Abstract
Through the study of recombinant synaptic proteins much has been discovered about
their structural, biophysical and pharmacological properties; however, there is less knowledge on
the properties of the native synaptic protein complexes. Because recombinant expression of
synaptic receptors is the interpretation of the cell wherein they are transcribed or translated, they
may differ from receptors in native membranes. In this study, we aimed to determine whether
microtransplantation of synaptic membranes (MSM) allows the study of native glutamate
metabotropic receptors (mGluRs). We microinjected synaptic membranes isolated from frozen
rat cortex samples into Xenopus oocytes and recorded ion currents elicited by 1 mM glutamate
using Two Electrode Voltage Clamp. These trials resulted in a fast ionotropic response of 5.61 ±
0.38 nA (n = 177 oocytes) followed by a delayed oscillatory metabotropic response of 48.59 ±
6.79 nA (n = 177 oocytes). We confirmed the presence of Group 1 mGluRs after observing
metabotropic oscillations during the administration of 100 μM of (±)-1-Aminocyclopentane-
trans-1,3-dicarboxylic acid (ACPD), a Group 1 specific mGluR agonist, and the loss of
glutamate metabotropic currents by 10 μM NPS 2390, a Group 1 specific mGluR antagonist.
Further investigation revealed that mGluR1 antagonism (LY 456236) showed little effect on
metabotropic oscillations when compared to either prior or following application of glutamate or
ACPD. However, antagonism of mGluR5 with 100 μM AZD 9272 showed a reduction of
metabotropic currents elicited by ACPD and glutamate. Finally, we confirmed expression of both
mGluR1 and mGluR5 in native synaptosomes by immunoblots, where we found protein
enhancement in the isolated synaptosomes when compared to rat cortex whole tissue lysate.
These results demonstrate the merit of native synaptosomes and their microtransplantation for
the study of mGluR5’s contribution to metabotropic glutamate signaling, which has been linked
with several neurological disorders such as Alzheimer’s disease, schizophrenia, and addiction.
their structural, biophysical and pharmacological properties; however, there is less knowledge on
the properties of the native synaptic protein complexes. Because recombinant expression of
synaptic receptors is the interpretation of the cell wherein they are transcribed or translated, they
may differ from receptors in native membranes. In this study, we aimed to determine whether
microtransplantation of synaptic membranes (MSM) allows the study of native glutamate
metabotropic receptors (mGluRs). We microinjected synaptic membranes isolated from frozen
rat cortex samples into Xenopus oocytes and recorded ion currents elicited by 1 mM glutamate
using Two Electrode Voltage Clamp. These trials resulted in a fast ionotropic response of 5.61 ±
0.38 nA (n = 177 oocytes) followed by a delayed oscillatory metabotropic response of 48.59 ±
6.79 nA (n = 177 oocytes). We confirmed the presence of Group 1 mGluRs after observing
metabotropic oscillations during the administration of 100 μM of (±)-1-Aminocyclopentane-
trans-1,3-dicarboxylic acid (ACPD), a Group 1 specific mGluR agonist, and the loss of
glutamate metabotropic currents by 10 μM NPS 2390, a Group 1 specific mGluR antagonist.
Further investigation revealed that mGluR1 antagonism (LY 456236) showed little effect on
metabotropic oscillations when compared to either prior or following application of glutamate or
ACPD. However, antagonism of mGluR5 with 100 μM AZD 9272 showed a reduction of
metabotropic currents elicited by ACPD and glutamate. Finally, we confirmed expression of both
mGluR1 and mGluR5 in native synaptosomes by immunoblots, where we found protein
enhancement in the isolated synaptosomes when compared to rat cortex whole tissue lysate.
These results demonstrate the merit of native synaptosomes and their microtransplantation for
the study of mGluR5’s contribution to metabotropic glutamate signaling, which has been linked
with several neurological disorders such as Alzheimer’s disease, schizophrenia, and addiction.
| Original language | English (US) |
|---|---|
| State | Published - Dec 22 2022 |
| Event | Society for Neuroscience: Neuroscience 2022 - San Diego Convention Center , San Diego, United States Duration: Nov 12 2022 → Nov 16 2022 https://www.sfn.org/meetings/neuroscience-2022 |
Conference
| Conference | Society for Neuroscience |
|---|---|
| Country/Territory | United States |
| City | San Diego |
| Period | 11/12/22 → 11/16/22 |
| Internet address |