Basal retinal neuron

In Bulla, CAPs are most abundant in the late subjective night and decrease nearly twofold in conductance at dawn. Circadian cycles in membrane potential, which are caused by rhythmic changes in membrane conductance, affect the membrane permeability of calcium ions in the BRNs. To test the necessity of increased calcium ion conductance, researchers treated BRNs with EGTA, an aminopolycarboxylic acid that “traps” calcium ions and prevents their movement into the cell. Previous experiments showed that light exposure and inducing potassium conductance were sufficient to shift circadian rhythm; however, when EGTA was used in addition to these methods, no circadian shift was observed. This experiment provided support for the necessity of calcium permeability in circadian pacemaker function and ability to match organism activity to environmental light cycles in the Bulla. Together with the ease of re-identification of BRNs, ease of recording, and reliability of circadian CAP production, Bullas are an excellent model to provide insight into the chronobiology, or “time-biology,” of molluscs.

Bulla, specifically, Bulla gouldiana, are model organisms to study circadian rhythms as they are easy to maintain in a laboratory settings, have re-identifiable large cells, and display measurable circadian behaviors. Compared to other mollusks and organisms of study in circadian biology, the retina of the Bulla contains a small number of relatively large neurons. This allows for easier surgical manipulation and distinction between populations of cells and longer intracellular recording time. Since chronobiological studies often require multiple days of recording, the ease of access facilitates data acquisition.

Discovery of cell-autonomous pacemaker

The eyes of the Bulla exhibit circadian rhythms in the firing of their optic nerves; however, up until the end of the 20th century, the mechanism was unknown. In the 1980s, Gene D. Block conducted extensive research on circadian rhythms in Bulla eyes. In 1982, he ruled out photoreceptor cells as regulators of circadian rhythms, and concluded the pacemaking cells must be in the retina. In 1984, Block and a team of students conducted 74 hour intracellular recordings of Bulla BRNs in constant darkness. This experiment was crucial in determining whether BRNs functioned as a circadian pacemaker, as a loss of rhythm in darkness would indicate the absence of an internal clock and that Bulla are simply following light cues. They found clear circadian rhythms in both membrane potential and frequency of action potential firing in constant darkness, which directly correlated with rhythmic CAP firing in the optic nerve. This indicated that the network that comprised the BRNs and produced CAPs contained a circadian oscillator. In 1993, Block’s lab isolated BRNs in vitro and found that they individually exhibited the same circadian rhythmicity in membrane conductance, thereby generating oscillations in membrane potential. Subsequently, the BRNs proved to be sufficient at photoentrainment, the process of matching an organism’s activity to the light and dark patterns in its environment. The findings that indicated BRNs were both individually intrinsically rhythmic and photo-entrainable led to the conclusion that BRNs function as cell-autonomous pacemakers in the Bulla. This was the first time individual neurons were found to be capable of functioning as circadian pacemakers.

The discovery of a cell autonomous pacemaker served as a stepping stone in the field of chronobiology, as there were few circadian pacemakers known to be localized to specific regions, let alone to specific autonomous cells in vitro. It would take another sixteen years before cell-autonomous pacemakers comprising the mammalian clock in the SCN were discovered.