The slow pathway in the electrosensory lobe of Gymnotus omarorum: Field potentials and unitary activity

Journal of Physiology - Paris 108 (2014) 71–83

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The slow pathway in the electrosensory lobe of Gymnotus omarorum: Field potentials and unitary activity Ana Carolina Pereira, Alejo Rodríguez-Cattáneo, Angel A. Caputi ⇑ Departamento de Neurociencias Integrativas y Computacionales, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay

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Article history: Available online 1 August 2014 Keywords: Sensory processing Point process Electric fish Neural networks Reafference

a b s t r a c t This is a first communication on the self-activation pattern of the electrosensory lobe in the pulse weakly electric fish Gymnotus omarorum. Field potentials in response to the fish’s own electric organ discharge (EOD) were recorded along vertical tracks (50 lm step) and on a transversal lattice array across the electrosensory lobe (resolution 50 lm  100 lm). The unitary activity of 82 neurons was recorded in the same experiments. Field potential analysis indicates that the slow electrosensory path shows a characteristic post-EOD pattern of activity marked by three main events: (i) a small and early component at about 7 ms, (ii) an intermediate peak about 13 ms and (iii) a late broad component peaking after 20 ms. Unit firing rate showed a wide range of latencies between 3 and 30 ms and a variable number of spikes (median 0.28 units/EOD). Conditional probability analysis showed monomodal and multimodal post-EOD histograms, with the peaks of unit activity histograms often matching the timing of the main components of the field potentials. Monomodal responses were sub-classified as phase locked monomodal (variance smaller than 1 ms), early monomodal (intermediate variance, often firing in doublets, peaking range 10–17 ms) and late monomodal (large variance, often firing two spikes separated about 10 ms, peaking beyond 17 ms). The responses of multimodal units showed that their firing probability was either enhanced, or depressed just after the EOD. In this last (depressed) subtype of unit the probability stepped down just after the EOD. Early inhibition and the presence of early phase locked units suggest that the observed pattern may be influenced by a fast feed forward inhibition. We conclude that the ELL in pulse gymnotiformes is activated in a complex sequence of events that reflects the ELL network connectivity. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction This study focuses on the analysis of the sensory activity in the electrosensory lobe (ELL) of Gymnotus omarorum, a weakly electric fish. These animals use their electric organ discharge (EOD) to probe their nearby environment (Bastian, 1986a; Bell, 1979; Lissmann, 1958, 1951; Lissmann and Machin, 1958) and communicate with conspecifics (Black-Cleworth, 1970; Hopkins, 1981; Kramer, 1990; Moller, 1995). The active electric sense relies on three parallel mechanisms for generating reafferent signals in sensory nerves: (a) the generation of the sensory carrier (the fish’s own EOD); (b) the active movements of the sensory surface to explore object features; and (c) prereceptor conditioning of reafferent images (Caputi, 2004). In some species the EOD is a brief pulse (‘‘pulse fish’’) whereas in other species it is a continuous wave (‘‘wave fish’’). Body shape ⇑ Corresponding author. E-mail address: [email protected] (A.A. Caputi). http://dx.doi.org/10.1016/j.jphysparis.2014.07.005 0928-4257/Ó 2014 Elsevier Ltd. All rights reserved.

and swimming movements have evolved together in these fish, with some species being carangiform1 and others balistiform2 swimmers (Blake, 1983). These electromotor and skeleton motor control characteristics are differently combined in the two largest groups of electric fish: African mormyriformes (comprising Mormyridae and Gymnarchidae) and America gymnotiformes (comprising Apteronotidae, Sternopygidae, Hypopomidae, Rhamphychthydae and Gymnotidae). African Mormyridae are carangiform fish. They emit a brief pulsatile EOD (on the order of ms) separated by irregular intervals. This pulse is generated by an electrogenic organ (EO) concentrated at the caudal peduncle. Because of this localized EO, in the absence of objects, the electroreceptor stimuli have the same temporal

1 Carangiform is a type of swimming practiced by fish in which undulation are limited to the caudal region with the body bending into less than one half of a sinusoidal wave form. 2 Balistiform is a type of swimming practiced by fish in which movement is effected by undulations of fins only.

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course all over the fish surface (Caputi et al., 1998; Pedraja et al., 2014). Moreover, resistive objects change the amplitude pattern but do not change the temporal course of the local EOD. In addition, tail movements change the relative location of the EO with regard to the fish’s body. Thus electric images evoked by the EOD on the surface of the fish’s skin change in amplitude with swimming movements. These changes in object’s polarization are cancelled at the first central electrosensory relay, the electrosensory lobe (Sawtell, 2010). Pulse gymnotiformes (Hypopomidae, Rhamphychthydae and Gymnotidae) also emit a pulsed EOD but separated by regular interval. In addition, these fish show a heterogeneous EO distributed over the caudal 90% of its fusiform body. Since the different body regions discharge different waveforms, the whole discharge consists of a complex but stereotyped spatio-temporal pattern (Caputi, 1999; Caputi et al., 2005, 1998, 1994, 1989). These fish are propelled by a single anal ribbon fin that undulates driven by a traveling wave. Speed and traveling direction of this wave are controlled by a motorneuron network morphologically and functionally independent from the main motor system controlling lateral movements of the fish’s body (Trujillo-Cenóz et al., 1986). African Gymnarchidae and American wave gymnotiformes (Apteronotidae, Sternopygidae) also have a fusiform body shape but emit a continuous sinewave-like carrier. Wave emitting fish show a distributed EO occupying the caudal 90% of the fish’s body in which regional EOD waveforms are shifted in phase (Pedraja et al., 2014). These differences in electromotor and skeleton-motor behaviors are associated with differences in electrosensory systems. The first neural electrosensory relay is the electrosensory lateral line lobe (ELL) located dorso-laterally at the medulla. This is a cerebellum-like structure showing a so called ‘‘passive or ampullary’’ electrosensory pathway originated on ampullary receptors, mainly sensitive to slow variations of transcutaneous potentials but also in minor extent to their own EOD and to mechanical stimuli (Bell, 1979, 1981; Kalmijn, 1974) and two paths originated in tuberous receptors sensing transient changes in transcutaneous electric fields. These are (a) the fast electrosensory pathway encoding fast transients or zero crossings of self- and allo-generated emitted fields (Bell, 1989; Bell and Grant, 1989, 1992; Bell et al., 1992, 1993; Castelló et al., 1998; Hopkins and Bass, 1981; Matsushita et al., 2013; Nogueira and Caputi, 2011, 2013; Nogueira et al., 2006; Sotelo et al., 1975; Szabo, 1967; Szabo et al., 1975) and (b) the slow electrosensory pathway encoding the changes in amplitude and waveform of the self- and allo-generated electric fields (Aguilera and Caputi, 2003; Bastian, 1986b, 1986c; Bell, 1979; Bell and Grant, 1992; Bell et al., 1992; Caputi et al., 2008, 2003; Pereira et al., 2005; Scheich and Bullock, 1974; von der Emde, 1990). The slow electrosensory pathway has been extensively explored in mormyriformes and wave gymnotiformes (for wave gymnotiformes see: Bastian, 1995, 1986a, 1986b, 1986c; Bastian et al., 1993, 2004; Berman and Maler, 1998a, 1998b, 1998c; Carr and Maler, 1986; Chacron et al., 2005, 2011; Clarke et al., 2013; Fernández et al., 2005; Khosravi-Hashemi and Chacron, 2014; Krahe and Maler, 2014; Maler, 1979; Maler et al., 1981, 1982; Marsat et al., 2012; Mehaffey et al., 2008; Turner et al., 1996; Turner and Maler, 1999; Turner et al., 2002 and for african pulse fish see: Bell and Grant, 1992; Bell et al., 1997a, 1997b, 1992; Engelmann et al., 2008; Han et al., 2000; Kennedy et al., 2014; Meek et al., 1999; Mohr et al., 2003a, 2003b; Sawtell and Bell, 2008; Sawtell and Williams, 2008; Sawtell et al., 2007). However, only a few studies have examined the ELL of pulse Gymnotiforms (Caputi et al., 2008; Pereira et al., 2005; Réthelyi and Szabo, 1973a, 1973b; Schlegel, 1973; Stoddard, 1998) or wave mormyriforms (Kawasaki, 2005, 1993; Matsushita and Kawasaki, 2004;

Kawasaki and Guo, 1996). This motivates the present study that addresses the question of how the ELL network responds to the naturally generated EOD in G. omarorum. We recorded field potentials and unitary activity evoked by the EOD in the ELL of decerebrated but spontaneously discharging fish. Three main sensory potentials at about 7, 13 and 23 ms after the EOD were identified. Unitary recordings show that individual neurons also fire following characteristic post-EOD patterns. In addition, some of these units show a clear reduction in firing probability immediately after the EOD, suggesting a fast feed forward inhibition in the ELL network that could precede the effects of slower afferent fibers.

2. Experimental procedures Twelve G. omarorum of 20–30 cm in length were used following the guidelines of the CHEA (Comisión Honoraria de Experimentación Animal, ordinance 4332-99, Universidad de la República Oriental del Uruguay). Experiments were approved by the Animal Ethics Committee of the Instituto de Investigaciones Biológicas Clemente Estable (protocol number 001/03/2011). Fish were gathered at Laguna del Cisne (Maldonado, Uruguay) 1–4 months before the experiment, kept in individual aquaria under a natural light cycle and fed with insect larvae. All physiological experiments were performed in decerebrate fish. With the exception of chronically implanted electrodes (Castelló et al., 1998; Pereira et al., 2005; Rodríguez-Cattáneo et al., 2012), the decerebrate preparation is the only way to record the responses of multiple ELL neurons under natural stimulation in pulse gymnotiformes. Furthermore, the current source density analysis performed in this study requires multiple penetrations, a procedure which cannot be done with chronically implanted preparations. At the end of the experiments animals were euthanized by an overdose of pentobarbital (10 mg, i/m).

2.1. Decerebration and surgery procedure Surgical procedures were conducted with the fish under deep anesthesia in which the EOD rate was unresponsive to visual, vibratory, electric or nociceptive stimuli. In those experiments in which the fish may undergo pain or discomfort, animals were anesthetized with Eugenol (0.01% in the aquarium water) until de-cerebration was completed. Once the fish stopped discharging, the skin over the upper cranial projection of the brain was removed and two small holes on the skull were made at the forebrain projection, one on each side of the midline. A small vacuum aspiration probe was introduced through one of the holes and the forebrain was completely aspirated. Once decerebration was completed the fish was moved into the recording chamber and anesthesia removed. This chamber consisted of a 26  45 cm tank filled up to 6 cm with of aquarium water (100 lS/cm) mounted on a vibration isolation table. A thin wire (150 lm diameter) was passed through both holes and the skull was firmly attached to a wood holder. These wires were then embedded in a dental cement structure binding the skull and the holder. A cotton thread was passed along the dorsal muscular mass leaving the body at about 5 cm from the tip of the tail. Using this thread firmly attached to the head holder and a caudal support we positioned the fish body straight along the long axis of the tank. Then, we removed a wide portion of the skull to expose the ELL taking care to maintain the water level below the border of the wound. Spontaneous EOD discharges resumed during this fixation procedure or shortly thereafter. The EOD rate changed in response to mechanical and electrical stimuli applied to the water, as in an intact fish.

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2.2. Electrophysiological recordings Extracellular field potentials were recorded between a reference electrode placed on the cistern magna and either one electrode or as many as 16 electrodes inserted in the ELL. We used either a true differential (AM system 1800) or a multiplexed differential (AM system 3600) amplifier (gain between 5000 and 20,000 adjusted during the experiment). To select population or unitary activity different filter settings were used (10 Hz–1 kHz for slow field potentials, and 300 Hz–5 kHz for neuron spikes). All signals were digitized at least at 20 kHz per channel (DataWave SciWorks, 8.0). The head to tail EOD, recorded with electrodes placed at the edges of the tank along the fish axis, was used as a time reference. ELL activity was recorded within 7 fish. In 4 fish we used glass pipettes filled with NaCl (3 M) stepped down every 50 or 100 lm long each tract and in the other 3 we used ‘‘Michigan type’’ multitrode bearing 16 channels on distributed along a line separated 50 lm. When using pipettes we used the two channels of the AM 1800 to record simultaneously unit and field potential signals with both filter settings. In 2 of these fish we injected Chicago sky blue at two points of the tract to identify its location in the fixed brain. After the experiment the brain was immersed in 4% paraformaldehyde for 24 h and vibrosectioned 100 lm thick. The blue marks were identified under light transmission microscopy. In the case of multitrodes, each recorded spot was separated 50 lm from the next covering span of 750 lm. These electrodes allowed us to make series of recording tracts equally spaced in the same plane. The electroreceptive surface is somatotopically represented on three maps oriented along parallel lines forming a 45° angle with the main axis (tuberous pisciculi). In one case we explored a plane transverse to the 3 pisciculi at the head projection level with 21 tracts separated every 100 lm. Field potentials were recorded first in epochs containing at least 300 EODs. Long lasting (1–3 min) recordings were obtained to study neuron spiking patterns.

2.3. Analysis of the field potentials EOD occurrence was detected by comparing continuous recorded traces with a threshold level (DataWave SciWorks, 8.0). Peri-EOD epochs (0.5 inter-EOD interval before, 1 interval after) were extracted and peri-EOD averages were calculated at each recording point as an estimate of the temporal pattern of electric potentials in resting conditions. The flow of current through an isotropic conductive extracellular medium is inextricably associated to local electric fields and potentials. Assuming a quasi-static extracellular electric field and an isotropic constant conductivity of the medium, the current density at every point of the extracellular space is proportional to the gradient of the potential, i.e., the electric field (Ohm’s law, Sears and Zemanski, 1954). In turn, the divergence of the electric field is a scalar function of time that measures the difference between the outward and inward flows of current through a closed surface surrounding a given point. It takes on values proportional to the current sources density or current sinks density created by local generators. If there are no local generators, divergence is null. Distant generators do not contribute to the divergence of the electric field because any current originated in distant sources and sinks enters and leaves any sphere around any given point (Nicholson, 1979). When systematic recordings were performed we estimated the activation of different regions of the ELL as the time course of the field divergence (Nicholson, 1979).

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In the case of a flat layer, lateral currents cancel out. Then the divergence is equivalent to the second derivative of the potential. The ELL layers are curved around the axis structure with an upward concavity but along the axis are relatively straight (see Section 3). However, the most medial tuberous pisciculum (centromedial map) is relatively flat at the rostral projection regions. Thus, we made systematic recordings from the centromedial map and calculated the second derivative of potential in every fish. In two of these fish we also made systematic recordings, placing the multitrode along equispaced lines on the plane perpendicular to the pisciculi longitudinal axes. The electrodes explored such plane following a lattice grid with points separated 50 lm in depth and 100 lm side to side. In this case for detecting the sources and sinks of current density we estimated the divergence by subtracting the average of the surrounding potentials to the potential at each recording point for each sampled time. 2.4. Analysis of the activity of the electrosensory units Spike waveforms were selected from the continuous recorded traces using DataWave SciWorks (version 8.0). More than 250 units were recorded in these experiments. This study was based on the analysis of those spikes clearly separable using the following algorithm. First, we inspected the signal and selected all visually identified events as separable using a digital voltage–time window. Window settings were determined taking into account the duration of the spike, and the direction and amplitude of the main peak. Extracted events consisted in epochs of 1–2 ms including the entire waveform of the visually identified spike. Then we refined the selection by excluding all waveforms that not match a set of 4–6 parameters chosen after a second inspection of the all selected spikes and the signal to background ratio. These parameters include (a) amplitude and duration of the peaks, (b) peak to peak amplitudes (in the case of triphasic waveforms we used both), (c) interpeak interval and (d) rising and falling slopes. The series of time stamps corresponding to each selected waveform was used to construct peri-EOD rasters, peri-EOD histograms, first order interspike histograms and autocorrelation histograms. In order to better dissect the structure of the peri-EOD histogram we calculated separate histograms for the three first consecutive spikes in the EOD cycle. This allowed us to refine spike type classification. Since we observed that many spikes had a relatively low probability of firing just after the EOD we calculated and compared for each spike the firing probability at the interval 4–8 ms after the EOD with the firing probability 8–4 ms before the EOD. 2.5. Anatomy Histological analysis of the ELL was made in 5 fish. In all these animals the vibratome sliced brain stems were Nissl stained to reveal the general structure of the ELL. In 3 of them Golgi-like staining of the efferent neurons was made using retrograde tracing. For this latter purpose, Neurobiotin (Vector Laboratories) was iontophoretically deposited at the lateral lemniscus decussation. We used glass pipettes filled with a 1% solution of the dye in KCL 2 M. The decussation is located about 2500 lm below the posterior k-shaped sutures of the skull bone. Target achievement was confirmed by a strong EOD-evoked potential. In all cases animals were deeply anesthetized with pentobarbital (10 mg, i/m), the cardiac region was surgically exposed and 50 ml of a mix of formaldehyde (1%) and glutaraldehyde (1%) was infused through a 22 gauge needle inserted through the heart deeply inside the aortic bulb. The brain was vibrosliced perpendicularly to the fish’s longitudinal axis in slices of 100 lm thick. In the case of retrograde labeling of efferent cells Neurobiotin was revealed with an avidin-peroxidase-diaminobencidine standard

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protocol (www.vectorlabs.com/protocols/PK-4000.pdf). Finally, sections were serially mounted in gelatin coated glass slides, dried, and immersed in a solution of 1% Methylene blue for 2 min, cleared and dehydrated in alcohol and mounted with Canada balsam. The samples were examined under light transmission microscope (Zeiss, Primo Star) and images were captured with a digital camera (Pixelink, 2040  1536 pixels). 3. Results 3.1. The anatomical structure of the ELL In order to provide the reader with a reference of the network under study we briefly summarize and illustrate the structure of the ELL of G. omarorum (to be described in detail elsewhere). Our data generally confirmed the findings of several authors in wave and pulse Gymnotiformes (Berman and Maler, 1999; Maler, 1979; Réthelyi and Szabo, 1973a; Sas and Maler, 1987, 1983; Schumway, 1989). As in all gymnotiformes the ELL shows 3 laminated tuberous maps (Fig. 1A, centromedial, CM; Centrolateral, CL; Lateral, L). A Nissl stained cross section at the level of the relay nucleus (Fig. 1

A marked as R) shows the ELL lamina and some cell types (Fig. 1A and B). From ventral to dorsal the laminas are: (1) Deep fiber layer (DFL) which in addition to the afferent fibers contains the somata of multipolar cells (white arrow in Fig. 1B). (2) Deep neuropil layer (DNL) where afferent terminals contact the basilar dendrites (Fig. 1C) from different types of cells located in different layers. The limit between DFL and DNL is often demarked by scattered ovoid cells which are also present at the DNL (black arrow in Fig. 1B). These cells as well as multipolar ones have been shown to convey feed forward inhibition to both the ipsi and contralateral sides (Berman and Maler, 1999; Maler and Mugnaini, 1994). Also unpublished data from Caputi and Castelló show immunohistochemical labeling for GABA in ovoid cells in the ELL of G. omarorum. The upper limit of this layer is demarked by the presence of spherical neurons (arrowhead in Fig. 1B). These receive a specific type of primary afferents (pulse markers) and represent the only neuron type belonging to the fast electrosensory pathway in the ELL. (3) Granule cell layer (GCL) contains two different types of granule inter-neurons (data not shown, Maler, 1979) and also efferent neurons having conspicuous basilar dendritic trees. Within these we found two different subtypes either lacking (‘‘bald basilar cells’’ Fig. 1C and D up pointing triangle) or showing (‘‘deep basilar

Fig. 1. (A) Nissl stained cross section at the level of the relay nucleus (R). Tuberous maps are indicated as CM (centromedial), CL (centrolateral) and L (lateral). (B) Multipolar (white arrow), ovoid (black arrow) and spherical neurons (double arrowhead) can be identified in the deepest layers. (C) Retrogradely labeled ‘‘bald’’ basilar neuron characterized by a round somata with a single basilar dendritic trunk extensively ramified at deep neuropil layer. (D) Cell layers and projection neurons. Avidine-di-aminobencidine procedure revealed neurobiotin-retrogradely-labeled neurons projecting out of the ELL through the lateral lemniscus. The methylene blue counterstain shows 8 different layers: deep fiber layer (DFL); deep neuropil layer (DNL); granule cell layer (GCL) containing two different subtypes of efferent cells – ‘‘deep basilar pyramids’’ with basilar dendrites, (down pointing triangle) and ‘‘bald basilar cells’’ with apical dendrites (up pointing triangle); plexiform layer (PlexL) containing intermediate basilar pyramids (circle); polymorphic layer (PNL) containing the somata of large pyramidal cells which can be classified as ‘‘basilar’’ (asterisk) or ‘‘non-basilar’’ (square) by of the presence or absence of basilar dendrites respectively; stratum fibrosum (StF) and ventral (VML). Dorsal molecular layers (DML) and the origin of the parallel fibers (EGP) are marked in (A).

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pyramids’’, Fig. 1D, down pointing triangle) apical dendrites. (4) Plexiform layer (PlexL) containing some intermediate basilar pyramids (Fig. 1D, circle), polymorphic neurons and efferent axons projecting to superior centers (Fig. 1D, white arrows). (5) Polymorphic layer (PNL) containing the somata of all pyramidal cells which can be classified as ‘‘basilar’’ (Fig. 1D, asterisk) or ‘‘non-basilar’’ (Fig. 1D, square) because of the presence or absence of basilar dendrites. (6) Stratum fibrosum (StF) composed by the descending axons projecting back from the praeminentialis nuclei (data not shown, Bastian and Courtright, 1991). (7) Ventral molecular layer (VML) in which according to the literature axon terminals from praeminentialis fibers and local inhibitory interneurons terminate on the initial branches of the apical pyramidal dendrites (Maler, 1979). (8) Dorsal molecular layer (DML) containing the apical dendritic arbors which are contacted by parallel fibers originated in a neighbor granule mass, the eminentia granularis posterior (EGP, Fig. 1A). 3.2. EOD related field potentials In spite of timing differences due to temperature (ranging between 17 and 23 °C), the sequence and main characteristics of the different components observed in the profile were qualitatively similar in all recorded fish. This suggests that the ELL network has a complex but stereotyped response to the EOD at resting conditions. However, the interpretation of the field potentials requires the knowledge and the corresponding correlation of the recording sites with the anatomy of the structure (Nicholson and Llinas, 1971). A current sink occurs when a group of neurons in a local population are jointly depolarized. This is because depolarization implies an inward flow of current from the extracellular space into the neuron, i.e., a current sink. According to the Gauss’ divergence theorem (Sears and Zemanski, 1954), the net flow of current through the low conductive neuron membrane is null. This means that in other regions of the neuron there should be an outward current, i.e., current sources. Hence, in a neural structure where neurons are organized following some anatomo-functional rule the divergence map as a function of time shows how the sources and sinks of the electric field move over such structure and consequently allow us to visualize how different regions of the neural network sequentially turn ‘‘on’’ and ‘‘off’’ (see Section 2 for a detailed explanation). As described in Section 3.1, the electrosensory lobe is a laminar structure in which afferent fibers project on the deepest layer of 4 different somatotopically arranged maps. Each map is oriented 45° (head rostral and medial) with respect to the main axis of the fish. The most medial map receives the primary afferents from ampullary electroreceptors dealing with low frequency electric fields. The other three maps, which are the focus of this article, receive primary afferents from tuberous receptors specifically tuned to the EOD of the species. The granule, plexiform and polymorphic layers, containing most neuron somata show an upper concavity centered along the main axis of the ELL. While at the centro-medial map these three layers are almost horizontal, at the lateral map they are almost vertical. This implies that when an electrode penetrates the structure it describes a line with different orientation with respect to the layers composed by neurons’ somata of different maps. Therefore, to analyze the time course of sources and sinks we had to perform parallel tracts along a plane perpendicular to the ELL axis where different somatotopic maps receive the projection from the same region of the skin. Peri-EOD averaged field potentials were recorded at different depths either simultaneously (3 fish) or sequentially (4 fish) at equally spaced points along a vertical line perpendicular to the cell layers. The color map of Fig. 2 shows the spatiotemporal profile of the field of potential along a tract (y axis) in the centro-medial map. The inter-EOD interval is on the x axis. The color code corresponds

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to the potential recorded between each point and the reference electrode. A few traces representing the recorded potential plotted vs. time are superimposed on the colormap at the corresponding recording depth. Note in the middle one the presence of three main peaks at about 7, 13 and 23 ms after the EOD. To have a biological spatial reference, the recording positions were aligned with the histological profiles of the ELL, and the layer’s limits were indicated by dotted horizontal lines. The color map typically showed a deep sharp negativity just 2 ms after the head positive peak of the EOD corresponding to fast electrosensory pathway. This is followed by a longer lasting positivity at the DNL corresponding to slow component of the primary afferent input (see bottom superimposed trace). During this period, between 4.7 and 8.3 ms, it is possible to identify a small negativity at the PlexL and lower PNL that we previously referred to as early slow pathway activity (Fig. 1A, labeled as N7, Pereira et al., 2005). The largest negativity occurs at the middle of the inter EOD cycle. This extends all along the PNL, VML and DML (Fig. 1A. labeled as N13). Finally, a third component starting at the DNL propagates up peaking at the PNL and extending widely in space and time toward the DML and the end of the cycle respectively (Fig. 2A, labeled as N23). A recording tract perpendicular to the centromedial map was chosen to exemplify the field potentials. When aiming at this map the electrode was moved perpendicularly to a relatively flat cell body layer and in this configuration only one dimension is relevant for calculating the divergence. The temporal activation of the ELL layers can be visualized in a single spatiotemporal color map. We calculated the 2nd spatial derivative of the potential along the track line. This approximation to the field divergence is valid because the perpendicular orientation of the electrode tract with respect to the cell layer implies that most current flows in the extracellular media along the direction of the track. Thus, in this particular geometry, the 2nd spatial derivative of the potential taken along the direction perpendicular to the layer allows us to show a two dimensional colormap of the sources (cold colors) and sinks (warm colors) as a function of time (Fig. 2B, the horizontal dimension corresponds to time and the vertical to the field gradient). The first sink (2.6 ms, arrow) is the fast electrosensory pathway. This is followed by a second deeper sink at the DNL, probably corresponding to afferent input of the slow electrosensory pathway. At about 7 ms after the EOD a new sink starts about the PNL moving down to the PlexL and then back up to the PNL peaking there at about 10 ms. These sinks have a main corresponding source at about the limit between the GCL and DNL. There is also a smaller source at the DML. The PNL sink remains active until about 15 ms. During this period a smaller sink moves up crossing the VML and progresses into the DML (double arrow). Finally, a third component originates in the GCL at about 20 ms and rapidly progresses up to the PNL peaking there at about 23 ms. As with the intermediate latency component, there is again a small sink progressing up to the DML. The two dimensional analysis suggests that the pattern of activation of the three maps is similar (Fig. 3). However, small differences as for example the intensity of the reddish color in the centro-medial map activation may be noted. The small number of fish explored with this technique (in one fish we explored the whole section and in other we did it over a partial extent) prevents us from reaching a general conclusion about difference between maps so far. 3.3. Firing patterns of electrosensory units We recorded from 84 unambiguously identified neurons. The mean spike rate showed a skewed distribution over the population. It was significantly smaller than the EOD rate in most cases (Wilcoxon test, p < 0.0001, N = 84) with median ratio between

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EOD

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Fig. 2. EOD related field potentials and their origin. (A) Color map of the spatiotemporal profile of the field of potential. Post EOD potentials were recorded every 50 lm along a track at the centro-medial map and averaged during an inter-EOD interval. For constructing the color map we interpolated the data in the spatial direction (the horizontal dimension corresponds to time and the vertical to the depth from the surface of the ELL). The color code indicates the potential recorded between each point and a distant reference electrode. As a spatial reference, the recording positions were aligned with the histological profiles of the ELL and the layers were indicated by dotted horizontal lines (DML: dorsal molecular layer; VML: ventral molecular layer; StF: stratum fibrosum; PNL: polymorphic layer; PlexL: plexiform layer; GLC: granule cell layer; DNL: deep neuropil layer). The superimposed black traces correspond to the time course of the recorded potentials at 600, 1350 and 2000 lm of depth. Four components can be identified during the EOD cycle. The first component is a sharp negativity occurring deep in the ELL at about 2 ms after the peak of the EOD and corresponds to the fast electrosensory pathway. This is followed by the other 3 negative components labeled as N7, N13 and N23. N7 occurs between 4.7 and 8.3 ms and corresponds to a small negativity at the PlexL and lower PNL (this is referred to as the early component of the slow pathway). N13 corresponds to the largest negativity, at the middle of the inter EOD cycle and extends all along the PNL, VML and DML. N23 starts at the DNL and propagates up peaking at the PL and extending widely in space and time toward the DML and the end of the cycle respectively. (B) Color map showing the second spatial derivative of the potential (equivalent in this case to the divergence of the electric field) along a track line perpendicular to the call layers of the ELL during the inter-EOD interval (the horizontal dimension corresponds to time and the vertical dimension to the second derivative of the potential). For constructing the color map we first calculated the spatial 2nd derivative for each time and then interpolated the data in the spatial direction. The arrow shows a first sink corresponding to the fast electrosensory pathway (2.6 ms). Presumably the corresponding source is in the deep fiber layer where fast electrosensory afferents are. A second deeper sink probably corresponding to the afferent of the slow electrosensory pathway appears at the DNL. At about 7 ms after the EOD a new sink starts about the PNL moving down to the PlexL and then moving up to the PNL peaking there at about 10 ms and remains active until about 15 ms. Its main corresponding source is at about the limit between the GCL and DNL. Concomitantly to this process, a smaller sink moves up crossing VML and further progresses along the DML (double arrow). Finally, a third component of the field potentials begins in the GLC at about 20 ms. It progresses up to the PNL, peaking at about 23 ms in this layer.

spikes and EODs of 0.28 spikes/EOD (inter-quartile range 0.18–0.50 units/EOD). Along the recording period the spikes did not fire uniformly in most cells. They fired repetitively in some EOD cycles and were silent in the others. The peri-EOD firing probability shows different patterns including post-EOD histograms characterized by (a) very low dispersion (phase locked units), (b) one or various modes clearly identifiable (monomodal and multimodal units) and (c) units having high firing probability just before the next EOD and a minimum either just after or at the middle of the EOD cycle (units depressed by the EOD without preferential activity phase). 3.3.1. Phase locked units A striking finding was that 6 out of 84 units were phase locked. Their histograms show short latencies and a very small standard deviation (latency range: 6.6–13.7 ms, standard deviation range: 0.02–0.7 ms, N = 6). In one fish we found a couple of phase locked spikes firing either only the earliest or in pairs (Fig. 4A and B). The shapes of these spikes were not the same. In addition, we observed a background hash of wavelets following the spike (Fig. 4B, individual traces), suggesting that they belong to a fiber bundle.

3.3.2. Monomodal units We recorded from 34 monomodal units, 15 early monomodal (firing about 14 ms) and 19 late monomodal units (firing about 24 ms after the EOD). Early monomodal units have a mean responsiveness rate of 0.3 per EOD (25–75% range = 0.19–0.43 per EOD). They show a postEOD histogram characterized by a sharp peak at about the timing of the main peak of the slow path field potentials (Fig. 5A and B). To compare the sharpness of the histogram peaks we calculated a ‘‘Q-ratio’’ between the number of spikes occurring within the interval mode ±2 ms and the number of spikes occurring within the interval mode ±4 ms (Q-ratio = 0.5 would imply a flat distribution, and Q-ratio = 1 would imply all spikes within the small interval). For early monomodal units Q-ratio was in average 0.75 which means that 3 out of 4 peri-modal spikes fell within the small interval. In accordance with this low latency variability, the autocorrelation histogram of mid-latency units typically showed periodic peaks reflecting the regularity of the EOD (Fig. 5C). Five out of the 15 early monomodal units fired in doublets (Fig. 5C, inset) with an inter-spike interval varying between 2 and 6 ms.

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Fig. 3. Current source density analysis after the EOD activation of different maps in the ELL. Color maps show the divergence of the electric field calculated in two dimensions over a cross section of the ELL perpendicular to the main axis of the lobe (divergence was calculated from the raw data for each time and then each color map was calculated by interpolation). As a spatial reference, a reconstruction of the recorded plane of the ELL was overlaid and vertical lines indicate the electrode tracks. Each map represents the activation of the ELL at significant times during the EOD cycle. The activation progresses in two waves. The first starts at 7 ms at CM and CL maps reaching the molecular layer at about 14 ms. The second wave starts at about 20 ms at about the GL and reaching the PNL and VML at about 23 ms. Small differences in the intensity and times of the map activations may be noted. In this fish, the centromedial is the first to be activated and the intensity of its activation is the greatest. The labels CM (centromedial), CL (centrolateral) and L (lateral) indicate the different maps. EGp corresponds to the eminentia granularis posterior. The pattern of activation of the three maps is similar.

Late monomodal units showed a non uniform response to the EOD. Although they have a mean responsiveness rate of 0.44 per EOD, a rather common finding was a few consecutive cycles showing responses of more than one spike, separated by about 10 ms (Fig. 6A red dots and B red stairs). This interspike interval was independent of the latency of the first spike and contributed to the broad profile of the post EOD histogram (Fig. 6C, median of modes = 24 ms, median Q-ratio = 0.63, N = 19). The broad excitation of these cells shown by the post EOD histogram corresponds to excitatory period between 10 and 17 ms in the current source density analysis. In accordance with the monomodal pattern the autocorrelation histogram showed periodic peaks. However, due to the larger variability and multiple firing per EOD these peaks were less marked and become unapparent at shorter analysis times, indicating that variability masked the regularity of the EOD (Fig. 6D). 3.3.3. Multimodal units Of the 35 multimodal recorded units, 15 were tri-modal and 20 bimodal. The very early and the intermediate peaks of the post EOD histogram was present in both bimodal and trimodal types.

Thirteen tri-modal units showed a similar pattern with modes at about 5, 11 and 28 ms (median values; a typical example is shown in Fig. 7A and B). The other two show a first mode at 11 and 14 ms respectively and additional late modes. Most tri-modal units fired more than one time during the same interval and at about the same latencies (compare blue and red histograms in Fig. 7B). Bimodal units showed early and intermediate latency modes (Fig. 7C and D). In these neurons the activity clearly alternated between epochs of high and low rate. Half of these units were recorded from one experiment performed at about 17 °C and the other half in various fish at temperatures between 20 and 22 °C. Statistics from the 10 units recorded at higher temperatures show modes located respectively at about 7 and 16 ms (median values). This suggests that bimodal units tends to fire a little after trimodal ones. At early and intermediate latencies statistical analysis confirmed these differences. The first mode occurs 1.6 ms after in bi than in tri modal units (ranges 4–7 ms, N = 13, and 6–8, N = 10 for trimodal and bimodal units respectively, Wilcoxon test comparing the modes p = 0.026) The second mode in bimodal units

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occurs in average 2.5 ms after the second mode of tri-modal units (ranges 9–19 ms, N = 13, and 14–21, N = 10 for trimodal and bimodal units respectively, Wilcoxon test comparing the modes, p = 0.032). Finally it is worth noting that in three pairs of trimodal–monomodal neurons recorded close to each other in the same electrode track in the centromedial and centrolateral maps, we found that monomodal neurons were deeper than trimodal neurons and the peak of the monomodal histogram filled up the valley between the second and third peak of the trimodal units suggesting that either one inhibits the other or that both receive mirror images entries (Fig. 8). 3.4. Evidence of inhibition and its potential relevance Eight units did not show a preferential phase. Instead these units showed a minimum (close to zero) firing rate at intermediate phases of the EOD cycle. These units had a relatively lower rate (0.26 firings per EOD, Fig. 9). In addition, one half of the multimodal units showed a drop in firing probability just after of the EOD. This suggests an inhibitory effect of the EOD on those units. In order to investigate this possibility, we first tested the null hypothesis that firing probability in a short window before the EOD, beginning 8 ms and ending 4 ms before the peak of the EOD was equal to firing probability to an equivalent short window after the EOD, beginning 4 ms after the peak of the EOD and ending 8 ms after. We also calculated the firing ratio before/after the EOD (B/A). We excluded the time window [ 4, 4] to avoid the potential interference of EOD artifact. In 15 out of 35 multimodal and 6 out of 8 ‘‘without preferential phase’’ units the probability to find the observed B/A ratio was smaller than 0.05 under the null hypothesis B/A = 1 (exact test for each unit, Figs. 7A and 9). In addition to rule out the possible effect of refractoriness induced by late firing spikes instead of inhibitory effect of the EOD, we also calculated the phase–cophase relationship of the spikes surrounding each

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EOD. We defined phase as the latency of the first spike after the EOD and cophase as the time between the same EOD and the closest spike preceding it. Favoring the lack of refractoriness, Spearman correlation coefficient between cophase and phase were in most cases positive (18/21), ranging between 0.03 and 0.15 suggesting that when a spike fires close before the EOD it will be most likely spiking early in the next cycle. Furthermore, when we studied the proportion of failures in the next EOD cycle for short (2–8 ms before EOD) and long (between the previous EOD and 8 ms before the EOD) cophases, we found that there was a higher probability of a lack of spikes when the previous spike faired at a long cophase in 7 out of 21 units (v2 test, p < 0.05 for each one of these units). Only in 1/21 unit the probability of failure was associated with a short cophase (v2 test, p < 0.05). In the other 13 units, there were no statistical association between failures and cophase (v2 test). 4. Discussion Our anatomical results confirm the structure of the electrosensory lobe of G. omarorum previously described by Réthelyi and Szabo (1973a), and its similarity with the well-studied cerebellum-like homologous structure of wave Gymnotiformes (Berman and Maler, 1999; Carr and Maler, 1986; Chacron et al., 2011; Krahe and Maler, 2014; Maler, 2009a, 2099b, 1979; Maler et al., 1991; Marsat et al., 2012; Schumway, 1989). In particular, we

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Fig. 6. Late monomodal units. Raster plot (A) and post-EOD histogram (B) of a typical late monomodal unit. These units frequently fire two or more times per EOD-cycle (blue, red, and green dots in the raster indicate the spike order within the same cycle). Superimposed to the post-EOD histogram in gray, blue and red histograms respectively illustrate the firing probability of the first and second spike within the cycle. Black line corresponds to the reference EOD, the yellow bar indicates the period where the spike firing cannot be evaluated due to the EOD artifact. (C) In this case, the first order histogram shows that the two spikes are separated by 7 ms. (D) The autocorrelation histogram shows periodic peaks that are less marked than in the early monomodal units.

confirmed recent findings (Maler, 2009a, 2099b) indicating the presence of efferent neurons somatas at different levels from the granule to pyramidal layers (see Fig. 1). We must stress the presence of a efferent neuron type that is not frequently referred to in the description of the cells of the ELL of wave fish. This is called ‘‘bald basilar cell’’ and is characterized by a large basilar dendritic tree and absence of apical tree. In spite of these histological similarities, the ELLs of wave and pulse fish deal with electrical images in quite different ways. High frequency wave fish (as genus Apteronotus and Eigenmania) have an EOD frequency comparable to the inverse spike-refractory period duration, whereas pulse fish have an EOD repetition rate one order of magnitude smaller. Whereas the EOD repetition rate of high frequency wave Gymnotifomes implies the need for extracting information from the envelope of a continuous signal (Chacron et al., 2011; Marsat et al., 2012; Metzen and Chacron, 2014), pulse Gymnotiformes appear to show a pulsatile frameby-frame image processing strategy (Aguilera and Caputi, 2003; Caputi et al., 2008, 2003; Pereira et al., 2005). Accordingly, our analysis of field potential and unitary recordings indicate that the slow electrosensory pathway of G. omarorum has a complex but precise activation pattern after each EOD. Thus, we show that a very similar neural network may adopt different activation patterns when stimulated under a different frequency regime. All the reported recordings were performed in a ‘‘basal condition’’ with the fish in the middle of a tank without surrounding objects. We used decerebrated fish in order to maintain the natural spatiotemporal pattern of activation of the afferent population. Preliminary studies had shown that the ELL activation patterns obtained with a bipolar artificial EOD were different from the patterns observed with a normal EOD. However, data obtained in freely moving fish show the same unitary patterns as those reported here (Rodríguez-Cattáneo et al., 2012). The activity of the slow path is summarized in Fig. 10. Current source density analysis shows that the main generators of the spatiotemporal pattern of ELL activation are between the GCL

Fig. 7. Multimodal units. (A and B) Raster plots (A) and peri-EOD histogram (B) of the most common trimodal type. An early and narrow peak of the histogram around 7 ms is followed by a firing probability depression that in turn precedes a sharp peak of activity (in this case at about 16 ms). In trimodal units a third broad peak of activity occurs at long latencies (25 ms in B). (C and D) Raster and peri-EOD histogram from a bimodal unit recorded at lower temperature (note the different time scale). Tri- and bi-modal units fire often more than one time per cycle (different colors in raster and histograms correspond to different spikes in the same cycle). Black line corresponds to the reference EOD, the yellow bar indicates the period where the spike firing cannot be evaluated due to the EOD artifact.

and PNL where most neuron somata are located. In most cells and in the current source density there is no activity in the somata layers until 7 ms after the EOD. At this latency there is a sink in the PNL layer in the current source density map and an early peak in many of the multimodal studied neurons. Following this activity there is frequent silence in both activities suggesting a strong inhibitory action (see for example unit 6-XI-002 in Fig. 7A & B and 5-XI-020 in Fig. 8). Comparative analysis suggest that this early excitation–inhibition sequence could be explained by a fast forward inhibition conveyed by the ovoid cells. In fact, Maler and Mugnaini (1994) showed several types of GABAergic interneurons in the lower layers, which receive direct or indirect tuberous electrosensory input (Maler, 1979; Maler et al., 1981; Mathieson et al., 1987). Double label experiments (retrograde transport and GABA immunohistochemistry) show that multipolar and ovoid cells of wave fish are GABAergic (Maler and Mugnaini, 1994). Similar unpublished experiments confirmed these findings in G. omarorum (Castelló,

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Pereira, and Caputi). These neurons form part of a commissural projection system providing reciprocal inhibition between homologous maps on the left and right sides of ELL and potentially supporting a common mode rejection mechanism (Bastian et al., 1993). According to Maler and Mugnaini (1994) ovoid cell axons projecting to the contralateral side ‘‘entered the ELL at its rostromedial edge. . . ran within the deep neuropil/deep fiber layers,

and ascended to ramify vertically within the granular/deep neuropil layers. These axons climbed upon and densely innervated deep basilar pyramidal cells and the basilar dendrites of basilar pyramidal cells’’. They conclude that ‘‘ovoid cells provide the vertically oriented GABAergic synaptic input to the basilar dendrites of basilar and deep basilar pyramidal cells and perhaps input to granular or polymorphic cell’’. Subsequent in vitro studies (Berman and Maler, 1998c) indicated that ‘‘basilar pyramidal cells differ3 only in that their basilar dendrites receive GABAergic input along its length from ovoid cells and direct excitatory (glutamatergic) input from the primary afferents’’. Moreover, basilar pyramidal neurons ‘‘responded to a single stimulus with a short latency (peak at 1.8 ms) EPSP, followed by a small slow IPSP’’. These authors also showed that basilar pyramids responded to tetanic stimulation with a summating EPSP followed by a slow IPSP. After CNQX/APV treatment, the depolarizing response was abolished completely, and the amplitude of the slow IPSP was increased clearly indicating a fast feed forward inhibitory effect driven by the same primary afferent activity (Berman and Maler, 1998c). In this framework, one must recall that 15 out of the 35 multimodal units show a statistically significant reduction of firing probability and most of them show a strong reduction in firing probability between 7.5 and 9.5 ms after the EOD. In addition 6/8 units without preferential phase show a relative maximum of the firing probability at the end of the EOD cycle. These and one third of the multimodal units likely showed early inhibition by the EOD since they dramatically reduce their firing probability right after the EOD and no evidence of refractoriness was found in our analysis. Taking into account the mentioned antecedents our findings suggest that the second period of silence may be driven by the ovoid input on the basilar dendrites of pyramidal neurons. The main peak of activity occurs between 10 and 17 ms. Within this period the post-EOD spike timing histograms of early monomodal and multimodal units show their main modes. This does not necessarily imply that modes corresponding to different unit types are synchronous. In fact, trimodal units peak earlier than bimodals. Furthermore, units presumably belonging to the same electrosensory column (Krahe and Maler, 2014; Maler, 2009a, 2099b) show mirror image patterns (Fig. 8). Trimodal units show a conspicuous reduction of firing probability after this main peak. This might be due to slower forward or recurrent projections inhibitory as for example granule cells or praeminentialis driven inhibition. During this period the sink of at the PNL progresses to the dorsal molecular layer. This feature and the presence of doublets in early monomodal units suggest that apical dendritic arbors of these neurons might be invaded by dendritic spikes. Firing in doublets is an important characteristic of basilar pyramidal neurons recorded in vitro in the ELL of the same species (Nogueira and Caputi, unpublished data) suggesting that at least some basilar pyramids may show a monomodal firing pattern. In basilar pyramids recorded in vitro, spikes are followed by a biphasic hyperpolarization–depolarization sub-threshold potential. This peculiar pattern of discharge suggests that basilar neurons share the burst mechanism described in wave fish (Fernández et al., 2005; Mehaffey et al., 2005; Turner and Maler, 1999; Turner et al., 2002, 1996). Finally, the late period of activation starts deeply in the GCL at about 20 ms and progresses up to the PNL peaking at about 24 ms. This is coincident with the modes of late monomodal units and the late mode of some multimodal units. In this study we did not evaluate the changes in pattern with changes in the local electric image. However, data obtained in freely moving fish (RodríguezCattáneo et al., 2012) suggest that the firing probability of units

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5. Conclusion A clear excitation–inhibition–excitation post EOD activation sequence is observed in field potentials and unitary recordings. This is strikingly different from post EOD effects in wave fish. Thus, it appears that a similar neural structure might process differently the sensory information when it is driven at a different frequency regime. Our data indicate that some of these differences may be due to feed forward activation of an inhibitory path. More work is necessary to specify the subthreshold and spiking activity of specific cell types and the relations between cells. Intracellular recording followed by anatomical labeling of recorded elements should be of great help in this effort. A major role of inhibitory interneurons in determining the activation sequence of different cell types is expected.

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spiking during this period was the most affected by changes in the electric image. The sequence of activation of different neurons types in response to the EOD follow a clear pattern summarized in Fig. 10. The ELL response to the EOD is, in this aspect, more similar to pulse mormyrids than to their closely genetically related wave gymnotiformes. This contrasts with anatomical commonalities between pulse and wave gymnotiformes. In fact, the anatomies of the ELL and neuron phenotypes of pulse and wave gymnotiforms are very much alike. The main difference between these two groups of gymnotiformes is the EOD rate. According to our data, there is approximately a 10 ms latency between the EOD and the main peak of activity in the ELL of G. omarorum. If the same ELL activation rules were occurring in wave fish, between 3 and 10 EOD cycles would have occurred before this main peak. Thus, it appears that similar structures activated differently might show different sensory processing.

Aguilera, P.A., Caputi, A.A., 2003. Electroreception in Gymnotus carapo: detection of changes in waveform of the electrosensory signals. J. Exp. Biol. 206, 989–998. Bastian, J., 1986a. Electrolocation: behavior, anatomy and physiology. In: Bullock, T.H., Heiligenberg, W. (Eds.), Electroreception. John Wiley and Sons, New York, pp. 577–612. Bastian, J., 1986b. Gain control in the electrosensory system mediated by descending inputs to the electrosensory lateral line lobe. J. Neurosci. 6, 553–556. Bastian, J., 1986c. Gain control in the electrosensory system: a role for the descending projections to the electrosensory lateral line lobe. J. Comp. Physiol. A 158, 505–515. Bastian, J., 1995. Pyramidal-cell plasticity in weakly electric fish: a mechanism for attenuating responses to reafferent electrosensory inputs. J. Comp. Physiol. A 176 (1), 63–78. Bastian, J., Courtright, J., 1991. Morphological correlates of pyramidal cell adaptation rate in the electrosensory lateral line lobe of weakly electric fish. J. Comp. Physiol. A 168 (4), 393–407. Bastian, J., Courtright, J., Crawford, J., 1993. Commissural neurons of the electrosensory lateral line lobe of Apteronotus leptorhynchus: morphological and physiological characteristics. J. Comp. Physiol. A 173, 257–274. Bastian, J., Chacron, M., Maler, L., 2004. Plastic and nonplastic pyramidal cells perform unique roles in a network capable of adaptive redundancy reduction. Neuron 41, 767–779. Bell, C.C., 1979. Central nervous system physiology of electroreception, a review. J. Physiol. Paris 75, 361–379. Bell, C.C., 1981. An efference copy which is modified by reafferent input. Science 214, 450–453. Bell, C.C., 1989. Sensory coding and corollary discharge effects in mormyrid electric fish. J. Exp. Biol. 146, 229–253. Bell, C.C., Grant, K., 1989. Corollary discharge inhibition and preservation of temporal information in a sensory nucleus of mormyrid electric fish. J. Neurosci. 9, 1029–1044. Bell, C.C., Grant, K., 1992. Sensory processing and corollary discharge effects in the mormyromast regions of the mormyrid electrosensory lobe. II. Cell type and corollary discharge plasticity. J. Neurophysiol. 68, 859–875. Bell, C.C., Grant, K., Serrier, J., 1992. Sensory processing and corollary discharge effects in the mormyromast regions of the mormyrid electrosensory lobe. I. Field potentials, cellular activity in associated structures. J. Neurophysiol. 68, 843–858. Bell, C.C., Caputi, A., Grant, K., Serrier, J., 1993. Storage of a sensory pattern by antiHebbian synaptic plasticity in an electric fish. Proc. Natl. Acad. Sci. USA 90, 4650–4654. Bell, C.C., Caputi, A.A., Grant, K., 1997a. Physiology and plasticity of morphologically identified cells in the mormyrid electrosensory lobe. J. Neurosci. 17, 6409–6423. Bell, C.C., Han, V.Z., Sugawara, S., Grant, K., 1997b. Synaptic plasticity in a cerebellum-like structure depends on temporal order. Nature 387, 278–281. Berman, N.J., Maler, L., 1998a. Distal versus proximal inhibitory shaping of feedback excitation in the electrosensory lateral line lobe: implications for sensory filtering. J. Neurophysiol. 80, 3214–3233. Berman, N.J., Maler, L., 1998b. Interaction of GABAA-mediated inhibition with voltage-gated currents of pyramidal cells: computational mechanism of a sensory searchlight? J. Neurophysiol. 80, 3197–3213. Berman, N.J., Maler, L., 1998c. Inhibition evoked from primary afferents in the electrosensory lateral line lobe of the weakly electric fish (Apteronotus leptorhynchus). J. Neurophysiol. 80, 3173–3196. Berman, N.J., Maler, L., 1999. Neural architecture of the electrosensory lateral line lobe: adaptations for coincidence detection, a sensory searchlight and frequency, dependent adaptative filtering. J. Exp. Biol. 202, 1243–1253. Black-Cleworth, P., 1970. The role of electrical discharges in the non-reproductive social behaviour of Gymnotus carapo (Gymnotidae, Pisces). Anim. Behav. Monogr. 3, 1–77. Blake, R.W., 1983. Fish Locomotion. Cambridge University Press, Cambridge.

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