Evidence for a physiological role of nerve growth factor in the central nervous system of neonatal rats

Neuron,

Vol. 3, 267-273,

September,

1989, Copyright

0 1989 by Cell Press

Evidence for a Physiological Role of Nerve Growth Factor in the Central Nervous System of Neonatal G. Vantini, N. Schiavo, A. Di Martino, I? Folato, C. Triban, 1. Callegaro, C. Toffano, and A. Leon Fidia Research Laboratories, Via Ponte della Fabbrica 3/A 35031 Abano Terme (PD) Italy

Summary Forebrain cholinergic neurons have been shown to respond in vivo to administration of nerve growth factor (NCF) with a prominent and selective increase of choline acetyltransferase (ChAT) activity. This has suggested that NCF can act as a trophic factor for these neurons. To test this hypothesis directly, anti-NGF antibodies (and their Fab fragments) were intracerebroventricularly injected into neonatal rats to neutralize endogenously occurring NCF. The anti-NGF antibody adminstration produced a decrease of ChAT activity in the hippocampus, septal area, cortex, and striatum of rat pups. This finding was substantiated by a concomitant decrease of immunopositive staining for ChAT in the septal area. These effects indicate that the occurrence of endogenous NGF in the CNS is physiologically relevant for regulating the function of forebrain cholinergic neurons. Introduction Among polypeptide growth factors capable of influencing survival and differentiation of neural cells during development, nerve growth factor (NGF) represents the most studied and characterized (Levi-Montalcini and Calissano, 1986; Levi-Montalcini, 1987). A physiological, trophic role for NGF on sympathetic and neural crestderived sensory neurons is well established, as indicated by the dramatic effects observed after its immunological, pharmacological, or surgical removal (Levi-Montalcini and Angeletti, 1968; Thoenen and Barde, 1980; Greene and Shooter, 1980). More recent investigations on newborn and adult rats have shown that cholinergic neurons in the corpus striatum and those in the basal forebrain projecting to the hippocampus and cortex respond to exogenous NGF with a selective and prominent increase of choline acetyltransferase (ChAT) activity (Cnahn et al., 1983; Mobley et al., 1985, 1986; Johnston et al., 1987; Fusco et al., 1988, Sot. Neurosci., abstract), the enzyme involved in the synthesis of acetylcholine. In addition, measurable levels of NGF, mRNA coding for NGF, NGF receptors, and mRNA coding for NGF receptors are present in rat brain, and their regional distribution suggests that NGF may regulate the function of the above-mentioned cholinergic neurons (for a review see Whittemore and Seiger, 1987; Johnson and Taniuchi, 1987). Taken together, the above observations have led to the

Rats

hypothesis that NGF may have a physiological role in the CNS, as well as in the PNS (Whittemore and Seiger, 1987; Thoenen et al., 1987). To date, however, no study has shown that specific removal or neutralization of endogenous NGF causes alterations in NGF-responsive CNS neurons. We have now measured ChAT activity in the hippocampus, septal area, cortex, and striatum of newborn rats after intracerebroventricular injections of immunoaffinity-purified rabbit anti-NGF antibodies (IgG) and their Fab fragments and have observed a decrease of ChAT activity in all of these regions. These results suggest that NGF regulates ChAT activity in forebrain cholinergic neurons, thereby performing a physiological role in the CNS.

Results and Discussion Newborn Sprague-Dawley rats of both sexes were intracerebroventricularly injected with either rabbit preimmune serum-derived IgG (6 Bg), immunoaffinitypurified anti-NGF IgG (6 pg), or the corresponding Fab fragments (3 pg), all dissolved in 10 ~1 of PBS. Different treatment schedules were followed in this study, as detailed in Table 1. To validate the injection procedure functionally, parallel experiments were also carried out by injecting NGF (5 pg) or cytochrome c (5 pg) intracerebroventricularly. In most cases, the experimental animals were used for the biochemical determination of ChAT activity in the septal area, hippocampus, cortex, and striatum. In the septal area and hippocampus, the activity of acetylcholinesterase (AChE) was also measured, as in these regions, it represents a reliable cholinergic marker (Eckenstein and Sofroniew, 1983; Levey et al., 1983). Some animals in the control, NGF-, and anti-NGF IgG-treated groups were randomly chosen for the immunocytochemical assessment of ChAT-positive neurons in the septal area and for AChE histochemistry in the hippocampus.

Anti-NGF and NGF Effects on ChAT Enzymatic Activity in Different Brain Areas Anti-NGF IgG and Fab administration produced significant decreases of ChAT activity in all of analyzed regions (Table 1). This effect was not observed after injections of either preimmune serum-derived IgC, their Fab fragments, or cytochrome c. In g-day-old animals, four injections of anti-NGF IgG on postnatal days (P) 2, 4, 6, and 8 decreased ChAT activity by 31% in hippocampus and 27% in cortex. This treatment schedule did not produce any significant effect in the striatum. A schedule of seven injections, daily, from P2 to P8, decreased ChAT activity by 30% in hippocampus, 30% in septum, 35% in cortex, and 16% in striatum. These data suggest that the observed anti-NGF IgG effects on ChAT are regionally specific. In particular, basal forebrain cholinergic neurons, projecting to hippocampus and cortex, seem more sensitive to anti-NGF IgG treatment than striatal

Table 1. Effects of Anti-NGF IgC, Cortex, and Striatum of Neonatal

Anti-NCF Rats

Fab Fragments,

and

NCF

on ChAT

Postnatal

Substance

Injected

Nonspecific

IgG

Brain

Region

Hippocampus Septal

Area

Cortex Striatum Anti-NGF

IgG

Hippocampus

Cytochrome

NGF

Fab Fab c

ChAT AChE ChAT AChE ChAT ChAT

2-a (Alternate 683

f

314 f 3873 f

Days) 16

5 206

Cortex Striatum Hippocampus Hippocampus

ChAT ChAT

698 f 23 588 f 22'

Hippocampus

ChAT AChE ChAT ChAT AChE ChAT

668 + 26

Area

Striatum Hippocampus Striatum

472

+ 40"

230 f 3520 f

Activity

in the

Hippocampus,

Septal

Area,

Days of Treatment

ChAT AChE ChAT AChE ChAT ChAT

Septal

Nonspecific Anti-NCF

Measure

and AChE

21b 156

3684 1061

f 218 + 49’

7607

f

318'

2-8 (Daily)

2-14 (Alternate

Days)

761 109 1479 166 371 3884

f f f f f &

42 3.2 108 8.3 14 121

2079

* 95

6119 f 353 f

231 17

533 106 1039 157 239 3270

f f f f * f

14h 4.1 79” 8 19b 61b

1556

9Eib

f

44% * 34ob 338 * 20

745 f 20 601 k lSd 725 a7 3598 1244 86 7921

f f i f f f

50 6.9 181 53’ 2.6 344’

Anti-NGF IgG (6 ug), preimmune serum-derived (nonspecific) IgG (6 ug), anti-NGF Fab fragments (3 pg), and nonspecific Fab fragments (3 ug) were all dissolved in 10 ul of PBS. Cytochrome c (5 ug) and NGF (5 ug) were dissolved as described (Mobley et al., 1985). All injection t otocols were started on P2. Animals were sacrificed 23-24 hr after the last injection. ChAT activity in noninjected rats and in animals receiving corresponding vehicles alone was virtually indistinguishable from that in rats treated with nonspecific IgG, nonspecific Fab fragments, or cytochrome c. Preincubation of the ChAT assay mixture from control animal hippocampi with anti-NGF IgG or Fab fragments (5 ug per 0.1 ml of final incubation volume) had no effect on enzyme activity. Values are means f SEM (n = 7-10). ChAT activity is expressed as nmollhr per 100 mg of protein; AChE activity is expressed as umollhr per 100 mg of protein. d P < 0.05 b P < 0.01 versus nonspecific IgG-treated animals. c P < 0.05. d P < 0.01 versus nonspecific Fab fragment-treated animals. p P < 0.01 versus cytochrome c-treated animals (Student’s t-test).

cholinergic interneurons. This may be due to either a different diffusion pattern of IgG in these brain regions or a differentially regulated sensitivity of diverse cholinergic neurons to endogenous NGF. Brain levels of both NGF and mRNA coding for NGF undergo remarkable regional and developmental changes (large et al., 1986; Shelton and Reichardt, 1986; Whittemore et al., 1986; Korsching et al., 1985). In addition, in hippocampus, NGF and ChAT levels show marked and parallel increases during the second postnatal week (Auburger et al., 1987). The coincidence of these changes support the notion that NGF may play an important role in the establishment of the cholinergic septo-hippocampal system. Given this background information, we explored whether, in septum and hippocampus of IS-day-old animals, anti-NGF IgG (schedule of seven injections on P2, P4, P6, P8, PlO, P12, and P14) could produce a more pronounced decrease in ChAT activity compared with that observed in g-day-old animals. By following this treatment protocol, in both septum and hippocampus, we observe significant decreases of ChAT activity (by 26% and 25%, respectively; see Table 1); the decreases were similar to those regis-

tered in g-day-old rats. These data suggest that in both 9- and IS-day-old animals the responsiveness of the septo-hippocampal cholinergic system to anti-NGF IgG treatment is about equivalent. However, this conclusion must be taken within the limits of the complete absence of data relative to diffusion kinetics of IgG in brain of 9- and 15-day-old rats. In agreement with previous reports, the administration of NGF elicited a prominent increase of ChAT activity (Table 1; Gnahn et al., 1983; Mobley et al., 1985, 1986; Johnston et al., 1987). In g-day-old animals, four injections of NGF on P2, P4, P6, and P8 increased ChAT activity by 59% in hippocampus and by 106% in striaturn. When NGF was administered daily from P2 to P8 the increase was 72% in hippocampus and 120% in striatum.

Anti-NGF and NGF Effects on Septal ChAT lmmunopositive Neurons The biochemical data on ChAT activity were paralleled by the finding that the immunostaining of ChAT-positive neurons in the septal complex was less evident following anti-NGF IgG treatment compared with controls (Figure

Ewdence 269

for a Physiological

Role of NCF

in CNS

1, compare

top and middle panels). On the contrary, NGF-treated animals showed a marked increase in this parameter (Figure 1, bottom panels; Figure 2). The consistency between the biochemical determinations of ChAT activity and the densitometric analysis of ChAT immunostaining strongly suggests that endogenous NGF is capable of controlling the expression of this enzyme. The difference in the anti-NGF IgG effect on ChAT activity (decreased by about 30%) and the ChAT immunostaining results (decreased by about 90%) in the septal area is not readily explainable. In general terms, it can be argued that, of the two measurements, determination of enzyme activity is much more quantitative, whereas quantitation of immunohistochemical results is more subjective. However, this discrepancy is not peculiar to the present work, but is a general one when considering the rat septo-hippocampal cholinergic system. For example, in the adult rat, complete transection of dorsal

Anti-NGF Effects on AChE Enzymatic and Histochemistry

septo-hippocampal cholinergic neurons produces more than 80% loss of ChAT immunopositive neurons in the septal complex (Kromer, 1987), whereas in the same area following the same type of lesion, ChAT activity is only transiently reduced by about 20% (Gasser et al., 1986; Gage et al., 1986). On the basis of this example,

Anti-NGF IgG treatment did not affect AChE activity in both the septal area and the hippocampus (Table 1), nor did it induce any substantial alteration in AChE-stained coronal sections through the hippocampus (data not shown). The lack of effect of these antibodies on AChE activity in the septal area and, more importantly, in the

it can be appreciated that we have provided not only biochemical but also immunocytochemical data, both of which are consistent with one another. We leave to further studies the explanation or the interpretation of how, in the same protein molecule (ChAT), the number of exposed and cryptic epitopes corresponds to the number of the catalytic sites.

hippocampus, where septal cholinergic neurons project, also indicates that the target innervation is not crucially altered. However, since in vivo adult sympathetic neurons have been shown to undergo transient functional impairment (Goedert et al., 1978) or death (Johnson et al., 1982) in relation to the duration of their exposure to anti-NGF antibodies, the limited and transient cholinergic deficit observed here may be attributed to unavoidable limitations inherent to injection schedules and route of antibody administration. For example, the minuteness of the penetration of intracerebrally administered IgG, which probably also decreases with the maturation of brain structures, and the discontinuous IgG treatment may result in only a partial and limited exposure of endogenous NGF to the antibodies. An alternative, non-mutually exclusive explanation for the lim-

Neuronal

Specificity

of Anti-NCF

Effects

Several observations have indicated that, in brain, NGF specifically affects striatal and basal forebrain cholinergic neurons without markedly affecting other neurotransmitter-related phenotypic traits (Gnahn et al., 1983; Mobley et al., 1985; Martinez et al., 1987; Montero and Hefti, 1988). To determine, at least partially, the specificity of the anti-NGF IgG effect, we measured dopamine content in striatum and noradrenaline content in hippocampus and cortex of g-day-old animals treated daily with either anti-NGF IgG or preimmune serum-derived IgG (controls) from P2 to P8. Using this treatment protocol, no effect was seen on either striatal dopamine (mean + SEM; controls = 2802 * 202 rig/g wet tissue [n = 71; anti-NGF = 2716 f 176 [n = 71) or hippocampal noradrenaline (mean f SEM; controls = 78.9 + 5.7 rig/g wet tissue [n = 71; anti-NGF = 74.7 f 4.0 [n = 71) or cortical noradrenaline (mean + SEM; controls = 52.8 + 2.3 rig/g wet tissue [n = 71; anti-NGF = 53.8 f 4.2 [n = 61). These findings directly support the concept that endogenous NGF plays a physiological trophic role in forebrain cholinergic neurons without substantially affecting nigro-striatal dopaminergic and central noradrenergic neurons.

Reversibility A major of NGF

of Anti-NCF

Effects

question with regard in the CNS is whether

to the physiological at this neonatal age

role NGF

might be essential for survival or crucially involved in target innervation processes of cholinergic neurons. To examine this question, we evaluated whether the effect elicited by anti-NGF IgG on ChAT activity in the septal area and hippocampus was permanent or transient. The results shown in Figure 3 indicate that this effect is transient. Upon discontinuation of anti-NGF IgG treatment, ChAT activity returned to normal values by P25. This suggests, within the limits of treatment protocols used in this study, that at this neonatal stage, NGF is probably not essential for the survival of these cholinergic neurons (see also below). That NGF plays a critical role for the survival and neurite elongation of these septal neurons in the fetal animal remains a possibility (Otten et al., 1985, Sot. Neurosci., abstract).

Activity

ited alterations observed in these neurons following anti-NGF IgG treatment is that NGF is only one of possibly two or several factors contributing physiologically to the trophic support of basal forebrain cholinergic neurons (Crutcher and Collins, 1982; Emerit et al., 1989).

Comparison of Anti-NGF Fab Fragments

IgG versus Anti-NCF

To overcome the poor diffusion of anti-NGF IgG in rat brain (Springer and Loy, 1985; Thoenen et al., 1987), we have prepared and biologically characterized anti-NGF Fab fragments (Callegaro et al., 1989), i.e., a lower molecular weight protein possessing, in principle, a better capability to penetrate CNS anatomical structures compared with the parent IgG. When compared with whole IgG, the anti-NGF Fab fragments were less effective, on a molar basis, in reducing ChAT activity (Table 1). This result could reflect differences in binding kinetics for the specific antigen, as well as differences in the diffusion or accumulation kinetics of the two antibody forms after in-

Neuron 270

Evidence

for a Physiological

Role of NGF in CNS

271

sensitive neuronal pathways is functionally similar for both of these protein molecules. Concerning the accessibility to NGF-sensitive central neurons by exogenous IgG-type molecules, it is noteworthy that, in adult rat brain, anti-NGF receptor mono-

clonal antibodies are taken up by basal forebrain neurons following intraventricular injection (Schweitzer, 1987). This implies that the basal forebrain is, to some extent, accessible to, and thus can be influenced by, relatively high molecular weight substances injected into cerebrospinal fluid. These same considerations underlie and support the present findings obtained in neonatal animals. Conclusion

anti-NGF

CONTROL

The present results are direct evidence that removal of endogenous NGF by immunological means produces specific deficits in the forebrain cholinergic neurons of neonatal rats and, as such, directly support the concept that NGF has a physiological role in the CNS. This reinforces the hypothesis that an altered production of endogenous NGF or a decreased sensitivity to this protein may be relevant in inducing forebrain cholinergic neuronal alterations and/or losses. Since deficits of cholinergic neurons represent one of the most consistent neuropathological features in the brains of Alzheimer’s patients, our data are compatible with the hypothesis that lack of the appropriate neuronotropohic factor(s)

NGF

Figure 2. Effect of Anti-NGF IgG and NGF on ChAT Immunostaining in the Septal Complex of g-Day-Old Rats Animals were treated with preimmune serum-derived IgG (CONTROL, n = 5), anti-NGF IgC (n = 41, and NCF (n - 5) daily from P2 to P8. For each an.imal, three coronal sections through the septal complex,

stained

to detect

ChAT

immunoreactive

neurons,

were

analyzed for quantitative morphology. Quantitative assessment of ChAT immunostaining was done by a computerized image analysis system (IBAS, Kontron, Zeiss) coupled to a photomicroscope (Zeiss) using a magnification of 40x. The area covered by ChAT immunostaining profiles analyzed region

is expressed as a percentage (dotted area in the schematic

of the total area of the diagram of a coronal

section). Each value represents the mean f SEM. Abbreviations: ac, anterior commissura; cc, corpus callosum; CP, caudate-putamen; MS, medial septum; VD8, vertical limb of the diagonal band.

is

loo-,

E z

80-

5Q

60-

ifi

40-

P ?J r;; 5

20-

0-

Figure

1. Coronal

important

Experimental

tracerebral injection (Cove11 et al., 1986; Holton et al., 1987). It is interesting to note that the extent of maximal decrease of ChAT activity obtained with either anti-NGF IgG or Fab fragments is about equivalent, suggesting that, at least in neonatal rats, the accessibility to NGF-

a z

an

and

Preparation and Purification of NCF, Anti-NGF IgG, and Fab Fragments NCF was isolated as the 2.5s subunit from submaxillary adult male mice according to the procedure of Bocchini

glands of and An-

Figure 3. ChAT Activity in the Septal Area and Hippocampus at Various Ages following an Anti-NGF IgC Treatment

'9

13

through

aspect underlying 1981; Hefti

disease (Appel,

Procedures

”

Sections

etiopathogenetic

this neurodegenerative Weiner, 1986).

the Septal

Complex

25

of g-Day-Old

AGE (days)

Rats Stained

to Visualize

Animals received daily injections of antiNGF IgG from P2 to P8, controls were injected with preimmune serum-derived IgG. Each value represents the mean * SEM (n = 7-10) expressed as a percentage of controls. ChAT activity values (nmollhr per 100 mg of protein) in the septal area (open bars) and the hippocampus (hatched bars) of control rats at 9 days of age were 1,406 f- 70 and 715 f 32 respectively; at 13 days of age, 3,984 f 365and 1,239 f 82; at 25daysofage. 12,290 f 699 and 6,168 f 145. The asterisks denote significant differences (P < 0.01, Student’s t-test) from age-matched controls.

ChAT

lmmunoreactive

(A) Specimen from an animal treated with anti-NGF IgG daily from P2 to P8. ChAT immunostaining is immunoreactive neurons in a specimen from an animal treated with preimmune serum-derived IgG as treated rat (daily, from P2 to P8). An extensive increase of ChAT immunostaining is visible. (B), (D), and of the regions indicated by the arrows in (A), (C), and (E), respectively. Magnification: 60x (A, C, and

Neurons

very low. (Cl Dldribution of ChAT in (A). (E) Specimen from an NGF(F) represent higher magnifications E); 600x (6, D, and F).

geletti (1969). Anti-mouse NCF antisera were raised in adult male New Zealand albino rabbits. The anti-NGF IgG fraction was purified from rabbit antisera by affinity chromatography according to the procedure described by Stoeckel et al. (1976). The anti-NGF Fab fragments were prepared from the whole serum rabbit IgG fraction by digestion with papain (Mage, 1980), and then purified by affinity chromatography. The IgC fraction was purified from the serum proteins by a procedure involving caprylic acid precipitation (Steinbuch and Audran, 1967). Fab fragments were separated from Fc fragments and intact IgG by affinity chromatography using a protein A-Sepharose column equilibrated with 0.01 M phosphate buffer (pH 8.0), 0.15 NaCI. The Fab fraction (eluting with the void volume) was subsequently incubated with mouse NGF coupled to Sepharose 46 overnight at room temperature. The uncoupled material was removed by washing with 0.01 M phosphate buffer (pH 7.4) containing 0.15 M NaCI, and the Sepharose 4B-NGF coupled with the antibodies was then applied to a column (1.6 x 20 cm). The specific Fab anti-NCF fraction was eluted using 4.5 M MgClz in 0.05 M acetate buffer (pH 5.0) and immediately dialyzed against 0.01 M phosphate buffer (pH 7.4) 0.15 M NaCI. Non-specific rabbit IgG and its Fab were prepared from whole serum obtained from the same rabbits prior to immunization with NCF. The intact IgG was purified by affinity chromatography on protein A-Sepharose equilibrated with 0.01 M phosphate buffer, 0.15 M NaCl (pH 8.0). The IgC fraction waseluted from the support usingO.O1 M citrate buffer (pH 3.5). This material, dialyzed against 0.01 M phosphate buffer, 0.15 M NaCl (pH 7.4), was used for the Fab preparation. All immunoglobulin fractions were stored in aliquots at -80°C until use.

Chemical and Biological Characterization of Anti-NGF IgG and Fab Fragments The chemical purity of anti-NCF IgG and corresponding Fab fragments was evaluated by SDS-polyacrylamide slab gel electrophoresis and by gel filtration on a Superose 12 column (Pharmacia, Sweden). Following electrophoresis and gel filtration, both antiNCF IgG and Fab fractions migrated or eluted, respectively, as single bands. In vitro, both anti-NGF IgG and Fab fragments blocked NGF biological activity as measured by using standardized microcultures of dorsal root ganglion neurons from B-day chicken embryos (Manthorpe et al., 1981; Skaper and Varon, 1983). Briefly, neuronal dissociates were prepared and enriched by preplating over a tissue culture plastic surface in culture medium for 2 hr at 37OC. Culture medium consisted of DMEM supplemented with 26.4 mM NaHCO,, 2 mM L-glutamine, 100 U/ml penicillin, and 10% (v/v) heat-inactivated fetal calf serum. Neurons were resuspended in medium, diluted to 40,000 per ml, and 50 PI of this cell suspension (2000 neurons) was added to microwells already containing 50 PI of test solution. After a 24 hr incubation at 37OC (5% CO?, 95% air), the cultures were fixed with 2% glutaraldehyde. The numbers of surviving neurons bearing neurites longer than two somal diameters was determined using phase-contrast microscopy. This quantitative approach was used to titrate the immune activity of both IgG and Fab fragments. In particular, both serially diluted anti-NGF IgG and Fab fragments blocked NGF biological activity with IDso values of 160 and 340 rig/ml, respectively, using 5 nglml NGF. In a complementary series of experiments, increasing amounts of NGF were incubated with constant concentrations of anti-NGF IgG of Fab fragments, followed by evaluation of the ability of NGF to support survival of the added sensory neurons. With 10 &ml of either immunoglobulin fraction, the IDso for NGF was 170 and 130 rig/ml for IgG and Fab, respectively. In vivo, anti-NGF IgG (4 mglkg) and Fab fragments (10 mg/kg), subcutaneously administered to neonatal rats on P2 and P3, produced a severe sympathectomy (about 90%). The degree of immunosympathectomy was evaluated by measuring the noradrenaline content in sympathetically innervated organs (heart and spleen) as previously described (Vantini et al., 1988). In all in vitro and in vivo experiments, anti-NGF IgG was, on a molar basis, more effective than the corresponding Fab fragments in antagonizing the biological activity of NGF. lmmunoglobulins and Fab fragments derived from the preimmune serum of the same rabbits were without effect.

Treatment Modalities, Enzyme Determinations, and lmmunocytochemistry To avoid self-aggregation processes causing decrease of antibody activity, both IgG and Fab fragments were prepared, stored, and used, in contrast to previous reports (Gnahn et al., 1983; Thoenen et al., 1987), at relatively low concentrations (below 1 mg/ml). In particular, anti-NGF IgG and Fab were used at 0.6 mg/ml and 0.3 mglml, respectively. lntracerebroventricular injections were done in ice-cold immobilized rat pups after transcutaneous puncture with a 27 gauge syringe needle placed approximately 0.5 mm caudal and 1.5 mm lateral to the bregma. Injections were well tolerated (see Results for dosage and treatment protocol utilized). At indicated times, the brains were rapidly removed, dissected on ice, and stored at -BOY until assays. In this study, the “septal area” refers to the medioventral portion of a brain slice obtained by coronal section of the forebrain at the level of the most rostra1 parts of genu corpus callosum (landmark of the rostra1 section) and optic chiasm (landmark of the caudal section). The “cortex” includes the cerebral frontoparietal cortex, which extends approximately 1 mm anterior and 1 mm posterior to the bregma. ChAT and AChE activities were measured by previously described procedures with minor modifications (Kaneda and Nagatsu, 1985; Fonnum, 1975; Johnson and Russel, 1975). Enzyme activity was expressed in relation to protein content (Lowry et al., 1951). Measurement ofcatecholamine levels in different brain regions was performed using an HPLC equipped with an electrochemical detector and following a published procedure (Vantini et al., 1984). The peroxidase-antiperoxidase (PAP) immunocytochemical procedure was used to stain for ChAT immunoreactive neurons. Rat pups were anesthetized with Pentothal and perfused transcardially with a solution containing 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were then removed, postfixed in the same solution for 5-6 hr, and sectioned at 40-50 pm on a vibratome. lmmunocytochemistry for ChAT was performed on free-floating sections with a primary monoclonal antibody concentration of 2.5 &ml. Acetylcholinesterase histochemistry was carried out according to Bear et al. (1985).

We thank D. Guidolin for performing the quantitative E. Bigon for preparing NGF, D. Benvegnir for doing the NGF, and E. Olivi and A. Bedeschi for their excellent sistance. We also thank S. D. Skaper, G. F. Azzone, Nunzi for helpful discussion during preparation of the Received

April

3, 1989;

revised

June

morphology, bioassay for technical asand M. C. manuscript.

26, 1989

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