Nitric oxide has been implicated in central autonomic control by a large number of studies. These indicate that both endothelial (eNOS) and neuronal (nNOS) isoforms play an important role in the nucleus tractus solitarii (NTS), the primary afferent relay of the autonomic nervous system. In 2002, we postulated that NO generated by eNOS located within the NTS endothelium is able modulate the activity of the surrounding neuronal network and affect cardio-vascular control (Paton et al., 2002). In support of this hypothesis, chronic inhibition of eNOS activity in NTS with an adenoviral vector expressing a dominant negative eNOS increased the sensitivity of baroreflex in normotensive animals (Waki et al., 2003) and also lowered arterial pressure in hypertensive rats (Waki et al., 2006). As the baroreflex pathway is under powerful inhibitory GABAergic control in NTS, we hypothesised that NO could increase release of GABA by local interneurones, which would explain why its blockade elevated baroreflex sensitivity. This was investigated in organotypic slice cultures where the interneurones were visualised using virally mediated gene expression with GAD67 promoter. We found that low nanomolar concentrations of NO were able to increase intracellular Ca2+ in these neurones via cADP-rybose-(cADPR)-mediated signalling. Moreover, the effect was particularly evident in axons, which are the sites primarily involved in GABA release (Wang et al., 2006). Thus, it is likely that NO -cADPR-mediated sensitisation of intracellular Ca2+ stores could lead to increased Ca2+ induced Ca2+ release thereby potentiating GABA exocytosis, and GABAergic inhibition. In addition, NO potentiated monosynaptically evoked inhibitory post-synaptic potentials (IPSPs) recorded intracellularly in acute NTS slices werealso dependent on cADPR. In contrast, much lower NO concentrations were required for enhancing evoked glutamatergic excitatory (E) PSP (threshold concentration ~0.4 nM) compared to GABAergic IPSP (threshold concentration ~ 3 nM), suggesting that GABAergic inhibition could be recruited under conditions of increased NO generation (Wang et al., 2007). These results are consistent with our hypothesis of vascular-neuronal signalling, but whether eNOS in the brain is exclusive to the endothelium or is also expressed at lower levels by glia is disputed. To properly address this issue a cell-specific gene knock-down experiment would be ideal. We have therefore generated and validated such a system which is based on lentiviral expression of miRNA-like transcripts. The system permits targeted gene knock-down in either astrocytes or neurones and expression can be switched off using administration of doxicycline into the drinking water. Up to ~85% knock-down of a target protein has been achieved in astrocytes and ~50% in neurones in vivo. As a first step we have tested the outcome of knocking down nNOS in NTS neurones. This resulted in a gradual elevation of the systolic blood pressure 14-21 days after virus injection. Crucially, this manipulation does not affect NO released from either afferents or other central structures projecting to NTS because these vectors are not transported retrogradely. We believe that this is the first clear demonstration of an endogenous role for nNOS in the chronic control of arterial pressure at the level of NTS.