The physiology and morphology of two types of electrosensory neurons in the weakly electric fishApteronotus leptorhynchus

J Comp Physiol A (1984) 154:199-209 Journal of

Sensory, Comparative .ou~,, and Physiology A Behavioral Physiology

�9 Springer-Verlag 1984

The physiology and morphology of two types of electrosensory neurons in the weakly electric fish Apteronotus leptorhynchus

James Saunders and Joseph Bastian Department of Zoology, University of Oklahoma, Norman, Oklahoma 73069, USA

Accepted October 12, 1983

Summary. Previous anatomical and physiological studies of the gymnotoid electrosensory lateral line lobe (ELLL) suggest that the anatomically identified basilar and non-basilar pyramidal cells correspond to the physiologically defined E and I cells. Intracellular injection of horseradish peroxidase (HRP) into physiologically identified E and I cells confirms this hypothesis. The morphologies and physiological responses of the basilar and non-basilar pyramidal cells were compared. Both types of pyramidal cells have extensive apical dendritic trees that interact with a parallel fiber network in the ELLL. The apical dendritic trees of the non- basilar pyramidal cells have a wider spread along the rostrocaudal axis of the ELLL than those of the basilar pyramidal cells. This difference is discussed in reference to the interaction of these cell types with the parallel fibers of the ELLL. The density of apical dendritic branches was measured and related to the distance of these branches from the cell body. No obvious differences were seen between the dendritic density patterns of basilar and non-basilar pyramidal cells. An interesting correlation, however, exists between the atypical physiological characteristics of two basilar pyramidal cells and their dendritic density patterns. Two cells of the medial (ampullary) segment of the ELLL were analyzed. Like the pyramidal cells of the three lateral (tuberous) regions of the ELLL, the physiology of these cells appears to be related to the presence of an extended basilar process. The ampullary cells, however, have apical dendritic trees that are oriented orthogonally to the dendritic trees of the pyramidal cells.

Abbreviations: A M amplitude modulation; D M L dorsal molecular layer; E L L L electrosensory lateral line lobe; EOD electric organ discharge; H R P horseradish peroxidase; LC lobus caudalis; Npd nucleus praeeminentialis dorsalis; P S T H post stimulus time histogram

Introduction

South American fish of the order Gymnotiformes have evolved specialized lateral line organs for electroreception and organs which emit an electrical discharge (for reviews see Szabo 1974; Heili- genberg 1977; Bullock 1982). Members of the gen- us Apteronotus generate a quasi-sinusoidal electric organ discharge (EOD) with a high constant frequency. The local amplitude of this EOD is a function of the conductivity of the animal's local envi- ronment. Thus an object near the fish with a conductivity different from the surrounding water creates an amplitude modulation (AM) of the EOD resulting in a transepidermal voltage change at a region of the fish's body. Transepidermal voltage changes are detected by tuberous electroreceptors on the body surface. One type of tuberous receptor in gymnotoids is the P-receptor or proba- bility coder, which responds to positive AMs (an increase in EOD amplitude) with a higher proba- bility of firing (Hagiwara etal. 1965a; Scheich et al. 1973; Hopkins 1976) and adapts slowly to stepwise changes in EOD amplitude (Hagiwara et al. 1965a; Bastian 1981 a). Each receptor organ is innervated by a single electrosensory afferent (Bennett 1967; Watson and Bastian 1979) that pro- jects to the Electrosensory Lateral Line Lobe (ELLL), formerly termed Posterior Lateral Line Lobe of the medulla (Maler et al. 1974).

Enger and Szabo (1965) found phasic cells in the ELLL of Apteronotus albifrons which were either excited or inhibited by the presence of a con- ductive metal plate in the EOD field (such a plate causes a local increase in the EOD amplitude or positive AM). Such cells were sensitive to the direc- tion of object movement within the field. Bastian (1981b) studied the response properties of two types of similar electrosensory neurons in the

200 J. Saunders and J. Bastian: Electrosensory neurons: physiology and morphology

A DML.

VML. St. Fi b. PoIy. L.

PI. L.

Gr. L.

DNL.

DFL.

M. " a

cx<2 Fig. 1. A Simplified summary of the neurocircuitry of the ELLL showing the laminae and major cell types (Maler et al. 1981). B Schematic 3-dimensional drawing of the ELLL depicting the orientation of the pyramidal cell apical dendritic tree and its associated fiber layers. The lamina in A from ventral to dorsal are the Deep Fiber Layer (DFL.), Deep Neuropil Layer (DNL.), Granule Layer (Gr. L.), Plexiform Layer (Pl. L.), Polymorphic Layer (Poly. L.), Stratum Fibrosum (St. Fib.), Ventral Molecular Layer (VML.), and Dorsal Molecular Layer (DML.). The receptor afferents (Rec. Aff), represented by large filled fibers, make synapses with Type I (Gr. i) and Type II (Gr. 2) granule cells (unfilled) and with the basilar pyramidal cells (stipled - B. Pyr.). This figure shows synapses between Type I granule cells and both types of pyramidal cells and between Type II granule cells and non-basilar pyramidal cells (stipled - Nb. Pyr.). Neither diagram is drawn to scale, and the axes shown in B are intrinsic to the ELLL. Dotted line in B: outline of the dendritic tree at a given dorsoventral level parallel to the horizontal plane of the brain

ELLL and used the terms 'E cells' and ' I cells' to describe their opposite responses to positive AMs. The E cells respond to positive AMs with an increase in spike frequency; the I cells respond to the same stimulus with a decrease in activity.

Neuroanatomical analysis of the ELLL of Ap- teronotus albifrons reveals two types of neurons, basilar and non-basilar pyramidal cells, whose synaptic connections suggest that they correspond to the physiologically differentiated E and I cells (Maler et al. 1981). The ELLL is a laminar structure (Fig. 1 A) with eight distinct layers and eleven cell types. The P-unit receptor afferents terminate in the most ventral lamina of the ELLL, the deep neuropil layer. The polymorphic layer contains the basilar pyramidal cells (probable E cells), the non- basilar pyramidal cells (probable I cells), and polymorphic cells (Maler 1979). Both types of pyramidal cells have extensive apical and somatic dendritic trees, but only the basilar pyramidal cells have a prominent basilar dendrite that terminates in the deep neuropil lamina. The basilar cells receive direct chemical synaptic input from the receptor afferents, whereas the non-basilar pyramidal cells receive a disynaptic input from the afferents via the granule cells (Maler et al. 1981). The two

types of granule cells, Type I and Type II, are similar except that the Type II granule cells have apical dendrites. Both types of granule cells receive gap junction and chemical synaptic input from the receptor afferent fibers. The morphology of these chemical synapses suggest that they are excitatory. The granule cells are connected to non-basilar pyramidal cells, basilar pyramidal cells and polymorphic cells by chemical synapses that are pre- sumed to be inhibitory, based on their microana- tomy (for review see Uchizono 1975; Maler et al. 1981). The chemical synaptic input to the non-basilar pyramidal cells via granule cells could, therefore, explain the depressed activity of I cells with increased receptor activity. The granule cells are also connected by gap junctions to the non-basilar pyramidal cells. Maler et al. (1981) suggest that the chemical synaptic inputs of the granule cells act to suppress basilar pyramid activity and the gap junction inputs of granule cells enhance non- basilar pyramid activity with a relatively long de- lay, thus, the granule cells could also be responsible for the phasic component of the response of both E cells and I cells to EOD amplitude modulations. Despite the close agreement between the anatomical and physiological models for the ELLL cir-

J. Saunders and J. Bastian: Electrosensory neurons: physiology and morphology 201

cuitry, there is no direct evidence linking the physiologically defined E and I cells and the anatomically identified basilar and non-basilar pyramidal cells.

The apical dendritic trees of both basilar and non-basilar pyramidal cells synapse in both divi- sions of the ELLL molecular layer: the Ventral Molecular Layer (VML) and the Dorsal Molecular Layer (DML). The orientation of the molecular layer fibers and of the apical dendritic tree is depicted in Fig. 1 B. The unmyelinated fibers of the VML arise from a layer of densely packed myelin- ated fibers dorsal to the polymorphic lamina, the stratum fibrosum. These fibers originate in the midbrain nucleus praeeminentialis dorsalis (Npd) (Maler et al. 1982). The fibers of the VML run mediolaterally at right angles to the apical dendrites of the pyramidal cells. The dorsal molecular layer (DML) consists of parallel and vertical fibers and is continuous with the molecular layer of the lobus caudalis of the cerebellum (Maler et al. 1974). The parallel fibers of this layer originate from granule cells of the lobus caudalis and are also orthogonal to the apical dendrites of the pyramidal cells (Maler 1979; Maler et al. 1981). The fibers of the DML, however, run rostrocaudal in the ELLL at right angles to those of the VML (Maler 1979).

The laminar structure of the lobe is disrupted by three discontinuities (breaks) that divide the lobe into four roughly equal segments: the lateral, centrolateral, centromedial, and medial segments (Carr et al. 1982). Each tuberous receptor afferent terminates in the lateral, centrolateral, and centromedial segments (Heiligenberg and Dye 1982). The ELLL, therefore, receives three complete somatotopic maps from the high frequency electroreceptors. The medial segment is the sole recipient of the ampullary (low frequency sensitive) receptor afferents (Heiligenberg and Dye 1982). The role of multiple somatotopic maps is not clear and no physiological or morphological comparisons have been made between pyramidal cells from different segments. The axons of both basilar and non-basilar pyramidal cells project to two midbrain struc- tures: the nucleus praeeminentialis dorsalis (Npd) and the torus semicircularis. The four separate maps of the ELLL converge to form a single somatotopic map in the torus (Carr et al. 1981). Each of the four maps, however, project to separate parts of the Npd (Maler et al. 1982).

This study demonstrates the relationship between the physiologically defined E and I cells and the anatomically identified basilar and non-basilar pyramidal cells by intracellular recording and

histological analysis. Light microscopy was used to compare the cellular morphologies of basilar and non-basilar pyramidal cells, as well as pyramidal cells in adjacent segments of the ELLL. These results are discussed with reference to the descending input to the ELLL from higher brain centers. In addition, the physiology and morphology of two cells in the medial (ampullary) segment are described.

Materials and methods

Weakly electric fish (Apteronotus leptorhynchus), 12-15 cm in length, were paralyzed with an injection of Flaxedil and sus- pended in a plexiglass tank that was 10 cm wide, 23 cm long and 5 cm deep with only the top of the animal 's head above the water surface. Artificial respiration and surgical procedures were identical to those described elsewhere (Bastian 1974, 1977). Water temperature was maintained between 23 and 25 ~ and the water conductivity was adjusted to 10 kOhms.cm.

EOD field measurement and stimulus generation. The electric organ discharge (EOD) field was measured with two 20 gauge wire electrodes that were insulated to within 5 mm of their tips and placed 1 cm apart perpendicular to the long axis of the fish at the pectoral fin. These electrodes were connected to a Grass model P15 preamplifier (filters set at 3 Hz and 10 kHz), whose output triggered a series of 5 V square wave pulses that were phase locked to the EOD cycle. These pulses were gated ' o n ' for 100ms, triggering an Exact model 509 function generator to produce single sine wave cycles with a period of about 1 ms. In this way, 100 ms tone bursts of an EOD-like waveform were produced in phase with the animal 's own EOD. The tone bursts were then applied to the fish via a 30 gauge wire electrode placed in the animal 's mouth and a 20 gauge wire placed near the fish's tail. The result of this stimulus was a 100 ms increase in the EOD amplitude, which was adjusted to approximately twice the original amplitude (usually between 1.5-2.0 mV/cm).

Recording and histological methods. Intracellular recordings were made with glass micropipettes tilled with a 10% horseradish peroxidase (HRP-Sigma Type VI) in a 0.5 mol/1 KC1 solution with a 0.1 mol/1 Tris-HC1 buffer (pH=7.4) . Typical electrode impedance was 40-60 MOhms. A WPI M701 bridge preamplifier was used for recording and HRP iontophoresis. Spike records and stimulus synchronization pulses were recorded on an F M magnetic tape recorder for later computer analysis. HRP was iontophoresed into the cell by passing a 2.5-5.0 nA current in the form of a 4 Hz positive D.C. offset sine wave for 2-10 min. The animal's skull was then packed with gelfoam and resealed with a histoacryl glue. The fish were respirated for 2-12 h and then perfused via a cannula in the bulhus arterio- sus. The animals were perfused with a Heparin-Ringer solution until all blood was removed, followed by perfusion with a buffered 4% glutaraldehyde solution (pH=7.4) for 10 min and a buffered 30% sucrose/4% glutaraldehyde solution for 20 rain. Transverse brain sections were cut at 60 ~m and processed with a modified Hanker-Yates protocol (Hanker et al. 1977; Bell et al. 1981). Sections were mounted on glass slides and counter- stained with cresyl violet.

Data collection and analysis. The dendritic morphology of HRP- filled cells was reconstructed from serial transverse sections in

202 J. Saunders and J. Bastian: Electrosensory neurons : physiology and morphology

A D 3 2 I

B D

Fig. 2. A Diagram of three consecutive transverse brain slices depicting the coordinate measurements made for horizontal contour maps. Slices are numbered from rostral to caudal with the cell body shown in slice 1. The distance of the most medial (X 1 and X4) and the most lateral (X 2 and Xa) dendritic branches from the midline of the brain was measured at several dorsoventral (z) levels (10 gm apart). The Y values were obtained by multiplying the section thickness (60 gm) by the number of sections between the dendritic branch and the cell body (Y1 = 60 ~tm, Y 2 = 120 gin). B Diagram of the relationship between the dendritic morphology within a transverse brain slice and a laminar-specific contour. Laminar-specific contours outline the dendritic tree at several levels parallel to the cell body lamina (labelled z). The x values were measured relative to a fixed line of reference (Ref.). A composite contour from several transverse sections is shown on the right, and its relationship to the illustrated section is indicated by the dotted lines and by the corresponding x values (X~ and Xz). Arrow at the left: aspect from which the contour is depicted. The vertical axis of the contour is rotated parallel to the lamina and is referred to as the laminar dorsoventral axis (D e and VL)

three ways. The first was by simply combining the labeled dendrites of successive sections into a single drawing with a projec- t ion microscope. The second and third ways were more quantitative and allowed the distribution of dendrites to be described at several levels that were parallel to either the horizontal plane (horizontal contour maps) or the lamination of the ELLL (laminar-specific contour maps). Horizontal contour maps define the perimeter of the dendritic tree at several dorsoventral levels parallel to the horizontal plane of the brain. Beginning at the most ventral level of each transverse section, the extent encom-

passed by the dendritic tree was determined as follows (see Fig. 2A) : the distance of the most medial and the most lateral dendritic branches from the midline of the brain was measured and entered into an array as the x coordinates limiting the medial and lateral boundaries of the dendritic tree. The y coordinate value in this array reflects the relative rostrocaudal posi- t ion of the branches and was obtained by multiplying the number of transverse sections from the cell body by the section thickness (60 gm). The z values at each x-y position within this array represent the dorsoventral elevation of each level above the cell body. This procedure was repeated for a series of dorsoventral levels 10 gm apart and for all transverse sections that contained a dendritic fragment. Contour maps of the arborization perimeters were made by linking the coordinates of all identical elevations (z values) by computer. Contour maps of this type were used to determine the orientation of the dendritic tree in the ELLL as well as the transverse and sagittal spreads of the arborization.

Laminar-specific contour maps were generated in a similar way except that the contour levels were parallel to the lamination of the port ion of the ELLL that contained the cell body. The x coordinates in this case correspond to the distance of the most distal branches from a fixed line of reference (REF of Fig. 2 B) perpendicular to the lamina of the lobe. The axis (D L - VL) is defined as being parallel to the intrinsic lamination of the lobe in the region of the cell under study. The relationship between a single laminar-specific contour and its corresponding level of the pyramidal cell dendritic tree may be seen in Fig. 2 B. The line connecting X~ and Xz of Fig. 2B runs along the DL--V L axis. These contours provide a measure of the cross- sectional area of the dendritic tree at several levels parallel to the specific lamina which contains the cell body. The number of branches at each laminar-specific level was determined from the serial reconstructions. Finally, the number of branches at each level was divided by the cross-sectional area at that level to yield a measure of the dendritic density.

The physiological response of a cell to the stimulus was determined immediately with an oscilloscope and analyzed sub- sequently with computer-generated post-stimulus time histo- grams (PSTH). Two types of PSTHs were analyzed: the first sampled spike activity for 500 ms and was used to determine the response type, the second type of PSTH only measured the first 50 ms of the response (with ten times the temporal resolution) and was used to measure response latencies. All measures made of the physiology and morphology of the cells studied were compared for differences between cell types (basilar or non-basilar) and between cell location (lateral or centrolateral zones).

R e s u l t s

Physiology of pyramidal cells

Direct observation and post-stimulus time histo- grams (PSTH) were used to classify the pyramidal cells as either E types or I types; PSTH's were made for 7 E types and 8 I types. E types respond to an increase in EOD amplitude with an increase in spike frequency; this response was observed for all basilar pyramidal cells. Figure 3A shows the cellular morphology of a basilar pyramidal cell, the intracellular record from this cell during a positive A M (Fig. 3 B) and a PSTH of its response to a 100 ms, 2 mV/cm increase in the EOD (Fig.


Fig. 3. A Basilar pyramidal cell reconstrncted from five 60 gm serial cross-sections of the ELLL. Scale bar 100 gm. B Re- corded intracellular activity of the cell shown in A. Stimulus is a 2-mV/cm increase in EOD amplitude. Tic mark represents stimulus duration (100 ms). Spike amplitude 27 mV. C Post Stimulus Time Histogram of the response of the cell shown in A to 25 replicates of a i00-ms, 2-mV/cm increase in EOD amplitude. D Biphasic response of another basilar pyramidal cell to a 100-ms, 2-mV/cm increase in EOD amplitude. Hori- zontal tic marks in C and D: stimulus duration. Vertical tic marks in C and D: 10 spikes/bin

13

C

i

Fig. 4. A Non-basilar pyramidal cell reconstructed from ten 60-~tm serial cross-sections. Scale bar 100 gin. B Recorded intracellular activity of the cell shown in A. Tic mark: duration (100 ms) of the stimulus, a 2-mV/cm increase in EOD amplitude. Spike amplitude approx. 20 inV. C Post Stimulus Time Histogram of response of the cell shown in A to 25 stimulus replicates (2 mV/cm increase in EOD amplitude). Horizontal tic mark: duration (100 ms) of the stimulus. Vertical tic mark 10 spikes/bin

3 C). A biphasic response to increased EOD amplitude was observed for two basilar cells (Fig. 3D). This response consisted of two distinct bursts of activity separated by a mean interburst interval of 80 ms. The basilar pyramidal cells which responded in this way also share morphological characteristics that will be discussed in the following section. I-types respond to an increase in EOD amplitude with a decrease in spike frequency. Most (90%) of the identified non-basilar cells were markedly inhibited by an increase in EOD amplitude; the remaining non-basilar pyramidal cell showed little or no response to the stimulus. Fi- gure 4A shows the cellular morphology, the intracellular record from this cell during a positive AM (Fig. 4 B), and a PSTH of its response to a 100 ms,

2 mV/cm increase in EOD amplitude (Fig. 4 C) for a non-basilar cell. The mean response latency of the basilar pyramidal cells (E types) was 3.74 ms (s.d. = 2.37); the mean response latency of the non- basilar pyramidal cells was longer (mean = 5.24 ms, s.d. =2.67), perhaps indicating more synaptic de- lays. The difference in response latencies is suggestive but not significant at the 0.05 level. The mean response latencies in this study are consistent with the modal response latencies reported for E and I cells by Bastian (1981 b).

Pyramidal cell morphology

The pyramidal cells in this study can be divided into two distinct categories, basilar and non-basi-


5 4

5

6

" 7

8

9

Fig. 5. Reconstruction of a pyramidal cell axonal termination in the torus semicircularis dorsalis (TSd), taken from eight 60-gin serial cross-sections. Laminae 3-9 of the TSd indicated at right. Scale bar 100 gm

lar. All of the cells studied were located in two of the three segments in the ELLL that receive tuberous receptor input, the lateral and centrolateral segments. All of the cells included in the morphological analysis were physiologically identified either directly or by PSTH's as E or I types. Ten basilar pyramidal cells are included, 4 of which were in the lateral and 6 in the centrolateral segment. Similarly, ten non-basilar cells were identified, 6 of which were lateral and 4 centrolateral. No morphological differences were found between either basilar or non-basilar pyramidal cells in adjacent segments of the ELLL. Complete dendritic arborizations were reconstructed for 6 cells of each

type . Segmental boundaries within the molecular lamina were estimated by extrapolating the boundaries visible in the polymorphic and granule layers. The pyramidal cell dendritic trees do not cross these extrapolated boundaries and remain in the same segment as the cell body. The cell bodies of all pyramidal cells studied were located in the polymorphic cell layer and have axons which remain lateral until they leave the ELLL at its rostral margin and enter the lateral lemniscus. No local collat- erals of pyramidal cell axons were found within the ELLL. Attempts to follow single axons from the ELLL to the torus semicircularis failed. One pyramidal cell axon was filled in the torus, however, and its arborization is shown in Fig. 5. This axonal process arborizes within six distinct laminae of the torus: Laminae 3, 4, 5, 7, 8 and 9. This

is consistent with the observations of Carr et al. (1981).

Cell bodies of basilar and non-basilar pyramidal cells are shown in Fig. 6 A, B respectively. The mean soma width of the basilar pyramidal cells was 18.4 gm (s.d. =7.7) and the mean soma width of the non-basilar pyramidal cells was 19.8 pm (s.d. = 7.7); no difference in mean soma width was found between the basilar and non-basilar cells. The mean length of the basilar cell bodies was significantly greater ( P < 0.05) than that of the non- basilar cells at 57.0 (s.d.=22.1) and 38.2 (s.d.= 10.9) gm respectively. The basilar dendrite of the basilar pyramids extends into the deep neuropil layer of the PLLL and the mean length of this process is 137 gm (s.d.=37). This figure is larger than that reported by Maler (1979), and the difference may be due to shrinkage or to the lack of serial reconstruction in the Golgi material.

In the gymnotoids, the ELLL extends out dor- sally and laterally from its fusion with the dorsal rhombencephalon. The intrinsic axes of the lobe are rotated by approximately 45 ~ from the brain- stem axes, moving the rostral border of the lobe relatively medial and the caudal border relatively lateral (Maler 1979). Throughout this paper, the terms ' rostrocaudal ' and 'mediolateral ' refer to the intrinsic axes of the ELLL unless otherwise stated. The apical dendritic trees of the pyramidal cells bend medially along the intrinsic mediolateral axis of the lobe. This bending can be seen in Fig. 7, which shows the extent of the horizontal arborization for each cell type at 5 dorsoventral levels. This type of contour map can be used to measure the extent of the dendritic trees along the intrinsic axes of the ELLL. The dendritic trees of both cell types reach the fourth ventricle, which defines the mediorostral boundary of the lobe, and no difference was found in the mediolateral spread of basilar and non-basilar pyramidal cells (mean of basilar = 696 txm, s.d. = 175; mean of non-basilar= 656 ~tm, s.d. =228). The mean rostrocaudal spread of the basilar cell trees (Fig. 7B), however, was 250 gm (s .d.=19) which is significantly less (P<0.01, Fischer's t-test) than the rostrocaudal spread of the non-basilar cell trees, Fig. 7A (365 gm, s.d. = 65). The values reported here for the mediolateral and rostrocaudal extents are 20-100% larger than comparable values given by Maler (1979). This dis- crepancy is probably due to the inavailability of completely reconstructed dendritic arbors and the considerable shrinkage of Golgi material in the previous work.

The cross-sectional area at various levels parallel to the cell body lamina and the dorsoventral


Fig. 6A, B. Photomicrographs of A Basilar pyramid cell body and B Non-basilar pyramid cell body. Scale = 100 ~ma

A NON-BASILAR B BASILAR

85%

>85%

Fig. 7A, B. Horizontal contour maps outlining the dendritic tree for Non-basilar A and Basilar B pyramidal cell dendritic trees at five dorsoventral levels, given as percentages of the total dorsoventral extent. Contours viewed from the dorsal aspect. Greater percentage values indicate more dorsal contours. Axes shown are intrinsic to the ELLL. Scale bar 100 gm

extent of each dendritic tree can be obtained from the laminar-specific contour maps. Examples of laminar-specific contour maps for basilar and non- basilar pyramids are given in Fig. 8. No significant difference was found between the mean maximal cross-sectional areas of basilar and non-basilar cells. The dorsoventral spread of the basilar cells (Fig. 8 B), however, was greater than that for the non-basilar cells, Fig. 8 A, (mean of basilar cells =

151 gm, s .d.=52.2; mean of non-basilar cells= 102 gm, s.d. =21.1). The difference between these means is suggestive, although not significant (P = 0.06). The fact that the dendritic trees of both cell types have comparable cross-sectional areas with different dorsoventral widths is consistent with a difference in the rostrocaudal spread (see Fig. 8). The parallel fibers of the dorsal molecular layer (DML) run orthogonal to the apical dendritic trees


D, A NON- BASILAR @ -' C R

VL

88%

65%

50%

B BASILAR '~,

~-~-

~ f 22%

Fig. 8A, B. Laminar specific contours outlining the dendritic tree for Non-basilar A and Basilar B Pyramidal cells taken at four levels parallel to the lamina that contains the cell body. Distances of each level from the cell body expressed as percentages of the total dendritic length. Scale bar 100 Ixm. The rostrocaudal axis shown is that of the brain stem, not of the ELLL. The dorsoventral axis given is parallel to the lamination of the lobe and is therefore referred to as the laminar dorsoventral axis (D L and VL)

d (OR

O9 I,I "I-

0 Z

rr~ 0o% x DENDRITIC LENGTH

Fig. 9. Relationship of dendritic density to the distance from the cell body (expressed as a percentage of the total dendritic length) for a typical pyramidal cell (open squares) and for an atypical type of basilar pyramidal cell that lacks a second peak of dendritic density (crosses)

in the intrinsic rostral-caudal axis; therefore the non-basilar pyramidal cells, having a relatively smaller dorsoventral extent, could contact with fewer D M L fibers than the basilar pyramidal cells. Since the non-basilar pyramidal cells have a large rostrocaudal spread, they have more potential contact sites per fiber than the basilar cells, if the dendritic branch densities are equivalent.

The dendritic density can be obtained by divid- ing the number of branches at a given laminar- specific level by the cross-sectional area of the dendritic tree at that level. A plot of the dendritic density of a typical pyramidal cell as a function of the distance from the cell body is shown in Fig. 9 (squares). Nine out of 12 of these plots show two distinct peaks o f dendritic density within the DML. There is no obvious correlation between the characteristics of these plots and either cell type or cell location. All of the arborizations show an increase in density, the first peak in Fig. 9, at a mean of 30% of the full extent of the tree (s.d.--10.6). The dendritic trees give off their major branches at this level. The density of this peak ranges from 1.05 x 103 to 3.28 x 103 branches/mm 2 with a mean of 2.06 x 103 (s.d. =0.68 x 103). The second peak is absent in three of the plots (e.g. Fig. 9, x's) and the dendritic density at this level is more variable; this second peak occurs at a mean of 80% of the full extent of the dendritic tree and ranges from 0.82 x 103 to 9.32 x 103 branches/mm 2 with a mean density of 4.52 x 103 branches/mm 2 (s.d. =2.88 x

103). Two of the three cells that did not show a second density peak were basilar pyramids with biphasic physiological responses mentioned earlier (Fig. 3 D). The second peak of dendritic density may reflect a compression of the dendritic tree at the rostromedial margin of the molecular layer.

Cells o f the medial segment

Two cells were identified in the medial segment of the ELLL. One of these cells had a prominent basilar process which extended into the deep neuropil layer (Fig. 10A); this cell responded to a 2 mV/cm increase in EOD amplitude with an increase in spike activity (Fig. 10 B). The width and length of this cell body were 20 and 40 gm respectively and the basilar process was 105 tam long. The second cell of the medial segment was 20 gm wide, 53 ~tm long and had a short basilar process which did not reach the deep neuropil layer (Fig. 10C). This cell was inhibited by a 2 mV/cm increase in EOD amplitude (Fig. 10D). The responses of these cells, which receive afferents from the low frequency ampullary receptors (Heiligen- berg and Dye 1982) is probably due to the low frequency components inherent in our A M of the EOD.

There are morphological characteristics that distinguish these medial cells from the pyramidal cells of the more lateral zones. The axons of the medial segment cells leave the lobe medially as op-


A

B D

Fig. 10. A Reconstruction of a cell from the medial segment of the PLLL with an extended basilar process taken from five 60-~tm serial cross-sections. Scale bar 100 gm. B Post Stimulus Time Histogram of 25 stimulus replications of a 2-mV/cm increase in EOD amplitude. Horizontal tic mark: stimulus duration (100 ms). Vertical tic mark: 10 spikes/bin. C Reconstruction of a cell from the medial segment of the PLLL with a brief basilar process taken from four 60-l~m serial cross-sections. D Post Stimulus Time Histogram of 75 stimulus replications of a 2-mV/cm increase in EOD amplitude. Horizontal tic mark: stimulus duration (100 ms). Vertical tic mark: 10 spikes/bin

posed to the more lateral pyramidal cell axons which exit mediorostrally. The shape of the medial cell with the basilar process is quite different from the fusiform shape of the basilar pyramidal cells. Furthermore, the dendritic trees of the two medial cells are quite different from those of the more lateral pyramidal cells. The dendritic trees of the medial cells extend rostral to the cell body and their main extent is along the intrinsic rostral-caudal axis of the lobe (orthogonal to that of the more lateral pyramidal cells) with a mean sagittal spread of 347 gm and a mean transverse spread of only 132 gm.

Discussion

The intracellular injection of HRP into physiologically identified cell types allows the physiology of a cell to be directly related to its anatomy. A de- tailed analysis of the morphology of identified cell types can then be used to predict aspects of the anatomy and physiology of other components in the system.

The neuroanatomical analysis of specific cell types in the ELLL of waveform gymnotoids led to predictions about the physiology of these cells (Maler 1979; Maler et al. 1981). Basilar pyramidal cells have prominent basilar dendrites which make synaptic contact with the P-unit receptor afferents (Maler et al. 1981). These synapses are anatomically characterized as excitatory synapses; therefore, Maler et al. (1981) predict that the spike activity of basilar pyramidal cells is directly related to electroreceptor activity. Non-basilar pyramids have no basilar dendrite and receive a chemical synaptic input from granule cells. This synapse is

symmetrical and contains pleomorphic vesicles (Maler et al. 1981), features which are considered characteristic of an inhibitory synapse (Uchizono 1975). The granule cells receive gap junction input and chemical synaptic input from the receptor afferents that is probably excitatory. On these anatomical grounds, Maler et al. (1981) predicted that the non-basilar pyramidal cell will be inhibited by increased electroreceptor activity. Such physiological cell types, termed E cells and I cells respectively, have been described in Apteronotus (Enger and Szabo 1965; Bastian 1981 b), Eigenmannia (Scheich 1977) and Sternopygus (Matsubara 1981). In this report, the intracellular injection of HRP into physiologically identified E cells and I cells confirms that these cell types correspond to the anatomically identified basilar and non-basilar pyramidal cells respectively. In contrast to the electroreceptor afferents, both E cells and I cells rapidly adapt to changes in the EOD amplitude (Enger and Szabo 1965; Scheich 1977; Bastian 1981b). This response was also observed for intracellular recordings from basilar and non-basilar pyramidal cells and could be due to delayed inhibitory synaptic input to basilar pyramids and delayed excitatory (gap junction) input to non-basilar pyramids by granule cells.

The observation that basilar pyramidal cells receive monosynaptic input and non-basilar pyramids receive a disynaptic input from the receptor afferents is consistent with the observed difference in response latencies. The mean response latency for I types is longer than that for E types. The mean latency of response values reported here are slightly greater than the modal response latencies given by Bastian (1981 b). The distribution of re-

208 J. Saunders and J. Bastian: Electrosensory neurons: physiology and morphology

sponse latencies for both E and I cells, however, is skewed towards longer latencies resulting in a mean that is greater than the mode.

The apical dendritic trees of both types of pyramidal cells receive input from three types of fibers in the molecular layers: the parallel and vertical fibers of the DML and the parallel fibers of the VML (Maler et al. 1981). The dendritic fields of both types of pyramidal cells in the VML are roughly circular, which is consistent with the observations of Maler (1979). The dendritic fields of basilar and non-basilar pyramids in the DML, however, are quite different. The non-basilar pyramidal cell dendritic field has an extended rostrocaudal spread and resembles a flattened ellipsoid (Fig. 8 A), whereas the dendritic field of the basilar pyramid has an extended dorsoventral spread and resembles a cigar-shaped (prolate) ellipsoid (Fig. 8B). Ramon-Moliner (1962) classified neurons according to the shape and orientation of the total dendritic volume and suggested that these pa- rameters were functionally significant in cells which interact with parallel fibers. The parallel fibers of the DML run longitudinally in the PLLL and orthogonal to the pyramidal cell arborization along their full extent (Maler 1979). Thus, the dendritic fields of basilar pyramids allow these cells to encounter more DML parallel fibers in the dorsoventral axis with a restricted rostrocaudal extent. Conversely, the broad rostrocaudal axis of non- basilar pyramid dendritic fields may allow these cells to integrate several synaptic inputs from a more restricted subset of parallel fibers.

The parallel fibers of the DML originate from granule cells in the caudal lobe (LC) of the cerebellum (Maler 1974). These cells receive electrosensory input from a variety of cell types in the nucleus praeeminentialis dorsalis (Npd) (Szabo et al. 1979; Sas and Maler, in preparation). The parallel fibers of the DML receive electrosensory input that is topographically organized. Because the greater dorsoventral extent of the basilar pyramids allows these cells to encounter more fibers than the non- basilar pyramids, the descending input to the basilar pyramids may include information from more cells of the Npd and, therefore, possibly a larger body surface than the non-basilar pyramids. It re- mains for the exact topography of the DML parallel fibers to be worked out before definite conclu- sions about the significance of the dendritic field differences of basilar and non-basilar pyramids can be made. Other possible inputs to the parallel fibers from the cerebellum include visual input (Bastian 1975) and proprioception (Bastian 1974).

The dendritic trees of both types of pyramidal cells are most dense at two distinct areas of the tree. The first of these areas occurs at the level of the dendritic tree which gives rise to the major branches. The second area of high dendritic density occurs at the distal branches of the tree near its medial boundary. The ELLL is bounded mediorostrally by the 4th ventricle and the volume avail- able to the dendritic arbor in the mediorostral ex- treme of the DML is much less than that of the more lateral regions of the molecular layer. The dendritic arbor of pyramidal cells taper in response to this reduced volume and this results in a compression of the most distal branches of the dendritic tree. This compression may provide the pyramidal cell with more dendritic interaction with a discrete set of fibers. A discrete interaction with these medial fibers may play a role in the nature of the pyramidal cell responses since two basilar pyramidal cells whose dendritic arbors did not taper into this reduced volume show atypical biphasic physiological responses.

The presence of cells in the medial segment of the ELLL that are sensitive to amplitude changes of the EOD is interesting. The medial segment of the ELLL is thought to receive only ampullary receptor afferents (Heiligenberg and Dye 1982), which are tuned to low frequency (0.05-50 Hz) voltage changes (Bullock 1982). The input to these cells was most likely from an ampullary receptor which responded to a low frequency component of the stimulus. Responses of ampullary receptor afferents to 'pulse' type EODs have been demonstrated in Electrophorus (Hagiwara et al. 1965b), Gymnotus carapo (Suga 1967) and the unrelated mormyrid Gnathonemus petersii (Bell and Russell 1978). Although the optimal frequency of the ampullary receptors is much lower than that of the tuberous organs, the sensitivity of ampullary receptors is greater (Bennett 1971). Another interesting feature of these medial cells is the orthogonal orientation of the main axis of the dendritic arbor to that of the lateral pyramidal cells. A difference in the dendritic arbor of medial cells from the more lateral pyramidal cells has also been reported in Gymnotus carapo (Rethelyi and Szabo 1973). It is possible that the apical dendritic trees of these cells interact with the parallel fibers of the VML and DML differently than of the pyramidal cells.

The ELLL of weakly electric fish is reasonably well understood both anatomically and physiologically. This system is, therefore, well suited for the study of the relationships between cell morphology and cell function.


Acknowledgements. The authors would like to thank Drs. H. Haines, S. O'Neal and L. Maler for many useful suggestions concerning this work and the preparation of this manuscript. Additional thanks go to H. Grau, N. Mulder, and Z. Marsh for their help in preparing this paper and to Dr. L. Maler for allowing us to preview his unpublished manuscripts. We are grateful to the Dept. of Zoology at the University of Okla- homa for support during this project and for the use of facilities. Supported by NIH grant RO1NS-123337 to Dr. J. Bastian.

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The physiology and morphology of two types of electrosensory neurons in the weakly electric fishApteronotus leptorhynchus