[Abstract Title]. - Society for Neuroscience

Authors: *P. RAMAMOORTHY, M. D. WHIM;
Dept Biol, Pennsylvania State Univ., State College, PA
Abstract: Neuropeptide Y is a widely distributed neuropeptide that is thought to modulate a
variety of physiological processes including blood pressure, feeding and the stress response. We
have previously found that cortical astrocytes can synthesize neuropeptide Y (NPY) and that the
newly synthesized peptide enters the regulated secretory pathway. However the pathway(s) that
control the secretion of neuropeptide Y are not clear. To address this question, cortical astrocytes
were isolated and maintained in vitro. At 8-12 DIV astrocytes were stimulated with glutamate for
1 min, then immediately fixed and stained for NPY-immunoreactivity (NPY-ir) without
permeablization. After stimulation many cells displayed surface NPY-ir puncta. Control
experiments indicated that the NPY-ir puncta only appeared after stimulation and were not due to
intracellular staining. Thus the NPY-ir puncta arose from the fusion of NPY-containing granules
with the cell membrane and subsequent display of the peptidergic core. Varying the time
between stimulation and fixation indicated that the NPY-ir puncta disappeared with a time
constant of ~ 1.6 mins. To clarify whether the disappearance of the puncta reflected NPY
secretion (rather than internalization) we monitored secretion from astrocytes expressing NPYred
fluorescent protein (NPY-RFP). In these cells following glutamate stimulation, the intensity
of individual NPY-RFP puncta declined in a step-like manner indicating a regulated secretion of
the granular contents. The glutamatergic-dependent appearance of the NPY-ir puncta was not
prevented by the presence of CPP and NBQX, antagonists of ionotropic glutamate receptors.
However t-ACPD (100 µM), an agonist of group I/II metabotropic glutamate receptors was as
effective as glutamate in causing secretion. (S)-3,5-DHPG, an agonist of group I mGluR‟s also
triggered the surface appearance of NPY-ir puncta, while (2R,4R)-APDC, an agonist of group II
mGluR‟s was ineffective. Group I mGluR‟s are coupled to calcium signaling and experiments
using fura-2 indicated that glutamate caused a transient rise in intracellular calcium. Collectively
these results suggest that activation of group I mGlu receptors leads to the elevation of
intracellular calcium which subsequently triggers the fusion of peptidergic granules and release
of NPY from cortical astrocytes. Since NPY is a widespread and potent regulator of synaptic
transmission, these results suggest that astrocytes could play a role in the peptidergic modulation
of synaptic signaling in the CNS.
Disclosures: P. Ramamoorthy , None; M.D. Whim, None.
Poster
241. Glial Neuronal Interactions
Time: Sunday, November 16, 2008, 1:00 pm - 5:00 pm
Program#/Poster#: 241.13/G7
Topic: B.11.b. Cell biology and signalling