The Temperature-sensitive Mechanism Of Temperature-sensitive Neurons In Parabrachial Lateral Nucleus

Previous study has identified the presence of warm-and cold-sensitive neurons in the lateral parabrachial nucleus(LPB),a crucial relay for afferent signals from the skin that promote defensive thermoregulatory responses before changes in environmental temperature affect body core temperature.However,the cellular mechanisms by which these thermosensitive neurons sense changes in temperature have not yet been determined.In this study,infrared visual patch clamp and immunofluorescence techniques were used to observe the effects of synaptic blockade on the activity and temperature sensitivity of LPB neurons,the electrophysiological properties of LPB thermosensitive neurons,the effects of temperature on A-type potassium channel-mediated IA currents,and HCN channel-mediated Ih currents of LPB neurons,so as to reveal their mechanisms for neuronal thermosensitivity of thermosensitive neurons in the LPB.Part ? The effects of synaptic blockade on the firing activity and temperature sensitivity of LPB neuronsObjectives To investigate whether synaptic transmission was involved in the neuronal thermosensitivity of LPB neurons.Methods Brain slices containing the LPB were prepared from SPF healthy male Sprague–Dawley rats(60~100g).Low Ca2+ high Mg2+ ACSF was perfused after identifying the temperature sensitivity to observe the effects of synaptic blockade on the firing activity and temperature sensitivity of LPB neurons.At last,the recovery of the activity and temperature sensitivity of LPB neurons after washout was observed.Results Synaptic blockade had no effect on the firing activity and temperature sensitivity of the 13 warm-sensitive neurons in the LPB(P > 0.05);the firing rate of the 11moderate-slope and 11 low-slope temperature-insensitive neurons in the LPB showed the variety of responses duing synaptic blockade,and the firing amplitude didn't change(P>0.05),but the temperature sensitivity coefficients incresed duing synaptic blockade(P< 0.05).The firing activity and temperature sensitivity of warm-sensitive and temperature-insensitive neurons in the LPB recovered after washout.Conclusions The intrinsic temperature sensitivity of the warm-sensitive neurons in LPB didn't depend on synaptic transmission.Part ? The electrophysiological properties of temperature-sensitive neurons in the LPBObjectives To identify the temperature-sensitive mechanism of LPB neurons at the cellular level by observing the effects of temperature on electrophysiological properties of the LPB neurons.Methods These experiments employed whole-cell intracellular recordings of warm-sensitive,cold-sensitive,and temperature-insensitive LPB neurons.The thermal effects on resting membrane potentials,action potential duration,firing amplitude,threshold potential,depolarizing prepotentials and afterhyperpolarizing potentials(AHP)were compared between different types of neurons.Results Intracellular spontaneous activity was recorded in 75 neurons during at least one cyclic temperature change to investigate the cellular mechanisms of lateral parabrachial neuronal thermosensitivity.Of the 75 spontaneously firing neurons,23 neurons were warm-sensitive,8 neurons were cold-sensitive,26 were moderate-slope temperature-insensitive,and 18 were low-slope temperature-insensitive.Warming increased the rate of rise of the depolarizing prepotential of 23warm-sensitive neurons in LPB(P<0.05)and then the firing frequency is accelerated,but there is no significant effect on the resting potential.Warming produced membrane hyperpolarization of 8 cold-sensitive neurons in LPB(P < 0.05),but there is no significant effect on the rate of rise of the depolarizing prepotential.Warming decreased action potential amplitudes and shortened the half-amplitude duration of LPB neurons(P < 0.01),but there were no differences between the cell types(P >0.05).Temperature had no effects on the threshold potential and AHP of LPB neurons(P>0.05).Conclusions1.The prepotential was the primary mechanism of LPB neuronal warm sensitivity.2.The neuronal cold sensitivity was primarily attributed to a thermally induced change in the resting potential.Part ? The ionic mechanism of LPB neuronal thermosensitivity Objectives To identify ionic mechanisms of LPB neuronal thermosensitivity by observing the expression of HCN2 channel in LPB neurons,the effects of temperature on IA current(mediated by HCN channel)and Ih current(mediated by A-type potassium channel)in the LPB neuronsMethods Immunofluorescence staining was used to detect the expression of HCN2 channels in LPB neurons.In the voltage clamp mode,the effects of temperature on the current amplitude,the time constant of inactivation or activation,and rate of inactivation or activation of IA or Ih currents in different types of LPB neurons were observed.Results Immunofluorescence experiments showed that HCN2 channels were expressed on the LPBc,LPBd and LPBel,the three subnuclei responsible for the transmission of cutaneous feedforward signals.In the voltage clamp mode,IAcurrent activity of 18 LPB neurons were recorded at 32??36??39?,including 6 warm-sensitive neurons,5 moderate-slopetemperature-insensitive neurons and 7 low-slope temperature-insensitiveneurons.And IA current activity was not recorded in the 6 cold-sensitive neurons.Warming increased the IA current amplitude of LPB neurons at membrane potential of +40m V(P<0.05);warming shortened the time constant of inactivation of IA currents,and increased the rate of inactivation of IA currents(P < 0.05).Temperature had no significant effect on the time constant of inactivation and the rate of inactivation of IA currents in the temperature-insensitive neurons(P>0.05).Ih current activity of 29 LPB neurons were recorded at 32?,36? and 39?,including 7 warm-sensitive neurons,8 moderate-slope temperature-insensitive neurons,9 low-slope temperature-insensitive neurons,and 5 cold-sensitive neurons.Warming increased the Ih currents amplitude of 7 warm-sensitive LPB neurons(P < 0.05)at membrane potential of-120 m V;warming shortened the time constant of activation of Ih currents,and increased the rate of activation of Ih currents(P<0.05).Temperature had no significant effect on the time constant of activation and the rate of activation of Ih current in the other three types of neurons(P>0.05).Conclusions1.The HCN2 channel expressed in the rat LPBc,LPBd and LPBel.2.The temperature-dependent inactivation of IA currents(mediated by HCN channel)was involved in the LPB neuronal warm sensitivity.3.The temperature-dependent activation of Ih currents(mediated by A-type potassium channel)was also involved in the LPB neuronal warm sensitivity.