Inhibition of nicotine-induced hippocampal norepinephrine release in rats by alpha-conotoxins MII and AuIB microinjected into the locus coeruleus

Neuroscience Letters 266 (1999) 113±116

Inhibition of nicotine-induced hippocampal norepinephrine release in rats by alpha-conotoxins MII and AuIB microinjected into the locus coeruleus Yitong Fu a, Shannon G. Matta a, J. Michael McIntosh b, Burt M. Sharp a,* a

Department of Pharmacology, University of Tennessee-Memphis, 874 Union Avenue, Memphis, TN 38163, USA b Department of Biology, University of Utah, Salt Lake City, UT 84112, USA Received 7 January 1999; received in revised form 10 March 1999; accepted 15 March 1999

Abstract Hippocampal norepinephrine (NE) is secreted by neurons projecting from the locus coeruleus (LC) to the hippocampus; LC nicotinic receptors (NAchRs) are involved in the effects of systemic nicotine on this pathway. To clarify the NAchR subtypes, NAchR antagonists, termed a -conotoxins, were microinjected into the LC before nicotine; MII and AulB were used to assess the potential involvement of a 3b 2 and a 3b 4 subunit-containing NAchRs, respectively. Nicotine dose-dependently stimulated hippocampal NE release (P , 0:01). MII (.0.25 pmol) reduced the NE response to nicotine (67% decrease; P , 0:05), as did AuIB (44% reduction by 25 pmol; P , 0:05). Administered together, however, MII and AuIB were no more effective than MII. Thus, MII and AuIB are capable of interacting with NAchR subtypes other than those previously de®ned as a 3b 2 and a 3b 4, respectively. NAchRs containing both b 2 and b 4subunits may be involved. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nicotine; Alpha-conotoxin MII; Alpha-Conotoxin AuIB; Norepinephrine; Hippocampus; Locus coeruleus; Nicotinic receptors; In vivo microdialysis

The CNS noradrenergic system is involved in stressrelated responses and memory function [1]. Systemically administered nicotine has been reported to stimulate norepinephrine (NE) release in the hippocampus [7], paraventricular nucleus of the hypothalamus [8], amygdala [7] and the cerebral cortex [19]. Therefore, nicotine-enhanced NE secretion in the limbic system, frontal cortex and hypothalamus may underlie some of the psychoactive effects of cigarette smoke. Previous studies have shown that systemic nicotine stimulates hippocampal NE release by binding to nicotinic cholinergic receptors (NAchRs) located within the locus coeruleus (LC) [7]. Hippocampal NE secretion was blocked by injecting the antagonists, mecamylamine (Mec), dihydro-b -erythroidine (DHbE) or methyllycaconitine into the cerebral aqueduct, immediately upstream from the LC [7]. Based on the differences observed in the potencies and ef®cacies of these nicotinic antagonists, hypotheses were advanced about the composition of the LC NAchR subtypes * Corresponding author. Tel.: 11-901-448-6000; fax: 11-901448-7206. E-mail address: [email protected] (B.M. Sharp)

involved in hippocampal NE release. All three antagonists had comparable IC50 values for NE secretion; oocyte transfection studies have shown that only a 3b 2 subunits have comparable sensitivity to these three antagonists [2,11]. Thus, these pharmacological studies point to the involvement of LC a 3b 2 receptors in hippocampal NE release. An additional contribution from a 3b 4 subunits was suggested by the greater ef®cacy of Mec (87% blockade of NE release) than DHb E (63% blockade); mecamylamine is known to be more effective than DHb E [2,11] at a 3b 4subunit-containing NAchRs. Both of these hypotheses are supported by in situ hybridization studies suggesting that a 3 is the major NAchR agonist subunit found in the LC, where moderate levels of b 2 and b 4subunit mRNAs are present [20]. Therefore, we postulated that a 3b 2 and a 3b 4 subunit-containing NAchRs within the LC are involved in hippocampal NE release in response to nicotine [7]. Studies utilizing NAchR antagonists with greater speci®city are required to evaluate this hypothesis further. Recently, NAchR antagonists speci®c for a 3b 2 and a 3b 4 subunit-containing NAchRs have been identi®ed. The a -conotoxins (isolated from the marine snail, Conus), MII and AuIB, selectively block a 3b 2 and a 3b 4 NAchRs,

0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 29 3- 1

114

Y. Fu et al. / Neuroscience Letters 266 (1999) 113±116

Fig. 1. Microinfusion of nicotine (60±600 pmol) into LC dosedependently stimulates hippocampal NE secretion. Peak NE levels were measured in the samples collected 20 min after nicotine infusion. *P , 0:05 or **, 0:01, respectively, compared with CSF infusion only (n ˆ 6 rats per group).

respectively [3,12,14,16]. Studies using these two a -conotoxins have shown that a 3b 4 and not a 3b 2 NAchRs are involved in nicotine-stimulated NE release from hippocampal synaptosomes. However, a 3b 2 and not a 3b 4 NAchRs mediated nicotine-stimulated striatal DA secretion [16]. In the present study, nicotine was directly microinjected into the LC region and hippocampal NE release was then measured, using in vivo microdialysis. To further determine the NAchR subunit(s) involved, MII and/or AuIB were microinjected into the LC prior to nicotine. Adult male Holtzman rats (250±300 g), given ad libitum access to standard rat chow and water, were individually housed on a 12 h reversed light cycle (lights off at 10:00, on at 22:00 h) for 14 days prior to the microdialysis experiments. After 7 days of acclimatization to the reversed light cycle, rats were equipped with guide cannulae stereotaxically implanted into both the hippocampus (AP, 23.0 mm; DV 22.6 mm; ML, 1.4 mm, from bregma with a ¯at skull) and the ipsilateral LC (AP, 29.7 mm; DV 25.6 mm; ML, 1.8 mm) [18]. Seven days thereafter, in vivo microdialysis was conducted on alert, freely moving rats. On the day of microdialysis, rats were moved into the alert-rat microdialysis chambers (CMA, Boston, MA) that were housed in a separate room lit by a red safe-light; all connections were made rapidly to minimize stress. A 2 mm concentric probe (MW cutoff: 13 000 Da) was perfused at 1 ml/min with a solution of Kreb's ringer buffer (147 mM NaCl, 4.0 mM KCl and 3.4 mM CaCl2; 0.2 mm ®lter sterilized and degassed). Then, the probe was inserted into the hippocampus, and was perfused continuously at 1 ml/min. Two hours after insertion of the probe, microdialysis samples were each collected over 20 min into vials containing 1 ml of 5% perchloric acid, and then NE levels were quanti®ed by HPLC with electrochemical detection (ESA, Chelmsford, MA), as previously described [7,8]. Chromatographic data were analyzed with PowerChrom (AD Instruments, Castle Hill, NSW, Australia) and NE values were

expressed as a percentage of basal NE levels. Basal NE levels were de®ned as the average of the three consecutive NE samples obtained immediately prior to the ®rst injection of nicotine or vehicle. Peak NE values were measured in the samples collected 20 min after the injection of nicotine; these were expressed as a percentage of the pre-infusion basal levels. Data were analyzed using one-way analysis of variance or unpaired t-tests (for experiments with two groups), by Statview. After the three 20 min, basal samples were collected, nicotine (6, 20, 60, 300 and 600 pmol; free base, Sigma) or arti®cial cerebrospinal ¯uid (CSF: 300 mg/ml BSA in 0.05 M phosphate buffer, pH 7.2) was microinjected in 200 nl over 60 s, directly into the LC. Thereafter, three additional 20 min samples were collected. Norepinephrine release (de®ned as peak NE in the ®gures) was only observed during the ®rst 20 min sample after the administration of nicotine. Fig. 1 shows that nicotine (60 pmol or higher) dose-dependently stimulated hippocampal NE secretion (F ˆ 4:08, P , 0:01). To identify the LC NAchR(s) involved in NE secretion, MII (0.025, 0.25, 0.5, 2.5 and 25 pmol) or AuIB (1, 2.5, 5 and 25 pmol) was microinjected directly into the LC over 60 s in 200 nl. Five minutes later, nicotine (60 pmol) was microinjected into the same site and hippocampal microdialysates were collected for another hour. The basal levels of hippocampal NE were not altered during the 5 min interval following the intra-LC injection of either dose of MII or AuIB. Fig. 2 demonstrated that MII (0.025±2.5 pmol) dose-dependently reduced NE secretion in response to nicotine. The maximal inhibition was 67% with an approximate IC50 of 0.15 pmol. Previous studies in our laboratory have shown that microinjection of 3H-nicotine into the LC resulted in a 1mm 3 spherical distribution [17]. Based on this radial diffusion, an IC50 of 0.15 pmol MII would be expected to yield a tissue concentration of approximately 150 nM (or less, since the molecular weight of MII is greater than nicotine). This IC50 value is higher than those reported

Fig. 2. Alpha-Conotoxin MII (0.025±25 pmol) dose-dependently reduced hippocampal NE secretion in response to nicotine. *P , 0:05, **P , 0:01, compared with CSF/Nicotine 60 pmol (n ˆ 5 rats per treatment).

Y. Fu et al. / Neuroscience Letters 266 (1999) 113±116

Fig. 3. Alpha-Conotoxin AuIB, at a dose of 25 pmol, inhibited hippocampal NE response to nicotine. *P , 0:05, compared with CSF/Nicotine 60 pmol (n ˆ 5 rats per treatment).

from 0.5±8.0 nM, based on studies using a 3b 2-containing NAchRs expressed in Xenopus oocytes [3,12,13]. These studies also established the speci®city of MII for a 3b 2containing NAchR; other subunit combinations were found to be at least 200-fold less sensitive to blockade by MII [3,12]. Fig. 3 showed that AuIB, at a dose of 25 pmol, signi®cantly inhibited nicotine-induced hippocampal NE secretion. The maximal inhibition was 44% with an approximate IC50 of 2.2 pmol. This translates into an estimated tissue concentration of 2.2 mM, a value similar to the IC50 value (0.75 mM) obtained with rat a 3b 4-containing NAchRs expressed in Xenopus oocytes [16]. It has been reported that AuIB is at least 100-fold more potent on a 3b 4-containing NAchRs compared to other nicotinic receptor combinations containing both a and b subunits. However, AuIB is only 10-fold more potent on a 3b 4 than on a 7 homonomeric NAchRs [16]. To exclude the possibility that the higher doses of AuIB (e.g. 25 pmol) blocked a 7 subunit-containing receptor, 25 pmol of a -bungarotoxin (a speci®c antagonist for a 7 subunit, [4]) was microinjected into the LC, 5 min prior to 60 pmol nicotine. The results indicated that a -bungarotoxin was completely ineffective (t ˆ 1:36, P . 0:05, n ˆ 5). This is consistent with our previous data which showed that an injection of a -bungarotoxin into the cerebral aqueduct had no effect on hippocampal NE release stimulated by systemic nicotine [7]. Figs. 2 and 3 show that there were no signi®cant differences in the inhibitory effects of the highest doses of either conotoxin on NE release. The partial effect of each conotoxin may re¯ect the fact that multiple NAchR subtypes in the LC are involved in the hippocampal NE response to nicotine. MII is reported to be a selective antagonist of a 3b 2 NAchRs, while AuIB is selective for a 3b 4 [16]. MII, at doses of 0.025±0.25 pmol, reduced nicotine-stimulated NE release by 17.7±49.9%. At higher doses, its effects were substantially less. Similarly, AuIB showed limited ef®cacy. These data suggest that a 3b 2- or a 3b 4-containing

115

NAchRs each account for only a fraction of nicotine-stimulated hippocampal NE secretion. Thus, the administration of MII and AuIB together would be expected to exhibit additive effects. Fig. 4 shows the results of an experiment in which 2.5 pmol MII and 25 pmol AuIB were coinjected into the LC 5 min prior to 60 pmol of nicotine. Administered together, these two conotoxins had no greater effect than MII alone. To determine the upper limits for blockade of nicotineinduced hippocampal NE secretion, mecamylamine (25 pmol) was injected into the LC 5 min prior to 60 pmol nicotine. Mecamylamine inhibited 85% of the NE response (P , 0:01 compared with CSF/nicotine, n ˆ 5), showing that NE secretion was largely mediated by NAchRs. This experiment also suggests that, in addition to NAchRs that are sensitive to a -conotoxins, other LC NAchRs are involved in hippocampal NE secretion by nicotine. The particular b subunit, along with the a 3 subunit, confers speci®city to the interaction with a -conotoxins. Thus, NAchRs with both b 2 and b 4 subunits, in the presence of a 3, may be capable of binding to both MII and AuIB. Indeed, NAchRs composed of a 3b 2b 4a 5 subunits have been found in chick ciliary ganglion [5]; moreover, a 3b 4b 2 NAchRs have been identi®ed in chick brain [6]. Therefore, in the present study the lack of additivity between MII and AuIB suggests the existence in the LC of NAchRs that contain both b 2 and b 4 subunits. The speci®city of MII and AuIB also suggest that these NAchRs contain a 3 subunits. However, it is possible that these receptors may contain a 6 subunits in addition to (or rather than) a 3 sununits. The major domain within the a 3 subunit (amino acid sequence 181±195) required for recognition by MII [12] is largely conserved in the a 6 subunit [9]. The activity of MII on a 6 subunits is unknown, but the higher

Fig. 4. Alpha-Conotoxin MII (2.5 pmol) and AuIB (25 pmol) administered simultaneously showed no additional blockade compared to 2.5 pmol MII alone (F ˆ 2:639, P ˆ 0:11). NE release was signi®cantly reduced in all a -conotoxin-treated groups compared to CSF/Nicotine 60 pmol (*P , 0:05; n ˆ 6 rats per treatment).

116

Y. Fu et al. / Neuroscience Letters 266 (1999) 113±116

IC50 observed for blockade by MII in the LC might be accounted for by the presence of a closely related non-a 3 subunit. In addition, high levels of a 6 mRNA have recently been detected in catecholaminergic neurons, including those in the LC and ventral tegmental area (VTA) [15]. Also, immunohistochemical studies indicate that VTA dopaminergic neurons express a 6-containing NAchRs [10]. Taken together, these studies suggest that nicotine acts on NAchRs containing both b 2 and b 4 subunits within the LC; these nicotinic receptors also contain a 3 and/or a 6 subunits. A small component of nicotine-induced hippocampal NE secretion is also mediated by mecamylamine-sensitive, a -conotoxin-insensitive NAchRs within the LC. This work is supported by NIDA DA03977 (to B.M.S.), MH53631 and GM48677 (to J.M.M.). [1] Bremner, J.D., Krystal, J.H., Southwick, S.M. and Charney, D.S., Noradrenergic mechanisms in stress and anxiety. I. preclinical studies. Synapse, 23 (1996) 28±38. [2] Cachelin, A.B. and Rust, G., b-Subunit co-determine the sensitivity of rat neuronal nicotinic receptors to antagonists. P¯uguers Arch.-Eur. J. Physiol., 429 (1995) 449±451. [3] Cartier, G.E., Yoshikami, D., Gray, W.R., Luo, S., Olivera, B.M. and McIntosh, J.M., A new alpha-conotoxin which targets alpha3beta2 nicotinic acetylcholine receptors. J. Biol. Chem., 271 (1996) 7522±7528. [4] Colquhoun, L.M. and Patrick, J.W., Pharmacology of neuronal nicotinic acetylcholine receptor subtypes. Adv. Pharmacol., 39 (1997) 191±220. [5] Conroy, W.G. and Berg, D.K., Neurons can maintain multiple classes of nicotinic acetylcholine receptors distinguished by different subunit compositions. J. Biol. Chem., 270 (1995) 4424±4431. [6] Conroy, W.G. and Berg, D.K., Nicotinic receptor subtypes in the developing chick brain: appearance of a species containing the a4, b2, and a5 gene products. Mol. Pharmacol., 53 (1998) 392±401. [7] Fu, Y., Matta, S.G., James, T.J. and Sharp, B.M., Nicotineinduced norepinephrine release in the rat amygdala and hippocampus is mediated through brainstem nicotinc cholinergic receptors. J. Pharmacol. Exp. Ther., 284 (1998) 1188±1196. [8] Fu, Y., Matta, S.G., Valentine, J.D. and Sharp, B.M., Adrenocorticotropin response and nicotine-induced norepinephrine secretion in the rat paraventricular nucleus are mediated through brainstem receptors. Endocrinology, 138 (1997) 1935±1943. [9] Fucile, S., Matter, J.M., Erkman, L., Ragozzino, D., Barabino,

[10]

[11] [12]

[13]

[14]

[15]

[16]

[17]

[18] [19]

[20]

B., Grassi, F., Alema, S., Ballivet, M. and Eusebi, F., The neuronal alpha6subunit forms functional heteromeric acetylcholine receptors in human transfected cells. Eur. J. Neurosci., 10 (1998) 172±178. Goldner, F.M., Dineley, K.T. and Patrick, J.W., Immunohistochemical localization of the nicotinic acetylcholine receptor subunit alpha6 to dopaminergic neurons in the substantial nigra and ventral tegmental area. NeuroReport, 18 (1997) 739±2742. Harvey, S. and Luetje, C.W., Determinats of competitive antagonist sensitivity on neuronal nicotinic receptor b subunits. J. Neurosci., 16 (1996) 3798±3806. Harvey, S.C., McIntosh, J.M., Cartier, G.E., Maddox, F.N. and Luetje, C.W., Determinants of speci®city for alphaconotoxin MII on a3 b2 neuronal nicotinic receptors. Mol. Pharmacol., 51 (1997) 336±342. Kaiser, S.A., Soliakov, L., Harvey, S.C., Luetje, C.W. and Wonnacott, S., Differential inhibition by alpha-conotoxinMII of the nicotinic stimulation of [ 3H]dopamine release from rat striatal synaptosomes and slices. J. Neurochem., 70 (1997) 1069±1076. Kulak, J.M., Nguyen, T.A., Olivera, B.M. and McIntosh, J.M., Alpha-conotoxin MII blocks nicotine-stimulated dopamine release in rat striatal synaptosomes. J. Neurosci., 17 (1997) 5263±5270. Le Novere, N., Zoli, M. and Changeux, J.P., Neuronal nicotinic receptor alpha 6subunit mRNA is selectively concentrated in catecholaminergic nuclei of the rat brain. Eur. J. Neurosci., 8 (1996) 2428±2439. Luo, S., Kulak, J.M., Cartier, G.E., Jacobsen, R.B., Yoshikami, D., Olivera, B.M. and McIntosh, J.M., Alpha-conotoxin AuIB selectively blocks a3 b4 nicotinic acetylcholine receptors and nicotine-evoked norepinephrine release. J Neurosci., 18 (1998) 8571±8579. Matta, S.G., McCoy, J.G., Foster, C.A. and Sharp, B.M., Nicotinic agonists administered into the fourth ventricle stimulate norepinephrine secretion in the hypothalamic paraventricular nucleus: an in vivo microdialysis study. Neuroendocrinology, 61 (1995) 383±392. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd ed., Academic Press, New York, 1986. Summers, K.L. and Giacobini, E., Effect of local and repeated systemic administration of (2)nicotine on extracellular levels of acetylcholine, norepinephrine, dopamine, and serotonin in rat cortex. Neurochem. Res., 6 (1995) 753± 759. Wada, E., Wada, K., Boulter, J., Deneris, E., Heinemann, S., Patrick, J. and Swanson, L., Distribution of a2 a3, a4, and b2 neuronal nicotinic receptor subunit mRNAs in the central nervous system: a hybridization histochemical study in the rat. J. Comp. Neurol., 284 (1989) 314±335.