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‘Systems-level’ sleep research – Genetic dissection of neural circuitry regulating behavioral state using conditional transgenics

Pharmacological approaches and transgenic animals with constitutive gene disruptions of genes were very valuable to clarify the overall picture of the molecular mechanisms of sleep-wake regulation. However, due to rapid progress in molecular biology, new techniques with higher spatial resolution are forthcoming and become available for sleep research. These techniques include a wide range of approaches from conditional deletion of genes based on the Cre/loxP technology to RNA interference to the in vivo reversible silencing (e.g., non-mammalian Cl channels) and activation (e.g., stimulatory, synthetic GPCRs) of neurons in freely-behaving animals

We have embarked on a program to identify the site of synthesis and action of somnogenic PGD₂ and generated mouse lines with the L-PGDS and DP1R genes amenable to conditional deletion using Crerecombinase. The functional role of the LPGDS/PGD₂/DP1R system in the leptomeninges can be critically tested by microinfusion of adeno-associated viral vectors (AAV) containing Crerecombinase under the cytomegalovirus (CMV) promoter into the subarachnoid space under the basal forebrain, as well as by microinjection of AAV-Cre directly into the CSF through the lateral ventricle of newborn mice. The conditional knockout mice for the L-PGDS/PGD₂/DP1R system are useful to study the spatial mechanisms governing the link between the humoral and neural network of sleep in the leptomeninges, choroid plexus, and oligodentrocytes and to identify the trigger site of PGD₂ for the induction of sleep.

We are also establishing a novel technology for systems somnology, called focal RNA interference (fRNAi), to silence the expression of sleep-active genes in rats by using AAV-carrying short hairpin (shRNA) elements (Fig. 1). We found that shRNA specific for various ‘sleep genes’ efficiently suppresses the expression of these genes in AAV-transduced neurons. Stereotaxic microinjections of AAV cause minimal tissue injury and produce widespread transduction of neurons. We are currently studying the effect of focal gene manipulation of adenosine A₂ᴀ (A₂ᴀR) and dopamine D₂ (D₂R) receptors in the basal ganglia, of vesicular ATP transporter (VNUT), adenosine deaminase, histidine decarboxylase in the tuberomammillary nucleus, and of vesicular transporters for GABA and glutamate in the midbrain, thalamus and frontal cortex on sleep-wake regulation. The focal knockout approach used in our studies has wide applicability for neuroanatomical dissection of the effects of different receptors with complex brain distributions (Lazarus, M. et al. Nat Neurosci10, 1131-1133, 2007).

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Fig. 1. AAV-mediated focal RNA interference of A2ARs in the rat striatum.

For instance, we are currently using fRNAi to identify neurons on which caffeine, the world’s most widely used psychoactive drug, acts to produce wakefulness. Our recent findings indicate that the loss of A₂ᴀRs specifically in the nucleus accumbens (NAc) results in abrogation of the arousal effect of caffeine. These results demonstrated for the first time that caffeine-induced wakefulness activates pathways that have traditionally been associated with locomotion and motivational behaviors (Fig. 2).

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Fig. 2. Neural circuitry regulating Caffeine-induced arousal

In a joint project of ‘translational research’ with the Corporation for Production and Research of Laboratory Primates, we are currently developing non-human primate (NHP) models by focal brain injection of AAVs carrying RNA interference elements as a model for the treatment of Parkinson‘s disease in humans. Knowing how to find the location of gene products linked with neural disorder may help with the development of highly specific gene therapy treatment. In contrast to many side effects of systemic drug treatment of neurological disease, blocking genes only in restricted areas of the brain open the possibility to keep the symptoms that help and eliminate those that do not. The use of AAVs carrying RNA interference may provide a focal gene therapy alternative to chronic systemic drug treatment in patients with severe neurological disorders.

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