Relationship Of M Currents Modulation And Neuronal Excitability In Rat SCG Neurons

The sympathetic nervous system is essential for homeostatic regulation of many critical physiological processes. Interactions with target tissues strongly influence morphological and electrophysiological properties of sympathetic neurons. The peripheral sympathetic nervous system of mammals can be divided into two anatomically distinct subdivisions: the paravertebral and prevertebral sympathetic ganglia. Neurons in both ganglia originate from common progenitors, but significant differences in the function and properties of neurons in the different ganglia arise during development. A significant fraction of neurons in the prevertebral ganglia receive synaptic input from both peripheral and central sources. These ganglia act as relatively sophisticated integrative centers controlling sympathetic outflow to the vasculature and enteric nervous system of the gastrointestinal tract. In contrast, most paravertebral ganglia transmit information from the spinal cord to the target organ without integrating other sources of information, although there is considerable convergence of preganglionic inputs. The superior cervical ganglia (SCG) are large fused ganglia located at the most rostral end of the paravertebral chain. Their target tissues include the vasculature of the head and neck, the submandibular gland and the iris of the eye.Two basic electrophysiological types of sympathetic neurons can be identified: one is phasic neurons, the other is tonic neurons. Phasic neurons receive one or a few large, suprathreshold synaptic inputs from preganglionic motor neurons located in the spinal cord. As a consequence, they function primarily as relay neurons connecting the central nervous system to peripheral organs. In contrast, tonic neurons receive multiple small, subthreshold synaptic inputs that have to summate in order to fire the cell. Synaptic input to tonic cells comes from both preganglionic spinal neurons and sensory neurons located in the periphery. There are different firing patterns between phasic neurons and tonic neurons. The former ones respond with transient bursts of action potentials, while the latter ones fire continuously in response to a maintained depolarizing stimulus.The different firing properties of sympathetic neurons appear to result from type and characteristics of voltage-gated ion channels. Many studies indicate"A current","M current"or"D2 current"may contribute to firing properties. Now it is generally accepted that"M current"is a key factor correlating with the firing properties of sympathetic neurons. M current is a low threshold, voltage- and time-dependent, slow-activated, slow–deactivated, non-inactivating outward potassium current first described in bullfrog sympathetic neurons by Brown and Adam in 1980. The ion channel underlying M current is called M channel. M channel is widespread in many types of tissues and cells including sympathetic ganglia, dorsal root ganglia, hippocampus, some neurons from center nervous system, NG108-15 and PC12 cell lines. M channel is one member of potassium channel superfamily encoded by KCNQ genes. Heteromeric assembly of KCNQ2 plus KCNQ3 subunits underlies the molecular composition of neuronal M channel and KCNQ5 contributes to its diversity. Mutation or functional defect of these subunits leads to neuronal disorders such as epilepsy, benign familial neonatal convulsion (BFNC), Alzheimer's disease and pain.M-current has a major impact on neuronal excitability because it is the only current active at voltages near the threshold for action potential initiation. This potassium current is slowly activated when the neuron is depolarized toward the threshold for action potential firing, hyperpolarizing the membrane back toward rest and reducing membrane excitability. Inhibiting M current makes membrane easy to depolarize and fire action potentials, then altering firing patterns and response to synaptic inputs. M current can be modulated by many neurotransmitters and hormones including acetylcholine, bradykinin, angiotensinⅡand luteinizing hormone releasing hormone (LHRH). Their up- and down-regulation of M current via activating their GPCRs or affecting downstream signal molecules result in modulating excitability of nervous system.Except GPCRs, little is known about other membrane receptors such as receptor tyrosine kinases (RTKs) on their role in the regulation of M current.Mammalian neurotrophins (NT) are a large family that includes nerve growth factor (NGF) which is the first member described, brain-derived neurotrophic factor (BDNF), NT3, NT4/5, NT6 and NT7. Expressed throughout the CNS and PNS, NGF plays a critical role by binding to its receptors in regulation of neuronal survival, proliferation, differentiation, axon and dendrite growth and patterning, expression of important functional proteins such as ion channels, neurotransmitter receptors. In addition to these effects, NGF also modulates synaptic structure and connections, neurotransmitter release, long-term potentiation (LTP) and synaptic plasticity.There are two types of receptors mediating the biological effects of NGF: Trk A receptor tyrosine kinase and the pan-neurotrophin receptor p75NTR. The former receptor belongs to receptor tyrosine kinases (RTKs) family and the latter is one of 26 members of the TNF receptor superfamily. NGF binding to Trk A receptor leads to receptor dimerization, autophosphorylation and subsequently activates several intracellular signaling events includind Ras-Raf-Erk, PI3 kinase-Akt/PKB, PLCγ-PI(4,5)P2 hydrolysis, non-receptor tyrosine phosphorylation, and NF-κB pathways. Activation of the common receptor p75NTR by NGF leads to activating Jun N-terminal kinase (JNK) which mediates the process of neuronal apoptosis, NF-κB pathways and generation of ceramide which can modulate cellular biological process.In addition to the long-term effects of NGF depending on regulation of genetic transcription and expression, there is more and more evidence showing that NGF also exerts acute effects such as modulation of synaptic transmission, ion channel function and neuronal excitability.In the present study, we studied the relationship of M currents modulation and excitability of rat SCG neurons. We first characterized neuronal M currents and excitability and the relationship between these two. Subsequently, we studied the effects of NGF, as well as classical M currents modulating agents such as M receptor agonist oxotremorine-M (Oxo-M) and M channel blocker linopirdine (LP), on neuronal M current and excitability, and investigated the molecular mechanism involved.Objective: By using perforated-patch clamp technique to record neuronal action potentials and M channel currents, we (1) studied the characteristics of action potentials and M currents of rat SCG neurons, (2) valuated the relationship between M currents and excitability, (3) studies effects of NGF, Oxo-M and LP on neuronal excitability and (4) investigated the action mechanisms of NGF on M currents.Methods: (1) cell culture: Primary cultures of neurons came from superior cervical ganglion (SCG) of 4-6 weeks old SD rat both sex. Briefly, SCG were removed and cut into several pieces, and then incubated with mixtures of collagenase (1.5mg/ml) and dispase (5mg/ml) to be digested for 30 min. After that, SCG were dissociated into a suspension of individual cells and planted onto poly-D-lysine coated glass coverslips in 24-well tissue culture plates. Cells were incubated at 37°C with 5% CO2 and 95% air atmosphere with proper medium and used for recording within 48 hr. (2) Electrophysiological recording: perforated- patch clamp technique were performed on SCG neurons to record action potentials and ion channel current at room temperature (22°C - 25°C). Perforated-patch clamp was carried out with amphotericin B (120ng/ml) in the pipette. The experiment is performed under a strictly controlled condition.Results: (1) According to the number of action potential elicited by maintained depolarizing current stimulus, rat SCG neurons were classified into three different electrophysiological types: phasic-1 (a single action potential), phasic-2 (transient 2~6 action potentials) and tonic (firing continuously). The proportion of these classes is 36.4%, 53.5% and 10.1%, respectively. These results indicate that phasic neurons are predominant types. Regarding number of spikes as the major determinant of neuronal excitability, we compared three types of neurons in aspects of resting potential, latency of spike firing, interval of spike and distance from resting potential to threshold. The results indicate that phasic-1, phasic-2 and tonic neurons are significantly different in number of spikes (p<0.01). The level of resting potential of tonic neurons (-49.7±1.73 mV) is significantly lower than phasic neurons (p<0.01), but that of phasic-1 (-54.8±6.94mV) and phasic-2 (-56.7±7.20mV) neurons are not different. Thus the excitability of tonic neurons is high than phasic neurons. (2) Action potential of phasic-1, phasic-2 and tonic neurons responded differently to increment step current. Phasic-1 neurons could not be elicited more than one action potential by injecting 0.2~0.3nA current, but the number of spikes of phasic-2 neurons was increased dramatically by injecting 0.01~0.1nA current and 30% of these cells were turned to tonic firing. Similar to phasic-2 neurons, tonic neurons needed only 20~30pA current to have more firing. These results suggest that tonic neurons are much more sensitive to depolarizing current. (3) Amplitudes and electrophysiological characteristics of M current are different in three types of neurons. The amplitude of M current in phasic-1, phasic-2 and tonic neurons is 118.4±10.01pA, 97.8±6.53pA and 33.7±3.36pA, respectively. Summary data indicate that M current in tonic neurons is much less than that of phasic neurons (p<0.01). I-V curves were established for three types of neurons. V1/2 of tonic neurons was right shifted compared with phasic neurons, which means tonic neurons need higher voltage to activate. Both activation and deactivation of tonic neurons are slower than that of phasic neurons (p<0.01). (4) Amplitude of M currents correlate well with firing properties of neurons, thus the bigger the M currents, the less the firing numbers. (5) The number of spikes of three types of SCG neurons were increased significantly by application of Oxo-M or LP (p<0.01). (6) 10μM Oxo-M inhibited M currents of phasic and tonic neurons to the same degree. (7) NGF is less potent than Oxo-M and LP in inhibiting M current of SCG neurons. Also, the current is difficult to recover after NGF action. (8) NGF increased excitability of tonic neurons, but it had no effect on phasic neurons. This might due to the fact that NGF can hardly inhibit M currents to the extent that will release control on excitability. (9) Application of Oxo-M on top of NGF increased excitability of phasic neurons. This result further indicates that amplitude of M current is a key determinant on the neuronal excitability.Conclusion: (1) Rat SCG neurons can be classified into three different electrophysiological types: phasic-1, phasic-2 and tonic. Phasic neurons are predominant types. (2) Tonic neurons have highest excitability and have highest sensitivity to depolarizing current stimulus. Phasic-2 neurons have lower excitability and less sensitivity than tonic neurons. Phasic-1 neurons have lowest excitability and the least sensitivity. (3) Amplitude and electrophysiological characteristics of M current are different in three types of neurons. Compared with phasic neurons, tonic neurons have larger M current, need higher voltage to activate and is slower in both activation and deactivation. (4) Number of spikes correlates closely with amplitude of M current, thus the bigger the M currents, the less the firing numbers. (5) Oxo-M and LP can inhibit M current of phasic and tonic neurons to the same degree. (6) NGF can also inhibits M current of phasic and tonic neurons equally, but NGF is much less potent than Oxo-M. Thus NGF can only increase excitability of tonic neurons, but has no effect on that of phasic neuron.