Neural Mechanisms Of AVPR1A RS3-RS1 Haplotypes Impacting Verbal Learning And Memory

Objective:Converging evidence from both animal and human studies has highlighted the pervasive role of neuropeptide arginine vasopressin(AVP)in both social and non-social learning and memory,which is mediated by arginine vasopressin receptor 1A(AVPR1A).However,the effect of genetic variants in AVPR1A on memory is unknown.To study the effects of RS3 and RS1 repeat polymorphisms in promoter region of AVPR1A on verbal learning and memory performance,we use multi-mode functional magnetic resonance imaging(f MRI)technology,and combine voxel-based morphometry(VBM)and resting-state functional connectivity(rs FC)to explore the underlying neural mechanism in a large sample of young healthy subjects(n=1028).Our hypothesis is that RS3 and RS1 repeat polymorphisms in the AVPR1A promoter region(i.e.genetic variation of RS3 and RS1 microsatellite haplotypes)may be regulated by the gray matter volume(GMV)and rs FC of the brain,especially the hippocampus(because the hippocampus is recognized as the core brain area of verbal learning and memory),it has a certain effect on the verbal learning and memory ability assessed by the California Verbal Learning Test Second Edition(CVLT-II).Subjects and Methods:In this study,a total of 1104 healthy young volunteers of Han nationality were recruited.They were selected as strong right handedness by Edinburgh Handedness scale.All the subjects were tested for RS3 and RS1 microsatellite polymorphisms in the promoter region of AVPR1A and the second version of California Verbal Learning Test-Chinese(California Verbal Learning Test-?,CVLT-?).We used a GE Discovery MR 750 3.0 Tesla scanner to collect structural images and resting-state f MRI(rs-f MRI)data of all subjects.A tight but comfortable foam padding was used to reduce head movement and earplugs were used to decrease the scanning noise.Subjects were asked to close their eyes and keep their bodies still.Try not to think about anything,don't fall asleep,and turn off the scanning room lights.1.Genotyping:10 ml of peripheral venous blood was drawn from all the subjects and DNA was extracted from white blood cells using the standard procedure of the EZgene TM Blood g DNA Miniprep Kit.The genotyping was determined by polymerase chain reaction(PCR).PCR products were analyzed using an ABI PRISM 3730 Genetic Analyzer(Applied Biosystems)and was detected the length difference between RS3 and RS1 repeat polymorphisms in the AVPR1A promoter region.Depending on the length of the RS3 and RS1 alleles,each microsatellite had three possible genotypes:long/long(LL),short/long(SL)or short/short(SS).For RS3-RS1 haplotypes,there were four possible combinations:RS3SL+SS-RS1SL+SS,RS3SL+SS-RS1LL,RS3LL-RS1SL+SS,RS3LL-RS1LL.2.Compare the differences in CVLT-II List A trial 1-5 total raw score among four genotypic groups of RS3 and RS1 in AVPR1A promoter region to evaluate the difference of total immediate free recall scores among different groups of subjects.3.Data preprocessing:All preprocessing steps for high-resolution structural image were carried out using the CAT12 software package(version r1364,http://dbm.neuro.uni-jena.de/cat)with a standard configuration based on Matlab 2014b platform and obtain the standardized GMV map.The preprocessing process included:bias correction,gray matter(GM)and white matter(WM) segmentation,creating population-specific tissue templates,spatial normalization and smoothing(8×8×8mm³).Rs-f MRI data preprocessing was performed using a toolbox for Data Processing&Analysis for Brain Imaging (DPABI,http://rfmri.org/dpabi)based on Matlab 2014b platform.The processing steps included:discarding unstable volumes,slice timing correction,head motion correction,spatial normalization,smoothing,regressing out sources of noise,scrubbing and filtering(0.01-0.08 Hz).4.Voxel-based whole brain GMV comparison was performed among the four genotypic groups using pre-processed high-resolution structural data,while controlling total intracranial volume(TIV),age,sex and years of education as nuisance variables.After finding that the GMV of bilateral hippocampi and other multiple brain regions were different in different groups(P<0.001,no multiple comparison correction),Anatomy v1.7 software was used to extract bilateral hippocampi as the mask,and one-way ANOVA was performed to evaluate the GMV differences between the four genotypic groups of subjects.Post-hoc analysis was used to determine any GMV difference between the two groups.Since different hippocampal subfields have different functions in learning and memory consolidation,and avpr1a is distributed in each subfield in rodents,we further explore the distribution of GMV in hippocampus with differences at group levels in subfields.5.Using the above VBM to obtain brain regions with significant group-level bilateral hippocampal GMV differences as ROIs,based on pre-processed resting-state functional image data,the correlation coefficients between the mean time series of each ROI and the mean time series of other voxels in the whole brain were calculated.And the brain regions with significant connectivity with each ROI were determined by single sample t-test.One-way ANOVA was used to compare whether there was a difference in rs FC between each ROI and other brain regions among the four genotypic groups in each mask.Finally,Post-hoc analysis was used to determine whether there was a significant difference in rs FC between each ROI and other brain regions between any two genotypic groups.6.The False Discovery Rate(FDR)method(P<0.05)was used to perform multiple comparison corrections on the above results(4 and 5).Finally,the corrected statistical parameters were mapped to the MNI standard three-dimensional template brain for display,and the statistically significant brain region cluster location,cluster size,and MNI were described and recorded coordinates and related strengths.7.The above-mentioned bilateral hippocampal GMV and rs FC with significant group level were extracted,and in all subjects,the correlation between AVPR1A RS3 and RS1 microsatellite polymorphisms and these image indexes was calculated by partial correlation method.8.Many studies have reported gender differences in the effects of AVP on memory.Therefore,the above analysis was completely repeated in the male and female subgroups,respectively,to explore whether there were gender differences in the effects of AVPR1A RS3 and RS1 microsatellite polymorphism on GMV,rs FC and verbal learning and memory ability in bilateral hippocampi.9.In addition to grouping according to the length of RS3 and RS1 alleles,many researchers also use target alleles to grouping.The same analysis method was used to compare GMV,rs FC and verbal learning and memory ability of the subjects with and without target alleles.Result:1.According to the AVPR1A gene microsatellite test,the distribution frequency of all alleles on each microsatellite accorded with Hardy-Weinberg distribution,and there were no differences in age,gender,and years of education among the groups.The results showed significant differences in the trials 1-5 total raw score in the list A of CVLT-II among the four genotypic groups.Post-hoc analysis revealed the trials 1-5 total raw score showed significant differences between some genotypic groups:RS3_SL+SS-RS1_SL+SS<RS3_LL-RS1_SL+SS,RS3LL-RS1SL+SS<RS3LL-RS1LL,RS3SL+SS-RS1SL+SS<RS3LL-RS1LL,and it showed a trend of allele-load-dependence throughout the four groups which indicated the total number of words recalled correctly was gradually increased with the decrease of the number of short-allele that the individuals carried.2.The whole-brain VBM analysis demonstrated that there were significant differences in GMV among the four genotypic groups in bilateral hippocampi and other brain regions,and hippocampal regions with differences in levels among groups involved almost all subfields of hippocampus.After Post-hoc analysis,we found that there were significant differences in bilateral hippocampal GMV between certain genotypic groups:RS3_SL+SS-RS1_SL+SS<RS3LL-RS1LL,RS3SL+SS-RS1SL+SS<RS3LL-RS1SL+SS,RS3LL-RS1SL+SS<RS3_LL-RS1_LL,and the decreased GMV of each hippocampus exhibited an allele-load-dependent trend which indicated the GMV of each hippocampus was gradually increased with the decrease of the number of short-allele that the individuals carried.3.Based on the analysis of voxel levels,we found that there were significant differences in rs FC between the right hippocampus and the bilateral thalami. ROI-based post-hoc comparisons revealed significant differences in rs FC between the right hippocampus and bilateral thalami between certain genotypic groups:RS3SL+SS-RS1SL+SS<RS3LL-RS1LL,RS3SL+SS-RS1SL+SS<RS3SL+SS- RS1_LL,RS3_SL+SS-RS1_LL<RS3_LL-RS1_LL,and the rs FC of right hippocampal- bilateral thalami exhibited an allele-load-dependent trend throughout the four groups which suggested the rs FC of right hippocampal-bilateral thalami was gradually increased with the decrease of the number of short-allele that the individuals carried.Because different thalamic subfields have different functions,it is necessary to further explore which thalamic subfields are more susceptible to involvement.The analysis found that the thalamus with group differences are mainly located in medial pre-frontal subfield.4.Partial correlation analysis found that the GMV of bilateral hippocampi and rs FC of right hippocampal-bilateral thalami were positively correlated with the trial 1-5 total raw score of CVLT list A.5.In male and female subjects,there was no significant difference between the subjects with different genotypes(long/short repeated groupings)in the trial1-5 total raw score and GMV of the whole brain.6.There were no significant differences between the subjects with target alleles and non-target alleles in trial 1-5 total raw scores and whole brain GMV.Conclusion:1.In normal young subjects,subjects with shorter alleles in the RS3-RS1 haplotype of the AVPR1A promoter region had poor verbal learning and memory abilities.2.Genetic variation in the RS3-RS1 haplotype of the AVPR1A promoter region may partially affect verbal learning and memory ability by regulating hippocampal structure and hippocampal-thalamic connectivity.3.AVPR1A RS3-RS1 haplotype polymorphisms affect GMV and rs FC in thehippocampus,which is of great significance for understanding the AVPR1A gene as a susceptibility gene for autism spectrum disorders(ASD).