Electroreceptive single units in the mesencephalic magnocellular nucleus of the weakly electric fish gymnotus carapo

Summary 1. Extra- and intracellular recordings from single units in the magnocellular mesencephalic nucleus (MMN) of the torus semicircularis, related to the fast electrosensory system are reported for the weakly electric fish Gymnotus carapo (Gymnotidae). 2. The non-spontaneously active units responded with single action potentials to the electric organ discharge (EOD) and to artificial electrical pulses with a very short latency of 0.8–1.5 ms. This strongly suggests, in agreement with morphological data, that transmission takes place through electrical synapses. 3. The dynamic range (probability and latency of the single action potential) of the response is extremely narrow and about the same as found in the relevant electrosensory fibres. Intracellular stimulation gives the same response characteristics and dynamic range. 4. The recovery of the response was studied in detail using different stimulus combinations of double pulses at varying delays. Under all conditions, the recovery period to evoke a test response after a conditioning stimulus and response increased in length with the strength of the conditioning stimulus. Inversely, the conditioning stimulus to prevent the unit from firing again had to be stronger as the delay between the two stimuli was increased. 5. Since there is no evidence of neural inhibition causing the long lasting and graded recovery characteristics for MMN units, an attempt was made to explain the findings by classical neurophysiological considerations adapted for electrical synaptic transmission (current sink theory). 6. This neural mechanism means that, if at all, the relatively weaker stimulus is not responded to, which protects the fish from being jammed by external pulses of physiological amplitude. In contrast, very strong foreign pulses can completely abolish responses to own EODs especially when timed appropriately. Both effects are discussed in view of their significance for the fish's electrosensory system and communication.     

Reciprocal connections between the preglomerular complex and the dorsolateral telencephalon in the weakly electric fish, Gymnotus carapo

The diencephalic preglomerular complex of gymnotiform fish receives inputs from several sensory areas. By employing anterograde and retrograde tracing techniques, we studied the afferent and efferent connections of the dorsolateral area (dorsal subdivision) of the telencephalon with the preglomerular nuclei in the weakly electric fish, Gymnotus carapo. Neurons of the medial preglomerular nucleus project to intermediate and deep portions of the middle (commissural) level of the dorsolateral telencephalon, and neurons located in the lateral preglomerular nucleus project to superficial portions of the middle levels of the dorsolateral telencephalon. Therefore, we observed a spatial distribution pattern of connectivity between dorsolateral telencephalon and preglomerular complex.     

Neurohistological analysis of the lateral lobe in a weakly electric fish,Gymnotus carapo (Gymnotidae,Pisces)

Summary The cyto- and fiber architecture of the lateral lobe (LL) of Gymnotus carapo was investigated using Nissl, Golgi and reduced silver stains as well as 1 semi-thin sections. The neurons and fiber tracts are distributed in six layers. The first layer is subdivided into two sublayers: 1A where the primary afferent fibers run in rostro-caudal direction and 1B where these fibers terminate and the large multipolar neurons can be found. The 2nd layer consists of a single row of adendritic, pear-shaped neurons. The axons of these neurons enter the 4th layer and leave the lateral lobe in medial direction. The 3rd layer contains the granular cells of two different types: granular cells with two dendritic trunks directed into dorsal and ventral directions respectively, and granular cells in which, instead of the dendritic trunk, the axon emerges from the dorsal pole of the perikaryon. The axon can be followed up to the 6th layer. The 4th layer consists of bundles of nerve fibers. Beside the axons of the pear-shaped neurons the bundles contain also the axons of the pyramidal neurons (5th layer) leaving the lateral lobe. The 5th layer contains the perikarya of the pyramidal neurons. They have two separate dendritic arborizations, one directed ventrally and entering layer 1B and another directed dorsally and penetrating into the 6th layer. Their axons join the 4th layer and run in medial, rostro-medial or rostral direction depending upon the localization of the neurons in the lateral lobe. The 6th layer (crista cerebellaris) consists of three sublayers. Sublayer 6A contains fine myelinated fibers of unknown origin; sublayer 6B contains fine, mainly non-myelinated fibers originating from the mesencephalon, sublayer 6C is built up of non-myelinated fibers originating from the cerebellum. — A preliminary diagram of the neuron circuits and of the synaptic arrangements involved in the relay of lateral-line organ impulses is suggested.Supported by Research Grant No. 659440 accorded to Dr. T. Szabo by the Direction de Recherches et Moyens d'Essais (D. R. M. E.).Visiting investigator, aided by Institut national de la Santé et de la Recherche Médical (I. N. S. E. R. M.).Address: Groupe des Laboratoires du C. N. R. S. 91190 Gif-sur-Yvette, France.     

Effect of drugs that alter alertness and emotionality on the novelty response of a weak electric fish, Gymnotus carapo

Weak field electric fish respond to alerting stimuli with a transient increase in the frequency of electric organ discharge (novelty response). In an attempt to demonstrate the influence of different degrees of alertness and of emotionality on the novelty response of Gymnotus carapo, we studied the variations in the magnitude of this response induced by the application of an electric stimulus to the water of the experimental box using a pair of electrodes, before and after intramuscular injections of d-amphetamine (1-2 and 4 mg/kg), sodium pentobarbital (10-20 and 30 mg/kg), diazepam (1-2 and 4 mg/kg), beta-carboline (2 mg/kg), and saline. After d-amphetamine injection the animal presented increased somatic motility but no changes in electric organ baseline firing rate or in response to the alerting electric stimulus. Sodium pentobarbital induced a partial loss of posture and a reduction of fin and operculum movements, as well as a reduction of baseline firing rate and of the response to the alerting electric stimulus, with frequent interruptions in electric organ firing. Beta-carboline caused increased motility, but no changes in basal firing rate or in response to the alerting stimulus. Diazepan-injected fishes remained still throughout the experiment, and some of those threated with the higher dose (4 mg/kg) presented interruptions on electric organ discharges in response to stimulation but no change on baseline firing rate. The data suggest that a reduction of the degree of alertness by the barbiturate and a decrease in emotionality and/or stress by the benzodiazepine interfere with the novelty response. The possible site of action of the drugs is discussed.     

Electrotonic coupling between neurones in the rat mesencephalic nucleus

Electrical stimulation of the trigeminal nerve evokes a ;short latency depolarization' (SLD) in the first order sensory neurones of the mesencephalic nucleus (MSN) of the Vth nerve in the rat. A series of experiments suggesting ;electrotonic coupling' as the mechanism for this SLD is provided.1. Electrical activity of MSN neurones was recorded intracellularly as action potentials were conducted from the periphery (somatopetally) to the masticatory nucleus. Typical sequential invasion of the initial segment and somatic region (IS-S) of the neurones was seen. The somatopetal activation of MSN neurones was characterized by the brevity, short refractoriness, high safety factor (IS-S), and short after-hyperpolarization of the spike potential.2. In twenty-three (10.5%) of the penetrated neurones, stimulation at levels subthreshold for somatopetal activation uncovered a SLD with a mean latency of 180 musec.3. The SLDs were found to be all-or-none in nature, and to have constant amplitude and latency for a given cell, plus a short half decay time.4. Hyperpolarization of a MSN neurone through the recording electrode produced a blockage of the IS-S spike and revealed M-spikes and SLDs which could be clearly separated, in every instance, as distinct all-or-none components. The amplitude of the SLD was found to be insensitive to the level of membrane potential within the ranges tested.5. In fifteen neurones the SLD generated action potentials which were conducted somatofugally as shown by their collision with somatopetally conducted action potentials in the same cell. The lack of collision between the SLD and somatopetal spikes demonstrated the independent origin of these two potentials.6. The independence of the SLD from the somatopetal invasion of the cell was also demonstrated by collision of a somatofugal action potential following direct stimulation through the recording micro-electrode and a somatopetal spike following trigeminal stimulation.7. Two possible mechanisms are considered for the genesis of the SLD: chemical synaptic transmission and electrotonic coupling between neighbouring cells. The conclusion is drawn that SLDs must be generated by the latter mechanism.     

Precision of the pacemaker nucleus in a weakly electric fish: network versus cellular influences

We investigated the relative influence of cellular and network properties on the extreme spike timing precision observed in the medullary pacemaker nucleus (Pn) of the weakly electric fish Apteronotus leptorhynchus. Of all known biological rhythms, the electric organ discharge of this and related species is the most temporally precise, with a coefficient of variation (CV = standard deviation/mean period) of 2 x 10(-4) and standard deviation (SD) of 0.12-1.0 micros. The timing of the electric organ discharge is commanded by neurons of the Pn, individual cells of which we show in an in vitro preparation to have only a slightly lesser degree of precision. Among the 100-150 Pn neurons, dye injection into a pacemaker cell resulted in dye coupling in one to five other pacemaker cells and one to three relay cells, consistent with previous results. Relay cell fills, however, showed profuse dendrites and contacts never seen before: relay cell dendrites dye-coupled to one to seven pacemaker and one to seven relay cells. Moderate (0.1-10 nA) intracellular current injection had no effect on a neuron's spiking period, and only slightly modulated its spike amplitude, but could reset the spike phase. In contrast, massive hyperpolarizing current injections (15-25 nA) could force the cell to skip spikes. The relative timing of subthreshold and full spikes suggested that at least some pacemaker cells are likely to be intrinsic oscillators. The relative amplitudes of the subthreshold and full spikes gave a lower bound to the gap junctional coupling coefficient of 0.01-0.08. Three drugs, called gap junction blockers for their mode of action in other preparations, caused immediate and substantial reduction in frequency, altered the phase lag between pairs of neurons, and later caused the spike amplitude to drop, without altering the spike timing precision. Thus we conclude that the high precision of the normal Pn rhythm does not require maximal gap junction conductances between neurons that have ordinary cellular precision. Rather, the spiking precision can be explained as an intrinsic cellular property while the gap junctions act to frequency- and phase-lock the network oscillations.     

Analysis of behavior-related excitatory inputs to a central pacemaker nucleus in a weakly electric fish

Gymnotid electric fish explore their environment and communicate with conspecifics by means of rhythmic electric organ discharges. The neural command for each electric organ discharge arises from activity of a medullary pacemaker nucleus composed of two neuronal types: pacemaker and relay cells. During different behaviors as in courtship, exploration and agonistic interactions, these species display specific electric organ discharge frequency and/or waveform modulations. The neural bases of these modulations have been explained in terms of segregation of inputs to pacemaker or relay cells, as well as differential activation of the glutamate receptors of these cells. One of the most conspicuous electric organ discharge frequency modulations in Gymnotus carapo results from the activation of Mauthner cells, a pair of reticulospinal neurons that are involved in the organization of sensory-evoked escape responses in teleost fish. The activation of Mauthner cells in these animals produces a prolonged increase in electric organ discharge rate, whose neural mechanisms involves the activation of both N-methyl-D-aspartate (NMDA) and metabotropic glutamatergic receptors of pacemaker cells. Here we provide evidence which indicates that pacemaker cells are the only cellular target of the synaptic inputs responsible for the Mauthner cell initiated electric organ discharge modulation at the medullary pacemaker nucleus. Additionally, although pacemaker cells express both NMDA and non-NMDA ionotropic receptors, we found that non-NMDA receptors are not involved in this synaptic action which suggests that NMDA and non-NMDA receptor subtypes are not co-localized at the subsynaptic membrane. NMDA receptor activation of pacemaker cells seems to be an efficient neural strategy to produce long-lasting enhancements of the fish sampling capability during Mauthner cell-initiated motor behaviors.     

Early development of the mesencephalic trigeminal nucleus.

The cells of the mesencephalic trigeminal nucleus (MTN) are the proprioceptive sensory neurons that innervate the jaw muscles. Interestingly, their evolution is generally thought to have been concomitant with that of the jaws. They are also the first born neurons of the mesencephalon, and their axons pioneer some of the major tracts within the brain. The cells of the MTN are also paradoxical in being the only group of intramedullary primary sensory neurons in amniotes. However, we know little about the early development of these important neurons, and we have analysed this here. To study the earliest stages of MTN development, we have used a battery of neural crest markers to try and pinpoint the progenitors of the MTN. We find that, contrary to current perceptions, the progenitors of the MTN are not highlighted by these markers, suggesting that they are not neural crest derived. However, the cells of the MTN are marked by means of their expression of Brn-3a. This gene labels cells that arise either side of the dorsal midline, extending rostrally from the isthmus across the roof of the mesencephalon. We have further demonstrated that the MTN develops under the influence of the Fgf-8 secreted by the isthmus. Ectopic Fgf-8 application promotes MTN development, whereas inhibiting Fgf-8 function in vivo drastically affects MTN development.     

Precision measurement of electric organ discharge timing from freely moving weakly electric fish

Physiological measurements from an unrestrained, untethered, and freely moving animal permit analyses of neural states correlated to naturalistic behaviors of interest. Precise and reliable remote measurements remain technically challenging due to animal movement, which perturbs the relative geometries between the animal and sensors. Pulse-type electric fish generate a train of discrete and stereotyped electric organ discharges (EOD) to sense their surroundings actively, and rapid modulation of the discharge rate occurs while free swimming in Gymnotus sp. The modulation of EOD rates is a useful indicator of the fish's central state such as resting, alertness, and learning associated with exploration. However, the EOD pulse waveforms remotely observed at a pair of dipole electrodes continuously vary as the fish swims relative to the electrodes, which biases the judgment of the actual pulse timing. To measure the EOD pulse timing more accurately, reliably, and noninvasively from a free-swimming fish, we propose a novel method based on the principles of waveform reshaping and spatial averaging. Our method is implemented using envelope extraction and multichannel summation, which is more precise and reliable compared with other widely used threshold- or peak-based methods according to the tests performed under various source-detector geometries. Using the same method, we constructed a real-time electronic pulse detector performing an additional online pulse discrimination routine to enhance further the detection reliability. Our stand-alone pulse detector performed with high temporal precision (<10 μs) and reliability (error <1 per 10(6) pulses) and permits longer recording duration by storing only event time stamps (4 bytes/pulse).     

Synaptology of the command (pacemaker) nucleus in the brain of the weakly electric fish, Sternarchus (Apteronotus) albifrons

The high frequency electric emission of the weakly electric fish Sternarchus (Apteronotus) albifrons depends on the pacemaker activity of a specific brainstem nucleus located in the ventral part of the rhombencephalic reticular formation. The general morphology and fine structure of this nucleus has been investigated, with particular reference to its synaptic connections.Three neuronal components could be distinguished in the nucleus; namely large cells of 80–100 μm diameter, small cells of 30–50 μm diameter and bundles of thin, myelinated fibres. These elements are embedded in a network of thick myelinated fibres. The large cells have a few small and short dendrites whereas the small neurons have long branching dendrites. Large and small neurons possess thick myelinated axons, but only those of the latter show branching patterns and send collaterals which have intranuclear courses only. Two types of synaptic terminals have been found on both neurons: large club endings exclusively with gap junctions and small bouton-like terminals with polarized chemical synapses. Serial semi-thin and ultra-thin sections revealed that the large club endings belong to the pacemaker cells, whereas the small terminals are found in the thin myelinated axons of extranuclear origin.The findings indicate that the small neurons are connected 1) to each other and 2) to the large neurons, by way of their large myelinated axons. Both, small (pacemaker) as well as large (relay), neurons receive chemical synapses from myelinated fine fibers probably originating from higher encephalic centers. Thus, electric organ discharge rhythm can be modulated at the level of pacemaker as well as of the relay cells. No somatosomatic, dendrodendritic or dendrosomatic connections were found between large, small or large and small cells.     

Androgen-dependent modulation of the electrosensory and electromotor systems of a weakly electric fish

The weakly electric fish, Sternopygus, generates an electric organ discharge (EOD) as a communicatory signal. This quasi-sinusoidal discharge is lower in frequency in mature males than in mature females and each fish discharges at its own individual frequency within its sex-specific range. EOD frequency is determined by the firing frequency of the medullary pacemaker nucleus, whereas EOD pulse duration is determined by the membrane properties of the cells of the electric organ, the electrocytes. These fish also possess sensory receptors, called tuberous electroreceptors, that detect their own EODs and those of neighboring fish. Electroreceptors are frequency tuned and the electroreceptors are best tuned to the fish's own EOD frequency. Administration of androgens to adult females or juveniles of either sex lowers EOD frequency and electroreceptor best frequency and broadens the EOD pulse, thus preserving the sinusoidal nature of the discharge. Experimental uncoupling of the pacemaker, receptors and electric organ by surgery or anesthetics suggests that androgens most probably affect each target organ independently. The androgen-dependent spike-broadening of the electrocyte response in Sternopygus occurs with no hypertrophy of the cell membrane. Current- and voltage-clamp of electrocytes indicates that these cells possess a Na⁺ and three types of K⁺ conductances. We propose that androgens modulate the duration of the EOD pulse by modulating the rate kinetics of one or more of these currents.     

Retinofugal projections in a weakly electric gymnotid fish (Apteronotus leptorhynchus)

The eyes of weakly electric gymnotid fish are poorly developed in comparison to those of most diurnal teleosts. The tectum and pretectum, despite their usual association with the visual system, are large and well differentiated in gymnotids. We have studied retinal projections in gymnotids in order to define the visual components of the mesencephalon and diencephalon and thus allow comparison with other teleosts in which retinofugal fibers have been extensively mapped. Retinofugal projections reported in this work are based on the anterograde transport of conjugated wheat germ agglutinin horseradish peroxidase, following injection into the posterior chamber of the eye of Apteronotus leptorhynchus (brown ghost knife fish). The results show a remarkable similarity to those of non-electroreceptive teleosts. Although the optic nerves appear to cross completely at the optic chiasm, close scrutiny shows a slender recrossing fascicle which continues from the contralateral tractus opticus medialis through the rostroventral hypothalamus to reach the ipsilateral side, providing a scanty projection to the n. opticus hypothalamicus, n. anterior periventricularis, n. dorsolateralis thalami, and n. commissurae posterioris. A few fibers ascend via the tractus opticus dorsomedialis to the rostral dorsomedial part of the stratum fibrosum et griseum superficiale of the ipsilateral tectum. The main body of the retinal projections in Apteronotus are to the following contralateral target areas: preoptic area, n. opticus hypothalamicus, n. anterior periventricularis, n. dorsolateralis thalami, n. pretectalis, area pretectalis, n. corticalis, n. commissurae posterioris, n. geniculatus lateralis, area and n. ventrolateralis thalami, caudal dorsal tegmentum and the tectum opticum. The retinotectal projection is modest in comparison to that of more vision dependent fish and terminates mainly in the upper half of the stratum fibrosum et griseum superficiale; hardly any retinal fibers reach the caudalmost tectum.     

Morphological correlates of triadic circuitry in the lateral geniculate nucleus of cats and rats

We used an in vitro slice preparation of the lateral geniculate nucleus in cats and rats to study morphological correlates of triadic circuitry in relay cells. The three triadic elements involve a retinal synapse onto a GABAergic dendritic terminal of an interneuron, a synapse from the same retinal terminal onto a relay cell dendrite, and a synapse from the same interneuron terminal onto the same relay cell dendrite. We made whole cell recordings and labeled cells with biocytin. Previous methods were used to identify triadic circuitry based on evidence that the retinal terminal activates a metabotropic glutamate receptor on the interneuronal terminal. Thus application of (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (an agonist to that receptor) increases the rate of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded in the relay cell, and if some of this increase remains with further addition of TTX (a TTX-insensitive response), a triad is indicated. We quantified the extent of the TTX-insensitive response and sought morphological correlates. In both rats and cats, this response correlated (negatively) with the number of primary dendrites and (positively) with polarity of the dendritic arbor. There was no correlation with cell size. Curiously, in cats, this response correlated with the presence of appendages at primary dendritic branches, but there was no such correlation in rats. These observations in cats map onto the X/Y classification, with X cells having triads, but it is not clear from our results if a comparable classification exists for rats.     

Evidence for electrotonic coupling between frog motoneurons in the in situ spinal cord.

A recurrent EPSP was observed on antidromic stimulation of motoneurons in the in situ spinal cord of Rana temporaria and R. esculenta at 20-22C. The EPSP was finely graded and not refractory following full or partial antidromic spike components in a given neuron. The EPSP amplitude varied in parallel with the antidromic field potential under different conditions, suggesting transmission of the EPSP to the recorded motoneuron depended on invasion of the somadendritic membrane or neighboring motoneurons by the antidromic spike. The latency of the EPSP with respect to antidromic invasion of the local motoneuron pool was too short for the EPSP to be mediated by chemical transmission. It was concluded the EPSP was electrically transmitted between the somadendritic membranes of the motoneurons. Under certain conditions, the EPSP magnitude could be made to vary with membrane potential in a direction opposite to that expected from a chemical EPSP. Dendritic spikes were sometimes associated with the EPSP.     

Synaptology of the medullary command (pacemaker) nucleus of the weakly electric fish (Apteronotus leptorhynchus) with particular reference to comparative aspects

Summary The general organization and synaptology of the medullary command (pacemaker) nucleus (MCN) was investigated in the high frequency weakly electric fish, Apteronotus leptorhynchus. This study was undertaken in order to establish differences and similarities between the MCN of A. leptorhynchus and that of the closely related species, Apteronotus albifrons which has been studied previously. The basic morphology and synaptology of the MCN in A. leptorhynchus is similar to that of A. albifrons. The MCN of A. leptorhynchus consists of large (relay) and small (pacemaker) cells; both cell types receive synaptic input or large club endings with electrotonic gap junctions and bouton-like terminals with polarized chemical synapses. Club endings originate from thick meyelinated fibres belonging to the small (pacemaker) cells, whereas the bouton-like terminals issue from thin myelinated fibers of extranuclear origin. Via their club endings, the small (pacemaker) cells are connected both to each other and to the large (relay) cells. Besides the similarities, there are distinct and characteristic differences between the MCN of the two species, which mainly concern the synaptology of the nucleus. In A. leptorhynchus, the large (relay) cells possess long dendritic processes, covered exclusively with bouton-like terminals; the axon initial segment of large (relay) cells receives boutons, in addition to club endings. Small (pacemaker) cells have short dendritic protrusions receiving input from club endings and boutons; furthermore, the small pacemaker cells axon initial segment receives both club endings and bouton-like terminals. These differences are discussed in terms of the functional organization of the MCN in certain gymnotoids and draw attention to the fact that the morphological and ultrastructural aspects of the central command of the electric organ discharge reveal several differences not only between different gymnotoid fish (Apteronotus and Eigenmannia) but also between closely related species such as A. albifrons and A. leptorhynchus.     

The synaptic organization of the prepacemaker nucleus in weakly electric knifefish, Eigenmannia: a quantitative ultrastructural study.

Summary Weakly electric knifefish (Eigenmannia sp.) produce continuous electric organ discharges at very constant frequencies. Modulations of the discharges occur during social interactions and are under control of the diencephalic prepacemaker nucleus. Abrupt frequency modulations, or chirps, which are observed predominantly during the breeding season, can be elicited by stimulation of neurons in a ventro-lateral portion of the prepacemaker nucleus, the so-called PPn-C. The PPn-C consists of approximately 100 loosely scattered large multipolar neurons which send dendrites into three territories, called dorso-medial, dorso-lateral, and Ventral. In the present ultrastructural investigation, the synaptic organization of these neurons, identified by retrograde labelling with horseradish peroxidase, was studied quantitatively.Somata and dendrites of the PPn-C receive input from two classes of chemical synapses. Class-1 boutons contain predominantly agranular, round vesicles and are believed to be excitatory. Class-2 boutons display predominantly flattened or pleiomorphic vesicles and are probably inhibitory. The action of the agranular vesicles in the synaptic boutons of these two classes may be modulated by the content of large dense-core vesicles. These comprise approximately 1% of the total vesicle population and are found predominantly in regions distant from the active zone of the synaptic bouton.The density of chemical synapses exhibits marked topographic differences. Class-1 boutons occur typically at densities of 3–12 synapses per 100 m of profile length on dendrites and cell bodies. No significant differences in density of class-1 boutons could be found between distal dendrites of the three territories, proximal dendrites and cell bodies. The density of class-2 synapses, on the other hand, increases significantly from usually less than 1 synapse per 100 m of profile length on distal dendrites to 2–3 synapses per 100 m of profile length on proximal dendrites and cell bodies.Such a topographic organization could enable the proximal elements to veto the depolarizing response of distal dendrites to excitatory inputs. The growth of dendrites in the dorso-medial territory during the breeding season, as shown in a previous study, and the concurrent doubling of excitatory input received by class-1 synapses, could overcome the inhibition caused on somata and proximal dendrites by class-2 synapses and thus account for the dramatic increase in the fish's propensity to chirp in the context of sexual maturity.