Loss of α7 nicotinic acetylcholine receptors in GABAergic neurons causes sex-dependent decreases in radial glia-like cell quantity and impairments in cognitive and social behavior

Abstract

The dentate gyrus (DG) is a unique brain structure in that neurons can be generated postnatally and integrated within existing circuitry throughout life. The maturation process of these newly generated neurons (granule cells) is modulated by nicotinic acetylcholine receptors (nAChRs) through a variety of mechanisms such as neural stem pool proliferation, cell survival, signal modulation, and dendritic integration. Disrupted nAChR signaling has been implicated in neuropsychiatric and neurodegenerative disorders, potentially via alterations in DG neurogenesis. GABAergic interneurons are known to express nAChRs, predominantly the α7 subtype, and have been shown to shape development, integration, and circuit reorganization of DG granule cells. Therefore, we examined histological and behavioral effects of knocking out α7 nAChRs in GABAergic neurons. Deletion of α7 nAChRs resulted in a reduction of radial glia-like cells within the subgranular zone of the DG and a concomitant trend towards decreased immature neurons, specifically in male mice, as well as sex-dependent changes in several behaviors, including social recognition and spatial learning. Overall, these findings suggest α7 nAChRs expressed in GABAergic neurons play an important role in regulating the adult neural stem cell pool and behavior in a sex-dependent manner. This provides important insight into the mechanisms by which cholinergic dysfunction contributes to the cognitive and behavioral changes associated with neurodevelopmental and neurodegenerative disorders.

Keywords: DCX; GFAP; Memory; Neurogenesis; Progenitor; Stem cell.

Fig. 1. Reduced radial glia-like cells within the dorsal DG of male cKO mice.

Images are representative samples from the dorsal DG at 40x from a hGFAP-GFP x α7nAChR^flox x GAD2-IRES-Cre mouse A. Expression of GFP was amplified using ck-antiGFP (Green; 1:10,000). Mice were also probed for immature neuron expression using gp-antiDCX (Red; 1:500) and dividing cells using rb-antiKi-67 (White; 1:400). Cell nuclei were marked using DAPI (Blue; 1:500). Scale bar = 100 μm. GFAP-positive with radial glia-like morphology, DCX-positive, and Ki-67-positive cell densities were averaged from four sections per mouse and plotted against sex and genotype B-D. **p < 0.01. # post-hoc Student’s t-test, p = 0.0362. Data expressed as mean ± SEM.

Fig 2.. Reduced distance traveled and rearing movements in cKO female mice during the open field test.

Distanced traveled and number of rearing movements during 60 min in an open field task A-D. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data expressed as mean ± SEM.

Fig. 3. Reduced social novelty preference in cKO male mice during the three chamber social approach test.

Measures were taken of time spent in proximity to each cage during the test for sociability A,B and preference for social novelty C,D. Data were lost from one male cKO mouse due to experimenter error. Data were removed for one outlier female cKO mouse that remained in proximity to novel mouse for 466 sec. **p < 0.01, ***p < 0.001, ****p < 0.0001. Data expressed as mean ± SEM.

Fig. 4. Normal acquisition learning in a hidden platform task but lack of quadrant preference in cKO mice during Morris water maze probe trials.

Criterion for learning was a group average of 15 sec to find the escape platform A-D. Mice were given a one-min probe trial without the platform following an initial 3-day acquisition of the hidden platform task E,F and a subsequent 4-day reversal phase G,H. ‘Target’ indicates the quadrant where the platform was located in each phase. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data expressed as mean ± SEM.

Fig. 5. Lack of differences in fear conditioning and context tests.

Measures were taken of total freezing during sixteen trials across training A. Total percentage freezing during context test was recorded B with baseline, tone, and post-tone freezing C. Data expressed as mean ± SEM.

Fig. 6. Decreases in object investigation in cKO mice during spatial and novel object recognition tests.

Measures were taken of total object investigation familiarization time during spatial object recognition as an average of time and percentage of time near novel versus familiar objects A,B. Total investigation time during novel object recognition was recorded and plotted as an average of time and percentage of time near novel versus familiar objects C,D. *p < 0.05, **p < 0.01. Data expressed as mean ± SEM.