Application Of Carbachol Research Articles

The mechanism of agonist induced Ca2+ signalling in intact endothelial cells studied confocally in in situ arteries

In endothelial cells there remain uncertainties in the details of how Ca2+ signals are generated and maintained, especially in intact preparations. In particular the role of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), in contributing to the components of agonist-induced signals is unclear.The aim of this work was to increase understanding of the detailed mechanism of Ca2+ signalling in endothelial cells using real time confocal imaging of Fluo-4 loaded intact rat tail arteries in response to muscarinic stimulation. In particular we have focused on the role of SERCA, and its interplay with capacitative Ca2+ entry (CCE) and ER Ca2+ release and uptake. We have determined its contribution to the Ca2+ signal and how it varies with different physiological stimuli, including single and repeated carbachol applications and brief and prolonged exposures.In agreement with previous work, carbachol stimulated a rise in intracellular Ca2+ in the endothelial cells, consisting of a rapid initial phase, then a plateau upon which oscillations of Ca2+ were superimposed, followed by a decline to basal Ca2+ levels upon carbachol removal. Our data support the following conclusions: (i) the size (amplitude and duration) of the Ca2+ spike and early oscillations are limited by SERCA activity, thus both are increased if SERCA is inhibited. (ii) SERCA activity is such that brief applications of carbachol do not trigger CCE, presumably because the fall in luminal Ca2+ is not sufficient to trigger it. However, longer applications sufficient to deplete the ER or even partial SERCA inhibition stimulate CCE. (iii) Ca2+ entry occurs via STIM-mediated CCE and SERCA contributes to the cessation of CCE. In conclusion our data show how SERCA function is crucial to shaping endothelial cell Ca signals and its dynamic interplay with both CCE and ER Ca releases.

Parvalbumin-Containing Fast-Spiking Basket Cells Generate the Field Potential Oscillations Induced by Cholinergic Receptor Activation in the Hippocampus

Gamma frequency oscillations in cortical regions can be recorded during cognitive processes, including attention or memory tasks. These oscillations are generated locally as a result of reciprocal interactions between excitatory pyramidal cells and perisomatic inhibitory interneurons. Here, we examined the contribution of the three perisomatic interneuron types--the parvalbumin-containing fast-spiking basket cells (FSBCs) and axo-axonic cells (AACs), as well as the cholecystokinin-containing regular-spiking basket cells (RSBCs) to cholinergically induced oscillations in hippocampal slices, a rhythmic activity that captures several features of the gamma oscillations recorded in vivo. By analyzing the spiking activities of single neurons recorded in parallel with local field potentials, we found that all three cell types fired phase locked to the carbachol-induced oscillations, although with different frequencies and precision. During these oscillations, FSBCs fired the most with the highest accuracy compared with the discharge of AACs and RSBCs. In further experiments, we showed that activation of μ-opioid receptors by DAMGO ([D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin acetate), which significantly reduced the inhibitory, but not excitatory, transmission, suppressed or even blocked network oscillations both in vitro and in vivo, leading to the desynchronization of pyramidal cell firing. Using paired recordings, we demonstrated that carbachol application blocked GABA release from RSBCs and reduced it from FSBCs and AACs, whereas DAMGO further suppressed the GABA release only from FSBCs, but not from AACs. These results collectively suggest that the rhythmic perisomatic inhibition, generating oscillatory fluctuation in local field potentials after carbachol treatment of hippocampal slices, is the result of periodic GABA release from FSBCs.

Cholinergic modulation of excitatory synaptic input integration in hippocampal CA1

During theta rhythm, the timing of inputs to hippocampal CA1 from the perforant path (PP) of the entorhinal cortex and the Schaffer collaterals (SCs) from individual CA3 pyramidal neurons can vary within an individual theta period. Importantly, during theta rhythms these interactions occur during elevated acetylcholine concentrations. Thus, I examined the effect that PP inputs have on SC inputs in hippocampal CA1 during cholinergic receptor activation. To do this I measured the impact that a single electrical stimulus of the stratum lacunosum-moleculare (SLM, which contains the PP) had on excitation evoked by stimulation of the stratum radiatum (SR, which contains the SC) using voltage-sensitive dye imaging, field excitatory postsynaptic potentials and whole cell patch clamping in rat hippocampal brain slices. My data showed that SLM stimulation one half a theta cycle or less (25-75 ms) before SR stimulation resulted in the summation of excitatory events in SR and SP of hippocampal CA1. The summation was unaffected by cholinergic receptor activation by carbachol. SLM stimulation one theta cycle (150-225 ms) preceding SR stimulation significantly suppressed excitatory events measured in SR and SP. This SLM stimulus inhibition of SR-driven excitatory events was augmented by carbachol application. The carbachol effect was blocked by atropine and SLM-driven suppression of excitatory events was blocked by the GABA(B) receptor antagonist CGP 54626. SR field EPSP slopes were unaffected by SLM prepulses. Carbachol increased the probability of SR input to drive action potential firing in CA1 pyramidal neurons, which was inhibited by SLM prepulses (150-225 ms). Together these data provide important information regarding the integration of inputs in hippocampal CA1 during theta rhythms. More specifically, SR inputs can be differentially gated by SLM feedforward inhibition at varying temporal intervals within a theta cycle.

GABAergic actions on cholinergic laterodorsal tegmental neurons: implications for control of behavioral state

Cholinergic neurons of the pontine laterodorsal tegmentum (LDT) play a critical role in regulation of behavioral state. Therefore, elucidation of mechanisms that control their activity is vital for understanding of how switching between wakefulness, sleep and anesthetic states is effectuated. In vivo studies suggest that GABAergic mechanisms within the pons play a critical role in behavioral state switching. However, the postsynaptic, electrophysiological actions of GABA on LDT neurons, as well as the identity of GABA receptors present in the LDT mediating these actions is virtually unexplored. Therefore, we studied the actions of GABA agonists and antagonists on cholinergic LDT cells by performing patch clamp recordings in mouse brain slices. Under conditions where detection of Cl − -mediated events was optimized, GABA induced gabazine (GZ)-sensitive inward currents in the majority of LDT neurons. Post-synaptic location of GABA A receptors was demonstrated by persistence of muscimol-induced inward currents in TTX and low Ca 2+ solutions. THIP, a selective GABA A receptor agonist with a preference for δ-subunit containing GABA A receptors, induced inward currents, suggesting the existence of extrasynaptic GABA A receptors. LDT cells also possess GABA B receptors as baclofen-activated a TTX- and low Ca 2+-resistant outward current that was attenuated by the GABA B antagonists CGP 55845 and saclofen. The tertiapin sensitivity of baclofen-induced outward currents suggests that a G IRK mediated this effect. Further, outward currents were never additive with those induced by application of carbachol, suggesting that they were mediated by activation of GABA B receptors linked to the same G IRK activated in these cells by muscarinic receptor stimulation. Activation of GABA B receptors inhibited Ca 2+ increases induced by a depolarizing voltage step shown previously to activate VOCCs in cholinergic LDT neurons. Baclofen-mediated reductions in depolarization-induced Ca 2+ were unaltered by prior emptying of intracellular Ca 2+ stores, but were abolished by low extracellular Ca 2+ and pre-application of nifedipine, indicating that activation of GABA B receptors inhibits influx of Ca 2+ involving L-type Ca 2+ channels. Presence of GABA C receptors is suggested by the induction of inward current by (E)-4- amino-2-butenoic acid (TACA) and its inhibition by 1,2,5,6-tetrahydropyridine-4-ylmethylphosphinic (TPMPA), a relatively selective agonist and antagonist, respectively, of GABA C receptors. All of these GABA-mediated actions were found to occur in histochemically-identified cholinergic neurons. Taken together, these data indicate for the first time that cholinergic neurons of the LDT exhibit functional GABA A, B and C receptors, including extrasynaptically located GABA A receptors, which may be tonically activated by synaptic overflow of GABA. Accordingly, the activity of cholinergic LDT neurons is likely to be significantly affected by GABAergic tone within the nucleus, and so, demonstrated effects of GABA on behavioral state may be mediated, in part, via direct actions on cholinergic neurons in the LDT.

Postnatal development enhances the effects of cholinergic inputs on recruitment threshold and firing rate of rat oculomotor nucleus motoneurons

Changes in the electrophysiological and morphological characteristics of motoneurons (Mns) of the oculomotor nucleus during postnatal development have been reported, however synaptic modifications that take place concurrently with postnatal development in these Mns are yet to be elucidated. We investigated whether cholinergic inputs exert different effects on the recruitment threshold and firing rate of Mns during postnatal development. Rat oculomotor nucleus Mns were intracellularly recorded in brain slice preparations and separated in neonatal (4–7 postnatal days) and adult (20–30 postnatal days) age groups. Stimulation of the medial longitudinal fasciculus evoked a monosynaptic excitatory potential in Mns that was attenuated with atropine (1.5 μM, a muscarinic antagonist). Mns were silent at their resting membrane potential, and bath application of carbachol (10 μM, a cholinergic agonist) induced depolarization of the membrane potential and a sustained firing rate that were more pronounced in adult Mns. Pharmacological and immunohistochemical assays showed that these responses were attributable to muscarinic receptors located in the membrane of Mns. In addition, compared to control Mns, carbachol-exposed Mns exhibited a higher firing rate in response to the injection of the same amount of current, and a decrease in the current threshold required to achieve sustained firing. These latter effects were more pronounced in adult than in neonatal Mns. In conclusion, our findings suggest that cholinergic synaptic inputs are already present in neonatal Mns, and that the electrophysiological effects of such inputs on recruitment threshold and firing rate are enhanced with the postnatal development in oculomotor nucleus Mns. We propose that cholinergic input maturation could provide a greater dynamic range in adult Mns to encode the output necessary for graded muscle contraction.

Persistent neural activity regulates Arc/Arg3.1 transcription in the dentate gyrus

The activity-regulated cytoskeleton-associated gene (Arc, also known as Arg3.1) is an effector immediate-early gene rapidly induced by strong neural activity. Although a number of studies have revealed significant functions of Arc and Arc has come into widespread use as a neural activity marker in behavioral studies, the mechanisms regulating Arc transcription remain unclear. Here, we examined the conditions of Arc transcription in acute slices of dentate gyrus. Surprisingly, kainic acid (1 μM to 10 mM) application to slices did not induce Arc transcription, although intraperitoneal injection of kainic acid (20 mg/kg) induced robust Arc transcription. No types of high-frequency stimulation examined induced Arc transcription in acute slices. These findings indicate that Arc transcription is dramatically suppressed in acute slices of the dentate gyrus, in which background neural activity is markedly reduced. Burst stimulation increased the number of Arc-expressing cells in the presence of picrotoxin, in which excitation was maintained even after the end of stimulation. Moreover, the involvement of background neural activity in Arc transcription was tested by application of carbachol, a muscarinic receptor agonist. Carbachol also increased the number of Arc-expressing cells, which was blocked by atropine, a muscarinic receptor antagonist. Taken together, these findings suggest that persistent background activity is critical for Arc transcription.

Carbachol-Induced Long-Term Synaptic Depression Is Enhanced During Senescence at Hippocampal CA3–CA1 Synapses

Dysregulation of the cholinergic transmitter system is a hallmark of Alzheimer's disease and contributes to an age-associated decline in memory performance. The current study examined the influence of carbachol, a cholinergic receptor agonist, on synaptic transmission over the course of aging. Extracellular excitatory postsynaptic field potentials were recorded from CA3-CA1 synapses in acute hippocampal slices obtained from young adult (5-8 mo) and aged (22-24 mo) male Fischer 344 rats. Bath application of carbachol elicited a transient depression of synaptic transmission, which was followed by a long-lasting depression (CCh-LTD) observed 90 min after carbachol cessation in both age groups. However, the magnitude of CCh-LTD was significantly larger in senescent animals and was attenuated by N-methyl-D-aspartate receptor blockade in aged animals. Blockade of L-type Ca(2+) channels inhibited CCh-LTD to a greater extent in aged animals compared to young adults. Finally, the expression of CCh-LTD was dependent on protein synthesis. The results indicate that altered Ca(2+) homeostasis or muscarinic activation of Ca(2+) signaling contribute to the enhanced CCh-LTD during senescence.

Regulator of G‐protein signalling 4 (RGS4) KO mice show increased susceptibility to atrial fibrillation

Susceptibility to atrial fibrillation (AF) depends strongly on parasympathetic activity. Since RGS4 inhibits parasympathetic signaling in the sinoatrial node, we explore whether changes in RGS4 levels altered the susceptibility of atrial fibrillation. Consistent with this possibility, RGS4 levels were decreased in atria of tachypaced dogs prior to their development of chronic AF. Moreover, in vivo ECG recordings of anaesthetized mice showed greater susceptibility to AF while optical mapping of isolated atrial preparations using a voltage‐sensitive dye revealed greatly increased susceptibility to rotor formation when rgs4 was ablated. Consistent with altered parasympathetic signaling in the myocardium of rgs4−/− mice, acetylcholine‐sensitive potassium currents (IKACh) evoked by carbachol application were greater in isolated atrial myocytes from rgs4−/− mice. These IKACh changes were, as expected, associated with marked enhancements of action potential duration shortening in response to parasympathetic activation, but not to slower conduction velocities. Together, our findings establish that RGS4 protects atrial tissues from excess parasympathetic signaling that predispose to atrial fibrillation.Technical and financial assistance for this work was provided by the Heart and Stroke Foundation of Ontario Grant‐in‐Aid Program grants T6799, and Canadian Institutes of Health Research (CIHR) Canada Graduate Scholarship Program.

Cell‐specific contribution to gamma oscillations

Gamma oscillations are a particularly prominent form of rhythmic activity occurring during wakefulness and attentive behaviour that result from synchronous activity of cortical neurons at frequencies ranging between 20 and 80 Hz. Abnormalities in these oscillations might underlie neuropsychiatric disorders such as schizophrenia (Uhlhaas et al. 2006). Therefore understanding the precise mechanisms underlying gamma oscillations – in particular the respective role of synaptic and intrinsic currents – represents a major goal in fundamental and clinical neuroscience. Gamma oscillations are prevalent in the hippocampus in vivo and they can be induced in the isolated CA3 region of the hippocampus in vitro with bath application of carbachol (Fisahn et al. 1998). They require both excitatory and inhibitory synaptic transmission. In particular, in their pioneering study Fisahn and co-workers highlighted the role of GABAergic activity by showing that prolongation of the GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) with barbiturate reduced the frequency of gamma oscillations. Models of leaky integrate-and-fire neurons suggest that synaptic recurrent feedback loops between interneurons and pyramidal cells are necessary and sufficient to explain gamma frequency oscillations (Brunel & Wang, 2003). In a recent study, gamma oscillation fluctuations in amplitude and frequency were found to be correlated with cycle-to-cycle changes in excitation and inhibition (Atallah & Scanziani, 2009). In this issue of The Journal of Physiology, Oren, Hájos and Paulsen (Oren et al. (2010) examined the relative contribution of synaptic and voltage-dependent action currents in pyramidal neurons during gamma oscillations induced with carbachol in the CA3 region of the rat hippocampus in vitro. They combined recordings of the local field potential (LFP) to monitor the oscillatory activity of the principal neurons with cell-attached recordings from subclasses of GABAergic interneurons. Changes in the oscillation amplitude were quantified by the amplitude of the wavelet transform. Oren et al. confirm in this study that inhibitory currents contribute significantly to LFP amplitude fluctuations as a consequence of an increased probability of firing in subclasses of interneurons that are responsible for perisomatic inhibition. Indeed when performing cell-attached recordings from various cell types, the authors could observe that, for a given oscillation cycle, the mean cycle amplitude was significantly higher when perisomatic (or interneuron-selective) interneurons discharged. In contrast, there was no such direct relationship between the discharge of a pyramidal cell and LFP amplitude. Therefore cycle-to-cycle variation amplitude appears to be the direct consequence of interneuron firing probability. However, the synchronized firing of pyramidal cells, which can be detected as a population spike in some cycles during gamma waves, affected the LFP. Indeed, the authors provide evidence that slow action currents, resulting from the activation of repolarisation conductances contribute in a significant manner to the early component of the LFP. Local field potentials are probably one of the oldest, least invasive and simplest ways to perform high sampling rate recordings of not only neuronal assemblies, but also the discharge activity of single neurons with the help of combined on- and offline signal processing analysis. The possibility of performing high rate recordings with almost no recording time limitation can then become an advantage in comparison with imaging techniques. Oren, Hájos and Paulsen have proven that these techniques should not yet be considered out of date since their use, in combination with other electrophysiological techniques (cell-attached and whole-cell recordings), can be useful and very informative for studying network activity and the contribution of different cell subtypes to the genesis or modulation of cortical oscillations. In this study, the firing probability of dendritic-targeting interneurons was reported not to be correlated with LFP amplitude fluctuations. This in vitro observation is in agreement with in vivo experiments showing that the discharge of these interneurons is not coupled to gamma oscillations (Tukker et al. 2007). Knowing that these interneurons exert specific inhibitory functions on EPSP integration and dendritic electrogenesis but not on action potential firing (Miles et al. 1996) leaves them free to serve distinct functions than the ‘sole’ contribution to gamma oscillations.

Cholinergic modulation of fast inhibitory and excitatory transmission to pedunculopontine thalamic projecting neurons.

The pedunculopontine nucleus (PPN) is part of the cholinergic arm of the reticular activating system, which is mostly active during waking and rapid-eye movement sleep. The PPN projects to the thalamus and receives cholinergic inputs from the laterodorsal tegmental nucleus and contralateral PPN. We employed retrograde labeling and whole cell recordings to determine the modulation of GABAergic, glycinergic, and glutamatergic transmission to PPN thalamic projecting neurons, and their postsynaptic responses to the nonspecific cholinergic agonist carbachol. M2 and M4 muscarinic receptor-modulated inhibitory postsynaptic responses were observed in 73% of PPN output neurons; in 12.9%, M1 and nicotinic receptor-mediated excitation was detected; and muscarinic and nicotinic-modulated fast inhibitory followed by slow excitatory biphasic responses were evident in 6.7% of cells. A significant increase in the frequency of spontaneous excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic currents during carbachol application was observed in 66.2% and 65.2% of efferent neurons, respectively. This effect was blocked by a M1 antagonist or nonselective muscarinic blocker, indicating that glutamatergic, GABAergic, and/or glycinergic neurons projecting to PPN output neurons are excited through muscarinic receptors. Decreases in the frequency of miniature EPSCs, and amplitude of electrical stimulation-evoked EPSCs, were blocked by a M2 antagonist, suggesting the presence of M2Rs at terminals of presynaptic glutamatergic neurons. Carbachol-induced multiple types of postsynaptic responses, enhancing both inhibitory and excitatory fast transmission to PPN thalamic projecting neurons through muscarinic receptors. These results provide possible implications for the generation of different frequency oscillations in PPN thalamic projecting neurons during distinct sleep-wake states.

Genetically Increased Cell-Intrinsic Excitability Enhances Neuronal Integration into Adult Brain Circuits

New neurons are added to the adult brain throughout life, but only half ultimately integrate into existing circuits. Sensory experience is an important regulator of the selection of new neurons but it remains unknown whether experience provides specific patterns of synaptic input or simply a minimum level of overall membrane depolarization critical for integration. To investigate this issue, we genetically modified intrinsic electrical properties of adult-generated neurons in the mammalian olfactory bulb. First, we observed that suppressing levels of cell-intrinsic neuronal activity via expression of ESKir2.1 potassium channels decreases, whereas enhancing activity via expression of NaChBac sodium channels increases survival of new neurons. Neither of these modulations affects synaptic formation. Furthermore, even when neurons are induced to fire dramatically altered patterns of action potentials, increased levels of cell-intrinsic activity completely blocks cell death triggered by NMDA receptor deletion. These findings demonstrate that overall levels of cell-intrinsic activity govern survival of new neurons and precise firing patterns are not essential for neuronal integration into existing brain circuits.

Muscarinic Receptor Subtypes Mediating Ca2+ Sensitization of Intestinal Smooth Muscle Contraction: Studies with Receptor Knockout Mice

In the present study, we have characterized muscarinic receptor subtypes that mediate carbachol-induced Ca2+ sensitization of contraction in intestinal smooth muscle, using mutant mice lacking M(2) or M(3) muscarinic receptors or both receptor subtypes. In alpha-toxin-permeabilized muscle strips from wild-type (WT) mice, isometric tension responses to Ca2+ applied cumulatively (pCa 7.0-5.0) were increased when the muscarinic agonist carbachol (100 microM) was added to the medium, as judged from shifts of pCa-tension curves in both 50% effective concentration (EC(50)) and maximum response (E(max)) of pCa-tension curve. In preparations from M(2)-knockout (KO) mice, pCa-tension curves were also shifted by carbachol (100 microM), and the extents of the EC(50) and E(max) changes resembled those observed in preparations from WT mice. In preparations from M(3)-KO or M(2)/M(3)-double KO mice, however, no significant changes in pCa-tension curves were obtained after carbachol application. The G(q/11)-type G-protein inhibitor YM-254890 (1 microM) completely blocked the Ca2+ sensitization of contraction induced by carbachol in M(2)-KO or WT preparations. The results strongly support the idea that the muscarinic activation of Ca2+ sensitization in intestinal smooth muscles is mediated by the M(3) muscarinic receptor coupled to G(q/11)-type G-proteins, without any significant involvement of the other muscarinic receptor subtypes including M(2).

Stabilization of visual responses through cholinergic activation

Neuronal processing of sensory information requires that rapidly changing synaptic inputs are continuously transformed into action potentials. Variability of spike firing is generally considered as noise and might therefore interfere with the reliability of synaptic transmission in sensory systems. In a system in which the number of spikes is a variable that determines the quality of neuronal transmission, variability of spike counts is a paradoxical attribute. In contrast, in a system in which precisely correlated spike firing can influence synaptic integration, response variability might be used as an additional mechanism for coding information. As acetylcholine has been shown to reduce spike-frequency adaptation and enhance gamma frequency (21–70 Hz) oscillations, we set out to study the influence of cholinergic modulation on the variability of spike counts and gamma oscillations. Iontophoretic application of carbachol, a cholinergic agonist, in cat primary visual cortex or electrical stimulation of the mesencephalic reticular formation reduced the spike count variability and stabilized gamma frequency oscillations of visually induced responses. Response stabilization was correlated with enhancement of gamma-frequency oscillations but not with averaged firing rates. Lowering variability of sensory responses might be a mechanism to stabilize and improve reliability of neuronal transmission. Cholinergic activation may therefore influence the efficacy of neuronal transmission by modulating the precise timing of neuronal responses.

Effect of carbachol on the expression of la on monocyte and lymphocyte apoptosis rate of the burned rats with delayed resuscitation

Objective To explore the changing trend of Ia on monocyte, lymphocyte apoptosis rate, TNF-α and IL-6 in abdominal aorta of burned rats with delayed resuscitation and the influence of application of carbachol on them. Methods Adult male Wistar rats were randomly divided into normal control group(n =8), scald group(n =48) and scald with carbachol treatment group(n =48). In latter two groups, rats were inflicted with 30% total body surface area (TBSA) full-thickness scald and delayed fluid resuscitation. All scald rats were sacrificed at the 6th hours or 1st, 2nd, 3rd, 7th, 14th day after scald, with 8 rats at each time point. Expression of Ia antigen on monocyte and lymphocyte apoptosis rate were determined by direct immunofluorescence on a flow cytometer, and TNF-α and IL-6 was measured by ELISA. Results Expression of la on monocyte was obviously lower than that of controls. The lowest levels were recorded on the 6th hours and 1st day after scald. Subsequently, Ia was elevated gradually, but still lower than that of normal rats(P <0. 01). After administration of carbachol, Ia expression was obviously promoted, compared with the simple scald group (P <0. 01). Lymphocyte apoptosis rate, TNF-α and IL-6 was higher than that of controls(P <0. 01). After administration of cavachol, , lymphocyte apoptosis rate and TNF-α and IL-6 was obviously down-regulated on the 6th hours, 1st day, 2nd day and 3rd day after scald injury, compared with the simple scald group (P < 0. 01 or 0. 05). Conclusion After severe burn with delayed fluid resuscitation, there is a low la expression, high lymphocyte apoptosis rate and increased releasing of proinflammatory cytokine. Immune function was suppressed. Carbacho] could improve the immune function of scald rats with delayed fluid resuscitation. Key words: CarbachoL/PD ; Bums/DT/IM

Role of Acetylcholine in the Modulation of Activity of Neocortical Neurons in Awake Animals Performing an Instrumental Conditioned Reflex

We studied modulatory effects of the cholinergic system on the activity of sensorimotor cortex neurons related to realization of an instrumental conditioned placing reflex. Experiments were carried out on awake cats; multibarrel glass microelectrodes were used for extracellular recording of impulse activity of neurons in the sensorimotor cortex and iontophoretic application of synaptically active agents within the recording region. The background and reflex-related activity was recorded in the course of realization of conditioned movements, and then changes of spiking induced by applications of the testing substances were examined. Applications of acetylcholine and carbachol resulted in increases in the intensity of impulse reactions of neocortical neurons evoked by presentation of an acoustic signal and in simultaneous shortening of the response latencies. An agonist of muscarinic receptors, pylocarpine, exerted a similar effect on the evoked activity of sensorimotor cortex neurons. Blockers of muscarinic receptors, atropine and scopolamine, vice versa, sharply suppressed impulse reactions of cortical neurons to afferent stimulation and simultaneously increased latencies of these responses. Applications of an agonist of nicotinic receptors, nicotine, was accompanied by suppression of impulse neuronal responses, an increase in the latency of spike reactions to presentation of a sound signal, and a corresponding increase in the latency of a conditioned motor reaction. In contrast, application of an antagonist of nicotinic receptors, tubocurarine, significantly intensified neuronal spike responses and shortened their latency. The mechanisms underlying the effects of antagonists of membrane muscarinic and nicotinic cholinoreceptors and the role of activation of these receptors in the modulation of activity of pyramidal and non-pyramidal neocortical neurons related to realization of the instrumental motor reflex are discussed.

Comparisons of the responses of anterior and posterior human adult male bladder neck smooth muscle to in vitro stimulation

To evaluate differing methods of stimulation on strips of human bladder neck smooth muscle and compare muscle taken from the anterior and posterior aspects. Samples of adult human male bladder neck muscle were obtained from patients undergoing open radical prostatectomy. Muscle was taken from either the anterior or posterior (nine and six patients, respectively) aspects of the bladder neck. Muscle strips dissected from these samples were suspended in the Brading-Sibley organ bath. The strips were superfused with 100 mm KCl-enriched Krebs' solution for 4 min to determine viability. This allowed experimentation on 17 strips from the anterior aspect of the bladder neck and 13 from the posterior bladder neck. These remaining strips were then superfused either with various concentrations (x10(-7) to x10(-3)m) of carbachol or noradrenaline in Krebs' solution, for 15 s. A further set of strips (eight from anterior, six from posterior) was suspended and responses to electrical field stimulation (EFS) with varying parameters were measured. Each EFS experiment was repeated after a 15 min exposure to 10(-3)m atropine, and again after a 15 min exposure 10(-7)m tetrodotoxin (TTX). Tension responses produced in these series of experiments were measured using strain gauges and analysed using data acquisition software. Student's t-test was used for the statistical analysis. All muscle strips included in the study responded to EFS. The magnitude of this contraction is frequency dependent. The contractions were abolished by superfusion of the muscle strips with atropine. There was no further suppression of the contractile response on addition of TTX. Posterior bladder neck samples had a greater mean contractile response per unit mass than anterior strips at all frequencies of >1 Hz, and significantly more at 20 and 30 Hz. There was a concentration-dependent response in bladder neck contraction to carbachol but only in the strips from the anterior bladder neck at concentrations of <10(-3)m. Posterior bladder neck strips did not significantly contract upon application of carbachol. Similarly, there was a concentration-dependent response to noradrenaline. Responses to noradrenaline were not uniform around the bladder neck, but not significantly different. Carbachol was the more 'potent' stimulator in anterior smooth muscle strips, but again the differences between agonists were not statistically significant. These experiments show physiological variability around the circumference of the human male bladder neck. The posterior bladder neck shows significantly stronger contraction to alpha-adrenergic agonists compared with cholinergic agonists; the anterior bladder neck does not have a similarly significant differential response. The uniform response to noradrenaline may underlie the bladder neck's role in the prevention of retrograde ejaculation. The differential responses to carbachol may reflect differences in the embryological derivation of the anterior and posterior bladder neck fibres or in their innervation. Some of these differences may have clinical importance through the action of therapeutic agents.

Muscarinic Modulation of Recruitment Threshold and Firing Rate in Rat Oculomotor Nucleus Motoneurons

Above recruitment threshold, ocular motoneurons (Mns) show a firing rate linearly related with eye position. Current hypothesis suggests that synaptic inputs are determinant for establishing the recruitment threshold and firing rate gain in these Mns. We investigated this proposal by studying the cholinergic modulation in oculomotor nucleus Mns by intracellular recordings in rat brain slice preparation. All recorded Mns were silent at their resting membrane potential. Bath application of carbachol (10 microm) produced a depolarization and a sustained firing that was not silenced on returning membrane potential to the precarbachol value via DC injection. In response to similar membrane depolarization or equal-current steps, carbachol-exposed Mns produced a higher firing rate and a shorter spike afterhyperpolarization phase with lower amplitude. The relationship between injected current and firing rate (I-F) was linear in control and carbachol-exposed Mns. The slope of these relationships (I-F gain) decreased with carbachol exposure. Bath application of agonist and antagonist of nicotinic and muscarinic acetylcholine receptors in addition to immunohistochemical studies support the notion that muscarinic receptors are primarily involved in the preceding responses. We conclude that muscarinic inputs play an important role in determining the recruitment threshold and firing rate gain observed in oculomotor Mns in vivo.

Cholinergic Responses in Crossed Tecto-Reticular Neurons of Rat Superior Colliculus

Neurons in the intermediate gray layer (SGI) of mammalian superior colliculus (SC) receive cholinergic innervation from the brain stem parabrachial region, which seems to modulate the signal processing in the SC. To clarify its role particularly in orienting behaviors, we studied cholinergic effects on the major output neuron group of the SGI, crossed tecto-reticular neurons (cTRNs), identified by retrograde labeling from the contralateral brain stem gaze center in SC slices obtained from rats (PND 17-22) by whole cell patch-clamp techniques. Bath application of carbachol induced either 1) nicotinic inward (nIN) + muscarinic inward (mIN) (11/24) or 2) nIN + mIN + muscarinic outward (mOUT) (13/24) current responses. Transient pressure application of 1 mM acetylcholine elicited nIN in all neurons tested (n = 58). In a majority of these neurons (52/58), the nIN was completely suppressed by dihydro-beta-erythroidine, a specific antagonist for alpha4beta2 nicotinic receptor subtype. The remaining 6/58 neurons exhibited not only the slower alpha4beta2 receptor-mediated component but also a faster component that was inhibited by a specific antagonist for alpha7 nicotinic receptor, alpha-bungarotoxin. cTRNs expressing alpha7 nicotinic receptors tended to be smaller in size than those lacking alpha7 receptors. Bath application of muscarine induced two response patterns: mIN only (17/38) and mIN+ mOUT (21/38). The mIN and mOUT were mediated by M3 (plus M1) and M2 muscarinic receptors, respectively. These results suggest that a major response to cholinergic inputs to cTRNs is excitatory. This would indicate the facilitatory role of the brain stem cholinergic system in the execution of orienting behaviors including saccadic eye movements.

Cholinergic modulation of neural activity in the telencephalon of the zebrafish

In this study, we have assessed the effects of muscarinic receptor activation on neural activity in the telencephalon of the zebrafish. Electrically evoked field potentials via the stimulation of the posterior dorsal telencephalon were recorded in the anterior dorsal telencephalon of the ipsilateral hemisphere, with a peak latency of 20-30ms. The application of carbachol (1-25microM) and oxotremorine-M (0.25-60microM) suppressed the amplitude of field potentials in a dose-dependent manner. This suppressive effect was blocked by a muscarinic receptor antagonist, atropine (20microM), and an M1 receptor antagonist, pirenzepine (5microM), but not by the M2 receptor antagonists, methoctramine (5microM) and gallamine (5microM). Oxotremorine-M (1microM) also suppressed the induction of field potentials via the stimulation of the contralateral telencephalon and the medial fiber bundle, which was blocked by atropine (20microM). An acetylcholine esterase inhibitor, physostigmine (20-30microM), reduced the peak amplitude of the evoked field potentials, while increasing late bursting activity. Furthermore, a high dose of physostigmine (100microM) induced spontaneous bursting activity. These results demonstrated the cholinergic influence on the neural activity in the telencephalon of the zebrafish.

Two Types of Cation Channel Activated by Stimulation of Muscarinic Receptors in Guinea-Pig Urinary Bladder Smooth Muscle

The present study, aiming to elucidate ion channel mechanisms underlying muscarinic receptor–induced depolarization, has characterized membrane currents induced by carbachol in single guinea-pig urinary bladder myocytes. Application of carbachol to cells that were voltage-clamped at − 50 mV produced an atropine-sensitive, biphasic inward current consisting of an initial peak followed by a smaller sustained phase. Replacing the extracellular Na+ and intracellular Cl− with impermeable tris+ and glutamate−, respectively, demonstrated that the biphasic current is entirely composed of cation currents. Its initial peak phase was abolished by buffering intracellular Ca2+ to a constant level of 100 nM or depleting intracellular Ca2+ stores, and it was mimicked by the Ca2+ releaser caffeine. Ca2+ entry evoked by voltage steps in the sustained phase induced no noticeable change, indicating that this phase of cation current is insensitive to a rise of [Ca2+]i. These results demonstrate that muscarinic receptor stimulation invokes the openings of two types of cation channel, a Ca2+-activated and a receptor-operated type; the former channels are gated by a rise in [Ca2+]i upon intracellular Ca2+ release, and the latter are gated through other muscarinic receptor–coupled signal transduction mechanisms.