Norepinephrine enables the induction of associative long-term potentiation at thalamo-amygdala synapses

Abstract

Emotional arousal, linked to a surge of norepinephrine (NE) in the amygdala, leads to creation of stronger and longer-lasting memories. However, little is known about the synaptic mechanisms of such modulatory NE influences. Long-term potentiation (LTP) in auditory inputs to the lateral nucleus of the amygdala was recently linked to the acquisition of fear memory. Therefore we explored whether LTP induction at thalamo-amygdala projections, conveying the acoustic conditioned stimulus information to the amygdala during fear conditioning, is under adrenergic control. Using whole-cell recordings from amygdala slices, we show that NE suppresses GABAergic inhibition of projection neurons in the lateral amygdala and enables the induction of LTP at thalamo-amygdala synapses under conditions of intact GABA(A) receptor-mediated inhibition. Our data indicate that the NE effects on the efficacy of inhibition could result from a decrease in excitability of local circuit interneurons, without direct effects of NE on release machinery of the GABA-containing vesicles or the size of single-quanta postsynaptic GABA(A) receptor-mediated responses. Thus, adrenergic modulation of local interneurons may contribute to the formation of fear memory by gating LTP in the conditioned stimulus pathways.

Fig. 1.

Norepinephrine gates LTP at thalamo-amygdala synapses under conditions of intact GABA_A receptor-mediated inhibition. (A) Schematic representation of a brain slice containing the LA that shows the position of the recording (R) and stimulation (S) pipettes. (B) EPSP-AP pairing-induced LTP of the thalamo-amygdala EPSP in the presence of PTX (50 μM) without NE (filled circles; n = 8; mean ± SEM) or with 10 μM NE (open circles; n = 8 neurons) in the bath solution. Traces are averages of three EPSPs obtained from individual experiments before (1) and after (2) LTP induction (arrow). (C) Summary graphs of LTP experiments in thalamo-amygdala pathway under conditions of intact inhibition without NE (filled circles; n = 9) or with 10 μM NE (open circles; n = 10) in the bath solution. Traces are averages of three EPSPs obtained before (1) and after (2) LTP induction. During LTP experiments, NE was applied throughout the recording period. (D) Summary of LTP experiments at the thalamo-amygdala pathway (mean ± SEM).

Fig. 2.

NE has no direct effect on baseline glutamatergic synaptic transmission in thalamo-amygdala pathway. (A) Values of amplitude of the NMDA receptor-mediated EPSCs (normalized by the pre-NE baseline) are plotted as a function of time (mean ± SEM; n = 10 neurons). Solid bar shows the duration of 10 μM NE application. Traces (top) show NMDAR EPSCs recorded before (1) and during (2) NE application. Synaptic currents were recorded in the presence of the AMPAR antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (20 μM) and PTX (50 μM) at a holding potential of +30 mV. (B) Current-voltage plot of the NMDA receptor EPSCs. Synaptic currents (Inset) were recorded in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione and PTX at holding potentials of −70 mV to +50 mV under baseline conditions (filled symbols) and ≈15 min after switching to the NE-containing solution (open symbols; n = 6 neurons). Traces are averages of three EPSCs recorded at each holding potential. (C) The AMPAR-mediated thalamo-amygdala EPSCs were recorded from pyramidal neurons at a holding potential of −70 mV (n = 6 neurons). Solid bar shows the duration of 10 μM NE application. Traces (top) are averages of three EPSCs recorded before (1) and during (2) NE application. (D) Application of NE induced potentiation of the thalamo-amygdala EPSC when α₂-adrenoreceptors were blocked by yohimbine (20 μM; n = 5). This potentiation was reversed by 10 μM propranolol. (E) NE application produced depression of the EPSC when β-adrenoreceptors were blocked by propranolol (10 μM; n = 4). Yohimbine partially blocked this potentiation. (F) Summary plot of the effects of NE on glutamatergic EPSCs in thalamic input to the LA. Error bars indicate SEM. (G) Responses of LA neurons to prolonged current injections (500 ms, 200 pA) recorded before (control) and during NE application. (H) Summary plots of the experiments as in G (n = 8; mean ± SEM).

Fig. 3.

NE suppresses GABAergic inhibition of neurons in the LA by decreasing excitability of local circuit interneurons. (A) Representative sIPSPs in the LA neuron at a holding potential of −70 mV recorded with or without 10 μM NE in the bath solution. (B) Cumulative interevent interval histograms of sIPSCs recorded under baseline conditions (filled symbols) and after NE was applied (open symbols) (n = 16). (C) Summary plots of sIPSC and mIPSC (recorded in the presence of 1 μM Tetrodotoxin (TTX; n = 4) data (mean ± SEM). (D) Recording from interneuron that showed the nonaccommodating firing pattern in response to depolarizing current injection. (E) An outward current in the LA interneuron recorded under whole-cell conditions at a holding potential of −70 mV in the presence of 10 μM NE in the bath solution. (F) NE does not depress monosynaptic GABA_AR IPSCs in the LA. The IPSCs were recorded from pyramidal neurons at a holding potential of −70 mV (n = 4 cells). Stimulation pipette was placed within the LA. Traces (top) are averages of three IPSCs recorded before (1) and during (2) NE application. Synaptic currents were recorded in the presence of 20 μM 6-cyano-7-nitroquinoxaline-2,3-dione.

Fig. 4.

NE-induced suppression of feedforward GABAergic inhibition is sufficient to gate LTP in thalamic input to the LA. (A) Effects of NE on biphasic synaptic responses evoked by stimulation of thalamo-amygdala pathway. The EPSP/IPSP sequences were recorded in the LA neuron under current-clamp conditions at −55 mV. (Inset) The averages of three responses recorded under baseline conditions (1) and during NE application (2). (B) Summary plot of the effects of NE on glutamatergic EPSPs and GABAergic IPSPs in thalamic input to the LA. The average amplitudes of synaptic responses recorded over the final 3 min of NE application were normalized by their baseline values (n = 8). (C) EPSP-AP pairing-induced LTP in thalamo-amygdala pathway in the presence of 1 μM PTX (n = 4). Traces show EPSPs recorded before (1) and after (2) the pairing procedure. Error bars indicate SEM.