Neural Pathways Of Nucleus Accumbens Adenosine A_2A Receptor Neurons Implicating The Role In The Regulation Of Sleep-wakefulness

Sleep is as important as water, air, and food for life organism. Sleep can not only relieve fatigue and reserve physical strength, protec brain and stabilize motion, enhance immunity and recover body, promote growth and development, but also participates in the process of memory. Enough sleep is an assurance of getting the best memory and mental agility. So far, regulation mechanism of physiological sleep-wakefulness is unknown. Prevention and treatment of clinical insomnia is also a lack of effective countermeasures.The nucleus accumbens (Acb), as an important nucleus of ventral striatum, is observed to comprise core region around the anterior commissural nucleus and shell region around the core region. Acb contains a lot of adenosine A2A receptors (A2AR) and is involved in the regulation of reward, addiction, and pain etc. Acb A2AR has recently been reported to be closely related with the regulation of sleep-wakefulness and sleep disorders. As you know, behaviour function triggered by a receptor is the result of the receptor neurons and their relative neural pathways. However, the efferent connections of Acb A2AR neurons remain to be elucidated.Why do we analyze the neuroanatomical pathway of Acb A2AR in the regulation of sleep-wakefulness? Analysis of this neural pathway is not only in favor of revealing the regulation mechanism of Acb A2AR in the regulation of sleep-wakefulness and perfecting and supplementing the theory and morphological basis of physiological sleep, but also provides neuroanatomical basis for exploring pathogeny nosogenesis and intervention study of clinical insomnia. The current data about projections of Acb neurons come from anatomical observation of traditional tracking technology (such as HRP retrograde tracing methos, BDA anterograde tracing methos, and fluorescein tracing method). Unfortunately, the traditional tracking technology cannot specifiaclly analyze the neural pathway of specific function. Thus, the neural pathways through that Acb A2ARs are to play the physilogical and pathophysiological functions are not yet clear.It is well known there are two major regulation systems of sleep and wakefulness in the brain, which are composed of numerous nuclei and transmitters (neuromodulation). Arousal system includes reticular formation of brainstem, noradrenergic neuron of locus coeruleus, serotonergic neuron of dorsal raphe nucleus, cholinergic neuron in the laterodorsal tegmental nucleus, pedunculopontine tegmental nucleus, and basal forebrain, histaminergic neurons in the tuberomammillary nucleus, orexin neuron in the lateral hypothalamus area. Sleep promoting system includes GABAergic neuron in the ventrolateral preoptic area, basal ganglion, cerebral cortex, limbic system, basal forebrain, preoptic area, brainstem, and thalamus. Hence, the brain regions through that Acb A2ARs play their regulatory role remains to be defined.Orexin neurons are located in the hypothalamus and send dense projections to the sleep-arousal related regions. Researches have found that the central orexin system plays a vital role in the regulation of arousal. Orexin gene knock-out or spontaneous mutation of orexin receptors gene will lead to mouse cataplexy or diseased rapid-eye-movement sleep, etc. narcolepsy like phenotype. Orexin neuros are not only as one of the strctures of arousal excited system to participate in promoting arousal, but also by means of the unified control of other sleep-arousal system to regulate the sleep-wakefulness. Reciprocal interactions between the Acb and lateral hypothalamus are hyothesized to regulate feeding, locomotion, and antinociception. Then, are there the reciprocal connections between the Acb and lateral hypothalamus? Evidences were previously presented to suggest that glutamate receptors in the Acb control feeding behavior via the hypothalamuse and orexin terminals could utilize glutamate in addition to orexin as a neurotransmitter in the ventral tegmental area and locus coeruleus. Whether can orexin terminals contain glutamate neurotransmitter in the Acb? All of these are the scientific problem for this study to solve.In regard to these problems, we focused on efferent projections of Acb A2AR neurons and the reciprocal fiber connections between Acb A2AR neurons and orexin neurons, exploring neural pathway of Acb A2AR in the regulation of sleep-wakefulness. The work had been done as follows:Part one Efferent projections of Acb A2AR neuronsPurpose:We applied an adeno-associated virus (AAV) that encodes a humanized Renilla green fluorescent protein (hrGFP) combined with A2AR Cre-mouse to selectively trace efferent projections of Acb A2AR neurons.Methods:The experiments were divided into the A2AR Cre-mouse group and wild type mouse group, and the virus was injected into the core (AcbC) and shell (AcbSh) of the nucleus accumbens of mice based on the coordinates (AcbC, AP: +1.1 mm; ML:1.0 mm; DV:3.8 mm; mAcbSh, AP:+0.98 mm; ML:0.5 mm; DV:4.0 mm; vAcb, AP:+1.10 mm; ML:1.0 mm; DV:5.0 mm) in a stereotaxic apparatus. After 4 weeks, the whole mouse brains were cut into 30 μm sections, and immunohistochemistry was then used to visualize hrGFP, highlighting the A2AR neurons in the injection sites, and their axons in projection regions. To determine the neurochemical property of Acb A2AR neurons, double-immunofluorescence stainings for hrGFP and GABA (gamma aminobutyric acid marker), hrGFP and DARPP32 (GABAergic projection neuron marker), and hrGFP and parvalbumin (GABAergic interneuron marker) were performed respectively.Results:The data showed that robust hrGFP was expressed in the Acb of the Cre-mouse group, but not in the wild type mouse. The hrGFP-immunoreactivity (hrGFP-IR) indicates that A2AR neurons can be found throughout the Acb and project their processes in various directions. The soma of hrGFP-IR neurons, which were measured 10-15μm in diameter, was round or oval. The processes, dendrites and axons, established a nerve fiber network around the perikaryon. Double immunofluorescences showed the majority hrGFP was co-localized with GABA, and the further results were hrGFP co-localization with DARPP32, but not with parvalbumin. These data indicated Acb A2AR neurons were GABAergic projection neuron, but not GABAergic interneuron. In addition, the serial section hrGFP-IR of whole brain with immunofluorescence or DAB staining allowed us to identify the projection regions of Acb A2AR neurons. The projections of from the Acb A2AR neuron distributed to nuclei in the forebrain, diencephalon, and brainstem. In the forebrain, A2AR neurons from all Acb sub-regions jointly projected to the ventral pallidum, the nucleus of the diagonal band, and the substantia innominata. Heavy projections from the AcbC and the ventral AcbSh, and weaker projections from the medial AcbSh, were observed in the lateral hypothalamus and lateral preoptic area. In the brainstem, Acb projections were found in the ventral tegmental area, while AcbC and ventral AcbSh also projected to the medial raphe nucleus, the dorsal raphe nucleus, and the ventrolateral periaqueductal gray.Conclusions:Acb A2AR neurons project to the aforementioned brain fields or nuclei, can form the inhibitory synapses, and play the corresponding role. The results supply a morphological base for understanding the roles of the A2AR and A2AR neurons in the Acb, especially in the regulatory mechanism of sleep.Part two Neural circuit between nucleus accumbens A2AR neurons and orexin neuronsPurpose:We aim to reveal neural circuit between neucleus accumbens A2AR neurons and orexin neurons.Methods:An adeno-associated virus (AAV) encoding humanized Renilla green fluorescent protein (hrGFP) was injected into Acb (AP:+1.1 mm; ML:1.0 mm; DV:3.8 mm) of A2AR Cre mouse. Immunohistochemistry was then used to visualize hrGFP, highlighting the A2AR neurons in the injection sites, and axons in the tuber part of lateral hypothalamus (LHt). Enzyme immunocytochemical double staining and double immunofluorescence staining were used to detect whether fiber termainals/axons of Acb A2AR neuron dominated LHt orexin neuron and fiber terminals/axons of orexin neurons dominated Acb A2AR neurons. Neurochemical property of neurotransmitter from orexin terminals or axons was detected by laser confocal microscope. Finally, immune-electron microscope was employed to observe synaptic connections of orexin terminals/axons within Acb.Results:Robust hrGFP positive neurons were observed in the Acb and sent fiber terminals or axons to the LHt. Fibers and/or axons of A2AR neurons appeared to dominate somas or proximal dendrites of orexin neurons. At the same time, Orexin terminals or axons appeared to contact somas and dendrites of GABAergic projection neurons and A2AR neurons in the Acb. In addition, Orexin terminals or axons contained the vesicular glutamate transporter (VGLuT2) and synaptophysin, but not the vesicular transporter for GABA (VGAT) in the Acb. Finally, Orexin terminals or axons formed excitatory synapse in the Acb by immuno-electron microscope.Conclusions:There are the reciprocal fiber connections between the Acb A2AR nrueons and orexin neurons. We suggest this reciprocal fiber connections and possible release of glutamate neurotransmitter from orexin terminals within the Acb can thus be important for Acb A2AR and orexin neurons in the commissural regulation of sleep-wakefulness, etc. functions.In conclusion, A2AR neurons in the AcbC, mAcbSh, and vAcbSh sent projections to various arousal related brain fields through which Acb sub-regions form various pathways to regulate sleep-wakefulness. There exist the reciprocal fiber connections between Acb A2AR neurons and orexin neurons in the morphology, and orexin terminals could have the capacity to release glutamate in adition to orexin in the Acb. The results provide a morphological base for understanding the roles of the A2AR and A2AR neurons in the Acb, especially in the regulation of sleep.