Interactions of the orexin/hypocretin neurones and the histaminergic system

Histaminergic and orexin/hypocretin systems are components in the brain wake‐promoting system. Both are affected in the sleep disorder narcolepsy, but the role of histamine in narcolepsy is unclear. The histaminergic neurones are activated by the orexin/hypocretin system in rodents, and the development of the orexin/hypocretin neurones is bidirectionally regulated by the histaminergic system in zebrafish. This review summarizes the current knowledge of the interactions of these two systems in normal and pathological conditions in humans and different animal models.     

Experience-dependent plasticity in hypocretin/orexin neurones: re-setting arousal threshold

The neuropeptide hypocretin is synthesized exclusively in the lateral hypothalamus and participates in many brain functions critical for animal survival, particularly in the promotion and maintenance of arousal in animals – a core process in animal behaviours. Consistent with its arousal-promoting role in animals, the neurones synthesizing hypocretin receive extensive innervations encoding physiological, psychological and environmental cues and send final outputs to key arousal-promoting brain areas. The activity in hypocretin neurones fluctuates and correlates with the behavioural state of animals and intensive activity has been detected in hypocretin neurones during wakefulness, foraging for food and craving for addictive drugs. Therefore, it is likely that hypocretin neurones undergo experience-dependent changes resulting from intensive activations by stimuli encoding changes in the internal and external environments. This review summarizes the most recent evidence supporting experience-dependent plasticity in hypocretin neurones. Current data suggest that nutritional and behavioural factors lead to synaptic plasticity and re-organization of synaptic architecture in hypocretin neurones. This may be the substrate of enhanced levels of arousal resulting from behavioural changes in animals and may help to explain the mechanisms underlying the changes in arousal levels induced by physiological, psychological and environmental factors.     

Hypocretin-2 (orexin-B) modulation of superficial dorsal horn activity in rat

The hypothalamic peptides hypocretin-1 (orexin A) and hypocretin-2 (Hcrt-2; orexin B) are important in modulating behaviours demanding arousal, including sleep and appetite. Fibres containing hypocretin project from the hypothalamus to the superficial dorsal horn (SDH) of the spinal cord (laminae I and II); however, the effects produced by hypocretins on SDH neurones are unknown. To study the action of Hcrt-2 on individual SDH neurones, tight-seal, whole-cell recordings were made with biocytin-filled electrodes from rat lumbar spinal cord slices. In 19 of 63 neurones, Hcrt-2 (30 n m to 1 μ m ) evoked an inward (excitatory) current accompanied by an increase in baseline noise. The inward current and noise were unaffected by TTX but were blocked by the P_2X purinergic receptor antagonist suramin (300–500 μ m ). Hcrt-2 (30 n m to 1 μ m ) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in the majority of neurones. The sIPSC increase was blocked by strychnine (1 μ m ) and by TTX (1 μ m ), suggesting that the increased sIPSC frequency was glycine and action potential dependent. Hcrt-2 increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in a few neurones but had no effect on dorsal root-evoked EPSCs in these or in other neurones. Neurones located in outer lamina II, particularly radial and vertical cells, were most likely to respond to Hcrt-2. We conclude that Hcrt-2 has excitatory effects on certain SDH neurones, some of which exert inhibitory influences on other cells of the region, consistent with the perspective that hypocretin has a role in orchestrating reactions related to arousal, including nociception, pain and temperature sense.     

Stimulation of orexin/hypocretin neurones by thyrotropin-releasing hormone

Central orexin/hypocretin neurones are critical for sustaining consciousness: their firing stimulates wakefulness and their destruction causes narcolepsy. We explored whether the activity of orexin cells is modulated by thyrotropin-releasing hormone (TRH), an endo-genous stimulant of wakefulness and locomotor activity whose mechanism of action is not fully understood. Living orexin neurones were identified by targeted expression of green fluorescent protein (GFP) in acute brain slices of transgenic mice. Using whole-cell patch-clamp recordings, we found that TRH robustly increased the action potential firing rate of these neurones. TRH-induced excitation persisted under conditions of synaptic isolation, and involved a Na⁺-dependent depolarization and activation of a mixed cation current in the orexin cell membrane. By double-label immunohistochemistry, we found close appositions between TRH-immunoreactive nerve terminals and orexin-A-immunoreactive cell bodies. These results identify a new physiological modulator of orexin cell firing, and suggest that orexin cell excitation may contribute to the arousal-enhancing actions of TRH.     

Dissociation between sensing and metabolism of glucose in sugar sensing neurones

Some of the neurones controlling sleep, appetite and hormone release act as specialized detectors of ambient glucose. Their sugar sensing is conventionally thought to involve glucokinase-dependent metabolism of glucose to ATP, which then alters membrane excitability by modulating ATP-dependent channels or transporters, such as ATP-inhibited K⁺ channels (K_ATP). However, recent studies also provide examples of both glucose-excited (GE) and glucose-inhibited (GI) neurones that sense glucose independently of such metabolic pathways. Two-thirds of hypothalamic GE neurones in primary cultures are also excited by the non-metabolizable glucose analogue α-methylglucopyranoside (α-MDG), which acts as a substrate for electrogenic (depolarizing) sodium–glucose cotransporter (SGLT). The excitatory responses to both glucose and α-MDG are abolished by arresting SGLT activity by sodium removal or the SGLT inhibitor phloridzin. Direct depolarization and excitation by glucose-triggered SGLT activity may ensure that GE neurones continue to sense glucose in 'high-energy' states, when K_ATP channels are closed. A major class of hypothalamic GI neurones, the orexin/hypocretin cells, also appear to use a non-metabolic sensing strategy. In these cells, glucose-induced hyperpolarization and inhibition are unaffected by glucokinase inhibitors such as alloxan, d -glucosamine, and N -acetyl- d -glucosamine, and mimicked by the non-metabolizable glucose analogue 2-deoxyglucose, but not by stimulating intracellular ATP production with lactate. The dissociation between sensing and metabolism of sugar may allow the brain to predict and prevent adverse changes in extracellular glucose levels with minimal impact on the flow of intracellular fuel.     

Predictive models of glucose control: roles for glucose‐sensing neurones

The brain can be viewed as a sophisticated control module for stabilizing blood glucose. A review of classical behavioural evidence indicates that central circuits add predictive (feedforward/anticipatory) control to the reactive (feedback/compensatory) control by peripheral organs. The brain/cephalic control is constructed and engaged, via associative learning, by sensory cues predicting energy intake or expenditure (e.g. sight, smell, taste, sound). This allows rapidly measurable sensory information (rather than slowly generated internal feedback signals, e.g. digested nutrients) to control food selection, glucose supply for fight‐or‐flight responses or preparedness for digestion/absorption. Predictive control is therefore useful for preventing large glucose fluctuations. We review emerging roles in predictive control of two classes of widely projecting hypothalamic neurones, orexin/hypocretin (ORX) and melanin‐concentrating hormone (MCH) cells. Evidence is cited that ORX neurones (i) are activated by sensory cues (e.g. taste, sound), (ii) drive hepatic production, and muscle uptake, of glucose, via sympathetic nerves, (iii) stimulate wakefulness and exploration via global brain projections and (iv) are glucose‐inhibited. MCH neurones are (i) glucose‐excited, (ii) innervate learning and reward centres to promote synaptic plasticity, learning and memory and (iii) are critical for learning associations useful for predictive control (e.g. using taste to predict nutrient value of food). This evidence is unified into a model for predictive glucose control. During associative learning, inputs from some glucose‐excited neurones may promote connections between the ‘fast’ senses and reward circuits, constructing neural shortcuts for efficient action selection. In turn, glucose‐inhibited neurones may engage locomotion/exploration and coordinate the required fuel supply. Feedback inhibition of the latter neurones by glucose would ensure that glucose fluxes they stimulate (from liver, into muscle) are balanced. Estimating nutrient challenges from indirect sensory cues may become more difficult when the cues become complex and variable (e.g. like human foods today). Consequent errors of predictive glucose control may contribute to obesity and diabetes.     

Paradoxical (REM) sleep deprivation in mice using the small‐platforms‐over‐water method: polysomnographic analyses and melanin‐concentrating hormone and hypocretin/orexin neuronal activation before,during and after deprivation

Studying paradoxical sleep homeostasis requires the specific and efficient deprivation of paradoxical sleep and the evaluation of the subsequent recovery period. With this aim, the small‐platforms‐over‐water technique has been used extensively in rats, but only rare studies were conducted in mice, with no sleep data reported during deprivation. Mice are used increasingly with the emergence of transgenic mice and technologies such as optogenetics, raising the need for a reliable method to manipulate paradoxical sleep. To fulfil this need, we refined this deprivation method and analysed vigilance states thoroughly during the entire protocol. We also studied activation of hypocretin/orexin and melanin‐concentrating hormone neurones using Fos immunohistochemistry to verify whether mechanisms regulating paradoxical sleep in mice are similar to those in rats. We showed that 48 h of deprivation was highly efficient, with a residual amount of paradoxical sleep of only 2.2%. Slow wave sleep and wake quantities were similar to baseline, except during the first 4 h of deprivation, where slow wave sleep was strongly reduced. After deprivation, we observed a 124% increase in paradoxical sleep quantities during the first hour of rebound. In addition, 34% of hypocretin/orexin neurones were activated during deprivation, whereas melanin‐concentrated hormone neurones were activated only during paradoxical sleep rebound. Corticosterone level showed a twofold increase after deprivation and returned to baseline level after 4 h of recovery. In summary, a fairly selective deprivation and a significant rebound of paradoxical sleep can be obtained in mice using the small‐platforms‐over‐water method. As in rats, rebound is accompanied by a selective activation of melanin‐concentrating hormone neurones.     

Physiological functions of glucose-inhibited neurones

Glucose-inhibited neurones are an integral part of neurocircuits regulating cognitive arousal, body weight and vital adaptive behaviours. Their firing is directly suppressed by extracellular glucose through poorly understood signalling cascades culminating in opening of post-synaptic K(+) or possibly Cl(-) channels. In mammalian brains, two groups of glucose-inhibited neurones are best understood at present: neurones of the hypothalamic arcuate nucleus (ARC) that express peptide transmitters NPY and agouti-related peptide (AgRP) and neurones of the lateral hypothalamus (LH) that express peptide transmitters orexins/hypocretins. The activity of ARC NPY/AgRP neurones promotes food intake and suppresses energy expenditure, and their destruction causes a severe reduction in food intake and body weight. The physiological actions of ARC NPY/AgRP cells are mediated by projections to numerous hypothalamic areas, as well as extrahypothalamic sites such as the thalamus and ventral tegmental area. Orexin/hypocretin neurones of the LH are critical for normal wakefulness, energy expenditure and reward-seeking, and their destruction causes narcolepsy. Orexin actions are mediated by highly widespread central projections to virtually all brain areas except the cerebellum, including monosynaptic innervation of the cerebral cortex and autonomic pre-ganglionic neurones. There, orexins act on two specific G-protein-coupled receptors generally linked to neuronal excitation. In addition to sensing physiological changes in sugar levels, the firing of both NPY/AgRP and orexin neurones is inhibited by the 'satiety' hormone leptin and stimulated by the 'hunger' hormone ghrelin. Glucose-inhibited neurones are thus well placed to coordinate diverse brain states and behaviours based on energy levels.     

Hypocretin/orexin and narcolepsy: new basic and clinical insights

Narcolepsy is a chronic sleep disorder, characterized by excessive daytime sleepiness (EDS), cataplexy, sleep paralysis and hypnagogic hallucinations. Both sporadic (95%) and familial (5%) forms of narcolepsy exist in humans. The major pathophysiology of human narcolepsy has been recently discovered based on the discovery of narcolepsy genes in animals; the genes involved in the pathology of the hypocretin/orexin ligand and its receptor. Mutations in hypocretin-related genes are rare in humans, but hypocretin ligand deficiency is found in a large majority of narcolepsy with cataplexy. Hypocretin ligand deficiency in human narcolepsy is probably due to the post-natal cell death of hypocretin neurones. Although a close association between human leucocyte antigen (HLA) and human narcolepsy with cataplexy suggests an involvement of autoimmune mechanisms, this has not yet been proved. Hypocretin deficiency is also found in symptomatic cases of narcolepsy and EDS with various neurological conditions, including immune-mediated neurological disorders, such as Guillain–Barre syndrome, MA2-positive paraneoplastic syndrome and neuromyelitis optica (NMO)-related disorder. The findings in symptomatic narcoleptic cases may have significant clinical relevance to the understanding of the mechanisms of hypocretin cell death and choice of treatment option. The discoveries in human cases lead to the establishment of the new diagnostic test of narcolepsy (i.e. low cerebrospinal fluid hypocretin-1 levels for ‘narcolepsy with cataplexy’ and ‘narcolepsy due to medical condition’). As a large majority of human narcolepsy patients are ligand deficient, hypocretin replacement therapy may be a promising new therapeutic option, and animal experiments using gene therapy and cell transplantations are in progress.     

Orexins/hypocretins excite basal forebrain cholinergic neurones.

The orexins (orexin A and B, also known as hypocretin 1 and 2) are two recently identified 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; 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 fibres coursing from the hypothalamus (Peyron et al., 1998) up to the preoptic area (POA) and basal forebrain (BF) suggests that they might influence 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 effect on the GABA sleep-promoting neurones of the POA, whereas they have a strong and direct excitatory effect on the cholinergic neurones of the contiguous BF. In addition, by comparing the effects of orexin A and B we demonstrate here that orexins' action depends upon orexin type 2 receptors (OX(2)), 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.     

A critical role of hypocretin deficiency in pregnancy

Hypocretin/orexin peptides are known for their role in the control of the wake–sleep cycle and narcolepsy–cataplexy pathophysiology. Recent studies suggested that hypocretin peptides also have a role in pregnancy. We tested this hypothesis by conducting a retrospective analysis on pregnancy complications in two different mouse models of hypocretin deficiency. We recorded 85 pregnancies of mice lacking either hypocretin peptides (knockout) or hypocretin‐releasing neurons (transgenic) and their wild‐type controls. Pregnancy was associated with unexplained dam death before delivery in 3/15 pregnancies in knockout mice, and in 3/23 pregnancies in transgenic mice. No casualties occurred in wild‐type pregnant dams (P < 0.007 versus hypocretin‐deficient mice as a whole). Hypocretin deficiency did not impact either on litter size or the number of weaned pups per litter. These data provide preliminary evidence of a critical role of hypocretin deficiency in pregnancy.     

Adenosine inhibits activity of hypocretin/orexin neurons by the A1 receptor in the lateral hypothalamus: a possible sleep-promoting effect

Neurons in the lateral hypothalamus (LH) that contain hypocretin/orexin have been established as important promoters of arousal. Deficiencies in the hypocretin/orexin system lead to narcolepsy. The inhibition of hypocretin/orexin neurons by sleep-promoting neurotransmitters has been suggested as one part of the sleep regulation machinery. Adenosine has been identified as a sleep promoter and its role in sleep regulation in the basal forebrain has been well documented. However, the effect of adenosine on arousal-promoting hypocretin/orexin neurons has not been addressed, despite recent evidence that immunocytochemical visualization of adenosine receptors was detected in these neurons. In this study, we examined the hypothesis that adenosine inhibits the activity of hypocretin/orexin neurons by using electrophysiological methods in brain slices from mice expressing green fluorescent protein in hypocretin/orexin neurons. We found that adenosine significantly attenuated the frequency of action potentials without a change in membrane potential in hypocretin/orexin neurons. The adenosine-mediated inhibition arises from depression of excitatory synaptic transmission to hypocretin/orexin neurons because adenosine depresses the amplitude of evoked excitatory postsynaptic potential and the frequency of spontaneous and miniature excitatory postsynaptic currents in these neurons. At the cell body of the hypocretin/orexin neurons, adenosine inhibits voltage-dependent calcium currents without the induction of GIRK current. The inhibitory effect of adenosine is dose dependent, pertussis toxin sensitive, and mediated by A1 receptors. In summary, our data suggest that in addition to its effect in the basal forebrain, adenosine exerts its sleep-promoting effect in the LH by inhibition of hypocretin/orexin neurons.     

Sleep and cardiovascular phenotype in middle‐aged hypocretin‐deficient narcoleptic mice

Narcolepsy with cataplexy (NC) is a lifelong disorder caused by loss of hypothalamic hypocretin/orexin (HCRT) neurones, often starting in childhood. NC patients show altered control of heart rate (HR) and a normotensive non‐dipper blood pressure (BP) profile, but the natural history and prognostic significance of these alterations remain unclear. Similar alterations have been observed in HCRT‐ataxin‐3 transgenic (TG) NC mice lacking HCRT neurones, but studies have been limited to young adult individuals <4 months of age. Here we evaluated long‐term effects of NC on derangements in the wake–sleep state and cardiovascular control by studying middle‐aged TG. We chronically instrumented TG and wild‐type mice aged 10–11 months with electrodes for sleep scoring and a telemetric transducer for BP and HR measurements. We then recorded mice in freely behaving conditions. TG showed a NC phenotype including fragmentation of wakefulness, reduced latency to rapid eye movement sleep (REMS) and cataplexy‐like events. TG also showed blunted BP decline on entering non‐rapid eye movement sleep (NREMS), enhanced BP increase on passing to REMS, increased HR, and blunted changes in HR upon arousal and awakening from NREMS. Histological and ultrastructural analysis of cardiovascular and renal tissue did not reveal evidence of subclinical hypertensive organ damage. These data indicate that HCRT neurone loss in TG causes alterations in wake–sleep behaviour and cardiovascular control that are not peculiar to the beginning of the disease but are maintained at least up to middle age. These alterations are similar to those in adult NC patients, but do not produce early subclinical damage to the heart and kidneys.     

Orexins (hypocretins) actions on the GHRH/somatostatin-GH axis

The secretion of growth hormone (GH) is regulated through a complex neuroendocrine control system that includes two major hypothalamic regulators, namely GH-releasing hormone (GHRH) and somatostatin (SST) that stimulate and inhibit, respectively, GH release. Classical experiments involving damage and electrical stimulation suggested that the lateral hypothalamic area (LHA) modulated the somatotropic axis, but the responsible molecular mechanisms were unclear. Evidence obtained during the last decade has demonstrated that orexins/hypocretins, a family of peptides expressed in the LHA controlling feeding and sleep, play an important regulatory role on GH, by inhibiting its secretion modulating GHRH and SST neurones. Considering that GH release is closely linked to the sleep–wake cycle and feeding state, understanding orexin/hypocretin physiology could open new therapeutic possibilities in the treatment of sleep, energy homeostasis and GH-related pathologies, such as GH deficiency.     

Hypocretin receptor expression in canine and murine narcolepsy models and in hypocretin-ligand deficient human narcolepsy

STUDY OBJECTIVE: To determine whether hypocretin receptor gene (hcrtR1 and hcrtR2) expression is affected after long-term hypocretin ligand loss in humans and animal models of narcolepsy. DESIGN: Animal and human study. We measured hcrtR1 and hcrtR2 expression in the frontal cortex and pons using the RT-PCR method in murine models (8-week-old and 27-week-old orexin/ataxin-3 transgenic (TG) hypocretin cell ablated mice and wild-type mice from the same litter, 10 mice for each group), in canine models (8 genetically narcoleptic Dobermans with null mutations in the hcrtR2, 9 control Dobermans, 3 sporadic ligand-deficient narcoleptics, and 4 small breed controls), and in humans (5 narcolepsy-cataplexy patients with hypocretin deficiency (average age 77.0 years) and 5 control subjects (72.6 years). MEASUREMENT AND RESULTS: 27-week-old (but not 8-week-old) TG mice showed significant decreases in hcrtR1 expression, suggesting the influence of the long-term ligand loss on the receptor expression. Both sporadic narcoleptic dogs and human narcolepsy-cataplexy subjects showed a significant decrease in hcrtR1 expression, while declines in hcrtR2 expression were not significant in these cases. HcrtR2-mutated narcoleptic Dobermans (with normal ligand production) showed no alteration in hcrtR1 expression. CONCLUSIONS: Moderate declines in hcrtR expressions, possibly due to long-term postnatal loss of ligand production, were observed in hypocretin-ligand deficient narcoleptic subjects. These declines are not likely to be progressive and complete. The relative preservation of hcrtR2 expression also suggests that hypocretin based therapies are likely to be a viable therapeutic options in human narcolepsy-cataplexy.     

Sleep/wake fragmentation disrupts metabolism in a mouse model of narcolepsy

Recent population studies have identified important interrelationships between sleep duration and body weight regulation. The hypothalamic hypocretin/orexin neuropeptide system is able to influence each of these. Disruption of the hypocretin system, such as occurs in narcolepsy, leads to a disruption of sleep and is often associated with increased body mass index. We examined the potential interrelationship between the hypocretin system, metabolism and sleep by measuring locomotion, feeding, drinking, body temperature, sleep/wake and energy metabolism in a mouse model of narcolepsy (ataxin-ablation of hypocretin-expressing neurons). We found that locomotion, feeding, drinking and energy expenditure were significantly reduced in the narcoleptic mice. These mice also exhibited severe sleep/wake fragmentation. Upon awakening, transgenic and control mice displayed a similar rate of increase in locomotion and food/water intake with time. A lack of long wake episodes partially or entirely explains observed differences in overall locomotion, feeding and drinking in these transgenic mice. Like other parameters, energy expenditure also rose and fell depending on the sleep/wake status. Unlike other parameters, however, energy expenditure in control mice increased upon awakening at a greater rate than in the narcoleptic mice. We conclude that the profound sleep/wake fragmentation is a leading cause of the reduced locomotion, feeding, drinking and energy expenditure in the narcoleptic mice under unperturbed conditions. We also identify an intrinsic role of the hypocretin system in energy expenditure that may not be dependent on sleep/wake regulation, locomotion, or food intake. This investigation illustrates the need for coordinated study of multiple phenotypes in mouse models with altered sleep/wake patterns.     

Insomnia following hypocretin2-saporin lesions of the substantia nigra

The neuropeptide hypocretin, also known as orexin, has been implicated in waking since its deletion leads to the sleep disorder narcolepsy. Hypocretin neurons project to major arousal areas, and in an effort to determine which region is responsible for the changes in sleep-wake architecture we have developed the neurotoxin hypocretin2-saporin, which lesions hypocretin receptor bearing neurons. Here, in rats, we investigate the effects of hypocretin2-saporin lesions of the substantia nigra and ventral tegmental area in the regulation of sleep and wakefulness. Bilateral injection of hypocretin2-sap into both the ventral tegmental area and substantia nigra (92 and 184 ng/microl, 0.25 microl in the ventral tegmental area and 0.5 microl in the substantia nigra) or into the substantia nigra alone (184 ng/microl, 0.5 microl) produced insomnia. The insomnia seemed to be associated with a large increase in locomotion on days 4 and 6 postinjection, as hyperactivity and stereotypic movements were consistently observed on the video recordings in all lesioned rats. In these rats, a nearly complete loss of both tyrosine hydroxylase and neuron-specific nuclear protein (neuronal nuclei) immunoreactive cells in the substantia nigra as well as diminution of tyrosine hydroxylase-immunoreactive fibers in the caudate putamen was found. Following bilateral injection of hypocretin2-sap at a lower concentration (46 ng/microl, 0.25 microl in the ventral tegmental area and 0.5 microl in the substantia nigra), very little reduction in the number of tyrosine hydroxylase- and neuronal nuclei-immunoreactive neurons and only a temporary increase in wakefulness (17.4% increase during light-off period on day 6 postinjection) were observed. Ventral tegmental area lesions (184 ng/mul of hypocretin2-sap, 0.25 microl, bilateral injections) did not produce significant changes in sleep, although most of the tyrosine hydroxylase- and neuronal nuclei-immunoreactive neurons in the ventral tegmental area were destroyed. Insomnia following hypocretin2-sap lesions of the substantia nigra could be secondary to increased motor activity resulting from reduction of tonic inhibitory control by the substantia nigra.