Increased uptake of [3H]choline by rat superior cervical ganglion: An effect of dexamethasone

Increased uptake of [3H]choline by rat superior cervical ganglion: An effect of dexamethasone

0028.3908/83/06071 l-06$03.00/0 Pergamon Press Ltd Neuropharmacolog!, Vol. 22, No. 6, Pp. 71l-716, 1983 Printed in Great Britain INCREASED SUPERIOR ...

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0028.3908/83/06071 l-06$03.00/0 Pergamon Press Ltd

Neuropharmacolog!, Vol. 22, No. 6, Pp. 71l-716, 1983 Printed in Great Britain

INCREASED SUPERIOR

UPTAKE OF t3H]CHOLINE BY RAT CERVICAL GANGLION: AN EFFECT OF DEXAMETHASONE

P. Y. SZE,* M. MARCHI,? A. C. TOWLE and E. GIACOBINI Department of Biobehavioral Sciences, The University of Connecticut, Storm, CT 06268, U.S.A. (Accepted 26 October 1982)

Summary-The high affinity uptake of [‘HIcholine by the superior cervical ganglion, isolated from the rat, was found to be increased by dexamethasone. Maximal increase (6@65% above control values) occurred at the steroid concentration of 5 x 10e5M. Other glucocorticoids (triamcinolone, corticosterone and hydrocortisone) were without an effect on the [sH]choline uptake. Following administration of dexamethasone (25 mg/kg, i.p.), there was a marked increase in the level of choline in the ganglion. The increase was 3-fold at 1 hr and IO-fold at 6 hr, and by 24 hr the choline levels still remained higher in the steroid-treated animals than in the controls. Levels of acetylcholine in the ganglion were also increased, beginning at I hr after the injection of steroid. The increase was 85% by 3 hr and 60% by 6 hr. Triamcinolone, a glucocorticoid that was without an effect on [‘HIcholine uptake in vitro, was also ineffective in altering the levels of choline and acetylcholine in uivo. It seems probable that the increase of choline uptake in the ganglion induced by dexamethasone may, at least in part, occur in the preganglionic cholinergic terminals, leading to increased synthesis of acetylcholine. Such an effect of dexamethasone provides another case of a selective steroid acting directly on nerve terminals by altering a transport mechanism. Key words: glucocorticoids, choline uptake, cholinergic activity, superior cervical ganglion

The superior cervical ganglion has been suggested as a target organ for adrenal corticosteroid hormones (Hanbauer, Guidotti and Costa, 1975a; Hanbauer, Lovenberg, Guidotti and Costa, 1975b; Otten and Thoenen, 1976a,b; Thoenen and Otten, 1978, McLennan, Hill and Hendry, 1980). This suggestion is based primarily on the observed effects of the synthetic steroid, dexamethasone, on tyrosine hydroxylase activity in the ganglion. For example, the activity of ganglionic tyrosine hydroxylase was increased following administration of a single dose of dexamethasone (Hanbauer et al., 1975a). In the induction of tyrosine hydroxylase by Nerve Growth Factor in cultured ganglia, the effect of the Nerve Growth Factor was potentiated by the addition of dexamethasone in the culture medium (Otten and Thoenen, 1976a). However, recent studies by autoradiography (Warembourg, Otten and Schwab, 1981) and biochemical characterization (Towle and Sze, 1982) indrcate that a cytoplasmic glucocorticoid receptor, similar to that in the liver and brain, is not detectable in the sympathetic ganglion. It is, there-

fore, unlikely that the effect of dexamethasone involves an intracellular receptor-mediated mechanism. Further, adrenalectomy resulted in a reduction of the activity of ganglionic tyrosine hydroxylase, but the reduction of enzyme activity was prevented by administration of epinephrine rather than corticosterone (Markey and Sze, 1980, 1981). Moreover, another study compared the effects of dexamethasone in uiuo with those of three other glucocorticoid steroids: corticosterone, hydrocortisone and triamcinolone (Sze and Hedrick, 1983). Only dexamethasone was found to be effective in increasing the activity of ganglionic tyrosine hydroxylase, and the effect was blocked by nicotinic receptor antagonists and by denervation of the ganglion. These findings suggest that the primary site of action of dexamethasone may involve preganglionic terminals. The present study was undertaken to examine the effects of glucocorticoids on cholinergic terminals in the superior cervical ganglion. The uptake of [3H]choline by isolated sympathetic ganglia as well as the levels of choline and acetylcholine in oiuo in the ganglia were determined.

*Send correspondence to: Dr Paul Y. Sze, Department of Biobehavioral Sciences, U-154, The University of Connecticut, Storrs, CT 06268, U.S.A. tDr M. Marchi was a visiting associate professor on leave from Istituto di Farmacologia e Farmacognosia, Universita degli Studi di Genova, 16146 Genova, Italy (his present address).

METHODS Materials

All animals from Charles 711

were 5-6 week old male Wistar rats River Breeders (Wilmington, MA),

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P. Y.

SZE

and were maintained in the colony on a 12 hr light/ dark cycle under controlled temperature and humidity. [Methyl-‘HIcholine chloride (sp. act. 8&85 Ci/mmol), [y-‘*P]adenosine triphosphate (sp. act. 1040 Ci/mmol) and Aquasol were obtained from New England Nuclear (Boston, MA). Choline chloride, choline kinase, acetylcholinesterase (type V-S), corticosterone, dexamethasone, hydrocortisone, triamicinolone, and other biochemicals were obtained from Sigma Chemical Co. (St Louis, MO). The steroids were dissolved in lOO’%ethanol prior to use in the [3H]choline uptake experiments. The final ethanol concentration was never more than lx, and this amount of ethanol had no effect on [‘HIcholine uptake. For injection, the steroids were prepared as fine suspensions in saline. Uptake of [3H]choline The superior cervical ganglia were dissected out under a stereomicroscope and the sheath surrounding the ganglion was removed. Uptake of [3H]choline was determined by a procedure previously described by Marchi, Hoffman, Giacobini and Fredrickson (1980a) and Marchi, Hoffman, Mussini and Giacobini (1980b). Briefly, the desheathed ganglia were pre-incubated (in the presence or absence of a glucocorticoid) at 37°C for 10 min in 0.5 ml of an incubation solution under an atmosphere of 95% 02-5% CO*. The incubation solution consisted of 119.8 mM NaCl; 4.7 mM KCl; 2.4 mM CaCI,; 0.6 mM KH,PO,; 0.6 mM MgSO,; 25.0mM NaHCO, and 5.0 mM glucose, and was oxygenated before use. [‘HIcholine (1 p Ci) was then added and the incubation continued for 5 min. The concentration of [3H]choline in the incubation medium was 0.64 PM. After the incubation, the ganglion was decanted onto a Whatman No. 1 filter paper disc and rinsed twice with 10ml of the incubation solution under gentle suction. Each ganglion was homogenized in 100 ~1 of 0. I N HCl and centrifuged at IOOOg at 4°C for IO min. An aliquot (40 ~1) of the supernatant was used for the determination of accumulation of [‘HIcholine. Duplicates were counted for each ganglion sample. Counting efficiency was 48-50x. All data were calculated after substracting a blank value representing uptake at OC for 5 min. Choline and acetylcholine assay Levels of choline and acetylcholine were assayed using the sensitive micromethod described by McCaman and Stetzler (1977). This method, a modification of the original method of Goldberg and McCaman (1973), allows the determination of subpicomole levels of choline and acetylcholine. Whole superior cervical ganglia were homogenized in 4&100 ~1 of ice-cold 0.1 N HCI in a glass microhomogenizer. Two ganglia were used for each assay. The homogenate was centrifuged (1000 g) at 4°C for IOmin. Aliquots (20-25 ~1) of the supernatant were lyophilized and stored at -90C until use. The

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lyophilized sample was resuspended in 10 ~1 of a buffer containing 0.1 M Na phosphate (pH 7.5) 4.5 mM MgCI,, and 2.5 pg (0.00125 unit) of choline kinase. After the addition of 5 ~1 of a solution consisting of 0.1 M Na phosphate (pH 7.5), 0.6 mM ATP and 2 PCi of [y-32P]adenosine triphosphate, the mixture was incubated at 37°C for 15 min. The reaction was stopped by the addition of 15 ~1 of 0.3 M barium acetate. The mixture was centrifuged (1OOOg) at 4°C for 20 min. An aliquot (25 ~1) of the supernatant was placed on an anion exchange column previously washed twice with 2 M NH, formate in 5N formic acid and regenerated with 50mM NaOH until the pH of the effluent was 12. The choline [32P]phosphate formed was eluted with 1.5 ml of 50 mM sodium hydroxide. The eluate was collected in a scintillation vial and 13 ml of Aquasol was added. The counting efficiency was 9&95x. The procedure for the determination of acetylcholine was based on the same principle as the choline assay. The lyophilized sample was resuspended in 10 ~1 of a buffer consisting of 0.1 M Na phosphate (pH 7.5), 4.5 mM MgC12, 0.6 mM ATP, and 2.5 pg (0.00125 units) of choline kinase. After an initial incubation at 37°C for 15 min, the mixture was cooled on ice and 5 ~1 of the following solution was added: 0.1 M Na phosphate (pH 7.5) 0.09 units of acetylcholinesterase, and 2 pCi of [y-32P]adenosine triphosphate. The mixture was re-incubated at 37°C for 15 min. The subsequent steps were the same as those described for choline assay. RESULTS

Uptake qf [‘HIcholine The rate of [3H]choline uptake by desheathed superior cervical ganglia is shown in Fig. 1. At a concentration of 0.6 PM [‘HIcholine, the accumulation of [3H]choline in the ganglion was linear with the incubation time for at least IOmin. The rate of uptake of [3H]choline at 37’C under the in vitro conditions used was calculated from the slope of the control curve, as 2.3 pmol/ 10 mimganglion. The uptake was virtually abolished by omitting NaCI from the incubation solution (substituted with an equimolar amount of KCl) or by reducing the temperature to 0°C (data not shown). Thus, the uptake of [3H]choline by the ganglion was Natand temperature-dependent. In several types of cholinergic nerve terminals, a high affinity [3H]choline uptake system has been characterized and its K,,, for [3H]choline is 0.5-10 PM (Murrin, 1980; Giacobini and Marchi, 1981). In the present experiments, the concentration of [‘HIcholine (0.6 PM) was chosen in order to determine such a high affinity uptake by the cholinergic terminals in the ganglion. When the ganglion was pre-incubated with 5 x 10e5 M dexamethasone for 5 min, a marked increase of the uptake of [3H]choline was found (Fig. I). Pre-incubation times longer than 5 min were used (up

Steroid

effect on [3H]choline

713

uptake Table 1. Effects of various glucocorticoid steroids on the uptake of [‘HIcholine by ganglia Steroid* Control Dexamethasone Corticosterone Hydrocortisone Triamcinolone

[3H]choline uptake? (% control) loo*4(10) **I65 & 5 (12) 96 f 5 (6) 93 k 7 (6) 107 k 6 (6)

*All steroid concentrations were 5 x lo-’ M. tIncubation with [‘HIcholine was 5 min. Each value is mean + SEM from the indicated number of ganglia. **P < 0.005.

Incubation

time

Fig. 1. Effects of dexamethasone on the uptake of [‘H]choline. The ganglia were pre-incubated in the presence (0-O) or absence (a-0) of 5 x lO-‘M dexamethasone. Each point is mean k SEM from at least 8 ganglia. *P < 0.005. The rate of uptake of [ZH]choline as calculated from the slope is 2.3 pmol/lO min per ganglion for the control, and 3.7 pmol/lO min per ganglion for the dexamethasone pre-incubated ganglion.

to 15 min), but no further increase of the uptake of [-‘HIcholine was produced. This effect was examined at various concentrations of the steroid (Fig. 2). The effect was not apparent at a steroid concentration of less than IO-‘M. At lo-‘M, there was a 22% increase of [‘HIcholine uptake. The maximum increase was obtained at a steroid concentration of 5 x 10m5M. The effects of three other glucocorticoid steroids (corticosterone, hydrocortisone and triamcinolone) were compared with that of dexamethasone. These three glucocorticoids were selected since another study (Sze and Hedrick, 1983) has shown that admin-

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i I

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10-5

Dexamethasone

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of these steroids did not produce an increase in the activity of tyrosine hydroxylase in the sympathetic ganglion, whereas administration of dexamethasone elicited a significant increase of the enzyme activity. As summarized in Table 1, among the 4 glucocorticoids compared, dexamethasone was the only glucocorticoid that could accelerate the uptake of [3H]choline by the ganglion. All four steroids were added to the incubation medium at 5 x lo-‘M, a concentration that was shown to produce the maximal effect in the case of dexamethasone (Fig. 2). Even at such a large concentration, corticosterone, the natural glucocorticoid hormone of the rat, was totally unable to produce an effect on the [3H]choline uptake. Thus, there is a correlation between the ability of dexamethasone to accelerate [3H]choline uptake by the ganglion in vitro, as seen in the data in Table 1, and the ability of the steroid in vivo to elicit an increase in the activity of ganglionic tyrosine hydroxylase. istration

(min)

I

10-3

(Ml

Fig. 2. The increase of [‘HIcholine uptake as a function of dexamethasone concentration. The ganglia were preincubated in the presence of dexamethasone at the indicated concentrations. Incubation with [‘HIcholine was 5 min. Uptake of [3H]choline in the control was taken as 100%. Each point represents the mean value from at least 6 ganglia.

Choline and acetylcholine levels In addition to the effect of dexamethasone on the uptake of [3H]choline in vitro, the effect of the steroid in vivo on levels of choline in the sympathetic ganglion was determined. Animals were injected intraperitoneally with a single dose of dexamethasone (25 mg/kg), a treatment that is known to increase the activity of tyrosine hydroxylase in the ganglion. Following administration of the steroid, there was a marked increase in the level of choline in the ganglion (Fig. 3). The increase was 3-fold at 1 hr and IO-fold at 6 hr after the steroid injection. The reason for the recovery to normal at 3 hr is not known. By 24 hr, levels of choline remained higher in the steroidtreated animals than in the controls. In contrast to dexamethasone, triamcinolone, a glucocorticoid that did not stimulate uptake of [‘HIcholine in vitro, was found to have no effect on the level of choline. Thus, the data indicates that the increase of choline uptake by dexamethasone could result in a prolonged elevation of levels of choline in the ganglion. If the increase in levels of choline found in the whole ganglion does indeed occur in the pregan-

P. Y. SZE et al.

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-z .-0 3000

triamcinolone, a steroid that has no effect on choline levels in the ganglion, was also without an effect in increasing preganglionic levels of acetylcholine.

-

DISCUSSION

0

I 3

I Time

(hr)

I 6 after

‘--k-

steroid

Fig. 3. Effects of dexamethasone and triamcinolone treatment on levels of choline. Dexamethasone or triamcinolone (both 25 mg/kg) was injected intraperitoneally and the ganglia were dissected at the indicated time periods after the injection. Control animals (0 time) were injected with saline. Each point is mean f SEM from 6 animals. *P < 0.005.

terminals, it should result in an increase in levels of acetylcholine. This was found to be the case. Figure 4 shows the level of acetylcholine in the ganglion following administration of dexamethasone. A significant increase was found 1 hr after the administration of steroid, and by 3 hr the increase was 85% above the control. The level remained high at 6 hr (60% above the control), and returned to normal by 24 hr. Thus, there was a significant increase in levels of acetylcholine for at least 6 hr following the administration of a single dose of dexamethasone. Since the metabolism of acetylcholine in the superior cervical ganglion is not well understood, it is difficult to compare the time course of the changes in levels of acetylcholine with that of the levels of choline. Nonetheless, it is clear that the increase in levels of choline induced by the steroid is, at least in part, reflected in the preganglionic levels of acetylcholine. Moreover, glionic

f

ol,/-0

I

Time

3 (hr)

6 after

24 steroid

Fig. 4. Effects of dexamethasone and triamcinolone ment on levels of acetvlcholine. Details are same Fig. 3.

treatas in

The primary purpose of this study was to examine the possibility of a direct effect of dexamethasone on cholinergic terminals in the sympathetic ganglion. The results demonstrate that the steroid exerted a concentration-dependent increase of high affinity uptake of [iH]choline by the isolated ganglion. Since an Na+-dependent, high affinity system for transport of [iH]choline has been identified in several types of central and peripheral cholinergic terminals (Yamamura and Snyder, 1972, 1973; Haga and Noda, 1973; Simon and Kuhar, 1976; Simon, Atweh and Kuhar, 1976; Suszkiw and Pilar, 1976; Kuhar and Murrin, 1978; Marchi et a/., 1980a,b), it is reasonable to infer that the increase in uptake of [3H]choline reflects an effect of the steroid on preganglionic terminals. This is supported by the fact that levels of acetylcholine in the ganglion were significantly elevated following an injection of the steroid. The elevation of levels of choline was even more remarkable, reaching a IO-fold increase at 6 hr after the administration of steroid. The increase in levels of acetylcholine, seen at 3 hr, which coincides with the return of choline levels to baseline values, is difficult to explain, since synthesis of acetylcholine in relation to choline pools is a complex phenomenon and has not been well studied in the cholinergic terminals of the sympathetic ganglia. Judging from the magnitude of the increase of acetylcholine (85-60x at 3-6 hr), which is much smaller than the marked increase of choline levels, it is unlikely that the increase of accumulation of choline seen in the whole ganglion occurs exclusively in the preganglionic terminals. It is possible that under in uivo conditions, the steroid may affect low affinity choline transport systems in the ganglionic cells as well as the high affinity uptake system in the cholinergic terminals. Regardless of this, the increase of levels of acetylcholine indicates that at least a portion of the increase of choline is related to the cholinergic terminals. The mechanism of the effects of dexamethasone on cholinergic terminals is not known. The synthesis of acetylcholine in cholinergic terminals can be modified directly or indirectly by several factors, including availability of the precursor choline. This is determined by the rate of transport across the neuronal membrane and the state of neuronal activity (Murrin, 1980). It is generally believed that the Na+-dependent high affinity uptake system supplies choline for the synthesis of acetylcholine under resting conditions (Goldberg, Wecker, Bierkamper, Millington and Kselzak-Redding, 1972). If the acceleration of choline transport by dexamethasone results in an increase of the choline pool directly linked to syn-

Steroid effect on [‘HIcholine uptake thesis of acetylcholine, an increase of levels of acetylcholine would be expected. Alternatively, an increase of uptake of choline may occur as a consequence of increased neuronal firing. This has been demonstrated in brain synaptosomal preparations (Antonelli, Beani, Bianchi, Pedata and Pepeu, 1981), diaphragm preparations (Goldberg et al., 1982) and isolated superior cervical ganglion (O’Regan and Collier, 198 1) following electrical, physiological or chemical stimulation. Although a depolarizing action of dexamethasone on cholinergic terminals in the sympathetic ganglia is yet to be shown, electrophysiological studies on neurons in the central nervous system have demonstrated changes in firing pattern after the application of steroids. For example, Teyler, Foy and Vardaris (1979) have reported a rapid and selective modulation of neural excitability in hippocampal slices, bathed in testosterone, or 178-estradiol. Certain hypothalamic and septal neurons respond to iontophoretic application of 17/I-estradiol by increasing unit activity, whereas these neurons do not respond to hydrocortisone (Kelly, Moss, Dudley and Fawcett, 1977a; Kelly, Moss and Dudley, 1977b). Other investigators have shown that application of glucocorticoids modulate the firing frequency of certain neurons in the hypothalamus, hippocampus and midbrain (Mandelbrod, Feldman and Weiman, 1974; Dafny, Phillips and Taylor, 1973; Ruf and Steiner, 1967). Therefore, the increase of [3H]choline uptake in vitro and levels of choline in oivo found in the present experiments could be due to a steroid-membrane interaction that resulted in an altered neuronal activity with a subsequent increased demand for choline uptake and synthesis of acetylcholine. The notion that glucocorticoids can act directly on synaptic terminals by altering the uptake of a neurotransmitter or its precursor is not new. The steroids are known to increase the uptake of tryptophan (Neckers and Sze, 1975) and y-amino butyric acid (GABA) (Miller, Chaptal, McEwen and Peck, 1978) by brain synaptosomes. Riker, Sastre, Baker, Roth and Riker (1979) have recently demonstrated that the rate of high affinity uptake of choline into cat brain synaptosomes was selectively increased in the caudate-putamen after acute or chronic treatment with glucocorticoids. In the ganglion, it is interesting that of the four glucocorticoid steroids examined, only dexamethasone was effective. Triamcinolone, a glucocorticoid which binds the cytoplasmic steroid receptor with a higher affinity than does corticosterone (the natural glucocorticoid of the rat), was without an effect on [3H]choline uptake in vitro or choline and acetylcholine levels in vivo. A recent study has identified and characterized the specific binding sites for a number of steroid hormones on synaptosomal plasma membranes derived from rat brain (Twole and Sze, 1983). The binding properties of the neuronal membranes are not the same as those of the cytoplasmic steroid receptors, and the syn-

715

patosomal membranes from different neuronal cell types may selectively bind different steroids. It is therefore possible that the type of cholinergic terminals in the superior cervical ganglion may have a selective affinity for dexamethasone. An important finding in the present study is that the lack of an effect from three other glucocorticoids (corticosterone, triamcinolone and hydrocortisone) was correlated with their inability to increase the activity of ganglionic tyrosine hydroxylase, reported in another study (Sze and Hedrick, 1983). Thus, among the glucocorticoids examined, an effect on the activity of tyrosine hydroxylase was found only with the synthetic steroid dexamethasone, and this effect appears to involve the preganglionic terminals rather than an intracellular receptor in the ganglionic neurons. The cytoplasmic glucocorticoid receptor in rat tissues binds corticosterone as well as synthetic gluincluding dexamethasone and tricocorticoids, amcinolone (Wrange, 1979). In fact, recent studies indicate that such a receptor is not detectable in the sympathetic ganglion (Warembourg et al., 1981; Towle and Sze, 1982). From this point of view, it seems that the superior cervical ganglion is not an ideal nervous tissue to study receptor-mediated actions of glucocorticoids in neurons. On the other hand, it is interesting that dexamethasone has a direct effect on cholinergic terminals in the sympathetic ganglion. While the effect appears to be only pharmacological, it provides another experimental system whereby the biochemical action of a selective steroid on synaptic terminals may be analyzed. Acknowledgement-This research was supported by USPHS Grant MH-29237 from the National Institute of Mental Health. We are grateful to Barbara Hedrick for her skill and patience

in dissecting

and desheathing

the ganglia.

REFERENCES Antonelli T., Beani L., Bianchi C., Pedata F. and Pepeu G. (1981) Changes in synaptosomal high affinity choline uptake following electrical stimulation of guinea-pig cortical slices: Effect of atropine and physostigmine. Br. J. Pharmac. 14: 525-53 1.

Dafny N., Phillips I. and Taylor A. N. (1973) Dose effects of cortisol on single unit activity in hypothalamus, reticular formation and hippocampus of freely behaving rats correlated with plasma steroid levels. Brain Res. 59: 251-272. Giacobini E. and Marchi M. (1981) Acetylcholine biosynthesis in developing cholinergic synapse. In: Cholinergic Mechanisms (Pepeu G. and Ladinsky H., Eds), pp. l-23. Plenum Press, New York. Goldberg A. M. and McCaman R. E. (1973) The determination of picomole amounts of acetylcholine in mammalian brain. J. Neurochem. 20: 1-8. Goldberg A. M., Wecker L., Bierkamper G., Millington W. and Kselzak-Redding H. (1982) Acetylcholine releaseinteraction of choline and neuronal activity. European Symposium on Cholinergic Transmission: Presynaptic Aspects, Strasbourg (abstract). Haga T. and Noda H. (1973) Choline uptake systems of rat brain synaptosomes. Biochim. biophys. Acta 291: 564-575. Hanbauer I., Guidotti A. and Costa E. (1975a) Dex-

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P. Y. SZE et al.

amethasone induces tyrosine hydroxylase in sympathetic ganglia but not in adrenal medulla. Brain Res. 85: 527-531. Hanbauer I., Lovenberg W., Guidotti A. and Costa E. (1975b) Role of cholinergic and glucocortico-steroid receptors in the tyrosine hydroxylase induction elicited by reserpine in superior cervical ganglion. Brain Res. 96: 197-200. Kelly M. J., Moss R. L., Dudley C. A. and Fawcett C. P. (I 977a) The specificity of the response of preoptic-septal area neurons to estrogen: 17cc-estradiol vs. 17p-estradiol and the response of extrahypothalamic neurons. Expl Brain Res. 30: 43-52. Kelly M. J., Moss R. L. and Dudley C. A. (1977b) Effects of microelectrophoretically applied estrogen, cortisol and acetylcholine on medial preoptic-septal unit activity throughout the estrous cycle of the female rat. Expl Bruin Res. 30: 53-64. Kuhar M. J. and Murrin L. C. (1978) Sodium-dependent, high affinity choline uptake. J. Neurochem. 30: ‘15-21. Mandelbrod J.. Feldman S. and Weiman R. (1974) Inhibition of firing is the primary effect of microelectrophoresis of cortisol to units in the rat tuberal hypothalamus. Brain Res. 80: 303-315. Marchi M., Hoffman D. W., Giacobini E. and Fredrickson T. (1980a) Age dependent changes in choline uptake of the chick iris. Brain Res. 195: 4233431. Marchi M., Hoffman D. W., Mussini I. and Giacobini E. (1980b) Development and aging of cholinergic synapses. III. Choline uptake in the developing iris of the chick. Devl Neurosci. 3: 185-198. Markey K. A. and Sze P. Y. (1980) Adrenal influence on tyrosine hydroxylase activity in superior cervical ganglion. Brain Res. 202: 347-356. Markey K. A. and Sze P. Y. (1981) Influence of adrenal epinephrine on postnatal development of tyrosine hydroxylase activity in the superior cervical ganglion. Devl Neurosci 4: 267-272. McCaman R. E. and Stetzler J. (1977) Radiochemical assay for ACh: modifications for subpicomole measurements. J. Neurochem. 28: 669-67 1. McLennan I. S., Hill C. E. and Hendry I. A. (1980) Glucocorticoids modulate transmitter choice in developing superior cervical ganglion. Nature 283: 306-307. Miller A. L., Chaptal C., McEwen B. S. and Peck E. J., Jr (1978) Modulation of high affinity GABA uptake into hippocampal synaptosomes by glucocorticoids. Psychoneuroendocrinology 3: 1555164. Murrin L. C. (1980) High-affinity transport of choline in neuronal tissue. Pharmacology 21: 132-140. Neckers L. and Sze P. Y. (1975) Regulation of 5-hydroxytryptamine metabolism in mouse brain by adrenal glucocorticoids. Brain Res. 93: 123-132. O’Regan S. and Collier B. (1981) Factors affecting choline transport by the cat superior cervical ganglion during and following stimulation, and the relationship between choline uptake and acetylcholine synthesis. Neuroscience 6: 511-520.

Otten U. and Thoenen H. (1976a) Modulatory role of glucocorticoids on nerve growth factor mediated enzyme induction in organ cultures of sympathetic ganglia. Brain Res. 111: 438-441. Otten U. and Thoenen J. (1976b) Selective induction of tyrosine hydroxylase and dopamine beta-hydroxylase in sympathetic ganglia in organ culture-role of glucocorticoids as modulators. Molec. Pharmac. 12: 353-361, Riker D. K., Sastre A., Baker T., Roth R. H. and Riker W. F. (1979) Regional high-affinity [jH]choline accumulation in cat forebrain: selective increase in the caudate-putamen after corticosteroid pretreatment. Molec. Pharmac. 16: 886899. Ruf K. and Steiner F. A. (1967) Steroid sensitive single neurons in rat hypothalamus and midbrain: Identification by microiontophoresis. Science 156: 667-669. Simon J. R. and Kuhar M. J. (1976) High affinity choline uptake: Ionic and energy requirements. J. Neurochem. 27: 93-99. Simon J. R., Atweh S. and Kuhar M. J. (1976) Sodium dependent high affinity choline uptake: A regulatory step in the synthesis of acetylcholine. J. Neurochem. 26: 909-922. Suszkiw J. B. and Pilar G. (1976) Selective localization of a high affinity choline uptake system and its role in ACh formation in cholinergic nerve terminals. J. Neurochem. 26: 1133-I 138. Sze P. Y. and Hedrick B. J. (1983) Effects of dexamethasone and other glucocorticoid steroids on tyrosine hydroxylase activity in the superior cervical ganglion. Bruin Res. (In press). Teyler T. J., Foy M. and Vardaris R. M. (1979) Modulation of hippocampal excitability in adult castrated and overectomized rats. Sot. Neurosci. Abstr. 5: 461. Thoenen H. and Otten U. (1978) Role of adrenocortical hormones in the modulation of synthesis and degradation of enzymes involved in the formation of catecholamines. In: Frontiers in Neuroendocrinology (Ganong W. F. and Martini L., Eds), Vol. 5, pp. 163-184. Raven Press, New York. Towle A. C. and Sze P. Y. (1982) ‘H-Corticosterone binding in rat superior cervical ganglion. Brain Res. 253: 221-229. Towle A. C. and Sze P. Y. (1983) Steroid binding to synaptic plasma membrane: Differential binding of glucocorticoids and gonadal steroids. J. Steroid Biochem. 18: 135-143. Warembourg M., Otten U. and Schwab M. E. (1981) of Schwann and satellite cells by Labelhng ‘H-dexamethasone in a rat sympathetic ganglion and sciatic nerve. Neuroscience 6: 1139-l143. Wrange 0. (1979) A comparison of the glucocorticoid receptor in cytosol from rat liver and brain. Biochim. biophys. Acfa 582, 346356. Yamamura H. 1. and Snyder S. H. (1972) Choline. High affinity uptake by rat brain synaptosomes. Science 178: 626628. Yamamura H. I. and Snyder S. H. (1973) High affinity transport of choline into synaptosomes of rat brain. J. Neurochem. 21: 1355-1374.