Proximate and ultimate causes of signal diversity in the electric fish Gymnotus.

A complete understanding of animal signal evolution necessitates analyses of both the proximate (e.g. anatomical and physiological) mechanisms of signal generation and reception, and the ultimate (i.e. evolutionary) mechanisms underlying adaptation and diversification. Here we summarize the results of a synthetic study of electric diversity in the species-rich neotropical electric fish genus Gymnotus. Our study integrates two research directions. The first examines the proximate causes of diversity in the electric organ discharge (EOD) - which is the carrier of both the communication and electrolocation signal of electric fishes - via descriptions of the intrinsic properties of electrocytes, electrocyte innervation, electric organ anatomy and the neural coordination of the discharge (among other parameters). The second seeks to understand the ultimate causes of signal diversity - via a continent-wide survey of species diversity, species-level phylogenetic reconstructions and field-recorded head-to-tail EOD (ht-EOD) waveforms (a common procedure for characterizing the communication component of electric fish EODs). At the proximate level, a comparative morpho-functional survey of electric organ anatomy and the electromotive force pattern of the EOD for 11 species (representing most major clades) revealed four distinct groups of species, each corresponding to a discrete area of the phylogeny of the genus and to a distinct type of ht-EOD waveform. At the ultimate level, our analyses (which emphasize the ht-EOD) allowed us to conclude that selective forces from the abiotic environment have had minimal impact on the communication component of the EOD. In contrast, selective forces of a biotic nature - imposed by electroreceptive predators, reproductive interference from heterospecific congeners, and sexual selection - may be important sources of diversifying selection on Gymnotus signals.

Electric organ discharge diversity in the genus Gymnotus: anatomo-functional groups and electrogenic mechanisms.

Previous studies describe six factors accounting for interspecific diversity of electric organ discharge (EOD) waveforms in Gymnotus. At the cellular level, three factors determine the locally generated waveforms: (1) electrocyte geometry and channel repertoire; (2) the localization of synaptic contacts on electrocyte surfaces; and (3) electric activity of electromotor axons preceding the discharge of electrocytes. At the organismic level, three factors determine the integration of the EOD as a behavioral unit: (4) the distribution of different types of electrocytes and specialized passive tissue forming the electric organ (EO); (5) the neural mechanisms of electrocyte discharge coordination; and (6) post-effector mechanisms. Here, we reconfirm the importance of the first five of these factors based on comparative studies of a wider diversity of Gymnotus than previously investigated. Additionally, we report a hitherto unseen aspect of EOD diversity in Gymnotus. The central region of the EO (which has the largest weight on the conspecific-received field) usually exhibits a negative-positive-negative pattern where the delay between the early negative and positive peaks (determined by neural coordination mechanisms) matches the delay between the positive and late negative peaks (determined by electrocyte responsiveness). Because delays between peaks typically determine the peak power frequency, this matching implies a co-evolution of neural and myogenic coordination mechanisms in determining the spectral specificity of the intraspecific communication channel. Finally, we define four functional species groups based on EO/EOD structure. The first three exhibit a heterogeneous EO in which doubly innervated electrocytes are responsible for a main triphasic complex. Group I species exhibit a characteristic cephalic extension of the EO. Group II species exhibit an early positive component of putative neural origin, and strong EO auto-excitability. Group III species exhibit an early, slow, negative wave of abdominal origin, and variation in EO auto-excitability. Representatives of Group IV generate a unique waveform comprising a main positive peak followed by a small, load-dependent negative component.

Roles of muscle activity and load on the relationship between muscle spindle length and whole muscle length in the freely walking cat.

The objective of this research was to compare the length of muscle spindles to the length of the whole muscle, during normal movements. Pairs of piezoelectric crystals were implanted near the origin and insertion of muscle fibres in the medial gastrocnemius (MG) muscle of cats. The distance between crystals was measured with pulsed ultrasound, the origin-to-insertion length of the MG muscle was measured with a transducer made of saline-filled silicone tubing, MG force was measured with a tendon force transducer and EMG activity was selectively recorded in the vicinity of implanted crystals. These signals were simultaneously recorded during posture or locomotion on a motorized treadmill. Three periods were identified in the step cycle, during which the relation between muscle length and spindle length changed dramatically. In period I (roughly corresponding to the late F and E1 phases of swing), the MG muscle and spindles followed similar length changes: both were stretched and then shortened by about 6 mm. In period II (corresponding to the stance phase, E2-E3) the MG muscle yielded under the weight of the body and was stretched by 1-3 mm, whereas the MG spindles typically continued shortening. In period III, the MG muscle shortened rapidly by 6-8 mm after the foot left the ground and then stretched again by about the same amount, whereas the spindles could remain nearly isometric. We attribute these large discrepancies in muscle and spindle length to the architecture of the MG muscle and the compliance of long tendinous elements in series with the spindles. We conclude that the length changes imposed on muscle spindles during voluntary movements are not simply related to the parent muscle length changes and cannot be estimated without taking into account the muscle architecture, the location of the spindle within the muscle, the level of muscle activation and the external load.

New transducers for measuring muscle length in unrestrained animals.

Muscle length in unrestrained, chronically implanted animals is conventionally measured with gauges consisting of a compliant silicone rubber tube filled with either hypertonic saline or mercury, the measurement principle being a continuous change in the electrical resistance of the fluid column inside the tubing with stretch. These gauges have two major disadvantages: (1) changes in resistance that are not related to changes in length, such as those produced by changes in temperature or osmotic dilution of the hypertonic saline, cause the measurements to drift, and (2) there is no direct and accurate way to calibrate the measurements. In this communication two new types of muscle length gauge are described that eliminate both problems. Both types make use of the principle of sonomicrometry, i.e., the measurement of distances with pulsed ultrasound. Both types have been successfully used to measure the length of the medial gastrocnemius muscle in chronically implanted cats during treadmill locomotion.

Velocity of ultrasound in active and passive cat medial gastrocnemius muscle.

The lengths and pinnation angles of muscle fibers in the medial gastrocnemius (MG) muscle have recently been measured in freely moving cats [Hoffer et al., Progr. Brain Res. 80, 75-85 (1989); Muscle Afferents and Spinal Control of Movement (1992)] using an ultrasound transit-time (USTT) technique. This method assumed that the velocity of ultrasound through intact muscles was constant, independent of fiber orientation, muscle activity, load, belly shape, or fiber movement. However, the velocity of ultrasound along and across the fibers has been reported to depend on the state of muscle activation in frog muscle experiments in vitro [Hatta et al., J. Physiol. 403, 193-209 (1988)]. In the present study, the assumption of constant velocity of ultrasound in the cat MG muscle was evaluated. In acute experiments, done in situ with intact blood supply, the USTT was measured along and across cat MG muscle fibers in the passive, reflexly activated and tetanically activated states, with and without changes in muscle fiber length, for situations that reproduced the length and force ranges normally used by cats during locomotion. The velocity of ultrasound was found to be independent of the state of activation or motion of the muscle, and independent of the direction of the measurement with respect to the fiber orientation, within a measurement uncertainty less than or equal to 0.2%. These results validate the use of the USTT technique for the measurement of intramuscular dimensions in freely moving animals.

Pharmacological study of the one spike spherical neuron phenotype in Gymnotus omarorum.

The intrinsic properties of spherical neurons play a fundamental role in the sensory processing of self-generated signals along a fast electrosensory pathway in electric fish. Previous results indicate that the spherical neuron's intrinsic properties depend mainly on the presence of two resonant currents that tend to clamp the voltage near the resting potential. Here we show that these are: a low-threshold potassium current blocked by 4-aminopyridine and a mixed cationic current blocked by cesium chloride. We also show that the low-threshold potassium current also causes the long refractory period, explaining the necessary properties that implement the dynamic filtering of the self-generated signals previously described. Comparative data from other fish and from the auditory system indicate that other single spiking onset neurons might differ in the channel repertoire observed in the spherical neurons of Gymnotus omarorum.

Peripheral electrosensory imaging by weakly electric fish.

Different species have developed different solutions to the problem of constructing a representation of the environment from sensory images projected onto sensory surfaces. Comprehension of how these images are formed is an essential first step in understanding the representation of external reality by a given sensory system. Modeling of the electrical sensory images of objects began with the discovery of electroreception and continues to provide general insights into the mechanisms of imaging. Progress in electric image research has made it possible to establish the physical basis of electric imaging, as well as methods to accurately predict the electric images of objects alone and as a part of a natural electric scene. In this review, we show the following. (1) The internal low resistance of the fish's body shapes the image in two different ways: by funneling the current generated by the electric organ to the sensory surface, it increases the fields rostrally, thus enhancing the perturbation produced by nearby objects; and by increasing the projected image. (2) The electric fish's self-generated currents are modified by capacitive objects in a distinctive manner. These modulations can be detected by different receptor types, yielding the possibility of "electric color." (3) The effects of different objects in a scene interact with each other, generating an image that is different from the simple addition of the images of individual objects, thus causing strong contextual effects.

The electric image in weakly electric fish: perception of objects of complex impedance.

Weakly electric fish explore the environment using electrolocation. They produce an electric field that is detected by cutaneous electroreceptors; external objects distort the field, thus generating an electric image. The electric image of objects of complex impedance was investigated using a realistic model, which was able to reproduce previous experimental data. The transcutaneous voltage in the presence of an elementary object is modulated in amplitude and waveform on the skin. Amplitude modulation (measured as the relative change in the local peak-to-peak amplitude) consists of a 'Mexican hat' profile whose maximum relative slope depends on the distance of the fish from the object. Waveform modulation depends on both the distance and the electrical characteristics of the object. Changes in waveform are indicated by the amplitude ratio of the larger positive and negative phases of the local electric organ discharge on the skin. Using the peak-to-peak amplitude and the positive-to-negative amplitude ratio of this discharge, a perceptual space can be defined and correlated with the capacitance and resistance of the object. When the object is moved away, the perceptual space is reduced but keeps the same proportions (homothetically): for a given object, the positive-to-negative amplitude ratio is a linear function of the peak-to-peak amplitude. This linear function depends on the electrical characteristics of the object. However, there are 'families' of objects with different electrical characteristics that produce changes in the parameters of the local electric organ discharge that are related by the same linear function. We propose that these functions code the perceptual properties of an object related to its impedance.

Electroreception in Gymnotus carapo: differences between self-generated and conspecific-generated signal carriers.

Local electric fields generated by the electric organ discharge of Gymnotus carapo were explored at selected points on the skin of an emitter fish ('local self-generated fields') and on the skin of a conspecific ('local conspecific-generated fields') using a specially designed probe. Local self-generated fields showed a constant pattern along the body of the fish. At the head, these fields were collimated, much stronger than elsewhere on the fish, and had a time waveform that was site-independent. This waveform consisted of a slow head-negative wave followed by a faster head-positive wave. In contrast, time waveforms in the trunk and tail regions were site-specific, with field vectors that changed direction over time. Local conspecific-generated fields were similar to the head-to-tail field, but their spatio-temporal pattern at the skin depended on the relative orientation between the receiving fish and the emitting fish. Because self-generated fields had a slow early component at the head region, they displayed a low-frequency peak in their power spectral density histograms. In contrast, the conspecific-generated fields had time waveforms with a sharper phase reversal, resulting in a peak at higher frequency than in the self-generated field. Lesions in emitting fish demonstrated that waveform components generated by the trunk and tail regions of the electric organ predominate in conspecific-generated fields, whereas waveform components generated by the abdominal region prevail in self-generated fields. Similar results were obtained from Brachyhypopomus pinnicaudatus. These results suggest that, in pulse-emitting gymnotids, electrolocation and electrocommunication signals may be carried by different field components generated by different regions of the electric organ.

Probability and amplitude of novelty responses as a function of the change in contrast of the reafferent image in G carapo.

Pulse electric fish evaluate successive electrosensory images generated by self-emitted electric discharges, creating a neural representation of the physical world. Intervals between discharges (system resolution) are controlled by a pacemaker nucleus under the influence of reafferent signals. Novel sensory stimuli cause transient accelerations of the pacemaker rate (novelty responses). This study describes quantitatively the effect of changes in contrast of reafferent electrosensory signals on the amplitude and probability of novelty responses. We found that: (i). alterations of a single image in an otherwise homogeneous series cause a novelty response; (ii). the amplitude of the elicited novelty response is a linear function of the logarithm of the change in image contrast; (iii). the parameters of this function, threshold and proportionality constant, allowed us to evaluate the transference function between change in stimulus amplitude and the amplitude of the novelty response; (iv). both parameters are independent of the baseline contrast; (v). the proportionality constant increases with the moving average of the contrast of hundreds of previous images. These findings suggest that the electrosensory system (i). calculates the difference between each reafferent electrosensory image and a neural representation of the past electrosensory input ('template'); (ii). creates the comparison template in which the relative contribution of every image decreases with the incorporation of successive images. We conclude that contrast discrimination in the electrosensory system of G. carapo obeys the general principle of appreciating any instantaneous input by the input's departure from a moving average of past images.

The electric organ discharge of Brachyhypopomus pinnicaudatus. The effects of environmental variables on waveform generation.

The electric organ discharge of Brachyhypopomus pinnicaudatus was studied by recording (1) the discharge field potentials in water at different conductivities and temperatures and (2) the spatiotemporal pattern of electromotive forces of the equivalent source. An early deflection, head positive (P wave), and a late deflection, head negative (N wave), are the major components of the discharge, however a striking double positive peak is generated at the abdominal level. Comparisons of this species with other pulse gymnotids provide evidence for common patterns of organization of the electrogenic system: (1) There is a head-to-tail activation wave along the fish; (2) the electromotive force increases exponentially from head to tail, but it is differentially attenuated by the passive tissues in male and females; (3) the abdominal region generates a complex species-specific waveform, whereas the tail discharge is similar across species. In B. pinnicaudatus the electric organ discharge waveform is sensitive to endocrine and environmental stimuli. The effect of seasonal sex differences on electrogenic and passive tissue, the changes in impedance matching between the fish's body and the environment, and the modulation of membrane properties by temperature, are able to modify the EOD waveform. Since these factors change during the breeding season, their appropriate combination might be crucial for reproduction.

The electric image in weakly electric fish: physical images of resistive objects in Gnathonemus petersii.

The present study describes a measurement-based model of electric image generation in the weakly electric mormyrid fish Gnathonemus petersii. Measurements of skin impedance, internal resistivity and fish body dimensions have been used to generate an electrical-equivalent model of the fish and to calculate electrical images and equivalent dipole sources for elementary resistive objects. These calculations allow us to understand how exafferent and reafferent signals are sensed by electroreceptors. An object's electric image consists of the modulation of the transcutaneous voltage profile generated by the fish's own discharge. The results suggest a set of rules for electrolocation: (1) the side of the fish where modulation is larger indicates the side on which the object is situated; (2) the object's position in the electroreceptive field is indicated by the point of maximum modulation of the transcutaneous voltage; (3) the degree of focus of the image indicates the distance to the object. In addition, center-surround opposition originating at pre-receptor level is proposed. Both experimental measurements and modeling indicate that fish skin impedance is relatively low (400-11 000 cm²) and mainly resistive. This low skin impedance appears to enhance the local electric organ discharge modulation, the center-surround effect, the signal-to-noise ratio for electrolocation and the active space for electrocommunication.

Electroreception in Gymnotus carapo: pre-receptor processing and the distribution of electroreceptor types.

This paper describes the peripheral mechanisms involved in signal processing of self- and conspecific-generated electric fields by the electric fish Gymnotus carapo. The distribution of the different types of tuberous electroreceptor and the occurrence of particular electric field patterns close to the body of the fish were studied. The density of tuberous electroreceptors was found to be maximal on the jaw (foveal region) and very high on the dorsal region of the snout (parafoveal region), decaying caudally. Tuberous type II electroreceptors were much more abundant than type I electroreceptors. Type I electroreceptors occurred exclusively on the head and rostral trunk regions, while type II electroreceptors were found along as much as 90 % of the fish. Electrophysiological data indicated that conspecific- and self-generated electric currents are 'funnelled' by the high conductivity and geometry of the body of the fish. These currents are concentrated at the peri-oral zone, where most electroreceptors are located. Moreover, within this region, field vector directions were collimated, constituting the most efficient stimulus for electroreceptors. It can be concluded that the passive properties of the fish tissue represent a pre-receptor device that enhances exafferent and reafferent electrical signals at the fovea-parafoveal region.