a subpopulation of striatal gabaergic neurons expresses the epidermal growth factor receptor

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Pergamon 0306-4522(95)00392-4 Neuroscience Vol. 69, No. 4, pp. 1025-1029, 1995 Elsevier Science Ltd Copyright 0 1995 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00 Letter to 5i&euroscience A SUBPOPULATION OF STRIATAL GABAERGIC NEURONS EXPRESSES THE EPIDERMAL GROWTH FACTOR RECEPTOR H. I. KORNBLUM,*t C. M. GALL,1 K. B. SEROOGYS and J. C. LAUTERBORNS *Departments of Molecular and Medical Pharmacology and Pediatrics, UCLA School of Medicine, Los Angeles, CA 90095, U.S.A. IDepartment of Anatomy and Neurobiology, UC. Irvine, Irvine, CA 92717, U.S.A. !jDepartment of Anatomy and Neurobiology, University of Kentucky School of Medicine, Lexington, KY 40536, U.S.A. Key words: transforming growth factor alpha, brain, Huntington’s disease, in situ hybridization, caudate, cerebral cortex. Epidermal growth factor and transforming growth factor alpha are mitogenic polypeptides that act at the epidermal growth factor receptor, a protein tyrosine hh~ase.~~~~~~~~” Studies have shown that epidermal growth factor and transforming growth factor alpha support the survival and promote the differentiation of central nervous system neurons in uitro.1u’3 Messen- ger RNAs for both transforming growth factor alpha and the epidermal growth factor receptor have been identified in the adult and developing mammalian cen- tral nervous system, particularly within the neostriatum of young animals. 11~15J’2’1,28* However, the cell types that synthesize these messenger RNAs in striatum are not well understood. The present study investigates the hypothesis that epidermal growth factor receptor and transforming growth factor alpha are synthesized by striatal GABAergic neurons using double-labeling in situ hybridization in the rat. Most neurons within the neostriatum that intensely expressed messenger RNA for the 67,000 mol. wt isoform of glutamate decarboxy- lase also expressed messenger RNA for the epidermal growth factor receptor. Scattered striatal cells with neurons1 morphology were humunoreactive for epidermal growth factor receptor protein, indicating that epidermal growth factor receptor messenger RNA expressed by striatal neurons is translated. Striatal neurons that expressed high levels of the 67,000 mol. wt isoform of glutamate decarboxylase messenger RNA did not appear to express transforming growth factor tTo whom correspondence should be addressed at: UCLA School of Medicine, Room 1246 JLNMRC, 700 West- wood Boulevard, Los Angeles, CA 90095, U.S.A. Abbreviations: EGF, epidermal growth factor; EGF-R, epi- dermal growth factor receptor; GAD, glutamate decar- boxylase; P, postnatal day; PB, phosphate buffer; TGFa, transforming growth factor alpha. alpha messenger RNA. The present study htdicates that epidermal growth factor receptor is synthesized by a subpopulation of GABAergic striatal ueurolLs, support- ing the hypothesis that transforming growth factor alpha and epidenual growth factor act directly upon neurons to produce their neurotrophic effects. These neurons may be GABAergic interneurons, which have been shown to be relatively resistant to degeneration in Huntington’s disease and excitotoxic models of this disease.“73 Transforming growth factor alpha (TGFa) and its receptor, epidermal growth factor receptor (EGF-R) are expressed in the developing and mature brain.‘i~‘5~27~30~32 Studies in vitro have demonstrated that TGFa and EGF enhance neuronal viability and maturation.‘3~21~33 However, there has been some con- troversy about whether these trophic factors act directly upon neurons via their common receptor or indirectly via glial cells. 2,2’ Studies have not, as of yet, defined the precise neuronal and/or glial phenotypes that express EGF-R in uivo. One particular brain area of interest with regard to TGFa and EGF is the neostriatum. TGFa mRNA is distributed relatively homogeneously within the neostriatum, with very high levels at young postnatal ages, which then decline with age.“p29 The distribution of striatal EGF- R mRNA is somewhat different, with expression in two apparent subpopulations when determined by in situ hybridization. First, many cells within the striatal subventricular, or proliferative, zone express EGF-R mRNA.28 Second, labeled cells are evenly distributed throughout the neostriatum and ventral striatum.27 While the cells in the first population probably rep- resent precursor or stem cells,22 the identity of the second population has yet to be determined. Like that of TGFa, the striatal expression of EGF-R mRNA 1025

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Page 1: A subpopulation of striatal gabaergic neurons expresses the epidermal growth factor receptor

Pergamon 0306-4522(95)00392-4

Neuroscience Vol. 69, No. 4, pp. 1025-1029, 1995 Elsevier Science Ltd

Copyright 0 1995 IBRO Printed in Great Britain. All rights reserved

0306-4522/95 $9.50 + 0.00

Letter to 5i&euroscience

A SUBPOPULATION OF STRIATAL GABAERGIC NEURONS EXPRESSES THE EPIDERMAL GROWTH

FACTOR RECEPTOR

H. I. KORNBLUM,*t C. M. GALL,1 K. B. SEROOGYS and J. C. LAUTERBORNS *Departments of Molecular and Medical Pharmacology and Pediatrics, UCLA School of Medicine,

Los Angeles, CA 90095, U.S.A. IDepartment of Anatomy and Neurobiology, UC. Irvine, Irvine, CA 92717, U.S.A.

!jDepartment of Anatomy and Neurobiology, University of Kentucky School of Medicine, Lexington, KY 40536, U.S.A.

Key words: transforming growth factor alpha, brain, Huntington’s disease, in situ hybridization, caudate, cerebral cortex.

Epidermal growth factor and transforming growth factor alpha are mitogenic polypeptides that act at the epidermal growth factor receptor, a protein tyrosine hh~ase.~~~~~~~~” Studies have shown that epidermal growth factor and transforming growth factor alpha support the survival and promote the differentiation of central nervous system neurons in uitro.1u’3 Messen- ger RNAs for both transforming growth factor alpha and the epidermal growth factor receptor have been identified in the adult and developing mammalian cen- tral nervous system, particularly within the neostriatum of young animals. 11~15J’2’1,28* However, the cell types that synthesize these messenger RNAs in striatum are not well understood. The present study investigates the hypothesis that epidermal growth factor receptor and transforming growth factor alpha are synthesized by striatal GABAergic neurons using double-labeling in situ hybridization in the rat. Most neurons within the neostriatum that intensely expressed messenger RNA for the 67,000 mol. wt isoform of glutamate decarboxy- lase also expressed messenger RNA for the epidermal growth factor receptor. Scattered striatal cells with neurons1 morphology were humunoreactive for epidermal growth factor receptor protein, indicating that epidermal growth factor receptor messenger RNA expressed by striatal neurons is translated. Striatal neurons that expressed high levels of the 67,000 mol. wt isoform of glutamate decarboxylase messenger RNA did not appear to express transforming growth factor

tTo whom correspondence should be addressed at: UCLA School of Medicine, Room 1246 JLNMRC, 700 West- wood Boulevard, Los Angeles, CA 90095, U.S.A.

Abbreviations: EGF, epidermal growth factor; EGF-R, epi- dermal growth factor receptor; GAD, glutamate decar- boxylase; P, postnatal day; PB, phosphate buffer; TGFa, transforming growth factor alpha.

alpha messenger RNA. The present study htdicates that epidermal growth factor receptor is synthesized by a subpopulation of GABAergic striatal ueurolLs, support- ing the hypothesis that transforming growth factor alpha and epidenual growth factor act directly upon neurons to produce their neurotrophic effects. These neurons may be GABAergic interneurons, which have been shown to be relatively resistant to degeneration in Huntington’s disease and excitotoxic models of this disease.“73

Transforming growth factor alpha (TGFa) and its receptor, epidermal growth factor receptor (EGF-R) are expressed in the developing and mature brain.‘i~‘5~27~30~32 Studies in vitro have demonstrated that TGFa and EGF enhance neuronal viability and maturation.‘3~21~33 However, there has been some con- troversy about whether these trophic factors act directly upon neurons via their common receptor or indirectly via glial cells. 2,2’ Studies have not, as of yet, defined the precise neuronal and/or glial phenotypes that express EGF-R in uivo. One particular brain area of interest with regard to TGFa and EGF is the neostriatum. TGFa mRNA is distributed relatively homogeneously within the neostriatum, with very high levels at young postnatal ages, which then decline with age.“p29 The distribution of striatal EGF- R mRNA is somewhat different, with expression in two apparent subpopulations when determined by in situ hybridization. First, many cells within the striatal subventricular, or proliferative, zone express EGF-R mRNA.28 Second, labeled cells are evenly distributed throughout the neostriatum and ventral striatum.27 While the cells in the first population probably rep- resent precursor or stem cells,22 the identity of the second population has yet to be determined. Like that of TGFa, the striatal expression of EGF-R mRNA

1025

Page 2: A subpopulation of striatal gabaergic neurons expresses the epidermal growth factor receptor

1026 H. I. Kornblum et al.

declines during postnatal development, but numerous hybridizing cells remain in the adult.

Neurons that express high levels of the 67,000 mol. wt isoform of glutamate decarboxylase (GAD6,) are distributed similar to those expressing EGF-R mRNA in the body of the striatum,*’ and are present at early postnatal ages.’ These cells may be GABAergic interneurons,4.17 which are among those relatively spared in Huntington’s disease and some animal models of this neurodegenerative disorder.‘x9 Expression of EGF-R by striatal GABAergic inter- neurons would be particularly intriguing because of the possibility that trophic mechanisms mediated by EGF-R may support the survival of these cells in the face of neurotoxic insults or injury. In the present study, we have examined the possibility that EGF-R is expressed by GABAergic neurons in the striatum and other brain areas by double-labeling in situ hybridization. Immunocytochemistry was also used to verify the presence of EGF-R protein in striatal perikarya. In addition, because numerous striatal cells express TGFa mRNA, the possible coexistence of TGFa and GAD,, mRNAs in the striatum was

examined. Because expression of EGF-R and TGFa mRNAs is maximal in the neonate, our analysis focused on the first week of postnatal development.

Hybridization to EGF-R mRNA and GAD6’ mRNA in the neostriatum on postnatal day 7 (P7) is shown in Fig. 1. For EGF-R mRNA, labeled cells were present within the striatal subventricular zone, and throughout the body of the caudateputamen (Fig. 1A). As previously described for adult animals,5’~20 the majority of striatal cells showed only light hybridization to GAD,, cRNA, barely above background levels, while a subpopulation of appar- ently larger cells displayed more intense hybridization as indicated by a darker blue reaction product. Many of the perikarya that were densely labeled with GAD,, cRNA were also labeled with EGF-R cRNA (Fig. lB-D). In counts of labeled cells collected from four rats, 94% of 360 neurons that were clearly labeled with GAD,’ cRNA were double-labeled. (Cells were considered to be positively labeled with [35S]EGF-R cRNA if the number of grains overlying the perikarya were more than ten times that of background density. Counts were made in four

Fig. 1. (A) Expression of EGF-R mRNA in the neostriatum of a P7 rat. Darkfield view of a section hybridized with 35S-labeled cRNA as described previously27,28 and counterstained with hematoxylin and eosin. Note the dense clusters of autoradiographic grains within the subventricular region (arrow) and the body of the neostriatum (arrowheads). Iv, lateral ventricle. (B,C) Co-expression of GAD,, mRNA (B, brightfield) and EGF-R mRNA (C, darkfield) in the neostriatum. Note cells that heavily hybridize digoxigenin(dig)-labeled GAD, cRNA also hybridize to ‘Wabeled EGF-R cRNA (arrows in B and C point to one example of a double-labeled cell; all cells shown that intensely hybridize GAD, mRNA also hybridize EGF-R cRNA). Open arrows in B and C indicate a single-labeled, EGF-R cRNA hybridizing cell. (D) High-power view of a double-labeled neuron within the striatum. (E, F) Expression of GAD,, mRNA (E) and TGFa mRNA (F) within the neostriatum. Arrow points to a cell that heavily labels with dig-GAD,, cRNA but not to [?j]TGFa cRNA. (G) EGF-R-immunoreactive cell within the neostriatum of a P7 rat. (H) Co-expression of EGF-R and GAD,, mRNAs in a cortical neuron of the superficial parietal cortex, as seen by the dark silver grains overlying the cell body. (I) Dig-labeled GAD,, neurons within the thalamic reticular nucleus do not also hybridize to 3SS-labeled EGF-R cRNA. Note a cluster of autoradiographic grains, indicating localization of EGF-R cRNA (arrow), that does not overlie GAD,, cRNA-containing cells. Scale bar = 200 pm (A), 40 nm (B,C,E,F), 20 nm (G), 10 pm (I), and 24 nm (D,H). Double-labeling in situ hybridization was performed as described previously’4 with the following modifications: Sprague-Dawley (Harlan) rat pups on P7 (n = 7) were killed by decapitation and their brains were frozen in isopentane at - 20°C and stored at - 70°C prior to use. The brains were sectioned on a cryostat at 20 pm thickness and thaw-mounted on to Superfrost Plus slides (Fisher). The slides were then fixed in 4% paraformaldehyde in phosphate buffer (PB) for 25 min, rinsed in PB, then water, and then air dried and stored at -70°C until use. Hybridization was carried out on slides for 16 h as described previously.” Following hybridization, slides were rinsed, incubated with alkaline phosphatase-conjugated anti digoxigenin antibody and color developed according to Lauterborn et ~1.‘~ The slides were then dipped in Ilford K.5d emulsion and developed in Kodak D-19 to reveal localization of 35S-labeled cRNAs. Digoxigenin-labeled GAD, cRNA was obtained by in vitro transcription of a 359 bp fragment of cat GAD,, using T7 RNA polymerase. ‘* EGF-R cRNA was obtained from a 2.9 kb cDNA encoding the extracellular domain of the mouse EGF-R using the SP6 RNA polymerase.” TGFa cRNA was obtained from a 2.1 kb fragment derived from the S-end of the rat TGFa cDNA using the T7 polymerase.” Some sections were hybridized to [‘?j]EGF-R alone. Controls consisted of sections hybridized with sense RNA strands and sections treated with RNAse A prior to hybridization with the cRNA probes. No cellular labeling was detectable under either control condition. For immunocytochemistry, rat pups aged two (n = 2), four (n = 2) and seven (n = 2) days were anesthetized with sodium pentobarbital and perfused through the heart with 4% parafonnaldehyde in 0.1 M phosphate buffer (PB). Cryostat-cut sections (20 pm) were thaw-mounted on to gelatincoated or Superfrost Plus slides. Immunocytochemistry was performed using the avidin-biotin peroxidase technique, as described previously.12 Purified sheep-anti- human EGF-R (Upstate Biotechnology Institute) was used at a dilution of 1: 100. Following washes in PB, slides were incubated in primary antibody dissolved in PB containing 4% normal horse serum for 1 h at 37°C and then for 12 h at room temperature. The sections were washed and incubated in biotinylated anti-sheep antibody (Vector labs) at a dilution of 1: 250. The sections were then incubated with avidin- biotin-peroxidase complex, followed by visualization with diaminobenzidine (Sigma) as chromogen and

hydrogen peroxide as substrate. Results were similar at the three ages studied.

Page 3: A subpopulation of striatal gabaergic neurons expresses the epidermal growth factor receptor

GABA/EGF-R colocalization in striatum 1027

Fig. 1.

Page 4: A subpopulation of striatal gabaergic neurons expresses the epidermal growth factor receptor

1028 H. I. Komblum.er al.

coronal sections from each brain, each section separ- ated by at least 200pm.) Conversely, 32% of the somata labeled with EGF-R cRNA appeared to be single-labeled (Fig. lB,C, open arrows). The size of these cells appeared to be similar to that of cells that also labeled with GAD,, cRNA.

As described previously,1’~30 TGFcr cRNA exhib- ited dense hybridization to numerous cells in P7 rat neostriatum (Fig. 1F). At this time-point, striatal cells that densely labeled with GAD6, cRNA were not found to be double-labeled with the TGFz cRNA (Fig. lE,F).

In sections processed for immunocytochemistry, a scattered population of cells throughout the body of the striatum displayed EGF-R-like immunoreactiv- ity. These immunostained perikarya exhibited neur- onal morphology (Fig. 1G) and were evenly distributed in a pattern similar to that observed for EGF-R mRNA-containing somata. In addition, as reported recently, 31,22 immunolabeled cells were pre- sent in the striatal subventricular zone. Sections incubated in the absence of the primary antibody did not show any cellular staining.

GAD,, cRNA colocalized with the EGF-R cRNA in areas outside striatum, although the extent of co-localization appeared to be lower. In neocortex (Fig. lH), hippocampus and olfactory tubercle (not shown), some cells were labeled with both GAD6, and EGF-R cRNAs, whereas many were not. There was no clear evidence for colocalization within the thal- amic reticular nucleus, despite the presence of EGF-R cRNA labeling of cells situated between GABAergic neurons (Fig. 11, arrow).

The present study demonstrates that the vast majority of striatal neurons that heavily label with GAD,, cRNA also express mRNA for EGF-R. The cell size and distribution suggest that the majority of these neurons are likely to be GABAergic striatal interneurons.4.‘7 These findings support the hypothesis that EGF and TGFb: can exert trophic actions directly upon neurons by interaction with EGF-R. Epidermal growth factor and TGFcl have been shown to enhance the survival and process outgrowth of subpopulations of both subcortical and cortical neurons in vitro.‘,2,‘3.2’ However, there has been conflicting data, using different culture systems, as to whether the effects of EGF and TGFu are mediated by direct actions on neurons2’ or mediated indirectly via glia.‘s2 We now demonstrate that at least a subpopulation of these neurons (striatal and cortical) express EGF-R mRNA in vivo. These results provide the first direct demonstration of

expression of EGF-R mRNA by postmitotic fore- brain neurons, although our previous study provided strong evidence for the expression of EGF-R mRNA by dopaminergic neurons of the ventral midbrain2’ It will be interesting to determine if the trophic effects of EGF and TGFcl on striatal and cortical neurons observed in vitro are exerted selectively upon GABA- ergic neurons. It must be noted that EGF-R mRNA is also expressed by some striatal cells that do not heavily label with GADb7 cRNA; these may be cholinergic interneurons, other GABAergic neurons or glia, although their size suggests that they are neurons.

It is not currently known if the GABAergic striatal neurons that express EGF-R mRNA are derived from precursor cells within the forebrain sub- ventricular zone that express this mRNA2’ or if the GABAergic cells initiate EGF-R expression after differentiation. It is interesting to note, however, that EGF-responsive stem cells derived from the striatal subventricular zone are capable of differentiating into GABAergic neurons” indicating that GABAergic neurons can be derived from cells that express EGF-R prior to their differentiation.

The present findings are consistent with the hy- pothesis that endogenous TGFcr plays a role in the normal development and/or function of striatal inter- neurons in vivo. TGFc( mRNA appears to be ex- pressed by a different population of striatal cells than is EGF-R mRNA since TGFc( cRNA did not label cells that intensely expressed GAD,, mRNA at the postnatal age studied. It is possible that TGFc( serves as a trophic (or other) signal to striatal interneurons by paracrine mechanisms. It seems likely that TGFc( mRNA is expressed by GABAergic projection neurons by virtue of the observation that TGFcl mRNA is expressed so intensely and ubiquitously within the neostriatum, and that the vast majority of neurons within this structure are GABAergic projection neurons.23,26

The expression of EGF-R mRNA by striatal inter- neurons also has implications for the study of Huntington’s disease. Striatal intemeurons (both GABAergic and cholinergic) are relatively spared in Huntington’s disease and some animal models of this neurodegenerative condition6*‘j9 One possible explanation for this is that the expression of EGF-R by these neurons, allowing for interaction with en- dogenous TGFa, or another EGF-R ligand confers protection upon them. These hypotheses are cur- rently being tested in animal models of Huntington’s disease.

REFERENCES

1. Alexi T. and Hefti F. (1993) Trophic actions of transforming growth factor alpha on mesencephalic dopaminergic neurons developing in culture. Neuroscience 55, 903-918.

2. Casper D., Mytilineou C. and Blum M. (1991) EGF enhances the survival of dopamine neurons in rat embryonic mesencephalon primary cell culture. J. Neurosci. Res. 30, 372-381.

Page 5: A subpopulation of striatal gabaergic neurons expresses the epidermal growth factor receptor

GABA/EGF-R colocalization in striatum 1029

3. Chesselet M.-F., Weiss L., Wuenschell C., Tobin A. J. and Affolter H.-U. (1987) Comparative distribution of mRNAs for glutamic acid decarboxylase, tyrosine hydroxylase, and tachykinins in the basal ganglia: an in situ hybridization study in the rodent brain. J. camp. Neural. 262, 125-140.

4. Cowan R. L., Wilson C. J., Emson P. C., Heizman and Heizman C. W. (1990) Parvalbumin-containing GABAergic interneurons in the rat neostriatum. J. camp. Neurol. 302, 197-205.

5. Esclapez M., Tillakaratne N. J., Kaufman D. L., Tobin A. J. and Houser C. R. (1994) Comparative localization of two forms of glutamic acid decarboxylase and their mRNAs in rat brain supports the concept of functional differences between the forms. J. Neurosci. 14, 1834-1855.

6. Figueredo C. G., Anderson K. D., Chen Q., Veenman C. L. and Reiner A. (1994) Relative survival of striatal projection neurons and interneurons after intrastriatal injection of quinolinic acid in rats. Expl Neurol. 129, 37-56.

7. Gonzles C., Lin T. C.-S. and Chesselet M.-F. (1992) Relative sparing of GABAergic interneurons in the striatum of gerbils with ischemia-induced lesions. Neurosci. Lert. 135, 53-58.

8. Greif K. F., Tillakaratne N. J., Erlander M. G., Feldblum S. and Tobin A. J. (1992) Transient increase in expression of a glutamate decarboxylase (GAD) mRNA during the postnatal development of the rat striatum. Deul Biol. 153, 1588164.

9. Harington K. M. and Kowall N. W. (1991) Parvalbumin immunoreactive neurons resist degeneration in Huntington’s disease striatum. J. Neuropath. exp. Neurol. 50, 309.

10. James R. and Bradshaw R. A. (1984) Polypeptide growth factors. A. Rev. Biochem. 53, 2599292. 11. Komblum H. I., Chugani H. T., Tatsukawa K. and Gall C. M. (1994) Cerebral hemidecortication alters expression

of transforming growth factor alpha mRNA in the neostriatum of developing rats. Molec. Brain Res. 21, 107-114. 12. Komblum H. I., Corwin J. T. and Trevarrow B. (1990) Selective labeling of sensory hair cells and neurons in auditory,

vestibular, and lateral line systems by a monoclonal antibody. J. camp. Neurol. 301, 162-170. 13. Komblum H. I., Raymon H. K., Morrison R. S., Cavanaugh K. P., Bradshaw R. A. and Leslie F. M. (1990) Epidermal

growth factor and basic fibroblast growth factor: effects on an overlapping population of neocortical neurons in vitro. Brain Res. 535, 2555263.

14. Lauterborn J. C., Tran T. M., Isackson P. J. and Gall C. M. (1993) Nerve growth factor mRNA is expressed by GABAergic neurons in rat hippocampus. NeuroReport 5, 273-276.

15. Lazar L. M. and Blum M. (1992) Regional distribution and developmental expression of epidermal growth factor and transforming growth factor-alpha mRNA in mouse brain by a quantitative nuclease protection assay. J. Neurosci. 12, 1688-1697.

16. Lee D. C., Rose T. M., Webb N. R., Todaro G. J. (1985) Cloning and sequence analysis of a cDNA for transforming growth factor alpha. Nature 313, 489491.

17. Lenz S., Perney T. M., Qin Y., Robbins E. and Chesselet M.-F. (1994) GABAergic interneurons of the striatum express the Shaw-like potassium channel Kv3.1. Synapse 18, 5566.

18. Leutz A. and Schachner M. (1981) Epidermal growth factor stimulates DNA-synthesis of astrocytes in primary cerebellar cultures. Cell. Tiss. Res. 220, 393404.

19. Luetteke N. C., Phillips H. K., Qiu T. H., Copeland N. G., Earp H. S., Jenkins N. A. and Lee D. C. (1994) The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase. Genes Dev. 8, 399413.

20. Mercugliano M., Soghomonian J. J., Qin Y., Nguyen H. Q., Feldblum S., Erlander M. G., Tobin A. J. and Chesselet M.-F. (1992) Comparative distribution of messenger RNAs encoding glutamic acid decarboxylases (Mr 65,000 and Mr 67,000) in the basal ganglia of the rat. J. camp. Neurol. 318, 245-254.

21. Morrison R. S., Komblum H. I., Leslie F. M. and Bradshaw R. A. (1987) Trophic stimulation of cultured neurons from neonatal rat brain by epidermal growth factor. Science 238, 72-75.

22. Morshead C. M., Reynolds B. A., Craig C. G., McBurney M. W., Staines W. A., Morassutti D., Weiss S. and van der Kooy D. (1994) Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells. Neuron 13, 1071-1082.

23. Mugnaini E. and Oertel W. H. (1985) An atlas of the distribution of GABAergic neurons and terminals in the CNS as revealed by GAD immunohistochemistry. In Handbook of Neurounatomy (eds Bjorklund A. H. and Hokfelt T.), pp. 436608. Elsevier, Amsterdam.

24. Pimentel E. (1994) Epidermal growth factor. In Handbook of Growth Factors: Peptide Growth Factors, pp. 97-185. CRC Press, Boca Raton.

25. Reynolds B. A., Tetzlaff W. and Weiss S. (1992) A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J. Neurosci. 12, 45654574.

26. Ribak C. E., Vaughn J. E. and Roberts E. (1979) The GABA neurons and their axon terminals in rat corpus striatum as demonstrated by GAD immunocytochemistry. J. camp. Neural. 187, 261-283.

27. Seroogy K., Numan S., Gall C. M., Lee D. C., Kornblum H. I. (1994) Expression of EGF receptor mRNA in rat nigrostriatal system. NeuroReport 6, 1055108.

28. Seroonv K. B.. Gall C. M.. Lee D. C. and Kornblum H. I. (1995) Proliferative zones of uostnatal rat brain exnress epide&ral growth factor receptor mRNA. Brain Res. 670, 157-164.

29. Seroogy K. B., Han V. K. and Lee D. C. (1991) Regional expression of transforming growth factor-alpha mRNA in the rat central nervous system. Neurosci. Lett. 125, 241-245.

30. Seroogy K. B., Lundgren K. H., Lee D. C., Guthrie K. M. and Gall C. M. (1993) Cellular localization of transforming growth factor-alpha mRNA in rat forebrain. J. Neurochem. 60, 1777-1782.

31. Weickert C. S. and Blum M. (1995) Striatal TGF-alpha: postnatal developmental expression and evidence for a role in proliferation of subependymal cells. Deal Brain Res. 86, 203-216.

32. Yeh J., Rosen K. M. and Villa K. L. (1992) Messenger ribonucleic acid for transforming growth factor-alpha, but not for epidermal growth factor, is expressed in fetal and neonatal mouse brain. Am J. Obstet. Gynecol. 167, 242-245.

33. Zhang M. B., Woo D. D. and Howard B. D. (1990) Transforming growth factor alpha and a PClZ-derived growth factor induce neurites in PC12 cells and enhance the survival of embryonic brain neurons. Cell. Regul. 1, 51 l-521.

(Accepted 29 August 1995)