The Effects Of Noradrenaline On Reticulospinal Neurons In Rat Caudal Pontine Reticular Nucleus

The central noradrenergic nervous system is one of the most important neurochemical networks in the brain.The neurons that produce noradrenaline(NA)are all located in several clusters within the brainstem,of which the locus coeruleus(LC)is the main source of central NA.Although the NA-producing neurons are located in the brainstem,they virtually innervate almost the whole central nervous system(CNS)including the spinal cord and thus work in diverse brain functions.In the CNS,the effect of NA in different target regions is mediated by mainly three categories of adrenoceptors,including ?1-,?2-,and ?-adrenoceptors.However,complex patterns of availability and distribution of the exact adrenoceptor in different regions have been revealed.In addition,different brain regions may use diverse downstream cellular effectors following adrenoceptor activation.As a result,even for a specific physiological function that is modulated by the noradrenergic system,such as sleep/wakefulness control,mood regulation,sensory processing,or motor regulation,its effects and mechanisms in the diverse brain structures involved may be different and complex and thus warrant interests.The noradrenergic system modulates multiple related functions primarily via the ascending and descending pathways.While many studies have dedicated to elucidating the effects and cellular bases that underlie the modulations of the ascending noradrenergic system on higher-order cognitive functions,studies concerning the effects of NA and mechanisms within the descending noradrenergic machineries have also garnered interest.Intriguingly,the descending noradrenergic fibers reach several important motor areas,with fairly high densities in the medial pontomedullary reticular formation(mPMRF)and spinal cord,suggesting a potent role for the descending noradrenergic system in central motor control.Consistently,the effects of NA and their underlying cellular bases in spinal motoneurons,the final common motor pathway,have been extensively demonstrated,and they indicate the prominent excitatory role of NA.Nevertheless,it is also widely accepted that the reticulospinal neurons within the mPMRF play important roles in diverse motor behaviors,such as locomotion,reaching,and posture control.In fact,the fibers from the reticulospinal neurons directly innervate the spinal cord motor circuitry.The related motor information from the reticulospinal neurons is thus sent to the spinal motoneurons to ultimately coordinate motor control.Intriguingly,a functional set of reticulospinal neurons that are located in the caudal pontine reticular nucleus(PnC)in the mPMRF has been reported to be specifically involved in a well-defined postural motor behavior called startle.In addition,the microinjection of NA into the mPMRF influences the startle.However,it remains largely unknown how the PnC reticulospinal neurons are modulated by the descending noradrenergic system.Therefore,the present study uses a combination of morphological and electrophysiological methods to systemically explore the effect and mechanisms of NA in PnC reticulospinal neurons.The main results are as follows,1.The effects of NA on whole-cell currents of PnC reticulospinal neurons are mediated by the post-synaptic ?1-and ?2-adrenoceptors.We first employed the whole-cell patch clamp method to directly explore the effect of NA on the whole cell currents of morpholocially and eletrophysiologically identified PnC reticulospinal neurons at the holding potential of-70 mV.Interestingly,bath application of 3 ?M NA induced an inward current in some(82/135,60.7 %)PnC reticulospinal neurons while an outward current in others(53/135,39.3 %).Besides,both the 3 ?M NA-induced inward(P = 0.364,n = 6)and outward(P = 0.511,n = 6)current in PnC reticulospinal neurons are tetrodotoxin(TTX)-resistent,suggesting that post-synaptic mechanisms are involved in the observed effects of NA.In 7 neurons exhibiting inward current responses to NA,0.01 ?M prazosin(Pra,a selective ?1-adrenoceptor antagonist)partially blocked the NA-elicited inward current(P < 0.05),indicating the involvement of ?1-adrenoceptors.In the presence of a higher concentration of Pra(10 ?M),the inward current was abolished,but an NA-elicited stable outward current was observed in the same 7 neurons.This reversed outward current was blocked by the application of 10 ?M yohimbine(Yh,a selective ?2-adrenoceptor antagonist)and 10 ?M Pra(P < 0.01).In 6 neurons exhibiting outward current responses to NA,0.1 ?M Yh partially blocked the NA-elicited outward current(P < 0.01).Next,in the presence of 10 ?M Yh,the outward current was abolished and NA elicited an inward current in the same 6 neurons.This NA-elicited inward current was abolished by further co-application of 10 ?M Pra with 10 ?M Yh(P < 0.01).These results demonstrate that the ?1-and ?2-adrenoceptors are co-expressed on the postsynaptic membrane of a single reticulospinal neuron to mediate the inward and outward current components,respectively,which are superposed to produce the overall NA-elicited current at the holding potential of –70 mV.Consistently,our immunofluresence staining revealed that the ?1A and ?2A adrenoceptors are indeed co-localized in individual PnC neurons with a diameter greater than 35 ?m.2.Activation of the post-synaptic ?1-and ?2-adrenoceptors exerts opposing modulations on the firing activities in the same PnC reticulospinal neuron.We next conducted current clamp recordings in I=0 mode to directly examine the modulations of the post-synaptic adrenoceptors in PnC reticulospinal neurons upon activation.Synaptic transmission was blocked by the addition of 10 ?M CNQX,20 ?M AP5,and 100 ?M picrotoxin into the ACSF.In this condition,all six of the recorded PnC reticulospinal neurons maintained regular spontaneous firing activities(mean firing rate,4.5±1.5 Hz;total range from 1.7 to 11.6 Hz).In these neurons,100 ?M phenylephrine(PE,a selective ?1-adrenoceptor agonist)increased the firing rate to 6.0 ± 1.4 Hz(P<0.05).In contrast,30 ?M UK 14304(UK,a selective ?2-adrenoceptor agonist)decreased the firing rate of these same neurons to 2.5 ± 1.7 Hz(P < 0.05).These results provide confirmation at the neuronal activity level for the functional co-expression of ?1-and ?2-adrenoceptors in a single PnC reticulospinal neuron and further suggest that these adrenoceptors can differentially influence the post-synaptic dynamics of the PnC reticulospinal neurons upon activation.3.Ionic mechanisms coupled to the post-synaptic ?1-and ?2-adrenoceptors in PnC reticulospinal neurons.To identify the ionic conductances,a slow linear voltage ramp(ranging from-125 to +5 mV,slope +10 mV/s)was employed to construct the I-V relationships of the respective adrenoceptor-mediated net current in the PnC reticulospinal neurons.(1)Activation of post-synaptic ?1-adrenoceptors activates the nonselective cationic conductances(NSCCs).In normal ACSF,the ?1-adrenoceptor-mediated net current was reversed near the equilibrium potential of the Na+-permeable NSCCs(mean:-29.1 ± 3.0 mV;n = 15).Moreover,all I-V relationships of the ?1-adrenoceptor-mediated net current displayed a positive slope over the voltage range around the reversal potentials,suggesting that the activation of post-synaptic ?1-adrenoceptors activates the NSCCs.Consistently,changing the Na+ concentration from 152.25 to 70 mM in the ACSF decreased the amplitude of the ?1-adrenoceptor-mediated inward current(P < 0.01,152.25 mM Na+: n = 40,70 mM Na+: n = 13)and shifted the reversal potential of the ?1-adrenoceptor-mediated net current to a more negative value,-42.9 ± 4.2 mV(P < 0.05,152.25 mM Na+: n = 15,70 mM Na+: n = 9).NSCCs mainly consisted of Na+ plus K+ conductances and the Na+/K+ permeability ratios at the Na+ concentrations of 152.25 and 70 mM were not significantly different(P = 0.702,152.25 mM Na+: n = 15,70 mM Na+: n = 9)calculated by using the reversal potentials and the Goldman-Hodgkin-Katz equation.These results indicate that activation of the post-synaptic ?1-adrenoceptors in the PnC reticulospinal neurons solely activates the NSCCs.(2)Activation of the post-synaptic ?2-adrenoceptors causes the inhibition of the NSCC and the activation of the K+ conductances in PnC reticulospinal neurons.In normal ACSF,the ?2-adrenoceptor-mediated net current was also reversed near the equilibrium potential of the NSCCs(mean-23.5 ± 4.3 mV;n = 10).In addition,a more negative reversal potential near the K+ equilibrium potential(calculated using the Nernst equation:-102.3 mV)was also observed from three of these neurons(mean-99.3 ± 4.3 mV).Moreover,all I-V relationships displayed a negative slope over the voltage range around the more positive reversal potentials and a positive slope over the voltage range around the more negative reversal potentials.These results suggest that the activation of the ?2-adrenoceptors causes the inhibition of the NSCCs and the activation of the K+ conductance in the PnC reticulospinal neurons.Consistently,changing the Na+ concentration from 152.25 to 70 mM in the ACSF decreased the amplitude of the ?2-adrenoceptor-mediated outwasrd current(P < 0.01,152.25 mM Na+: n = 25,70 mM Na+: n = 10).In this condition,the majority of the tested neurons(n = 6 / 7)had two reversal potentials: one at the mean of-109.9 ± 4.7 mV,still near the K+ equilibrium potential,and the other at the mean of-28.2 ± 1.0 mV which was obviously higher than the expected equilibrium potential of the NSCCs in this condition.This expected equilibrium potential of the NSCCs can be evaluated by the reversal potential of the ?1-adrenoceptor-mediated current in 70 mM Na+ ACSF(-42.9 ± 4.2 mV,n = 9).Adding 1 mM Ba2+ into the 70 mM Na+ ACSF further attenuated the ?2-adrenoceptor-mediated outwasrd current(P < 0.05,70 mM Na+: n = 10,1 mM Ba2+: n = 6)and all tesed neurons displayed only one reversal potential at-44.7 ± 1.3 mV(n = 6),which was not significantly different from the expected equilibrium potential of the NSCCs in 70 mM Na+ ACSF(P = 0.370).These data all indicate that K+ and NSCCs conductance is indeed coupled to the ?2-adrenoceptors in the PnC reticulospinal neurons and the activity of K+ conductance may largely contribute to affect the more positive reversal potentials in 70 mM Na+ ACSF.Moreover,the Na+/K+ permeability ratio of the ?2-adrenoceptor-mediated current without the influence of the K+ conductance was not significantly different from the ?1-adrenoceptor-mediated current(P = 0.671,?1-adrenoceptor: n = 6,?2-adrenoceptor: n = 24),further confirming that the same NSCCs is coupled to the ?1-and ?2-adrenoceptors.4.?2-adrenoceptors are also located in excitatory pre-synaptic terminals to mediate the NA-induced inhibition of the mEPSC in PnC reticulospinal neurons.We next isolated the mEPSCs or mIPSCs of the PnC reticulospinal neurons to test whether NA could also affect neurotransmission to these neurons.NA did not affect the mIPSC frequency(P = 0.428,n = 6)or mIPSC amplitude(P = 0.508,n = 6)in PnC reticulospinal neurons.Intriguingly,it inhibited the mEPSC frequency(P < 0.01,n = 7)but not the mEPSC amplitude(P = 0.335,n = 7).After pharmacologically blocking the ?2-adrenoceptor,the inhibition of the mEPSC frequency induced by NA was completely abolished(P = 0.145,n = 6).The mEPSC amplitude was also unaffected(P = 0.994,n = 6).Additionally,pharmacologically activation of the ?2-adrenoceptor mimicked the NA-induced inhibition of the mEPSC frequency on all five of the tested neurons(P < 0.05),and it also did not influence the mEPSC amplitude(P = 0.347).These results demonstrate that ?2-adrenoceptors may also be located in the excitatory pre-synaptic terminals targeting the PnC reticulospinal neurons,where they exert inhibitory control over the excitatory release onto these neurons.5.The amplitude of the evoked EPSC onto PnC reticulospinal neurons is attenuated by the pre-synaptic effect of NA.To further determine whether the inhibitory effect on excitatory release mediated by the pre-synaptic ?2-adrenoceptors ultimately participated in functionally modulating the excitatory input onto the PnC reticulospinal neurons,we evoked the EPSCs in PnC reticulospinal neurons using a stimulation electrode set near the recorded neurons.3 ?M NA decreased the eEPSC amplitude by 47.1 ± 4.9 %(n = 10).Meanwhile,this depression was accompanied by an increase in the PPR(eEPSC2 / eEPSC1)(P < 0.05,n = 10).On the same neurons,Yh nearly completely abolished the 3 ?M NA-induced depressive modulation of the eEPSC(P < 0.01,n = 10).An accompanying increase of the PPR in ACSF was not observed in the presence of 10 ?M Yh(P = 0.572,n = 10).These results confirm that the depressive modulation of NA on the eEPSC is indeed and solely mediated by the activation of the pre-synaptic ?2-adrenoceptors.We further explored the characteristics of this pre-synaptic ?2-adrenoceptor-mediated inhibitory modulation on the eEPSC.After activating the pre-synaptic ?2-adrenoceptors with various concentrations of NA(1–10 ?M),we demonstrated a homogeneous and concentration-dependent depressive modulation of the eEPSC.1,3,and 10 ?M NA decreased the normalized amplitude of eEPSC by 29.2 ± 2.2 %(n = 8),45.9 ± 3.9 %(n = 10),and 51.2 ± 3.3 %(n = 10),respectively.Meanwhile,this depression was accompanied by an increased PPR(eEPSC2 / eEPSC1)in these neurons and the increase in the PPR also occurred in a concentrationdependent manner.Furthermore,we found that the degrees of depressive modulation of NA(1–10 ?M)on the eEPSC did not correlate with the amplitudes of the NA-elicited post-synaptic current(r =-0.0219,P = 0.621,n = 36).Thus,these results may further exclude the possible involvement of postsynaptic mechanisms and suggest that these concentration dependent modulations are solely mediated by the presynaptic ?2-adrenoceptors.