Choline acetyltransferase messenger RNA expression in developing and adult rat brain: regulation by nerve growth factor

Molecular Brain Research, 9 (1991) 319-325 Eisewer

319

BRESM 70273

Choline acetyltransferase messenger RNA expression in developing and adult rat brain: regulation by nerve growth factor Lucio Cavicchioli 1, Thomas P. Flanigan 2, John G. Dickson 2, Guido Vantini 1, Roberto Dal Toso 1, Mariella Fusco 1, F r a n k S. Walsh 2 and A l b e r t a L e o n 1 1CNS Department, Ftdta Research Laboratories, Abano Terme (Italy) and 21nstttute of Neurology, National Hospital, London (U K.)

(Accepted 23 October 1990) Key words" Chohne acetyltransferase; mRNA regulation; Nerve growth factor, Polymerase chain reaction

The polymerase chain reaction (PCR) was used to develop a method for detection and relatwe quantification of the chohne acetyltransferase (CHAT) mRNA m neonatal and adult rat CNS. Oligonucleotlde primers derived from a porcine ChAT eDNA sequence were used m coupled reverse transcriptase (RT)-PCR to amplify a eDNA sequence of 206 bp which arises in a cycle- and RNA-dependent manner and which hybndlzes with both an internal ohgonucleotide and a ChAT cDNA probe. ChAT mRNA was detected in spinal cord, septal area, stnatum, cortex and h~ppocampus but not m cerebellum and cardiac or skeletal muscle. In the septal area, relatwe quantitative evaluation of ChAT mRNA levels by RT-PCR mdtcates that this transcript is developmentally regulated and increased following mtracerebral admimstration of nerve growth factor (NGF) to both neonatal and young adult rats. This suggests that the increases of ChAT acUvlty observed m basal forebrain during development or after NGF administration are, at least m part, associated with an increase in corresponding levels of mRNA. INTRODUCTION The synthesis of the neurotransmitter acetylcholine is catalyzed by the enzyme choline acetyltransferase (CHAT; acetyl-Coa: choline-O-acetyltransferase, E C 2.3.1.6.). This enzyme is a specific marker for cholinergic neurons in both the peripheral and central nervous system (CNS). Therefore, determinations of C h A T immunoreactivity or enzyme activity have been widely used to localize cholinergic neurons and to study their functions in different experimental paradigms 9"17'26'31. Nonetheless, almost no information is available, at transcriptional, translational or posttranslatlonal levels, concerning possible mechanisms of C h A T regulation. Recent evidence suggests that nerve growth factor (NGF) plays a prominent role in regulating the function of forebrain cholinergic neurons 1'15. For example, intracerebroventricular (i.c.v.) administration of N G F (i) stimulates C h A T activity in the forebrain of neonatal and adult rats2°'21'32; (ii) prevents the loss of C h A T activity and ChAT-immunopositive neurons in the septal area followmg specific lesions to the septohippocampal or septocortlcal neuronal pathwaysl°'24'as; and (iil) decreases atrophy in forebrain cholinergic neurons and cognitive impairments in aged rats 18. Furthermore, the physiologically significant nature of this NGF-induced

regulation of C h A T activity (at least in the basal forebrain) was clearly demonstrated in a recent study in which anti-NGF antibodies were administered i.c.v, in developing rats to neutralize endogenous trophic activity 35. However, the molecular mechanisms underlying trophic regulation of the basal forebrain cholinergic system by N G F still remain largely undefined. The recent cloning and sequencing of porcine 2 and rat 4 C h A T genes have made it possible to investigate the regulation of C h A T gene expression and thereby to gain some insight into the molecular events underlying developmental, physiological and pharmacological changes in C h A T activity. Since the C h A T gene transcript is present at relatively low levels, requiring large amounts of poly(A) + R N A for detection, by conventional Northern blot procedures, even in ventral spinal cord 2, we chose to use a more sensitive R N A - b a s e d technique to measure C h A T m R N A in multiple samples from discrete brain areas. Recently, the polymerase chain reaction (PCR) coupled to reverse transcription (RT) and with the use of either physiological 8 or synthetic 36 internal standards, has been used as a quantitative assay to detect and quantify m R N A species of low abundance from limiting amounts of tissue. Accordingly, in the present study we utilized a R T - P C R procedure (i) to quantify the relative levels of

Correspondence. L Cavicchloh, CNS Department, Fidia Research Laboratories, Via Ponte della Fabbnca 3/A, 1-35031 Abano Terme (PD), Italy

0169-328X/91/$03 50 © 1991 Elsevier Science Publishers B.V (Biomedical Division)

320 ChAT mRNA

m v a r i o u s a r e a s o f t h e rat b r a i n ,

(U) to

characterize the developmental pattern of ChAT mRNA in t h e b a s a l f o r e b r a m a n d (iii) t o i n v e s t i g a t e t h e p o s s i b l e N G F - m d u c e d r e g u l a t t o n o f C h A T g e n e t r a n s c r i p t i o n in b o t h n e o n a t a l a n d a d u l t rats t r e a t e d w i t h 1.c.v. a d m i n i s tration of NGF.

MATERIALS AND METHODS

NGF purtficatton and charactertzatton NGF was isolated as the 2 5S subumt from submaxillary gland of the adult male mouse, as previously reported3. Sodmm dodecyl sulfate (SDS) slab gel electrophoresls, in non-reducing conditions, showed a single band of approxamately 25 kDa The biological actlvaty of the punfled NGF, evaluated uUhzlng fetal chick dorsal root ganglion cells m vitro 13, was in the range of 0 1-0 2 ng proteln/troph~c unit

NGF admmtstratton NGF, or cytochrome c (Sigma, type VI) were dissolved in phosphate-buffered arUficml cerebrospmal fluid with 0 01% bovme albumm37 Neonatal rats (Sprague-Dawley of both sexes) were treated dady from postnatal day (P) 2 to P8 with an LC v mlecUon32 of NGF or cytochrome c (5 ag m a total volume of 5 al over a penod of 30 s) and sacrificed by decapitation at P9 At all times, neonatal rats were anesthetized on ice pnor to rejection Adult male rats (Sprague-Dawley, 250 g b wt ) were anesthetized with sodmm pentobarbital (50 mg/kg b w t , i p ) followed by stereotaxlc lmplantatton of an intraventncular cannula posmoned m the right lateral ventricle The cannula was connected to an osmotic minipump (Alzet model 2002; flow rate 0 5 al/h) filled with 25 ag of NGF or cytochrome c (total volume 200 al) which was placed subcutaneously in the neck of the animal Rats were decapitated 14 days after lmplantaUon The basal forebram region of neonatal and adult ammals was rapidly dissected29, and total RNA extracted. In addition, m order to monitor the effect of mtraventrlcular NGF admmlstration, ChAT actlwty was assessed19 in the septal area of rats randomly chosen from each experimental group

Isolatton of RNA Total RNA was extracted accordmg to previous reports ~2, wath minor modlficataons Briefly, tissue was somcated untd disruption m 3 M LICI, 6 M urea, 0 1% w/v SDS, 0.02% (w/v) heparm and 0 01 M sodmm acetate, pH 5 2, transfer RNA (20 ag) was added to each sample as a carrier After overmght precipatatlon at 4 °C, followed by centrffugatlon (30 mm at 10,000 g), RNA pellets were suspended in 4 M LtCI, 8 M urea, recentrlfuged as above and resuspended in 0 01 M Tns-HCI pH 7 4, and 0.001 M EDTA contammg protemase K (Sigma) at a concentration of 400 ag/ml. Samples were incubated for 1 h at 25 °C and then extracted twice m phenol/chloroform 1 1 and once tn chloroform Finally, RNA was precipitated m ethanol, lyophyhzed and resuspended in distilled water Optical density at 260 nm was utlhzed to estimate RNA concentranon3°

Productton of ohgonucleottdes and CHAT-1 cDNA Forward (sense) and reverse (anti-sense) synthetic 24-mer ohgonucleotide primers were derived from both porcine ChAT2 and rat cyclophdm H cDNA sequences (Table I) In addition, a 230 base pair (bp) CHAT-1 cDNA was synthetlzed by British Biotechnology Ltd usmg the porcine sequence (bases 1811-2041) 2 The synthetic CHAT-1 cDNA was mserted mto the EcoRI-HmdIII site of p-GEM 3 plasmld and amplified m the JM109 strain of E colt The ChAT-I cDNA probe was prepared as an insert following EcoRI-BarnHI digestion of CHAT-1 p-GEM 3 plasmld DNA

Lmked RT-PCR procedure Total RNA was reverse transcribed at 42 °C for 60 rain in 10 al reaction volume contammg ChAT and cyclophlhn speofic reverse primers or olago(dT)18 primer (5 aM), AMV reverse transcrlptase (10 U Angllan Baotec Ltd ), 20 mM Tns-HCl, pH 8.3, 2 5 mM MgCl 2, 50 mM KCI, 12 5 U human placental nbonuclease mhibator (Amersham) and 1 mM dNTPs (PharmacIa). The reactaon was mhlbited by the addition of 5 mM EDTA, and RNA was hydrolyzed m the presence of 50 mM NaOH at 65 °C for 60 mm Followmg neutralization with HC1, volume was increased to 100 al with H20 and samples utilized lmmedmtely PCR was performed using a Perkm Elmer Cetus DNA Thermal cycler Ten al of RT products were diluted to 50 al wath 67 mM Tns-HCl pH 8 8 (25 °C), 16 6 mM (NH4)2SO 4, 6 7 mM MgCl2, 10 mM 2-mercaptoethanol, 170 mg/ml bovine serum albumin, 150 aM of each dNTP, 1 aM of each primer, and 1 U recombmant Taq DNA polymerase (Perkm Elmer) in polypropylene mlcrocentnfuge tubes (0 5 ml, Sigma) Samples were then overlaad with 50 al mmeral od to prevent evaporataon, and incubated at 94 °C for 7 rain, 55 °C for 1 mm and 72 °C for 2 rain to allow second strand synthesis to occur Cycle parameters for amphficaUon were 94 °C for 1 man, 55 °C for l0 s, and 72 °C for 1 rain To avoid substrate saturation of the Taq DNA polymerase, ChAT and cyclophlhn amphflcatlons were performed m separate test tubes wath the same volume (10 al) of RT products, and with PCR primers always present in large excess Cycle numbers for ChAT and cyclophdin samples were as reported in the text Reactions were stopped by the addmon of EDTA (20 mM final concentrataon)

Analysts of PCR products PCR products were analyzed m 15% (w/v) neutral ethldmm agarose gels, the 206 bp electrophoretlc band was electroeluted from the gel, subcloned m M13 mp 19 RF and sequenced as previously reported 34 For Southern blot analysis followmg electrophoresls m 1 5% (w/v) agarose gels, PCR products were transferred to Hybond-N (Amersham) usmg a LKB Novablot apparatus following treatment with alkali and neutrahzatlon Transfer buffer consisted of 0 15 M sodmm citrate, 1 5 M NaCI, pH 7 0 (10 × SSC) DNA was fixed to Hybond-N by UV cross-hnkmg Filter hybridization was performed at 60 °C (CHAT-1 or cyclophllln cDNA probes) or 42 °C (ohgonucleotlde probes) in presence of rapid hybridization solution (Amersham) ChAT and cyclophllln probes were labelled with 32p to a specific activity of at least 5 × 10s cprn/ag DNA using a multlpnme DNA labelling system (Amersham) Ohgonycleotlde probes were 5"-end labelled to a speofic activity of l0 s cpm/ag ohgonucleotlde, using T4 polynucleotlde klnase (Amersham) as previously reported3° Filters were rAsed to a stringency of 0 4 × SSC, 0 1 % SDS at 60°C (cDNA probes) or 1 × SSC at room temperature (oligonucleotlde probes) and then exposed to Kodak X-Omat films with mtenslfylng screens at -70 °C For semlquantltatlve analyses, autorad~ograms of the same blot at different exposure times (typically from 15 mm to 4 h to avmd film saturation) were scanned using a scanning densitometer (Ultroscan Laser Densltometer, LKB 2202)

RESULTS AND DISCUSSION

Charactertzation o f R T - P C R analysts f o r detectton o f ChAT mRNA In early s t a g e s o f this s t u d y , use o f c o n v e n t i o n a l N o r t h e r n b l o t p r o c e d u r e s to e x a m m e t o t a l R N A s a m p l e s f r o m p o s t n a t a l d a y 14 rat s e p t a l r e g i o n s f o r t h e p r e s e n c e o f C h A T m R N A w a s a t t e m p t e d D e s p i t e t h e fact t h a t t h e septum represents one of the richest sources of chohnergtc n e u r o n a l cell b o d i e s m rat b r a i n 16, filter h y b r i d t z a -

321 tion of septal R N A (up to 20/~g) with the 230 bp synthetic CHAT-1 c D N A probe (see methods) failed to produce a detectable signal even following autoradiographic exposure of up to 2 weeks (data not shown). Consequently, we examined the possibility of enhancing detection of ChAT m R N A by using RT-coupled PCR amplification. Based on the porcine cDNA sequence, a set of forward (sense) and reverse (antisense) PCR primers (oligonucleotides 1 and 2 respectively) flanking a m R N A segment which overlaps the CHAT-1 synthetic cDNA, and an internal oligonucleotide probe (oligonucleotide 3), were synthesized (Table I). The antisense primer, oligonucleotide 2, was first used to prime reverse transcription of total R N A from postnatal day 14 rat septum as well as from cardiac and skeletal muscle tissue. The cDNAs so produced were then used as templates to initiate a PCR using both oligonucleotldes 1 and 2 in the presence of Taq polymerase. After 28 cycles of PCR, products were analyzed by electrophoresis m ethidium-agarose gels and by Southern blot hybridization with both the CHAT-1 cDNA and internal oligonucleotide 3. Only in the case of septal RNA was an ethidium-staining band of 206 bp observed. Skeletal and cardiac muscle tissue, which do not express ChAT enzyme, yielded several ethidium stained bands. However, though the latter products were RNA-dependent and could be shown to incorporate PCR primers (data not shown), in Southern blots they did not hybridize to either the CHAT-1 cDNA or internal ohgonucleotide 3. In contrast, the major 206 bp amplification product from septal R N A hybridized specifically with both probes (Fig. la,b). In addition, when the corresponding electrophoretic band was eluted and sequenced, the sequence corresponded to that of rat ChAT m R N A 4 with the expression of the incorporated primers derived from the porcine sequence (data not shown). Thus, the combination of R T - P C R amplification and Southern blot hybrid-

123

123

a

b

Fig 1. PCR amplification (28 cycles) of ChAT mRNA derived from selected postnatal day 14 rat tissues. (1) basal forebram; (2) cardiac muscle; (3) skeletal muscle; specific hybridization with CHAT-1 cDNA probe (a) or internal oligonucleotlde 3 (b).

ization allowed for specific detection of ChAT m R N A from rat septum. Detection o f C h A T m R N A in different C N S regions

To assess ChAT m R N A expression in different CNS regions, equivalent quantities of total R N A from spinal cord, septum, striatum, cortex, hippocampus and cerebellum of adult rat were subjected to RT. The resultant cDNAs were amplified over 28 PCR cycles and products were analyzed by Southern blot hybridization with the CHAT-1 cDNA and internal oligonucleotide 3. As shown in Fig. 2, higher levels of ChAT m R N A were detected in striatum, septum and spinal cord, when compared to cortex and hippocampus. Cerebellum, which is devoid of cholinergic neurons, was negative. The amount of cyclophilin m R N A amplified after 25 PCR cycles was similar

1

2

3

4

5

6

7

TABLE I Primers and ohgonucleottde probes used m amphficatton analyses of ChA T and cyclophthn mRNA Ohgonucleoude code*

1 2 3 4 5 6

ChAT Position m cDNA sequence

Complementary relauveto mRNA

1712-1735 1895-1918 1811-1834 44-67 392-415 338-361

sense ant~sense sense sense antlsense antisense

* Ohgonucleotides 1-3 refer to ChAT sequence2 and 4-6 refer to cyclophihn sequencer i

O Fig. 2. Hybridization intensities with CHAT-1 eDNA probe on RT-PCR products denved form 500 ng RNA of different CNS regions of young adult rats Spinal cord (lane 1); basal forebrain (lane 2), strlatum (lane 3); hippocampus (lane 4), cortex (lane 5); and cerebellum (lanes 6 and 7).

322 in all samples (see below). These data are m agreement with the anatomical location of central cholinergic pathways, indicating that relatively high amounts of ChAT m R N A are present m those regions which contain abundant cholinergic neuronal cell bodies. Thus, the hlppocampus and the cerebral cortex, two brain regions which receive abundant cholinergic terminals from extrinsic neurons, contain high levels of C h A T activity but relatively low levels of the corresponding m R N A . The latter is most probably associated with the presence of rare intrinsic cholinergic interneurons 16'17.

PCR conditions for relattve quantitation of ChAT-mRNA m rat septum We next determined whether the R T - P C R procedures

123

4

5

1

2

3

4

5

could be used to quantify the level of C h A T m R N A relative to the m R N A levels of cyclophilin, which was chosen as internal standard. Cyclophihn is an abundant and constltutwely expressed 17 k D a cyclosporm-binding protein which is present in several rat tissues including the CNS neuronal tissue. The levels of cyclophilin m R N A have been reported to be expressed at constant levels in the rat postnatal developmental period 11 (see also Fig 4b) and appear to be unmodified by a variety of experimental conditions 33. For relative quantitatlon, different amounts of total R N A from postnatal day 14 rat septum were used as template to initiate R T reactions. C h A T and cyclophilin PCR amplification products were then assessed by Southern blot hybridtzatlon and densltometric scanning of autoradlographs. As shown in Fig. 3a-c, a linear correlation was observed, for both C h A T and cyclophilin, between the R T - P C R amplification products obtained and the amount of input septal R N A , when the latter was used in the range of 200-750 ng. This linear relationship was maintained for up to 25 or 30 cycles for cyclophilin

1

a

2

3

4

5

4

5

b C h A T .-.-~

2f

8

i I

i I o 5 ,!

1

O~

0

3

//~

I [

2

/"

~ I

t

h

200

~

L

I

L

400

I

I

I

t

600

I

~

[ 000

I

I

I

I 1000

L

I

I

1200

ng of RNA input

Cyclophilin . - , 0

C Fig 3 Examples of PCR amplification of cyclophihn (a) and ChAT (b) cDNAs (25 and 30 cycles respectively) derived from increasing amounts of total RNA extracted from basal forebram of postnatal day 14 rats 200 ng RNA (lane 1), 400 ng (lane 2); 500 ng (lane 3), 750 ng (lane 4); 1000 ng (lane 5) (b) filter hybndization with CHAT-1 cDNA probe, (a) filter hybridization with cyclophllln cDNA probe, (c) densltometric evaluation of hybridization intensines related to RT-PCR amplification of ChAT (D--U]) or cyclophihn ( I - - I I ) cDNAs Assays were run in tnphcate and specific hybridization intensities for each amount of RNA input represent mean value +_ S.E.M Note maintenance of linear relationship within the 200-750 ng range of RNA input

b Fig 4 ChAT (a) and cyclophihn (b) hybridization bands in the septal area of rats at different ages postnatal day (P) 3 (P3, lane 1), P7 (lane 2); P14 (lane 3); P25 (lane 4); 2 months (lane 5) Identical ahquots of the same RT reaction products (500 ng RNA input) were used for PCR amplification of ChAT mRNA (30 cycles) and cyclophihn mRNA (25 cycles)

323 or CHAT, respectively, and verified several times during the course of the experiments. Saturation of cyclophilin amplification occurred between 26 and 28 P C R cycles with 500 ng of total R N A input. A c c o r d i n g l y , all subsequent analyses of C h A T and cyclophilin m R N A levels were p e r f o r m e d within the a b o v e range of R N A input and P C R cycles to obtain a relative quantitative d e t e r m i n a t i o n of C h A T gene expression in the rat septal region under different experimental conditions.

A

1

ChAT

-

Cyclophilin--

2

3

4

5

6

o

e

e

O

o

7

6

900900

91)

8

O

o

Developmental pattern of ChAT mRNA levels in the basal forebram T h e expression of C h A T enzyme activity in the rat s e p t u m is clearly regulated during development. We thus e x a m i n e d the regulation at the pretranslatlonal level by assessing C h A T m R N A in the basal forebrain region of rats at different ages. Figs. 4a and 5 show that at P3 and P7 the a m o u n t of C h A T m R N A is a p p r o x i m a t e l y 20% of the adult level, whereas at P14 the C h A T gene transcript is 75% of the adult level. A d u l t levels of C h A T m R N A are reached b e t w e e n P14 and P25. This postnatal profile of C h A T m R N A expression closely correlates, both quantitatively and t e m p o r a l l y , with enzyme activity d e t e r m i n e d at the same ages 26.

Effects of in vivo NGF admintstration Since the R T - P C R p r o c e d u r e p r o v e d to be a sensitive m e t h o d for identification and relative quantification of C h A T m R N A , we next e x a m i n e d whether or not N G F - i n d u c e d activation of C h A T acitivity was associated with modifications in C h A T gene transcript. Extensive studies on the distribution of NGF, N G F receptors, their respective m R N A s , and on the influence

1 < zrr E 0.8

=I r

ChAT

--

1

2

3

e

e

0

###

4

5

~D ~

6

e

ooo

Fig. 6. ChAT and cyclophilin hybridization bands in the septal area of neonatal (A) and adult (B) rats following treatment with cytochrome c (A, lanes 1-4, B, lanes 1-3) or NGF (A, lanes 5-8; B, lanes 4-6). Each lane corresponds to 500 ng RNA input derived from septal area of individual rats. See text for treatment protocols

of N G F on C h A T activity in basal forebrain cholinergic neurons, have clearly defined these neurons as N G F sensitive. In particular, n e o n a t a l and adult rats r e s p o n d to i.c.v. N G F administration with a selective and p r o m -

250

~

*

:o ,ooiA_

0.6

O

o •~ <

0.4 z P 0.2

_/ I

el

o

o

~ 100l . ~ ~ IZ

1

E

i

t

3 d

7 d

14 d

26 d

2 mo

a g e of r a t s

Fig. 5 Relative quanuflcatlon of postnatal expression of ChAT mRNA in rat septum (see Fig. 4) Values are expressed as the ratio between autoradlographlc intensities of ChAT and cyclophihn mRNA, respectively.

8

so-~

,

0 newborn

/

adult

Fig 7. Relative quantification of ChAT mRNA levels in newborn (n = 4) or adult (n = 3) rats treated with cytochrome c (open bars) or NGF (hatched bars) (see Fig. 5). Results are expressed as percentage of the respective control values. *P < 0.01, **P < 0.05 (Student's t-test).

324 ment increase of C h A T activity. Thus, to examine the effect of N G F treatment on ChAT m R N A expression, we chose treatment protocols which have proved to be highly efficacious in activating C h A T enzyme activity in neonatal and adult rats (see Materials and Methods) In addition, results were normalized by means of parallel amplification of cyclophdin m R N A , which is not modtfled following such treatment protocols 7 As shown m Figs. 6a and 7, N G F induced a pronounced increase (+122% with respect to control) m C h A T m R N A in neonatal rat basal forebraln. In young adult rats (Figs. 6b and 7) N G F also significantly mcreased ChAT gene transcripts ( + 3 6 % vs control) These data indicate that the NGF-induced actlvatton of ChAT enzyme in basal forebraln neurons of both developing and adult rats is paralleled by an mcrease in ChAT mRNA Interestingly, m neonatal rats, the increase m ChAT gene transcript induced by N G F is quantltattvely similar to increases in C h A T enzyme actwity (+120%, data not shown) and lmmunostaining 35, thereby suggesting a close association between ChAT m R N A and correspondmg protein levels. Furthermore, in young adult rats, we recently reported that continuous infusion of N G F ~s capable of inducing a dose-dependent increase in acttwty in the septal area 2°. Consistent with this result, the chosen schedule of 25/~g mfused over a two-week period yielded on average a 50% mcrease in C h A T actwlty (data not shown). C h A T gene transcript in the same region appears to be similarly increased, thereby confirming that NGF-mduced changes in C h A T enzymatic actwtty are assocmted with increases in C h A T m R N A steady-state levels. Consistent with our data, an NGF-induced increase of C h A T m R N A in adult septum has been reported using in situ hybridization procedures 25. Whether this increase reflects an enhanced rate of ChAT gene transcription or stabilization of ChAT m R N A remains an open question. In addition, post-translational mechanisms, such as phosphorylation, have recently been suggested to play a role in the physiological action of ChAT 5. However, the involvement of such post-translational changes in the NGF-induced activation of ChAT enzyme must still be ascertained. The molecular mechanisms by which N G F regulates cholinergic function m the basal forebram are not fully defined. In vitro studies have shown that at least two general mechanisms are mvolved in N G F action: (1) REFERENCES 1 Barde, Y.-A , Trophlc factors and neuronal survival, N e u r o n . 2 (1989) 1525-1534 2 Berrard, S , Brlce, A , Lottspelch, F, Braun, A , Barde, Y A and Mallet, J , cDNA cloning and complete sequence of porcine

induction of short-term, transcription-independent changes, most probably reflecting activation of second messenger system(s) and (2) medium- to long-term, transcription-dependent events, involving expression of several proto-oncogenes and other proteins 22'27. For example, expression of N G F receptors has been shown to be enhanced, m PC12 cells or septal cultures, upon exposure to NGF 6A4'23. Furthermore, it has been shown that N G F also amplifies expression of N G F receptors in basal forebrain of developing and adult rats in VlVO7'25. This increase in N G F receptor expression following N G F treatment could possibly reflect the existence of an auto-regulatory mechanism driven by target-derived N G E Furthermore, the increase in C h A T m R N A transcripts found in the present study using the same experimental model, raises the possibility of a causal and temporal interrelationship between NGF-lnduced activation of N G F receptors and enhanced C h A T expression. CONCLUSION ChAT m R N A has been assessed in discrete areas of the rat bram. Results, obtained with R T - P C R methodology, indicate that C h A T m R N A is present only in those areas possessing chohnergic cell bodies: striatum, septum and spmal cord have relatively high levels with respect to cortex or hippocampus. The amount of C h A T m R N A in the septum ts developmentally regulated, reaching adult levels between postnatal days 14 and 25. Furthermore, investigations mto mechanisms underlying regulation of ChAT gene expression have shown that i.c.v, administration of N G F mduces a sigmficant increase in ChAT gene transcrtpt m the basal forebrain neurons of both developing and adult rats. The further characterization of these and other molecular aspects in the regulation of CNS chohnergtc function may help in elucidating features underlying cholinerglc deficits in neurodegeneratlve diseases and may provide lnslght into pharmacological modulation of discrete molecular determmants m CNS cholinerglc neurons.

Acknowledgements The authors thank E Bigon for NGF purification. D Benvegnfl for assays of NGF activity m vitro, N Schlavo for ChAT assays and A Bedeschl, P Lentola and L Pohto for secretarml assistance We are also extremely grateful to A Herb and P H Seeburg for their assistance during the sequencing of the ChAT-PCR fragment

chohne acetyltransferase m vitro translation of the correspondmg RNA yields active protein, P r o c N a t l A c a d Sct U S A , 84 (1987) 9280-9284 3 Bocchml, V and Angelettl, P U , The nerve growth factor purification of a 30000 molecular weight protein, P r o c N a i l A c a d Sct U S A . 64 (1969) 787-794

325 4 Brice, A., Berrard, S , Raynaud, B., Ansleau, S , Coppola, T., Weber, M.J. and Mallet, J., Complete sequence of a cDNA encoding an active rat choline-acetyltransferase, a tool to investigate the plasticity of cholinerglc phenotype expression, J Neuroscl. Res, 23 (1989) 266-273. 5 Bruce, G and Hersh, L.B., The phosphorylat~on of Choline Acetyltransferase, Neurochem Res., 14 (1989) 613-620. 6 Cahssano, P and Shelanskl, M., Interacuon of nerve growth factor with pheochromocytoma cells. Evidence for tight binding and sequestration, Neuroscience, 5 (1980) 1033-1039 7 Cavicchloli, L , Flamgan, T.P., Vantlm, G , Fusco, M., Polato, P., Toffano, G., Walsh, FS. and Leon, A., NGF amplifies expression of NGF receptor messenger RNA in forebraln chollnerglc neurons of rats, Eur. J. Neurosci., 1 (1989) 258-262 8 Chelly, J., Kaplan, J C , Malre, P., Gautron, S and Kahn, A , Transcription of the dystrophm gene in human muscle and non-muscle Ussues, Nature, 333 (1988) 858-860 9 Cuello, A C and Sofromew, M.V, The anatomy of the CNS chohnerglc neurons, Trends Neurosct, 7 (1984) 74. 10 Cuello, A C , Garofalo, L., Kenigsberg, R L and Maysmger, D , Ganghosides potentmte in vwo and in vitro effects of nerve growth factor on central cholmerglc neurons, Proc. Natl Acad Scl. U.S.A, 86 (1989) 2056-2060. 11 Danielson, P.E., Forss-Petter, S., Brow, M A , Calavetta, L., Douglass, J., Milner, R J and Sutcllffe, J.G., plB15 a cDNA clone of the rat mRNA encoding cyclophdm, DNA, 7 (1988) 261-267. 12 Dlckson, G , Prentice, H.M., Kenimer, J G and Walsh, FS., Identification and characterization of neuron-specific and developmentally-regulated gene transcripts in the chick embryo spinal cord, J. Neurochem., 46 (1986) 787-793 13 Doherty, P., Dickson, J G , Flanigan, T.P and Walsh, F.S., The effect of nerve growth factor and its antibodies on neurofilament protein expression in primary cultures of sensory and spinal neurons, Neuroscz Len., 51 (1984) 55-60 14 Doherty, P, Seaton, P , Flamgan, T.P. and Walsh, F S , Factors controlling the expression of the NGF receptor in PC12 cells, Neurosct Lett, 92 (1988) 222-227 15 Dreyfus, C F., Effects of nerve growth factor on chohnerglc bram neurons, TIPS, 10 (1989) 145-149 16 Eckenstem, F and Thoenen, H , Production of specific antisera and monoclonal antibodies to chohne acetyltransferase: characterlzaUon and use for identification of chollnergic neurons, EMBO J , 1 (1982) 363-368. 17 Fiblger, H C., The organization and some projecUons of cholinerglc neurons of the mammalian forebram, Brain Res. Rev, 4 (1982) 327-388 18 Fischer, W , Wlctorln, K , Bjorklund, A., Wilhams, L . R , Varon, S and Gage, F H , Amelioration of chohnerglc neurons atrophy and spatial memory impairment in aged rats by nerve growth factor, Nature, 329 (1987) 65-68. 19 Fonnum, F , A rapid radlochemical method for the determination of choline acetyltransferase, J Neurochem, 40 (1975) 407-409. 20 Fusco, M., Oderfeld-Nowak, B., Vantml, G , Schmvo, N , Gradkowska, M , Zaremba, M and Leon, A , NGF affects uninjured, adult rat septohlppocampal chollnerg~c neurons, Neurosclence, 33 (1989) 47-52 21 Gnahn, H , Heftl, F, Heumann, R., Schwab, M and Thoenen,

22

23

24

25

26

27

28 29

30

31

32

33

34

35

36

37

H , NGF-mediated increase m chohne-acetyltransferase (CHAT) in the neonatal forebram, evidence for a physiological role of NGF m the brain? Dev Brain Res., 9 (1983) 45-52 Greene, L A. and Shooter, E M , The nerve growth factor: biochemistry, synthesis and mode of action, Annu Rev. Neurosct., 3 (1980) 353-402. Harukka, J. and Hefti, F., Development of septal cholinerglc neurons m culture: plating density and ghai cells modulate effects of NGF on survival, fiber growth and expression of transmitter-specific enzymes, J. Neurosct., 8 (1988) 2967-2985. Heftl, F., Dravld, A and Hartikka, J., Chronic mtraventricular rejections of nerve growth factor elevate chohne acetyltransferase actlwty m adult rats with parUal septo-hippocampal lesion, Brain Res, 293 (1984) 305-311. Higglns, G.A., Koh, S., Chen, K.S. and Gage, EH., NGF induction of NGF receptor gene expression and cholinergic neuronal hypertrophy within the basal forebram of the adult rat, Neuron, 3 (1989) 247-256. Large, T . H , Bodary, S . C , Clegg, D.O., Weskamp, G , Otten, U and Relchardt, L.E, Nerve growth factor gene expression m the developing rat brain, Science, 234 (1986) 352-355. Levi, A , Biocca, S , Cattaneo, A and Cahssano, P, The mode of action of nerve growth factor on PC12 cells, Mol. Neurobtol., 2 (1988) 201-226 Kromer, L.E, Nerve growth factor treatment after brain inJury prevents neuronal death, Science, 235 (1987) 214-216. Johnston, M V , Rutkowskl, J.L., Wainer, B.H., Long, J.B. and Mobley, W C., NGF effects on developmg forebrain cholinerglc neurons are regionally specific, Neurochem. Res, 12 (1987) 985-994 Maniatis, T , Fritsch, E.F and Sambrook, J., Molecular Cloning A Laboratory Manual, Cold Spnng Harbour Laboratory, Cold Spring Harbour, New York, 1982 McGeer, P.L., McGeer, E.G. and Peng, J H , Mimrevlew: chohne-acetyltransferase: purification and immunohistochemical locahzatlon, Life Sci, 34 (1984) 2319-2338. Mobley, W.C., Rutkowski, J.L., Tennekoon, G I , Buchanan, K and Johnston, M V , Chohne acetyltransferase activity in strlatum of neonatal rats increased by nerve growth factor, Sctence, 229 (1985) 284-287 Mocchettl, I. and Costa, E., In vlvo studies of the regulation of neuropeptlde stores in structures of the rat brain, Neuropharmacology, 26 (1987) 855-862. Sanger, F , Nlcklen, S and Coulson, A R , DNA sequencing with chain terminating mhibltors, Proc Natl Acad. Scl. U.S A., 74 (1977) 5436-5467. Vantml, G , Schiavo, N., Di Martlno, A., Polato, P , Triban, C., Callegaro, L , Toffano, G and Leon, A., Evidence for a physiological role of nerve growth factor in the central nervous system of neonatal rats, Neuron, 3 (1989) 267-273. Wang, A M , Doyle, M.V and Mark, D F , Quantltation of mRNA by the polymerase chain reaction, Proc. Natl. Acad Sct U.S A , 86 (1989) 9717-9721 Wllhams, L R., Varon, S., Peterson, G , Wlctorin, K., Fischer, W, Bjorklund, A and Gage, F N., Continuous mfusion of nerve growth factor prevents basal forebram neuronal death after fimbrm-formx transecUon, Proc Natl Acad Scl. U.S.A., 83 (1986) 9231-9235