Putting the theory into 'burstlet theory': A biophysical model of bursts and burstlets in the respiratory preBötzinger complex

Inspiratory breathing rhythms arise from synchronized neuronal activity in a bilaterally distributed brainstem structure known as the preBötzinger complex (preBötC). In in vitro slice preparations containing the preBötC, extracellular potassium must be elevated above physiological levels (to 7-9mM) to observe regular rhythmic respiratory motor output in the hypoglossal nerve to which the preBötC projects. Reexamination of how extracellular K+ affects preBötC neuronal activity has revealed that low amplitude oscillations persist at physiological levels. These oscillatory events are sub-threshold from the standpoint of transmission to motor output and are dubbed burstlets. Burstlets arise from synchronized neural activity in a rhythmogenic neuronal subpopulation within the preBötC that in some instances may fail to recruit the larger network events, or bursts, required to generate motor output. The fraction of subthreshold preBötC oscillatory events (burstlet fraction) decreases sigmoidally with increasing extracellular potassium. These observations underlie the burstlet theory of respiratory rhythm generation. Experimental and computational studies have suggested that recruitment of the non-rhythmogenic component of the preBötC population requires intracellular Ca2+ dynamics and activation of a calcium-activated non-selective cationic current. In this computational study, we show how intracellular calcium dynamics driven by synaptically triggered Ca2+ influx as well as Ca2+ release/uptake by the endoplasmic reticulum in conjunction with a calcium-activated non-selective cationic current can explain all of the key observations underlying the burstlet theory of respiratory rhythm generation. Thus, we provide a mechanistic basis to unify the experimental findings on rhythm generation and motor output recruitment in the preBötC.

­­Differential contribution of the subthreshold operating currents IT, Ih and IKir to the resonance of thalamocortical neurons

Membrane potential oscillations of thalamocortical (TC) neurons are believed to be involved in the generation and maintenance of brain rhythms that underlie global physiological and pathological brain states. These membrane potential oscillations depend on the synaptic interactions of TC neurons and their intrinsic electrical properties. These oscillations may be also shaped by increased output responses at a preferred frequency, known as intrinsic neuronal resonance. Here we combine electrophysiological recordings in mouse brain slices, modern pharmacological tools, dynamic clamp and computational modeling to study the ionic mechanisms that generate and modulate TC neuron resonance. We confirm findings of pioneering studies showing that most TC neurons display resonance which results from the interaction of the slow inactivation of the low threshold calcium current IT with the passive properties of the membrane. We also show that the hyperpolarization activated cationic current Ih is not involved in the generation of resonance, instead, it plays a minor role in the stabilization of TC neuron impedance magnitude due to its large contribution to the steady conductance. More importantly, we also demonstrate that TC neuron resonance is amplified by the inward rectifier potassium current IKir by a mechanism that hinges on its strong voltage dependent inward rectification (i.e. a negative slope conductance region) Accumulating evidence indicate that the ion channels that control the oscillatory behavior of TC neurons participate in pathophysiological processes. Results presented here points to IKir as a new potential target for therapeutic intervention.

Suppression of mICAT in Mouse Small Intestinal Myocytes by General Anaesthetic Ketamine and its Recovery by TRPC4 Agonist (-)-englerin A

A better understanding of the negative impact of general anesthetics on gastrointestinal motility requires thorough knowledge of their molecular targets. In this respect the muscarinic cationic current (mICAT carried mainly via TRPC4 channels) that initiates cholinergic excitation-contraction coupling in the gut is of special interest. Here we aimed to characterize the effects of one of the most commonly used “dissociative anesthetics”, ketamine, on mICAT. Patch-clamp and tensiometry techniques were used to investigate the mechanisms of the inhibitory effects of ketamine on mICAT in single mouse ileal myocytes, as well as on intestinal motility. Ketamine (100 µM) strongly inhibited both carbachol- and GTPγS-induced mICAT. The inhibition was slow (time constant of about 1 min) and practically irreversible. It was associated with altered voltage dependence and kinetics of mICAT. In functional tests, ketamine suppressed both spontaneous and carbachol-induced contractions of small intestine. Importantly, inhibited by ketamine mICAT could be restored by direct TRPC4 agonist (-)-englerin A. We identified mICAT as a novel target for ketamine. Signal transduction leading to TRPC4 channel opening is disrupted by ketamine mainly downstream of muscarinic receptor activation, but does not involve TRPC4 per se. Direct TRPC4 agonists may be used for the correction of gastrointestinal disorders provoked by general anesthesia.

Further characterization of the synergistic activation mechanism of cationic channels by M2 and M3 muscarinic receptors in mouse intestinal smooth muscle cells

In mouse ileal myocytes, muscarinic receptor-mediated cationic current ( mIcat) occurs mainly through synergism of M2 and M3 subtypes involving Gi/o-type GTP-binding proteins and phospholipase C (PLC). We have further studied the M2/M3 synergistic pathway. Carbachol-induced mIcat was markedly depressed by YM-254890, a Gq/11 protein inhibitor. However, the mIcat was unaffected by heparin, calphostin C, or chelerythrine, suggesting that mIcat activation does not involve signaling molecules downstream of phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown. M2-knockout (KO) mice displayed a reduced mIcat (~10% of wild-type mIcat) because of the lack of M2-Gi/o signaling. The impaired mIcat was insensitive to neuropeptide Y possessing a Gi/o-stimulating activity. M3-KO mice also displayed a reduced mIcat (~6% of wild-type mIcat) because of the lack of M3-Gq/11 signaling, and the mIcat was insensitive to prostaglandin F2α possessing a Gq/11-stimulating activity. These results suggest the importance of Gq/11/PLC-hydrolyzed PIP2 breakdown itself in mIcat activation and also support the idea that the M2/M3 synergistic pathway represents a signaling complex consisting of M2-Gi/o and M3-Gq/11-PLC systems in which both G proteins are special for this pathway but not general in receptor coupling.

High Efficacy by GAL-021: A Known Intravenous Peripheral Chemoreceptor Modulator that Suppresses BKCa-Channel Activity and Inhibits IK(M) or Ih

GAL-021 has recently been developed as a novel breathing control modulator. However, modifications of ionic currents produced by this agent remain uncertain, although its efficacy in suppressing the activity of big-conductance Ca2+-activated K+ (BKCa) channels has been reported. In pituitary tumor (GH3) cells, we found that the presence of GAL-021 decreased the amplitude of macroscopic Ca2+-activated K+ current (IK(Ca)) in a concentration-dependent manner with an effective IC50 of 2.33 μM. GAL-021-mediated reduction of IK(Ca) was reversed by subsequent application of verteporfin or ionomycin; however, it was not by that of diazoxide. In inside-out current recordings, the addition of GAL-021 to the bath markedly decreased the open-state probability of BKCa channels. This agent also resulted in a rightward shift in voltage dependence of the activation curve of BKCa channels; however, neither the gating charge of the curve nor single-channel conductance of the channel was changed. There was an evident lengthening of the mean closed time of BKCa channels in the presence of GAL-021, with no change in mean open time. The GAL-021 addition also suppressed M-type K+ current with an effective IC50 of 3.75 μM; however, its presence did not alter the amplitude of erg-mediated K+ current, or mildly suppressed delayed-rectifier K+ current. GAL-021 at a concentration of 30 μM could also suppress hyperpolarization-activated cationic current. In HEK293T cells expressing α-hSlo, the addition of GAL-021 was also able to suppress the BKCa-channel open probabilities, and GAL-021-mediated suppression of BKCa-channel activity was attenuated by further addition of BMS-191011. Collectively, the GAL-021 effects presented herein do not exclusively act on BKCa channels and these modifications on ionic currents exert significant influence on the functional activities of electrically excitable cells occurring in vivo.

Response to coincident inputs in electrically coupled primary afferents is heterogeneous and is enhanced by H-current (IH) modulation

Electrical synapses represent a widespread modality of interneuronal communication in the mammalian brain. These contacts, by lowering the effectiveness of random or temporally uncorrelated inputs, endow circuits of coupled neurons with the ability to selectively respond to simultaneous depolarizations. This mechanism may support coincidence detection, a property involved in sensory perception, organization of motor outputs, and improvement signal-to-noise ratio. While the role of electrical coupling is well established, little is known about the contribution of the cellular excitability and its modulations to the susceptibility of groups of neurons to coincident inputs. Here, we obtained dual whole cell patch-clamp recordings of pairs of mesencephalic trigeminal (MesV) neurons in brainstem slices from rats to evaluate coincidence detection and its determinants. MesV neurons are primary afferents involved in the organization of orofacial behaviors whose cell bodies are electrically coupled mainly in pairs through soma-somatic gap junctions. We found that coincidence detection is highly heterogeneous across the population of coupled neurons. Furthermore, combined electrophysiological and modeling approaches reveal that this heterogeneity arises from the diversity of MesV neuron intrinsic excitability. Consistently, increasing these cells’ excitability by upregulating the hyperpolarization-activated cationic current ( IH) triggered by cGMP results in a dramatic enhancement of the susceptibility of coupled neurons to coincident inputs. In conclusion, the ability of coupled neurons to detect coincident inputs is critically shaped by their intrinsic electrophysiological properties, emphasizing the relevance of neuronal excitability for the many functional operations supported by electrical transmission in mammals. NEW & NOTEWORTHY We show that the susceptibility of pairs of coupled mesencephalic trigeminal (MesV) neurons to coincident inputs is highly heterogenous and depends on the interaction between electrical coupling and neuronal excitability. Additionally, upregulating the hyperpolarization-activated cationic current ( IH) by cGMP results in a dramatic increase of this susceptibility. The IH and electrical synapses have been shown to coexist in many neuronal populations, suggesting that modulation of this conductance could represent a common strategy to regulate circuit operation supported by electrical coupling.

Capsaicin causes robust reduction in glycinergic transmission to rat hypoglossal motor neurons via a TRPV1-independent mechanism

The effect of capsaicin on glycinergic synaptic transmission to juvenile rat hypoglossal motor neurons in acute brainstem slices was evaluated in the presence of TTX. Capsaicin caused a robust decrease in miniature IPSC frequency, amplitude, and half-width, showing that this effect is independent of action potential generation. In the presence of capsazepine, a classic TRPV1 antagonist, capsaicin was still able to reduce spontaneous inhibitory postsynaptic current (IPSC) amplitude and frequency. We further investigated whether the effect of capsaicin on glycinergic transmission to hypoglossal motor neurons is pre- or postsynaptic in nature by recording pairs of evoked IPSCs. Interestingly, capsaicin also reduced evoked IPSC amplitude without affecting paired-pulse ratio, indicating a postsynaptic mechanism of action. Significant reduction was also observed in evoked IPSC half-width, rise time, and decay tau. We also show that capsaicin does not have any effect on either transient (It) or sustained (Is) potassium currents. Finally, we also show that the hyperpolarization-activated cationic current (Ih) also remains unchanged after capsaicin application. NEW & NOTEWORTHY Capsaicin reduces the amplitude of quantal and evoked glycinergic inhibitory neurotransmission to brainstem motor neurons without altering activity-dependent transmitter release. This effect of capsaicin is not due to activation of TRPV1 receptors, as it is not blocked by capsazepine, a TRPV1 receptor antagonist. Capsaicin does not alter voltage-dependent potassium current or the hyperpolarization-activated cationic current in brainstem motor neurons.

Biophysical mechanisms in the mammalian respiratory oscillator re-examined with a new data-driven computational model

An autorhythmic population of excitatory neurons in the brainstem pre-Bötzinger complex is a critical component of the mammalian respiratory oscillator. Two intrinsic neuronal biophysical mechanisms—a persistent sodium current (INaP) and a calcium-activated non-selective cationic current (ICAN)—were proposed to individually or in combination generate cellular- and circuit-level oscillations, but their roles are debated without resolution. We re-examined these roles in a model of a synaptically connected population of excitatory neurons with ICAN and INaP. This model robustly reproduces experimental data showing that rhythm generation can be independent of ICAN activation, which determines population activity amplitude. This occurs when ICAN is primarily activated by neuronal calcium fluxes driven by synaptic mechanisms. Rhythm depends critically on INaP in a subpopulation forming the rhythmogenic kernel. The model explains how the rhythm and amplitude of respiratory oscillations involve distinct biophysical mechanisms.

Biophysical mechanisms in the mammalian respiratory oscillator re-examined with a new data-driven computational model

An autorhythmic population of excitatory neurons in the brainstem pre-Bötzinger complex is a critical component of the mammalian respiratory oscillator. Two intrinsic neuronal biophysical mechanisms—a persistent sodium current (INaP) and a calcium-activated non-selective cationic current (ICAN)—were proposed to individually or in combination generate cellular-and circuit-level oscillations, but their roles are debated without resolution. We re-examined these roles with a new computational model of an excitatory population with randomly distributed INaP and ICAN conductances and synaptic connections. This model robustly reproduces experimental data showing contrary to previous hypotheses, rhythm generation is independent of ICAN activation, which instead determines population activity amplitude. The novel insight is that this occurs when ICAN is primarily activated by neuronal calcium fluxes driven by synaptic mechanisms. Rhythm depends critically on INaP in a subpopulation forming the rhythmogenic kernel. The model explains how the rhythm and amplitude of respiratory oscillations involve distinct biophysical mechanisms.