Eric Murillo-Rodriguez

The diurnal rhythm of adenosine levels in the basal forebrain of young and old rats

There are significant decrements in sleep with age. These include fragmentation of sleep, increased wake time, decrease in the length of sleep bouts, decrease in the amplitude of the diurnal rhythm of sleep, decrease in rapid eye movement sleep and a profound decrease in electroencephalogram Delta power (0.3-4 Hz). Old rats also have less sleep in response to 12 h-prolonged wakefulness (W) indicating a reduction in sleep drive with age. The mechanism contributing to the decline in sleep with aging is not known but cannot be attributed to loss of neurons implicated in sleep since the numbers of neurons in the ventral lateral preoptic area, a region implicated in generating sleep, is similar between young (3.5 months) and old (21.5 months) rats. One possibility for the reduced sleep drive with age is that sleep-wake active neurons may be stimulated less as a result of a decline in endogenous sleep factors. Here, we test this hypothesis by focusing on the purine, adenosine (AD), one such sleep factor that increases after prolonged W. In experiment 1, microdialysis measurements of AD in the basal forebrain at 1 h intervals reveal that old (21.5 months) rats have more extracellular levels of AD compared with young rats across the 24 h diurnal cycle. In experiment 2, old rats kept awake for 6 h (first half of lights-on period) accumulated more AD compared with young rats. If old rats have more AD then why do they sleep less? To investigate whether changes in sensitivity of the AD receptor contribute to the decline in sleep, experiments 3 and 4 determined that for the same concentration of AD or the AD receptor 1 agonist, cyclohexyladenosine, old rats have less sleep compared with young rats. We conclude that even though old rats have more AD, a reduction in the sensitivity of the AD receptor to the ligand does not transduce the AD signal at the same strength as in young rats and may be a contributing factor to the decline in sleep drive in the elderly.

Relationship between CSF hypocretin levels and hypocretin neuronal loss

The sleep disorder narcolepsy may now be considered a neurodegenerative disease, as there is a massive reduction in the number of neurons containing the neuropeptide, hypocretin (HCRT). Most narcoleptic patients have low to negligible levels of HCRT in the cerebrospinal fluid (CSF), and such measurements serve as an important diagnostic tool. However, the relationship between HCRT neurons and HCRT levels in CSF in human narcoleptics is not known and cannot be directly assessed. To identify this relationship in the present study, the neurotoxin, hypocretin-2-saporin (HCRT2-SAP), was administered to the lateral hypothalamus (LH) to lesion HCRT neurons. CSF was extracted at circadian times (ZT) 0 (time of lights-on) or ZT8 at various intervals (2, 4, 6, 12, 21, 36, 60 days) after neurotoxin administration. Compared to animals given saline in the LH, rats with an average loss of 73% of HCRT neurons had a 50% decline in CSF HCRT levels on day 60. The decline in HCRT levels was evident by day 6 and there was no recovery or further decrease. The decline in HCRT was correlated with increased REM sleep. Lesioned rats that were kept awake for 6 h were not able to release HCRT to match the output of saline rats. As most human narcoleptics have more than 80% reduction of CSF HCRT, the results from this study lead us to conclude that in these patients, virtually all of the HCRT neurons might be lost. In those narcoleptics where CSF levels are within the normal range, it is possible that not all of the HCRT neurons are lost and that the surviving HCRT neurons might be increasing output of CSF HCRT.

The anandamide membrane transporter inhibitor, VDM-11, modulates sleep and c-Fos expression in the rat brain

Endogenous cannabinoids or endocannabinoids are lipid molecules that have a variety of biological actions, most notably via activation of the cannabinoid receptors. The family of endocannabinoids includes arachidonoylethanolamide (ANA) which modulates different behaviors, such as sleep. However, it is unknown whether pharmacological elevation of ANA endogenous levels might induce sleep. VDM 11 [(5 Z,8 Z,11 Z,14 Z)-N-(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide] is commonly used as an inhibitor of ANA cellular uptake, and thereby to potentiate its actions. In this study we have examined whether VDM-11 exerts any effect on the sleep-wake cycle and c-Fos expression in brain areas. When assayed alone in rats, VDM-11 (10 or 20 microg/5 microL, i.c.v.) at the beginning of the lights-off period, reduced wakefulness and increased sleep. The CB(1) cannabinoid receptor antagonist, SR141716A, partially reversed the effects of VDM-11 on sleep. Additionally, VDM-11 enhanced c-Fos expression in sleep-related brain areas such as the anterior hypothalamic area, paraventricular thalamic nucleus, and pedunculopontine tegmental nucleus. It is concluded that VDM-11 displays sleep-inducing properties and these effects slightly, albeit significantly, are reversed using SR141716A. Furthermore, c-Fos data suggest a possible underlying neuroanatomical substrate of the sleep-inducing properties of VDM-11. We report evidence suggesting that VDM-11 might be considered for the development of new pharmacological and pharmaceutical approaches to treat sleep disorders such as insomnia.

Effects of hypocretin (orexin) neuronal loss on sleep and extracellular adenosine levels in the rat basal forebrain

Neurons containing the neuropeptide hypocretin (HCRT, orexin) are localized only in the lateral hypothalamus, from where they innervate multiple regions implicated in arousal, including the basal forebrain. HCRT activation of downstream arousal neurons is likely to stimulate release of endogenous factors. One such factor is adenosine, which in the basal forebrain increases in level with wakefulness and decreases with sleep, and is hypothesized to regulate the waxing and waning of sleep drive. Does loss of HCRT neurons affect adenosine levels in the basal forebrain? Is the increased sleep that accompanies HCRT loss a consequence of higher adenosine levels in the basal forebrain? In the present study, we investigated these questions by lesioning the HCRT neurons with HCRT‐2–saporin (HCRT‐2–SAP) and measuring sleep and extracellular levels of adenosine in the basal forebrain. In separate groups of rats, the neurotoxin HCRT‐2–SAP or saline was administered locally to the lateral hypothalamus, and 80u2003days later adenosine and sleep were assessed. Rats given the neurotoxin had a 94% loss of HCRT neurons. These rats woke less at night, and had more rapid eye movement sleep, which is consistent with HCRT hypofunction. These rats also had more sleep after brief periods of sleep deprivation. However, in the lesioned rats, adenosine levels did not increase with 6u2003h of sleep deprivation, whereas an increase in adenosine levels occurred in rats without lesion of the HCRT neurons. These findings indicate that adenosine levels do not increase with wakefulness in rats with a HCRT lesion, and that the increased sleep in these rats occurs independently of adenosine levels in the basal forebrain.

The Use of Saporin Conjugates to Dissect Neurons Responsible for Sleep and Wakefulness

Virtually all organisms manifest regular periods of behavioral quiescence and activity. In mammals and birds, these periods have evolved into regular episodes of wakefulness and sleep. The sleep period itself has further differentiated into two distinct states, slow wave sleep (SWS) and rapid eye movement (REM) sleep. In humans, SWS has further differentiated into four distinct stages, each identified by a specific pattern on the electroencephalogram (EEG). REM sleep is very similar to wakefulness in many ways; the main difference is that there is behavioral quiescence during REM sleep. For this reason, REM sleep is often referred to as paradoxical sleep.