The importance of increased cholinergic activity in triggering REM sleep or REM sleep atonia is well established (see ref. 32). Similarly, activation of the cholinergic systems using the acetylcholinesterase inhibitor physostigmine also greatly exacerbates cataplexy 33. This cholinergic effect is mediated via muscarinic receptors since muscarinic stimulation aggravates cataplexy, while its blockade suppresses it, and nicotinic stimulation or blockade has no effect.33
Monoaminergic transmission is also critical for the control of cataplexy. All therapeutic agents currently used to treat cataplexy (i.e., antidepressants or monoamine oxidase inhibitors [MAOIs]), are known to act on these systems. Furthermore, whereas a subset of cholinergic neurons are activated during REM sleep, the firing rate of monoaminergic neurons in the brainstem (such as in the locus coeruleus (LC) and the raphe magnus) are well known to be dramatically depressed during this sleep stage.34,35 Using canine narcolepsy, it was recently demonstrated that adrenergic LC activity is also reduced during cataplexy.36 In contrast, dopaminergic neurons in the ventral tegmental area (VTA) and substantia nigra (SN) do not significantly change their activity during natural sleep cycles.37,38
Since cataplexy in dogs can be easily elicited and quantified, the canine narcolepsy model has been intensively used to dissect the mode of action of currently used anticataplectic medications. The most commonly prescribed anticataplectic mediations in humans are tricyclic antidepressants. These compounds, however, have a complex pharmacological profile that includes monoamine uptake inhibition (dopamine [DA], epinephrine, norepinephrine [NE] and serotonin [5-HT]), anticholinergic, alpha-1 adrenergic antagonistic and antihistaminergic effects. It is thus difficult to conclude which one of these pharmacological properties is actually involved in their therapeutic effects.
We therefore first studied the effects of a large number (a total of 17 compounds) of uptake blockers/release enhancers specific for the adrenergic, serotonergic or dopaminergic system, and adrenergic uptake inhibition was found to be the key property involved in the anticataplectic effect.39 Serotonergic uptake blockers were only marginally effective at high doses and the dopaminergic uptake blockers were completely ineffective. Interestingly, it was later found that these dopamine uptake inhibitors had potent alerting effects in canine narcolepsy.1
We also compared the effects of several antidepressants with those of their demethylated metabolites. Many antidepressant compounds (most typically tricyclics) are known to metabolize significantly by a hepatic first pass into their demethylated metabolites that have longer half-lives and higher affinities for adrenergic uptake sites.40 During chronic drug administration, these demethylated metabolites accumulate40 and can thus be involved in the drug's therapeutic action. The effects of 5 available antidepressants (amitriptyline, imipramine, clomipramine, zimelidine, and fluoxetine) were compared with those of their respective demethylated metabolites (nortriptyline, desipramine, desmethylclomipramine, norzimelidine and norfluoxetine).41 In all cases, the demethylated metabolites were found to be more active on cataplexy than were the parent compounds. We also found that the active dose of all anticataplectic compounds tested positively, correlated with the in vitro potency of each compound to the adrenergic transporter but not with that of the serotonergic transporter.41 In fact, the anticataplectic effects were negatively correlated with the in vitro potency for serotonergic uptake inhibition, but this may be a bias since potent adrenergic uptake inhibitors included in the study have a relatively low affinity to serotonergic uptake sites.
Although the results presented were obtained from inbred Hcrtr-2 mutated narcoleptic Dobermans, similar findings have been obtained in more diverse cases of sporadic canine narcolepsy in various breeds donated to our colony (see ref. 33). Protryptiline and desipramine (two compounds with no significant serotonergic uptake blocking properties), have also been shown to be very effective for the treatment of human cataplexy.42-45 Thus preferential involvement of adrenergic system for the modes of action of anticataplectic effects of antidepressants are suggested regardless of the form of deficit in hypocretin neurotransmission (receptor mutation vs, hypocretin ligand deficiency).
In order to dissect receptor subtypes that significantly modify cataplexy, more than 200 compounds with various pharmacological properties (cholinergic, adrenergic, dopaminergic, serotonergic, prostaglandins, opioids, benzodiazepines, GABA-ergics and adenosinergics) have also been studied in the narcoleptic canine model (see1 for details). Although many compounds (such as M2 antagonists, alpha-1 agonists, alpha-2 antagonists, dopaminergic D2/D3antagonists, 5HT1a agonists, TRH analogs, prostaglandin E2, and L type Ca2+ channel blockers reduce cataplexy), very few compounds significantly aggravate cataplexy 1. Since cataplexy can be easily and non-specifically reduced such as by unpleasant drug side effects, we assume the cataplexy-aggravating effects are more specific. Stimulation of muscarinic M2 (non-M1) receptors significantly aggravates cataplexy. Among the monoaminergic receptors, stimulation of the postsynaptic adrenergic alpha-1b receptors46 and presynaptic alpha-2 receptors47 was also found to aggravate cataplexy, a result consistent with a primary adrenergic control of cataplexy. We also found that small doses of DA D2/D3 agonists significantly aggravated cataplexy and induced significant behavioral sedation/drowsy state in these animals.48,49 These pharmacological findings parallel neurochemical abnormalities previously reported in canine narcolepsy, namely significant increases in alpha-2 receptors in the LC,50 D2 receptors in the amygdala, nucleus accumbens51 and M2 receptors in the pons.52 To date, no other receptor ligands (i.e., adenosinergic, histaminergic or GABA-ergic) have been found to aggravate cataplexy, but thalidomide (an old hypnotic with an immunomodulatory property) significantly aggravates cataplexy, but the mechanisms involved in this effect are not known.53
The sites of action of D2/D3 agonists were also investigated by local drug perfusion experiments, and a series of experiments identified acting sites for these compounds. These include dopaminergic nuclei or cell groups, such as the VTA,49 SN54 and A1155 (a diencephalic DA cell group that directly project to the spinal ventral horn), suggesting a direct involvement of DA cell groups and DA cell body autoreceptors for the regulation of cataplexy. The cataplexy-inducing effects of D2/D3agonists are, however, difficult to reconcile considering the fact that dopaminergic uptake blockers have absolutely no effect on cataplexy.39 We believe that D2/D3 receptor mechanisms are more specifically involved in the control of sleep-related motor tonus (i.e., cataplexy or muscle atonia without phasic REM events) than those for active REM sleep. A recent finding in canine narcolepsy that sulpiride (a D2/D3 antagonist) significantly suppresses cataplexy but has no effect on REM sleep, also supports this notion.31 It should be also noted that D2/3 agonists are clinically used for the treatment of human periodic leg movements during sleep (PLMS).56 Involuntary leg movements during sleep are often associated with restless leg syndrome (RLS) and disturbed nighttime sleep. As reported in human,57 narcoleptic Dobermans often exhibit PLMS-like movements.58 It thus appears that there is an overlap in pathophysiological mechanisms between between cataplexy and PLMS, and dopaminergic system (i.e., D2/D3 receptor mechanisms) may be specifically involved both symptoms.
The effects on cataplexy by cholinergic stimulation in various brain regions were also examined in narcoleptic and in control canines. Local injection or perfusion of carbachol (a predominantly muscarinic agonist) into the pontine reticular formation (PRF) was found to aggravate canine cataplexy in a dose-dependent fashion.59 The results obtained in the PRF with cholinergic agonists were somewhat expected considering the well established role of the pontine cholinergic systems in the regulation of REM sleep and REM sleep atonia. Surprisingly, however, we also found that the local injection/perfusion of carbachol unilaterally or bilaterally into the BF (rostral to the preoptic area, in the vertical or horizontal limbs of the diagonal band of Broca and medial septum) dose-dependently aggravated cataplexy and induced long-lasting episodes of muscle atonia accompanied by desynchronized EEG in narcoleptic canines.60 Physostigmine was also found to aggravate cataplexy when injected into the same site, thus suggesting that fluctuations in endogenous levels of acetylcholine in this structure may be sufficient to induce cataplexy.60 The carbachol injections did not induce cataplexy in normal animals, but rather induced wakefulness.
The BF is anatomically connected with the limbic system, which is regarded as a critical circuit for integrating emotions. Furthermore, BF neurons are known to respond to the arousing property of appetitive stimuli,61 which we use to induce cataplexy in narcoleptic dogs. Considering the fact that emotional excitation is an alerting stimulus in normal animals but induces cataplexy in narcoleptic animals, the BF may be involved in triggering a paradoxical reaction to emotions-atonia rather than wakefulness, in narcoleptic animals. These results also suggest that more global brain structures (than those for REM sleep generation) are involved in the induction of cataplexy. Cataplexy is now demonstrated to be tightly associated with the loss of hypocretin neurotransmission. Global and persistent cholinergic/monoaminergic imbalance due to the loss of hypocretin neurotransmission may be required for the occurrence of cataplexy, and cataplexy could not be induced simply by an increase in REM sleep propensity and/or vigilance state instability that occurs in various disease conditions (such as depression) or in some physiological conditions (such as REM sleep deprivation). The findings that REM sleep abnormalities and sleep fragmentation are often seen in other sleep disorders such as narcolepsy without cataplexy, sleep apnea, and even in healthy subjects when their sleep patterns are disturbed, but these subjects who never develop cataplexy further supports this hypothesis. The mechanism for emotional triggering for cataplexy remains to be studied, but multiple brain sites and multiple functional and anatomical systems are likely to be involved.
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