The Roles Of Cerebellum And Brainstem In The Laryngeal Chemoreflex Of Rats And The Eye Blink Conditioning Of Mice

Chapter 1 The distribution of Fos-like immunoreactivity neurons activated by the laryngeal chemoreflex in the medulla oblongata of ratsObjective:The effects of laryngeal chemoreflex(LCR)include closure of the glottis,apnea,bradycardia and hypertension,which is one of the fatal induces of the sudden infant death syndrome[SIDS].The LCR is evoked by food or digestive juice accidentally entering the laryngeal lumen,which has been revealed that it is associated to poor development of infant brain.As a simple reflex,the center of LCR locates in the medulla oblongata,while the involved nuclei are still unclear.Cerebellar fastigial nucleus participates in diverse reflexes,including respiration,but little report shows its involvement in the LCR,The study aims to explore the medullary and cerebellar nuclei that contribute to the LCR of rats.Methods:Adult male SD rats were grouped in experimental and control.Experimental animals were infused with KCl-HCl in the laryngeal lumen unconsciously to elicit the LCR,in the mean time,muscle tension diagram(MTD)of the trunk musculature was recorded before and after the LCR elicitation.Control animals were underwent the same operation except chemical infusion.After sacrifice,the medulla and cerebellum were cut,slices were stained subsequently with c-fos indicating the activated neurons during the LCR.Fos-like immunoreactivit(FLI)on slices were observed and analyzed under fluorescence microscopy to determine the distribution and number variability between experimental and control.Results:In comparison,the MTD of the trunk musculature significantly weakened by eliciting the LCR.Number of regions of Fos-like immunoreactivity(FLI)that were activated by the LCR significantly increased in the nucleus of the solitary tract(NTS),the vestibular nuclear complex(VNC),the loose formation of the nucleus ambiguus(AmbL),the rostral ventral respiratory group(RVRG),the ventrolateral reticular complex(VLR),the pre-Botzinger complex(PrBot),the Botzinger complex(Bot),the spinal trigeminal nucleus(SP5),and the raphe obscurus nucleus(ROb)bilaterally from the medulla oblongata.However,no FLI was found in cerebellum of experimental or control animals.Conclusion:medulla oblongata is the center of LCR,various medullary nucleiare involved in the LCR.Cerebellum may not contribute the LCR.Chapter 2 The medullary catecholaminergic,serotoninergic and cholinergic neurons involve in modulation of the laryngeal chemorelex of ratsObjective:Catecholaminergic,serotoninergic and acetylcholinergic neurons are widely distributed in the medulla oblongata.Our previous studies demonstrates catecholaminergic and serotoninergic direct inputs to the laryngeal motor nucleus(LMN),while the LMN itself is observed acetlycholinergic.Furthermore,studies of SIDS pathology have emphasized the functions of catecholamines and the significance of the medullary serotonin pathway.In consequence we assume medullary catecholaminergic,serotoninergic and acetylcholinergic neurons contribute to the laryngeal chemorelex.The aim of the study is to find out the distribution of these neurotransmittergic neurons which are activated by the laryngeal chemoreflex in the medulla oblongata.Methods:In experimental animals,FLI-VAchT-ir double labeled neurons are observed in the nucleus ambiguous loose formation;12.71%of neurons with FLI in the dorsolateral part of the nucleus of the solitary tract(SolDL)showed tyrosine hydroxylase-immunoreactivity(TH-ir,catecholaminergic),and 70.87%of neurons with FLI in the ROb were serotoninergic.No co-labeled neurons as described above show in control animals.Results:In experimental animals,12.71%of neurons with FLI in the dorsolateral part of the nucleus of the solitary tract((SolDL)showed tyrosine hydroxylase-immunoreactivity(TH-ir,catecholaminergic),and 70.87%of neurons with FLI in the ROb were serotoninergic.However,no double labeled neurons were found in control animals.Conclusion:Acetylcholinergic LMN of the AmbL,part of catecholaminergic neurons in the SolDL and most part of serotoninergic neurons in the Rob are activated during LCR,indicating that disfunction of these neuronsmay response to the sudden infant death syndrome.Chapter 3 The cerebellar fastigial nucleus regulates eye blink conditioning of mice:behavioral studiesObjective:Eye blink conditioning is one of the most typical Pavlovian reflexe that has been intensively used to study the neural mechanism of associative learning.In cerebellar circuits,mossy fiber and climbing fiber inputs from pons and inferior olive respectively project to cerebellar cortex and deep cerebellar nuclei,being organized and transfer outputs to evoke conditioned and unconditioned responses.Previous studies demonstrate that deep cerebellar nuclei play an essential role in the eye blink conditioning.Chronic lesion of deep cerebellar nuclei of naive mice causes learning disability or elimination of conditioned response in well-trained mice.However,little information is available about the effects of temporary or acute block of them on mice eye blink conditioning.Fastigial nucleus is the earliest developed deep cerebellar nuclei phylogenetically and involved in diverse physical functions and reflexes,while its role in eye blink conditioning is still unknown.In consequence,the aims of studies are:1.Investigating roles of the fastigial nucleus in the eye blink conditioning;2.Studying the effects on learning by blocking the fastigial nuclei during eye blink conditioning on naive mice;3.Observing the behavioral alteration of trained animals by chronic or temporary shutting down the fastigial during eye blink conditioning.Methods:1.Trained adult C57BL/6 mice were microinjected with muscimol in the fastigial nucleus.Behavioral changes were observed,recorded and compared before,5 min after and 1 day after injection.2.Designed receptor exclusively activated by designed drugs(DREADD)system was applied to temporarily shut down the fastigial nucleus.Adult C57BL/6 mice were injected with clozapine-N-oxide intraperitoneally everyday and trained with eye blink conditioning paradigm subsequently for 10 days;from 11th-17th days,animals were well trained without prior clozapine-N-oxide injection;animals were re-administrated with clozapine-N-oxide before eye blink conditioning trials for 2 days.3.Purkinje cell terminals of trained adult L7cre-Ai27 mice were optogenetically activated to inhibit fastigial nucleus.Eye lid movement was recorded during optogenetical inhibition of the fastigial nucleus in 0-250 ms,0-125 ms and 125-250 ms of conditioned stimulus.Results:1.The conditioned responses are eliminated by 5 min after muscimol injection in the fastial nucleus;while the conditioned response percentage out of all trials and amplitude after 1-day's muscimol injection show no significant difference compared to before muscimol injection.2.Compared to control animals,DREADD-interposed/fatigial mice also acquire conditioned response,but the amplitude of conditioned response is significantly lower than control.3.Conditioned responses are eliminated by shutting down the fastigial nucleus in 0-250 ms and 125-250 ms,while the conditioned response percentages out of optogenetial trials are no difference with non-optogenetical trials.Conclusion:The fastigial nucleus contributes to the eye blink conditioning of mice.Blocking it results in unacquisition in naive animals,but the roles of fastigial in conditioned mice need to further study.Chapter 4 In vivo electrophysiology study of interposed and fastigial nuclei in the eye blink conditioning of miceObjective:Electrophysiology studies encoded with Pavlovian reflex reveal the neural mechanism of cerebellar motor learning.The electrophysiology patterns in cerebellar cortex during eye blink conditioning have been elaborated.As the only output relay in the cerebellum,the deep cerebellar nuclei play important roles in information integration,transition and efferent in the eye blink conditioning,while little has been known about cellular activity.The fastigial nucleus composes diverse types of neurons,including GABAergic neurons which its contribution is still not clear yet in the eye blink conditioning.Both the interposed and fastigial nuclei are known to be involved in the eye blink conditioning in different pathways.Whether they receive the common inputs from brain stem needs to further study.Here we aim to:1.Study the cellular activities of interposed and fastigial nuclei in the eye blink conditioning mice;2.Identify the GABAergic neurons in the fastigial nucleus by optogenetical approach,and explore the electrophysiological characters of these cells during the eye blink conditioning;3.Investigate the common inputs to both the interposed and fastigial nuclei through retrograde tracing method.Methods:All mice are trained with eye blink conditioning paradigm to generate solid and well-performed conditioned responses.1.The interposed and fastigial neuron activities are extracellular record during eye blink conditioning of trained C57BL/6 mice,in the meantime,the eyelid movement is recorded.In the last recording day,cholera toxin B subunit is microinjected to localize the recording site.2.Adult Gad2-cre mice are microinjected with hSyn1-AAV-flex-chrimson-tdtomato in the fastigial nucleus.A 2 mm optical fiber is implanted in the same drilled tract to optogenetically activated the GABAergic neurons.After three-weeks' viral expression,cells which response to opto light are identified and tested activities encoding eye blink conditioning.Results:1.In total 42 interposed cells are recorded,25(60%)cells show facilitation during conditioned response and the others show suppression;trial-by-trial spike-eyelid correlation is observed in 6(14%)cells.270 interposed cells are recorded in total,certain part(60%)cells show facilitation during conditioned response and the others show suppression;trial-by-trial spike-eyelid correlation is observed in 20(7.4%)cells.2.In total 31 opto light-responding cells of fastigial nucleus are recorded in the Gad2-cer mice,10 cells show increased firing frequency during the opto light stimulation(GABAergic neurons),and they do not modulate during eye blink conditioning;the rest 21 cells show decreased firing frequency(local inhibited cells)and they all modulate as facilitation during conditioned response.Conclusion:1.Cerebellar interposed and fastigial neurons perform variable modulation in eye blink conditioning;some neuron spike frequency correlate to eye lid amplitude,indicating these neurons directly govern eye lid movement during eye blink conditioning,may play roles in motor learning.2.Fastigial GABAergic cells may not contribute to conditioned response;while the local GABAergically inhibited cells participate.