Marcela Miranda-Morales

Transient receptor potential ankyrin-1 has a major role in mediating visceral pain in mice

The excitatory ion channel transient receptor potential ankyrin-1 (TRPA1) is prominently expressed by primary afferent neurons and is a mediator of inflammatory pain. Inflammatory agents can directly activate [e.g., hydroxynonenal (HNE), prostaglandin metabolites] or indirectly sensitize [e.g., agonists of protease-activated receptor (PAR(2))] TRPA1 to induce somatic pain and hyperalgesia. However, the contribution of TRPA1 to visceral pain is unknown. We investigated the role of TRPA1 in visceral hyperalgesia by measuring abdominal visceromotor responses (VMR) to colorectal distention (CRD) after intracolonic administration of TRPA1 agonists [mustard oil (MO), HNE], sensitizing agents [PAR(2) activating peptide (PAR(2)-AP)], and the inflammatory agent trinitrobenzene sulfonic acid (TNBS) in trpa1(+/+) and trpa1(-/-) mice. Sensory neurons innervating the colon, identified by retrograde tracing, coexpressed immunoreactive TRPA1, calcitonin gene-related peptide, and substance P, expressed TRPA1 mRNA and responded to MO with depolarizing currents. Intracolonic MO and HNE increased VMR to CRD and induced immunoreactive c-fos in spinal neurons in trpa1+/+ but not in trpa1(-/-) mice. Intracolonic PAR(2)-AP induced mechanical hyperalgesia in trpa1+/+ but not in trpa1(-/-) mice. TNBS-induced colitis increased in VMR to CRD and induced c-fos in spinal neurons in trpa1(+/+) but not in trpa1(-/-) mice. Thus TRPA1 is expressed by colonic primary afferent neurons. Direct activation of TRPA1 causes visceral hyperalgesia, and TRPA1 mediates PAR(2)-induced hyperalgesia. TRPA1 deletion markedly reduces colitis-induced mechanical hyperalgesia in the colon. Our results suggest that TRPA1 has a major role in visceral nociception and may be a therapeutic target for colonic inflammatory pain.

BACTERIAL CELL PRODUCTS SIGNAL TO MOUSE COLONIC NOCICEPTIVE DORSAL ROOT GANGLIA NEURONS

This study examined whether bacterial cell products that might gain access to the intestinal interstitium could activate mouse colonic nociceptive dorsal root ganglion (DRG) neurons using molecular and electrophysiological recording techniques. Colonic projecting neurons were identified by using the retrograde tracer fast blue and Toll-like receptor (TLR) 1, 2, 3, 4, 5, 6, 9, adapter proteins Md-1 and Md-2, and MYD88 mRNA expression was observed in laser-captured fast blue-labeled neurons. Ultrapure LPS 1 microg/ml phosphorylated p65 NF-kappaB subunits increased transcript for TNF-alpha and IL-1beta and stimulated secretion of TNF-alpha from acutely dissociated DRG neurons. In current-clamp recordings from colonic DRG neurons, chronic incubation (24 h) of ultrapure LPS significantly increased neuronal excitability. In acute studies, 3-min superfusion of standard-grade LPS (3-30 microg/ml) reduced the rheobase by up to 40% and doubled action potential discharge rate. The LPS effects were not significantly different in TLR4 knockout mice compared with wild-type mice. In contrast to standard-grade LPS, acute application of ultrapure LPS did not increase neuronal excitability in whole cell recordings or afferent nerve recordings from colonic mesenteric nerves. However, acute application of bacterial lysate (Escherichia coli NLM28) increased action potential discharge over 60% compared with control medium. Moreover, lysate also activated afferent discharge from colonic mesenteric nerves, and this was significantly increased in chronic dextran sulfate sodium salt mice. These data demonstrate that bacterial cell products can directly activate colonic DRG neurons leading to production of inflammatory cytokines by neurons and increased excitability. Standard-grade LPS may also have actions independent of TLR signaling.

Cross-inhibitory interactions between GABAA and P2X channels in myenteric neurones

Inhibitory interactions between GABAA[induced by γ‐aminobutyric acid (GABA)] and P2X [activated by adenosine 5′‐triphosphate (ATP)] receptors of myenteric neurones from the guinea pig small intestine were characterized using whole‐cell recordings. Currents induced by GABA (IGABA) or ATP (IATP) were inhibited by picrotoxin or pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid, respectively. Currents induced by GABA + ATP (IGABA+ATP) were only as large as the current induced by the most effective transmitter, revealing current occlusion. This occlusion requires maximal activation of at least one of these receptors. Sequential applications of neurotransmitters, and kinetic and pharmacological properties of IGABA+ATP indicate that they are carried through both GABAA and P2X channels. ATP did not affect IGABA in neurones: (i) in which P2X channels were not present; (ii) after inhibiting P2X channels with Ca2+ (iii) in the presence of pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid, a P2X receptor antagonist; (iv) after P2X receptor desensitization or (v) at IATP reversal potential. Similarly, GABA did not affect P2X‐mediated currents in neurones: (i) in which GABAA channels were not present; (ii) in the presence of picrotoxin, a GABAA channel blocker; (iii) after GABAA receptor desensitization or (iv) at the IGABA reversal potential. Current occlusion occurred as fast as current activation and it was still present in the absence of Ca2+, at 11 °C, after adding to the pipette solution a cocktail of protein kinase inhibitors (staurosporine + genistein + K‐252a), after substituting the GTP in the pipette with GDP‐β‐S and after treating the cells with N‐ethylmaleimide. Taken together, all of these results are consistent with a model of cross‐inhibition between GABAA and P2X.

Locus Ceruleus Norepinephrine Release: A Central Regulator of CNS Spatio-Temporal Activation?

Norepinephrine (NE) is synthesized in the Locus Coeruleus (LC) of the brainstem, from where it is released by axonal varicosities throughout the brain via volume transmission. A wealth of data from clinics and from animal models indicates that this catecholamine coordinates the activity of the central nervous system (CNS) and of the whole organism by modulating cell function in a vast number of brain areas in a coordinated manner. The ubiquity of NE receptors, the daunting number of cerebral areas regulated by the catecholamine, as well as the variety of cellular effects and of their timescales have contributed so far to defeat the attempts to integrate central adrenergic function into a unitary and coherent framework. Since three main families of NE receptors are represented—in order of decreasing affinity for the catecholamine—by: α2 adrenoceptors (α2Rs, high affinity), α1 adrenoceptors (α1Rs, intermediate affinity), and β adrenoceptors (βRs, low affinity), on a pharmacological basis, and on the ground of recent studies on cellular and systemic central noradrenergic effects, we propose that an increase in LC tonic activity promotes the emergence of four global states covering the whole spectrum of brain activation: (1) sleep: virtual absence of NE, (2) quiet wake: activation of α2Rs, (3) active wake/physiological stress: activation of α2- and α1-Rs, (4) distress: activation of α2-, α1-, and β-Rs. We postulate that excess intensity and/or duration of states (3) and (4) may lead to maladaptive plasticity, causing—in turn—a variety of neuropsychiatric illnesses including depression, schizophrenic psychoses, anxiety disorders, and attention deficit. The interplay between tonic and phasic LC activity identified in the LC in relationship with behavioral response is of critical importance in defining the short- and long-term biological mechanisms associated with the basic states postulated for the CNS. While the model has the potential to explain a large number of experimental and clinical findings, a major challenge will be to adapt this hypothesis to integrate the role of other neurotransmitters released during stress in a centralized fashion, like serotonin, acetylcholine, and histamine, as well as those released in a non-centralized fashion, like purines and cytokines.

Axon reflexes evoked by transient receptor potential vanilloid 1 activation are mediated by tetrodotoxin-resistant voltage-gated Na+ channels in intestinal afferent nerves.

Capsaicin-sensitive nerves mediate axon vasodilator reflexes in the intestine, but the ion channels underlying action potential (AP) propagation are poorly understood. To examine the role of voltage-gated Na+ channels underlying these reflexes, we measured vasomotor and electrophysiological responses elicited by capsaicin in guinea pig and mouse dorsal root ganglia (DRG) neurons, submucosal arterioles, and mesenteric arteries in vitro. Transient receptor potential vanilloid 1 (TRPV1) agonists dilated guinea pig ileal submucosal arterioles and were blocked by capsazepine and ruthenium red. In double-chamber baths, capsaicin-evoked activation of TRPV1 on proximal perivascular nerves in the left chamber evoked dilations of the distal segment of the submucosal arteriole in the right chamber. Dilations were tetrodotoxin (TTX) (1 μM)-resistant, but reducing extracellular Na+ (10% solution) or applying the Nav 1.8 antagonist A-803467 [5-(4-chlorophenyl-N-(3,5-dimethoxyphenyl)furan-2-carboxamide] (1 μM) in the proximal chamber blocked capsaicin-evoked dilations in the distal chamber (88%; P = 0.01 and 75% and P < 0.02, respectively). In mouse mesenteric arteries, electrical field stimulation and capsaicin (2 μM) evoked dilations that were also TTX-resistant. In perforated patch-clamp recordings, APs in mouse and guinea pig capsaicin-sensitive DRG neurons were TTX-resistant but blocked by 10% extracellular Na+. When capsaicin-evoked AP conduction was studied in in vitro ileal multiunit afferent nerve preparations, capsaicin responses were elicited in the presence of TTX, whereas distention-evoked responses were almost completely blocked by TTX. Together, these data provide evidence for TTX-resistant AP conduction in extrinsic sensory neurons that innervate guinea pig and mouse intestine and suggest this neural propagation is sufficient to mediate axon reflexes in the intestine.

Functional interactions between nicotinic and P2X receptors in celiac ganglia neurons

Here we characterized the cross-inhibitory interactions between nicotinic and P2X receptors of celiac neurons from the guinea pig by recording whole-cell currents induced by 1mM ACh (I(ACh)), 1mM ATP (I(ATP)) and by the simultaneous application of both agonists (I(ACh)(+ATP)). I(ACh) and I(ATP) were inhibited by hexamethonium (nicotinic channel blocker) and PPADS (P2X receptor antagonist), respectively. The amplitude of I(ACh)(+ATP) was equal to the current induced by the most effective agonist, indicating a current occlusion. Various observations indicate that I(ACh)(+ATP) is carried out through both nicotinic (nACh) and P2X channels: i) I(ACh)(+ATP) desensitisation kinetics were in between that of I(ACh) and I(ATP); ii) application of ATP+ACh, decreased I(ACh) and I(ATP), whereas no cross-desensitisation was observed between nACh and P2X receptors; iii) ATP did not affect I(ACh) in the presence of PPADS or after P2X receptor desensitisation; and iv) ACh did not affect I(ATP) when nACh channels were blocked with hexamethonium or after nACh receptor desensitisation. Current occlusion is not mediated by activation of metabotropic receptors as it is: i) voltage dependent (was not observed at + 5 mV); ii) present at low temperature (10 degrees C) and after inhibition of protein kinase activity (with staurosporine); and iii) absent at 30 microM ATP and 30 microM ACh (concentrations that should activate metabotropic receptors). In conclusion, current occlusion described here is similar to the previously reported myenteric neurons. This occlusion is likely the result of allosteric interactions between these receptors.

Interleukin 6 trans-signaling regulates basal synaptic transmission and sensitivity to pentylenetetrazole-induced seizures in mice

The pro‐inflammatory cytokine interleukin 6 (IL‐6) interacts with the central nervous system in a largely unknown manner. We used a genetically modified mouse strain (GFAP‐sgp130Fc, TG) and wild type (WT) mice to determine whether IL‐6 trans‐signaling contributes to basal properties of synaptic transmission. Postsynaptic currents (PSCs) were studied by patch‐clamp recording in cortical layer 5 of a mouse prefrontal cortex brain slice preparation. TG and WT animals displayed differences mainly (but not exclusively) in excitatory synaptic responses. The frequency of both action potential‐independent (miniature) and action potential‐dependent (spontaneous) excitatory PSCs (EPSCs) were higher for TG vs. WT animals. No differences were observed in inhibitory miniature, spontaneous, or tonic inhibitory currents. The pair pulse ratio (PPR) of electrically evoked inhibitory as well as of excitatory PSCs were also larger in TG animals vs. WT ones, while no changes were detected in electrically evoked excitatory‐inhibitory synaptic ratio (eEPSC/eIPSC), nor in the ratio between the amino‐propionic acid receptor (AMPAR)‐mediated and N‐methyl D aspartate‐R (NMDAR)‐mediated components of eEPSCs (IAMPA/INMDA). Evoked IPSC rise times were shorter for TG vs. WT animals. We also compared the sensitivity of TG and WT animals to pentylenetetrazole (PTZ)‐induced seizures. We found that TG animals were more sensitive to PTZ injections, as they displayed longer and more severe seizures. We conclude that the absence of basal IL‐6 trans‐signaling contributes to increase the basal excitability of the central nervous system, at the system level as well at the synaptic level, at least in the prefrontal cortex.

Heteromeric channels with different phenotypes are generated when coexpressing two P2X2 receptor isoforms

To investigate if channels with different stoichiometry are formed from P2X2 receptor isoforms during their heterologous co-expression. The two-electrode voltage-clamp technique was used to measured ATP induced currents in Xenopus laevis oocytes. We used a mutant (P2X2-2bm) because its ATP sensitivity is lower than P2X2-2b receptors, which highlights the differences with its splice variant P2X2-1a.Currents through homomeric channels had significantly different Hill coefficients. P2XR are trimeric proteins with three agonist binding sites; therefore, only two homomeric and two heteromeric stoichiometries are possible when both P2X2 isoforms are coexpressed, the heteromeric channels might be formed by: i) 2(P2X2-1a)+1(P2X2-2bm); or ii) 1(P2X2-1a)+2(P2X2-2bm). Because P2X2 channels open when two binding sites are occupied, these stoichiometries are expected to have different ATP sensitivities. Thus, co-expressing both P2X2 isoforms, two oocyte populations were distinguished based on their sensitivity to ATP and Hill coefficients. For the first population (P2X2-1a like), the ATP EC50 and the Hill coefficient were not different than those of homomeric P2X2-1a channels similarly, for the second population (P2X2-2bm like), these variables were also not different than for those of homomeric P2X2-2bm channels. Various findings indicate that homomeric channel expression is not responsible for such differences. Our observations indicate that two heteromeric channels can be assembled from two P2X2 receptor isoforms. Our data support a current model, according to which, ATP activation of two subunits can open P2X2 channel. However, PPADS appears to bind to all three subunits in order to inhibit ATP effects on P2X2 receptors.

Cholinergic signaling plasticity maintains viscerosensory responses during Aspiculuris tetraptera infection in mice small intestine

Intestinal parasites alter gastrointestinal (GI) functions like the cholinergic function. Aspiculuris tetraptera is a pinworm frequently observed in laboratory facilities, which infests the mice cecum and proximal colon. However, little is known about the impact of this infection on the GI sensitivity. Here, we investigated possible changes in spontaneous mesenteric nerve activity and on the mechanosensitivity function of worm-free regions of naturally infected mice with A. tetraptera. Infection increased the basal firing of mesenteric afferent nerves in jejunum. Our findings indicate that nicotinic but not muscarinic receptors, similarly affect spontaneous nerve firing in control and infected animals; these axons are mainly vagal. No difference between groups was observed on spontaneous activity after nicotinic receptor inhibition. However, and contrary to the control group, during infection, the muscarinic signaling was shown to be elevated during mechanosensory experiments. In conclusion, we showed for the first time that alterations induced by infection of the basal afferent activity were independent of the cholinergic function but changes in mechanosensitivity were mediated by muscarinic, but not nicotinic, receptors and specifically by high threshold nerve fibers (activated above 20mmHg), known to play a role in nociception. These plastic changes within the muscarinic signaling would function as a compensatory mechanism to maintain a full mechanosensory response and the excitability of nociceptors during infection. These changes indicate that pinworm colonic infection can target other tissues away from the colon.