Cholinergic Inhibition Research Articles

Inhibiting medial septal cholinergic neurons with DREADD alleviated anxiety-like behaviors in mice

Cholinergic neurons in the medial septum (MS) participate in a variety of cognitive and emotional behaviors. Some studies but not others show that lesions or inhibition of the MS reduce anxiety-like behaviors and locomotive exploration in rats. However, these conclusions come from manipulations that are either irreversible or non-specific to cholinergic neurons, casting doubt on their validity. With DREADD (designer receptors exclusively activated by designer drugs), we temporarily and reversibly inhibited cholinergic neurons in the MS. We observed consistent anxiolytic effects of MS cholinergic inhibition in the novelty-suppressed feeding test, the marble burying test and the elevated plus-maze test, as well as increased exploratory activities in the open field test. These findings confirm an excitatory role of the MS cholinergic neurons in the control of innate anxiety, and reconcile conflicting findings from previous studies using irreversible lesions or non-specific inhibition.

AB287. SPR-14 High fat diet leads to impaired mitochondrial respiration, increased hydrogen peroxide emission and greater nerve-mediated purinergic contraction in mouse bladder

ObjectiveChronic high fat diet (HFD) is associated with the development of metabolic syndrome and type 2 diabetes, known risk factors for lower urinary tract symptoms, overactive bladder, and incontinence. HFD induced increases in oxidative stress and impaired mitochondrial integrity is a possible mechanism underlying this pathology. Our objectives were to determine bladder function following long-term HFD and characterize mitochondrial oxidant emission and respiratory function. We hypothesized that chronic HFD would lead to increased cholinergic mediated contraction and impaired mitochondrial respiration.MethodsMale C57BL/6J mice were maintained on a control diet (CD: 4% fat, 60% carbohydrates, 26% protein) or a HFD (45% fat, 35% carbohydrates, 20% protein) from 6 weeks of age for 20 weeks. A week prior to sacrifice, lower urinary tract function was assessed via void spot assay. Animals were euthanized and bladders and visceral fat depots were collected and weighed. Bladders were denuded of urothelium, cut into 8×2 mm strips and placed in tissue baths. Increasing concentrations of carbachol (cholinergic mediated) contraction and adrenergic relaxation to norepinephrine (NE) were measured. Electric field stimulated (EFS) contractions with and without atropine (cholinergic antagonist) were assessed. A separate group of bladders were denuded, homogenized and mitochondria quantified via citrate synthase activity. High-resolution respirometry measured oxygen consumption of individual electron transport chain complexes. Mitochondrial hydrogen peroxide (H2O2) emission was measured fluorimetrically using Amplex Ultra Red. Data were compared using a student t-test.ResultsHFD mice had a greater number of voids (P<0.005) and a larger void area (P<0.005). Blood glucose was greater in HFD (CD: 85±6.1; HFD: 139±5.9) as were body and overall visceral fat weights (P<0.0005). In contrast, HFD did not impact bladder weight. HFD did not change detrusor cholinergic contraction or adrenergic relaxation. However, detrusor contraction to EFS was markedly increased with HFD in both the presence and absence of atropine. Cholinergic inhibition blunted EFS contraction to a greater extent in CD bladders (CD: 75.7%±2.65; HFD: 42.3%±2.82, P<0.05) indicating that HFD had shifted EFS contraction to greater purinergic release. Citrate synthase activity indicated similar amounts of intact mitochondria. HFD resulted in a decreased respiratory capacity across 4 of 6 mitochondrial complexes investigated (P<0.05). Complex I and Complex II in state 4 conditions were unchanged. Additionally, mitochondrial H2O2 emission was increased in HFD bladders (CD: 11.4 pmols/min/mg ±0.53; HFD: 18.6±0.66, P<0.05).ConclusionsProlonged HFD leads to increased urinary frequency and volume, increased detrusor purinergic EFS contraction and mitochondrial dysfunction including decreased respiratory capacity and increased H2O2 emission. Further investigation of the role of mitochondrial dysfunction and resulting detrusor dysfunction following HFD is merited.Funding Source(s)None

63. Phase dependency of the human primary motor cortex and cholinergic inhibition cancellation during beta tACS

Growing evidence suggests that human motor cortex has a tendency to resonant activity at about 20 Hz, and therefore stimulation near this frequency can synchronize the activity of neuronal populations. However, do interneuronal circuits contribute to such resonance and if so are they selectively entrained? Is the balance between inhibitory and excitatory interneurons maintained throughout? We investigated how different interneuronal circuits react to exogenously driven rhythmic activities using TMS during transcranial alternating current stimulation (tACS) at motor and non-motor resonance frequencies. We tested different TMS interneuronal protocols and triggered TMS pulses at different phases of tACS. The effect of short-latency afferent inhibition was abolished by tACS at 20 Hz regardless of phase of stimulation. tACS phase selectively modulated MEP size during single pulse, GABAAergic short-interval intracortical inhibition (SICI), and glutamatergic intracortical facilitation (ICF). In the case of ICF and SICI this phase effect was more marked during stimulation at 20 Hz. Moreover, the phase dependency of ICF and SICI was relatively balanced so that net effects on cortical excitability were attenuated. The present study provides in vivo evidence linking cortical beta activity to sensorimotor integration, and for dynamic but balanced glutamatergic and GABAAergic effects during beta oscillations in motor cortex.

Phase Dependency of the Human Primary Motor Cortex and Cholinergic Inhibition Cancelation During Beta tACS.

The human motor cortex has a tendency to resonant activity at about 20 Hz so stimulation should more readily entrain neuronal populations at this frequency. We investigated whether and how different interneuronal circuits contribute to such resonance by using transcranial magnetic stimulation (TMS) during transcranial alternating current stimulation (tACS) at motor (20 Hz) and a nonmotor resonance frequency (7 Hz). We tested different TMS interneuronal protocols and triggered TMS pulses at different tACS phases. The effect of cholinergic short-latency afferent inhibition (SAI) was abolished by 20 Hz tACS, linking cortical beta activity to sensorimotor integration. However, this effect occurred regardless of the tACS phase. In contrast, 20 Hz tACS selectively modulated MEP size according to the phase of tACS during single pulse, GABAAergic short-interval intracortical inhibition (SICI) and glutamatergic intracortical facilitation (ICF). For SICI this phase effect was more marked during 20 Hz stimulation. Phase modulation of SICI also depended on whether or not spontaneous beta activity occurred at ~20 Hz, supporting an interaction effect between tACS and underlying circuit resonances. The present study provides in vivo evidence linking cortical beta activity to sensorimotor integration, and for beta oscillations in motor cortex being promoted by resonance in GABAAergic interneuronal circuits.

Heterosynaptic modulation of evoked synaptic potentials in layer II of the entorhinal cortex by activation of the parasubiculum.

The superficial layers of the entorhinal cortex receive sensory and associational cortical inputs and provide the hippocampus with the majority of its cortical sensory input. The parasubiculum, which receives input from multiple hippocampal subfields, sends its single major output projection to layer II of the entorhinal cortex, suggesting that it may modulate processing of synaptic inputs to the entorhinal cortex. Indeed, stimulation of the parasubiculum can enhance entorhinal responses to synaptic input from the piriform cortex in vivo. Theta EEG activity contributes to spatial and mnemonic processes in this region, and the current study assessed how stimulation of the parasubiculum with either single pulses or short, five-pulse, theta-frequency trains may modulate synaptic responses in layer II entorhinal stellate neurons evoked by stimulation of layer I afferents in vitro. Parasubicular stimulation pulses or trains suppressed responses to layer I stimulation at intervals of 5 ms, and parasubicular stimulation trains facilitated layer I responses at a train-pulse interval of 25 ms. This suggests that firing of parasubicular neurons during theta activity may heterosynaptically enhance incoming sensory inputs to the entorhinal cortex. Bath application of the hyperpolarization-activated cation current (Ih) blocker ZD7288 enhanced the facilitation effect, suggesting that cholinergic inhibition of Ih may contribute. In addition, repetitive pairing of parasubicular trains and layer I stimulation induced a lasting depression of entorhinal responses to layer I stimulation. These findings provide evidence that theta activity in the parasubiculum may promote heterosynaptic modulation effects that may alter sensory processing in the entorhinal cortex.

Muscarinic control of rostromedial tegmental nucleus GABA neurons and morphine-induced locomotion.

Opioids induce rewarding and locomotor effects by inhibiting rostromedial tegmental GABA neurons that express μ-opioid and nociceptin receptors. These GABA neurons then strongly inhibit dopamine neurons. Opioid-induced reward, locomotion and dopamine release also depend on pedunculopontine and laterodorsal tegmental cholinergic and glutamate neurons, many of which project to and activate ventral tegmental area dopamine neurons. Here we show that laterodorsal tegmental and pedunculopontine cholinergic neurons project to both rostromedial tegmental nucleus and ventral tegmental area, and that M4 muscarinic receptors are co-localized with μ-opioid receptors associated with rostromedial tegmental GABA neurons. To inhibit or excite rostromedial tegmental GABA neurons, we utilized adeno-associated viral vectors and DREADDs to express designed muscarinic receptors (M4D or M3D respectively) in GAD2::Cre mice. In M4D-expressing mice, clozapine-N-oxide increased morphine-induced, but not vehicle-induced, locomotion. In M3D-expressing mice, clozapine-N-oxide blocked morphine-induced, but not vehicle-induced, locomotion. We propose that cholinergic inhibition of rostromedial tegmental GABA neurons via M4 muscarinic receptors facilitates opioid inhibition of the same neurons. This model explains how mesopontine cholinergic systems and muscarinic receptors in the rostromedial tegmental nucleus and ventral tegmental area are important for dopamine-dependent and dopamine-independent opioid-induced rewards and locomotion.

Integrated regulation of AMPA glutamate receptor phosphorylation in the striatum by dopamine and acetylcholine

Dopamine (DA) and acetylcholine (ACh) signals converge onto protein kinase A (PKA) in medium spiny neurons of the striatum to control cellular and synaptic activities of these neurons, although underlying molecular mechanisms are less clear. Here we measured phosphorylation of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) at a PKA site (S845) as an indicator of AMPAR responses in adult rat brains in vivo to explore how DA and ACh interact to modulate AMPARs. We found that subtype-selective activation of DA D1 receptors (D1Rs), D2 receptors (D2Rs), or muscarinic M4 receptors (M4Rs) induced specific patterns of GluA1 S845 responses in the striatum. These defined patterns support a local multitransmitter interaction model in which D2Rs inhibited an intrinsic inhibitory element mediated by M4Rs to enhance the D1R efficacy in modulating AMPARs. Consistent with this, selective enhancement of M4R activity by a positive allosteric modulator resumed the cholinergic inhibition of D1Rs. In addition, D1R and D2R coactivation recruited GluA1 and PKA preferentially to extrasynaptic sites. In sum, our in vivo data support an existence of a dynamic DA-ACh balance in the striatum which actively modulates GluA1 AMPAR phosphorylation and trafficking.This article is part of the Special Issue entitled ‘Ionotropic glutamate receptors’.

Inhibition of airway surface fluid absorption by cholinergic stimulation.

In upper airways airway surface liquid (ASL) depth and clearance rates are both increased by fluid secretion. Secretion is opposed by fluid absorption, mainly via the epithelial sodium channel, ENaC. In static systems, increased fluid depth activates ENaC and decreased depth inhibits it, suggesting that secretion indirectly activates ENaC to reduce ASL depth. We propose an alternate mechanism in which cholinergic input, which causes copious airway gland secretion, also inhibits ENaC-mediated absorption. The conjoint action accelerates clearance, and the increased transport of mucus out of the airways restores ASL depth while cleansing the airways. We were intrigued by early reports of cholinergic inhibition of absorption by airways in some species. To reinvestigate this phenomenon, we studied inward short-circuit currents (Isc) in tracheal mucosa from human, sheep, pig, ferret, and rabbit and in two types of cultured cells. Basal Isc was inhibited 20–70% by the ENaC inhibitor, benzamil. Long-lasting inhibition of ENaC-dependent Isc was also produced by basolateral carbachol in all preparations except rabbit and the H441 cell line. Atropine inhibition produced a slow recovery or prevented inhibition if added before carbachol. The mechanism for inhibition was not determined and is most likely multi-factorial. However, its physiological significance is expected to be increased mucus clearance rates in cholinergically stimulated airways.

Dynamic changes in scope for heart rate and cardiac autonomic control during warm acclimation in rainbow trout.

Time course studies are critical for understanding regulatory mechanisms and temporal constraints in ectothermic animals acclimating to warmer temperatures. Therefore, we investigated the dynamics of heart rate and its neuro-humoral control in rainbow trout ( ITALIC! Onchorhynchus mykissL.) acclimating to 16°C for 39 days after being acutely warmed from 9°C. Resting heart rate was 39 beats min(-1)at 9°C, and increased significantly when fish were acutely warmed to 16°C ( ITALIC! Q10=1.9), but then declined during acclimation ( ITALIC! Q10=1.2 at day 39), mainly due to increased cholinergic inhibition while the intrinsic heart rate and adrenergic tone were little affected. Maximum heart rate also increased with warming, although a partial modest decrease occurred during the acclimation period. Consequently, heart rate scope exhibited a complex pattern with an initial increase with acute warming, followed by a steep decline and then a subsequent increase, which was primarily explained by cholinergic inhibition of resting heart rate.

Activation of BK and SK channels by efferent synapses on outer hair cells in high-frequency regions of the rodent cochlea.

Cholinergic neurons of the brainstem olivary complex project to and inhibit outer hair cells (OHCs), refining acoustic sensitivity of the mammalian cochlea. In all vertebrate hair cells studied to date, cholinergic inhibition results from the combined action of ionotropic acetylcholine receptors and associated calcium-activated potassium channels. Although inhibition was thought to involve exclusively small conductance (SK potassium channels), recent findings have shown that BK channels also contribute to inhibition in basal, high-frequency OHCs after the onset of hearing. Here we show that the waveform of randomly timed IPSCs (evoked by high extracellular potassium) in high-frequency OHCs is altered by blockade of either SK or BK channels, with BK channels supporting faster synaptic waveforms and SK channels supporting slower synaptic waveforms. Consistent with these findings, IPSCs recorded from high-frequency OHCs that express BK channels are briefer than IPSCs recorded from low-frequency (apical) OHCs that do not express BK channels and from immature high-frequency OHCs before the developmental onset of BK channel expression. Likewise, OHCs of BKα(-/-) mice lacking the pore-forming α-subunit of BK channels have longer IPSCs than do the OHCs of BKα(+/+) littermates. Furthermore, serial reconstruction of electron micrographs showed that postsynaptic cisterns of BKα(-/-) OHCs were smaller than those of BKα(+/+) OHCs, and immunofluorescent quantification showed that efferent presynaptic terminals of BKα(-/-) OHCs were smaller than those of BKα(+/+) OHCs. Together, these findings indicate that BK channels contribute to postsynaptic function, and influence the structural maturation of efferent-OHC synapses.

Synaptic calcium regulation in hair cells of the chicken basilar papilla.

Cholinergic inhibition of hair cells occurs by activation of calcium-dependent potassium channels. A near-membrane postsynaptic cistern has been proposed to serve as a store from which calcium is released to supplement influx through the ionotropic ACh receptor. However, the time and voltage dependence of acetylcholine (ACh)-evoked potassium currents reveal a more complex relationship between calcium entry and release from stores. The present work uses voltage steps to regulate calcium influx during the application of ACh to hair cells in the chicken basilar papilla. When calcium influx was terminated at positive membrane potential, the ACh-evoked potassium current decayed exponentially over ∼100 ms. However, at negative membrane potentials, this current exhibited a secondary rise in amplitude that could be eliminated by dihydropyridine block of the voltage-gated calcium channels of the hair cell. Calcium entering through voltage-gated channels may transit through the postsynaptic cistern, since ryanodine and sarcoendoplasmic reticulum calcium-ATPase blockers altered the time course and magnitude of this secondary, voltage-dependent contribution to ACh-evoked potassium current. Serial section electron microscopy showed that efferent and afferent synaptic structures are juxtaposed, supporting the possibility that voltage-gated influx at afferent ribbon synapses influences calcium homeostasis during long-lasting cholinergic inhibition. In contrast, spontaneous postsynaptic currents ("minis") resulting from stochastic efferent release of ACh were made briefer by ryanodine, supporting the hypothesis that the synaptic cistern serves primarily as a calcium barrier and sink during low-level synaptic activity. Hypolemmal cisterns such as that at the efferent synapse of the hair cell can play a dynamic role in segregating near-membrane calcium for short-term and long-term signaling.

A 'calcium capacitor' shapes cholinergic inhibition of cochlear hair cells.

Efferent cholinergic neurons project from the brainstem to inhibit sensory hair cells of the vertebrate inner ear. This inhibitory synapse combines the activity of an unusual class of ionotropic cholinergic receptor with that of nearby calcium-dependent potassium channels to shunt and hyperpolarize the hair cell. Postsynaptic calcium signalling is constrained by a thin near-membrane cistern that is co-extensive with the efferent terminal contacts. The postsynaptic cistern may play an essential role in calcium homeostasis, serving as sink or source, depending on ongoing activity and the degree of buffer saturation. Release of calcium from postsynaptic stores leads to a process of retrograde facilitation via the synthesis of nitric oxide in the hair cell. Activity-dependent synaptic modification may contribute to changes in hair cell innervation that occur during development, and in the aged or damaged cochlea.

M4 mAChR-Mediated Modulation of Glutamatergic Transmission at Corticostriatal Synapses

The striatum is the main input station of the basal ganglia and is extensively involved in the modulation of motivated behavior. The information conveyed to this subcortical structure through glutamatergic projections from the cerebral cortex and thalamus is processed by the activity of several striatal neuromodulatory systems including the cholinergic system. Acetylcholine potently modulates glutamate signaling in the striatum via activation of muscarinic receptors (mAChRs). It is, however, unclear which mAChR subtype is responsible for this modulatory effect. Here, by using electrophysiological, optogenetic, and immunoelectron microscopic approaches in conjunction with a novel, highly selective M4 positive allosteric modulator VU0152100 (ML108) and M4 knockout mice, we show that M4 is a major mAChR subtype mediating the cholinergic inhibition of corticostriatal glutamatergic input on both striatonigral and striatopallidal medium spiny neurons (MSNs). This effect is due to activation of presynaptic M4 receptors, which, in turn, leads to a decrease in glutamate release from corticostriatal terminals. The findings of the present study raise the interesting possibility that M4 mAChR could be a novel therapeutic target for the treatment of neurological and neuropsychiatric disorders involving hyper-glutamatergic transmission at corticostriatal synapses.

Activity-Dependent Modulation of Layer 1 Inhibitory Neocortical Circuits by Acetylcholine

Layer 1 neocortical GABAergic interneurons control the excitability of pyramidal neurons through cell-class-specific direct inhibitory and disynaptic disinhibitory circuitry. The engagement of layer 1 inhibitory circuits during behavior is powerfully controlled by the cholinergic neuromodulatory system. Here we report that acetylcholine (ACh) influences the excitability of layer 1 interneurons in a cell-class and activity-dependent manner. Whole-cell recordings from identified layer 1 interneurons of the rat somatosensory neocortex revealed that brief perisomatic application of ACh excited both neurogliaform cells (NGFCs) and classical-accommodating cells (c-ACs) at rest by the activation of nicotinic receptors. In contrast, under active, action potential firing states, ACh excited c-ACs, but inhibited NGFCs through muscarinic receptor-mediated, IP3 receptor-dependent elevations of intracellular calcium that gated surface-membrane calcium-activated potassium channels. These excitatory and inhibitory actions of ACh could be switched between by brief periods of NGFC action potential firing. Paired recordings demonstrated that cholinergic inhibition of NGFCs disinhibited the apical dendrites of layer 2/3 pyramidal neurons by silencing widespread, GABA(B) receptor-mediated, monosynaptic inhibition. Together, these data suggest that the cholinergic system modulates layer 1 inhibitory circuits in an activity-dependent manner to dynamically control dendritic synaptic inhibition of pyramidal neurons.

Ultrastructure of cisternal synapses on outer hair cells of the mouse cochlea.

C (cisternal) synapses with a near membrane postsynaptic cistern are found on motor neurons and other central neurons, where their functional role is unknown. Similarly structured cisternal synapses mediate cholinergic inhibition of cochlear hair cells via α9α10-containing ionotropic receptors and associated calcium-activated (SK2) potassium channels, providing the opportunity to examine the ultrastructure of genetically altered cisternal synapses. Serial section electron microscopy was used to examine efferent synapses of outer hair cells (OHCs) in mice with diminished or enhanced cholinergic inhibition. The contact area of efferent terminals, the appositional area of the postsynaptic cistern, the distance of the cistern from the plasma membrane, and the average width of the cisternal lumen were recorded. The synaptic cisterns of wild-type OHCs were closely aligned (14-nm separation) with the hair cell membrane and coextensive with the micrometers-long synaptic terminals. The cisternal lumen averaged 18 nm so that the cisternal volume was approximately 30% larger than that of the cytoplasmic space between the cistern and the plasma membrane. Synaptic ultrastructure of α9L9'T knockin OHCs (acetylcholine receptor gain of function) were like those of wild-type littermates except that cisternal volumes were significantly larger. OHCs of SK2 knockout mice had few small efferent terminals. Synaptic cisterns were present, but smaller than those of wild-type littermates. Taken together, these data suggest that the cistern serves as a sink or buffer to isolate synaptic calcium signals.

Direct Quantitation of Methyl Phosphonate Adducts to Human Serum Butyrylcholinesterase by Immunomagnetic-UHPLC-MS/MS

Hydrolysis of G- and V-series organophosphorus nerve agents (OPNAs) containing a phosphorus-methyl bond yields a methylphosphonic acid (MeP) product when adducted to human butyrylcholinesterase (BChE). The MeP adduct is considered a sign of "aging" and results in loss of the o-alkyl identifier specific to each nerve agent. After aging has occurred, common therapeutics such as oximes cannot reactivate the cholinesterase enzyme and relieve cholinergic inhibition. Until now, a direct, quantitative method for determination of the MeP adduct to BChE was unavailable. Aged adducts in serum samples were processed by immunomagnetic separation of BChE by antibody conjugated bead, isotope-dilution, pepsin digestion, followed by UHPLC separation and detection by conventional electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Ions were detected in selected reaction monitoring (SRM) mode, and transition m/z 874.3 → 778.3 was used for quantitation. The analytical response ratio was linearly proportional to the serum concentration of MeP-adducted peptide (MeP-P) over the nominal concentration range of 2.0-250 ng/mL, with a coefficient of determination of R(2) ≥ 0.997. Intrarun accuracy, expressed as %Relative Error (%RE), was ≤13.5%, 16.3%, and 3.20% at 2.0, 16, and 250 ng/mL, respectively; the corresponding precision expressed as %RSD was ≤11.9%, 6.15%, and 3.39%. Interday %RSD was ≤7.13%, 5.69%, and 1.91%. Recovery of MeP-P from serum was ≥68% across the validated concentration range, and contributions from matrix effects were minimal. The method provides a direct, quantitative measurement of MeP-P found in clinical samples suspected of nerve agent exposure and subjected to such post-sampling stresses as elevated temperature and extended shipping.

Lipid-rich enteral nutrition regulates mucosal mast cell activation via the vagal anti-inflammatory reflex

Nutritional stimulation of the cholecystokinin-1 receptor (CCK-1R) and nicotinic acetylcholine receptor (nAChR)-mediated vagal reflex was shown to reduce inflammation and preserve intestinal integrity. Mast cells are important early effectors of the innate immune response; therefore modulation of mucosal mast cells is a potential therapeutic target to control the acute inflammatory response in the intestine. The present study investigates intestinal mast cell responsiveness upon nutritional activation of the vagal anti-inflammatory reflex during acute inflammation. Mucosal mast cell degranulation was induced in C57/Bl6 mice by administration of Salmonella enterica LPS. Lipid-rich enteral feeding prior to LPS significantly decreased circulatory levels of mouse mast cell protease at 30 min post-LPS compared with isocaloric low-lipid nutrition or fasting. CCK-1R blockage reversed the inhibitory effects of lipid-rich feeding, whereas stimulation of the peripheral CCK-1R mimicked nutritional mast cell inhibition. The effects of lipid-rich nutrition were negated by nAChR blockers chlorisondamine and α-bungarotoxin and vagal intestinal denervation. Accordingly, release of β-hexosaminidase by MC/9 mast cells following LPS or IgE-ovalbumin complexes was dose dependently inhibited by acetylcholine and nicotine. Application of GSK1345038A, a specific agonist of the nAChR α7, in bone marrow-derived mast cells from nAChR β2-/- and wild types indicated that cholinergic inhibition of mast cells is mediated by the nAChR α7 and is independent of the nAChR β2. Together, the present study reveals mucosal mast cells as a previously unknown target of the nutritional anti-inflammatory vagal reflex.

Inositol 1,4,5-Triphosphate Drives Glutamatergic and Cholinergic Inhibition Selectively in Spiny Projection Neurons in the Striatum

The striatum is critically involved in the selection of appropriate actions in a constantly changing environment. The spiking activity of striatal spiny projection neurons (SPNs), driven by extrinsic glutamatergic inputs, is shaped by local GABAergic and cholinergic networks. For example, it is well established that different types of GABAergic interneurons, activated by extrinsic glutamatergic and local cholinergic inputs, mediate powerful feedforward inhibition of SPN activity. In this study, using mouse striatal slices, we show that glutamatergic and cholinergic inputs exert direct inhibitory regulation of SPN activity via activation of metabotropic glutamate receptors (mGluRs) and muscarinic acetylcholine receptors. While pressure ejection of the group I mGluR (mGluR1/5) agonist DHPG [(S)-3,5-dihydroxyphenylglycine] equally engages both mGluR1 and mGluR5 subtypes, the mGluR-dependent component of IPSCs elicited by intrastriatal electrical stimulation is almost exclusively mediated by the mGluR1 subtype. Ca(2+) release from intracellular stores specifically through inositol 1,4,5-triphospahte receptors (IP(3)Rs) and not ryanodine receptors (RyRs) mediates this form of inhibition by gating two types of Ca(2+)-activated K(+) channels (i.e., small-conductance SK channels and large-conductance BK channels). Conversely, spike-evoked Ca(2+) influx triggers Ca(2+) release solely through RyRs to generate SK-dependent slow afterhyperpolarizations, demonstrating functional segregation of IP(3)Rs and RyRs. Finally, IP(3)-induced Ca(2+) release is uniquely observed in SPNs and not in different types of interneurons in the striatum. These results demonstrate that IP(3)-mediated activation of SK and BK channels provides a robust mechanism for glutamatergic and cholinergic inputs to selectively suppress striatal output neuron activity.

Olivocochlear suppression of outer hair cells in vivo: evidence for combined action of BK and SK2 channels throughout the cochlea

Cholinergic inhibition of cochlear hair cells via olivocochlear (OC)-efferent feedback is mediated by Ca(2+) entry through α9-/α10-nicotinic receptors, but the nature of the K(+) channels activated by this Ca(2+) entry has been debated (Yoshida N, Hequembourg SJ, Atencio CA, Rosowski JJ, Liberman MC. J Neurophysiol 85: 84-88, 2001). A recent in vitro study (Wersinger E, McLean WJ, Fuchs PA, Pyott SJ. PLoS One 5: e13836, 2010) suggests that small-conductance (SK2) channels mediate cholinergic effects in the apical turn, whereas large-conductance (BK) channels mediate basal turn effects. Here, we measure, as a function of cochlear frequency, the magnitude of BK and SK2 expression in outer hair cells and the strength of in vivo OC suppression in BK(+/+) mice vs. BK(-/-) lacking the obligatory α-subunit (Meredith AL, Thorneloe KS, Werner ME, Nelson MT, Aldrich RW. J Biol Chem 279: 36746-36752, 2004). Except at the extreme apical tip, we see immunostaining for both BK and SK2 in BK(+/+). Correspondingly, at all testable frequencies (8-45 kHz), we see evidence for both SK2 and BK contributions to OC effects evoked by electrically stimulating the OC bundle: OC-mediated suppression was reduced, but not eliminated, at all frequencies in the BK(-/-) ears. The suppression remaining in BK nulls was blocked by strychnine, suggesting involvement of α9-/α10-cholinergic receptors, coupled to activation of the remaining SK2 channels.

Role of M-Type Potassium Channels Glutamatergic Synaptic Transmission and Status Epilepticus (IN5-1.003)

Objective: Whether M-currents modulate glutamatergic synaptic transmission and whether the drugs that open these channels could terminate seizures induced by muscarinic stimulation. Background Muscarinic activation by pilocarpine and anologs result in seizures presumably by the inhibition of M-currents. Muscarinic stimulation modulates glutamatergic synaptic transmission but the mechanisms remain uncertain. Design/Methods: Whole-cell patch clamp recordings from CA3 and CA1 hippocampal pyramidal neurons and video EEG recoprdings from rats. Results: In voltage clamp recordings, XE-991 an M channel blocker increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs), but had no effect on their amplitude. XE-991 and its anolog linopordine also increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) but had no effect on their amplitude. M1 receptor agonist NcN-A-343 also increased the frequency of mEPSCs. The M-channel opener flupirtine had the effect opposite of XE-991. The XE-991 enhancement of mEPSCs frequency was not observed in calcium free external ACSF. When P/Q- or N-type calcium channel was blocked, the effect of XE-991 on mEPSCs was partially prevented. These data suggest that M-channel blockage increases presynaptic calcium dependent glutamate release in CA1 neurons. Current clamp recordings performed in CA3 neurons revealed that XE-991 depolarized membrane potential and increased burst firing. McN-A-343 had similar effect on CA3 neurons as XE-991. We then tested whether M-channel opener flupirtine could terminate status epilepticus (SE). Administration of flupirtine (50 mg/kg) and diazepam (10 mg/kg) individually 10 minutes after continuous EEG activity of first grade seizure was observed did not terminate SE; however, co-administration both drugs significantly reduced the length of SE, with 80% (4/5) rats returning to normal EEG within 90 minutes of treatment. Conclusions: Cholinergic activation causes depolarization of presynaptic CA3 neurons via M-channels, which activates calcium channels to increase glutamate release. Cholinergic inhibition of M-channel contributes to seizure generation.Drugs targeting M currents can terminate status epilepticus. Supported by: Congessionaly Directed Medical Research Program of the Department of Defense. NIH. Disclosure: Dr. Kapur has received (royalty or license fee or contractual rights) payments from a patent. Dr. Sun has nothing to disclose.