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Previous Article | Next Article
Volume 16, Number 18, Issue of September 15, 1996 pp. 5830-5843
Copyright ©1996 Society for NeuroscienceCellular Organization of an Antennal Mechanosensory Pathway in the Cockroach, Periplaneta americana
John A. Burdohan and Christopher M. Comer Neuroscience Group, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60607
ABSTRACT
Escape responses of cockroaches, Periplaneta americana, can be triggered by wind and mediated by a group of ``giant interneurons'' that ascend from cercal mechanoreceptors to motor centers. Recently it has been observed that escape also can be triggered by tactile stimulation of the antennae, and it is then independent of the giant interneurons. Here we identify a descending antennal mechanosensory pathway that may account for escape. Cobalt backfills demonstrated that a limited number of cells in the head ganglia have axons that project through all three thoracic ganglia. Comparison with known wind-sensory pathways indicated that wind is not a reliable stimulus for activating descending antennal pathways. However, direct touch stimulation of an antenna reliably evoked short-latency responses in cells with axons in the cervical connectives. Intracellular recording and dye injection revealed members of this pathway, referred to as descending mechanosensory interneurons (DMIs). The two axons of largest diameter in the cervical connectives were found to belong to DMIs, and these large-caliber interneurons were studied in detail. One had a soma in the supraesophageal ganglion, and the other in the subesophageal ganglion. Both had extensive neuritic arborizations at the same level as the soma and axonal arbors in all three thoracic ganglia. Each of these DMIs exhibited short-latency responses to small antennal movements, demonstrated a degree of directional sensitivity, and rapidly conducted impulses to thoracic levels. These cells have properties suggesting that they play a role in a short-latency behavior such as touch-evoked escape.
Key words: antennae; cockroach, escape behavior; interneurons; sensory coding; touch
INTRODUCTION
Recent experiments suggest that many orienting behaviors can be controlled by more than one neural pathway. Usually, this is related to the need to integrate information from more than one sensory modality. Some clear examples are found in the control of orienting movements of head and body by visual or somatosensory inputs in frogs (Comer and Grobstein, 1981), the control of head movements in owls by visual or acoustic inputs (DuLac and Knudsen, 1990
Several decades of work have shown that, when the escape response of cockroaches is triggered by wind, it is mediated by several pairs of giant interneurons (GIs), which transmit signals from cercal wind receptors to thoracic motor centers (Roeder, 1967
Despite the evidence for control of escape by the wind-sensory GIs, cockroaches are capable of producing escape responses after elimination of all GI axons ascending to motor centers (Comer et al., 1988
Preliminary work revealed the existence of descending interneurons (Burdohan and Comer, 1990
MATERIALS AND METHODS
All animals were adult male Periplaneta americana, obtained from our own breeding colonies or purchased from suppliers. For both anatomical and physiological experiments, an animal first was anesthetized with carbon dioxide and, after removal of its legs and wings, pinned out on a paraffin-coated platform. An incision was made along the dorsal midline extending from the abdomen to the anterior edge of the pronotum. The cuticle was spread apart and pinned to expose the thoracic cavity. The gut was excised, and muscles and connective tissue overlying the ventral nerve cord (VNC) were removed. During the course of an experiment, the VNC was perfused continuously with saline (Callec and Sattelle, 1973Anatomical demonstration of descending interneurons. Cobalt backfills were performed to determine whether significant numbers of cells in the brain (supraesophageal ganglion, Sa) and the subesophageal ganglion (Sb) had axons projecting to thoracic levels. The nerve cord was cut between the second and third thoracic ganglia (T2 and T3), and the anterior cut end of either one or both connectives was placed in a Vaseline well constructed inside the thoracic cavity. A few drops of a 4% solution of cobalt hexamine chloride were placed in the well, which was then covered with Vaseline. After allowing time for transport, the presence of cobalt was demonstrated as described below.
In experiments to reveal anatomical details of individual descending interneurons, a wax-coated metal platform was placed under the cervical connectives [or the connectives between the first thoracic ganglion (T1) and T2], and individual axons were impaled with microelectrodes made from glass capillary tubing. The electrodes were filled with a 4% cobalt hexamine chloride solution containing 0.4% fast green and had resistances of 20-60 M
. Electrical signals were amplified and displayed via conventional methods.
After a cell was penetrated, it was tested for responses to mechanical stimulation of the antennae (see below). Responsive cells were pressure-injected with cobalt. Pressure was applied in pulses (Picospritzer, General Valve, Fairfield, NJ) until a green longitudinal profile was visible within the cord. After allowing time for dye travel appropriate to a single-cell fill (2-3 hr at room temperature) or a connective backfill (1-2 d at 5°C), the two head ganglia and the three thoracic ganglia were removed and fixed in Carnoy's or 10% formalin. In the case of single-cell fills, silver intensification of the tissue was performed according to the method of Bacon and Altman (1977)
In all descriptions that follow, references to the laterality of stimulation, the position of soma, and the location and extent of major arborizations are with respect to the side of the nerve cord on which the axon of a cell is located. All drawings are represented by the orientation scheme advocated by Strausfeld et al. (1984)
Physiological analysis of descending interneurons. Extracellular recordings were made with hook electrodes constructed from Teflon-coated silver wire. A region of the VNC was lifted onto the hook, and the recording site was insulated with a mixture of petroleum jelly and mineral oil. Electrical signals were amplified and displayed on an oscilloscope for on-line observation and stored on magnetic tape for later analysis. Intracellular recordings were made with glass microelectrodes, filled with 4% Lucifer yellow, and backfilled with 1 lithium chloride, having final resistances of 60-120 M
Wind stimuli. Two types of wind-generating devices were used. Both devices were built in our laboratory. The first type was used initially to examine descending unit activity driven by wind. It consisted of a piston encased in a plastic housing (22 cm inner diameter). The piston was held in an upward position by a set of springs. A lever arm connected to a rotary solenoid depressed the piston when the solenoid was triggered by an electrical pulse. This apparatus produced wind puffs with a peak velocity of 1.8-2.0 m/sec and an acceleration of 22 m/sec2. Wind velocity was measured with a hot-wire anemometer (Flowtronic 55A1).
The second device was similar in design to the wind machine described by Westin et al. (1977)
Touch stimuli. Some mechanical stimuli were delivered by hand-held glass probes. However, to tap (and deflect) the flagellar segments of an antenna by controlled amounts, a piezoelectric device [rectangular bimorph (PZT-5H, Vernitron)] was used (Zill and Moran, 1981
Because the antennae actively move, it was necessary to restrain them partially to stimulate them in a standardized manner during intracellular recording experiments. Plaster of Paris was applied to cover the joint between the head and the first basal segment (the scape) and between the scape and the second segment (the pedicel). The joint between the pedicel and the flagellum was not covered. After the plaster dried, each flagellum was oriented at an angle of ~45° with respect to the horizontal and midline vertical axes, a position the flagella often assume in alert animals. The levels of descending activity evoked by mechanical stimulation seemed to be reduced somewhat by eliminating active movements in this way, because higher levels of activity can be evoked under experimental conditions that lack such restraint (Ye and Comer, 1996
In addition to touch stimulation of the antennae, cells were tested for their responsiveness when other sites were touched with a fine-tipped camel hair brush. Structures tested in this manner included the head hairs, mouthparts, dorsally and laterally situated hairs on the thorax and abdomen, and the cerci.
Other stimuli. Cells also were tested, although less rigorously, for their responsiveness to visual stimuli. These tests included room lights ON and OFF, as well as hand-held stimuli (black cardboard squares mounted at the end of a Plexiglas rod) passed in various directions within an animal's visual field. These same stimuli have been effective at eliciting visual responses from other descending interneurons in previous studies (Ye and Comer, 1994
Data analysis. Most routine spike counting was accomplished by digitizing electrical activity from recording sessions previously stored on VHS tape, with the aid of a data acquisition board (Model TL-1, Axon Instruments, Foster City, CA) and programs written in our lab (by A. Keegan). The board sampled at 10 kHz per channel. The analysis programs counted the number of impulses in each record and determined the latency to onset of the first impulse after stimulus presentation. In extracellular recording experiments, units were counted only if they occurred within 70 msec of the arrival of a wind puff at the animal and only if they were of large amplitude (>25% of the maximal amplitude observed).
RESULTS
The descending interneuron population
Because cockroach escape can be controlled by receptors associated with the antennae, we sought to identify neural pathways that might transmit information from the head to thoracic motor centers. The distribution of cell bodies in the head ganglia with descending axons was mapped after application of cobalt to the connectives between the second and third thoracic ganglia (T2-T3). Successful fills were reconstructed in three different animals.All backfills displayed the same distribution of labeled cell bodies; Figure 1 (top) shows a reconstruction from the preparation that demonstrated the largest number of labeled cell bodies. There were ~50 labeled somata in the supraesophageal ganglion (Sa) and ~35 somata in the subesophageal ganglion (Sb). Both connectives were exposed to cobalt in this preparation, so a minimum estimate would be that ~40-45 axons on each side of the VNC arise from cells of the head ganglia and project as far as T3. In Sa, most labeled cells were grouped together in the protocerebrum, approximately at the level of the posterior aspect of the optic lobes. Only a few other cell bodies were labeled, and these were scattered widely in the deutocerebrum and the tritocerebrum. In Sb, labeled cell bodies were concentrated near the ventral midline of that ganglion, with just a few positioned laterally and toward the dorsal surface.
Fig. 1. Axons in the cervical connectives include a population of interneurons with somata in the head ganglia and projections through the thoracic ganglia. Top, Camera lucida reconstruction of position of cobalt-filled cell bodies seen in whole mounts of head ganglia viewed from the dorsal surface. Anterior is toward the top. Cobalt was applied to both left and right connectives below the mesothoracic ganglion. Sa, Supraesophageal ganglion; Sb, subesophageal ganglion; a.n., antennal nerve. Bottom, Cross section through the cervical connectives viewed in phase contrast. The two largest axons (labeled a1 and b1) are those of individually identifiable interneurons that respond to antennal mechanosensory stimulation, as described in the text. Scale bar in both panels, 100 µm.
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The physiological analysis of descending pathways was performed at the level of the cervical connectives. Figure 1 (bottom) shows a cross section through the connectives; each contains a large number of axons, including profiles of numerous large-caliber axons (diameters between 20 and 60 µm). The two largest axons (labeled a1 and b1) belong to cells of the descending antennal pathway and will be described below in detail.
Descending mechanosensory pathways differ from ascending wind-sensory pathways
Under certain conditions, wind can activate descending interneurons. However, there are several features that distinguish this wind-evoked activity from that of the GIs. Typically, a burst of large amplitude multiunit activity can be evoked in the cervical connectives at short latency in response to a wind puff of high velocity and acceleration (see Materials and Methods). This could be descending activity, ascending activity, or both. To clarify the origin of the activity, GI input was eliminated either by covering the cerci with a mixture of petroleum jelly and mineral oil or by transecting the VNC between the thorax and the abdomen. After these procedures, impulse activity still could be evoked by wind at the cervical level (Fig. 2, top, VNC Cut). The remaining activity essentially was abolished after removal of both antennae (Fig. 2, top, Ant X). The results from six animals are summarized in Figure 2 (bottom). Note that eliminating GI (or GI-related) input was associated with an increase in the latency of the response to wind (from ~5 to ~25 msec). These results suggest that wind puffs (at least those of high velocity and acceleration) activated at least two systems passing through the cervical connectives: an ascending system (on the basis of the cercal covering results, this may include the rostral-most projections of the GIs) and a descending (antennal) system.
Fig. 2. Many units recorded at the cervical level represent descending signals related to the antennae. Top, Wind-evoked multiunit activity recorded extracellularly from the cervical connective. Wind had a peak velocity from 1.8 to 2.0 m/sec. VNC Cut, Response after transecting the ventral nerve cord at the level of A1-A2; Ant X, response after cutting off both antennae at the pedicel. Calibration: vertical, 2 mV; horizontal, 10 msec. Bottom, Summary of all experiments performed on six animals. Histogram on left gives mean number of impulses recorded at cervical level. Height of bar for each condition was derived by averaging responses for each animal and then calculating the grand mean across animals ± SEM. Only large amplitude impulses were counted, i.e., those25% of the maximum amplitude observed, and only those that occurred within 70 msec of wind onset. Histogram on right gives mean latency in milliseconds to first impulse across the same six animals ± SEM. Total number of trials averaged = 230. Conditions: I, intact; CC, after covering the cerci; VC, after cutting the VNC between abdomen and thorax; AX, after removing both antennae.
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To distinguish better the activity of ascending and descending interneurons elicited by wind, we made simultaneous extracellular recordings at the level of the cervical and abdominal connectives of the VNC. (The large amplitude wind-evoked impulses observed in extracellular recordings from the abdominal connectives are known from numerous studies to be those of the GIs.) Activity, then, was evoked either with a standard wind similar to that already used (high intensity wind; high peak velocity and acceleration as indicated in Materials and Methods) or a low intensity wind (lower in both peak velocity and acceleration).
Figure 3 (top) shows that, after isolating thoracic and abdominal portions of the VNC, activity was recorded at both levels in response to an intense wind. However, after lowering the wind intensity, activity at the cervical level essentially was abolished. Also note that direct comparison of the latencies at these two sites demonstrates that the GIs respond to wind much more quickly than descending interneurons. The results from three animals are summarized in Figure 3 (bottom). The low intensity wind used here is one that is still substantially above threshold for escape behavior (Camhi and Nolen, 1981
Fig. 3. Descending units are less responsive to wind than units of the ascending cercal wind-sensory system. Top, Wind-evoked, multiunit activity recorded simultaneously at cervical (Ce) and abdominal (Ab) levels. Nerve cord was cut between abdomen and thorax. Wind puffs of two different peak velocities were used. High (left), 1.8-2.0 m/sec; Low (right), 0.3-0.7 m/sec. Calibration: vertical, 2 mV; horizontal, 20 msec. Bottom, Summary of all experiments performed on three animals. Height of bar for each condition gives mean number of large impulses recorded simultaneously at cervical (black bars) and abdominal (open bars) level. Averages for each condition were derived by averaging responses for each animal and then calculating the grand mean across animals ± SEM. Total number of trials averaged = 93. No. Impulses, Number of large impulses counted (criteria as in Fig. 2). Conditions: Intact, Before cutting the VNC; XCord cut
, after cutting the VNC; High Wind, Low Wind, wind puffs with peak velocities as described above.
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Fig. 4. The largest diameter cervical axons are those of interneurons that produce short-latency phasic responses to antennal touch. Intracellular recordings were made from DMI axons in the cervical connective. The records show the response of two cells (identified as DMIs, corresponding to the axons labeled in Fig. 1, bottom) to a 1.0 mm deflection of an antenna lasting 1 sec (top trace, monitor of voltage to piezoelectric crystal). For DMIa-1, the contralateral antenna was tapped from the lateral direction (deflecting the flagellum medially). For DMIb-1, the contralateral antenna was tapped from the front direction (deflecting the flagellum backward). These directions can be considered optimal for each cell (see Fig. 12). Calibration: vertical, 40 mV (intracellular records only); horizontal, 10 msec.
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The structure of individual descending interneurons
To determine the location and structure of interneurons with these touch-sensory properties, the thoracic connectives were probed with microelectrodes. Cells that were activated by tapping an antenna were filled with cobalt. A number of cells were encountered that had axons descending from the head ganglia and responded to direct mechanical stimulation of one or both antennae. They will be referred to, operationally, as descending mechanosensory interneurons (DMIs). These interneurons will be differentiated further on the basis of their ganglion of origin: DMIa for those with a cell body located in Sa, and DMIb for those with a soma in Sb.The largest DMIs
The main focus of this study was to identify neurons that could play a role in generating antennal touch-evoked evasive behavior. Hence, attention was concentrated on mechanosensory cells that were capable of rapid conduction to thoracic motor centers. As seen in Figure 1 (bottom), two axons in each cervical connective are conspicuously larger in diameter than any others. When cross sections were examined from preparations with individual cells filled with cobalt, it was immediately clear that these axons are those of DMIs. The more medial axon is that of a DMIa, and the lateral axon is that of a DMIb. Because these are the first DMIs of each class to be studied in detail, they will be referred to as DMIa-1 and DMIb-1, respectively (labeled in Fig. 1, bottom). Each of these DMIs has been recorded from, and filled, in at least 40 separate experiments. As described below, each interneuron is uniquely identifiable, based on anatomical criteria, and each responds in a characteristic way to touching one or both antennae. [As indicated in Materials and Methods, references to the laterality of stimulation and to anatomical features are with respect to the side of the nerve cord on which the axon of a cell is located. Other descending interneurons, with smaller axons and various sensory properties, will be described elsewhere (Burdohan et al., unpublished data).]General sensory properties and structure of DMIa-1
DMIa-1 exhibited a short-latency phasic response to direct mechanical stimulation of the contralateral antenna (Fig. 4). Mechanical stimulation of the ipsilateral antenna and other sensory structures of the body, including the cerci and head hairs, failed to elicit a response. Visual stimuli also were ineffective in activating this interneuron.The cell body of a-1 is located in Sa near the medial surface of the contralateral protocerebrum midway between the dorsal and ventral surfaces (Fig. 5). The primary neurite extends posteriorly toward the dorsal surface into the deutocerebrum, giving off numerous fine branches along its extent. On the cell body side of the ganglion, the neurite gives off an extensive arborization that encompasses much of the posterior portion of the deutocerebrum, dorsal to, but not including, the antennal lobe.
Fig. 5. Anatomy of DMIa-1 in the head ganglia. Camera lucida drawings show dorsal (left) and lateral (right) whole-mount views in both head ganglia (Sa and Sb as indicated). Cell in this and subsequent figures labeled by intracellular injection of cobalt hexamine. Anterior is toward the top. In lateral view, dorsal is to the left. a.n., Antennal nerve. Scale bars: Sa, 150 µm; Sb, 100 µm.
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The neurite continues its course near the dorsal surface to the ipsilateral side of Sa, where it becomes thicker and gives off a number of ventrolaterally directed branches on its course to the circumesophageal connective. In Sb, the axon gives off primarily ventrolaterally directed branches as it continues posteriorly into the cervical connective. In the thorax (Fig. 6), the branching pattern is similar in all three ganglia. A prominent lateral branch is given off that arborizes extensively in the peripheral portion of each ganglion, and a number of shorter lateral branches are also given off. As seen in lateral view, all of the branches are confined to the dorsal half of the ganglia. The axon has been observed to course at least as far as the first abdominal ganglion (data not shown).
Fig. 6. Anatomy of DMIa-1 in the thoracic ganglia. Camera lucida drawings show dorsal (left) and lateral (right) whole-mount views in each of the three thoracic ganglia (T1-T3, as indicated). Anterior is toward the top. In lateral view, dorsal is to the left. Scale bar, 100 µm.
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General sensory properties and structure of DMIb-1
DMIb-1 also exhibited a short-latency phasic response to direct mechanical stimulation of the antennae (Fig. 4). However, unlike a-1, b-1 received input from both antennae. In addition, mechanical stimulation of the head hairs and mouthparts also elicited a response in b-1 (data not shown). Mechanical stimulation of other body regions, including the cerci, failed to activate b-1, as did visual stimuli.The cell body of b-1 is located on the ventrolateral surface of Sb near the posterior limit of the contralateral hemiganglion (Fig. 7), corresponding to the labial neuromere. The primary neurite courses dorsally and medially to cross the midline. During this course, it gives off a number of branches on either side of the midline that arborize extensively throughout the dorsal region of the ganglion. As the neurite approaches the ipsilateral peripheral extent of the ganglion, it splits into ascending and descending branches. The narrow ascending branch courses along the lateral margin of the ganglion, giving off a number of medially directed branches. The descending branch expands in caliber and courses posteriorly. In the thorax (Fig. 8), the branching pattern is similar in all three ganglia. Three prominent lateral branches are given off that arborize extensively along their course to the peripheral limits of each ganglion, and numerous medioventrally directed branches are given off that approach, but do not cross, the ganglionic midline. The axon of this cell also has been observed to continue posteriorly at least as far as the first abdominal ganglion (data not shown).
Fig. 7. Anatomy of DMIb-1 in the subesophageal ganglion. Camera lucida drawings show dorsal (left) and lateral (right) whole-mount views in Sb. Anterior is toward the top. In lateral view, dorsal is to the left. Scale bar, 100 µm.
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Fig. 8. Anatomy of DMIb-1 in the thoracic ganglia. Camera lucida drawings show dorsal (left) and lateral (right) whole-mount views in each of the three thoracic ganglia (T1-T3). Anterior is toward the top. In lateral view, dorsal is to the left. Scale bar, 100 µm.
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Anatomical uniqueness of DMIa-1 and DMIb-1
Although a-1 typically exhibited slight variations in structure in different animals, the overall branching pattern of this neuron was the same in all animals; this was true of b-1, as well. An example of this is shown for the pattern of each cell in T2 (Fig. 9). This anatomical similarity of each DMI, in different animals, was seen at all levels (Sa-T3) of the VNC examined. It was always possible to distinguish these two DMIs from each other and from other descending interneurons on the basis of their anatomy. In addition, their sensory properties (e.g., which antenna provided input) always corresponded with their morphology. For these reasons, DMIa-1 and DMIb-1 are considered to be individually identifiable neurons.
Fig. 9. Anatomical constancy of DMIa-1 and DMIb-1. Camera lucida drawings of whole-mount dorsal views of three separate cobalt hexamine fills of each DMI in the mesothoracic ganglion (T2). As an aid for comparing the branching patterns, the midline of each ganglion is shown as a dashed line. Anterior is toward the top. Scale bar, 100 µm.
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Additional distinguishing features of each DMI could be observed in cross section. As shown in Figure 10, the axon of a-1 courses through the medial aspect of the medial dorsal tract (MDT) in T1 and T2 (also in Sb; data not shown). However, in T3 the axon shifts ventrally to the dorsal intermediate tract (DIT). Confirming what was seen in whole mount, arborizations occurred in and among the dorsal tier of fiber tracts. In addition, arborizations from the prominent lateral branch occurred in the periphery of each ganglion. The axon of b-1 courses through the middle of the lateral dorsal tract (LDT) in T1-T3 (Fig. 10). The ascending branch in Sb courses within the same tract (data not shown). Similar to a-1, arborizations of b-1 occurred in and among the dorsal tier of fiber tracts, as well as in the lateral aspect of each ganglion.
Fig. 10. Distinguishing details of DMIa-1 and DMIb-1 seen in sections. Camera lucida reconstructions from transverse sections of DMIa-1 (left) and DMIb-1 (right) in the thoracic ganglia. Each drawing was constructed from five to six serial sections (12 µm thick). The level of each drawing is indicated to the left on a dorsal view of the ganglia (A, T1; B, T2; C, T3). Axonal arbors are shown in relation to the fiber tracts described by Tyrer and Gregory (1982)
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Characteristics of responses to antennal touch
As noted above, impulses were recorded at the cervical level from both DMIs at very short latency after antennal stimulation (Fig. 4). The average response latencies (± SD) for each DMI (under optimal conditions; see Fig. 12) were 7.1 ± 1.0 msec for a-1, and 8.2 ± 0.3 msec (contralateral antenna) or 9.9 ± 2.4 msec (ipsilateral antenna) for b-1. Within the range tested (10 msec-1 sec), stimulus duration had no effect on the number of impulses evoked (data not shown). In addition to their rapid response to antennal stimulation, both DMIs could transmit this information quickly to thoracic levels. The average estimated conduction velocity (± SD) calculated (from a minimum of 20 trials conducted in 4 different animals) for each DMI were: 4.6 ± 0.5 m/sec for a-1, and 4.7 ± 0.2 m/sec for b-1.
Fig. 12. Directional sensitivity of DMIs. Directionality is plotted as average number of impulses recorded for taps delivered from each of the four directions tested: front (Front), back (Back), medial (Med), lateral (Lat). Front and back represent stimuli moving the flagellum parallel to the long axis of the body, as shown in the inset; medial and lateral were movements at right angles to those shown. Taps were delivered to deflect the antenna by the same amplitude (1.0 mm) and at the same rate (0.1 mm/msec) on all trials. These values were chosen because they were optimal for activating the cells. Means for each direction (black squares) were derived by averaging responses for each animal and then calculating the grand mean across animals ± SEM. Top, Response of DMIa-1 to mechanical stimulation of the contralateral antenna. Data from 11 cells, each from a different animal. Total number of trials = 277. Bottom, Response of DMIb-1 to mechanical stimulation of each antenna. Data from eight cells, each from a different animal. Total number of trials = 329. Left side shows responsiveness of cell to tapping the contralateral (Contra) antenna. Right side shows responsiveness to tapping the ipsilateral (Ipsi) antenna.
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Neurons involved in the detection of predators via antennal contact might be expected to exhibit a high degree of sensitivity to movements of the antennae. DMIs were tested for this ability by observing their response at different amplitudes of antennal deflection. The amount of antennal deflection was controlled by varying the voltage to the bimorph, which, in turn, altered the displacement of the probe attached to its free end. However, it should be noted that the antennae were not attached to the probe, so it is possible that their actual displacement was greater than that of the probe. Hence, these results should not be interpreted as indicating an absolute threshold for response.
The responsiveness of both DMIs (measured as the mean number of impulses evoked across trials for each animal and at each displacement tested) varied as the antennae were displaced by different amounts (Fig. 11). DMIa-1 exhibited the greatest sensitivity, responding occasionally at the smallest displacement tested (0.01 mm) and very typically firing one spike in response to 0.05 mm deflections. As the amplitude of antennal deflection was increased further, the responsiveness of a-1 also increased. At the largest displacements tested (0.5 and 1.0 mm), the cell responded reliably with two impulses.
Fig. 11. Sensitivity of DMIa-1 and DMIb-1 to stimulus amplitude. Histogram summarizes all experiments on sensitivity performed with 19 animals. Antenna was tapped to deflect it by varying amounts. Horizontal axis (Stimulus Displacement) in mm; vertical axis, mean number of impulses counted (Average No. Impulses) for each displacement tested. Open bars, Response of a-1 to tapping the contralateral antenna (no response was observed to tapping the ipsilateral antenna); black and gray bars, response of b-1 to tapping the ipsilateral or contralateral antenna, respectively. Height of bar for each condition was derived by averaging responses for each animal and then calculating the grand mean across animals ± SEM. Total number of trials averaged = 309 (for 11 a-1 recordings) and 336 (for 8 b-1 recordings). Stimuli were presented from the optimal direction of each cell (see Fig. 12).
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DMIb-1 seemed to be somewhat less sensitive than a-1. It did not respond to 0.01 mm deflections and occasionally fired for 0.05 and 0.10 mm deflections. The responsiveness of b-1 also increased with increases in antennal deflection, responding reliably for 0.5 and 1.0 mm deflections. There were no marked differences in the responsiveness of DMIb-1 that depended on which antenna was deflected. It should be noted that, at the greatest deflections tested, a characteristic difference between the two cells was maintained: there was a consistent difference in average spike rate, with a-1 firing twice as many as b-1 (Fig. 11). The latency of the response in both DMIs varied only slightly with changes in antennal displacement (data not shown).
In most experiments, mechanical stimuli were delivered to the antennae at the fastest rate attainable with the bimorph (0.1 mm/msec; see Materials and Methods). It is our impression that the responsiveness of both DMIs was optimized by such abrupt stimulus application. However, to describe how the DMIs are influenced by rate of antennal deflection, it will be necessary to test interneurons under conditions in which the trajectory of antennal movement is controlled more completely.
Neurons involved in generating movements directed away from a threat might be expected to exhibit some form of directional sensitivity in their responses to antennal deflection. Both DMIs were tested for this ability by observing their response to mechanical stimuli delivered to the antennae from the four basic directions around the animal (Fig. 12). The inset emphasizes that the term ``direction,'' as used here, indicates that from which a stimulus was delivered, and it is thus opposite to the actual direction in which the antenna was displaced.
Both DMIs varied in their response to stimuli from different directions (Fig. 12). DMIa-1 was responsive for all directions from which the contralateral antenna was tapped. There was, however, a slight bias in its sensitivity. It responded with approximately two impulses, on average, to taps delivered from the front, lateral side, or rear; but responsiveness dropped to approximately one impulse for stimuli approaching from the medial side. No response was ever elicited from the ipsilateral antenna, even if tapped vigorously with a hand-held probe.
DMIb-1 responded to tapping of either antenna and was much more directional than a-1 (Fig. 12). It fired near its maximal rate (under these conditions) only to deflections from the front. With the other directions tested, the responsiveness was much lower. Deflections from the rear rarely activated the cell, so that the average spike rate was barely 0.1 impulses per trial. Deflections from the medial and lateral directions were more effective than those from the rear but still produced an average number of spikes approaching 0.5 per trial. There were no readily apparent differences in latency as a function of direction for either DMI (data not shown).
DISCUSSION
This set of experiments begins the characterization of a descending antennal mechanosensory pathway in Periplaneta americana that has some organizational and functional parallels with the ascending wind-sensory GI system. With the exception of a preliminary report (Burdohan and Comer, 1990Sensory inputs and the DMI pathway
DMI inputs seem to be exclusively mechanosensory. Both were activated by a tap to the appropriate antenna, and b-1 also was responsive to mechanical stimulation of cephalic hairs and mouthparts. Visual stimuli had no apparent effect. There are two possible sources for antennal mechanosensory input to the DMIs. First, tactile sensory receptors on flagellar segments of the antennae (Toh, 1977Some perspective on likely contributions from different receptor types can be provided from a consideration of the morphology of individual DMIs and the central organization of antennal projections. Although few details are known, mechanoreceptors on the flagellum are believed to project primarily into the antennal lobes (Rospars, 1988
Both DMIs, however, do project into regions of the head ganglia that have been shown, in related insects, to receive input from mechanoreceptors associated with basal antennal segments. Gewecke (1979)
Motor outputs and the DMI pathway
Both DMIs have axonal arborizations in dorsal and lateral regions of all three thoracic ganglia. This suggests that DMIs can access the leg motor circuitry because a number of neurons related to leg control reside within or project into these regions. These include the following: (1) leg motor neurons and premotor interneurons (Ritzmann and Pollack, 1990Similarities between DMIs and other insect interneurons
First, the DMIs show some similarities with previously described neurons of cockroach CNS. In particular, DMIa-1 has a similar branching pattern in the thoracic ganglia to that seen for GI-1 (Stubblefield and Comer, 1989Descending interneurons that may play a role in generating evasive responses also have been identified in a number of other insects. The giant descending neuron (GDN) of flies (Wyman et al., 1984
Another prominent function for descending interneurons of insects is in aspects of flight control (Rowell, 1989
DMIs in relation to escape behavior
Comer et al. (1988)Direct mechanical stimulation of the antennae activates the descending pathway at latencies considerably shorter than does wind stimulation (5-10 vs 20-30 msec; Burdohan and Comer, 1990
The present findings suggest that DMIs and GIs function as separate sensory-processing pathways. Camhi et al. (1978)
Cockroach DMIs may be viewed as a sensory-processing pathway for the control of short-latency evasive behavior (Ye and Comer, 1996
FOOTNOTES
Received Feb. 21, 1996; revised June 21, 1996; accepted June 25, 1996.
This work was supported by grants from the National Science Foundation (BNS-8909051 and IBN-9222619) to C.M.C. We thank Dr. Sasha Zill for kindly providing samples of piezoelectric bimorphs; J. King, J. Larimer, and W. J. Thompson for reading an earlier version of this manuscript; and S. B. for encouragement.
Correspondence should be addressed to Dr. Christopher M. Comer, Department of Biological Sciences (M/C 066), University of Illinois at Chicago, 845 West Taylor Street, Chicago, IL 60607.
Dr. Burdohan's present address: Department of Neurobiology and Anatomy, University of Texas Medical School, Houston, TX 77225.
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