Complex trait analysis of the mouse striatum: independent QTLs modulate volume and neuron number

Abstract

The striatum plays a pivotal role in modulating motor activity and higher cognitive function. We analyzed variation in striatal volume and neuron number in mice and initiated a complex trait analysis to discover polymorphic genes that modulate the structure of the basal ganglia. ) and is associated with differences of up to 400,000 neurons among animals. We have discovered remarkable numerical and volumetric variation in the mouse striatum, and we have been able to map two QTLs that modulate independent anatomic parameters.

Background

The striatum plays a pivotal role in modulating motor activity and higher cognitive function. We analyzed variation in striatal volume and neuron number in mice and initiated a complex trait analysis to discover polymorphic genes that modulate the structure of the basal ganglia.

Results

) and is associated with differences of up to 400,000 neurons among animals.

Conclusion

We have discovered remarkable numerical and volumetric variation in the mouse striatum, and we have been able to map two QTLs that modulate independent anatomic parameters.

Background

].

] are therefore of considerable importance in ensuring adaptive behavior at maturity.

].

].

We report here both neuroanatomic and genetic quantitative evidence that the size of the striatum and the number of neurons contained within it are modulated independently.

Strain differences

) strains.

= 2.0. ns).

).

As a result of the reciprocal relation between volume and density, there is no significant difference in striatal neuron number among the five strains. Total striatal neuron numbers ranges over a very modest range - from a low of 1.72 ± .015 million in C57BL/6J to a high of 1.93 ± .035 million in BXD5.

Correlational Statistics

). In this crucial respect, results from the genetically heterogeneous F2 animals differ from those of inbred strains.

= 0.73, df = 2, ns) are all normally distributed (Kolmogorov-Smirnov Normality Test).

P < .05.

QTL Analysis

). This last parameter was estimated by subtracting the estimated bilateral striatal weight (assuming a specific gravity of 1.0) from that of the whole brain.

Linkage Statistics for Striatal Volume and Neuron Number

or numbers of cells). The two bold loci marked with asterisks achieve genome-wide significance in this sample population.

.

. Plot for striatal neuron number on Chr. 19. Peak values for the LRS are around 50 cM.

, and the LRS is reduced to 6.9, a value which still has a point-wise probability of only 0.03, indicating a significant independent effect.

). Allelic differences in this interval account for up to 30% of the total variance in striatal neuron number. As the heritability of this trait is 0.64, this trait can be said to account for over 80% of the genetic variance. Residual neuron counts have a higher LRS than the total neuron counts (LRS of 15.0 vs. 11.9). This indicates that the Chr 19 interval is likely to have selective effect on the striatum. Consistent with this hypothesis, the LRS for brain weight on distal Chr 19 is under 1.0, and weights of all three genotypes average 480 ± 5 mg. Linkage on Chr 19 is not affected at all by remapping with control for the striatal volume locus on Chr 10. Thus, Chr 10 and Chr 19 intervals do not interact or cooperate in controlling striatal volume or neuron number.

Between-strain variability

]. Taken together, these results point to a high level of variability in neuron number in the CNS of mice.

], a finding supporting the notion that packing density varies significantly among mouse strains.

]. Given the relatively small number of strains that we have sampled, our hypothesis of lower variation in striatal cell populations requires a more extensive test, a problem which we are now pursuing using the large numbers of strains in the Mouse Brain Library .

Verification of QTL Results

.

].

].

].

Brain volume and neuron number

], we speculate that while total neuron number in the cerebrum may relate to total brain weight, the relationship of these two variables is flexible at the regional level.

Subjects

= 6, formally this recombinant inbred strain is known as BXD-5/Ty). One of the ten C57BL/6J subjects was removed from the analysis because values for striatal neuron number were anomalous with Z scores more than 2.5.

] using Jackson Laboratory foundation stock. The F2 mice ranged in age from 35 to 143 days. The standard inbred strains ranged in age from 51 to 365 days. We studied approximately equal numbers of males and females.

Histological Preparation

All brains analyzed in this study are part of the Mouse Brain Library (MBL). The MBL is both a physical and Internet resource. High-resolution digital images of sections from all cases are available at .

-pentyl alcohol in water, 0.5-1.0 ml ip) and perfused through the left ventricle with 0.9% sodium phosphate buffered (PB) saline (pH 7.4) followed by 1.25% glutaraldehyde/1.0% paraformaldehyde in 0.1 M PB (pH 7.40) over a period of 2 to 4 min. An additional 10-ml of double-strength fixative (2.5% glutaraldehyde/2.0% paraformaldehyde) was injected for 1 to 2 min at an increased flow rate. The head with brain was placed a vial with the last fixative and stored at 4°C until dissection.

]. Brains were cut on a sliding microtome at 30 μm in either horizontal or coronal planes. Free-floating sections were stained with cresylechtviolett and four series of every tenth section were mounted on slides and coverslipped (see for further details).

Total Brain Volume

]. Subsequent measurements of striatal volume and neuron packing density were corrected for volumetric shrinkage. The average shrinkage was 62.2 ± 0.4% (a mean residual volume of 37.8%).

Striatal Volume

Volume of the striatum was also determined from serial section analysis using point counting and Cavalieri's rule. Images from the sections were captured at 12.5 x and were projected onto a video monitor. Point counting was performed as above. Volume was computed separately for the right and left sides and corrected for shrinkage.

Striatal Neuron-Packing Density and Neuron Number

corrected for shrinkage. Multiplying the volume of the striatum by its cell-packing density permitted estimation of the number of neurons in that nucleus.

Reliability

We determined test-retest reliability by having an observer blindly re-measure striatal volume on a subset of 10 brains from the collection. The observer not only re-measured the striatal volume from the same series of sections as the original measure, but also estimated volume from a second series of 1 in 10 sections offset by 5 sections from the previous series. The correlations among the three estimations ranged from .95 to .99 (P < .05), indicating a high degree of reliability for this dependent variable.

].

Regression Analysis of Trait Values

The unadjusted striatal estimates vary to a large extent as a result of variation in total brain weight. However, one of our goals in this study is to map QTLs with relatively intense effects on the striatum. For this reason we also have corrected all of the parameters used in the mapping analysis for variation in brain weight using linear regression analysis. We have mapped data with and without compensation for variance in brain weight. The corrected values are referred to as residuals.

Analysis

for original data used to perform this analysis). A Bonferroni/Dunn correction was used for post hoc examination of significant main effects in the ANOVA. This post-hoc test is functionally identical to a Fisher PLSD, but the alpha level is more conservative (.005).

StrMap.qtx

This file contains anatomic data for each of the subjects used in the current experiment.

Click here to download StrAnatData.xls

format.

Click here to download StrMap.qtx

Acknowledgements

This work was supported, in part, by grants HD20806 and NS35485 from the Public Health Service of the USA. The authors wish to thank Dr. Jing Gu, Aaron Levine, Anna Ohlis, and Stefany Palmieri for technical assistance. We thank Richelle Strom for generating the F2 intercross mice.