Further evidence for ACh GABA cotransmission

Further evidence for ACh/GABA cotransmission had also been observed in the cortex. Extensive axonal arborization from cholinergic BF neurons exists throughout the cortical layers, allowing direct observation of cholinergic synaptic terminals. One study in the visual cortex of cat examined immunolabeling of ChAT and GABA in electron microscopy cryosections, for the purpose of identifying the extent of cholinergic innervations of cortical GABAergic interneurons. This study reported not only an enrichment for cholinergic terminals on GABA-immunoreactive postsynaptic cells, suggesting a preference of cholinergic Bax inhibitor peptide V5 sale for inhibitory interneurons, but also substantial co-labeling of presynaptic cholinergic terminals for both GABA and ChAT (Beaulieu and Somogyi, 1991). In addition, retrograde labeling of cortically projecting BF neurons with cholera toxin showed co-staining with markers for GABA release (Gritti et al., 1997). While many of these most likely represent a separate population of cortically-projecting GABAergic neurons, they are consistent with cortically projecting ChAT+ neurons also expressing GABA. In addition to receiving long-range cholinergic projections from basal forebrain, the cortex contains a small population of local, apparently cholinergic interneurons. These ChAT-expressing cells express the vasointestinal peptide (VIP), a common marker for a subclass of cortical interneuron, and share similar stereotyped morphology and laminar distribution (Consonni et al., 2009, Cauli et al., 2014). Only one study to date has attempted to directly address the function of these neurons, using paired recordings between cortical ChAT-expressing cells and neighboring pyramidal neurons (von Engelhardt et al., 2007). The authors found that these cells can influence signaling in adjacent pyramidal neurons indirectly by increasing spontaneous EPSCs via presynaptic nicotinic receptors. This study does not directly report a GABAergic output of these intrinsic cortical ChAT cells, but did report that a subset of them express GAD67. It seems likely therefore that these cells also release GABA, either onto a cell-type not well sampled by this study or with sparse connectivity not easily detected using paired recordings between presynaptic cholinergic neurons and postsynaptic pyramidal neurons. Beyond analysis of presynaptic markers, colocalization of postsynaptic receptors or scaffolding proteins could suggest individual synapses that are sensitive to both ACh and GABA. For example, in chick ciliary ganglion, ultrastructural analysis shows discrete clusters of both nAChRs and glycine receptors (GlyRs) directly opposing individual presynaptic specializations and overlapping with gephryin, an important postsynaptic scaffolding molecule for GABA receptors and GlyRs (Tsen, 2000). In cultured hippocampal neurons, nAChRs labeled by fluorophore-conjugated alpha-bungarotoxinin cluster around GABAergic synapses, both presynaptically and postsynaptically, and extensive codistrubution of nAChRs with GABA receptors can be observed at synapses and developing filopodia (Kawai et al., 2002, Zago et al., 2006). In the cortex, costaining of labeled basal forebrain axonal projections not only shows presynaptic terminals that contain both VGAT and VAChT protein, but also adjacent gephyrin-positive postsynaptic specializations abutting ∼30% of VAChT-labeled terminals, suggesting the possibility of dual ACh/GABA synapses (Henny and Jones, 2008). Similarly, GFP-labeled terminals of cortically-projecting cholinergic GP neurons also colocalize with gephryin, but not with PDS-95 (Saunders et al., 2015b). Despite these promising examples, however, very little ultrastructural evidence exists comparing the relative distribution of ACh and GABA receptors at central synapses which could provide more definitive support for cotransmission.
Evidence from optogenetics In the past, paired recordings between presynaptic and postsynaptic neurons were the gold standard to unambiguously demonstrate neurotransmitter corelease, ensuring that the different neurotransmitters were originating from the same presynaptic source. However, finding connected pairs of neurons can be very difficult, and this method is not feasible for long-range projections. In contrast, electrical stimulation of axonal fibers, which allows for activation of far more presynaptic cells, increases the chances of observing a postsynaptic response, but cannot distinguish between axons of different cell populations, making it difficult to determine whether distinct neurotransmitters came from the same or different cells. Cre-dependent optogenetic stimulation using ChR2 or its variants can activate many neurons of a genetically-defined subpopulation at once (Zhao et al., 2011), identifying classes of neurons that release multiple neurotransmitters but not proving that any individual neuron does so.