The TRPV1 channel in rodents is a major target for antinociceptive effect of the probiotic Lactobacillus reuteri DSM 17938.

Certain probiotic bacteria have been shown to reduce distension-dependent gut pain, but the mechanisms involved remain obscure. Live luminal Lactobacillus reuteri (DSM 17938) and its conditioned medium dose dependently reduced jejunal spinal nerve firing evoked by distension or capsaicin, and 80% of this response was blocked by a specific TRPV1 channel antagonist or in TRPV1 knockout mice. The specificity of DSM action on TRPV1 was further confirmed by its inhibition of capsaicin-induced intracellular calcium increases in dorsal root ganglion neurons. Another lactobacillus with ability to reduce gut pain did not modify this response. Prior feeding of rats with DSM inhibited the bradycardia induced by painful gastric distension. These results offer a system for the screening of new and improved candidate bacteria that may be useful as novel therapeutic adjuncts in gut pain. Certain bacteria exert visceral antinociceptive activity, but the mechanisms involved are not determined. Lactobacillus reuteri DSM 17938 was examined since it may be antinociceptive in children. Since transient receptor potential vanilloid 1 (TRPV1) channel activity may mediate nociceptive signals, we hypothesized that TRPV1 current is inhibited by DSM. We tested this by examining the effect of DSM on the firing frequency of spinal nerve fibres in murine jejunal mesenteric nerve bundles following serosal application of capsaicin. We also measured the effects of DSM on capsaicin-evoked increase in intracellular Ca(2+) or ionic current in dorsal root ganglion (DRG) neurons. Furthermore, we tested the in vivo antinociceptive effects of oral DSM on gastric distension in rats. Live DSM reduced the response of capsaicin- and distension-evoked firing of spinal nerve action potentials (238 ± 27.5% vs. 129 ± 17%). DSM also reduced the capsaicin-evoked TRPV1 ionic current in DRG neuronal primary culture from 83 ± 11% to 41 ± 8% of the initial response to capsaicin only. Another lactobacillus (Lactobacillus rhamnosus JB-1) with known visceral anti-nociceptive activity did not have these effects. DSM also inhibited capsaicin-evoked Ca(2+) increase in DRG neurons; an increase in Ca(2+) fluorescence intensity ratio of 2.36 ± 0.31 evoked by capsaicin was reduced to 1.25 ± 0.04. DSM releasable products (conditioned medium) mimicked DSM inhibition of capsaicin-evoked excitability. The TRPV1 antagonist 6-iodonordihydrocapsaicin or the use of TRPV1 knock-out mice revealed that TRPV1 channels mediate about 80% of the inhibitory effect of DSM on mesenteric nerve response to high intensity gut distension. Finally, feeding with DSM inhibited perception in rats of painful gastric distension. Our results identify a specific target channel for a probiotic with potential therapeutic properties.

The gut-brain axis rewired: adding a functional vagal nicotinic "sensory synapse".

It is generally accepted that intestinal sensory vagal fibers are primary afferent, responding nonsynaptically to luminal stimuli. The gut also contains intrinsic primary afferent neurons (IPANs) that respond to luminal stimuli. A psychoactive Lactobacillus rhamnosus (JB-1) that affects brain function excites both vagal fibers and IPANs. We wondered whether, contrary to its primary afferent designation, the sensory vagus response to JB-1 might depend on IPAN to vagal fiber synaptic transmission. We recorded ex vivo single- and multiunit afferent action potentials from mesenteric nerves supplying mouse jejunal segments. Intramural synaptic blockade with Ca(2+) channel blockers reduced constitutive or JB-1-evoked vagal sensory discharge. Firing of 60% of spontaneously active units was reduced by synaptic blockade. Synaptic or nicotinic receptor blockade reduced firing in 60% of vagal sensory units that were stimulated by luminal JB-1. In control experiments, increasing or decreasing IPAN excitability, respectively increased or decreased nerve firing that was abolished by synaptic blockade or vagotomy. We conclude that >50% of vagal afferents function as interneurons for stimulation by JB-1, receiving input from an intramural functional "sensory synapse." This was supported by myenteric plexus nicotinic receptor immunohistochemistry. These data offer a novel therapeutic target to modify pathological gut-brain axis activity.-Perez-Burgos, A., Mao, Y.-K., Bienenstock, J., Kunze, W. A. The gut-brain axis rewired: adding a functional vagal nicotinic "sensory synapse."

Psychoactive bacteria Lactobacillus rhamnosus (JB-1) elicits rapid frequency facilitation in vagal afferents.

Mounting evidence supports the influence of the gut microbiome on the local enteric nervous system and its effects on brain chemistry and relevant behavior. Vagal afferents are involved in some of these effects. We previously showed that ingestion of the probiotic bacterium Lactobacillus rhamnosus (JB-1) caused extensive neurochemical changes in the brain and behavior that were abrogated by prior vagotomy. Because information can be transmitted to the brain via primary afferents encoded as neuronal spike trains, our goal was to record those induced by JB-1 in vagal afferents in the mesenteric nerve bundle and thus determine the nature of the signals sent to the brain. Male Swiss Webster mice jejunal segments were cannulated ex vivo, and serosal and luminal compartments were perfused separately. Bacteria were added intraluminally. We found no evidence for translocation of labeled bacteria across the epithelium during the experiment. We recorded extracellular multi- and single-unit neuronal activity with glass suction pipettes. Within minutes of application, JB-1 increased the constitutive single- and multiunit firing rate of the mesenteric nerve bundle, but Lactobacillus salivarius (a negative control) or media alone were ineffective. JB-1 significantly augmented multiunit discharge responses to an intraluminal distension pressure of 31 hPa. Prior subdiaphragmatic vagotomy abolished all of the JB-1-evoked effects. This detailed exploration of the neuronal spike firing that encodes behavioral signaling to the brain may be useful to identify effective psychoactive bacteria and thereby offer an alternative new perspective in the field of psychiatry and comorbid conditions.

Upregulation of D2-class signaling in dopamine-denervated striatum is in part mediated by D3 receptors acting on Ca V 2.1 channels via PIP2 depletion.

The loss of dopaminergic neurons in the substantia nigra compacta followed by striatal dopamine depletion is a hallmark of Parkinson's disease. After dopamine depletion, dopaminergic D(2) receptor (D(2)R)-class supersensitivity develops in striatal neurons. The supersensitivity results in an enhanced modulation of Ca(2+) currents by D(2)R-class receptors. However, the relative contribution of D(2)R, D(3)R, and D(4)R types to the supersensitivity, as well as the mechanisms involved, have not been elucidated. In this study, whole cell voltage-clamp recordings were performed to study Ca(2+) current modulation in acutely dissociated striatal neurons obtained from rodents with unilateral 6-hydroxydopamine lesions in the substantia nigra compacta. Selective antagonists for D(2)R, D(3)R, and D(4)R types were used to identify whether the modulation by one of these receptors experiences a selective change after dopaminergic denervation. It was found that D(3)R-mediated modulation was particularly enhanced. Increased modulation targeted Ca(V)2.1 (P/Q) Ca(2+) channels via the depletion of phosphatidylinositol 4,5-bisphosphate, an intracellular signaling cascade hard to detect in control neurons and hypothesized as being amplified by dopamine depletion. An imbalance in the striatal expression of D(3)R and its splice variant, D(3)nf, accompanied enhanced D(3)R activity. Because Ca(V)2.1 Ca(2+) channels mediate synaptic GABA release from the terminals of striatal neurons, reinforcement of their inhibition by D(3)R may explain in part the profound decrease in synaptic strength in the connections among striatal projection neurons observed in the dopamine-depleted striatum.

Capsaicin produces antidepressant-like effects in the forced swimming test and enhances the response of a sub-effective dose of amitriptyline in rats.

Transient receptor potential vanilloid 1 (TRPV1) channels have been implicated in depression and anxiety. The aim of this study was to evaluate the antidepressant-like properties of the TRPV1 agonist capsaicin using the forced swimming test (FST) in rats. Capsaicin (0.001-0.25 mg/kg, i.p.) produced a reduction of immobility in the FST. A maximally effective dose of the tricyclic antidepressant amitriptyline (12 mg/kg) reduced immobility as well. Notably, doses of capsaicin (1 pg/kg, 1 ng/kg, and 0.001 mg/kg) that were ineffective when applied alone produced a significant decrease in immobility when combined with a subthreshold dose of amitriptyline (5 mg/kg). Rats treated with capsaicin (0.01 mg/kg) + amitriptyline (5 mg/kg) displayed less immobility than those treated with a maximally effective dose of amitriptyline. The non-pungent TRPV1 channel agonist palvanil (0.05-0.1 mg/kg, i.p.) also decreased immobility in the FST. Capsaicin (0.05 mg/kg) did not affect general locomotion in the open field test, nor performance in the elevated plus maze, or skeletal muscle contraction strength measured in vitro after the FST (at 0.25 mg/kg). Altogether, our results imply that low doses of capsaicin produce antidepressant-like effects, and enhance the effect of a subthreshold dose of amitriptyline in the FST.

Modulation of direct pathway striatal projection neurons by muscarinic M4-type receptors.

Models of basal ganglia (BG) function posit a dynamic balance between two classes of striatal projection neurons (SPNs): direct pathway neurons (dSPNs) that facilitate movements, and indirect pathway neurons (iSPNs) that repress movement execution. Two main modulatory transmitters regulate the output of these neurons: dopamine (DA) and acetylcholine (ACh). dSPNs express D1-type DA, M1-and M4-type ACh receptors, while iSPNs express D2-type DA and M1-type ACh receptors. Actions of M1-, D1-, and D2-receptors have been extensively reported, but we still ignore most actions of muscarinic M4-type receptors. Here, we used whole-cell recordings in acutely dissociated neurons, pharmacological tools such as mamba-toxins, and BAC D(1 or 2)-eGFP transgenic mice to show that activation of M4-type receptors with bath applied muscarine enhances Ca(2+)-currents through CaV1-channels in dSPNs and not in iSPNs. This action increases excitability of dSPNs after both direct current injection and synaptically driven stimulation. The increases in Ca(2+)-current and excitability were blocked specifically by mamba toxin-3, suggesting mediation via M4-type receptors. M4-receptor activation also increased network activity of dSPNs but not of iSPNs as seen with calcium-imaging techniques. Moreover, actions of D1-type and M4-type receptors may add to produce a larger enhancement of excitability of dSPNs or, paradoxically, oppose each other depending on the order of their activation. Possible implications of these findings are discussed.

Circadian modulation of the Cl(-) equilibrium potential in the rat suprachiasmatic nuclei.

The suprachiasmatic nuclei (SCN) constitute a circadian clock in mammals, where γ-amino-butyric acid (GABA) neurotransmission prevails and participates in different aspects of circadian regulation. Evidence suggests that GABA has an excitatory function in the SCN in addition to its typical inhibitory role. To examine this possibility further, we determined the equilibrium potential of GABAergic postsynaptic currents (E(GABA)) at different times of the day and in different regions of the SCN, using either perforated or whole cell patch clamp. Our results indicate that during the day most neurons in the dorsal SCN have an E(GABA) close to -30 mV while in the ventral SCN they have an E(GABA) close to -60 mV; this difference reverses during the night, in the dorsal SCN neurons have an E(GABA) of -60 mV and in the ventral SCN they have an E(GABA) of -30 mV. The depolarized equilibrium potential can be attributed to the activity of the Na(+)-K(+)-2Cl(-) (NKCC) cotransporter since the equilibrium potential becomes more negative following addition of the NKCC blocker bumetanide. Our results suggest an excitatory role for GABA in the SCN and further indicate both time (day versus night) and regional (dorsal versus ventral) modulation of E(GABA) in the SCN.

A reconfiguration of CaV2 Ca2+ channel current and its dopaminergic D2 modulation in developing neostriatal neurons.

The modulatory effect of D(2) dopamine receptor activation on calcium currents was studied in neostriatal projection neurons at two stages of rat development: postnatal day (PD)14 and PD40. D(2)-class receptor agonists reduced whole cell calcium currents by about 35% at both stages, and this effect was blocked by the D(2) receptor antagonist sulpiride. Nitrendipine partially occluded this modulation at both stages, indicating that modulation of Ca(V)1 channels was present throughout this developmental interval. Nevertheless, modulation of Ca(V)1 channels was significantly larger in PD40 neurons. omega-Conotoxin GVIA occluded most of the Ca(2+) current modulation in PD14 neurons. However, this occlusion was greatly decreased in PD40 neurons. omega-Agatoxin TK occluded a great part of the modulation in PD40 neurons but had a negligible effect in PD14 neurons. The data indicate that dopaminergic D(2)-mediated modulation undergoes a change in target during development: from Ca(V)2.2 to Ca(V)2.1 Ca(2+) channels. This change occurred while Ca(V)2.2 channels were being down-regulated and Ca(V)2.1 channels were being up-regulated. Presynaptic modulation mediated by D(2) receptors reflected these changes; Ca(V)2.2 type channels were used for release in young animals but very little in mature animals, suggesting that changes took place simultaneously at the somatodendritic and the synaptic membranes.

5-HT1A, 5-HT2, and GABAB receptors interact to modulate neurotransmitter release probability in layer 2/3 somatosensory rat cortex as evaluated by the paired pulse protocol.

Activation of gamma-aminobutyric acid B (GABA(B)) and 5-hydroxytryptamine (5-HT) receptors produces presynaptic inhibition at glutamatergic terminals in the rat neocortex. To evaluate interactions between these metabotropic receptors, field potentials were recorded in layer 2/3 of somatosensory cortex. In addition, the paired pulse (PP) protocol was used to measure changes in the ratio of the second/first extracellular synaptic potentials (S(2)/S(1) ratio) as an index of glutamate release probability in the area. Lowering extracellular [Ca(2+)](o) to 0.5 mM, increased the S(2)/S(1) ratio by 318 +/- 134%. 5-HT (1 microM) increased the S(2)/S(1) ratio by 61 +/- 15%. In presence of the GABA(A) antagonist bicuculline (10 microM), 5-HT increased the S(2)/S(1) ratio by 98 +/- 15%. This effect did not desensitize after two consecutive applications of the amine, and was dose dependent in the concentration range between 0.03-1 microM (EC(50) = 2.36 x 10(-7) mol/L). The increase of S(2)/S(1) ratio induced by 5-HT (1 microM) was blocked reversibly by the 5-HT(1A) antagonist NAN-190 (10-30 microM), but was unaffected by the selective GABA(B) antagonist CGP 52432 (1 microM). The action of 5-HT was mimicked by the 5-HT(1A/7) agonist 8OH-DPAT (10 microM), increasing the S(2)/S(1) ratio by 84 +/- 2%, a response that was unaffected by the 5-HT(2/7) antagonist ritanserin (2 microM). The 5-HT(1B) agonist CP93129 (10 microM) had no effect. The GABA(B) agonist baclofen (1 microM) increased the S(2)/S(1) ratio up to 308 +/- 33%, which is similar to that produced by low [Ca(2+)](o). When the effect of baclofen was maximal, application of 5-HT (1 microM) reversed the S(2)/S(1) ratio back to 78 +/- 27%, a result that was blocked by the 5-HT(2/7) antagonist ritanserin (100 nM). Notably, the interaction between the GABA(B) agonist and 5-HT was order dependent, because enhancement of the S(2)/S(1) ratio elicited by baclofen was not inhibited if 5-HT was applied first. These results suggest a complex interaction between GABA(B), 5-HT(1A), and 5-HT(2) receptors in layer 2/3 of rat somatosensory cortex. Activation of GABA(B) receptors induces PP facilitation (inhibits glutamate release) more efficiently than does activation of 5-HT(1A) receptors. When the effect of GABA(B) receptor activation is maximal, however, the influence of 5-HT changes to the opposite direction, inhibiting PP facilitation (increasing glutamate release) through activation of 5-HT(2) receptors.