Noradrenergic neurons of the locus coeruleus: inhibition by epinephrine and activation by the α-antagonist piperoxane

1. Brain Res. 1976 Aug 13;112(2):413-9. Noradrenergic neurons of the locus coeruleus: inhibition by epinephrine
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Category: Cognitive Science, Brain, Epinephrine, Animals, Male, Norepinephrine, Iontophoresis, Rats, Locus coeruleus, Neurosciences, Gamma-Aminobutyric Acid, Piperidines, Isoproterenol, Clonidine, Norepinephrine, Iontophoresis, Rats, Locus coeruleus, Neurosciences, Gamma-Aminobutyric Acid, Piperidines, Isoproterenol, Clonidine

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Brain Research, 112 (1976) 413-419


© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands

Noradrenergic neurons of the locus coeruleu$: inhibition by epinephrine and activation by the a-antagonist piperoxane

JESSE M. CEDARBAUM and GEORGE K. AGHAJANIAN Departments of Psychiatry and Pharmacology, Yale University School of Medicine, Connecticut Mental Health Center, New Haven, Conn. 06508 (U.S.A.)

(Accepted April 28th, 1976)

The a-adrenergic agonist drug clonidine has been shown to decrease the stimulation-induced release of norepinephrine (NE) from both peripheral a5 and centrallt, 37 noradrenergic neurons, to reduce the turnover of NE in brain 1 and to inhibit the spontaneous firing of the noradrenergic neurons of the nucleus locus coeruleus (LC) 3s. On the other hand, several a-adrenolytic drugs produce opposite effects: increasing stimulation-induced release of NE s,11,~6,37, increasing NE turnover in brain 1,~,4, and antagonizing the hypotension, bradycardia and decreased sympathetic tone produced by the action of clonidine in the CNS 4,7,12,29-al. In particular, the a-adrenergic blocking drug piperoxane 10 appears unique in that it most consistently antagonizes the physiological, biochemical and behavioral effects of clonidine4,7,12, 29-31. Piperoxane has also been shown to increase the percentage of EEG waking time in the rat la, an effect opposite to that produced by the administration of clonidine 20 or by destruction of the LC or its ascending projections19, 23 which give rise to the noradrenergic innervation of the cerebral cortex 40. Recently the LC has been demonstrated to receive an input from immunohistochemically identified epinephrine (E) containing neurons located in the medulla oblongata ts. It has been suggested that piperoxane may be a specific antagonist and that clonidine may be a specific agonist at E-mediated synapses in the CNS 4. If this is so, and if piperoxane blocks a tonic inhibitory E input to LC neurons, piperoxane should produce an increase in the rate of firing of these cells. It was therefore felt to be of interest to study the effects of piperoxane on the firing rate of LC neurons alone and in combination with clonidine, NE and E. In addition, an attempt was made to characterize further the receptor involved, using the fl-agonist isoproterenol (ISO) and the fl-antagonist sotalol (MJ1999) 24, both of which have been shown to exert effects at certain NE-mediated synapses in rat brain16, a2. Single-unit recording and microiontophoretic techniques were employed as described previouslySa. Male albino (Sprague-Dawley) rats, weighing 220--300 g, were anesthetized with chloral hydrate (400 mg/kg, i.p.); additional injections were given as needed. The animals were mounted in a stereotaxic apparatus and a 3-mm burr hole was drilled in the occipital bone with its center located 1.1 mm lateral to the

414 midline and 1.1 mm posterior to lambda. For single barrel unit recording, micropipettes with a tip diameter of 1 /~m were used. They were filled with 2 M NaCI saturated with Fast green. In vitro impedances were 3-7 M ~ , measured in 0.9"~i saline at 60 Hz. The electrode signals were passed through a high input impedance amplifier into an oscilloscope, and finally into an electronic rate meter triggered by individual neuronal spikes. Integrated firing rate wasg raphicallyr ecorded as consecutive 10-sec samples from the analog output of the counter. The output from the oscilloscope also drove an audiomonitor. Spike amplitude was continuously monitored using the storage function of the oscilloscope. In the microiontophoretic studies, 5-barrel micropipettes were used. All barrels contained a few strands of fiberglass to allow rapid filling of the tips by capillary action ~9. The tips of the micropipettes were broken back to 4-5 /zm. The central, recording barrel was filled with 2 M NaC1 saturated with Fast green. In vitro impedances of the recording barrel were 4-7 M ~ . One of the 4 side barrels, filled with 4 M NaCI, was used for automatic current balancing. The other 3 barrels each contained a solution of one of the following drugs, all at pH 3.8-4.1:0.1 M piperoxane hydrochloride; 0.1 M sotalol hydrochloride; 0.1 Mepinephrine bitartrate; 0.1 M norepinephrine bitartrate; 0.1 M isoproterenol hydrochloride; 0.01 M clonidine hydrochloride, ionically diluted 10-fold by 0.1 M NaC! (ref. 38) or 0.01 M 7-aminobutyric acid (GABA) ionically diluted in 0.2 M NaC1. A retaining current of 8 nA was maintained in all drug barrels between periods of ejection, and current balance was automatically kept within ~ 2 nA. Intravenous doses of drugs, expressed as the free base, were injected into a lateral tail vein. The animals' body temperature was maintained at 36 ± 1 °C by means of a rectal thermistor probe coupled to a thermostatically controlled heating pad placed beneath the animal. The recording sites were marked at the end of each experiment by passing a 20 #A cathodal current through the recording electrode for 15 min, thereby depositing a spot of Fast green at the location of the electrode tip. The rats were then perfused through the heart with 10 O//obuffered formalin and serial 50-/~m frozen sections of the brain were cut, mounted, stained with cresyl violet and counterstained with neutral red. The location of the LC cells in all cases was found to be consistent with that described previously 1~,21. Only cells which were subsequently confirmed by histology to be in the LC were included in this study. The cells of the LC were found to be firing spontaneously with a rate of 0.5-3 spikes/sec 15. Occasional units, later identified histologically as being in the caudal pole of the nucleus, appeared to be firing in short bursts synchronously with, or just preceding the inspiratory phase of respiration. Two general characteristics of LC and surrounding neurons were of great help in locating the tiny nucleus at the time of recording. Cells of the mesencephalic nucleus of the Vth nerve lie just lateral to the LC, and respond to proprioceptive stimulation of the facial region (e.g. jaw stretch) with brief bursts of firing. In addition the cells of the LC themselves respond to noxious stimuli (e.g. compression of the contralateral hind paw) with a brief increase in firing followed by a quiescent intervalZl. Piperoxane, injected intravenously, produced a sustained increase in the rate of


PIPEROXANE mg/kg i.v. 0.5 1.0 1.0 2.0 2.0 2.0



V V-


PIP4 NE E 12 12

NE 12

E 12



\ 144

SOT 20

E 16



E 16


E 16

E 16


Fig. 1. Top trace: effect of intravenous piperoxane on spontaneous firing of a noradrenergic neuron

in the LC. Numbers above arrows indicate doses given in mg/kg at intervals of approximately 2 rain. Middle trace: inhibition of LC unit by iontophoretically applied NE and E, which is markedly attenuated during concurrent application of piperoxane (PIP). Note the increase in baseline firing during piperoxane application. In this and bottom trace, the bars above the record indicate times of ejection; numbers above the bars indicate current in nA. Bottom trace: failure of sotalol (SOT) to block Einhibbition of a unit in which piperoxane completely prevented any E-induced decrease in firing. Note recovery of inhibitory response to E after cessation of piperoxane. spontaneous firing o f L C neurons in all animals studied (n = 12). Doses as low as 0.5 m g / k g caused a doubling in the mean rate o f firing f r o m 1.1 -4- 0.1 (mean 4S.E.M.) to 2.0 :[: 0.3 spikes/sec (n = 4; P < 0.05, paired t-test). Additional doses produced further increases in firing until a maximal effect was attained at 2-3 mg/kg, with rates o f up to 7.5/sec occasionally being observed (top trace, Fig. 1). In agreement with previous results 88, clonidine, in doses o f 4-8/~g/kg i.v., substantially reduced or completely inhibited the firing o f L C neurons. Piperoxane, in a dose as low as

416 0.5 mg/kg i.v., promptly returned cell firing to the original rate or greater (n ...... 9). Additional doses produced further stepwise increases in firing, following which clonidine, in doses several times that originally used to inhibit the cells, was no longer able to substantially reduce activity. In contrast to piperoxane, the fl-blocking drug sotalol in doses up to 15 mg/kg i.v. had no consistent effect on spontaneous firing of LC neurons, and failed to prevent or reverse their inhibition by clonidine (n ...... 5). The results of the microiontophoretic experiments are summarized in Table 1. NE or E, when applied to LC cells with ejection currents of 12-20 hA, reduced spontaneous firing to low levels, with a half-maximal effect being achieved in 10-20 sec (Fig. 1, middle trace). 1SO, ejected with similar currents, had identical effects. Clonidine ejected with currents of 5-8 nA also markedly inhibited LC units, but the inhibition tended to develop much more gradually, over 30-40 sec, and to have a much longer duration 38. GABA ejected with currents of 10-20 nA caused an abrupt decrease in cell firing followed by a prompt return to pre-ejection levels of activity when ejection was halted (in = 8). Piperoxane, applied iontophoretically with a current of 4-6 nA, either abolished or markedly attenuated the inhibitory response to all 4 adrenergic agonist drugs tested (Table 1 ; Fig. 1, middle and bottom traces). The blockade developed rapidly with the onset of piperoxane ejection and dissipated within 1-2 min following its termination. No local anesthetic effects of piperoxane (i.e. reduction in spike amplitude and firing rate) were noted at the currents employed to produce blockade. In fact, several LC cells appeared to be slightly activated by iontophoretic piperoxane (e.g. Fig. 1, middle trace). Piperoxane did not antagonize inhibition of LC cells by GABA (n :-- 7). Sotalol, on the other hand, failed to block the inhibitory effects of E (Fig. 1, bottom trace), NE, cl...

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