Genetic Dissection Of Neural Circuitry

Understanding the principles organizing the complex neuron circuits in mammalian brain and thereby deciphering how they process biological information and guide various behaviors is the ultimate goal of neuroscience.Recent developed genetic analysis combined with pharmacogenetic and optogenetic approaches play a prominent role in dissecting neural circuitry.In this thesis,we bring experimental access to the complex neural systems from two aspects.In the first part,we use genetic approaches to establish a diagram to reveal that a heterogeneity exist in hypothalamic POMC(Pro-opiomelanocortin)neurons which is responsible for the control of body weight versus blood glucose,respectively.Specifically,our findings are:a)glucose and leptin active different subsets of POMC neurons,b)that this is due to differential expression of KATP channels(ATP-sensitive potassium channel)versus leptin receptors(LEPRs),c)that the "glucose sensing"versus "leptin-responding" subsets of POMC neurons control blood glucose versus body weight,respectively.Anatomical and functional studies using genetic manipulations(to produce loss or gain of function)in different POMC subpopulations(leptin-responsive versus glucose responsive)are required to fully investigate the significance of this heterogeneity.To this end,genetic reagents are generated to manipulate the gene expression in leptin-responsive versus glucose-responsive POMC neurons.Lepr-ires-cre mice are used to manipulate leptin-responsive POMC neurons.For the glucose-excited neurons,we generate Surl-ires-cre mice.Finally,to reveal the contribution to body weight versus blood glucose of individual group of POMC neurons with precise loss-of-function(LOF)and gain-of-function(GOF)experiments,we generate lox-STOP-lox-POMC-flp mice.Combined with flp-dependent adeno-associate virus(AAV),we are able to define the anatomy(cell bodies and projections),establish and assess function(based on sites of projections)of glucose/leptin-responding POMC neurons.For the second part,in order to dissect the individual neuron's property from molecular level,we demonstrate that microRNA,a post-transcriptional regulator,can be secreted from the synaptic terminals under physiological stimulation and functionally active after being taken up by the synaptic fraction via endocytic pathway.In this study,we first characterized synaptosome microRNA(miRNA)profiles using microarray and qRT-PCR.MicroRNAs were detected in isolated synaptic vesicles,and Ago2 immunoprecipitation studies revealed an association between miRNAs and Ago2.Second,we found that miR-29a,miR-99a,and miR-125a were significantly elevated in synaptosome supernatants after depolarization.MiRNA secretion by the synaptosome was Ca2+-dependent and was inhibited by the exocytosis inhibitor,okadaic acid.Furthermore,application of nerve growth factor increased miRNA secretion without altering the spontaneous release of miRNAs.Conversely,kainic acid decreased miRNA secretion and enhanced the spontaneous release of miRNAs.These results indicate that synaptosomes could secrete miRNAs.Finally,synthesized miRNAs were taken up by synaptosomes,and the endocytosis inhibitor Dynasore blocked this process.After incubation with miR-125a,additional miR-125a was bound to Ago2 in the synaptosome,and expression of the miR-125a target gene(PSD95 mRNA)was decreased;these findings suggest that the ingested miRNAs were assembled in the RNA-induced silencing complex,resulting in the degradation of target mRNAs.When can we say we have understood a neural circuitry?Our understanding of the circuitry mechanisms underlying behavior is relatively advanced in select simple system with identified neurons.First,all neurons in the circuitry can be unambiguously identified using genetic makers or electrophysiological parameters,including soma distribution,upstream inputs as well as downstream projections.Second,multiple intracellular and extracellular recordings can be used to construct a circuit diagram,which is critical to correlate firing patterns with output and to probe the effects of activating or silencing neurons on the network.Third,individual neurons can be selectively turned on/off to manipulate the GOF/LOF functions with genetic approaches.Finally,by exploring the individual neurons in molecular level,we can modify the circuitry element and predict related effect on neural functions and thus related animal behavior.In conclusion,by combing molecular,neuroanatomical,physiological,and genetic manipulations,we give a pilot study to reveal the logical of the neural circuitry in complex brains that guide behaviors.