The Microfluidic device

The microfluidic devices (Fig. 1 and Supp. Fig. 1) were fabricated by micro-milling onto transparent acrylic (PMMA) slabs. The fluidic channels, 100 μm in width and 25 to 200 μm in height, were sealed by 250 μm-thick transparent PMMA sheets using chemically-assisted thermal bonding. The circuit outlets are directed towards an open pool designed such that the larva, partially embedded in agarose, has its mouth precisely positioned in front of the delivery channel (Fig. 1d,e). The microfluidic device consists of two mostly independent circuits. The first one, driven by a push-pull syringe pump, imposes a continuous buffer flow circulating around the larva’s face (blue arrows in Fig. 1e). The second circuit controls the delivery of two independent stimuli (A and B) via two electromagnetic microvalves. In the resting state, the stimulus reservoirs are at atmospheric pressure and the solutions are continuously pumped through a V-shaped channel to an underpressurized waste container. Switching a valve (injection state) overpressurizes one reservoir, triggering the instant release of the stimulus solution through the injection channel (Fig. 1b,c). Both stimuli share the same delivery outlet, thus guaranteeing that no spatial clue is associated with switching between stimuli. The device can be positioned under a two-photon microscope for simultaneous recording of neuronal activity (Fig. 1a), while a fast camera monitors the larva’s tail movements from below, taking advantage of the chip’s transparency.

Figure 1 Microfluidic device for precise delivery of chemical stimuli. (a) Setup scheme: an epifluorescence two-photon microscope images neural activity while pulse-like stimuli are delivered to a zebrafish larva. An IR-sensitive camera images tail behavior from below. The microfluidic chip is connected to a computer-controlled manifold for the delivery of two different stimuli, S A and S B . (b) Infrared images of the device around the larva’s face during injection of stimulus S A (infrared dye, left channel). S B (right channel) is buffer. The red rectangle indicates the region used in the kinematic view of Fig. 2a,b (c) Sketch of the fluid flow in the device during the three stationary states: at rest, during injection of S A and during injection of S B . (d) Side-view scheme of the larva’s head resting on the movable slider. The animal’s face is gently cleared before positioning. (e) Scheme of the microfluidic device: the larva’s head embedded in agarose lying on the mobile slider is positioned in the pool in front of the delivery channel. Two electro-microvalves control pressures in stimuli reservoirs S A and S B and trigger injection. p 0 is the atmospheric pressure and p in > p 0 . Full size image

A crucial feature of this design is the negative pressure imposed on the waste container, which drives a co-flow of the stimulus solution and the chamber fluid in the downstream arm of the V-shaped channel. Although the flow rate is relatively low (of the order of 1μL.s−1), it prevents any cross-pollution between the buffer and the stimulus solution in the resting state, while guaranteeing through constant renewal that the stimulus solution at the entrance of the injection channels (i.e. ≈ 600 μm from the targeted sensory receptors) remains at the desired concentration.

Flow kinematics of the microfluidic device

To characterize the performances of the microfluidic device we monitored the delivery of a dye-containing solution using high-speed videography. For this purpose, we switched one of the microvalves to instantly increase the pressure to p in in one of the stimuli reservoirs. The time-evolution of the relative concentration, probed along a line tangential to the animal’s mouth (Fig. 2a,b), exhibited a rapid transition from zero (no dye) to one (nominal dye concentration) within a few tens of milliseconds (Fig. 2c). This transition dynamics was found to be highly reproducible and showed no significant dependence on the imposed pressure p in for p in ≥300 mbar. For each value of p in , we computed the onset and offset time-delays (noted τ r and τ d , respectively) defined as the delays between the valve switching and the time at which the relative concentration reached half the maximal concentration (inset of Fig. 2c). We computed the evolution of the jet width (Fig. 2d), which showed a similar dynamics towards a plateau but with a plateau value that increased quasi-linearly with p in . It is worth noting that despite the jet is one order of magnitude thiner than the mouth’s size, it splits in two branches close to the head surface and flows symmetrically along the exposed parts of the head. The real surface of contact is thus determined by the amount of agarose removed around the larva’s mouth. Tracking the position of the dye/water interface during injection onset, we also estimated the velocity of the propagation front as a function of the distance to the mouth, for various values of p in (inset of Fig. 2d).

Figure 2 Flow characterization in the microfluidic device. (a–b) Left: kinematic view of the chamber during injection onset (a) and offset (b), for stimuli S A and S B , recorded with a high-speed camera. For (a–d), S A and S B are an IR-dye used to characterize the flow dynamics. The region of interest is highlighted on Fig. 1b: the outlet of the delivery channel is at the top while the larva’s head tip is at the bottom of each frame. Right: Profiles of the relative concentration along a cross section of the chamber close to the larva’s mouth (purple and green lines on the kinematic view) at different moments after closing (a) or opening (b) the micro-valves. Jet’s width is defined as the width of the profile at half-maximum relative concentration. (c) Evolution of the relative concentration of the profiles shown in (a-b) for different reservoir pressures p in , averaged over 5 trials. Inset: rising (▲ τ r ) and decay (▼ τ d ) times such that the system reaches half the maximal relative concentration after microvalve switch, as a function of p in . (d) Evolution of jet width for different values of p in , averaged over 5 trials. Inset: Speed of the stimulus front during injection onset as a function of the distance to the larva’s mouth d m , for different p in . Same color code as in (c). (e) Raster (top) and average trace (bottom) of the normalized neuronal activity ΔF/Fσ in the vagal lobe during alternate presentations of citric acid (CA, first stimulus S A ) and buffer (second stimulus S B ) at p in = 500 mbar. Neurons are ordered by decreasing responses to CA pulses. Similar responses were obtained for n = 10 larvae. For each channel, three trains of five 300 ms-pulses separated by 15 s were delivered. Error bars: standard deviations. Full size image

These hydrodynamic recordings revealed several important features of the device. First, the stimulation onset and offset delays were small compared to typical gustatory stimulation times and were weakly dependent on injection overpressure. The valve switching sequence consistently drove the effective stimulus presentation sequence, albeit with a constant time delay. This device thus allows for near-millisecond precision on the stimulus presentation time, i.e. when the stimulus actually contacts the animal. The shortest stimulus duration that can be delivered by the device was ≈35 ms, as it corresponds to the time needed for the injection flows to reach a stationary state. We could also generate reproducible pulses of less than 10 ms, but in this regime the maximal concentration did not reach the nominal concentration. Second, the flow velocity on the fish face was independent of the injection pressure: it was imposed by the continuous buffer flow that pinches the stimulus jet and drags it to the animal. This process guarantees that the hydromechanical stress imposed on the larva is strictly invariant during and between stimuli presentations, with no modification at pulse onset and offset, such that no artifactual mechanosensory clues should accompany the chemical stimulation.

Gustatory neuronal responses

To test whether the gustatory-stimulus delivery could also stimulate mechanosensory receptors, we examined gustatory-evoked neuronal responses elicited by pulse-like exposure to distinct flavours. We probed in particular the specificity of the neuronal response to gustatory inputs and the absence of associated activation of mechanosensory receptors around the larva’s mouth induced by change in the flow pattern. Two-photon calcium imaging was perfomed on six-to-seven day old Huc:GCaMP3 transgenic larvae2 first restrained in low-melting agarose (see Sample preparation in Methods). Prior to positioning them within the stimulation device, we carefully removed the agarose around the larva’s mouth in order to expose the lips and mouth cavity, a region rich in taste buds11,12,13.

The two stimulation channels were first used to alternatively deliver series of five 300 ms-long pulses of sour (citric acid, 10 mM) or tasteless (buffer, control) stimuli. The most responsive primary gustatory centers in teleost fish are the facial (VII), glossopharyngeal (IX) and vagal (X) lobes14. At the developmental stage for which the recordings were made the glossopharyngeal lobe is not yet fully-developed and is rather difficult to identify. In large-field imaging experiments (not shown) we could observe some activity in the vagal and facial lobes with acute citric acid stimulations, which suggests that most probably the stimuli activated both external (lips) and intraoral taste buds. The strongest responses where evoked in specific neuronal populations of the vagal lobe (Fig. 2e). Importantly, the control solution elicited no measurable activity, thus indicating that the measured evoked activity in the vagal lobe is solely representative of gustatory inputs rather than other variables associated with the stimulus (e.g. hydromechanical cues)

Neuronal representation of gustatory stimuli with different hedonic values

The possibility offered by the microfluidic device to rapidly alternate the presentation of two stimuli allowed us to compare between the gustatory-induced activity patterns associated with distinct gustants in a single experiment. We illustrated this capability using two stimuli of distinct hedonic values: sour aversive, citric acid (CA) and appetitive umami, L-proline (LP). We sequentially delivered series of 300 ms-long pulses of either compound while recording neuronal activity in the vagal lobe (Fig. 3) . The mean signal, averaged over all identified neurons of the vagal lobe, show important responses at the population level (Fig. 3b), while at the single-cell level neuronal responses to either or both tastants have been observed (Fig. 3c). Although the topology of the gustatory-induced activity patterns appeared intermingled, we observed for three different planes (dorsal, medial and ventral) that a large fraction of neurons responded exclusively to either CA or LP, while a few responded to both (see Fig. 3d).

Figure 3 Neuronal responses to chemical stimulation. (a) Scheme of the larval brain and average fluorescence image of the vagal lobe. Neurons ① to ③, whose traces are shown in (c), are pinpointed. (b) Trace of the normalized neuronal activity averaged across all identified neurons of the vagal lobe. For each tastant, three trains of five 300 ms pulses separated by 15 s were delivered. (c) Normalized neuronal activity ΔF/Fσ of three neurons of the vagal lobe during alternate presentations of CA (stimulus S A ) and LP (stimulus S B ). Neuron ① responds exclusively to CA, ② responds exclusively to LP and ③ responds to both. (d) (Left, Middle) Maximum of ΔF/Fσ measured in the first 3 s following stimulus onset, averaged over 15 identical pulses, overlaid on the imaged plane (gray). (Right) Neurons responding to CA only (red), LP only (green) or both (yellow). p in = 500 mbar. Full size image

In the zebrafish larva, ascending projections carrying taste-related signals reach diencephalon and telencephalic areas. We thus similarly monitored CA and LP-induced responses in the dorsal telencephalic area (Supp. Fig. 3), a region known to respond to gustatory stimuli15. Interestingly, we found that even in higher gustatory centers, both CA and LP-induced responses remained relatively segregated.

Effect of stimulus duration on gustatory-induced neuronal responses and motor behavior

In aquatic species, the gustatory system is not only responsible for the decision of swallowing or rejecting ingested objects, but it also enables the animal to detect the location of potential food sources6 or to trigger escape from dangerous chemicals and water conditions7. Large amplitude tail deflections were robustly evoked by brief exposures to CA, an aversive tastant for the larva (Supp. Movie. 3 and 4). We took advantage of the unique time-precision of the microfluidic device and its suitability for simultaneous two-photon imaging and motor behavior video-monitoring to investigate the neuronal mechanisms underlying gustatory-induced motor behaviors (Fig. 4a).

Figure 4 Effect of stimulus duration on behavioral and neuronal responses. (a) Time-traces of the tail angle and average normalized neuronal activity in the vagal lobe during trains of five similar stimuli of citric acid. The trains contained stimuli of increasing duration (50–500 ms). (b) Probability of observing at least one tail flip in a time interval of 3 s following the stimulus onset. The probability of a spontaneous tail movement in a random 3 s interval far from stimulation is close to zero (dashed line). (c) Average number of tail flips in a time interval of 3 s follwowing stimulus onset. (d) Number of neurons responding to the stimulation in trials for which no motor response (blue) or a motor response (orange) was observed. (e) Amplitude of the response (defined as the maximum of ΔF/Fσ in the 3 s post-stimulation) averaged across the responsive neurons in trials associated (orange) or not associated (blue) with a motor response. (f) Topographical organization of the gustatory-evoked neuronal response patterns in the vagal lobe circuit. The color code reflects the amplitude of the response averaged across trials where no motor response was evoked (left) and trials where a motor response was observed (right) for the five different stimuli durations. For (b–f), data were pooled from 11 larvae (22 half vagal lobes) to which 25 stimuli were presented as shown in (a). The recorded area is the same as in Fig. 3. Error bars: standard deviations. p in = 500 mbar. Full size image

We observed that acute CA stimulation induced discrete, one-sided tail flips (see Supp. Fig. 4c, Supp. Movie. 3 and Supp. Movie. 4) sometimes followed by short series of weaker oscillations, a behavior that is reminiscent of escape responses (C-turn). These responses occured with a probability that consistently increased with the stimulus duration (Fig. 4b). The average number of tail flips immediately following the stimulus presentation were also positively correlated with the stimulus duration (Fig. 4c). We found that the overall neuronal evoked response in the vagal lobe was larger when this was associated with a tail motor behavior. Thus, to probe the neural activity directly evoked by the gustatory inputs we separately analyzed the events that did not induce a tail flip.

For trials not associated with a motor response, we observed a quasi-linear relationship between the number of activated neurons and the duration of the chemical pulses (Fig. 4d, blue). Interestingly, the Ca2+ transient amplitude of the responding neurons were much less affected by the stimulus duration (Fig. 4e). When the stimuli induced a tail flip, an intense and extended neuronal response was measured as shown in Fig. 4d,f. The differences in topography for gustatory-induced responses associated or not associated with motor behaviors suggest that rostro-lateral regions are associated with the sensory response while more caudo-medial ones are associated with the gustatory-induced motor response, as illustrated in Fig. 4f.

These results demonstrate that the stimulus duration is mainly encoded by the number of activated neurons in specific regions of the vagal lobe. The latter drives in a probabilistic manner a discrete transition towards a neuronal-circuit state capable of inducing a tail-flip response.

Olfactory-induced neuronal responses in the olfactory bulb

As a proof of concept, we performed experiments to validate the suitability of the microfluidic device for olfactory stimulation in zebrafish larvae. For this experiment, we presented to the larva’s nostril two odor-specific aminoacids (lysine and phenylalanine, 300 ms pulses, 10 mM) while monitoring changes in calcium dynamics for three different focal planes of the olfactory bulb along the dorso-ventral axis (dorsal, medial and ventral). We observed that the two odorants induced distinct neuronal response patterns (Supp. Fig. 4) as previously reported16.