| Neuronal excitability is one of the basic characteristics of neurons. Neurons will fire action potentials when they receive adequate stimulus. Generally, the whole period of neuronal excitation can be divided into three processes, depolarization period (membrane potential was reduced in very short time, a process forming the upslope of the action potential), repolarization period (membrane potential was increased negatively in short time, a process forming the downslope of the action potential) and recovery period or resting period (membrane potential maintains relatively stable, normally called resting potential).Neuronal excitation is founded on the activity of ion channels. In general, sodium ions flux into the cells when sodium channels are open, which leads to depolarization of neurons and forms the upslope of action potential. Soon after, sodium channels become inactivated and closed, and at the same time potassium channels are activated and open, which allow potassium ions flux out of the cell, which results in repolarization of neurons and forms the downslope of action potential. The upslope and downslope of action potential make a spike potential. After each action potential, displaced ions are recovered to their original disproportional distribution across the membrane by the action of sodium-potassium pump. This disproportional distribution of sodium and potassim across the mebrane prepares the cell to fire next action potential when the cell receives again a suprathreshold stimulus. Apart from sodium currents, other inward currents can also induce membrane depolarization action potential. For example, in sensory neurons like dorsal root ganglion (DRG) neurons, capsaicin can lead to the activation and opening of TRPV1, which are also sensitive to thermal stimulus and pH. M-type potassium channel or M/KCNQ channel is a voltage-gated channel with characteristics of slow-activation and deactivation and non-inactivation. Activation of muscranic receptor strongly inhibitits this potassium channel, hence, it is called M-type potassium channel. The molecular basis of M/KCNQ channel is the tetraheterologus of KCNQ2 and KCNQ3 channels. Mutations of KCNQ2/3 lead to benign familial neonatal convulsions. M/KCNQ channel distributes widely in the nerve system in mammalian, including the central nerve system and peripheral nerve system. Generally, M/KCNQ channel is activated around– 60 mV, a membrane potential near the resting membrane potential. Thus M/KCNQ channels are believed to play a key role in stabilizing membrane potential and regulating neuronal excitability. M/KCNQ channel can be regulated by many factors such as G protein-coupled receptors, receptor tyrosine kinase, and other factors.Voltage-gated sodium channels (VGSC) are composed of a complex of aαsubunit that forms the voltage-sensitive and ion-selective pore, in association with one or more auxiliaryβsubunits. To date, nineαsubunits of the VGSC superfamily, Nav1.1-Nav1.9, have been identified, which are widely distributed in mammalian tissue. Among these VGSC, Nav1.7 channel is dominantly expressed in superior cervical ganglion (SCG) neurons. Nav1.7 channel is coded by gene, SCN9A, a missense mutation in which underlies the primary erythermalgia. Many factors modulate VGSC, including G-protein coupled receptors and tyrosine kinases.Transient receptor potential vanillicacid 1 channel, simply called TRPV1 channel, is a kind of non-selective cation channels, which allows calcium, magnesium and sodium ions flux into cells. TRPV1 channel mainly distributes in nociceptive neurons such as the nociceptive neurons in DRG. TRPV1 channel can be activated responding to nociceptive stimulus. TRPV1 channel also is the receptor of capsaicin, a kind of extractive agent from capsicum chilli. Capsaicin binds to TRPV1 channel and then activates this channel and induces influx of cations, which enhances neuronal excitability. TRPV1 channel is also sensitive to thermal stimulus, pH and machinery pressure. Many factors can modulate the activatiy of TRPV1 channel, such as membrane PI(4,5)P2, Ca2+ and Calmodulin, etc.The present study will investigate the functional regulation of M/KCNQ channel, voltage-gated sodium channel and TRPV1 channel, and related neuronal excitability using primary cultured neurons from rat SCG and DRG. We also study the intrcellular calcium signals induced by activation several G protein-coupled receptors expressed in DRG neurons.1. The regulation of M/KCNQ currents and neuronal excitability by NGF. Aim: (1) To record M/KCNQ currents and neuronal action potential in rat SCG neurons, and to study the mechanism of regulation of M/KCNQ currents and neuronal exctibality produced by nerve growth factor (NGF).Methods: (1) Cell culture. Primary cultures of neurons were prepared from SCG from 3- to 5-week-old Sprague-Dawley rats. Briefly, ganglia were digested with collagenase (1 mg/ml) and next trypsin (2.5 mg/ml), and then ganglia were dissociated into a suspension of individual cells and planted on poly-lysine coated glass coverslips. Cellas were incubated at 37°C with a 5% CO2 + 95% air. The DMEM medium plus 10% serum were changed to neurobasal A medium plus 2% B27 supplement after 12 hr and cells were used within 48 hr. (2) Electrophysiology. Perforated patch and conventional whole-cell patch were used to record the neuronal M/KCNQ currents under voltage-clamping mode. Pipettes were pulled from borosilicate glass capillaries and had resistances of 3-5 M? when filled with pipette solution. Currents and action potentials were recorded using an Axon 200B amplifier and pClamp 9.0 software, and were filtered at 2 KHz. The protocol for recording of M/KCNQ currents is as follows: SCG neurons were held at -20 mV followed a 0.8 s hyperpolarization step to -60 mV every 4 s. The amplitude of M/KCNQ currents was defined as the outward currents sensitive to 30μM linopirdine, a specific M/KCNQ channel blocker, and was measured from deactivation currents records at -60 mV as the difference between the average of an initial 10-ms segment, taken 10-20 ms into the hyperpolarizing step, and the average during the last 10 ms of that step. Perforated patch was used to record neuronal action potentials under current-clamping mode. The protocol to record action potential is as follows: SCG neurons were held at 0 current level and the action potentials were elicited by injection of a depolarizing current for 2 s. The external solution used to record M/KCNQ currents contained (in mM): NaCl 120, KCl 3, HEPES 5, NaHCO3 23, Glucose 11, MgCl2 1.2, CaCl2 2.5 and TTX 0.00005, (adjusted to pH 7.4 with NaOH). The pipette solution for perforated patch recording consisted of (in mM): KAc 90, KCl 40, HEPES 20 and MgCl2 3 (adjusted to pH 7.3-7.4 with KOH). Na2ATP (3 mM) and EGTA (5 mM) were added to the above internal solution for conventional whole-cell recording. The external solution used to record neuronal action potentials was the same as that used for M/KCNQ currents recording, but did not contain TTX. The internal solution for action potential recording was also the same solution as that used for M/KCNQ currents using perforated patch recording. Single M/KCNQ current was recorded under cell-attached patch. The protocol for recording single M/KCNQ current is as follows: Cells bath perfused with high K+ external solution (63 mM) had a resting membrane potential near -20 mV. Membrane potentials (Vm) were therefore calculated as Vm=Vrest -Vpipette, where Vrest was taken to be -20 mV and Vpipette was the voltage applied. When applied 0 mV, the patch membrane will be clamped at -20 mV and at this level, single M/KCNQ channel will be open. The data were sampled at 5-10 kHz after filtered at 0.5-2 kHz. Transitions between open and closed states were detected by setting the threshold to 50% of the open channel level. Sylgard-coated pipettes had resistances of 8-10 MΩwhen filled with pipette solution consisting of (mM): NaCl 125, KCl 3, MgCl2 1.2, HEPES 10, Glucose 11 and apamin 200 nM, charybdotoxin 100 nM,αandβdendrotoxins 300 nM, tetrodotoxin 250 nM (pH 7.3). The extracellular solution consisted of (mM): NaCl 65, KCl 63, CaCl2 0.5, MgCl2 1.2, HEPES 10, Glucose 11 and CgTx-GVIA 250 nM, nifedipine 10μM, tetrodotoxin 250 nM (pH 7.3 with KOH). (3) Data were analyzed using clamfit and origin software. Results: (1) Rat SCG neurons can be divided into three electrophysiological phenotypes based on their action potential firing patterns: Phasic-1, Phasic-2 and Tonic neurons. Phasic-1 neurons, making up 36% of total SCG neurons studied, fired only one spike during the period of stimulation even with increased current injection. Phasic-2 neurons, seen in 54% of neurons, fired two to six spikes, but fired more frequently in response to the increased current injection, and the phasic firing pattern could be converted to a tonic firing pattern. Tonic neurons were seen in 10% of SCG neurons. Tonic neurons fired action potentials in a sustained manner even with a minimal stimulus, and the number of spikes increased with increased current injection. The resting potential between phasic-1 and phasic-2 neurons had no significant difference, but they were higher than that of tonic neurons and there had a significant difference between phasic and tonic neurons. The spike number of tonic neurons was much more than that of phasic-1 and phasic-2 neurons, and there had significant difference between tonic and phasic neurons, whereas, there was no significant diferrence between the two phasic neurons. (2) Three kinds of neuronal excitatory pattern in SCG neurons were tightly related to their M/KCNQ currents. We first identified the excitatory pattern of SCG neurons under current-clamping mode, and then use voltage-clamping mode to record the neuronal M/KCNQ currents in the same cell. The shape and amplitude of M/KCNQ currents were similar in phasic-1 and phasic-2 neurons, and the amplitude of tail M/KCNQ current at -60 mV was relatively big. On other hand, the amplitude of M/KCNQ current from tonic neurons was relatively small. The density of tail M/KCNQ currents at -60 mV from phasic-1, phasic-2 and tonic neurons were 2.8±0.2,2.3±0.2 and 0.9±0.1 pA/pF, respectively. The two phasic neurons were significantly different from tonic neurons but no significant difference existed between two phasic neurons. The current-voltage (I-V) relationship curves of the three type neurons were also different. The half-activation voltages for phasic-1, phasic-2 and tonic neurons were -30±1, -29±1 and -15±3 mV, respectively. Compared with phasic neurons, the I-V curve of tonic neurons was positively shifted. A detailed analysis of the relationship between spike number and the density of M/KCNQ tail currents was made. It appeared that the density of M/KCNQ tail currents was diagnostic in separating SCG neurons into either phasic or tonic neurons. Specifically, a line of demarcation was located at an M/KCNQ current density level of -1 pA/pF. We used single exponential equation to fit the kenitics of M/KCNQ currents activation (at -20 mV) and deactivation (at -60 mV) and obtained the the corresponding time constants, respectively. The time constants of activation for phasic-1, phasic-2 and tonic neurons were 60±5, 64±4 and 99±12 ms, respectively. The time constants of deactivation for phasic-1, phasic-2 and tonic neurons were 51±2, 60±6 and 96±5 ms, respenctively. Both activation and deactivation of tonic neurons were significantly slower than phasic neurons, but no significant differences were found between phasic-1 and phasix-2 neurons. (3) NGF inhibited M/KCNQ currents in rat SCG neurons. NGF at concentration of 20 ng/ml (within the range of reported physiological concentration of NGF in mammalians) inhibited the M/KCNQ currents of SCG neurons both in phasic and tonic neurons. NGF inhibited M/KCNQ currents by 25±2% and 26±3% in phasic and tonic neurons, respectively, and there had no significant differences between them. Application of NGF in the presence of 5μM linopirdine after the M/KCNQ currents was inhibited by linopirdine did not further inhibit the currents. The inhibition was 86.3±3.2% and 86.6±3.3% before and after application of NGF, and these were not significantly different. Oxo-M, a muscranic receptor agonist, also strongly inhibited M/KCNQ currents. NGF also inhibited M/KCNQ currents by 34±4% under the conventional whole-cell patch mode. Under this condition, we established the concentration-response curve for NGF-induced inhibition of M/KCNQ currents and fitted this curve by Hill function. The half-maximal inhibition (IC50) was 0.7±0.1 ng/ml and the coefficient was 0.9±0.1. AG879 (50μM), a specific inhibitor of Trk A receptor (one of the two NGF receptors, Trk A receptor and p75 receptor), when applied in pipette solution, siginificantly reduced NGF-induced inhibition of M/KCNQ currents from 34±4% to 17± 3%, whereas it had no effect on oxo-M-dediated inhibition. These results suggested that NGF inhibited M/KCNQ currents through activation of TrkA receptor. Bath applied genistein (100μM), a broad spectrum inhibitor for cellular protein tyrosine kinase, reduced significantly NGF-induced inhibition of M/KCNQ currents from 34±4% to 7±4%. Bath application of U73122, a commonly used inhibitor of phospholipase C (PLC), also significantly reduced the inhibitory effect on M/KCNQ currents mediated by NGF from 34±4% to 12±5%. These data suggested that NGF inhibited M/KCNQ currents through Trk A receptor and its downstream signal pathways, possibly involving both tyrosine phosphorylation and PI(4,5)P2 hydrolysis. (4) NGF inhibited single M/KCNQ current in cell-attached patches. NGF significantly reduced M/KCNQ channel Po by 29±3%. Oxo-M (3μM) strongly reduced the M/KCNQ channels Po by 89±2%. These data were consisitent with the whole-cell experiment. NGF did not change the conductance of the single M/KCNQ channel. The conductances were 6.1±0.2 and 6.5±0.3 pS, before and after applied NGF and had no significant difference. Oxo-M did not change the channel conductance either. The conductance was 6.4±0.3 pS in the presence of oxo-M. Both NGF and oxo-M reduced the M/KCNQ channel Po at each voltage level but only NGF significantly decreased the half-activation voltage (V1/2) from -32±3 mV to -25±2 mV. (5) NGF increased the excitability of tonic neurons but not phasic neurons. NGF significantly increased the number of spikes fired in tonic neurons from 12±2 to 20±2. NGF did not significantly change the resting potential of tonic neurons (the resting potential are -47±3 mV and -47±4 mV, before and after NGF, respectively). In the same batch of tonic neurons, oxo-M (10μM) and linopirdine (30μM) rapidly and significantly increased the spike number from 12±2 and 12±5 to 28±4 and 30±4, respectively. Oxo-M and linopirdine also induced small depolarization, but the changes did not reach the statistical significance. In the case of phasic-1 and phasic-2 neurons, NGF neither significantly changed their excitability nor significantly changed their resting potential level. On other hand, oxo-M and linopirdine significantly increased the excitability of both phasic-1 neurons (oxo-M and linopirdine enhanced the spike number from 1 to 4.7±1.2 and 3.2±1, respectively) and phasic-2 neurons (oxo-M and linopirdine enhanced the spike number from 3.2±0.7 and 3.1±0.5 to 31±6 and 16±2, respectively). Both Oxo-M and linopirdine significantly depolarized the resting potential of phasic-2 neurons (the two agent depolarized membrane from -58±2 mV and -50±3 mV to -49±2 mV and -43±2 mV, respectively); they did not affect the resting membrane potentials of phasic-1 neurons. (6) Modulation of neuronal excitability was tightly related to function of neuronal M/KCNQ channels. In phasic neurons, NGF (20 ng/ml) alone inhibited M/KCNQ currentd by 20±2%, whereas following adminstration of oxo-M (10μM) inhibited M/KCNQ currents by 62±7% in the presence of NGF. Under this condition, NGF alone did not change the excitability of phasic neurons but a significant enhancement of the neuronal excitability was produced by oxo-M in the presence of NGF; Oxo-M increased the spike number from 4.4±0.7 to 22±5 in the presence of NGF. In a reversed sequential application of these two drugs, oxo-M alone inhibited M/KCNQ currents by 72±8% and following adminstration of NGF did not further inhibit M/KCNQ currents in the presence of oxo-M. Oxo-M alone increased the spike number of phasic-2 neurons from 3.4±1.2 to 12±5, whereas, additional application of NGF did not show a significant effect. (7) Small inhibition of M/KCNQ currents by low concentration of linopirdine mimiced the effect of NGF on neuronal excitability. That NGF failed to modulate the excitability of phasic neurons may be due to its insufficient inhibition of the relative large M/KCNQ current densities. We choose linopirdine, a specific M/KCNQ channel blocker, to verify this hypothesis. We first established the concentration-response relationship curve of linopirdine-induced inhibition of M/KCNQ currents to find a proper concentration of linopirdine with a similar inhibitory effect to that seen with NGF. Linopirdine began to inhibit M/KCNQ currents at 0.3μM and reached its maximal inhibition at 30μM. The half-maximum inhibitory concentration was 2.1±0.2μM. According to this curve, 0.7μM linopirdine would inhibit M/KCNQ current by 25%, similar to the inhibition mediated by NGF (20 ng/ml). Linopirdine at concentration of 0.7μM significantly enhanced the excitability of tonic neurons by increasing its spike number from 12±1 to 18±2. However, this concentration of linopirdine did not alter the excitability of phasic-1 and phasic-2 neurons. Thus, the selective modulation of excitability of tonic neurons by NGF was likely due to ite moderate capability in inhibiting M/KCNQ current.Conclusion: (1) Rat SCG neurons can be divided into three electrophysiological phenotypes based on their action potential firing patterns: phasic-1, phasic-2 and tonic neurons. Phasic-1 and phasic-2 neurons expressed relatively large M/KCNQ current density, which will render the cell low excitability and difficulty to fire action potentials. Tonic neurons expressed relatively small M/KCNQ current density, thus will have high excitability and easy to fire action potentials. (2) NGF at physiological concentration can significantly inhibit M/KCNQ currents of SCG neurons. NGF may inhibit M/KCNQ current through activation of Trk A receptor and its downstream signal pathways, possibly involving both tyrosine phosphorylation and PI(4,5)P2 hydrolysis. NGF inhibited M/KCNQ currents in a similar degree among phasic-1, phasic-2 and tonic neurons. (3) NGF only increased neuronal excitability of tonic neurons. Phasic neurons may still have relative large M/KCNQ currents left after inhibition induced by NGF, and the residual M/KCNQ currents were sufficient to stabilize cellular membrane potential. (4) Low concentration of linopirdine, who had the similar inhibitory effect to NGF, mimiced the effects of NGF on neuronal excitability.2. The regulation of voltage-gated sodium currents and neuronal excitability by genistein.Aim: To study the mechanisms involve in the regulation of VGSC currents induced by genistein, a broad spectrum inhibitor of cellular protein tyrosine kinase.Method: Voltage-gated sodium currents (VGSC) and neuronal action potentials were recorded from rat SCG neurons. (1) Cell culture. SCG neurons were primary cultured with the same procedur describered in part one. (2) Electrophysiology. Using perforated patch clamp technique to record VGSC currents under voltage-clamping mode and record neuronal action potentials under current-clamping mode. Pipettes were pulled from borosilicate glass capillaries and had a resistance of 3-5 M? when filled with internal solution. Series resistance compensation has always been used and up to 80-90% compensation can be reached in our condition. Under this condition, the maximum access resistance was about 2 M?. The sampling rate was 10 KHz for membrane currents and was 2.5 KHz for membrane potential recordings. The protocol used to record the VGSC currents was as follows: the cells were held at -70 mV and a 20 ms depolarizing step to 0 mV was applied every 3 s. The action potentials were elicited by an approximate two-fold threshold depolarizing current of 0.1 nA. The external solution used to record the VGSC currents contained (in mM): NaCl 120, KCl 3, HEPES 5, NaHCO3 23, glucose 11, MgCl2 1.2, CaCl2 2.5, BaCl2 0.2, CdCl2 0.2, (adjusted to pH 7.4 with NaOH). The internal solution for VGSC currents recording consisted of (in mM): CsCl 90, KCl 40, HEPES 20, MgCl2 3 (adjusted to pH 7.3-7.4 with CsOH). The external solution used to record neuronal action potentials contained (in mM): NaCl 120, KCl 3, HEPES 5, NaHCO3 23, glucose 11, MgCl2 1.2, CaCl2 2.5, (adjusted to pH 7.4 with NaOH). The internal solution for action potential recording contained (in mM): KAC 90, KCl 40, HEPES 20, MgCl2 3 (adjusted to pH 7.3-7.4 with KOH). (3) Cell immunofluorescence and confocal imaging. Briefly, neurons were incubated with primary rabbit anti-Nav1.7 Ab or anti-Nav1.1 Ab overnight at 4°C, followed by three washes with PBS and incubation with goat anti-rabbit IgG-TRITC sencondary Ab for 30 min at 37°C. Cells were examined on an inverted laser-scanning microscope. TRITC was excited at 564 nm and the emitted fluorescence signal was at 570 nm. (4) Data were analyzed using clamfit and origin software.Results: (1) Predominantly expressed voltage-gated sodium channel in rat SCG neurons was Nav1.7 channel. Cell immunofluorescence and confocal imaging experiment indicated that, Nav1.7 channel was dominantly expressed in rat SCG neurons. Nav1.1 channel, another voltage-gated sodium channel, was also weakly expressed in SCG neurons. (2) Genistein significantly inhibited the voltage-gated sodium currents in SCG neurons. 100μM genistein strongly inhibited VGSC currents by 73.3±5.4%. A double phase inhibition was evident for genistein-induced inhibition: an initial fast inhibition followed by a relatively slow inhibition. These two processes were fitted with a double exponential function and time constants were obtained. The fast time constant was 10.6±1.2 s, and the slow time constant was 55.9±2.4 s, respectively. Genistein inhibited VGSC currents in a concentration dependent manner. The concentration of half-maximal (IC50) inhibition was 9.1±0.9μM and the coefficient was 1.1±0.2. VGSC currents in SCG neurons were TTX-sensitive currents since 0.05μM TTX reversally and compeletly inhibited them. Voltage-dependence of genistein-indcued inhibtion was tested. 50μM genistein obviously inhibited VGSC currents at all voltages tested, more significantly at the range from -20 mV to 0 mV, the range of maximal activation. Genistein significantly shifted the voltage-dependent activation of VGSC current to the right, but did not affect voltage-dependent inactivation. The half-maximal activation voltage (V1/2) was significantly more positive for genistein-treated neurons (-20.6±0.1 mV) than in control cells (-32.4±0.2 mV). The half-maximal inactivation voltage were -41.3±0.4 mV for genistein and -41.8±0.3 mV for controls, respectively. (3) Genistein-indeuced inhibition of VGSC currents involved two mechanisms: PTK-independent and PTK-dependnent mechanisms. Daidzein is an inactive structural analog of genistein. Daidzein also inhibited VGSC currents in a concentration dependent manner and reached its maximal inhibitory effect at 100μM (the inhibition is 28.5±3.1 %). Fitting the curve with Hill function produced an IC50 of 20.7±0.1μM and a coefficient of 1.2±0.2. When daidzein and genistein were co-applied, daidzein (100μM) alone inhibited VGSC currents by 27.8±3.6%, additional applied genistein (100μM) inhibited VGSC currents by 57.3±10.1%. These data suggested that genistein inhibited VGSC currents through PTK-independent and other menchanisms. We tested the effect of Vanadate, a broadly used inhibitor of cellular protein tyrosine phosphotase. The effect of vanadate will antagonize the effect of genistein. Genistein alone inhibited VGSC currents by 57±5.4%, additional application of vanadate (1 mM, a saturate concentration) partly rescued the genistein-induced inhibition from 57±5.4% to 39.5±4.7%. Pretreatment with vanadate significantly reduced the genistein-induced inhibition of VGSC current by 18.7±3.3%. These data suggested that anti-PTK activity were partly responsible for the VGSC current inhibition produced by genistein. We also tested other three functional analog of genistein, tyrphotin 23, erbstatin analog and PP2 in their saturate concentration of 100μM, 100μM and 1μM, respectively. The three agents all weakly but significantly inhibited VGSC currents, and their inhibitory effect were 14.6±0.5%, 19.3±1.0 and 17.8±1.9%, respectively. At parallel experiments, gensitein inhibited VGSC currents by 54-58%. These data further suggested that genistein inhibited VGSC currents partly involved the PTK-dependent mechanism. (4) Genistein depressed the neuronal excitability and reduced the depolarized rate of action potential but did not change the resting potential of SCG neurons. Bath application of genistein (100μM) immediately suppressed the neuronal excitability by reducing the action potential number from 8.7±2.1 to 1.3±0.3. However, genistein did not significantly change the neuronal resting potential; the resting potentials were 56.1±3.1 and 54.7±4.1 before and after genistein application, respectively. Genistein significantly reduced the depolarization rate of action potential from 7.0±1.3 to 4.4±0.9 V/s.Conclusion: (1) Genistein inhibited VGSC currents through PTK-dependent and PTK-independent mechanisms. (2) Genistein suppressed neuronal excitability and reduced the depolarization rate of SCG neurons.3. Detection of intracellular Ca2+ signals in DRG neurons induced by G(q) protein-coupled receptors.Aim: Do study the intracellular Ca2+ signals induced by activation of G(q) protein-coupled receptors expressed in rat DRG neurons.Methods: (1) Cell culture. The procedur of primary culture for rat DRG neurons is similar to SCG neurons described above. DRG neurons were prepared from 14 day-old Sprague-Dawley rats. (2) Ca2+ imaging. Neurons were loaded with fura-2 AM (5μM) in the presence of pluronic F-127 (20%) and imaged using a Nikon TE-2000 microscope equipped with a Hamamatsu photonics Ca2+ imaging system. Fura-2 was excited at 340 and 380 nm and images were analyzed with simplePCT 6.0. (3) Solutions. Bath solution of DRG neurons contained (mM): NaCl 145, KCl 5, MgCl2 2, CaCl2 2, HEPES 10, Glucose 10 and adjusted to pH 7.4 with NaOH. In the Ca2+-free bath solution, Ca2+ was omitted and 3 mM EGTA was added. (4) Drugs. In this part of eaperiment, bradykinin (BK, 100 nM) was used as a positive control, capsaicin (CAP, 1μM) and acrolein (ACR, 0.1 mM) was used to identify the noceptive DRG neurons. KCl (50 mM) was used to identify the DRG neurons.Results: (1) Identification of the DRG neurons. When bathed with normal extracellular solution (with Ca2+), 349 DRG neurons (identified by 50 mM KCl) were studied. In the following description of the results, the number of neurons which show an intracellular Ca2+ rising in responding to a particular agonist will be called as the agonist-sensitive neurons. Among 349 DRG neurons, 109 neurons were BK-sensitive neurons, making up 31.2% of the total neurons. 74 neurons were CAP-sensitive neurons, or 21.2% of the total DRG neurons. 62 neurons were ACR-sensitive neurons, 17.8% of the total DRG neurons. Among BK-sensitive DRG neurons, CAP- and ACR-sensitive neurons were 31 and 29, making up 28.4% and 26.6% of the BK-sensitive neurons, respectively. When bathed with Ca2+-free external solution, 57 DRG neurons were studied. Among the 57 neurons, 4 neurons, making up 7% of the total, were sensitive to BK; 1 neuron, making up of 1.8%, was sensitive to CAP; 1 neuron, making up of 1.8%, was sensitive to ACR, respectively. Among the 4 BK-sensitive DRG neurons, CAP- and ACR-sensitive neurons were 1 and 1, making up 25% and 25%, respectively. (2) Cholecystokinin receptors (CCK receptors). CCK receptors were activated by CCK-8 (1μM). In 131 DRG neurons bathed with normal external solution, CCK-8 elicited Ca2+ signals in 46 neurons, or 35.1% of the total neurons. Under Ca2+-free condition, No neurons out of 57 DRG neurons studied were sensitive to CCK. (3) Endothelin (ET) receptors. ET receptors were activated by ET-1 (100 nM). In Ca2+ extracellular solution, ET-1 elicited intracellular Ca2+ signals in 49 neurons out of 101 DRG neurons studied, making up a ratio of 48.5%. Among 50 DRG neurons bathed in Ca2+-free condition, 5 neurons were sensitive to ET-1. (4) Mrg D receptors. Mrg D receptors, a kind of orphan proteins, were abundantly expressed in DRG neurons and were activatied byβ-Alanine (500μM). In Ca2+ extracellular solution, 12 neurons out of 134 DRG neurons studied were sensitive toβ-Alanine. In Ca2+-free condition, No neurons were sensitive toβ-Alanine. (5) Histamine (H1) receptors. H1 receptors were activatied by Histamine (100μM). In normal external solution, Histamine only elicited Ca2+ signals in 10 neurons out of 95 DRG neurons. (6) Substance P receptors. Substance P receptors, also called NK1 receptors, were activated by substance P (SP, 1μM). When bathed with normal extracellular solution, 55 DRG neurons (identified by 50 mM KCl) were studied. Only one neuron was sensitive to SP. (7) 5-HT receptors. 5-HT receptors were activated by 5-HT (10μM).When bathed with normal solution, 55 DRG neurons were studied. No neurons were sensitive to 5-HT. (8) Angiotensin II receptors (AT1 receptors). AT1 receptors were activated by angiotensin II (100 nM). When bathed with normal external solution, 44 DRG neurons were studied. Among the detected 44 neurons, only 2 neurons were sensitive to angiotensin II. (9) Purinergic Y receptors (P2Y receptors). P2Y receptors were activated by adenosine triphsphate (ATP, 100 nM). In normal extracellular solution, 43 DRG neurons were studied, but no neurons were sensitive to ATP.Conlusion: Among agonists of Gq-copupled receptors we stuided, agonits of BK receptors, CCK receptors, ET receptors, MrgD receptors and histamine receptors elicited intracellular Ca2+ signals, whereas agonists of substance P receptors, angiotensin II receptors, 5-HT receptors and purinergic receptors could not elicit the intracellular Ca2+ signals.4. Modulation of TRPV1 currents in DRG neurons by capsaicin and CCK.Aim: To study modulation of TRPV1 currents in DRG neurons by GPCR.Methods: (1) Cell culture. Primary culture of rat DRG neurons was described above. (2) Electrophysiology. Perforated whole-cell patch was used to record currents. The protocol used to record TRPV1 and other inward currents was as follows: neurons were held at -60 mV constantly, BK and other GPCRs agonists were applied to elicit inward currents, and specific blockers of TRPV1 and Ca2+-activated chloride currents (CaCC) were used to verify the identity of the inward currents. Pipettes were pulled from borosilicate glass capillaries and had a resistance of 3-5 M? when filled with internal solution. The sampling rate was 2.5 KHz after filtered at 0.5 KHz. The extracellular solution contained (mM): NaCl 145, KCl 5, MgCl2 2, CaCl2 2, HEPES 10, Glucose 10 and adjusted to pH 7.4 with NaOH. In the Ca2+-free bath solution, Ca2+ was replaced by 0.1 mM EGTA.Results: (1) Capsaicin elicited fast desensitizing TRPV1 currents in DRG neurons. Capsaicin (CAP, 1μM) elicited TRPV1 currents in CAP-sensitive nociceptive DRG neurons. Application of capsazepine (CZP, 100μM), a specific TRPV1 channel blocker, eliminated the capsaicin-induced inward currents at -60 mV. TRPV1 current reached its maximal value within 20 ~ 30 s, and then desensitized rapidly. The current amplitude at 5min was significantly smaller than the initial TRPV1 currents, about 30.5%±3.6% of the initial amplitude of the TRPV1 currents. The peak currents of TRPV1 from two sequential 30 s applications of CAP interrupted by a 5-6 min period of no CAP gave very different values The amplitude of the second CAP-induced TRPV1 currents was only about 50.7%±3.8% of that of the first CAP-induced TRPV1 currents. (2) CCK-8 elicited a small inward current in DRG neurons but antagonized...
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