Neural Mapping of Direction and Frequency in the Cricket Cercal Sensory System

The Journal of Neuroscience, March 1, 1999, 19(5):1771–1781

Sussan Paydar,2 Caitlin A. Doan,2 and Gwen A. Jacobs1 1Center for Computational Biology, Montana State University, Bozeman, Montana 59717, and 2Department of Molecular and Cell Biology, University of California, Berkeley, California 94720

Primary mechanosensory receptors and interneurons in the segregation was investigated, by calculating the relative over- cricket cercal sensory system are sensitive to the direction and lap between the long and medium-length hair afferents with the frequency of air current stimuli. Receptors innervating long dendrites of two interneurons that are known to have different mechanoreceptor hairs (Ͼ1000 ␮m) are most sensitive to low- frequency sensitivities. Both interneurons were shown to have frequency air currents (Ͻ150 Hz); receptors innervating nearly equal anatomical overlap with long and medium hair medium-length hairs (900–500 ␮m) are most sensitive to higher afferents. Thus, the differential overlap of these interneurons frequency ranges (150–400 Hz). Previous studies demonstrated with the two different classes of afferents was not adequate to that the projection pattern of the synaptic arborizations of long explain the observed frequency selectivity of the interneurons. hair receptor afferents form a continuous map of air current Other mechanisms such as selective connectivity between direction within the terminal abdominal ganglion (Jacobs and subsets of afferents and interneurons and/or differences in Theunissen, 1996). We demonstrate here that the projection interneuron biophysical properties must play a role in establish- pattern of the medium-length hair afferents also forms a con- ing the frequency selectivities of these interneurons. tinuous map of stimulus direction. However, the afferents from the long and medium-length hair afferents show very little spa- Key words: sensory maps; sensory system; insect; functional tial segregation with respect to their frequency sensitivity. The neuroanatomy; three-dimensional reconstruction; mech- possible functional signiﬁcance of this small degree of spatial anosensory receptors

A basic organizational principle of most sensory systems is the current direction (Bacon and Murphey, 1984; Troyer et al., 1994; representation of stimulus parameters within neural maps. Neural Jacobs and Theunissen, 1996). Neurons with similar directional maps are ensembles of neurons in which specific parameters of tuning overlap anatomically. The spatial segregation between the sensory stimulus are represented in a continuous manner arbors increases within the map as the difference in directional across the neural ensemble. Many neural maps represent multiple tuning increases. The sensory neurons make excitatory synaptic stimulus parameters. In the visual cortex, for example, maps of connections onto interneurons that project to higher levels of the orientation selectivity, ocular dominance, and direction selectiv- nervous system. These interneurons encode information about ity coexist in the same regions of cortex. How do such complex the direction and frequency of air currents (Kanou and Shi- neural maps emerge from the anatomical projection pattern of an mozawa, 1984; Jacobs et al., 1986; Miller et al., 1991; Theunissen ensemble of neurons? How do the anatomical features of such et al., 1996). maps constrain and/or simplify the extraction of information by Although the afferents of the sensory receptors are known to the neurons forming the next processing stage? form a neural map of stimulus direction, it is not known how We addressed these questions in the cercal sensory system of stimulus frequency is represented within the terminal ganglion. the cricket. In this system, primary sensory neurons innervate Are afferent arborizations segregated with respect to their fre- mechanoreceptor hairs on two abdominal sensory appendages, quency selectivities? Does a continuous map of stimulus fre- called cerci. The sensory neurons are activated by air current quency coexist with the map of stimulus direction within the stimuli and display differential sensitivity to variation in direction ganglion? To address these questions, the anatomical projection and frequency parameters (Kamper and Kleindienst, 1990; War- patterns of two populations of sensory neurons were analyzed land et al., 1991; Landolfa and Jacobs, 1995; Landolfa and Miller, quantitatively. Both populations of neurons project to the same 1995; Roddey and Jacobs, 1996). The axons of the primary general region of the terminal ganglion, and both populations are sensory neurons project in an orderly array into the terminal sensitive to the same range of air current directions. However, the abdominal ganglion, forming a continuous representation of air two afferent populations are sensitive to different ranges of air current frequency. One population is most sensitive to air cur- Received July 27, 1998; revised Dec. 7, 1998; accepted Dec. 9, 1998. rents having low frequencies (0–150 Hz), whereas the other is This work was supported by National Science Foundation Grant IBN-9796117 tuned to higher frequencies (150–400 Hz) (Roddey and Jacobs, and National Institutes of Health Grant R29 NS29847 to G.A.J. We thank Sandy Pittendrigh and Kelli Hodge for expert help with the graphics and John Miller for 1996). In this paper, we determined how multiple stimulus pa- critically reading this manuscript. rameters are represented anatomically within the ganglion by Correspondence should be addressed to Dr. Gwen A. Jacobs, Center for Com- comparing the projection patterns of these two populations of putational Biology, 30 AJM Johnson Hall, Montana State University, Bozeman, MT 59717. sensory neurons. We also tested the hypothesis that the Copyright © 1999 Society for Neuroscience 0270-6474/99/191771-11$05.00/0 frequency-tuning properties of interneurons, which receive input 1772 J. Neurosci., March 1, 1999, 19(5):1771–1781 Paydar et al. • Direction and Frequency Representation in the Cercal System from these sensory neurons, depend on specific anatomical rela- RESULTS tionships between the interneurons and the two populations of To clarify the presentation of the results, the following is a short sensory neurons. description of the functional tuning properties of the primary MATERIALS AND METHODS sensory neurons described in this paper. The location of each mechanoreceptor as well as its functional charac- Tuning properties of primary sensory neurons in the teristics are relatively invariant among animals (Landolfa and Jacobs, 1995). Thus it is possible to identify hairs and measure their functional cercal sensory system characteristics by direct observation through the dissecting microscope. Each wind-sensitive sensory neuron in the cercal system inner- The length of the mechanoreceptor, which determines its dynamic sen- vates a single filiform mechanoreceptive hair. The biomechanical sitivity, was measured with a calibrated ocular micrometer. Its directional sensitivity was determined by measuring the angle of the hair’s move- properties of the mechanoreceptor determine how it moves in ment axis with respect to the cercal axis and translating it to the body response to air currents and, in turn, determines the firing pattern coordinate system (Landolfa and Jacobs, 1995). For each identified of the sensory neuron. The mechanoreceptor hair is lodged in a mechanoreceptor, the length and directional tuning was measured for 10 socket that constrains its motion to a single plane. The directional examples of that identified hair. The angular measurements across ani- tuning properties of the sensory neuron are thereby determined mals had an SD of Ͻ2.5 ␮m. Histological staining techniques. Adult female crickets were used within by the orientation of this plane of motion with respect to the 24 hr of their imaginal molt (Bassetts Cricket Ranch, Visalia, CA). Each cercus. Sensory neurons are also broadly tuned to air current individual afferent was stained with cobalt chloride and silver-intensified frequency. The length of the mechanoreceptor determines the using methods developed by Bacon and Altman (1977), further modified frequency-tuning properties of the sensory neurons (Kamper and by Johnson and Murphey (1985) and Jacobs and Nevin (1991). The interneurons were stained with cobalt chloride and developed using the Kleindienst, 1990; Landolfa and Jacobs, 1995; Landolfa and same methods. Miller, 1995). The mechanoreceptor hairs vary in length from Computer reconstruction of stained neurons. The microscopy and com- ϳ50 ␮m to 1.5 mm. puter techniques used to digitize stained cells were developed in our In this study we compared the anatomical projection patterns of laboratory and have been described in detail elsewhere (Jacobs and two populations of afferents: those innervating the longest hairs Nevin, 1991). Briefly, a computer-controlled, digitizing light microscope is used as the data entry device. Tissue containing a single dye-filled on the cercus (the “L afferents,” associated with mechanorecep- nerve cell is mounted on the microscope stage, and the operator controls tor hairs Ͼ1000 ␮m in length) and those innervating medium- the precise movement of the neuron in three dimensions by means of length hairs (the “M afferents,” associated with hairs 700–950 ␮m three precision stepper motors, each mounted on a different axis of the in length). Several aspects of the anatomical projection patterns mechanical microscope stage. The neuron is moved under the microscope so that its branches are traced under a video cursor, superimposed were analyzed quantitatively: arbor size, arbor position in the over a frame-grabbed video image of the tissue. Movements of the terminal abdominal ganglion, and amount of anatomical overlap microscope stage are monitored by linear encoders mounted to the stage, with neighboring arbors. The goal of these experiments was to each encoder having a resolution of 0.1 ␮m. A set of x, y, and z determine how stimulus direction and frequency parameters are coordinates are recorded for the end points of each dendritic segment, represented anatomically within a neural map by testing whether along with the mean diameter of the segment between the end points. To compare neurons from different animals, three to five samples of the terminal arborizations of the L and M afferents are system- each of the 13 representative M afferents and one example of each atically and continuously segregated within the ganglion. interneuron [interneuron 10-3 (IN10-3) and IN9-3] were digitized. All digitized neurons were scaled and aligned to a common coordinate Ensemble projection pattern of the medium system using a suite of software programs called NeuroSys, developed in hair afferents our laboratory. NeuroSys contains a relational database and a suite of morphological analysis and computational tools (http://www.nervana- The axonal arborizations of 13 uniquely identified M afferents .montana.edu/NeuroSys). The information currently in the database were stained, digitally reconstructed, scaled, and aligned to our consists of Ͼ200 receptors associated with two functional classes of database of identified neurons. Figure 1 shows the axonal ar- Ͼ ␮ afferents: those innervating the longest hairs on the cercus ( 500 min borization of each identified M afferent within the terminal length) and those innervating medium-length hairs (900–500 ␮min length). We have constructed a three-dimensional model of this system, ganglion. All M afferents project into the terminal abdominal in the form of a probabilistic atlas, by combining three key morphological ganglion and arborize in specific locations according to their and functional characteristics of the receptor neurons: (1) their optimal directional tuning characteristics. Both the location on the cercus stimulus directions, (2) their frequency-tuning characteristics, and (3) and the directional sensitivities of these M afferents correspond the spatial distribution of each afferent’s synaptic sites within the cercal to identified L afferents with the same locations and directional glomerulus. The database also contains reconstructions of a subset of the identified primary interneurons. sensitivities described in previous work (Jacobs and Theunissen, Estimating the total surface area of an afferent’s synaptic field. Jacobs and 1996), with one exception. The exception is that one additional Theunissen (1996) developed a method for quantifying the spatial dis- identified M afferent (Fig. 1, #13) was reconstructed in the tribution of the surface area of the terminal varicosities of primary current study, whereas only 12 afferents were reconstructed in the sensory afferents. The same method was used here. Briefly, for each identified afferent type, three to five examples were taken from different initial study of the L afferents. Each corresponding pair of iden- animals. The terminal varicosities of each cell were transformed into a tified L and M afferents had the same directional tuning. continuous function representing the anatomical location and density The location and branching structure of the axonal arbors of distribution of the varicosities of a given identified afferent. These each identified M afferent was compared with identified L affer- density functions were visualized as three-dimensional space-filling ents with the same directionality. Figure 2 shows the branching clouds of points and used to calculate the location and spatial extent of the arborization of a single afferent. Details of statistical methods used patterns of three examples of M afferents (blue) and three L for this procedure are described by Jacobs and Theunissen (1996). afferents of the same directional tuning (red). Thus the projection Overlap measurements. The anatomical overlap between pairs of affer- pattern of the population M afferents is very similar to the ents or between interneurons and afferents was calculated as the inter- projection pattern of L afferents. Each pair of corresponding section between the density functions of the two cells. For the interneurons, the density distribution of their terminal dendritic branches (Ͻ2 afferents projects to the same general area, according to their ␮m in diameter) was calculated in the same way as the afferent varicos- common directional tuning characteristics. ities (for details, see Jacobs and Theunissen, 1996). However, there were small but significant differences in the Paydar et al. • Direction and Frequency Representation in the Cercal System J. Neurosci., March 1, 1999, 19(5):1771–1781 1773

Figure 1. Terminal arborizations of 13 types of identified cercal sensory afferents within the terminal ganglion. A, Diagram of a cricket showing the location of the terminal abdominal ganglion and cerci. B, Computer reconstruction of the outline of the terminal ganglion showing afferent type #1 as an example, dorsal view. The vertical and horizontal loops inside the ganglion are fiducial marks and are used for scaling and aligning. Afferent type #12 (also referred as X) is the only afferent that projects bilaterally. All other panels show the remaining 12 classes of afferent types shown from the same view as B. The afferent type numbers are shown in the top left corners. Scale bar (in A), 50 ␮m. branching patterns of each pair when measured on a finer scale. ily to the larger sizes of the L afferent arbors. Third, there were First, as shown in Table 1, the total surface area of varicosities regions where L and M afferents were completely segregated associated with each identified M afferent was one-half to one- from each other. third as large as the total varicosity surface area of the corre- Figure 3 shows the combined projection patterns of all the M sponding L afferent. Second, although M and L afferents of and L afferents. Each afferent arbor is represented by a cloud of similar directional tuning projected to very similar neuropil loca- points, color coded according to the peak directional tuning angle tions, the arborizations did not overlap completely. In general, a of the sensory neuron. Note that afferents with similar directional greater proportion of each M afferent overlaped with each L tuning overlap extensively and those with different directional afferent than vice versa. That is, the M afferent terminal arboriza- tuning are spatially segregated. The arbors form a continuous tions were constrained to a subset of the total volume occupied by representation of air current direction. The maps had a three- the corresponding afferent terminals. This is attributable primar- dimensional, spiral-like shape in which directional tuning 1774 J. Neurosci., March 1, 1999, 19(5):1771–1781 Paydar et al. • Direction and Frequency Representation in the Cercal System

Figure 2. Left. Spatial relationships between pairs of L and M afferents. Shown is a computer reconstruction of the terminal arbors of three M afferents (blue)in the correct spatial relationships to L afferents of the same directional tuning (red). From top to bottom these are afferent types 10 (A),8(B), and 6 (C). All afferents are shown from the dorsal view (the same view as in Fig. 1B) with the midline of the ganglion being the right edge of each panel. Note that the terminal ﬁelds of the M afferents are much smaller compared with the L afferents, and their terminals project to the same regions as the L afferents. There is some segregation between each pair of L and M afferents. The degree of such segregation varies among afferent types.

Figure 3. Right. Functional representation of direction in the terminal ganglion. A, Functional representation of direction formed by L afferents terminal arborizations. Each color cloud represents the average probability distribution of the terminal fields of afferents tuned to a particular wind direction. The directional tuning of each density cloud is represented by its color (shown also by the color wheel at the top right corner). The color wheel shows the direction with respect to the animal’s body in the horizontal plane; yellow indicates air currents directed at the animal’s head. B, Functional representation of direction by the M afferents (same view as in A). This figure demonstrates that the direction map of the M afferents is similar in structure to the L afferent map. The terminal fields of the M afferents overlap less with each other than the L afferents, making the map appear patchy and less tightly organized. C, Rep- resentation of direction by both L and M afferents within the terminal ganglion. M and L afferents tuned to the same direction are spatially in register with one another, allowing direction as a whole to be represented continuously within the terminal ganglion. changed continuously around the spiral. The neural map of di- Figure 4, the amounts of anatomical overlap between all paired rection formed by the M afferents had the same continuous combinations of M afferent arborizations were plotted as a func- functional organization as the L afferent map (Fig. 3B). However, tion of the difference in their peak directional tuning and com- because of the smaller size of their arborizations, the M afferents pared with the equivalent data from L afferents. Both plots show formed a more patchy representation of air current direction. The that there is approximately an inverse relationship between the projection patterns of L and M afferents are shown superimposed amount of anatomical overlap and the separation in directional together in Figure 3C. Note that the directional maps formed by tuning for L and M afferents. There is significant anatomical the long and medium-length hairs are aligned with one another overlap between many pairs of M afferents having peak direc- and together form one continuous map of air current direction. tional tunings separated by Ͻ90°. This relationship holds in gen- An important feature of the neural map of direction formed by eral for both L and M afferents; however, the amount of overlap the L afferents is the extensive anatomical overlap between af- for M afferents is smaller than that for L afferents. ferent arbors from neurons with similar directional tuning (Jacobs and Theunissen, 1996). This relationship also held true in general Alignment of the L and M afferent direction maps for the M afferents, except that the degree of overlap was smaller. Previous studies have shown that the terminals of the L afferents This is attributable to the smaller size of the M afferent arbors. In are distributed nonuniformly throughout the map of air current Paydar et al. • Direction and Frequency Representation in the Cercal System J. Neurosci., March 1, 1999, 19(5):1771–1781 1775

Table 1. Anatomical characteristics of the identiﬁed sensory afferents

Percent area of the Percent area of the medium hair afferent long hair afferent Area of the long hair Area of the medium hair that overlaps with the that overlaps with the Afferent type afferent (␮m2) afferent (␮m2) long hair afferent medium hair afferent 1 1029.05 Ϯ 167.4 714.14 Ϯ 16.31 59.07 Ϯ 0.84 40.95 Ϯ 0.57 2 1319.70 Ϯ 266.4 1002.4 Ϯ 132.23 51.36 Ϯ 0.70 39.01 Ϯ 0.53 3 1113.04 Ϯ 277.8 676.68 Ϯ 156.54 45.00 Ϯ 0.89 27.36 Ϯ 0.54 4 1392.86 Ϯ 147.9 1052.13 Ϯ 204.45 44.38 Ϯ 0.66 33.52 Ϯ 0.50 5 1052.15 Ϯ 227.0 847.55 Ϯ 102.03 67.30 Ϯ 1.07 54.21 Ϯ 0.86 6 1062.8 Ϯ 273.5 829.72 Ϯ 210.10 49.02 Ϯ 0.72 38.27 Ϯ 0.56 7 1314.72 Ϯ 201.6 643.78 Ϯ 71.71 54.05 Ϯ 0.99 26.74 Ϯ 0.49 8 1275.6 Ϯ 340.4 747.22 Ϯ 158.63 49.16 Ϯ 0.86 28.80 Ϯ 0.50 9 1254.34 Ϯ 322.8 955.76 Ϯ 91.88 61.27 Ϯ 0.91 46.68 Ϯ 0.69 10 1492.97 Ϯ 265.06 648.61 Ϯ 85.65 49.02 Ϯ 0.95 21.30 Ϯ 0.41 11 1072.70 Ϯ 94.9 708.69 Ϯ 149.64 63.91 Ϯ 0.90 42.22 Ϯ .060 13 1488.9 Ϯ 303.9 835.35 Ϯ 92.05 78.80 Ϯ 0.94 43.65 Ϯ 0.72

The total surface area of the L and M afferents as well as the amount of anatomical overlap between each pair were measured for each identiﬁed afferent type. Values are in square micrometers Ϯ SD. The surface area of each M afferent is signiﬁcantly smaller than the L afferent with the same directionality. In most cases larger less area of each M afferent overlaps with the L afferent of similar directional tuning. The percent overlap that each L afferent overlaps with each M afferent is much lower than the opposite case.

Figure 5. Nonuniform representation of direction within different regions of the L and the M hair direction maps. Each point is the average magnitude of all directional vectors that are tuned to the same direction at that point in the map (shown on the x-axis). Directional tuning is represented with respect to body coordinates. There are ﬁve locations within the L direction map (dotted line) that have a greater magnitude compared with the rest of the map (data modiﬁed from Jacobs and Theunissen, 1996). Similar nonuniformity is seen for the M direction map (solid line). There are six locations in this map that have larger terminal density compared with the rest of the map. The peak density for the M afferents, in general, is much less than for the L afferents.

Figure 4. Correlation between anatomical overlap and differences in directional tuning. A, B, Relationship between the amount of anatomical direction (Jacobs and Theunissen, 1996). The distribution of M overlap and angular separation among pairs of L and M afferents, respec- afferent terminals, within the same neural space, was also non- tively. Both graphs show that percent anatomical overlap between affer- uniform. Figure 5 shows the normalized magnitude of the afferent ents decreases as the distance between their directional tuning increases. density distributions plotted as a function of net directional There is essentially no anatomical overlap between afferents whose directional tuning is Ͼ90°. Note that percent anatomical overlap is less tuning angle within the map. The distribution of L afferent between pairs of M hair afferents compared with the same pairs of L terminals (dotted line) was quite nonuniform, forming ﬁve large afferents. peaks of increased terminal density. The distribution of M afferents (solid line) follows this pattern very closely, with the exception of the presence of an additional peak between Ϫ90 and Ϫ135°. This peak corresponds to an afferent (number 13) that was 1776 J. Neurosci., March 1, 1999, 19(5):1771–1781 Paydar et al. • Direction and Frequency Representation in the Cercal System

Figure 6. Anatomical segregation between two functional representations. A, Functional representation of stimulus direction by L and M afferents, color coded according to peak directional tuning with respect to body coordinates. Inset,The color wheel corresponds to the peak directional tuning in body coordinates of the subsets of afferents shown in C–F. B, Functional representation of stimulus frequency, represented by the L (red) and M (blue) afferent arborizations. C–F,Ar- borization patterns of subsets of L and M afferents tuned to different air current directions. C, Three L afferents and three M afferents with peak directional sensitivities near Ϫ45°. D, Three L afferents and three M afferents with peak directional sensitivities near 45°. E, Three L afferents and three M afferents with peak directional sensitivities near Ϫ135°. F, Three L afferents and three M afferents with peak directional sensitivities near 135°. not included in the L afferent sample. Thus, both maps show a spatial relationships between the L and M afferent populations similar nonuniform distribution of terminal varicosities. In addi- can be seen more easily. Figure 6B shows the projection pattern tion, Figure 5 demonstrates quantitatively the high degree of of all L afferents in red and all M afferents in blue. As indicated spatial alignment between the two maps of stimulus direction. in the previous sections, there is a great deal of anatomical Is a representation of air current frequency overlap between L and M afferents within the combined projec- mapped anatomically? tion. However, there are small regions in which the L and M Figure 6 illustrates the degree of anatomical segregation between afferent populations are clearly segregated. By examining subsets the L and M afferents with respect to their frequency sensitivity. of afferents that arborize in similar regions of the map, these The neural map of stimulus direction formed by all L and M regions of segregation can be seen more clearly. Figure 6C shows afferents is shown in Figure 6A. In this panel, color is used to the arborizations of three L afferents (red) and three M afferents indicate peak directional sensitivity, as in the previous ﬁgures. (blue) that have peak directional sensitivities near Ϫ45° (as indi- The color wheel in the bottom right of this panel indicates the cated in Figure 6A, small C on the color wheel in the bottom right correspondence between the color in the map and the direction of corner). The arborizations of three more subsets of afferents, with peak sensitivity of the afferents projecting to that region. The peak directional sensitivities clustered at 45, Ϫ135, and 135°, are remaining panels use a different color-coding scheme, so that shown in Figure 6D–F, respectively. Paydar et al. • Direction and Frequency Representation in the Cercal System J. Neurosci., March 1, 1999, 19(5):1771–1781 1777

Figure 7. Distribution pattern of dendritic arbors of 10-3 and 9-3 interneurons with respect to the bilateral M and L hair direction maps. A, Stereo pair image of three-dimensional reconstructions of interneurons 10-3 (red) and 9-3 (blue)inthe correct spatial relationships to each other. The dendritic arbors of these two interneurons overlap extensively and occupy similar regions within the cercal glomerulus. B, D, Anatomical relationships between 10-3 dendrites X, Y, and Z and different directional regions of the M and L direction maps, respectively. Each color cloud represents the probability density function of afferents tuned to a given direction. The directional sensitivity of each cloud is represented by its color shown along the direction wheel (B, top right corner). The branching patterns of the interneurons are shown in black. The soma of each interneuron has been removed so as not to obscure the branching patterns. Note that the distribution pattern of 10-3 dendrites with respect to different directional regions are different between the two direction maps. For example, the portion of the M afferent direction map tuned to lateral wind directions ( green, top left corner of each panel) is spatially expanded over a larger area and falls outside dendrite X territory in the M afferent direction map. Note that there is more anatomical overlap between the same dendrite and the same directional region in the L afferent direction map. C, E, The anatomical relationships between dendritic arbors of 9-3 labeled W, X, Y, and Z and different directional regions of bilateral L hair (C) and M hair (E) direction maps. The amount and pattern of anatomical overlap between IN9-3 and the direction maps varies across directional regions. For example, the distribution pattern of dendrite Y in the region representing anterior wind direction (orange, top right corner) is different with respect to each of the two direction maps. Expansion of this region in the M afferent map allows more anatomical overlap with dendrite Y compared with that in the L afferent direction map.

Although there are regions of segregation between M and L and each responds to a specific range of air current frequencies afferents, there is no consistent pattern or axis of segregation (Jacobs and Murphey, 1987; Miller et al., 1991; Theunissen et al., indicating a continuous representation of stimulus frequency 1996). Previous studies suggest that the directional tuning char- within the ensemble projection pattern. The continuous repre- acteristics of each interneuron are determined by its anatomical sentation of direction is conserved in the combined projection structure: the position of each neuron’s dendritic branches within pattern of L and M afferents, yet there does not appear to be a the afferent map of air current direction determines the set of significant, systematic representation of stimulus frequency excitatory inputs it can receive and thus shapes its directional through any dimension. tuning characteristics (Bacon and Murphey, 1984; Jacobs and Are similar mechanisms used by primary sensory Murphey, 1987; Troyer et al., 1994). Could a similar anatomy- interneurons to extract directional and frequency based mechanism determine the differential frequency sensitivi- tuning parameters from the neural map? ties of the interneurons? That is, could the anatomy of the The primary sensory neurons synapse with a group of ϳ20 dendritic arbors of the different interneurons allow differential uniquely identified sensory interneurons. Each of these interneu- connectivity with afferents tuned to different frequency rons has a unique morphology and a unique directional tuning, sensitivities? 1778 J. Neurosci., March 1, 1999, 19(5):1771–1781 Paydar et al. • Direction and Frequency Representation in the Cercal System

Figure 8. Quantitative analysis of anatomical relationships between INs 10-3 and 9-3 and directional regions of the L and M afferent direction maps (triangles, L afferents; squares, M afferents). Left column, Percent anatomical overlap among X, Y, and Z dendrites of the 10-3 interneuron and different regions of the direction maps. The peak directional sensitivity of each dendrite corresponds closely with the amount of anatomical overlap it has with the afferents (whether L or M) that have the same directional sensitivities. Note that dendrites of the 10-3 overlap slightly more with L afferents than with the M afferents with the same directional sensitivity. Right column, Percent anatomical overlap among each dendrite of the 9-3 interneuron (X, Y, W, Z) and different regions of the direction maps. The peak directional sensitivity of each dendrite correlates closely with the amount of its anatomical overlap with afferents that have the same peak directional sensitivity.

To answer this question, the amount of anatomical overlap similar to that of the M afferents (Miller et al., 1991; Theunissen between L and M afferents with two different identiﬁed interneu- et al., 1996). Other studies have suggested that these differences rons was measured. These two interneurons are known to have in frequency tuning might arise from differential connectivity similar directional tuning properties but are sensitive to different between M and L afferents (Chiba et al., 1992). ranges of air current frequency. Interneuron 10-3 (Fig. 7A, red) The dendritic arborizations of these two interneurons overlap has a frequency sensitivity range similar to the L afferents, and extensively, occupying similar areas of neuropil (Fig. 7A, stereo interneuron 9-3 (Fig. 7A, blue) has a frequency sensitivity range pair). The pattern of anatomical overlap between interneurons Paydar et al. • Direction and Frequency Representation in the Cercal System J. Neurosci., March 1, 1999, 19(5):1771–1781 1779

10-3 and 9-3 and the M and L direction maps is shown in Figure organization results in a unique combination of stimulus param- 7B–E. Although both interneurons appear to sample very similar eters mapped at any one neuropil location. In the nucleus mag- areas of the two maps, there are subtle differences between the nocellularis of the barn owl, interaural phase difference is two arborization patterns. For example, dendrite X of 10-3 over- mapped along one axis, and stimulus frequency is mapped along laps almost equally with areas representing lateral and anterior another axis (Carr and Konishi, 1990). This arrangement is directions ( green and red, respectively) in the L direction map. functionally similar to the cricket system, in that a map of inter- However, that same dendrite overlaps less with these two regions aural time differences exists for each range of stimulus in the M direction map. Conversely, dendrite Y of interneuron frequencies. 9-3 overlaps more with areas representing lateral wind directions The functional organization of the cercal cricket system is in the M direction map than in the L map. perhaps more similar to V1 in the cat visual cortex. Several To quantify these anatomical observations, the amount of researchers have demonstrated the existence of multiple func- anatomical overlap between each of the interneuron dendrites tional maps in the cat visual cortex (Hubener et al., 1997; Shoham and each of the afferents was measured (Fig. 8). The results of et al., 1997). They and others have demonstrated a continuous these calculations demonstrate two points. First, corresponding functional map of orientation preference (Arieli et al., 1995; dendrites of each interneuron do, indeed, overlap with the same Bosking et al., 1997) and, more recently, maps of spatial fre- sets of afferents. Second, the anatomical overlap between the quency. The maps of spatial frequency are not continuous repre- interneurons and both populations of afferents is statistically sentations but are patchy and divided into two domains, one indistinguishable. Thus, the degree of differential connectivity tuned to low spatial frequencies and one to high spatial frequen- emerging from the physical segregation illustrated in Figure 7 is cies. We predict a very similar functional organization in the not significant, and this small degree of segregation could not be cricket system. The afferent projection scheme in the cricket responsible for the observed frequency selectivity of the cercal system results in a continuous map of stimulus direction interneurons. that has structural attributes very similar to those of the pinwheels seen in the visual cortical map (Bonhoeffer and Grinvald, 1993). DISCUSSION In contrast, the representation of stimulus frequency in the Representation of multiple sensory parameters cricket cercal map does not show any clear anatomical segrega- Representations of multiple sensory stimulus parameters have tion. The cercal map of stimulus frequency is very likely to be been demonstrated in a variety of systems (Middlebrooks et al., patchy and discontinuous. Low-frequency air current stimuli 1980; Middlebrooks and Pettigrew, 1981; Shipp and Zeki, 1985; should elicit large patches of activity, whereas high stimulus Hubel and Livingstone, 1987; Clarey et al., 1994). This paper frequencies should elicit relatively small patches of activity. This demonstrates how two different sensory stimulus parameters, prediction is based on the fact that afferents sensitive to low air direction and frequency, are organized anatomically in the cricket current velocities have large overlapping arborizations, and affer- cercal sensory system. Afferents tuned to similar air current ents sensitive to high stimulus frequencies have much smaller directions segregate from one another to form a continuous nonoverlapping arborizations. Thus, in both the visual system representation of stimulus direction within the terminal ganglion and the cercal system, one functional parameter is mapped con- (Jacobs and Theunissen, 1996). However, the extensive overlap tinuously (visual stimulus orientation/air current direction) and between L and M afferents indicates that there is no obvious another functional parameter is not (visual spatial frequency/air segregation of their arbors according to frequency sensitivity. current frequency). Thus, stimulus frequency is not mapped in any recognizable Extraction of information from map by interneurons: coordinate system. Rather, information about stimulus frequency two different mechanisms is distributed throughout the neural map of stimulus direction. This anatomical organization has several functional conse- There is good evidence, based on physiological studies, that quences. Given that direction and frequency parameters are interneuron 10-3 receives a majority of its afferent input from the encoded by the same sets of afferents, the same regions of neu- longest hairs on the cercus (Shimozawa and Kanou, 1984a,b; ropil will be activated by air currents from a given direction Chiba et al., 1992; Theunissen et al., 1996). Interneuron 10-3 is regardless of the stimulus frequency. Thus the direction of the tonically active under ambient conditions (as are the L afferents) stimulus is preserved throughout the entire frequency range of and tuned to a frequency range very similar to that of the L stimuli. In contrast, for a given frequency, the area of neuropil afferents. Conversely, interneuron 9-3 is insensitive to the lowest activated by a sensory stimulus will change systematically as a frequency ranges (as are M afferents) and is silent under ambient function of stimulus direction. Most biologically relevant stimuli conditions, suggesting that the bulk of its afferent inputs may are composed of a range of stimulus frequencies; however, fewer come from M afferents (Miller et al., 1991). may be composed of stimuli in which direction is changing However, our results indicate that anatomical relationships dynamically. between afferents and interneurons do not play a significant role in determining the frequency-tuning characteristics of the inter- Comparison with other sensory systems neurons. Two interneurons, interneuron 10-3 (tuned to low fre- The anatomical organization of the cricket system is quite differ- quencies) and interneuron 9-3 (tuned to higher frequencies), have ent from some neural maps found in the auditory system. In owls, nearly equal anatomical access to L and M afferents. Thus, if cats, and bats different stimulus parameters are mapped in or- frequency tuning of interneurons depends on selective connec- thogonal coordinate systems (Knudsen and Konishi, 1978; tions with afferents tuned to a specific dynamic range of stimuli, O’Neill and Suga, 1982; Middlebrooks and Knudsen, 1984; for then mechanisms other than anatomical segregation must play a review, see Knudsen et al., 1987; Carr, 1993). For example, in the role. primary auditory cortex, stimulus intensity is mapped orthogonal Possible mechanisms that could account for these differences in to stimulus frequency (Middlebrooks et al., 1980). This functional frequency tuning fall into two general categories. First, selective 1780 J. Neurosci., March 1, 1999, 19(5):1771–1781 Paydar et al. • Direction and Frequency Representation in the Cercal System connectivity between interneurons and restricted subsets of af- in area 18 of cat visual cortex: optical imaging reveals a pinwheel-like ferents having specific frequency-tuning characteristics (e.g., be- organization. J Neurosci 13:4157–4180. Borst A, Egelhaaf M (1992) In vivo imaging of calcium accumulation in tween 10-3 and L afferents) could be achieved through mecha- fly interneurons as elicited by visual motion stimulation Proc Natl Acad nisms other than differential overlap with segregated inputs. Sci USA 89:4139–4143. Presumably, mechanisms for the recognition of molecular labels Borst A, Egelhaaf M (1994) Dendritic processing of synaptic informa- or for activity-dependent strengthening of certain synapses would tion by sensory interneurons Trends Neurosci 12:257–263. need to exist to allow such specificity of connection. 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