Electrophysiological,Morphological And Modulatory Properties Of Descending Serotonergic Neurons In The Brainstem Of EPet-EYFP Mice

The 5-HT neurons in the brain-stem play a crucial role in initiating locomotion, but there is no way to systematically study 5-HT neurons because of the limitations of the technology. The model of ePet-EYFP mouse were helpful, so this paper will use this model to investigate 5-HT neurons.the morphological and electrophysiological properties of 5-HT neurons were not studied comprehensive, and this kind of investigate on the mechanism of morphological and electrophysiological properties of 5-HT neurons was importent. Neurotransmitters (NMDA,5-HT, ACh etc.) involved in the regulation of locomotion, but this kind of neurotransmitters (NMDA and ACh) regulation on ePet-EYFP mouse of brain-stem 5-HT neurons is not clear, so we further studied the effects of NMDA and ACh on the membrane characteristics of 5-HT neurons, and discussion of the possible mechanisms for initiating locomotion.Objective:In this study,we investigated the morphological and electrophysiological properties of 5-HT neurons in the parapyramidal region (PPR) and midline raphe nuclei (MRN) of the neonatal ePet-EYFP mice.We also studied the effect of electrophysiological by NMDA and ACh on these 5-HT neurons with discussion of the possible mechanisms for initiating locomotion.Methods:Brain-stem slices were prepared from ePet-EYFP mice,5-HT neurons were selected by fluorescence microscopy and whole cell patch clamp recording were made from 5-HT neurons of ePet-EYFP mice.The resting membrane potential (Em)> action potential (AP)、time constant (Tm) and voltage threshold (Vth) were recorded to study the changes of these parameters before and after bath application of agents (NMDA+ACh).5-HT neurons were stained intracellularly by cascade blue(10%) during whole cell patch clamp recording for purpose of morphological study of 5-HT neurons.Results:(1) The ePet-cre and R26-stop-EYFP mice were bred and screened by agarose gel electrophoresis and PCR techniques, and the model of ePet-EYFP homozygous transgenic mice were obtained by four generation.(2) The experimental results were obtained from intracellular recordings of 87 neurons (67 5-HT neurons and 20 non-5-HT neurons).The 5-HT neurons located in the PPR(33) and MRN(34).5-HT neurons that in response to maintained depolarizing currents could be classified into three categories:1-2spikes(11/67), phasic firing(11/67) and tonic firing(45/67).(3)PICs (31/67) and H-current (5/67) were expressed in 5-HT neurons of the PPR and MRN,while bursting activity and membrane oscillations(EPSP,14/67;IPSP,1/67) were observed in 5-HT neurons.(4) There was no significant difference between MRN and PPR 5-HT neurons in their membrane properties(P>0.05). Membrane potential were 60.5±5.0 mV and 62.7±7.4 mV respectively. Compared with the non-5-HT neurons neurons,the Rin and Tm of 5-HT neurons were significantly higher. The Rin were 944.0±315.9MΩ and 1402.8±944.1MΩ, and time constant were 32.7±11.8 ms and 58.9±25.6ms, respectively.(5) The three types of 5-HT neurons are clearly different in their membraneproperties. Compared with type 15-HT neurons, the Ith of type 3 were significantly lower (P<0.05). TheirIth were 16.9±15.6 pA and 31.7±20.3 pA respectively. The Vth and Rin of type 3 were significantly lower than those of type 2(P<0.05), where Vth were -36.5±6.6 mV and -29.9±5.9 mV respectively and Rin were 1279.7±827.2 MΩ and 2124.7±1176.2 MΩ respectively. Among the three types of 5-HT neurons, the Vth of type 2 (-29.9±5.9 mV) were the biggest and that of type 3 (-36.5±6.6 mV) were the smallest.There was no significant difference among these three types of 5-HT neurons in their AP height、width and AHP height、width (P>0.05).(6) 15 μM NMDA and a cocktail of 15 μM NMDA plus ACh were applied in proper order to recording chamber with slices of 110-250 μm thickness taken from the brain-stem of ePet-EYFP transgenic mice. NMDA lowed (n= 12) input resistance and the depth of AHP (P < 0.05) with no significant difference in voltage threshold and the height of AP(P>0.05). A cocktail of NMDA plus ACh lowed (n= 12) input resistance, the depth of AHP and the height of AP (P< 0.05). and depolarized the voltage threshold of the 5-HT neurons (P< 0.05).(7) 5-HT neurons (n=29) were labelled with Cascade Blue (10%). The 5-HT neurons could be divided into oval (n=22) and stellate (n=7) categories according to somatic shapes or pseudounipolar (n=4)、bipolar (n=9) and multipolar (n=13) groups based on the number of stem dendrites. Different from morphology 5-HT neurons were not substantially different in morphological parameters (somatic diameter and spherical area). The proportion of the(8) bipolar and multipolar 5-HT neurons was 84.6%. There was no significant difference between bipolar and multipolar 5-HT neurons in their membrane properties (P>0.05), while membrane time constant of pseudounipolar 5-HT neuron was lower than that of multipole 5-HT neurons (P<0.05). There was no significant difference (P>0.05) between the electrophysiological parameters of the oval and stellate 5-HT neurons.Conclusion:(1) There was no significant different in neuron excitability between 5-HT neurons and non-5-HT neurons in the brain-stem of ePet-EYFP transgenic mice, suggesting that the excitability of 5-HT neurons might not be the major factor to induce the locomotion. However, the descending projections of these 5-HT neurons to spinal cord and their regulation of excitability of spinal neurons may be the main factors for initiation of locomotion.(2) The morphological characteristics of brain-stem 5-HT neurons might not be the mechanism for the 5-HT neurons generating locomotion. However, their morphological deffference might provide a basis with themselves to receive different neuromodulations from other system such as MLR.(3) The membrane properties of the 5-HT neurons of brain-stem with modulation of NMDA and ACh suggested that neuromoudaltions of the 5-HT neurons by neurotransmitters could be one of the mechanisms that motor system uses to generate or mediate locomotion.