Loss Of A Membrane Guanylyl Cyclase Expressed On Midbrain Dopamine Neurons Produce ADHD-like Behavior In Mice

Midbrain dopamine system is an important neuronal circuit system in the brain, regulating diverse of important behavioral processes. This system includes the dopamine neurons locating in ventral tegmental area (VTA)and substantia nigra compacta (SNc) and their projection areas dorsal striatum (caudate and putamen), nucleus accumbens (NAc), hippocampus, prefrontal cortex (PFC) and amygdala. The midbrain dopamine neurons release neurotransmmiter dopamine to regulate their postsynptic areas. And their dysfunctions are associated with many human neurobehavior disorders such as Parkinson Disease, Attention Deficit Hyperactivity Disorder (ADHD) and schizophrenia. Thus it is highly valuable to identify cellular targets that selectively regulate their activity.Here, we report for the first time that these neurons selectively express guanylyl cyclase-C (GC-C), a membrane receptor previously considered to be intestine-specific. This expression patterns are only observed in the soma and dendrites of dopamine neurons, but not the axon parts in striatum.In vitro, GC-C in midbrain dopamine neurons can be activated by GC-C' hormone ligands in intestin-G and UG. But this activation cannot directly change spontaneous activity of midbrain dopamine neurons, neither can impact on the excitatory synaptic currents, nor the inhibitory currents mediated by GABAa receptors. Unexpected, GC-C activation greatly potentiates the excitatory responses mediated by metabotropic glutamate receptors and acetylcholine receptors on midbrain dopamine neurons by 30% in vitro. This activity depends on the activity of cGMP-dependent protein kinase (PKG):while the PKG's activator can mimic the potentiating effects of G/UG, PKG's inhibitor can abolish this one. In addition, in GC-C knockout mice no potentiating effects can be observed with the presence of G/UG.We also test a serial of behavior assay in GC-C knockout mice, including open-field test, odorant habituation test, go/no go paradigm, and circadian rhythm test. GC-C knockout mice shows ADHD-like phenotype:exhibit hyperactivity, reduced odorant habituation, lacking of sustained attention and behavior inhibition. Moreover, their behavioral phenotypes are rescued by ADHD therapeutics. These results indicate important behavioral and physiological functions for the GC-C/PKG signaling pathway within the brain and suggest novel therapeutic targets for psychiatric disorders. the necklace olfactory system is formed by a group of specialized olfactory sensory neurons and their corresponding post-synaptic neurons, like mitral cells. These olfactory sensory neurons project their axons specifically to glomeruli in the caudal part of the olfactory bulbs. These glomeruli line up in a circular way at the caudal part of the olfactory bulbs, thus are called necklace glomeruli. The olfactory sensory neurons of the necklace olfactory system have their unique molecular markers, including GC-D, CAII,CNGA3 and PDE2A, while they don't express GPCRs or CNGA2 channels that commonly used in the classical olfactory sensory neurons, suggesting a different functional role of them.The previous studies of our lab revealed that these olfactory sensory neurons can be activated by low concentrations (0.1%) of CO₂, and the activation is depending on the activity of CAII and CNGA3. In vivo physiology study also show that in anesthetized mouse neurons that are sensitive to low concentrations of CO₂ are detected near the necklace glomeruli. Mice can learn to respond to 0.5% CO₂ correctly after two weeks training under go/no-go paradigm, shows that they can truly detect low concentrations of CO₂. Contrararily CAII mutant or CNGA3 knockout mice cannot detect low concentrations of CO₂. Recent biochemical study further confirmed that Bi-carbonate can dirrectly activate GC-D to produce intracellular massager cGMP and then evoke neuronal responses to CO₂.The fact that mouse can detect low concentrations of CO₂ via necklace system is quite different from the case in human. Primates can't detect low concentrations of CO₂ because GC-D is a pseudo-gene in primates. Then what's the function of the CO₂ detection by mouse necklace system? We plan to resolve this question by two genetic methods. First, we express the human DTR in necklace olfactory sensory neurons by knock-in DTR gene directly after the GC-D gene locus. Then we could specifically remove the necklace olfactory sensory neurons at any stage of the mouse lifespan by injection of DT and observe the behavioral effect of such manipulation. Second, we could also express the barley lectin (BL) in necklace olfactory sensory neurons by knock-in BL gene directly after the GC-D gene locus. BL can be anterogradely and trans-synaptically transported to the mitral cells that innervate the necklace glomeruli, and then we could observe the projection area of these mitral cells by staining against BL, thus provide anatomical clues for the function of the necklace system.