Purinergic modulation of neuronal gap junction circuits in the CNS of the leech

Abstract

Gap junctions (GJs) are widely distributed in brains across the animal kingdom. To visualize the GJ-coupled networks of two major mechanosensory neurons in the ganglia of medicinal leeches, we injected these cells with the GJ-permeable tracer Neurobiotin. When diffusion time was limited to only 30 min, tracer coupling was highly variable for both cells, suggesting a possible modulation of GJ permeability. In invertebrates the innexins (homologs of vertebrate pannexins) form the GJs.Because extracellular adenosine triphosphate (ATP) modulates pannexin and leech innexin hemichannel permeability and is released by leech glial cells following injury, we tested the effects of bath application of ATP after the injection of Neurobiotin and observed a significant increase in the number of neurons tracer coupled to the sensory neurons. This effect required the elevation of intracellular Ca 2+ and could be produced by bath application of caffeine. Conversely, scavenging endogenous extracellular ATP with the ATPase apyrase decreased the number of coupled cells.ATP also increased electrical conductance and tracer permeability between the bilateral Retzius neurons. This modulatory effect of ATP on GJ coupling was blocked by siRNA knockdown of a P1-like adenosine receptor. Finally, exposure of leech ganglia to extracellular ATP induced a characteristic low frequency (<0.3 Hz) rhythmic bursting activity that was roughly synchronous among multiple neurons, a behavior that was significantly attenuated by the GJ blocker octanol. These findings highlight the mediation by ATP of a robust physiological mechanism for modifying neuronal circuits by rapidly recruiting neurons into active networks and entraining synchronized bursting activity.

Initial Variability and Time-Dependent Changes of Neuronal Response Features Are Cell-Type-Specific

“…In our dataset, the effect of the network on the T3 cell was clearly visible as postsynaptic potentials during the zero stimulation periods in the repeated control protocol ( Supplementary Figure 2 ). P cells also display neurobiotin coupling via gap junctions with several interneurons ( Segura et al, 2020 ), but we did not see any postsynaptic potentials in our P1 cell recordings. Rz cells are only coupled to their homolog Rz cell in the same ganglion ( Segura et al, 2020 ).…”

“…P cells also display neurobiotin coupling via gap junctions with several interneurons ( Segura et al, 2020 ), but we did not see any postsynaptic potentials in our P1 cell recordings. Rz cells are only coupled to their homolog Rz cell in the same ganglion ( Segura et al, 2020 ).…”

“…T cells also receive inhibitory and excitatory synaptic input from P and N cells via polysynaptic connections ( Burgin and Szczupak, 2003 ), as well as reafferent inhibition from the motor system during crawling ( Alonso et al, 2020 ). Furthermore, several interneurons are known to be connected with T cells via electrical synapses, many of which can be seen in neurobiotin stainings ( Kretzberg et al, 2016 ; Segura et al, 2020 ), and some of which are rectifying ( Firme et al, 2012 ). In our dataset, the effect of the network on the T3 cell was clearly visible as postsynaptic potentials during the zero stimulation periods in the repeated control protocol ( Supplementary Figure 2 ).…”

Cell anatomy and network input explain differences within but not between leech touch cells at two different locations

“…Gap junction permeable tracer injection into dorsal T cells revealed a highly variable number of electrically coupled neurons. This number depends on extracellular ATP, suggesting 10.3389/fncel.2023.1186997 modulation of network interactions (Segura et al, 2020). Hence, the modulation of gap junctions between pairs of T cells and other cell types probably contributes to the response variability between individual cells and maybe also over recording time.…”

“…Applying electrophysiological double recordings, dye injections for anatomical studies, and compartmental modeling, we investigate three potential factors for the observed response variability within and between T cell locations: (1) The time-dependent activity changes reported by Meiser et al (2019) and Scherer et al (2022) . (2) Synaptic input that the T cells receive from the unstimulated network ( Baylor and Nicholls, 1969b ; Scherer et al, 2022 ), and from a coupled T cell in the same ganglion ( Baylor and Nicholls, 1969b ; Meiser et al, 2019 ) (3) The anatomical structure ( Muller and McMahan, 1976 ; Segura et al, 2020 ), which we also compare to T1 cells. While all of them contribute to the variability within the T cells recorded at each of the soma locations, we rule out that any of these factors can exclusively explain the systematic difference in excitability between T2 and T3 cells.…”