Research Laboratories and Divisions

Overview

The lab’s research strategy is to identify the neural circuitry responsible for the alternation between wake, sleep and REM sleep.  Sleep and circadian rhythms of body temperature and activity are measured in rodents (rats and mice).  Molecular (Dr. Meng Liu), pharmacological (Dr. Carlos Blanco-Centurion), anatomical (Dr. Satvinder Kaur) and electrophysiology (Dr. Stephen Thankachan) methods are used to trace the network, record activity of neurons, identify gene expression in specific neuronal populations and whether destruction of specific neurons or loss of specific genes affects sleep. To destroy  orexin receptor bearing neurons we created a novel compound linking saporin to the ligand orexin. This compound provides a rapid, cost effective method to test specific hypotheses regarding the role of specific brain regions in sleep-wake regulation.  Once it is found that loss of a gene or neurons results in a sleep abnormality then novel methods are used to repair the network and determine whether sleep returns to normal. Recently, we demonstrated the first ever use of gene transfer to treat a sleep disorder.  We found that orexin gene transfer significantly improved sleep and cataplexy in the mice model of narcolepsy (Liu et al., European J Neuroscience, 2008). 


The primary focus of the research is on the sleep disorder, narcolepsy.  Dr. Shiromani’s research career was started in 1986 by a small grant from the American Narcolepsy Association.  Since then the laboratory has been continuously funded by the NIH, the VA and the DoD.

This lab provides training in long-term (months) recording of sleep and circadian rhythms in rodents.  When the lab was established in 1990 it was the first one in the Northeast area to do long-term recording of both sleep and circadian rhythms (via telemetry) in rodents.  Dr. Shiromani has trained many students who have now established their own labs.  Training is also provided in molecular biology, immunohistochemistry, microdialysis, HPLC and single cell recording of neurons in freely-behaving rodents.

Dr. Carlos Blanco-Centurion was awarded the Young Investigator Prize from the Sleep Research Society in 2007.  He began as a trainee in the lab and now is an Instructor.

Orexin Gene Transfer (Meng Liu)

Soon after the discovery that narcolepsy is a neurodegenerative sleep disorder our lab began to devise ways to repair the circuit.  Initial approach was to graft orexin neurons into the brain in a manner similar to that employed by researchers in Parkinson’s disease.  However, we quickly realized that only 1-5% of the grafted neurons survived (Arias et al., 2004) and that more neurons would be required to influence sleep behavior.  Therefore, we undertook the strategy of gene transfer.  Dr. Meng Liu in the lab inserted the gene for orexin into an inactivated herpes simplex-1 virus (supplied by Dr. Rachael Neve) and did the preliminary experiments demonstrating that the virus transferred the gene for orexin into neurons and also made the gene product.  Then the gene was delivered to the brains of mice that lacked the gene, i.e., orexin knockout mice.  In these mice the neurons in the targeted brain region began to make the peptide and also released it into the cerebrospinal fluid.  Most importantly, when the gene was transfered into the orexin knockout mice their narcoleptic behavior was significantly reduced (by 60%)(see Liu et al., 2008). We have now improved the efficacy of the virus so that the gene can be expressed for at least a month (see photo).  


Neuronal Circuit Mapping (Carlos Blanco-Centurion and Satvinder Kaur)

In order to repair circuits we need to first understand the wiring.  One approach is to destroy specific brain neurons and then determine the change in sleep.  We have refined this approach so that several phenotypes of brain neurons can be selectively lesioned simultaneously.  Using this approach Carlos Blanco-Centurion (Blanco-Centurion et al., 2007) found that selective destruction of the basal forebrain cholinergic neurons, the noradrenergic locus coeruleus neurons and the histamine neurons resulted in the rats waking up less frequently and they had longer bouts of sleep.  We concluded that these three arousal neurons serve a very specific purpose with regard to sleep: they rapidly awaken a sleeping brain, and with it turn on cognitive function, attention and vigilance.  These arousal neurons are triggers that rapidly terminate a sleep bout.  They monitor the internal and external environment and rapidly stop the sleep neurons in the event of an alarm, either internal (rising carbon dioxide, for example with sleep apnea) or external (intruder, baby crying).  If these neurons are depressed (which might occur with too many hypnotics) then there is the risk of death.  However, if these neurons are too sensitive then there would be frequent arousals and difficulty falling asleep, which may be the case in post-traumatic stress syndrome (see Shiromani, Keane and LeDoux, 2009).

Another project is tracing the network underlying REM sleep.  This stage of sleep is triggered from the pontine brainstem.  However, it is still not known which pontine neurons inhibit it and which neurons generate it.   We have hypothesized that the neurons in the pons that inhibit it contain the orexin-2 receptor.  We have focused on this receptor because when this receptor is mutated or knocked out then there is an increase in REM sleep and narcoleptic behavior.  To lesion the orexin-2 receptor bearing neurons we created a neurochemical compound by binding the ligand orexin-B to the ribosomal inhibiting protein saporin (see Gerashchenko et al., 2001).  When this compound was administered locally to the pons, the rats had significant (+56%) increase in REM sleep, which supports our hypothesis that pontine neurons that contain the orexin-2 receptor inhibit REM sleep (see Blanco-Centurion et al., 2004). 


We hypothesize that there is another site in the pons that is also inhibitory to REM sleep (see model).  This region is called the ventral lateral periaqueductal gray (vlPAG) area, and it was initially identified by Jouvet’s group as playing an important role in REM sleep generation (Petitjean et al., Brain Res. 88, 439–453; Sastre et al., Neuroscience Vol. 74, No. 2, pp. 415–426, 1996).  Pierre Luppi’s group has expanded on Jouvet’s research (Luppi et al., J Physiol Paris 100: 271-283, 2006). 


There are GABA neurons in the vlPAG and to facilitate research we are using mice that express green fluorescent protein in GABA neurons (see Oliva et al., J Neuroscience, 20:3354, 2000) (see photo).  These neurons contain the orexin-2 receptor (see photo).  Satvinder Kaur is lesioning the GABA vlPAG neurons and has found significant increase (+179%) in REM sleep in orexin knock-out mice (research in progress).

Abbreviations: DA=Dopamine; HCRT=hypocretin, also known as orexin; LC=locus coeruleus; mnPOA=median preoptic area; NE=norepinephrine; VLPOA=ventral lateral preoptic area; vlPAG=ventral lateral periaqueductal gray area; 5-HT=serotonin; Red=inhibition; Green=excitation

Hypothesized model underlying REM sleep regulation.  This model was publicly presented at the SLEEP’05 meeting by Dr. Shiromani.  Hypothesized group of GABA neurons in the pons that receive input from the hypothalamic sleep and wake active neurons and gate REM sleep. Wake-active neurons activate the GABA neurons which then inhibit REM sleep.  Decrease in the strength of the wake-active signal to the GABA neurons would hasten REM sleep as in narcolepsy or in depression.  Once the activity of the wake active neurons subsides the activity of the sleep active neurons begins to inhibit the pontine GABA neurons.  REM sleep ensues when the REM sleep on neurons become disinhibited.

Electrophysiology (Stephen Thankachan)

As part of our strategy to identify the network underlying sleep, we monitor the activity of neurons in areas implicated in sleep.  In the past we have used c-FOS as a marker of neuronal activity, but it is at best an indirect indicator of the behavior of the neuron.  To provide real-time assessment of the behavior of neurons it is best to use electrophysiology.  Stephen Thankachan monitored the activity of neurons in the vlPAG in orexin knockout mice (Thankachan et al., 2009).  These mice are an animal model of narcolepsy and Stephen is the first to record the activity of single neurons in this model (see photo).  He discovered that the majority of neurons in the vlPAG are active during wake which supports ours and Luppi’s hypothesis that activity of these neurons during wake inhibits REM sleep.  However, he also found that 10% of neurons are selectively active only during REM sleep indicating that REM sleep generator neurons are also present in the vlPAG.

Action potential of wake active neurons in the pons of orexin knockout mice. The figure represents the summation of seven action potentials of the same neuron.

Sampling of Publications

1.    Shiromani P, Armstrong D, Berkowitz A, Jeste D, and Gillin JC.  Distribution of choline acetyltransferase immunoreactive somata in the feline brainstem: Implications for REM sleep generation.  Sleep 11:1-16,1988.
2.    Siegel J, Nienhuis R, Fahringer H, Paul R, Shiromani P, Dement WC, Mignot E, and Chiu C.  Neuronal activity in narcolepsy: Identification of cataplexy related cells in the medial medulla.  Science, 252:1315-1318, 1991.
3.    Shiromani, PJ, Kilduff, TC, Bloom, FE and McCarley, RW.  Cholinergically-induced REM sleep triggers Fos-like immunoreactivity in dorsolateral pontine regions associated with REM sleep.  Brain Research,580:351-357, 1992.
4.    Shiromani, PJ, Malik, M, Winston, S, and McCarley, RW.  Time course of Fos-like immunoreactivity associated with cholinergically-induced REM sleep.  J Neuroscience, 15:3500-3508, 1995.
5.    Sherin, JE, Shiromani, PJ, McCarley, RW, and Saper, CB.  Activation of ventrolateral preoptic neurons during sleep.  Science, 271:216-219,1996.
6.    Shiromani PJ, Basheer R, Thakkar J, Wagner D, Greco MA, Charness ME. Sleep and wakefulness in c-fos and fos B gene knockout mice. Molecular Brain Res, 80: 75-87, 2000.
7.    Gerashchenko D, Kohls MD, Greco MA, Waleh NS, Salin-Pascual R, Kilduff  TS, Lappi DS, Shiromani PJ  Hypocretin2-saporin induced lesion of the lateral hypothalamus produces narcoleptic-like sleep behavior in the rat, J Neuroscience, 51:7273-7283, 2001.
8.    Shiromani, PJ, Xu, M, Winston, EM, Shiromani, SN, Gerashchenko, D and Weaver, DR.  Sleep rhythmicity and homeostasis in mice with targeted disruption of mPeriod genes  Am J Physiol Regul Integr Comp Physiol (March 18, 2004).  0.1152/ajpregu.00138.2004 (http://ajpregu.physiology.org/papbyrecent.shtml). 287:R47-R57, 2004
9.    Blanco-Centurion, C, Gerashchenko, D, Salin-Pascual, R, and Shiromani, PJ.  Effects of hypocretin2-saporin and anti-dopamine--hydroxylase-saporin neurotoxic lesions of the dorsolateral pons on sleep and muscle tone.  European Journal of Neuroscience, 19:2741-2752, 2004 
10.    Desarnaud, F, Murillo-Rodriguez, E, Lin, L, Xu, M, Gerashchenko, D, Shiromani, SN, Nishino, S, Mignot, E, and Shiromani, PJ.  The diurnal rhythm of hypocretin in young and old F344 rats, Sleep, 27: 851-856, 2004.
11.    Arias, O, Murillo-Rodriguez, E, Xu, M, Blanco-Centurion, C, Drucker-Colin, R.R, and Shiromani, PJ. Transplant of hypocretin neurons into the pontine reticular formation: A preliminary study.  Sleep, 27: 1465-1470, 2004.
12.    Blanco-Centurion, C, Xu, M, Murillo-Rodriguez, E, Gerashchenko, D, Shiromani, AM, Salin-Pascual, R, Hof, PR, and Shiromani, PJ.  Adenosine and sleep homeostasis in the basal forebrain.  J Neuroscience 26 (31):8092-8100, 2006.
13.    Zhang, S, Lin, L, Kaur, S, Thankachan, S, Blanco-Centurion, C, Yanagisawa, M, Mignot, E, Shiromani, PJ.  The development of hypocretin deficiency in ataxin-hypocretin transgenic rats, Neuroscience, 148(1):34-43, 2007.
14.    Blanco-Centurion, C, Gerashchenko, D, and Shiromani, PJ. Effects of saporin-induced lesions of three arousal populations on daily levels of sleep and wake, J Neuroscience, Dec 19; 27(51):14041-8, 2007.
15.    Geraschenko, D, Wisor, JP, Burns, D, Reh, RK, Shiromani, PJ, Sakurai, T, de la Iglesia HO, Kilduff, TS.  Identification of a population of sleep-active cerebral cortex neurons.  Proceedings National Academy of Sciences (USA) 105 (29):10227-10232, 2008.
16.    Liu, M, Thankachan, S. Kaur, S, Begum, S, Blanco-Centurion, C, Yanagisawa, M, Sakurai, T, Neve, R and Shiromani, PJ.  Hypocretin gene transfer improves narcoleptic symptoms in mice.  European Journal of Neuroscience, 28: 1382-1393, 2008.
17.    Thankachan, S, Kaur, S, Shiromani, PJ.  Activity of pontine neurons in orexin knockout mice.  J Neuroscience, Feb 11, 2009.
18.    Shiromani, PJ, Keane, T and Le Doux, J (editors).  Post-traumatic Stress Disorder: Basic Science and Clinical Practice.  Humana/Springer Press, New York, 2009 (Library of Congress Control Number: 2008942054)

Meng Liu, PhD (trainee)
Satvinder Kaur, PhD (trainee)
Stephen Thankachan, PhD (trainee)