Calcium homeostasis in cultured embryonic rat septohippocampal neurons is altered by ethanol and nerve growth factor before and during depolarization

BRAIN RESEARCH ELSEVIER

Brain Research 729 (1996) 176-189

Research report

Calcium homeostasis in cultured embryonic rat septohippocampal neurons is altered by ethanol and nerve growth factor before and during depolarization B. Webb

~,d S . S . S u a r e z c M . B . H e a t o n ~,d D . W . W a l k e r

a,b.d,*

" Department of Nearoscience, College of Medicine, UniversiO, of Florida, Gainesville, FL 32610 USA b V.A. Medical Center, Gainesuille, FL 32610, USA ~ Department of Anatomy, College of Veterinary Medicine, Cornell UniuersiO', Bhaca, NY 14853, USA a Universi~_' of Florida Brain Institute and Center for Alcohol Research, Box 100244, JHMHSC, Gaineseille, FL 32610-0244, USA

Accepted 26 March 1996

Abstract Ethanol and nerve growth factor (NGF) affect the survival of septohippocampal (SH) neurons. The effect of ethanol and NGF on calcium (Ca 2+) homeostasis in these neurons was investigated in this study. Changes in intracellular-free C a 2 + c o n c e n t r a t i o n ([Ca 2+ ]i) were measured using indo-1 in cultured embryonic (E21) SH neurons before stimulation (basal) and during stimulation with 30 mM potassium cloride (KCI+). SH neurons were treated with 0, 100, 200, 400, or 800 rag% ethanol with NGF ( + N G F ) or without NGF ( - N G F ) . NGF treatment decreased, while ethanol did not affect basal [Ca2+ ]~. The combination of ethanol and NGF treatment led to increases in basal [Ca 2+ ]i. While [Ca 2+ ]i was lower during stimulation with KC1 + following ethanol or NGF treatment, ethanol and NGF treatment together led to significantly greater increases or decreases in [Ca 2+ ]i compared to similarly treated NGF neurons. Responses of SH neurons were compared to those of medial septal (MS) neurons. Changes in [Ca2+ ]i during treatment with ethanol and/or NGF were reduced in SH neurons compared with MS neurons. We conclude that changes in Ca 2+ homeostasis can occur in SH neurons in the presence of ethanol and/or NGF. The changes following ethanol treatment are enhanced by NGF. By altering Ca 2+ homeostasis, NGF may enhance the survival of SH neurons during ethanol-induced neurotoxicity. Keywords: Intracellular-free calcium; Neurotrophin; Septohippocampal; Indo-1; Ethanol neurotoxicity; Nerve growth factor

1. Introduction The septohippocampal (SH) pathway in the rat is comprised of projections to the hippocampus from medial septal cholinergic and G A B A e r g i c neurons [2]. Damage to the septohippocampal pathway from chronic ethanol treatment (CET) has been implicated in deficient memory and learning in rodents [19] and this has been attributed to a cholinergic deafferentation of the hippocampus [1,32]. Deafferentation of the hippocampus is believed to occur following a loss of cholinergic neurons in the medial septum as a result of CET [3-5]. Ethanol has been shown to modify numerous processes involved in calcium (Ca 2+ ) signalling and cellular Ca 2+ homeostasis in neural cells [13,21,22,38,42,43,49]. These processes include those involved in Ca 2+ influx, efflux,

* Corresponding author. Department of Neuroscience, College of Medicine, University of Florida, Box 100244, JHMHSC, Gainesville, FL 32610-0244, USA. Fax: + 1 (352) 392-8347.

and intracellular mobilization. However, it is difficult to know to what extent any particular cellular effect of ethanol accounts for the effects observed at higher levels of neuronal integration [23]. Therefore, the decreased survival of SH neurons following CET may result from the effects of ethanol on the processes regulating Ca 2+ homeostasis in these neurons. The hippocampus and cortex contain the highest level of nerve growth factor (NGF) protein and its m R N A in the mature brain [58]. The fact that N G F is a target-derived protein and the hippocampus is the target tissue for SH neurons, suggests that N G F may be required for the survival, function and maintenance of rat basal forebrain cholinergic neurons [7,16,28]. How N G F enhances survival of some neuronal populations has not been determined. It appears that trophic factors, such as NGF, play an important role in neuronal Ca 2 + homeostasis [29,33]. Therefore, N G F may enhance the survival of neuronal populations by stabilizing [Ca :+ ]i during events which lead to a disruption of Ca 2+ homeostasis. Since the processes of Ca 2~ sig-

0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved PII S0006-8993(96)0041 9-2

B. Webb et al. / Brain Research 729 (1996) 176-189

nailing and cellular Ca 2+ homeostasis are among the targets for the effects of ethanol, it is possible that NGF contributes to Ca 2+ homeostasis in SH neurons during exposure to ethanol. NGF has been shown to have a moderate ameliorative effect on ethanol neurotoxicity in septal cultures [25]. Thus, the question of how Ca 2+ homeostasis is maintained in NGF-dependent SH neurons following ethanol exposure is of major interest. In this study we explore the ways in which NGF and ethanol affect [Ca 2+ ]i in cultured embryonic rat SH neurons. To identify the SH neurons in cultures of medial septal (MS) neurons, we retrogradely labelled medial septal projection neurons with a fluorescent tracer prior to culture [55]. Therefore, our cultures contained labelled (SH) and unlabelled MS neurons (mixed populations which could include unlabelled SH neurons). The results of our studies on the MS neurons are reported elsewhere [56]. Those results indicated that, in E21 rat MS neurons, ethanol or NGF stabilized Ca 2+ homeostasis by preventing increases in basal [Ca-,+ ]~. During high K+-stimulation either treatment increased [Ca2+] i. Together, ethanol and NGF increased basal [Ca2+]i and either decreased or did not change high K+-induced changes in [Ca 2+ ]i depending on the ethanol dose. The purpose of this study was; first, to determine the effect of ethanol or NGF on Ca 2+ homeostasis in unstimulated (basal) and high K+-stimulated SH neurons; second, to determine whether NGF modulates Ca 2+ homeostasis differently in unstimulated and high K+-stimulated SH neurons during ethanol treatment: and third, to compare these results with previous studies in MS neurons. Our hypotheses were: (1) treatment with ethanol alters Ca 2* homeostasis in SH neurons by causing transient and persistent changes in [Ca2-]i; (2) NGF modulates Ca 2+ homeostasis in SH neurons by stabilizing [Ca 2+ ]i; and (3) NGF modulates Ca 2+ homeostasis in SH neurons differently in the absence of ethanol than it does during ethanol treatment.

2. Methods

177

(95%) and carbon dioxide (5%). The Tyrode solution consisted of 125.6 mM NaC1; 3.1 mM KC1; 1.2 mM NaH2PO4; 2.0 mM Mg SO4,7H20; 25.9 mM N a H C Q ; 10.0 mM glucose; 1.9 mM CaCIz.2H20 in distilled water. This preparation is similar to that originally described for isolated chick spinal cord-hind limbs by Landmesser [35]. 2.2. Fluorescence labelling The procedure for the in vitro labelling of SH neurons was as previously described [55]. Briefly, the meninges were carefully removed from the brain surface and the hippocampi were exposed. Each hippocampus was injected 4 times with 0.1 ILl DiIC ~8(3) (1, l'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate, Molecular Probes). Following injection of the tracer, the fetal brain was maintained in Tyrode solution aerated with oxygen and carbon dioxide, at room temperature for 24 h. Then MS regions were dissected and prepared for culture. 2.3. General culture Details of these methods have been described previously [56]. Briefly, each MS region was placed into a vacutainer tube, incubated at 37°C for 20 rain, centrifuged, the solution removed, and replaced with culture medium. The tissue was dissociated and the dissociated neurons were plated. NGF (20 n g / m l ) was added to all cultures in order to facilitate the survival of SH neurons. The cultures were placed into a tissue culture incubator. After allowing 24 h for the neurons to adhere to the dish, an additional 1 ml of medium was added. Every 3 days, the culture medium in each dish was replaced with new medium containing NGF. After 6 days in culture, the medium was replaced and the cultures were divided into two groups: group I continued to receive NGF ( + NGF); group II was NGF-withdrawn and received 0.7 txg/ml anti-NGF ( - N G F ) . Previously, we have shown that this amount of anti-NGF is sufficient to block the action of NGF [54]. Alter 24 h the neurons were loaded with the fluorescent Ca 2- indicator dye indo-I and changes in [Ca 2 +]j at the cell body observed with a low light video system [51,56].

2.1. Animal model 2.4. In Htro ethanol exposure Brains from Long-Evans rat fetuses were removed on embryonic day 21 (E21). The day of insemination was considered to be embryonic day 0. Timed pregnant female rats (Charles River) were anesthetized with methoxyflurane and the uterus removed through a transverse abdominal incision. The intact uterus containing the fetuses was immersed in ice cold sterile saline. The female rat was euthanized by decapitation. Each fetus was individually removed from the uterus, freed from the amniotic sac, and decapitated. The entire brain was removed quickly and placed into an iced bath containing cold (4°C), sterile filtered, Tyrode solution (pH 7.4) aerated with oxygen

The culture dish containing the loaded neurons was secured on the microscope using soft dental wax. DiI labelled neurons were located in the field. An initial measurement of basal [Ca2+]i for each neuron was measured at room temperature and is referred to as the baseline [Ca~+]i for that neuron. The buffer for group I cultures was exchanged for buffer containing 20 n g / m l NGF. Group II cultures continued to be NGF-withdrawn but did not receive anti-NGF. Cultures from both groups received one of five treatments. An individual culture received either buffer lacking EtOH (0 rag%) or buffer with a

178

B. Webbet al. / Brain Research 729 (1996) 176-189

varying concentration (100, 200, 400, or 800 m g / d l ) of alcohol (95% ethanol, U.S.P.). Measurements of basal [Ca2+ ]i were taken as the buffer was exchanged (Start), at 1.6, 3.2, 4.8 and 6.4 s, and at 10, 30, 45 and 60 rain. At 60 min (which served as the new baseline [Ca 2+ ]i), the buffer was exchanged for new buffer containing the same amount of NGF, EtOH, and in addition 30 mM KC1. Measurements of high K+-stimulated changes in [Ca2+] i were taken during the buffer exchange (Start), at 1.6 and 3.2 s and at 5 and 10 min. The dilutions of EtOH used in this study were determined using an EtOH testing kit (Sigma Diagnostic). Cultures were tested for possible evaporation of EtOH during the experiment. EtOH levels of 100, 400, 800 mg% were tested at 0, 30, and 60 rain.

2.8. Validation of calcium measurements

The imaging system was validated using calibration solutions. The results indicated that a linear relationship existed between log [ ( R - R m i n ) / ( R m ~ X - R ) ] and log [Ca 2+] within the physiological range of [Ca 2~ ]i. The value for [Ca 2+ ]i in the neurons was determined using the formula [Ca 2+ ]i = Kd B(R - R min)/(Rm,× - R) [24,46,53]. Rmi n and Rm~X were determined from measurements taken in the cultured neurons at zero and maximum Ca 2+. Zero calcium was achieved by quenching the fluorescent signal in the neurons (addition of 10 mM Mn 2+ and 10 ~ M 4-bromo A-23187). High Ca 2+ levels were achieved by adding 10 ~ M 4-bromo A-23187 to buffer already containing 2 mM Ca 2+.

2.5. Calcium indicator loading 2.9. Statistical analysis

Details of these methods have been previously described [56]. Briefly, the neurons were incubated with the 2 p~M indo-1 AM/loading buffer. Extracellular dye was removed by rinsing and gentle agitation of the cultures with fresh loading buffer. Then the cultures were incubated for an additional 0.5 h at 37°C to allow for deesterification of the dye to it's Ca2+-sensitive form, indo-1. Complete hydrolysis of the AM ester was tested for immediately after the incubation period in some of the cultures and in different cultures at the end of the experiment.

Treatment effects of EtOH and NGF (and interactions) were analyzed by analysis of variance (ANOVA). When dependent variables were measured across time in the same culture, 'overall' treatment effects were determined by repeated measure ANOVAs followed by post-hoc comparisons within significant variables (Bonferroni/Dunn post-hoc test). Ca 2+ concentrations are reported as mean _+S.D. Percentage of control data is presented as + S.E.M. Significance level was P < 0.05.

2.6. Imaging system and optics 3. Results

Changes in levels of [Ca 2+ ]i were measured using 360 nm excitation of the Ca2+-sensitive dye, indo-1. Emission images at 409 nm (Ca2+-bound dye) and 490 nm (Ca2+-free dye) were acquired simultaneously using separate CCD cameras. The system consisted of a Zeiss Axiovert microscope; a xenon stroboscopic light source; two intensified CCD cameras; and two sets of two image digitization boards. The system has been described in greater detail elsewhere [51 ]. 2.7. Fluorescence data for indo-I

The fluorescence ratio data was interpreted as an indication of relative Ca 2+ increase or decrease. Calibration solutions were used to insure that the system was detecting Ca 2+ increases in the physiological range of [Ca2+]~. Sixty-four sequential paired images were collected or 10 paired images were collected and the averaged image used to determine the ratio R. To determine R, the mean background signal in the region surrounding the cell was subtracted from the mean signal obtained from the cell itself for each member of the image pair, F1 (obtained at 490 rim) and F2 (obtained at 405 nm). The mean background-subtracted signal from F2 was divided by that of FI to obtain the ratio R [56].

3.1. Basal intracellular calcium

To determine the effects of EtOH a n d / o r NGF on basal [Ca2+ ]i, a baseline ratio representing unstimulated [Ca 2+ ]i was acquired for each neuron measured. In order to control for individual differences in baseline [Ca 2+ ]i for the neurons studied, the data was normalized. Within each experiment, the ratio R representing [Ca 2+ ]i at each time poinl for an individual neuron (Rtime x) was divided by that neuron's baseline ratio representing the starting [Ca2+]~ (Rbaseline). The data in this study, reported as percentage of control, uses these values (Rtime x / R baseline)" The mean baseline level of Ca 2= in the SH neurons was determined by converting the ratio to the actual concentration of Ca 2+ [56]. [Ca2+]i is reported as mean S.D. The data from group I or + N G F SH neurons (no. of neurons n = 18) and group II or - N G F SH neurons (n = 14) were combined as there was no significant difference in baseline [Ca2+ ]i between the two groups (Student's t-test). Baseline [Ca2+] i was 98 4- 14 nM. 3.1.1. Effect of experimental protocol on [ C a 2 + ], Following a baseline measurement of [Ca2+] i in each neuron, the buffer was replaced using a transfer pipette.

B. Webb et al. / Brain Research 729 (1996) 176-189

Measurements of basal [Ca 2+ ]i were taken as soon as the buffer exchange was completed (start) and after 1.6, 3.2, 4.8, 6.4 s and 10, 30, 45, and 60 rain. Buffer exchange was completed within 30 s. Measurements from two to five cultures were averaged for analysis. The number of neurons measured varied from culture to culture and depended on the number of neurons located in a given field of study. To determine the effect of our experimental protocol on basal [Ca2+]i, buffer lacking EtOH (0 mg%) was added randomly to cultures of group II, - N G F SH neurons. There was a significant overall effect of experimental protocol ( P < 0.014) on [Ca2+]~ in these neurons in that simply replacing more of the same buffer increased [Ca 2~ ]i by 30% to 136.5 _+ 2 nM. This effect will be referred to as 'buffer replacement'. Significant treatment effects were determined by comparing individual treatment effects on [Ca2+ ]i with the effect of buffer replacement on [Ca 2+ ]i.

160%

-m-- 0 mg% EtOH +NGIF -"-'0 mg% EIOH -NGF I - o - - 2 0 0 rag% EtOH +NGF

(a)

140%

T

g r..) 120%

--T

t

ControlStert

24o% 1

+ - ~ l l

~

+-

1.6

3.2 Sec

4.8

6.4 Time

"T

r

~

10

30 Min

45

(b)

m0% EtOH -NGF

~100

m0% EIOH -NGF

200% o

te0%

r~ 100% e

140% 120% 100% -i

..... 7

Control

Stlrt

i 1.6 Sec

140%

-~ + 0 rag% EtOH +NGF 1 ~200 rag% EIOH +NGF

il/

- ~ -

i

3.2

5

10

Time

.

Min (C ) i

•

+ -i

i

T

120% 100% 80%

o n [ C a 2 + ]i

60

O

220%

160%

Each group II, - N G F culture randomly received replacement buffer containing one level of EtOH, either 100, 200, 400, or 800 rag% to determine the effect of EtOH on b a s a l [Ca2+]i . These levels of EtOH were chosen since they have been used for other experiments in our laboratory and so we could make comparison to these earlier studies [56]. Levels of 100 and 200 mg% EtOH represent EtOH levels that are similar to blood EtOH levels commonly measured in humans after consuming several alcohol containing drinks. While a blood EtOH level of 800 rag% is not normally observed, a blood EtOH level of 400 rag% is commonly observed in chronic alcoholics. There was no effect of EtOH treatment on basal [Ca 2+ ]i in these neurons. To be sure that the EtOH in our cultures did n o t evaporate from our buffer during the 60 min experimental period, the cultures were sealed with dental wax. The levels of EtOH in the culture dishes were also analyzed at O, 30, and 60 min. The results of these analyses showed that the initial levels of EtOH added to the cultures were accurate and that the levels of EtOH present in the cultures were stable for 60 min.

Baseline [Ca 2* ]i was measured in + NGF-treated neurons (group I neurons continuously cultured in the pres-

80%

240% 220%

3.1.2. Effect of EtOH on [Ca: +]i

3.1.3. Effect of NGF

100%

80%

179

+ Control

---7

i Start

T/I--

~

1.8

3.2

5

Sec

Time

MIn

----I 10

Fig. 1. Effect of nerve growth factor (NGF) and ethanol (EtOH) on [ C a 2 + ] i in cultured E21 rat septohippocampal (SH) neurons. After I week in culture in the presence of NGF (20 n g / m l ) , the medium was changed for medium containing NGF ( + NGF) or without NGF and with a monoclonal antibody to NGF ( - NGF) for an additional 24 h. Baseline {Ca2+ ]i was measured, a: the buffer was replaced with buffer lacking ethanol (0 rag% or - E t O H ) with or without 20 n g / m l NGF, or containing 200 mgCTc EtOH with NGF. Basal [Ca 2+ ]i was measured as the buffer was exchanged (start) and at 1.6, 3.2.4.8, 6.4 s and 10. 30, 45. 60 rain and expressed as ratio percentage of control (Rmn e x/Rba~eli,e)" NGF treatment (n = 6) prevented the overall rise in basal [Ca 2 ]i that was observed in similarly treated - N G F neurons (n = 5) ( P < 0.0001). Overall, treatment with 200 mgC~ + E t O H / + N G F (n = 4) increased [Ca2+ ]i compared to - E t O H / + NGF (n = 6) until 30 rain ( P < 0.0001 ). b: buffer was replaced with buffer containing 30 mM KCl and 100 mg% EtOH without NGF or lacking ethanol (0 mg% EtOH) with or without NGF. The [Ca 2. ]i in each neuron at the end of 60 min was used as the new baseline [Ca 2 + ]i for that neuron. Measurements of stimulated [Ca 2+ ]i were acquired as the buffer was changed (start) and at 1.6 and 3.2 s, and 5 and 10 min. High K+-stimulated [Ca 2+ ]i was significantly lower in 100 rage} + E t O H / - N G F neurons (n = 4) compared with 0 mg% - E t O H / - NGF-treated neurons (n = 4) ( P < 0.0001 ). Overall. high K+-stimulated [Ca2+ ]i was significantly increased in - E t O H / - N G F neurons (n = 4) compared to - E t O H / + NGF neurons (n = 3) until 5 min ( P < 0.04). c: buffer was replaced with buffer containing 30 mM KCl, 20 n g / m l NGF, containing 200 rag% EtOH, or lacking ethanol (0 rag% or - E t O H ) . Measurements were made as mentioned in lb. Overall, high K+-stimulated [Ca 2+ ]i was significantly higher in 200 m g ~ + E t O H / + NGF neurons ( n = 4) compared to E t O H / + NGF neurons ( P < 0.0001). Statistical analysis: ANOVA with Bonferoni/Dunn posthoc test.

180

B. Webb et al. / Brain Research 729 (1996) 176-189 180%

~

"*"400

(a)

rag% EtOH +NGF

140% i

Table 1 Summary of EtOH a n d / o r NGF effects on basal and high K %stimulated changes in [Ca 2+ ]i in SH neurons Treatment

120%

Effect on [Ca 2+

]i

Basal

Group 1: NGF-treated neurons

100%

+ NGF with ETOH EtOH concentration 0 rag%

80%

NC

100 mg% 00%

240%

I I Control Start

--

220%

~400 ~400

I 1.0

; 3.2 SOO

r 4.8

i II 8.4 Time

•

10

30 MIn

i

i

45

60

m0% StOH *NGF mg% EtOH -NGF

200 mg% 400 mg% 800 mg%

EtOH concentration 0 mg% 100 mg% 200 rag% 400 mg% 800 mg%

200% 180% 0

100% u

140% 120%

NC NC fi"

NC NC

Group Ii: NGF-withdrawn Neurons

(b)

o

~[ NC NC NC NC

NC NC NC

NC = no change in [Ca 2+ ]i- Overall significant treatment effects were determined by repeated measure ANOVA with Bonferroni/Dunn post-hoc test. Significance level was P < 0.05.

100% 80%

High K +

~-

i

Conlr~

Start

--~

1.6 SO¢

i I}

$.2 Time

T - - - I

S MIn

10

Fig. 2. Comparison of the effect of ethanol (EtOH) on [Ca 2+ ]~ in nerve growth factor treated ( + N G F ) and nerve growth factor withdrawn ( - NGF) cultured E21 rat septohippocampal (SH) neurons, a: Buffer was replaced with buffer containing 400 mg% EtOH with or without 20 n g / m l NGF. [Ca 2+ ]i measurements were acquired and expressed as described in Fig. la. Basal [Ca 2+ ]i was significantly decreased in the 400 mg% + E t O H / + N G F neurons ( n = 4 ) compared to the 400 rag% + E t O H / - N G F - t r e a t e d neurons ( n = 5) for 60 min ( P < 0.0001). b: Buffer was replaced with buffer containing 400 mg% EtOH and 30 mM KC1, with or without 20 n g / m l NGF. Measurements of stimulated [Ca2÷ ]i were acquired and expressed as described in Fig lb. Overall, high K+-stimulated changes in [Ca 2+ ]i was significantly decreased over 10 rain in 400 mg% + E t O H / + NGF neurons (n = 4) compared to 400 rng% + E t O H / - N G F neurons (n = 5) ( P < 0.0001). Statistical analysis: ANOVA with Bonferoni/Dunn post-hoc test.

ence of NGF). Then the buffer was replaced with buffer containing 20 n g / m l . IGF lacking EtOH. The ANOVA revealed a significant overall effect of NGF on basal [Ca2+] i in these neurons ( P <0.0001). + N G F neurons (no. of neurons, n = 6), did not increase b a s a l [ C a 2 + ] i , while [Ca 2+ ]i increased in group II, NGF-withdrawn neurons (n = 5) (Fig. la). These results indicate that NGF treatment can prevent increases in b a s a l [Ca2+] i under these experimental conditions. 3.1.4. Effect o f EtOH and N G F on [Ca2+] i in + NGFtreated SH neurons

To test whether the combined treatment of EtOH and NGF ( + E t O H / + NGF) would affect [Ca 2+ ]i differently than treatment with EtOH or NGF alone, baseline [Ca 2+ ]~ in + N G F neurons was measured. Then the buffer was replaced with buffer containing 20 n g / m l NGF and one of four levels of EtOH (either 100, 200, 400 or 800 rag%).

Significant overall treatment effects of EtOH were observed in + NGF neurons ( P < 0.0001). Post-hoc comparisons revealed that treatment with 100 mg% ( P < 0.0003), 200 mg% ( P < 0 . 0 0 0 1 ) and 800 rag% ( P < 0 . 0 0 0 1 ) + E t O H / + NGF increased basal [Ca 2÷ ]i above levels present before the buffer replacement. The increase in basal [Ca2÷ ]i was not dose-dependent. Treatment with 200 mg% EtOH resulted in the largest increase in [Ca 2+ ]i (Fig. la). The data for 100 mg% and 800 mg% EtOH are not shown. A significant interaction (ANOVA) occurred between EtOH and NGF treatment in this neuronal population ( P < 0.0054) which resulted in an increase in basal [Ca 2+ ]i within the first 10 min of treatment.

Table 2 Comparison summary of the effects of EtOH on basal and high K +stimulated changes in [Ca 2÷ ]i in NGF-treated compared with NGFwithdrawn SH neurons [Ca2+ ]i in NGF-treated neurons relative to [Ca 2+ ]i in NGF-withdrawn neurons Treatment

Basal

High K +

EtOH concentration 0 mg%

•

11

1O0 mg%

g

200 mg% 400 mg% 800 mg%

1~ g ND

]~ ND

= [ Ca2+ ]i in + N G F neurons > - N G F neurons. N D = [ C a 2+ ]i in + NGF neurons = - N G F neurons. Overall significant treatment effects were determined by repeated measure ANOVA with Bonferroni/Dunn post-hoe test. Significance level was P _< 0.05.

B. Webb et al. / Brain Research 729 (1996) 176-189

240%

•-~-'-I00 mo% EtOH +NGF ÷NGF +NGF --e--!

For all time points, the ratio R representing [Ca2+]~ for each neuron was divided by each neuron's ratio representing its new baseline (60 rain pre-high K + stimulation) [ Ca2+ ]i (Rti . . . . . /Rbaseline) and is referred to as percentage of control.

.,-~-200 m0% EtOH --~--&OO ma% EtQH

I

220%

200% •" 180%

lSI

i~

180% 140% 120% 100%

eo%

~

Control

. . . . . . .

Start

T

1,6 Soo

....

-4'

3.2 Time

5

10 MIn

Fig. 3. Comparison of the different ethanol (EtOH) treatment levels on high K+-stimulated changes in [Ca 2. ]i in +NGF-treated cultured E21 rat septohippocampal (SH) neurons. + N G F neurons were treated and basal [Ca 2+ ]i was measured as previously described in Fig. 1. Buffer was replaced with buffer containing 30 mM KCl, 20 n g / m l NGF, and 100 mg% EtOH. 200 mg% EtOH, 400 mg% EtOH, or 800 mg% EtOH. Overall, stimulated [Ca2+]i was significantly higher in 200 mg% + E t O H / + NGF neurons (n = 4) compared to 100 mg% + E t O H / + NGF or 400 mg% + E t O H / + NGF neurons. Statistical analysis: ANOVA with Bonferoni/Dunn post-hoe test (P < 0.0001).

3.1.5. Comparison of EtOH effects on [Ca2 + ]i in + N G F and - N G F SH neurons After determining the effect of + E t O H / + NGF on basal [Ca 2+]i, we compared + N G F - with -NGF-treated neurons that had received the same level of EtOH treatment. Based on our previous results, it was predicted that [Ca2+] i would be higher following EtOH treatment in +NGF-treated neurons, but this was not observed. Basal [Ca2÷] i was lower in NGF-treated than NGF-withdrawn neurons treated with 100 rag% ( P < 0.0023) and 200 rag% EtOH (p < 0.0296) and is not shown. Treatment with 400 rag% EtOH ( P < 0 . 0 0 0 1 ) resulted in lower [Ca2+] i in NGF-treated neurons compared with NGF-withdrawn neurons almost continually throughout the entire 60 min (Fig. 2a). In summary, NGF, but not EtOH, treatment prevented the rise in b a s a l [Ca2+]i observed in NGF-withdrawn neurons (Table 1). When SH neurons were exposed to + E t O H / + NGF, changes in Ca 2+ homeostasis occurred which resulted in an increase in basal [Ca 2+ ]i at early time points (Table l). These increases were still below the levels of [Ca 2 + ]i observed in + E t O H / - NGF neurons (Table 2). 3.2. High potassium stimulated changes in intracelhdar calcium In this study, the effect of EtOH a n d / o r NGF on high K+-stimulated changes in [Ca2+]i in the cell body of cultured E21 rat SH neurons was investigated. Previously studied SH neurons were stimulated after 60 rain with high K +. Measurements of [Ca 2+ ]i were made as the buffer was exchanged (start) and at 1.6, and 3.2 s and at 5 and 10 rain.

3.2.1. Effect of experimental protocol on stimulated [ Cu2 + ]i High K*-stimulated changes in [Ca2-], were determined by changing the buffer at 60 rain to replacement buffer containing 30 mM KC1 and applying it to group II - N G F SH neurons that had been treated similarly. [Ca 2. ]~ immediately increased to 665.4 + 44 nM after stimulation with 30 mM KCI for the entire 10-rain period (Fig. I B). 3.2.2. Effect of EtOH on stimulated/Ca 2 ~], To study the effect of EtOH on high K*-stimulated changes in [Ca 2+ ]i, buffer containing 30 mM KC1 and one of the four levels of EtOH (100, 200, 400, or 800 rag%) was added to group II - N G F SH neurons previously treated with the same EtOH level. There was an overall significant effect of EtOH treatment in these neurons ( P < 0.0001). Treatment with 100 rag% EtOH resulted in lower [Ca2~]i ( P < 0 . 0 0 0 1 ) compared to treatment with buffer lacking EtOH (Fig. lb). 3.2.3. Effect of N G F on stimulated [Ca: + ], The effect of NGF on high K+-stimulated changes in [Ca2+]i was studied by changing the buffer of group I + N G F neurons, at 60 rain from. + N G F / - E t O H to buffer containing 30 mM KCI and + N G F / - EtOH. There

240%

,

220% i _ 200%

•----100 rag% EtOH +NGF --::--100 mg% EtOH -NGF

o

~.

1,0%1 14o%

120% 100%

80% -1 Control

StIrt

1,6 Sec

3.2 Time

5

10

MIn

Fig. 4. Comparison of the effects of ethanol (EtOH) on high K+-stinmlated changes in [Ca 2+ ]i in + NGF and - N G F treated cultured E21 rat septohippocampal (SH) neurons. + NGF and - NGF neurons were treated as previously described in Fig. 1. The basal [Ca 2+ ]i in each neuron at the end of 60 min was used as the new baseline [Ca 2+ ]i for that neuron. Buffer was replaced with buffer containing 100 mg% EtOH and 30 mM KC1, with or without 20 n g / m l NGF. Measurements of stimulated [Ca2+ ]i were acquired and expressed as described in Fig 4. Overall, stimulated [Ca 2+ ]i was significantly higher in 100 rag% + E t O H / + NGF neurons (n = 4) compared to 100 rag% + E t O H / - NGF neurons for 10 min. Statistical analysis: ANOVA with Bonferoni/Dunn post-hoe test (P < 0.0001).

182

B. Webb et al. / Brain Research 729 (1996) 176-189

Table 3 Comparison summary of the effects of EtOH on basal and high K +stimulated changes in [Ca- ]i in SH neurons compared with MS neurons [Ca2+ ]i in NGF-treated SH neurons relative to [Ca2+ ]i in NGF-treated MS neurons Treatment

Basal

EtOH concentration 0 rag% 100 mg% 200 mg% 400 mg% 800 mg%

~ 1l ~ ~ II

High K +

ND ND

mg% EtOH ( P < 0.0001) and higher [Ca2*]~ compared with 400 mg% + E t O H / + NGF ( P < 0.0001) and 800 mg% + E t O H / + NGF ( P < 0.0001). Overall, treatment with 200 rag% + E t O H / + NGF resulted in higher levels of [Ca2+] i immediately after the buffer change which lasted for 10 min. Treatment with 200 mg% + E t O H / + NGF induced the highest rise in [Ca 2+ ]i observed during high K + stimulation in the + N G F neurons, although all EtOH treatments produced significant effects ( P < 0.0001) (Fig. 3).

ND

[Ca2+ ]i in NGF-withdrawn SH neurons relative to [Ca 2+ ]i in NGF-withdrawn MS neurons Treatment

Basal

High K +

EtOH concentration 0 mg% 100 mg% 200 mg% 400 rag% 800 mg%

ND ND ND ND ND

fl"

3.2.5. Comparison o f EtOH effects on stimulated [Ca2 + ]i in + N G F and - N G F - t r e a t e d SH neurons After determining the effect of + E t O H / + NGF on high K+-stimulated changes in [Ca2+] i in group I + N G F neurons, we compared the results of + NGF neurons with group II - N G F neurons that had received the same EtOH

ND ND ND 180%

=[Ca2+ ]i in SH neurons > MS neurons, ND = [ C a 2+ ]i in SH neurons = MS neurons. Overall significant treatment effects were determined by repeated measure ANOVA with Bonferroni/Dunn post-hoc test. Significance level was P < 0.05.

"-e--S. --o-MS

100 rag% E t O . +NGF 100 rag% EtOH +NGF

(a)

140%

i

120%

was a significant overall effect of NGF treatment on [Ca2+ ]i during high K + stimulation ( P < 0.0001). [Ca 2+ ]i increased 27% by 1.6 s and increased 61% (from 72.5 + 1.5 nM to 245.4+ 13.6 nM) by 10 min. The increase in [Ca2+] i in + N G F neurons (n = 3) was not as great ( P < 0.04) as that observed in -NGF neurons (n = 4) for the first 5 min (Fig. lb). 3.2.4. Effect o f EtOH and N G F on stimulated [Ca: +]i in + NGF-treated SH neurons We were interested in determining whether EtOH treatment had a different effect on high K+-stimulated changes in [Ca 2+ ]i when neurons were simultaneously treated with NGF. To test this, the buffer was changed after 60 min in group I + NGF neurons to buffer containing 30 mM KCI, 20 n g / m l NGF, and one of four EtOH treatments (100, 200, 400 or 800 rag%). This buffer was applied to group I + E t O H / + NGF neurons treated with a similar EtOH level and NGF during the previous 60 min. There was a significant overall effect of + E t O H / + NGF ( P < 0.0001) on high K+-stimulated [Ca 2+ ]i in + N G F neurons. When + E t O H / + N G F treatments were c o m p a r e d to E t O H / + NGF, 200 mg% + E t O H / + NGF increased high K+-stimulated [Ca 2+ ]~ ( P < 0.0001), but other levels of EtOH produced no significant effects ( P < 0.05) (Fig. lc). Comparison of the effects of the different levels of +EtOH/+NGF on high K+-stimulated changes in [Ca2+]~ showed that, overall, 100 mg% + E t O H / + NGF resulted in lower [Ca 2+ ]i compared to treatment with 200 -

100% i

80%

~

[

Control Start

180%t

[

I

I

1.8

3.2 Seo

4.8

I e/

r

8.4 Time

10

i ---" ~ SO MIn

I

45

O0

•"-~,--SH 200 mo% EtOH +NGF •- ~ - M 8 200 rag% EtOH +NGF

(b)

140% "6

_

120%

100%

80%

~--T-Control S t a r t

r

r

I

- i tl

I

1,6

3,2 Sic

4.8

6,4 Time

10

F

30

- ' 7

45

60

MIn

Fig. 5. Comparison of the effects of ethanol (EtOH) and NGF on [Ca 2+ ]i in +NGF-treated cultured E21 rat septohippocampal (SH) and medial septal (MS) neurons. + N G F SH and MS neurons were treated as described in Fig. 1. Buffer was replaced with experimental buffer containing 20 n g / m l NGF and either 100 mg% EtOH or 200 mg% EtOH. Basal [Ca2+ ]i measurements were acquired and expressed as described in Fig. 1. a: overall, basal [Ca 2+ ]i was significantly higher in the 100 mg% +EtOH/+NGF MS neurons ( n = 7 ) compared to 100 rag% + E t O H / + N G F SH neurons (n = 3) for 60 min ( P < 0.0001). b: overall, basal [Ca 2+ ]i was also significantly higher in the 200 mg% +EtOH/+NGF MS neurons ( n = l l ) compared to 200 rag% + E t O H / + NGF SH neurons (n = 4) for 60 rain ( P < 0.0001). Statistical analysis: ANOVA with Bonferoni/Dunn post-hoc test.

B. Webb et al. / Brain Research 729 (1996) 176 189 240%

o

(a)

-..B-SH 0 m0% EtOH -NGF 4 "~-MS 0 m0% EtOH -NGF

220%

3.3. Comparison of SH neurons with MS neurons

The effect of EtOH a n d / o r NGF treatment on basal (unstimulated) and high K+-stimulated [Ca2+ ]~ in E21 rat MS neurons was previously reported by our laboratory [56]. Ethanol or NGF prevented increases in basal [Ca 2+ ]i, while in combination, ethanol and NGF increased basal [ C a 2 + ] i . Ethanol or NGF increased [Ca :~ ]i during high K + stimulation while the combined treatment of ethanol and NGF either decreased or did not change [Ca2+]i depending on the ethanol dose. The MS neurons were unlabelled neurons from similar or the same cultures that contained DiI-labelled SH neurons. The MS neurons were studied under identical experimental conditions or at the same time the SH neurons were studied. Data from that

200%

4 100% 18o%

±

~_ 140% 120% 100% 80%

...... Start

r

Control

24o%

~ I # - - - - - T - 3.2 5 Time Mln

:i •.,.a.-SH 100 m0% EtOH -NGF

220,, ::::I~ M S o

T

1.8 Seo

(b)

100 m0% EtOH -NGF

]83

200% 100% ~1

i

a

T

F

240% ~ 220% ~

Z 140% 120% -

-

~

.

~

o

200% 180% ~

100%

L

00% ~ Control

- --

7. . . . . Start

10

1.6 See

8,2 Time

7

7

5 MIn

10

Fig. 6. Comparison of the effects of replacement buffer lacking ethanol (EtOH) and EtOH treatments on high K+-stimulated changes in [Ca2+ ]i in cultured E21 rat septohippocampal (SH) and medial septal (MS) neurons. - NGF SH and MS neurons were treated as described in Fig. 1. The basal [Ca 2 + ]i in each neuron at the end of 60 min was used as the new baseline [Ca 2 + ]i for that neuron. Buffer was replaced with buffer containing 30 mM KCI, without NGF, and either without EtOH (0 mg% or - E t O H ) or containing 100 rag% EtOH. Measurements of stimulated [Ca24 ]i were acquired and expressed as described in Fig lb. a: overall, stimulated [Ca -~ ], was significantly higher in the - E t O H / - NGF SH neurons (n = 4) compared to - E t O H / - NGF MS neurons (n = 19) for 10 rain (P < 0.0001). b: overall, stimulated [Ca 2+ ]i was significantly higher in the 100 rag% + E t O H / - N G F MS neurons (n = 10) compared to 100 mg% + E t O H / - N G F SH neurons ( n = 4 ) for 10 min ( P < 0.0005). Statistical analysis: ANOVA with Bonferoni/Dunn post-hoc test.

180% i ~.

(a)

---m--SH 0 mg% EtOH +NGF -~--M$ 0 rag% EtOH +NQF

T

T

Start

1.6 Sec

~

140% 120% 100% 80%

- Control

-

-I II

3,2 Time

-

--~-

. . . . . .

240% ~ --~-'-$H 400 m0% EtOH +NGF 220% ~ --o--MS 400 m0% EtOH ÷NGF -o 200%

7

5 Min

10

(b)

[

~o 10o%

L

180% o

~. 14o% 120%

treatment. Based on our previous results, it was predicted that EtOH would induce the highest levels of [Ca:+]~ during high K+ stimulation in + NGF compared to - NGF neurons. EtOH treatment induced overall significant differences in [Ca2+]~ in high K+-stimulated neurons treated with NGF compared to NGF-withdrawn neurons. There was a significant interaction between EtOH (100, 200, and 400 rag%) and NGF treatment ( P < 0 . 0 0 0 1 ) . Overall, treatment with 100 mg% (Fig. 4) and 200 rag% + E t O H / + NGF increased ( P < 0.0001) while 400 mg% + E t O H / + NGF decreased [Ca2+ ]i compared to similarly treated - N G F neurons ( P < 0.0001) (Fig. 2B). In summary, treatment of SH neurons with physiologically relevant levels of EtOH induced greater changes in Ca 2+ homeostasis during high K + stimulation when NGF was present (Table 2).

100% 80% Control

Start

1.8 Seo

3.2 Time

5 Min

10

Fig. 7. Comparison of the effects of ethanol (EtOH) a n d / o r NGF on high K+-stimulated changes in [Ca 2+ ]i in +NGF-lreated cultured E21 rat septobippocampal (SH) and medial septal (MS) neurons. + NGF SH and MS neurons were treated as described in Fig. I. The basal [Ca 2+ ]i in each neuron at the end of 60 min was used as the new baseline [Ca 2+ ]i for that neuron. Buffer was replaced with buffer containing 30 mM KCI, 20 n g / m l NGF, and without EtOH (0 mg% or - E t O H ) or with 400 mg% EtOH. Measurements of stimulated [Ca -`+ ]i were acquired and expressed as described in Fig lb. a: overall, stimulated [Ca 2+ ], was significantly higher in the - E t O H / + N G F MS neurons (n = 11) compared to - E t O H / + NGF SH neurons (n = 3) for 5 min ( P < 0.047). b: overall, stimulated [Ca 2+ ]i was also significantly higher in the 400 mg% + E t O H / + N G F MS neurons ( n = 2 0 ) compared to 400 m g ~ + E t O H / + NGF SH neurons (n = 4) for 10 min ( P < 0.0005). Statistical analysis: ANOVA with Bonferoni/Dunn posbhoc test.

184

B. Webb et al. / Brain Research 729 (1996) 176 189

study were compared to data from this study to determine: (1) whether SH neurons were more sensitive to EtOH a n d / o r NGF treatment than unlabelled MS neurons; and (2) whether Ca 2+ homeostasis was affected differently by EtOH a n d / o r NGF treatment in the two neuronal populations.

3.3.1. Comparison of effects on basal [Ca: +]i The mean baseline [Ca2+] i was 6 6 + 14 nM for MS neurons (n = 246) and 98 + 14 nM for SH neurons (n = 32). The data from MS neurons were combined from group I + N G F and group II -NGF MS neurons as it had been combined in the group I + NGF and group II - N G F SH neurons, because there was no significant difference in baseline [Ca2+] i between the two groups. No significant difference was found in baseline [Ca 2+ ]~ between MS and SH neurons (Student's t-test). There was a significant effect of NGF treatment on basal [Ca2+]i in both SH and MS neurons ( P < 0.0001). Significant effects of NGF on [Ca 2+ ]i were also observed in SH neurons treated with 100 rag% ( P < 0.0023), 200 rag% ( P < 0.03), and 400 mg% EtOH ( P < 0.0001) and in MS neurons treated with 100 rag% ( P < 0.0001), 200 mg% ( P < 0.0004), 400 mg% ( P < 0.034) and 800 mg% EtOH ( P < 0.0008). The interaction between EtOH and NGF was significant in SH neurons ( P < 0.0054) and MS neurons ( P < 0.0001) and resulted in higher levels of basal [Ca2 + ]i. The effects of buffer replacement and EtOH treatments on basal [Ca2+]i were compared in + N G F and - N G F treated neurons from the two neuronal populations. There were no significant differences in [Ca2+]i in - N G F / S H and - N G F / M S neurons (Table 3). When the effects of control and EtOH treatments were compared in + N G F / S H and + N G F / M S neurons, there were overall significant differences in basal [Ca2+]i with 0 mg% ( P < 0.0001), 200 rag% ( P < 0.0001), 400 rag% ( P < 0.0008), and 800 mg% ( P < 0.0054) EtOH treatment. [Ca 2+ ]~ was lower in + N G F / S H compared to + N G F / M S neurons during all treatments (Fig. 5a, Table 3). These results suggest that NGF modulated Ca 2+ homeostasis differently in SH and MS neurons during all treatments. 3.3.2. Comparison of effects on high K+-stimulated changes in [Ca 2 +]i There was a significant effect of NGF treatment on high K+-stimulated changes in [Ca2+]i in SH ( P <0.04) and MS neurons ( P < 0.0001), Significant effects of NGF on stimulated [Ca2+] i were also observed in SH neurons treated with 100, 200 and 400 mg% EtOH ( P < 0.0001) and in MS neurons treated with 200 rag% ( P < 0.0001), 400 mg% ( P < 0.0001) and 800 mg% EtOH ( P < 0.0033). The interaction between EtOH and treatment with NGF was significant in both neuronal populations ( P < 0.0001). The effects of buffer replacement and EtOH treatments on high K+-stimulated changes in [Ca 2+ ]~ were compared

in + N G F and -NGF-treated neurons from the two neuronal populations. There were overall significant differences in stimulated [Ca2+]i when - N G F / S H and - N G F / M S neurons were compared during treatment with 0 mg% ( P < 0.0001) and 100 rag% ( P < 0.0005) EtOH. Stimulated [Ca2+]i was higher in SH neurons during control treatment but lower during treatment with 100 rag% EtOH than in MS neurons (Fig. 6a,b). When the effects of control and EtOH treatments on high K*-stimulated changes in [Ca 2 +li were compared in + N G F / S H and + N G F / M S neurons, overall, [Ca2+] i was significantly lower during 0 rag% ( P < 0.047) and 400 rag% ( P < 0,0001) EtOH treatment in + N G F / S H neurons (Fig. 7a,b, Table 3).

4. Discussion The experiments described here explore questions concerning the effect of EtOH and NGF treatment on Ca 2+ homeostasis in cultured E21 rat SH neurons. We observed that NGF, but not EtOH, prevented changes in basal [Ca2+] i that occurred in SH neurons under our culture conditions. When SH neurons were treated with EtOH in the presence of NGF, a significant interaction between the two treatments resulted in the elevation of basal [Ca 2+ ]i (Table 1). Since neuronal survival is greatly influenced by electrical activity during certain stages of development, the way in which EtOH and NGF treatment would affect [Ca2+ ]i during high K + stimulation was investigated. High K + stimulation resulted in a rapid increase in [Ca2+],. EtOH or NGF treatment resulted in less of a change in stimulated [Ca 2+]i. During high K + stimulation, a significant interaction occurred between the two treatments which further altered Ca 2+ homeostasis (Table 2). The SH pathway is considered to play an essential role in the formation of many types of memory [44]. CET in rodents can result in a progressive decline in learning and memory [19] and chronic ethanol abuse in humans is known to result in brain damage and associated functional abnormalities. Chronic alcoholism with dementia is accompanied by a degeneration of the cholinergic neurons in the basal forebrain (SH neurons) [3,4,6]. The mechanism underlying this effect is unknown. Following fimbria/fornix lesions [18] in the rat, there is a loss of cholinergic projection neurons and a downregulation of phenotype-specific genes which can be reversed following intraventricular injection of NGF [18,27,59]. To study the role that trophic factors may have on survival and growth of SH neurons, our laboratory developed a technique to identify projection neurons of the SH pathway [55]. In this study SH neurons were retrogradely labelled with DiI using that technique. During buffer replacement, NGF-deprived neurons increased basal [Ca2+]i from 98 _+ 14 nM to 136.5-t-2 nM during 60 min. Other investigators have shown that tran-

B. Webb et al. / Brain Research 729 (1996) I76-189

sient or longlasting increases in [Ca 2* ]i often follow cellular disturbances [8-10]. This change in Ca ,"+ homeostasis may have been the result of cellular damage occurring from: repeated exposure to ultraviolet excitation, the duration of the experiment, a change in pH, or the accumulation of harmful metabolic products resulting from metabolism of indo-l. We interpret the rise in [Ca2+]i in SH neurons to be a response to the disturbance of normal cellular activities from a yet to be defined source. NGF ameliorated this effect in SH neurons by stabilizing [Ca 2+ ]i (Fig. 1A). 4.1. The e ~ ' c t o f EtOH or N G F treatment on basal [ C a 2 + I,

Treatment with EtOH did not alter Ca 2+ homeostasis in NGF-deprived neurons. A lack of effect of EtOH in this study may be explained by the fact that the neurons were treated acutely with ethanol (AET). AET affects the mechanisms involved in regulating Ca 2+ homeostasis differently than does CET [12,22,42]. Longer periods of EtOH treatment may be required before changes in Ca 2+ homeostasis can be observed. The embryonic neurons in this study could also be more resistant to the effects of EtOH than older, more mature neurons [57]. NGF stabilized basal [Ca 2+ ]i in SH neurons (Fig. 1A). This indicates that, under certain conditions, the action of NGF is to prevent [Ca z+ ]i from increasing. This action of NGF could explain the dependency that SH neurons have on NGF for their survival and maintenance. SH neurons may need NGF in order to regulate basal [Ca2+]i thereby maintaining Ca 2+ homeostasis during changing environmental conditions. In other NGF-dependent neuronal populations, it has been found that young dependent neurons have lower [Ca2+]i than older less NGF-dependent neurons. NGF apparently functions to maintain Ca 2+ at its optimal level in these NGF-dependent neurons [29,33]. At optimal intracellular concentration, Ca 2+ can lead to neurite outgrowth in some neurons [30,36,40], while higher levels, for extended periods, can induce severe pathological damage [9,10,31]. While NGF functions to enhance neuronal survival and neurite outgrowth [41], it also protects CNS neurons from toxic insults by stabilizing intracellular Ca 2~ [8,10,26,34]. SH neurons have a relatively low basal [Ca2+]~ indicating that they may also have problems regulating Ca :~. NGF may protect SH neurons from Ca 2 t-mediated neuronal damage by maintaining basal

[Ca2~ ]i. 4.2. The e~'ect (?[" E t O H and N G F treatment on basal [Ca2 + ]i in + NGF-treated neurons

NGF-treated SH neurons responded to the combined treatment of EtOH and NGF with elevations in [Ca 2' ]i. The mechanism(s) of action of acute EtOH and NGF on Ca 2+ homeostasis in SH neurons is unknown. Previous

185

studies on the modulation of EtOH neurotoxicity by NGF in chick dorsal root ganglion (DRG) neurons showed that NGF protected against the suppression of neurite outgrowth by EtOH, and enhanced survival by effectively raising the threshold for EtOH neurotoxicity [26]. In EtOH-treated rat septal neurons, NGF enhanced process outgrowth but did not enhance survival [25]. CET may suppress neurite outgrowth and survival by altering [Ca 2÷ ], at the growth cone or cell body. NGF may ameliorate the neurite-suppressing and decreased-survival action of EtOH by stabilizing basal [Ca 2+ ]~ at the higher levels necessary for neurite outgrowth. AET causes a decrease in Ca 2' influx and effiux by inhibiting NMDA channels; voltage sensitive Ca 2 + channels (VSCC) [14,42], usually of the L-type [38]; Na ~ / C a 2 exchange [43]; and Ca2+/Mg2*-ATPase activity [21,22] These cellular mechanisms are involved in the maintenance of Ca 2+ homeostasis in neurons. In the SH neurons we studied, [Ca 2" ]~ was measured at the cell body, which represents the integrated action of all of the cellular components that regulate [Ca 2+ ]~. There was no clear dose-dependent response to EtOH treatment. While EtOH clearly acts on Ca2+-regulating mechanisms [20], its effect on the individual components that maintain [Ca2*]~ at a critical level is complex. This may be related to the fact that different cellular mechanisms involved in Ca 2+ homeostasis may have different sensitivities and time courses for the inhibitory action of EtOH. 4.3. The effect o f EtOH or N G F treatment on high K ~stimulated changes in [Ca 2 + ]i

The neurotoxic effects of EtOH may be targeted at events which occur at the neuronal cell body or at the level of synaptic activity. This study concerned the possible implications of the effect of EtOH on depolarization-induced changes in C a 2+ homeostasis at the cell body of SH neurons. Although stimulation with 30 mM KC1 is not a physiological signal experienced by SH neurons during normal synaptic activity [11], this type of stimulation allowed us to look at the effects of EtOH and NGF on [Ca2+ ]i when the SH neurons are experiencing a sustained elevation in [Ca2+]~. K*-induced depolarization of NGFdeprived SH neurons increased [Ca :+ ]i to 665.4 _+ 44 nM. Overall, EtOH tended to inhibit the change in [Ca 2+ ]i following high K + stimulation of NGF-deprived SH neurons, although there was no clear dose-dependent effect of EtOH on stimulated [Ca 2+ ]i. This observation is consistent with what is known about the inhibitory effect of AET on VSCCs [23,38]. Nothing is known about the direct effect of NGF on [Ca2+]i during high K + in cultured SH neurons. In the present study, NGF-treated neurons responded to stimulation with 30 mM KC1 by increasing [Ca :+ ]i although the increase in [Ca2+]i was not as great as that observed in NGF-deprived neurons until after 10 min (Fig. I B). NGF

186

B. Webb et al. / Brain Research 729 (1996) 176 - 189

may act to slow rapid changes in [Ca2+ ]i, thereby ameliorating adverse effects that rapid rises in [Ca 2+ ]i may cause. This may allow neurons to experience sustained levels of [Ca2+ ]i without experiencing the neurotoxicity which often follows elevations in [Ca2+]i. Similar responses to those observed in this study occurred in sympathetic neurons stimulated with high K + in the presence of NGF [45], suggesting that the high K+-induced elevation of [Ca2+]i was not producing toxic effects when NGF was present. We hypothesize that the delayed elevation of [Ca 2+ ]i was a consequence of an NGF-dependent neuroprotective mechanism. 4.4. The effect o f E t O H and N G F treatment on high K +-stimulated changes in [Ca 2 + ]i in + N G F - t r e a t e d neurons.

While the action of EtOH or NGF tended to inhibit changes in [Ca2+]i at the cell body in NGF-deprived SH neurons during K+-induced depolarization, the interaction of EtOH and NGF further increased or decreased [Ca 2+ ]i depending on the dose of EtOH used. Physiologically relevant levels of EtOH and NGF increased stimulated [Ca2+] i while the higher doses lowered [Ca2+] i. During normal cellular activity, neurons respond to depolarization with changes in [Ca2+] i. In neurons, failure to achieve ideal levels of [Ca2+] i necessary for Ca2+-dependent mechanisms to occur, may affect normal cellular processes, such as neufite outgrowth and neuronal survival. In this study, we observed a reduction in [Ca2+]i in EtOHtreated SH neurons during high K + stimulation. If SH neurons respond similarly to EtOH exposure during normal activity, Ca2+-dependent processes necessary for the maintenance and survival of this neuronal population may be adversely affected. These results are consistent with the hypothesis that NGF protects neurons from neurotoxicity by maintaining Ca 2+ homeostasis during cellular activity. In future experiments we will determine the specific cellular Ca2+-dependent processes which are the targets for the action of EtOH and NGF. Our data suggest that NGF affects Ca 2+ homeostasis differently in neurons during EtOH exposure (or other neurotoxic insults) than during normal conditions. This could involve two different transduction pathways or EtOH may interfere with the ability of NGF to modulate neuronal Ca 2+ homeostasis. Several mechanisms that are compatible with our data can be proposed to explain how NGF and EtOH may be interacting. First, NGF may negate the inhibitory action of EtOH on L-type Ca 2+ channels or stabilize the events which occur following EtOH-induced membrane disorder [48]. Second, NGF may have a direct affect on Ca 2+ channels in SH neurons. The effective number of functional Ca 2+ channels [47] and the proportion of current carried through L-type Ca 2+ channels [50] can be influenced by NGF. Third, EtOH and NGF may have common effects on second messengers such as cyclic AMP (cAMP) {37,39] and inositol phosphate (IP). EtOH

treatment can increase [Ca2+]i in neurons by increasing cAMP and IP turnover [19], thereby, facilitating an increase in [Ca 2~-]i [13,49]. NGF also increases cAMP levels [45,52]. While the action of EtOH may be altered in the presence of NGF, conversely, NGF's action could be altered by the presence of EtOH. NGF binds to the p75 receptor and to the tyrosine kinase receptor (trkA). The binding of NGF to trkA induces rapid changes in protein phosphorylation and gene expression [15]. EtOH could alter second messenger systems [37,39] that regulate kinase activity. EtOH may also interfere with G-protein signal transduction pathways that play a role in the receptor-induced modulation of neuronal VSCCs and in the signal transduction pathway of neurotrophins [17]. This might explain how treatment with NGF prevents increases in [Ca 2. ]~ in the absence of EtOH while [Ca 2+ ]i increases immediately in NGF-treated SH neurons when EtOH is present. 4.5. Comparison o f S H neurons with medial septal neurons

Our dissociated medial septal cultures contained two distinguishable populations of neurons, DiI-labelled and unlabelled neurons. The unlabelled neurons could have been SH neurons which had not become labelled with Dil, interneurons with local projections, or a population of neurons with projections to other areas of the brain, such as the cortex. In this study, the effects of EtOH a n d / o r NGF on Ca 2~ homeostasis in DiI-labelled SH neurons were compared with the effects previously obtained by us lbr unlabelled MS neurons [56]. The comparison of NGF-deprived neurons indicated that there were no significant differences in baseline [Ca 2+ ]i in the two neuronal populations. EtOH prevented increases in [Ca2+]i in MS neurons, but not in SH neurons. NGF did prevent the rise in basal [Ca2+] i in a similar manner in both MS and SH neurons. The SH neurons response to NGF treatment resulted in lower [Ca 2+ ]~ compared to MS neurons. This suggests that NGF may play a greater role in the regulation of Ca 2+ homeostasis in SH neurons and is consistent with other reported observations that NGF is required for the survival and maintenance of these neurons [7,16,28]. What is new from the results of this study, is the indication that the survival-promoting activity of NGF may involve the modulation of Ca 2~- homeostasis, NGF and EtOH interacted to increase [Ca 2+]i in both SH and MS neurons, with the highest [Ca 2+ ]i occurring in MS neurons (Fig. 5A,B). Our results indicated that MS neurons were more sensitive to EtOH treatment, while SH neurons were more sensitive to NGF treatment. It will be necessary to design future experiments to investigate how these two sensitivities are weighted during the interaction and what effect the increase in [Ca2+]~ has on neuronal function. During high K ÷ stimulation, NGF-deprived SH neurons responded with greater changes in [Ca 2+ ]i than did NGFdeprived MS neurons (Fig. 6A). These results are difficult

B. Webb et al. / Brain Research 729 (1996) 176-189

to interpret. In our previous study, the N G F - d e p r i v e d MS neurons failed to respond to high K + stimulation with an additional increase in [Ca 2+ ]i [56]. [Ca 2+ ]i increased from 66 _+ 14 nM to 276 _+ 77 nM within the first 60 min prior to K + stimulation, and destabilization of Ca R+ homeostasis probably occurred in those neurons. Therefore, the MS neurons may have been unable to respond to high K + stimulation with additional increases in [Ca2+] i. EtOH affected high K+-stimulated changes in [Ca 2+ ]i in NGFdeprived MS neurons by increasing [Ca R+ ]i above control, while similar treatment resulted in [Ca2+]i levels below control in N G F - d e p r i v e d SH neurons (Fig. 6B). This implies that SH neurons are more sensitive to the inhibition of changes in [Ca2+] i by EtOH during high K + stimulation. Therefore, EtOH may affect those processes that require changes in [Ca2+]i in SH neurons during normal neuronal activity, such as neurotransmitter release and the maintenance of synaptic efficacy. This could have profound effects on the survival of these neurons. N G F affected high K+-stimulated changes in [Ca 2+ ]i by slowing the increase in [Ca 2+ ]~ in SH neurons but not in MS neurons (Fig. 7A). This observation is consistent with our hypothesis that N G F protects neurons from neurotoxicity by maintaining Ca 2+ homeostasis during celllular activity and that SH neurons are more responsive to N G F than MS neurons. The interaction between N G F and EtOH treatments led to higher [Ca2+]i during high K + stimulation in MS neurons compared with SH neurons (Fig. 7B). While N G F was able to reverse the inhibitory action of EtOH at lower EtOH levels, it was unable to do so during treatment with higher EtOH levels. The response of the SH neurons could be due to a high sensitivity to EtOH inhibition. The failure of N G F to ameliorate this potentially neurotoxic effect of EtOH in SH neurons, could affect the ability of SH neurons to maintain appropriate synaptic connections with target neurons. In this manner, the normal targets of the SH neurons, hippocampal neurons, could be adversely affected by EtOH treatment indirectly. The SH neurons were labelled with DiI. In a previous study, DiI did not appear to interfere with normal cellular processes [55]. Nevertheless, we cannot rule out the possibility that the presence of Dil within the plasma membrane of SH neurons altered their response to treatment with EtOH a n d / o r NGF. DiI is lipid soluble and may affect the response of proteins within the membrane, such as receptors, G-proteins and second messengers, to specific stimuli. It is unlikely that there was any interaction between Dil fluorescence and indo fluorescence in this study. The excitation for indo was at 360 nm with emissions at 409 and 490 nm, while the excitation for DiI was at 510 nm with emission at 590 rim. 4.6. S u m m m T

Current research indicates that N G F and EtOH act on several cellular mechanisms actively involved in the regu-

187

lation of cytoplasmic Ca 2+. In this study, the effect of EtOH and N G F on Ca R+ homeostasis was investigated in cultured SH neurons by measuring changes in basal and high K+-induced [Ca 2+ ]i at the cell body. N G F maintained Ca 2+ homeostasis by preventing increases in basal and high K+-stimulated changes in [Ca2+]i. EtOH had no effect on basal [Ca~-+] i. EtOH did act in a manner similar to N G F during high K + stimulation by inhibiting the change in [Ca2+] i. Together, EtOH and N G F increased basal [Ca2+]i and altered high K % i n d u c e d [Ca2+]i . The Ca2+-dependent processes that were mediating these responses are yet to be determined. The results of this study show that EtOH and N G F affect Ca 2- homeostasis differently in SH neurons than other MS neurons. Determining the ways in which EtOH and N G F act in concert to influence homeostasis in cultured SH and MS neurons could greatly extend our understanding of how trophic factors protect neurons from the neurotoxic effects of substances and events which disturb normal cellular processes.

Acknowledgements W e thank Dr. Eugene Johnson, Jr. for his gift of NGF, Stephen M. Varosi for technical assistance with the image analysis, and Xiaobing Dai for assistance with the calcium imaging and system calibration. This research has been supported by the Medical Research Service of the Department of Veterans Affairs, N I A A A grants AA00200, AA09128, and AA10480. B. W e b b was supported by a predoctoral Fellowship from the University of Florida Center for Neurobiological Sciences (PHS Grant T 32 M H I 5 7 3 7 A ) and is currently supported by an individual predoctoral Fellowship grant 1F31AA05372-01 from NIAAA.

References [1] Abraham, W.C., Hunter, B.E., Zornetzer, S.F. and Walker. D.W., Augmentation of short-term plasticity in CA1 of rat hippocampus after chronic ethanol treatment. Brain Res., 221 ( 1981) 271-287. [2] Amaral, D.J. and Kurz, J., An analysis of the origins of the cholinergic and noncholinergic septal projections to the hippocampal tbrmation in the rat, J. Comp. Neurol., 240 (1985) 37-59. [3] Antuono, P., Sorbi, S., Bracco, L., Fusco, T. and Amakucci, L.A.. Discrete sampling technique in senile dementia of the alzheimer type and alcoholic dementia: Study of the cholinergic system. In L Amaducci, A.N. Davison and P. Antuono (Eds.), Aging of the Brain and Dementia, Vol. 13, Raven Press, New York, 1980, pp. 151-158. [4] Arendt. T., Allen, Y., Marchbanks, R.M., Schugens, M.M., Sinden. J., Lantos, P.L. and Gray, J.A., Cholinergic system and memory in the rat: effects of chronic ethanol, embryonic basal forebrain brain transplants and excitotoxic lesions of cholinergic basal forebrain projection system, Neuroscience. 33 (1989) 435-262. [5] Arendt, T., Hennig, D., Gray, J.A. and Marchbanks, R., Loss of neurons in the rat basal forebrain cholinergic system after prolonged intake of ethanol, Brain Res. Bull.. 21 (1988a) 563-570.

188

B. Webb et al. / Brain Research 729 (1996) 176-189

[6] Arendt, T., Allen, Y., Sinden, J., Schugens, M.M., Marchbanks, R.M., Lantos, P.L. and Gray, J.A., Cholinergic-rich brain transplants reverse alcohol-induced memory deficits, Nature, 332 (1988b) 448450. [7] Arimatsu, Y. and Miyamoto, M., Survival-promoting effect of NGF on in vitro septohippocampal neurons with cholinergic and GABAergic phenotypes, Dec. Brain Res., 58 (1991) 189-201. [8] Cheng, B. and Mattson, M.P., NGF and bFGF protect rat hippocampal and human cortical neurons against hypoglycemic damage by stabilizing calcium homeostasis, Neuron, 7 (1991) 1031-1041. [9] Cboi, I.W., Cerebral hypoxia, some new approaches and unanswered questions, J. Neurosci., 10 (1990) 2493-2501. [10] Choi, D.W., Calcium mediated neurotoxicity: relationship to specific channel types and role in ischemic damage, TINS, 11 (1988) 465469. [1 l] Cohen, L.B., Salzberg, B.M., Davila, H.V., Ross, W.N., Landowne, D., Waggoner, A.S. and Wang, C.H., Changes in axon fluorescence during activity: Molecular probes of membrane potential, J. Membrane Biol., 19:1 (1974) 1-36. [12] Daniell, L.C. and Harris, R.A., Effect of chronic ethanol treatment and selective breeding for hypnotic sensitivity to ethanol on intracellular ionized calcium concentration in synaptosomes, Alcohol. Clin. Exp. Res., 12 (1988) 179. [13] Daniell, L.C. and Harris, R.A., Ethanol and inosital 1,4,5-trisphosphate release calcium from separate stores of brain microsomes, J. Pharmacol. Exp. Ther., 250 (1989) 875. [14] Dildy-Mayfield, J.E., Machu, T. and Leslie, S.W., Ethanol and voltage-or receptor-mediated increases in cytosolic Ca 2+ in brain cells, Alcohol, 9 (1991) 63-69. [15] Downen, M., Mudd, L., Roback, J.D., Palfrey, H.C. and Wainer, B.H., Early nerve growth factor-induced event in developing rat septal neurons, Dev. Brain Res., 74 (1993) 1-13. [16] Ernfors, P., Wictorin, A., Bjorklund, L.R., Williams, L.R., Varon, S. and Gage, F.H., Amelioriation of cholinergic atrophy and spatial memory in aged rats by nerve growth factor, Nature, 329 (1987) 65-68. [17] Ewald, D.A., Sternweis, P.C. and Miller, R.J., Guanine nucleotidebinding protein Go-induced coupling of neuropeptide Y receptors to Ca 2+ channels in sensory neurons, Proc. Natl. Acadl. Sci., 85 (1988) 3633-3637. [18] Fischer, W. and Bjorklund, A., Loss of ACHE- and NGFr labeling precedes neuronal death of axotomized septal-diagonal band neurons: Reversal by intraventricular NGF infusion, Exp. Neurol., 113 (1991) 93-108,. [19] Freund, F. and Walker, D.W., Impairment of avoidance learning by prolonged ethanol consumption in mice, J. Parmacol. Exp. Ther., 179 (1971) 284-292. [20] Gandhi, C.R. and Ross, D.H., Influence of ethanol on calcium, inositol phospbolipids and intracellular signalling mechanisms, Experimentia, 45 (1989) 407-413. [21] Gandhi, C.R. and Ross, D.H., Characterization of high-affinity MgZ+-independent CaZ+-ATPase from rat brain synaptosomal membranes, J. Neurochem., 50 (1988) 248-256. [22] Garret, K.M. and Ross, D.H., Effects of in vivo ethanol administration on Ca2+/Mg2+-ATPase and ATP-dependent Ca 2+ uptake activity in synaptosomal membranes, Neurochem. Res., 8 (1983) 1013-1028. [23] Greenberg, D.A. and Chan. A.W.K., Ethanol, calcium channels, and intracellular calcium in cultured neural cells. In R.R. Watson (Ed.), Alcohol and Neurobiology; Receptors, Membranes and Channels, CRC Press, Boca Raton, 1992, pp. 141-160. [24] Grynkiewicz, G., Poenie, M. and Tsien, R.Y., A new generation of Ca 2+ indicators with greatly improved fluorescence properties, J. Biol. Chem., 260 (1985) 3440. [25] Heaton, M.B., Paiva, M., Swanson, D.J. and Walker, D.W., Responsiveness of cultured septal and hippocampal neurons to ethanol and neurotrophic substances, J. Neurosci. Res., 39 (1994) 305-319.

[26] Heaton, M.B., Paiva, M., Swanson. D.J. and Walker, I).W., Modulation of ethanol neurotoxicity by nerve growth factor, Brain Res.. 620 (1993) 78-85. [27] Hefti, F., Dravid, A. and Hartikka, J., Chronic intraventricular injections of nerve growth factor elevate hippocampal choline acetyltransferase activity in adult rats with partial septo-hippocampal lesions, Brain Res., 293 (1984) 305-311. [28] Higgins, G.A., Koh, S., Chen, K.S. and Gage, F.H., NGF induction of NGF receptor gene expression and cholinergic neuronal hypertrophy within the basal forebrain of the adult rat, Neuron. 3 (1989) 247-256. [29] Johnson, E.M., Jr, Koike, T. and Franklin, J.L., A calcium set point hypothesis of neuronal dependence on neurotrophic factors, Exp. Neurol., 115 (1992) 163-166. [30] Kater, S.B., Mattson, M.P., Cohen, C. and Connor, J., Calcium regulation of the neuronal growth cone, Trends Neurosei., I I (1988) 315-321 [31] Khachaturian. Z.S., In J.F. Disterhoft, W.H. Gispen, J. Traber and Z.S. Khachaturian (Eds.), Calcium Hypothesis of Aging and Dementia, Ann. N Y Acad. Sci., Vol. 747, New York Academy of Sciences, New York, 1994, pp. l - 12. [32] King, M.A., Hunter, B.E. and Walker, D.W., Alterations and recovery of dendritic spine density in rat hippocampus following long-term ethanol ingestion, Brain Res., 459 (1988) 381-385. [33] Koike, T. and Tanaka, S., Evidence that NGF dependence of sympathetic neurons for survival in vitro may be determined by levels of cytoplasmic flee Ca 2+, Proc. Natl. Acad. Sci. USA, 88 (1991) 3892-3996. [34] Kromer, L.F., Nerve growth factor treatment after brain injury prevents neuronal death, Science, 235 (1987) 214-216. [35] Landmesser, L., The distribution of motoneurons supplying chick hind limb muscles, J. Physiol. (Lond.), 28 (1978) 371-389. [36] Lankford, K.L. and Letourneau, P.C., Evidence that calcium may control neurite outgrowth by regulting the stability of actin illaments, J. Cell Biol., 109 (I 989) 1229-1243. [37] Legendre, P., Rosenmund, C. and Westbrook, G.L., Inactivation of NMDA channels in cultured hippocampal neurons by intracellular calcium, J. Neurosci.. 13 (1993) 674-685. [38] Leslie, S.W., Brown, L.M., Dildy, J.E. and Sims J.S., Ethanol and neuronal calcium channels, Alcohol, 7 (1990) 233-236. [39] Luthin, G.R. and Tabakoff, B., Activation of adenylate cyclase by alcohol requires the nucleotide-binding protein, Exp. Ther., 228 (1984) 579. [40] Mattson, M.P. and Kater, S.B., Calcium regulation of neurite elongation and growth cone motility, J. Neurosci., 7 (1987) 4034-4043. [41] Mattson, M.P., Excitatory amino acids, growth factors and calcium: a teeter-totter model for neuronal plasticity and degeneration, Adz. Exp. Med. Biol., 268 (1990) 2t 1-220. [42] Michaelis, M.L., Michaelis, E.K. and Tehan, T., Alcohol effects oll synaptic membrane calcium ion fluxes, Pharmacol. Biochem. Behat., 18 (Suppl.1) (1983) 19-23. [43] Michaelis. M.S., Michaelis, E.K., Nunley, E.W. and Galton, N., Effects of chronic alcohol administration on synaptic membrane Na+-Ca 2~ exchange activity, Brain Res., 414 (1987) 239-244. [44] Milner, B., The memory deficit in bilateral hippocampal lesions, Psychiatr. Res. Rep., 11 (1959) 43-52. [45] Murrell, R.D. and Tolkovsky, A.M., Role of voltage-gated Ca 2+ channels and intracellular Ca 2+ in rat sympathetic neuron survival and function promoted by high K + and cyclic AMP in the presence of absence of NGF, Eur. J. Neurosci., 5 (1993) 1261-1272. [46] Popov, E.G., Gavrilov, I.Y., Pozin E.Y. and Gabbasov, Z.A., Multiwavelength method for measuring concentration of free cytosolic calcium using the fluorescent probe indo-1, Arch. Biochem. Biophys., 261 (1988) 91-96. [47] Rausch, D.M., Lewis, D.L., Barker, J.L. and Eiden, L.E., Functional expression of dihydropyridine-insensitive calcium channels during

B. Webb et al. / Brain Research 729 (1996) 176-189

[48] [49]

[50]

[51]

[52]

[53]

[54]

PCI2 cells by nerve growth factor (NGF), oncogene ras, or src tyrosine kinase, Cell. Mol. Neurobiol., 10 (1990) 237-255. Seemen, P., The membrane action of anesthetics and tranquilizers, Pharmac. Ree.. 24 (1972) 583-655. Shah, J. and Pant, H.C., Spontaneous calcium release induced by ethanol in the isolated rat brain microsomes, Brain Res. 474 (1988) 94. Streit, J. and Lux, H.D., Calcium current inactivation during nerve growth factor-induced differentiation of PCI 2 cells, Pflugers Arch., 416 (19911) 368-374. Suarez, S.S., Varosi, S,M, and Dai, X., Intracellular calcium increases hyperactivation in intact, moving hamster sperm and oscillates with the flagellar beat cycle, Proc. Natl. Acad. Sci., 90 (1993) 4660-4664. Tanaka, S. and Koike, T., Caffeine promotes survival of cultured sympathetic neurons deprived of nerve growth factore through a cAMP-dependent mechanism, Biochim. Biophys. Acta, 1175 (1992) 114-122. Tsien, R. and Pozyan, T., Measurement of cytosolic free Ca 2+ with quirt 2. In S. Fleischer, B. Fleischer (Eds.), Methods in Enzymology, Vol. 172. Academic Press Inc., San Diego, 1992, pp. 230-261. Walker. D.W.. Lee, N., Heaton, M.B., King, M.A. and Hunter, B.E.,

[55]

[56]

[57]

[58]

[59]

189

Chronic ethanol consumption reduces the neurotrophic activity in rat hippocampus, Neurosci. Lett., 147 (1995) 77-80. Webb, B., Heaton, M.B., King, M.A. and Walker, D.W., A method for labeling embryonic rat medial septal region projection neurons. in vitro, using fluorescent tracers, Brain Res, Bull., 37 (1995) 317-323. Webb, B., Suarez, S.S,, Heaton, M.B. and Walker, D.W., Ethanol and nerve growth factor effects on calcium homeostasis in cultured embryonic rat medial septal neurons before and during depolarization, Brain Res,, 701 (1995) 61-74. West, J.R. and Pierce, D.R., Perinatal alcohol exposure and neuronal damage. In J.R. West (Ed.), Alcohol and Brain Det,elopment, Oxford University Press, New York, 1986, pp. 121-157. Whittemore, S.R., Ebendal, T., Larkfors, L., Olson, L., Seiger, A., Stromberg, I. and Persson, H., Developmental and regional expression of nerve growth factor messenger RNA and protein in the rat central nervous system, Proc. Natl. Acad. Sci. USA, 83 (1986) 817-821. Williams, L.R., Varon, S., Peterson, G.M.. Wictorin. K., Fischer, W., Bjorkund, A and Gage, F.H.. Continuous infusion of nerve growth factor prevents basal forebrain neuronal death after fimbria fomix transection, Proc. Natl. Acad. Sci.. 11 (1986) 216-219.