Kainate receptor auxiliary subunit NETO2 is required for normal fear expression and extinction

Abstract

NETO1 and NETO2 are auxiliary subunits of kainate receptors (KARs). They interact with native KAR subunits to modulate multiple aspects of receptor function. Variation in KAR genes has been associated with psychiatric disorders in humans, and in mice, knockouts of the Grik1 gene have increased, while Grik2 and Grik4 knockouts have reduced anxiety-like behavior. To determine whether the NETO proteins regulate anxiety and fear through modulation of KARs, we undertook a comprehensive behavioral analysis of adult Neto1^-/- and Neto2^-/- mice. We observed no differences in anxiety-like behavior. However, in cued fear conditioning, Neto2^-/-, but not Neto1^-/- mice, showed higher fear expression and delayed extinction compared to wild type mice. We established, by in situ hybridization, that Neto2 was expressed in both excitatory and inhibitory neurons throughout the fear circuit including the medial prefrontal cortex, amygdala, and hippocampus. Finally, we demonstrated that the relative amount of synaptosomal KAR GLUK2/3 subunit was 20.8% lower in the ventral hippocampus and 36.5% lower in the medial prefrontal cortex in Neto2^-/- compared to the Neto2^+/+ mice. The GLUK5 subunit abundance was reduced 23.8% in the ventral hippocampus and 16.9% in the amygdala. We conclude that Neto2 regulates fear expression and extinction in mice, and that its absence increases conditionability, a phenotype related to post-traumatic stress disorder and propose that this phenotype is mediated by reduced KAR subunit abundance at synapses of fear-associated brain regions.

Fig. 1

No difference in anxiety-like behavior between Neto1^−/− and Neto1^+/+ or Neto2^−/− and Neto2^+/+ mice. Results from the elevated plus maze (a, b), the elevated zero maze (c), the light/dark box test (d, e), the open field test (f), the stress-induced hyperthermia (g), the stress-induced plasma corticosterone (CORT) levels (h), the saccharin preference (i), and the forced swim test (j). Each dot represents one animal, M males and F females. Mean ± 1 standard error is shown. Genotype effect calculated using t-test. KO knockout, WT wild type

Fig. 2

Contextual fear conditioning in Neto1^−/−, Neto1^+/+, Neto2^−/−, and Neto2^+/+ mice. Schematic of the protocol (a). Freezing levels of Neto1 male (b) and female (c), and Neto2 male (d) and female (e) mice during contextual fear acquisition and contextual fear memory retrieval test. Mean ± 1 standard error is shown. Genotype effect calculated by mixed ANOVA (acquisition) or t-test (context test). P-values surviving multiple testing correction are shown. M males, F females, KO knockout, WT wild type, US unconditioned stimulus, Cx ret context retrieval

Fig. 3

Cued fear conditioning and extinction in Neto1^−/−, Neto1^+/+, Neto2^−/−, and Neto2^+/+ mice, and Morris water maze, displaced and novel object recognition, and spontaneous alternation in Neto2^−/− and Neto2^+/+ mice. Protocol for investigating fear conditioning and extinction memory a. Cx ret context retrieval, Cue ret cue retrieval, Ex ret extinction retrieval. Percent time freezing for Neto2 males (b) and females (c), and Neto1 males (d) and females (e). Genotype effect calculated by t-test (Cx ret) or mixed ANOVA. Percentage of extinguished mice (f, g, h, and i). Genotype effect calculated by log rank (mantel-cox) comparison. j Time spent in quadrants of the Morris water maze during the probe trial. NE northeast, SE southeast, SW southwest, NW northwest. (T) indicates the quadrant that contained the escape platform during training. Genotype effect calculated by t-test. (k) Time spent around objects during displaced and novel object recognition tasks. DO displaced object, NDO non-displaced object, NO novel object and FO familiar object. Genotype effect calculated by Wilcoxon test. (l) Alternation score in the T-maze task. Genotype effect calculated by t-test. Each dot represents one animal and dashed line in (l) indicates chance level. Mean ± 1 standard error is shown. P-values surviving multiple testing correction are shown. M males, F females, KO knockout, WT wild type

Fig. 4

Neto2 is expressed in the medial prefrontal cortex (mPFC), dorsal, and ventral hippocampus (dHpc and vHpc), and amygdala (Amg). Atlas representation of brain regions analyzed by in situ hybridization: a mPFC (Cg1 cingulate cortex 1, PL prelimbic cortex, and IL infralimbic cortex), b dHpc (CA1, CA3, and DG dentate gyrus), c vHpc (vCA1, vCA3, and vDG), and d Amg (LA lateral amygdala, BLA basolateral amygdala, CE central amygdala, and ITCs intercalated cells) [51]. In situ hybridization (ISH) of Neto2 in e Cg1, f PL, and g IL subregions of mPFC; h CA1, i CA3, and j DG subregions of dHpc; k vCA1, l vCA3, and m vDG subregions of vHpc and n LA, o BLA, and p ITCs subregions of Amg. Neto2 probe specificity was tested using Neto2 knockout tissue and a sense probe (Figure S3)

Fig. 5

Neto2 is expressed in both excitatory and inhibitory neurons in fear-related brain regions. High magnification representative images of Neto2 (red) and Gad1 (marker of inhibitory neurons; green) or Vglut1 (marker of excitatory neurons; green) mRNA expression in a Cg1, b PL, and c IL subregions of mPFC; d percentage of Neto2-expressing cells that also express Gad1 or Vglut1 in mPFC. Neto2, Gad1, and Vglut1 mRNA expression in e CA1, f CA3, and g DG subregions of dHpc; h percentage of Neto2-expressing cells that also express Gad1 or Vglut1 in dHPC. Neto2, Gad1, and Vglut1 mRNA expression in i vCA1, j vCA3, and k vDG subregions of vHpc; percentage of Neto2-expressing cells that also express Gad1 or Vglut1 in l vHPC. Neto2, Gad1, and Vglut1 mRNA expression in m LA, n BLA, and o ITC subregions of Amg; percentage of Neto2-expressing cells that also express Gad1 or Vglut1 in p Amg. Cg1 cingulate cortex 1, PL prelimbic cortex, IL infralimbic cortex, DG dentate gyrus, LA lateral amygdala, BLA basolateral amygdala, and ITCs intercalated cells

Fig. 6

GLUK2/3 and GLUK5 kainate receptor subunit abundance in lysates and crude synaptosomes from cerebellum (Cb), ventral hippocampus (vHpc), medial prefrontal cortex (mPFC), and amygdala (Amg) of Neto2^−/− and Neto2^+/+ mice. a Brain regions dissected for the immunoblot staining. b Representative bands from immunoblots of brain lysates from Neto2^−/− and Neto2^+/+ mice using antibodies against NETO2, GLUK2/3, and GLUK5. c Validation of synaptosomal enrichment: NETO2 (synaptic marker), synaptophysin (SYP; presynaptic marker), PSD-95 (post-synaptic marker), and KAR subunits GLUK2/3 and GLUK5 from Cb homogenate (H, n = 3) and synaptosomes (SYN, n = 3). For quantification, each lane was first normalized to the b-actin signal and then to the homogenate level. P-values derived from t-test. d Representative bands from synaptosomal immunoblots from Neto2^−/− and Neto2^+/+ mice using antibodies against NETO2, GLUK2/3, and GLUK5. e Ratio of GLUK2/3 and GLUK5 in Neto2^−/− (Cb, n = 7; vHpc, n = 7; mPFC, n = 4; Amg, n = 6) vs WT mice (Cb, n = 7; vHpc, n = 7; mPFC, n = 5; Amg, n = 6) calculated from three replicate immunoblots. Prior to calculating the ratio, protein level from each lane was normalized to b-actin loading control. The significance of Neto2 ablation on GLUK2/3 and GLUK5 protein levels measured using t-test. *p < 0.05, **p < 0.01, ***p < 0.001. For uncropped blots, see Figure S4