Ference) by atropine in the PBS and AAV2nNOSshRNA groups.Discussion The current study, utilizing interference RNA technology, makes the following novel contributions. First, the AAV2nNOSshRNA, used for the first time in this study, downregulated expression of nNOS locally while not influencing expression of eNOS, which, being found in immediate proximity of neurons expressing nNOS (Lin et al. 2007), could also contribute NO?to cellular transmission at the site. The shRNA affected nNOS in NTS with no discernible pathological changes in the tissue as a result of injections. As further evidence of selectivity of the shRNA, we found that, in AZD3759 site addition to eNOS, other neuronal markers in NTS were not affected. Thus there was no change in glutamate receptors, in vesicular glutamate transporters, in a marker of catecholamine neurons, in neurofilament, or in immunolabelling for NTS neurons with PGP9.5. Furthermore, there was no sign of inflammation in NTS after treatment. Decreased expression of nNOS in NTS led to attenuated baroreflex activity suggesting that NO?from nNOS serves an excitatory function in baroreflex transmission at the NTS level. However, at the site in NTS where we altered expression of nNOS only reflex tachycardia, seen when blood pressure was acutely lowered, was attenuated while reflex bradycardia in response to acutely raised arterial pressure was not. Attenuation of reflex tachycardia was to the same degree as that achieved by systemic administration of propranolol, which blocks sympathetic chronotropic cardiac effects. In addition, propranolol did not further reduce reflex tachycardic effects in animals treated with nNOSshRNA. This finding suggests that nNOSshRNA had reduced sympathetic chronotropic effects on the heart to such an extent that pharmacological blockade of sympathetic effects mediated through cardiac -receptors produced no further reduction in those sympathetic chronotropic events. Thus, although some effect mediated through altered withdrawal of parasympathetic toneCwhen baroreceptors were unloaded cannot be completely excluded, the data are most consistent with loss of nNOS in the NTS having maximally, if not exclusively, affected the sympathetic limb of the baroreflex. In analysing baroreflex function we utilized both linear regression analysis (reported in this paper) and logistic analysis with sigmoid curve Necrosulfonamide price fitting (not shown in this paper). For each method we analysed changes in HR with respect to changes in MAP given that plotting changes as opposed to plotting raw data for HR and MAP provides better regression and curve fitting (Head McCarty, 1987). With the former analysis we found that animals treated with nNOSshRNA manifested significant changes in baroreflex responses over the full range of decreases in AP with resulting reflex increases in HR and of increases in AP with resulting reflex decreases in HR. With sigmoid curve fitting we found a similar trend that did not reach significance. However, sigmoid curve fitting after logistic analysis of data did not allow separation of the reflex tachycardic from the reflex bradycardic limbs of the reflex as did linear regression and thus failed to identify changes that only occurred in reflex tachycardia. Although Head McCarty (1987) urged use of sigmoid curve fitting applied when they had achieved a full range of changes in blood pressure (60 to 160 mmHg) and heart rate, we avoided such full range intentionally in our study given that other publicati.Ference) by atropine in the PBS and AAV2nNOSshRNA groups.Discussion The current study, utilizing interference RNA technology, makes the following novel contributions. First, the AAV2nNOSshRNA, used for the first time in this study, downregulated expression of nNOS locally while not influencing expression of eNOS, which, being found in immediate proximity of neurons expressing nNOS (Lin et al. 2007), could also contribute NO?to cellular transmission at the site. The shRNA affected nNOS in NTS with no discernible pathological changes in the tissue as a result of injections. As further evidence of selectivity of the shRNA, we found that, in addition to eNOS, other neuronal markers in NTS were not affected. Thus there was no change in glutamate receptors, in vesicular glutamate transporters, in a marker of catecholamine neurons, in neurofilament, or in immunolabelling for NTS neurons with PGP9.5. Furthermore, there was no sign of inflammation in NTS after treatment. Decreased expression of nNOS in NTS led to attenuated baroreflex activity suggesting that NO?from nNOS serves an excitatory function in baroreflex transmission at the NTS level. However, at the site in NTS where we altered expression of nNOS only reflex tachycardia, seen when blood pressure was acutely lowered, was attenuated while reflex bradycardia in response to acutely raised arterial pressure was not. Attenuation of reflex tachycardia was to the same degree as that achieved by systemic administration of propranolol, which blocks sympathetic chronotropic cardiac effects. In addition, propranolol did not further reduce reflex tachycardic effects in animals treated with nNOSshRNA. This finding suggests that nNOSshRNA had reduced sympathetic chronotropic effects on the heart to such an extent that pharmacological blockade of sympathetic effects mediated through cardiac -receptors produced no further reduction in those sympathetic chronotropic events. Thus, although some effect mediated through altered withdrawal of parasympathetic toneCwhen baroreceptors were unloaded cannot be completely excluded, the data are most consistent with loss of nNOS in the NTS having maximally, if not exclusively, affected the sympathetic limb of the baroreflex. In analysing baroreflex function we utilized both linear regression analysis (reported in this paper) and logistic analysis with sigmoid curve fitting (not shown in this paper). For each method we analysed changes in HR with respect to changes in MAP given that plotting changes as opposed to plotting raw data for HR and MAP provides better regression and curve fitting (Head McCarty, 1987). With the former analysis we found that animals treated with nNOSshRNA manifested significant changes in baroreflex responses over the full range of decreases in AP with resulting reflex increases in HR and of increases in AP with resulting reflex decreases in HR. With sigmoid curve fitting we found a similar trend that did not reach significance. However, sigmoid curve fitting after logistic analysis of data did not allow separation of the reflex tachycardic from the reflex bradycardic limbs of the reflex as did linear regression and thus failed to identify changes that only occurred in reflex tachycardia. Although Head McCarty (1987) urged use of sigmoid curve fitting applied when they had achieved a full range of changes in blood pressure (60 to 160 mmHg) and heart rate, we avoided such full range intentionally in our study given that other publicati.