Preliminary Study Of The Somatic Secretion In Rat Mesencephalic Trigeminal Neurons

The information transmission between neurons usually occurs in synapses. After formed in the neuronal body, the vesicles which containing neurotransmitters are to be transported to the nerve endings, and released by the exocytosis in pre-synapse. Recent studies have found that neuronal soma also has the function of neurotransmitter, such as hippocampal neurons, locus coeruleus neurons, DRG neurons and TG. neurons. However, limited by the conventional experimental methods, there still have many outstanding issues to be identified. For example, there have inconsistent conclusions about the role of calcium ions (Ca2+) in somatic secretion between central and peripheral neurons.Mesencephalic trigeminal nucleus neurons (Mes V neurons) are a special class of primary sensory neurons, and they received proprioception from pulp, periodontal ligament. Mes V neurons have the similar structures, functions and origin with peripheral nerves, but their soma are located in CNS. Compared with other primary sensory neurons, Mes V neurons are surrounded by a large number of nerve fibers and terminals came from different brain regions. Morphological studies confirmed that there were synaptic connections between Mes V neuron soma and these fibers and terminals. In addition, the contact between the Mes V neuron bodies is very close, suggesting that Mes V neuron soma may play an important role in the intercelluar information transmission.Previous studies confirmed that P2 purine receptors are involved in the generation and transmission of sensory information. Among the total, ionotropic P2X receptors are closely related with nociceptive pain, while the metabolic types of P2Y receptors, especially P2Y1 receptors are located in large-diameter sensory neurons, and related with mechanical sensory. It has been reported that P2X receptors can be expressed in Mes V neurons. In view of the distribution and function characteristic of P2Y1 receptors, P2Y1 receptors could be expressed in Mes V neurons and its possible mechanism remains to be further studied.In this study, whole-cell patch clamp recording, membrane capacitance measurements, carbon fiber electrode detection, fluorescence measurement of calcium, immunohistochemistry and other methods were applicated to identify whether Mes V neurons have somatic secretion and its possible relationship with Ca2+. In addition, we also observed and evaluated the expression and function of P2Y1 receptors in Mes V neurons.Results:Part I1. In the depolarizing stimulus, membrane capacitance measurement suggested that ?Cm was increased both in brain slices and single isolated Mes V neurons. At the same time, the increase of ?Cm was correlated with the stimulus duration and intensity.2. Under the action of high [K+]o (80mmol/L), the quantum release of "false neurotransmitters" in Mes V neurons was recorded by micro-carbon fiber electrodes.3. Trypsin could significantly inhibit the increase of depolarization-induced ?Cm in Mes V neurons.4. Calcium imaging showed that the depolarization-induced ?Cm could be increased accompanied by the increase of [Ca2+]i.5. After 0 mM [Ca2+]o ACSF perfusion or by using BAPTA (10 mmol/L) chelation of intracellular Ca2+, depolarization did not induce the increase of ?Cm in Mes V neurons.Part II:1. In the hyperpolarized stimulus, membrane capacitance measurement suggested that ?Cm was increased both in brain slices and single isolated Mes V neurons. Meanwhile, the increase of ?Cm was correlated with the stimulus duration and intensity.2. TeNT could significantly inhibit the increase of hyperpolarization-induced ?Cm in Mes V neurons.3. Fluorescence measurements showed hyperpolarization could not induce the increase of [Ca2+]i. On the contrary, [Ca2+]i was decreased in some cells (13:3).4. After 0 mM [Ca2+]o ACSF perfusion,hyperpolarization stimulus could significantly increase the ?Cm of Mes V neurons.Part III:1. Immunohistochemical staining results showed that Mes V neurons could express the P2Y1 receptor protein.2. P2Y1 receptor agonist ADP-β-S application could induce the Ih-activated current curve significantly shifted to the right, and the half-activation voltage moved to the depolarization direction.Conclusion:1. Mes V neurons have the character of somatic secretion.2. Depolarization-induced somatic secretion of Mes V is calcium- dependent, while the hyperpolarization-induced somatic secretion of Mes V is calcium- independent.3. Physiological concentrations of [Ca2+]o has the inhibit effects on the somatic secrertion induced by hyperpolarization.4. There are P2Y1 receptors on the surface of Mes V neurons. The activated P2Y1 receptor can regulate the opening of HCN channels, which may be involved in the sensory information transmission of Mes V neurons.In summary, our study demonstrated that Mes V neurons have the character of somatic secretion. Subsequently, we preliminarily studied the relationship between somatic secretion and calcium-dependent, and identified the expression of P2Y1 receptors on Mes V neurons. Our study also suggested that P2Y1 receptors might mediate the information transmission of Mes V neurons through Ih adjusting.