Increases in Microvascular Perfusion and Tissue Oxygenation via Vasodilatation After Anodal Transcranial Direct Current Stimulation in the Healthy and Traumatized Mouse Brain.

Traumatic brain injury (TBI), causing neurological deficit in 70% of survivors, still lacks a clinically proven effective therapy. Transcranial direct current stimulation (tDCS) has emerged as a promising electroceutical therapeutic intervention possibly suitable for TBI; however, due to limited animal studies the mechanisms and optimal parameters are unknown. Using a mouse model of TBI we evaluated the acute effects of the anodal tDCS on cerebral blood flow (CBF) and tissue oxygenation, and assessed its efficacy in long-term neurologic recovery. TBI was induced by controlled cortical impact leading to cortical and hippocampal lesions with reduced CBF and developed hypoxia in peri-contusion area. Sham animals were subjected to craniotomy only. Repetitive anodal tDCS (0.1 mA/15 min) or sham stimulation was done over 4 weeks for four consecutive days with 3-day intervals, beginning 1 or 3 weeks after TBI. Laser speckle contrast imaging (LSCI) revealed that anodal tDCS causes an increase in regional cortical CBF in both traumatized and Sham animals. On microvascular level, using in-vivo two-photon microscopy (2PLSM), we have shown that anodal tDCS induces arteriolar dilatation leading to an increase in capillary flow velocity and tissue oxygenation in both traumatized and Sham animals. Repetitive anodal tDCS significantly improved motor and cognitive neurologic outcome. The group with stimulation starting 3 weeks after TBI showed better recovery compared with stimulation starting 1 week after TBI, suggesting that the late post-traumatic period is more optimal for anodal tDCS.

Adenosine receptor activation is responsible for prolonged depression of synaptic transmission after spreading depolarization in brain slices.

Spreading depolarization (SD) is a slowly propagating, coordinated depolarization of brain tissue, which is followed by a transient (5-10min) depression of synaptic activity. The mechanisms for synaptic depression after SD are incompletely understood. We examined the relative contributions of action potential failure and adenosine receptor activation to the suppression of evoked synaptic activity in murine brain slices. Focal micro-injection of potassium chloride (KCl) was used to induce SD and synaptic potentials were evoked by electrical stimulation of Schaffer collateral inputs to hippocampal area Cornu Ammonis area 1 (CA1). SD was accompanied by loss of both presynaptic action potentials (as assessed from fiber volleys) and field excitatory postsynaptic potentials (fEPSPs). Fiber volleys recovered rapidly upon neutralization of the extracellular direct current (DC) potential, whereas fEPSPs underwent a secondary suppression phase lasting several minutes. Paired-pulse ratio was elevated during the secondary suppression period, consistent with a presynaptic mechanism of synaptic depression. A transient increase in extracellular adenosine concentration was detected during the period of secondary suppression. Antagonists of adenosine A1 receptors (8-cyclopentyl-1,3-dipropylxanthine [DPCPX] or 8-cyclopentyl-1,3-dimethylxanthine [8-CPT]) greatly accelerated fEPSP recovery and abolished increases in paired-pulse ratio normally observed after SD. The duration of fEPSP suppression was correlated with both the duration of the DC shift and the area of tissue depolarized, consistent with the model that adenosine accumulates in proportion to the metabolic burden of SD. These results suggest that in brain slices, the duration of the DC shift approximately defined the period of action potential failure, but the secondary depression of evoked responses was in large part due to endogenous adenosine accumulation after SD.

Contribution of astrocyte glycogen stores to progression of spreading depression and related events in hippocampal slices.

Spreading depression (SD) is a wave of coordinated cellular depolarization that propagates slowly throughout brain tissue. SD has been associated with migraine aura, and related events have been implicated in the enlargement of some brain injuries. Selective disruption of astrocyte oxidative metabolism has previously been shown to increase the propagation rate of SD in vivo, but it is currently unknown whether astrocyte glycogen stores make significant contributions to the onset or propagation of SD. We examined SD in acutely-prepared murine hippocampal slices, using either localized microinjections of KCl or oxygen and glucose deprivation (OGD) as stimuli. A combination of glycogenolysis inhibitors 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) and 1-deoxynojirimycin (DNJ) increased the propagation rates of both high K(+)-SD and OGD-SD. Consistent with these observations, exposure to l-methionine-dl-sulfoximine (MSO) increased slice glycogen levels and decreased OGD-SD propagation rates. Effects of glycogen depletion were matched by selective inhibition of astrocyte tricarboxylic acid (TCA) cycle activity by fluoroacetate (FA). Prolonged exposure to reduced extracellular glucose (2 mM) has been suggested to deplete slice glycogen stores, but significant modification SD of propagation rate was not observed with this treatment. Furthermore, decreases in OGD-SD latency with this preexposure paradigm appeared to be due to depletion of glucose, rather than glycogen availability. These results suggest that astrocyte glycogen stores contribute to delaying the advancing wavefront of SD, including during the severe metabolic challenge of OGD. Approaches to enhance astrocyte glycogen reserves could be beneficial for delaying or preventing SD in some pathologic conditions.

Intracellular Zn2+ increases contribute to the progression of excitotoxic Ca2+ increases in apical dendrites of CA1 pyramidal neurons.

Sustained intracellular Ca(2+) elevation is a well-established contributor to neuronal injury following excessive activation of N-methyl-d-aspartic acid (NMDA)-type glutamate receptors. Zn(2+) can also be involved in excitotoxic degeneration, but the relative contributions of these two cations to the initiation and progression of excitotoxic injury is not yet known. We previously concluded that extended NMDA exposure led to sustained Ca(2+) increases that originated in apical dendrites of CA1 neurons and then propagated slowly throughout neurons and caused rapid necrotic injury. However the fluorescent indicator used in those studies (Fura-6F) may also respond to Zn(2+), and in the present work we examine possible contributions of Zn(2+) to indicator signals and to the progression of degenerative signaling along murine CA1 dendrites. Selective chelation of Zn(2+) with N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) significantly delayed, but did not prevent the development and progression of sustained high-level Fura-6F signals from dendrites to somata. Rapid indicator loss during the Ca(2+) overload response, which corresponds to rapid neuronal injury, was also not prevented by TPEN. The relationship between cytosolic Zn(2+) and Ca(2+) levels was assessed in single CA1 neurons co-loaded with Fura-6F and the Zn(2+)-selective indicator FluoZin-3. NMDA exposure resulted in significant initial increases in FluoZin-3 increases that were prevented by TPEN, but not by extracellular Zn(2+) chelation with Ca-EDTA. Consistent with this result, Ca-EDTA did not delay the progression of Fura-6F signals during NMDA. Removal of extracellular Ca(2+) reduced, but did not prevent FluoZin-3 increases. These results suggest that sustained Ca(2+) increases indeed underlie Fura-6F signals that slowly propagate throughout neurons, and that Ca(2+) (rather than Zn(2+)) increases are ultimately responsible for neuronal injury during NMDA. However, mobilization of Zn(2+) from endogenous sources leads to significant neuronal Zn(2+) increases, that in turn contribute to mechanisms of initiation and progression of progressive Ca(2+) deregulation.

Modulation of the amplitude of NAD(P)H fluorescence transients after synaptic stimulation.

In brain slices, excitatory synaptic stimulation results typically in transient initial decreases in NAD(P)H fluorescence, followed by longer-lasting NAD(P)H increases that overshoot pre-stimulus NAD(P)H levels before returning slowly to baseline. We concluded recently that mitochondrial metabolism (rather than NADH generation by glycolysis) was responsible for the "overshoot" phase of responses evoked in murine hippocampal slices. The present study examined factors that may influence the amplitude of the overshoot phase, without necessarily directly influencing mitochondrial or glycolytic metabolism. The amplitudes of overshoots were influenced strongly by changes in pre-stimulus NAD(P)H fluorescence levels produced by a prior electrical stimulus. In contrast, these changes in pre-stimulus redox state had little effect on the amplitude of evoked initial NAD(P)H decreases. Resting NAD(P)H fluorescence levels differed significantly across sub-regions of each slice, however, this is not due to differences in resting redox state, and the relative amplitude of NAD(P)H overshoots were not different in different slice regions. Exposure to an A1 receptor agonist (CPA) reduced the amplitude of postsynaptic potentials, and preferentially reduced the amplitude of NAD(P)H overshoots, before initial oxidizing components of biphasic transients were reduced significantly. These results suggest that amplitudes of NAD(P)H overshoots may not be good quantitative measures of the intensity of a discrete stimulus, under some conditions where the stimulus is small relative to recent activity in the slice. Comparison of flavoprotein autofluorescence with NAD(P)H levels seems useful when making quantitative comparisons of responses from different regions of slices, where optical properties and ongoing activity may be substantially different.

Calcium-dependent NMDA-induced dendritic injury and MAP2 loss in acute hippocampal slices.

Excessive glutamate receptor stimulation can produce rapid disruption of dendritic morphology, including dendritic beading. We recently showed that transient N-methyl-d-aspartic acid (NMDA) exposure resulted in irreversible loss of synaptic function and loss of microtubule associated protein 2 (MAP2) from apical dendrites. The present study examined the initiation and progression of dendritic injury in mouse hippocampal slices following this excitotoxic stimulus. NMDA exposure (30 microM, 10 min) produced irregularly shaped dendritic swellings, evident first in distal apical dendrite branches, and later (20-90 min) involving most proximal dendrites. Over the same time course, immunoreactivity for the microtubule-associated protein MAP2 was progressively lost from apical dendrites, and increased in CA1 somata. This damage and MAP2 loss was Ca2+-dependent, and was not reversible within the time course of these experiments (90 min post-NMDA washout). Formation of regularly-spaced, spherical dendritic varicosities (dendritic beading) was rarely observed, except when NMDA was applied in Ca2+-free ACSF. Under these conditions, beading appeared predominant in interneurons, as assessed from experiments with GAD67-GFP (Deltaneo) mice. Ca2+-removal was associated with significantly better preservation of dendritic structure (MAP2) following NMDA exposure, and other ionic fluxes (sensitive to Gd3+ and spermine) may contribute to residual damage occurring in Ca2+-free conditions. These results suggest that irregularly shaped dendritic swelling is a Ca2+-dependent degenerative event that may be quite different from Ca2+-independent dendritic beading, and can be a predominant type of injury in CA1 pyramidal neurons in slices.

GABA(B)-receptor modulation of short-term synaptic depression at an excitatory input to murine hippocampal CA3 pyramidal neurons.

GABA(B) agonists inhibit excitatory transmission to hippocampal CA3 neurons during low frequency stimulation. We examined whether GABA(B) receptor activation can also enhance synaptic efficacy, when investigated at an input with high initial release probability. Short-term depression of field excitatory postsynaptic potential (EPSP) amplitude was observed during trains of stimuli applied to associational/commissural inputs (10-50 Hz; 22 degrees C). Baclofen (10 microM) reduced the amplitude of initial EPSPs in a train, and also reduced the degree of short-term depression. EPSPs recorded late in a train were significantly larger in baclofen than those recorded in control solution. These dual effects were mimicked by another selective GABA(B) agonist (SKF 97541, 10 microM), and abolished by a GABA(B)-selective antagonist (SCH 50911, 20 microM). The effects of baclofen were similar at a higher recording temperature (32 degrees C), where short-term depression was observed at higher stimulation frequencies. These results are consistent with the idea that a reduction of transmitter release probability could increase the fidelity of high-frequency transmission at this input, an effect that could help account for excitatory effects of GABA(B) agonists in some seizure models.

Isolation and characterization of resident macrophages from the smooth muscle layers of murine small intestine.

Macrophages within the murine tunica muscularis were isolated and cultured for physiological studies. Following dispersion, macrophages were identified by phagocytotic activity of fluorescein isothiocyanate (FITC)-dextran. Immediately following isolation, macrophages were rounded and possessed fluorescent granula but developed a ramified shape after 3-4 days in culture. Resident and cultured macrophages were immunopositive for F4/80 and I-Ad/I-Ed. Greater than 90% of F4/80 positive cultured cells were FITC-dextran positive. Macrophages had resting membrane potentials (RMP) of -33.3 +/- 1.5 mV after 1 day in culture, which increased to -53.9 +/- 4.4 mV after 3-4 days. The change in RMP was associated with the development of an inward rectifying K+ current, and a decrease in a voltage-dependent, inactivating outward current. After 3-4 days in culture the inflammatory mediated substances adenosine triphosphate (ATP), platelet-activating factor and bacterial lipopolysaccharide induced increases in cytoplasmic Ca2+ ([Ca2+]i). Forskolin suppressed the ATP-induced increase in [Ca2+]i. Macrophages exhibited oxidative bursts, measured by oxidation of dihydrorhodamine-123 to rhodamine-123. Oxidative bursts coincided with a reduction in intracellular pH. Macrophages expressed a proton conductance that may participate in pH maintenance during reactive oxygen production. These results suggest that resident macrophages in the intestine may play a role in the immunological protection of the gut.

Strain-dependent differences in calcium signaling predict excitotoxicity in murine hippocampal neurons.

Commonly used inbred murine strains differ substantially in their vulnerability to excitotoxic insults. We investigated whether differences in dendritic Ca(2+) signaling could underlie the differential vulnerability of C57BL/6 (resistant to kainate excitotoxicity) and C57BL/10 strains (vulnerable). A striking difference was found in fine dendrite Ca(2+) responses after kainate exposure. Ca(2+) signals in distal dendrites were large in C57BL/10 neurons, and, if a threshold concentration of approximately 1.5 microm was reached, a region of sustained high Ca(2+) was established in the distal dendritic tree. This region then served as an initiation site for a degenerative cascade, producing high Ca(2+) levels that slowly spread to involve the entire neuron and led to cell death. Dendritic Ca(2+) signals in C57BL/6 neurons were much smaller and did not trigger these propagating secondary responses. Strain differences in dendritic Ca(2+) signaling were also evident after tetanic stimulation of Schaffer collaterals. Ca(2+) responses were much larger and peaked earlier in distal dendrites of C57BL/10 compared with those in C57BL/6. Neurons from both strains had similar membrane properties and responded to kainate with intense action potential firing. Degenerative Ca(2+) responses were seen in both strains if soma Ca(2+) could be sustained above 1.5 microm. The early phases of secondary Ca(2+) responses were attributable to Ca(2+) influx and were abolished rapidly by buffered zero Ca(2+) saline. Taken together, these data indicate that the substantial difference in Ca(2+) signals in fine distal dendrites and in the initiation of spreading secondary responses may underlie the selective vulnerability of these neurons to excitotoxic insults.

Ca2+ signaling in gerbil CA3 hippocampal neurons following transient in vivo ischemia.

Using the gerbil model of post-ischemic neuron death in the hippocampal CA1 region, it was recently shown that there is a strong down-regulation of voltage-gated Ca2+ influx in neurons examined at 2 days after the ischemic insult (Connor, J.A., Razani-Boroujerdi, S., Greenwood, A.C., Cormier, R.J., Petrozzino, J.J. and Lin, R.C., Reduced voltage-dependent Ca2+ signaling in CA1 neurons after brief ischemia in gerbils, J. Neurophysiol., 81 (1999) 299-306). The aim of the present study was to determine whether a similar change occurs in pyramidal neurons of the CA3 region that are relatively resistant to transient ischemia. In vitro intracellular recordings and fluorometric Ca2+ measurements were made from CA3 neurons in coronal slices prepared from controls and 1 or 2 days following in vivo ischemia. In slices from control and post-ischemic animals, the electrophysiological properties of CA3 neurons were consistent with significant voltage-gated Ca2+ influx, leading to spike frequency adaptation. Quantitative results indicated no significant difference in Ca2+ transients evoked by action potential trains. This Ca2+ signaling was compared with responses in CA1 neurons from the same preparations, which showed substantially diminished Ca2+ influx at 2 days post-ischemia. These findings suggest that diminished Ca2+-signaling is not a general feature of pyramidal neurons following ischemia, but is characteristic of neurons destined to die.

Rebound excitation and alternating slow wave patterns depend upon eicosanoid production in canine proximal colon.

1. We tested the hypothesis that eicosanoid production could be related to the long-duration slow waves that occur after brief periods of inhibitory neurotransmission (rebound excitation) and the alternating patterns of long- and short-duration slow waves observed in the canine proximal colon. 2. Electrical field stimulation of colonic muscles inhibited slow waves during the stimulus and a long-duration slow wave occurred after the stimulus. Indomethacin reduced the post-stimulus response without affecting the inhibitory response. 3. ATP or 2-methylthio-ATP produced post-stimulus rebound responses similar to the response to field stimulation. Indomethacin inhibited the rebound response caused by ATP or 2-methylthio-ATP. 4. Alternating patterns consisting of long- and short-duration slow waves occurred spontaneously in some colonic muscles. These patterns could also be induced with acetylcholine. 5. Indomethacin, acetylsalicylic acid and ibuprofen abolished the alternating pattern and shifted the bimodal distribution of slow wave durations toward an intermediate duration. 6. Patch clamp experiments on isolated colonic myocytes showed that indomethacin blocked L-type Ca2+ currents. The effects of indomethacin on rebound excitation and alternating slow waves were accomplished at concentrations that blocked cyclooxygenase activity without significantly inhibiting L-type Ca2+ currents. 7. The results demonstrate that rebound excitation and alternating slow wave patterns in the canine colon have similar dependence on endogenous eicosanoid production. Rebound excitation may result from reduced production of an inhibitory eicosanoid during inhibitory nerve stimulation, and the alternating pattern may result from oscillations in eicosanoid production as a function of changes in cytoplasmic Ca2+ during long and short slow waves.

Evidence that nitric oxide acts as an inhibitory neurotransmitter supplying taenia from the guinea-pig caecum.

Nitric oxide synthase-containing nerve fibres are abundant within taenia of the guinea-pig caecum, but there is little previous evidence supporting a direct role for nitric oxide (NO) in responses to enteric inhibitory nerve stimulation. In this study we have attempted to identify an NO-dependent component of inhibitory transmission in isolated taenia coli. Isometric tension was recorded in the presence of atropine and guanethidine (both 1 microM). Tone was raised with histamine (1 microM), and intrinsic inhibitory neurons stimulated using either a nicotinic agonist (1,1-dimethyl-4-phenylpiperazinium iodide; DMPP) or electrical field stimulation (EFS). DMPP (1-100 microM) produced concentration-dependent biphasic relaxations, comprising an initial peak relaxation followed by a sustained relaxation. Responses to DMPP were antagonized by tetrodotoxin (1 microM) or apamin (0.3 microM) and abolished by hexamethonium (300 microM). L-nitro-arginine (L-NOARG; 100 microM) and oxyhaemoglobin (2%) both significantly reduced sustained relaxations produced by DMPP. EFS (5 Hz, 30 s) also produced biphasic relaxations. Both L-NOARG and an inhibitor of soluble guanylate cyclase (ODQ, 1-10 microM) reduced the sustained component of EFS responses. Two NO donors, sodium nitroprusside (SNP) and diethylenetriamine-nitric oxide adduct (DENO), produced concentration-dependent relaxations. Responses to SNP and DENO were antagonized by ODQ (1 microM) and by apamin (0.3 mM). These results suggest that NO contributes directly to a component of inhibitory transmission in guinea-pig taenia coli. The actions of NO appear to be mediated via cyclic GMP synthesis, and may involve activation of small conductance calcium activated K+ channels. A role for NO is most evident during sustained relaxations evoked by longer stimulus trains or chemical stimulation of intrinsic neurons.

Action potential-dependent calcium transients in myenteric S neurons of the guinea-pig ileum.

Simultaneous intracellular microelectrode recording and Fura-2 imaging was used to investigate the relationship between intracellular calcium ion concentration ([Ca2+]i) and excitability of tonic S neurons in intact myenteric plexus of the guinea-pig ileum. S neurons were impaled in myenteric ganglia, at locations near connections with internodal strands. The calcium indicator Fura-2 was loaded via the recording microelectrode. The estimated [Ca2+]i of these neurons was approximately 95 nM (n = 25). Intracellular current injection (200 ms pulses, 0.2 nA, delivered at 0.05 Hz) resulted in action potential firing throughout the stimulus pulse, accompanied by transient increases in [Ca2+]i (to approximately 240 nM, n = 12). Increasing the number of evoked action potentials by increasing stimulus duration (100-500 ms) or intensity (0.05-0.3 nA) produced correspondingly larger [Ca2+]i transients. Single action potentials rarely produced resolvable [Ca2+]i events, while short bursts of action potentials (three to five events) invariably produced resolvable [Ca2+]i increases. Some neurons demonstrated spontaneous action potential firing, which was accompanied by sustained [Ca2+]i increases. Action potential firing and [Ca2+]i increases were also observed by activation of slow synaptic input to these neurons, in cases where the slow depolarization initiated action potential firing. Action potentials (evoked or spontaneous) and associated [Ca2+]i transients were abolished by tetrodotoxin (1 microM). Omega-conotoxin GVIA (100 nM) reduced [Ca2+]i transients by approximately 67%, suggesting that calcium influx through N-type calcium channels contributes to evoked [Ca2+]i increases. The S neurons in this study showed prominent afterhyperpolarizations following bursts of action potential firing. The time-course of afterhyperpolarizations was correlated with the time-course of evoked [Ca2+]i transients. Afterhyperpolarizations were blocked by tetrodotoxin and reduced by omega-conotoxin GVIA, suggesting that calcium influx through N-type channels contributes to these events. The electrical properties of Fura-2-loaded neurons were not significantly different from properties of neurons recorded without Fura-2 injection, suggesting that Fura-2 injection alone does not significantly influence the electrical properties of these cells. These data indicate that myenteric S neurons in situ show prominent, activity-dependent increases in [Ca2+]i. These events can be generated spontaneously, or be evoked by intracellular current injection or synaptic activation. [Ca2+]i transients in these neurons appear to involve action potential-dependent opening of N-type calcium channels, and the elevation in [Ca2+]i increase may underlie afterhyperpolarizations and regulate excitability of these enteric neurons.

Topographical and electrophysiological characteristics of highly excitable S neurones in the myenteric plexus of the guinea-pig ileum.

1. Most intracellular electrical recordings from myenteric neurones have been made from the centre of large ganglia. In this study, we examined the electrophysiological properties of neurones at the corners of large ganglia close to internodal strands and in microganglia. 2. Of 150 neurones in these locations: 111 were tonic S neurones; 9 were phasic S neurones and 30 were AH neurones. 3. Tonic S neurones were characterized by: (i) low resting membrane potentials (-50 +/- 1 mV, mean +/- s.e.m.); (ii) high input impedance (522 +/- 23 MOmega); (iii) low threshold for action potential (AP) generation (0.012 +/- 0.004 nA); (iv) firing of APs throughout a depolarizing pulse (duration <= 1 s) and one to four APs following a hyperpolarizing pulse and (v) spontaneous fast excitatory postsynaptic potentials (FEPSPs). A substantial proportion of tonic S neurones (43 %) also fired APs spontaneously (7.6 +/- 0.6 Hz; range, 0.3-19 Hz). All APs were blocked by tetrodotoxin (1 microM). 4. Tonic S neurones were subclassified, according to their post-stimulus responses, as SAH or SAD neurones. Following a burst of APs, SAH neurones exhibited a prominent after-hyperpolarization (duration, 711 +/- 10 ms) and SAD neurones an after-depolarization (duration, 170 +/- 10 ms). The after-hyperpolarization was reduced in four of ten neurones by apamin (0.3 microM). 5. FEPSPs were evoked in 20 of 38 S neurones by electrical stimulation applied both oral and anal to the recording site. Repetitive stimuli evoked slow excitatory postsynaptic potentials (SEPSPs) in some tonic S neurones. 6. Three functional classes of S neurones were identified after injection of neurobiotin through the recording microelectrode: (i) longitudinal muscle motor neurones, (ii) short circular muscle motor neurones, and (iii) ascending interneurones. 7. In conclusion, there appears to be topographical organization of highly excitable, tonic S neurones within the myenteric plexus, since, in contrast to other S neurones, they can be readily impaled in myenteric ganglia close to internodal strands and in microganglia.

Effects of a novel guanylate cyclase inhibitor on nitric oxide-dependent inhibitory neurotransmission in canine proximal colon.

1. Previous studies suggested that nitric oxide (NO) may cause hyperpolarization and relaxation of canine colonic smooth muscle by both cGMP-dependent and cGMP-independent mechanisms. This hypothesis was tested using 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a novel inhibitor of NO-stimulated guanylate cyclase. 2. In the presence of histamine (30 microM), atropine and indomethacin (both at 1 microM), electrical field stimulation of intrinsic neurons (EFS; 5 Hz) produced inhibition of phasic contractile activity that is due to NO synthesis. ODQ caused a concentration-dependent block of this response (10 nM to 10 microM). 3. Inhibitory junction potentials (IJPs) due to NO synthesis were recorded from muscle cells located near the myenteric border of the circular muscle layer, using intracellular microelectrodes. IJPs were abolished by ODQ (1-10 microM). 4. EFS (10-20 Hz) produced frequency-dependent inhibition of electrical slow waves recorded from cells located near the submucosal surface of the circular muscle layer. This inhibition is due to NO synthesis, and it was abolished by ODQ (1-10 microM). 5. Hyperpolarization and relaxation produced by an NO donor, sodium nitroprusside, were abolished by ODQ pretreatment (1-10 microM). In contrast, inhibitory responses to 8-Br-cGMP (1 mM) were unaffected by ODQ. 6. ODQ alone (1-10 microM) had no significant effect on spontaneous electrical or phasic contractile activity. In tissues pre-treated with L-NAME (300 microM), ODQ decreased the amplitude of spontaneous or histamine-stimulated phasic contractile activity. 7. These results suggest that electrical and mechanical effects of endogenously released and exogenously applied NO in canine colon are largely due to cGMP synthesis by ODQ-sensitive soluble guanylate cyclase. No evidence to support a direct (cGMP-independent) mechanism of NO action was found. ODQ also appears to cause a non-specific inhibition of muscle contractile activity; however, this effect does not contribute to block of NO-dependent effects.

Regulation of citrulline recycling in nitric oxide-dependent neurotransmission in the murine proximal colon.

1. We investigated the contribution of nitric oxide (NO) to inhibitory neuromuscular transmission in murine proximal colon and the possibility that citrulline is recycled to arginine to maintain the supply of substrate for NO synthesis. 2. Intracellular microelectrode recordings were made from circular smooth muscle cells in the presence of nifedipine and atropine (both 1 microM). Electrical field stimulation (EFS, 0.3-20 Hz) produced inhibitory junction potentials (i.j.ps) composed of an initial transient hyperpolarization (fast component) followed by a slow recovery to resting potential (slow component). 3. L-Nitro-arginine-methyl ester (L-NAME, 100 microM) selectively abolished the slow component of i.j.ps. The effects of L-NAME were reversed by L-arginine (0.2-2 mM) but not by D-arginine (2 mM). Sodium nitroprusside (an NO donor, 1 microM) reversibly hyperpolarized muscle cells. This suggests that NO mediates the slow component of i.j.ps. 4. L-Citrulline (0.2 mM) also reversed the effects of L-NAME, and this action was maintained during sustained exposures to L-citrulline (0.2 mM). This may reflect intraneuronal recycling of L-citrulline to L-arginine. 5. Higher concentrations of L-citrulline (e.g. 2 mM) had time-dependent effects. Brief exposure (15 min) reversed the effects of L-NAME, but during longer exposures (30 min) the effects of L-NAME gradually returned. In the continued presence of L-citrulline, L-arginine (2 mM) readily restored nitrergic transmission, suggesting that during long exposures to high concentrations of L-citrulline, the ability to generate arginine from citrulline was reduced. 6. Aspartate (2 mM) had no effect on i.j.ps, the effects of L-NAME, or the actions of L-citrulline in the presence of L-NAME, L-Citrulline (0.2-2 mM) alone had no effect on i.j.ps under control conditions. 7. S-methyl-L-thiocitrulline (10 microM), a novel NOS inhibitor, blocked the slow component of i.j.ps. The effects of this inhibitor were reversed by L-arginine (2 mM), but not by L-citrulline (2 mM). 8. These results suggest that i.j.ps in the murine colon result from release of multiple inhibitory neurotransmitters. NO mediates a slow component of enteric inhibitory neurotransmission. Recycling of L-citrulline to L-arginine may sustain substrate concentrations in support of NO synthesis and this pathway may be inhibited when concentrations of L-citrulline are elevated.

Involvement of nitric oxide in inhibitory neuromuscular transmission in equine jejunum.

OBJECTIVES: To evaluate the role of nitric oxide (NO), vasoactive intestinal peptide (VIP), and a transmitter acting through an apamin-sensitive mechanism in mediating inhibitory transmission in the equine jejunal circular muscle, and to determine the distribution of VIP-and NO-producing nerve fibers in the myenteric plexus and circular muscle. PROCEDURE: Circular muscle strips were suspended in tissue baths containing an oxygenated modified Krebs solution and attached to isometric force transducers. Responses to electrical field stimulation (EFS), tetrodotoxin, the NO antagonists L-N-nitro-arginine-methyl-ester (L-NAME) and N-nitro-L-arginine, apamin, VIP, authentic NO, and the NO donar sodium nitroprusside were tested. Immunostaining for VIP-like and NADPH diaphorase histochemical staining were performed on paraformaldehyde fixed tissue. RESULTS: Subpopulations of myenteric neurons and nerve fibers in the circular muscle were positive for NADPH diaphorase and VIP-like staining. EFS caused a frequency-dependent inhibition of contratile activity. Tetrodotoxin prevented the EFS-induced inhibition of contractions. L-NAME (200 microM) and apamin (0.3 microM) significantly (P < 0.01) reduced EFS-stimulated inhibition of contractile activity at most frequencies tested. The effects of L-NAME and apamin were additive. In their combined presence, EFS induced excitation instead of inhibition (196.7% increase at 5 Hz, n = 28, P < 0.01). Inhibition of contractile activity by EFS was mimicked by sodium nitroprusside. Authentic NO (3-6 microM) abolished contractile activity. VIP induced a dose-dependent inhibition of contractile activity (89.1 +/- 6.3% reduction at approximately 0.3 microM, n = 16). Antagonism of NO synthesis did not alter the response to VIP. CONCLUSION: NO, VIP, and a substance acting through an apamin-sensitive mechanism appear to comediate inhibitory transmission in the equine jejunal circular muscle. CLINICAL RELEVANCE: These findings may suggest new therapeutic targets for motility disorders, such as agents that inhibit the synthesis or actions of NO.

Activation of delayed rectifier potassium channels in canine proximal colon by vasoactive intestinal peptide.

1. Vasoactive intestinal peptide (VIP) inhibits phasic contractions and tone of gastrointestinal smooth muscles. This study examines electrical mechanisms that may mediate the inhibitory actions of VIP. 2. Electrical slow waves were recorded from canine proximal colon circular muscles. VIP (0.1 microM) decreased basal slow wave frequency but had no effect on amplitude or duration. When slow waves were enhanced with Bay K 8644 (1 microM), VIP decreased slow wave duration and inhibited contractions. 3. VIP inhibited slow waves and phasic contractions stimulated by tetraethylammonium chloride (TEA; 10 mM), but did not significantly reduce events stimulated by 4-amino-pyridine (4-AP; 10 mM). 4. Whole-cell outward currents were recorded from isolated myocytes, using the amphotericin B perforated patch technique. VIP (1 microM) increased charybdotoxin-insensitive outward currents. 5. Single voltage-dependent K+ channels were recorded in cell-attached patches. VIP increased reversibly the open probability, mean open time and mean burst duration of 4-AP-sensitive, charybdotoxin-insensitive K+ channels (KDR1). Two additional 4-AP- and charybdotoxin-insensitive K+ channels (approximately 90 pS and < 4 pS) were also observed in these patches, but were not significantly affected by VIP. 6. In summary, the effects of VIP on electrical slow waves may be due, in part, to activation of 4-AP-sensitive, 'delayed rectifier' K+ channels. Activation of these channels may contribute to premature slow wave repolarization, reduced Ca2+ entry, and inhibition of contractile force.

Involvement of nitric oxide in neuromuscular transmission in canine proximal colon.

The evidence is very strong that NO serves as a neurotransmitter in some autonomic neurons. In the canine proximal colon, NOS is localized in fibers and varicosities of inhibitory motor neurons that course through the muscle layers. Excitation of inhibitory neurons enhances Ca2+ entry into varicosities and activates NOS. In the GI tract enteric inhibitory neurons not only possess the ability to synthesize NO, they may also recycle the by-product, citrulline, back to arginine, thus sustaining inhibitory neurotransmission. NO appears to diffuse freely from nerve terminals and into nearby postjunctional cells. Interstitial cells appear to be innervated by nerves that release NO, and postjunctional effects in these cells include production of cGMP and synthesis of additional NO. In smooth muscle cells NO signals are transduced by guanylate cyclase, the production of cGMP, and activation of protein kinase G, but direct stimulation by NO of some cellular effectors, such as K+ channels also appears to play a role. Responses of smooth muscle cells include activation of K+ channels, inhibition of Ca2+ channels, and a reduction in the sensitivity of the contractile apparatus to Ca2+. All of these factors may contribute to the reduction in mechanical activity produced by stimulation of enteric inhibitory neurons. NO-dependent neurotransmission is critical for many of the physiological processes of the GI tract, such as relaxation of sphincters, gastric accommodation, and receptive relaxation during feeding, and the descending inhibition arc of the peristaltic reflex.

Detection of nitric oxide release from canine enteric neurons.

Previous pharmacological and immunohistochemical studies have suggested that nitric oxide (NO) is a mediator of enteric neuromuscular transmission in the canine proximal colon. The present study demonstrates the release of nitric oxide (NO) and/or its oxidation products (collectively termed NOx) following stimulation of intrinsic neurons with the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP). Strips of muscularis externa, including the ganglionated myenteric and submucosal plexuses were suspended under tension in modified Krebs solution. DMPP (1-100 microM) produced concentration-dependent inhibition of circular muscle contractile activity and this effect was antagonized by L-nitroarginine methyl ester or L-nitroarginine (100 microM). Tetrodotoxin (TTX, 1 microM) significantly reduced mechanical responses to DMPP (10 microM) but had no effect on responses to high concentrations of DMPP (100 microM). Aliquots of the bathing medium were assayed for NOx after regeneration of NO from NO2- and/or NO3-. NO was measured as chemiluminescence produced by reaction with ozone. Detection was linear over the range 6-25,000 pmol of added NO2- or NO3-. In the absence of drugs, a basal release of 1113.5 +/- 100.4 pmol NOx/g tissue (n = 27) was detected after a 30-min incubation period. DMPP (1-100 microM) stimulated a concentration-dependent increase in NOx to 5099.9 +/- 430 pmol/g per 30 min (n = 17) at 100 microM. NOx release was inhibited by an inhibitor of nitric oxide synthase activity (N(G)-monomethyl-L-arginine) or by reduction in extracellular Ca2+ concentration. DMPP-stimulated NOx accumulation was significantly reduced, but not abolished by TTX (1 microM). These results provide further evidence that NO is released following stimulation of intrinsic neurons in canine proximal colon and support previous suggestions that nicotinic agonists may directly stimulate terminals of NO-releasing neurons.