Postweaning Development Influences Endogenous VPAC1 Modulation of LTP Induced by Theta-Burst Stimulation: A Link to Maturation of the Hippocampal GABAergic System

Abstract

Long-term potentiation (LTP) induced by theta-burst stimulation (TBS) undergoes postweaning developmental changes partially linked to GABAergic circuit maturation. Endogenous vasoactive intestinal peptide (VIP) acting on its VPAC₁ receptor strongly influences LTP induced by theta-burst stimulation (TBS), an effect dependent on GABAergic transmission. Although VPAC₁ receptor levels are developmentally regulated during embryogenesis, their variation along postweaning development is unknown, as is the VPAC₁ modulation of LTP or its relation to hippocampal GABAergic circuit maturation. As such, we investigated how VPAC₁ modulation of LTP adjusts from weaning to adulthood along with GABAergic circuit maturation. As described, LTP induced by mild TBS (5 bursts, 4 pulses delivered at 100 Hz) was increasingly greater from weaning to adulthood. The influence of the VPAC₁ receptor antagonist PG 97-269 (100 nM) on TBS-induced LTP was much larger in juvenile (3-week-old) than in young adult (6-7-week-old) or adult (12-week-old) rats. This effect was not associated with a developmental decrease in synaptic VPAC₁ receptor levels. However, an increase in pre and post-synaptic GABAergic synaptic markers suggests an increase in the number of GABAergic synaptic contacts that is more prominent than the one observed in glutamatergic connections during this period. Conversely, endogenous VPAC₂ receptor activation did not significantly influence TBS-induced LTP. VPAC₂ receptor levels enhance pronouncedly during postweaning development, but not at synaptic sites. Given the involvement of VIP interneurons in several aspects of hippocampal-dependent learning, neurodevelopmental disorders, and epilepsy, this could provide important insights into the role of VIP modulation of hippocampal synaptic plasticity during normal and altered brain development potentially contributing to epileptogenesis.

Keywords: PSD-95; VGAT; VGlut1; VIP; VPAC1 receptor; gephyrin; hippocampus; theta-burst LTP.

Figure 1

Endogenous inhibition of hippocampal CA1 long-term mild-TBS induced LTP elicited by VIP acting on VPAC₁ receptors is attenuated from weaning to adulthood. (A) Time-course of changes in fEPSP slope caused by mild theta-burst stimulation (5 bursts at 5 Hz, each composed of four pulses at 100 Hz, mild TBS) in a typical experiment showing both the control pathway (–○–, S1, absence of added drugs) and the test pathway (–●–, S2) to which the VPAC₁ receptor antagonist PG 97-269 (100 nM) was added 30 min before LTP induction, in the same slice obtained from a juvenile rat (3 weeks). Mild TBS was delivered after obtaining a stable baseline for at least 16 min. Inset: Traces of fEPSPs obtained in the same experiment before (time point 1) and 50–60 min after (time point 2) theta burst stimulation in control conditions and before (time point 3) and 50–60 min after (time point 4) theta burst stimulation in the presence of PG 97-269 (100 nM). Traces are the average of eight consecutive responses and are composed of the stimulus artifact, the presynaptic volley, and the fEPSP. Averaged time-course of changes in fEPSP slope caused by theta-burst stimulation (mild TBS) in the absence (–○–) and in the presence (–●–) of the selective VPAC₁ receptor antagonist PG 97-269 (100 nM) in juvenile rats (3-week-old, (B)), young adult rats (6–7-week-old, (C)) and adult rats (12-week-old, (D)). Control and test conditions (absence and presence of added drugs) were evaluated in independent pathways in the same slice. Inset: LTP magnitude estimated from the averaged enhancement of fEPSP slope observed 50–60 min, after mild TBS in the absence of added drugs (–○–) and in the presence of PG 97-269 (–●–, 100 nM) in juvenile rats (B), young adult rats (C) and adult rats (D). Values (B–D) are the mean ± S.E.M. * p < 0.05 (Student’s t-test) as compared to the null hypothesis. † p < 0.05 (paired Student’s t-test) as compared to the LTP obtained in control conditions (absence of added drugs) for the same age.

Figure 2

Activation of VPAC₂ receptors is not involved in the endogenous control of hippocampal CA1 long-term potentiation of synaptic transmission by VIP from weaning to adulthood. (A) Time-course of changes in fEPSP slope caused by theta-burst stimulation (5 bursts at 5 Hz, each composed of four pulses at 100 Hz, mild TBS) in a typical experiment showing both the control pathway (–○–, S1, absence of added drugs) and the test pathway (–●–, S2), to which the VPAC₂ receptor antagonist PG 99-465 (100 nM) was added 30 min before LTP induction, in the same slice obtained from a juvenile rat (3 weeks). Mild TBS was delivered after obtaining a stable baseline for at least 16 min. Inset: Traces of fEPSPs obtained in the same experiment before (time point 1) and 50–60 min after (time point 2) theta burst stimulation in control conditions and before (time point 3) and 50–60 min after (time point 4) theta burst stimulation in the presence of PG 99-465 (100 nM). Traces are the average of eight consecutive responses and are composed of the stimulus artifact, the presynaptic volley, and the fEPSP. Averaged time-course of changes in fEPSP slope caused by theta-burst stimulation (mild TBS) in the absence (–○–) and in the presence (–●–) of the selective VPAC₂ receptor antagonist PG 99-465 (100 nM) in juvenile rats (3-week-old, (B)), young adult rats (6–7-week-old, (C)) and adult rats (12-week-old, (D)). Control and test conditions (absence and presence of added drugs) were evaluated in independent pathways in the same slice. Inset: LTP magnitude estimated from the averaged enhancement of fEPSP slope observed 50–60 min, after mild TBS in the absence of added drugs (–○–) and in the presence of PG 99-465 (–●–, 100 nM) in juvenile rats (B), young adult rats (C), and adult rats (D). Values (B–D) are the mean ± S.E.M. * p < 0.05 (Student’s t-test) as compared to the null hypothesis. † p < 0.05 (paired Student’s t-test) as compared to the LTP obtained in control conditions (absence of added drugs) for the same age.

Figure 3

Endogenous inhibition of hippocampal CA1 strong-TBS induced LTP elicited by VIP acting on VPAC₁ receptors is abolished reaching adulthood. (A) Time-course of changes in fEPSP slope caused by moderate theta-burst stimulation (5 bursts at 5 Hz, each composed of four pulses at 100 Hz, mild TBS in a typical experiment showing both the control pathway (–○–, S1, absence of added drugs) and the test pathway (–●–, S2), to which the VPAC₁ receptor antagonist PG 97-269 (100 nM) was added 30 min before LTP induction, in the same slice obtained from a young adult rat (6–7 weeks). Moderate TBS was delivered after obtaining a stable baseline for at least 16 min. Inset: Traces of fEPSPs obtained in the same experiment before (time point 1) and 50–60 min after (time point 2) moderate theta burst stimulation in control conditions and before (time point 3) and 50–60 min after (time point 4) moderate theta burst stimulation in the presence of PG 97-269 (100 nM). Traces are the average of eight consecutive responses and are composed of the stimulus artifact, the presynaptic volley, and the fEPSP. Averaged time-course of changes in fEPSP slope caused by theta-burst stimulation (moderate TBS) in the absence (–○–) and in the presence (–●–) of the selective VPAC₁ receptor antagonist PG 97-269 (100 nM) in young-adult rats (6–7-week-old, (B)) and adult rats (12-week-old, (C)). Control and test conditions (absence and presence of added drugs) were evaluated in independent pathways in the same slice. Inset: LTP magnitude estimated from the averaged enhancement of fEPSP slope observed 50–60 min, after moderate TBS in the absence of added drugs (–○–) and in the presence of PG 97-269 (–●–, 100 nM) in young-adult rats (B) and adult rats (C). Values (A,B) are the mean ± S.E.M. * p < 0.05 (Student’s t-test) as compared to the null hypothesis. † p < 0.05 (paired Student’s t-test) as compared to the LTP obtained in control conditions (absence of added drugs) for the same age.

Figure 4

Impact of post-weaning development into adulthood on the levels of structural, GABAergic, and glutamatergic synaptic proteins. (A,E,I). Western-blot immunodetection of PSD-95, VGlut1, Gephyrin, VGAT, and synaptophysin as obtained in one individual experiment in which total hippocampal membranes were subjected to WB analysis. Averaged total PSD-95 (B), VGlut1 (C), Gephyrin (F), VGAT (G), and synaptophysin (J) immunoreactivities. VGlut1/PSD-95 (D) ratio and VGAT/Gephyrin ratio (H) for each animal were also plotted as an estimate of synaptic growth. VGlut1/synaptophysin (K) ratio and VGAT/Synaptophysin ratio (L) are also depicted and were used as an estimate of GABAergic vs glutamatergic synaptic enhancement. Values are mean ± S.E.M of five independent experiments performed in duplicate and were normalized to the immunoreactivity of α-tubulin, used as a loading control. A total of 100%—averaged target immunoreactivity was obtained for 3-week-old animals. * Represents p < 0.05 ** represents p < 0.01 (ANOVA, Tukey’s multiple comparison test) as compared to protein levels in 3-week-old animals or as otherwise indicated. Original figures can be found in Supplementary Materials.

Figure 5

Influence of post-weaning development into adulthood on the global hippocampal levels VPAC₁ and VPAC₂ receptors. Western-blot immunodetection of VIP VPAC₁ (A) and VPAC₂ (D) receptors as obtained in one individual experiment in which hippocampal synaptosomes were subjected to WB analysis. Averaged total VPAC₁ (B) and VPAC₂ (E) immunoreactivity. Values are mean ± S.E.M of five independent experiments performed in duplicate and were normalized to the immunoreactivity of α-tubulin, used as a loading control. 100%–averaged VPAC₁ or VPAC₂ immunoreactivity obtained for 3-week-old animals. VPAC₁/synaptophysin (C) ratio and VPAC₂/Synaptophysin ratio (F) are also depicted. * Represents p < 0.05 and *** represents p < 0.001 (ANOVA, Tukey’s multiple comparison test) as compared to protein levels in 3-week-old animals or as otherwise indicated. Original figures can be found in Supplementary Materials.

Figure 6

Influence of post-weaning development into adulthood on the VIP synaptic content and synaptic VPAC₁ and VPAC₂ receptors. (A,C,E). Western-blot immunodetection of VIP, VPAC₁ and VPAC₂ receptors as obtained in one individual experiment in which hippocampal synaptosomes were subjected to WB analysis. Averaged total VPAC₁ (B), VPAC₂ (D), and VIP (F) immunoreactivities. Values are mean ± S.E.M of five independent experiments performed in duplicate and were normalized to the immunoreactivity of α-tubulin, used as a loading control. 100%–averaged VIP, VPAC₁ or VPAC₂ immunoreactivity obtained for 3-week-old animals. * Represents p < 0.05 (ANOVA, Tukey’s multiple comparison test) as compared to protein levels in 3-week-old animals. Original figures can be found in Supplementary Materials.