Baroreflex transmission did so after inhibition of the NMDA-type glutamate receptor while sympathetic elements of baroreflex transmission were spared, thus suggesting that the latter was mediated through XAV-939 site actions at non-NMDA receptors in NTS. However, as noted we have found that cardiovascular responses to local application of NMDA itself in the NTS are blocked by pharmacological inhibition of nNOS in NTS. Thus, our studies cannot eliminate the possibility that alteration of sympathetic effects by nNOS shRNA occurs through effects on neurons expressing NMDA receptors. In fact, it is likely that is the case in that we have found a high degree of colocalization of nNOS and NMDA receptors in NTS neurons (Lin Talman, 2002). The physiological effects of nNOSshRNA in NTS are likely due to a local effect rather than an effect of the shRNA at a distant site. We know from our earlier studies (Lin et al. 2011) that AAV2 is retrogradely transported to the NG where it may transduce signals uniformly in neurons within that ganglion. Indeed in this study nNOS was downregulated in ganglionic neurons. Thus the decrease in nNOS expression in the NTS after shRNA application could have happened at both presynapticand postsynaptic sites. Although we cannot completely exclude a contribution to the physiological effects by changes in nNOS in baroreceptor afferents, it would be unlikely that altering function of those NG neurons would differentially affect one element of baroreflex transmission at the primary neuron. Such differentiation would be more likely at the second order neuronal level in the NTS. The absence of changes in nNOS expression at other brainstem sites that share reciprocal connections with NTS likewise supports the local action in NTS. Our studies further show that upregulation of nNOS in NTS does not enhance baroreflex responses to changes in arterial pressure. We interpret that finding as indicating that, in the basal state, NO?production through nNOS is already optimal and further enhancement of the capacity for NO?synthesis does not then alter physiological responses that are under NO?control. Our findings do not conflict with those from other labs that suggested opposite (inhibitory) baroreflex effects of NO?when the bioactive molecule is synthesized by eNOS. However, such differences in responses when the same freely diffusible (Garthwaite, 1995; Lancaster, 1996) agent is released from two separate sources in close proximity to each other do raise a question about the mechanism that could mediate the two effects. Given that nNOS and eNOS containing structures lie immediately adjacent to each other in the NTS it is unlikely that those differences can be explained simply by a different site of action of NO?released from one vs. the other enzyme. As we and others have pointed out, physiological actions of NO?may depend upon packaging of the molecule into a larger bioactive Necrosulfonamide web substance such as a nitrosothiol (Ohta et al. 1997; Lipton et al. 2001). If that were the case, one could conjecture that different S-nitrosothiols may be the mediators of differing effects of NO?in NTS control of baroreflex functions. In summary, our findings provide anatomical, neurochemical and physiological validation of a newly developed shRNA for nNOS and with that new tool they provide support for an excitatory role of NO?C2012 The Authors. The Journal of PhysiologyC2012 The Physiological SocietyJ Physiol 590.nNOS and the baroreflexsynthesized by nNOS in modula.Baroreflex transmission did so after inhibition of the NMDA-type glutamate receptor while sympathetic elements of baroreflex transmission were spared, thus suggesting that the latter was mediated through actions at non-NMDA receptors in NTS. However, as noted we have found that cardiovascular responses to local application of NMDA itself in the NTS are blocked by pharmacological inhibition of nNOS in NTS. Thus, our studies cannot eliminate the possibility that alteration of sympathetic effects by nNOS shRNA occurs through effects on neurons expressing NMDA receptors. In fact, it is likely that is the case in that we have found a high degree of colocalization of nNOS and NMDA receptors in NTS neurons (Lin Talman, 2002). The physiological effects of nNOSshRNA in NTS are likely due to a local effect rather than an effect of the shRNA at a distant site. We know from our earlier studies (Lin et al. 2011) that AAV2 is retrogradely transported to the NG where it may transduce signals uniformly in neurons within that ganglion. Indeed in this study nNOS was downregulated in ganglionic neurons. Thus the decrease in nNOS expression in the NTS after shRNA application could have happened at both presynapticand postsynaptic sites. Although we cannot completely exclude a contribution to the physiological effects by changes in nNOS in baroreceptor afferents, it would be unlikely that altering function of those NG neurons would differentially affect one element of baroreflex transmission at the primary neuron. Such differentiation would be more likely at the second order neuronal level in the NTS. The absence of changes in nNOS expression at other brainstem sites that share reciprocal connections with NTS likewise supports the local action in NTS. Our studies further show that upregulation of nNOS in NTS does not enhance baroreflex responses to changes in arterial pressure. We interpret that finding as indicating that, in the basal state, NO?production through nNOS is already optimal and further enhancement of the capacity for NO?synthesis does not then alter physiological responses that are under NO?control. Our findings do not conflict with those from other labs that suggested opposite (inhibitory) baroreflex effects of NO?when the bioactive molecule is synthesized by eNOS. However, such differences in responses when the same freely diffusible (Garthwaite, 1995; Lancaster, 1996) agent is released from two separate sources in close proximity to each other do raise a question about the mechanism that could mediate the two effects. Given that nNOS and eNOS containing structures lie immediately adjacent to each other in the NTS it is unlikely that those differences can be explained simply by a different site of action of NO?released from one vs. the other enzyme. As we and others have pointed out, physiological actions of NO?may depend upon packaging of the molecule into a larger bioactive substance such as a nitrosothiol (Ohta et al. 1997; Lipton et al. 2001). If that were the case, one could conjecture that different S-nitrosothiols may be the mediators of differing effects of NO?in NTS control of baroreflex functions. In summary, our findings provide anatomical, neurochemical and physiological validation of a newly developed shRNA for nNOS and with that new tool they provide support for an excitatory role of NO?C2012 The Authors. The Journal of PhysiologyC2012 The Physiological SocietyJ Physiol 590.nNOS and the baroreflexsynthesized by nNOS in modula.
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