Calcium-dependent Potassium Conductance Research Articles

The contribution of intermediate conductance calcium-dependent potassium channels to hypothalamic vasopressin neuronal excitability

Vasopressin (VP) magnocellular neurosecretory neurons of the supraoptic nucleus (SON) in the hypothalamus are critical regulators of renal water retention and vascular tone. VP neurons undergo detrimental plastic changes in cardiovascular diseases such as heart failure (HF), resulting in hyperexcitability and thus altered fluid/electrolyte balance. Hyperexcitability is caused in part by changes in intrinsic excitability mechanisms, including the slow afterhyperpolarization (sAHP), a phenomenon underlain by a calcium-dependent K+ current ( I sAHP). The sAHP is activated by Ca2+ and results in an efflux of K+ from the cell, hyperpolarizing it, and throttling firing. Importantly, we found that the sAHP is greatly inhibited in VP neurons from HF rats, contributing to increased neuronal excitability in this condition. While the features of the sAHP are well characterized, the identification of the channel underlying the I sAHP is unknown. Combining patch clamp electrophysiology and pharmacology in Wistar rats, we investigated Intermediate conductance Ca2+-dependent K+ (IK) channels as a potential candidate responsible for carrying the I sAHP. We generated and measured I sAHPs in voltage clamp via fixed steps of twenty spikes at twenty Hz and measured the the I sAHP as the resulting tail current at the end of the pulse. To account for rundown of the current over the recording period and for the pharmacological blocker’s slow mechanism of action, we measured I sAHPs every 60 seconds for at least 15 minutes. Bath application of TRAM-34 (2 μM) at 7 minutes, a specific IK channel blocker, failed to inhibit I sAHP peak amplitude or area (p > 0.05; n=36 SON neurons). We used biocytin-filled pipettes to identify neurons post hoc and run immunohistochemistry for oxytocin (OT) and VP to identify cell type within the SON. We found no significant difference in I sAHP amplitude or area between VP and OT cells under either control or TRAM-34 application. Taken together, our studies indicate that IK channels do not contribute to the I sAHP in SON neurosecretory neurons, despite robust IK expression in this region. Future studies will aim at identifying the role that IK channels play in SON, as well as targeting other potential K+ channel candidates for this NHLBI F32 HL158172 (MKK) NHLBI HL090948 (JES) NINDS NS094640 (JES) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Characterization of effector ion channels that mediate excitatory cholinergic modulation of XII motoneurons in neonatal mice in vitro

Obstructive sleep apnea is a sleep disorder characterized by repeated stops and starts of breathing during sleep. During an apneic episode there is a decrease in upper airway muscle tone, particularly of the genioglossus muscle (primary tongue protruder). Recent data suggest that this sleep specific loss of muscle tone is due to muscarinic modulation of hypoglossal (XII) motoneurons that innervate genioglossus. In adult rats, during REM sleep, activation of muscarinic acetylcholine receptors depresses genioglossus inspiratory output. By contrast, in neonatal mouse brainstem slices, muscarine potentiates inspiratory output when locally applied to the XII motor nucleus. To determine whether these different effects of muscarine are due to postnatal maturation, we first need to determine the effector ion channels that mediate the excitatory response to muscarine in neonatal mice. In individual XII motoneurons recorded from neonatal brainstem slices, muscarinic receptor activation inhibited an apamin‐sensitive small conductance calcium dependent potassium current (SK) and potentiated a hyperpolarized‐activated mixed cation current (Ih). It was hypothesized that both effector ion channels contribute to the net potentiation of inspiratory drive by muscarine in neonatal mice. Therefore, we aimed to determine 1) the effect of inhibition of SK, and 2) the contribution of Ih modulation to the modulation of inspiratory drive by muscarinic acetylcholine receptor activation at XII motoneurons in neonatal mice. Using rhythmically medullary slice preparations from neonatal CD1 mice (postnatal day P0–5), we first investigated the effects of blocking SK on inspiratory burst output. Local application of apamin (30s) to the XII motor nucleus had a net effect of increasing inspiratory burst amplitude (n=3) to 134% of baseline (range 118–143%) or 132% (range 111–169%) of baseline (2 and 20 mM respectively). These data are important to establish the contributions of SK to inspiratory burst amplitude before testing how muscarine may modulate SK during inspiratory bursts. We then confirmed that muscarine had a net excitatory effect on inspiratory burst amplitude in neonatal mice (128 ± 6% of baseline, n = 9). Next, we aimed to investigate if muscarinic excitation is mediated by blocking SK channels. In one P3 preparation, local application of apamin attenuated the muscarinic‐mediated increase in XII inspiratory burst amplitude from 157% to 137%. Lastly, in one P2 brainstem slice preparation, contrary to our hypothesis, blocking Ih with ZD7288 caused the potentiation of inspiratory burst amplitude by muscarine to increase from 130% to 166%. Future experiments will be needed to confirm these results. Our next steps will focus on characterization of developmental changes in muscarinic modulation of XII motoneuron inspiratory burst output.

Role of BKCa Channels in Mesenteric Artery Vascular Smooth Muscle Tone of Diabetic Rats

High conductance calcium‐dependent potassium (BKCa) channels are broadly distributed in different tissues where their activity is regulated by depolarization and calcium. The BKCa channels are abundant in vascular smooth muscle and their role regulating contractile tone has been widely discussed. Several studies have reported that in diabetes mellitus (DM) potassium currents mediated by these channels are diminished, especially in those located in small resistant vessels. BKCa channels are present in conducting arteries as well but their role in muscle tone seems to be small if any.The objective of this work was to study the role of BKCa channels in muscle tone of diabetic rat mesenteric arteries.Male Wistar rats (190 ± 10 gr in weight) were used and DM was induced by injecting streptozotocin (65 mg/kg) into de caudal artery. At 4, 8 and 10 weeks after streptozotocin or vehicle injection, rats were sacrificed and the mesenteric artery excised. We conducted isometric tension experiments using 10 micromolar of the selective channel activator NS1619 or 100 nM Iberiotoxin, a specific blocker of the BKCa channels applied at the peak of the maximum contraction induced by 10 microM phenylephrine (PE). We also took confocal fluorescence images to identify the localization of the alpha subunit ‐BKCa channel in mesenteric artery rings.Tension experiments show that NS1619 had no significant effect on the contraction induced by PE neither in control or DM rats, no matter the time of DM evolution (for example, at 10 weeks the percentage of relaxation was of 11.93 ± 4.73 in DM and of 9.0 in control group). Similarly, Iberiotoxin by itself had a weak contractile effect on the vascular tone of control and DM groups (for example, DM of 10 weeks the percentage of contraction was of 2.65 ± 1.89 and of 6.07 ± 3.34 in control rats). Immunofluorescence shows that BKCa alpha subunit channel is present within the endothelium, the smooth muscle cells and the adventitia. However, it is noteworthy that the alpha subunit is more abundant in endothelial cells and to a lesser extent in muscle cells where it can be seen mainly at the cytoplasm.We concluded that in spite that the BKCa channels are expressed in mesenteric artery smooth muscle, they do not participate in maintaining the muscle tone because they are present mainly intracellulary. At present we are conducting experiments to know the localization of the BKCa beta subunit and see if DM affect it.Support or Funding InformationSupported by CONACYT Grant 223350 to RETThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

BK Channel Gating-Ring Voltage Dependence Motions

The open probability of Large Conductance Voltage and Calcium dependent potassium (BK) channels is regulated allosterically by change in the transmembrane voltage and intracellular concentration of divalent ions (Ca2+ and Mg2+). BK channels are formed as tetramers of alpha subunits. The “divalent sensor” resides within the gating-ring, a large tetrameric structure formed by eight Regulator of Conductance of Potassium (RCK) domains, two from each subunit. Each RCK domain (RCK1 and RCK2) contains a high affinity- Ca2+binding site. Using Patch-clamp fluorometry, we have shown large changes in FRET signals within the gating-ring in response to Ca2+ and voltage. Here we investigate the origins of the voltage dependent motions. We tested manipulations that mostly change the BK channels’ relative probability of opening and in all cases the voltage dependence of the FRET signals remained within the voltage range of wild-type. However, mutations in the voltage sensor that are known to shift the charge displacement currents have a parallel shift of the gating ring's motions. These result suggest that the conformational changes of the gating ring are coupled to the voltage sensor, and could be relevant in the allosteric modulation of the BK channels.

Thalamic reticular impairment underlies attention deficit in Ptchd1(Y/-) mice.

Developmental disabilities, including attention-deficit hyperactivity disorder (ADHD), intellectual disability (ID), and autism spectrum disorders (ASD), affect 1 in 6 children in the United States. Recently, PTCHD1 (Patched-domain containing protein 1) gene mutations have been found in ~1% of patients with ID and ASD. PTCHD1 deletion patients show symptoms of ADHD, sleep disruption, hypotonia, aggression, ASD, and ID. Although PTCHD1 is likely critical for normal development, the connection between its deletion and the ensuing behavioral defects is poorly understood. Here, we report that during early postnatal development, mouse Ptchd1 is selectively expressed in the thalamic reticular nucleus (TRN), a group of GABAergic neurons that regulate thalamo-cortical transmission, sleep rhythms, and attention. Ptchd1 deletion attenuates TRN activity by reducing calcium-dependent potassium currents (SK). Restricted TRN deletion of Ptchd1 leads to attention deficits and hyperactivity, both of which are rescued by pharmacological augmentation of SK channels. Global Ptchd1 deletion recapitulates learning impairment, hyper-aggression, and motor defects, all of which are insensitive to SK pharmacological targeting and not found in the TRN-restricted deletion mouse. This study maps clinically-relevant behavioral phenotypes onto TRN dysfunction in a human disease model, while also identifying molecular and circuit targets for intervention.

Selective Cation Binding to the Gating-Ring Triggers Independent RCK Motions in the BK Channel

Large conductance voltage and calcium dependent potassium channels (BK channels) are important controllers of cell excitability. Functional BK channels are formed as tetramers of alpha subunits. Their open probability is regulated synergistically by changes in transmembrane voltage and intracellular concentration of divalent cations including Ca2+, Mg2+, Cd2+ and Ba2+. The voltage sensor resides within the transmembrane region of the alpha subunit, whereas a large C-terminal intracellular region called ‘gating ring’ senses divalent cation binding. The gating ring assembles as a large tetrameric structure that contains a total of eight Regulator of Conductance of K (RCK) domains, two from each subunit. In each RCK domain resides one high-affinity Ca2+ binding site. Even though the two RCK domains of each subunit (RCK1 and RCK2) are similar, their Ca2+ binding sites are not, particularly in their selectivity to divalent ions other than Ca2+. For example, Cd2+ effect is mediated exclusively through the RCK1 domains, while Ba2+ and Mg2+ (at low concentrations) prefer solely the site from the RCK2 domains. Using patch clamp fluorometry, we have shown previously that large rearrangements of the gating-ring occur in response to Ca2+ and voltage. By themselves, Cd2+, Mg2+ and Ba2+ induce movements of the gating-ring that are substantially smaller than those triggered by Ca2+. Full extent motions are achieved once binding sites from RCK1 and RCK2 domains are occupied by Cd2+ and Ba2+, or Cd2+ and Mg2+. These results suggest that the gating ring structure is flexible, potentially allowing independent movements of the RCK1 and RCK2 domains.

The Effect of Variations in Ionic Conductance Values on the Suppression of Repetitive Spiking in a Mathematical Model of Type-A Medial Vestibular Nucleus Neurons

A previous study has proposed a mathematical model of type-A medial vestibular nucleus neurons (mVNn). This model is described by a system of nonlinear ordinary differential equations, which is based on the Hodgkin-Huxley formalism. The type-A mVNn model contains several ionic conductances, such as the sodium conductance, calcium conductance, delayed-rectifier potassium conductance, transient potassium conductance, and calcium-dependent potassium conductance. The previous study revealed that spontaneous repetitive spiking in the type-A mVNn model can be suppressed by hyperpolarizing stimulation. However, how this suppression is affected by the ionic conductances has not been clarified in the previous study. The present study performed numerical simulation analysis of the type-A mVNn model to clarify how variations in the different ionic conductance values affect the suppression of repetitive spiking. The present study revealed that the threshold for the transition from a repetitive spiking state to a quiescent state is differentially sensitive to variations in the ionic conductances among the different types of ionic conductance.

A detailed, conductance-based computer model of intrinsic sensory neurons of the gastrointestinal tract.

Intrinsic sensory neurons (ISNs) of the enteric nervous system respond to stimuli such as muscle tension, muscle length, distortion of the mucosa, and the chemical content in the lumen. ISNs form recurrent networks that probably drive many intestinal motor patterns and reflexes. ISNs express a large number of voltage- and calcium-gated ion channels, some of which are modified by inflammation or repeated physiological stimuli, but how interactions between different ionic currents in ISNs produce both normal and pathological behaviors in the intestine remains unclear. We constructed a model of ISNs including voltage-gated sodium and potassium channels, N-type calcium channels, big conductance calcium-dependent potassium (BK) channels, calcium-dependent nonspecific cation channels (NSCa), intermediate conductance calcium-dependent potassium (IK) channels, hyperpolarization-activated cation (Ih) channels, and internal calcium dynamics. The model was based on data from the literature and our electrophysiological studies. The model reproduced responses to short or long depolarizing current pulses and responses to long hyperpolarizing current pulses. Sensitivity analysis showed that Ih, IK, NSCa, and BK have the largest influence on the number of action potentials observed during prolonged depolarizations. The model also predicts that changes to the voltage of activation for Ih have a large influence on excitability, but changes to the time constant of activation for Ih have a minor effect. Our model identifies how interactions between different iconic currents influence the excitability of ISNs and highlights an important role for Ih in enteric neuroplasticity resulting from disease.

Parvalbumin Tunes Spike-Timing and Efferent Short-Term Plasticity in Striatal Fast Spiking Interneurons

Striatal fast spiking interneurons (FSI) modulate the output of the striatum by synchronizing medium-sized spiny neurons (MSN). FSI are the only striatal neurons to express the calcium-binding protein parvalbumin (PV). This selective expression of PV raises questions about the functional role of this calcium buffer in controlling FSI calcium dynamics, and, consequently, the FSI spiking mode and neurotransmission. To study the functional involvement of FSI in striatal microcircuit activity and the role of PV in FSI function, we performed perforated patch recordings on EGFP-expressing FSI in brain slices from control and PV-/- mice. Our results revealed that PV-/- FSI fired more regularly and were more excitable than control FSI by a mechanism in which calcium buffering is linked to spiking activity as the result of the activation of small conductance (SK) calcium-dependent potassium channels. A modeling approach of striatal FSI supports our experimental results. Furthermore, PV deletion modified frequency-specific short-term plasticity at inhibitory FSI to MSN synapses. Our results therefore support the hypothesis that in FSI, PV is crucial for the fine-tuning of the temporal responses of the FSI network and for the orchestration of MSN populations. This, in turn, may play a direct role in the generation and pathology-related worsening of motor rhythms.

A critical re‐evaluation of the specificity of action of perivagal capsaicin

Perivagal application of capsaicin (1% solution) is considered to cause a selective degeneration of vagal afferent C fibres and has been used extensively to examine the site of action of many gastrointestinal (GI) neuropeptides. The actions of both capsaicin and GI neuropeptides may not be restricted to vagal afferent fibres, however, as other non-sensory neurones have displayed sensitivity to capsaicin and brainstem microinjections of these neuropeptides induce GI effects similar to those obtained upon systemic application. The aim of the present study was to test the hypothesis that perivagal capsaicin induces degeneration of vagal efferents controlling GI functions. Experiments were conducted 7-14 days after 30 min unilateral perivagal application of 0.1-1% capsaicin. Immunohistochemical analyses demonstrated that, as following vagotomy, capsaicin induced dendritic degeneration, decreased choline acetyltransferase but increased nitric oxide synthase immunoreactivity in rat dorsal motor nucleus of the vagus (DMV) neurones. Electrophysiological recordings showed a decreased DMV input resistance and excitability due, in part, to the expression of a large conductance calcium-dependent potassium current and the opening of a transient outward potassium window current at resting potential. Furthermore, the number of DMV neurones excited by thyrotrophin-releasing hormone and the gastric motility response to DMV microinjections of TRH were decreased significantly. Our data indicate that perivagal application of capsaicin induced DMV neuronal degeneration and decreased vagal motor responses. Treatment with perivagal capsaicin cannot therefore be considered selective for vagal afferent C fibres and, consequently, care is needed when using perivagal capsaicin to assess the mechanism of action of GI neuropeptides.

Changes in Calcium-Dependent Potassium Conductance of Isolated Smooth Muscle Cells of the Rat Bladder in Experimental Diabetes

Activation of large conductance calcium-dependent potassium channels (BK channels) affects the membrane repolarization at action potential generation and the resting potential of detrusor smooth muscle cells (SMC). Overactive bladder syndrome (OBS) is one of the complications of diabetes mellitus. Using patch-clamp technique, we have found a significant decrease in potassium current (IBK) through BK channels of isolated detrusor SMC, and a decrease in the amplitude and frequency of BK-mediated spontaneous transient outward currents (STOCs) in rats with streptozotocin-induced diabetes mellitus compared with control animals. Under the diabetes, IBK density at step depolarization to +50 mV averaged (10.0 ± 0.5) pA / pF (the control values were (15.0 ± 0.4) pA / pF), whereas the mean values of the STOC frequency and amplitude at holding potential of -20 mV decreased from (12.0 ± 1.5) Hz to (2.4 ± 0.6) Hz and (0.9 ± 0.1) to 0.5 pA / pF ± 0.1 pA / pF, respectively. Using real time polymerase chain reaction (RT-PCR), it was found that mRNA expression of primary pore-forming KCa1.1-subunit of the BK channel increased in diabetes, whilst that for the auxiliary BKCaβ1-subunit decreased. It has been concluded that the observed changes in BK-channel currents in diabetes may enhance excitability of detrusor SMC, thereby promoting myogenic OBS. However, more studies are needed to determine how a decrease in BKCa β1 expression affects the functional properties of BK channels in diabetes, and to identify the type and direction of calcium signal changes modulating channel activation.

Glyco-Phospho-Glycero Ether Lipids (GPGEL): synthesis and evaluation as small conductance Ca2+-activated K+ channel (SK3) inhibitors

The SK3 channel, a member of the small conductance calcium-dependent potassium channels (SKCa), plays a central role with respect to the motility of highly metastatic cancerous cells (e.g. MDA-MB435s – breast cancer cell). Edelfosine, an ether lipid derivative, is able to partially inhibit this channel activity and thus reduce the SK3-dependent cell motility, but, due to its toxicity, analogues of this compound were highly desired. Ohmline, an edelfosine analogue that possesses a lactose unit in its polar domain, was the first efficient and non-toxic SK3 inhibitor that exhibits an amphiphilic structure. In the present work, we have modified the polar head of Ohmline by placing a disubstituted phosphate group between a disaccharide unit (lactose, maltose, and melibiose) and the glycerol ether-lipid moiety. It was first observed that this modification increases the water solubility of these compounds. All these novel compounds are efficient SK3 channel inhibitors with an activity comparable to Ohmline (patch-clamp measurements). These compounds are also able to reduce the SK3-dependent cell motility with similar efficacies to Ohmline. In a broader perspective it is shown that the presence of one anionic charge (coming from the presence of a phosphate group) in the polar head group does not alter the SK3 channel inhibition and provides insights into the future development of a class of migration-targeted anticancer agents.

Influences of membrane properties on phase response curve and synchronization stability in a model globus pallidus neuron

The activity patterns of the globus pallidus (GPe) and subthalamic nucleus (STN) are closely associated with motor function and dysfunction in the basal ganglia. In the pathological state caused by dopamine depletion, the STN-GPe network exhibits rhythmic synchronous activity accompanied by rebound bursts in the STN. Therefore, the mechanism of activity transition is a key to understand basal ganglia functions. As synchronization in GPe neurons could induce pathological STN rebound bursts, it is important to study how synchrony is generated in the GPe. To clarify this issue, we applied the phase-reduction technique to a conductance-based GPe neuronal model in order to derive the phase response curve (PRC) and interaction function between coupled GPe neurons. Using the PRC and interaction function, we studied how the steady-state activity of the GPe network depends on intrinsic membrane properties, varying ionic conductances on the membrane. We noted that a change in persistent sodium current, fast delayed rectifier Kv3 potassium current, M-type potassium current and small conductance calcium-dependent potassium current influenced the PRC shape and the steady state. The effect of those currents on the PRC shape could be attributed to extension of the firing period and reduction of the phase response immediately after an action potential. In particular, the slow potassium current arising from the M-type potassium and the SK current was responsible for the reduction of the phase response. These results suggest that the membrane property modulation controls synchronization/asynchronization in the GPe and the pathological pattern of STN-GPe activity.

A computational model of the moth macroglomerular complex

The macroglomerular complex (MGC) is known as the olfactory sub-system processing pheromonal information. In the moth Manduca sexta, the projection neurons (PNs) arborizating in the MGC exhibit two types of responses to pheromone stimulation [1]: a simple monophasic long and tonic excitation (+) and a complex multiphasic pattern (excitation-inhibition (+/-) or inhibition-excitation-inhibition (-/+/-)). The PNs connected to the same glomerulus are synchronized and the level of synchrony is modulated by lateral inhibition [2]. Here we studied the role of inhibition and intrinsic properties of PNs on their synchrony and their response patterns. We developed a computational model of the MGC involving two types of inhibitory local neurons (86 LN-IIa, 68 LN-IIb) connected randomly to 41 excitatory PNs [3]. LN-IIa’s have very dense branching in the MGC and are excited briefly by the pheromone blend, whereas LN-IIb’s possess less dense arborizations and respond by a long-lasting excitation. Single cells are modeled as conductance-based neurons with similar intrinsic currents (Na+, K+, Ca2+, A, SK) and leak current but different parameter values for PNs and LNs. First, we examined the effect of inhibition from LNs. In PN responses, the after-hyperpolarization (AHP) was preserved when the LN-IIa were removed but prior inhibition vanishes so that triphasic patterns (-/+/-) are changed to biphasic (+/-). However, removing LN-IIb made the duration of the AHP phase significantly shorter. The synchrony rate was also disturbed by removing one or another of LNs but loss in synchrony remained more important without LN-IIa (fig.1.A). Then we analyzed the effect of the small conductance calcium-dependent potassium (SK) current on the AHP phase. Blocking the SK channel in the PN model disrupted AHP as found experimentally with local injection of Bicuculline. We also investigated whether the latency of the PN response could be due to the A current: its blocking in the model (experimentally this is done by 4-aminopyridine for example) reduced both PN synchrony (fig.1.B) and latency (fig.1.C). As LNs respond significantly faster than PNs, the onset of the first inhibition in the model coincides with inhibition from LNs. Lateral inhibition then plays the role of a “reset” by eliminating the influence of initial conditions. Taken together, these observations suggest that PN synchrony and firing patterns result from a complex interplay between interglomerular inhibition and neuronal intrinsic properties. Figure 1 Role of LN inhibition and intrinsic properties on PN synchrony and latency. A- Both LN-IIa and LN-IIb are involved in PN-PN synchrony. B- Blocking IA current decreases PN-PN synchrony. C- Response latencies of LNs (IIa and IIb) and PNs (intact and IA ...

Dendritic spikes mediate negative synaptic gain control in cerebellar Purkinje cells

Dendritic spikes appear to be a ubiquitous feature of dendritic excitability. In cortical pyramidal neurons, dendritic spikes increase the efficacy of distal synapses, providing additional inward current to enhance axonal action potential (AP) output, thus increasing synaptic gain. In cerebellar Purkinje cells, dendritic spikes can trigger synaptic plasticity, but their influence on axonal output is not well understood. We have used simultaneous somatic and dendritic patch-clamp recordings to directly assess the impact of dendritic calcium spikes on axonal AP output of Purkinje cells. Dendritic spikes evoked by parallel fiber input triggered brief bursts of somatic APs, followed by pauses in spiking, which cancelled out the extra spikes in the burst. As a result, average output firing rates during trains of input remained independent of the input strength, thus flattening synaptic gain. We demonstrate that this "clamping" of AP output by the pause following dendritic spikes is due to activation of high conductance calcium-dependent potassium channels by dendritic spikes. Dendritic spikes in Purkinje cells, in contrast to pyramidal cells, thus have differential effects on temporally coded and rate coded information: increasing the impact of transient parallel fiber input, while depressing synaptic gain for sustained parallel fiber inputs.

Lactobacillus reuteri ingestion and IKCa channel blockade have similar effects on rat colon motility and myenteric neurones

We have previously shown that ingestion of Lactobacillus reuteri may modulate colonic enteric neuron activity but with unknown effects on colon motility. The aim of the present report was to elucidate the neuronal mechanisms of action of the probiotic by comparing the effects on motility of L. reuteri ingestion with blockade of a specific ionic current in enteric neurons. We have used intraluminal pressure recordings from ex vivo rat colon segments and whole cell patch clamp recordings from neurons of rat longitudinal muscle myenteric plexus preparations to investigate the effects of L. reuteri and TRAM-34 on colon motility and neurophysiology. The effects of daily feeding of 10(9) L. reuteri bacteria or acute application of TRAM-34 on threshold fluid filling pressure or pulse pressure was measured. Lactobacillus reuteri increased intraluminal fluid filling pressure thresholds for evoking pressure pulses by 51% from 0.47 +/- 0.17 hPa; the probiotic also decreased the pulse pressure amplitudes, but not frequency, by 18% from 3.91 +/- 0.52 hPa. The intermediate conductance calcium-dependent potassium (IK(Ca)) channel blocker TRAM-34 (3 micromol L(-1)) increased filling threshold pressure by 43% from 0.52 +/- 0.22 hPa and reduced pulse pressure amplitude by 40% from 2.63 +/- 1.11 hPa; contraction frequency was unaltered. TRAM-34 (3 micromol L(-1)) reduced membrane polarization, leak conductance and the slow afterhyperpolarization current in 16/16 myenteric rat colon AH cells but 19/19 S cells were unaffected. The present results are consistent with L. reuteri enhancing tonic inhibition of colon contractile activity by acting via the IK(Ca) channel current in AH cells.

Commentary on “Factors influencing clinical features of absence seizures”

Commentary on “Factors influencing clinical features of absence seizures”

Dopamine induced switch in the subthreshold dynamics of the striatal cholinergic interneurons: A numerical study

The striatum is a part of the basal ganglia, which are a group of nuclei in the brain associated with motor control, cognition and learning. Striatal cholinergic interneurons (AchNs) play a crucial role in these functions. AchNs are tonically active in vivo and in vitro, and are able to fire in the absence of synaptic inputs. AchNs respond to sensory stimuli and sensorimotor learning by transiently suppressing their firing activity. This pause is dopamine signal sensitive, but the neurophysiological mechanism of the dopaminergic influence is under debate. Both the regular spiking response as well as the pause response are influenced by the inwardly rectifying outward G kir , a slow hyperpolarization activated noninactivating G h , and calcium and calcium-dependent potassium conductances [Wilson, C., Goldberg, J., 2006. Origin of the slow afterhyperpolarization and slow rhythmic bursting in striatal cholinergic interneurons. J. Neurophysiol. 95(1), 196–204; Wilson, C., 2005. The mechanism of intrinsic amplification of hyperpolarizations and spontaneous bursting in striatal cholinergic interneurons. Neuron 45(4), 575–585]. Recent experimental evidence has shown that dopaminergic modulations on G h , G kir and calcium conductances influence the AchN's excitability [Deng, P., Zhang, Y., Xu, Z., 2007. Involvement of I( h) in dopamine modulation of tonic firing in striatal cholinergic interneurons. J. Neurosci. 27(12), 3148–3156; Aosaki, T., Kiuchi, K., Kawaguchi, Y., 1998. Dopamine D 1 -like receptor activation excites rat striatal large aspiny neurons in vitro. J. Neurosci. 18(14), 5180–5190]. We employed computational models of the AchN to analyze the conductance based dopaminergic changes. We analyzed the robustness of these subthreshold oscillations and how they are affected by dopaminergic modulation. Our results predict that these conductances allow the dopamine to switch the AchN between stable oscillatory and fixed-point behaviors. The present approach and results show that dopamine receptors ( D 1 and D 2 ) mediate opposing effects on this switch and therefore on the suprathreshold excitability as well. The switching effect of the dopaminergic signal is the major qualitative feature that can serve as a building block for higher network-level descriptions. To our knowledge this is the first paper that synthesizes the growing body of experimental literature about the dopaminergic modulation of the AchNs into a modelling framework.

Functional coupling of TRPV4 cationic channel and large conductance, calcium-dependent potassium channel in human bronchial epithelial cell lines

Calcium-dependent potassium channels are implicated in electrolyte transport, cell volume regulation and mechanical responses in epithelia, although the pathways for calcium entry and their coupling to the activation of potassium channels are not fully understood. We now show molecular evidence for the presence of TRPV4, a calcium permeable channel sensitive to osmotic and mechanical stress, and its functional coupling to the large conductance calcium-dependent potassium channel (BK(Ca)) in a human bronchial epithelial cell line (HBE). Reverse transcriptase polymerase chain reaction, intracellular calcium imaging and whole-cell patch-clamp experiments using HBE cells demonstrated the presence of TRPV4 messenger and Ca(2+) entry, and outwardly rectifying cationic currents elicited by the TRPV4 specific activator 4alpha-phorbol 12,13-didecanoate (4alphaPDD). Cell-attached and whole-cell patch-clamp of HBE cells exposed to 4alphaPDD, and hypotonic and high-viscosity solutions (related to mechanical stress) revealed the activation of BK(Ca) channels subsequent to extracellular Ca(2+) influx via TRPV4, an effect lost upon antisense-mediated knock-down of TRPV4. Further analysis of BK(Ca) modulation after TRPV4 activation showed that the Ca(2+) signal can be generated away from the BK(Ca) location at the plasma membrane, and it is not mediated by intracellular Ca(2+) release via ryanodine receptors. Finally, we have shown that, unlike the reported disengagement of TRPV4 and BK(Ca) in response to hypotonic solutions, cystic fibrosis bronchial epithelial cells (CFBE) preserve the functional coupling of TRPV4 and BK(Ca) in response to high-viscous solutions.

Withdrawal From Intermittent Ethanol Exposure Increases Probability of Burst Firing in VTA Neurons In Vitro

Changing the activity of ventral tegmental area (VTA) dopamine neurons from pacemaker to burst firing is hypothesized to increase the salience of stimuli, such as an unexpected reward, and likely contributes to withdrawal-associated drug-seeking behavior. Accordingly, pharmacological, behavioral, and electrophysiological data suggest an important role of the VTA in mediating alcohol-dependent behaviors. However, the effects of repeated ethanol exposure on VTA dopamine neuron ion channel function are poorly understood. Here, we repeatedly exposed rats to ethanol (2 g/kg ethanol, ip, twice per day for 5 days), then examined the firing patterns of VTA dopamine neurons in vitro after 7 days withdrawal. Compared with saline-treated animals, the function of the small conductance calcium-dependent potassium channel (SK) was reduced in ethanol-treated animals. Consistent with a role for SK in regulation of burst firing, NMDA applied during firing facilitated the transition to bursting in ethanol-treated but not saline-treated animals; NMDA consistently induced bursting only in saline-treated animals when SK was inhibited. Also, enhanced bursting in ethanol-treated animals was not a result of differences in NMDA-induced depolarization. Further, I(h) was also reduced in ethanol-treated animals, which delayed recovery from hyperpolarization, but did not account for the increased NMDA-induced bursting in ethanol-treated animals. Finally, repeated ethanol exposure and withdrawal also enhanced the acute locomotor-activating effect of cocaine (15 mg/kg, ip). Thus withdrawal after repeated ethanol exposure produced several alterations in the physiological properties of VTA dopamine neurons, which could ultimately increase the ability of VTA neurons to produce burst firing and thus might contribute to addiction-related behaviors.