orexin a preferentially excites glucose-sensitive neurons...

Orexin A Preferentially Excites Glucose-SensitiveNeurons in the Lateral Hypothalamus of the RatIn VitroXiao Hui Liu,

1Richard Morris,

2David Spiller,

3Michael White,

3and Gareth Williams

1

Falls in blood glucose induce hunger and initiate feed-

ing. The lateral hypothalamic area (LHA) contains glu-

cose-sensitive neurons (GSNs) and orexin neurons,

both of which are stimulated by falling blood glucoseand are implicated in hypoglycemia-induced feeding. Wecombined intracellular electrophysiological recordingwith fluorescein labeling of GSNs to determine theirneuroanatomic and functional relationships with orexinneurons. Orexin A (1 �mol/l) caused a 500% increase(P < 0.01) in spontaneous firing rate and rapid andlasting depolarization that was tetrodotoxin-resistantand thus a direct postsynaptic effect. Orexin A alteredthe intrinsic neuronal properties of GSNs, consistentwith increased excitability. Confocal microscopy showedthat GSNs were intimately related to orexin neurons:orexin-immunoreactive axons were frequently en-twined around GSN dendrites, establishing close andputatively synaptic contacts. Orexin-cell axons alsopassed in close proximity to glucose-responsive neu-rons, which are inhibited by low glucose, but orexin Acaused smaller depolarization than on GSNs and only a200% increase in spontaneous firing rate (P < 0.05 vs.GSN). We conclude that GSNs are specific target neu-rons for orexin A and suggest that they may mediate, atleast in part, the acute appetite-stimulating effect oforexin A. Orexin neurons may regulate GSNs so as tocontrol the onset and termination of hypoglycemia-induced feeding. Diabetes 50:2431–2437, 2001

Reduced availability of glucose, the brain’s mainmetabolic fuel, causes intense hunger (1). Thelateral hypothalamic area (LHA) is crucial to thehyperphagia induced by hypoglycemia and glu-

coprivation, as this feeding response is abolished by LHAlesions (1). The LHA neuronal systems that drive gluco-privic feeding are unknown, but promising candidates

include the glucose-sensing neurons and orexin (hypocre-tin) neurons that are prominent in this region.

Glucose-sensing neurons, which alter their firing behav-ior in response to changes in ambient glucose concentra-tion, are found in the hypothalamus and in several othercentral nervous system regions (2). Glucose-sensitive neu-rons (GSNs) are inhibited by rising glucose concentrationsbut excited when glucose falls, whereas glucose-respon-sive neurons (GRNs) are stimulated as glucose rises andare inhibited by hypoglycemia (3,4). GSNs are particularlyabundant in the LHA, where they account for 30–40% of allneurons (3,5). These cells are stimulated directly by lowglucose in vitro (3,6) but are also regulated indirectly invivo, being inhibited by rising glucose levels in the hind-brain and viscera and by gastric distension (1,7). Theseindirect signals are presumed to be relayed to the LHAfrom the nucleus of the solitary tract (NTS) in the medulla,which contains glucose-sensing neurons and also receivesvagal afferents from visceral glucose sensors and gastricstretch receptors (7). In view of these properties, lateralhypothalamic GSNs are assumed to participate in trigger-ing and controlling glucoprivic feeding (1–3). The glucose-sensing neurons of the LHA have not been characterizedmorphologically or neurochemically, and their place in thehierarchy of the many hypothalamic systems that regulatefeeding is not known.

Orexins A and B are 33- and 28-residue peptides derivedfrom prepro-orexin; this precursor is also termed “prepro-hypocretin,” and hypocretins 1 and 2 include the se-quences of orexin A and B, respectively (8,9). Prepro-orexin is expressed exclusively in a discrete neuronalpopulation in the LHA and adjacent zona incerta (8,9).Orexin neurons project within the LHA itself and to otherhypothalamic nuclei involved in feeding and also sendheavy projections to distant central nervous system re-gions, including the NTS and the locus ceruleus in themedulla (9,10). Orexins are implicated in various auto-nomic processes, including feeding and wakefulness (11).Orexin A stimulates feeding acutely when injected intra-cerebroventricularly (8,12) or into the LHA (13), butchronic administration does not induce sustained hyper-phagia or obesity; this suggests a role in short-term feedingregulation (8,11,14). Prepro-orexin expression increasesduring fasting and acute hypoglycemia (8,15,16), and re-cent work suggests that a subset of orexin neurons arestimulated by falls in blood glucose (15–17). During acutehypoglycemia, 14% of orexin neurons display the earlyactivation marker Fos, whereas 9% of all Fos-positive LHA

From the 1Diabetes and Endocrinology Research Group, Department ofMedicine University of Liverpool, Liverpool, U.K.; the 2Department of Preclin-ical Veterinary Science University of Liverpool, Liverpool, U.K.; and the3School of Biological Science, University of Liverpool, Liverpool, U.K.

Address correspondence and reprint requests to Gareth Williams, Diabetesand Endocrinology Research Group, Department of Medicine, University ofLiverpool, Liverpool, L69 3GS, U.K. E-mail: [email protected].

Received for publication 2 April 2001 and accepted in revised form 19 July2001.

G.W. has received research support from SmithKline Beecham.aCSF, artificial cerebrospinal fluid; GRN, glucose-responsive neuron; GSN,

glucose-sensitive neuron; LHA, lateral hypothalamic area; NTS, nucleus of thesolitary tract; PBS, phosphate-buffered saline; Vm, resting membrane poten-tial.

DIABETES, VOL. 50, NOVEMBER 2001 2431

neurons contain orexin (15). Hypoglycemia-induced acti-vation of orexin and nonorexin LHA cells is abolished byfeeding (17). It is interesting that neuronal activation in theNTS followed the same pattern, suggesting that events inthe LHA might be mediated, at least in part, indirectly viathe ascending projection from the NTS (17).

The striking similarities between orexin neurons andGSN in the LHA suggest that they may interact function-ally. The possible relationships of GSNs with other appe-tite-regulating neuronal systems have not been explored,and the LHA neurons that mediate orexin A’s feeding effectare unknown. We hypothesized that orexin neurons regu-late GSNs through the release of orexin A. We investigatedthis possibility by using intracellular electrophysiologicalrecording in rat brain slices in vitro, first to identify GSNsand then to characterize their responses to exogenousorexin A. To visualize their anatomic relationship withorexin neurons, we filled GSNs with neurobiotin for sub-sequent staining with streptavidin-fluorescein and immu-nostained the tissue for orexin A.

RESEARCH DESIGN AND METHODS

Hypothalamic slice preparation. Wistar rats aged 2–3 weeks were anesthe-tized with sodium pentobarbital (40 mg/kg intraperitoneally) and decapitatedin accordance with U.K. Home Office procedures. For optimization of tissueviability (18), the brain was immediately immersed in chilled sucrose-basedartificial cerebrospinal fluid (aCSF) and bubbled with 95% O2/5% CO2, thecomposition of the medium being (in mmol/l) 2.5 KCl, 1.2 NaH2PO4, 26NaHCO3, 0.1 CaCl2, 1.3 MgCl2, 10 glucose, and 189 sucrose. Transversehypothalamic slices (200–400 �m) at the level of the median eminence werecut using a tissue slicer (Vibroslice 752/M; Campden Instruments, Cambridge,U.K.) and then incubated for 1 h in the same medium. One slice was thenimmersed in a recording chamber and continually perfused (6–8 ml/min) atroom temperature with gassed aCSF that had the following composition (inmmol/l): 120 NaCl2, 2.5 KCl, 1.2 NaH2PO4, 26 NaHCO3, 2.4 CaCl2, 1.3 MgCl2,and 10 glucose. The slice was allowed to equilibrate in the recording chamberfor an additional 1 h before study.Intracellular electrophysiological recording and data analysis. Intracel-lular recording microelectrodes were made from borosilicate glass on aFlaming/Brown model P-87 micropipette puller (Sutter Instruments, Novato,CA). The electrodes had resistances of 120–180 M� when filled with 2 mol/lKCH3SO4. Neurobiotin (Vector Laboratories, Burlingame, CA) 2% wt/vol wasincluded in the electrode filling solution. Standard current clamp recordingswere made using an Axoprobe-1A amplifier (Axon Instruments, Burlingame,CA). Current and voltage signals were digitized (sample rate �5 kHz, filteredat 3 kHz) and analyzed using SPIKE2 software (Cambridge Electronic Design,Cambridge, U.K.). Input resistance of the neuron was monitored by measuringthe amplitude of the voltage transient in response to a 0.1- to 0.3-nAhyperpolarizing current step (500 ms duration). The change in number ofevoked action potentials was tested using a 0.1- to 0.5-nA depolarizing currentpulse (500–1,000 ms duration).

Bath glucose concentration was altered to 3 or 15 mmol/l from basal 10mmol/l, for 3-min periods, to identify glucose-sensing neurons as describedbelow. After re-equilibration in 10 mmol/l glucose for 30 min, orexin A(GlaxoSmithKline, Harlow, U.K.; 1 �mol/l in aCSF) was applied to the slicesfor 2–4 min by switching between reservoirs containing standard aCSF andthe orexin solution. Switching artifacts were rarely apparent.

Changes in membrane potential and intrinsic neuronal properties inresponse to changes in glucose concentration or addition of orexin A wereexpressed as mean � SE and compared versus baseline and among the threecell types, using the paired or unpaired Student’s t test as appropriate. Achange in input resistance of �20% was regarded as significant. Changes infiring frequency in response to addition of orexin A were measured during100-s intervals and expressed as percentage increase above baseline. Thesedata were analyzed initially using two-way analysis of variance with cell typeand time as independent variables, followed by the unpaired t test to examinedifferences between cell types at individual time points.Immunocytochemistry. For visualization of neurobiotin-filled neurons,slices were fixed overnight in 4% paraformaldehyde and washed in fourchanges of 0.1 mol/l phosphate-buffered saline (PBS). Slices were thenincubated in 1% Triton X-100 in PBS for 2 h and in 1:100 streptavidin-

dichlorotriazinylaminofluorescein (Jackson, West Grove, PA) in 1% Triton foranother 2 h before being washed four times in PBS. Orexin A immunoreac-tivity was identified using standard indirect immunofluorescence techniques.The slice was incubated with rabbit polyclonal antisera raised against nativeorexin A (SmithKline Beecham; 1:250 to 1:500 dilution with 1% Triton X-100)for 24 h at room temperature. Slices were washed four times in PBS, thenincubated for 4–6 h at room temperature with a conjugated Cy3-taggedsecondary antibody (Jackson) 1:100 in PBS containing 1% Triton X-100. Afterfour additional washes in PBS, slices were floated onto gelatin-coated slidesand mounted with Vectashield mounting medium (Vector Laboratories).

Dual fluorescence studies were performed using a Zeiss LSM510 confocalmicroscope. Dichlorotriazinylaminofluorescein (neurobiotin) was excited us-ing the 488-nm light from an argon ion laser, and emitted light was collectedoff a 545-nm dichroic mirror through a 530- to 550-nm band-pass filter. Cy3fluorescence (orexin A) elicited by the 543-nm line of a helium-neon laser wascollected through a 545-nm dichroic mirror and a 560-nm long-pass filter.Spillover between fluorochromes was minimized by sequential scanning withthe alternate excitation light sources and photomultiplier tubes were pro-tected by appropriate filters. With the use of the supplied Zeiss software,overlapping confocal slices were combined to create three-dimensionalrenders of the stack of images.

Axons and dendrites showed classical features (19) and were discrimi-nated as follows. Dendrites arose from the neuron soma and taperedgradually; they branched typically into two, and these second-order dendriteswere reduced in diameter. By contrast, the axon arose from a cone-shapedhillock on the soma or a principal dendrite, as a small-caliber process thatmaintained the same diameter as it ran from the soma. At branch points, theaxonal branches had the same diameter as the parent axon. Axons of orexinneurons typically carried varicosites and terminated in boutons.

RESULTS

Electrophysiological responses of GSNs and GRNs.

GSNs. GSNs comprised 13 of the 29 (45%) LHA neuronscharacterized in detail. GSNs were identified by showingan increased spontaneous firing rate (�100% for �2 min)when the bath glucose concentration was lowered from 10to 3 mmol/l (Fig. 1A). At 10 mmol/l glucose, restingmembrane potential (Vm) of the GSNs was 64.7 � 1.7 mV,spontaneous firing rate was 0.13 � 0.01 Hz, and inputresistance was 212 � 25 M�. Exposure to 3 mmol/lglucose caused prompt depolarization of 5.2 � 1.0 mV anda 150% increase in firing rate (0.29 � 0.02 Hz; P � 0.01).Conversely, 15 mmol/l glucose inhibited spontaneous fir-ing (0.09 � 0.03 Hz; P � 0.01 vs. 10 mmol/l glucose). Thesecells were then subjected to one or more additional tests,described below.

Addition of orexin A (1 �mol/l) caused rapid, marked,and prolonged excitation of GSNs. When tested at 10mmol/l glucose, orexin A caused prompt (within 40 s)depolarization of 9.9 � 2.2 mV (n � 13) and an immediate500% increase in spontaneous firing rate (P � 0.01) thatdeclined gradually over several minutes (Figs. 1B and 2).Further addition of orexin A within 60 min elicited smallerdepolarizations of 5–8 mV, indicating desensitization.Orexin A also altered the intrinsic neuronal properties ofGSNs, consistent with increased excitability. When theGSNs were clamped at their baseline Vm, the input resis-tance was significantly increased by 34% (284 � 18 vs.212 � 25 M�, n � 8; P � 0.05). In addition, the depolar-izing threshold current needed to trigger an action poten-tial was reduced by 37% (0.07 � 0.01 vs. 0.11 � 0.2 nA, n �8; P � 0.01), whereas the number of spikes induced by astepped depolarizing current was doubled (4.2 � 0.7 vs.2.2 � 0.4, n � 7; P � 0.01) (Fig. 1C).

The magnitude and time course of depolarization sug-gested that orexin A acted directly on the impaled GSNs.For verification of this, GSNs (n � 3) were rechallengedwith orexin A in the presence of tetrodotoxin (1 �mol/l).

OREXIN A EXCITES GSN IN HYPOTHALAMUS OF RATS

2432 DIABETES, VOL. 50, NOVEMBER 2001

FIG. 1. Electrophysiological responses of typical GSNs. A: Lowering the bath glucose concentration from 10 to 3 mmol/l causes increased firing.B: Orexin A (1 �mol/l) excites a GSN, causing prompt depolarization and a sustained increase in spontaneous firing rate (see also Fig. 2). C:Injecting a depolarizing current pulse during depolarization induced by orexin A caused a marked increase in the number of evoked actionpotentials. This effect was sustained when the Vm was clamped at the control level. These recordings were from three different cells.

X.H. LIU AND ASSOCIATES


No significant reduction in the depolarization induced byorexin A was seen (data not shown), confirming a directpostsynaptic action. Orexin A also antagonized the inhib-itory effects of hyperglycemia on GSNs, restoring thespontaneous firing rate at a glucose concentration of 15mmol/l (0.39 � 0.15 vs. 0.09 � 0.03 Hz, n � 3; P � 0.01).GRNs and glucose-indifferent neurons. GRNs weredefined as cells showing �50% reduction in spontaneousfiring rate when glucose was decreased from 10 to 3mmol/l, and these accounted for 10 (34%) of the LHAneurons sampled. At 10 mmol/l glucose, GRNs had a Vm of61.2 � 1.9 mV and spontaneous firing rate of 0.19 � 0.04Hz, which fell to 0.06 � 0.01 Hz (P � 0.01) in 3 mmol/lglucose (Fig. 3A). Five of these cells were also tested at aglucose concentration of 15 mmol/l, which increased theirfiring rate to 0.46 � 0.05 Hz (P � 0.01).

Orexin A caused excitation of GRNs, but these effectswere significantly less marked than on GSNs and werecomparable to those elicited by orexin A in the glucose-indifferent neurons that constituted the remaining six(21%) neurons studied (Figs. 2, 3A, and 3C). At 10 mmol/lglucose, orexin A–induced depolarization of GRNs was6.3 � 1.4 mV (n � 10; P � 0.05 vs. GSNs) and inglucose-indifferent neurons was 4.2 � 1.4 mV (n � 6; P �0.05 vs. GRNs). Two-way analysis of variance showedsignificant effects of cell type (P � 0.024) and of time (P �0.02) on firing rate after application of orexin A (see Fig.2). The spontaneous firing rate of GRNs increased to apeak of only 200% above baseline (P � 0.05 vs. GSNs). Themagnitude of this response to orexin A was closely similarto that of glucose-indifferent cells, and the firing rates ofthese two cell types did not differ significantly at any timepoint (Fig. 2).Imaging of glucose-sensing and orexin neurons. Con-focal microscopy of fluorescein-labeled GSNs revealedfusiform or pyramidal cell bodies with complex ramifyingprocesses, some extending up to 800 �m from theperikaryon (Fig. 4A). Cells identified electrophysiologi-cally as GRNs showed broadly similar structural features.In some sections, two to five contiguous cells of similarmorphology were labeled (Fig. 4B), even though only asingle GSN or GRN had been injected with neurobiotin.

As in previous reports (20–22), immunostaining fororexin A showed rounded or elongated cell bodies in the

LHA and perifornical area, with abundant intensely la-beled fibers, many carrying the characteristic varicosities.None of the labeled GSNs or GRNs contained orexin A orhad varicose processes. However, both GSNs and GRNswere intimately related to orexin neurons (Fig. 4). Vari-cose orexin-cell axons were frequently entwined aroundand in close contact with the dendrites of GSNs (Fig. 4C).Conversely, axons and dendrites of GSNs were seen inclose contact with dendrites of orexin neurons (Fig. 4A).Orexin axons also passed close to GRNs processes, whiledendrites and axons of GRNs were in close proximity todendrites of orexin neurons (Fig. 4D).

DISCUSSION

This is the first demonstration of the detailed morphologyof the glucose-sensing neurons of the LHA and of theiranatomic and functional relationship to another hypotha-lamic neuronal system implicated in autonomic regulation.Both GSNs and GRNs have extensive and complex den-dritic systems and long axons that could interact withother neuronal systems in and possibly beyond the LHA.The spread of intracellularly injected label into chains ofmorphologically similar neurons (Fig. 4B) suggests thatglucose-sensing neurons are coupled through gap junc-tions and thus may form a functional network.

Particularly striking are the intimate reciprocal relation-ships between orexin neurons and glucose-sensing cells.We did not find any glucose-sensing neurons (either GSNsor GRNs) to contain orexin A. It has been reported thatintracellular labeling can mask �-aminobutyric acid–likeimmunoreactivity in hippocampal neurons (23), and it ispossible that orexin immunoreactivity was similarly con-cealed here. However, none of the fluorescein-labeled cellsin our study seemed to have the varicose processes typicalof orexin neurons (20–22). We therefore conclude that atleast some GSNs and GRNs are distinct from orexinneurons, although because orexin neurons are relativelysparse, we cannot exclude the possibility that some glu-cose-sensing cells may express orexin. We previouslyreported that acute hypoglycemia induced Fos positivity ina population of LHA neurons of which only 9% wereorexin-immunoreactive (17); we presume that the non-orexin-containing remainder of these hypoglycemia-acti-vated cells includes the GSNs.

Our findings also indicate that orexin neurons makesynaptic contact on GSNs and excite them via release oforexin A. Our imaging method cannot identify synapsesdirectly, but the juxtaposition of orexin axons and GSNsprocesses (Fig. 4C) and the powerful, tetrodotoxin-resis-tant depolarization of GSNs induced by orexin A stronglysuggest synaptic contact. Thus, GSNs seem to be targetneurons for orexin in the LHA. It is intriguing that some ofour images suggest that GSNs may also synapse ontoorexin neurons; this raises the possibility that these twoneuronal populations regulate each other in a reciprocalmanner, but this must remain speculative until the neuro-chemical identity of GSNs has been established. Orexin Aalso caused excitation of GRNs; however, this was signif-icantly less than in GSNs, being no greater than thatelicited in LHA neurons indifferent to glucose and consis-tent with the peptide’s effects on neurons elsewhere, e.g.,in the locus ceruleus (24).

FIG. 2. Orexin A (1 �mol/l) preferentially excites GSNs more thanGRNs or glucose-indifferent cells in the LHA. Increases in firing rateare expressed as the percentage change above baseline during 100-speriods after orexin A application. *P < 0.05; **P < 0.01.



FIG. 3. Electrophysiological responses of a typical GRNs. A: Lowering the bath glucose concentration from 10 to 3 mmol/l abolished firing in aGRN. B: Orexin A (1 �mol/l) induced only modest depolarization and a slight increase in firing rate (see also Fig. 2). C: Injecting a depolarizingcurrent pulse caused a modest increase in the number of evoked action potentials. These recordings were from three different cells.



The orexins have been attributed various physiologicalfunctions, and it is likely that this population of neurons isfunctionally heterogeneous. There is sound circumstantialevidence that both orexin neurons (11–17) and GSNs (1,2)could be involved in hypoglycemia-induced feeding. Wenow suggest that a subset of orexin neurons are respon-sible for regulating certain lateral hypothalamic GSNs andthat this interaction is important in controlling aspects offeeding behavior. GSNs may mediate the acute appetite-stimulating effect of orexin A injected into the LHA (13).Moreover, GSNs may drive feeding during hypoglycemia,which is known to excite orexin neurons (15–17) andwould be predicted to enhance orexin A release.

In theory, the orexin neurons could also help to termi-nate glucoprivic feeding by relaying inhibitory satietysignals to the GSNs. It is not known whether the projection

from the NTS that conveys inhibitory signals to the GSNsterminates directly on these cells. Our recent studies showthat a subset of orexin neurons are stimulated by hypogly-cemia but promptly inhibited by eating and that theiractivation parallels that of neurons in the NTS (17). Wepropose that the orexin neurons are the primary point ofimpact of the inhibitory pathway relayed via the NTS andthat they regulate the GSNs accordingly. At present, wecannot determine whether the glucose sensitivity of orexinneurons is an intrinsic property of these cells or resultsfrom inputs from glucose-sensing cells elsewhere, notablyin the LHA or NTS.

Finally, it is possible that orexin neurons and GSNs alsoplay a role in normal feeding, which is also regulated bychanges in glucose availability, although much more sub-tle than those induced by hypoglycemia or glucose anti-

FIG. 4. Morphology of glucose-sensing neurons and orexin neurons in the LHA. A: Typical GSNs labeled with fluorescein (green), showingextensive ramification of the dendritic system and its intimate relationship with surrounding orexin neuronal cell bodies (red). Both the axon(arrowhead) and dendrites (arrow) of GSNs have close contacts with orexin neurons. B: Three contiguous neurons labeled with fluorescein. Thecell originally injected showed the electrophysiological characteristics of a GRN. Close contact with an orexin cell body is shown (arrow). C:Close, putatively synaptic contact between an orexin axon bearing varicosities (red) and a dendrite of a GSN (green). D: Putative synapticcontacts between the axon (arrowhead) and dendrites of a GRN and the processes of orexin neurons. All close contacts were verified by rotationof the three-dimensional confocal image stack.



metabolites. It has been shown that small glucose falls(�0.3–0.5 mmol/l) precede spontaneous feeding episodesin normal rats and that administration of exogenousglucose to abolish these dips can delay feeding (25). Invivo electrophysiological studies indicate that such smallglucose dips can be detected by some “class 1” GSNs in theLHA (5). Immunoneutralization of orexin A (26) or admin-istration of an orexin 1 receptor antagonist (27) inhibitsshort-term food intake in normal rats. We speculate thatthis is due at least in part to reduced excitation by orexinA of GSNs, thus effectively lowering the blood glucoselevel at which eating is triggered.

ACKNOWLEDGMENTS

X.L. was supported by the Scottish Executive and addi-tional funding was from GlaxoSmithKline U.K. and theLiverpool Diabetes Research Action Fund.

We are indebted to Drs. Martyn Evans, Jon Arch, andShelagh Wilson (GlaxoSmithKline, Harlow, Essex, U.K.)for generous gifts of reagents and much useful and stim-ulating discussion and to Profs. Ian Silver and MariaErecinska and Dr. Gul Erdemli for helpful comments onthe manuscript.

REFERENCES

1. Bernardis LL, Bellinger LL: The lateral hypothalamic area revisited:ingestive behavior. Neurosci Biobehav Rev 20:189–287, 1996

2. Levin BE, Dunn-Meynell AA, Routh VH: Brain glucose sensing and bodyenergy homeostasis: role in obesity and diabetes. Am J Physiol 276:R1223–R1231, 1999

3. Oomura Y, Ono T, Ooyama H, Wayner MJ: Glucose and osmosensitiveneurons of the rat hypothalamus. Nature 222:282–284, 1969

4. Ashford MLJ, Boden PR, Treherne JM: Glucose-induced excitation ofrat hypothalamic neurones is mediated by ATP-sensitive K channels.Pflugers Arch 415:479–483, 1990

5. Silver IA, Erecinska M: Glucose-induced intracellular ion changes insugar-sensitive hypothalamic neurons. J Neurophysiol 79:1733–1745, 1998

6. Shibata S, Oomura Y, Kita H: Ontogenesis of glucose sensitivity in the ratlateral hypothalamus: a brain slice study. Dev Brain Res 5:114–117, 1982

7. Shimizu N, Oomura Y, Novin D, Grijava CV, Cooper PH: Functionalcorrelations between lateral hypothalamic glucose-sensitive neurons andhepatic portal glucose-sensitive units in rat. Brain Res 265:49–54, 1983

8. Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H,Williams SC, Richardson JA, Kozlowski GP, Wilson S, Arch JRS, Bucking-ham RE, Haynes AC, Carr SA, Annan RS, McNulty DE, Liu WS, Terrett JA,Elshourbagy NA, Bergsma DJ, Yanagisawa M: Orexins and orexin recep-tors—a family of hypothalamic neuropeptides and G protein-coupledreceptors that regulate feeding behavior. Cell 92:573–585, 1998

9. De Lecea L, Kilduff TS, Peyron C, Gao XB, Foye PE, Fukuhara C,Battenberg ELF, Gautvik KM, Bartlett FS, Frankel WN, van den Pol AN,Bloom FE, Gautvik VT, Sutcliff JG: The hypocretins: hypothalamus-specific

peptides with neuroexcitatory activity. Proc Natl Acad Sci U S A 95:322–327, 1998

10. Peyron C, Tighe DK, van Den Pol AN, De Lecea L, Heller HC, Sutcliff JG,Kilduff TS: Neurons containing hypocretin (orexin) project to multipleneuronal systems. J Neurosci 18:9996–10015, 1998

11. Arch JRS: Orexins, feeding and the big picture. Br J Nutr 84:401–403, 200012. Sweet DC, Levine AS, Billington CJ, Kotz CM: Feeding response to central

orexins. Brain Res 821:535–538, 199913. Mullett MA, Billington CJ, Levine AS, Kotz CM: Hypocretin I in the lateral

hypothalamus activates key feeding-regulatory brain sites. Neuroreport

11:103–108, 200014. Haynes AC, Jackson B, Overend P, Buckingham R, Wilson S, Tadayyon M,

Arch JS: Effects of single and chronic intracerebroventricular administra-tion of the orexins on feeding in the rat. Peptides 20:1099–1105, 1999

15. Cai XJ, Widdowson P, Harrold J, Wilson S, Buckingham R, Arch J,Tadayyon M, Clapham JC, Wilding J, Williams G: Hypothalamic orexinexpression: modulation by blood glucose and feeding. Diabetes 48:2132–2137, 1999

16. Griffond B, Risold PY, Jacquemard C, Colard C, Fellmann D: Insulin-induced hypoglycemia increases preprohypocretin (orexin) mRNA in therat lateral hypothalamic area. Neurosci Lett 262:77–80, 1999

17. Cai XJ, Evans ML, Lister CA, Leslie RA, Arch JRS, Wilson S, Williams G:Hypoglycemia activates orexin neurons and selectively increases hypotha-lamic orexin-B levels: responses inhibited by feeding and possibly medi-ated by the nucleus of the solitary tract. Diabetes 50:105–112, 2001

18. Aghajanian GK, Rasmussen K: Intracellular studies in the facial nucleusillustrating a simple new method for obtaining viable motoneurons in adultrat brain slices. Synapse 3:331–338, 1989

19. Cheunsuang O, Morris R: Spinal lamina I neurons that express neurokinin1 receptors: morphological analysis. Neuroscience 97:335–345, 2000

20. Date Y, Ueta Y, Yamashita H, Yamaguchi H, Matsukura S, Kangawa K,Sakurai T, Yanagisawa M, Nakazato M: Orexins, orexigenic hypothalamicpeptides, interact with autonomic, neuroendocrine and neuroregulatorysystems. Proc Natl Acad Sci U S A 96:748–753, 1999

21. Horvath TL, Peyron C, Diano S, Ivanov A, Aston-Jones G, Kilduff TS, vanden Pol AN: Hypocretin (orexin) activation and synaptic innervation of thelocus coeruleus noradrenergic system. J Comp Neurol 415:145–159, 1999

22. Cutler DJ, Morris R, Sheridhar V, Wattam TAK, Holmes S, Patel S, ArchJRS, Wilson S, Buckingham RE, Evans ML, Leslie SA, Williams G: Differ-ential distribution of orexin A and orexin B immunoreactivity in the ratbrain and spinal cord. Peptides 20:1455–1470, 1999

23. Scharfman HE, Kunkel DD, Schwartzkroin PA: Intracellular dyes maskimmunoreactivity of hippocampal interneurons. Neurosci Lett 96:23–28,1989

24. Hagan JJ, Leslie RA, Patel S, Evans ML, Wattam TA, Holmes S, BenhamCD, Taylor SG, Routledge C, Hemmati P, Munton RP, Jones DNC, SmithMI, Piper DC, Hunter AJ, Porter RA, Upton N: Orexin A activates locuscoeruleus cell firing and increases arousal in the rat. Proc Natl Acad Sci

U S A 96:10911–10916, 199925. Louis-Sylvestre J, Le Magnen J: A fall in blood glucose level precedes meal

onset in free-feeding rats. J Neurosci Biohev Rev 4 (Suppl. 1):13–15, 198026. Yamada H, Okumura T, Motomura W, Kobayashi Y, Kohgo Y: Inhibition of

food intake by central injection of anti-orexin antibody in fasted rats.Biochem Biophys Res Commun 267:527–531, 2000

27. Haynes AC, Jackson B, Chapman H, Tadayyon M, Johns A, Porter RA, ArchJR: A selective orexin-1 receptor antagonist reduces food consumption inmale and female rats. Regul Pept 96:45–51, 2000



orexin a preferentially excites glucose-sensitive neurons...

Documents