Visualising vagal afferent neurons and their terminals whilst silencing TRPV1

Vagal afferent neuronal cell bodies are located within the nodose or jugular ganglia and have central projections which terminate in the nucleus tractus solitarii (NTS) within the brainstem. These vagal neurons also have peripheral projections which innervate diverse thoracic structures including the oesophagus and respiratory tract. These peripheral projections terminate within different layers of these structures and are composed of morphological specialisations, including intraganglionic laminar nerve endings (IGLEs) and intramuscular arrays. Functionally these vagal afferents can be distinguished based on nerve conduction velocities as distinct subtypes of C-fibres and A-fibres. Furthermore, these afferents can also be subcategorised by distinct responses to various stimuli, including low-threshold stretch and capsaicin application (Kollarik et al. 2010a).

Previous investigations have generally approached such studies either morphologically or functionally. For morphological studies neuronal tracing dyes have been utilized, as they are transported along nerve fibres, allowing visualization of nerve structures after tissue processing with immunohistochemical techniques. As such neuronal tracers have been instrumental in specifically identifying visceral innervating neurons and their projections.

In vivo ‘retrograde’ tracing, via injections of neuronal tracers into visceral organs, have been utilised to investigate central projections of vagal afferents in the NTS (Wank & Neuhuber, 2001). Furthermore, such tracing techniques have also allowed the identification of vagal afferent cell bodies within the nodose or jugular ganglia which innervate specific regions of the viscera (Wank & Neuhuber, 2001; Brierley et al. 2009). In vivo‘anterograde’ tracing, via injections of various tracers into the nodose ganglia, has allowed the morphology of vagal afferent peripheral projections to be established (Berthoud et al. 1997; Wang & Powley 2000). More recently a functional approach, followed immediately by an in vitro‘anterograde’ tracing approach advanced the field by allowing a direct correlation of the function and structure of vagal afferent peripheral endings (Zagorodnyuk & Brookes, 2000). For example, this allowed a determination that functional low-threshold stretch sensitive vagal oesophageal mechanoreceptors appear anatomically as IGLEs (Zagorodnyuk & Brookes, 2000). Furthermore, the utilization of gene knockout mice has been particularly useful in demonstrating roles of select ion channels in vagal afferents, particularly where selective antagonists have been lacking (Brierley et al. 2009). In this issue of The Journal of Physiology, Kollarik et al. (2010b) demonstrate that with one technique, recombinant viral technology, the benefits of these other studies can be combined to label vagal afferent neurons and their central and peripheral projections whilst down-regulating specific ion channels.

Firstly, the authors demonstrated that using safe non-replicating adeno-associated viral vectors (AAVs) coupled with enhanced green fluorescent protein (eGFP) they could transfect vagal afferent neurons in vivo. Once effectively transfected these neurons and fibres express eGFP, detected as a green fluorescence signal, allowing for morphological identification and assessment. Injections of these AAV directly into the nodose ganglion resulted in strong eGFP production in the majority of nodose ganglion neurons 4–5 weeks following injection. Using a neurofilament marker the authors showed that both myelinated A-fibres (neurofilament positive) and unmyelenated C-fibres (neurofilament negative) were transfected by this AAV technique.

Secondly, the authors demonstrated that by injecting AAVs into the oesophageal wall, the viral genetic material could be transported from the peripheral terminals back to the neuronal cell body residing in the vagal ganglia. Indeed the authors demonstrated similar levels of neurons expressing eGFP 7 weeks after injection as they did in their previous studies using a traditional (DiI) neuronal tracer. Thirdly, the authors demonstrated that by using this AAV technique, individual afferent nerve fibres could be followed to their endings in the oesophageal and tracheal wall. In particular, IGLEs were visualised within the myenteric plexus of the oesophagus. What is perhaps most remarkable is that these afferent peripheral nerve terminals were readily visualized in living tissues, without the need for histological processing. Moreover, this fluorescence signal was stable without a reduction in intensity, despite fluorescence light exposure of over 30 min! The authors also showed that AAV injections into the nodose ganglia revealed the central terminations of these vagal neurons in the brainstem, as demonstrated by bright fluorescence of terminals in the NTS. Interestingly, no cell bodies in the vagal motor nuclei or any other area of the brainstem displayed eGFP, indicating efferent fibres were not labelled by this technique. Furthermore, no fluorescent oesophageal motor endplates were observed in the oesophageal striated muscle. Taken together these two findings led the authors to conclude that this AAV technique selectively transfected vagal sensory afferent neurons, without transfecting autonomic efferent nerves.

Having successfully demonstrated the validity of this elegant AAV technique the authors then set about determining if selected genes, in this case TRPV1, could be knocked down in these vagal afferent neurons using AAV vectors encoding shRNA. To do this they injected AAV with a TRPV1-targeting shRNA, or one with a negative control (shRNA) into the nodose ganglia. After 5 weeks they harvested the nodose ganglia, dissociated the neurons and collected only the neurons expressing eGFP, which were identified by green fluorescence. Using quantitative RT-PCR they determined that TRPV1 mRNA expression was reduced by greater than 95% in the neurons transfected with the TRPV1-targeting shRNA compared with the control eGFP neurons transfected with control shRNA. Critically this reduction in TRPV1 expression did not result in the altered expression of the other channels investigated, namely Nav1.3, Nav1.7 and Nav1.8 or ASIC3.

The authors then elegantly demonstrated that reduced TRPV1 mRNA expression translated to a reduced functional role for TRPV1. Using patch clamp techniques, the authors demonstrated that the percentage of capsaicin responsive neurons was greatly reduced in neurons transfected with TRPV1-targeting shRNA (9%) compared with control shRNA (46%). They also showed that in the neurons that did respond to capsaicin there was an 85% reduction in the response to capsaicin in nodose neurons transfected with AAV encoding TRPV1-targeting shRNA. Importantly, the TRPV1-targeting shRNA did not alter the resting membrane potential, action potential threshold, or input resistance of these neurons compared with control shRNA. Furthermore, responses to KCl and bradykinin were comparable between control and TRPV1-targeting shRNA, indicating that TRPV1-targeting shRNA had a select effect only on TRPV1. Taken together these results clearly demonstrated the effectiveness of AAV TRPV1-targeting shRNA to suppress TRPV1 expression in vagal afferent neurons.

Overall, these findings document how one novel approach can have multifaceted benefits in assessing visceral afferent structure and function. Although, it appears that AAV tracing studies take longer (weeks to months) compared with other neuronal tracing techniques (days or 1 week), being able to selectively target a fluorescently identified neuronal cell body or afferent fibre which also has down-regulated channel function is particularly advantageous. This AAV coupled shRNA technique also enables such studies of down-regulated channel function to be conducted in any species, in this case the guinea-pig. However, it does come with the caveat that AAV coupled shRNA is applicable so long as the specific gene sequence for that particular species is known and is therefore able to be targeted via shRNA. Finally, as this AAV transfection method does not appear to label efferent fibres, it provides an ideal platform for combined morphological and functional studies of visceral afferent endings in numerous species. As such it may have some benefits over other established approaches (Zagorodnyuk & Brookes, 2000) as with the AAV technique afferent labelling is present before conducting the functional studies. The current study also highlights the need to embrace and implement new and developing technologies.