A lineage-related reciprocal inhibition circuitry for sensory-motor action selection
Summary (3 min read)
INTRODUCTION
- The selection and maintenance of a behavioural action is the outcome of specific neural interactions mediated by circuits that have evolved to coordinate adaptive behaviours.
- Thus, within the context of the proposed homology of CX and BG circuitries, the many tools available for targeted cell and circuitspecific manipulations of the EB in Drosophila28, makes the invertebrate system ideal for investigating neural mechanisms and computations underlying action selection.
- The authors show that targeted manipulations of subtype-specific R neurons reveal that EB microcircuitry is involved in sensory-motor transformation required for the selection and maintenance of motor actions and their organisation into action sequences.
R neurons derive from embryonic neuroblast ppd5
- Their layerspecific projections form a toroidal neuropil located dorsoanteriorly straddling the midline of the protocerebrum29-31.
- Based on their layer-specific projections, R neurons are classified into four subtypes (R1-4) each resolved by subtype-specific Gal4 lines30,31 (Fig. 1 and Supplementary Fig. 1).
- Protocerebral poxn+ lineages derive from embryonic stem cells, neuroblasts ppd5 and ppd8, that are distinguished by en and Dachshund (Dac) expression33, with Dac restricted to ppd8 (Fig. 2a-f).
- Similarly, poxn-Gal4 tracing identified poxn>tub>mCD8::GFP labelled R neurons and their neuropil, all of which are immunopositive for anti-Poxn (Fig. 2i).
- In contrast, the authors did not detect anti-Dac immunoreactivity in poxn+ cells (Supplementary Fig. 3e-n).
R neurons form a connectivity network among sister cells
- EB cells multiply during development and, similar to observations on grasshopper CX development35, establish an orthogonal axon scaffold that is later reshaped by morphogenesis into the EB’s characteristically toroidal architecture29-31.
- Co-labelling revealed apposed Syt::GFP and DenMark punctae across layers and modules (Fig. 3b-d and Fig. 4a-d; Supplementary Movie 1).
- More rarely, tetrad synapses terminating onto four postsynaptic profiles contain Kottler et al., 2017 – preprint version dense core vesicles, indicating that neuromodulatory elements likely participate in EB functionality (Supplementary Fig. 4h).
- In contrast, super-resolution microscopy of GRASP signals revealed punctate (Fig. 4i-k) and elongated, grid-type GFP expression extending across R2-R4 layers and modules (Fig. 4l,m and Supplementary Movies 3 and 4).
- Taken together, these data suggest connectivity among R neurons that extends across layers and modules of the EB neuropil.
GABAergic inhibition of R neurons
- The authors therefore tested whether poxn+ R neurons are GABAergic.
- Together, these data suggest that c232 and EB1 R neurons are inhibitory GABAergic interneurons that are inhibited by GABAA receptor signalling.
- To further address this, the authors made use of temperature-related behaviour which in Drosophila is known to elicit goal-directed locomotion41 (Fig. 6).
- Spiking occurred instantaneously and across the whole population, with GCaMP-fluorescence intensity 4-7-fold above baseline for ring neuropil and cell bodies, respectively, and rapidly declined over 2 minutes, followed by inactivity even at noxious 35°C (Fig. 5g).
- These data suggest that GABAergic inhibition of R neurons coordinates voluntary motor actions and their assembly into action sequences.
R neurons mediate the selection and maintenance of behavioural actions
- A Action selection circuitry is readily engaged by external stimuli that drive an animal’s behaviour49.
- (a) Raster plots and 10min movement trajectories are shown for respective EB ring layers (colour coded).
- (b) Top row, average activity over 60min recordings, including response to mechanical stimulation given after 30min (orange dashed line); bottom row, action measures of mean activity, speed while active and distance travelled.
- Recordings at 31°C revealed behavioural alterations of Kottler et al., 2017 – preprint version c105>TNT flies that initiated more, albeit slower, actions of shorter duration (bout length) which were intermitted by longer pauses (Interbout Interval, IBI length); these alterations, however, were unrelated to their arousal state which remained unchanged (Fig. 8 and Supplementary Fig. 9).
- In contrast, TRPA1-mediated acute activation of EB-R neurons at 31°C identified behavioural alterations almost exclusively restricted to R3 neurons (Fig. 9).
A computational model of EB action selection circuitry
- The sum of their data supports the proposition that R neurons form GABAergic inhibition circuitry among inhibitory sister cells that underlies the selection and maintenance of behavioural actions.
- To further explore the computational features of such circuitry within the CX, the authors developed a neural model relying on leaky integrator units to simulate the mean field activity of neural populations55 (see supplementary methods).
- The features characterizing these stimuli, such as signal intensity and localization in space, are then propagated in parallel to EB modules, activating them dependent on the strength of the signal, as has been shown for visual cues26, and weighed by the relative connections.
- The authors opted for fixed lateral inhibition between R neurons, while exploring the consequences of several different configurations of excitatory inputs from the PB and FB to the EB, and of inhibitory outputs from EB to LAL (Supplementary Fig. 12).
- The authors simulations show that parallel input connectivity, coupled with internal lateral inhibitions are sufficient to realize sensory integration, to detect the highest saliency, to cancel competing signals and noise, and to adapt to changes in the environment such as novel sensory input (Fig. 10b-e and Supplementary Fig. 12).
DISCUSSION
- The authors results identify a pair of bilateral symmetric, engrailedexpressing embryonic neuroblasts, ppd5, that generate all four subtypes of ellipsoid body R neurons (R1-R4) in the central complex of the adult brain in Drosophila.
- A key feature of action selection is the evaluation and transformation of sensory representations into motor representations49.
- The predictions of their model are supported by functional imaging studies showing that specific EB module activity correlates with the azimuth of a salient visual cue26.
- The R neuron circuitry is embedded in a connectivity network with the PB, FB and the LAL45,56-58, the latter a premotor command centre projecting to descending neurons that innervate central pattern generators executing motor actions62.
- In mice, optogenetic manipulations have shown that direct pathway stimulation facilitates motor actions, whereas indirect pathway stimulation decreases motor activity2, both of which are cooperatively active during voluntary movement3,4.
METHODS SUMMARY
- Drosophila stocks and genetics Flies were maintained on standard cornmeal medium at 25°C in a 12hr:12hr light/dark cycle.
- For lineage tracing, w1118 and Gal4 strains were crossed to UAS-mCD8::GFP, tub-FRTCD2-FRT-Gal4, UAS-FLP/CyO GMR Dfd YFP.
- For behavioural experiments, UAS-Rdl-IR, UAS-TNT, UAS-NaChBac, UAS-ShibireTS, or UAS-TRPA1 were crossed to R neuron-specific Gal4 lines, and controls generated by crossing driver and responder lines to w1118.
- Further details are provided in the extended methods section of Supplementary Information.
Functional imaging and electrophysiology
- Bioluminescence imaging was performed in open brain preparations of tethered flies.
- GCaMP6 imaging and electrophysiology were carried out on isolated brains maintained in saline solution.
- Whole cell current clamp recordings were performed using glass electrodes with 8-12 MΩ resistance filled with intracellular solution and an Axon MultiClamp 700B amplifier, digitised with an Axon DigiData 1440A (sampling rate: 20kHz; filter: Bessel 10kHz) and recorded using pClamp 10 (Molecular Devices, CA, USA).
Behavioural analysis
- Single, 3-5 day old flies were placed in an open-field arena, and their behaviour was recorded with a Logitech c920 camera at 10 frames per second.
- After 30 minutes, mechanical stimulation was delivered through shaft-less motors (Precision Microdrives) controlled by custom-made DART software70.
- These behavioural experiments were carried out in a temperature-controlled chamber at either 25°C or 31°C.
- Thermal preference behaviour was carried out using a linear temperature gradient from 18°C to 31°C.
Neural network modelling
- Ellipsoid body R neuron modelling was performed in MATLAB using custom scripts.
- Network dynamics were captured with a firing rate model based on a leaky integrator defined by a continuous-time differential equation.
- Supplementary Information includes twelve figures, two tables and four movies, as well as Detailed Methods and Supplementary References.
AUTHOR CONTRIBUTIONS
- Z.N.L. & D.C.D. performed and analysed anatomical experiments.
- G.V. & J.R.M. performed and analysed bioluminescence imaging.
- R.F. designed DART software including custom-made MATLAB scripts; J.D. & D.M.H. wrote earlier versions of MATLAB scripts.
- F.H. directed the study and prepared the manuscript to which the authors contributed.
COMPETING FINANCIAL INTEREST
- B.K. & R.F. are co-founders of Burczyk/Faville/Kottler LTD.
- All remaining authors declare no conflict of interest.
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Additional excerpts
...R2–R4 neurons, like TL2/3 neurons, are GABAergic (Kottler et al., 2017; Table 1)....
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Frequently Asked Questions (17)
Q1. What contributions have the authors mentioned in the paper "A lineage-related reciprocal inhibition circuitry for sensory-motor action selection" ?
Here, the authors show that paired embryonic neuroblasts generate central complex ring neurons that mediate sensory-motor transformation and action selection in Drosophila. A computational model substantiates genetic and behavioural observations suggesting that R neuron circuitry functions as salience detector using competitive inhibition to amplify, maintain or switch between activity states.
Q2. What is the role of the EB-LAL interface?
As a node of convergence, situated several synapses downstream of sensory neurons and several synapses upstream of motor ganglia, the position and connectivity of the EB-LAL interface implicates it in the translation of sensory representations into motor representations.
Q3. What is the effect of the EB modules on the behavioural alterations of c232?
The features characterizing these stimuli, such as signal intensity and localization in space, are then propagated in parallel to EB modules, activating them dependent on the strength of the signal, as has been shown for visual cues26, and weighed by the relative connections.
Q4. Why do the authors identify R1-4 neurons as lineage-related sister cells?
Because Dac expression distinguishes between neuroblasts ppd5 and ppd8, their data identify R1-4 neurons as lineage-related sister cells derived from a pair of bilateral symmetric stem cells, embryonic neuroblast ppd5.
Q5. What is the role of the EB in initiation and termination of motor actions?
In mice, optogenetic manipulations have shown that direct pathway stimulation facilitates motor actions, whereas indirect pathway stimulation decreases motor activity2, both of which are cooperatively active during voluntary movement3,4.
Q6. What is the role of the EB in initiation, maintenance and termination of motor actions?
In Drosophila, their genetic manipulations and neural computations identify facilitation, inhibition and disinhibition of motor activity as differential R neuron functions underlying the initiation, maintenance and termination of actions and their organisation into action sequences.
Q7. What is the role of the EB in the behavioural manifestations of the fly?
Layers and modules in the ellipsoid body have been implicated in a variety of behavioural manifestations that identify key roles of the EB in response not only to visual cues for spatial orientation15,19,22,24,26 and memory formation16,17,21, but also in response to olfactory cues for ethanol-induced locomotion20 and tactile cues for arousal18 and turning behaviour23,25 as well as in relation to sleep drive27.
Q8. What is the effect of c232 on the EB1-Gal4?
Rdl-RNAi with stimulation protocol 1 at V40, indicating that GABAergic c232-neurons are inhibited by c232-mediated GABA-A receptor signalling.
Q9. How many spikes did the authors inject into EB1 R neurons?
To test their stimulus-salience response, the authors injected increasing amounts of depolarising current (up to 40pA) into EB1 R neurons, which evoked repetitive firing with successively more spikes triggered by higher currents, up to a maximum frequency of 80Hz (Fig. 5f), suggesting that spike frequency increased proportional to stimulus salience/strength.
Q10. What is the inverse correlation between Rdl-IR expression and behavioural activity?
Disinhibited behavioural activity was observed with Rdl-IR expression for each of the EB's concentric layers, which was inversely correlated with action initiation as flies less frequentlyinitiated behavioural activity.
Q11. What is the role of the EB module in the behavioural manifestations of short-term?
The availability of an EB module-specific activity for a prolonged time can guide successive behaviours and in this sense exhibits essential features of short-term memory61, which in the case of visual short-term memory has been shown to require R neuron activity16.
Q12. What was the EB's position in relation to the visual cue?
The observed activity "bump" was largely confined to one module but its position within the EB ring did not match a retinotopic position of the visual cue.
Q13. What are the main causes of the failure of EB1 R neurons to select motor actions?
The failure of such selectivity is illustrated by pathologies of human motor control and motivational disorders, such as the indecisiveness of motor actions seen in Parkinsonism, and the diminution of voluntary motor actions seen in abulia47.
Q14. What is the effect of the EB on the selection process?
As a consequence, inhibitory projections from the EB towards the LAL can reverberate the selection process and perform a surround gating of the LAL.
Q15. What is the effect of noise on the PB?
In four simulations used as case studies (Fig. 10), noisy information about sensory cues is converted into the activation of PB modules.
Q16. What alterations in the behaviour of c232> TRPA1 flies were not?
c232>TRPA1 flies were characterized by significantly decreased basal activity that contrasted with more actions initiated which were not maintained very long and intermitted by extended pauses; these behavioural alterations were unrelated to changes in the arousal state of c232>
Q17. What is the role of a gABAergic inhibition of R neurons?
These data suggest that GABAergic inhibition of R neurons coordinates voluntary motor actions and their assembly into action sequences.