Effects of corticosterone on neurones of the locus coeruleus, in the rat

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Neuroscience Letters, 80 (1987) 85-88


Elsevier Scientific Publishers Ireland Ltd.

NSL 04782

Effects of corticosterone on neurones of the locus coeruleus, in the rat Gian Luigi Avanzino, Rosa Ermirio, Carla E. Cogo, Piero Ruggeri and Claudio Molinari Istituto di Fisiologia Umana, Universitd degli Studi, Genova (Italy) (Received 10 February 1987; Revised version received 29 April 1987; Accepted 8 May 1987)

Key words. Glucocorticoid hormone; Corticosterone; Microelectrophoresis; Locus coeruleus; Stress; Brainstem The effects of microelectrophoretic application of corticosterone (CS) on single neurones of the locus coeruleus (LC) were investigated in rats under urethane anaesthesia. Ejecting currents generally ranged from 10 to 60 nA. CS produced an excitatory effect in 73% of the neurones and no effect in 27%. The prevailingly excitatory effects induced by CS on LC neurones may be related to the regulation of those complex events, which constitute the 'stress response'.

Our previous studies demonstrated that neurones of the brainstem reticular formation (RF) and of raphe nuclei (RN) respond to the microelectrophoretic application of adrenal glucocorticoid hormones by varying their basal firing rate [3-5]. These effects appear to be specific in the single areas of the brainstem studied and may be related to the regulatory activity of glucocorticoids on a number of nervous functions, such as regulation of adrenocorticotropic hormone (ACTH) secretion, control of sensory inputs, including pain, regulation of paradoxical sleep, expression of mood, adaptation of the organism to the environment and modulation of enzymatic processes involved in the secretion of catecholamines in the central nervous system (CNS) [9, 13]. In the present study we have investigated the effects of microelectrophoretically applied corticosterone (CS) on the neurones of locus coeruleus (LC), a pontine nucleus that contains noradrenergic neurones [8] and that is implicated in several of the nervous functions, which are also regulated by glucocorticoids [1, 7, 12]. Experiments were performed on 20 male Wistar rats, weighing 280-320 g, and anaesthetized with urethane (i.p. 180 rag/100 g body wt.; Sigma St. Louis, MO, U.S.A.). The rats were positioned in a stereotaxic instrument and body temperature was maintained at 37°C. A 3-mm burr hole was drilled in the occipital bone and the Correspondence: G.L. Avanzino, Istituto di Fisiologia Umana, Universit~i degli Studi, Viale Benedetto XV, 3, 1-16132 Genova, Italy. 0304-3940/87/$ 03.50 O 1987 Elsevier Scientific Publishers Ireland Ltd.

86 transverse sinus was exposed, The sinus was subsequently isolated and ligated at its ends, laterally to the confluence of sinuses and sectioned together with the surrounding dura. Sciatic nerve was dissected free from connective tissue and kept in a pool of warm paraffin oil. Dipolar hook electrodes were used for the electrical stimulation of nerves. Multi-barreled micropipettes were stereotaxically introduced into the brainstem between AP - 0 . 3 mm and AP - 0 . 8 mm posterior to the interaural line and from 1.1 to 1.2 mm lateral to the midline, according to the stereotaxic atlas of Paxinos and Watson [16]. An operating microscope (SOM 6, Karl Kaps Asslar, F.R.G.) was used for positioning the microelectrode tip on stereotaxic reference points. Standard record procedures were followed and the neuronal spikes were counted by a DI30 Digitimer Spike Processor. The multi-barreled micropipettes were prepared and filled according to previously detailed methods [2]. The central barrel, filled with 4 M NaC1, was used for extracellular recording, a second one was filled with 0.013 M corticosterone sodium hemisuccinate, pH 7, (Parke-Davis, S.p.A. Milano, Italy). A third barrel was filled with Pontamine sky blue (2.5% solution in sodium acetate buffer, pH 5.6, according to Boakes et al. [6]) and used both as a balancing current barrel and as a marker of the position of the electrode tip. A fourth barrel was filled with NaC! 1M for checking of artifacts due to the electrophoretic current. CS was applied by a negative current ranging from 10 to 60 nA. The backing current ranged from 5 to 8 nA. LC neurones were tentatively identified during recording sessions according to (i) their spontaneous discharge rate (1-4 Hz), (ii) long duration action potential (2 ms) and (iii) the biphasic excitatory-inhibitory response to noxious stimuli or sciatic nerve stimulation, that consisted of a burst of spikes followed by a period of suppressed activity [7]. In addition, at the end of the experiments, the position of the electrode tip was marked with Pontamine sky blue. The brain was fixed in formalin and following histological examination the location of each recording site was determined by calculating the calibrated movements of the micromanipulator as related to the marked point. The critical ratio (CR) test [14] was used to statistically evaluate the changes in the neuronal firing rate, induced by CS. Values of CR > I1.961 are considered significant and correspond to an approximately 30% change of baseline firing rate [17]. Changes were confirmed by at least 3 consecutive applications of CS for each unit studied. CS was tested on 48 LC neurones. 35 neurones (73%) were excited, and 13 (27%) were unaffected by CS. The excitatory effects were maximal within 1-5 s and lasted up to 10 s after the end of the application (Fig. 1). We did not include in the total number of cells studied 7 neurones (1 excited, 2 inhibited, 4 unaffected by CS), whose position was histologically uncontrolled or found near the LC. The present results show that the majority o f L C neurones increase their firing rate, following microelectrophoretic application of CS. These data further support the previously reported notion that CS exerts a specific action on brainstem structures [3-5]. In fact, in some anatomical districts CS causes prevailingly inhibitory effects

87 C S 50 nA

C S 3 0 nA !

NaCI ( - ) 5 0 n A










3 0 sec

Fig. 1. Effects of corticosterone (CS) on spontaneously active neurones of locus coeruleus. The firing mean rate in spikes/s, in successive 2.5-s epochs, is plotted against time. Microelectrophoretic applications are indicated by the horizontal bars. A 50-nA current applied to the NaCI barrel did not mimic the effects of CS.

such as the case of the neurones of rostral portion of the RF, while in other sites the effects are exclusively or prevailingly excitatory. This is the case of the neurones of the RN and of the caudal portion of the RF, respectively. It has been demonstrated that LC, which contains approximately half of all noradrenergic neurones in the rat brain [18] and innervates by its widely ramifying axonal projections nearly the entire brain and spinal cord [11, 15], is involved in autonomic, endocrine and behavioural responses [1, 12]. It is also known that an increase of plasma CS levels occurs in response to stressful stimuli [10, 20] and stress also leads both to increased central noradrenaline turnover [19] and to enhanced neuronal activity of the LC [7, 12]. Thus, one of the possible functional significances of the activation of LC neurones in response to local application of CS could be the regulation of those complex events which constitute the 'stress response'. The effects of steroid hormones on the CNS have been divided into genomic and non-genomic [13]. The effects obtained by microelectrophoretic application of CS on LC neurones may be considered as non-genomic, based upon their brief onset latency and short duration after the end of application. This type of effects could account for the rapid changes caused by CS in various nervous functions, in contrast with a slow, genomic action of glucocorticoid hormones, which may induce only long-lasting changes. This study was supported in part by a Grant for Scientific Research (60%) of MPI. Corticosterone hemisuccinate was a gift from Parke-Davis S.p.A. Milano. 1 Aston-Jones, G. and Bloom, F.E., Norepinephrine-containing locus coeruleus neurons in behaving rats exhibit pronounced responses to non-noxious environmental stimuli, J. Neurosci. 1 (1981) 887 900. 2 Avanzino, G.L., Bradley, P.B. and Wolstencroft, J.H., Actions of prostaglandins E~, E2 and F2~ on brain stem neurones, Br. J. Pharmacol. Chemother., 27 (1966) 152163. 3 Avanzino, G.L., Celasco, G., Cogo, C.E., Ermirio, R. and Ruggeri, P., Actions of microelectrophoretically applied glucocorticoid hormones on reticular formation neurones in the rat, Neurosci. Lett., 38 (1983) 45-49.

88 4 Avanzino, G.L., Ermirio, R., Ruggeri, P. and Cogo, C.E., Effect of microelectrophoretically applied corticosterone on raphe neurones in the rat, Neurosci. Lett., 50 (1984) 307 31 I. 5 Avanzino, G.L., Ermirio, R., Ruggeri, P. and Cogo, C.E., Effects of corticosterone on neurones of reticular formation in rats, Am. J. Physiol., 252 (1987) in press. 6 Boakes, R.J., Bramwell, G.J., Briggs, I., Candy, J.M. and Tempesta, E., Localization with Pontamine sky blue of neurones in the brainstem responding to microiontophoretically applied compounds, Neuropharmacology, 13 (1974) 475~479. 7 Cedarbaum, J.M. and Aghajanian, G.K., Activation of locus coeruleus neurons by peripheral stimuli: modulation by a collateral inhibitory mechanism, Life Sci., 23 (1978) 1383--1392. 8 Dathstrom, A. and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta Physiol. Scand., 62 Suppl. 232 (1964) 1 55. 9 De Kloet, E.R., Adrenal steroids as modulators of nerve cell function, J. Steroid Biochem., 20 (1984) 175-181. 10 Feldman, S., Siegel, R.A., Weidenfeld, J., Conforti, N. and Melamed, E., Adrenocortical responses to ether stress and neural stimuli in rats following the injection of 6-hydroxydopamine into the medial forebrain bundle, Exp. Neurol., 83 (1984) 215-220....

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