BACKGROUND
bioluminescence imaging  is a powerful research technique based on detection of light emission produced by oxidation of luciferin by luciferase  <cit> .
the advantages of this technique in studies of small living animals are the following: high signal-to-noise  ratio with low background signal intensities, low limit and simplicity of detection, wide dynamic range of signal intensities, and applicability to genetic manipulation as well as continuous and quantitative analyses.
bli can also be applied repeatedly to the same animal, thus contributing to the reduction in the number of animals used.
bli can be applied to the detection of the biological signal intensity changes that affect the up- and down-regulations of transcription.
luciferase reporter animals are used in the research fields of inflammation  <cit> , toxicology  <cit> , oncology  <cit> , angiogenesis  <cit> , nutrition  <cit> , and neurobiology  <cit> .
furthermore, split luciferase and bioluminescence resonance energy transfer  techniques have recently been developed for the detection of protein–protein interaction  <cit> .
previously, we established a novel reporter transgenic  mouse strain, namely, arc-luc tg mice, in which the firefly luciferase gene  is driven by the arc promoter  <cit> .
arc, that is, the activity-regulated cytoskeleton-associated protein gene, is one of the immediate early genes , which are highly responsive to various external stimuli in the central nervous system   <cit> .
using arc-luc tg mice, we have successfully detected the bioluminescence signals of arc-luc in the adult mouse brain.
the intensity of bioluminescence signals changed after physiological or pharmacological manipulations and the change well correlated with neuronal-activity-dependent arc expression.
our arc-luc tg mice were established using the c57bl/ <dig> strain because this strain is one of the inbred strains most widely used in the fields of genetical engineering and neuroscience.
however, its black fur, skin pigmentation, and hair regrowth after depilation interfere with bli during development.
thus, the periods of bli were restricted less than 24 h and at an interval of more than 4 weeks  <cit> .
to extend the use of bli at various mouse developmental stages, we have introduced a mutant hl gene into the original arc-luc tg mice to overcome the above-mentioned problems.
here, we examined the availability of arc-luc tg hl mice for bli at various developmental stages under physiological and pathological conditions.
RESULTS
in vivo imaging of arc-luc tg hl mice
previously, we successfully generated arc-luc tg mice for the bli of arc expression  <cit> .
however, the genetic background of the arc-luc tg mice is c57bl/ <dig>  and the black fur and skin pigmentation of this strain attenuate photon emission at various developmental stages .
thus, we introduced the mutant hl gene into the original arc-luc tg mice by crossing them with the hl mice.
the established arc-luc tg hl mice showed rapid induction of iegs after kainic acid  injection .
the fold changes of these iegs after ka injection in arc-luc tg hl mice were the same as those in the c57bl/ <dig> mouse strain .
using cooled ccd camera systems, we detected bioluminescence signals in areas around the nose, brain, paws, kidneys, and testes in the arc-luc tg hl mice .
the distribution of these signals was consistent with those previously reported for arc expression and public database from transcriptome analysis  <cit> .
the continuous bli of an individual animal showed that the bioluminescence signal intensity in the brain gradually decreased from  <dig> to 8 weeks of age .
furthermore, we examined the expression levels of luc and endogenous arc proteins by western blot analysis and found that they were slightly higher in the cerebral cortex at 4 weeks of age than at 8 weeks of age .
these developmental changes in protein expression levels correlated with the changes in bioluminescence signal intensity.
these findings demonstrate that changes in the arc expression level in the entire body during mouse development can be noninvasively detected by the visualization of arc-luc expression in arc-luc tg hl mice.fig.
<dig> comparison of pigmentation of head between c57bl/ <dig> and arc-luc tg hl mouse strains.
a dorsal views of head of c57bl/ <dig>  and arc-luc tg hl  mice at  <dig>  and 8 weeks of age .
the photographs of the c57bl/ <dig> mouse were taken after depilation.
the c57bl/ <dig> mouse at 8 weeks of age  and the arc-luc tg hl mouse at both ages  are examples of suitable samples for bli because of little pigmentation.
the c57bl/ <dig> mouse at 4 weeks of age  is an example of an unsuitable sample for bli with skin pigmentation enclosed in a white dotted circle.
scale bar 10 mm.
b expression of iegs in c57bl/ <dig> and arc-luc tg hl mice.
total rna was extracted from the hippocampus 30 min after ka injection .
the changes in arc, egr- <dig>  and c-fos expression levels were determined by quantitative rt-pcr analysis.
the data represent mean ± sd
fig.
<dig> noninvasive bli using arc-luc tg hl mouse.
a bright field  and bioluminescence signal images  of an individual arc-luc tg hl mouse from  <dig> to 8 weeks of age.
bioluminescence signals in the arc-luc tg hl mouse were obtained after luciferin injection  under anaesthesia induced with isoflurane.
scale bar 20 mm.
b western blot analysis of proteins in the cerebral cortex was performed using anti-luc , anti-arc , and anti-actin  antibodies .
location of protein size markers are indicated on the right side.
the optical density of each band was quantified using nih imagej.
the luc and arc protein expression levels normalized to actin protein expression level at  <dig> and 8 weeks of age are shown in the bar graph .
the data represent mean ± sd.
*p <  <dig> ; two-tailed student’s t test
monitoring responses to sensory stimuli
arc is one of the iegs, which are induced by various sensory stimuli.
we previously reported the activity-dependent and plastic changes in arc expression in the visual cortex of adult arc-luc tg mice  <cit> .
in this experiment, we extended the application of arc-luc tg hl mice to continuous imaging at an interval of less than 7 days.
we examined the pattern of changes in bioluminescence signal intensity during the development of mice in visual-critical period   <cit>  with visual operation .
the arc-luc tg hl mice on p <dig> were monocularly deprived by eyelid suture.
one day after monocular deprivation , the bioluminescence signal intensity significantly decreased in the brain region contralateral to the sutured eye after 3 h exposure to an intense light stimulus.
seven days after continuous monocular deprivation , the bioluminescence signal intensity in this region slightly increased .
furthermore, the reopening  of the sutured eye followed by light exposure resulted in a marked increase in the bioluminescence signal intensity in this region .
for the quantitative assessment of hemispheric dominance of arc expression in the brain, we calculated lateralization index , which is often used in functional magnetic resonance imaging  studies  <cit> .
li decreased to − <dig>   on md <dig>  and it slightly increased on md <dig> but remained below zero .
moreover, li further increased to + <dig>   after ro and returned to about  <dig> one day after ro .fig.
<dig> laterality of bioluminescence signals from visual cortex after monocular deprivation  by suturing and reopening surgery.
a experimental schedule for monocular deprivation and reopening and bli.
the time points of bli are indicated with black triangles.
b the obtained bioluminescence images  before eye suture , 1 day after md , 7 days after md , immediately after reopening , and 1 day after ro .
mice were exposed to an intense light for 3 h before each imaging time point.
the locations of regions of interest  are enclosed in dotted circles in the control image, and the obtained bioluminescence signal intensities are used for the calculation of lateralization index  shown in .
scale bar 10 mm.
c plots showing the li under each condition shown in  .
li was calculated using the formula /, where l and r are the bioluminescence signal intensities of roi in the regions contralateral  and ipsilateral  to the sutured eye, respectively.
li was nearly zero under the control condition , decreased below zero under md conditions , and increased immediately at ro , and finally returned close to the baseline at ro <dig> .
the data represent mean ± sd.
*p <  <dig> ; two-tailed student’s t test
spatial exploration in a novel environment induces arc expression, and object and place recognition tasks induce the expression of some iegs including arc .
we carried out the evaluation of bioluminescence signal intensity changes associated with an object recognition  task.
individually housed mice at 4 weeks of age were provided a novel plastic cube in their home cages .
the object provided induced various exploratory behaviors of mice, such as approaching, sniffing, climbing, biting, and turning them over for several minutes.
no statistically significant differences were detected in the total intensity of bioluminescence signals from the entire forebrain between the or and control groups .
however, we observed marked differences in the distribution of bioluminescence signals from the brain between the two groups.
we divided rois in the forebrain into  <dig> rectangular blocks in the rostral to caudal direction .
the distribution of relative bioluminescence signal intensity normalized to the signal intensity corresponding to the entire-forebrain photon density showed a down-regulation of the signals from the rostral region  and the up-regulation of those from the posterior brain regions including the somatosensory, barrel, and visual cortices  .
therefore, we concluded that the detection of bioluminescence signals in the arc-luc tg hl mice clearly provided detailed neuronal-activity-dependent arc expression changes induced by sensory stimuli during development.fig.
<dig> bioluminescence signal intensity changes associated with or task.
a experimental schedule for or task and bli.
bioluminescence signal intensity was measured in each mouse 6 h after putting a plastic cube into their home cage .
for the control, no object was presented.
bli was also performed without any treatment 1 day before the or task .
the time points of bli are indicated with black triangles.
b representative images of bioluminescence signals  in the arc-luc tg hl mouse brain at 4 weeks of age for the object and control.
location of rois in the forebrain is indicated in the control image.
scale bar 10 mm.
c subdivision of roi in forebrain into smaller rectangles at  <dig>  mm intervals in rostral to caudal direction.
d distribution of bioluminescence signals associated with or .
profiles of photon density throughout the forebrain for object presentation  and control condition .
each value obtained from rois in  was normalized to the mean signal intensity  in the whole forebrain.
significant differences between the control and or conditions are shown in gray  in the graph.
the data represent mean ± sd.
*p <  <dig> ; two-tailed student’s t test
bioluminescence signal intensity changes after ka treatment
ka is derived from a natural marine product from digenea simplex  <cit>  and is an excitatory amino acid receptor agonist selective to ka-type glutamate receptors.
acute treatment with ka is often carried out to produce a neurodegenerative disease model using laboratory animals and also induces arc expression through synaptic activation.
we reported the continuous increase in arc-luc expression level over 6 h after ka injection at the adult stage  <cit> .
here, we extend the examination of the temporal pattern of ka-induced arc expression in the arc-luc tg hl mice at 4 weeks of age .
bli was conducted after intraperitoneal ka injection  .
the bioluminescence signal intensity increased three to fourfold within 3 h after ka injection, and then decreased to about twofold at 6 h. furthermore, a second increase was detected in the posterior region at 12 h and the intensity finally returned close to the baseline at 24 h; this signal distribution pattern was different from that in the control saline-treated group.
these results obtained from the arc-luc tg hl mice suggest the advantages of temporal and quantitative evaluations of the effect of pharmacological manipulation in vivo at various developmental stages without interferences from fur and skin pigmentation.fig.
<dig>
arc-luc expression level changes after ka injection at 4 weeks of age.
a experimental schedule for bli after ka injection.
the time points of bli are indicated with black triangles.
b images  from representative mice taken at 0 h  and  <dig>   <dig>   <dig> and 24 h after ka  or saline  injection.
the locations of rois are indicated in the images at 0 h. scale bar, 10 mm.
c changes in fold induction of bioluminescence signals within 24 h after ka injection .
*p <  <dig> ; two-tailed student’s t test
discussion
bli is a noninvasive, simple, and quantitative method to evaluate brain function.
however, the bli of the mouse brain using the c57bl/ <dig> strain at various developmental stages has some problems such as light attenuation due to its black fur and skin pigmentation and the limited imaging period and interval caused by the hair regrowth after depilation.
to overcome these problems, we established the arc-luc tg hl mouse strain for imaging neuronal-activity-dependent arc expression at various developmental stages.
we examined the reliability of this new mouse strain for investigating neuronal-activity-dependent arc expression processes.
the arc-luc tg hl mice have several advantages that make them useful for investigating the neuronal-activity-dependent changes during development.
first, we can acquire stable bioluminescence signals from the brain without interferences after 3 weeks of age.
albino mice are also useful for bli without shaving, but there remains the problem of light absorbance by white fur  <cit> .
the application of our novel mouse strain to bli is valuable in terms of the increase in the intensity of bioluminescence signals per specified time interval.
as shown in fig.
<dig>  comparison of bioluminescence signal intensities at various developmental stages of the same animal enabled the detection of age-related changes in arc expression level.
second, we can track bioluminescence signals daily and weekly.
because skin pigmentation occurs 5 days after depilation of the c57bl/ <dig> mouse strain, we cannot measure bioluminescence signals in the original arc-luc tg mice for more than five consecutive days.
we have to wait at least 4 weeks for the full regrowth of hair for the next bli.
the arc-luc tg hl mice on the other hand can be used daily and weekly to detect time-dependent changes in arc expression level in the brain by bli .
third, because the use of arc-luc tg hl mice enables bli for extended periods using the same animal, we can investigate the life-long effects of damage incurred from the early stages to the adulthood stage by bli of arc expression using fewer mice.
however, despite these advantages of applying the arc-luc tg hl mice to bli, imaging at  <dig> or 2 weeks of age remains unfeasible because the hair loss in arc-luc tg hl mice starts approximately on p <dig>  there are several reports about the genes related to the regulation of hair growth and hair cycle , but the molecular mechanism underlying the initiation of first hair growth is as yet not fully understood.
further improvement of bli to overcome this problem is necessary.
the arc expression level in the brain slightly decreased from the early to adult stages .
furthermore, we observed that the bioluminescence signal intensity of arc-luc in the submandibular gland increased with growth .
these findings suggest that arc expression in mice was driven in age-dependent and tissue-specific manners.
in this study, bli along with surgical monocular deprivation showed rapid changes in the bioluminescence signal intensity in the region contralateral to the deprived eye on md <dig> and the subsequent recovery on md <dig> .
furthermore, continuous bli showed the transient up-regulation of signals in the region contralateral to the deprived eye by reopening .
these changes in bioluminescence signal intensity can be explained by reduced neuronal activity in contralateral visual cortex , recovery of neuronal activity in contralateral visual cortex by increase of the input from open eye , and increased neuronal activity in contralateral visual cortex by reopening of deprived eye .
some reports indicate the changes in arc expression during visual-critical period in the visual cortex  <cit> .
taken together with these previous reports, our results suggest that bioluminescence signal intensity changes caused by visual operation are induced by up- and down regulation of neuronal activity in visual cortex at a critical period.
arc is induced by the or task and spatial exploration in novel environments .
on the basis of this finding, we examined the signal intensity changes of arc associated with the or task.
we successfully detected an increase in bioluminescence signal intensity in the posterior region of the brain including the somatosensory, barrel, and visual cortices of the mice after exposure to the novel object in their home cages .
our findings suggest that a novel object is mainly processed using the senses of touch and sight.
although it is generally believed that mice mainly use the senses of smell and touch for or, visual information also must be used for or.
furthermore, the use of arc-luc tg hl mice in combination with behavioral analysis may lead to the development of a technique for monitoring the neuronal activity in higher brain functions, such as recognition, emotion, and learning and memory.
in fig.
<dig>  we detected biphasic bioluminescence signal changes with a 9 h interval between the early  and late phases  after ka injection in the arc-luc tg hl mice.
the rapid and protein-synthesis-independent transcription of arc as an ieg is based on ‘promoter proximal pol ii stalling’  <cit>  and a specific enhancer with highly responsive to neuronal activity is located in the arc promoter  <cit> .
on the other hand, arc expression under the regulation of new protein synthesis resulted in a late response  <cit> .
actually, several studies showed a biphasic arc induction with an 8–10 h interval and the necessity of protein synthesis for late induction of arc after an electroconvulsive shock  <cit>  and contextual fear conditioning  <cit> .
considering the regulation of arc expression, the same molecular mechanism may explain our results.
however, an increase in bioluminescence signal intensity after ka injection might be a result of greater entry of d-luciferin caused by the disruption of blood brain barrier.
further study is required for the evaluation of d-luciferin penetrance after status epilepticus.
CONCLUSIONS
we have shown the advantages of an application of the hl gene to bli of the mouse brain in this study.
without interferences, the arc-luc tg hl mice exhibited clear and stable photon signals and enabled the tracking of arc expression under physiological and pathological conditions during development and at a short time interval.
these results indicate that this mouse strain can be used for rapid and quantitative assessments of neuronal-activity-dependent processes in the mouse brain over a wide range from a focal area to the entire brain area with various time windows.

 bioluminescence imaging is a powerful research technique based on detection of light emission produced by oxidation of luciferin by luciferase. the advantages of this technique in studies of small living animals are the following: high signal-to-noise ratio with low background signal intensities, low limit and simplicity of detection, wide dynamic range of signal intensities, and applicability to genetic manipulation as well as continuous and quantitative analyses. bli can also be applied repeatedly to the same animal, thus contributing to the reduction in the number of animals used. bli can be applied to the detection of the biological signal intensity changes that affect the up- and down-regulations of transcription. luciferase reporter animals are used in the research fields of inflammation, toxicology, oncology, angiogenesis, nutrition, and neurobiology. furthermore, split luciferase and bioluminescence resonance energy transfer techniques have recently been developed for the detection of protein–protein interaction. previously, we established a novel reporter transgenic mouse strain, namely, arc-luc tg mice, in which the firefly luciferase gene is driven by the arc promoter. arc, that is, the activity-regulated cytoskeleton-associated protein gene, is one of the immediate early genes, which are highly responsive to various external stimuli in the central nervous system. using arc-luc tg mice, we have successfully detected the bioluminescence signals of arc-luc in the adult mouse brain. the intensity of bioluminescence signals changed after physiological or pharmacological manipulations and the change well correlated with neuronal-activity-dependent arc expression. our arc-luc tg mice were established using the c57bl/ strain because this strain is one of the inbred strains most widely used in the fields of genetical engineering and neuroscience. however, its black fur, skin pigmentation, and hair regrowth after depilation interfere with bli during development. thus, the periods of bli were restricted less than 24 h and at an interval of more than 4 weeks. to extend the use of bli at various mouse developmental stages, we have introduced a mutant hl gene into the original arc-luc tg mice to overcome the above-mentioned problems. here, we examined the availability of arc-luc tg hl mice for bli at various developmental stages under physiological and pathological conditions.  in vivo imaging of arc-luc tg hl mice previously, we successfully generated arc-luc tg mice for the bli of arc expression. however, the genetic background of the arc-luc tg mice is c57bl/ and the black fur and skin pigmentation of this strain attenuate photon emission at various developmental stages. thus, we introduced the mutant hl gene into the original arc-luc tg mice by crossing them with the hl mice. the established arc-luc tg hl mice showed rapid induction of iegs after kainic acid injection. the fold changes of these iegs after ka injection in arc-luc tg hl mice were the same as those in the c57bl/ mouse strain. using cooled ccd camera systems, we detected bioluminescence signals in areas around the nose, brain, paws, kidneys, and testes in the arc-luc tg hl mice. the distribution of these signals was consistent with those previously reported for arc expression and public database from transcriptome analysis. the continuous bli of an individual animal showed that the bioluminescence signal intensity in the brain gradually decreased from to 8 weeks of age. furthermore, we examined the expression levels of luc and endogenous arc proteins by western blot analysis and found that they were slightly higher in the cerebral cortex at 4 weeks of age than at 8 weeks of age. these developmental changes in protein expression levels correlated with the changes in bioluminescence signal intensity. these findings demonstrate that changes in the arc expression level in the entire body during mouse development can be noninvasively detected by the visualization of arc-luc expression in arc-luc tg hl mice.fig.  comparison of pigmentation of head between c57bl/ and arc-luc tg hl mouse strains. a dorsal views of head of c57bl/ and arc-luc tg hl mice at and 8 weeks of age. the photographs of the c57bl/ mouse were taken after depilation. the c57bl/ mouse at 8 weeks of age and the arc-luc tg hl mouse at both ages are examples of suitable samples for bli because of little pigmentation. the c57bl/ mouse at 4 weeks of age is an example of an unsuitable sample for bli with skin pigmentation enclosed in a white dotted circle. scale bar 10 mm. b expression of iegs in c57bl/ and arc-luc tg hl mice. total rna was extracted from the hippocampus 30 min after ka injection. the changes in arc, egr- and c-fos expression levels were determined by quantitative rt-pcr analysis. the data represent mean ± sd fig.  noninvasive bli using arc-luc tg hl mouse. a bright field and bioluminescence signal images of an individual arc-luc tg hl mouse from to 8 weeks of age. bioluminescence signals in the arc-luc tg hl mouse were obtained after luciferin injection under anaesthesia induced with isoflurane. scale bar 20 mm. b western blot analysis of proteins in the cerebral cortex was performed using anti-luc, anti-arc, and anti-actin antibodies. location of protein size markers are indicated on the right side. the optical density of each band was quantified using nih imagej. the luc and arc protein expression levels normalized to actin protein expression level at and 8 weeks of age are shown in the bar graph. the data represent mean ± sd. *p < ; two-tailed student’s t test monitoring responses to sensory stimuli arc is one of the iegs, which are induced by various sensory stimuli. we previously reported the activity-dependent and plastic changes in arc expression in the visual cortex of adult arc-luc tg mice. in this experiment, we extended the application of arc-luc tg hl mice to continuous imaging at an interval of less than 7 days. we examined the pattern of changes in bioluminescence signal intensity during the development of mice in visual-critical period with visual operation. the arc-luc tg hl mice on p were monocularly deprived by eyelid suture. one day after monocular deprivation, the bioluminescence signal intensity significantly decreased in the brain region contralateral to the sutured eye after 3 h exposure to an intense light stimulus. seven days after continuous monocular deprivation, the bioluminescence signal intensity in this region slightly increased. furthermore, the reopening of the sutured eye followed by light exposure resulted in a marked increase in the bioluminescence signal intensity in this region. for the quantitative assessment of hemispheric dominance of arc expression in the brain, we calculated lateralization index, which is often used in functional magnetic resonance imaging studies. li decreased to − on md and it slightly increased on md but remained below zero. moreover, li further increased to + after ro and returned to about one day after ro.fig.  laterality of bioluminescence signals from visual cortex after monocular deprivation by suturing and reopening surgery. a experimental schedule for monocular deprivation and reopening and bli. the time points of bli are indicated with black triangles. b the obtained bioluminescence images before eye suture, 1 day after md, 7 days after md, immediately after reopening, and 1 day after ro. mice were exposed to an intense light for 3 h before each imaging time point. the locations of regions of interest are enclosed in dotted circles in the control image, and the obtained bioluminescence signal intensities are used for the calculation of lateralization index shown in. scale bar 10 mm. c plots showing the li under each condition shown in. li was calculated using the formula /, where l and r are the bioluminescence signal intensities of roi in the regions contralateral and ipsilateral to the sutured eye, respectively. li was nearly zero under the control condition, decreased below zero under md conditions, and increased immediately at ro, and finally returned close to the baseline at ro. the data represent mean ± sd. *p < ; two-tailed student’s t test spatial exploration in a novel environment induces arc expression, and object and place recognition tasks induce the expression of some iegs including arc. we carried out the evaluation of bioluminescence signal intensity changes associated with an object recognition task. individually housed mice at 4 weeks of age were provided a novel plastic cube in their home cages. the object provided induced various exploratory behaviors of mice, such as approaching, sniffing, climbing, biting, and turning them over for several minutes. no statistically significant differences were detected in the total intensity of bioluminescence signals from the entire forebrain between the or and control groups. however, we observed marked differences in the distribution of bioluminescence signals from the brain between the two groups. we divided rois in the forebrain into rectangular blocks in the rostral to caudal direction. the distribution of relative bioluminescence signal intensity normalized to the signal intensity corresponding to the entire-forebrain photon density showed a down-regulation of the signals from the rostral region and the up-regulation of those from the posterior brain regions including the somatosensory, barrel, and visual cortices. therefore, we concluded that the detection of bioluminescence signals in the arc-luc tg hl mice clearly provided detailed neuronal-activity-dependent arc expression changes induced by sensory stimuli during development.fig.  bioluminescence signal intensity changes associated with or task. a experimental schedule for or task and bli. bioluminescence signal intensity was measured in each mouse 6 h after putting a plastic cube into their home cage. for the control, no object was presented. bli was also performed without any treatment 1 day before the or task. the time points of bli are indicated with black triangles. b representative images of bioluminescence signals in the arc-luc tg hl mouse brain at 4 weeks of age for the object and control. location of rois in the forebrain is indicated in the control image. scale bar 10 mm. c subdivision of roi in forebrain into smaller rectangles at  mm intervals in rostral to caudal direction. d distribution of bioluminescence signals associated with or. profiles of photon density throughout the forebrain for object presentation and control condition. each value obtained from rois in was normalized to the mean signal intensity in the whole forebrain. significant differences between the control and or conditions are shown in gray in the graph. the data represent mean ± sd. *p < ; two-tailed student’s t test bioluminescence signal intensity changes after ka treatment ka is derived from a natural marine product from digenea simplex and is an excitatory amino acid receptor agonist selective to ka-type glutamate receptors. acute treatment with ka is often carried out to produce a neurodegenerative disease model using laboratory animals and also induces arc expression through synaptic activation. we reported the continuous increase in arc-luc expression level over 6 h after ka injection at the adult stage. here, we extend the examination of the temporal pattern of ka-induced arc expression in the arc-luc tg hl mice at 4 weeks of age. bli was conducted after intraperitoneal ka injection. the bioluminescence signal intensity increased three to fourfold within 3 h after ka injection, and then decreased to about twofold at 6 h. furthermore, a second increase was detected in the posterior region at 12 h and the intensity finally returned close to the baseline at 24 h; this signal distribution pattern was different from that in the control saline-treated group. these results obtained from the arc-luc tg hl mice suggest the advantages of temporal and quantitative evaluations of the effect of pharmacological manipulation in vivo at various developmental stages without interferences from fur and skin pigmentation.fig.  arc-luc expression level changes after ka injection at 4 weeks of age. a experimental schedule for bli after ka injection. the time points of bli are indicated with black triangles. b images from representative mice taken at 0 h and and 24 h after ka or saline injection. the locations of rois are indicated in the images at 0 h. scale bar, 10 mm. c changes in fold induction of bioluminescence signals within 24 h after ka injection. *p < ; two-tailed student’s t test discussion bli is a noninvasive, simple, and quantitative method to evaluate brain function. however, the bli of the mouse brain using the c57bl/ strain at various developmental stages has some problems such as light attenuation due to its black fur and skin pigmentation and the limited imaging period and interval caused by the hair regrowth after depilation. to overcome these problems, we established the arc-luc tg hl mouse strain for imaging neuronal-activity-dependent arc expression at various developmental stages. we examined the reliability of this new mouse strain for investigating neuronal-activity-dependent arc expression processes. the arc-luc tg hl mice have several advantages that make them useful for investigating the neuronal-activity-dependent changes during development. first, we can acquire stable bioluminescence signals from the brain without interferences after 3 weeks of age. albino mice are also useful for bli without shaving, but there remains the problem of light absorbance by white fur. the application of our novel mouse strain to bli is valuable in terms of the increase in the intensity of bioluminescence signals per specified time interval. as shown in fig.  comparison of bioluminescence signal intensities at various developmental stages of the same animal enabled the detection of age-related changes in arc expression level. second, we can track bioluminescence signals daily and weekly. because skin pigmentation occurs 5 days after depilation of the c57bl/ mouse strain, we cannot measure bioluminescence signals in the original arc-luc tg mice for more than five consecutive days. we have to wait at least 4 weeks for the full regrowth of hair for the next bli. the arc-luc tg hl mice on the other hand can be used daily and weekly to detect time-dependent changes in arc expression level in the brain by bli. third, because the use of arc-luc tg hl mice enables bli for extended periods using the same animal, we can investigate the life-long effects of damage incurred from the early stages to the adulthood stage by bli of arc expression using fewer mice. however, despite these advantages of applying the arc-luc tg hl mice to bli, imaging at or 2 weeks of age remains unfeasible because the hair loss in arc-luc tg hl mice starts approximately on p there are several reports about the genes related to the regulation of hair growth and hair cycle, but the molecular mechanism underlying the initiation of first hair growth is as yet not fully understood. further improvement of bli to overcome this problem is necessary. the arc expression level in the brain slightly decreased from the early to adult stages. furthermore, we observed that the bioluminescence signal intensity of arc-luc in the submandibular gland increased with growth. these findings suggest that arc expression in mice was driven in age-dependent and tissue-specific manners. in this study, bli along with surgical monocular deprivation showed rapid changes in the bioluminescence signal intensity in the region contralateral to the deprived eye on md and the subsequent recovery on md. furthermore, continuous bli showed the transient up-regulation of signals in the region contralateral to the deprived eye by reopening. these changes in bioluminescence signal intensity can be explained by reduced neuronal activity in contralateral visual cortex, recovery of neuronal activity in contralateral visual cortex by increase of the input from open eye, and increased neuronal activity in contralateral visual cortex by reopening of deprived eye. some reports indicate the changes in arc expression during visual-critical period in the visual cortex. taken together with these previous reports, our results suggest that bioluminescence signal intensity changes caused by visual operation are induced by up- and down regulation of neuronal activity in visual cortex at a critical period. arc is induced by the or task and spatial exploration in novel environments. on the basis of this finding, we examined the signal intensity changes of arc associated with the or task. we successfully detected an increase in bioluminescence signal intensity in the posterior region of the brain including the somatosensory, barrel, and visual cortices of the mice after exposure to the novel object in their home cages. our findings suggest that a novel object is mainly processed using the senses of touch and sight. although it is generally believed that mice mainly use the senses of smell and touch for or, visual information also must be used for or. furthermore, the use of arc-luc tg hl mice in combination with behavioral analysis may lead to the development of a technique for monitoring the neuronal activity in higher brain functions, such as recognition, emotion, and learning and memory. in fig.  we detected biphasic bioluminescence signal changes with a 9 h interval between the early and late phases after ka injection in the arc-luc tg hl mice. the rapid and protein-synthesis-independent transcription of arc as an ieg is based on ‘promoter proximal pol ii stalling’ and a specific enhancer with highly responsive to neuronal activity is located in the arc promoter. on the other hand, arc expression under the regulation of new protein synthesis resulted in a late response. actually, several studies showed a biphasic arc induction with an 8–10 h interval and the necessity of protein synthesis for late induction of arc after an electroconvulsive shock and contextual fear conditioning. considering the regulation of arc expression, the same molecular mechanism may explain our results. however, an increase in bioluminescence signal intensity after ka injection might be a result of greater entry of d-luciferin caused by the disruption of blood brain barrier. further study is required for the evaluation of d-luciferin penetrance after status epilepticus.  we have shown the advantages of an application of the hl gene to bli of the mouse brain in this study. without interferences, the arc-luc tg hl mice exhibited clear and stable photon signals and enabled the tracking of arc expression under physiological and pathological conditions during development and at a short time interval. these results indicate that this mouse strain can be used for rapid and quantitative assessments of neuronal-activity-dependent processes in the mouse brain over a wide range from a focal area to the entire brain area with various time windows. 
thus, the use of the arc-luc tg hl mice enabled the tracking of neuronal-activity-dependent processes over a wide range from a focal area to the entire brain area with various time windows. however, the black fur, skin pigmentation and hair regrowth after depilation of mouse interfere with bli during developmental and daily examination. the aim of this study was to extend the application of arc-luc transgenic mice to the bli of neuronal activity in the mouse brain by introducing the hairless gene and to examine arc-luc expression at various developmental stages without interference from black fur, skin pigmentation, and hair regrowth. this study showed the advantages of using the mutant hl gene in bli of arc expression in the mouse brain at various developmental stages. under physiological and pathological conditions, bli was performed to detect the signal intensity changes at various developmental stages and at an interval of <7 days. after surgical monocular deprivation during visual-critical period, large signal intensity changes in bioluminescence were observed in the mouse visual cortex. the temporal pattern of kainic-acid-induced arc-luc expression showed biphasic changes in signal intensity over 24 h. bioluminescence imaging is a powerful technique for monitoring the temporal and spatial dynamics of gene expression in the mouse brain. the established arc-luc tg hl mice exhibited clear and stable photon signals from the brain without interference during development. the arc-luc tg hl mice were established by crossing the tg c57bl/ mouse strain with the hl mouse strain.
 bioluminescence imaging is a powerful technique for monitoring the temporal and spatial dynamics of gene expression in the mouse brain. however, the black fur, skin pigmentation and hair regrowth after depilation of mouse interfere with bli during developmental and daily examination. the aim of this study was to extend the application of arc-luc transgenic mice to the bli of neuronal activity in the mouse brain by introducing the hairless gene and to examine arc-luc expression at various developmental stages without interference from black fur, skin pigmentation, and hair regrowth.  the arc-luc tg hl mice were established by crossing the tg c57bl/ mouse strain with the hl mouse strain. under physiological and pathological conditions, bli was performed to detect the signal intensity changes at various developmental stages and at an interval of <7 days. the established arc-luc tg hl mice exhibited clear and stable photon signals from the brain without interference during development. after surgical monocular deprivation during visual-critical period, large signal intensity changes in bioluminescence were observed in the mouse visual cortex. exposure of mice to a novel object changed the photon distribution in the caudal and rostral cerebral areas. the temporal pattern of kainic-acid-induced arc-luc expression showed biphasic changes in signal intensity over 24 h.  this study showed the advantages of using the mutant hl gene in bli of arc expression in the mouse brain at various developmental stages. thus, the use of the arc-luc tg hl mice enabled the tracking of neuronal-activity-dependent processes over a wide range from a focal area to the entire brain area with various time windows. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords hairlessarcdevelopmentbioluminescence imagingcabinet office, government of japan the food safety commission of japan, no.1001mori hisashi the ministry of education, science, sports and culture of japanscientific research on innovation area, comprehensive brain science network, no.221s003mori hisashi issue-copyright-statement© the author 2017 