using in vivo time - lapse confocal and two - photon imaging of the visual center optic tectum in living larval zebrafish , we first monitored changes in the morphology of microglia under resting state .
similar to the typical morphological properties of resting microglia in the mouse cortex , resting microglia in zebrafish are also highly branched with dynamic processes ended with either stick - like or bulbous tips ( fig .  1 ) .
these two differently shaped tips can be inter - converted as the process moves around .
the bulbous ending forms rapidly through expansion from a stick - like ending , and stalls on the contacted neuronal soma for several minutes before gradually shrinking back again .
the existence of resting microglia - neuron contact is further confirmed by using 3-dimensional reconstruction and transmission electron microscopy technique .
series images of a resting microglial cell acquired every 5 with in vivo time - lapse confocal imaging of the optic tectum in a 6-dpf tg(apo - e : egfp ) zebrafish larva , in which egfp is specifically expressed in microglia .
images at different time points are shown in different colors . in previous studies , global increase or decrease of neural activity in vivo by using the gabaa receptor inhibitor bicuculline or the voltage - gated sodium channel blocker ttx , respectively , could oppositely regulate the dynamics of resting microglia . in zebrafish
, we further proved that neural activity plays an instructive role in steering the motility of resting microglial processes and the formation of microglia - neuron contact . using glutamate uncaging , we locally upregulated neuronal activity in a small brain region of the intact zebrafish larvae .
glutamate uncaging , a non - invasive approach , can efficiently induce a local increase of neural activity .
we applied repetitive glutamate uncaging about 20 m away from microglial soma , and found that the processes of resting microglia can gradually navigate toward the uncaging side at about 10 min after the uncaging onset .
furthermore , more bulbous tips are formed at the uncaging side , indicating an elevated formation of microglia - neuron contact in responding to the local increase of neuronal activity in vivo .
different from the microglia in culture and mammalian brain slices , we found no ionotropic glutamate receptors expressed on microglia in the optic tectum of zebrafish larvae using in vivo whole - cell recording , excluding the possibility that uncaged glutamate directly acts on resting microglia .
importantly , the formation of resting microglial bulbous endings can also be regulated by natural sensory inputs , e.g. , visual stimuli , which can globally increase neuronal activity in the optic tectum . however , after global downregulation of neural activity via pre - incubation of larvae with ttx , the number of bulbous endings is markedly decreased .
all these results further confirm an instructive role of neuronal activity in regulating the dynamics of resting microglial processes and the formation of microglia - neuron contact . having shown that local elevation of neuronal activity can induce the formation of microglial bulbous endings wrapping neuronal somata
, we next asked what might be mechanisms by which neurons with high activity  talk  to resting microglia .
we found that this process requires the membrane depolarization - activated pannexin-1 hemichannels on tectal neurons and the atp / p2 purinergic receptor signaling between neurons and microglia .
pannexin-1 , a large pore - like hemichannel , is widely expressed in the central nervous system .
the opening of pannexin-1 is gated by some cellular signals , such as membrane depolarization , intracellular calcium and so on .
small molecules , including atp , nad and pge2 , can be released through these hemichannels .
using whole - mount in situ hybridization and in vivo whole - cell recording , we found that the functional pannexin-1 hemichannels are expressed in tectal neurons but not microglia .
after impairing the function of pannexin channels by drug treatment or morpholino - mediated genetic downregulation , glutamate uncaging - induced orientated movement of microglial processes and formation of bulbous contacts were prevented .
similar results were observed when we applied the atp - hydrolyzing enzyme apyrase or the p2 purinergic receptor blocker suramin .
neuronal activity steers resting microglial processes by facilitating the redistribution of the cytoskeleton protein small rho gtpase rac .
rac is known to be required for the membrane protrusion and migration of many types of cells , including zebrafish germ cells .
using confocal fret imaging , we found that the rac fret intensity is significantly increased in microglial processes , especially at bulbous endings , at the uncaging side after repetitive glutamate uncaging .
it is immediately followed by the oriented movement of microglial processes and the formation of bulbous endings . however , if we genetically inhibited endogenous rac activity in microglia , those neuronal activity - induced microglial morphological changes could not be observed .
taken together , we identify the underlying mechanisms by which neurons with high activity signal to resting microglia . considering the facilitated formation of microglia - neuron contact induced by neuronal activity increase
by simultaneously monitoring changes in both the morphology of microglial processes and ca activity of tectal neurons , we found that microglial processes preferentially contact with neurons that exhibit high levels of spontaneous activity before contact .
we then mosaically overexpressed the human inward rectifier k channel kir2.1 ( kir ) , which is used to reduce the excitability of neurons , and a non - conducting mutant version of kir2.1 ( mkir ) in tectal neurons . in comparison with mkir - expressing neurons ,
these results further indicate that resting microglia preferentially contact neurons with higher activities , consistent with the data obtained with glutamate uncaging .
while accumulating evidence shows that resting microglia respond to neural activity , we surprisedly noticed that resting microglia can also in turn regulate neuronal activity . in the zebrafish optic tectum
, we found that such resting microglia - neuron contact can downregulate both spontaneous and visually evoked activities in contacted neurons .
interestingly , the decreases in both the frequency and magnitude of spontaneous ca activity are positively correlated with the duration of microglia - neuron contact .
what is the consequence on neuronal firing after loss of resting microglia ? in response to two - photon laser - induced focal injury , some microglia quickly trans - locate to the injury site .
consistent with the speculation based on our previous findings , the spontaneous activity of neurons within microglia pre - existing territories is significantly elevated after the translocation of microglia to the injury site . meanwhile , in other cases with the similar injury but no microglial translocation , the neuronal activity is not significantly changed .
thus , our study reveals a functional regulation of neuronal activity by resting microglia under physiological conditions , offering a new way for homeostatic regulation of neuronal activity .
collectively , our work brings forth and demonstrates a novel reciprocal regulation between resting microglia and neurons in vivo .
we not only demonstrate an instructive role of neuronal activity in resting microglial motility , but also reveal , for the first time , a previously unappreciated function of microglia in homeostatic regulation of neuronal activity in the healthy brain ( fig .  2 ) .
considering the bi - directional modulation between neurons and microglia , this study also represents a new perspective in understanding the physiological function of resting microglia .
highly active neurons attract resting microglial processes and induce the formation of microglia - neuron contact via atp signaling ( top ) . such neuronal activity - driven microglia - neuron contact in turn
using in vivo time - lapse confocal and two - photon imaging of the visual center optic tectum in living larval zebrafish , we first monitored changes in the morphology of microglia under resting state .
similar to the typical morphological properties of resting microglia in the mouse cortex , resting microglia in zebrafish are also highly branched with dynamic processes ended with either stick - like or bulbous tips ( fig .  1 ) .
these two differently shaped tips can be inter - converted as the process moves around .
the bulbous ending forms rapidly through expansion from a stick - like ending , and stalls on the contacted neuronal soma for several minutes before gradually shrinking back again .
the existence of resting microglia - neuron contact is further confirmed by using 3-dimensional reconstruction and transmission electron microscopy technique .
series images of a resting microglial cell acquired every 5 with in vivo time - lapse confocal imaging of the optic tectum in a 6-dpf tg(apo - e : egfp ) zebrafish larva , in which egfp is specifically expressed in microglia .
in previous studies , global increase or decrease of neural activity in vivo by using the gabaa receptor inhibitor bicuculline or the voltage - gated sodium channel blocker ttx , respectively , could oppositely regulate the dynamics of resting microglia . in zebrafish
, we further proved that neural activity plays an instructive role in steering the motility of resting microglial processes and the formation of microglia - neuron contact . using glutamate uncaging , we locally upregulated neuronal activity in a small brain region of the intact zebrafish larvae .
glutamate uncaging , a non - invasive approach , can efficiently induce a local increase of neural activity .
we applied repetitive glutamate uncaging about 20 m away from microglial soma , and found that the processes of resting microglia can gradually navigate toward the uncaging side at about 10 min after the uncaging onset .
furthermore , more bulbous tips are formed at the uncaging side , indicating an elevated formation of microglia - neuron contact in responding to the local increase of neuronal activity in vivo .
different from the microglia in culture and mammalian brain slices , we found no ionotropic glutamate receptors expressed on microglia in the optic tectum of zebrafish larvae using in vivo whole - cell recording , excluding the possibility that uncaged glutamate directly acts on resting microglia .
importantly , the formation of resting microglial bulbous endings can also be regulated by natural sensory inputs , e.g. , visual stimuli , which can globally increase neuronal activity in the optic tectum . however , after global downregulation of neural activity via pre - incubation of larvae with ttx , the number of bulbous endings is markedly decreased .
all these results further confirm an instructive role of neuronal activity in regulating the dynamics of resting microglial processes and the formation of microglia - neuron contact .
having shown that local elevation of neuronal activity can induce the formation of microglial bulbous endings wrapping neuronal somata , we next asked what might be mechanisms by which neurons with high activity  talk  to resting microglia .
we found that this process requires the membrane depolarization - activated pannexin-1 hemichannels on tectal neurons and the atp / p2 purinergic receptor signaling between neurons and microglia .
pannexin-1 , a large pore - like hemichannel , is widely expressed in the central nervous system .
the opening of pannexin-1 is gated by some cellular signals , such as membrane depolarization , intracellular calcium and so on .
small molecules , including atp , nad and pge2 , can be released through these hemichannels . using whole - mount in situ hybridization and in vivo whole - cell recording
, we found that the functional pannexin-1 hemichannels are expressed in tectal neurons but not microglia . after impairing the function of pannexin channels by drug treatment or morpholino - mediated genetic downregulation , glutamate uncaging - induced orientated movement of microglial processes and formation of bulbous contacts
similar results were observed when we applied the atp - hydrolyzing enzyme apyrase or the p2 purinergic receptor blocker suramin .
neuronal activity steers resting microglial processes by facilitating the redistribution of the cytoskeleton protein small rho gtpase rac .
rac is known to be required for the membrane protrusion and migration of many types of cells , including zebrafish germ cells .
using confocal fret imaging , we found that the rac fret intensity is significantly increased in microglial processes , especially at bulbous endings , at the uncaging side after repetitive glutamate uncaging .
it is immediately followed by the oriented movement of microglial processes and the formation of bulbous endings .
however , if we genetically inhibited endogenous rac activity in microglia , those neuronal activity - induced microglial morphological changes could not be observed .
taken together , we identify the underlying mechanisms by which neurons with high activity signal to resting microglia .
considering the facilitated formation of microglia - neuron contact induced by neuronal activity increase , we wondered what the physiological function of such contact is . by simultaneously monitoring changes in both the morphology of microglial processes and ca activity of tectal neurons
, we found that microglial processes preferentially contact with neurons that exhibit high levels of spontaneous activity before contact .
we then mosaically overexpressed the human inward rectifier k channel kir2.1 ( kir ) , which is used to reduce the excitability of neurons , and a non - conducting mutant version of kir2.1 ( mkir ) in tectal neurons . in comparison with mkir - expressing neurons ,
these results further indicate that resting microglia preferentially contact neurons with higher activities , consistent with the data obtained with glutamate uncaging .
while accumulating evidence shows that resting microglia respond to neural activity , we surprisedly noticed that resting microglia can also in turn regulate neuronal activity . in the zebrafish optic tectum , we found that such resting microglia - neuron contact can downregulate both spontaneous and visually evoked activities in contacted neurons .
interestingly , the decreases in both the frequency and magnitude of spontaneous ca activity are positively correlated with the duration of microglia - neuron contact .
what is the consequence on neuronal firing after loss of resting microglia ? in response to two - photon laser - induced focal injury , some microglia quickly trans - locate to the injury site .
consistent with the speculation based on our previous findings , the spontaneous activity of neurons within microglia pre - existing territories is significantly elevated after the translocation of microglia to the injury site . meanwhile , in other cases with the similar injury but no microglial translocation , the neuronal activity is not significantly changed .
thus , our study reveals a functional regulation of neuronal activity by resting microglia under physiological conditions , offering a new way for homeostatic regulation of neuronal activity .
collectively , our work brings forth and demonstrates a novel reciprocal regulation between resting microglia and neurons in vivo .
we not only demonstrate an instructive role of neuronal activity in resting microglial motility , but also reveal , for the first time , a previously unappreciated function of microglia in homeostatic regulation of neuronal activity in the healthy brain ( fig .  2 ) .
considering the bi - directional modulation between neurons and microglia , this study also represents a new perspective in understanding the physiological function of resting microglia .
highly active neurons attract resting microglial processes and induce the formation of microglia - neuron contact via atp signaling ( top ) . such neuronal activity - driven microglia - neuron contact in turn
 microglia are the primary immune cells in the brain . under pathological conditions , 
 they become activated and participate in scavenging , inflammation and tissue repair in response to brain injury . while the function and underlying mechanism of activated microglia have been intensively studied in the past decades , physiological functions of resting microglia 
 remain largely underestimated . in our recent work , by simultaneously monitoring both the motility of resting microglial processes and the activity of surrounding neurons in intact zebrafish optic tectum , we examined the interaction between resting microglia and neurons . local increase in neuronal 
 activity attracts resting microglial processes and drives them to contact neurons with high levels of activity . 
 this process is mediated by neuronal release of  find - me  signals such as atp via pannexin-1 hemichannels and requires small rho gtpase rac in microglia . 
 reciprocally , the microglia - neuron contact reduces both the spontaneous and visually evoked activities of contacted neurons . we here summarize and explain the key results in the context of our previous work 
 . 


Demonstration of Contact between Resting Microglial Process and Neuronal Soma
Neuronal Activity Steers the Motility of Resting Microglial Processes and Induces the Formation of Microglia-Neuron Contact
Molecular and Cellular Mechanisms of Neuronal Activity-Induced Changes in Microglial Dynamics
Physiological Significance of Resting Microglia-Neuron Contact