Retinal horizontal cells of goldfish (Carassius auratus) display subtype‐specific differences in spontaneous action potentials in situ

Abstract

Horizontal cells (HCs) are neurons of the outer retina, which provide inhibitory feedback onto photoreceptors and contribute to image processing. HCs in teleosts are classified into four subtypes (H1-H4), each having different roles: H1-H3 feed back onto different sets of cones, H4 feed back onto rods, and only H1 store and release the inhibitory neurotransmitter, γ-aminobutyric acid (GABA). Dissociated HCs exhibit spontaneous Ca 2+-based action potentials (APs), yet it is unclear if APs occur in situ, or if all subtypes exhibit APs. We measured intracellular Ca 2+ and report APs in slice preparations of the goldfish retina. In HCs furthest from photoreceptors (i.e., H3/H4), APs were less frequent, with greater duration and area under the curve (a measure of Ca 2+ flux). Next, we classified acutely dissociated HCs into subtypes by integrating the ratio of dendritic field size vs. soma size (r d/s). H1 and H2 subtypes had low r d/s values (<8); H3/H4 had high r d/s (>12). To verify this model, H1s were identified by immunoreactivity for GABA and 95% of these cells had an r d/s < 4. In Ca 2+ imaging experiments, as r d/s increased, AP duration and area under the curve increased, while frequency decreased. Our results demonstrate the presence of Ca 2+-based APs in the goldfish retina in situ and show that HC subtypes H1 through H4 exhibit progressively longer and less frequent spontaneous APs. These results suggest that APs may play an important role in inhibitory feedback, and may have implications for understanding the relative contributions of HC subtypes in the outer retina.

Seasonal changes in membrane structure and excitability in central neurons of goldfish (Carassius auratus) under constant environmental conditions

“…1A-D) based on morphological description from previous studies (Dowling et al, 1985;Tachibana, 1981;Country et al, 2021) and observed no apparent differences in morphology between summer and winter. Plotting the distribution of the ratio of dendritic field area to soma area provided an estimate of the subtypes present in the dissociation (Country et al, 2021). Calculation and plotting of the rd/s values of 80 neurons showed that the distribution of cells isolated during the summer and winter were similar, with median rd/s values of 7.2 and 6.2, respectively (Fig.…”

“…We identified all subtypes of horizontal cells (Fig. 1A-D) based on morphological description from previous studies (Dowling et al, 1985;Tachibana, 1981;Country et al, 2021) and observed no apparent differences in morphology between summer and winter. Plotting the distribution of the ratio of dendritic field area to soma area provided an estimate of the subtypes present in the dissociation (Country et al, 2021).…”

“…Ca 2+ -based action potentials in horizontal cells have been described in a number of species, and are present in isolated cells, tissue slices and intact retina (Johnston and Lam 1981; Tachibana, 1981; Shingai and Christensen, 1983; Lasater et al 1984; Murakami and Takahashi, 1987; Dixon et al, 1993; Takahashi et al 1993; Kreitzer et al, 2012; Country et al, 2019, 2021). Although a specific role for action potentials in horizontal cells has not been firmly established, they may be involved in Ca 2+ -dependent regulation of pH (Kreitzer et al, 2012), they may be important for light-to-dark transitions in membrane potential, or play a role in feedback inhibition of pre-synaptic photoreceptors (Country et al, 2019).…”

“…Morphometry of specific subtypes (i.e. H1, H2, H3 and H4) was performed following procedures described by Country et al (2021), which identifies subtypes based on the ratio of dendritic field area to soma area ( r d/s ). Briefly, an ellipse representing the approximate dendritic spread was calculated using the formula for area ( A ): This process was repeated for the soma, and the r d/s of the ellipse representing the dendritic field versus the ellipse representing the soma was measured in all cells.…”

“…Morphometry of specific subtypes (i.e. H1, H2, H3 and H4) was performed following procedures described by Country et al (2021), which identifies subtypes based on the ratio of dendritic field area to soma area (rd/s). Briefly, an ellipse representing the approximate dendritic spread was calculated using the formula for area (A):…”