Time coding in the midbrain of mormyrid electric fish. II. Stimulus selectivity in the nucleus exterolateralis pars posterior

The anterior and posterior exterolateral nuclei (ELa and ELp) of the mormyrid midbrain are thought to play a critical role in the temporal analysis of the electric discharge waveforms of other individuals. The peripheral electroreceptors receiving electric organ discharges (EODs) of other fish project through the brainstem to ELa via a rapid conducting pathway. EODs, composed of brief, but stereotyped waveforms are encoded as a temporal pattern of spikes. From previous work, we know that phase locking is precise in ELa. Here it is shown that evoked potentials recorded from ELp show a similar high degree of phase locking, although the evoked potentials last much longer. Single-unit recordings in ELp reveal two distinct populations of neurons in ELp: type I cells are responsive to voltage step functions, and not tuned for stimulus duration; type II cells are tuned to a specific range of stimulus durations. Type II cells are less responsive than type I cells, tend to respond with bursts of action potentials rather than with single spikes, have a longer latency, show weaker time locking to stimuli, and are more sensitive to stimulus polarity and amplitude. The stimulus selectivity of type II cells may arise from convergence of type I cell inputs. Despite the loss of rapid conduction between ELa and ELp, analysis of temporal features of waveforms evidently continues in ELp, perhaps through a system of labeled lines. Accepted: 25 June 1997     

Central auditory neurophysiology of a sound-producing fish: the mesencephalon of Pollimyrus isidori (Mormyridae)

This paper describes the auditory neurophysiology of the mesencephalon of P. isidori, a soundproducing mormyrid fish. Mormyrids have a specialized pressure-sensitive auditory periphery, and anatomical studies indicate that acoustic information is relayed to the mesencephalic nucleus MD. Fish were stimulated with tone bursts and clicks, and responses of MD neurons were recorded extracellularly. Auditory neurons had best frequencies (BF) and best sensitivities (BS) that fell within the range of frequencies and levels of the natural communication sounds of these fish. BSs were in the range of 0 to — 35 dB (re. 1.0 dyne/cm²). Many of the neurons were tuned (Q_{10 dB}: 2–6), and had BFs in the range of 100–300 Hz where the animal's sounds have their peak energy. A range of different physiological cell types were encountered, including phasic, sustained, and complex neurons. Some of the sustained neurons showed strong phase-locking to tones. Many neurons exhibited non-monotonic rate-level functions. Frequencies flanking the BF often caused a reduction in spontaneous activity suggesting inhibition. Many neurons showed excellent representation of click-trains, and some showed a temporal representation of inter-click-intervals with errors less than 1 ms.Abbreviations BF best frequency - BS best sensitivity - ELa anterior exterolateral toral nucleus - ELp posterior exterolateral toral nucleus - EOCD electric organ command discharge - FFT fast Fourier transform - HRP horseradish peroxidase - ICI inter-clickinterval - MD mediodorsal toral nucleus (=auditory nucleus) - OR onset response rate - PSTH peri-stimulus-time-histogram - R synchronization coefficient - RA response area - SS steady state response rate     

Spatial distribution of the medullary command signal within the electric organ of Gymnotus carapo

The pacemaker nucleus of Gymnotus carapo contains two types of neurons: pacemaker cells which set up the frequency of the electric organ discharge (EOD) and relay cells which convey the command signal to the spinal cord. Direct activation of a single relay cell provides enough excitation to discharge a pool of spinal electromotor neurons and electrocytes, generating a small EOD (unit EOD). Different relay cells generate unit EODs of variable size and waveform, indicating the involvement of different groups of electrocytes. A special technique of EOD recording (multiple air-gap) was combined with intracellular stimulation of relay cells to study the spatial distribution within the electric organ (EO) of the command signal arising from different relay cells. Three types of relay cells could be identified: type I commanding the rostral 10% of the EO, type II which distribute their command all along the EO and type III driving the caudal 30%. Waveform analysis of unit EODs indicates that doubly innervated electrocytes which are the most relevant for attaining the specific EOD waveform, receive a favored command from the pacemaker nucleus.Abbreviations CV conduction velocity - EMF electromotive force - EMN electromotor neuron - EO electric organ - EOD electric organ discharge - PN pacemaker nucleus - uEOD unit electric organ discharge     

Electrical stimulation of the preoptic area in Eigenmannia: evoked interruptions in the electric organ discharge

The functional role of the basal forebrain and preoptic regions in modulating the normally regular electric organ discharge was determined by focal brain stimulation in the weakly electric fish, Eigenmannia. The rostral preoptic area, which is connected with the diencephalic prepacemaker nucleus, was examined physiologically by electrical stimulation in a curarized fish. Electrical stimulation of the most rostral region of the preoptic area with trains of relatively low intensity current elicits discrete bursts of electric organ discharge interruptions in contrast to other forebrain loci. These responses were observed primarily as after-responses following the termination of the stimulus train and were relatively immune to variations in the stimulus parameters. As the duration and rate of these preoptic-evoked bursts of electric organ discharge interruptions (approximately 100 ms at 2 per s) are similar to duration and rate of natural interruptions, it is proposed that these bursts might be precursors to natural interruptions. These data suggest that the preoptic area, consistent with its role in controlling reproductive behaviors in vertebrates, may be influencing the occurrence of electric organ discharge courtship signals by either direct actions on the prepacemaker nucleus or through other regions that are connected with the diencephalic prepacemaker nucleus. Accepted: 16 October 1999     

Ontogeny of the electric organ discharge and the electric organ in the weakly electric pulse fish Brachyhypopomus pinnicaudatus (Hypopomidae, Gymnotiformes)

I recorded the electric organ discharges (EODs) of 331 immature Brachyhypopomus pinnicaudatus 6–88 mm long. Larvae produced head-positive pulses 1.3 ms long at 7 mm (6 days) and added a second, small head-negative phase at 12 mm. Both phases shortened duration and increased amplitude during growth. Relative to the whole EOD, the negative phase increased duration until 22 mm and amplitude until 37 mm. Fish above 37 mm produced a “symmetric” EOD like that of adult females. I stained cleared fish with Sudan black, or fluorescently labeled serial sections with anti-desmin (electric organ) or anti-myosin (muscle). From day 6 onward, a single electric organ was found at the ventral margin of the hypaxial muscle. Electrocytes were initially cylindrical, overlapping, and stalk-less, but later shortened along the rostrocaudal axis, separated into rows, and formed caudal stalks. This differentiation started in the posterior electric organ in 12-mm fish and was complete in the anterior region of fish with “symmetric” EODs. The lack of a distinct “larval” electric organ in this pulse-type species weakens the hypothesis that all gymnotiforms develop both a temporary (larval) and a permanent (adult) electric organ. Accepted: 1 March 1997     

Efficient Production and Characterization of Recombinant Human NELL1 Protein in Human Embryonic Kidney 293-F Cells

NELL1 is a secretory protein that induces osteogenic differentiation and bone formation by osteoblastic cells. Because of its potent osteoinductive activity, NELL1 may be useful for bone regeneration therapy. However, at present, we have little knowledge regarding NELL1 receptors and NELL1-mediated signaling pathways. We have previously produced NELL1 using an insect’s cell expression system; however, the protein was relatively unstable and was degraded by proteases released from dead cells. In the present study, NELL1 protein was expressed in human embryonic kidney 293-F cells. Stable cell lines expressing NELL1 fused to a C-terminal hexahistidine-tag were obtained by G418 selection of transfected cells. Cells grown in serum-free medium showed high levels of NELL1 protein production (approximately 4 mg/l cell culture) for up to 6 months. NELL1 protein was purified from culture medium using a one-step nickel-chelate affinity chromatography protocol. Purified NELL1 protein immobilized onto culture dishes induced the expression of both early and late osteogenic markers on mouse mesenchymal C3H10T1/2 cells. When NELL1-expressing 293-F cells were grown on gelatin-coated glass cover slips, recombinant NELL1 was deposited in the extracellular matrix after detachment of cells. These results suggest that NELL1 acts as an extracellular matrix component. Recombinant NELL1 formed multimers and was glycosylated. An abundant source of functionally active NELL1 protein will be useful for more advanced studies, such as the development of novel techniques for bone regeneration.     

Directional characteristics of tuberous electroreceptors in the weakly electric fish,Hypopomus (Gymnotiformes)

This paper is an electrophysiological study of the directionality of the tuberous electroreceptors of weakly electric fish. We recorded from two classes of tuberous electroreceptors known for pulse gymnotiforms: Burst Duration Coders (BDCs), and Pulse Markers (PMs). Both code for stimulus amplitude, although the dynamic range for BDCs is greater, and both exhibit strong directional preferences. Polar plots of spike number (for BDCs) or spike threshold (for PMs) versus electric field azimuth, are figure-8 shaped with two asymmetrical, elliptical lobes separated by 180°. The best azimuth of these two types of receptors from a given body region correlate with each other and with measures of best azimuth for transepidermal current flow. The shape and asymmetry of the directionality profiles appear to be caused by filter dynamics of the receptors. Pulse Markers are located on the anterior part of the body surface while Burst Duration Coders are located all over. The best directions of receptors in the anterior third of the body vary systematically with location from 0° to 180°. This region is probably critical for determining the direction of local electric fields. Together these receptors provide the CNS with sufficient information to construct a map of horizontal plane electric field directions.Abbreviations BDC Burst Duration Coder - ELL electrosensory lateral line lobe - EOD electric organ discharge - nALL anterior lateral line nerve - PM Pulse Marker     

Mauthner cell-evoked synaptic actions on pacemaker medullary neurons of a weakly electric fish

The present study was designed to examine the synaptic events in neurons of the pacemaker nucleus of Gymnotus carapo during the increase in rate of the electric organ discharge following activation of Mauthner cells. Pacemaker and relay cells were investigated using intracellular recordings which were performed under two different conditions: (1) with the pacemaker nucleus spontaneously discharging and (2) after its activity was abolished by anesthesia. Mauthner axon activation induced an increase in the rate of pacemaker cell discharges. This response was accompanied by an increase in the slope of the pacemaker potential (up to 110%) and a depolarization of these cells. The discharges of relay cells followed one to one those of pacemaker cells. In contrast to that observed in pacemaker cells, only brief depolarizing antidromic effects could be evoked in relay cells after Mauthner axon activation. In quiescent pacemaker cells, Mauthner cell activation induced a prolonged (up to 500 ms) depolarizing potential with an average amplitude of 1.92 ± 0.82 mV; its latency was 4.43 ± 1.14 ms. Our data indicate that, within the pacemaker nucleus, the population of pacemaker cells is the only target for Mauthner cell-evoked, short-latency excitatory synaptic actions. Accepted: 1 March 1997     

Commissural neurons of the electrosensory lateral line lobe of Apteronotus leptorhynchus: morphological and physiological characteristics

Extracellular injections of horseradish peroxidase were used to label commissural cells connecting the electrosensory lateral line lobes of the weakly electric fish Apteronotus leptorhynchus. Multiple commissural pathways exist; a caudal commissure is made up of ovoid cell axons, and polymorphic cells' axons project via a rostral commissure. Intracellular recording and labeling showed that ovoid cells discharge spontaneously at high rates, fire at preferred phases to the electric organ discharge, and respond to increased receptor afferent input with short latency partially adapting excitation. Ovoid cell axons ramify extensively in the rostro-caudal direction but are otherwise restricted to a single ELL subdivision. Polymorphic cells are also spontaneously active, but their firing is unrelated to the electric organ discharge waveform. They respond to increased receptor afferent activity with reduced firing frequency and response latency is long. Electrical stimulation of the commissural axons alters the behavior of pyramidal cells in the contralateral ELL. Basilar pyramidal cells are hyperpolarized and nonbasilar pyramidal cells are depolarized by this type of stimulation. The physiological results indicate that the ovoid cells participate in common mode rejection mechanisms and also suggest that the ELLs may function in a differential mode in which spatially restricted electrosensory stimuli can evoke heightened responses.Abbreviations ccELL caudal commissure of the ELL - CE contralaterally excited - DML dorsal molecular layer - ELL electrosensory lateral line lobe - EOD electric organ discharge - HRP horseradish peroxidase - IE ipsilaterally excited - MTI mouth-tail inverted - MTN mouth-tail normal - rcELL rostral commissure of the ELL - TRI transverse inverted - TRN transverse normal     

Neuronal Types in the Human Anterior Ventral Thalamic Nucleus: A Golgi Study

Neurons in the anterior ventral (AV) thalamic nucleus of human adults were impregnated by Golgi-Kopsch impregnation method. Results showed that at least three morphological types of neurons could be recognized in the human AV thalamic nucleus. Type I neurons were medium to large with rich dendritic arborization. Both tufted and radiating dendritic branching patterns were seen in almost every neuron of this type. Only the initial axonal segments of these cells were impregnated suggesting that these axons were heavily myelinated. Type II neurons were medium in size with poor to moderate dendritic arborization. Many of these cells possess a few dendritic grape-like appendages. Long segments (up to 300 μm) of their axons were impregnated suggesting that these axons were either unmyelinated or thinly myelinated. These axons change their direction and form loops very often. No local branches were seen for these axons suggesting that they could be projection axons. Type III neurons were small with only one or two dendrites with poor arborization. No axons for these cells were seen in this study. The three neuronal types in the human AV thalamic nucleus were compared with neuronal types already described in other thalamic nuclei of human and non-human species. The results of this study might provide a morphological basis for further electrophysiological and / or pathological studies.     

Plasticity of the electric organ discharge waveform of the electric fish Brachyhypopomus pinnicaudatus I. Quantification of day-night changes

The electric organ discharge of the gymnotiform fish Brachyhypopomus pinnicaudatus is a biphasic waveform. The female's electric organ discharge is nearly symmetric but males produce a longer second phase than first phase. In this study, infrared-sensitive video cameras monitored the position of unrestrained fish, facilitating precise measurement of electric organ discharge duration and amplitude every 2 h for 24 h. Males (n=27) increased electric organ discharge duration by 37 ± 12% and amplitude by 24 ± 9% at night and decreased it during the day. In contrast, females (n=8) exhibited only minor electric organ discharge variation over time. Most of a male's increase occurred rapidly within the first 2–3 h of darkness. Electric organ discharge values gradually diminished during the second half of the dark period and into the next morning. Modulation of the second phase of the biphasic electric organ discharge produced most of the duration change in males, but both phases changed amplitude by similar amounts. Turning the lights off at mid-day triggered an immediate increase in electric organ discharge, suggesting modification of existing ion channels in the electric organ, rather than altered genomic expression. Exaggeration of electric organ discharge sex differences implies a social function. Daily reduction of duration and amplitude may reduce predation risk or energy expenditure. Accepted: 12 September 1998     

Electric organ discharges of the gymnotiform fishes: III. Brachyhypopomus

We measured and mapped the electric fields produced by three species of neotropical electric fish of the genus Brachyhypopomus (Gymnotiformes, Rham phichthyoidea, Hypopomidae), formerly Hypopomus. These species produce biphasic pulsed discharges from myogenic electric organs. Spatio-temporal false-color maps of the electric organ discharges measured on the skin show that the electric field is not a simple dipole in Brachyhypopomus. Instead, the dipole center moves rostro-caudally during the 1st phase (P1) of the electric organ discharge, and is stationary during the 2nd phase (P2). Except at the head and tip of tail, electric field lines rotate in the lateral and dorso-ventral planes. Rostro-caudal differences in field amplitude, field lines, and spatial stability suggest that different parts of the electric organ have undergone selection for different functions; the rostral portions seem specialized for electrosensory processing, whereas the caudal portions show adaptations for d.c. signal balancing and mate attraction as well. Computer animations of the electric field images described in this paper are available on web sites http://www.bbb.caltech.edu/ElectricFish or http://www.fiu.edu/∼stoddard/electricfish.html. Accepted: 22 September 1998     

The SV2 Protein of Synaptic Vesicles Is a Keratan Sulfate Proteoglycan

Abstract: We have determined that synaptic vesicles contain a vesicle-specific keratan sulfate integral membrane proteoglycan. This is a major proteoglycan in electric organ synaptic vesicles. It exists in two forms on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, i.e., the L form, which migrates like a protein with an M_r of 100, 000, and the H form, with a lower mobility that migrates with an M_r of ∼250, 000. Both forms contain SV2, an epitope located on the cytoplasmic side of the vesicle membrane. In addition to electric organ, we have analyzed the SV2 proteoglycan in vesicle fractions from two other sources, electric fish brain and rat brain. Both the H and L forms of SV2 are present in these vesicles and all are keratan sulfate proteoglycans. Unlike previously studied synaptic vesicle proteins, this proteoglycan contains a marker specific for a single group of neurons. This marker is an antigenically unique keratan sulfate side chain that is specific for the cells innervating the electric organ; it is not found on the synaptic vesicle keratan sulfate proteoglycan in other neurons of the electric fish brain.     

Social electric signals in freely moving dyads of <Emphasis Type="Italic">Brachyhypopomus pinnicaudatus</Emphasis>

Brachyhypopomus pinnicaudatus (pulse-type weakly electric fish) is a gregarious species that displays reproductive behavior and agonistic encounters between males only during the breeding season. During social interactions, in addition to its basal electric organ discharge (EOD), fish emit social electric signals (SESs) in the contexts of reproduction and intrasexual aggression. We reproduced natural behavior in laboratory settings: SESs recorded in the field are indistinguishable from those observed in our experimental setup. SESs are nocturnal, change seasonally and exhibit sexual dimorphism. This study provides an exhaustive characterization and classification of SESs produced by males and females during the breeding season. In male–female dyads, males produce accelerations and chirps while females interrupt their EODs. The same SESs are observed in male–male dyads. We present a novel, thorough classification of male chirps into four independent types (A, B, C, and M) based on their duration and internal structure. The type M chirp is only observed in male–male dyads. Chirps and interruptions, both in male–female and male–male dyads, are emitted in bouts, which are also grouped throughout the night. Our data suggest the existence of a sophisticated electric dialog during reproductive and aggressive interaction whose precise timing and behavioral significance are being investigated.     

Electrosensory modulation of escape responses

Once initiated, rapid escape responses of teleost fishes are thought to be completed without additional sensory modification. This suggests that the motor program for a particular response is selected for by the constellation of sensory cues existing at the time of the releasing stimulus. This paper presents initial evidence that a highly specialized, phylogenetically recent electrosensory system is integrated with a primitive motor system and allows an animal to continuously monitor its environment for producing accurate escape behaviors.Behavioral testing for directed startle responses in a Y-maze demonstrates that when presented immediately before an acoustic startle stimulus, electric fish (Eigenmannia virescens), direct their response away from the cue (a transient shorting of their electric field). Thus, electrosensory cues as brief as 100 ms provide directional information to the escape motor network.In electric fish that are curarized to facilitate intracellular recording, the normal electric organ discharge is attenuated. When an electronically generated replacement field of the same frequency and amplitude as the fish's normal signal is shorted, a fast-rising, 7 ms latency post-synaptic potential is evoked from the Mauthner cell. Similar PSPs are generated by turning the replacement stimulus on and off. In some recordings, removing the S1 replacement field elicits a rebound of other afferent activity to the Mauthner cell; replacing the field suppresses this activity.Abbreviations EHP extrinsic hyperpolarizing potential - EOD electric organ discharge - JAR jaming avoidance response - LED light emitting diode - PSP postsynaptic potential     

Region- and neuronal phenotype-specific expression of NELL2 in the adult rat brain

NELL2, a neural tissue-enriched protein, is produced in the embryo, and postembryonically in the mammalian brain, with a broad distribution. Although its synthesis is required for neuronal differentiation in chicks, not much is known about its function in the adult mammalian brain. We investigated the distribution of NELL2 in various regions of the adult rat brain to study its potential functions in brain physiology. Consistent with previous reports, NELL2-immunoreactivity (ir) was found in the cytoplasm of neurons, but not in glial fibrillary acidic protein (GFAP)-positive glial cells. The highest levels of NELL2 were detected in the hippocampus and the cerebellum. Interestingly, in the cerebellar cortex NELL2 was observed only in the GABAergic Purkinje cells not in the excitatory granular cells. In contrast, it was found mainly in the hippocampal dentate gyrus and pyramidal cell layer that contains mainly glutamatergic neurons. In the dentate gyrus, NELL2 was not detected in the GFAP-positive neural precursor cells, but was generally present in mature neurons of the subgranular zone, suggesting a role in this region restricted to mature neurons.     

Non-pyramidal neurons of layers I-III in the dog's cerebral cortex (parietal lobe). A Golgi survey

In an attempt to classify neurons in the upper layers of the cerebral cortex according to modern nomenclature based on Golgi impregnations, non-pyramidal neurons in layers II and III of the dog's cerebral cortex have been categorized into thirteen types: large double-bouquet cells with long ascending and descending axons (type I double-bouquet cells); bipolar neurons; multipolar neurons with long tufted descending axons (type II double-bouquet cells); neurons with long ascending axons; neurons with superficial axon plexuses; elongated large multipolar neurons with extended generalized axonal arborizations; neurons with long descending axons; small bi-tufted neurons with short ascending, descending or local axons; small multipolar neurons with short ascending, descending or local axons; multipolar neurons with local or extended axonal arborizations usually forming arcades (some of them also with a long descending axon); basket cells; neurogliaform neurons, and chandelier cells. Neurons in the molecular layer were horizontal cells and multipolar neurons with short axons. These data have been compared with those described in other species to provide a provisional classification of non-pyramidal neurons located in the upper layers of the cerebral cortex.     

The Glutamatergic Neurons in the Spinal Cord of the Sea Lamprey: An In Situ Hybridization and Immunohistochemical Study

Glutamate is the main excitatory neurotransmitter involved in spinal cord circuits in vertebrates, but in most groups the distribution of glutamatergic spinal neurons is still unknown. Lampreys have been extensively used as a model to investigate the neuronal circuits underlying locomotion. Glutamatergic circuits have been characterized on the basis of the excitatory responses elicited in postsynaptic neurons. However, the presence of glutamatergic neurochemical markers in spinal neurons has not been investigated. In this study, we report for the first time the expression of a vesicular glutamate transporter (VGLUT) in the spinal cord of the sea lamprey. We also study the distribution of glutamate in perikarya and fibers. The largest glutamatergic neurons found were the dorsal cells and caudal giant cells. Two additional VGLUT-positive gray matter populations, one dorsomedial consisting of small cells and another one lateral consisting of small and large cells were observed. Some cerebrospinal fluid-contacting cells also expressed VGLUT. In the white matter, some edge cells and some cells associated with giant axons (Müller and Mauthner axons) and the dorsolateral funiculus expressed VGLUT. Large lateral cells and the cells associated with reticulospinal axons are in a key position to receive descending inputs involved in the control of locomotion. We also compared the distribution of glutamate immunoreactivity with that of γ-aminobutyric acid (GABA) and glycine. Colocalization of glutamate and GABA or glycine was observed in some small spinal cells. These results confirm the glutamatergic nature of various neuronal populations, and reveal new small-celled glutamatergic populations, predicting that some glutamatergic neurons would exert complex actions on postsynaptic neurons.     

Sensory cues for the gradual frequency fall responses of the gymnotiform electric fish, Rhamphichthys rostratus

The sensory cues for a less known form of frequency shifting behavior, gradual frequency falls, of electric organ discharges (EODs) in a pulse-type gymnotiform electric fish, Rhamphichthys rostratus, were identified. We found that the gradual frequency fall occurs independently of more commonly observed momentary phase shifting behavior, and is due to perturbation of sensory feedback of the fish's own EODs by EODs of neighboring fish. The following components were identified as essential features in the signal mixture of the fish's own and the neighbor's EOD pulses: (1) the neighbor's pulses must be placed within a few millisecond of the fish's own pulses, (2) the neighbor's pulses, presented singly at low frequencies (0.2–4 Hz), were sufficient, (3) the frequency of individual pulse presentation must be below 4 Hz, (4) amplitude modulation of the sensory feedback of the fish's own pulses induced by such insertions of the neighbor's pulses must contain a high frequency component: sinusoidal amplitude modulation of the fish's own EOD feedback at these low frequencies does not induce gradual frequency falls. Differential stimulation across body surfaces, which is required for the jamming avoidance response (JAR) of wave-type gymnotiform electric fish, was not necessary for this behavior. We propose a cascade of high-pass and low-pass frequency filters within the amplitude processing pathway in the central nervous system as the mechanism of the gradual frequency fall response.Abbreviations EOD electric organ discharge - f frequency of EOD or pacemaker command signal - JAR jamming avoidance response - S ₁ stimulus mimicking fish's own EOD - f ₁ frequency of S₁ - S ₂ stimulus mimicking neighbor's EOD - f ₂ frequency of S₂