Narcoleptic Canines Research Articles

History of narcolepsy at Stanford University.

Although narcolepsy was first described in the late nineteenth century in Germany and France, much of the research on this disorder has been conducted at Stanford University, starting with Drs. William C. Dement and Christian Guilleminault in the 1970s. The prevalence of narcolepsy was established, and a canine model discovered. Following the finding in Japan that almost all patients with narcolepsy carry a specific HLA subtype, HLA-DR2, Hugh Mac Devitt, F. Carl Grumet, and Larry Steinman initiated immunological studies, but results were generally negative. Using the narcoleptic canines, Dr. Nishino and I established that stimulants increased wakefulness by stimulating dopaminergic transmission while antidepressants suppress cataplexy via adrenergic reuptake inhibition. A linkage study was initiated with Dr. Grumet in 1988, and after 10 years of work, the canine narcolepsy gene was cloned by in 1999 and identified as the hypocretin (orexin) receptor 2. In 1992, studying African Americans, we also found that DQ0602 rather than DR2 was a better marker for narcolepsy across all ethnic groups. In 2000, Dr. Nishino and I, in collaboration with Dr. Lammers in the Netherlands, found that hypocretin 1 levels in the cerebrospinal fluid (CSF) were undetectable in most cases, establishing hypocretin deficiency as the cause of narcolepsy. Pursuing this research, our and Dr. Siegel’s group, examining postmortem brains, found that the decreased CSF hypocretin 1 was secondary to the loss the 70,000 neurons producing hypocretin in the hypothalamus. This finding revived the autoimmune hypothesis but attempts at demonstrating immune targeting of hypocretin cells failed until 2013. At this date, Dr. Elisabeth Mellins and I discovered that narcolepsy is characterized by the presence of autoreactive CD4+ T cells to hypocretin fragments when presented by DQ0602. Following reports that narcolepsy cases were triggered by vaccinations and infections against influenza A 2009 pH1N1, a new pandemic strain that erupted in 2009, our groups also established that a small epitope of pH1N1 resembles hypocretin and is likely involved in molecular mimicry. Although much remains to be done, these achievements, establishing hypocretin deficiency as the cause of narcolepsy, demonstrating its autoimmune basis, and showing molecular mimicry between hypocretin and sequences derived from a pandemic strain of influenza, are likely to remain classics in human immunology.

Narcoleptic orexin receptor knockout mice express enhanced cholinergic properties in laterodorsal tegmental neurons

Pharmacological studies of narcoleptic canines indicate that exaggerated pontine cholinergic transmission promotes cataplexy. As disruption of orexin (hypocretin) signaling is a primary defect in narcolepsy with cataplexy, we investigated whether markers of cholinergic synaptic transmission might be altered in mice constitutively lacking orexin receptors (double receptor knockout; DKO). mRNA for Choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT) and the high-affinity choline transporter (CHT1) but not acetylcholinesterase (AChE) was significantly higher in samples from DKO than wild-type (WT) mice. This was region-specific; levels were elevated in samples from the laterodorsal tegmental nucleus (LDT) and the fifth motor nucleus (Mo5) but not in whole brainstem samples. Consistent with region-specific changes, we were unable to detect significant differences in Western blots for ChAT and CHT1 in isolates from brainstem, thalamus and cortex or in ChAT enzymatic activity in the pons. However, using ChAT immunocytochemistry, we found that while the number of cholinergic neurons in the LDT and Mo5 were not different, the intensity of somatic ChAT immunostaining was significantly greater in the LDT, but not Mo5, from DKO than from WT mice. We also found that ChAT activity was significantly reduced in cortical samples from DKO compared with WT mice. Collectively, these findings suggest that the orexins can regulate neurotransmitter expression and that the constitutive absence of orexin signaling results in an up-regulation of the machinery necessary for cholinergic neurotransmission in a mesopontine population of neurons that have been associated with both normal rapid eye movement sleep and cataplexy.

Hypocretin/Orexin: A Molecular Link Between Sleep, Energy Regulation, and Pleasure

Hypocretin (Hcrt) is a neurotransmitter of the dorsal and lateral hypothalamus that regulates sleep, appetite, and energy consumption. Recent evidence indicates that it is also involved in pleasure/reward-seeking. Mutation of the Hcrt-receptor gene causes narcolepsy in canines, and Hcrt knockout mice exhibit narcolepsy-like symptoms. Human narcoleptics do not commonly have mutations in the ligand or receptor but do have degeneration of Hcrt-containing neurons, possibly through an autoimmune mechanism. When Hcrt neurons degenerate in mice, hypophagia and obesity are observed, symptoms that are also present in some human narcoleptics. This article reviews the recent literature with regard to the many functions of this single molecule. The authors suggest that eating habits and impulsivity may be topics worth exploring in the evaluation of narcoleptic patients.

Narcoleptic canines display periodic leg movements during sleep

Periodic leg movements during sleep (PLMS) is a high prevalent sleep disorder of unknown etiology. The disease is pharmacologically treated with dopaminergic agonists (i.e. D2/D3 agonists) and opiates. Periodic leg movements during sleep often occur in narcoleptic patients. We observed that narcoleptic canines, like narcoleptic humans, also exhibit jerky, unilateral or bilateral slow leg movements during sleep. The movements in dogs are characterized by repetitive dorsiflexions of the ankle, lasting 0.5-1.5 s, and occur at regular intervals of 3-20 s, thus showing similarities to PLMS in humans. The observation that D2/D3 agonists aggravate cataplexy in narcoleptic dogs suggests that altered dopaminergic regulation in canine narcolepsy may play a critical role in both cataplexy and PLMS. Our canines may therefore be an invaluable resource in PLMS research.

Sulpiride, a D2/D3 Blocker, Reduces Cataplexy but not REM Sleep in Canine Narcolepsy

Cataplexy, an abnormal manifestation of REM sleep atonia, is currently treated with antidepressants. These medications also reduce physiological REM sleep and induce nocturnal sleep disturbances. Because a recent work on canine narcolepsy suggests that the mechanisms for triggering cataplexy are different from those for REM sleep, we hypothesized that compounds which act specifically on cataplexy, but not on REM sleep, could be developed. Canine studies also suggest that the dopamine D2/D3 receptor mechanism is specifically involved in the regulation of cataplexy, but little evidence suggests that this mechanism is important for REM sleep regulation. We therefore assessed the effects of sulpiride, a commonly used D2/D3 antagonist, on cataplexy and sleep in narcoleptic canines to explore the possible clinical application of D2/D3 antagonists for the treatment of human narcolepsy. Both acute and chronic oral administration of sulpiride (300 mg/dog, 600 mg/dog) significantly reduced cataplexy without noticeable side effects. Interestingly, the anticataplectic dose of sulpiride did not significantly reduce the amount of REM sleep. Sulpiride (and other D2/D3 antagonists) may therefore be an attractive new therapeutic indication in human narcolepsy.

Neuropharmacological Characterization of Basal Forebrain Cholinergic Stimulated Cataplexy in Narcoleptic Canines

Basal forebrain (BF) cholinergic regulation of cataplexy was investigated in narcoleptic canines. Specific cholinergic agonists and antagonists, and excitatory or inhibitory amino acid neurotransmitter receptor agonists, were perfused through microdialysis probes implanted bilaterally in the BF of narcoleptic canines. Cataplexy was monitored using the food-elicited cataplexy test (FECT) and recordings of electroencephalogram, electrooculogram, and electromyogram. In narcoleptic canines, carbachol and oxotremorine (10−5–10−3M), but not McN-A-343 or nicotine (10−4–10−3M), produced a dose-dependent increase in cataplexy. In addition,N-methyl-d-aspartate (10−4–10−3M) and kainic acid (10−5–10−4M) did not have any effects, while muscimol (10−3M) produced a weak (P<0.10) increase in cataplexy. In control canines, carbachol (10−5–10−3M), but not oxotremorine (10−4–10−3M), produced muscle atonia after the highest concentration in one of three animals. Carbachol (10−3M)-induced cataplexy in narcoleptic canines was blocked by equimolar perfusion with the muscarinic antagonists atropine, gallamine, and 4-DAMP but not pirenzepine. These findings indicate that carbachol-stimulated cataplexy in the BF of narcoleptic canines is mediated by M2, and perhaps M3, muscarinic receptors. The release of acetylcholine in the BF was also examined during FECT and non-FECT behavioral stimulation in narcoleptic and control canines. A significant increase in acetylcholine release was found in both narcoleptic and control BF during FECT stimulation. In contrast, simple motor activity and feeding, approximating that which occurs during an FECT, did not affect acetylcholine release in the BF of narcoleptic canines. These findings indicate that BF acetylcholine release is enhanced during learned emotion/reward associated behaviors in canines.

Neurobiology of Narcolepsy

Narcolepsy is characterized by excessive daytime sleepiness and abnormal rapid eye movement sleep. It affects about 0.05% of the Caucasian population. Human narcolepsy involves the interaction of environmental factors with a specific immunogenetic background. It is tightly associated with a major histocompatibility complex allele, human leukocyte antigen (HLA) DQB1*0602. Genetic factors other than HLA are also involved. In contrast, narcolepsy in Dobermans is transmitted as a single autosomal recessive trait. This canine narcolepsy gene is unlinked to the major histocompatibility complex class II but co-segregates with a DNA segment with high homology to the human immunoglobulin μ-switch sequence, further suggesting immunopathology in narcolepsy. However, attempts to demonstrate that narcolepsy is an autoimmune disease have been unsuccessful. Narcolepsy is treated with antidepressants for rapid eye movement sleep-related symptoms and with amphetamine-like stimulants for sleepiness. Pharmacological studies using narcoleptic canines indicate that monoaminergic and cholinergic systems are involved in the pathophysiology of narcolepsy. Dopaminergic uptake mechanisms and D2(3) autoreceptors are involved in the control of alertness, whereas adrenergic uptake mechanisms, α-1 and α-2/dopaminergic D2(3) receptors, are involved in the control of cataplexy, suggesting that amphetamine-like stimulants act via the dopaminergic system and that antidepressants exhibit their anticataplectic effects via the adrenergic system. Local drug perfusion studies indicate that D2(3) agonists in the ventral tegmental area induce cataplexy and sleepiness in narcoleptic dogs but not in control dogs. Furthermore, perfusion of M2 agonists in the pontine reticular formation and the basal forebrain induces cataplexy in narcoleptic dogs. Extracellular single-unit and acetylcholine measurement studies suggest that basal forebrain cholinoceptive sites mediate the emotional trigger for cataplexy. Although narcolepsy does not seem to be a classical autoimmune disease, concomitant increases in microglial HLA class II expression with the development of the disease occur in canine narcolepsy. A neuroimmune-related process at an early age is thus likely to contribute to the neurochemical imbalance seen in narcolepsy. NEUROSCIENTIST 4:133–143, 1998

Effects of Thyrotropin-Releasing Hormone and Its Analogs on Daytime Sleepiness and Cataplexy in Canine Narcolepsy

The therapeutic potential of thyrotropin-releasing hormone (TRH) and TRH analogs in narcolepsy, a sleep disorder characterized by abnormal rapid eye movement (REM) sleep and daytime sleepiness, was examined using the canine model. The effects of TRH and the biologically stable TRH analogs CG3703, CG3509, and TA0910 on daytime sleep and cataplexy, a symptom of abnormal REM sleep, were assessed using polysomnographic recordings and the food elicited cataplexy test (FECT), respectively. CG3703 (100 and 400 microg/kg, i.v.) and TA0910 (100 and 400 microg/kg, i.v.) significantly increased wakefulness and decreased sleep in narcoleptic canines, whereas TRH (400 and 1600 microg/kg, i.v.) had no significant effect. TRH (25-1600 microg/kg, i.v.) and all three TRH analogs, CG3703 (6. 25-400 microg/kg, i.v., and 0.25-16 mg/kg, p.o.), CG3509 (25-1600 microg/kg, i.v.), and TA0910 (25-1600 microg/kg, i.v.), significantly reduced cataplexy in canine narcolepsy. These compounds did not produce any significant side effects during behavioral assays, nor did they alter free T3 and T4 levels in serum even when used at doses that completely suppressed cataplexy. Although more work is needed to establish the mode of action of TRH analogs on alertness and REM sleep-related symptoms, our results suggest a possible therapeutic application for TRH analogs in human sleep disorders.

Local administration of dopaminergic drugs into the ventral tegmental area modulates cataplexy in the narcoleptic canine

Cataplexy in the narcoleptic canine may be modulated by systemic administration of monoaminergic compounds. In the present study, we have investigated the effects of monoaminergic drugs on cataplexy in narcoleptic canines when perfused locally via microdialysis probes in the amygdala, globus pallidus/putamen, basal forebrain, pontine reticular formation and ventral tegmental area of narcoleptic and control Doberman pinchers. Cataplexy was quantified using the Food-Elicited Cataplexy Test and analyzed by electroencephalogram, electrooculogram and electromyogram. Local perfusion with the monoaminergic agonist quinpirole, 7-OH-DPAT and BHT-920, into the ventral tegmental area produced a dose-dependent increase in cataplexy without significantly reducing basal muscle tone. Perfusion with the antagonist raclopride in the same structure produced a moderate reduction in cataplexy. Local perfusion with quinpirole, 7-OH-DPAT and BHT-920 into the globus pallidus/putamen also produced an increase, while raclopride produced a decrease, in cataplexy in narcoleptic canines. In control animals, none of the above drugs produced cataplexy or muscle atonia when perfused into either the ventral tegmental area or the globus pallidus/putamen. Other monoaminergic drugs tested in these two brain areas; prazosin, yohimbine, amphetamine, SKF 38393 and SCH 23390 had no effects on cataplexy. Local perfusion with each of the above listed drugs had no effect on cataplexy in any of the other brain regions examined. These findings show that cataplexy may be regulated by D2/D3 dopaminergic receptors in the ventral tegmental area and perhaps the globus pallidus/putamen. It is suggested that neurons in the mesolimbic dopamine system of narcoleptics are hypersensitive to dopaminergic autoreceptor agonists.

Cholinergic Regulation of Cataplexy in Canine Narcolepsy in the Pontine Reticular Formation is Mediated by M2 Muscarinic Receptors

Both rapid eye movement sleep and cataplexy in the narcoleptic canine have been shown to increase after both systemic and local administration of cholinergic agonists in the pontine reticular formation. Furthermore, binding studies indicate an increase in the number of M2 muscarinic receptors in the pontine reticular formation of narcoleptic canines. In the present study we have investigated the receptor subtypes involved in mediating the cholinergic stimulation of cataplexy, as defined by brief periods of hypotonia induced by emotions, within the pontine reticular formation of narcoleptic canines. Specific cholinergic and monoaminergic agonists and antagonists, and excitatory or inhibitory amino-acid neurotransmitter receptor agonists, were perfused through microdialysis probes implanted bilaterally in the pontine reticular formation of narcoleptic canines, and cataplexy was monitored using the Food-Elicited Cataplexy Test and recordings of electroencephalogram, electrooculogram and electromyogram. In narcoleptic canines, bilateral perfusion with oxotremorine (M2 muscarinic) (10(-5)-10(-3) M) in the pontine reticular formation produced a dose-dependent increase in cataplexy, which reached complete muscle atonia (status cataplecticus) during the highest concentration. In control canines bilateral perfusion with oxotremorine (10(-5)-10(-3) M) did not produce any cataplectic attacks, but did produce muscle atonia after the highest concentration. Bilateral perfusion with either McN-A-343 (M1 muscarinic) or nicotine (both 10(-5)-10(-3) M) did not have any effect on cataplexy in either narcoleptic or control canines. The increase in cataplexy in narcoleptic canines produced by local perfusion with carbachol (10(-4) M) followed by equimolar perfusion with a muscarinic antagonist was rapidly reversed by atropine (muscarinic) and gallamine (M2 muscarinic), partially reversed by 4-DAMP (M3/M1 muscarinic) and completely unaffected by pirenzepine (M1 muscarinic). Bilateral perfusion with excitatory, glutamatergic receptor agonists N-methyl-D-aspartate, AMPA (both at 10(-4)-10(-3) M) and kainic acid (10(-5)-10(-4) M) did not have any effect on cataplexy, whereas bilateral perfusion with the inhibitory GABAergic receptor agonist muscimol (10(-4)-10(-3) M) produced a moderate increase in cataplexy in the narcoleptic canines. Bilateral perfusion with numerous monoaminergic compounds, BHT-920 (alpha-2 agonist), yohimbine (alpha-2 antagonist), propranolol (beta antagonist) and prazosin (alpha-1 antagonist), did not have any effect on cataplexy. These findings demonstrate that cholinergic regulation of cataplexy in the narcoleptic canine at the level of the pontine reticular formation is mediated by M2, and possibly M3, muscarinic receptors. The effects of muscimol indicate that the stimulation of cataplexy might be elicited by local neuronal inhibition.

Modafinil binds to the dopamine uptake carrier site with low affinity.

Modafinil is a stimulant compound with an unknown mechanism of action that has been used successfully to control excessive daytime sleepiness in narcoleptic patients in Europe (1,2). The compound also has a minimal effect on cataplexy and other accessory symptoms of the narcolepsy tetrad (1,2). It is well tolerated and is generally preferred to amphetamine and related compounds in the countries where it is available. The mode of action of modafinil is still not understood. The compound has been shown to decrease sleep and increase wakefulness in cats (3) and monkeys (4), and increase spontaneous locomotion in mice and to a lesser extent in rats (5). It also decreases barbitaland phenobarbital-induced sleep in mice and rats and does not produce stereotypies below very high doses (5). The compound is suggested to have no peripheral effects in rodents, dogs and monkeys (5-7). Surprisingly, the compound is also devoid of any sympathomimetic effects, including those at the cardiovascular level (2,6). The current hypothesis regarding the mode of action of modafinil involves the stimulation of alpha-l adrenergic mechanisms. The modafinil-induced increase in motor activity in mice is antagonized by central alpha-l antagonists such as prazosin or phenoxybenzamine, but not by dopamine antagonists (5). Similarly, the awakening properties of the compound (in monkeys and cats) were antagonized by prazosin, although antagonisms were only partial (3). Other experiments, however, have suggested a different mode of action. First, the compound does not bind alpha-l receptors in vitro (up to 10-3 Musing [3H]-prazosin in canine cortical membranes, data not shown). Secondly, our experiments in narcoleptic canines have shown that the compound does not modify canine cataplexy even at doses promoting alertness (7). Because canine cataplexy is very sensitive to compounds acting presynaptically and postsynaptically on adrenergic transmission (see reference 8 for review), the results suggest a nonadrenergic mode of action. Electrophysiological and volta metric data also do not support the hypothesis of catecholaminergic mechanism of action. High doses of modafinil have been shown not to modify the firing rate of noradrenergic neurons of the locus coeruleus or dopaminergic neurons of the ventral tegmental area or substantia nigra in rats (9). Similarly, modafinil does not modify catechol oxidation peaks as measured by voltametry in the mouse caudate nucleus and rat striatum or nucleus accumbens, including after pargyline treatment (10). To further study the profile of modafinil, the compound was evaluated using the NIMH/Novascreen® Psychotherapeutic Drug Discovery and Development Program. Binding assay experiments included receptors for adenosine, dopamine, ,),-aminobutyric acid (GABA), serotonin, N-methyl-D-aspartate, kainate, quisqualate, glycine (strychnine sensitive and insensitive), benzodiazepine, phencyclidine (PCP), MK-801, angiotensine, Arg-vasopressine, bombesin, cholecystokinin (central and peripheral), neurospeptide Y (NPY), substance K and P, neurotensin, somatostatin, vasoactive intestinal polypeptide (VIP), atrial natriuretic factor 1 (ANF1), epidermal growth factor, nerve growth factor, various ion channels (calcium channels N, T and L; chloride channels and low conduction potassium channels) and second messenger systems (adenylate cyclase, phorbol ester and inositol triphosphate). All results were negative when a concentration of 10-3 M was tested. In a second set of studies, we decided to explore the affinity of modafinil for various neurotransmitter uptake sites. The concentration tested was 10-4 M and uptake sites studied included adenosine CH-nitrobenzylthioinosine in rat cortex), choline CH-choline in rat brain), GABA CH-GABA in rat cortical membranes), dopamine CH-WIN in guinea pig stratum), norepinephrine CH-desmethylimipramine in rat cortex) and

Cholinergic mechanisms in canine narcolepsy—II. Acetylcholine release in the pontine reticular formation is enhanced during cataplexy

Cataplexy in the narcoleptic canine has been shown to increase after local administration of carbachol into the pontine reticular formation. Rapid eye movement sleep has also been shown to increase after local administration of carbachol in the pontine reticular formation, and furthermore, acetylcholine release in the pontine tegmentum was found to increase during rapid eye movement sleep in rats. Therefore, in the present study we have investigated acetycholine release in the pontine reticular formation during cataplexy in narcoleptic canines. Extracellular acetylcholine levels were measured in the pontine reticular formation of freely moving narcoleptic and control Doberman pinschers using in vivo microdialysis probes. Cataplexy was induced by the Food-Elicited Cataplexy Test and monitored using recordings of electroencephalogram, electrooculogram and electromyogram. Basal levels of acetylcholine in the microdialysis perfusates were approximately 0.5 pmol/10min in both control and narcoleptic canines. Local perfusion with tetrodotoxin (10 −5M) or artificial cerebrospinal fluid without Ca 2+ produced a decrease, while intravenous injections of physostigmine (0.05 mg/kg) produced an increase in acetylcholine levels, indicating that the levels of acetylcholine levels measured are derived from neuronal release. During cataplexy induced by the Food-Elicited Cataplexy Test, acetylcholine levels increased by approximately 50% after four consecutive tests in narcoleptic canines, but did not change after four consecutive tests in control canines. Motor activity and feeding behavior, similar to that occurring during a Food-Elicited Cataplexy Test, had no effect on acetycholine levels in the narcoleptic canines. These findings show that acetylcholine release in the pontine reticular formation is enhanced during Food-Elicited Cataplexy Tests in narcoleptic canines, and that this increase is specifically linked to the incidence of cataplectic attacks. Thus, cataplexy in the narcoleptic canine is associated with an increase in acetylcholine release in the pontine reticular formation. Furthermore, this study provides evidence supporting the hypothesis that narcolepsy is a disorder of rapid eye movement sleep regulation.

Cholinergic mechanisms in canine narcolepsy—I. Modulation of cataplexy via local drug administration into the pontine reticular formation

Cataplexy in the narcoleptic canine has been shown to increase after systemic administration of cholinergic agonists. Furthermore, the number of cholinergic receptors in the pontine reticular formation of narcoleptic canines is significantly elevated. In the present study we have investigated the effects of cholinergic drugs administered directly into the pontine reticular formation on cataplexy, as defined by brief episodes of hypotonia induced by emotions, in narcoleptic canines. Carbachol and atropine were perfused through microdialysis probes implanted bilaterally in the pontine reticular formation of freely moving, narcoleptic and control Doberman pinschers. Cataplexy was quantified using the Food-Elicited Cataplexy Test, and analysed using recordings of electroencephalogram, electrooculogram and electromyogram. Cataplexy was characterized by a desynchronized electroencephalogram and a drop in electromyogram and electrooculogram activity. In narcoleptic canines, both unilateral and bilateral carbachol (10 −5 to 10 −3 M) produced a dose-dependent increase in cataplexy, which resulted in complete muscle tone suppression at the highest concentration. In control canines, neither bilateral nor unilateral carbachol (10 −5 to 10 −3 M) produced cataplexy, although bilateral carbachol, did produce muscle atonia at the highest dose (10 −3). The increase in cataplexy after bilateral carbachol (10 −4 M) was rapidly reversed when the perfusion medium was switched to one containing atropine (10 −4 M). Bilateral atropine (10 −3 to 10 −2 M) alone did not produce any significant effects on cataplexy in narcoleptic canines; however, bilateral atropine (10 −2 M) did reduce the increase in cataplexy produced by systemic administration of physostigmine (0.05 mg/kg, i.v.). These findings demonstrate that cataplexy in narcoleptic canines can be stimulated by applying cholinergic agonists directly into the pontine reticular formation. The ability of atropine to inhibit locally and systemically stimulated cataplexy indicates that the pontine reticular formation is a critical component in cholinergic stimulation of cataplexy. Therefore, it is suggested that the pontine reticular formation plays a significant role in the cholinergic regulation of narcolepsy.

Monoaminergic uptake in synaptosomes prepared from frozen brain tissue samples of normal and narcoleptic canines

Canine narcolepsy, a model of the human disorder, is associated with altered catecholamine but not serotonin (5-HT) metabolism in some brain areas, particularly the amygdala. A possible explanation for these global changes could be the existence of specific defects in monoamine uptake processes. We have studied the uptake of [ 3H]norepinephrine (NE), [ 3H]5-HT in synaptosomes prepared from cortex and amygdala of narcoleptic and control Doberman pinscher brains. Since narcoleptic canines are relatively few in number, we have used a specific brain freezing procedure that has been reported to allow restoration of metabolically functional tissue upon thawing. Preliminary studies comparing monoamine uptake in fresh and frozen brain samples of both groups of dogs were carried out and demonstrated that this procedure significantly altered serotoninergic but not noradrenergic and dopaminergic uptake. All further investigations were then done on synaptosomes prepared from frozen samples. Our results demonstrate that synaptosomal uptake of [ 3H]NE, [ 3H]DA and [ 3H]5-HT in cortex and amygdala are not altered in narcolepsy.

CNS monoamines and their metabolites in canine narcolepsy: a replication study

In two separate studies a significantly greater concentration of DA (dopamine) and its metabolite, DOPAC (3,4-dihydroxyphenylacetic acid), was observed in the amygdala of narcoleptic canines. DOPAC was also significantly elevated in the reticularis parvicellularis, whereas NE (norepinephrine) was significantly elevated in the reticularis oralis, but depressed in the preoptic hypothalamus. No changes were observed in concentrations of serotonin or its metabolite, 5-HIAA (5-hydroxyindoleauric acid) in any region in the narcoleptic canine brain. Results of the two studies were similar, except that previously observed differences between narcoleptic and control canines in DOPAC levels in the caudate and reticularis oralis failed to replicate. Thus, steady state measures of neurotransmitter/metabolite tissue concentrations suggest region-specific alterations in DA and NE metabolism, rather than a global deficit in catecholamine neurotransmission in canine narcolepsy.

Biogenic amine concentrations in the brains of normal and narcoleptic canines: current status.

Biogenic amine concentrations in the brains of normal and narcoleptic canines: current status.

Electroencephalographic correlates of cataplectic attacks in narcoleptic canines

Cataplectic attacks were monitored behaviorally and polygraphically in 4 narcoleptic dogs, of which three inherited the disorder. The recorded EEG signals were evaluated by power spectral analysis. We found 3 distinct stages of cataplexy: an initial stage which resembled wakefulness with tonic suppression of EMG activity, a later stage which was highly similar to REM sleep, and a final transitional stage to wakefulness or NREM sleep. The first stage of cataplexy was characterized by full postural collapse, a waking-like EEG spectrum, visual tracking, and a hypotonic EMG. The second stage of cataplexy differed electrographically from the previous stage by the onset of hypersynchronous hippocampal theta activity, a REM-like EEG spectrum, larger amplitude EEG signals, and a higher peak theta frequency. Glazed eyes, sporadic rapid eye movements and muscle twitches were also present. The final stage of cataplexy was characterized by mixed amplitude, mixed frequency EEG activity, and by the absence of rapid eye movements, visual tracking, directed movements, and muscle twitches. The EEG spectra of two other narcoleptic phenomena, sleep-onset REM periods and NREM sleep onsets from cataplexy, were nearly identical to the spectra of the normally occurring REM and NREM sleep periods.