- Email: [email protected]
hypocretins excite basal forebrain cholinergic neurones
PII: S 0 3 0 6 - 4 5 2 2 ( 0 1 ) 0 0 5 1 2 - 7
Neuroscience Vol. 108, No. 2, pp. 177^181, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 01 $20.00+0.00
www.neuroscience-ibro.com
Letter to Neuroscience OREXINS/HYPOCRETINS EXCITE BASAL FOREBRAIN CHOLINERGIC NEURONES E. EGGERMANN,a M. SERAFIN,a L. BAYER,a D. MACHARD,a B. SAINT-MLEUX,a B. E. JONESb ë HLETHALERa * and M. MU a b
De¨partement de Physiologie, Centre Me¨dical Universitaire, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland
Department of Neurology and Neurosurgery, McGill University, Montreal Neurological Institute, 3801 University Street, Montreal, QC, Canada H3A 2B4 Key words: arousal, cortical activation, sleep, ventrolateral preoptic nucleus, wakefulness.
The orexins (orexin A and B, also known as hypocretin 1 and 2) are two recently identi¢ed neuropeptides (de Lecea et al., 1998; Sakurai et al., 1998) which are importantly implicated in the control of wakefulness (for reviews see Hungs and Mignot, 2001; Kildu¡ and Peyron, 2000; Sutcli¡e and de Lecea, 2000; van den Pol, 2000; Willie et al., 2001). Indeed, alteration in these peptides' precursor, their receptors or the hypothalamic neurones that produce them leads to the sleep disorder narcolepsy (Chemelli et al., 1999; Lin et al., 1999; Peyron et al., 2000; Thannickal et al., 2000). The mechanisms by which the orexins modulate wakefulness, however, are still unclear. Their presence in ¢bres coursing from the hypothalamus (Peyron et al., 1998) up to the preoptic area (POA) and basal forebrain (BF) suggests that they might in£uence the important sleep and waking neural systems situated there (Jones, 2000). The present study, performed in rat brain slices, demonstrates, however, that the orexins have no e¡ect on the GABA sleep-promoting neurones of the POA, whereas they have a strong and direct excitatory e¡ect on the cholinergic neurones of the contiguous BF. In addition, by comparing the e¡ects of orexin A and B we demonstrate here that orexins' action depends upon orexin type 2 receptors (OX2 ), which are those lacking in narcoleptic dogs (Lin et al., 1999). These results suggest that the orexins excite cholinergic neurones that release acetylcholine in the cerebral cortex and thereby contribute to the cortical activation associated with wakefulness. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.
We ¢rst recorded in rat brain slices from identi¢ed sleep-promoting neurones, which were selected in the ventrolateral preoptic nucleus (VLPO, Fig. 1A) on the basis of their triangular form (Fig. 1B) and their inhibition by noradrenaline (Gallopin et al., 2000) (Fig. 1C). In 10 out of 11 experiments, performed in a loose-attached patch con¢guration (four under synaptic blockade by 10 mM Mg2 /0.1 mM Ca2 ), orexin A and B (applied at 0.1 or 1.0 WM) had no e¡ect (Fig. 1D) and in one experiment (at 1.0 WM) a minor excitatory action. The complete absence of an inhibitory response indicates that the orexins' role in maintaining wakefulness cannot depend upon a direct inhibition of the sleep-promoting neurones of the POA. We then tested the e¡ect of orexins on neurones of the magnocellular preoptic nucleus (MCPO, Fig. 1A), which is a major component of the cortical-projecting cholinergic system of the BF (Gritti et al., 1993). For that purpose, we ¢rst established, by ¢lling large-sized neurones (large diameter v23 Wm) with Neurobiotin (green £uorescent cell in Fig. 2A, lower inset), that those cells which were characterised electrophysiologically by the presence of a short-lasting recti¢cation (arrow in Fig. 2A, upper inset) were all cholinergic (12/12) as indicated by their immuno£uorescent staining for choline acetyltransferase (red cell in Fig. 2A). Neurones were next tested for their response to orexins while recording in a loose-attached patch con¢guration and then identi¢ed as cholinergic by switching to the whole-cell mode (see Experimental procedures) for electrophysiological characterisation (n = 10) and Neurobiotin ¢lling for subsequent immunohistochemical con¢rmation (n = 4). All cholinergic neurones were excited by orexin A (n = 10/10, Fig. 2B) and even more potently by orexin B (n = 7/7, Fig. 2C), when applied at concentrations ranging from 0.01 to 0.1 WM. This excitatory action was shown to be postsynaptic since it persisted under conditions of synaptic blockade (n = 3/3). Finally, whereas at 1 nM (n = 2) both peptides had no e¡ect, at 0.1 WM orexin B had a stronger e¡ect
*Corresponding author. Tel.: +41-22-7025807; fax: +41-227025402. E-mail address: [email protected] (M. Mu«hlethaler). Abbreviations : ACSF, arti¢cial cerebrospinal £uid; BF, basal forebrain; HEPES, N-(2-hydroxy ethyl)piperazine-NP-(2-ethanesulphonic acid); LC, locus coeruleus; MCPO, magnocellular preoptic nucleus ; OX1 , orexin type 1; OX2 , orexin type 2; POA, preoptic area; VLPO, ventrolateral preoptic nucleus. 177
NSC 5351 26-11-01
Cyaan Magenta Geel Zwart
178
E. Eggermann et al.
Fig. 1. Absence of e¡ect of orexins on VLPO neurones. (A) The BF/POA. (B, C) VLPO cells' identi¢cation by their shape and their inhibition by noradrenaline (NA). Scale bar = 5 Wm. (D) Such cells were una¡ected by orexins (Ox A: illustrated here). 3V, third ventricle; ac, anterior commissure; MCPO, magnocellular preoptic nucleus ; oc, optic chiasm ; POA, preoptic area; SIa, substantia innominata pars anterior ; VLPO, ventrolateral preoptic nucleus.
than orexin A (mean increase þ S.E.M. in ¢ring rate for orexin A 3.4 þ 1.0 Hz and for orexin B 5.7 þ 1.3 Hz; n = 4, paired t-test: t = 4.25, P = 0.024). The same was true at 0.01 WM (orexin A 0.4 þ 0.1 Hz and orexin B 1.9 þ 0.4 Hz; n = 4, paired t-test: t = 3.37, P = 0.043). In two cases the orexins had no e¡ect at 0.01 WM. These ¢ndings suggest (see below) that the e¡ects of the orexins are not due to an action upon orexin type 1 (OX1 ) but rather on OX2 receptors (Sakurai et al., 1998). It is noteworthy that in transfected cells expressing human OX2 receptors (Sakurai et al., 1998), orexin A and B demonstrated the same potency. Our results, indicating a stronger e¡ect of orexin B, could possibly re£ect di¡erences between rat and human orexin receptors. VLPO sleep-promoting neurones are part of a larger group of cells scattered in the BF/POA (Szymusiak, 1995) and which, by their GABAergic projections, are thought to inhibit the waking neuronal systems and thus enable sleep (Shiromani et al., 1999). During waking, such VLPO cells should be directly inhibited by major transmitters of arousal including noradrenaline and acetylcholine (Gallopin et al., 2000). The presence of orexin ¢bres (Peyron et al., 1998) and receptors (Marcus et al., 2001) in the POA has thus naturally led to the suggestion (for reviews see Hungs and Mignot, 2001; Willie et al., 2001) that the orexins could maintain wakefulness through a direct inhibition of sleep-promoting neurones. Our study, showing a complete absence of response of VLPO neurones to the orexins, does not support this hypothesis. The lack of a direct inhibition of VLPO neurones is congruent with a number of other recent studies commonly demonstrating a depolarising action of the orexins (Bourgin et al., 2000; Brown et al., 2001; de Lecea et al., 1998; Horvath et al., 1999; Ivanov and Aston-Jones, 2000; van den Pol et al., 1998). Interestingly, histamine, another waking transmitter that also appears to exert uniquely excitatory postsynaptic
NSC 5351 26-11-01
e¡ects (Hill et al., 1997), is similarly devoid of action on VLPO neurones (Gallopin et al., 2000). It must be stressed, however, that both histamine and the orexins might still inhibit VLPO neurones, although by an indirect path. Indeed, both histamine (Khateb et al., 1995) and the orexins (discussed below) can, for example, excite BF cholinergic neurones which in turn could inhibit VLPO neurones (Gallopin et al., 2000). In contrast to the absence of e¡ect of the orexins on VLPO sleep-promoting neurones, BF cholinergic neurones were always strongly excited by these peptides. This action, which was shown to be postsynaptic, appears to be mediated by a certain sub-type of orexin receptor known as the OX2 receptor. Indeed, the original studies on the orexins (A and B) and their receptors (OX1 and OX2 , also known as Hcrtr1 and Hcrtr2) have concluded that both peptides have a similar a¤nity for the OX2 receptor, whereas orexin A has approximately a 10 times higher a¤nity than orexin B for the OX1 receptor (Sakurai et al., 1998). Our results showing that orexin B is signi¢cantly more e¡ective than orexin A in depolarising the cholinergic cells thus indicate that OX2 and not OX1 receptors are the ones responsible for the e¡ects of the orexins on BF cholinergic neurones. As such, our results obtained in young rats are in agreement with the recent in situ hybridisation study comparing OX1 and OX2 receptor distribution in the adult rat brain and demonstrating indeed that OX2 receptors are more heavily expressed than OX1 receptors in the BF (Marcus et al., 2001). Among other systems through which the orexins may act, the noradrenergic locus coeruleus (LC) neurones have been strongly implicated. Indeed, the LC, which plays an important role in waking and arousal (Jones, 2000), receives a relatively dense orexin innervation (Peyron et al., 1998). Multiple in vitro and in vivo studies have provided evidence that LC neurones are excited by
Cyaan Magenta Geel Zwart
Orexin excites basal forebrain cholinergic neurones
179
Fig. 2. Excitatory action of orexins on BF cholinergic neurones of the MCPO. (A) Electrophysiological identi¢cation (upper left inset) and immunohistochemical con¢rmation (white arrowhead indicating a choline acetyltransferase-immunopositive cell) of a BF cholinergic neurone injected with Neurobiotin (lower right inset). Scale bar = 25 Wm. (B, C) Such cells were excited by both orexin A (Ox A) and B (Ox B). Extracellular action potentials (lower panel B) before (1), during (2) and after (3) orexin e¡ect are demonstrated.
the orexins (Bourgin et al., 2000; Hagan et al., 1999; Horvath et al., 1999; Ivanov and Aston-Jones, 2000). The functional signi¢cance of these results with respect to the control of wakefulness has, however, been debated (Hungs and Mignot, 2001; Marcus et al., 2001) because narcolepsy in dogs has been shown to depend on a de¢cit of OX2 receptors (Lin et al., 1999) whereas, at least in rats, the LC is endowed mostly with OX1 receptors (Marcus et al., 2001). In conclusion, given the well documented role of the BF cholinergic neurones in the cortical activation associated with wakefulness (for reviews see Jones, 2000; Jones and Mu«hlethaler, 1999), the excitation of these cells by the orexins ¢ts well with the view that the BF represents an important site for the arousing action of the orexins. In agreement with this view, it is noteworthy that the local perfusion of orexins in the BF induces an increase in wakefulness (Methippara et al., 2000), although the receptor type involved in the e¡ect was not determined since only orexin A was used in that study. The implication of OX2 receptors suggested by our study is of signi¢cance as it is precisely a de¢cit of this type of orexin receptors that was shown to underlie narcolepsy in dogs (Lin et al., 1999). The exact contribution to the symptoms of narcolepsy of a decreased orexin in£uence upon cholinergic neurones remains however to be established.
EXPERIMENTAL PROCEDURES
The optimal slices for recording were chosen according to published atlases for the VLPO (Sherin et al., 1998) and the MCPO (Gritti et al., 1993) in the rat. Coronal brain slices
NSC 5351 26-11-01
(300^400 Wm thick) were obtained from young rats (15^20 days, provided by the animal facility of the Geneva Medical Center and treated according to the Swiss Federal Veterinary O¤ce). They were incubated at room temperature in arti¢cial cerebrospinal £uid (ACSF) which contained (in mM): NaCl 130, KCl 5, KH2 PO4 1.25, MgSO4 1.3, NaHCO3 20, glucose 10 and CaCl2 2.4, bubbled with a mixture of 95% O2 and 5% CO2 . The same solution was used throughout the experiments on VLPO neurones. In order to induce spontaneous activity of BF cholinergic neurones, which were recorded in the looseattached mode, CaCl2 and MgSO4 were reduced (respectively to 0.7 and 0.3 mM) whereas KCl was increased to 6.2 mM. Individual slices were transferred to a thermoregulated (32³C) chamber on a Zeiss Axioskop equipped with an infrared camera (Dodt and Zieglgansberger, 1994) for extracellular or whole-cell recordings of identi¢ed cells. Slices were maintained immersed and continuously superfused at 4^5 ml/min with ACSF. Patch electrodes were pulled on a DMZ universal puller (Zeitz Instrumente, Germany) from borosilicate glass capillaries (GC150F10, Clark Instruments, UK). For whole-cell recordings, the pipettes (5^12 M6) contained the following solution (in mM): 126 KMeSO4 , 8 phosphocreatine, 4 KCl, 5 MgCl2 , 10 HEPES, 3 Na2 ATP, 0.1 GTP and 0.1 1,2-bis-(2-aminophenoxylethaneN,N,NP,NP-tetraacetic acid (pH 7.4; 290^310 mOsm). When needed, Neurobiotin (0.2%) was added to the intra-pipette solution. For extracellular recordings in the loose-attached con¢guration, patch electrodes (5^7 M6) were ¢lled with ACSF. In the VLPO, the neurones of interest were chosen on the basis of their shape and inhibition by noradrenaline (Gallopin et al., 2000), whereas for cholinergic neurones we used a two-stage approach: we ¢rst recorded them extracellularly and then, after the full pharmacological study was done, we switched to the wholecell mode for their electrophysiological and in a few cases their subsequent immunohistochemical identi¢cation. This approach was chosen to insure a maximum yield of immunostained cells by avoiding excessive internal washout while simultaneously providing for an unimpaired responsiveness to the orexins. After electrophysiological recordings, slices of BF were immediately immersed successively in an ice-cold ¢xative with 3% paraformaldehyde for 30 min and in 30% sucrose for 24 h,
Cyaan Magenta Geel Zwart
180
E. Eggermann et al.
frozen between two coverslips and stored at 380³C to be later cut with a cryostat in 45 Wm thick sections. To reveal cholinergic neurones, sections were subjected to immuno£uorescent staining using a rat monoclonal IgG against choline acetyltransferase (1:4; Boehringer Mannheim Biochemica, USA) as primary antibody and an anti-rat IgG-Cy3 (1:200; Jackson ImmunoResearch, USA) as a secondary antibody. A Cy2-conjugated streptavidin (1:200; Jackson ImmunoResearch) was used to reveal Neurobiotin. As a result, Neurobiotin-¢lled neurones
appear in green and choline acetyltransferase-positive cells in red under £uorescence microscopy.
AcknowledgementsöThis study was supported by grants from the Swiss Fonds National to M.M. and M.S., the Canadian Medical Research Council to B.E.J. and a Roche fellowship to L.B.
REFERENCES
Bourgin, P., Huitron-Resendiz, S., Spier, A.D., Fabre, V., Morte, B., Criado, J.R., Sutcli¡e, J.G., Henriksen, S.J., de Lecea, L., 2000. Hypocretin-1 modulates rapid eye movement sleep through activation of locus coeruleus neurons. J. Neurosci. 20, 7760^7765. Brown, R.E., Sergeeva, O., Eriksson, K.S., Haas, H.L., 2001. Orexin A excites serotonergic neurons in the dorsal raphe nucleus of the rat. Neuropharmacology 40, 457^459. Chemelli, R.M., Willie, J.T., Sinton, C.M., Elmquist, J.K., Scammell, T., Lee, C., Richardson, J.A., Williams, S.C., Xiong, Y., Kisanuki, Y., Fitch, T.E., Nakazato, M., Hammer, R.E., Saper, C.B., Yanagisawa, M., 1999. Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation. Cell 98, 437^451. de Lecea, L., Kildu¡, T.S., Peyron, C., Gao, X., Foye, P.E., Danielson, P.E., Fukuhara, C., Battenberg, E.L., Gautvik, V.T., Bartlett, F.S., Frankel, W.N., van den Pol, A.N., Bloom, F.E., Gautvik, K.M., Sutcli¡e, J.G., 1998. The hypocretins: hypothalamus-speci¢c peptides with neuroexcitatory activity. Proc. Natl. Acad. Sci. USA 95, 322^327. Dodt, H.U., Zieglgansberger, W., 1994. Infrared videomicroscopy : A new look at neuronal structure and function. Trends Neurosci. 17, 453^458. Gallopin, T., Fort, P., Eggermann, E., Cauli, B., Luppi, P.H., Rossier, J., Audinat, E., Mu«hlethaler, M., Sera¢n, M., 2000. Identi¢cation of sleeppromoting neurons in vitro. Nature 404, 992^995. Gritti, I., Mainville, L., Jones, B.E., 1993. Codistribution of GABA- with acetylcholine-synthesizing neurons in the basal forebrain of the rat. J. Comp. Neurol. 329, 438^457. Hagan, J.J., Leslie, R.A., Patel, S., Evans, M.L., Wattam, T.A., Holmes, S., Benham, C.D., Taylor, S.G., Routledge, C., Hemmati, P., Munton, R.P., Ashmeade, T.E., Shah, A.S., Hatcher, J.P., Hatcher, P.D., Jones, D.N., Smith, M.I., Piper, D.C., Hunter, A.J., Porter, R.A., Upton, N., 1999. Orexin A activates locus coeruleus cell ¢ring and increases arousal in the rat. Proc. Natl. Acad. Sci. USA 96, 10911^10916. Hill, S.J., Ganellin, C.R., Timmerman, H., Schwartz, J.C., Shankley, N.P., Young, J.M., Schunack, W., Levi, R., Haas, H.L., 1997. International Union of Pharmacology. XIII. Classi¢cation of histamine receptors. Pharmacol. Rev. 49, 253^278. Horvath, T.L., Peyron, C., Diano, S., Ivanov, A., Aston-Jones, G., Kildu¡, T.S., van Den Pol, A.N., 1999. Hypocretin (orexin) activation and synaptic innervation of the locus coeruleus noradrenergic system. J. Comp. Neurol. 415, 145^159. Hungs, M., Mignot, E., 2001. Hypocretin/orexin, sleep and narcolepsy. BioEssays 23, 397^408. Ivanov, A., Aston-Jones, G., 2000. Hypocretin/orexin depolarizes and decreases potassium conductance in locus coeruleus neurons. NeuroReport 11, 1755^1758. Jones, B.E., 2000. Basic mechanisms of sleep-wake state. In: Kryger, M.H., Roth, T., Dement, W.C. (Eds.), Principles and Practice of Sleep Medicine. Saunders, PA, pp. 134^154. Jones, B.E., Mu«hlethaler, M., 1999. Cholinergic and GABAergic neurons of the basal forebrain : Role in cortical activation. In: Lydic, R., Baghdoyan, H.A. (Eds.), Handbook of Behavioral State Control: Cellular and Molecular Mechanisms. CRC, New York, pp. 213^233. Khateb, A., Fort, P., Pegna, A., Jones, B.E., Mu«hlethaler, M., 1995. Cholinergic nucleus basalis neurons are excited by histamine in vitro. Neuroscience 69, 495^506. Kildu¡, T.S., Peyron, C., 2000. The hypocretin/orexin ligand-receptor system : implications for sleep and sleep disorders. Trends Neurosci. 23, 359^ 365. Lin, L., Faraco, J., Li, R., Kadotani, H., Rogers, W., Lin, X., Qiu, X., de Jong, P.J., Nishino, S., Mignot, E., 1999. The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene. Cell 98, 365^376. Marcus, J.N., Aschkenasi, C.J., Lee, C.E., Chemelli, R.M., Saper, C.B., Yanagisawa, M., Elmquist, J.K., 2001. Di¡erential expression of orexin receptors 1 and 2 in the rat brain. J. Comp. Neurol. 435, 6^25. Methippara, M.M., Alam, M.N., Szymusiak, R., McGinty, D., 2000. E¡ects of lateral preoptic area application of orexin-A on sleep-wakefulness. NeuroReport 11, 3423^3426. Peyron, C., Faraco, J., Rogers, W., Ripley, B., Overeem, S., Charnay, Y., Nevsimalova, S., Aldrich, M., Reynolds, D., Albin, R., Li, R., Hungs, M., Pedrazzoli, M., Padigaru, M., Kucherlapati, M., Fan, J., Maki, R., Lammers, G.J., Bouras, C., Kucherlapati, R., Nishino, S., Mignot, E., 2000. A mutation in a case of early onset narcolepsy and a generalized absence of hypocretin peptides in human narcoleptic brains. Nature Med. 6, 991^997. Peyron, C., Tighe, D.K., van den Pol, A.N., de Lecea, L., Heller, H.C., Sutcli¡e, J.G., Kildu¡, T.S., 1998. Neurons containing hypocretin (orexin) project to multiple neuronal systems. J. Neurosci. 18, 9996^10015. Sakurai, T., Amemiya, A., Ishii, M., Matsuzaki, I., Chemelli, R.M., Tanaka, H., Williams, S.C., Richardson, J.A., Kozlowski, G.P., Wilson, S., Arch, J.R., Buckingham, R.E., Haynes, A.C., Carr, S.A., Annan, R.S., McNulty, D.E., Liu, W.S., Terrett, J.A., Elshourbagy, N.A., Bergsma, D.J., Yanagisawa, M., 1998. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 92, 573^585. Sherin, J.E., Elmquist, J.K., Torrealba, F., Saper, C.B., 1998. Innervation of histaminergic tuberomammillary neurons by GABAergic and galaninergic neurons in the ventrolateral preoptic nucleus of the rat. J. Neurosci. 18, 4705^4721. Shiromani, P.J., Scammell, T., Sherin, J.E., Saper, C.B., 1999. Hypothalamic regulation of sleep. In: Lydic, R., Baghdoyan, H.A. (Eds.), Handbook of Behavioral State Control: Cellular and Molecular Mechanisms. CRC, New York, pp. 311^325. Sutcli¡e, J.G., de Lecea, L., 2000. The hypocretins: excitatory neuromodulatory peptides for multiple homeostatic systems, including sleep and feeding. J. Neurosci. Res. 62, 161^168. Szymusiak, R., 1995. Magnocellular nuclei of the basal forebrain: substrates of sleep and arousal regulation. Sleep 18, 478^500. Thannickal, T.C., Moore, R.Y., Nienhuis, R., Ramanathan, L., Gulyani, S., Aldrich, M., Cornford, M., Siegel, J.M., 2000. Reduced number of hypocretin neurons in human narcolepsy. Neuron 27, 469^474. van den Pol, A.N., 2000. Narcolepsy : a neurodegenerative disease of the hypocretin system ? Neuron 27, 415^418.
NSC 5351 26-11-01
Cyaan Magenta Geel Zwart
Orexin excites basal forebrain cholinergic neurones
181
van den Pol, A.N., Gao, X.B., Obrietan, K., Kildu¡, T.S., Belousov, A.B., 1998. Presynaptic and postsynaptic actions and modulation of neuroendocrine neurons by a new hypothalamic peptide, hypocretin/orexin. J. Neurosci. 18, 7962^7971. Willie, J.T., Chemelli, R.M., Sinton, C.M., Yanagisawa, M., 2001. To eat or to sleep ? orexin in the regulation of feeding and wakefulness. Annu. Rev. Neurosci. 24, 429^458. (Accepted 1 October 2001)
NSC 5351 26-11-01
Cyaan Magenta Geel Zwart
Neuroscience Vol. 108, No. 2, pp. 177^181, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 01 $20.00+0.00
www.neuroscience-ibro.com
Letter to Neuroscience OREXINS/HYPOCRETINS EXCITE BASAL FOREBRAIN CHOLINERGIC NEURONES E. EGGERMANN,a M. SERAFIN,a L. BAYER,a D. MACHARD,a B. SAINT-MLEUX,a B. E. JONESb ë HLETHALERa * and M. MU a b
De¨partement de Physiologie, Centre Me¨dical Universitaire, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland
Department of Neurology and Neurosurgery, McGill University, Montreal Neurological Institute, 3801 University Street, Montreal, QC, Canada H3A 2B4 Key words: arousal, cortical activation, sleep, ventrolateral preoptic nucleus, wakefulness.
The orexins (orexin A and B, also known as hypocretin 1 and 2) are two recently identi¢ed neuropeptides (de Lecea et al., 1998; Sakurai et al., 1998) which are importantly implicated in the control of wakefulness (for reviews see Hungs and Mignot, 2001; Kildu¡ and Peyron, 2000; Sutcli¡e and de Lecea, 2000; van den Pol, 2000; Willie et al., 2001). Indeed, alteration in these peptides' precursor, their receptors or the hypothalamic neurones that produce them leads to the sleep disorder narcolepsy (Chemelli et al., 1999; Lin et al., 1999; Peyron et al., 2000; Thannickal et al., 2000). The mechanisms by which the orexins modulate wakefulness, however, are still unclear. Their presence in ¢bres coursing from the hypothalamus (Peyron et al., 1998) up to the preoptic area (POA) and basal forebrain (BF) suggests that they might in£uence the important sleep and waking neural systems situated there (Jones, 2000). The present study, performed in rat brain slices, demonstrates, however, that the orexins have no e¡ect on the GABA sleep-promoting neurones of the POA, whereas they have a strong and direct excitatory e¡ect on the cholinergic neurones of the contiguous BF. In addition, by comparing the e¡ects of orexin A and B we demonstrate here that orexins' action depends upon orexin type 2 receptors (OX2 ), which are those lacking in narcoleptic dogs (Lin et al., 1999). These results suggest that the orexins excite cholinergic neurones that release acetylcholine in the cerebral cortex and thereby contribute to the cortical activation associated with wakefulness. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.
We ¢rst recorded in rat brain slices from identi¢ed sleep-promoting neurones, which were selected in the ventrolateral preoptic nucleus (VLPO, Fig. 1A) on the basis of their triangular form (Fig. 1B) and their inhibition by noradrenaline (Gallopin et al., 2000) (Fig. 1C). In 10 out of 11 experiments, performed in a loose-attached patch con¢guration (four under synaptic blockade by 10 mM Mg2 /0.1 mM Ca2 ), orexin A and B (applied at 0.1 or 1.0 WM) had no e¡ect (Fig. 1D) and in one experiment (at 1.0 WM) a minor excitatory action. The complete absence of an inhibitory response indicates that the orexins' role in maintaining wakefulness cannot depend upon a direct inhibition of the sleep-promoting neurones of the POA. We then tested the e¡ect of orexins on neurones of the magnocellular preoptic nucleus (MCPO, Fig. 1A), which is a major component of the cortical-projecting cholinergic system of the BF (Gritti et al., 1993). For that purpose, we ¢rst established, by ¢lling large-sized neurones (large diameter v23 Wm) with Neurobiotin (green £uorescent cell in Fig. 2A, lower inset), that those cells which were characterised electrophysiologically by the presence of a short-lasting recti¢cation (arrow in Fig. 2A, upper inset) were all cholinergic (12/12) as indicated by their immuno£uorescent staining for choline acetyltransferase (red cell in Fig. 2A). Neurones were next tested for their response to orexins while recording in a loose-attached patch con¢guration and then identi¢ed as cholinergic by switching to the whole-cell mode (see Experimental procedures) for electrophysiological characterisation (n = 10) and Neurobiotin ¢lling for subsequent immunohistochemical con¢rmation (n = 4). All cholinergic neurones were excited by orexin A (n = 10/10, Fig. 2B) and even more potently by orexin B (n = 7/7, Fig. 2C), when applied at concentrations ranging from 0.01 to 0.1 WM. This excitatory action was shown to be postsynaptic since it persisted under conditions of synaptic blockade (n = 3/3). Finally, whereas at 1 nM (n = 2) both peptides had no e¡ect, at 0.1 WM orexin B had a stronger e¡ect
*Corresponding author. Tel.: +41-22-7025807; fax: +41-227025402. E-mail address: [email protected] (M. Mu«hlethaler). Abbreviations : ACSF, arti¢cial cerebrospinal £uid; BF, basal forebrain; HEPES, N-(2-hydroxy ethyl)piperazine-NP-(2-ethanesulphonic acid); LC, locus coeruleus; MCPO, magnocellular preoptic nucleus ; OX1 , orexin type 1; OX2 , orexin type 2; POA, preoptic area; VLPO, ventrolateral preoptic nucleus. 177
NSC 5351 26-11-01
Cyaan Magenta Geel Zwart
178
E. Eggermann et al.
Fig. 1. Absence of e¡ect of orexins on VLPO neurones. (A) The BF/POA. (B, C) VLPO cells' identi¢cation by their shape and their inhibition by noradrenaline (NA). Scale bar = 5 Wm. (D) Such cells were una¡ected by orexins (Ox A: illustrated here). 3V, third ventricle; ac, anterior commissure; MCPO, magnocellular preoptic nucleus ; oc, optic chiasm ; POA, preoptic area; SIa, substantia innominata pars anterior ; VLPO, ventrolateral preoptic nucleus.
than orexin A (mean increase þ S.E.M. in ¢ring rate for orexin A 3.4 þ 1.0 Hz and for orexin B 5.7 þ 1.3 Hz; n = 4, paired t-test: t = 4.25, P = 0.024). The same was true at 0.01 WM (orexin A 0.4 þ 0.1 Hz and orexin B 1.9 þ 0.4 Hz; n = 4, paired t-test: t = 3.37, P = 0.043). In two cases the orexins had no e¡ect at 0.01 WM. These ¢ndings suggest (see below) that the e¡ects of the orexins are not due to an action upon orexin type 1 (OX1 ) but rather on OX2 receptors (Sakurai et al., 1998). It is noteworthy that in transfected cells expressing human OX2 receptors (Sakurai et al., 1998), orexin A and B demonstrated the same potency. Our results, indicating a stronger e¡ect of orexin B, could possibly re£ect di¡erences between rat and human orexin receptors. VLPO sleep-promoting neurones are part of a larger group of cells scattered in the BF/POA (Szymusiak, 1995) and which, by their GABAergic projections, are thought to inhibit the waking neuronal systems and thus enable sleep (Shiromani et al., 1999). During waking, such VLPO cells should be directly inhibited by major transmitters of arousal including noradrenaline and acetylcholine (Gallopin et al., 2000). The presence of orexin ¢bres (Peyron et al., 1998) and receptors (Marcus et al., 2001) in the POA has thus naturally led to the suggestion (for reviews see Hungs and Mignot, 2001; Willie et al., 2001) that the orexins could maintain wakefulness through a direct inhibition of sleep-promoting neurones. Our study, showing a complete absence of response of VLPO neurones to the orexins, does not support this hypothesis. The lack of a direct inhibition of VLPO neurones is congruent with a number of other recent studies commonly demonstrating a depolarising action of the orexins (Bourgin et al., 2000; Brown et al., 2001; de Lecea et al., 1998; Horvath et al., 1999; Ivanov and Aston-Jones, 2000; van den Pol et al., 1998). Interestingly, histamine, another waking transmitter that also appears to exert uniquely excitatory postsynaptic
NSC 5351 26-11-01
e¡ects (Hill et al., 1997), is similarly devoid of action on VLPO neurones (Gallopin et al., 2000). It must be stressed, however, that both histamine and the orexins might still inhibit VLPO neurones, although by an indirect path. Indeed, both histamine (Khateb et al., 1995) and the orexins (discussed below) can, for example, excite BF cholinergic neurones which in turn could inhibit VLPO neurones (Gallopin et al., 2000). In contrast to the absence of e¡ect of the orexins on VLPO sleep-promoting neurones, BF cholinergic neurones were always strongly excited by these peptides. This action, which was shown to be postsynaptic, appears to be mediated by a certain sub-type of orexin receptor known as the OX2 receptor. Indeed, the original studies on the orexins (A and B) and their receptors (OX1 and OX2 , also known as Hcrtr1 and Hcrtr2) have concluded that both peptides have a similar a¤nity for the OX2 receptor, whereas orexin A has approximately a 10 times higher a¤nity than orexin B for the OX1 receptor (Sakurai et al., 1998). Our results showing that orexin B is signi¢cantly more e¡ective than orexin A in depolarising the cholinergic cells thus indicate that OX2 and not OX1 receptors are the ones responsible for the e¡ects of the orexins on BF cholinergic neurones. As such, our results obtained in young rats are in agreement with the recent in situ hybridisation study comparing OX1 and OX2 receptor distribution in the adult rat brain and demonstrating indeed that OX2 receptors are more heavily expressed than OX1 receptors in the BF (Marcus et al., 2001). Among other systems through which the orexins may act, the noradrenergic locus coeruleus (LC) neurones have been strongly implicated. Indeed, the LC, which plays an important role in waking and arousal (Jones, 2000), receives a relatively dense orexin innervation (Peyron et al., 1998). Multiple in vitro and in vivo studies have provided evidence that LC neurones are excited by
Cyaan Magenta Geel Zwart
Orexin excites basal forebrain cholinergic neurones
179
Fig. 2. Excitatory action of orexins on BF cholinergic neurones of the MCPO. (A) Electrophysiological identi¢cation (upper left inset) and immunohistochemical con¢rmation (white arrowhead indicating a choline acetyltransferase-immunopositive cell) of a BF cholinergic neurone injected with Neurobiotin (lower right inset). Scale bar = 25 Wm. (B, C) Such cells were excited by both orexin A (Ox A) and B (Ox B). Extracellular action potentials (lower panel B) before (1), during (2) and after (3) orexin e¡ect are demonstrated.
the orexins (Bourgin et al., 2000; Hagan et al., 1999; Horvath et al., 1999; Ivanov and Aston-Jones, 2000). The functional signi¢cance of these results with respect to the control of wakefulness has, however, been debated (Hungs and Mignot, 2001; Marcus et al., 2001) because narcolepsy in dogs has been shown to depend on a de¢cit of OX2 receptors (Lin et al., 1999) whereas, at least in rats, the LC is endowed mostly with OX1 receptors (Marcus et al., 2001). In conclusion, given the well documented role of the BF cholinergic neurones in the cortical activation associated with wakefulness (for reviews see Jones, 2000; Jones and Mu«hlethaler, 1999), the excitation of these cells by the orexins ¢ts well with the view that the BF represents an important site for the arousing action of the orexins. In agreement with this view, it is noteworthy that the local perfusion of orexins in the BF induces an increase in wakefulness (Methippara et al., 2000), although the receptor type involved in the e¡ect was not determined since only orexin A was used in that study. The implication of OX2 receptors suggested by our study is of signi¢cance as it is precisely a de¢cit of this type of orexin receptors that was shown to underlie narcolepsy in dogs (Lin et al., 1999). The exact contribution to the symptoms of narcolepsy of a decreased orexin in£uence upon cholinergic neurones remains however to be established.
EXPERIMENTAL PROCEDURES
The optimal slices for recording were chosen according to published atlases for the VLPO (Sherin et al., 1998) and the MCPO (Gritti et al., 1993) in the rat. Coronal brain slices
NSC 5351 26-11-01
(300^400 Wm thick) were obtained from young rats (15^20 days, provided by the animal facility of the Geneva Medical Center and treated according to the Swiss Federal Veterinary O¤ce). They were incubated at room temperature in arti¢cial cerebrospinal £uid (ACSF) which contained (in mM): NaCl 130, KCl 5, KH2 PO4 1.25, MgSO4 1.3, NaHCO3 20, glucose 10 and CaCl2 2.4, bubbled with a mixture of 95% O2 and 5% CO2 . The same solution was used throughout the experiments on VLPO neurones. In order to induce spontaneous activity of BF cholinergic neurones, which were recorded in the looseattached mode, CaCl2 and MgSO4 were reduced (respectively to 0.7 and 0.3 mM) whereas KCl was increased to 6.2 mM. Individual slices were transferred to a thermoregulated (32³C) chamber on a Zeiss Axioskop equipped with an infrared camera (Dodt and Zieglgansberger, 1994) for extracellular or whole-cell recordings of identi¢ed cells. Slices were maintained immersed and continuously superfused at 4^5 ml/min with ACSF. Patch electrodes were pulled on a DMZ universal puller (Zeitz Instrumente, Germany) from borosilicate glass capillaries (GC150F10, Clark Instruments, UK). For whole-cell recordings, the pipettes (5^12 M6) contained the following solution (in mM): 126 KMeSO4 , 8 phosphocreatine, 4 KCl, 5 MgCl2 , 10 HEPES, 3 Na2 ATP, 0.1 GTP and 0.1 1,2-bis-(2-aminophenoxylethaneN,N,NP,NP-tetraacetic acid (pH 7.4; 290^310 mOsm). When needed, Neurobiotin (0.2%) was added to the intra-pipette solution. For extracellular recordings in the loose-attached con¢guration, patch electrodes (5^7 M6) were ¢lled with ACSF. In the VLPO, the neurones of interest were chosen on the basis of their shape and inhibition by noradrenaline (Gallopin et al., 2000), whereas for cholinergic neurones we used a two-stage approach: we ¢rst recorded them extracellularly and then, after the full pharmacological study was done, we switched to the wholecell mode for their electrophysiological and in a few cases their subsequent immunohistochemical identi¢cation. This approach was chosen to insure a maximum yield of immunostained cells by avoiding excessive internal washout while simultaneously providing for an unimpaired responsiveness to the orexins. After electrophysiological recordings, slices of BF were immediately immersed successively in an ice-cold ¢xative with 3% paraformaldehyde for 30 min and in 30% sucrose for 24 h,
Cyaan Magenta Geel Zwart
180
E. Eggermann et al.
frozen between two coverslips and stored at 380³C to be later cut with a cryostat in 45 Wm thick sections. To reveal cholinergic neurones, sections were subjected to immuno£uorescent staining using a rat monoclonal IgG against choline acetyltransferase (1:4; Boehringer Mannheim Biochemica, USA) as primary antibody and an anti-rat IgG-Cy3 (1:200; Jackson ImmunoResearch, USA) as a secondary antibody. A Cy2-conjugated streptavidin (1:200; Jackson ImmunoResearch) was used to reveal Neurobiotin. As a result, Neurobiotin-¢lled neurones
appear in green and choline acetyltransferase-positive cells in red under £uorescence microscopy.
AcknowledgementsöThis study was supported by grants from the Swiss Fonds National to M.M. and M.S., the Canadian Medical Research Council to B.E.J. and a Roche fellowship to L.B.
REFERENCES
Bourgin, P., Huitron-Resendiz, S., Spier, A.D., Fabre, V., Morte, B., Criado, J.R., Sutcli¡e, J.G., Henriksen, S.J., de Lecea, L., 2000. Hypocretin-1 modulates rapid eye movement sleep through activation of locus coeruleus neurons. J. Neurosci. 20, 7760^7765. Brown, R.E., Sergeeva, O., Eriksson, K.S., Haas, H.L., 2001. Orexin A excites serotonergic neurons in the dorsal raphe nucleus of the rat. Neuropharmacology 40, 457^459. Chemelli, R.M., Willie, J.T., Sinton, C.M., Elmquist, J.K., Scammell, T., Lee, C., Richardson, J.A., Williams, S.C., Xiong, Y., Kisanuki, Y., Fitch, T.E., Nakazato, M., Hammer, R.E., Saper, C.B., Yanagisawa, M., 1999. Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation. Cell 98, 437^451. de Lecea, L., Kildu¡, T.S., Peyron, C., Gao, X., Foye, P.E., Danielson, P.E., Fukuhara, C., Battenberg, E.L., Gautvik, V.T., Bartlett, F.S., Frankel, W.N., van den Pol, A.N., Bloom, F.E., Gautvik, K.M., Sutcli¡e, J.G., 1998. The hypocretins: hypothalamus-speci¢c peptides with neuroexcitatory activity. Proc. Natl. Acad. Sci. USA 95, 322^327. Dodt, H.U., Zieglgansberger, W., 1994. Infrared videomicroscopy : A new look at neuronal structure and function. Trends Neurosci. 17, 453^458. Gallopin, T., Fort, P., Eggermann, E., Cauli, B., Luppi, P.H., Rossier, J., Audinat, E., Mu«hlethaler, M., Sera¢n, M., 2000. Identi¢cation of sleeppromoting neurons in vitro. Nature 404, 992^995. Gritti, I., Mainville, L., Jones, B.E., 1993. Codistribution of GABA- with acetylcholine-synthesizing neurons in the basal forebrain of the rat. J. Comp. Neurol. 329, 438^457. Hagan, J.J., Leslie, R.A., Patel, S., Evans, M.L., Wattam, T.A., Holmes, S., Benham, C.D., Taylor, S.G., Routledge, C., Hemmati, P., Munton, R.P., Ashmeade, T.E., Shah, A.S., Hatcher, J.P., Hatcher, P.D., Jones, D.N., Smith, M.I., Piper, D.C., Hunter, A.J., Porter, R.A., Upton, N., 1999. Orexin A activates locus coeruleus cell ¢ring and increases arousal in the rat. Proc. Natl. Acad. Sci. USA 96, 10911^10916. Hill, S.J., Ganellin, C.R., Timmerman, H., Schwartz, J.C., Shankley, N.P., Young, J.M., Schunack, W., Levi, R., Haas, H.L., 1997. International Union of Pharmacology. XIII. Classi¢cation of histamine receptors. Pharmacol. Rev. 49, 253^278. Horvath, T.L., Peyron, C., Diano, S., Ivanov, A., Aston-Jones, G., Kildu¡, T.S., van Den Pol, A.N., 1999. Hypocretin (orexin) activation and synaptic innervation of the locus coeruleus noradrenergic system. J. Comp. Neurol. 415, 145^159. Hungs, M., Mignot, E., 2001. Hypocretin/orexin, sleep and narcolepsy. BioEssays 23, 397^408. Ivanov, A., Aston-Jones, G., 2000. Hypocretin/orexin depolarizes and decreases potassium conductance in locus coeruleus neurons. NeuroReport 11, 1755^1758. Jones, B.E., 2000. Basic mechanisms of sleep-wake state. In: Kryger, M.H., Roth, T., Dement, W.C. (Eds.), Principles and Practice of Sleep Medicine. Saunders, PA, pp. 134^154. Jones, B.E., Mu«hlethaler, M., 1999. Cholinergic and GABAergic neurons of the basal forebrain : Role in cortical activation. In: Lydic, R., Baghdoyan, H.A. (Eds.), Handbook of Behavioral State Control: Cellular and Molecular Mechanisms. CRC, New York, pp. 213^233. Khateb, A., Fort, P., Pegna, A., Jones, B.E., Mu«hlethaler, M., 1995. Cholinergic nucleus basalis neurons are excited by histamine in vitro. Neuroscience 69, 495^506. Kildu¡, T.S., Peyron, C., 2000. The hypocretin/orexin ligand-receptor system : implications for sleep and sleep disorders. Trends Neurosci. 23, 359^ 365. Lin, L., Faraco, J., Li, R., Kadotani, H., Rogers, W., Lin, X., Qiu, X., de Jong, P.J., Nishino, S., Mignot, E., 1999. The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene. Cell 98, 365^376. Marcus, J.N., Aschkenasi, C.J., Lee, C.E., Chemelli, R.M., Saper, C.B., Yanagisawa, M., Elmquist, J.K., 2001. Di¡erential expression of orexin receptors 1 and 2 in the rat brain. J. Comp. Neurol. 435, 6^25. Methippara, M.M., Alam, M.N., Szymusiak, R., McGinty, D., 2000. E¡ects of lateral preoptic area application of orexin-A on sleep-wakefulness. NeuroReport 11, 3423^3426. Peyron, C., Faraco, J., Rogers, W., Ripley, B., Overeem, S., Charnay, Y., Nevsimalova, S., Aldrich, M., Reynolds, D., Albin, R., Li, R., Hungs, M., Pedrazzoli, M., Padigaru, M., Kucherlapati, M., Fan, J., Maki, R., Lammers, G.J., Bouras, C., Kucherlapati, R., Nishino, S., Mignot, E., 2000. A mutation in a case of early onset narcolepsy and a generalized absence of hypocretin peptides in human narcoleptic brains. Nature Med. 6, 991^997. Peyron, C., Tighe, D.K., van den Pol, A.N., de Lecea, L., Heller, H.C., Sutcli¡e, J.G., Kildu¡, T.S., 1998. Neurons containing hypocretin (orexin) project to multiple neuronal systems. J. Neurosci. 18, 9996^10015. Sakurai, T., Amemiya, A., Ishii, M., Matsuzaki, I., Chemelli, R.M., Tanaka, H., Williams, S.C., Richardson, J.A., Kozlowski, G.P., Wilson, S., Arch, J.R., Buckingham, R.E., Haynes, A.C., Carr, S.A., Annan, R.S., McNulty, D.E., Liu, W.S., Terrett, J.A., Elshourbagy, N.A., Bergsma, D.J., Yanagisawa, M., 1998. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 92, 573^585. Sherin, J.E., Elmquist, J.K., Torrealba, F., Saper, C.B., 1998. Innervation of histaminergic tuberomammillary neurons by GABAergic and galaninergic neurons in the ventrolateral preoptic nucleus of the rat. J. Neurosci. 18, 4705^4721. Shiromani, P.J., Scammell, T., Sherin, J.E., Saper, C.B., 1999. Hypothalamic regulation of sleep. In: Lydic, R., Baghdoyan, H.A. (Eds.), Handbook of Behavioral State Control: Cellular and Molecular Mechanisms. CRC, New York, pp. 311^325. Sutcli¡e, J.G., de Lecea, L., 2000. The hypocretins: excitatory neuromodulatory peptides for multiple homeostatic systems, including sleep and feeding. J. Neurosci. Res. 62, 161^168. Szymusiak, R., 1995. Magnocellular nuclei of the basal forebrain: substrates of sleep and arousal regulation. Sleep 18, 478^500. Thannickal, T.C., Moore, R.Y., Nienhuis, R., Ramanathan, L., Gulyani, S., Aldrich, M., Cornford, M., Siegel, J.M., 2000. Reduced number of hypocretin neurons in human narcolepsy. Neuron 27, 469^474. van den Pol, A.N., 2000. Narcolepsy : a neurodegenerative disease of the hypocretin system ? Neuron 27, 415^418.
NSC 5351 26-11-01
Cyaan Magenta Geel Zwart
Orexin excites basal forebrain cholinergic neurones
181
van den Pol, A.N., Gao, X.B., Obrietan, K., Kildu¡, T.S., Belousov, A.B., 1998. Presynaptic and postsynaptic actions and modulation of neuroendocrine neurons by a new hypothalamic peptide, hypocretin/orexin. J. Neurosci. 18, 7962^7971. Willie, J.T., Chemelli, R.M., Sinton, C.M., Yanagisawa, M., 2001. To eat or to sleep ? orexin in the regulation of feeding and wakefulness. Annu. Rev. Neurosci. 24, 429^458. (Accepted 1 October 2001)
NSC 5351 26-11-01
Cyaan Magenta Geel Zwart