Corticostriatal circuit defects in Hoxb8 mutant mice

Abstract

Hoxb8 mutant mice exhibit compulsive grooming and hair removal dysfunction similar to humans with the obsessive-compulsive disorder (OCD)-spectrum disorder, trichotillomania. As, in the mouse brain, the only detectable cells that label with Hoxb8 cell lineage appear to be microglia, we suggested that defective microglia cause the neuropsychiatric disorder. Does the Hoxb8 mutation in microglia lead to neural circuit dysfunctions? We demonstrate that Hoxb8 mutants contain corticostriatal circuit defects. Golgi staining, ultra-structural and electrophysiological studies of mutants reveal excess dendritic spines, pre- and postsynaptic structural defects, long-term potentiation and miniature postsynaptic current defects. Hoxb8 mutants also exhibit hyperanxiety and social behavioral deficits similar to mice with neuronal mutations in Sapap3, Slitrk5 and Shank3, reported models of OCD and autism spectrum disorders (ASDs). Long-term treatment of Hoxb8 mutants with fluoxetine, a serotonin reuptake inhibitor, reduces excessive grooming, hyperanxiety and social behavioral impairments. These studies provide linkage between the neuronal defects induced by defective Hoxb8-microglia and neuronal dysfunctions directly generated by mutations in synaptic components that result in mice, which display similar pathological grooming, hyperanxiety and social impairment deficits. Our results shed light on Hoxb8 microglia-driven circuit-specific defects and therapeutic approaches that will become essential to developing novel therapies for neuropsychiatric diseases such as OCD and ASDs with Hoxb8-microglia being the central target.

Figure 1. Altered cortical and striatal synapses in Hoxb8 mutants

a) Representative dendritic spines from 6 months old WT and Hoxb8 female mutant mice from frontal cortical and dorsal striatal regions. Scale bar: 1 µm. b) Representative electron microscopic images of cortical-asymmetric, cortical-symmetric, striatal-asymmetric and striatal-symmetric synapses from 6 months old WT and Hoxb8 mutant brains and visualized using Viking software ^, . Scale bar: 100 nm c, d) Significantly increased cortical (P=0.0034, F=9.118, 28 WT and 29 mutant neurons, 2–3 healthy dendrites per neuron) (c) but decreased (d) striatal spine density (P=0.001, F=10.488, 13 WT and 18 mutant neurons, 2–3 healthy dendrites per neuron) in female Hoxb8 mutants (3 litters per group, 8 months old mice) using golgi staining (Fd Neurotechnologies inc). ‘n’ represents the total dendrites analyzed per genotype per brain region. e, f) Bar plot displaying significantly increased synapse length at cortical asymmetric (P=0.00012, F=14.8310) and symmetric (P<0.0001, F=19.8205) (e) but a significantly decreased striatal asymmetric (P<0.0001, F=138.0321) and symmetric synapses (P<0.0001, F=37.9242) (f). (g–j) Cumulative probability plot demonstrating a significant rightward shift in the PSD length in Hoxb8 mutants compared to WT mice (g, P<0.0001, D=0.2815; h, P<0.0001, D=0.1992) but a significant leftward shift in PSD thickness for cortical asymmetric and cortical symmetric synapses (i, P<0.0001, D=0.3187; j, P<0.0001, D=0.2045). (k–n) A contrasting significant decrease (leftward shift) within striatal-asymmetric (k, P<0.0001, D=0.1595) and symmetric (l, P<0.0001, D=0.1156) synapses for PSD length and PSD thickness (m, P<0.0001, D=0.28; n, P<0.0001, D=0.15). (o–p) Bar graph representation of significantly increased PSD length at cortical-asymmetric (P<0.0001, F=93.2869) and cortical-symmetric (P<0.0001, F=53.6979) (o) synapses of Hoxb8 mutants but a contrasting decrease within striatal-asymmetric (p) (P<0.0001, F= 22.1849) and striatal-symmetric synapses (p) (P<0.0001, F= 18.1742). (q–r) Bar graph representation of significantly decreased PSD thickness at cortical-asymmetric (P<0.0001, F=192.3888) and cortical-symmetric (P=0.014, F=5.9328) (q) synapses of Hoxb8 mutants but a contrasting increase within striatal-asymmetric (P<0.0001, F=30.8782) and striatal-symmetric (P<0.0001, F=17.4861) (r) synapses. Red arrow represents post-synaptic density in individual synapse. All analysis was conducted on WT and Hoxb8 mutant female mice brains. One-way ANOVA and Tukey’s posthoc test (c–f, o–r). Kolmogorov-Smirnov test, (g-n).

Figure 2. Altered CNQX independent form of LTP and miniature EPSCs in Hoxb8 mutants at corticostriatal synapses

a) Paired-pulse ratio is unchanged in mutants (P=0.3453, F (28, 1680)=1.086 for interaction; P<0.0001, F (28,1680)=10.09 for interpulse interval, P=0.9560, F(1, 60)=0.003074 for genotype; P<0.0001, F(60,1680)=76.89 for subjects matching). Paired-pulse ratio of the fEPSP amplitudes was assessed using inter-pulse intervals between 20–300 ms in 10 ms intervals. Single stimuli were given to slices every 30 s for a 30 min to collect baseline. Representative traces and measurements are the average from five consecutive traces. b) Representative trace of field potentials in ±CNQX condition (black and red trace). S1, non-synaptic and S2, synaptically mediated component. Overlaid traces show the sensitivity of S1 and S2 component to CNQX. c) Overlaid trace of field potential recording under control (black) and LTP (red) condition showing S1 and S2 components. Scale bar, 0.1mV (Y-axis) and 2 ms (X-axis). d) Increased CNQX-insensitive LTP for WT (black) and mutants (red) (P=0.1665, F (60, 3300)=1.177 for interaction; P=0.0014, F(60, 3300)=1.642 for interpulse interval, P=0.0378, F(1, 55)=4.531 for genotype; P<0.0001, F (55, 3300) = 524.5 for matched subjects). LTP was induced by high frequency stimulation (1 second/100 Hz). Low frequency stimulation was resumed for 60 min to quantify LTP relative to baseline. e) Normal CNQX-sensitive LTP for WT (black) and mutants (red) (P=0.9477, F(60, 3060)=0.7213 for interaction; P=0.0316, F(60, 3060)=1.371 for time; P=0.558, F(1, 51)=0.3471 for genotype and P<0.0001, F(51, 3060)=476.7 for matched subjects, Two-way ANOVA repeated measures). Significance was determined at P<0.05. Data were excluded if the slope of fEPSPs during the 30 min baseline changed by >20%. f, g) AMPA receptor mediated mEPSC recording traces at −70 mV from dorsomedial striatal MSNs from parasiggital corticostriatal acute brain slices for WT (left) and Hoxb8 mutants (n=3 mice per genotype, 3 week old) isolated in the presence of TTX (1 µM), DL-2-amino-5-phosphonovaleric acid (50 µM), D-Serine (10 µM), Picrotoxin (100 µM) and Gabazine (5 µM) to block action potential, NMDA, GABA and GABAA receptors, respectively in whole cell voltage clamp recording configuration. Based on kinetics, the electrophysiological property of mEPSC events such as decay time and frequency, the responses from Hoxb8 mutants were classified into group 1 and 2. Orange and green bars on the traces represent time periods where no mEPSC activity was detected. Cartoon on the top shows the location of pipette positioning and recording within striatum h) mEPSC individual representative events for WT and Hoxb8 mutants from group 1 and group 2 type of MSN neuronal responses. The amplitude varied among individual events. Small, medium and large amplitude events were recorded and analyzed. (i–l) Cumulative probability plot of WT and Hoxb8 mutants showing rightward shift in the curve for Hoxb8 mutants for inter-event interval and mEPSC amplitudes for group 1 (i, P=0.002, D= 0.08375; P=0.00029, F=13.18906 for inset; k, P<0.0001, D=0.6917; P<0.0001, F=2654.712 for inset) and group 2 neurons (j, P<0.0001, D=0.7023; P<0.0001, F=949.241; l, P<0.0001, D=0.2040; P<0.0001, F=111.279 for inset). Inset represents bar graph from the same data set. ‘n’ represents the number of neurons patched per experimental group obtained from 3 WT and 3 Hoxb8 mutants. Atleast 100 events were sampled per neuron. Series and input resistance were monitored continuously and neuronal recordings were discarded if these parameter changed by > 20%. All experiments were conducted blindly in which the experimenter was unaware of the electrophysiological outcome of different cell types within dorsal striatum. mEPSC’s were analyzed using Minianalysis Synaptosoft software. One-way ANOVA and Tukey’s posthoc test for bar graphs, Kolmogorov-Smirnov test, (i–l). 6 months old WT and Hoxb8 mutant brain slices were used for the slice electrophysiological experiments.

Figure 3. Altered Grooming, anxiety and social behaviors in Hoxb8 mutants

a) Mutants show increased grooming (P<0.0001, F=59.115628) in 24 hour grooming assay as compared to WT mice (4–12 months old). b) Representative track plots of male WT, male mutants, female WT and female mutants in 5 minute elevated plus maze test under ambient light conditions. Note the movement of mutants pertained to closed as compared to open chamber. c) Hoxb8 mutants (6–8 months old) spend reduced time in open (P=0.00474, F=9.10139) and increased time in closed arm (P=0.0005, F=14.7216) (left panel). d) Representative track plots of male WT and male mutant (upper panel), female WT and female mutant (lower panel) in the open field test. e, f) Mutants (6–8 months old) spend significantly reduced (P=0.02369, F=5.70155) time- at the center of the open field in a 30 minute open field test and within the light zone (P=0.00027, F=15.4018) (right panel) in 5 minute light-dark test (illumination 600 Lx). g) Representative heat maps of individual male and female WT and Hoxb8 mutants (Case 1–2) during three-chambered social interaction assay with an empty left chamber and an intruder in the right chamber (same sex as the test mouse). h) Female mutants (6–8 months old) spent significantly reduced time with the intruder mouse in the right chamber (P=0.1148, F=15.40186 for left chamber non-contact duration; P=0.3004, F=2.6407275 for the total duration in the center; P=0.0222, F=5.8829406 for total contact duration with the intruder) in social interaction assay. Data represents Mean±SEM, uses one-way ANOVA and Tukey’s HSD posthoc test for comparison. ns, not significant P>0.05, *P<0.05). i) Bar graph representation of the comparison of social interaction pattern of female Hoxb8 mutants and WT mice (6–8 months old) with an empty chamber (left) and a cage-mate (right) (P=0.0861, F=3.27635 for the non contact duration with the left chamber; P=0.427, F=0.65771 for total duration in the center; P=0.0095, F=8.325 for total contact duration with intruder). The cage-mate was of same age and sex as of experimental mice. All bar graph data represents mean±SEM. Data comparison, one-way ANOVA and Tukey’s HSD posthoc test was used for group comparison. ns, P>0.05 not significant, *P<0.05, **P<0.001. 4–12 months old mice were used for grooming behavior, 6–8 months old mice for elevated plus maze, open field and social behavioral tests. All test mice were conditioned for 5–15 minutes in specific experimental rooms prior to the experiments. All behavioral experiments were conducted during day light period of the light/dark cycle. All experiments were conducted blindly without the knowledge of experimenter to genotypes.

Figure 4. Rescue of grooming, anxiety and social behaviors by chronic fluoxetine treatment

a) Rescue of grooming behavior in age- and sex-matched Hoxb8 mutants (6–12 months) treated with 5 mg/kg fluoxetine in post 5 week treated Hoxb8 mutants (P=0.0001, F(1, 36)=18.95 for interaction; P<0.0001, F(1, 36)= 32.43 for drug; P<0.0001, F(1,36)=54.23 for genotype). b, c) 5-week treatment did not affect locomotion (b, P=0.8259, F(1, 36)= 0.04911 for interaction; P=0.0434, F(1, 36)=4.385 for drug; P=0.2486, F(1,36)=1.375 for genotype) and immobility duration (c, P=0.8108, F(1, 36)=0.05817 for interaction; P=0.1552, F(1,36)=2.108 for drug; P=0.1074, F(1,36)=2.726 for genotype) significantly in mutants. d, e) 5-week treated mutants show increased time in the open arm and the center of the open field. Complete rescue was observed in the plus maze test (d, P=0.0595, F(1,36)=3.788 for interaction; P=0.0170, F(1,36)=6.261 for drug; P= 0.0675, F(1,36)= 3.554 for genotype) but a partial rescue was noted in the open field test (e, P=0.0022, F(1,36)=10.71 for interaction; P= 0.9404, F(1,36)=0.005653 for drug; P=0.1790, F(1,36)=1.871 for genotype) f) 13 week treated Hoxb8 mutants spend more time like WT mice in the light zone as compared to untreated mutants in 5 minute light-dark test (P=0.0013, F(1, 49)=11.56 for interaction; P= 0.0014, F(1,49)=11.50 for drug; P=0.3651, F(1,49)=0.8359 for genotype). g) Representative heat maps of saline- and fluoxetine-treated male and female WT and Hoxb8 mutants (Case 1–2) during three-chambered social interaction assay with an empty left chamber and an intruder in the right chamber (same sex as test mouse). Rescue of social interaction is more prominent in female Hoxb8 mutants. h)Hoxb8 mutants show WT-like interaction time with the intruder mouse post two-week fluoxetine treatment in three-chambered social interaction test. The experimental female mouse was placed in the center chamber. The left chamber was left empty and the right chamber consisted of an intruder mouse of same sex as the experimental mouse (P=0.7438, F(1, 43)=0.1082 for interaction; P=0.0002, F(1,43)=16.45 for drug; P=0.1429, F(1,49)=2.227 for genotype for the time in left chamber; P=0.7295, F(1, 43)=0.1211 for interaction; P=0.3615, F(1,43)=0.8509 for drug; P=0.5411, F(1,49)=0.3796 for genotype for the time in the center chamber; P=0.1147, F(1, 43)=2.593 for interaction; P=0.5048, F(1,43)=0.4523 for drug; P= 0.0126, F(1,49)=6.776 for genotype for the time in the right chamber). 6–12 months old age-matched WT and Hoxb8 mutants were used for the experiments.