Excitatory Effect Of Corticotropin-Releasing Factor On Cerebellar Interposed Nucleus And Lateral Vestibular Nucleus And The Underlying Mechnisms

The cerebellum and the brainstem vestibular nuclear complex are two important subcortical motor centers,which hold key positions in movement coordination and balance control.Previous reports have revealed a wide distribution of corticotropin-releasing factor(CRF)in the cerebellum and the vestibular nuclear complex,and these two brain regions are also endowed with CRF receptors.However,their functional roles are still unknown.It has been well known that CRF is mainly synthesized and secreted by the neurons of the hypothalamic paraventricular nucleus(PVN),and serves as an important component of the hypothalamic-pituitary-adrenal axis.The involvement of CRF in stress-related responses has been studied for a long time,while little is known about its role in motor control.In fact,CRF is synthesized not only in the PVN but also in many other brain regions,including the inferior olivary nucleus(IO),a critical brain region sending afferents to both the cerebellum and the vestibular nuclear complex.Cerebellar climbing fibers,originating from the IO,constitute one of the two classical cerebellar afferent systems and hold a key position in cerebellar circuit-mediated motor control.Furthermore,as transitional nuclei and an important target for the cerebellar outputs,the vestibular nuclear complex also receives abundant inputs from the IO.Therefore,in this study,by using retrograde-tracing techniques,real-time quantitative PCR,immunofluorescence,in vitro patch clamp recordings and behavioral tests,we investigate the effect of CRF on neuronal activity of the cerebellar interposed nucleus(IN)and the lateral vestibular nucleus(LVN)as well as the underlying receptor and ionic mechanisms,and particularly,the function of CRFergic afferents in cerebellar circuit-mediated motor behaviors and cerebellar ataxia.The study will not only contributes to clarifying the role of CRFergic afferents in cerebellar and vestibular circuitry as well as motor control,but also helps to understand and treat motor disorders such as ataxia in clinic.1.CRF promotes cerebellar IN-mediated motor behaviors and improves motor disorders of a rat model of ataxiaThe cerebellum,the largest subcortical motor structure,plays an important role in the control of balance,regulation of muscle tone,and coordination of voluntary movement.Previous studies have revealed an extensive distribution of CRF-immunoreactive fibers in the cerebellum,and an expression of CRF receptors in cerebellar cortical Purkinje cells as well as the cerebellar nuclear neurons.Interestingly,the IO,the origin of the climbing fibers,shows immunoreactivities for CRF from neuronal somata to fiber terminals,indicating that CRF may act as a neurotransmitter in the olivocerebellar system for modulating the cerebellar circuitry and the related motor behaviors.In the cerebellar circuitry,the cerebellar nuclei receive collateral afferents from mossy fibers and climbing fibers,which constitute the primary cerebellar circuit,and makes the ultimate outputs of the cerebellum together with the flocculonodular lobe.Therefore,cerebellar nuclei are not the relay station simply receiving outputs from cerebellar Purkinje cells,but the key node for integrating the cerebellar inputs with the cerebellar cortical circuit.Among the cerebellar nuclei,the IN is one of the final output nuclei of the spinocerebellum and precisely controls distal muscles of the limbs and digits.Lesions of the IN lead to limb ataxia.In this study,we examine the effect of CRF on the IN neurons as well as the underlying receptor and ionic mechanisms.Particularly,the functional role of CRFergic afferents in the cerebellar circuit-mediated motor behaviors and cerebellar ataxia is investigated.By using retrograde-tracing techniques,real-time quantitative PCR,immunofluorescence,in vitro patch clamp recording combined with behavioral methods,we report that:(i)10 sends direct CRFergic projections to the IN,in which both CRFR1 and CRFR2 are expressed and co-localized;(ii)CRF selectively excites the glutamatergic projection neurons in the IN,but not the GABAergic projection neurons and interneurons;(iii)CRF concentration-dependently excites the IN projection neurons and the effect is a direct postsynaptic one;(iv)CRFR1 and CRFR2 co-mediate the CRF-induced excitatory effect on the IN projection neurons;(v)a dual ionic mechanism,including hyperpolarization-activated cyclic nucleotide-gated(HCN)channels and inward rectifier K+ channels,is involved in the excitation induced by CRF on the IN projection neurons;(vi)the CRFergic afferent inputs from the IO to IN significantly promote motor coordination and motor balance;(vii)downregulation of IO crf mRNAs lead to ataxia-like motor disorders,and microinjection of CRF into the IN rescues the motor dysfunctions of ataxia caused by downregulation crf mRNAs in the IO or intraperitoneal injection of 3-acetylpyridine(3-AP).In summary,CRFergic afferents,originating from the IO,promote rat motor coordination and balance,and improve motor dysfunctions of ataxia by selectively exciting the IN glutamatergic proection neurons.2.Effect of CRF on LVN neurons and the underlying receptor mechanismCentral vestibular system holds a key position in body balance and posture control.Vestibular nuclear complex includes four subnuclei:LVN,medial vestibular nucleus,superior vestibular nucleus and inferior vestibular nucleus.Among the vestibular nuclei,the LVN receives most abundant climbing fiber afferents from the IO,and plays a key role in adjustment of muscle tone and postural maintenance.By using immunofluorescence and in vitro patch clamp recordings,we investigate the effect of CRF on LVN neuronal activity.Our results show that:(i)CRF excites both spontaneous firing and silent LVN neurons;and(ii)the excitatory effect of CRF on LVN neurons is a direct postsynaptic one;(iii)CRF excites the LVN neurons via co-activation of CRFR1 and CRFR2;(iv)both CRFR1 and CRFR2 are expressed and co-localized in the LVN neurons.These results suggest that CRFergic afferents,originating from the IO,regulate LVN neuronal activity by co-activation of CRFR1 and CRFR2.In conclusion,the central CRF system participates in not only the classical non-somatic functions,such as autonomic and emotional responses,but also in somatic motor control via its direct modulations on the cerebellar and vestibular nuclear neurons.