DEVELOPMENTAL BRAIN

RESEARCH

E L S E V I E R Developmental Brain Research 91 (1996) 140-142

Short communication

Co-expression of tyrosine hydroxylase and glutamic acid decarboxylase in dopamine differentiation factor-treated striatal neurons in culture

Stephen R. Max, Adrienne Bossio, Lorraine Iacovitti * Departments of Anatomy and Neurobiology, Neurology and Biochemistry, Medical College of Pennsylvania and Hahnemann Uni~,ersity, Broad and Vine

Streets, Philadelphia, PA 19102, USA

Accepted 28 September 1995

Abstract

We have previously shown that dopamine differentiation factors (DDF) can stimulate the novel expression of tyrosine hydroxylase (TH) in the phenotypically plastic neurons of the embryonic mouse striatum (Du et al., J. Neurosci., 14 (1994) 7688-7694; Du and Iacovitti, J. Neurosci., 15 (1995) 5420-5427). The present study sought to determine whether TH induction required down-regulation of an existing GABAergic trait in striatal neurons or whether enzymes of both neurotransmitter systems were simultaneously expressed. Immunocytochemical analysis revealed that, following treatment with DDFs, TH and the GABA synthesizing enzyme glutamic acid decarboxylase (GAD) were co-expressed in the same neurons. Moreover, GAD enzyme activity was not affected by the dramatic increase in TH. Thus, the induction of a novel neurotransmitter phenotype in brain neurons does not appear to occur at the expense of the existing phenotype.

Keywords: Dopamine; Differentiation; Growth factor; Tyrosine hydroxylase; Tissue culture; GABA

During differentiation, neurons acquire cell-specific traits that, taken together, constitute their phenotype. One critical 'decision' developing neurons must make is the neurotransmitter(s) they will synthesize and use at their synapses. Work from this laboratory has focused on the development of catecholamine traits in neurons from the central nervous system. We have shown that dopamine differentiation factors (DDF) can stimulate the expression of tyrosine hydroxylase (TH) in neurons that normally do not express this rate-limiting enzyme for catecholamine synthesis [2-4,7]. Specifically, treatment of murine striatal neurons in culture with acidic fibroblast growth factor (aFGF) and an accompanying catecholamine induced the novel appearance of TH [3]. Since the majority of neurons in the adult striatum synthesize GABA [11], our results suggested that the DDF-responsive cells in CNS may be GABAergic. We therefore sought to determine: (i) whether catecholamine- and GABA traits can be co-expressed in striatal neurons; and, (ii) if so, whether expression of catecholamine traits is accompanied by diminution of GABA traits. To this end, we assessed, both by quantita-

* Corresponding author. Fax: (1) (215) 762-3127.

0165-3806/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSD1 0165-3806(95)00163-8

tive enzyme assay and immunocytochemical staining, the transmitter synthesizing enzymes TH and glutamic acid decarboxylase (GAD) in cultured striatal neurons after treatment with DDF.

To do so, the developing striatum was dissected, disso- ciated and plated as described previously [2,11]. The cellu- lar plating density was approximately 0.5-1 X 10 6

cells/dish. After a one day stabilization period in standard media, cultures were incubated for 3 -5 days in media containing test reagents (treatment at 48 h intervals). The following day, cultures were harvested for biochemical assay of TH and GAD. Cultures were rinsed in PBS, harvested and cells were disrupted by sonication in 5 mM potassium phosphate buffer containing 0.2% Triton X-100 ( v / v ) at a dilution which insures that the reaction is linear with enzyme concentration and time. Aliquots were then assayed for TH activity by conversion of 14C-labeled tyro- sine to DOPA as described previously [1]. GAD activity was assayed according to Wilson et al. [16]. Measured GAD activity was almost completely inhibited by 1 mM aminooxyacetic acid. Protein levels were measured using the Biorad protein reagent. Corresponding sister cultures were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and processed with antibodies to (1) TH

S.R. Max et al. /Developmental Brain Research 91 (1996) 140-142 141

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Fig. 1. Immunocytochemical localization of GAD (A) and TH (B) in cultured striatal neurons incubated in media containing 10 ng /ml aFGF + 10 /~M DA. Note that while virtually all neurons are GAD +, only a subset co-express TH (arrow denotes unstained TH-neurons). Not shown are control (untreated) cultures where all neurons are GAD + as in Panel A but are devoid of the TH labeling seen in Panel B. Bar = 10 /zm.

(1:5000 dilution, kind gift of Dr. T.H. Joh, NY, NY) or (2) GAD67 (1:1000 dilution, kind gift of Dr. Allan J. Tobin, Los Angeles, CA) using the immunoperoxidase/ABC method of staining (Elite Vectakit). Data were statistically analyzed by the two-tailed unpaired Students t-test to compare the statistical significance between control and experimental groups. Differences were considered signifi- cant only when the P value was less than 0.05.

As anticipated, under control culture conditions, virtu- ally all striatal neurons contained immunoreactive GAD (Fig. 1A) and were devoid of TH labeling [2,4,6]. How- ever, treatment with DDFs (aFGF (10 n g / m l ) + DA (10 brM); aFGF + DA + mazindol (20 /~M); aFGF + mazindol) resulted in the induction of TH in a subset of neurons (Fig. 1B). Importantly, the novel appearance of TH in these cells did not alter their existing GAD expres-

sion. Consequently, immunocytochemical analysis re- vealed overlapping subsets of stained neurons; essentially all striatal neurons were GAD-positive, while approxi- mately 40% were TH-positive. The co-expression of TH and GAD was confirmed and quantified by enzyme assay of extracts of treated cultures. As expected, TH activity was undetectable in control cultures, but increased to measurable levels in DDF-treated cultures (Fig. 2A). In contrast, GAD activity was present in control cultures and remained at comparable levels following DDF treatment (Fig. 2B).

These results resolve two important questions concern- ing differentiation of CNS neurons. Thus, it is clear that GAD and TH are co-expressed in DDF-treated striatal neurons in culture, confirming our hypothesis that TH is being induced in GABAergic neurons. The second ques-

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Fig. 2. Effects of treatment of cultured caudate neurons with various dopamine differentiation factors on TH (Panel A) and GAD (Panel B) enzyme activity levels. Cultures were incubated in control media (Ctl) or in media supplemented with 10 ng /ml aFGF _+ 10 /zM DA ± 20 /xM mazindol. Note that GAD activity remains unchanged despite significant increases in TH.* = P < 0.01; * * = P < 0.001. Values represent the mean + S.E.M. of (4-12 cultures noted in parenthesis) in 2-3 separate platings.

142 S.R. Max et al. / Developmental Brain Research 91 (1996) 140-142

t ion concerns the relat ive levels o f G A D in treated vs.

untreated cells. In the peripheral nervous system, the ex-

pression o f a nove l neurotransmit ter trait is accompanied

by a concomi tan t decrease in the transmitter e n z y m e nor-

mal ly expressed in untreated cells [8,10,12,13]. In this

study, we have shown that G A D act ivi ty does not change

in striatal neurons in culture after D D F treatment, in the

face of a dramat ic increase in T H activity. Thus, in the

brain, in at least this one system, induct ion o f a novel

neurotransmit ter phenotype in not accompl i shed at the

expense o f exis t ing transmitter functions.

Our prior studies indicated that T H express ion is short-

l ived in brain neurons unless exposure to D D F s is main-

ta ined [3,6]. In this case, it is p resumed that co-express ion

of T H and G A D wil l persist indefini tely. A l though the

phys io logica l re levance of this dual phenotype is not yet

clear, it is no tewor thy that T H is expressed by a number of

G A B A e r g i c structures dur ing deve lopmen t (e.g. /3 cel ls of

pancreat ic islets; [14,15]). Moreove r , in a number of in-

stances, co-express ion o f T H and G A B A traits is main-

tained, even in adult structures, such as, the cerebral cor tex

[9], retina [17] and hypotha lamus [5]. Whe the r G A B A e r g i c

neurons are unique in their abili ty to s imul taneously ex-

press the T H gene or whe ther neurons of other t ransmitter

phenotype also share this capaci ty awaits further invest iga-

tion.

Acknowledgements

This research was supported by N I H Grant NS 24204-07

awarded to L.I. W e thank Ms. Natal ie Stull for expert

technical assistance.

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