Suppression of conditioning to ambiguous cues by pharmacogenetic inhibition of the dentate gyrus

Abstract

Serotonin receptor 1A knockout (Htr1a(KO)) mice show increased anxiety-related behavior in tests measuring innate avoidance. Here we demonstrate that Htr1a(KO) mice show enhanced fear conditioning to ambiguous conditioned stimuli, a hallmark of human anxiety. To examine the involvement of specific forebrain circuits in this phenotype, we developed a pharmacogenetic technique for the rapid tissue- and cell type-specific silencing of neural activity in vivo. Inhibition of neurons in the central nucleus of the amygdala suppressed conditioned responses to both ambiguous and nonambiguous cues. In contrast, inhibition of hippocampal dentate gyrus granule cells selectively suppressed conditioned responses to ambiguous cues and reversed the knockout phenotype. These data demonstrate that Htr1a(KO) mice have a bias in the processing of threatening cues that is moderated by hippocampal mossy-fiber circuits, and suggest that the hippocampus is important in the response to ambiguous aversive stimuli.

Figure 1

Increased response to partially conditioned cues in Htr1a^KO mice. (a) Training protocol for simultaneous conditioning to partial and perfect cues. Knockout (KO) mice showed greater freezing responses 24 h after training to the partial (T, tone), but not perfect (L, light), conditioned stimulus when compared with wild-type (WT) littermates (S, shock; WT: N = 18, KO: N = 18; ** P < 0.01; n.s., not significant). (b) Same training protocol as in a, but unpaired cue presentations have been omitted. In the absence of partial cue conditioning, wild-type and knockout mice showed indistinguishable freezing responses to both conditioned stimuli during testing (WT: N = 10, KO: N = 10). (c) Mice treated with WAY100635 from P13 to P34 showed greater freezing responses to the partial, but not perfect, conditioned stimulus when compared with saline-treated littermates (saline: N = 10, WAY: N = 12; * P < 0.05). Error bars, s.e.m.

Figure 2

Transgenic lines expressing Htr1a in the CeA and DG. (a) Tissue- and cell type–specific expression of Htr1a was achieved by making transgenic mice with the Htr1a coding sequences under the control of the Nrip2 promoter. (b–g) Crossing of transgenic founder lines to mice lacking the endogenous receptor (Htr1a^KO) resulted in receptor expression restricted to cells in the central nucleus of the amygdala (Htr1a^CeA; b,e) and granule cells of the dentate gyrus (Htr1a^DG; c,f), as detected by autoradiography using the Htr1a ligand ¹²⁵I-MPPI (dDG, dorsal DG; vDG, ventral DG). No receptor binding was detected in brains from Htr1a^KO mice (d,g).

Figure 3

Electrophysiological characterization of Htr1a^CeA line. (a) Magnitude of membrane hyperpolarization by application of serotonin (5-HT, 100 μM) to neurons in brain slices from adult Htr1a^CeA mice. Error bars, s.e.m. (b,c) CeA neurons were characterized as type I (b) or type II (c) by the presence or absence of a depolarizing after-potential seen following a depolarizing current–induced action potential, respectively. Significant hyperpolarization was seen in type I, but not type II, CeA neurons (9/16 and 7/16 neurons, respectively; t-test, P = 0.02), and no hyperpolarization response was detected in hippocampal DG neurons (N = 3) from the same mice, or CeA neurons from knockout mice (N = 8). (d) Raw data trace of the membrane potential of a representative type I CeA neuron showing a hyperpolarizing response to 5-HT application. The line depicts the duration of time that 5-HT was in the perfusion buffer. (e) Silencing of depolarizing current–induced spontaneous neural activity in a type I CeA neuron following application of 5-HT. (f) Photomicrograph of a biocytin-labeled type I CeA neuron.

Figure 4

Electrophysiological characterization of Htr1a^DG line. (a) Magnitude of membrane hyperpolarization by application of serotonin (5-HT, 100 μM)) to neurons in brain slices from adult Htr1a^DG mice. Significant hyperpolarization was seen in hippocampal DG granule neurons (N = 24), but not in hippocampal CA1 (N = 8; t-test, P = 0.0003) or type I neurons from the CeA (N = 3; t-test, P = 0.003). Similarly, no hyperpolarization response was detected in DG neurons from knockout mice (N = 5). Error bars, s.e.m. (b) Membrane potential response to current injection into a representative DG neuron from a Htr1a^DG mouse. (c) Raw data trace of the membrane potential response of a DG neuron showing hyperpolarization response to 5-HT application. The line depicts the duration of time that 5-HT was in the perfusion buffer. (d) Silencing of depolarizing current–induced spontaneous neural activity in a DG neuron after application of 5-HT. (e) No inhibition of current-induced spontaneous neural activity was detected in a DG neuron from a Hrt1a^KO mouse. (f) Photomicrograph of a biocytin-labeled DG neuron.

Figure 5

Suppression of conditioned responses by inhibition of neurons in the CeA and DG. (a) Administration of 8-OH-DPAT (0.2 or 0.5 mg per kg, subcutaneous) to Htr1a^CeA mice before ambiguous-cue fear-conditioning testing suppressed freezing to both partial and perfect cues (saline, N = 12; 0.2 mg per kg, N = 12; 0.5 mg per kg, N = 11; * P < 0.05, *** P < 0.001). Similar treatment of knockout mice was ineffective (saline, N = 11; 0.2, N = 9; 0.5, N = 11). (b) Administration of 8-OH-DPAT (0.2 or 0.5 mg per kg, subcutaneous) to Htr1a^DG mice before ambiguous-cue fear-conditioning testing suppressed freezing to the partial, but not perfect, cue (saline, N = 12; 0.2 mg per kg, N = 9; 0.5 mg per kg, N = 10; * P < 0.05, ** P < 0.01). Similar treatment of knockout mice was ineffective (Saline, N = 9; 0.2 mg per kg, N = 10; 0.5 mg per kg, N = 10). Error bars, s.e.m.