 2 0 ° C in absolute methanol, 1 min in 0.2% PF, 7 min in 0.07 M N a O H and 5 min in 4% PF. Cells were then incubated overnight in undiluted anti-BrdU. The cells were washed, incubated 30 min in K-2 (1:501 or overnight at 4°C in anti-GABA (1:3001 antiserum, followed by a 30 min incubation in a mixture of RITC-donkey anti-rabbit IgG (BrdU and G A D ) or RITC-goat anti-guinea pig IgG (Brdu and G A B A ) and FITC- rat anti-mouse IgG (1:40), then washed 5 times. No Triton X-100 was used in conjunction with the PF-alcohol fixation. A2B5 and GALC. Cells were fixed 10 min in 2% PF, rinsed and incubated 15 min in a mixture of primary antisera (1:300). The cells were washed and incubated 15 min in a mixture of FITC-goat anti-mouse IgM and RITC-rat anti-mouse lgG (1:401, then washed 5 times. Triton was not used in labeling of cell surface antigens. Neurofilament and GFAP. Cells were fixed 7 min at - 2 0 ° C in 95% E T O H and 5% acetic acid, rinsed 3 times in M E M with I(1% fetal calf serum, then washed 3 times. Cells were incubated 15 rain in a mixture of a n t i - G F A P (1:3001 and anti-NF (1:81, washed 3 times, and incubated in a mixture of FITC-goat anti-mouse IgM and R1TC-rat anti-mouse IgG (1:40), then washed 5 times. No Triton X-100 was used in conjunction with the acid-alcohol fixation. A2B5 and GFAP. Ceils were fixed 10 min in 2% PF, rinsed, inca-

268 bated in A2B5 ascites (1:300), washed, fixed in acid-alcohol as described above, and incubated 15 min in anti-GFAP (1:300), washed, and incubated in F1TC-goat anti-mouse IgM and R1TC-rat antimouse lgG (1:40). No Triton X-100 was used in conjunction with the acid-alcohol fixation. Controls. Controls were incubated in buffer without primary antibody, followed by incubation in the mixtures of secondary antisera. In addition, the conjugated secondary antibodies were screened for species cross-reactivity. Control cells incubated in secondary antibody alone did not fluoresce. No cross-reactivity was seen with the conjugated secondary antibodies. Specificity of GABA staining was checked by pre-absorbing the GABA antibody for 1 h at room temperature, with 100 /xg/ml soluble GABA. Ceils incubated with pre-absorbed anti-GABA antibodies did not fluoresce. Microscopy. Cells immersed in PBS were examined on a Zeiss photomicroscope equipped with epifluorescence and appropriate filters for visualization of fluoresceine and rhodamine. The percentage of positively stained cells was determined by dividing the number of fluorescent cells in a field by the total number of cells in the same field (visualized under phase contrast with a water immersion 25 x Planapo objective). Five random fields were counted per dish, averaging 500-1,000 cells per plate. Duplicate plates were run for each sample. Initial time course data was subjected to analysis of variance (ANOVA) to determine variation within groups. No significant difference between replicate plates was detected. Measurement of amino acids Samples of whole spinal cords (presumably containing both cell bodies and fibers) were homogenized with 5% trichloroacetic acid (TCA) containing amino ethyl cysteic acid (external standard). The proteins were separated by centrifugation. The TCA in the supernatant was removed by four back extractions with water-saturated ethyl ether. The aqueous phase containing the amino acids was evaporated to dryness using a Savant Evapomix. The samples were resuspended in lithium citrate buffer pH 2.6 and analyzed in a Beckman 7300 (Palo Alto, CA) high performance amino acid analyzer with post column derivatization to o-phthalaldehyde as described by Benson and Hare 4. The peaks were integrated using a Beckman 7000 Data System (Palo Alto, CA). The unknown samples were quantitated by comparison to standards and expressed on a per milligram protein basis. Proteins were measured as described by Lowry et al. :~°.

tive a b u n d a n c e

at E l 9 a n d s h o w i n g the s a m e d r o p al

E21. P r o g r e s s i v e , p a r a l l e l i n c r e a s e s in b o t h G A B A and G A B A + cell n u m b e r o c c u r r e d d u r i n g t h e first 10 postnatal

days.

GABA

Thus,

measured

changes

in

the

concentration

in w h o l e tissue h o m o g e n a t e s

of

gener-

ally p a r a l l e l e d c h a n g e s in t h e r e l a t i v e n u m b e r of cells i m m u n o r e a c t i v e for G A B A

throughout the embryonic

a n d e a r l y p o s t n a t a l p e r i o d o f spinal c o r d d e v e l o p m e n t .

GABA is only expressed postmitotically Since GABA was detected in cells derived from El3 spinal cord, a time at which many cells are still dividing, we analyzed El3 cord for both GABA and GAD67 expression in mitotically active cells. El3 cells were allowed to incorporate BrdU over a 4 h period, then fixed and stained for the presence of GABA or GAD and BrdU. Although 18% of the ceils contained BrdU-labeled nuclei, GABA was never detected in BrdU + cells. Parallel experiments, in which El3 cells were exposed to BrdU and then labeled for GAD, revealed that 75% of the cells were GAD + and 20% of all GAD + cells had BrdU-labeled nuclei (Fig. 4). While 18% of all acutely adherent cells derived from El3 spinal cords were mitotic (BrdU+), 45% of the cells expressed neurofilament (NF) (Fig. 5). The percentage of neurons increased over the subsequent 8 days, such that by E21, 93% of all cells were NF +. Thereafter the percentage of total cells expressing NF declined, as the relative number of glial cells increased (detected by double labeling with NF and GFAP, A2BS, or GalC; not shown). By the end of the first postnatal

RESULTS 100

GABA concentration and GABAergic cell number change in a parallel manner GABA concentration in spinal cord homogenates between E13 and PNI0 was determined by HPLC. The corresponding percentage of GABAergic cells in dissociates of E13-PN10 rat spinal cords was determined by immunocytochemistry. At El3, the earliest age examined, trace levels of GABA ( ~ 0.5 nmol/mg protein) were detected in cord homogenates and 0.1% of all

o

spinal

m

cord

cells w e r e

G A B A + (Fig. 3). M e a s u r e d

levels of the amino acid increased 3-fold between El3 a n d E l 5 a n d G A B A + cells i n c r e a s e d 10-fold ( a l t h o u g h t h e y still r e p r e s e n t e d a v e r y s m a l l f r a c t i o n o f t o t a l cells, i.e., f r o m 0 . 1 % to 1%). Howe~,er, w h i l e n o s i g n i f i c a n t change El5

in G A B A

and

El7,

concentration

the percentage

c r e a s e d 8-fold. B e t w e e n

El7

was noted

between

o f G A B A + ceils inand birth both GABA

c o n c e n t r a t i o n a n d t h e p e r c e n t o f G A B A + cells e x h i b i t e d p a r a l l e l i n c r e a s e s a n d d e c r e a s e s , p e a k i n g in r e l a -

I

,11 j 10

D. D)

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O

E

0.1

,

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E13

E17

,

E21

,

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,

J

~

PNS

J

,

~

I

PN1O

Age in Days Fig. 3. GABA, detected in dissociated spinal cord cells or whole tissue homogenates by immunocytochemistry and HPLC respectively, progressively increases during spinal cord development. The immunocytochemical results represent 4 complete time-course experiments in which 10 fields in duplicate plates were analysed for each age indicated. The HPLC analysis was performed on pooled homogenates from each age (n = 3 separate experiments). Bars represent S.E.M.

269

A

B

Jk~

Fig. 4. E13 cells that incorporate BrdU are never GABA+; however some stain with the K-2 anti-GAD antibody. Acutely adherent cells were exposed to BrdU for 4 h, then fixed and stained for BrdU and GABA or GAD immunoreactivity. A: a cell immunoreactive for GABA (arrow). B: a different cell fluoresces with a fluorescein-conjugated antibody, indicating uptake of BrdU (asterisk). C: phase-contrast microscopy of the cells in A and B showing that two different cells exhibit GABA and BrdU staining. D: a small group of cells immunoreactive for K-2. E: a GAD ~ cell also exhibits BrdU staining (asterisk in D and E). F: phase-contrast microscopy of the cells in D and E with the double-labeled cell indicated by the asterisk.

week, 55-65%

o f all cells w e r e n e u r o n s ( N F + ) , w h i l e

3 0 % o f t h e cells w e r e a s t r o c y t e s ( G F A P + ) .

The

re-

m a i n i n g 5 - 1 5 % o f t h e cells w e r e e i t h e r o l i g o d e n d r o cytes ( G a l C +) o r O - 2 A p r o g e n i t o r cells ( A 2 B 5 +, N F - ,

GFAP-).

W e o b t a i n e d s i m i l a r p e r c e n t a g e s w h e n cells

were stained with the m a r k e r , t e t a n u s toxin 4-~.

early

postmitotic

neuronal

270 100

u} m. O >

80

60

O

E

40

20 )

E13

ElS

E17

E19

E21

PN7

PN14

PN21

Age in Days

Fig. 5. GABA immunoreactivity is detected in neurons throughout development. The dark grey blocks in the histogram are the relative number of neurofilament-positive (NF + ) cells present in populations dissociated at embryonic and early postnatal ages and briefly (1 h)cultured. The light gray blocks represent the relative number of GABA + cells present in the same dissociates. The number in parentheses is the percent of NF ÷ cells that were GABA+. The data are the result of 3 complete time-course experiments in which 10 fields on duplicate cultures were read for each age indicated. Bars represent S.E.M.

GABA is only expressed in neurons Double labeling experiments for G A B A and NF revealed that G A B A was only detected in cells that expressed N F (Fig. 6). While 0.1% of total cells expressed G A B A at E13, only 0.5% of neurons at E l 3 were G A B A + (Fig. 5). Neuronal expression of G A B A increased markedly during the remainder of the embryonic period, and peaked at the end of the first postnatal week (Fig. 4). Surprisingly, by PN7, > 50% of all neurons acutely dissociated from the spinal cord were G A B A +. Thereafter, the percentage of neurons expressing G A B A a p p e a r e d to plateau until PN14 after which G A B A e r g i c neurons declined to about 18%. The relative n u m b e r of GABAergic cells in both time course experiments (represented in Figs. 3 and 4) are very similar. The cell preparations consisted mainly of cell bodies which had b e e n stripped of their processes during dissociation. It was possible that decreases in the percentage of G A B A + neurons between PN14 and PN21 could have been due to transport of G A B A from cell bodies to terminals, which we were unable to detect. Therefore, in one set of experiments, PN20 pups were injected with colchicine into the fourth ventricle 18 h prior to sacrifice, in order to interrupt axonal transport of G A B A . Table I illustrates the effects of colchicine treatment on the percentage of G A B A + neurons. The number of neurons expressing G A B A increased 29% following treatment with colchicine. The percentage of cells that expressed G A D proteins labeled with the 1440 antibody (preferentially recognizes GAD65 protein) increased 33%. However this increase corresponded to a change in G A B A + neurons from 22% to

about 29% and G A D ~ cells from 18% to 24%. Thus, although pretreatment with colchicine resulted in a modest but detectable increase in the relative number of GABAergic cells, the decrease noted between PN14 and PN21 was still statistically significant.

GABAergic expression emerges in embryonic neurons along clear anatomical gradients Emergence of GABAergic neurons followed positive rostro-caudal and ventro-dorsal gradients meaning that more G A B A ÷ cells were present in rostral than in caudal regions and more G A B A + cells were present in ventral than in dorsal regions. This p h e n o m e n o n was consistently apparent in the E17 spinal cord (Table II and Fig. 7, see asterisks in each panel). The trend continued through E19 as the overall percentage of G A B A + cells gradually increased. Within the cervical and lumbosacral regions of the cord there was a distinct reversal of the ventral-to-dorsal gradient at E2t, as the dorsal halves of the cords in these regions contained a relatively greater percentage of G A B A + ceils than their corresponding ventral halves (Table II and Fig. 7, see top and bottom panel). At this stage, the rostral to caudal gradient was no longer evident. By PN5, there was a marked decrease in the percentage of GABAergic neurons in both cervical regions. Throughout the time course, differences in the relative ratios of G A B A + cells in the ventral versus dorsal halves were most pronounced in cervical and lumbosacral regions. From E19 onward, both halves of the thoracic cord contained similar ratios of G A B A + cells (Table 2 and Fig. 7, middle panel).

271 TABLE I

The effect o f 24 h pretreatment with colchieine on the relatit'e number of GABA + and GAD +cells (recognized by 1440 anti-GAD antibody) m 3-week-old rat spinal cord Treatment

Control

With colchicine

O'c hwrease

% GABA ~

22.2 (2.0)

28.9 (2.9)

29.0

%GAD +

18.2 (0.8)

24.2 (2.3)

33.0

peared to be diffusely distributed throughout the cytoplasm and residual processes of dissociated cells (Fig. 8B). Between PN7 and PN14, some cells exhibited a punctate pattern of GABA immunoreactivity. By PN21, GABA nearly always appeared punctate (Fig. 8D). At this time, clusters of detached dissociated processes as well as adherent boutons on cell surfaces were also intensly GABA +

\ Postnatal GABA co-localizes with synaptophysin Cells from E21 and PN21 pups were further analyzed by indirect immunofluorescence for co-localiza-

151

tion of synaptophysin and GABA. While GABA was diffusely distributed throughout the cytoplasm at E21, synaptophysin appeared in more discrete patches (Fig. 8A,B). Although both antigens appeared in the same general areas of the cell there was not a point-for-point co-distribution. However, in cells derived from PN21 spinal cords, both synaptophysin and GABA ira-

TABLE 1I

The gradient of GABA + cells in d(fferent regions of the dez'eloping rat spinal cord

Fig. 6. GABA immunoreactivity is detected only in neurofilamentpositive ( N F * ) cells. A: phase micrograph of acutely dissociated El9 spinal cord cells. B: same field as in A stained ,~,hh anti-NF and FlTC-conjugated secondary antibody. Most of the cells at this age fluoresce indicating that they are NF ÷. C: same field as in A and B, double-stained with anti-GABA and a RITC-conjugated secondary antibody. All the fluorescent (GABA +) cells in the field are also NF +. The asterisks in A. and B. indicate cells that are N F * but GABA . The arrows in A indicate 2 cells that lack both NF and GABA immunoreactivity.

GABA exhibits distinctive intracellular distributions during det,elopment During the course of the study, it became evident that the intracellular distribution of GABA immunoreaction products also changed during the period of differentiation. In the embryonic period, GABA ap-

Regional peaks of GABA expresskm at each of the 5 ages examined are underlined in bold type. Asterisks indicate the age at which the ventral and dorsal gradients reverse. Values are given+ S.E.M for El7 and El9 where n = 3 and n = 2, respectively; for all other ages n - 1. Means were determined from duplicate plates where five fields were counted per plate.

El7

El9

E21

Cervical ventral

El5 8.51

35.71 (5.67)

41.25 (4.41)

211.34

PN5 3.811

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Fig. 7. Quantitative analysis of GABA immunoreactivityshows characteristic temporal and spatial gradients during the late embryonic and early postnatal period. Each graph depicts the relative number of GABA+ cells in the six different regions of the spinal cord at four selected ages. A ventral-to-dorsal gradient emerges at El5, is quite pronounced at El7 and continues through El9. At the latter two ages there is also a distinct cervical-to-lumbosacralgradient. At E21 there is a reversal of the gradient such that the dorsal regions in cervical and lumbosacrat cord have more GABA+ cells. At PN5 the dorsal-to-ventral gradient appears only in lumbosacral cord. Error bars (S.E.M.) are displayed only for El7 and El9 where n = 3 and n = 2 experiments, respectively. For other ages n = 1. For all experiments means were determined from duplicate plates where five fields were counted per plate. munoreactivity appeared punctate and entirely colocalized (Fig. 8C,D). DISCUSSION Biochemically and immunocytochemically detectable G A B A was present in acutely dissociated

spinal cord cells at embryonic day 13 (El3), the earliest age examined. At this stage of development, some neuroblasts within the cord have yet to undergo terminal cell division 49. We found that E l 3 cells incorporating the thymidine analog BrdU were not G A B A ~, indicating only postmitotic cells express GABA. Furthermore, only those cells that stained for neurofilament protein or bound tetanus toxin were GABA*. Thus, GABAergic expression in the developing spinal cord was restricted to postmitotic neurons. The observation that some E13 cells were K-2 + (GAD +) but not G A B A + may be due to the fact that early G A D appears to be a low molecular weight, enzymatically inactive peptide incapable of synthesizing G A B A 2. The relative number of G A B A + neurons increased from 0.1% at E13 to 20% at E21. Surprisingly, by PN7, over 50% of acutely dissociated spinal neurons were G A B A +. By PN21 the GABAergic phenotype had decreased to just under 20% of all neurons. A large number of neurons in the embryonic and newborn spinal cord contain G A B A when cells are differentiating and before significant synaptogenesis has occurred 38. Ongoing work in our laboratory- (Suzan Nadi, manuscript in preparation) indicates that embryonic spinal cord cells are capable of spontaneous and evoked release of GABA. In addition, neonatal growth cones have been shown to release G A B A via Ca~,+-indepen dent mechanisms which appear to involve Na + entry ~'3. It is thus interesting to speculate that G A B A may be a paracrine or autocrine factor with a t r o p h i c or developmental role. G A B A and G A B A A agonists have been shown to induce the expression of low affinity G A B A receptors on cultured cerebellar neurons 39. Examination of sections of developing rat brain and spinal cord indicates that GABA-containing fibers from extra-spinal ( D R G ) and supraspinal (brain) areas appear in ventral regions of the cord as early as El3 and closely correspond to the location of newly formed GABA-containing cells 2'31. It is possible that G A B A secreted by fibers in vivo provides an induction mechanism or the necessary stimulus for preprogrammed development of GABAergic neurons a n d / o r receptors for GABA. Interestingly, many cells derived from the E14-16 rat spinal cord also exhibit bicuculline-sensitive, depolarizing responses to G A B A and the G A B A A receptor agonist, muscimo154'55. GABAAreceptor expression emerges in the same rostro-caudal/ventro-dorsal gradient described here for the appearance of GABAergic neurons in the cord. There was a slight discrepancy between G A B A detectable by H P L C and the G A B A + n e u r o n s visualized by immunocytochemistry at the early embryonic ages

273

A

C

B

D

Fig. 8. Spinal cord ceils from E21 and PN21 are immunoreactive for both GABA and synaptophysin but the distribution of immunoreacfivity changes with age. A: an E21 cell was doubleqabeled for both GABA and synaptophysin. The cell exhibits patchy synaptophysin staining. The same cell in B exhibits diffuse GABA staining. C: PN21 cell double-labeled for GABA and synaptophysin exhibits a more punctate staining for synaptophysin. The same cell in D exhibits punctate GABA staining. The synaptophysin and GABA puncla appear h) be entirely co-localized. ( E 1 3 - 1 5 ) . T h e r e was d e t e c t a b l e G A B A in tissue hom o g e n a t e s b e t w e e n E l 3 a n d E l 5 w h e n very few cells were G A B A +. T h e e a r l i e r a p p e a r a n c e of G A B A det e c t e d by H P L C may reflect G A B A in G A B A - a n d G A D - c o n t a i n i n g fibers that o r i g i n a t e from e x t r a s p i n a l and s u p r a s p i n a l a r e a s at E 1 3 - 1 5 2'14"31. D i f f e r e n c e s in the sensitivity a n d r e s o l u t i o n of the b i o c h e m i c a l and i m m u n o c y t o c h e m i c a l m e t h o d s may also c o n t r i b u t e . H P L C of single cell s u s p e n s i o n s (as o p p o s e d to hom o g e n a t e s of cells and fibers) at E l 3 r e v e a l e d the p r e s e n c e of p i c o m o l a r a m o u n t s of G A B A ( d a t a not shown). T h e level of G A B A d e t e r m i n e d b i o c h e m i c a l l y a n d the relative n u m b e r o f G A B A + cells d e t e r m i n e d i m m u n o c y t o c h c m i c a l l y closely p a r a l l e l e d each o t h e r from E l 5 onwards. Interestingly, we f o u n d that the e m b r y o n i c d e v e l o p m e n t of G A B A followed a v e n t r o - d o r s a l and rostroc a u d a l g r a d i e n t that p a r a l l e l e d t e r m i n a l cell division in the spinal cord L4'~. This was most p r o n o u n c e d at E l 7 . T h e large n u m b e r o f G A B A + cells in the ventral regions b e f o r e birth could be d u e to the t r a n s i e n t

expression of G A B A in m o t o n e u r o n s and o t h e r cell types. E v i d e n c e for the p r e s e n c e of G A B A and G A I ) in m o t o n e u r o n s of several species has b e e n docum e n t e d in the l i t e r a t u r e 73t'455~'. T h e d i s a p p e a r a n c e of G A B A i m m u n o r e a c t i v i t y in ventral regions is p r o b a b l y

not d u e to cell d e a t h o f e m b r y o n i c m o t o n e u r o n s since the G A B A i m m u n o r e a c t i v i t y d i s a p p e a r s l a t e r than the m o t o n e u r o n d e a t h period 1<24"~1"5~. By E21 the g r a d i e n t had r e v e r s e d in the cervical and l u m b o s a c r a l regions such that the dorsal halves c o n t a i n e d m o r e G A B A ~ cells. T h e d r a m a t i c d r o p in the n u m b e r of G A B A cells in the cervical region at PN5 could f o r e s h a d o w d e c r e a s e s in the m o r e c a u d a l regions that occur between PN5 a n d PN21. D e c r e a s e s in the n u m b e r of G A B A + cells in all regions could be the result of the loss of c a p a c i t y of n e u r o n s to synthesize the a m i n o acid. T h e r e may be functional r e a s o n s for the persist e n c e o f g r a d i e n t s in the cervical a n d lumbosacra[ cord as o p p o s e d to t h o r a c i c cord. T h e first two a f o r e m e n t i o n e d regions a r e r e s p o n s i b l e fl)r nearly all cholinergic i n p u t to limb m u s c u l a t u r e as well as p a r a s y m p a t h e t i c -

274 cholinergic input to the viscera. GABA could be specifically modulating differentiation of neurons in these pathways. There are a number of parallels between the present study and recent reports on the embryonic development of G A B A in other systems. Several regions of the developing mammalian and avian CNS including rat neocortex 9, rabbit retina 42, primate telencephalon 41 and chick cranial nerve undergo transient GABA expression. In the rat neocortex GABA expression follows an 'inside-out' gradient within the cortical plate, i.e. the cells that mature and settle first are the first to develop the GABAergic phenotype. By birth many of the GABA + cells have disappeared. The first cells to mature in the spinal cord are motoneurons in ventral cervical regions ~'49. They are also the first to express the GABAergic phenotype. In the rabbit retina, a large and diverse population of neonatal (PN1) neurons are GABA-immunoreactive but this expression is greatly reduced by PN20. The decline appears to be a 'downregulation' of the GABAergic phenotype, not the result of massive cell death. Transient GABA immunoreactivity is also present in cranial motor nerves of the chick embryo and periods of cell death only partly coincide with transient GABA expression ~. We see transient expression of G A B A in spinal cord cells dissociated from whole cord or in subpopulations isolated from discrete anatomical regions. Loss of GABA immunoreactivity in ventral regions does not coincide with the cell death period and must be accounted for by some other mechanism. Peak expression of GABA in transient subplate zone cells in the primate telencephalon coincides with the ingrowth of major cortical afferents suggesting that G A B A may play a role in the organization of synapses in the overlying cortical plate. The period of peak, albeit transient, expression of G A B A in the spinal cord occurs just before and after birth, also a period of intense neurite outgrowth and synapse formation t'3~. The aforementioned observations are consistent with the hypothesis that G A B A has developmental and trophic functions throughout the developing CNS. A change in the distribution of intracellular GABA from diffuse to punctate occurred during the second postnatal week. The significance of this change is not known. It may reflect differences in the trafficking pattern of the G A D enzymes responsible for G A B A synthesis during successive stages of development and may be associated with changes in the functional role of the amino acid. Changes could also be related to the increased synaptic activity of mature neurons and the ability of the neurons to synthesize products of the GAD65 gene that appear at birth 2 and are preferen-

tially detected by the 1440 antibody. The punctate G A B A staining seen in more mature neurons co-localizes with the 38 kD synaptic vesicle protein, synaptophysin ~. Co-localization of GABA with synaptophysin suggests that the GABA immunoreactivity we see is not solely due to GABA uptake since it appears to be localized in synaptic vesicles. G A B A immunoreactivity within the cell body implies that GABA is synthesized at the cell body level and not just at synaptic terminals. We did not see synaptophysin immunoreactivity before E21. Bergmann et al. s have reported expression of synaptophysin by in situ hybridization histochemistry and immunocytochemistry as early as El2 in the rat spinal cord. Since there are data to suggest that synaptophysin is glycosylated before transport to nerve endings 64 it is possible that the antibody we used recognizes only a glycosylated form of the protein. There have been several accounts of a disparity between the presence of G A B A (detected biochernically or immunocytochemically) and GAD, the ratelimiting enzyme responsible for its synthesis 19'47'59~62. We were able to detect G A D as early as we could detect GABA but this was dependent on the G A D antibody used. Initially, we were unable to detect G A D prenatally 3 when we used the 1440 antibody that preferentially recognizes proteins encoded by the GADs5 gene 12. We assumed, as did others, that GABA was synthesized through an alternative pathway, i.e., the polyamines 4~''~7'~s'67. In fact, studies in our laboratory have shown that when acutely cultured El5 cells were assayed for G A B A production from t~C-labeled precursors, approximately half the counts incorporated into G A B A were from ornithine (Suzan Nadi, personal communication), suggesting some synthesis via the polyamine pathway. However, when we used the commercially available K-2 antiserum that identifies proteins encoded by the GAD67 gene, we could detect G A D protein as early as El3 when we first detected G A B A 2. Some of the disparity between immunocytochemically detectable G A B A and GAD may thus be a result of the type of GAD antiserum used. In summary, GABA expression in the embryonic spinal cord differs markedly from that in the adult. While most GABAergic neurons are located in the dorsal horn of the mature spinal cord 33"44'65, embryonic GABAergic neurons initially predominate in ventral regions. However, this expression appears transient. During the first postnatal week there is a clear dorsoventral gradient of G A B A + cells in cervical and lumbar regions. The emergence of GABAergic cells loosely parallels the ability of embryonic cells to respond to exogenously applied GABA. In addition, the diffuse

275

distribution of G A B A within embryonic cells is not consistent with the vesicular distribution of a fast-acting neurotransmitter released at synapses. These results provide further evidence that the amino acid has neurotrophic activity during CNS development.

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