Introduction

As evidenced by the narcoleptic syndrome that occurs in mice following knock out of the gene for the peptide hypocretin/orexin (Hcrt/Orx),1 in dogs following knock out of the gene for Hcrt/Orx receptors2 and in humans in association with the loss of Hcrt/Orx peptide and neurons,3' 4 Hcrt/Orx appears to be essential for the maintenance of waking. This role may be fulfilled through the widespread projections and influence of the Hcrt/Orx neurons on multiple systems including the hypothalamo-pituitary-adrenal (HPA) axis, the sympathetic nervous system and central arousal systems, as reviewed in this volume. Indeed, Hcrt/Orx appears to have an excitatory influence upon all the brainstem arousal systems, including the noradrenergic locus coeruleus neurons,5, 6 the cholinergic pontomesencephalic neurons7 and the histaminergic tuberomammillary neurons (Fig. 1).8, 9 As we will elaborate in the present chapter, Hcrt/Orx also activates the major subcortical relays from the brainstem reticular formation to the cerebral cortex, the diffuse thalamo-cortical projection system and the cholinergic basalo-cortical projection system. Lastly, it also acts directly upon a select group of cortical neurons that may in turn together with the latter systems stimulate widespread cortical activation that supports the waking state. Interestingly, as we will also mention, Hcrt/Orx does not directly inhibit presumed sleep-promoting neurons in the forebrain.

B.E. Jones, Department of Neurology and Neurosurgery, McGill University, Montreal Neurological Institute, 3801 University Street, Montreal, Quebec, Canada H3A 2B4 M. Muhlethaler, Département de Neurosciences fondamentales, Centre Médical Universitaire, 1 rue Michel-Servet, 1211 Genève 4, Switzerland

Hypocretin Orexin Brainstem

Figure 1. Hcrt/Orx neurons amid arousal systems. Schematic sagittal view of rat brain showing the major neuronal systems and major excitatory pathways (light gray arrows) involved in promoting the EEG fast activity (upper left) and EMG high muscle tone and activity (lower right) characteristic of the waking state. Hcrt/Orx neurons are located in the perifornical and lateral, tuberal and posterior hypothalamus (PH), where they are situated amongst ascending pathways from the brainstem arousal systems. The major ascending pathways emerge from the brainstem reticular formation (RF, most densely from the mesencephalic (RF Mes) and oral pontine (PnO) fields) to ascend along 1) a dorsal trajectory into the thalamus (Th) where they terminate upon (midline, medial and intralaminar) nuclei of the nonspecific thalamo-cortical projection system, which in turn projects in a widespread manner to the cerebral cortex (Cx) and 2) a ventral trajectory through the lateral hypothalamus up to the basal forebrain where they terminate upon magnocellular basal neurons, shown in the substantia innominata (SI), which also in turn project in a widespread manner to the cerebral cortex.10 Descending projections collect from multiple levels of the reticular formation (though most densely from the caudal pontine, PnC, and medullary gigantocellular, Gi, fields) to form the reticulo-spinal pathways. The major transmitter systems that promote waking and contribute to these ascending and descending systems are represented by symbols where their cell bodies are located. Glutamatergic (Glu) neurons comprise the vast population of neurons of the reticular formation (not shown) and the diffuse thalamo-cortical projection system. Cholinergic neurons, containing acetylcholine (ACh), are located in the laterodorsal and pedunculopontine tegmental (LDTg and PPTg) nuclei in the brainstem from where they project along with other reticular neurons dorsally to the thalamus and ventrally to the posterior hypothalamus and basal forebrain, as well as to the brainstem reticular formation. Cholinergic neurons in the forebrain (SI) comprise the basalo-cortical projection. Noradrenergic (NA) neurons of the locus coeruleus (LC) send axons along the major ascending and descending pathways to project in a diffuse manner to the cortex, the subcortical relay stations, brainstem and spinal cord. Histaminergic (H) neurons of the tuberomammillary nucleus (TM) also project in a diffuse manner to the forebrain and cortex. The Hcrt/Orx neurons receive input from glutamatergic RF neurons, noradrenergic LC neurons and cholinergic LDTg/PPTg neurons of the brainstem. They also receive input from nearby histaminergic neurons. And they receive inhibitory input from GABAergic neurons of the basal forebrain (represented by a small dark gray arrow ending in a block to represent inhibition). Like other neurons of the arousal systems, Hcrt/Orx neurons project diffusely to spinal cord, the brainstem and the forebrain along the same major pathways utilized by other arousal systems. Through these projections (shown as black lines ending in arrowheads to indicate excitation), they excite many neurons, including the noradrenergic LC neurons and cholinergic LDTg/PPTg neurons in the brainstem and histaminergic neurons in the hypothalamus. In the forebrain, they directly excite the glutamatergic neurons of the nonspecific thalamo-cortical projection system (in Th) and the cholinergic neurons of the basalo-cortical projection system (in SI), which in turn stimulate widespread cortical activation. And they also directly excite cortical neurons within the deepest layer of the cortex, which in turn influence other cortical neurons. Anatomical abbreviations: 7g, 7th nerve genu; ac, anterior commissure; CPu, caudate-putamen; Cx, cortex; Gi, gigantocellular reticular formation; GP, globus pallidus; Hi, hippocampus; ic, internal capsule; LC, locus coeruleus; LDTg, laterodorsal tegmental nucleus; opt, optic tract; PH, posterior hypothalamus; POA, preoptic area; PnC, pontis caudalis reticular formation; PnO, pontis oralis reticular formation; PPTg, pedunculopontine tegmental nucleus; RF Mes, mesencephalic reticular formation; Rt, reticularis nucleus of the thalamus; s, solitary tract; scp, superior cerebellar peduncle; SI, substantia innominata; Sol, solitary tract nucleus; SN, substantia nigra; Th, thalamus; TM, tuberomammillary nucleus; VTA, ventral tegmental area. Other abbreviations: ACh, acetylcholine; EEG, electroencephalogram; EMG, electromyogram; Glu, glutamate; H, histamine; Hcrt/Orx, hypocretin/orexin; NA, noradrenaline. Copied and modified with permission from Jones (2002) In Biological Psychiatry (D'Haenen, D., den Boer, J. A. & Willner, P., eds.), Vol. 2, pp. 1215-1228.11

2. MODULATION AND ACTIVITY OF HCRT/ORX NEURONS

The Hcrt/Orx neurons are situated within the lateral hypothalamus where fibers ascend within the medial forebrain bundle (MFB) from the brainstem arousal systems, including the reticular formation, the locus coeruleus noradrenergic neurons and the cholinergic pontomesencephalic neurons (Fig. 1).10, 12, 13 They can thus be activated by these constituents of the ascending reticular and brainstem activating systems. Indeed like most neurons, they are excited by glutamate or its agonists14 that would be released by neurons of the reticular formation.10 They would also act in series with the noradrenergic and cholinergic neurons which discharge during waking,15-17 if excited by these transmitters. Yet, surprisingly in electrophysiological studies performed in hypothalamic slices of transgenic mice expressing green fluorescent protein (GFP) through the Hcrt/Orx promoter, noradrenaline (NA) was found to inhibit and acetylcholine (ACh) to have no effect upon the GFP fluorescent neurons,14, 18 In contrast, however, we recently found in hypothalamic slices of rats on immunohistochemically identified Hcrt/Orx neurons that both NA and ACh (or its agonist, carbachol) exerted a direct depolarizing and excitatory effect.19 These results indicate that the Hcrt/Orx neurons would be excited in tandem with the major arousal systems of the brainstem. By their reciprocal projections and excitatory actions, they would in turn sustain the excitation of the brainstem arousal systems, while influencing in parallel the relay neurons in the thalamus and basal forebrain to promote cortical activation.

From studies examining release of Hcrt/Orx and c-Fos expression in association with waking and sleep in rats, cats or primates, the Hcrt/Orx neurons appear to be most active during the period of the day when the animals are most active and thus also during the waking state.20-26 Moreover, they would appear to be most active during periods of maximal motor activity. Only by recording from identified Hcrt/Orx neurons in the hypothalamus will it be known whether they discharge or not during rapid eye movement sleep (REMS).27 Given their excitation by NA and ACh (above), they would be excited and active during waking when both the noradrenergic and cholinergic brainstem neurons are active, and depending upon the balance of inputs could also be excited during REMS when the cholinergic neurons discharge16,28 or alternatively they could be inhibited by GABAergic inputs (below). C-Fos expression is maximal during waking and minimal during sleep, including REMS, in Hcrt/Orx neurons, in contrast to that in surrounding melanin concentrating hormone (MCH) neurons.25, 29

From in vitro studies, we have discovered that Hcrt/Orx neurons are endowed with particular membrane properties that provide them with an intrinsic mechanism for sustained tonic discharge independent of synaptic input.30 Identified by Neurobiotin labeling combined with immunohistochemical staining, Hcrt/Orx cells show a low threshold spike (LTS), after-depolarization potential (ADP) and inward rectification upon hyperpolarization (indicative of an h current, Ih, Fig. 2). They are spontaneously active with an average spike rate of ~3 Hz and tonically depolarized with a membrane potential of ~-46 mV in the presence of tetrodotoxin (TTX, which eliminates sodium action potentials). These properties are not found in adjacent MCH neurons that are not spontaneously active nor depolarized (having a resting membrane potential of ~-62 mV, Fig. 2). The relatively depolarized membrane potential of the Hcrt/Orx neurons was found to be independent of synaptic input, as evident under conditions of synaptic uncoupling (with TTX, block of glutamatergic and GABAergic receptors or high Mg2+/low Ca2+, Fig. 3). It was found to be maintained by a calcium-activated nonselective cation current (ICAN) that was manifest by the ADP. The Hcrt/Orx cells could accordingly sustain tonic discharge and influence upon their target neurons during the waking state independent of other arousal systems. Indeed, given the innervation and excitation of the brainstem arousal systems by Hcrt/Orx, the Hcrt/Orx neurons may represent the central generator for eliciting and maintaining activity in the arousal systems of the brain during waking (Fig. 1).

In order to stop their spontaneous discharge, Hcrt/Orx neurons must be actively inhibited as can be achieved by the inhibitory neurotransmitter, GABA (Fig. 3).30 It is thus presumed that GABAergic neurons that would become active during slow wave sleep (SWS) could turn off the Hcrt/Orx neurons. Since no neurons have been recorded in the lateral hypothalamus that are active during SWS,31 such SWS-active GABAergic neurons are most likely located in the basal forebrain or preoptic area. Indeed, GABAergic neurons have been shown by retrograde and most recently anterograde transport to project from basal forebrain into the posterior lateral hypothalamus and directly onto the Hcrt/Orx neurons.32-34 Electrophysiological and c-Fos studies have identified GABAergic SWS-active and promoting neurons in the basal forebrain35-38 that could thus have the capacity to directly inhibit Hcrt/Orx neurons and remove an otherwise tonic excitatory influence upon all other brainstem arousal systems (Fig. 1).

3. EXCITATORY INFLUENCE OF HCRT/ORX UPON THE DIFFUSE THALAMO-CORTICAL PROJECTION SYSTEM

The Hcrt/Orx neurons project to the thalamus to provide a particularly dense innervation to the midline and intralaminar nuclei.39 These thalamic neurons give rise in turn to widespread projections to the cerebral cortex40 via which they stimulate widespread cortical activation.41-43 This system thus also serves as the dorsal relay of the ascending reticular activating system for stimulating widespread and prolonged fast cortical activity.44 Input to this system by the Hrct/Orx neurons thus provides an important route by which they may influence cortical activation (Fig. 1).

We have found that Hcrt/Orx depolarizes and excites neurons in the rhomboid (Rh) and centromedial (CM) nuclei of the thalamus, which give rise to very widespread projections to the cerebral cortex (Fig. 4).45 This action is likely mediated by Orx2 receptors, given the potent effect of Orx B relative to Orx A on these cells. Interestingly, the peptide has no effect upon neurons of the specific visual (dorsal lateral geniculate, DLG) or somatic sensory relay nuclei (ventral posterior lateral, VPL, Fig. 4). Hcrt/Orx would thus not alter transmission of specific sensory information but could prolong the cortical response to sensory input through the nonspecific projection system. The selective excitation of the nonspecific thalamo-cortical projection system would also maintain widespread fast cortical activity during the waking state.43

After Hyperpolerization

Figure 2. Characterization of neurons expressing Hcrt/Orx or MCH. (Ai) Tonic firing in response to a depolarizing current pulse delivered from the level of resting potential (arrowhead). (A2-3) Low threshold spike (LTS, arrow) and after-depolarization (ADP, star) triggered by a depolarizing current pulse delivered from a hyperpolarized level. Further hyperpolarization eliminates the LTS and the ADP (lower trace in A3). (B) Superimposed responses to hyperpolarizing pulses suggesting the presence of an h current (Ih, dot). Note that only the trace with the deepest hyperpolarization is shown in full. (C) Tonic firing at rest and its elimination by tetrodotoxin (TTX, 1.0 |M) to determine resting potentials. (D - F) Immunohistochemical identification of a Hcrt/Orx neuron injected with Neurobiotin (arrow in D) and expressing immunoreactivity (Ir) for Hcrt/Orx (E) but not for the melanin-concentrating hormone (MCH, F). (G1) Firing with accommodation triggered by a depolarizing current pulse delivered from the resting potential level. (G2-3) Absence of either LTS or ADP in response to depolarizing pulses applied from more hyperpolarized levels. (H) Responses to hyperpolarizing current pulses demonstrating the absence (dot) of any sag that could have indicated the presence of an Ih. (I) Absence of spontaneous firing in such neurons and their mean resting potential. (J - L) Immunohistochemical identification of an MCH neuron injected with Neurobiotin (arrow in J) and expressing immunoreactivity for MCH (L) and not for Hcrt/Orx (K). Membrane potentials (arrowheads): -47 mV (A), -44 mV (B), -48 mV (C), -61 mV (G), -61 mV (H, I). Copied with permission from Eggermann, E. et al. (2003) J. Neurosci. 23, 15571562.30

Figure 3. Spontaneous activity and GABAergic inhibition of Hcrt/Orx neurons. (A) Persistence of the membrane depolarization in TTX (1.0 |M), ionotropic blockers (MK801 at 20 |M, NBQX at 10 |M and bicuculline at 10 |M) and in a solution containing 10 mM Mg2+ and 0.1 mM Ca2+. (B) Persistence of the spontaneous activity in presence of synaptic blockade (right panel showing an enlargement of the area identified by a star in the left panel). (C) Inhibition by a brief application of the GABAA agonist, muscimol (5 s at 100 | M). Membrane potentials (arrowheads): -42 mV (A), -43 mV (B), -44 mV (C). Copied with permission from Bayer et al. (2002) J. Neurosci. 22, 7835-7839.30

Figure 3. Spontaneous activity and GABAergic inhibition of Hcrt/Orx neurons. (A) Persistence of the membrane depolarization in TTX (1.0 |M), ionotropic blockers (MK801 at 20 |M, NBQX at 10 |M and bicuculline at 10 |M) and in a solution containing 10 mM Mg2+ and 0.1 mM Ca2+. (B) Persistence of the spontaneous activity in presence of synaptic blockade (right panel showing an enlargement of the area identified by a star in the left panel). (C) Inhibition by a brief application of the GABAA agonist, muscimol (5 s at 100 | M). Membrane potentials (arrowheads): -42 mV (A), -43 mV (B), -44 mV (C). Copied with permission from Bayer et al. (2002) J. Neurosci. 22, 7835-7839.30

4. EXCITATORY INFLUENCE OF HCRT/ORX UPON THE CHOLINERGIC BASALO-CORTICAL PROJECTION SYSTEM

Hcrt/Orx neurons also project through the MFB up to the basal forebrain.39 We have found that Hcrt/Orx directly depolarizes and excites cholinergic basal forebrain neurons (Fig. 5).46 As in the thalamus, the effect of the peptide appears to be mediated in the basal forebrain through Orx2 receptors. By direct widespread projections to the cerebral cortex and excitatory actions therein, the cholinergic basal forebrain neurons have the capacity to stimulate widespread and prolonged cortical activation, marked by fast gamma activity.47 Hcrt/Orx could thus activate the cholinergic basalo-cortical system during waking for the elicitation and maintenance of cortical activation associated with behavioral arousal and/or attention. Injections of this peptide into the region of the cholinergic basal forebrain neurons have been shown to stimulate cortical activation along with a behaviorally active state.48

Basal Forebrain Cholinergic System

Figure 4. Actions of Hcrt/Orx on nonspecific and specific thalamo-cortical projection neurons. Nuclei of nonspecific systems (left, including CM, where units were also recorded, and Rh, as shown) and specific systems (right, somatosensory relay nuclei, VPM and VPL, where units were recorded as shown and) where neurons were tested for their response to the orexin (Orx) peptide and some labeled with Neurobiotin. (A) Red triangles correspond to injected cells shown in B and C. (B, C) Neurobiotin-filled neurons in the Rh and VPL nuclei (insets showing characteristic responses to hyperpolarizing pulses). (D) Depolarizing and excitatory effect of Orx B on Rh neurons. (E) Absence of effect of Orx B on VPL neurons. Calibrations: 500 |m in A and 20 |m in B and C. Abbreviations: IAM, interanteromedial thalamic nucleus; ic, internal capsule; CM, centromedial nucleus; MD, mediodorsal thalamic nucleus; Sub, submedius thalamic nucleus; VPL, ventral posterolateral thalamic nucleus; VPM, ventral posteromedial thalamic nucleus; Rh, rhomboid nucleus; Rt, reticular thalamic nucleus. Copied with permission from Bayer et al. (2002) J. Neurosci. 22, 7835-7839.45

Figure 4. Actions of Hcrt/Orx on nonspecific and specific thalamo-cortical projection neurons. Nuclei of nonspecific systems (left, including CM, where units were also recorded, and Rh, as shown) and specific systems (right, somatosensory relay nuclei, VPM and VPL, where units were recorded as shown and) where neurons were tested for their response to the orexin (Orx) peptide and some labeled with Neurobiotin. (A) Red triangles correspond to injected cells shown in B and C. (B, C) Neurobiotin-filled neurons in the Rh and VPL nuclei (insets showing characteristic responses to hyperpolarizing pulses). (D) Depolarizing and excitatory effect of Orx B on Rh neurons. (E) Absence of effect of Orx B on VPL neurons. Calibrations: 500 |m in A and 20 |m in B and C. Abbreviations: IAM, interanteromedial thalamic nucleus; ic, internal capsule; CM, centromedial nucleus; MD, mediodorsal thalamic nucleus; Sub, submedius thalamic nucleus; VPL, ventral posterolateral thalamic nucleus; VPM, ventral posteromedial thalamic nucleus; Rh, rhomboid nucleus; Rt, reticular thalamic nucleus. Copied with permission from Bayer et al. (2002) J. Neurosci. 22, 7835-7839.45

5. EXCITATORY INFLUENCE OF HCRT/ORX UPON CORTICO-CORTICAL PROJECTION NEURONS

Like other diffuse projecting neurons of the arousal systems, Hrct/Orx neurons project directly to the cerebral cortex in addition to projecting onto subcortical relay neurons.39 In the cortex, Hcrt/Orx fibers are distributed in a widespread manner and through all layers, although most densely in the deeper layers. In a systematic study of postsynaptic effects of Hcrt/Orx in the cortex, we recently discovered that the peptide has no direct effect upon cortical neurons in layers 1 through 6a, even though it has indirect effects through presynaptic terminals of afferent inputs to the cortical neurons (Fig. 6). Such presynaptic effects were demonstrated upon layer 5 pyramidal cells through actions upon terminals of the nonspecific thalamo-cortical afferents.49 On the other hand, we found that Hrct/Orx does have a postsynaptic depolarizing action upon cortical neurons located exclusively in layer 6b (Fig. 6).50 This action appears to be mediated by an Orx2

receptor. Neurons in layer 6b give rise to widespread projections to layer 1 of surrounding cortical areas51' 52 and could thus propagate widespread cortical activation.

60 s

Figure 5. Excitatory action of Hcrt/Orx on cholinergic basal forebrain neurons. (A) Electrophysiological identification (upper left inset) and immunohistochemical confirmation (white arrowhead indicating a ChATimmunopositive cell) of a basal forebrain cholinergic neuron labeled with Neurobiotin (lower right inset). Calibration: 25 |m. (B, C) Such cells were excited by both orexin (Orx) A and B. Extracellular action potentials (lower panel B) before (1) during (2) and after (3) Orx effects are demonstrated. Copied with permission from Eggermann et al. (2001) Neuroscience 108, 177-181.46

60 s

Figure 5. Excitatory action of Hcrt/Orx on cholinergic basal forebrain neurons. (A) Electrophysiological identification (upper left inset) and immunohistochemical confirmation (white arrowhead indicating a ChATimmunopositive cell) of a basal forebrain cholinergic neuron labeled with Neurobiotin (lower right inset). Calibration: 25 |m. (B, C) Such cells were excited by both orexin (Orx) A and B. Extracellular action potentials (lower panel B) before (1) during (2) and after (3) Orx effects are demonstrated. Copied with permission from Eggermann et al. (2001) Neuroscience 108, 177-181.46

Figure 6. Exclusive action of Hcrt/Orx on cortical neurons of sublayer 6b in cortex. (A) Toluidine blue counter-stained cortical slice slab containing two recorded neurons (arrowheads) labeled with Neurobiotin in sublayers 6a and 6b (separated by a horizontal band of fibers, *) and whose responses to OrxB are shown in panels D and E. (B, C) Absence of responses to Orx B of neurons in layers 2/3 and 4/5. (D) Absence of response to OrxB of a neuron in layer 6a. (E) Depolarizing response to OrxB of a neuron in layer 6b. (Fi-2) Enlargement of neurons 1 and 2 from panel A. Calibrations: 15 |m. (G) Increase in post-synaptic potentials (PSPs) in a layer 5 neuron in the presence of OrxB, which is impeded in the presence of a high Mg2+/low Ca2+ solution (not shown). Calibrations: 5 mV/2 sec. Abbreviation: cc, corpus callosum. Copied and modified with permission from Bayer (In press) J. Neurosci.50

6. INDIRECT INFLUENCE OF HCRT/ORX UPON SLEEP-PROMOTING NEURONS

Given the importance of Hcrt/Orx for maintaining a waking state, it was thought likely that the peptide could have a direct inhibitory influence upon sleep-promoting neurons. We tested this possibility on the presumed GABAergic sleep-promoting neurons of the ventrolateral preoptic area (VLPO), identified by their inhibitory response to NA.53 Somewhat to our surprise, there was no evidence for a direct inhibitory effect of Hcrt/Orx upon these neurons (Fig. 7).46 Yet, no direct inhibitory action of Hcrt/Orx has been found to date in the central nervous system. Although no indirect effect of Hcrt/Orx was evident in the slice upon the VLPO neurons, it is very possible that Hcrt/Orx could excite particular GABAergic neurons that would in turn inhibit sleep-promoting neurons. Without this, Hcrt/Orx would act indirectly to inhibit sleep-promoting neurons through its excitatory action upon the noradrenergic locus coeruleus neurons,5, 6 since NA inhibits GABAergic sleep-promoting neurons in the preoptic region and basal forebrain (Fig. 7).35,38,53

Figure 7. Absence of effect of Hcrt/Orx on ventrolateral preoptic area (VLPO) neurons. (A) The basal forebrain/preoptic area. (B, C) VLPO cells' identification by their shape (calibration, 5 |m) and their inhibition by noradrenaline (NA). (D) Such cells were unaffected by Orx A (or B, not shown). Abbreviations: ac, anterior commissure; oc, optic chiasm; MCPO, magnocellular preoptic nucleus; POA, preoptic area; SIa, substantia innominata pars anterior; VLPO, ventrolateral preoptic nucleus; 3V, third ventricle. Copied with permission from Eggermann et al. (2001) Neuroscience 108, 177-181.46

Figure 7. Absence of effect of Hcrt/Orx on ventrolateral preoptic area (VLPO) neurons. (A) The basal forebrain/preoptic area. (B, C) VLPO cells' identification by their shape (calibration, 5 |m) and their inhibition by noradrenaline (NA). (D) Such cells were unaffected by Orx A (or B, not shown). Abbreviations: ac, anterior commissure; oc, optic chiasm; MCPO, magnocellular preoptic nucleus; POA, preoptic area; SIa, substantia innominata pars anterior; VLPO, ventrolateral preoptic nucleus; 3V, third ventricle. Copied with permission from Eggermann et al. (2001) Neuroscience 108, 177-181.46

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