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.
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Introduction
Grooming is an evolutionarily well-conserved innate behavior of rodents and other mammalian species that is essential for survival. The grooming circuit induces hierarchically ordered set of actions, which in Hoxb8 mutants are characterized by compulsive excessive grooming, hair removal and lesions at the sites of overgrooming.1,2,3,4,5 Hoxb8 mutant analysis suggested that defective microglia underlie the behavioral deficits.4 How Hoxb8 gene disruption alters neural circuit, induces behavioral dysfunctions and cause neuronal pathology has neither been addressed nor has such ensuing neural damage been previously defined. Functional imaging in humans6,7,8 and genetic mutational studies in SAPAP3, Slitrk5 and Shank3 mutant9,10,11 mice has pointed to corticostriato-thalamo-cortical circuit defects as a basis for obsessive-compulsive disorder (OCD) pathogenesis.12,13,14,15,16,17
To address the role of Hoxb8 gene function in neuronal pathology we examined neuronal integrity of Hoxb8 mutants and evaluated corticostriatal structural and functional synaptic impairments. We further demonstrate that Hoxb8 mutants exhibit anxiety and social interaction behavioral dysfunction in addition to pathological grooming. These behaviors in mutants are rescued by long-term fluoxetine treatment similar to humans.18 This work ties together Hoxb8 gene-induced neural pathology with the neural SAPAP3, SlitrK5 and Shank3 mutant9,10,11 mice models of OCD and autism spectrum disorder (ASD). We infer that the Hoxb8 gene in the Hoxb8 cell lineage normally modulate the corticostriatal circuit and controls grooming behavior. The absence of Hoxb8 function in microglia leads to aberrant modulation and physical impairment of these neural circuits leading to OCD-like compulsive behavior in mice.
Materials and methods
All experiments were approved by the The Institutional Animal Care and Use Committee, University of Utah.
Electron microscopy
Tissues were fixed (24 h) in 1% formaldehyde, 2.5% glutaraldehyde, 3% sucrose and 1 mM MgSO4 in 0.1 M cacodylate buffer, osmicated (60 min) in 0.5% OsO4 in 0.1 M cacodylate buffer, processed in maleate buffer for en bloc staining with uranyl acetate and processed for resin embedding.19 Sixty to 90 nm sections were mounted onto Formvar Films (Ted Pella, Redding, CA, USA) and imaged (GATAN Ultrascan 4000, Pleasanton, CA, USA) at 80 KeV (JEOL, Peabody, MA, USA, JEM-1400-EM, × 5000 magnification and nanometer resolution) from 1 × 1 × 1 mm3 tissue volume. IR tools mosaicked transmission electron microscopy data and corrected aberrations and electron-optical distortions after mosaic construction on individual tiles. All statistics used for electron microscopy analysis per sample per genotype and grouped analysis is summarized in Supplementary Table 2. Sample size for data analysis was determined by power analysis and literature.9,11 Spine and synapse quantification was performed by an experimenter blinded to genotype and the brain region.
Slice electrophysiology
Slices from isolated brains were placed in ice-cold (4 °C) oxygenated sucrose-based artificial cerebral spinal fluid (ACSF) (95% O2/5% CO2) containing (in mM): sucrose (180.0), KCl (3.0), Na2PO4 (1.4), MgSO4 (3.0), NaHCO3 (26.0), glucose (10.0) and CaCl2 (0.5). ACSF contained (in mM): NaCl (126.0), KCl (3.0), Na2PO4 (1.4), MgSO4 (1.0), NaHCO3 (26.0), glucose (10.0) and CaCl2 (2.5) (pH 7.3–7.4, 290–300 mOsm).
Corticostriatal long-term potentiation
Field excitatory postsynaptic potentials were recorded (30–31 °C) from slices perfused with oxygenated ACSF (2.5 ml min−1). Concentric bipolar stimulating electrodes were placed in dorsomedial striatum at its interface with corpus callosum.20,21,22 Recording microelectrodes were placed near (250 μm) stimulating electrodes in dorsomedial striatum. Field excitatory postsynaptic potentials were evoked with 100 μs stimuli (1–40 V, stimulation strength 50% of minimal and maximal field excitatory postsynaptic potential amplitudes).
Whole-cell recordings
Acute brain slices (300 μm thickness) were cut and recovered (45–60 min) in a submerged chamber (31 °C) with ACSF (in mM) 125 NaCl, 2.5 KCl, 2.0 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 25 NaHCO3 and 15 D-glucose (pH 7.4, 300–310 mOsm), and perfused with oxygenated (95% O2/5% CO2) ACSF at 2 ml min−1 at 31 °C. Internal solution was (in mM) 107 CsMeSO3, 10 CsCl, 3.7 NaCl, 5 TEA-Cl, 20 HEPES, 0.2 EGTA, 5 lidocaine, 4 ATP-magnesium and 0.3 GTP-sodium salt (pH 7.3, 298–301 mOsm). Data were sampled at 10 kHz with low- and high-pass filter set to 1 kHz and 3 Hz. Sample size for data analysis was determined by power analyiss and literature. Sample size for all electrophysiological experiments was determined based on the literature.9,10,11,20,21,22,23
Grooming behavior
Grooming assay used vibration-sensitive platforms and Laboras software (Hoofddorp, The Netherlands) for data analysis. Parameters extracted from grooming assay is shown in Supplementary Table 3 and statistical summary is provided in Supplementary Table 5.
Elevated plus maze, open-field and light-dark test
Anxiety was tested in plus maze (5 × 35 × 15 × 40 cm), open-field (40 × 40 × 35 cm) and light-dark arena (40 × 40 × 35 cm). Mouse movement was tracked using ANY-Maze software (Stoelting, Wood Dale, IL, USA). Parameters extracted from anxiety assay are shown in Supplementary Table 4 and statistical summary is provided in Supplementary Table 5.
Three-chambered social assay
Thirty-minute social assay was performed in three-chambered compartment. Test mouse was placed in center while intruder mouse in right with left chamber being empty. After placing test mouse at the center for 10 min habituation, left and right doors were opened for test mouse to interact with empty or intruder chamber. Test mice movements were tracked using Laboras. Statistical summary is provided in Supplementary Table 5.
Fluoxetine treatment
Mice that underwent fluoxetine or saline treatment were acclimated in home cages for >7 days. Wild-type (WT) and mutants were intraperitoneally injected (5 mg kg−1) with fluoxetine once a day for 1 day, 1, 5 and 13 weeks. Sample size for all behavioral and drug treatment conditions were determined by literature9,10,11 and power analysis. Statistical summary is provided in Supplementary Table 5.
All behavioral experiments were conducted blindly with the experimenter blind to genotype and drug treatment conditions.
Results
Hoxb8 mutants show altered pre- and postsynaptic structures
We first determined whether Hoxb8 mutants show altered corticostriatal synapse morphology similar to those shown to be defective in SAPAP3 and Slitrk5 mutant mice, well-studied mouse models of OCD. We measured dendritic spine density in frontal cortical and striatal neurons using Golgi–Cox staining. Mutants exhibit significantly higher spine density in the frontal cortex but lower density in the striatum (Figures 1a, c and d) implying distinct effects of Hoxb8 mutation on spine maintenance within cortex and striatum. Spines averaged per mouse reproduced the average value (Supplementary Table 1d–e). To identify the dendritic spine density changes in OCD-specific striatal brain regions, as opposed to global spine density changes in the entire striatum, we quantified spine density from dorso and ventro-medial striatum of WT and Hoxb8 mutants. Spine density in Hoxb8 mutants increased significantly in both dorso- and ventro-medial striatal regions (Supplementary Figures 1c, e, d and f) without affecting spine density from the visual cortex (Supplementary Figures 1b and g), a control brain region that is independent of OCD circuit. These data suggests that distinct striatal sub-regions show diverse dendritic spine phenotype.
To identify whether the Hoxb8 mutation affects synaptic structures, we examined asymmetric (excitatory) and symmetric (inhibitory) cortical and striatal synapses using electron microscopy. Representative synapses are displayed and modeled (Figure 1b and Supplementary Figure 3a). At asymmetric and symmetric cortical synapses, synaptic length increased significantly compared with WT (Figure 1e and Supplementary Figures 2m and n). Contrastingly, synaptic length decreased significantly at striatal asymmetric and symmetric synapses (Figure 1f and Supplementary Figures 2o and p), suggesting defective but contrasting cortical and striatal structural synapse in mutants. To determine postsynaptic alteration, we quantified postsynaptic density (PSD) length and thickness. PSD length in mutants shifted rightward (Figures 1g and h) in cumulative distribution and was significantly longer at asymmetric and symmetric cortical synapses (Figure 1o). Contrastingly, PSD thickness of asymmetric and symmetric cortical synapses of mutants shifted leftward in cumulative distribution (Figures 1i and j) and were significantly smaller than WT (Figure 1q). Synaptic expansion in mutants concurrently increased pre-synaptic diameter at asymmetric and symmetric synapses, but expanded postsynaptic diameter only at asymmetric synapses (Supplementary Figures 3b–e, j and l, and Supplementary Table 1). To identify whether synaptic structural changes affect active zone, we quantified active zone length and thickness at cortical synapses. Mutants exhibit longer active zones at asymmetric and symmetric synapses (Supplementary Figures 2a, b and i, and Supplementary Table 1). Changes in the active zone thickness were insignificant at either of the synapses (Supplementary Figure 2k), although cumulative distribution showed statistical significance for symmetric synapses (Supplementary Figures 2d and c, and Supplementary Table 1). Together the data imply that the Hoxb8 mutation affects the cortical pre- and postsynapses and results in synaptic expansion at asymmetric and symmetric synapses. As we detected synaptic expansion within frontal cortical synapses, we asked whether such structural alteration result in more or stronger synapses? We analyzed total excitatory and inhibitory synapses within frontal cortex and observed that mutants exhibit significantly increased mono- and total asymmetric but not symmetric synapses (Supplementary Figures 2q–t) implying increased synaptic density in mutants in consistence with synaptic structural modification.
In contrast to cortex, Hoxb8 mutants showed a significant reduction of PSD length (Figure 1p) and shifted cumulative distribution leftward at asymmetric (Figure 1k) and symmetric (Figure 1l) striatal synapses. PSD thickness of asymmetric and symmetric striatal synapses in mutants was significantly greater than WT (Figure 1r) and shifted cumulative distribution rightward (Figures 1m and n). Synaptic contraction concurrently reduced pre and postsynaptic diameter with leftward shifting of cumulative distribution (Supplementary Figures 3f–i, k and m, and Supplementary Table 1). Similar to pre- and postsynaptic contraction, mutants exhibit smaller active zones at asymmetric and symmetric synapses (Supplementary Figures 2e, f and j, and Supplementary Table 1). Contrast to cortex, active zone thickness shrunk at asymmetric but expanded significantly at symmetric striatal synapses (Supplementary Figures 2g, h and l, and Supplementary Table 1), indicating that Hoxb8 mutation affects striatal and cortical synapses differentially (Supplementary Tables 1c and 2). Synaptic analysis revealed a significant increase in mono and total synapses at asymmetric excitatory but reduced mono and total synapses at symmetric synapses (Supplementary Figures 2u–x) in mutants consistent with the synaptic structural modification at cortical and striatal synapses in Hoxb8 mutants, leading to an increased excitation at the corticostriatal circuit as has been reported for SAPAP3, Slitrk5 and Shank3 mutant mice.9,10,11
Altered CNQX-insensitive long-term potentiation and miniature excitatory currents in Hoxb8 mutants
We investigated whether altered pre- and postsynaptic structure observed in frontal cortex and striatum affected corticostriatal neurotransmission in Hoxb8 mutants. The input–output curves, slope normalized to peak response, field potential amplitudes and paired-pulse ratio at striatal synapses revealed insignificant difference between mutant and WT slices (Supplementary Figures 4a–c and Figure 2a) in response to layer 5/6 cortical fiber stimulation implying that the electrophysiological properties and short-term plasticity is intact in mutants.
To probe presynaptic plasticity, population spike responses were measured in dorsal striatum. Single pulse layer 5/6 cortical stimulation evoked a population spike that was dependent on synaptic glutamate release and postsynaptic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor)/kainate receptors.20 Population spike consisted of non-synaptic (S1, source current) presynaptic component and synaptically mediated postsynaptic component (S2, sink current)20,21,22,24 (Figures 2b and c). Because of non-laminar striatal organization, sink and source currents (a) overlap in space, (b) arise from action potentials with shorter latency and (c) are CNQX-insensitive and interchangeable measures of presynaptic depolarization.
In control experiments, CNQX, (AMPA/kainate receptor antagonist) blocked the S2 but not S1 component (Figure 2b), indicating that the action potential dependent component is CNQX-sensitive (S2), whereas the independent component is CNQX-insensitive (S1). To investigate whether mutants show changes in S2 versus S1, tetanic stimulation was applied to dorsomedial striatum and long-term potentiation was measured. Strikingly, the S1 increased significantly in mutants compared with WT (Figure 2d) without affecting the S2 (Figure 2e), suggesting that the loss of the Hoxb8 gene affects CNQX-independent (cortical driven) but not CNQX-dependent (intra-striatal) long-term potentiation. S1 versus S2 field potential amplitudes did not differ significantly (Supplementary Figure 4d), implying that the postsynaptic striatal output is smaller despite presynaptic changes consistent with the reduced spine density, synapse and PSD length of mutants (Figures 1d, f and p) at striatal synapses analyzed.
As cortical and striatal spine density and PSD morphology was altered, we questioned whether electrophysiological signature of such synaptic change exist in dorsomedial striatum. We recorded AMPA-receptor-mediated excitatory miniature evoked postsynaptic currents (mEPSCs) from dorsal medium spiny neurons (MSNs). Two distinct mEPSC types were detected in MSNs of mutants that were distinguishable by response kinetics and decay time, and categorized into slow (Group 1) and fast (Group 2) miniature events (Figures 2g and h, and Supplementary Figure 4f). Representative events are displayed in Figure 2h. Group 1 MSNs of mutants exhibit significantly longer inter-event interval with higher amplitudes (Figures 2g, i and k), whereas group 2 MSNs showed significantly reduced inter-event interval and mEPSC amplitudes relative to WT mice (Figures 2g, j and l), implying that the probability of the presynaptic neurotransmitter release and postsynaptic AMPA receptors might be affected in opposite way. These effects were prominent when rise and decay times (Supplementary Figures 4g–j), mEPSC charge, event half-width (Supplementary Figures 4k–n) and frequency (Supplementary Figures 5a and b) were analyzed. The relationship between (a) mEPSC amplitude and 10–90% rise time (Supplementary Figures 5c and d), (b) amplitude and average rise rate (Supplementary Figures 5e and f) and (c) event half width and 10–90% rise time (Supplementary Figures 5g and h) were distinguishable between WT and mutants. These data imply that mEPSC events of MSNs are affected in mutants at single neuronal level.
Hoxb8 mutants show impaired grooming, anxiety and social behaviors
To determine the role of Hoxb8 mutation in grooming, we used 24 h automatic Laboras platform containing sensitive vibration detectors to detect grooming and non-grooming behaviors.23 Reproducibly,3,4 Laboras detected significantly higher self-grooming behavior in mutants compared to WT mice (Figure 3a and Supplementary Tables 3).
Two features common to OCD and ASD are hyperanxiety and social behavioral deficits.9,10,11,15 We investigated whether such behaviors in mutants are altered. In plus maze test, mutants spent significantly more time in closed arm compared with WT mice (Figures 3b and c) without altering distance in closed arm (Supplementary Figure 6a), implying that mutants are more anxious in plus maze. Mutants spent significantly decreased time in open arm under different light intensities (1.7-fold at 75 lx, 2.7-fold at 100 lx; Supplementary Figure 6b), traveled for shorter distances (1.5-fold, 75 lx; 3.2-fold, 100 lx; Supplementary Figure 6c) and had longer latencies (3.7-fold, 75 lx; 4.9-fold, 100 lx; Supplementary Figure 6d) than controls.
In open-field test, an additional indicator of anxiety level, mutants exhibit significantly reduced- time at the center (Figures 3d and e), line crossings, entries and circling numbers (Supplementary Figures 6f and e), implying higher anxiety levels relative to controls. Both male and female mutants showed higher anxiety levels (Supplementary Table 4). In light–dark test, mutants spent reduced time in light (Figure 3f) and more time in dark chamber (Supplementary Figure 6h) compared with WT mice without affecting the total light zone entries (Supplementary Fig. 6i). All these anxiety tests suggested that Hoxb8 mutants exhibit hyperanxiety levels relative to controls.
We tested social behaviors in Hoxb8 mutants using a three-chambered social assay in which the test subject (WT or mutant, male or female) is placed at the center, with an empty left chamber and an intruder mouse in the right chamber. Mutants spent significantly reduced time socially interacting with the intruder (Figure 3h). Heat maps derived from male and female mutants (Figure 3g) during social assay show evidence that female mutants exhibit significantly higher social impairment compared with males. To determine whether mutants interact normally with cage-mates, test mice were challenged to interact with a cage-mate in social assay. Mutants showed a significant social deficit even with a cage mate, a defect that was confined to the female mutants (Figure 3i and Supplementary Figure 6j), indicating that female mutants show higher detectable social impairment than males.
Fluoxetine treatment of Hoxb8 mutants alleviates grooming, anxiety and social impairment behaviors
We evaluated whether fluoxetine ameliorates behavioral abnormalities in Hoxb8 mutants. One week, but not 1-day treatment reduced grooming time of mutants without significantly affecting the WT mice (Supplementary Figure 7a). No sedative effect was detected as measured by locomotion and immobility between saline or fluoxetine-treated groups, implying the appropriateness of drug dosage (Figures 4b and c). To determine long-term effects of fluoxetine on anxiety, mutants and WT mice were chronically treated for five weeks. In plus maze test, treated mutants spent a 3.4-fold increased time in open-arm compared with saline-treated mutants (Figure 4d). Treated mutants that spent more time at the center of the open field compared with saline treated mutants did not reach WT levels (Figure 4e), implying a partial rescue. When exposed to a novel intruder, treated mutants compared with WT mice spent a reduced time in empty chamber and similar time at the center or with the intruder mouse (Figures 4g and h), implying that the treatment resulted in improved social behaviors. To investigate whether the alleviated behaviors are sustained during chronic fluoxetine treatment, mutants and WT mice were treated with fluoxetine upto thirteen weeks. Anxiety and exploratory locomotion were tested using light–dark and open-field tests. The distance at the center or periphery of open field (Supplementary Figures 7b and c) and the time in light zone (Supplementary Figure 7d) reached WT-like levels implying extensive rescue. Amount of time spent by mutants in light zone during light–dark exploration increased to WT levels (Figure 4f). These data demonstrate that fluoxetine treatment alleviates pathological grooming, anxiety and aberrant social behaviors in Hoxb8 mutants.
Discussion
Targeted disruption of Hoxb8 gene causes brain-specific3,4 compulsive hair removal behavior closely resembling the OCD spectrum disorder Trichotillomania in humans.24 We now show that Hoxb8 loss of function results in synaptic and physiological defects within corticostriatal circuit. This corticostriatal defect in mutants resulted in frontal cortical synaptic expansion and striatal synaptic contraction. The increased cortical synapse and spine density within frontal cortex in conjunction with increased dendritic spines in dorso- and ventro-medial sub-regions of striatum implicate a potential increase in excitatory corticostriatal synapse.
These synaptic modifications were also detectable electrophysiologically. At circuit level we detected impaired CNQX-independent striatal synaptic long-term potentiation. This plasticity may emerge from changes in presynaptic medial prefrontal cortical neurons synapsing onto striatal MSNs during action potential firing at frequencies that induce synaptic plasticity. mEPSC recordings from individual MSNs showed two distinct mEPSCs, higher amplitude, low-frequency and longer inter-event interval (group 1) and the lower amplitude, high-frequency and reduced inter-event interval (group 2), implying the sensitivity of whole-cell recordings to measure synaptic properties of MSNs.
Mutants exhibit grooming, anxiety and social behavioral impairment. Fluoxetine alleviates grooming, anxiety and social deficits in mutants similar to Sapap3-, Slitrk5- and Shank3-based OCD/ASD mouse models and human OCD patients.7,25,26,27,28,29,30,31,32
A direct correlation of immune dysfunction and neuropsychiatric disorders is observed in major depression, OCD, autism, schizophrenia and Alzheimer’s disease.33,34,35,36,37,38,39,40,41,42,43,44 Consequences of Hoxb8 gene deficiency that results in corticostriatal synaptic aberration provides an independent way that the brain appear to utilize, to mediate and modulate repetitive behaviors. Interestingly, the loss of Hoxb8 gene function that results in excessive grooming behavior was not restricted to repetitive behaviors since we detected fluoxetine-sensitive hyperanxiety and social behavioral deficit in Hoxb8 mutants similar to trichotillomania-type OCD patients.31,32,44,45,46
Although the causative agents of the neuropsychiatric disorder in Hoxb8-mutant mice versus Sapap3, Slitrk5 and Shank3 mutant mice are very different, defective microglia versus defective synaptic components, the end results, both in terms of behavioral deficits, including high levels of anxiety and the affected neural circuits, the corticostriatal interface, are very similar. These results strongly support the hypothesis that in the absence of proper maintenance of circuit modulation by Hoxb8-microglia, very similar neural circuit damage ensues in Hoxb8, Sapap3, Slitrk5 and Shank3 mutant mice, resulting in very similar behavioral pathology.
Hoxb8 gene thus appears to play an important role in maintaining brain homeostasis in regulating corticostriatal circuit function and behavioral output. Hoxb8 gene dysfunction would alter synaptic morphology and physiological properties and thereby affect behaviors, the output of which would depend on the constellation of genetic and environmental insults pertinent to individual patient. Such models tie together immune dysfunctions, particularly pertaining to Hoxb8 gene function through microglia,25,47,48,49,50,51,52 the brain’s immune system, with repetitive and anxiety behaviors and a spectrum of neuropsychiatric disorders.
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Acknowledgments
We thank A Boulet and K Higgins for insightful discussions on the manuscript and editing, SK Chen for experimental discussion, H Eldering, BV Metris, for technical support, S Matsuoka for genotyping, and A Gosdis and V Krishnegowda for their participation in early experiments. This work was supported by Seed grant and research instrumentation funding from University of Utah to NN and MRC, HHMI, NIH/NEI, EY02576-36, EY015128-8 and EY014800-08 (P30) to BWJ and RM.
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Nagarajan, N., Jones, B.W., West, P.J. et al. Corticostriatal circuit defects in Hoxb8 mutant mice. Mol Psychiatry 23, 1868–1877 (2018). https://doi.org/10.1038/mp.2017.180
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DOI: https://doi.org/10.1038/mp.2017.180
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