Neurotensin and dynorphin Bi-Directionally modulate CeA inhibition of oval BNST neurons in male mice

Abstract

Neuropeptides are often co-expressed in neurons, and may therefore be working together to coordinate proper neural circuit function. However, neurophysiological effects of neuropeptides are commonly studied individually possibly underestimating their modulatory roles. Here, we triggered the release of endogenous neuropeptides in brain slices from male mice to better understand their modulation of central amygdala (CeA) inhibitory inputs onto oval (ov) BNST neurons. We found that locally-released neurotensin (NT) and dynorphin (Dyn) antagonistically regulated CeA inhibitory inputs onto ovBNST neurons. NT and Dyn respectively increased and decreased CeA-toovBNST inhibitory inputs through NT receptor 1 (NTR1) and kappa opioid receptor (KOR). Additionally, NT and Dyn mRNAs were highly co-localized in ovBNST neurons suggesting that they may be released from the same cells. Together, we showed that NT and Dyn are key modulators of CeA inputs to ovBNST, paving the way to determine whether different conditions or states can alter the neuropeptidergic regulation of this particular brain circuit.

Keywords: CeA; Dynorphin; GABA; Neuropeptides; Neurotensin; ovBNST.

Figure 1.. Endogenous neuropeptides modulation of electrically-evoked ovBNST GABA_A-IPSCs.

A, Schematic illustrating stimulating and recording electrodes placement in mice brain slices containing the ovBNST. Recordings were restricted to the displayed shaded oval area. B-D, Effects of postsynaptic depolarization (double arrow symbol) on binned (1 minute, 6 events) electrically-evoked GABA_A-IPSCs in (B) aCSF (n=10 cells/6 mice), (C) the presence of the non-selective NTR antagonist SR142948 (10μM, n=8 cells/5 mice) or (D) SR142948 + the KOR antagonist norNBI (100nM, n=11 cells/4 mice). Insets in B-D are representative electrically-evoked GABA_A-IPSCs before and after postsynaptic depolarization (double arrows). E, Bar graphs summarizing the proportion of responding neurons to postsynaptic depolarization across different pharmacological treatments. Blue LTP, grey no change and orange LTD. Asterisks, p<0.05.

Figure 2.. Effect of postsynaptic depolarization on optically-evoked Vgat^CeA➜ovBNSTGABA_A-IPSCs.

A, Illustration demonstrating ChR2 bi-lateral injections into the CeA of Vgat-Cre mice. B, Illustration demonstrating optically stimulated recordings of ovBNST neurons. C-E, Postsynaptic depolarization in male Vgat-Cre mice injected with ChR2 in the CeA and recorded from ovBNST brain slices in (C) aCSF (n=13 cells/6 mice), (D) in the presence of the non-selective NTR antagonist SR142948 (10μM, n=12 cells/5 mice), (E) SR142948 + the KOR antagonist norNBI (100nM, n=8 cells/2 mice). Insets in C-E are representative optically-evoked GABA_A-IPSCs before and after postsynaptic depolarization (double arrows). F, Bar graphs summarizing the proportion of responding neurons to postsynaptic depolarization across different pharmacological treatments. Blue LTP, grey no change and orange LTD. Asterisks, p<0.05.

Figure 3.. Expression and co-localization of Pdyn and Nts mRNA in the ovBNST.

A, Representative image of dual fluorescent in situ hybridization for Nts/Pdyn (Nts (green), Pdyn (purple), and DAPI (blue)). B, Ratio of the total number of Nts mRNA-expressing cells that are positive for Pdyn (co-localizing) (average ±SEM % n=4 mice, 4 slices/mouse).

Figure 4.. Expression and co-localization of NT and NTRs mRNA in the ovBNST and CeA.

Representative image of dual fluorescent in situ hybridization (FISH) for Nts/Ntsr1 (Nts (purple), Ntsr1 (green), DAPI (blue) in the ovBNST (A) and CeA (B). Ratio of the total number of Nts mRNA-expressing cells that are positive for Ntsr1 (co-localizing) (average ±SEM % n=2 mice, 2 slices/mouse) in the ovBNST (C) and CeA (D). Representative image of dual FISH for Nts/Ntsr2 (Nts (purple), Ntsr2 (green), DAPI (blue)) in the ovBNST (E) and CeA (F). Ratio of the total number of Nts mRNA-expressing cells that are positive for Ntsr2 (co-localizing) (average ±SEM % n=2 mice, 2 slices/mouse) in the ovBNST (G) and CeA (H).

Figure 5.. Contribution of neurotensin receptors 1 and 2 on exogenous NT-induced modulation of electrically-evoked ovBNST GABA_A-IPSCs.

A, Effect of a 5-minute bath application of NT (1uM) (black horizontal bar) on the peak amplitude of eIPSCs (n=8 cells/4 mice). Effect of NT (1μM, black horizontal bar) on eIPSCs in the presence of (B) the NTR2-selective antagonist NTRC844 (100 nM, n=7 cells/3 mice) or the (C) NTR1-selective antagonist SR48692 (1 μM, n=5 cells/3 mice) D, Effect of a 5-minute bath application of NTR2 agonist JMV431 (100nM, n=8 cells/4 mice) (black horizontal bar) on the peak amplitude of eIPSCs in the ovBNST. Insets in B-D are representative electrically-evoked GABA_A-IPSCs before and after bath application of NT or JMV431 for 5 minutes. Bar graphs summarizing the proportion of responding neurons to different pharmacological treatments (E). Blue LTP, grey no change and orange LTD. Asterisks, p<0.05.

Figure 6.

Illustration of NT and Dyn bi-directional modulation of CeA inputs onto ovBNST neurons.