Role of the locus coeruleus in the noradrenergic response to a systemic administration of nicotine

Neuropharmacology Vol. 32, No. IO, PP. 931-949, 1993 Printedin Great Britain

0028-3908/93 $6.00 + 0.00 PergamonPressLtd

ROLE OF THE LOCUS COERULEUS IN THE NORADRENERGIC RESPONSE TO A SYSTEMIC ADMINISTRATION OF NICOTINE S. N. MITCHELL* Department

of Psychology, Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF, U.K. (Accepted I July 1993)

Summary-Experiments were conducted using in uioo microdialysis to ascertain the role of nicotinic receptors in the terminal, or the cell body area, in the hippocampal noradrenaline response provoked by a systemic administration of nicotine. These experiments combined systemic administration of nicotine with local administration of antagonists into the hippocampus via the microdialysis probe, or close to the LC via a cannula, while continuously monitoring extracellular levels of NA in the hippocampus. Systemic administration of nicotine (0.4 mg/kg, s.c.) produced a rapid and prolonged increase in extracellular levels of noradrenaline in the hippocampus of conscious animals, reaching a maximum in the first 10 min sample. In anaesthetised animals the maximum occurred 20 min after administration, but the subsequent response profile was similar. In both anaesthetised and freely moving animals nicotine increased extracellular levels of dihydroxyphenylacetic acid and homovanillic acid in the hippocampus, but failed to alter levels of dopamine or 5hydroxyindoleacetic acid. In anaesthetised animals intrahippocampal administration of nicotine (250pM over 10 min via the dialysis probe) significantly increased extracellular levels of noradrenaline; the response was shortlasting, being evident only in the 10 min sample during exposure to the drug. Local administration of nicotine failed to alter extracellular levels of any other amine or metabohte measured. Mecamylamine (25 PM), a nicotinic channel blocker, administered intrahippocampally 10 min prior to an intrahippocampal administration of nicotine completely blocked the increase in noradrenaline. However, intrahippocampal administration of mecamylamine (25 PM) for 10 min. or for the duration of recording, failed to antagonise the effect of a systemic administration of nicotine (0.4 mg/kg, s.c.) on extracellular levels of noradrenaline, dihydroxyphenylacetic acid or homovanilhc acid. In contrast administration of mecamylamine (50 PM) close to the locus coeruleus abolished the increase in noradrenaline levels in the ipsilateral hippocampus following a systemic administration of nicotine (0.4 mg/kg, s.c.), while trimethaphan (SOPM), a nicotinic receptor antagonist, significantly reduced the response. Administration of mecamylamine also attenuated increases in dihydroxyphenylacetic acid and homovanilhc acid, suggesting that the response of these metabolites may be associated with the functional metabolism of noradrenergic neurones. Locus coeruleus administration of kynurenic acid (I mM), a non-specific excitatory amino acid antagonist, was without effect. Finally, application of nicotine (50 PM) close to the locus coeruleus significantly increased extracellular levels of noradrenaline in the ipsilateral hippocampus. It is concluded that although terminals within the hippocampus are sensitive to nicotine this is not the route by which a systemic administration of nicotine increases extracellular levels of noradrenaline; instead, the majority of the response appears to be mediated by a direct effect at the cell

body area, the locus coeruleus. Key words-noradrenaline,

Previous

studies

have shown

locus coeruleus, hippocampus, microdialysis, nicotine.

that systemic

adminis-

tration of nicotine increases catecholamine (CA) synthesis, determined ex viuo (Mitchell, Brazell, Alavijeh, Joseph and Gray, 1989), and noradrenaline (NA) release in the hippocampus, determined in vivo in conscious rats (Brazell, Mitchell and Gray, 1991). These responses were blocked by prior treatment with the nicotinic antagonist mecamylamine, but not hexamethonium (Mitchell et al., 1989; Braze11 et al., 1991), indicating a central site of action, as only mecamylamine can penetrate the CNS. Moreover, *Present address: Sandoz Research Institute Berne Ltd., Monbijoustrasse 115, P.O. Box, CH-3001, Beme, Switzerland.

lesions to the ascending dorsal noradrenergic bundle (DNAB), which projects from the locus coeruleus (LC) and provides the major noradrenergic innervation of the hippocampus (Ungerstedt, 1971) blocked the effect of nicotine on CA synthesis (Mitchell, Brazell, Schugens and Gray, 1990). However, the precise site of action of nicotine, i.e. at the level of the nerve terminals or cell body, remains unknown. In vitro studies have indicated a sensitivity of both sites to nicotine. In particular nicotine increased NA release from isolated preparations of the hypothalamus (Hall and Turner, 1972; Goodman, 1974) and hippocampus (Arqueros, Naquiro and Zunino, 1978; Snell and Johnson, 1989) and increased the firing rate of LC neurons (Egan and North, 1986) 937

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in a brain slice preparation. Recent studies using in vivo microdialysis have confirmed in freely moving rats, that nicotine administered locally via the dialysis probe can increase extracellular levels of NA, implying that the response to a systemic administration may include an action of the drug at these sites (Mitchell, Smith, Joseph and Gray, 1993). In anaesthetised rats, LC neurons have also been shown to respond in a dose dependent manner following intravenous administration of nicotine (Engberg and Svensson, 1980; Svensson and Engberg, 1980) producing marked, but shortlasting increases in firing rate. These responses have revealed an alternative route through which nicotine may activate central noradrenergic neurons in vivo. This is a peripheral route, through the stimulation of primary sensory afferents (Hajos and Engberg, 1988); it is indirect, and is mediated by the release of excitatory amino acids (EAAs) from terminals projecting from the nucleus paragigantocellularis (Chen and Engberg, 1989; Engberg, 1989). Experiments were therefore conducted using in vivo microdialysis in anaesthetised animals to ascertain the role of nicotinic receptors in the terminal, or the cell body area, in the hippocampal noradrenaline response provoked by a systemic administration of nicotine, and the contribution of EAAs in this response. These experiments combined systemic administration of nicotine with local administration of nicotinic or excitatory amino acid antagonists into the hippocampus via the microdialysis probe, or close to the LC via a cannula, while continuously monitoring extracellular levels of NA in the hippocampus. METHODS

Animals Male Sprague-Dawley rats (29&32Og, B & K Universal Ltd.) used in these experiments were housed on a IO/14 hr dark/light cycle; food and water were available ad libitum. Diaiysis probe preparation and surgical implantation A concentric dialysis probe (made using AN69 Hospal membrane, 3-4mm in length; o.d. 290 pm when wet) was continuously perfused at 2.1 pl/min with Krebs’ solution containing (in mM) NaCl (120), KCI (3.8), KH,PO, (1.2), CaCl, (1.25), MgSO, (1.2), glucose (10) and NaHCO, (26), and the uptake inhibitor nomifensine (1 p M). Inclusion of nomifensine allowed the measurement of both NA and DA, as previously reported (Braze11 et al., 1991). In vitro recovery was assessed by immersion in Krebs’ solution containing a mixture of catecholamine standards (100 nM) and ascorbic acid (50 p M). All dialysis samples were collected in tubes containing 1 ~1 glacial acetic acid (1 M). Typical recoveries were between 12 and 14%. In anaesthetised animals, surgical implantation of calibrated dialysis probes were conducted under chlo-

ral hydrate anaesthesia (600 mg/kg, i.p.) in a Kopf stereotaxic frame (flat head; from bregma and dura surface: caudal - 5.8 mm, lateral -4.9 mm, vertical - 6.5 mm; Paxinos and Watson, 1982). Maintenance doses were administered when required; animals were placed on a heated pad (Vetko, Harvard) and body temperature was maintained between 35537’C. In experiments with freely moving animals, guide cannulae were implanted 5-7 days earlier under equithesin anaesthesia (3 ml/kg, i.p.; caudal - 5.8 mm, lateral -4.9 mm; vertical -2.5 mm) and on the test day probes were implanted under brief sedation (Brietal 10 mg, i.p.). Freely moving animals were then placed in a cage (dimensions: 26 x 38 x 18 cm) and connected to the infusion pump using a rodent jacket, liquid swivel and tether (Harvard). Thirty minutes following probe implantation in both anaesthetised and freely moving animals, samples were collected every 10 min in tubes containing glacial acetic acid and immediately assayed using HPLC with electrochemical detection. Samples were collected for two hours before drugs were administered, either systemically by subcutaneous (s.c.) injection (0.4 mg/kg), or locally into the hippocampus or close to the LC. For intrahippocampal administration, drugs were delivered via the dialysis probe, by switching in on-line Krebs’ solution containing the drug using a liquid switch (CMA 110, Carnegie Medicin). For LC administration, drugs were delivered through a cannula (33-gauge) implanted close to the region (from bregma and dura surface: caudal -9.5 mm, lateral - 1.4 mm, vertical -6.0 mm). Thirty minutes after cannula implantation drugs were delivered in 1~1 over I min, and the cannula was removed at the end of the experiment. All drug-induced changes in extracellular levels were expressed as a percentage of a preinjection control period (obtained by averaging the last four samples prior to drug administration, t = 90-120; 100%). Within the same dialysis sample levels of dopamine (DA), and the DA metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) and the 5-hydroxytryptamine metabolite, 5-hydroxyindoleacetic acid (5-HIAA) were also measured. The limit of detection was 5-10 fmol/sample (IO-20 fmol for HVA). Basal levels (fmol/sample) of compounds of interest were: (1) in free moving animals-NA 16.2 f 1.9, DA 4.7 &-0.4, DOPAC 58.5 f 6.4, HVA 64.9 k4.2, 05-HIAA 2859.0 f 111.5; (2) in anaesthetised animals-NA 25.0 k 1.2, DA 6.0 + 0.5, DOPAC 28.4 f 1.9, HVA 38.5 f 2.5, 5-HIAA 2024.5 + 74.5. Under both experimental conditions, 2 hr after probe implantation, extracellular levels of NA were Ca2 + -dependent. Replacement of Ca2 + with Mg2 + (1.25 mM) at this time caused extracellular levels of NA to fall below detection limits within 30min; this was observed in both freely moving and anaesthetised animals. Following the experiment histological verification of probe and cannula placements were conducted either by perfusion (for 5 min via probe) or injection

Nicotine and noradrenaline release

(0.2 ~1 through the cannula) of ink. Brains were fixed in formal-saline then sectioned to observe location. HPLC details

The HPLC system consisted of an ACS 351 series pump (HPLC Technology), on-line degasser (ERC 3510, Erma Inc.), Chromspher C,, cartridge column (5 pm particle size), guard column and saturation pre-column (all from Chrompack UK Ltd). Electrochemical detection was accomplished with an LC-4 detector (BAS Inc.); working electrode maintained at +0.73 V with respect to an Ag/AgCl reference electrode. Chromatographic separation and electrochemical detection were performed at room temperature. The mobile phase consisted of a 0.1 M/0.2 M citrate/phosphate buffer containing 1.4 mM octane sulphonic acid, 7% methanol and 1 mM ethylenediaminetetraacetic acid, at pH 2.65; the flow-rate was 0.75 ml/min. Peaks were displayed, integrated and stored using a Shimadzu CR3A coupled to an FDD-1A disk drive (Dyson Instruments Ltd). Materials

All chemicals for HPLC were AnalaR or HPLC grade (BDH). Nicotine di-tartrate (Sigma) was dissolved in isotonic saline for subcutaneous administration (0.4mg/kg, given as free base; neutralised with a few drops of 2 M NaOH) or in Krebs’ solution for local administration (250 p M). Other agents: mecamylamine hydrochloride (Merck Sharp & Dohme), trimethaphan camsylate (Roche) and kynurenic acid (Sigma). Statistical analysis

Differences in response profiles produced by systemic nicotine administration under different conditions of pretreatment were analysed by analysis of variance with repeated measures using areas under the curve. The response to local administration of nicotine were tested for significance using Student’s t-test by comparing the response to that produced by a controlled Krebs’ administration. RESULTS

Eflect of systemic administration of nicotine in anaesthetised rats: comparison with conscious animals

In freely moving animals systemic administration of nicotine (0.4 mg/kg, s.c.) produced a rapid increase in extracellular levels of NA in the hippocampus within the first 10min. This response was maximal for 10-30 min before falling, but levels remained elevated above saline controls (1 ml/kg, s.c.) for the duration of recording [saline n = 6, nicotine n = 11, comparison of area under the curve, AUC, 130-270 min: F,,:,+ = 29.47, P < 0.01; Fig. l(a)]. In these animals mcotine administration also significantly increases extracellular levels of DOPAC [AUC 130-270: &4) = 9.36, P < 0.01; Fig. l(b)] and HVA [AUC 130-270: F,,,,4) = 17.88, P < 0.01; Fig. l(c)].

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Extracellular levels of DA were barely detectable above baseline noise, and there was no significant effect of nicotine. In anaesthetised animals, systemic nicotine (0.4 mg/kg, s.c.) had a more delayed effect on extracellular levels of NA, producing a maximal increase in the second collection sample-20 minafter injection. Levels were maximal 20-40 min after administration, and again remained elevated above saline controls for the duration of recording [saline n = 5, nicotine n = 6; AUC 130-270 min: F(,,*)= 24.90, P < 0.01; Fig2(a)]. Extracellular levels of DOPAC [AUC 130-270: Fur) = 8.53, P < 0.05; Fig. 2(b)] were significantly increased, while levels of HVA were only marginally affected [AUC 13&270: Fc,,8)= 4.01, NS; Fig. 2(c)]. Extracellular levels of DA were again barely discernable from background noise, and there was no clear effect of nicotine at this dose. Extracellular levels of 5-HIAA were unaffected by nicotine in both freely moving and anaesthetised animals. The response to systemic nicotine in five consecutive samples from an anaesthetised rat is shown in Fig. 3. Eflect of local administration of nicotine in anaesthetised animals: effect of mecamylamine pretreatment

In anaesthetised animals, intrahippocampal administration of nicotine (250 PM perfused through the dialysis probe for 10 min) significantly increased extracellular levels of NA to 195 f 13% of control (P < 0.001, n = 8). This effect was only evident in the collection sample during exposure to the drug; levels returned to control levels in the following IO min collection sample (Fig. 4). There was no significant effect of nicotine treatment on extracellular levels of DA, DOPAC, HVA of 5-HIAA. Assuming that the physical limitations determining the extent to which nicotine diffuses across the membrane from the probe are the same as those for the catecholamine standards (i.e. 12-14%) perfusion with 250pM nicotine over 10 min at a flow-rate of 2.1 pl/min is equivalent to a delivery of 100-120 ng. Intrahippocampal administration of potassium (15 mM for 10 min) also significantly increased extracellular levels of NA to 142 + 10% (P < 0.005, n = 5) in the first 10 min of exposure; again levels had returned to control in the following 10 min sample. Intrahippocampal administration of mecamylamine (25 FM for IO min), given via the dialysis probe in the 10min sample prior to an intrahippocampal administration of nicotine (250 PM), completely inhibited the response seen in the first 10min sample (n = 8; see Fig. 4). Administration of the nicotinic agonist DMPP (dimethylphenylpiperazine), for IO min via the dialysis probe at 250 PM also significantly increased extracellular levels of NA (192 + 18, n = 3). DMPP produced a more prolonged effect, with levels still being elevated in the second 10 min period (201 k 13, P < 0.001); levels returned close to control in the third (123 k 6%. P < 0.05).

S. N. MITCHELL

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Fig. 1. Freely-moving animals. The effect of nicotine (0,4mg/kg, s.c.; 0) or saline (1 ml/kg, s.c.; 0) on extracellular levels of: (A) NA, (B) DOPAC and (C) HVA in the hippocampus of conscious animals. The data represent the mean & SEM relative to a pre-injection control period. Injection given at arrow.

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Fig. 2. Anaesthetised animals. The effect of nicotine (0.4mg/kg, s.c.; 0) or saline (I ml/kg, SC.; 0) on extracellular levels of (A) NA, (B) DOPAC and (C) HVA in the hippocampus of anaesthetised animals. The data represent the mean + SEM relative to a pre-injection control period. Injection given at arrow.

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Fig. 3. Representative chromatograms showing the response to a S.C. administration of nicotine (0.4 mg/kg) in five consecutive samples. Drug given at arrow. 1 = NA, 2 = DOPAC, 3 = SHIAA, 4 = DA and 5 = HVA. Run-time 9 min; 0.32 nA/V full scale deflection.

Effect of intrahippocampal administration of mecamylamine on the hippocampal response to a systemic administration of nicotine in anaesthetised animals

To determine whether nicotinic receptors in the hippocampus were responsible for the increase in

extracellular levels of NA (or DOPAC and HVA), mecamylamine was included in the Krebs’s solution perfusing the hippocampus in a dose which was effective (as shown above) at blocking the effects of a localised response to nicotine. In these experiments,

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Fig. 4. Intrahippocampal administration of drugs on extracellular levels of NA. The figure shows the responses in two consecutive 10min collection periods. Drug was present only during the first. Data represent the mean + SEM relative to a pre-injection control period. NIC = nicotine 250 PM; K = potassium 15 mM; MEC = mecamylamine 25pM (given 10min prior to NIC). *P i 0.005, **P < 0.001 vs control.

@

Nicotine and noradrenaline release mecamylamine was administered via the dialysis probe at 25 PM for 10 min, either 10 min prior to a systemic administration of nicotine (0.4 mg/kg, s.c.), or continuously for the duration of recording, and the result compared to that of a control response. Both regimes of mecamylamine administration into the hippocampus (10 min, n = 6; continuous, n = 4; control n = 6) failed to inhibit the effects of nicotine on extracellular levels of NA [AUC 140-280: F (z ,zJ= 0.49, NS; Fig. 5(a)]. Intrahippocampal administration of mecamylamine for either periods of pretreatment also failed to block the effect of systemic nicotine administration on extracellular levels of DOPAC [AUC 140-280: Fu,r, = 0.84, NS; Fig. S(b)] and HVA [AUC 140-280: Fc2,,zj= 0.71, NS; Fig. S(c)]. Eflect of LC administration of nicotinic and excitatory amino acid antagonists on the hippocampal response to systemic administration of nicotine in anaesthetised animals

In these experiments a cannula loaded with drug or Krebs’ solution was placed close to the LC 2 hr after onset of recording. Thirty minutes later, antagonists or control Krebs’ solution were administered in a volume of 1 ~1 over 1 min, followed 10 min later by a systemic administration of nicotine (0.4 mg/kg, s.c.). Placement of the cannula close to the LC by itself produced a transient rise in extracellular levels of NA in the ipsilateral hippocampus (only seen in a single 10 min collection sample), while administration of antagonists alone failed to alter extracellular levels of NA. Administration of nicotine following a control LC administration of Krebs’ solution produced a typical response profile for NA, DOPAC and HVA in the ipsilateral hippocampus (NA and DOPAC n = 9; HVA n = 8). LC administration of mecamylamine (50 PM) abolished the NA response to a systemic administration of nicotine [n = 6; AUC 170-240: F,,.,,, = 12.24, P < 0.01; Fig. 6(a)] and significantly reduced the drug’s effects on extracehular levels of DOPAC [n = 5; AUC 170-280: Fc1,,2j= 6.19, P < 0.05; Fig. 6(b)] and HVA [n = 5; AUC 170-280: F (,,,,,) = 7.31, P < 0.05; Fig. 6(c)]. LC administration of trimethaphan (50 PM), a nicotinic receptor antagonist (Ascher, Large and Rang, 1979; Rang, 1982), significantly reduced the effects of nicotine on extracellular levels of NA [n = 7; AUC 170-240: F (,.,3j = 7.07, P < 0.05; Fig. 6(a)]. From the figure the effect of trimethaphan was more apparent on the initial response to nicotine, with the remaining response profile being comparable to control. Trimethaphan also reduced the extent of both the DOPAC and HVA response to nicotine, however, these effects were not significant [DOPAC, n = 7; AUC 170-280: F,,.,,, = 2.44, NS; Fig. 6(b); HVA, n = 6; AUC 170-280: F(,,,,) = 2.56, NS; Fig. 6(c)]. In order to examine the conribution of EAAs in the response provoked by systemic nicotine, kynurenic acid (1 mM), a non-specific EAA antagonist (Perkins and Stone, 1982) was administered close to the LC.

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This drug failed to significantly alter the NA response to a systemic injection of nicotine (n = 10; AUC 170-280: Fc,.,6b= 1.09, NS). Following its administration, a S.C.injection of nicotine still evoked a large increase in extracellular levels of NA at the outset (20 min after the drug). However, over the remaining period levels were always lower than the control response [Fig. 7(a)], but the difference was not significant (n = 10; AUC 190-280: Fc1,16j= 2.58, NS). Administration of kynurenic acid close to the LC also failed to alter the hippocampal DOPAC response [n = 10; AUC 190-280: Fc,6) = 0.54, NS; Fig. 7(b)], although levels were lower than controls towards the end of the sampling period. The HVA response was unaffected by kynurenic acid pretreatment [n = 10; AUC 190-280: Fc,.,6j = 0.22, NS; Fig. 7(c)]. Effect of nicotine administered close to the LC on hippocampal NA release in anaesthetised animals

Local application of nicotine (1 ~1 of a 50 pM solution; equivalent to 8.1 ng) close to the LC significantly elevated extracellular levels of NA in the hippocampus to 281 + 65% (n = 5; P < 0.02) compared to control administration of Krebs’ solution. This response was only seen in the first 10min following administration, and levels of NA had returned to control levels in the following 10min sample. DEXXJSSlON

In both anaesthetised and freely moving animals, acute systemic administration of nicotine increases extracehular levels of NA in the hippocampus. However, one notable difference in the response profiles between the two groups of animals was that in the former group the maximum response was delayed, being evident IO-20 min following nicotine administration, rather than within the first 10min. In both groups of animals extracellular levels of DA were unaffected at this dose, but increases were observed in the levels of DA metabolites DOPAC and HVA. Extracellular levels of the 5-HT metabolite 5-HIAA were similarly unaffected by nicotine administration, confirming previous findings (Braze11 et al., 1991). Increases in extracellular levels of NA, DA, DOPAC and HVA following a systemic administration of a higher dose of nicotine have been reported to be blocked by prior treatment with mecamylamine, but not by hexamethonium. Both drugs are nicotinic channel blockers at autonomic ganglia (Ascher et al., 1979; Rang, 1982), but only mecamylamine can penetrate the CNS (Ashgar and Roth, 1971; Martin, Onaivi and Martin, 1989). The results therefore suggested that the increases represented an effect at central nicotinic receptors. In the present study, the observation that nicotine, as well as the nicotinic agonist DMPP, when administered via the dialysis probe into the hippocampus, increased extracellular levels of NA, suggested that

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Fig. 5. Effect of intrahipp~mpa~ administration of m~amylamine (MEC, 25 PM) given 10 min prior to a 0.4 mg/kg, S.C.injection of nicotine on extracellular levels of: (A) NA, (B) DOPAC and (C) HVA in the hippocampus of anaesthetised animals. Mecamylamine was delivered for either IO min (0) or for the duration of recording (a) as indicated by bars; control response (0). The data represent mean & SEM and expressed in relation to a pre-injection control period. Nicotine given at arrow.

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Fig. 6. Effect of mecamylamine (50pM; n ) or trimethaphan (50pM; A) delivered close to the LC on the response evoked in the ipsilateral hippocampus to a systemic administration of nicotine: comparison with control LC administration of Krebs’ solution (0). Cannula loaded with drug or vehicle lowered to lie close to the LC at I, 1~1 administered 30 min later at 2, and nicotine (0.4 mg/kg. s.c.) injected at 3. The data represent the mean f SEM relative to a pre-injection control period. (A) NA; (B) DOPAC; (C) HVA.

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Fig. 7. Effect of kynurenic acid (1 mM; n ) delivered close to the LC on the response evoked in the ipsilateral hippocampus: comparison with control LC administration of Krebs’ solution (0). Details as in legend to Fig. 6.

Nicotine and noradrenaline release the response to a systemic administration may be mediated by an action of the drug in the terminal region. The local effect of nicotine also appeared to represent a specific interaction with nicotinic receptors as it was blocked by an intrahippocampal administration of mecamylamine. The observation that the response to nicotine was shorter than that to DMPP, may be related to the ease with which it densitises the receptor through persistent depolarisation; a phenomenon which is commonly reported for nicotine, but not DMPP (Taylor, 1980). Although mecamylamine blocked terminal-induced release of NA, administration of the antagonist via the dialysis probe failed to antagonise the response to a systemic administration, even when administered continuously. The increase in extracellular levels of NA seen in anaesthetised animals following a systemic administration of nicotine were therefore not a consequence of an interaction with presynaptic nicotinic receptors regulating, directly or indirectly, the activity of noradrenergic neurones. Furthermore, neither regimes of mecamylamine pretreatment affected the DOPAC or HVA response, implying that these responses were similarly unrelated to a presynaptic action of the drug on dopaminergic neurones. In vitro electrophysiological studies have shown that neurones are sensitive to nicotine, causing them to depolarise (Egan and North, 1986). In the present study, the observation that first, administration of mecamylamine close to the LC inhibited the majority of the NA response in the ipsilateral hippocampus provoked by a systemic administration of nicotine, and second, that administration of trimethaphan significantly reduced the response, suggested that nicotinic receptors in the vicinity of the LC mediated the response. Moreover, the sensitivity of LC neurones was further indicated by the effect of a local application of nicotine. The source of acetylcholine which normally acts on these nicotinic receptors may arise from neighbouring pedunculopontine cell groups (McNaughton and Mason, 1980; Armstrong, Saper, Levey, Wainer and Terry, 1983). Although the LC has been reported to be rich in acetylcholinesterase (AChE; Albanese and Butcher, 1980), a more recent report has indicated that only a few neurones in the proximity of the LC are AChE-positive (Ruggiero, Giuliano, Anwar, Stornetta and Reis, 1990). The observation that mecamylamine (and to some extent, trimethaphan) attenuated the DOPAC and HVA response to a systemic administration, indicated that levels of both may be more representative of the functional metabolism of noradrenergic, rather than dopaminergic neurones, in this brain area. Although there is a well defined dopaminergic innervation of the hippocampus (Verney, Bauklac, Berger, Alvarez, Vigny and Helle, 1985), an alternative source of DOPAC and HVA may arise from overspill metabolism of DA in its conversion to NA in noradrenergic neurones. As nicotine increases NA synthesis (Mitchell et al., 1989) the formation of DA

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may occur at a rate and extent beyond the storage capacity of the neurone, with excess being metabolised. Extracellular levels of DQPAC have been used as an index of noradrenergic metabolic activity in the LC (Gillon, Richard, Quintin, Pujol and Renaud, 1990; Brun, Suaud-Chagny, Lachuer, Gonon and Buda, 1991); these results extend this to suggest that levels of both DOPAC and HVA may be representative of the functional metabolic state of noradrenergic neurones in the hippocampus. The absence of an increase in hippocampal levels of DOPAC and HVA following a local administration of nicotine into either the hippocampus or close to the LC, may be related to the time course of the response-being relatively short-lived compared with that provoked by a systemic challenge. Consequently, the demands set on NA release may be accommodated by the size of the releasable pool following a local but not a systemic administration. NA release provoked by the latter may be more dependent on newly synthesised transmitter i.e. the rate of tyrosine hydroxylation; as described above, increases in DOPAC and HVA may then be a consequence of an increase in NA synthesis. It has previously been shown that systemic administration of nicotine increases tyrosine hydroxylase (TH) in the LC and sequentially in terminal areas served by the DNAB in a time-dependent manner (Smith, Mitchell and Joseph, 1991). It is now apparent that this phenomenon reflects induction of TH, and in the hippocampus these biochemical changes are accompanied by an increase in the potential to release NA (Mitchell et al., 1993) and have behavioural effects associated with an increase in noradrenergic function (Mitchell, Grigoryan, Smith, Joseph, Sinden and Gray, 1991). The induction of TH may be a consequence of an effect of the drug at the LC, or in response to terminal release; this can now be tested directly. In addition to a direct cholinergic input (Armstrong et al., 1983), the LC also receives an excitatory input from the paragigantocellularis (PGC), located in the ventrolateral medulla (Aston-Jones, Ennis, Pieribone, Nickel1 and Shipley, 1986; Ennis and Aston-Jones, 1988) which acts as a relay station in the transmission of sensory information from the periphery through the release of excitatory amino acids. In anaesthetised rats, intravenous (iv.) administration of nicotine produces an instantaneous and shortlasting increase in the firing of LC cells, and it has been proposed that heightened LC activity may be mediated indirectly via this route, following the stimulation of primary sensory afferents (Chen and Engberg, 1989; Engberg, 1989). However, it is unclear whether such an increase in firing rate is responsible for the observed increase in NA release reported here. Results to date do not clarify the situation. First, local application of nicotine failed to increase firing rate of LC neurones in anaesthetised animals (Engberg and Svensson, 1980)

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S.N. MITCHELL

yet it increased release of NA in the hippocampus (present study), and caused LC neurones to depolarise in an in vitro slice preparation (Egan and North, 1986). Second, administration of the peripheral nicotinic channel blocker, hexamethonium, can inhibit the response of LC cells to an i.v. administration of nicotine in anaesthetised rats (Hajos and Engberg, 1988), but not the increase in NA synthesis and release in the hippocampus of conscious animals evoked by a S.C.administration (Mitchell et al., 1989; Braze11 et al., 1991). These latter responses can be inhibited by the peripheral and central nicotinic blocker, mecamylamine (Braze11er nl., 1991; Mitchell et al., 1989). However, mecamylamine also blocks NMDA stimulated NA release in vitro (Snell and Johnson, 1989), suggesting that it may act as a non-specific ligand-gated channel blocker. Conversely, MK-801 can block some peripheral and central effects of nicotine (Ramoa, Alkondon, Aracava, Irons, Lunt, Desphande, Wonnacott, Aronstam and Albuquerque, 1990). However, the effectiveness of a nicotinic receptor antagonist and the relative ineffectiveness of a non-specific EAA antagonist, kynurenic acid, implies that mecamylamine may be acting predominantly at nicotinic sites. Kynurenic acid was chosen specifically as it had been shown to block peripheral activation of the LC, e.g. following iv. nicotine (Engberg, 1989) and footshock (Ennis and Aston-Jones, 1988), and to reduce (but not completely abolish) activation of noradrenergic metabolic activity in the LC following sciatic nerve stimulation (Brun et al., 1991). In the present experiments, kynurenic acid was relatively ineffective on the initial NA response, but it did appear to have some effect on the latter stages. This finding, together with the observation that trimethaphan was not as effective as mecamylamine (assuming that at stimilar doses, the drugs are equi-potent at blocking nicotinic responses mediating this response; Ascher et al., 1979; Rang, 1982), suggests that an EAA component to the response may exist, but that it is small compared to the nicotinic component. The extent to which a peripheral-PGC-mediated increase in LC firing translates into enhanced terminal release of NA in the hippocampus, or another region predominantly served by the DNAB, needs to be evaluated. For the effects of nicotine, the extent to which a peripheral mechanism is involved may depend on the route of administration. Peripheral nerve stimulation-known to indirectly alter the activity of LC neurones (Brun et al., 1991tmay occur to a greater extent after an i.v. than a S.C. administration. The LC is also a heterogenous group of neurones with distinct spatially organised afferent projection areas (Loughlin, Foote and Bloom, 1986a; Laughlin, Foote and Grzanna, 1986b) particularly those giving rise to projections to the hippocampus, hypothalamus and spinal cord; PGCmediated excitation of the LC through a peripheral effect of nicotine may be localised to a sub-region of

the LC which is not sensitive to a direct effect of the drug. The present results show that systemic administration of nicotine increases extracellular levels of NA in the hippocampus of both conscious and anaesthetised rats. In anaesthetised animals, noradrenergic neurones in the hippocampus are sensitive to local application of nicotine, but the response provoked by a systemic administration of the drug is mediated primarily by an effect at the noradrenergic cell bodies, the LC. The similarity of response in anaesthetised and conscious animals suggests that responses to systemic nicotine will also be mediated at cell bodies in freely moving animals. Acknowledgements-This work was supported in part by The Council for Tobacco Research. U.S.A. MY thanks to Dr M. H. Joseph for comments on the manus&ipt.

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