The distribution of neuropeptides and neurotransmitters within the rostral nucleus of the solitary tract of mice
Stratford, J.M, Parnes, J.A. and Finger, T.E.
Rocky Mountain Taste and Smell Center, Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA, [email protected]
NPY and Galanin are localized to the medial nTS and DMX
Background
 Feeding is a complex behavior that relies on the coordination of multiple
sensory systems
 Gustatory, trigeminal and post-oral signals control acceptance or rejection of food
 The nucleus of the solitary tract (nTS) is the first site for convergence of gustatory,
trigeminal and vagal (post-oral) afferents
 The nTS also receives modulatory inputs from various areas of the CNS that express
diverse peptidergic and neurotransmitter systems related to regulation of food intake:
neuropeptide Y (NPY) and galanin stimulate food intake in part via inhibition of
vagal activity
calcitonin gene-related peptide (CGRP) is found in trigeminal pain fibers
acetylcholine is involved in the coordination of motor reflexes, such as those
involved in swallowing
 The role of these neuropeptides in gustatory-dominated (rostral) regions of the
nTS is unknown
-
Goal
Examine the distribution of NPY, galanin, CGRP, and choline
acetyltransferase (the enzyme necessary for the synthesis of
acetylcholine) within the rostral nTS of mice
Methods
r1
galanin were similar, which is
consistent with their common
function in stimulating hunger
through inhibition of vagal
activity
r3
 ChAT was present in lateral and
r4
 CGRP was found not only in
r5
medial nTS, which receives input
from the trigeminal and vagus
nerves
4v
lateral
medial
ChAT expression is greatest in
vagal terminal fields and medial nTS
CGRP is prominent in the
lateral and central nTS
r1
Expression Mapping
 Protein expression quantified using a custom-made
MATLAB program
 Spatial distribution of expression was estimated by
thresholding the pixel intensity of fluorescent labels based
on 2X standard deviation of background level
Images modified from Paxinos
and Franklin (2001)
Nissl false colored B & W
r5
Thus, gustatory regions of
the nTS have diverse
neuropeptides that may
relate to feedback control of
food intake
 Correlate neuropeptide
distribution with activity marker
(e.g. c-Fos)
 Neuropeptide distribution in
feeding-related transgenic mice:
─ P2X2/3 dbl KO mice
(lack taste function)
─ Ob/Ob; db/db mice
(lack leptin or the leptin
receptor, respectively)
r2
r4
deep-red) to confirm nTS boundaries
 n. of the Solitary Tract (nTS) divided into five coronal levels
extending from rostral (r1) to caudal (r5)
putative trigeminal areas (lateral
nTS) but also in gustatoryresponsive central nTS
Future Directions
r3
 Cases counterstained with Nissl (NeuroTrace® 640/660
Composite:
 The distribution of NPY and
r2
Immunohistochemistry
 C57BL/J6 mice (n = 1 - 3 for each stain) or mice in which expression of green fluorescent protein (GFP)
is driven by the choline acetyltransferase promoter (ChAT-tauGFP; n = 3)
 Perfused transcardially with 4% paraformaldehyde, brain removed, post- fixed for 3 h, coronally
sectioned (40 µm) and processed for neuropeptides (see below) using standard florescent
immunohistochemical procedures
 Expression of all neuropeptides was visualized using primary antibodies (see table below), except for
ChAT GFP, which was expressed endogenously
 Sections co-stained for the purinergic P2X2 receptor for reference between stains
Summary
Acknowledgements
 ChAT-tauGFP mice were provided by
4v
Dorsal
Medial
Each row represents one level of the nTS (r1 – r5). The left most column for each stain set (P2X2/NPY, P2X2/Galanin, P2X2/ChAT or P2X2/CGRP) contains photomicrographs of
immunofluorescence staining. The other two columns for each stain set represent the spatial distribution of pixels that are 2X standard deviation of background. Each heat map is color
coded (blue = minimal; red = maximal) with red denoting the maximum number of thresholded pixels for each stain set. DMX= dorsal medial nucleus of the vagus nerve; 4V= 4th ventricle
Sukamar Vijayaraghavan (U C
Denver AMC)
 Special thanks to John Thompson (U
C Denver; john.a.thompson
@ucdenver.edu) for creation of the
heat map imaging MATLAB program
 Funded by NIH Grants D007495,
DC000147-29A1, and P30 DC04657