Activation of neurokinin 3 receptor increases Na(v)1.9 current in enteric neurons.

The intrinsic primary afferent neurons (IPANs) of the guinea pig enteric nervous system express Na(v)1.9 sodium channels that produce a persistent TTX-resistant current having a low activation threshold and slow gating kinetics. These neurons receive slow EPSPs induced mainly by the activation of neurokinin 3 receptors (NK3r). Here, we demonstrate that senktide, a specific NK3r agonist, potentiates the Na(v)1.9 current (I(Nav1.9)) in IPANs. Using whole-cell patch-clamp recordings from IPANs in duodenum longitudinal muscle/myenteric plexus preparations, we show that short (1-5 s) and long (up to 1 min) applications of senktide, increase the I(Nav1.9) peak current up to 13-fold. The effect, blocked by a NK3r antagonist SB235375 is transient, lasting approximately 2 min and is due to a negative shift of the activation voltage by approximately 20 mV and of fast inactivation by approximately 10 mV. As a consequence, the window current resulting from the product of the activation and fast inactivation curves is shifted and enlarged. The transient effect of senktide is likely to be due to the fast desensitization of NK3r. Protein kinase C (PKC) activation with phorbol or oleoyl acetylglycerol also increases I(Nav1.9), although persistently, by inducing similar voltage-dependent changes. Current-clamp experiments showed that I(Nav1.9) modulation by senktide lowers action potential threshold and increases excitability. The increase in I(Nav1.9) by NK3r activation is also likely to amplify slow EPSPs generated in the IPANs. These changes in excitability potentially have a profound effect on the entire enteric synaptic circuit and ultimately on gut motility and secretion.

Selective expression of a persistent tetrodotoxin-resistant Na+ current and NaV1.9 subunit in myenteric sensory neurons.

Voltage-gated Na(+) currents play critical roles in shaping electrogenesis in neurons. Here, we have identified a TTX-resistant Na(+) current (TTX-R I(Na)) in duodenum myenteric neurons of guinea pig and rat and have sought evidence regarding the molecular identity of the channel producing this current from the expression of Na(+) channel alpha subunits and the biophysical and pharmacological properties of TTX-R I(Na). Whole-cell patch-clamp recording from in situ neurons revealed the presence of a voltage-gated Na(+) current that was highly resistant to TTX (IC(50), approximately 200 microm) and selectively distributed in myenteric sensory neurons but not in interneurons and motor neurons. TTX-R I(Na) activated slowly in response to depolarization and exhibited a threshold for activation at -50 mV. V(1/2) values of activation and steady-state inactivation were -32 and -31 mV in the absence of fluoride, respectively, which, as predicted from the window current, generated persistent currents. TTX-R I(Na) also had prominent ultraslow inactivation, which turns off 50% of the conductance at rest (-60 mV). Substituting CsF for CsCl in the intracellular solution shifted the voltage-dependent parameters of TTX-R I(Na) leftward by approximately 20 mV. Under these conditions, TTX-R I(Na) had voltage-dependent properties similar to those reported previously for NaN/Na(V)1.9 in dorsal root ganglion neurons. Consistent with this, reverse transcription-PCR, single-cell profiling, and immunostaining experiments indicated that Na(V)1.9 transcripts and subunits, but not Na(V)1.8, were expressed in the enteric nervous system and restricted to myenteric sensory neurons. TTX-R I(Na) may play an important role in regulating subthreshold electrogenesis and boosting synaptic stimuli, thereby conferring distinct integrative properties to myenteric sensory neurons.

Myelin basic protein-reactive T cells induce conduction failure in vivo but not in vitro.

The ability of myelin basic protein (MBP)-reactive T cells to induce conduction failure was investigated and. With the model, somatosensory evoked potentials (SEP) were recorded before and during adoptively transferred experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Maximum amplitude SEP were reached within 15 min of anesthesia. During EAE, the SEP decreased considerably and their onset was delayed. However, the compound action potentials (CAPs) recorded from Lewis rat optic nerves incubated with encephalitogenic T cells were not affected, emphasizing the importance of environmental factors. This study shows that the model described here is an useful means of investigating the neurological disorders associated with EAE.

Controlling the excitability of IPANs: a possible route to therapeutics.

Recent studies have shown that intrinsic primary afferent neurons (IPANs) express a much larger range of ionic currents than non-sensory neurons of the enteric nervous system. These ionic currents can be modulated by neurotransmitters that are synaptically released onto the soma (unlike cranial and spinal sensory neurons). The membrane receptors and ionic channels that are involved in the sensory transduction processes of IPANS are beginning to be defined. IPANS can move between a large range of excitability states that are influenced by neurotransmitters and hormones. An additional cause of variability in excitability is the actions of inflammatory mediators. It is becoming apparent that the variation in excitability of IPANS might play a critical role in determining the physiological state of the intestine.

Effects of K+ channel blockers on developing rat myelinated CNS axons: identification of four types of K+ channels.

Four blockers of voltage-gated potassium channels (Kv channels) were tested on the compound action potentials (CAPs) of rat optic nerves in an attempt to determine the regulation of Kv channel expression during the process of myelination. Before myelination occurred, 4-aminopyridine (4-AP) increased the amplitude, duration, and refractory period of the CAPs. On the basis of their pharmacological sensitivity, 4-AP-sensitive channels were divided in two groups, the one sensitive to kaliotoxin (KTX), dendrotoxin-I (DTX-I), and 4-AP, and the other sensitive only to 4-AP. In addition, tetraethylammonium chloride (TEA) applied alone broadened the CAPs. At the onset of myelination, DTX-I induced a more pronounced effect than KTX; this indicates that a fourth group of channels sensitive to 4-AP and DTX-I but insensitive to KTX had developed. The effects of KTX and DTX-I gradually disappeared during the period of myelination. Electron microscope findings showed that the disappearance of these effects was correlated with the ongoing process of myelination. This was confirmed by the fact that DTX-I and KTX enlarged the CAPs of demyelinated adult optic nerves. These results show that KTX- and DTX-sensitive channels are sequestrated in paranodal regions. During the process of myelination, KTX had less pronounced effects than DTX-I on demyelinated nerves, which suggests that the density of the KTX-sensitive channels decreased during this process. By contrast, 4-AP increased the amplitude, duration, and refractory period of the CAPs at all the ages tested and to a greater extent than KTX and DTX-I. The effects of TEA alone also gradually disappeared during this period. However, effects of TEA on CAPs were observed when this substance was applied after 4-AP to the adult optic nerve; this shows that TEA-sensitive channels are not masked by the myelin sheath. In conclusion, the process of myelination seems to play an important part in the regulation and setting of Kv channels in optic nerve axons.

Analysis of whole-cell currents by patch clamp of guinea-pig myenteric neurones in intact ganglia.

Whole-cell patch-clamp recordings taken from guinea-pig duodenal myenteric neurones within intact ganglia were used to determine the properties of S and AH neurones. Major currents that determine the states of AH neurones were identified and quantified. S neurones had resting potentials of -47 +/- 6 mV and input resistances (R(in)) of 713 +/- 49 MOmega at voltages ranging from -90 to -40 mV. At more negative levels, activation of a time-independent, caesium-sensitive, inward-rectifier current (I(Kir)) decreased R(in) to 103 +/- 10 MOmega. AH neurones had resting potentials of -57 +/- 4 mV and R(in) was 502 +/- 27 MOmega. R(in) fell to 194 +/- 16 MOmega upon hyperpolarization. This decrease was attributable mainly to the activation of a cationic h current, I(h), and to I(Kir). Resting potential and R(in) exhibited a low sensitivity to changes in [K(+)](o) in both AH and S neurones. This indicates that both cells have a low background K(+) permeability. The cationic current, I(h), contributed about 20 % to the resting conductance of AH neurones. It had a half-activation voltage of -72 +/- 2 mV, and a voltage sensitivity of 8.2 +/- 0.7 mV per e-fold change. I(h) has relatively fast, voltage-dependent kinetics, with on and off time constants in the range of 50-350 ms. AH neurones had a previously undescribed, low threshold, slowly inactivating, sodium-dependent current that was poorly sensitive to TTX. In AH neurones, the post-action-potential slow hyperpolarizing current, I(AHP), displayed large variation from cell to cell. I(AHP) appeared to be highly Ca(2+) sensitive, since its activation with either membrane depolarization or caffeine (1 mM) was not prevented by perfusing the cell with 10 mM BAPTA. We determined the identity of the Ca(2+) channels linked to I(AHP). Action potentials of AH neurones that were elongated by TEA (10 mM) were similarly shortened and I(AHP) was suppressed with each of the three omega-conotoxins GVIA, MVIIA and MVIIC (0.3-0.5 microM), but not with omega-agatoxin IVA (0.2 microM). There was no additivity between the effects of the three conotoxins, which indicates the presence of N- but not of P/Q-type Ca(2+) channels. A residual Ca(2+) current, resistant to all toxins, but blocked by 0.5 mM Cd(2+), could not generate I(AHP). This patch-clamp study, performed on intact ganglia, demonstrates that the AH neurones of the guinea-pig duodenum are under the control of four major currents, I(AHP), I(h), an N-type Ca(2+) current and a slowly inactivating Na(+) current.