Herein we provide strong support for the hypothesis that the pathological grooming behavior observed in Hoxb8 mutant mice results from a deficiency in microglia. In support of this hypothesis we have shown that the only detectable Hoxb8 labeled cell lineage in the brain, (the source of the complex, innate behavioral grooming syntax), is microglia. Second, disruption of Hoxb8 function results in the reduction of the total number of microglia in adult mouse brains (i.e. a microglia phenotype). Further, the excessive pathological grooming behavior in Hoxb8 mutant mice can be rescued by transplantation with normal bone marrow. Finally, restricted deletion of Hoxb8 in the hematopoietic system (Tie2Cre) recapitulates the excessive pathological grooming behavior in these mice, while restricted disruption of Hoxb8 in the spinal cord (Hoxc8Cre) does not.
There appear to be two principle sources of microglia in the mouse, a resident population that is present in the brain early during embryogenesis prior to vascularization (Alliot et al., 1999
), and a second population of bone marrow origin, derived from circulating monocytes, that migrate into the brain through the vascular system shortly after birth (Kaur et al., 2001
; Ransohoff and Perry, 2009
). The kinetics of infiltration of Hoxb8
labeled microglia into the brain is consistent with this population being the bone marrow-derived subpopulation. As such, the Hoxb8
lineage provides a useful molecular marker for distinguishing between these two microglial subpopulations. Do they have similar or different roles in the brain? Molecular markers allow genetic interrogation of the system. What would be the consequences of selectively ablating only one population? Interestingly, although Hoxb8
labeled microglia represent only 40% of the total microglial population present within the adult brain, selective inactivation of Hoxb8
in this subpopulation, is sufficient to induce the pathological grooming behavior. This fact would favor the hypothesis that the two microglial subpopulations present in the brain are performing distinguishable roles.
Microglia could affect neuronal activity and behavior by a number of mechanisms, including the secretion of cytokines that stimulate or inhibit neuronal activity, and work in parallel with neurotransmitters. Microglia have also been reported to function in regulating neuronal cell death during embryogenesis (Frade and Barde, 1998
; Marin-Teva et al., 2004
). Absence of appropriate cell death during neurogenesis could manifest itself later as aberrant behavior. Finally, the experiments of Wake et al. (2009)
illustrating that microglia processes are very dynamic and engage in intimate contacts with synapses are particularly intriguing. They observed that the duration of contact at synapses is dependent on neuronal activity. From the above, it is becoming apparent that due to their mobility and dynamic contacts with synapses, microglia could represent an additional system for stabilizing and managing neural networks. By virtue of their high abundance in the cortex, including the frontal orbital regions and basal ganglia, the microglia of Hoxb8
lineage are positioned in close proximity to the pathways controlling repetitive behavior.
Obsessive compulsive disorder in human patients is associated with three principal brain regions: the prefrontal cortex, particularly the orbitofrontal cortex and anterior cingulated cortex; basal ganglia, including dorsal striatum and globus pallidus; and thalamus, namely the dorsal medial nucleus (Graybiel and Rauch, 2000
; Huey et al., 2008
) . Excessive grooming in rodents is widely believed to mimic the key traits of obsessive compulsive disorders. Although the syntactic grooming chain can be fully executed in rats decerebrated at various mesencephalic levels, indicating that neural circuits specifying the basic sequential structure are all present within the brainstem (Berridge, 1989
), cortex and striatum play important roles in modulating the initiation and completion of grooming bouts (Berridge and Whishaw, 1992
). Dopamine is a prominent neurotransmitter for implementation of the grooming pattern (Taylor et al. 2010
). However, Welch, et al. (2007)
have reported OCD-like behaviors in Sapap3
mutant mice that have reduced synaptic transmission in glutamanergic cortico-striatal circuits, and they further showed that the compulsive grooming in these mutants is alleviated with serotonin reuptake blockers. Thus, it is apparent that multiple brain regions and signaling pathways control the frequency of repetitive behaviors.
An alternative hypothesis has been put forward that the pathological grooming observed in Hoxb8
mutant mice is due to the sensory defects resulting from impaired formation of the spinal cord (Holstege et al., 2008
). However, all of the experiments that we have presented contradict this hypothesis and have instead indentified a defect in the hematopoietic system, and more specifically a deficiency in microglia as likely causative for the aberrant grooming behavior. Our experiments have clearly separated the pathological grooming behavior from the sensory spinal cord defects to distinct cellular compartments. However, our experiments do not rule out the possibility that at later stages of the pathology the spinal sensory defects could exacerbate the consequences of excessive grooming.
Some of the apparent differences in the interpretation of Hoxb8 mutant phenotype by the Deschamps and our laboratories may result from monitoring different behavioral features. We monitor the time spent grooming (on Laboras platforms) whose excesses in Hoxb8 mutant mice leads to pathological behavior and very broad hair removal and skin lesion patterns. Grooming can be distinguished from scratching by the Laboras platforms. We did not observe increased scratching in our Hoxb8 mutant mice except modest increases at the very late stages of the pathology when lesions become apparent and the animals would normally be euthanized. The Deschamps laboratory reports very localized hair removal and skin lesions which appear more consistent with a response to a localized chronic itch. What accounts for the differences in phenotypic outcomes between the two Hoxb8 mutant mice? Differences in genetic backgrounds is not likely to be a strong contributor since both lines have been crossed to a predominantly C57Bl/6J background.
Notably, the Hoxb8
mutant alleles are different. This is a concern because the density of genes is very high within the Hox
complex, and there are many non-coding RNA transcripts as well as protein encoding transcripts transcribed within this complex (Mainguy et al., 2007
). The Deschamps allele is a LacZ knockin into the first exon of Hoxb8
. Our allele was generated by introduction of a nonsense codon in the first exon and a loxP
site into the second. Both inserts are small relative to the LacZ insert. We have shown that a knockin allele of neor
into the Hoxb8
locus shows additional phenotypes due to perturbation of neighboring Hox
gene expression, which disappear upon removal of the neor
gene (Greer and Capecchi, 2002
). Similarly, the LacZ gene could perturb neighboring RNA and/or protein expression, and thereby altering phenotypes. Consistent with this interpretation, in mice homozygous for a knockin allele of CreER™
inserted into the first exon of Hoxb8
(supplemental Figure S1E
), we have observed the localized hair removal and skin lesion phenotype described by Holstege et al, 2008
in their Hoxb8
mutant (supplemental Figure S4D
). This new phenotype shows up at a low penetrance (~10%) in addition to our characteristic Hoxb8
mutant grooming phenotype.
We have demonstrated that a deficiency in hematopoiesis of Hoxb8 mutant mice is causal to the pathological grooming deficit observed in these mutant animals. This deficit is correctable by normal bone marrow transplants. We have argued that the grooming malbehavior is primarily manifested by a deficit in microglia derived from bone marrow. However, a deficiency in T and/or B cells may also contribute to the severity of the behavioral pathology. Also, we have not ruled out other cellular members of the hematopoietic system as potential contributors to Hoxb8 pathological grooming.
Why couple behavior such as grooming to the host's immune system? From an evolutionary perspective it may make perfect sense to couple a behavior such as grooming, whose purpose is to reduce pathogen count with the cellular machinery, the innate and adaptive immune systems, used to eliminate pathogens.
In summary, we have provided strong support for the hypothesis that the excessive pathological grooming behavior exhibited by Hoxb8 mutant mice is caused by a defect in microglia. That a behavioral deficit could be corrected by bone marrow transplantation is indeed surprising. The therapeutic implications of our study on amelioration of neurological behavioral deficits in humans have not escaped us. This mouse model provides an opportunity to determine how impaired microglia results in generating such a distinct compulsive behavioral anomaly. Further, since the Hoxb8 lineage specifically marks the microglia subpopulation derived from bone marrow, this mouse can also be used to genetically interrogate this cell subpopulation relative to the resident microglial subpopulation.