Sa2 Case Study

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SA2 Expected outcomes and potential difficulties.
The results of the experiments proposed in SA2 are expected to determine if POMC and AgRP neurons form synapses with MC3R VTA neurons, determine the identity of MC3R-expressing VTA neurons, and to map afferent and efferent projections of VTA MC3R neurons. GABA neurons are the second most numerous neuronal population in the VTA. Therefore, it is expected that a large proportion of TH negative MC3R-expressing VTA neurons will colocalize with GAD67. Observations to the contrary would suggest glutamatergic identity of TH negative MC3R VTA neurons. If this is the case, colocalization of MC3R and Vglut2 will be analyzed in mice expressing tdTomato under the control of MC3R promoter and GFP under
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If retrograde labeling with SAD D G-GFP(EnvA) does not reveal any AgRP or POMC neurons co-expressing GFP, the possibility of non-synaptic release of α-MSH and AgRP in the VTA will be examined. Both AgRP and α-MSH are present in neural fibers116,117, permitting IHC labeling of these neuronal fibers.VTA sections of MC3R-eYFP mice will be subject to IHC using primary antibodies targeted against AgRP or alpa-MSH. Confocal images of VTA sections will be examined to assess the anatomical association of AGRP or α-MSH containing fibers and MC3R-expressing neurons.
Although CTB is a well established retrograde tracer 11,118-120, it has also been used for anterograde labeling121. However, we do not expect anterograte transport to interfere with the ability to identify retrogradively labeled neurons because the signal produced by anterograde labeling is weak and requires IHC amplification121,122. CTB uptake by fibers of passage or damaged axons has been reported to be minimal11,120 thus, non-specific labeling is not
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Optogenetic stimulation of LH neurons expressing vesicular GABA transporter (LHVGat ; putative GABA neurons) induces feeding and optical self-stimulation, whereas selective ablation of these neurons reduces feeding, body weight gain, and motivation to obtain palatable reward124. VTA MC3R-GABA may decrease feeding and/or food reward by direct inhibition of LHVgat neurons. Additionally, VTA MC3R expressing neurons may affect feeding and food reward by indirect action on LH GABA neurons. Activation of AcbSh D1R neurons projecting to LH GABA neurons results in feeding bout termination101. VTA MC3R-DA neurons may decrease feeding and food reward by indirect inhibition of LHVGat neurons via activation of AcbSh D1R

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