Cultured basal forebrain cholinergic neurons in contact with cortical cells display synapses, enhanced morphological features, and decreased dependence on nerve growth factor

THE JOURNAL OF COMPARATIVE NEUROLOGY 373451-465 (1996)

Cultured Basal Forebrain Cholinergic Neurons in Contact With Cortical Cells

Display Synapses, Enhanced Morphological Features, and Decreased

Dependence on Nerve Growth Factor

DUN H. HA, RICHARD T. ROBERTSON, CHARLES E. RIBAK, AND JOHN H. WEISS Departments of Anatomy and Neurobiology (D.H.H., R.T.R., C.E.R., J.H.W.), Neurology

(J.H.W.), and Psychobiology (J.H.W.), University of California, Irvine, California 92697-4290

ABSTRACT Prior studies examining the dependence of basal forebrain cholinergic neurons (BFCNs) on

nerve growth factor (NGF) for survival have reached differing conclusions depending on the experimental paradigm employed, suggesting the importance of environmental and developmental variables. The present study examined the NGF dependence of BFCNs and modulatory effects of target (cortical) neurons under the controlled conditions of dissociated cell cultures. Initial experiments found BFCNs (identified by using choline acetyltransferase immunocytochemistry) in pure basal forebrain (BF) cultures to be dependent on NGF between the 2nd and 4th week in vitro. During that developmental period, NGF deprivation for 3 days, induced by application of anti-NGF antibody, resulted in degeneration of over 80% of BFCNs, whereas at earlier or later times, BFCNs were largely resistant to NGF deprivation. When BF neurons were plated together with cortical neurons (as dissociated co-cultures), the BFCNs grew neuritic processes (labeled with acetylcholinesterase histochemistry) that appeared to specifically target cortical neurons; electron microscopy revealed that synapses formed between these cells. BFCNs in co-cultures were more resistant to NGF deprivation, were larger, and had much more extensive neuritic growth than BFCNs in pure BF cultures. The resistance of BFCNs to NGF deprivation provided by cortical neurons could be largely reproduced by addition of other trophic factors (brain-derived neurotrophic factor, BDNF; neurotrophin 3, NT3; neurotrophin 415, NT415; or glial-derived neurotrophic factor, GDNF) during NGF deprivation in pure BF cultures. These results suggest that developing BFCNs undergo a critical period requiring trophic influences that may be provided by NGF or other trophic factors, as well as unknown factors derived from cortical neurons. , 1996 Wilcy-Liss, Inc.

Indexing terms: neurotrophins, electron microscopy, co-culture, acetglcholinesterasc, cholinc acetyltransferase

Basal forebrain cholinergic neurons (BFCNs) send a topographically organized axonal projection to the cerebral cortex (Divac, 1975; Wenk et al., 1980; McKinney et al., 1983; Saper, 1984; Calarco and Robertson, 1995). This cholinergc projection is believed to regulate cortical function and to be involved in learning and memory (Bartus and Johnson, 1976; Bartus et al., 1982; Lo Conte et al., 1982; Buzsaki et al., 1988; Buzsaki and Gage, 1989). The observation that BFCNs degenerate in Alzheimer’s disease (Davies and Maloney, 1976; Whitehouse et al., 1982; Coyle et al., 1983), as indicated by atrophy of their cell bodies and the decrease in cholinergic markers in the cerebral cortex, has initiated considerable scientific interest in factors that

might influence their survival. One candidate is nerve growth factor (NGF). Although it has long been known that NGF critically influences growth and survival of sympathetic and dorsal root ganglia neurons in the peripheral nervous system (Levi-Montalcini and Angeletti, 1963; Greene, 1977a,b; Gorin and Johnson, 1979; Eichler and Rich, 1989J, recent studies have found NGF to have important central effects on BFCNs.

Accepted May 24, 1996 Address reprint requests to Dr. John H. Weiss, Department of Neurology,

University of California, Invine, CA 92697-4290. E-mail: jweissiu uci.edu

( 1996 WILEY-LISS, INC.

452 D.H. HA ET AL.

The ability of NGF to induce phenotypic changes in BFCNs, most notably the up-regulation of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE; Hefti et al., 1985; Martinez et al., 1987; Hartikka and Hefti, 1988), suggests its involvement in regulating cellular function. However, the role of NGF in determining BFCN survival has been uncertain, as effects of NGF appear to vary depending upon experimental conditions. On one hand, a variety of evidence has suggested that BFCNs may be dependent upon NGF for survival. Removal of NGF from dissociated cultures of BFCNs, for even brief periods, has been reported to trigger irreversible degeneration (Har- tikka and Hefti, 1988; Svendsen et al., 1994; Kew et al., 1996). Also, placement of fimbria-fornix lesions in vivo, a maneuver that likely disrupts retrograde transport of NGF from the hippocampus, caused apparent degeneration of septa1 BFCNs that could be prevented by NGF treatment after lesion placement (Hefti, 1986; Gage et al., 1986, 1988; Williams et al., 1986; Kromer, 1987). On the other hand, however, other evidence suggests that although important for regulating phenotype, NGF may not be necessary for BFCN survival. For example, in cases of fimbria-fornix lesions, if treatment with NGF was delayed until BFCNs were presumed to be dead, significant numbers of ChAT- positive neurons could still be recovered (Hagg et al., 1988, 1989 ). Furthermore, infusion of anti-NGF antibodies into lateral ventricles at early postnatal ages induced a loss in ChAT activity that was reversible upon discontinuation of the anti-NGF treatment (Vantini et al., 1989). Finally, recent in vivo studies using transgenic mice lacking the NGF gene reported that BFCNs were not lost, whereas other neurons, such as developing sympathetic and sensory ganglia, were significantly degenerated (Crowley et al., 1994). Thus, results obtained from in vitro and in vivo studies differ, likely reflecting differences between simplified culture systems and the highly complex in vivo environment.

The present study utilized a novel culture system to test the hypotheses that 1) BFCNs are critically dependent on NGF for survival during development and 2) cortical (target) neurons modulate that dependence. First, we examined NGF dependence of BFCNs in pure basal forebrain (BF) cultures as a function of age. Second, because BFCNs in vivo project their axons to cerebral cortex at ages when cultured BFCNs are dependent on NGF for survival (Har- tikka and Hefti, 1988; Svendsen et al., 1994), we investi- gated whether the presence of cortical cells in BF cultures would alter NGF dependence. Finally, we explored the potential for other trophic factors to enhance the survival of BFCNs in the absence of NGF.

Abbreuiations

Ab antibody AChE acetylcholinesterase BDNF brain-derived neurotrophic factor BF basal forebrain BFC-CNs BFCNh basal forebrain cholinergic neurons cu‘r choline acetyltransferase C N T F ciliary neurotrophic factor DIV days in vitro EGF epidermal growth factor FGF fibroblast growth factor G D N F &-derived neurotrophic factor N G F nerve growth factor N T 3 neurotrophin 3 NT4 5 neurotrophin 415

BFCNs contacting cortical neurons in tandem cultures

MATERIALS AND METHODS Cell culture preparation

Timed-pregnant Sprague-Dawley rats were killed by le- thal injections of sodium pentobarbital. The fetuses (gesta- tional age 16-17 days) were rapidly removed from these pregnant rats, and their brains harvested, stripped of meninges, and positioned on their ventral surfaces. Cortical hemispheres were spread laterally to expose the basal forebrain region. The septum and diagonal band regions were removed and plated largely as described by Hartikka and Hefti (1988), with minor modifications (Weiss et al., 1994). Neocortical tissue was removed as described by Rose et al. (1993). The tissues from the two brain regions were kept separated, minced, and then incubated in trypsin for 30 minutes at 37°C. After removing the trypsin, the tissues were gently triturated by using large and small bore glass pipettes, and the resulting cell suspensions were diluted in plating medium consisting of Eagle’s minimal essential medium (MEM-Earle’s salts, supplied glutamine-free) supplemented with 10% heat-inactivated horse serum, 10% fetal bovine serum, glutamine (2 mM), and glucose (total 25 mM). Neurons were plated on previously established monolayers of cortical astrocytes (see below) in 24-well tissue culture plates, and maintained in a 37”C/5% COz incubator. For most experiments, non-neuronal cell division was halted after 5-7 days in vitro (DIV) by exposure to M cytosine arabinoside for 1-3 days. Cultures were shifted into a maintenance medium (growth medium, GM) identical with the plating medium but lacking fetal serum, and supplemented with exogenous NGF (50 ngiml of 2.5s) until the time of experimental manipulation, when the maintenance medium was switched to a defined medium consisting of MEM + glucose.

Pure BF cultures consisted of BF neurons at low densities (0.5-1.0 x lo5 viable cells per cm”. To make sure that these cultures were as pure as possible, sharp forceps were used during the microdissection to carefully dissect and isolate the cylindrical structures containing cholinergic neurons, followed by removal of any cortical tissue that was still attached. Mixed BF-cortical co-cultures were prepared by plating BF and cortical neurons together at approximately a 1:1 ratio, yielding a density of about 2.0 x 10” viable cells per cm2. “Tandem cultures,” containing regions that had only BF neurons as well as regions with both BF and cortical neurons in the same culture well and sharing the same medium, were prepared by first dividing each well into two compartments by using glass rings (8 mm diameter; Bellco Glass, Vineland, NJ) coated with a fine layer of sterile vacuum grease. Cortical neurons were then plated either in the center or the perimeter compartment and allowed to adhere to the bottom (0.5-1.0 x lo5 viable cells per cm”. After 2-3 DIV, the glass rings were removed, and BF neurons were plated into the entire well (0.5-1.0 x lo5 viable cells per cm”. Viable cell densities were determined at time of fixation.

Astrocyte cultures were prepared by using the same protocol as cortical cultures, except that cortices were removed from early postnatal rats (1-3 days postnatal) and plated directly on Falcon Primaria culture plates in media supplemented with epidermal growth factor (final concen- tration 10 ng/ml). Neurons were plated directly onto the astrocytic monolayers once they became confluent (about 2 weeks).

CORTICAL CELLS ALTER NGF DEPENDENCE OF BFCNS 453

Nerve growth factor deprivation Two different techniques were used to induce 3-day

periods of NGF deprivation. The more vigorous method involved essentially complete ( > 99.5%) media exchanges, into MEM supplemented only with glucose. The less vigorous technique (partial media exchange) involved replacing about 65% of the media, and therefore caused less disruption to the environment at the bottom of the wells. Both paradigms employed anti-NGF antibodies (125 ngiml; Boeh- ringer Mannheim, Indianapolis, IN) to neutralize residual exogenous NGF as well as NGF that may be produced endogenously. Following NGF deprivation, the cultures were washed of antibody and readministered exogenous NGF (50 ngiml) for a minimum of 3 days prior to ChAT staining. Studies by several investigators (Hefti et al., 1985; Hartikka and Hefti, 1988; Svendsen et al., 1994) indicate that such exposures to NGF markedly increase ChAT expression in BFCNs and thus should permit good ChAT staining in surviving cells.

Choline acetyltransferase immunocytochemistry

Cultures were fixed for 45 minutes in 4% paraformaldehyde and then washed ( x 3) in phosphate-buffered saline (PBS). They were then preincubated in a blocking solution consisting of 10% horse serum in PBS for 1 hour at 25”C, followed by exposure to a monoclonal antibody to ChAT (1:2,000; Chemicon, Temecula, CA) in fresh blocking solution (24 hours, 4°C). Cultures were then washed in PBS ( x 3), and incubated in secondary antibody (biotinylated horse anti-mouse; 1:200, 1 hour, 25°C). After washout, avidin-horseradish peroxidase (ABC solution; Vector Labo- ratories, Burlingame, CA) was added (1 hour, 25”C), and labeled cells were visualized using a metal-enhanced diami- nobenzidine (DAB) substrate kit (Pierce, Rockford, IL).

Acetylcholinesterase histochemistry AChE staining was carried out as described by Tag0 et al.

(19861, with minor modifications. After 45 minutes of fixation in 4% paraformaldehyde, cultures were washed three times in 0.1 M maleate buffer (pH 6.0) then incubated in a fresh solution of 300 pM CuS04, 500 pM sodium citrate, 50 pM potassium ferricyanide, and 30 pM acetylthio- choline iodide in 0.1 M maleate buffer for 1-2 hours. Cultures were then rinsed five times in 0.05 M Tris buffer (pH 7.6) and exposed to intensification solution (O.04Tj DAB, 0.3% nickel ammonium sulfate, and 0.003% H202 in 0.05% Tris buffer) until cells were clearly stained (5-30 minutes).

Electron microscopy To determine whether BFCNs formed synapses with

cortical cells, mixed BF-cortical co-cultures were prepared, for electron microscopy, on “Transwell-COL” cell culture inserts (Costar, Pleasanton, CA) previously coated with astrocytes. After fixation and AChE-histochemistry as described above, cultures were postfixed in a solution of 4% paraformaldehyde and 1% glutaraldehyde for 30 minutes in 0.1 M PBS, and then in 1% Os04 for 30 minutes at 4°C. The cultures were then dehydrated in graded ethanol. The use of propylene oxide detached the intact membrane from the wall unit of the insert and facilitated the flat embedding in Medcast of the entire membrane, including the labeled neurons with processes attached to cortical cell clusters.

Ultrathin serial sections were prepared, counterstained with uranyl acetate and lead citrate, and examined with a Philips CMlO electron microscope.

Neurotrophic factors All neurotrophic factors were used at 50 ngiml. Nerve

growth factor (NGF), ciliary neurotrophic factor (CNTF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and acidic fibroblast growth factor (aFGF) were purchased from Boehringer Mannheim (Indianapolis, IN); GDNF and NT3 were purchased from Alomone Labs (Jerusalem, Israel); BDNF and NT4 were purchased from Promega (Madison, WI).

Quantitative analysis The total number of ChAT-positive neurons per culture

well was determined from counts in 52 consecutive, non- overlapping microscope fields, covering over 9 5 8 of the well area, using low-power ( 1 0 0 ~ ) brightfield optics. Thus, virtually all stained cells in each culture well were counted. Neurons were identified as ChAT-positive if they were clearly stained and if at least two neurites could be dis- tinctly identified. In control conditions, neurons were treated identically to those in experimental conditions except that they were exposed continuously to exogenous NGF; control culture wells generally contained 100-300 CUT-positive neurons. In experimental conditions, ChAT-positive neurons were excluded from counts if they were degenerated, as indicated by possession of truncated processes, atrophic cell bodies, and disrupted cell membranes. In each experiment, the percent loss of ChAT-positive neurons was calculated as the difference between the mean number of intact stained cells in several (three to four) control (un- treated) cultures and the mean number in several cultures that were NGF deprived, and expressed as a percentage of the former. In all experiments, control cultures and treated cultures were on the same multi-well culture plate and derived from the same plating.

For morphologic measurements (in tandem cultures), AChE-labeled neurons were randomly selected (under brightfield microscopy at 200 x from four sister cultures for each condition in each experiment. Two-dimensional video images of the selected neurons were imported into a computer for morphometric analysis using COMOS software from BioRad. Three parameters were measured: cell area, total neuritic length, and number of first-, second-, and third-order branches. A first-order neurite is distinguished from the soma by its extension beyond a line which traces completely the circumference of the soma surface.

All values are given as the mean 2 standard error of the mean (SEM), normalized to control conditions in each experiment. Significance of data was determined by Stu- dent’s t-test or by ANOVA (as indicated), with the Bonfer- roni post-hoc test, using Instat software (Graph Pad Inc.; San Diego, CA).

RESULTS BFCNs depend on NGF for survival during a

period of development in vitro BFCNs in dissociated cultures retain many of their in

vivo morphological characteristics and, when supplemented with exogenous NGF, are able to survive in vitro for at least 45 days. Figure 1A illustrates the numerous BFCNs, as

Fig. 1. BFCNs develop NGF dependence in culture. ChAT-positive neurons in pure BF cultures were photographed after 21 DIV, after being exposed to NGF (50 ngiml) either continuously (control condition; A,B), or with a 3-day period of NGF deprivation from 3-6 DIV tC,D), 9-12 DIV (E,F), or 15-18 DIV (G,H). Note that deprivation

from 3 to 6 DIV had little effect on BFCN morphology, whereas deprivation from 9 to 12 DIV or from 15 to 18 DIV caused severe atrophy of surviving BFCNs. Arrows indicate cells illustrated at both low and high magnification. Scale bars = 200 ym in A,C,E,G and 50 ym inB,D,F,H.

CORTICAL CELLS ALTER NGF DEPENDENCE OF BFCNS -155

Fig. 2. BFCNs lose dependence on NGF at late stages after culturing. BFCNs were photographed after 39 DIV, after being exposed to N G F (50 ng/ml) either continuously (control condition; A,Bl, or with a 3-day period of NGF deprivation from 33 to 36 DIV (C,D). Note that

deprivation from 33 to 36 DIV had little effect on BFCN morphology. Arrows indicate cells illustrated at both low and high magnification. Scale bars = 200 pm in A,C and 50 pm in B,D.

identified by ChAT immunocytochemistry, seen at 21 DIV under these conditions. These neurons appeared healthy and commonly had large cell bodies with two to four primary neurites (Fig. 1B). In conditions of continuous exposure to NGF for 3 weeks, approximately 0.5-2.0% of the BF neurons are cholinergic (as assessed by ChAT staining). These numbers are similar to ones previously reported (Hartikka and Hefti, 1988; Svendsen et al., 1994; Weiss et al., 1994).

The effects of NGF deprivation on survival of BFCNs were examined during early (0-7 DIV), intermediate (9-30 DIV), and late (33-42 DIV) periods after plating of neurons into cultures. NGF deprivations were for 3-day periods and were accomplished by complete ( > 99%)) media exchanges with serum-free media lacking NGF and supplemented with anti-NGF antibodies (125 ngiml). Whereas NGF deprivation during the early period appeared to have little effect on the BFCNs (Fig. 1C,D), identical treatment during the intermediate period resulted in a significant loss of CUT-positive neurons; the majority of those that re- mained appeared atrophic with only one to two poorly stained neurites (Fig. 1E-H). Interestingly, deprivation during the late period, like the early period, had little apparent effect on either BFCN number or morphology (Fig. 2 ).

Quantification of BFCN survival, as assessed by counts of CUT-immunoreactive neurons in NGF-deprived cultures

compared to numbers present in control conditions receiv- ing continuous NGF, confirmed these qualitative impres- sions: BFCNs were particularly vulnerable to NGF deprivation during the intermediate period, whereas younger and older BFCNs were less dependent on NGF (Fig. 3 ) . In control experiments BF cultures were grown in the absence of any added NGF until the last 3 days prior to staining. Under these conditions, loss of ChAT-positive neurons (86.4 2 0.9% lost; mean i- SEMI, was comparable to that observed after the 3-day periods of deprivation during the critical period described above, whereas less than 3% of the background (unstained) neurons were lost, consistent with their presumed non-cholinergic identity.

BFCNs project their axons specifically toward cortical clusters in mixed co-cultures

Because BFCNs send axonal projections to the cerebral cortex in vivo, we next sought to determine whether they would develop analogous projections in vitro. Basal forebrain and cortical neurons, dissected separately from the same brains, were plated together in the same wells and grown as “mixed co-cultures” (see Materials and Methods). Cortical neurons could be distinguished from BF neurons by their tendency to aggregate into clusters (Fig. 4A). BFCNs in these mixed co-cultures were identified by ChAT immunocytochemistry, which stained mainly cell bodies

D.H. HA ET AL. 456

4 loo - 2 15 e a

50

5 25

h

v +

A *

B

*

I d I 0 ~ ? n

3-6 9-12 15-18 21-24 27-30 33-36 39-42

Period of NGF deprivation (days after plating)

Fig. 3. BFCNs undergo a period of NGF dependence between the 2nd and 4th weeks after culturing. A Sister BF cultures were exposed to NGF for 21 days except for a 3-day period of deprivation using complete ( > 99.5% 1 media exchanges with addition of anti-NGF antibodies, as described, followed by assessment of BFCN loss (see Materials and Methods). B: BF cultures were exposed continuously to NGF except during 3-day periods of deprivation, as above. Because of the long duration of these experiments, each deprivation episode was treated as a separate experiment with its own control. BFCN loss was assessed after 3 days of NGF replacement following the deprivation period. n = 12 cultures compiled from three repetitions of the experiment for each condition. * indicates significant difference from control values; P < .01 (ANOVA with Bonferroni test).

and proximal neurites, followed by AChE histochemistry, which stained the extensive neuritic tree displayed by many of these neurons. No specific ChAT or AChE staining was observed in pure cortical cultures (Fig. 4A), indicating the AChE-stained neurons in co-cultures were of basal forebrain origin. BFCNs in co-cultures generally appeared to have larger cell bodies and more complex neuritic arboriza- tion (Fig. 4C,D) than those found in pure BF cultures (Fig. 4B). In addition, AChE-stained cholinergic axons often appeared to project specifically toward aggregated clusters of cortical neurons (Fig. 4C,D), in many cases extending long distances to target the clusters (Fig. 4E).

BFCNs form synapses with cortical neurons in dissociated co-cultures

We next employed electron microscopic (EM) analysis to examine AChE-positive cholinergic projections to cortical neuronal clusters and search for evidence of synapse formation. As indicated by the light microscopic data, cholinergic projections appeared to appose cortical somata. Thus, several hundred cortical cell bodies were examined, but no synapses between cortical somata and AChE-labeled terminals were observed. Instead, all of the observed 40 synapses formed by AChE-positive axon terminals were located on cortical neurites that were adjacent to (Fig. 5A,B) or distant from (Fig. 5C-E) cortical somata. Thirty-eight of the 40 observed synapses were of the symmetric type. In certain cases, synaptic identity was further confirmed by serial sections (Fig. 5D,E).

Detailed morphologic examination of presynaptic structures, including synaptic vesicles and mitochondria, was impaired by the dense labeling of the cholinergic terminals with the AChE reaction product. As observed in organotypic co-cultures (Distler and Robertson, 1993), AChE reaction product was also found in the extracellular space and in the synaptic cleft (Fig. 5D,E).

Enhanced survival of BFCNs in mixed co-cultures during NGF deprivation

The observation that BFCNs in mixed co-cultures appeared more robust than BFCNs in pure BF cultures led us to examine more closely whether the presence of cortical neurons might have trophic effects on BFCNs. We first examined the effect of cortical neurons on the vulnerability of BFCNs to NGF deprivation. Mixed co-cultures were either kept continuously exposed to NGF or were deprived of NGF between 14 and 17 DIV, a period during which BFCNs in pure BF cultures were found to be critically dependent on NGF for survival (Fig. 3).

As observed in pure BF cultures, about 80% of BFCNs in co-cultures were lost following NGF deprivation using complete media exchanges and anti-NGF antibodies (Fig. 6Al,A2). Because such a complete media exchange could remove any trophic factors derived from the cortical cells, we next induced NGF deprivation using anti-NGF antibodies, but with only a partial (about 65%) media exchange. Under these conditions, NGF deprivation in co-cultures resulted in only a 40% loss of BFCNs; in identically treated pure BF cultures, close to 809 of BFCNs were lost (Fig. 6A1 ,A2).

Two control experiments were utilized to test the specificity of the survival enhancing effects of cortical neurons. First, as BFCNs in co-cultures only showed resistance to NGF deprivation after partial media exchanges but not after complete media exchanges, we considered the possibility that the observed resistance is due to inadequate neutralization of residual NGF by available antibodies. Arguing against this possibility, tripling the amount of antibody used (from 125 ngiml to 375 ngiml) resulted in no greater degree of damage to the BFCN population, suggesting that the lower antibody dosage was sufficient to remove all neutralizable NGF (Fig. 6A3). Second, because co- cultures had greater cell density than pure cultures, it was possible that the increased survival might simply reflect the greater number of cells present in co-cultures, perhaps producing greater amounts of endogenous NGF or other protective factors. To examine this possibility, pure BF cultures that had either greater (1 x lo5 viable cells/cm2) or lower (1 x lo4 viable cells/cm2) neuronal densities than mixed BF-cortical co-cultures (5 x lo4 viable cells/cm2) were prepared. NGF deprivation in these pure BF cultures resulted in similar high levels of BFCN loss, irrespective of the neuronal density, whereas the same treatment in the mixed co-cultures caused significantly less damage to BFCNs (Fig. 6B).

Enhanced survival of BFCNs in co-cultures requires close association

with cortical neurons Enhanced resistance to NGF deprivation of BFCNs in

co-cultures could depend upon physical contacts between BFCNs and cortical neurons or could be mediated through soluble factors produced by the cortical neurons. To help differentiate these possibilities, the effects of NGF deprivation on BFCNs were studied in “tandem cultures” containing distinct regions with BF neurons in potential contact with cortical neurons, and regions with only BF neurons. Neurons from different regions in the same well shared the same medium and astrocyte substratum. Control sister cultures exposed to NGF showed the expected BFCN morphology (Fig. 7A-D). The effects of NGF deprivation

CORTICALCELLSALTERNGFDEPENDENCEOFBFCNS 457

Fig. 4. Cholinergic neurites specifically target clusters of cortical neurons. Photomicrographs show cortical neuronal clusters in pure cortical cultures (A); AChE-labeled BFCNs in pure BF cultures (B); and AChE-labeled BFCNs in mixed BF-cortical co-cultures (C-E). Note the

apparent random behavior of cholinergic projections in pure BF cultures in comparison to the long distances they traverse specifically toward cortical clusters in co-cultures. Arrowheads indicate typical cortical clusters; arrows indicate AChE labeled BFCNs. Scale bars = 200 pn.

D.H. HA ET AL.

Fig. 5. Electron micrographs of synapses between neurites of cortical neurons and axons of BFCNs labeled with AChE reaction product in dissociated co-cultures. A: A portion of an AChE-positive axon terminal IT! that contains synaptic vesicles and forms a symmetric synapse (arrow! with an unlabeled cortical neurite. Note that the synapse is formed with the cortical neurite and not with the adjacent cortical soma IS) that shows a nucleus (n) . ~ 3 1 , 0 0 0 . B: Higher magnification of the symmetric synapse in A to illustrate the presence

of synaptic vesicles in the densely stained axon terminal. ~59 ,000 . C: Another AChE-positive axon terminal with synaptic vesicles forms a symmetric synapse (arrow) with a long cortical neurite. ~ 4 0 , 0 0 0 . D,E: Adjacent serial sections show a symmetric synapse (large arrows) formed between an AChE-positive axon terminal and a branched neurite. Note that both sections show the presence of another synapse (small arrows) formed between an unlabeled axon terminal and the same neurite. ~ 4 0 , 0 0 0 .


Complete media exchange deprivation after such media replacement still resulted in the loss of over 805% of BFCNs (Fig. 8B), suggesting that only small amounts of factors are produced and/or that they are not freely diffusable.

E S Partial media exchange A v1 2 1001 1 2 3

Pure cultures Co-cultures (125nghnI Ab) (125ngIml Ab)

Cortical neurons enhance morphological characteristics of BFCNs in tandem cultures In addition to conferring enhanced resistance to NGF

deprivation, potential cortical contacts also appeared to dramatically alter the appearance of the BFC-CNs. Irrespec- tive of the presence of NGF, BFC-CNs from tandem cultures displayed much larger cell bodies and longer processes than isolated BFCNs (Fig. 7A-H). Also, the neurites of BFC-CNs appeared to be wider in diameter than those of isolated BFCNs and were covered with spinous structures. This qualitative assessment was confirmed by quantitative measurements showing soma area and total neuritic length per cell of BFC-CNs to be substantially greater than those of isolated BFCNs (Fig. 9A,B). In the presence of NGF, BFC-CNs and isolated BFCNs possessed similar numbers of primary neurites, but with regard to second- and third-order neurites, the BFC-CNs had markedly greater numbers (Fig. 9C). Also, unlike BFC-CNs, which maintained their morphologic characteristics after NGF deprivation, isolated BFCNs deprived of NGF showed significantly decreased values of all parameters examined (Fig. 9A-C).

Other trophic factors can enhance survival of BFCNs in the absence of NGF

co-cultures (375ng/ml Ab)

tjj " Low density Low density High density Co-cultures Pure cultures Pure cultures

Fig. 6. Presence of cortical neurons decreases vulnerability of BFCNs to NGF deprivation injury. A Pure BF cultures or BF-cortical co-cultures (14 DIVJ were deprived of NGF for 3 days either by complete i > 99.5%) or partial (about 65%) media exchanges with addition of anti-NGF antibodies as indicated, followed by assessment of ChAT-positive neuronal loss a t DIV 20. B: Low-density co-cultures ( 5 x lo1 viable cells/cm2j, low-density pure BF cultures (1 x lo4 viable cellsicm' j , and high-density pure BF cultures (1 x 10;'viable cellsicm" were prepared and NGF deprived with partial media exchanges and anti-NGF antibody (125 ng/ml), and injury was assessed exactly as above. n = 11-16 cultures compiled from three to four repetitions of the experiment for each condition. P < ,001 (ANOVA with Bonferroni test) for all significant differences: Asterisk indicates significant difference from pure BF cultures with partial media exchanges. Number sign indicates significant difference from co-cultures with complete media exchanges. Ampersand indicates significant difference from low- and high-density pure BF cultures.

between 14 and 17 DIV in each region of these tandem cultures were identical with those previously observed for that type of culture in isolation: BFCNs in apparent direct contact with cortical neurons (BFC-CNs) generally appeared healthy (Fig. 7E,F), whereas those not in evident contact with cortical neurons (isolated BFCNs) were atrophic (Fig. 7G,H). Quantitative assessment showed a loss of over 80% of isolated BFCNs, whereas less than 40% of BFC-CNs were lost when compared to their respective controls (Fig. 8A). Because cortical factors could be released into the medium at levels insufficient for adequate diffusion to pure BF regons of the tandem cultures, we examined the effects of medium, taken from either cortical cultures or from co-cultures, on BFCNs in pure BF cultures. NGF

Because of the apparent ability of cortical neurons to enhance morphological characteristics of BFCNs as well as their survival during NGF deprivation, we examined the ability of exogenous trophic factors to reproduce those effects. Pure BF cultures were deprived of NGF by means of complete media exchanges between 14 and 17 DIV as described above, using either media with antibody to NGF alone or with antibody plus another neurotrophic factor. Of all trophic factors tested (at 50 ngiml), only the neurotrophins (BDNF, NT3, NT4/5) and glial-derived neurotrophic factor (GDNF) significantly reduced the loss of BFCNs (Fig. 10). However, none of these neurotrophins, either alone or in combinations, reproduced the enhanced morphologic characteristics of the BFC-CNs under the exposure paradigm employed.

DISCUSSION Review of primary findings

The aim of the present study was to examine certain behaviors of developing BFCNs that are not easily studied in either simple culture models or in vivo. We have thus developed a culture system that facilitates long-term maintenance of BFCNs and allows for outgrowth of their processes, which were observed to project toward cortical clusters and to form synapses with cortical neurites. Using this system, we first found BFCNs in pure BF cultures to undergo a period, between the 2nd and 4th weeks in culture, during which they are dependent upon NGF for survival. Second, the presence of cortical neurons was found to diminish the dependence of BFCNs on NGF during this period while inducing morphologic enhance- ments of the BFCNs that could not be reproduced by NGF alone.

Fig. 7. NGF deprivation differentially affects BFCNs contacting partial media exchanges and anti-NGF antibodies (125 ngiml). Note cortical neurons (BFC-CNs) and isolated BFCNs in tandem cultures. that isolated BFCNs are deficient morphologically compared to BFC- Photomicrographs show AChE-stained BFC-CNs (A,B) and isolated CNs, irrespective of the presence of NGF. Arrows indicate cells BFCNs (C,D) exposed continuously to NGF, or BFC-CNs (E,F) and illustrated a t both low and high magnification. Scale bars = 200 pm in isolated BFCNs (G,H) deprived of NGF between days 14 and 17 by A,C,E,G and 50 pm in B,D,F,H.

CORTICAL CELLS ALTER NGF DEPENDENCE OF BFCNS

A

3 loo- I c 75- g 50-

2 25- V

n +

* c l

461

*

A

" BFC-CN BFCN

B 100-

75 -

50-

25 -

# # #

U

GM BF Cortex BF-cortex

Type of medium Fig. 8. Resistance of BFC-CNs in tandem cultures to NGF depriva-

tion injury requires close association with cortical neurons. A Tandem cultures were NGF deprived as described in Figure 7, followed by assessment of survival of ChAT-positive BFC-CNs and BFCNs. n = 12 sister cultures per condition compiled from three repetitions of the experiment. B BFCNs in 11 DIV pure BF cultures were exposed to growth media (GM) or media taken from age-matched cultures of the indicated type for 3 days, followed by 3 days of NGF deprivation induced by partial media exchange with the same media type, but containing anti-NGF antibodies (experimental group). Controls for each media type received NGF only. n = 12-15 sister cultures per condition compiled from five repetitions of the experiment. Asterisk indicates significant difference from isolated BFCNs deprived of NGF; P < ,0001 (two-tailed t-test). Number sign indicates no significant difference from GM; P > .05 (ANOVA with Bonferroni test).

Technical considerations The culture system utilized in this study provided the

high degree of control over the biochemical milieu and accurate monitoring of responses in individual neurons permitted by dissociated cultures. In addition, to model the in vivo environment and provide a substrate for extension of BF projections, the dissociated cells were plated on pre-established astrocyte monolayers. Although proliferating astrocytes have been shown to produce NGF (Lu et al., 1991), we believe that the presence of the astrocytes in our

#

v +NGF +NGF +Ab +Ab

BFC-CN BFCN BFC-CN BFCN

* ---- # *

OJ +NGF +NGF +Ab +Ab $3 BFC-CN BFCN BFC-CN BFCN

C 0 primary branches ES3 secondary branches -tertiary branches

# #

+NGF +NGF +Ab +Ab BFC-CN BFCN BFC-CN BFCN

Fig. 9. BFC-CNs in tandem cultures show enhanced morphologic features. Tandem cultures were either exposed continuously to NGF, or were deprived of NGF for 3 days by means of partial media exchanges and anti-NGF antibodies. After 3 days of NGF replacement, AChE- positive BFC-CNs and BFCNs were randomly selected for morphometric measurement of cell size (A), extent of neuritic outgrowth (B), and frequency of branching (C). n = 50-55 neurons for each condition compiled from three separate platings. P < .01 (ANOVA with Bonfer- ronni test) for all significant differences: Number sign indicates significant difference from isolated BFCNs with the same treatment. Asterisk indicates significant difference from NGF treatment of same cell types.

cultures has not altered our primary conclusions for two reasons. First, all of our experimental conclusions are based on comparisons with control cultures plated on identical

162 D.H. HA ET AL.

+BDNF

+NT4

+GDNF

+BDNF+NT3+NT4

+GDNF+NT3+NT4

r - 7

0 25 50 75 100

%ChAT(+) Neuronal Loss

Fig. 10. Exogenous trophic factors can enhance survival of BFCNs after NGF deprivation. Pure BF cultures were deprived of NGF from 14 to 17 DIV by means of complete media exchanges with anti-NGF antibody alone, or with the addition of indicated trophic factorb) (all at 50 ng ,ml) , followed, after 3 days of NGF replacement, by ChAT staining and assessment of injury as described. n = 8-16 cultures for each condition compiled from four repetitions of the experiment. Asterisk indicates significant difference from antibody only condition; P < .01 (ANOVA with Bonferronni test).

astrocyte beds. Second, any endogenous NGF production by astrocytes would be unlikely to confound the NGF deprivation experiments, as the endogenous NGF should have been neutralized by the antibody to NGF.

BFCNs undergo a critical period of NGF dependency during development in vitro

A major aim of the present study has been to examine the dependence of BFCNs upon NGF for survival, as a function of age after plating. However, as NGF is known to regulate expression of ChAT and AChE activity by BFCNs as well as their survival, it is important to consider whether loss of ChAT-stained neurons reflects death of BFCNs or alter- ations in ChAT expression. The paradigm employed, in which BFCN survival is assessed by ChAT staining, but only after continuous exposure to NGF for a minimum of 3 days, should reliably reflect BFCN survival for the following reasons. First, as discussed above, several days of exposure to NGF markedly increase ChAT expression in BFCNs, and thus should permit good ChAT staining in surviving cells (Hefti et al., 1985; Hartikka and Hefti, 1988; Svendsen et al., 1994). In addition, culture studies have provided compelling evidence that loss of BFCNs after NGF deprivation reflects cell death and not simply a phenotypic change. Supporting this conclusion, estimated BFCN loss was the same whether assessed by ChAT or p75 (low- affinity nerve growth factor receptor) immunocytochemistry, or by AChE histochemistry, and could not be reversed by several days of NGF re-exposure (Svendsen et al., 1994).

Further indicating that the lost cells had undergone apoptotic death, their loss was accompanied by evidence of DNA fragmentation (Kew et al., 19961, and was prevented by protein synthesis inhibitors (Svendsen et al., 1994; Kew et al., 1996).

The primary finding of these studies is that BFCNs in low-density dissociated cultures appear to be dependent upon NGF for survival only during a discrete period between the 2nd and 4th week in culture; BFCNs from younger or older cultures showed little NGF dependence. The loss of NGF dependence observed as BFCNs matured beyond 33 DIV is consistent with recent in vitro results from Svendsen et al. (1994) and with in vivo findings that adult BFCNs were resistant to degeneration after ablation of hippocan:pal or cortical target neurons (Sofroniew et al., 1987, 1990, 1993; Minger and Davies, 1992a,b). Taken together with our finding that young ( < 7 DIV) BFCNs also lack NGF dependence in vitro, the critical period of NGF dependence observed in this study roughly parallels the period in vivo (postnatal weeks 1-3) during which BFCNs are developing their axonal projections to cortex (Dinopou- 10s et al., 1989; Gould et al., 1991; Calarco and Robertson, 1995; De Carlos et al., 1995). During this same period, an increase in NGF production as well as increases in NGF and NGF-receptor mRNA expression have been shown to occur in cortex (Korsching et al., 1985; Large et al., 1986; Shelton and Reichardt, 1986; Whittemore et al., 1986; Koh and Loy, 1989). The temporal concordance of these events suggests the possibility that cortical NGF may play a critical role in the establishment of normal cholinergic projections in cortex. Thus, an inability of BFCNs to respond to NGF during this period, as shown in mice carrying a disrupted TrK-A receptor gene, may result in abnormal loss of cholinergic projections (Smeyne et al., 1994). This time- dependent switching of neurotrophin requirements has been demonstrated in other neuronal systems (Buchman and Davies, 1993) and could play a role in determining the density and distribution of cortical innervation by BF cholinergic axons.

The finding of a critical period in vitro, however, is in apparent contradiction with the observation that early postnatal (3-28 days) BFCNs were shrunken but not lost in transgenic mice entirely lacking the NGF gene (Crowley et al., 1994). The shrunken BFCNs could be in early stages of degeneration and destined to die, or they may be kept alive in the absence of NGF by target-derived factors that have been shown to influence BFCN survival, such as BDNF (Alderson et al., 1990; Morse et al., 1993; Nonomura et al., 1995), NT3 (Friedman et al., 1993), and NT415 (Friedman et al., 1993; Nonomura et al., 1995). Alternatively, BF- derived factors, including FGF (Anderson et al., 1988; Grothe et al., 1989; Bizon et al., 1996) and GDNF (Schaar et al., 1993), may be able to promote survival through direct or indirect paracrine pathways.

A culture model of in vivo interactions between cortical neurons and BFCNs

A technique to co-culture dissociated BF and cortical neurons on an astrocyte substratum was developed to model in vivo interactions between BFCNs and cortical neurons. A striking finding in these co-cultures was the apparent specificity with which cholinergic neurites appeared to target clusters of cortical neurons. Two factors suggest that the targeting of cortical clusters is specific. First, while AChE-positive neurites in pure BF cultures are


generally short and do not appear to project toward other cells, AChE-positive fibers in co-cultures often project over long distances to contact cortical clusters. Second, electron microscopy revealed the formation of symmetric synapses between AChE-positive axon terminals and neurites within the targeted cortical clusters. These synaptic features (symmetric morphology and location on neurites rather than somata) are similar to those described for BFCN-cortical synapses in vivo (Houser et al., 1985; Umbriaco et al., 1994) and in organotypic co-cultures (Distler and Robertson, 1993).

Previous control experiments carried out in our labora- tory further support the specificity of the effects of cortical cells on BFCNs. First, when basal forebrain explants were placed between cortical and cerebellar explants in triple- organotypic cultures, cholinergic axons only grew into the cortical tissue (Distler and Robertson, 1992). In addition, dissociated BFCNs seeded upon organotypic slices of cortex survived, whereas those that were seeded onto cerebellar slices did not (unpublished observations).

Cortical neurons enhance BFCN survival and morphology

BFCNs in co-cultures were able to survive NGF deprivation much better than BFCNs in pure BF cultures. The additional observation in “tandem cultures” that the survival-enhancing effects of cortical neurons appeared to require contact, or at least close association, between BFCNs and cortical neurons suggests the possibility that factors released by cortical neurons may be taken up directly by BFCN terminals at synapses and retrogradely transported to help maintain BFCN survival in the absence of NGF (DiStefano et al., 1992). However, these survival- enhancing effects were only observed if the bathing medium was not completely replaced prior to NGF deprivation, raising the concern that the enhanced BFCN survival might be due to residual NGF incompletely neutralized by the antibody. Arguing against this possibility, however, was the observation that deprivation induced by the antibody appeared to be maximal, as tripling the amount of antibodies did not result in any further cell loss. Thus, the survival-enhancing effects of cortical neurons could be mediated by NGF only if it is present in sites, such as synaptic clefts, where it might be inaccessible to neutralization by antibody. Alternatively, the survival-enhancing effects could be induced by cortically derived trophic mol- ecules other than NGF. Further, bioelectric activity has been shown to enhance neuronal survival in vitro (Bergey et al., 1981; Baker and Ruijter, 1991; Ramakers and Boer, 1991) and could be contributory. Whatever the factor(s) is (are), the apparent lack of enhanced survival when the medium is completely exchanged suggests that the presence of soluble chemical factors is critically important.

The survival-enhancing effects of cortical neurons on survival of BFCNs showed specificity, as the enhancement could not be reproduced in pure BF cultures of greater neuronal densities than BF-cortical co-cultures. However, the density of these “high-density’’ pure BF cultures was still less than that previously reported to confer a decreased NGF dependence to BFCNs in pure BF cultures (Hartikka and Hefti, 1988), suggesting the possibility that BF neurons might produce survival-enhancing factors in smaller quantities than cortical neurons. The appearance of enhanced morphologic features (increased soma size and neuritic branching and outgrowth) of BFCNs in apparent

contact with cortical neurons provides further evidence for the specificity of the effects of cortical neurons on BFCNs and strongly suggests roles for factors other than NGF. These features were not observed in high-density pure BF cultures, and were not reproduced by the continuous presence of NGF in pure BF cultures. Further supporting the involvement of other cortically derived factors, enhanced morphologic features of surviving BFC-CNs per- sisted after NGF deprivation, whereas the few isolated BFCNs surviving NGF deprivation were markedly atrophic.

Enhancement of BFCN survival by some exogenous trophic factors

Of the variety of trophic factors tested, only the neurotrophins (BDNF, NT3, NT415) and GDNF significantly enhanced BFCN survival during NGF deprivation. This finding is consistent with previous studies indicating that neurotrophins have trophic effects on BFCNs (Alderson et a]., 1990; Friedman et al., 1993; Morse et al., 1993; Nonomura et al., 1995). In contrast, the effects of GDNF, a putative survival factor for striatal-dopaminergic neurons and motor neurons (Beck et al., 1995; Oppenheim et al., 1995; Tomac et al., 1995; Yan et al., 19951, have not been previously documented for BFCNs. The trophic effects of these exogenous factors, taken together with their expression in cortex (Ernfors et al., 1990; Maisonpierre et al., 1990; Wetmore et al., 1990; Lauterborn et al., 1994; Schmidt-Kastner et al., 1994; Springer et al., 1994), suggest their candidacies as endogenous factors produced by cortical neurons that contribute to the enhanced resistance to NGF deprivation and morphologic features of BFC-CNs in tandem cultures. However, the inability of these factors, alone or in combinations, to reproduce the morphology- enhancing effects of cortical neurons on BFC-CNs suggests either that other cortical factors are involved, or that the 3-day exposures employed are insufficient to significantly influence morphology.

The lack of effect of bFGF in the present paradigm contrasts with previous studies (Anderson et al., 1988; Grothe et al., 1989) that showed bFGF to enhance BFCN survival. However, bFGF may be acting indirectly, possibly by stimulating NGF production in actively proliferating astrocytes (Lu et al., 1991; Perkins and Cain, 1995). Therefore, the addition of excess NGF-neutralizing antibodies during bFGF treatment would be expected to directly counteract the survival-enhancing effects of bFGF on BFCNs.

CONCLUSIONS The present study has two primary conclusions. First,

BFCNs appear to be dependent on NGF for survival during a specific critical developmental period. This period roughly corresponds to the period when BFCNs are projecting their axons to innervate cortex in vivo. Second, this study provides direct evidence in support of the idea that cortical neurons have important trophic effects on developing BFCNs, a phenomenon which has long been suspected. Specifically, cortical neurons provide a trophic influence on BFCNs during the critical period that enhances their survival as well as their morphological features. This influence occurs in the absence of NGF and cannot be fully reproduced by the continuous presence of NGF. Con- versely, neither the presence of cortical neurons nor any of the other exogenous neurotrophic factors are able to fully

464 D.H. HA ET AL.

maintain BFCN survival during NGF deprivation. Thus, these results suggest that multiple trophic factors may act in concert to regulate the survival and growth of BFCNs during a critical period of development. Different factors may have different roles. For example, whereas NGF might be a particularly potent survival-promoting factor, other cortically derived factors may be more important for growth, branching, and navigation of axons in the target regions.

The culture system established in this study may help to bridge differences between studies done in simplified culture systems and those undertaken in the more complex environment of the brain. This culture system will be useful for future studies aimed at understanding how BFCNs and cortical neurons influence each other during development and how their survival or function may be altered as a result of their interaction.

ACKNOWLEDGMENTS We acknowledge and appreciate the help of the late

Yashoda Jhurani with electron microscopy. We thank Kiet Lieu for technical assistance. This work was supported by NIH grants NS 30884 (J.H.W.), NS 30109 (R.T.R.), and NS 15669 (C.E.R.) and by grants from the Alzheimer’s disease and related disorders association (J.H.W.) and the PEW Scholars Program in the Biomedical Sciences (J.H.W.).

LITERATURE CITED Alderson, R.F., A.L. Alterman, Y.A. Barde, and RM. Lindsay (1990)

Brain-derived neurotrophic factor increases survival and differentiated functions of rat septal cholinergic neurons in culture. Neuron 5297- 306

Anderson, K.J., D. Dam, S. Lee, and C.W. Cotman (1988) Basic fibroblast growth factor prevents death of lesioned cholinergic neurons in vivo. Nature 332t360-361

Baker, R.E., and J.M. Ruijter (1991) Chronic blockade of bioelectric activity in neonatal rat neocortex in uztrot physiological effects. Int. J. Dev. Neurosci. 9:321-329.

Bartus, R.T., and H.R. Johnson (1976) Short-term memory in the rhesus monkey. disruption by the anti-chohnergic scopolamine. Pharmac. Bio- chem. Behav. 5t3946.

Bartus, R.T., R.L. Dean, B. Beer, and A.S. Lippa (1982) The cholinergic hypothesis of geriatric memory dysfunction. Science 21 7:408417.

Beck, K.D., J. Valverde, T. Alexi, K. Poulsen, B. Moffat, R.A. Vandlen, A. Rosenthal, and F. Hefti (1995) Mesencephalic dopaminergic neurons protected by GDNF from axotomy-induced degeneration in the adult brain. Nature 373:339-341.

Bergey, G.K., S.C. Fitzgerald, B.K. Schrier, and P.G. Nelson (1981) Neuro- rial maturation in mammalian cell culture is dependent on spontaneous electrical activity. Brain Res. 207.49-58.

Bizon, J.L., J.C. Lauterborn, P.J. Isackson, and C.M. Gall (1996) Acidic fibroblast growth factor mRNA is expressed by basal forebrain and striatal cholinergic neurons. J. Comp. Neurol. 366:379-389.

Buchman, V.L., and A.M. Davies (1993) Different neurotrophins are expressed and act in a developmental sequence to promote the survival of embryonic sensory neurons. Development 118:989-1001.

Buzsaki, G., and F.H. Gage (1989) The cholinergic nucleus basalis: a key structure in neocortical arousal. Exs 57:159-171.

Buzsaki, G., T.G. Bickford, G. Ponomareff, L.J. Thal, R.J. Mandel, and F.H. Gage (1988) Nucleus basalis and thalamic control of neocortical activity in the freely moving rat. J. Neurosci. 26t735-744.

Calarco, C A., and R.T. Robertson (1995) Development of basal forebrain projections to visual cortex: DiI studies in rat. J. Comp. Neurol. 354:608-626.

Coyle, J.T., D.L. Price, and M.R. DeLong (1983) Alzheimer’s disease: a disorder of cortical cholinergic innervation. Science 219:1184-1190.

Crowley, C., S.D. Spencer, M.C. Nishimura, K.S. Chen, S. Pitts-Meek, M.P Armanini, L.H. Ling, S.B. MacMahon, D.L. Shelton, A.D. Levinson, and H.S. Phillips (1994) Mice lacking nerve growth factor display perinatal

loss of sensory and sympathetic neurons yet develop basal forebrain cholinergic neurons. Cell 76:1001-1011

Davies, P., and A J. Maloney (1976) Selective loss of central cholinergic neurons in Alzheimer’s disease. Lancet 2.1403.

De Carlos, J.A., B.L. Schlaggar, and D.D.M. O’Leary (1995) Development of acetylcholinesterase-positive thalamic and basal forebrain afferents to embryonic rat neocortex. Exp. Brain Res. 104:385-401.

Dinopoulos, A., L.A. Eadie, I. Dori, and J.G. Parnavelas (1989) The development of basal forebrain projections to the rat visual cortex. Exp Brain Res. 76t563-571.

DiStefano, P.S., B. Friedman, C. Radziejewski, C. Alexander, P. Boland, C.M. Schick, R.M. Lindsay, and S.J. Wiegand (1992) The neurotrophins BDNF, NT-3, and NGF display distinct patterns of retrograde axonal transport in peripheral and central neurons. Neuron 8t983-993.

Distler, P.G., and R.T. Robertson (1992) Development of AChE-positive neuronal projections from basal forebrain to cerebral cortex in organotypic tissue slice cultures. Dev. Brain Res. 67.181-196.

Distler, P.G., and R.T. Robertson (1993) Formation of synapses between basal forebrain afferents and cerebral cortex neurons: an electron microscopic study in organotypic slice cultures. J. Neurocytol. 22t627- 643.

Divac, I. (1975) Magnocellular nuclei of the basal forebrain project to neocortex, brain stem, and olfactoly bulb. Review of some functional correlates. Brain Res. 93:385-398.

Eichler, M.E., and K.M. Rich (1989) Death of sensory ganglion neurons after acute withdrawal of nerve growth factor in dissociated cell cultures. Brain Res. 482.340-346.

Ernfors, P , C Wetmore, L. Olson, and H. Persson (1990) Identification of cells in rat brain and peripheral tissues expressing mRNA for members of the nerve growth factor family. Neuron 551 1-526.

Friedman, W.J., C.F. Ibanez, F. Hallbrook, H. Persson, L.D. Cain, C.F. Dreyfus, and I.B. Black (1993) Differential actions of neurotrophins in the locus coeruleus and basal forebrain. Exp. Neurol. 119.72-78.

Gage, F.H., K. Wictorin, W. Fischer, L.R. Williams, S. Varon, and A. Bjorklund (1986) Retrograde cell changes in medial septum and diagonal band following fimbria-fornix transection: quantitative temporal analysis. Neuroscience 19:241-255.

Gage, F.H., D.M. Armstrong, L.R. Williams, and S. Varon (1988) Morphologi- cal response of axotomized septal neurons to nerve growth factor. J. Comp. Neurol. 269:147-155.

Gorin, P.D., and E.M. Johnson (1979) Experimental autoimmune model of nerve growth factor deprivation: effects on developing peripheral sympathetic and sensory neurons. Proc. Natl. Acad. Sci. U.S.A. 76:5382-5386.

Gould, E., N.J. Woolf, and L.L. Butcher (1991) Postnatal development of cholinergic neurons in the rat: I. Forebrain. Brain. Res. Bull. 27:767- 789.

Greene, L.A. i 1977a) Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons: I. Sympathetic neurons. Dev. Biol. 58:96-105.

Greene, L.A. (197713) Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons: 11. Sensory neurons. Dev. Biol. 58:106-113.

Grothe, C., D. Otto, and K. Unsicker (1989) Basic fibroblast growth factor promotes in vitro survival and cholinergic development of rat septal neurons: comparison with the effects of nerve growth factor. Neurosci- ence 31:649-661.

Hagg, T., M. Manthorpe, H.L. Vahlsmg, and S. Varon (1988) Delayed treatment with nerve growth factor reverses the apparent loss of cholinergic neurons after acute brain damage. Exp. Neurol. 101:303- 312.

Hagg, T., B. Fass-Holmes, H.L. Vahlsing, M Manthorpe, J.M. Conner, and S. Varon (1989) Nerve growth factor (NGF) reverses axotomy-induced decreases in choline acetyltransferase, NGF receptor and size of medial septum cholinergic neurons. Brain Res. 505:29-38.

Hartikka, J., and F. Hefti (1988) Development of septal cholinergic neurons in culture: plating density and glial cells modulate effects of NGF on survival, fiber growth, and expression of transmitter-specific enzymes. J. Neurosci. 8:2967-2985.

Hefti, F. (1986) Nerve growth factor promotes survival of septal cholinergic neurons after fimbrial transections. J. Neurosci. 62155-2162.

Hefti, F., J. Hartikka, F. Eckenstein, H. Gnahn, R. Heumann, and M. Schwab (19851 Nerve growth factor increases choline acetyltransferase but not survival or fiber outgrowth of cultured fetal septal cholinergic neurons. Neuroscience 14:55-68.

Houser, C.R., G.D. Crawford, P.M. Salvaterra, and J E Vaughn (1985) Immunocytochemical localization of acetyltransferase in rat cerebral

CORTICALCELLSALTERNGFDEPENDENCEOFBFCNS 465

cortex: a study of cholinergic neurons and synapses. J. Comp. Neurol. 234: 17-34.

Kew, J.N.C.. D.W. Smith, and M.V. Sofroniew (19961 Nerve growth factor withdrawal induces the apoptotic death of developing septal cholinergc neurons in vitro-protection by cyclic AMP analogue and high potassium. Neuroscience 70.329-339.

Koh. S., and K. Loy (1989) Localization and development of nerve growth factor-sensitive rat basal forebrain neurons and their afferent projections to hippocampus and neocortex. J . Neurosci. 9.2999-0318.

Korsching, S.. G. Auburger, R. Heumann, J. Scott, and H. Thoenen (19851 Levels of nerve growth factor and its mKNA in the central nervous system of the rat correlate with cholinerac innervation. EMBO J. 4:1389-1393.

Kromer. 1 3 (1987) Nerve growth factor treatment after brain injury prevents neuronal death. Science 235:214-216.

Large, T.H.. S.C. Bodary, D.O. Clegg, G. Weskamp, U. Otten, and L.F. Reichardt i 1986) Nerve growth factor gene expression in the developing rat brain. Science 234.352-355.

Lauterborn, J.C.. P.J. Isackson, and C.M. Gall (19941 Cellular localization of NGF and NT-3 mRNAs in postnatal rat forebrain. Mol. Cell Neurosci. 5: 46-62.

Levi-Montalcini, R.. and P.U. Angeletti (19631 Essential role of the nerve growth factor in the survival and maintenance of dissociated sensory and sympathetic embryonic nerve cells in vitro. Dev. Biol. 7:653-659.

Lo Conte, G., E. Casamenti, V. Bigl, E. Milaneschi, and G. Pepeu (1982) Effect of magnocellular forebrain nuclei lesions on acetylcholine output from the cerebral cortex, electrocorticogram and behavior. Arch. Ital. Biol. 120:176-188.

Lu, B.. M. Yokoyama, C.F. Dreyfus. and I.B. Black (1991) NGF gene expression in actively growing brain glia. J. Neurosci. 1 I:318-326.

Maisonpierre, P .C, L. Belluscio, B. Friedman, R.F. Alderson, S.J. Wiegand, M.E. Furth, K.M. Lindsay, and G.D. Yancopoulos (19901 NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression. Neuron 5501-509.

Martinez, H.J. , C.F. Dreyfus, G.M. Jonakait, and I.B. Black (1987) Nerve growth factor selectively increases cholinergic markers but not neuropep- tides in rat basal forebrain in culture. Brain Res. 412.295-301.

McKinney. M., J .T. Coyle, and J.C. Hedreen (19831 Topographic analysis of the innervation of the rat neocortex and hippocampus by the basal forebrain cholinergic system. J. Comp. Neurol. 21 7:103-121.

Minger, S.L., and P. Davies t1992a) Persistent innervation of the rat neocortex by basal forebrain cholinergic neurons despite the massive reduction of cortical target neurons: I . Morphometric analysis. Exp. Neurol. 117.124-138.

Minger, S.L., and P. navies (1992b) Persistent innervation of the rat neocortex by basal forebrain cholinergic neurons despite the massive reduction of cortical target neurons: 11. Neurochemical analysis. Exp. Neurol. I 1 7.139-150.

Morse, J.K., S.J. Wiegand, K. Anderson, Y. You, N. Cai, J. Carnahan, J. Miller. P.S. DiStefano, C.A. Altar, R.M. Lindsay, and R.F. Alderson ( 1993) Brain-derived neurotrophic factor (BDNF) prevents the degeneration of medial septal cholinergic neurons following fimbria transection. J. Neurosci. 13:4 146-4156.

Nonomura, T., C. Nishio, R.M. Lindsay, and H. Hatanaka (1995) Cultured basal forebrain cholinergic neurons from postnatal rats show both overlapping and non-overlapping responses to the neurotrophins. Brain Kes. 683:129-139.

Oppenheim, R.W.. L.J. Houenou, J .E. Johnson, L.F. Lin, L. Li,A.C. L0,A.L. Newsome, D.M. Prevette, and S. Wang (19951 Developing motor neurons rescued from programmed and axotomy-induced cell death by GDNF. Nature 373,344-346.

Perkins, LA., and L.D. Cain (1995) Basic fibroblast growth factor tbFGF) increases the survival of embryonic and postnatal basal forebrain cholinerec neurons in primary culture. Int. J . Dev. Neurosci. 13:51-61.

Kamakers, G.J.A.. and G.J. Boer (1991) Chronic suppression of bioelectric activity and cell survival in primary cultures of rat cerebral cortex: biochemical observations. Eur. J. Neurosci. 3154-161.

Rose, K.. M.P. Goldberg, and D.W. Choi (1993) Cytotoxicity in murine cortical cell culture. In C.A. Tyson and J.M. Frazier (eds): In Vitro Biological Methods. San Diego: Academic Press, pp. 46-60.

Saper, C.B. (1984) Organization of cerebral cortical afferents systems in the rat. I. Magnocellular basal nucleus. J . Comp. Neurol. 222313-342.

Schaar, D.G., B.A. Sieber, C.F. Dreyfus, and I.B. Black (1993) Regional and cell-specific expression of GDNF in rat brain. Exp. Neurol. 124:368-371

Schmidt-Kastner, K., A. Tomac, B. Hoffer, S. Bektesh, B. Rosenzweig, and L. Olson (1994) Glial cell-line derived neurotrophic factor (GDNFI mRNA upregulation in triatum and cortical areas after pilocarpine-induced status epilepticus in rats. Brain Res. Mol. Brain Res. 26:325-330.

Shelton, D.L., and L.F. Reichardt (1986) Studies on the expression of the nerve growth factor (NGF) gene in the central nervous system: level and regional distribution of NGF mRNA suggest that NGF functions as a trophic factor for several distinct populations of neurons. Proc. Natl. Acad. Sci. U.S.A. 83.2714-2718.

Smeyne, R.J., R. Klein, A. Schnapp, L.K. Long, S. Bryant, A. Lewin, S.A. Lira, and M. Barbacid 11994) Severe sensory and sympathetic neuropa- thies in mice carrymgdisrupted TrkiNGF receptor gene. Nature368:24& 249.

Sofroniew, M.V., R.C.A. Pearson, and T.B.S. Powell (19871 The cholinergc nuclei of the basal forebrain of the rat: normal structure, development and experimentally induced degeneration. Brain Res. 41 I:310-331

Sofroniew, M.V., N.P. Galletly, 0. Isacson, and C.N. Svendsen (1990) Survival of adult basal forebrain cholinergc neurons after loss of target neurons. Science 247.338-342.

Sofroniew, M.V., J.D. Cooper, C.N. Svendsen, P. Crossman, N.Y. Ip, R.M. Lindsay, F. Zafra, and D. Lindholm (1993) Atrophy but not death of adult septal cholinergic neurons after ablation of target capacity to produce mRNAs for NGF, BDNF, and NT3. J. Neurosci. 13.5263-5276.

Springer, J.E., X. Mu, L.W. Bergmann, and J.Q. Trojanowski (1994) Expression of GDNF mRNA in rat and human nervous tissue. Exp. Neurol. 127.167-170.

Svendsen, C.N., J .N. Kew, K. Staley, and M.V. Sofroniew (1994) Death of developing septal cholinergic neurons following NGF withdrawal in vitro: protection by protein synthesis inhibition. J. Neurosci. 14:75-87.

Tago, H., H. Kimura, and T. Maeda (19861 Visualization of detailed acetylcholinesterase fiber and neuron staining in rat brain by a sensitive histochemical procedure. J . Histochem. Cytochem. 34: 1431-1438.

Tomac, A., E. Lindqvist, L.F. Lin, S.O. Ogren, D. Young, B.J. Hoffer, and L. Olson I19951 Protection and repair of the nigrostriatal dopaminergic system by GDNP in vivo. Nature 373:335-339.

Umbriaco, D., K.C. Watkins, L. Descarries, C. Cozzari, and B.K. Hartman (19941 Ultrastructural and morphometric features of the acetylcholine innervation in adult rat parietal cortex: an electron microscopic study in serial sections. J . Comp. Neurol. 348,351-373.

Vantini, G., N. Schiavo, A. Di Martino, P. Polato, C. Triban, L. Callegaro, G. Toffano, and A. Leon ( 19891 Evidence for a physiological role of nerve growth factor in the central nervous system of neonatal rats. Neuron 3:267-273.

Weiss, J .H., H.Z. Yin, and D.W. Choi (1994) Basal forebrain cholinergic neurons are selectively vulnerable to AMPA/kainate receptor-mediated neurotoxicity. Neuroscience 60:659-664.

Wenk, H., V. Bigl, and U. Meger (1980) Cholinergic projections from magnocellular nuclei of the basal forebrain to cortical areas in rats. Brain Res. Rev. 2:295-316.

Wetmore, C., P. Ernfors, H. Persson, and L. Olson (1990) Localization of brain-derived neurotrophic factor mRNA to neurons in the brain by in situ hybridization. Exp. Neurol. 109:141-152.

Whitehouse, P.J., D.1,. Price, R.G. Struble, A.W. Clark, J .T. Coyle, andM.R. Delon (1982) Alzheimer's disease and senile dementia: loss of neurons in the basal forebrain. Science 215.1237-1239.

Whittemore, S.R., T . Ebendal, L. Larkfors, L. Olson, A. Seiger, I. Stromberg, and H. Persson (19861 Development and regional expression of beta nerve g o w t h factor messenger KNA and protein in the rat central nervous system. Proc. Natl. Acad. Sci. U.S.A. 83:817-821.

Williams, L.R., S. Varon, G.M. Peterson, K. Wictorin, W. Fischer, A. Bjorklund, and F.H. Gage (1986) Continuous infusion of nerve growth factor prevents basal forebrain neuronal death after fimbria fornix transection. Proc. Natl. Acad. Sci. U.S.A. 839231-9235.

Yan, Q., C. Matheson, and O.T. Lopez (1995) In vivo neurotrophic effects of GDNF on neonatal and adult facial motor neurons. Nature 373,341-344.

Documents

Cultured basal forebrain cholinergic neurons in contact with cortical cells display synapses, enhanced morphological features, and decreased dependence on nerve growth factor