MDHn Activity Is Positively Correlated with Forewing Motor Output.

The MDHns arborize throughout the dorsal aspect of the mesothoracic neuromere, which along with the metathoracic neuromere, house flight central pattern-generating circuitry, including wing sensory input (36⇓–38). To characterize the relationship between the MDHns and wing-motor output, we developed an approach that leaves the entire central nervous system intact, exposing the mesothoracic neuromere where all sensory motor nerves emanating from the thoracic neuromeres were sectioned for stability; this also allowed us to make intracellular recordings from individual MDHns while simultaneously recording forewing depressor and elevator motor neuron output from the trunk IIN1b fiber using a suction electrode (39) (Fig. 1A). Intracellular electrode guidance to MDHns was visually blind, but spike shape and a tonic firing pattern guided selection of specific cells for recording. Each recorded cell was dye-filled, and HA-immunolabeling was used to confirm that an MDHn was recorded (Fig. 1B).

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Fig. 1.

MDH activity is correlated with flight-motor patterns. (A) Schematic of key components of the moth CNS, including the AL, subesophageal zone (SEZ), and AMMC and the pterothoracic ganglion (PTG), which includes the fused mesothoracic and metathoracic neuromeres. Also highlighted is our experimental approach, which included the simultaneous intracellular recording of an MDHn (blue) and suction electrode recording of the IIN1b nerve fiber (red), while wing-motor output is driven from the flight central pattern generator (red circle) via bath-applied chlordimeform (50 µM). (B) HA immunolabeling (green) of the MDHns with the intracellularly recorded MDHn filled with Alexa 568 (magenta; magnification = 20×). Inset below is a zoom-in of two distinct cell bodies labeled. (Left) Both laser channels. (Center) Alexa 568 channel showing a single filled cell body and primary neurite. (Right) HA channel showing the two cell bodies and primary neurites of the MDHn pair. Complete spatial overlap confirms the recording was of a MDHn. (C_i) Superimposition of the smoothed instantaneous spike rate of the recorded MDHn (blue) and the raw extracellular recording of the IIN1b fiber (red). Dashed rectangle highlights the time sample shown in C_ii, which shows the raw spike trains for both traces. Note that the MDH spike rate always increases just before and during bouts of wing motor output. (D) Plot of z-score normalized spike rate for MDH (blue) and IIN1b (red) across 10 min of continuous recording, demonstrating that as IIN1b activity increases over time, so too does MDH spike rate (r = 0.71). (E) Scatterplot of z-score normalized spike rate of MDH and IIN1b. Linear regression (n = 4 recordings/738 points; R² = 0.09, r = 0.30). (F) Mean spike rate from epochs where the IIN1b was quiet versus producing wing-motor output from the recording highlighted in C. Error bars represent the SE. Statistical comparisons between states indicates corresponding significant increase in both IIN1b and MDH (Welch’s t test; n = 8 recording segments; *P < 0.05). (G) Cross-correlation between MDH and IIN1b firing rates using Gaussian smoothing windows ranging in width from 2 ms to 1,000 ms. Note that for smoothing widows within typical spike integration times (2–5 ms), there is no correlation between measures.

All recorded MDHns (n = 5) produced highly stereotyped spike waveforms and were tonically active even in the absence of motor output (Fig. 1C). In four of the five animals we were able to the hold intracellular recording long enough to induce wing-motor output via bath application of chlordimeform (10⁻⁵ M), an effective and selective octopamine agonist known to reliably induce fictive flight in insects, including M. sexta (36). In all cases MDHn tonic spike frequency was positively correlated with the presence and strength of wing-motor output (Fig. 1 C–G). This correlation could indicate that the MDHns receive input from motor circuitry or that chlordimeform directly affects the MDHns in parallel with motor circuitry. However, increases in MDHn firing rate were coupled to individual brief bouts of wing-motor output (Fig. 1C), suggesting that MDHn activity was coupled to motor output per se and not necessarily chlordimeform application. This also suggests that MDHns were driven by network components that produce and regulate the initiation and cessation of wing-motor output. In cases where wing-motor output increased or otherwise remained tonically active on a time scale of minutes, MDH activity increased in coordination with gradual increases in motor output (Fig. 1D). Mean normalized spike rate of both MDHn and IIN1b were positively correlated across all recordings (Fig. 1E) and manually segmenting recordings into epochs of wing-motor output versus “quiescence” (SI Materials and Methods) demonstrated a significant increase in MDHn spike rate during wing-motor output (Fig. 1F). Thus, the activity of MDHns represents a corollary of wing-motor output.

MDHn activity could provide two types of information about wing-motor output. MDHn spiking activity could be a precise efference copy of wing-motor function (indicative of a lower-order CDC), or rather than encoding precise wing movement, MDHn spiking activity could reflect the current behavioral state of the flight-motor network (observed in higher-order CDCs). Cross-correlation analysis revealed no temporally precise spiking relationships between the recorded motor-output fiber and MDHn (Fig. 1G). Rather, MDHn activity preceded bouts of motor activity by ∼100 ms and the correlation between MDHn and IIN1b spiking was only evident when data were smoothed across 25 ms or more (Fig. 1G), indicating that MDHn spiking activity and flight-motor output were correlated on a slower timescale. Thus, while MDHn and wing-motor output appear to be driven by an at least partially overlapping circuitry, the MDHns do not represent a precise efference copy per se. This is further supported by the observation that in all MDHn recordings, there was persistent tonic spiking in the absence of motor output. Thus, MDHns appear to encode changes in behavioral state.

AL Neural Entrainment to Stimulus Temporal Structure Is Modulated by Histamine.

MHDns increase their spiking activity during wing-motor output, thus it stands to reason that HA release in the AL increases as well. We next asked whether HA release from the MDHns, which are the exclusive source of HA in the AL of M. sexta (30), affects neural responses to temporally patterned odor stimulation that simulates the periodic flow effects induced by wing beating (23). We therefore used extracellular tetrodes to record simultaneously from multiple individual AL neural units (40) while stimulating the antenna with a single odorant (either 2-hexanone or 2-octanone). Odor was presented in blocks of five 500-ms-long 20-Hz pulse trains using a 50% duty cycle (i.e., 25-ms on and 25-ms off) and 10 s between each train of a block. This was repeated every 2 min for 30 min. After the first block, the moth received one of the three treatments (Fig. 2A). On average 18–22% of AL neural units within each group entrained to odor pulse trains. Based on their spiking characteristics, these units can be putatively classified as PNs (41). First, to disrupt HA function, we bath-applied the HA-r antagonist cimetidine (500 µM) (Fig. 2A). In many units that were initially able to entrain to 20-Hz pulsed stimuli, cimetidine application decreased their ability to entrain to stimulus temporal structure. For example, the unit depicted in Fig. 2B initially responded reliably to all 10 pulses of the pulse train across all five repeats, as indicated by 10 prominent peaks in the histogram. After cimetidine application, the same unit failed to reliably entrain to the stimulus; it failed to respond to the first two pulses then consistently responded to three, perhaps four subsequent pulses. To evaluate the ability of units to entrain to the 20-Hz pulse trains, we calculated power spectral densities for each unit in response to each stimulus block, then calculated the integrated power from 18 to 22 Hz (25). Cimetidine application significantly decreased the mean integrated power across units over time indicating that, relative to time matched controls, the ability of units to entrain to pulsed odor had degraded within 6 min of application (Fig. 2C). If blocking HA-r function reduces the ability of AL neurons to entrain to pulsed stimuli, it stands to reason that removing input from the sole source of HA in the AL (the MDHns) (30) should have the same effect. Therefore, our second approach was to sever the neck connective in a second group of moths, thus axotomizing the two MDHn axons therein (Fig. 2A). As with cimetidine application, we observed that in moths where the neck connectives were cut, units that were initially able to reliably track pulsed odor were less able to track over time relative to time-matched sham surgery controls (e.g., Fig. 2D). Across the population this manifests as a significant reduction in integrated power around the pulsing frequency within 16 min (Fig. 2E). It is important to note that entrainment across the population was not completely lost in either case. Rather, there was a loss of responses to individual pulses of a train (Fig. 2B, red box) and the relative degradation in ability of the cell to produce discrete bursts to individual pulses separated by interstitials with no spiking (Fig. 2D, before vs. after). Finally, if disrupting the MDH circuit degrades the temporal fidelity of odor encoding, bath application of HA should have the opposite effect. Therefore, in a final group of moths HA (50 µM) was bath-applied during pulsed odor stimulation (Fig. 2A). Within 4 min of initiating HA application, the ability of individual units within the ensemble to entrain to the stimulus temporal structure increased, and in some instances units that did not initially entrain to odor pulses were recruited into the population of entrained units (e.g., Fig. 2F). Across the population, we observed a significant increase in mean integrated power at the pulsing frequency relative to controls (Fig. 2G). This HA-induced increase in power only occurs at the pulse frequency and does so as the overall population spiking response to the pulse trains increases as well (Fig. S1). These results collectively indicate that MDHn release of HA within the AL enhances entrainment to the stimulus temporal structure as opposed to filtering out the reafference. This again supports the notion that this circuit be classified as a higher-order CDC (2).

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Fig. 2.

HA enhances entrainment of AL PNs to rapidly pulsed odor. (A) To evaluate the effect of MDHn HA release on the ability of AL PNs to entrain to pulsed stimuli, we performed three experiments, each in separate groups of animals. For all experiments, multichannel electrodes were placed into the AL and multiunit recordings were made while the ipsilateral antenna was stimulated with a block of five 500-ms-long stimulation at 20-Hz pulse trains every 2 min for a total of 15 presentations. After the first block of pulse trains, animals were challenged with an experimental treatment. (Left) In the first experiment, to disrupt HA-r function we bath-applied 50 µM cimetidine (CIM) in saline vehicle continuously over the course of the experiment. (Center) In the second experiment of animals, to remove intrinsic HA input from the MDHns the neck connective was cut, thereby axotomizing the MDHns. (Right) In the third experiment of animals, direct bath application of HA (50 µM) in saline vehicle was used to simulate increased MDHn output during flight. Exemplar peristimulus rasters and histograms for the baseline responses (before) and during/after cimetidine (B), neck connective cut (D), and HA (F) treatments. Mean integrated power from 18 to 22 Hz by time across all recorded neurons that entrained to the pulsed odor at some point during cimetidine (C), neck connective cut (E), and HA (G) treatments. Error bars represent the SE. Results plotted as a function of time since treatment. Power was normalized by dividing mean power from each block by the mean baseline (pretreatment block) power. Arrows indicated the first block where there was a significant difference in power between experimental and control treatments (Welch’s t test for two samples with unequal variance; P < 0.05). Regressions are second order polynomials. Red rectangle (B) highlights the loss of responses to the first two pulses as a consequence of cimetidine relative to pretreatment.

Olfactory Acuity Is Histamine Dependent.

The capacity for the olfactory system to guide behavior is fundamentally dependent on its ability to detect and identify (i.e., discriminate) important odors. Simulating wing-beat–induced flows enhances both separation of neural representations of different odors (18) and olfactory acuity in behavioral assays (25, 42). Therefore, we predict that because this CDC enhances odor processing, it will likewise enhance behavioral measures of odor detection and discrimination. Two behavioral assays were used to determine if HA circuit function contributes to the detection and discrimination of odors (SI Materials and Methods). Both assays were based on a well-established Pavlovian olfactory learning approach (43⇓⇓⇓⇓–48), where moths were first conditioned (for detection assays) or differentially conditioned (for discrimination assays) to respond to target odors. Twenty-four hours postconditioning, moths were randomly assigned into drug or control injection treatments and tested in a blind format. Based on initial control experiments (Fig. S2), an effective dose of ∼1 nL of 50 µM cimetidine was injected into each AL. Fifteen minutes following injection, moths were challenged with a blank stimulus, then a series of increasing concentrations of the conditioned odor to determine the concentration at which they detected the odor as measured by a significant increase in conditioned feeding response relative to the response to blanks. Both groups acquired the conditioned response (Fig. 3A). However, tests subsequent to injection indicated that cimetidine-injected moths displayed an order-of-magnitude higher detection threshold relative to controls (Fig. 3B). This was replicated using a different HA-r antagonist, ranitidine (SI Materials and Methods, SI Behavioral Results, and Fig. S3). Collectively, these results suggest that HA within the AL enhances olfactory sensitivity.

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Fig. 3.

HA-r blockade disrupts behavioral measures of olfactory acuity. (A) Acquisition of the conditioned feeding response to a single odor (2-hexanone) as a function of conditioning trial for groups of moths in the detection threshold assay. Twenty-four hours later, one group of moths was bilaterally injected with either 50 µM cimetidine (CIM) in saline vehicle or the saline vehicle without drug (saline) in a blind manner, then tested. (B) Conditioned feeding response as a function of odor concentration for the CIM and saline groups. Open and filled arrowheads indicate detection threshold concentrations, for the saline and CIM groups, respectively, as defined by the lowest concentration odor yielding a significant increase in response relative to the blank (one-tailed paired t test; n = 60; P < 0.001). (C) Acquisition of the differential conditioned feeding response to the CS⁺ and CS⁻ stimuli for CIM- and saline-injected groups. Moths were first differentially conditioned to one of the two odorants (2-hexanone or 2-octanone). Both odors were used as the CS⁺ and CS⁻ in separate but equally sized groups to counterbalance odor-dependent effects; for display, data were pooled by CS⁺ and CS⁻. (D) Discrimination index [(CS⁻) − (CS⁺)] displayed by concentration for the CIM- and saline-injected groups. Open and filled arrowheads indicate discrimination threshold, the concentration at which there was a significant differential response to the CS⁺ and CS⁻ odors using a one-tailed paired t tests (saline controls: P = 0.03; n = 46; CIM injected: P = 0.05; n = 43). All Regression lines are third-order polynomials and all error bars represent the SE.

As detection is a prerequisite for identification (44, 47), it stands to reason that increased detection thresholds would also impact the moths ability to identify target odors. Here we observed that discrimination thresholds also increased when HA-r function was blocked. Moths in both drug-treated and control groups learned to differentially respond to the CS⁺ (odor paired with 0.75 M sucrose solution) and CS⁻ (odor only) odors (Fig. 3C). Again, 24 h after conditioning, moths were injected with either cimetidine or the saline vehicle, this time in a double-blind format. We then tested with both the CS⁺ and CS⁻ odors across a dilution series of increasing concentration to determine the odor discrimination threshold, the lowest odor concentration at which moths responded significantly more to the CS⁺ relative to the CS⁻ (i.e., a “conditioned differential response”) (SI Materials and Methods). The discrimination threshold for saline-injected moths occurred at an odor concentration of 0.1 µg/2 µL, but when injected with cimetidine, a significant differential response was observed at 10.0 µg/2 µL. Thus, disruption of HA-r function decreases both the ability to detect (Fig. 3B) and identify (Fig. 3D) odors.