Microtransplantation of Postmortem Native Synaptic mGluRs Receptors into Xenopus Oocytes for Their Functional Analysis

Abstract

Metabotropic glutamate receptors (mGluRs) are membrane receptors that play a central role in the modulation of synaptic transmission and neuronal excitability and whose dysregulation is implicated in diverse neurological disorders. Most current understanding about the electrophysiological properties of such receptors has been determined using recombinant proteins. However, recombinant receptors do not necessarily recapitulate the properties of native receptors due to the lack of obligated accessory proteins, some of which are differentially expressed as function of developmental stage and brain region. To overcome this limitation, we sought to microtransplant entire synaptosome membranes from frozen rat cortex into Xenopus oocytes, and directly analyze the responses elicited by native mGluRs. We recorded ion currents elicited by 1 mM glutamate using two electrodes voltage clamp. Glutamate produced a fast ionotropic response (6 ± 0.3 nA) in all microtransplanted oocytes (n = 218 oocytes) and a delayed oscillatory response (52 ± 7 nA) in 73% of them. The participation of Group 1 mGluRs was confirmed by the presence of metabotropic oscillations during the administration of (±)-1-Aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD; Group 1 mGluR agonist), and the absence of oscillations during co-administration of N-(1-adamantyl)quinoxaline-2-carboxamide (NPS 2390; Group 1 mGluR antagonist). Since both mGluR1 and mGluR5 belong to Group 1 mGluRs, further investigation revealed that mGluR1 antagonism with LY 456236 has little effect on metabotropic oscillations, while mGluR5 antagonism with 100 µM AZD 9272 has significant reduction of metabotropic currents elicited by ACPD and glutamate. We confirmed the expression of mGluR1 and mGluR5 in native synaptosomes by immunoblots, both of which are enhanced when compared to their counterpart proteins in rat cortex tissue lysates. Finally, these results demonstrate the merit of using microtransplantation of native synaptosomes for the study of mGluRs and the contribution of mGluR5 to the metabotropic glutamate signaling, providing a better tool for the understanding of the role of these receptors in neurological disorders.

Keywords: G-protein coupled receptors; mGluR; microtransplantation of synaptic membranes; phospholipase C; post-mortem.

Figure 1

The effects of 1 mM glutamate (Glu) on oocytes microtransplanted with rat cortex synaptosome membranes. Representative two electrodes voltage clamp responses to 1 mM Glu on a non-injected (A) and a microtransplanted oocyte (B). Recordings were done 24–28 h after injection. A subset of microtransplanted oocytes showed an immediate fast-activating inward current followed by a delayed oscillatory current. Arrows indicate the beginning of the response. Oscillatory currents appeared after a delay within a range of 3–38 s. (C) Amplitude of fast and oscillatory Glu responses in microtransplanted oocytes showing both responses (52.2 ± 7.3 nA; n = 159 oocytes from 17 frogs; Mean ± SEM). Paired t-test.

Figure 2

Oscillatory responses are mediated by Group 1 mGluRs. (A) Representative ion currents elicited by 100 μM ACPD and 1 mM Glu in the same microtransplanted oocyte. (B) Concentration response curve for ACPD normalized by the maximum amplitude of the response elicited by 1 mM Glu in each oocyte (each point represents the mean ± S.E.M from 4 to 13 oocytes tested for each concentration).

Figure 3

Antagonism of Group 1 receptors inhibits oscillatory glutamate responses. (A) Glutamate currents from the same microtransplanted oocyte; each application was separated by a 4 min washing period of Ringer’s solution. A complete loss of oscillations is shown during the application of 10 µM NPS 2390, a mGluR group 1 antagonist (n = 6 oocytes from 1 frog). (B) ACPD currents of a microtransplanted oocyte with each application type separated by a wash period of 4 min of Ringer’s solution. (C,D) 10 µM of NPS 2390 co-applied with glutamate or ACPD resulted in a complete loss of oscillations (n = 6 and 5 oocytes from 2 frogs, for C and D, respectively).

Figure 4

Antagonism of mGluR1 has no effect on metabotropic oscillations. (A) Glutamate currents of a microtransplanted oocyte in response to application of the mGluR1 antagonist, LY 456236, with each application separated by 4 min of wash by Ringer’s solution. No significant effect on metabotropic responses was shown (C) when LY 456236 was co-applied with glutamate (n = 13 oocytes from 4 frogs). (B) ACPD currents of a single microtransplanted oocyte in response to application of the mGluR1 antagonist, LY 456236, with each application separated by 4 min of wash by Ringer’s solution. There was no significant change in amplitudes (D), although a slight increase in current was observed in some oocytes after a wash period (n = 6 oocytes from 2 frogs).

Figure 5

Inhibition of mGluR5 affects presence of metabotropic glutamate oscillations. (A) Glutamate currents of a single microtransplanted oocyte in response to application of the mGluR5 antagonist, AZD 9272, with each method of application separated by 4 min of wash by Ringer’s solution. Antagonism of mGluR5 alongside glutamate agonism displayed a significant reduction of responses (C), a trend which would prolong into the washing period (n = 9 oocytes from 2 frogs). (B) ACPD currents of a single microtransplanted oocyte in response to application of the mGluR1 antagonist, AZD 9272, with each method of application separated by 4 min of wash by Ringer’s solution. Blocking of mGluR5 with ACPD (D) showed a significant reduction in response that persisted into the post-antagonist application (n = 9 oocytes from 2 frogs).

Figure 6

Enrichment of mGluR5 in cortical synaptosomes. Representative immunoblots for mGluR1α (A) and mGluR5 (B) of lysates from rat cortex synaptosomes (“S”), cortex (“C”) and cerebellum (“Cb”) probed with anti-mouse mGluR1α and anti-rabbit mGluR5 antibodies. Bands for mGluR1α and mGluR5 ran at ∼150 kDa. β3-tubulin (∼50 kDa) was used as loading control. L, molecular mass marker. Electrophysiological recordings were made (C) 24–28 h after injection using synaptic membranes isolated from rat cerebellum with amplitudes (D) of oscillatory Glu responses in microtransplanted oocytes showing responses (35.74 ± 8.38 nA; n = 9 oocytes from 1 frog; Mean + SEM). Paired t test.