Abstract
GABA neurons in the VTA and SNc play key roles in reward and aversion through their local inhibitory control of dopamine neuron activity and through long-range projections to several target regions including the nucleus accumbens. It is not clear whether some of these GABA neurons are dedicated local interneurons or if they all collateralize and send projections externally as well as making local synaptic connections. Testing between these possibilities has been challenging in the absence of interneuron-specific molecular markers. We hypothesized that one potential candidate might be neuronal nitric oxide synthase (nNOS), a common interneuronal marker in other brain regions. To test this, we used a combination of immunolabelling (including antibodies for nNOS that we validated in tissue from nNOS-deficient mice) and cell type-specific virus-based anterograde tracing in mice. We found that nNOS-expressing neurons, in the parabrachial pigmented (PBP) part of the VTA and the SNc were GABAergic and did not make detectable projections, suggesting they may be interneurons. In contrast, nNOS-expressing neurons in the rostral linear nucleus (RLi) were mostly glutamatergic and projected to a number of regions, including the lateral hypothalamus (LH), the ventral pallidum (VP), and the median raphe (MnR) nucleus. Taken together, these findings indicate that nNOS is expressed by neurochemically- and anatomically-distinct neuronal sub-groups in a sub-region-specific manner in the VTA and SNc.
Highlights
Around one third of neurons in the VTA and SNc are GABAergic (Olson and Nestler, 2007; Nair-Roberts et al., 2008)
It is well established that there is a large population of neuronal nitric oxide synthase (nNOS)-expressing neurons in the interpeduncular nucleus (IPN), which lies just ventral to the VTA and was well suited to act as a positive control (Vincent and Kimura, 1992; Rodrigo et al, 1994; Ascoli et al, 2008)
The second antibody (Cell Signaling; 4234; AB_10694499) displayed some sparse immunoreactivity “spots” that could be mistaken for cell bodies within the VTA and SNc (Fig. 1B), which were present in the nNOS-deficient tissue, suggesting that they were non-specific
Summary
Around one third of neurons in the VTA and SNc are GABAergic (Olson and Nestler, 2007; Nair-Roberts et al., 2008). September/October 2018, 5(5) e0381-18.2018 1–19 sumption (Omelchenko and Sesack, 2009; Tan et al, 2012; van Zessen et al, 2012) They send long-range axonal projections to several target regions, including the nucleus accumbens where they can regulate associative learning (Brown et al, 2012; Taylor et al, 2014). It is not clear whether a subset of these GABA neurons are dedicated local interneurons or if they all collateralize and send projections externally as well as making local synaptic connections. We hypothesized that some of these discrepant findings may have arisen because of non-specific immunolabelling To address this directly, we tested three different nNOS antibodies for reliable immunolabelling in the VTA and SNc, using tissue from nNOS-deficient mice as a control.
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