Orexin Depolarizes Central Amygdala Neurons via Orexin Receptor 1, Phospholipase C and Sodium-Calcium Exchanger and Modulates Conditioned Fear

Abstract

Orexins (OX), also known as hypocretins, are excitatory neuropeptides with well-described roles in regulation of wakefulness, arousal, energy homeostasis, and anxiety. An additional and recently recognized role of OX is modulation of fear responses. We studied the OX neurons of the perifornical hypothalamus (PeF) which send projections to the amygdala, a region critical in fear learning and fear expression. Within the amygdala, the highest density of OX-positive fibers was detected in the central nucleus (CeA). The specific mechanisms underlying OX neurotransmission within the CeA were explored utilizing rat brain slice electrophysiology, pharmacology, and chemogenetic stimulation. We show that OX induces postsynaptic depolarization of medial CeA neurons that is mediated by OX receptor 1 (OXR1) but not OX receptor 2 (OXR2). We further characterized the mechanism of CeA depolarization by OX as phospholipase C (PLC)- and sodium-calcium exchanger (NCX)- dependent. Selective chemogenetic stimulation of OX PeF fibers recapitulated OXR1 dependent depolarization of CeA neurons. We also observed that OXR1 activity modified presynaptic release of glutamate within the CeA. Finally, either systemic or intra-CeA perfusion of OXR1 antagonist reduced the expression of conditioned fear. Together, these data suggest the PeF-CeA orexinergic pathway can modulate conditioned fear through a signal transduction mechanism involving PLC and NCX activity and that selective OXR1 antagonism may be a putative treatment for fear-related disorders.

Keywords: central amygdala; chemogenetic; fear conditioning; orexin (hypocretin); orexin receptor 1 (OX1R).

FIGURE 1

OX mediates postsynaptic depolarization of CeA neurons. (A) Representative 20× images revealed OX A positive cell bodies in the PeF (f is fornix, -2.80 mm bregma) and OX A positive fibers in the amygdala (-2.40 mm bregma). Boxed area is enlarged to show individual OX A-positive PeF neurons. (B) Schematic of PeF projections to the amygdala and (C) summary quantification of fluorescence, n = 7 animals. (D) Depolarization response to OX A was observed for CeA neurons (n = 9–15 cells). (E) CeA neurons depolarized to applications of either OX A or OX B (n = 10–12 cells). (F) Input resistance of CeA neurons before and during OX perfusion. (G) Representative traces of OX mediated depolarization. Solid bars under traces indicate perfusion of OX or vehicle. (H) Depolarization persisted following 1 μM TTX treatment indicating a postsynaptic effect (n = 9–11 cells). (I) Mapping of OX sensitive (blue, 23/51 recordings) and insensitive (brown) neurons in the CeA shows a pattern of sensitivity in the medial CeA. Data are means ± SEM, symbols indicate significance by Sidak’s post hoc test, p < 0.05, ^∗ between subjects, # within subjects.

FIGURE 2

CeA depolarization to OX occurs via OXR1 but not OXR2. (A) Summary data for the application of OX A, or (B) OX B, in the absence or presence of receptor antagonists (OXR1 antagonist C 56 in red, and OXR2 antagonist JNJ10397049 in orange, n = 9–13 cells per condition). (C) Representative traces of OX A conditions and (D) OX B conditions. Solid bars under traces indicate perfusion of OX following 10 min perfusion of antagonist. (E) Schematics illustrating that OXR1 antagonism prevents OX mediated depolarization and OXR2 antagonism does not affect OX mediated depolarization. Data are means ± SEM, symbols indicate significance by Sidak’s post hoc test, p < 0.05, ^∗ between subjects, # within subjects.

FIGURE 3

Stimulated release of endogenous OX produces OXR1 dependent CeA neuron depolarization. (A) Representative PeF (20×) and CeA (40×) images demonstrate DREADD-mCitrine-positive cell bodies and fibers of cells expressing prepro-OX-DREADD-mCitrine virus. Boxed area is enlarged to show individual mCitrine-positive PeF neurons. (B) Schematic of regions that were imaged (-2.40 mm bregma for CeA, -2.80 mm bregma for PeF). (C) Summary data of Clozapine-N-oxide (CNO), DREADD agonist, effects for the indicated conditions (n = 9–13). (D) Representative traces from CNO treated neurons. CNO stimulated depolarization in animals expressing PeF-targeted DREADD virus except when OXR1 was antagonized by C 56. Solid bars under traces indicate perfusion of CNO. Data are means ± SEM, symbols indicate significance by Sidak’s post hoc test, p < 0.05, ^∗ between subjects, # within subjects.

FIGURE 4

CeA depolarization to OX occurs via sodium and calcium conductance mediated by PLC and NCX activity. (A) Representative traces of CeA neurons and (B) summary data for OX A treated cells with altered ionic gradients. Solid bars under traces indicate perfusion of OX. CeA depolarization did not occur when sodium or calcium conductance was reduced, but was maintained when potassium conductance was eliminated (n = 8–11 cells). (C) Summary data for OX and antagonist treated cells. PLC antagonist 10 μM U73122 and NCX antagonists 80 μM KB-R7943 and 3 mM NiCl prevented OX mediated CeA depolarization (n = 7–10 cells). (D) Schematic of putative signaling pathway by which OX mediates postsynaptic depolarization via PLC and NCX without contribution to depolarization by potassium channels. Data are means ± SEM, symbols indicate significance by Sidak’s post hoc test, p < 0.05, ^∗ between subjects, # within subjects.

FIGURE 5

OXR1 modifies presynaptic glutamate release in the CeA. (A) Schematic and image depicting location of stimulation and recording electrodes during evoked excitation experiments. (B) Representative potential traces evoked from CeA neurons. (C) Summary data of potentials evoked from CeA neurons from a holding potential of -70 mV and (D) their recorded input resistance (n = 11). (E) Representative paired-pulse potential traces for indicated treatments. (F) Summary data of paired pulse ratios from CeA neurons treated with 200 nM OX A in the presence and absence of OXR antagonists (C 56 OXR1 antagonist and JNJ10397049 OXR2 antagonist) and (G) their recorded input resistance. Treatments were applied to the same cells in randomized orders (n = 5). Data are means ± SEM, symbols indicate significance by Sidak’s post hoc test, p < 0.05, ^∗ between subjects.

FIGURE 6

OXR1 antagonism reduces expression of cued fear in rodents. (A) Summary data of auditory fear conditioning and expression following systemic I.P. injections of vehicle, C 56 at 10 and 30 mg/kg, and JNJ10397049 at 30 mg/kg. (B) Summary data of auditory fear conditioning and expression following cannulae injection of 100 nl vehicle or 300 pmole C 56. None of the treatment conditions affected fear acquisition. OXR1 antagonism, but not OXR2 antagonism, reduced expression of cued fear. Data are means ± SEM, symbols indicate significance by Fisher’s LSD post hoc test, p < 0.05, # treatment effect.