Electrophysiological properties and modeling of murine vomeronasal sensory neurons in acute slice preparations

The vomeronasal system is involved in the detection of pheromones in many mammals. Vomeronasal sensory neurons encode the behaviorally relevant information into action potentials that are directly transmitted to the accessory olfactory bulb. We developed a model of the electrical activity of mouse basal vomeronasal sensory neurons, which mimics both the voltage-gated current properties and the firing behavior of these neurons in their near-native state, using a minimal number of parameters. Data were obtained by recordings with the whole-cell voltage-clamp or current-clamp techniques from mouse basal vomeronasal sensory neurons in acute slice preparations. The resting potential ranged from -50 to -70 mV, and current injections of less than 2-10 pA induced tonic firing in most neurons. The experimentally determined firing frequency as a function of injected current was well described by a Michaelis-Menten equation and was exactly reproduced by the model, which could be used in combination with future models that will include details of the mouse vomeronasal transduction cascade.     

Calcium-activated chloride channels in the apical region of mouse vomeronasal sensory neurons

The rodent vomeronasal organ plays a crucial role in several social behaviors. Detection of pheromones or other emitted signaling molecules occurs in the dendritic microvilli of vomeronasal sensory neurons, where the binding of molecules to vomeronasal receptors leads to the influx of sodium and calcium ions mainly through the transient receptor potential canonical 2 (TRPC2) channel. To investigate the physiological role played by the increase in intracellular calcium concentration in the apical region of these neurons, we produced localized, rapid, and reproducible increases in calcium concentration with flash photolysis of caged calcium and measured calcium-activated currents with the whole cell voltage-clamp technique. On average, a large inward calcium-activated current of -261 pA was measured at -50 mV, rising with a time constant of 13 ms. Ion substitution experiments showed that this current is anion selective. Moreover, the chloride channel blockers niflumic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid partially inhibited the calcium-activated current. These results directly demonstrate that a large chloride current can be activated by calcium in the apical region of mouse vomeronasal sensory neurons. Furthermore, we showed by immunohistochemistry that the calcium-activated chloride channels TMEM16A/anoctamin1 and TMEM16B/anoctamin2 are present in the apical layer of the vomeronasal epithelium, where they largely colocalize with the TRPC2 transduction channel. Immunocytochemistry on isolated vomeronasal sensory neurons showed that TMEM16A and TMEM16B coexpress in the neuronal microvilli. Therefore, we conclude that microvilli of mouse vomeronasal sensory neurons have a high density of calcium-activated chloride channels that may play an important role in vomeronasal transduction.     

Chemosensitive conductance and inositol 1,4,5-trisphosphate-induced conductance in snake vomeronasal receptor neurons

Snake vomeronasal receptor neurons in slice preparations were studied using the patch-clamp technique in the conventional and nystatin-perforated whole-cell configurations. The mean resting potential was approximately -70 mV; the average input resistance was 3 GOmega. Neurons required current injection of only 1-10 pA to display a variety of spiking patterns. Intracellular dialysis of 100 microM inositol 1,4,5-trisphosphate (IP(3)) evoked an inward current in 38% of neurons, with an average peak amplitude of 16.4 +/- 2.8 pA at a holding potential of -70mV. Application of 100 microM 3-deoxy-3-fluoro-D-myo-inositol 1,4,5-trisphosphate (F-IP(3)), a derivative of IP(3), also evoked an inward current in 4/8 (50%) neurons (32.6 +/- 58 pA at -70 mV, n = 4). The reversal potentials of the induced components were estimated to be -14 +/- 5 mV for IP(3) and -17 +/- 3 mV for F-IP(3). Bathing the neurons in 10 microM ruthenium red solution greatly reduced the IP(3)-evoked inward current to 1.6 +/- 1.1 pA at -70 mV (n = 6). With Cs(+)-containing internal solution, neither the Ca(2+)-ATPase inhibitor thapsigargin (1-50 microM) nor the Ca(2+)-ionophore ionomycin (10 microM) evoked a significant current response, suggesting that IP(3) can elicit current response in the neurons without mediation by intracellular Ca(2+) stores. Intracellular application of 1 mM cAMP evoked no detectable current response. Extracellular application of chemoattractant for snakes evoked a very large inward current. The reversal potential of the chemoattractant-induced current was similar to that of the IP(3)-induced current. The present results suggest that IP(3) may act as a second messenger in the transduction of chemoattractants in the garter snake vomeronasal organ.     

Role of the vomeronasal organ on the estral cycle reduction by pheromones in the rat

The role of he vomeronasal organ on the estral cycle reduction induced by pheromones is studied in adult female wistar rats. The animals were divided in three groups: I, intact rats; II, vomeronasalectomized rats (VNX); and III, sham operated rats (sham). Each group was submitted to another three distinct conditions from the day they were weaned (21 days old): Isolated female rats; with male odors from two adult males of tested sexual potency, and isolated rats again. The isolated intact rats show mainly 5 day length cycles. The groups I and III (intacts and sham) with male odors, show 4 day length cycles. The VNX animals show 5 day cycles in any one experimental conditions. These results support the idea that the vomeronasal organ is the receptor of the male reducing cycle pheromone in the female rat.     

A voltage-dependent persistent sodium current in mammalian hippocampal neurons

Currents generated by depolarizing voltage pulses were recorded in neurons from the pyramidal cell layer of the CA1 region of rat or guinea pig hippocampus with single electrode voltage-clamp or tight-seal whole-cell voltage-clamp techniques. In neurons in situ in slices, and in dissociated neurons, subtraction of currents generated by identical depolarizing voltage pulses before and after exposure to tetrodotoxin revealed a small, persistent current after the transient current. These currents could also be recorded directly in dissociated neurons in which other ionic currents were effectively suppressed. It was concluded that the persistent current was carried by sodium ions because it was blocked by TTX, decreased in amplitude when extracellular sodium concentration was reduced, and was not blocked by cadmium. The amplitude of the persistent sodium current varied with clamp potential, being detectable at potentials as negative as -70 mV and reaching a maximum at approximately -40 mV. The maximum amplitude at -40 mV in 21 cells in slices was -0.34 +/- 0.05 nA (mean +/- 1 SEM) and -0.21 +/- 0.05 nA in 10 dissociated neurons. Persistent sodium conductance increased sigmoidally with a potential between -70 and -30 mV and could be fitted with the Boltzmann equation, g = gmax/(1 + exp[(V' - V)/k)]). The average gmax was 7.8 +/- 1.1 nS in the 21 neurons in slices and 4.4 +/- 1.6 nS in the 10 dissociated cells that had lost their processes indicating that the channels responsible are probably most densely aggregated on or close to the soma. The half-maximum conductance occurred close to -50 mV, both in neurons in slices and in dissociated neurons, and the slope factor (k) was 5-9 mV. The persistent sodium current was much more resistant to inactivation by depolarization than the transient current and could be recorded at greater than 50% of its normal amplitude when the transient current was completely inactivated. Because the persistent sodium current activates at potentials close to the resting membrane potential and is very resistant to inactivation, it probably plays an important role in the repetitive firing of action potentials caused by prolonged depolarizations such as those that occur during barrages of synaptic inputs into these cells.     

Gender difference in BK channel expression in amygdala complex of rat brain

The expression of large-conductance Ca²⁺-activated K⁺ (BK) channel protein in amygdala complex was higher in adult (8-10 weeks old) male rats than in female. Castration at 4-6 weeks old significantly reduced BK channel expression in amygdala to the level similar to that in female. Immunocytochemical analyses of pyramidal-like neurons isolated from amygdala revealed that somas with relatively large size were highly immunoreactive to both anti-androgen receptor (AR) and anti-BK channel antibodies, while those with smaller size were not. The double-immunopositive neurons were dominant (60%) among pyramidal-like neurons isolated from amygdala of male rats but rare among those from female. The membrane current sensitive to penitrem A, a BK channel blocker, was the major K⁺ current component in large neurons and showed higher current-density than that in smaller ones. These results suggest the gender-dependent cell population expressing BK channels in amygdala complex and its up-regulation by AR stimulation.     

Subtypes of dorsal root ganglion neurons based on different inward currents as measured by whole-cell voltage clamp

Electrophysiological and pharmacological properties distinguished subtypes of adult mammalian dorsal root ganglion neurons (DRGn) in monolayer dissociated cell culture. By analogy of action potential waveform and duration, neurons with short duration (SDn) and long duration (LDn) action potentials resembled functionally distinct subtypes of DRGn in intact ganglia. Patch clamp and conventional intracellular recording techniques were combined here to elucidate differences in the ionic basis of excitability of subtypes of DRGn in vitro. Both SDn and LDn were quiescent at the resting potential. Action potentials of SDn were brief (less than 2 msec), sensitive to tetrodotoxin (TTX, 5-10 nM), exhibited damped firing during long depolarizations, and did not respond to algesic agents applied by pressure ejection. Action potentials of LDn were 2-6 msec in duration, persisted in 30 microM TTX, and fired repetitively during depolarizing current pulses or exposure to algesic agents (e.g., capsaicin, histamine and bradykinin). Whole-cell recordings from freshly dissociated neurons revealed two inward sodium currents (INa; variable with changes in sodium but not calcium concentration in the superfusate) in various proportions: a rapidly activating and inactivating, TTX-sensitive current; and, a slower, TTX (30 microM)-resistant INa. Large neurons, presumable SDn, had predominantly TTX-sensitive current and little TTX-resistant current. The predominant inward current of small neurons, presumably LDn, was TTX-resistant with a smaller TTX-sensitive component. By analogy to findings from intact ganglia, these results suggest that fundamentally different ionic currents controlling excitability of subtypes of DRGn in vitro may contribute to functional differences between subtypes of neurons in situ.     

脊神经损伤后钠电流的变化及其离子通道机制

目的：检测脊神经切断大鼠背根节（DRG）神经元重复放电能力和钠电流的变化,并研究介导其电流变化的钠通道亚型的表达情况。方法：脊神经切断术后2～8d慢性痛大鼠模型背根节急性分离,对中等直径DRG神经元运用全细胞膜片钳技术记录神经元放电和钠电流的变化。对背根节神经元进行RT-PCR检测,分析其钠通道亚型的表达情况。结果：电流钳下,实验组DRG神经元在电流刺激下产生重复放电,而对照组神经元多诱发单个动作电位,电压钳记录发现实验组背根节神经元快钠电流和持续性钠电流幅值均明显大于对照组,PCR结果显示,Nav1.3、Nav1.7和Nav1.8通道亚型mRNA表达显著增高。结论：钠通道介导了脊神经受损模型的DRG神经元兴奋性增高,持续性钠电流可能通过调节阈下膜电位振荡的产生调节神经元兴奋性。     

The feasibility and limitation of patch-clamp recordings from neonatal rat cardiac ventricular slices

Examine the feasibility of whole-cell patch-clamp recordings from the cardiac ventricular slices of newborn (P(3)-P(7)) Sprague-Dawley rats to identify a better substitute for single cardiac myocytes prepared using enzymatic treatment. High resistance seals (>1 G?) were obtained from cardiac ventricle tissues prepared without using enzymatic treatment. Thereafter, cell-attached and whole-cell patch-clamp techniques were used on thin cardiac slices (200 μm thick) in 2009 in the Institute of Molecular Medicine of Peking University. An averaged sodium current (n=11 cells) was recorded in the cell-attached mode, and this displayed features similar to those previously reported for isolated rat ventricular myocytes. The outward potassium current, hyperpolarization-activated cation channel or I (f) channel (HCN channel), and action potential were recorded in the whole-cell mode (n=2 cells), and the identical properties were observed from the cardiac slices. The cell-attached mode is stable and reliable for recording the ion current. The resting potential for cardiac slices measured using current-clamp recording in the whole-cell mode was -50 to -70 mV. The resting potential of cardiac slices has properties similar to those of enzyme-prepared cardiomyocytes, with the exception that it is positive. We achieved whole-cell patch-clamp recordings from cardiac slices and affirmed the feasibility and values of both cell-attached and whole-cell recording modes using this technique. Nevertheless, there remain difficulties and limitations associated with the application of whole-cell patch-clamping to cardiac slices, due primarily to the existence of large amounts of connective tissue even in newborn rats.     

Female snake sex pheromone induces membrane responses in vomeronasal sensory neurons of male snakes

The vomeronasal organ (VNO) is important for activating accessory olfactory pathways that are involved in sexually dimorphic mating behavior. The VNO of male garter snakes is critically important for detection of, and response to, female sex pheromones. In the present study, under voltage-clamp conditions, male snake VNO neurons were stimulated with female sexual attractiveness pheromone. Thirty-nine of 139 neurons exhibited inward current responses (reversal potential: -10.6 +/- 2.8 mV). The amplitude of the inward current was dose dependent, and the relationship could be fitted by the Hill equation. Under current-clamp conditions, application of pheromone produced membrane depolarizing responses and increases in firing frequency. These results suggest that the female pheromone directly affects male snake VNO neurons and results in opening of ion channels, thereby converting the pheromone signal to an electrical signal. The response to female pheromone is sexually dimorphic, that is, the pheromone does not evoke responses in VNO neurons of female snakes. An associated finding of the present study is that the female sex pheromone, which is insoluble in aqueous solutions, became soluble in the presence of Harderian gland homogenate.     

蝎毒耐热蛋白对大鼠海马神经元钠通道的抑制作用

应用全细胞膜片钳技术观察蝎毒耐热蛋白（scorpion venom heat resistant protein,SVHRP）对急性分离大鼠海马神经元电压依赖性钠通道的影响。结果表明,急性分离大鼠海马神经元产生的河豚毒素（tetrodotoxin,TTX）敏感的电压依赖性钠电流被SVHRP浓度依赖性地抑制,半数抑制浓度为（0．0034±0．0004）μg／mL,Hill常数为0．4361±0．0318;SVHRP可使钠通道稳态激活曲线向电压的正方向移动,正常TTX敏感的钠通道的半数激活电压（V1/2）为（-34．38±0．62）mV（n=16）,给予0．1μg／mL的SVHRP后V1/2为（-23．96±0．41）mV（n=8,P〈0．05）,斜坡因子（κ）由正常的4．52±0．52变为3．73±0．08（n=8,P〈0．05）。SVHRP亦能改变电压依赖性钠通道的稳态失活曲线,使其向电位的负方向移动,SVHRP处理前钠通道半数失活电压（V1/2）为（-32．60±1．52）mV,κ为6．73±0．51（n=16）;0．1μg／mL的SVHRP处理后V1/2变为（-50．69±2．55）mV（n=8,P〈0．01）,κ为5．49±0．72（n=8,P〈0．05）。结果提示,SVHRP能抑制电压依赖性钠电流,改变钠通道的动力学特性,抑制其激活,促进其失活,从而影响神经元的兴奋性,这可能是其抗癫痫的机制之一。     

Voltage-dependent and odorant-regulated currents in isolated olfactory receptor neurons of the channel catfish.

The electrical properties of olfactory receptor neurons, enzymatically dissociated from the channel catfish (Ictalurus punctatus), were studied using the whole-cell patch-clamp technique. Six voltage-dependent ionic currents were isolated. Transient inward currents (0.1-1.7 nA) were observed in response to depolarizing voltage steps from a holding potential of -80 mV in all neurons examined. They activated between -70 and -50 mV and were blocked by addition of 1 microM tetrodotoxin (TTX) to the bath or by replacing Na+ in the bath with N-methyl-D-glucamine and were classified as Na+ currents. Sustained inward currents, observed in most neurons examined when Na+ inward currents were blocked with TTX and outward currents were blocked by replacing K+ in the pipette solution with Cs+ and by addition of 10 mM Ba2+ to the bath, activated between -40 and -30 mV, reached a peak at 0 mV, and were blocked by 5 microM nimodipine. These currents were classified as L-type Ca2+ currents. Large, slowly activating outward currents that were blocked by simultaneous replacement of K+ in the pipette with Cs+ and addition of Ba2+ to the bath were observed in all olfactory neurons examined. The outward K+ currents activated over approximately the same range as the Na+ currents (-60 to -50 mV), but the Na+ currents were larger at the normal resting potential of the neurons (-45 +/- 11 mV, mean +/- SD, n = 52). Four different types of K+ currents could be differentiated: a Ca(2+)-activated K+ current, a transient K+ current, a delayed rectifier K+ current, and an inward rectifier K+ current. Spontaneous action potentials of varying amplitude were sometimes observed in the cell-attached recording configuration. Action potentials were not observed in whole-cell recordings with normal internal solution (K+ = 100 mM) in the pipette, but frequently appeared when K+ was reduced to 85 mM. These observations suggest that the membrane potential and action potential amplitude of catfish olfactory neurons are significantly affected by the activity of single channels due to the high input resistance (6.6 +/- 5.2 G omega, n = 20) and low membrane capacitance (2.1 +/- 1.1 pF, n = 46) of the cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

HRP uptake by olfactory and vomeronasal receptor neurons: use as an indicator of incomplete lesions and relevance for non-volatile chemoreception

The transport of HRP (horseradish peroxidase) from the nasalcavity to the brain by intact olfactory receptor axons was usedto investigate the effectiveness of methods commonly used inbehavioral studies for deafferenting nasal chemoreceptor systems.The HRP experiments demonstrated that routine intranasal lavagewith zinc sulfate solution fails to destroy all olfactory receptorneurons in hamsters, in spite of the distinct behavioral deficitthat this treatment can cause in the male hamster. The intracranialdeafferentation of the accessory olfactory bulb by surgicalsection of the vomeronasal nerves was generally effective butthere was much incidental damage to main olfactory nerves thatwould probably not be detected without the HRP tracer. The distribution pattern of HRP molecules introduced into themammalian nasal cavity, as shown by the uptake of HRP by nasalchemoreceptors and its transport to the brain, was also usedto identify potential pathways for non-volatile stimulus moleculeswithin the nose. HRP reaction product was reliably detectedin the glomeruli of the main olfactory bulb after HRP was depositedat the nostril, demonstrating that nonvolatile materials, oncethey have entered the nasal cavity, can reach the main olfactoryreceptor neurons in the posterior nasal epithelium. Significantamounts of HRP reaction product were never observed in the accessoryolfactory bulbunlessa large dose of epinephrine had been givento activate the vomeronasal organ pumping mechanism, which drawssubstances into the vomeronasal organ lumen. Thus, it seemsthat stimulus access to vomeronasal receptor neurons is controlledindependently of access to main olfactory receptor neurons.     

Effects of dragon’s blood resin and its component loureirin B on tetrodotoxinsensitive voltage-gated sodium currents in rat dorsal root ganglion neurons

Using whole-cell patch clamp technique on the membrane of freshly isolated dorsal root ganglion (DRG) neurons, the effects of dragon’s blood resin and its important component loureirin B on tetrodotoxin-sensitive (TTX-S) voltage-gated sodium currents were observed. The results show that both blood resin and loureirin B could suppress TTX-S voltage-gated sodium currents in a dose-dependent way. The peak current amplitudes and the steady-state activation and inactivation curves are also made to shift by 0.05% blood resin and 0.2 mmol/L loureirin B. These results demonstrate that the effects of blood resin on TTX-S sodium current may contribute to loureirin B in blood resin. Perhaps the analgesic effect of blood resin is caused partly by loureirin B directly interfering with the nociceptive transmission of primary sensory neurons.     

人参皂甙Rd通过调节SNI大鼠背根神经节钠、钾电流抑制痛敏

目的：观察人参皂甙Rd（ginsenoside Rd）对大鼠坐骨神经分支选择性损伤（spared sciatic nerve injury,SNI）引起的痛敏的影响及其作用机制。方法：坐骨神经分支选择性损伤术后7天,观察腹腔注射不同浓度人参皂甙Rd后大鼠后足的机械性缩足反应阈值（paw withdrawl mechanical threshold,PWMT）的变化;在术后7天,急性分离并取出大鼠腰4和腰5段背根节,对整节DRG上的中小型神经元运用全细胞膜片钳技术进行记录。结果：坐骨神经分支选择性损伤术后7天,大鼠出现明显的机械性痛敏,腹腔注射5 mg/ml和10 mg/ml的人参皂甙Rd能剂量依赖性的翻转大鼠机械性痛敏;坐骨神经分支选择性损伤能明显地增大SNI大鼠DRG中小型神经元上的钠电流以及减小电压依赖性钾电流,而100μM人参皂甙Rd能有效翻转该钠、钾电流的变化。结论：人参皂甙Rd能有效地改善坐骨神经分支选择性损伤引起的机械性痛敏,其机制可能与人参皂甙Rd明显地调节SNI大鼠DRG中小型神经元上的电压依赖性钠、钾电流有关。     

Effects of dragon’s blood resin and its component loureirin B on tetrodotoxin-sensitive voltage-gated sodium currents in rat dorsal root ganglion neurons

Copyright by Science in China Press 2004 Dragons blood resin is one of famous precious Traditional Chinese Medicine (TCM), which has been widely applied in clinical treatment of cardiovascular disease, cervical spondylosis, gynecological disease, etc., due to its actions of dissipating blood stasis, eas-ing pain, arresting bleeding, promoting tissue regen-eration and wound healing[1]. At present, the investi-gation on the pharmacological mechanism of blood resin is concentrated on promoting…     

Effects of dragon’s blood resin and its component loureirin B on tetrodotoxin-sensitive voltage-gated sodium currents in rat dorsal root ganglion neurons

Using whole-cell patch clamp technique on the membrane of freshly isolated dorsal root ganglion (DRG) neurons, the effects of dragon’s blood resin and its important component loureirin B on tetrodotoxin-sensitive (TTX-S) voltage-gated sodium currents were observed. The results show that both blood resin and loureirin B could suppress TTX-S voltage-gated sodium currents in a dose-dependent way. The peak current amplitudes and the steady-state activation and inactivation curves are also made to shift by 0.05% blood resin and 0.2 mmol/L loureirin B. These results demonstrate that the effects of blood resin on TTX-S sodium current may contribute to loureirin B in blood resin. Perhaps the analgesic effect of blood resin is caused partly by loureirin B directly interfering with the nociceptive transmission of primary sensory neurons.     

P物质对大鼠分离的DRG细胞GABA激活电流的抑制作用

本文就用全细胞膜片箝技术,在新鲜分离的大鼠ＤＲＧ细胞上证明,在部分细胞Ｐ物质（１０＾－７－１０＾－５ｍｏｌ／Ｌ）可引起浓度依赖性的内向流（４／２６）;在多数细胞虽未检测到ＳＰ引起的膜电流,但却能对ＧＡＢＡＡ受体激活介导的膜内向流产生抑制效应（１８／２２）,并有加速去敏感的作用。本文就有关ＳＰ以ＧＡＢＡ激活电流抑制效应的可能意义进行了讨论。     

Molecular and functional characterization of the electroneutral Na/HCO3 cotransporter NBCn1 in rat hippocampal neurons

We examined molecular and electrophysiological properties of the electroneutral sodium/bicarbonate cotransporter (NBCn1) that is present in rat hippocampal neurons. By PCR, a deletion variant (NBCn1-E) that lacks 123 amino acids in the cytoplasmic N-terminal domain was found in adult neurons. The previously characterized NBCn1-B, which does not have the deletion, was detected in embryonic neurons. In Xenopus oocytes, NBCn1-E raised the intracellular pH in the presence of HCO(3) without significantly affecting the membrane potential. Despite this electroneutral cotransport activity, the transporter mediated a steady-state current that positively shifted the resting potential by almost 30 mV. The mean reversal potential of the steady-state current was -21.2 mV, close to the resting potential of -21.4 mV. The reversal potential shifted 26 mV in response to a 10-fold increase of external Na(+) for concentrations above 10 mm. The current activity mediated by the transporter was unaffected by K(+), Mg(2+), Ca(2+), or Cl(-). Stable expression of NBCn1-E in human embryonic kidney cells also evoked an inward current that shifted the resting potentials more positive compared with the sham-transfected controls. In primary cultures of embryonic hippocampal neurons, the NBCn1 protein was localized in somatodendrites and synapses. NBCn1 protein was partially colocalized with the postsynaptic density protein PSD-95. Single-cell PCR showed that NBCn1 mRNA expression was present in both gamma-aminobutyric acid (GABA)ergic and non-GABAergic neurons. We propose that NBCn1 in hippocampal neurons may affect neuronal activity by regulating local pH as well as steady-state inward currents at synapses.