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1 Department of Physiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK
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Abstract |
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0.4 nM (EC50
0.9 nM) for potentiating glutamatergic EPSPs but 3 nM for monosynaptic GABAergic IPSPs. Bath application of the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) abolished NOaq- and DEA/NO-induced potentiation of evoked EPSPs, IPSPs and depolarization. All NO actions were mimicked by the non-NO-dependent guanylate cyclase activator Bay 41-2272. The effects of NO on EPSPs and IPSPs persisted in cells where postsynaptic sGC was blocked by ODQ and therefore were presynaptic, owing to a direct modulation of transmitter release combined with depolarization of presynaptic neurones. Therefore, while lower concentrations of NO may be important for fine tuning of glutamatergic transmission, higher concentrations are required to directly engage GABAergic inhibition. This differential sensitivity of excitatory and inhibitory connections to NO may be important for determining the specificity of the effects of this freely diffusible gaseous messenger.
(Received 12 October 2006;
accepted after revision 20 November 2006; first published online 20 November 2006)
Corresponding author S. Kasparov: Department of Physiology, School of Medical Sciences, Bristol Heart Institute, University of Bristol, Bristol, BS8 1TD, UK. Email: sergey.kasparov{at}bristol.ac.uk
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Introduction |
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Further progress in understanding the functions of NO in the NTS is hampered by the lack of information about its intracellular effects on excitatory and inhibitory transmission. Microdialysis studies demonstrated that NO may increase glutamate release in the NTS (Lin et al. 2000) and other brain regions (Lawrence & Jarrott, 1993; Ohta et al. 1996; Watts et al. 2005), although in some studies the effect of NO on glutamate release was biphasic, e.g. excitatory/inhibitory (Segieth et al. 1995; Sequeira et al. 1997). In addition, a blocker of nitric oxide synthase (L-NAME) reduced the discharge of NTS neurones triggered by iontophoretic application of 
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; Dias et al. 2003). Although these observations suggest that NO facilitates glutamatergic transmission, the mechanism of this effect remains unknown.
The principal inhibitory transmitter in the brain is GABA, and in hypothalamus NO has been reported to potentiate GABAergic transmission (Li et al. 2003; Stern & Zhang, 2005). Recently, we have found that NO enhances transmitter release from NTS GABAergic neurones via an evolutionarily conserved cascade involving cGMP production and potentiation of Ca2+ release from cyclic adenosine diphosphate ribose (cADPR)/ryanodine-sensitive stores (Wang et al. 2006b).
So far, very few studies have attempted to relate specific cellular actions to NO concentrations. In almost all cases, NO donors have been used, but it is extremely difficult to deduce how much NO was actually released because, on the one hand, NO release is temperature- and time dependent and on the other, NO degradation and loss through diffusion are extremely rapid.
Taken together, currently available data indicate that in the NTS NO might be able to potentiate both excitatory and inhibitory transmission. If this is the case, are there any mechanisms which could help to translate its action into a physiologically relevant signal? We hypothesized that excitatory and inhibitory neurotransmission in the NTS might be differentially sensitive to NO concentrations. We addressed this possibility using patch clamp recordings from NTS neurones in acute slices using carefully controlled aqueous concentrations of NO.
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Methods |
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Experiments were performed using brainstem slices from young rats (Wistar; postnatal day 12–15) prepared as previously described (Kasparov & Paton, 1999). Briefly, a Wistar rat of either sex was terminally anaesthetized with halothane. The brain was quickly removed and immersed in ice-cold artificial cerebrospinal fluid (ACSF; for composition see Kasparov & Paton, 1999) saturated with carbogen (95% O2 and 5% CO2). The tissue block containing the NTS was glued onto the platform of a Vibroslice cutter (Campden Instruments Ltd). Transverse 250 µm thick slices were cut at the level of area postrema and just caudal to it and stored in ACSF bubbled continuously with carbogen at room temperature. Approximately 1 h later, a slice was transferred into the recording chamber where it was superfused at a continuously monitored speed of 2 ml min–1 at 31 ± 0.5°C. All recordings and calibrations were performed at this temperature. It took approximately 2 min to completely exchange the solution in the recording chamber.
Electrophysiological recordings
Recordings were made in a whole-cell patch clamp configuration from caudal NTS using pipettes pulled to a resistance of 3–5 M
filled with the following intracellular solution (mM): 130 potassium gluconate, 10 Hepes, 11 EGTA, 4 NaCl, 2 MgCl2, 1 CaCl2, 2 ATP, 0.5 GTP and 5 glucose, pH 7.3 adjusted using KOH and HCL. Recorded signals were amplified using an SEC-05LX amplifier (NPI, Tamm, Germany), and the data acquired and analysed using Spike2 software (CED, Cambridge, UK). Recordings were made in bridge mode. Positions of the recorded neurones were documented on predrawn schematic diagrams from Paxinos & Watson (1986).
The membrane resistance was calculated from the slopes of the middle portions of I–V plots using a series of hyperpolarizing and depolarizing current pulses (range, –0.12 to +0.02 nA; 0.5 s width; 0.3 Hz) delivered via the recording pipette.
In some of the experiments, synaptic potentials were evoked by electrical stimulation within the ipsilateral solitary tract (TS) via a concentric bipolar stimulating electrode (0.2 ms pulse width; 0.5–5 V; 0.3 Hz). The distance between the stimulating electrode and the recording site ranged from 0.3 to 1.0 mm. In some cells, TS stimulation evoked only EPSPs without discernable IPSPs. To avoid action potentials and voltage-related changes in evoked EPSPs, neurones were examined at holding potentials of –80 to –75 mV. Ipsilateral solitary tract-evoked EPSPs had a mean latency of 2.2 ± 0.1 ms (range, 1–4 ms; n = 65). Ipsilateral solitary tract-evoked IPSPs were recorded at holding potentials of –60 to –40 mV. Once chosen, holding potentials were kept unchanged for each individual cell for the duration of the recording. Under these conditions, GABAergic events appeared hyperpolarizing. Ipsilateral solitary tract-evoked IPSPs were, in most cases, polysynaptic (mean latency, 4.6 ± 0.3 ms; range, 1.9–7 ms; n = 79) and reversed at roughly –65 mV. Monosynaptic IPSP connections (invariable latency = 2 ms) were apparent in only < 10% of neurones recorded in this manner. Therefore, TS-evoked IPSPs usually disappeared in the presence of the 6-cyano-7-nitroxaline-2,3-dione (CNQX, a blocker of non-NMDA receptor-mediated fast glutamatergic transmission). To record monosynaptic IPSPs we applied 20 µM CNQX, stimulated electrically within the medial part of the NTS and blindly searched for neurones with CNQX-resistant IPSPs (typically 1 in 5–6 recorded neurones). Since evoked EPSPs recorded in the dorsal and medial NTS were mediated almost exclusively by non-NMDA receptors (Andresen & Yang, 1990), only CNQX was used to block polysynaptic connections. (See Supplementary information for more detail.) According to previous data from our laboratory, IPSPs recorded in the NTS were sensitive to the GABAA receptor blocker, which was bicuculline in most cases (Butcher et al. 1999). Therefore, in this study, we assumed that IPSPs were all mediated by GABAA receptors.
After a stabilization period of > 10 min, baseline cell activity was recorded for 3–5 min, followed by 10 min episodes of treatment with drugs and at least 10 minute washout periods. Measurements of average amplitude of 10 consecutive EPSPs or IPSPs were taken every 3 min (3rd, 6th and 9th minute after addition of drugs) for analysis using scripts written in Spike2 software.
Drug application, and preparation and application of NO aqueous solution
Pure NO gas was purchased from the British Oxygen Company Ltd. Diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO), a NO donor, was dissolved in alkaline solution (10 mM NaOH) as concentrated stock and stored at –80°C. Thawing was allowed only before use when it was diluted into ACSF and used immediately. The half-life of DEA/NO at 37°C is 2 min and at 22–25°C it is 16 min in 0.1 M phosphate buffer (pH 7.4). During the period of DEA/NO application, it was continuously added into the ACSF flow from a syringe which was at room temperature, so it may be expected that the degradation of DEA/NO in the syringe was moderate, while the bulk of NO release must have occurred already in the recording chamber. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), DEA/NO and CNQX were purchased from Sigma. Bay 41-2272 was kindly provided by Pharma Research Center, Bayer (Wuppertal, Germany).
Aqueous NO solution (NOaq) was prepared in a sealed flask (50 ml) which was first bubbled for 50 min with nitrogen to deoxygenate, followed by pure NO gas for 20 min. The concentration of the saturated NOaq at room temperature is about 2 mM (Archer et al. 1995; Hogg & Kalyanaraman, 1997; Ohkawa et al. 2001). Serial dilutions were then made in deoxygenated saline. Care was taken to minimize the risk of NO turning into gas phase by using sealed, airtight glass syringes containing deoxygenated saline (for more detail, see Wang et al. 2006a).
Statistical analysis
All values are expressed as means ± S.E.M. Differences were examined using Student's paired t test, Student's unpaired t test with Welch's correction, and one-way analysis of variance (ANOVA) with Tukey's post hoc test, as appropriate and indicated in the text. P < 0.05 was considered significant. Statistical evaluation was carried out using Microsoft Excel and GraphPad Prism.
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Results |
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One of the key objectives of this study was to establish whether different concentrations of NO may have different effects on excitatory and inhibitory transmission in the NTS. In order to estimate the effective concentration of NO at the recording site, we calibrated the recording chamber using an electrochemical NO sensor (Amino-700, Innovative Instruments, Inc., Tampa, FL, USA). This sensor is not pH dependent but sensitive to temperature. Before the measurements, the sensors (Amino-700 and Amino700-XL) were calibrated at room temperature using the standard calibration solution to generate NO, followed by calibration at the temperature used for recording. Owing to the limited sensitivity of the NO sensors, the lowest bath concentrations of NO (< 3 nM) could not be reliably measured directly. To overcome this problem, NO concentrations in the bath were assessed using higher nominal concentrations of NOaq stock (100, 500, 1000 and 10000 nM; Table 1) under identical conditions. The measurements were repeated several times on different experimental days and, in most cases, with more than one electrode. The measured NO concentrations in the chamber were plotted against the nominal concentrations of NOaq (as calculated using dilution factors) and fitted with a monoexponential function (Fig. 1). This function was used to forecast the values of bath NO concentrations at the lower end of the nanomolar range (Table 2) where NO sensors failed to detect NO reliably. All the concentrations quoted in the following text are based on the values obtained using this correction. It is important to realize that the function forecasted that at zero nominal infused NO, the NO concentration in the chamber should be close to nil, indicating that it correctly predicts the end point of the curve. In addition, because the higher four data points have been measured experimentally while the left end point of the curve can be postulated to be nil, the estimates within the shallow (left) end of this curve should be fairly accurate. We therefore acknowledge the limitations of the indirect estimates of the active concentration of NO, but believe that our calculations are fairly accurate. As for the exponential shape of the curve, it is consistent with the predictably non-linear loss of NO in the open-ended chamber through loss into the air and through NO oxidation when it contacts ACSF (see Supplementary material for further details).
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Overview of the neuronal populations recorded in this study
The NTS consists of multiple neuronal phenotypes belonging to a variety of reflex pathways, but to date no commonly accepted criteria may be applied to functionally identify them in a brain slice in vitro. When stimulating within TS, we subdivided the recorded cells (n
= 214) into two subsets: those which only displayed TS-evoked EPSPs (both mono- and polysynaptic) without detectable IPSPs and those with a mixture of TS-evoked IPSPs and EPSPs. All neurones tested here were located within the medial parts of caudal NTS. They had an average resting membrane potential (RMP) of –61.5 ± 3.8 mV (–50 to –72 mV), input resistance of 366 ± 28 M (209–707 M) and action potential amplitude > 50 mV.
Effects of NO on ‘passive’ membrane properties.
A depolarization of 3–4 mV was observed in many tested cells following exposure to both NOaq and DEA/NO (Table 3). The presence of NO-evoked depolarization did not appear to correlate with either TS-evoked EPSPs or EPSPs–IPSPs. For example, 1.1 nm NO caused a depolarization in five of eight neurones with EPSPs only and in four of five neurones with EPSPs–IPSPs. In addition, this effect of NOaq was not clearly concentration dependent (Table 3). Consistent with these observations, 0.1, 1 and 10 µM DEA/NO (corresponding to 15, 55 and 980 nm NO in perfusate) also induced a depolarization in 10 of 19, five of 13 and eight of 13 neurones, respectively (Table 3). However, NO-induced depolarization was not an artefact because it was repeatable (data not shown; also see Wang et al. 2006b) and could be prevented by the soluble guanylate cyclase (sGC) blocker ODQ (see subsection ‘Effects of NO on EPSPs and IPSPs in NTS are mediated by sGC’). In addition, a decrease in input resistance was observed in some neurones after application of NOaq and DEA/NO. This effect was not specific for either subpopulation of cells (i.e. those displaying evoked EPSPs only versus EPSPs–IPSPs) and appeared at a threshold of 1 nm NO. For example, 1.1 and 3 nM NO (using NOaq) decreased membrane input resistance by 10% (n
= 11/12 neurones, P < 0.01) and 11% (n
= 7/11 neurones, P < 0.05), respectively, while 0.4 and 0.8 nM had no effect. We could not establish any correlation between changes in input resistance and other effects of NO or DEA/NO.
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0.4 nM, NOaq significantly and concentration-dependently increased the amplitude of TS-evoked EPSPs (Fig. 2A and C) and IPSPs (Fig. 2B and C). As mentioned in the Methods, IPSPs recorded in this way were essentially in all cases polysynaptic. The EC50 for potentiation of EPSPs was 0.9 nM, and for polysynaptic IPSPs it was 1.0 nM (Fig. 2C). DEA/NO also potentiated evoked EPSPs and IPSPs significantly (Fig. 3A–C). Note, however, that the effects were essentially maximal already with 0.1 µM DEA/NO and did not significantly increase in spite of the large increase in DEA/NO (and end-point NO) concentrations. This indicates that the effects of NO on polysynaptic postsynaptic potential saturate somewhere near 15 nM.
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15 nM NO) increased the amplitude of evoked monosynaptic IPSPs by 27 ± 4% (P < 0.01; Fig. 4A) and 30 ± 6%, respectively (n
= 3, data not shown). Thus, compared with EPSPs, higher concentrations of NO are required for a direct effect on GABA release (Fig. 4A).
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55 nM NO, n
= 3; data not shown) on monosynaptic IPSPs were abolished by ODQ, indicating that these effects were all mediated by sGC.
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Presynaptic mechanisms are responsible for effects of NO on EPSPs and IPSPs.
Nitric oxide may potentiate evoked postsynaptic potential by enhancing transmitter release from presynaptic neurones (Wang et al. 2006b), but a change in the responsiveness of the postsynaptic receptors is also a possibility. To address this issue, we introduced ODQ into postsynaptic neurones via a patch pipette to block postsynaptic activation of sGC/cGMP signalling. Following 15 min diffusion of ODQ from the pipette into the neurone (10 µM in the pipette solution; note that this concentration completely eliminated all NO effects when used by bath), application of 1 µM DEA/NO (15 nm NO) still caused an increase in the amplitude of EPSPs by 30 ± 4% (n
= 5, P < 0.01; Fig. 6A), although no depolarization occurred in any of the cells. To further verify that the potentitation of monosynaptic IPSPs does not result from a postsynaptic action of NO, in similar experiments we applied ODQ in a group of cells recorded under the same conditions as described in subsection ‘Effect of NO on monosynaptic IPSPs’. In these cells, 1 µM DEA/NO still increased the amplitude of CNQX-resistant IPSPs by 22 ± 6% (n
= 7/8 neurones, P < 0.01; Fig. 6A and B) although, again, no depolarization occurred in these cells (not shown). Hence, blockade of sGC selectively in the postsynaptic cell removed its postsynaptic depolarizing action but did not prevent NO-induced potentiation of EPSPs and IPSPs. Moreover, any traces of ODQ in the extracellular media, released either from the patch electrode or from the recorded postsynaptic cell, have not affected these effects, leaving us with the conclusion that this effect is presynaptic.
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Discussion |
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We have established at least three distinct effects of NO in the NTS. First, it potentiates glutamatergic EPSPs; second, it potentiates GABA release via a direct action on GABAergic neurones but also via potentiation of polysynaptic excitatory connections. Third, NO can depolarize NTS neurones. It is generally accepted that low NO concentrations act primarily through the sGC/cGMP signal cascade. Consistent with this concept, all effects described in this study could be abolished by a bath-applied sGC blocker, ODQ (Fig. 5A–D). Further, the non-NO-dependent sGC activator Bay 41-2272 potentiated EPSPs and IPSPs in a manner similar to NOaq or NO donor (Fig. 5E), supporting a role for sGC in mediating both types of enhanced postsynaptic potential.
Mechanisms of NO potentiation of glutamatergic transmission
Activation of glutamatergic transmission in the NTS can induce baroreflex-like responses, such as a decrease in blood pressure and heart rate (Ohta & Talman, 1994; Dias et al. 2005). Microinjections of the NO precursor L-arginine evoked comparable responses which could be antagonized by glutamate receptor blockers (Lo et al. 1997). Moreover, microdialysis of NO solution elicited > 10-fold increases in the extracellular level of glutamate, although the concentrations of NO used in that study were extremely high (400 µM) and their dependence on cGMP was not demonstrated (Lin et al. 2000). Similar results were also published by Matsuo et al. (2001). These studies illustrate the ability of NO to potentiate glutamate release. The NO-induced increase in glutamatergic EPSPs in our experiments was reversible, concentration dependent, sGC/cGMP mediated, and it required extremely low NO concentrations (threshold < 1 nM). Interestingly, it reached plateau at as little as 15–50 nM NO, as may be concluded from the experiments with DEA/NO (Fig. 3). This suggests that extremely small quantities of NO may be released to carry out retro- and anterograde signalling in excitatory synapses under physiological conditions. These findings could explain why real-time visualization of NO release in the brain is still a major challenge.
It is unlikely that the NO-induced potentiation of EPSPs resulted primarily from presynaptic depolarization (e.g. increased excitability of glutamatergic connections) because potentiation of EPSPs was clearly dose dependent, while the depolarizing action of NO was not (Table 3). Moreover, 0.4 nM NO significantly potentiated EPSPs, at which concentration depolarization was overall negligible. Therefore, depolarization does not seem to be essential for the NO effect on EPSPs, although it may contribute to it to some extent. Most probably, potentiation of EPSPs in the NTS depends largely on the same mechanism as that described in hippocampus, where NO may release Ca2+ from ryanodine-sensitive stores (Lu & Hawkins, 2002), an action which may be expected to facilitate transmitter release. Indeed, we have recently found that NO effects on GABA release in the NTS are also mediated via such a mechanism (Wang et al. 2006b).
Nitric oxide potentiation of GABAergic transmission
Two types of IPSPs were recorded in this study. The TS-evoked IPSPs had a mean latency of 4.6 ± 0.3 ms and reversed at about –65 mV, consistent with the activation of GABAA receptors, and were similar to GABAergic IPSPs registered in NTS neurones by our previous reports (Butcher et al. 1999; Kasparov & Paton, 1999) and other laboratories (Jordan et al. 1988; Feldman & Felder, 1991; Nakagawa et al. 1991; Brooks et al. 1992; Andresen & Yang, 1995). In the absolute majority of cases, such TS-evoked IPSPs were polysynaptic because they could be abolished by CNQX. Therefore, potentiation of TS-evoked polysynaptic IPSPs by low concentrations of NO in our experiments most probably resulted from strengthening glutamatergic polysynaptic connections, and it is not surprising that the apparent EC50 for TS-evoked EPSPs and polysynaptic IPSPs was almost identical (Fig. 2C).
However, much higher NO concentrations (3 nM or more) were required to directly potentiate monosynaptic GABAergic IPSPs (Fig. 4). We did not establish the full concentration–response curve for the monosynaptic IPSPs because only one of five or six neurones exhibited these events, and this would require a very large number of recordings. Importantly, corrections used in this study to estimate effective bath NO concentrations may not account for the differences in threshold concentrations for potentiation of EPSPs and IPSPs. Without these corrections, the gap between these thresholds would have appeared to span two orders of magnitude (e.g. 1 versus 100 nM; Fig. 4A and Table 2).
Effects of NO on postsynaptic potential are presynaptic
We confirmed that the NO-induced potentiation of EPSPs and monosynaptic IPSPs are solely presynaptic by blocking sGC selectively in the postsynaptic cell. ODQ was introduced into the pipettes at the same concentration which blocked all NO effects when applied in the bath. In this case, ODQ failed to inhibit NO-induced potentiation of TS-evoked EPSPs or monosynaptic IPSPs. We are confident that the sGC inhibitor diffused adequately from the patch pipette into the neurones, since it prevented NO-induced depolarization. This result, together with the fact that NO releases Ca2+ from cADPR/ryanodine-sensitive stores in NTS GABAergic neurones (Wang et al. 2006b), demonstrates that the potentiation of synaptic transmission in the NTS is presynaptic. We did not use analysis of spontaneous miniature EPSPs or IPSPs for demonstration of the presynaptic action of NO in this study because the relative contribution of Ca2+ entry from the extracellular space versus release from the internal stores, such as those sensitive to cADPR, in generation of these potentials is unclear.
Nitric oxide-induced depolarization
As mentioned in Table 3, NO depolarized many cells, an action mediated by sGC. If NO tonically depolarizes NTS neurones in vivo, this might contribute to an enhanced excitability of neuronal circuits. For example, removal of such an effect may account for the reported attenuation in cardiovascular effects triggered by NTS injections of excitatory amino acids after NOS blockade in vivo (Lin et al. 1999). However, in our experiments depolarization was not clearly concentration dependent over the range of NO and DEA/NO concentrations used. Nevertheless, the depolarization appears genuine because it was reversible, and could be prevented by blockade of sGC (Fig. 5B) and mimicked by a non-NO-based NO activator, Bay 41-2272 (Fig. 5E). Lack of a clear dose dependency of this action might result from its very high sensitivity to NO, meaning that it was already close to maximum with the lowest doses used in our experiments. Alternatively, it could be a mixture of different actions which have different concentration dependencies or might only be specific to a proportion of the NTS neurones and these effects, when combined, would make revealing dose dependency difficult. Finally, it is possible that the ability of NO to depolarize an NTS neurone depends on the concentration of the sGC in its soma. The available immunohistochemical data indicate that, while sCG is abundant in the NTS, not all somata are positive for sGC (Lin & Talman, 2005). Nitric oxide-induced depolarization of only some NTS neurones but not others, coupled with strong voltage dependency of NMDA receptor-mediated currents, can explain why the in vivo effects of a NOS inhibitor, L-NAME, on NMDA-evoked discharge were so variable (Dias et al. 2003). It may be speculated that removal of NO-induced depolarization in only some of these cells affected their responsiveness to NMDA.
Concentration dependency of the action of NO: potential implications for pathway-specific modulation in the NTS
In light of our data, the physiological outcome of NO signalling may be quite complex, because within the same pool of neurones NO could potentiate both glutamatergic and GABAergic transmission. Some studies suggest that under conditions of maximal stimulation of endogenous NO production by nNOS from cerebellar neurones in vitro, the local NO concentration did not rise above 4 nM (Bellamy et al. 2002). If the same applies to the NTS then, according to the present results, this would be enough to reach almost maximal stimulation of EPSPs and polysynaptic IPSPs but have relatively little direct effect on GABA release. It is important to note that polysynaptic IPSPs are essentially as sensitive to NO as EPSPs (Fig. 2). Therefore, the reflex pathways where polysynaptic GABAergic connections are prominent might become inhibited, while pathways with little polysynaptic GABAergic inhibition might become strongly activated. Interestingly, barosensitive NTS neurones, which are involved in blood pressure homeostasis, typically have ample inhibitory inputs (McWilliam & Shepheard, 1988; Mifflin & Felder, 1988), consistent with unpublished observations from this laboratory. A considerable fraction (40%) of NTS neurones with inputs from pharyngo-oesphageal receptors also had clear IPSPs (Paton et al. 1999b). In contrast, IPSPs were detectable in only one of 39 cells which received inputs from abdominal viscera (Paton et al. 1999a) and in 12 of 58 (20%) of NTS chemoreceptive neurones (Mifflin, 1992). Hence, it is possible that when NO is released in the NTS after microinjection of an NO donor (Vitagliano et al. 1996; Lin et al. 1999; Paton et al. 2001), some reflex pathways, such as those activated from the gastrointestinal afferents, might be activated, while others, such as baroreflex pathway, might be inhibited (Paton et al. 2001).
Considering the effects of NO released from its endogenous sources in the NTS, one needs to take into account localization of nNOS and eNOS. Neuronal NOS is present in some NTS neurones and is also quite abundant in axonal fibres, although the origin of these axons is unclear at the present time (reviewed by Paton et al. 2005). Neuronal NOS and sGC colocalize in many NTS neurones (Lin & Talman, 2005), and inhibition of nNOS in the NTS has been reported to depress baroreceptor reflex (Talman & Dragon, 2004). These observations would be consistent with the primarily autocrine role of nNOS-derived NO in NTS. Given the very high sensitivity of glutamatergic transmission to NO as demonstrated by this study, this would require generation of only minute quantities of this gas. The bulk of eNOS in the NTS definitely resides in endothelium, while its presence in central neurones (Paton et al. 2001) remains somewhat elusive. Given the very high capillary density in the NTS (Gross et al. 1990) and the fact that in the brain NO only needs to diffuse 1–2 µm to reach a process of a GABA-containing cell (Rabhi et al. 1987; Ovtscharoff, 1992; Benagiano et al. 2001), it is possible that recruitment of this additional and distributed source of NO may be enough to reach local NO concentrations sufficient for direct activation of GABA exocytosis. When angiotensin II is microinjected into the NTS, it depresses the baroreceptor reflex, and this effect requires release of eNOS-derived NO (Paton et al. 2001). Moreover, when eNOS was chronically disabled in the NTS of spontaneously hypertensive rats, this resulted in an increase in the baroreceptor reflex sensitivity and a decrease in blood pressure (Waki et al. 2006). These observations are consistent with the ‘vascular–neuronal signalling’ in NTS, which we postulated in 2002 (Paton et al. 2002). We speculate that activation of the endothelial production of NO under some conditions might bring the local NO concentration to a level sufficient to activate GABAergic inhibition. This could shift the excitatory–inhibitory balance and have differential effects of multiple reflex pathways integrated within this nucleus.
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