Extended Data Fig. 7: Characterization of the influence of abdominal ganglion circuitry (Crz neurons and dopaminergic neurons) on the CDNs.
From: Dopamine biases decisions by limiting temporal integration
a) The Crz neurons project throughout the abdominal ganglion, with processes closely apposed to those of the CDNs, both near their axons (left) and dendrites (right), though synaptic connectivity cannot be concluded. (b) Optogenetic stimulation of the CDNs while the Crz neurons are silenced results in termination of the mating, demonstrating that the CDNs operate downstream of the Crz neurons in determining the motivational state of the fly. (c) Silencing the Crz neurons reduces the response to sustained stimulation of the CDNs by selectively decreasing the gain on the input (~8-fold), leaving the time constant of integration largely unaffected. (d) Cumulative distribution functions used for estimating the parameters of panel (c). (e) For the first ~6 min of mating, high CaMKII activity in the Corazonin neurons prevents the network eruption that triggers sperm transfer2,3. Before the eruption, males that have not mated recently are impervious to challenges of apparently all varieties and severities. At 6 min, the eruption increases \({p}_{0}\), allowing threat information to be delivered to the Copulation Decision Neurons (CDNs), through mechanisms not yet understood. After the eruption, dopaminergic inputs to the CDNs increase intracellular CaMKII levels to restrict \(\tau \), the timescale of competing information retention. (f) Dopaminergic neurons (orange) send projections throughout the abdominal ganglion, often forming varicosities near CDN (green) processes (indicated by white arrowheads). Images are obtained from a single optical plane. (g) Silencing the dopaminergic neurons does not affect overall copulation duration. (h) Warmth alone decreases τ but increases p0 showing that heat cannot account for the effects of stimulation the dopaminergic neurons. (i) Data as in Fig. 4b but plotting lifetime instead of fluorescence. The measurement was more variable but there is a consistent ~200 picosecond increase in lifetime with thermogenetic stimulation. (j) Thermogenetic stimulation of the dopaminergic neurons of the abdominal ganglion results in an increase in fluorescence and fluorescence lifetime. Note that 1) the lifetime is not linearly related to the increase in fluorescence (and so the two measures have differential sensitivity across concentration) and 2) both signals begin to decrease before the temperature is decreased. Because bath application of dopamine resulted in stable fluorescence, this seems unlikely to be bleaching of the indicator. We speculate it results either from habituation of the TrpA1 channel or rapid depletion of the dopaminergic neurons, at least at the scale of the sensor’s dynamic range. Allowing several minutes of recovery at 20 °C permitted a second stimulation to be equally efficacious (not shown). (k) Warming the abdominal ganglion without expression of TrpA1 in dopaminergic neurons resulted in a small but consistent decrease in both fluorescence lifetime and fluorescence itself. The GRAB-DA3m protein itself is likely temperature sensitive, but the effect of temperature produces a change in fluorescence signal opposite to that observed during stimulation of dopaminergic neurons, arguing that signals as in Fig. 4b are not artifacts of the temperature ramp. (l) Bath application of dopamine in the concentration range used in Fig. 4 results in increases in fluorescence lifetime and fluorescence quantitatively similar to that evoked by thermogenetic stimulation of dopaminergic neurons, arguing that these bath concentrations result in physiologically-plausible exposure to dopamine at the CDN axons. These values differ substantially from the reported sensitivity of GRAB-DA3m30 which we speculate arises from the protective glial sheath surrounding the abdominal ganglion, which may buffer the exogenous dopamine levels or rapidly degrade it. Supporting this conclusion, in unpublished experiments, we found that pipette administration of dopamine to a still bath (rather than continuous perfusion) only transiently increased the excitability of CaMKII, unlike the sustained excitability increase observed in Fig. 4.
