Dynorphin inhibits basal forebrain cholinergic neurons by pre- and postsynaptic mechanisms.

KEY POINTS: The basal forebrain is an important component of the ascending arousal system and may be a key site through which the orexin neurons promote arousal. It has long been known that orexin-A and -B excite basal forebrain cholinergic neurons, but orexin-producing neurons also make the inhibitory peptide dynorphin. Using whole-cell recordings in brain slices, we found that dynorphin-A directly inhibits basal forebrain cholinergic neurons via κ-opioid receptors, and decreases afferent excitatory synaptic input to these neurons. While the effects of dynorphin-A and orexin-A desensitize over multiple applications, co-application of dynorphin-A and orexin-A produces a sustained response that reverses depending on the membrane potential of basal forebrain cholinergic neurons. At -40 mV the net effect of the co-application is inhibition by dynorphin-A, whereas at -70 mV the excitatory response to orexin-A prevails. ABSTRACT: The basal forebrain (BF) is an essential component of the ascending arousal systems and may be a key site through which the orexin (also known as hypocretin) neurons drive arousal and promote the maintenance of normal wakefulness. All orexin neurons also make dynorphin, and nearly all brain regions innervated by the orexin neurons express kappa opiate receptors, the main receptor for dynorphin. This is remarkable because orexin excites target neurons including BF neurons, but dynorphin has inhibitory effects. We identified the sources of dynorphin input to the magnocellular preoptic nucleus and substantia innominata (MCPO/SI) in mice and determined the effects of dynorphin-A on MCPO/SI cholinergic neurons using patch-clamp recordings in brain slices. We found that the orexin neurons are the main source of dynorphin input to the MCPO/SI region, and dynorphin-A inhibits MCPO/SI cholinergic neurons through κ-opioid receptors by (1) activation of a G protein-coupled inwardly rectifying potassium current, (2) inhibition of a voltage-gated Ca(2+) current and (3) presynaptic depression of the glutamatergic input to these neurons. The responses both to dynorphin-A and to orexin-A desensitize, but co-application of dynorphin-A and orexin-A produces a sustained response. In addition, the polarity of the response to the co-application depends on the membrane potential of BF neurons; at -40 mV the net effect of the co-application is inhibition by dynorphin-A, whereas at -70 mV the excitatory response to orexin-A prevails. This suggests that depending on their state of activation, BF cholinergic neurons can be excited or inhibited by signals from the orexin neurons.

Carbachol excites sublaterodorsal nucleus neurons projecting to the spinal cord.

Considerable electrophysiological and pharmacological evidence has long suggested an important role for acetylcholine in the regulation of rapid-eye-movement (REM) sleep. For example, injection of the cholinergic agonist carbachol into the dorsomedial pons produces an REM sleep-like state with muscle atonia and cortical activation, both of which are cardinal features of REM sleep. Located within this region of the pons is the sublaterodorsal nucleus (SLD), a structure thought to be both necessary and sufficient for generating REM sleep muscle atonia. Subsets of glutamatergic SLD neurons potently contribute to motor inhibition during REM sleep through descending projections to motor-related glycinergic/GABAergic neurons in the spinal cord and ventromedial medulla. Prior electrophysiological and pharmacological studies examining the effects of acetylcholine on SLD neurons have, however, produced conflicting results. In the present study, we sought to clarify how acetylcholine influences the activity of spinally projecting SLD (SLDsp) neurons. We used retrograde tracing in combination with patch-clamp recordings and recorded pre- and postsynaptic effects of carbachol on SLDsp neurons. Carbachol acted presynaptically by increasing the frequency of glutamatergic miniature excitatory postsynaptic currents. We also found that carbachol directly excited SLDsp neurons by activating an Na(+)-Ca(2+) exchanger. Both pre- and postsynaptic effects were mediated by co-activation of M1 and M3 muscarinic receptors. These observations suggest that acetylcholine produces synergistic, excitatory pre- and postsynaptic responses on SLDsp neurons that, in turn, probably serve to promote muscle atonia during REM sleep.

Regulation of daily locomotor activity and sleep by hypothalamic EGF receptor signaling.

The circadian clock in the suprachiasmatic nucleus (SCN) is thought to drive daily rhythms of behavior by secreting factors that act locally within the hypothalamus. In a systematic screen, we identified transforming growth factor-alpha (TGF-alpha) as a likely SCN inhibitor of locomotion. TGF-alpha is expressed rhythmically in the SCN, and when infused into the third ventricle it reversibly inhibited locomotor activity and disrupted circadian sleep-wake cycles. These actions are mediated by epidermal growth factor (EGF) receptors on neurons in the hypothalamic subparaventricular zone. Mice with a hypomorphic EGF receptor mutation exhibited excessive daytime locomotor activity and failed to suppress activity when exposed to light. These results implicate EGF receptor signaling in the daily control of locomotor activity, and identify a neural circuit in the hypothalamus that likely mediates the regulation of behavior both by the SCN and the retina.

Melanopsin in cells of origin of the retinohypothalamic tract.

All known eukaryotic organisms exhibit physiological and behavioral rhythms termed circadian rhythms that cycle with a near-24-hour period; in mammals, light is the most potent stimulus for entraining endogenous rhythms to the daily light cycle. Photic information is transmitted via the retinohypothalamic tract (RHT) to the suprachiasmatic nucleus (SCN) in the hypothalamus, where circadian rhythms are generated, but the retinal photopigment that mediates circadian entrainment has remained elusive. Here we show that most retinal ganglion cells (RGCs) that project to the SCN express the photopigment melanopsin.

Wakefulness: an eye-opening perspective on orexin neurons.

Orexin-containing neurons regulate wakefulness, and loss of orexin produces narcolepsy. Recent studies of mice lacking orexin neurons have shown that these cells also play essential roles in the control of feeding and energy balance.

The sleep switch: hypothalamic control of sleep and wakefulness.

More than 70 years ago, von Economo predicted a wake-promoting area in the posterior hypothalamus and a sleep-promoting region in the preoptic area. Recent studies have dramatically confirmed these predictions. The ventrolateral preoptic nucleus contains GABAergic and galaninergic neurons that are active during sleep and are necessary for normal sleep. The posterior lateral hypothalamus contains orexin/hypocretin neurons that are crucial for maintaining normal wakefulness. A model is proposed in which wake- and sleep-promoting neurons inhibit each other, which results in stable wakefulness and sleep. Disruption of wake- or sleep-promoting pathways results in behavioral state instability.

Mechanisms of CNS response to systemic immune challenge: the febrile response.

The acute-phase reaction is the multisystem response to acute inflammation. The central nervous system (CNS) mediates a coordinated set of autonomic, endocrine and behavioral responses that constitute the cerebral component of the acute-phase reaction. However, the mechanisms of immune signaling of the CNS remain controversial. Emerging evidence indicates that different parts of the acute-phase reaction are initiated by distinct mechanisms and in different brain regions. Cytokines produced as a result of local infections (for example, in the abdominal or thoracic cavities) might activate vagal sensory fibers, resulting in sickness behavior and fevers. Additionally, circulating immune stimuli might activate meningeal macrophages and perivascular microglia along the borders of the brain, eliciting the local production of prostaglandins and responses such as fever, anorexia, sleepiness, and activation of the hypothalamo-pituitary-adrenal (HPA) axis. The biological importance of these responses might favor the existence of multiple parallel CNS pathways that are engaged by cytokines.

Fos expression in orexin neurons varies with behavioral state.

The neuropeptide orexin (also known as hypocretin) is hypothesized to play a critical role in the regulation of sleep-wake behavior. Lack of orexin produces narcolepsy, which is characterized by poor maintenance of wakefulness and intrusions of rapid eye movement (REM) sleep or REM sleep-like phenomena into wakefulness. Orexin neurons heavily innervate many aminergic nuclei that promote wakefulness and inhibit REM sleep. We hypothesized that orexin neurons should be relatively active during wakefulness and inactive during sleep. To determine the pattern of activity of orexin neurons, we recorded sleep-wake behavior, body temperature, and locomotor activity under various conditions and used double-label immunohistochemistry to measure the expression of Fos in orexin neurons of the perifornical region. In rats maintained on a 12 hr light/dark cycle, more orexin neurons had Fos immunoreactive nuclei during the night period; in animals housed in constant darkness, this activation still occurred during the subjective night. Sleep deprivation or treatment with methamphetamine also increased Fos expression in orexin neurons. In each of these experiments, Fos expression in orexin neurons correlated positively with the amount of wakefulness and correlated negatively with the amounts of non-REM and REM sleep during the preceding 2 hr. In combination with previous work, these results suggest that activation of orexin neurons may contribute to the promotion or maintenance of wakefulness. Conversely, relative inactivity of orexin neurons may allow the expression of sleep.

Hypothalamic arousal regions are activated during modafinil-induced wakefulness.

Modafinil is an increasingly popular wake-promoting drug used for the treatment of narcolepsy, but its precise mechanism of action is unknown. To determine potential pathways via which modafinil acts, we administered a range of doses of modafinil to rats, recorded sleep/wake activity, and studied the pattern of neuronal activation using Fos immunohistochemistry. To contrast modafinil-induced wakefulness with spontaneous wakefulness, we administered modafinil at midnight, during the normal waking period of rats. To determine the influence of circadian phase or ambient light, we also injected modafinil at noon on a normal light/dark cycle or in constant darkness. We found that 75 mg/kg modafinil increased Fos immunoreactivity in the tuberomammillary nucleus (TMN) and in orexin (hypocretin) neurons of the perifornical area, two cell groups implicated in the regulation of wakefulness. This low dose of modafinil also increased the number of Fos-immunoreactive (Fos-IR) neurons in the lateral subdivision of the central nucleus of the amygdala. Higher doses increased the number of Fos-IR neurons in the striatum and cingulate cortex. In contrast to previous studies, modafinil did not produce statistically significant increases in Fos expression in either the suprachiasmatic nucleus or the anterior hypothalamic area. These observations suggest that modafinil may promote waking via activation of TMN and orexin neurons, two regions implicated in the promotion of normal wakefulness. Selective pharmacological activation of these hypothalamic regions may represent a novel approach to inducing wakefulness.

Orexin (hypocretin) neurons contain dynorphin.

Orexins (also called hypocretins) are peptide neurotransmitters expressed in neurons of the lateral hypothalamic area (LHA). Mice lacking the orexin peptides develop narcolepsy-like symptoms, whereas mice with a selective loss of the orexin neurons develop hypophagia and severe obesity in addition to the narcolepsy phenotype. These different phenotypes suggest that orexin neurons may contain neurotransmitters besides orexin that regulate feeding and energy balance. Dynorphin neurons are common in the LHA, and dynorphin has been shown to influence feeding; hence, we studied whether dynorphin and orexin are colocalized. In rats, double-label in situ hybridization revealed that nearly all (94%) neurons expressing prepro-orexin mRNA also expressed prodynorphin mRNA. The converse was also true: 96% of neurons in the LHA containing prodynorphin mRNA also expressed prepro-orexin mRNA. Double-label immunohistochemistry confirmed that orexin-A and dynorphin-A peptides were highly colocalized in the LHA. Wild-type mice and orexin knock-out mice showed abundant prodynorphin mRNA-expressing neurons in the LHA, but orexin/ataxin-3 mice with a selective loss of the orexin neurons completely lacked prodynorphin mRNA in this area, further confirming that within the LHA, dynorphin expression is restricted to the orexin neurons. These findings suggest that dynorphin-A may play an important role in the function of the orexin neurons.

Deletion of presynaptic adenosine A1 receptors impairs the recovery of synaptic transmission after hypoxia.

Adenosine protects neurons during hypoxia by inhibiting excitatory synaptic transmission and preventing NMDA receptor activation. Using an adeno-associated viral (AAV) vector containing Cre recombinase, we have focally deleted adenosine A(1) receptors in specific hippocampal regions of adult mice. Recently, we found that deletion of A(1) receptors in the CA1 area blocks the postsynaptic responses to adenosine in CA1 pyramidal neurons, and deletion of A(1) receptors in CA3 neurons abolishes the presynaptic effects of adenosine on the Schaffer collateral input [J Neurosci 23 (2003) 5762]. In the current study, we used this technique to delete A(1) receptors focally from CA3 neurons to investigate whether presynaptic A(1) receptors protect synaptic transmission from hypoxia. We studied the effects of prolonged (1 h) hypoxia on the evoked field excitatory postsynaptic potentials (fEPSPs) in the CA1 region using in vitro slices. Focal deletion of the presynaptic A(1) receptors on the Schaffer collateral input slowed the depression of the fEPSPs in response to hypoxia and impaired the recovery of the fEPSPs after hypoxia. Delayed responses to hypoxia linearly correlated with impaired recovery. These findings provide direct evidence that the neuroprotective role of adenosine during hypoxia depends on the rapid inhibition of synaptic transmission by the activation of presynaptic A(1) receptors.

Modafinil more effectively induces wakefulness in orexin-null mice than in wild-type littermates.

Narcolepsy-cataplexy, a disorder of excessive sleepiness and abnormalities of rapid eye movement (REM) sleep, results from deficiency of the hypothalamic orexin (hypocretin) neuropeptides. Modafinil, an atypical wakefulness-promoting agent with an unknown mechanism of action, is used to treat hypersomnolence in these patients. Fos protein immunohistochemistry has previously demonstrated that orexin neurons are activated after modafinil administration, and it has been hypothesized that the wakefulness-promoting properties of modafinil might therefore be mediated by the neuropeptide. Here we tested this hypothesis by immunohistochemical, electroencephalographic, and behavioral methods using modafinil at doses of 0, 10, 30 and 100 mg/kg i.p. in orexin-/- mice and their wild-type littermates. We found that modafinil produced similar patterns of neuronal activation, as indicated by Fos immunohistochemistry, in both genotypes. Surprisingly, modafinil more effectively increased wakefulness time in orexin-/- mice than in the wild-type mice. This may reflect compensatory facilitation of components of central arousal in the absence of orexin in the null mice. In contrast, the compound did not suppress direct transitions from wakefulness to REM sleep, a sign of narcolepsy-cataplexy in mice. Spectral analysis of the electroencephalogram in awake orexin-/- mice under baseline conditions revealed reduced power in the theta; band frequencies (8-9 Hz), an index of alertness or attention during wakefulness in the rodent. Modafinil administration only partly compensated for this attention deficit in the orexin null mice. We conclude that the presence of orexin is not required for the wakefulness-prolonging action of modafinil, but orexin may mediate some of the alerting effects of the compound.

Distribution of Fos-like immunoreactivity in the rat brain following intravenous lipopolysaccharide administration.

The central nervous system, particularly the hypothalamus, is intimately involved in the coordination of various aspects of the inflammatory response, including the generation of fever. We used intravenous injections of bacterial cell wall lipopolysaccharide (LPS; 5 or 125 micrograms/kg) to stimulate the acute phase response and mapped the resultant distribution of Fos-like immunoreactivity in the rat brain. In addition, we compared the patterns of Fos distribution with the thermoregulatory responses elicited by the LPS. Administration of LPS resulted in a dose- and time-dependent pattern of Fos-like immunoreactivity throughout the rat brain consistent with a coordinated autonomic, endocrine, and behavioral response to the LPS challenge that was most pronounced 2 hours following injection. Specifically, Fos-like immunoreactivity was observed in key autonomic regulatory nuclear groups, including the insular and prelimbic cortices, paraventricular hypothalamic nucleus, parabrachial nucleus, nucleus of the solitary tract, and the rostral and caudal levels of the ventrolateral medulla. In addition, a significant sustained elevation of Fos-like immunoreactivity was observed in a cell group adjacent to the organum vasculosum of the lamina terminalis, which we termed the ventromedial preoptic area. This sustained elevation of Fos-like immunoreactivity coupled with the alterations in body temperature elicited by LPS leads us to hypothesize that the ventromedial preoptic area may be a key site for the initiation of fever during endotoxemia.

Intravenous lipopolysaccharide induces cyclooxygenase 2-like immunoreactivity in rat brain perivascular microglia and meningeal macrophages.

Production of prostaglandins is a critical step in transducing immune stimuli into central nervous system (CNS) responses, but the cellular source of prostaglandins responsible for CNS signalling is unknown. Cyclooxygenase catalyzes the rate-limiting step in the synthesis of prostaglandins and exists in two isoforms. Regulation of the inducible isoform, cyclooxygenase 2, is thought to play a key role in the brain's response to acute inflammatory stimuli. In this paper, we report that intravenous lipopolysaccharide (LPS or endotoxin) induces cyclooxygenase 2-like immunoreactivity in cells closely associated with brain blood vessels and in cells in the meninges. Neuronal staining was not noticeably altered or induced in any brain region by endotoxin challenge. Furthermore, many of the cells also were stained with a perivascular microglial/macrophage-specific antibody, indicating that intravenous LPS induces cyclooxygenase in perivascular microglia along blood vessels and in meningeal macrophages at the edge of the brain. These findings suggest that perivascular microglia and meningeal macrophages throughout the brain may be the cellular source of prostaglandins following systemic immune challenge. We hypothesize that distinct components of the CNS response to immune system activation may be mediated by prostaglandins produced at specific intracranial sites such as the preoptic area (altered sleep and thermoregulation), medulla (adrenal corticosteroid response), and cerebral cortex (headache and encephalopathy).

Relationship of EP(1-4) prostaglandin receptors with rat hypothalamic cell groups involved in lipopolysaccharide fever responses.

The action of prostaglandin E(2) (PGE(2)) in the preoptic area is thought to play an important role in producing fever. Pharmacologic evidence suggests that, among the four subtypes of E-series prostaglandin (EP) receptors, i.e., EP(1), EP(2), EP(3), and EP(4), the EP(1) receptor mediates fever responses. In contrast, evidence from mice with EP receptor gene deletions indicates that the EP(3) receptor is required for the initial (<1 hour) fever after intravenous (i.v.) lipopolysaccharide (LPS). To investigate which subtypes of EP receptors mediate systemic infection-induced fever, we assessed the coexpression of Fos-like immunoreactivity (Fos-IR) and EP(1-4) receptor mRNA in nuclei in the rat hypothalamus that have been shown to be involved in fever responses. Two hours after the administration of i.v. LPS (5 microg/kg), Fos-IR was observed in the ventromedial preoptic nucleus, the median preoptic nucleus, and the paraventricular hypothalamic nucleus. In these nuclei, EP(4) receptor mRNA was strongly expressed and the Fos-IR intensely colocalized with EP(4) receptor mRNA. Strong EP(3) receptor mRNA expression was only seen within the median preoptic nucleus but Fos-IR showed little coexpression with EP(3) receptor mRNA. EP(2) receptor mRNA was not seen in the PGE(2) sensitive parts of the preoptic area. Although approximately half of the Fos-immunoreactive neurons also expressed EP(1) receptor mRNA, EP(1) mRNA expression was weak and its distribution was so diffuse in the preoptic area that it did not represent a specific relationship. In the paraventricular nucleus, EP(4) mRNA was found in most Fos-immunoreactive neurons and levels of EP(4) receptor expression increased after i.v. LPS. Our findings indicate that neurons expressing EP(4) receptor are activated during LPS-induced fever and suggest the involvement of EP(4) receptors in the production of fever.

Narcolepsy and low CSF orexin (hypocretin) concentration after a diencephalic stroke.

Idiopathic narcolepsy usually results from a loss of the hypothalamic neuropeptide orexin (hypocretin), but the cause of secondary narcolepsy resulting from focal brain lesions is unknown. The authors describe a young man who developed narcolepsy after a large hypothalamic stroke. His lesion included much of the hypothalamic region in which orexin is produced, and his CSF concentration of orexin was low. The authors hypothesize that a loss of orexin neurons or their relevant targets may be the specific neuropathology causing this and many other cases of secondary narcolepsy.

Polymorphisms in hypocretin/orexin pathway genes and narcolepsy.

The neuroexcitatory peptide hypocretin and its receptors are central to the pathophysiology of both human and animal models of the disease. In this study of American and Icelandic patients with narcolepsy, the authors found no significant association between narcolepsy and single-nucleotide polymorphisms in the genes for hypocretin or its two known receptors, hypocretin receptor-1 and hypocretin receptor-2.

An adenosine A2a agonist increases sleep and induces Fos in ventrolateral preoptic neurons.

Considerable evidence indicates that adenosine may be an endogenous somnogen, yet the mechanism through which it promotes sleep is unknown. Adenosine may act via A1 receptors to promote sleep, but an A2a receptor antagonist can block the sleep induced by prostaglandin D(2). We previously reported that prostaglandin D(2) activates sleep-promoting neurons of the ventrolateral preoptic area, and we hypothesized that an A2a receptor agonist also should activate these neurons. Rats were instrumented for sleep recordings, and an injection cannula was placed in the subarachnoid space just anterior to the ventrolateral preoptic area. After an 8-10-day recovery period, the A2a receptor agonist CGS21680 (20 pmol/min) or saline was infused through the injection cannula, and the animals were killed 2 h later. The brains were stained using Fos immunohistochemistry, and the pattern of Fos expression was studied in the entire brain. CGS21680 increased non-rapid eye movement sleep and markedly increased the expression of Fos in the ventrolateral preoptic area and basal leptomeninges, but it reduced Fos expression in wake-active brain regions such as the tuberomammillary nucleus. CGS21680 also induced Fos in the shell and core of the nucleus accumbens and in the lateral subdivision of the central nucleus of the amygdala. To determine whether these effects may have been mediated through A1 receptors, an additional group of rats received subarachnoid infusion of the A1 receptor agonist N(6)-cyclopentyladenosine (2 pmol/min). In contrast to CGS21680, infusion of N(6)-cyclopentyladenosine into the subarachnoid space produced only a small decrease in rapid eye movement sleep, and the pattern of Fos expression induced by N(6)-cyclopentyladenosine was notable only for decreased Fos in regions near the infusion site. These findings suggest that an adenosine A2a receptor agonist may activate cells of the leptomeninges or nucleus accumbens that increase the activity of ventrolateral preoptic area neurons. These ventrolateral preoptic area neurons may then coordinate the inhibition of multiple wake-promoting regions, resulting in sleep.

Effects of adenosine on gabaergic synaptic inputs to identified ventrolateral preoptic neurons.

The ventrolateral preoptic nucleus (VLPO) is a key regulator of behavioral state that promotes sleep by directly inhibiting brain regions that maintain wakefulness. Subarachnoid administration of adenosine (AD) or AD agonists promotes sleep and induces expression of Fos protein in VLPO neurons. Therefore, activation of VLPO neurons may contribute to the somnogenic actions of AD. To define the mechanism through which AD activates VLPO neurons, we prepared hypothalamic slices from 9 to 12-day-old rat pups and recorded from 43 neurons in the galaninergic VLPO cluster; nine neurons contained galanin mRNA by post hoc in situ hybridization. Bath application of AD (20 microM) to seven of these neurons had no direct effect but caused a significant decrease in the frequency of spontaneous miniature inhibitory postsynaptic currents in the presence of tetrodotoxin, indicating a presynaptic site of action. We conclude that AD-mediated disinhibition increases the excitability of VLPO neurons thus contributing to the somnogenic properties of AD.

No evidence for a circadian rhythm of protein synthesis in the rat suprachiasmatic nuclei.

The mammalian suprachiasmatic nuclei contain an endogenous circadian pacemaker. A quantitative autoradiographic tracer method that measures L-[1-14C]leucine incorporation into protein was used to determine whether the overall rate of protein synthesis in the nuclei varies in a circadian fashion. Unlike the robust circadian rhythms of electrical activity and energy metabolism previously recorded from the nuclei at the two time points sampled, protein synthesis remained constant.