Chronic substance P (NK1) receptor antagonist and conventional antidepressant treatment increases burst firing of monoamine neurones in the locus coeruleus

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Neuroscience Vol. 109, No. 3, pp. 609^617, 2002 ß 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00

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CHRONIC SUBSTANCE P (NK1 ) RECEPTOR ANTAGONIST AND CONVENTIONAL ANTIDEPRESSANT TREATMENT INCREASES BURST FIRING OF MONOAMINE NEURONES IN THE LOCUS COERULEUS K. A. MAUBACH,a * K. MARTIN,a G. CHICCHI,b T. HARRISON,c A. WHEELDON,a C. J. SWAIN,c M. J. CUMBERBATCH,a N. M. J. RUPNIAKa and G. R. SEABROOKa a

Department of Pharmacology, Merck Sharp p Dohme Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, UK b

c

Merck Research Laboratories, Rahway, NJ, USA

Department of Medicinal Chemistry, Merck Sharp p Dohme Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, UK

AbstractöThe mechanism of action of conventional antidepressants (e.g. imipramine) has been linked to modulation of central monoamine systems. Substance P (NK1 ) receptor antagonists may have antidepressant and anxiolytic e¡ects in patients with major depressive disorder and high anxiety but, unlike conventional antidepressants, are independent of activity at monoamine reuptake sites, transporters, receptors, or monoamine oxidase. To investigate the possibility that substance P receptor antagonists in£uence central monoamine systems indirectly, we have compared the e¡ects of chronic administration of imipramine with that of the substance P receptor antagonist L-760735 on the spontaneous ¢ring activity of locus coeruleus neurones. Electrophysiological recordings were made from brain slices prepared from guinea-pigs that had been dosed orally every day for 4 weeks with either L-760735 (3 mg/kg), imipramine (10 mg/kg), or vehicle (water), or naive animals. Chronic, but not acute, treatment with the substance P receptor antagonist L-760735, induced burst ¢ring of neurones in the locus coeruleus. This e¡ect resembles that of the conventional antidepressant imipramine. However, their e¡ects are dissociable since, in contrast to chronic imipramine treatment, chronic L-760735 treatment does not cause functional desensitisation of somatic K2 adrenoceptors. The mechanism by which chronic substance P receptor antagonist or conventional antidepressant treatment in£uences the pattern of ¢ring activity of norepinephrine neurones remains to be elucidated. However, an indirect action in the periphery or distant brain nuclei has been excluded by the use of the in vitro slice preparation, suggesting a local site of action in the locus coeruleus. ß 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: tachykinin, imipramine, substance P, norepinephrine.

of depression in animals. One viable approach is to compare the e¡ect of SP antagonists and established antidepressants on the function of brain areas associated with mood. The intrinsic ¢ring patterns of norepinephrine (NE)containing neurones of the locus coeruleus (LC) are intimately linked with behavioural arousal (Aston-Jones et al., 1999) and are a¡ected in stress-related animal paradigms linked to depression (Simson and Weiss, 1988; Pavcovich et al., 1990). Patients with depression display abnormalities in NE function, including a decrease in the metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) in the brain and urine, a blunted growth hormone response to the K2 adrenoreceptor agonist clonidine, up-regulation of postsynaptic L adrenoreceptors and a reduction in neuronal NE transporter binding sites (Klimek et al., 1997; Schatzberg, 1998). Furthermore, chronic treatment with conventional antidepressants that block NE reuptake, for example the tricyclic antidepressant (TCA) imipramine, has a profound e¡ect on NE systems within the brain (Mongeau et al., 1997). The LC constitutes the largest group of NE neurones in the brain, which innervate many regions of the CNS

There is a pressing need for improved antidepressant therapies, given the prevalence of mood disorders and the limited utility of existing medications (Maubach et al., 1999). The demonstration that the substance P (SP) receptor antagonist MK-869 has therapeutic utility in depression and acts via a novel mechanism (Kramer et al., 1998), provides an exciting new possibility for the treatment of a¡ective disorders. Understanding the mechanism of action of SP antagonists is hampered by the ill-de¢ned aetiology of mood disorders, the poorly understood mechanism of action of established antidepressants, and the inability to model the pathophysiology *Corresponding author. Tel.: +44-1279-440000; fax: +44-1279440390. E-mail address: [email protected] (K. A. Maubach). Abbreviations : aCSF, arti¢cial cerebrospinal £uid; DR, dorsal raphe; EGTA, ethylene glycol-bis (2-aminoethyl-ether)N,N,NP,NP-tetraacetic acid ; HEPES, N-(2-hydroxyethyl)piperazine-NP-(2-ethanesulphonic acid); LC, locus coeruleus ; MHPG, 3-methoxy-4-hydroxyphenylglycol ; NE, norepinephrine; NK, neurokinin; p.o., parenteral oral; SP, substance P; TCA, tricyclic antidepressant. 609

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(Dahlstrom and Fuxe, 1964). Acute TCA treatment in animal models depresses LC ¢ring, via activation of K2 adrenoceptors, which mediate inhibitory feedback on the NE neurones (Svensson and Usdin, 1978). Chronic TCA treatment has been shown to decrease cAMP-dependent protein kinase activity (Melia et al., 1995) and tyrosine hydroxylase expression (Brady et al., 1991), and increase NE release (Mongeau et al., 1994; Scho¡elmeer and Mulder, 1983) from the LC; e¡ects invariably ascribed to K2 adrenoceptor desensitisation. The reduced inhibitory e¡ects of clonidine, an K2-adrenergic partial agonist, on the spontaneous ¢ring of LC neurones (Mongeau et al., 1998; Lacroix et al., 1991), as well as reduced [3 H]clonidine binding (Koslow et al., 1983) following chronic TCA treatment, is consistent with this hypothesis. It is possible that MK-869 and related SP antagonists may in£uence central monoamine systems by blocking the actions of SP in the LC. LC neurones are innervated by SP-containing ¢bres (Halliday et al., 1988), express NK1 receptors (Shults et al., 1984) and are activated by SP (Cheeseman et al., 1983). In the present study we have investigated the e¡ect of chronic treatment with the SP antagonist L-760735 on NE neurones in the LC using electrophysiological recording from guinea-pig brain slices in vitro. This preparation has the advantage in that the area under study is devoid of tonic inputs from distal loci. The e¡ects of this treatment on ¢ring activity and K2 adrenoceptor function were compared to that of imipramine.

In brief, the whole brain was rapidly removed and placed in ice-cold `arti¢cial cerebrospinal £uid, (aCSF)' where NaCl had been switched for sucrose to minimise neuronal damage (Lipton et al, 1995), aerated with 95% O2 plus 5% CO2 . The sucrosebased aCSF had the following composition (mM): sucrose (248), KCl (2.5), NaH2 PO4 (1.2), CaCl2 W2H2 O (2.4), MgCl2 W6H2 O (1.3), NaHCO3 (26) and D-glucose (10), pH 7.4 prior to chilling. A block of the mid-brain containing the LC was prepared with a transverse cut to the cerebellum and cortex. Coronal slices (350 Wm thick) were cut using a vibratome and then transferred to a holding chamber where they were placed in an aCSF mixture (sucrose replaced with 124 mM sucrose and 63 mM NaCl mixture) for 5 min to prevent cell damage due to osmotic stress. Subsequently the tissue was transferred to aCSF (with 126 mM NaCl) in an aerated atmosphere (95% O2 plus 5% CO2 ) at room temperature until required. Brain slices were transferred to the recording chamber where they were maintained in vitro held on a nylon net ¢tted to the bath and submerged in a continuous £ow of aCSF (V2 ml/min) aerated with 95% O2 plus 5% CO2 at 34³C for extracellular recordings and at room temperature for patch clamp recordings. Slices were allowed to equilibrate in the recording chamber for approximately 1 h before recordings were made. Extracellular electrophysiology

A¤nity for the cloned human and guinea-pig NK1 receptor expressed in intact CHO or COS cells was determined as described previously (Cascieri et al., 1992). A¤nity for more than 90 other neurotransmitter receptors and ion channels was determined by Panlabs DiscoveryScreen. A¤nity for monoamine receptors were determined using methods based on previous assays (Berger et al., 1990).

Conventional techniques were used to make extracellular microelectrode recordings from neurones within the LC. Microelectrodes were pulled from Starbore capillary glass (Radnoti, USA, 1.2 mm outer diameter) using a Brown Flaming micropipette puller (Sutter instruments, Model P-87). The electrode tips were ¢lled by capillary action using bicarbonate-free aCSF solution. Electrodes with resistances of approximately 5 M6 were used. Electrodes were placed onto the surface of the slice in the region of the LC under visual control (crescent-shaped translucent area) and slowly advanced down through the slice until action potentials were detected. The recording was monitored using an oscilloscope and run through a spike processor which permits the dissociation of action potentials from background noise by only counting events that pass through a window discriminator, which is set above the noise level and below the action potential peak. On-line and o¡-line data acquisition and analysis was accomplished using a CED 1401plus (Cambridge Electronic Design, CED) and an Axoclamp-2A ampli¢er and a personal computer using a software support Spike 2 version 2.23 (CED). Only cells that were spontaneously active, ¢red characteristically broad action potentials (Williams et al., 1984) and were sensitive to the application of the K2 adrenoceptor partial agonist, clonidine, and were therefore thought to be the NE-containing cells, were selected for analysis.

Chronic dosing protocol

Whole-cell patch clamp electrophysiology

Dunkin Hartley guinea-pig pups (male, 7 days old at the start of the study) were dosed daily for 28 days. Four treatment groups were investigated: the SP antagonist, L-760735 (3 mg/ kg/day, p.o.), imipramine (10 mg/kg/day p.o.), vehicle (distilled water p.o.) and naive. Doses were based on those required to give complete inhibition of neonatal vocalisations in this species (Kramer et al., 1998). The animals were individually labelled with transponders and ear tag identi¢cation numbers. It was necessary to implement a staggered dosing protocol over a period of 6 months to permit the delivery of one guinea-pig per day for use by electrophysiologists who performed the study, blind to the treatment group, on day 29 for each animal. 34^38-day-old guinea-pigs were utilised for the naive group and acute application studies.

Whole-cell patch clamp recordings were made from visually identi¢ed neurones using an infrared microscope (Zeiss Axioskop). Microelectrodes were pulled from borosilicate ¢lament glass (CEI, 1.2 mm outer diameter) using a Brown Flaming puller. The electrode tips were ¢lled with a solution containing in mM: K-gluconate (150), MgCl2 (2), CaCl2 (0.01), HEPES (10), EGTA (0.1), ATP^Mg (2) and GTP^Tris (0.5) bu¡ered to pH 7.3 with KOH. Electrode resistance ranged from 4 to 5 M6 and neurones were either voltage-clamped at 360 mV to study their current^voltage relationship or current-clamped to study ¢ring activity at resting membrane potential. On-line data acquisition was accomplished using pClamp software (Axon Instruments) and analysed using Clampex8, Clamp¢t8 and Peakscan (Jarolimek and Misgeld, 1997). The resting membrane potential was corrected to account for the liquid junction potential.

EXPERIMENTAL PROCEDURES

A¤nity determinations

LC slice preparation Guinea-pigs were decapitated without prior anaesthesia, in accordance with the UK Animals (Scienti¢c Procedures) Act 1986, and brain slices were prepared as follows.

Analysis of ¢ring patterns To detect the interval between events for further analysis of

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¢ring patterns, the raw potential waveforms were acquired using a CED1401plus AD converter and analysed using a Spike2 script. This script allows a cursor to be placed over the level of background noise to detect all events occurring within a selected time period. The variation coe¤cient, a measure of the regularity of the cell ¢ring patterns, was calculated as the ratio between the standard deviation and the mean interval value of events, and expressed as a percentage. Bursts were de¢ned by a minimum of three events with an inter-event interval shorter than 90 ms and an inter-burst interval exceeding 180 ms. Burst ¢ring was calculated as the ratio between events in bursts and the total number of events within a sample population, expressed as a percentage (Tung et al., 1989). Statistical analysis Data are reported as the mean þ S.E.M., the sample size (n) represents the number of brain slices, where no greater than two recordings were made from any one animal. When comparisons were made within groups an unpaired Student's t-test was used to test for signi¢cance, whereas when comparisons were made across treatment groups the non-parametric unpaired Kruskal Wallis test was used followed by post hoc comparison in pairs with Dunn's Multiple Comparison test. A P value of 6 0.05 was considered to be signi¢cant. Materials SP, NE, clonidine and imipramine were purchased from Sigma (UK). The SP antagonist, L-760735 (Kramer et al., 1998) was synthesised at Merck Sharp p Dohme. All solutions were applied by changing the perfusion solution to the bath, usually for 2-min periods. SP was dissolved in distilled water and made up to a stock concentration of 100 WM and stored as aliquots at 34³C. Clonidine was dissolved in 100% dimethylsulfoxide (DMSO; ¢nal bath concentration of 0.1%) and made up to a stock concentration of 100 mM and stored as aliquots at 34³C.

RESULTS

The bis(tri£uoromethyl) morpholine SP antagonist L-760735, an analogue of MK-869, was used as a research tool in this and previous pre-clinical studies (Kramer et al., 1998). L-760735 has high a¤nity and selectivity for the human and guinea-pig SP (NK1 ) receptor (mean inhibitory concentration IC50 0.27 and 0.34 nM respectively). In contrast, L-760735 displays no signi¢cant a¤nity for K2 adrenoceptors (K2A IC50 6.1 WM, K2B 36% inhibition at 40 WM and K2C IC50 23 WM) or L adrenoceptors in vitro (L1 15%, L2 12% and L3 8% inhibition at 1 WM). E¡ect of acute application of NE and tachykinin ligands The e¡ect of acute application of NE and tachykinin ligands on the ¢ring rate of LC neurones was studied in brain slices in vitro prepared from naive guinea-pigs. Neurones were spontaneously active and ¢red characteristically broad action potentials ( s 2.5 ms) at a rate of 2.8 þ 0.7 Hz (n = 14, range 0.64^6.36 Hz). NE (3 WM) decreased the ¢ring rate of LC neurones (398 þ 1% compared to control, n = 3, P 6 0.01, Fig. 1a), whereas the TCA imipramine (3 WM) abolished ¢ring of LC neurones altogether (n = 4, Fig. 1b). SP (100 nM) increased the ¢ring rate of LC neurones (+117 þ 38% compared to

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control, n = 5, P 6 0.01, Fig. 1c), and the SP antagonist L-760735 (100 nM) had no e¡ect on the ¢ring rate of LC neurones (0 þ 11% compared to control, n = 4, P = 0.94, Fig. 1d). Chronic SP receptor antagonist treatment stimulates burst ¢ring of LC neurones The ¢ring activity of LC neurones was then investigated in brain slices in vitro prepared from guinea-pigs that had been chronically dosed (28 days) with vehicle (water), L-760735 (3 mg/kg/day p.o.) or imipramine (10 mg/kg/day p.o.). There was a trend towards a reduction in the average ¢ring rate for LC neurones from L-760735-treated and imipramine-treated animals (1.02 þ 0.23 Hz, range 0.09^3.71 Hz, n = 22 and 1.09 þ 0.23 Hz, range 0.08^4.09 Hz, n = 22 respectively) compared with vehicle-treated animals (1.60 þ 0.57 Hz, range 0.09^6.92 Hz, n = 19), however this did not reach signi¢cance (P = 0.95), and can be attributed to the range of ¢ring rates observed. Although many neurones displayed a characteristic tonic ¢ring pattern of activity (Fig. 1ei), the presence of bursts and quiescent periods in the ¢ring pattern of many LC neurones (Fig. 1eii), made comparisons based on the average ¢ring rate inappropriate and so a more detailed analysis of the ¢ring patterns was made. LC neurones from both L-760735-treated and imipramine-treated animals displayed a marked increase in the degree of burst ¢ring (72 þ 7% burst ¢ring events, n = 22, P 6 0.001 and 60 þ 9%, n = 22, P 6 0.05 respectively) compared to vehicle-treated animals (at 31 þ 9%, n = 19). Similarly, LC neurones from both L-760735treated and imipramine-treated animals displayed an increase in the irregularity of ¢ring (variation coe¤cient of 296 þ 55%, n = 22, P 6 0.001 and 269 þ 77%, n = 22, P 6 0.05 respectively) compared to vehicle-treated animals (105 þ 19%, n = 19). This bursting pattern of activity was a result of chronic treatment with these compounds since it was not observed following acute administration of the same dose of either L-760735 or imipramine in naive animals (Fig. 1b,d). It has previously been reported that stress can alter the ¢ring rate and pattern of LC neurones (repeated inescapable footshock paired with the forced swim task, Simson and Weiss, 1988; repeated immobilisation, Pavcovich et al., 1990; cold stress, Mana and Grace, 1997). Therefore, to determine whether any mild stress that may result from the dosing procedure in£uenced the pattern of ¢ring activity of LC neurones in vitro, we conducted a second study including a group of naive animals. The average ¢ring rate for neurones in this naive group were 3.01 þ 0.61 Hz (n = 20 from 10 animals, range 0.11^4.77 Hz). The percentage of burst ¢ring events and coe¤cient of variation from naive animals (39 þ 10% and 119 þ 40% respectively, n = 20) was not di¡erent from that of vehicle-treated guinea-pigs (40 þ 24% and 133 þ 63% respectively, n = 5 from three animals, P = 0.66 and 0.38 respectively). Since there was no di¡erence in the ¢ring patterns between the

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of burst ¢ring and irregularity of ¢ring (66 þ 6% and 286 þ 45% respectively, n = 43 from 23 animals) compared to vehicle-treated animals (33 þ 9% and 111 þ 19% respectively, n = 24 from 18 animals, P 6 0.05). Chronic SP receptor antagonist treatment does not desensitise K2 adrenoceptors To investigate whether chronic treatment alters the expression of K2 adrenoceptors in the LC, we also determined responses to the application of the K2 adrenoceptor partial agonist, clonidine. Clonidine concentrationdependently (0.1^100 WM) reduced the ¢ring rate of LC neurones (IC50 0.23 WM). Chronic imipramine treatment reduced the potency of clonidine (IC50 21.7 WM) compared to vehicle treatment (P 6 0.001), consistent with a down-regulation of K2 adrenoceptors in this nucleus (Fig. 2). In contrast, chronic L-760735 treatment did not a¡ect either the potency or e¤cacy of clonidine (IC50 0.31 WM, Fig. 2) and therefore the e¡ect of the SP antagonist on the NE system was distinct from that of the NE reuptake inhibitor imipramine. These ¢ndings also show that a change in K2 adrenoceptor function in this nucleus does not underlie the changes in ¢ring pattern caused by SP antagonist treatment. Burst ¢ring induced by chronic SP receptor antagonist treatment is membrane potential-independent The possibility that the burst ¢ring pattern of activity may be elicited by changes in spontaneous synaptic activity was investigated using whole-cell patch clamp recordings in brain slices prepared from the same animals as used in the second extracellular recording study. In voltage clamp mode (membrane potential held at 360 mV), there was no di¡erence in the current^voltage relationship of neurones in the di¡erent treatment groups, indicating that there were no gross changes in input resistance. The frequency of occurrence of spontaneous excitatory postsynaptic potentials in LC neurones in the L-760735 and imipramine treatment groups (0.57 þ 0.28 Hz, n = 6 and 0.31 þ 0.11 Hz, n = 3 respectively) was not di¡erent from LC neurones in the naive and vehicle treatment groups (0.37 þ 0.15 Hz, n = 4 and 0.49 þ 0.17 Hz, n = 4 respectively).

vehicle-treated groups from the two studies (burst ¢ring P = 0.71, irregularity of ¢ring P = 0.57) these data were pooled, and again LC neurones from L-760735-treated animals displayed a marked increase in the degree

Fig. 1. E¡ect of acute application and chronic dosing of NE and SP ligands. Continuous chart records of extracellular voltage recordings from LC neurones in slices prepared from naive guinea-pig showing the e¡ect of acute application of NE and SP ligands on ¢ring rate. The vertical de£ections are action potentials which occur spontaneously both in the whole animal and in this brain slice preparation in vitro. (a) Acute application of NE (3 WM) decreased the ¢ring rate. (b) Acute application of imipramine (3 WM) abolished ¢ring. (c) Acute application of SP (100 nM) increased the ¢ring rate. (d) Acute application of L-760735 (100 nM) had no e¡ect on ¢ring rate. (e) Continuous chart records of extracellular voltage recordings from LC neurones in slices prepared from guinea-pigs chronically dosed with vehicle (ei) or L-760735 (3 mg/kg/day p.o., eii).

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Fig. 2. Chronic SP receptor antagonist treatment does not desensitise K2 adrenoceptors. Concentration-response curves showing increasing inhibition of the ¢ring of LC neurones with increasing clonidine concentration (0.1^100 WM) in vehicle, L-760735 (3 mg/ kg/day p.o.) or imipramine (10 mg/kg/day p.o.) treated guineapigs. Chronic imipramine treatment (¢lled circle) reduced the suppressant e¡ect of clonidine on ¢ring rate (IC50 21.7 WM, n = 4, P 6 0.001) compared to vehicle treatment (¢lled square, IC50 0.23 WM, n = 6). In contrast, chronic L-760735 treatment (open circle) did not a¡ect the suppressant e¡ect of clonidine on ¢ring rate (IC50 0.31 WM, n = 6).

Since burst ¢ring can be generated by activation of sodium channels (Azouz et al., 1996) and voltage-activated calcium channels (Wong and Prince, 1978), the ¢ring pattern of activity can simply alter with membrane potential. In current clamp mode (in the absence of additional current) there was no di¡erence in the resting membrane potential (364.8 þ 1.2, 367.4 þ 1.7 and 365.3 þ 2.6 mV; P = 0.73), action potential amplitude (78.8 þ 3.9, 86.9 þ 2.8 and 79.0 þ 9.4 mV; P = 0.15) or afterhyperpolarisation amplitude (11.5 þ 1.2, 11.2 þ 1.5 and 9.9 þ 2.4 mV; P = 0.09) of neurones in the control (n = 15; naive, n = 8 and vehicle, n = 7 pooled), L-760735 (n = 10) or imipramine (n = 4) treatment groups, respectively. The amplitude and duration of afterhyperpolarisations is known to re£ect Ca2‡ -activated K‡ conductances and has been reported to in£uence the ¢ring pattern of neurones (Williams et al., 1984). There appeared to be a trend towards a more transient afterhyperpolarisation in the two antidepressant treatment groups (afterhyperpolarisation amplitude 8 ms after peak as % of peak amplitude, 62.1 þ 11.1% and 48.8 þ 18.5% for L-760735 and imipramine respectively) compared to control (69.7 þ 7.2% and 71.3 þ 11.1% for naive and vehicle respectively), however this did not reach signi¢cance (Fig. 3a,b). However, when afterhyperpolarisations were measured in all neurones that showed 6 20% burst ¢ring (n = 9) or s 80% burst ¢ring (n = 13), the duration of afterhyperpolarisations was shorter in burst

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¢ring neurones, demonstrating that changes in such K‡ conductances may underlie the change in ¢ring pattern (Fig. 3c). We went on to con¢rm that the ¢ring patterns were also changed during this study. Since there was no di¡erence in the degree of burst ¢ring (38 þ 13% vs 15 þ 7%, P = 0.16) or irregularity of ¢ring (80 þ 15% vs 60 þ 6%, P = 0.27) in LC neurones from naive (n = 8) and vehicletreated animals (n = 7), these data were pooled into one control data set (n = 15). LC neurones from both L-760735-treated and imipramine-treated animals displayed an increase in the degree of burst ¢ring (90 þ 5%, P 6 0.0001, n = 10 and 88 þ 9%, P = 0.002, n = 4, respectively) compared to control animals (27 þ 8%, n = 15) using this recording technique. Similarly, LC neurones from both L-760735 treated and imipramine treated animals displayed an increase in the irregularity of ¢ring (157 þ 36%, P = 0.009, n = 10 and 129 þ 23%, P = 0.011, n = 4 respectively) compared to control animals (71 þ 8%, n = 15).

DISCUSSION

The novel ¢ndings from this study are that chronic treatment with a conventional antidepressant imipramine, and the SP antagonist L-760735, both induce burst ¢ring of neurones in the LC, but importantly that their e¡ects can be di¡erentiated in that the SP antagonist, unlike imipramine, did not alter somatic adrenoceptor function. E¡ect of acute application of NE and SP ligands on LC ¢ring characteristics LC neurones recorded from naive guinea-pigs were spontaneously active and ¢red characteristically broad action potentials at a rate of V3 Hz consistent with previous studies (Williams et al, 1984). Acute application of NE decreased the ¢ring rate of LC neurones consistent with the activation of K2 adrenoceptors and subsequent membrane hyperpolarisation (Williams et al., 1985). Acute application of imipramine abolished ¢ring of LC neurones as would be expected following NE reuptake inhibition and activation of K2 adrenoceptors by endogenous NE within the slice preparation (Dahlstrom and Fuxe, 1964). Acute application of SP increased the ¢ring rate of LC neurones, as has been reported previously following activation of NK1 receptors and subsequent membrane depolarisation (Cheeseman et al., 1983). The mechanism responsible for this excitation is thought to be due to a cation conductance increase and an inwardly rectifying potassium conductance decrease (Shen and North, 1992). In contrast, acute application of L-760735 had no e¡ect on the ¢ring rate of LC neurones, indicating the absence of any endogenous SP tone within the in vitro slice preparation. Unlike some other classes of SP antagonists (Schmidt et al., 1992), L-760735 would not be expected to display ancillary pharmacology at the concentration tested.

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E¡ect of chronic administration of NE and SP ligands on LC ¢ring characteristics Given both the presence of NK1 receptors and the excitatory e¡ects of SP in the LC, SP antagonists may reduce spontaneous LC activity. Whilst there was a trend towards a reduction in the average ¢ring rate in LC neurones from the L-760735 and imipramine chronic treatment groups compared to vehicle, this did not reach signi¢cance. These ¢ndings are consistent with results observed for other TCAs, e.g. desipramine (Valentino et al., 1990; Melia et al., 1995), but not with some antidepressants, e.g. milnacipran, where a reduction in ¢ring rate has been reported (Brady et al., 1991; Melia et al., 1995). Chronic treatment with L-760735 or imipramine did, however, elicit a functional change in the LC by increasing the degree of burst ¢ring. This observation is consistent with a recent report that acute i.v. administration of the SP antagonist GR 205171 increased the ¢ring rate of rat LC neurones in vivo (Millan et al., 2001). Moreover, acute i.v. administration of the SP antagonists RP 67580 or LY 306740 have been shown to enhance restraint stress-induced c-fos in rat LC in vivo (Hahn and Bannon, 1998). Both groups suggest that a local action on LC neurones is unlikely, postulating an indirect action on a proximal population of NK1 receptors on neurones inhibitory to NE neurones, either in the periphery or distant brain nuclei. However, in our study, the ability of chronic SP antagonist treatment to increase burst ¢ring was observed in the isolated in vitro slice preparation, enabling us to narrow the ¢eld considerably. Mechanism underlying increase in LC neurone burst ¢ring

Fig. 3. Burst ¢ring induced by chronic SP receptor antagonist treatment may be due to a conductance change. (a) Whole-cell recording of an action potential recorded from a vehicle-treated guinea-pig (ai) and an L-760735-treated guinea-pig (aii). (b) Graph showing the decay in the amplitude of the afterhyperpolarisation (AHP) at 1, 2, 3, 4 and 8 ms after the maximum. Recordings from animals chronically treated with the SP receptor antagonist L-760735 (open circle, n = 10) and the TCA imipramine (¢lled circle, n = 4) had afterhyperpolarisations of shorter duration than control animals (vehicle and naive, ¢lled square, n = 16). (c) Recordings from animals that showed greater than 80% burst ¢ring (n = 13, ¢lled circle) had afterhyperpolarisations of signi¢cantly shorter duration than animals that showed 6 20% burst ¢ring (n = 9, ¢lled square).

A change in the spontaneous activity of synaptic inputs may contribute to the increase in burst ¢ring seen in the present study. The nucleus paragigantocellularis (a major excitatory amino acid pathway) and dorsal raphe (DR) nuclei both send major projections to the LC. SP is coexpressed with serotonin in approximately 50% of DR neurones in human brain (Baker et al., 1991) and genetic disruption and pharmacological blockade of the NK1 receptor increases the ¢ring rate of DR neurones (Santarelli et al., 2001) and desensitises 5-HT1A autoreceptors in this nucleus (Froger et al., 2001). In addition, burst ¢ring patterns of activity have previously been reported in the DR (Hajos and Sharp, 1996). Whilst it is possible that there may be some synchronisation between the LC and raphe in vivo, these connections are not intact in the isolated in vitro slice preparation in the present study. We did record spontaneous synaptic excitatory activity within the LC slice preparation, however there was no di¡erence in the frequency of this activity in the drug treatment groups, indicating that these inputs are unlikely to underlie the observed change in LC ¢ring characteristics. Alternative explanations for the burst ¢ring may relate to an interaction between NK1 and K2-adrenergic receptors within NE neurones, as has previously been observed with SP and somatostatin or [met]enkephalin

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in the LC (Velimirovic et al., 1995), or a change in the intrinsic biophysical properties of LC neurones following chronic SP antagonist treatment. A change in Ca2‡ -activated K‡ conductances, may explain the trend towards afterhyperpolarisations of shorter duration following action potentials in the drug treatment groups. A link between Ca2‡ -activated K‡ conductances as a target for psychiatric intervention is not unprecedented, as a trinucleotide expansion repeat in hSKCa3, a gene that encodes one of the subtypes of Ca2‡ -activated K‡ channels, has been implicated in some patients with bipolar disorder (Chandy et al., 1998). Further studies are needed to determine the mechanism by which chronic SP antagonist or TCA treatment increases burst ¢ring in the guinea-pig LC. E¡ect of chronic TCA and SP receptor antagonist treatment on K2 adrenoceptor desensitisation Chronic treatment with imipramine reduced the sensitivity of LC neurones to the K2 adrenoceptor agonist, clonidine, consistent with prolonged inhibition of NE uptake and functional desensitisation of K2 adrenoceptors. This ¢nding is consistent with previous studies with chronic desipramine treatment (Scuve¨e-Moreau and Svensson, 1982; Brady et al., 1991; Melia et al., 1995). In contrast, chronic SP antagonist treatment did not reduce the sensitivity of LC neurones to clonidine and therefore did not desensitise somatic K2 adrenoceptors. This observation indicates that NE reuptake is not blocked by SP antagonist treatment, and is supported by evidence that L-760735 has no signi¢cant a¤nity for monoamine reuptake sites, transporters, or monoamine oxidase (Kramer et al., 1998). In contrast, Haddjeri and Blier (2000) have shown that acute i.v. administration of the SP antagonists, WIN 51,708 and CP-96,345, attenuates somatic K2 adrenoceptor function in rat LC. This apparent discrepancy most likely re£ects the use of different species and methodologies in these studies. WIN 51,708 and CP-96,345 display only moderate a¤nity for the rat NK1 receptor (both IC50 6 20 nM, Saria, 1999), whereas L-760735 has high a¤nity for the guinea-pig NK1 receptor (IC50 0.34 nM). Furthermore, WIN 51,708 and CP-96,345 show poor selectivity vs ion channels (Caeser et al., 1993; McLean, 1996; Schmidt et al., 1992) and enantiomeric controls were not utilised to eliminate non-speci¢c e¡ects of this compound in those studies. Relevance of LC ¢ring characteristics The activity of the LC is intimately linked with behavioural arousal. Previous studies have shown a concomitant increase in ¢ring rate of LC neurones with the level of alertness in animal studies ranging from rapid-eye movement (REM) sleep where LC neurones are virtually

615

silent (Aston-Jones and Bloom, 1981), to fully awake where LC neurones ¢re at a highly regular low rate (Foote et al., 1980). Phasic burst ¢ring activity in the LC has been associated with focused attention, electroencephalogram (EEG) spindling and orienting behaviour whereas very high tonic ¢ring rates have been associated with scanning labile attention (Aston-Jones et al., 1999). Although chronic SP antagonist treatment increases burst ¢ring in the LC when isolated in vitro, it is di¤cult to predict what e¡ect this treatment will have on the LC ¢ring pattern in an awake or sleeping animal, since LC ¢ring characteristics change with level of consciousness. The ¢ring pattern of LC neurones is known to be a¡ected by acute and chronic stress. Previous studies have shown an increase in tonic ¢ring rates by acute stressors [single inescapable footshock (Simson and Weiss, 1988), immobilisation (Pavcovich et al., 1990), cold stress (Mana and Grace, 1997)]. It has also been demonstrated that chronic stressors result in further increases in LC ¢ring rate and also change the ¢ring pattern to more phasic activity with bursts of very high frequency ¢ring [repeated inescapable footshock paired with the forced swim task (Simson and Weiss, 1988), repeated immobilisation (Pavcovich et al., 1990), 17^21 days of cold stress (Mana and Grace, 1997)]. The ability of imipramine and L-760735 to change the ¢ring pattern of LC neurones may therefore facilitate a reactive coping mechanism that allows adaptation to stress, or may redress imbalances caused by stressful situations or re£ect changes in arousal.

CONCLUSION

SP antagonists are likely to have many sites of action, since SP and NK1 receptors are localised in other brain regions that orchestrate stress responses (Shults et al., 1984). There is evidence for direct actions of SP antagonists in the amygdala (Smith et al., 1999), hypothalamus (Shaikh et al., 1993) and reticulopontine nucleus (Krase et al., 1994). However, the present study shows that both a SP antagonist and a conventional antidepressant in£uence the intrinsic pattern of ¢ring activity of LC neurones, suggesting that this may contribute to their antidepressant mechanism of action. AcknowledgementsöWe would like to thank Wolfgang Jarolimek for his expert assistance in electrophysiology, Fred Kuenzi for the preparation of scripts to permit analysis of data captured within Spike 2 and Andrew Jack for his expert advice on statistical analyses. We would also like to thank Robert Frankshun, Fintan Kelleher and Karen Haworth for NK1 receptor chemistry and also Marc Kurtz, Maria-Luisa Candelore and Margaret Cascieri for receptor binding data. We would like to acknowledge the support of Susan Boyce, Glenn Mason, Tony Davidge, Mellissa Nixon and Angela Williams in the dosing of animals.

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K. A. Maubach et al. REFERENCES

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