The Function Of Glial Cells And ADF Neuron In Sensory Modulation In C. Elegans

Neurons and glial cells constitute our nervous system,and glial cells occupy 90 percent,the majority of them.But few studies are focused on glial cells than on neurons.In mammalian central nervous system,there are three kinds of glial cells:astrocyte,oligodendrocyte and microglia.Astrocytes make up the majority.However,they were seen as negative cells because they could not introduce nerve pulse.They can only afford neurons necessary circumstance,including regulating the concertration of neurotransmitters nearby the neurons and protecing neurons.Now,more and more evidence show that astrocytes actually actively pariticipate in neurotransmission.Firstly,astrocyte can enclose the synapse and form so called "tripartite synapse in morphology,this suggests that they may have a modulation role in neurotansmission in synapse.Then,astrocytes also represent calcium siganling and release of gliotransmitters.Finally,these gliotransmitters can regulate the expression and activity of receptors both in presynaptic and postsynaptic membrane and have a feedback regulation on neutransmission between neurons.However,for the moment,little is known about the source of calcium signaling in astrocyte.Prevalent perspectives believe that astrocyte activity extremely depend on neuronal activation.Notably,most reults are gathered from cultured cells in vitro or seperated brain slices,and few in vivo studies support this opinion.Here,we adopt C.elegans as our in vivo study model to explore whether glial cells(OLQ socket glia,OLsoc)could respond to external mechanical stimulation and their potential functional outputs.In this study,using proper mechanical stimulation given to the nose tip of the worm can induce robust calcium increase in OLsoc glia.We can clearly observe calcium propagation from cilium to cell body of OLsoc glia,and this increment could last for quite a long time(sometimes even last for above 20 mins).However,calcium signaling also induced by mechanical stimulation in OLQ neurons wrapped by OLsoc glia increases much faster and returns to basal level within 10 seconds.Long-lasting calcium signaling in OLsoc indicates that they may participant in modulating the sensory transduction,such as adaptation and plastivity in neuronal system.To futher understand where calcium elevation originate from,we used a primary culture system to achieve isolated OLsoc glial cells,which allows us to analysis OLsoc glia in a "circuit-free" state.We both performed electrophysiological recording and calcium imaging experiments on isolated OLsoc glia.And mechanical stimulation can induce robust inward currents and obvious calcium elevation,which indicates OLsoc glia have an endogenous acticvity responding to external stimulation.Meanwhile,we set OLQ neurons and OLsoc glia as a model to unveil the potential crosstalk between neurons and their corrsponding glial cells.OLQ neuronal activity shows no influence on OLsoc glia touch sensation.Taken together,these results demonstrate that OLsoc can cell-autonomously respond to mechanical stimulus and this inherent response is not affected by neurons.We also detected calcium variation in isolated AMsh responding to odor stimuli.Our previous in vivo data showed that AMsh could be activated by a series of repulsive odors,but whether this response was endogenous still remained confirmed,so we adopted cultured primary AMsh.Calcium imaging results showed that isolated AMsh could also be activated by odor stimuli.Both OLsoc and AMsh can directly respond to external stimuli demonstrated that this cell-autonomous sensation in glial cells could be universal mechanisms in C.elegans.Since both OLQ neurons andOLsoc glia can respond to mechanical stimulation on their own,there should be mechao-gated channels expressed themselves.However,only several calcium imaging results proposed some probable candidate channels but no direct electrophysiological evidence.Here,we screen out DEG-1 as the mechano-gated channel expressed in OLQ neurons,but not in OLsoc glial cells.Also blocking any single defined channel in OLsoc represent no obvious effect on calcium elevation.These results suggest a novel mechanosensitive channel should be expressed in OLsoc glia.Next we want to know whether this touch-induced calcium increase in OLsoc may have any functional outputs or influence on behaviors.Firstly,we activate OLsoc glia by optogenetics and analysis worm behavior.Tracking movie results show that activated OLsoc glia lead to no obvious behavioral changes.Considering longer delay of calcium signaling in OLsoc,we extended our recording time up to 3 mins,but no difference observed.Then,we tried to deactivate OLosc glia by expressing a histamine-gated chlorine channel(HisCll).Instant deactivated OLsoc glial activity conferred severe behavioral defects including nose touch-induced reversal response and anterior body touch-induced suppression of foraging behavior.These results effectively prove that OLsoc glial cells in C.elegans participant in real time functional outputs.The above mentioned reversal and suppresion of foraing behaviors are related to OLQ neurons,so we suggest that behavioral defects caused by OLsoc inactivation could be a consequence of affects on OLQ neuronal function.To confirm this hypothesis,we recorded mechanical stimulation induced calcium transients in OLQ neurons when OLsoc glial activity was inhibited.While calcium increments in OLQ neurons induced by two consecutive nose touch stimuli in wild type worms were of similar amplitude,the calcium transients induced by the second touch was significantly decreased in HisCll transgenic worms.OLQ neuronal response to repeated stimulation was inhibited.We have already mentioned above that calcium signaling in OLsoc shows longer period,which suggests their modulation roles in sensory transduction.Combined with this calcium imaging results,we believe that OLsoc glia indeed are involved instanr behavioral outputs in C.elegans.All together,we report here that OLsoc glia in C.elegans can independently respond to outside mechanical stimulation and this response is not affected by OLQ neurons.We also define DEG-1 as the major mechano-gated channel in OLQ neurons,but a novel channel in OLsoc glia.Our behavioral experiments show deactivated OLsoc activity significantly infects reversal response and suppression of foraging behavior.The underlying mechanism is that behavioral defects result from reduced touch sensation of OLQ neurons when OLsoc glial activity are inhibited.Our study afford evidence that glial cells could also respond to outside stimulation and assist neurons to code information,which should provide new insights into discovering more roles of glial cells in brain fucntion.In the second part of this dissertation we explored the function of ADF neuron in sensory modulation in C.elegans.Serotonin modulates many behaviors important for survival and development including egg-laying,feeding,movement and normal neural migration in the developing nervous system in C.elegans.Previous studies showed that there are several kinds of serotoninergic neurons in C.elegans,such as ADF,NSM and HSN.And ADF neurons are the sole serotonergic sensory neurons in hermaphrodite worms.They are reported to be involved in a residual response to cAMP.However,their in vivo characteristics,such as whether they can be activated by chemical or mechanical stimulation when exposed to enviromental stimulation or changes,are largly unknown.Here,by adopting in vivo calcium imaging,genetic manipulation and neural activity regulation,we report that ADF neurons can be reliably activated by nociceptive stimuli such as repulsive odors and mechanical stimulation.Interestingly,none of those attractive odors can activate them.We also approve that neurotransmitters play a vital role in copper and IAA induced ADF calcium promotion.Using cell-specific slicing or activation,we further confirm that ADF neurons affect ASH-induced backward locomotion.And calcium imaging data show that ADF preactivation depresses ASH neural maximun calcium transients responding to those aversive stimuli.Togeter,our data afford evidence that ADF neurons are also active participators in enviromental experience and fine-tune worm behavior.