Distribution of the cholinergic nuclei in the brain of the weakly electric fish, Apteronotus leptorhynchus: Implications for sensory processing.

Acetylcholine acts as a neurotransmitter/neuromodulator of many central nervous system processes such as learning and memory, attention, motor control, and sensory processing. The present study describes the spatial distribution of cholinergic neurons throughout the brain of the weakly electric fish, Apteronotus leptorhynchus, using in situ hybridization of choline acetyltransferase mRNA. Distinct groups of cholinergic cells were observed in the telencephalon, diencephalon, mesencephalon, and hindbrain. These included cholinergic cell groups typically identified in other vertebrate brains, for example, motor neurons. Using both in vitro and ex vivo neuronal tracing methods, we identified two new cholinergic connections leading to novel hypotheses on their functional significance. Projections to the nucleus praeeminentialis (nP) arise from isthmic nuclei, possibly including the nucleus lateralis valvulae (nLV) and the isthmic nucleus (nI). The nP is a central component of all electrosensory feedback pathways to the electrosensory lateral line lobe (ELL). We have previously shown that some neurons in nP, TS, and tectum express muscarinic receptors. We hypothesize that, based on nLV/nI cell responses in other teleosts and isthmic connectivity in A. leptorhynchus, the isthmic connections to nP, TS, and tectum modulate responses to electrosensory and/or visual motion and, in particular, to looming/receding stimuli. In addition, we found that the octavolateral efferent (OE) nucleus is the likely source of cholinergic fibers innervating the ELL. In other teleosts, OE inhibits octavolateral hair cells during locomotion. In gymnotiform fish, OE may also act on the first central processing stage and, we hypothesize, implement corollary discharge modulation of electrosensory processing during locomotion.

Cellular and Network Mechanisms May Generate Sparse Coding of Sequential Object Encounters in Hippocampal-Like Circuits.

The localization of distinct landmarks plays a crucial role in encoding new spatial memories. In mammals, this function is performed by hippocampal neurons that sparsely encode an animal's location relative to surrounding objects. Similarly, the dorsolateral pallium (DL) is essential for spatial learning in teleost fish. The DL of weakly electric gymnotiform fish receives both electrosensory and visual input from the preglomerular nucleus (PG), which has been hypothesized to encode the temporal sequence of electrosensory or visual landmark/food encounters. Here, we show that DL neurons in the Apteronotid fish and in the Carassius auratus (goldfish) have a hyperpolarized resting membrane potential (RMP) combined with a high and dynamic spike threshold that increases following each spike. Current-evoked spikes in DL cells are followed by a strong small-conductance calcium-activated potassium channel (SK)-mediated after-hyperpolarizing potential (AHP). Together, these properties prevent high frequency and continuous spiking. The resulting sparseness of discharge and dynamic threshold suggest that DL neurons meet theoretical requirements for generating spatial memory engrams by decoding the landmark/food encounter sequences encoded by PG neurons. Thus, DL neurons in teleost fish may provide a promising, simple system to study the core cell and network mechanisms underlying spatial memory.

A time-stamp mechanism may provide temporal information necessary for egocentric to allocentric spatial transformations.

Learning the spatial organization of the environment is essential for most animals' survival. This requires the animal to derive allocentric spatial information from egocentric sensory and motor experience. The neural mechanisms underlying this transformation are mostly unknown. We addressed this problem in electric fish, which can precisely navigate in complete darkness and whose brain circuitry is relatively simple. We conducted the first neural recordings in the preglomerular complex, the thalamic region exclusively connecting the optic tectum with the spatial learning circuits in the dorsolateral pallium. While tectal topographic information was mostly eliminated in preglomerular neurons, the time-intervals between object encounters were precisely encoded. We show that this reliable temporal information, combined with a speed signal, can permit accurate estimation of the distance between encounters, a necessary component of path-integration that enables computing allocentric spatial relations. Our results suggest that similar mechanisms are involved in sequential spatial learning in all vertebrates.

Hippocampal-like circuitry in the pallium of an electric fish: Possible substrates for recursive pattern separation and completion.

Teleost fish are capable of complex behaviors, including social and spatial learning; lesion studies show that these abilities require dorsal telencephalon (pallium). The teleost telencephalon has subpallial and pallial components. The subpallium is well described and highly conserved. In contrast, the teleost pallium is not well understood and its relation to that of other vertebrates remains controversial. Here we analyze the connectivity of the subdivisions of dorsal pallium (DD) of an electric gymnotiform fish, Apteronotus leptorhynchus: superficial (DDs), intermediate (DDi) and magnocellular (DDmg) components. The major pathways are recursive: the dorsolateral pallium (DL) projects strongly to DDi, with lesser inputs to DDs and DDmg. DDi in turn projects strongly to DDmg, which then feeds back diffusely to DL. Our quantitative analysis of DDi connectivity demonstrates that it is a global recurrent network. In addition, we show that the DD subnuclei have complex reciprocal connections with subpallial regions. Specifically, both DDi and DDmg are reciprocally connected to pallial interneurons within the misnamed rostral entopeduncular nucleus (Er). Based on DD connectivity, we illustrate the close similarity, and possible homology, between hippocampal and DD/DL circuitry. We hypothesize that DD/DL circuitry can implement the same pattern separation and completion computations ascribed to the hippocampal dentate gyrus and CA3 fields. We further contend that the DL to DDi to DDmg to DL feedback loop makes the pattern separation/completion operations recursive. We discuss our results with respect to recent studies on fear avoidance conditioning in zebrafish and attention and spatial learning in a pulse gymnotiform fish. J. Comp. Neurol. 525:8-46, 2017. © 2016 Wiley Periodicals, Inc.

Subsecond Sensory Modulation of Serotonin Levels in a Primary Sensory Area and Its Relation to Ongoing Communication Behavior in a Weakly Electric Fish.

Serotonergic neurons of the raphe nuclei of vertebrates project to most regions of the brain and are known to significantly affect sensory processing. The subsecond dynamics of sensory modulation of serotonin levels and its relation to behavior, however, remain unknown. We used fast-scan cyclic voltammetry to measure serotonin release in the electrosensory system of weakly electric fish, Apteronotus leptorhynchus. These fish use an electric organ to generate a quasi-sinusoidal electric field for communicating with conspecifics. In response to conspecific signals, they frequently produce signal modulations called chirps. We measured changes in serotonin concentration in the hindbrain electrosensory lobe (ELL) with a resolution of 0.1 s concurrently with chirping behavior evoked by mimics of conspecific electric signals. We show that serotonin release can occur phase locked to stimulus onset as well as spontaneously in the ELL region responsible for processing these signals. Intense auditory stimuli, on the other hand, do not modulate serotonin levels in this region, suggesting modality specificity. We found no significant correlation between serotonin release and chirp production on a trial-by-trial basis. However, on average, in the trials where the fish chirped, there was a reduction in serotonin release in response to stimuli mimicking similar-sized same-sex conspecifics. We hypothesize that the serotonergic system is part of an intricate sensory-motor loop: serotonin release in a sensory area is triggered by sensory input, giving rise to motor output, which can in turn affect serotonin release at the timescale of the ongoing sensory experience and in a context-dependent manner.

Cryptic laminar and columnar organization in the dorsolateral pallium of a weakly electric fish.

In the weakly electric gymnotiform fish, Apteronotus leptorhynchus, the dorsolateral pallium (DL) receives diencephalic inputs representing electrosensory input utilized for communication and navigation. Cell counts reveal that, similar to thalamocortical projections, many more cells are present in DL than in the diencephalic nucleus that provides it with sensory input. DL is implicated in learning and memory and considered homologous to medial and/or dorsal pallium. The gymnotiform DL has an apparently simple architecture with a random distribution of simple multipolar neurons. We used multiple neurotracer injections in order to study the microcircuitry of DL. Surprisingly, we demonstrated that the intrinsic connectivity of DL is highly organized. It consists of orthogonal laminar and vertical excitatory synaptic connections. The laminar synaptic connections are symmetric sparse, random, and drop off exponentially with distance; they parcellate DL into narrow (60 µm) overlapping cryptic layers. At distances greater than 100 µm, the laminar connections generate a strongly connected directed graph architecture within DL. The vertical connectivity suggests that DL is also organized into cryptic columns; these connections are highly asymmetric, with superficial DL cells preferentially projecting towards deeper cells. Our experimental analyses suggest that the overlapping cryptic columns have a width of 100 µm, in agreement with the minimal distance for strong connectivity. The architecture of DL and the expansive representation of its input, taken together with the strong expression of N-methyl-D-aspartate (NMDA) receptors by its cells, are consistent with theoretical ideas concerning the cortical computations of pattern separation and memory storage via bump attractors.

Sex-specific role of a glutamate receptor subtype in a pacemaker nucleus controlling electric behavior.

Electric communication signals, produced by South American electric fish, vary across sexes and species and present an ideal opportunity to examine the bases of signal diversity, and in particular, the mechanisms underlying sexually dimorphic behavior. Gymnotiforms produce electric organ discharges (EOD) controlled by a hindbrain pacemaker nucleus (PN). Background studies have identified the general cellular mechanisms that underlie the production of communication signals, EOD chirps and interruptions, typically displayed in courtship and agonistic contexts. Brachyhypopomus gauderio emit sexually dimorphic signals, and recent studies have shown that the PN acquires the capability of generating chirps seasonally, only in breeding males, by modifying its glutamatergic system. We hypothesized that sexual dimorphism was caused by sexual differences in the roles of glutamate receptors. To test this hypothesis, we analyzed NMDA and AMPA mediated responses in PN slice preparations by field potential recordings, and quantified one AMPA subunit mRNA, in the PNs of males and females during the breeding season. In situ hybridization of GluR2B showed no sexual differences in quantities between the male and female PN. Functional responses of the PN to glutamate and AMPA, on the other hand, showed a clear cut sexual dimorphism. In breeding males, but not females, the PN responded to glutamate and AMPA with bursting activity, with a temporal pattern that resembled the pattern of EOD chirps. In this study, we have been successful in identifying cellular mechanisms of sexual dimorphic communication signals. The involvement of AMPA receptors in PN activity is part of the tightly regulated changes that account for the increase in signal diversity during breeding in this species, necessary for a successful reproduction.

Dendritic SK channels convert NMDA-R-dependent LTD to burst timing-dependent plasticity.

Feedback and descending projections from higher to lower brain centers play a prominent role in all vertebrate sensory systems. Feedback might be optimized for the specific sensory processing tasks in their target brain centers, but it has been difficult to connect the properties of feedback synapses to sensory tasks. Here, we use the electrosensory system of a gymnotiform fish (Apteronotus leptorhynchus) to address this problem. Cerebellar feedback to pyramidal cells in the first central electrosensory processing region, the electrosensory lateral line lobe (ELL), is critical for canceling spatially and temporally redundant electrosensory input. The ELL contains four electrosensory maps, and we have previously analyzed the synaptic and network bases of the redundancy reduction mechanism in a map (centrolateral segment; CLS) believed to guide electrolocation behavior. In the CLS, only long-term depression was induced by pairing feedback presynaptic and pyramidal cell postsynaptic bursts. In this paper, we turn to an ELL map (lateral segment; LS) known to encode electrocommunication signals. We find remarkable differences in synaptic plasticity of the morphologically identical cerebellar feedback input to the LS. In the LS, pyramidal cell SK channels permit long-term potentiation (LTP) of feedback synapses when pre- and postsynaptic bursts occur at the same time. We hypothesize that LTP in this map is required for enhancing the encoding of weak electrocommunication signals. We conclude that feedback inputs that appear morphologically identical in sensory maps dedicated to different tasks, nevertheless display different synaptic plasticity rules contributing to differential sensory processing in these maps.

Expression of the cannabinoid CB1 receptor in the gymnotiform fish brain and its implications for the organization of the teleost pallium.

Cannabinoid CB1 receptors (CB1R) are widely distributed in the brains of many vertebrates, but whether their functions are conserved is unknown. The weakly electric fish, Apteronotus leptorhynchus (Apt), has been well studied for its brain structure, behavior, sensory processing, and learning and memory. It therefore offers an attractive model for comparative studies of CB1R functions. We sequenced partial AptCB1R mRNAs and performed in situ hybridization to localize its expression. Partial AptCB1R protein sequence was highly conserved to zebrafish (90.7%) and mouse (81.9%) orthologs. AptCB1R mRNA was highly expressed in the telencephalon. Subpallial neurons (dorsal, central, intermediate regions and part of the ventral region, Vd/Vc/Vi, and Vv) expressed high levels of AptCB1R transcript. The central region of dorsocentral telencephalon (DC(core) ) strongly expressed CB1R mRNA; cells in DC(core) project to midbrain regions involved in electrosensory/visual function. The lateral and rostral regions of DC surrounding DC(core) (DC(shell) ) lack AptCB1R mRNA. The rostral division of the dorsomedial telencephalon (DM1) highly expresses AptCB1R mRNA. In dorsolateral division (DL) AptCB1R mRNA was expressed in a gradient that declined in a rostrocaudal manner. In diencephalon, AptCB1R RNA probe weakly stained the central-posterior (CP) and prepacemaker (PPn) nuclei. In mesencephalon, AptCB1R mRNA is expressed in deep layers of the dorsal (electrosensory) torus semicircularis (TSd). In hindbrain, AptCB1R RNA probe weakly labeled inhibitory interneurons in the electrosensory lateral line lobe (ELL). Unlike mammals, only few cerebellar granule cells expressed AptCB1R transcripts and these were located in the center of eminentia granularis pars posterior (EGp), a cerebellar region involved in feedback to ELL.

Organization of the gymnotiform fish pallium in relation to learning and memory: IV. Expression of conserved transcription factors and implications for the evolution of dorsal telencephalon.

We have cloned the apteronotid homologs of FoxP2, Otx1, and FoxO3. There was, in the case of all three genes, good similarity between the apteronotid and human amino acid sequences: FoxP2, 78%; Otx1, 54%; FoxO3, 71%. The functional domains of these genes were conserved to a far greater extent, on average: FoxP2, 89%; Otx1, 76%; FoxO3, 82%. This led us to hypothesize that the cellular functions of these genes might also be conserved. We used in situ hybridization to examine the distribution of the mRNA transcripts of these genes in the apteronotid telencephalon. We confined our analysis to the pallial regions previously associated with learning about social signals, whose circuitry has been closely examined in the other articles of this series. We found that AptFoxP2 and AptOtx1 transcripts were expressed predominantly in the dorsocentral division of the pallium (DC); the dorsolateral division of the pallium (DL) contained only weakly labeled neurons. In both cases, the distribution of labeled neurons was very heterogeneous, and unlabeled neurons could be found adjacent to strongly labeled ones. In contrast, we found that most neurons in DL strongly expressed AptFoxO3 mRNA, although there was only weak expression in a small number of cells within DC. We briefly discuss the relevance of our results regarding the functional roles of AptFoxP2/AptOtx1-expressing neurons in DC for communication vs. foraging behavior. We extensively discuss the implications of our results for possible homologies between DL and DC and medial and dorsal pallium of tetrapods, respectively.

Organization of the gymnotiform fish pallium in relation to learning and memory: I. Cytoarchitectonics and cellular morphology.

The present article examines the anatomical organization of the dorsal telencephalon of two gymnotiform fish: Gymnotus sp. and Apteronotus leptorhynchus. These electric fish use elaborate electrical displays for agonistic and sexual communication. Our study emphasizes mainly pallial divisions: dorsolateral (DL), dorsodorsal (DD), and dorsocentral (DC), previously implicated in social learning dependent on electric signals. We found that the pallial cytoarchitectonics of gymnotiformes are similar to those reported for the commonly studied goldfish, except that DC is larger and better differentiated in gymnotiformes. We identified a new telencephalic region (Dx), located between DL and DC, and describe the morphological and some biochemical properties of its neurons. Most neurons in DL, DD, and DC are glutamatergic with spiny dendrites. However, the size of these cells as well as the orientation and extent of their dendrites vary systematically across these regions. In addition, both DD and DL contained numerous small GABAergic interneurons as well as well-developed GABAergic plexuses. One important and novel observation is that the dendrites of the spiny neurons within all three regions remain confined to their respective territories. We confirm that DL and DC express very high levels of NMDA receptor subunits as well as CaMKIIα, a key downstream effector of this receptor. In contrast, this enzyme is nearly absent in DD, while NMDA receptors are robustly expressed, suggesting different rules for synaptic plasticity across these regions. Remarkably, GABAergic pallial neurons do not express CaMKIIα, in agreement with previously reported results in the cortex of rats.

Frequency-tuned cerebellar channels and burst-induced LTD lead to the cancellation of redundant sensory inputs.

For optimal sensory processing, neural circuits must extract novel, unpredictable signals from the redundant sensory input in which they are embedded, but the detailed cellular and network mechanisms that implement such selective cancellation are presently unknown. Using a combination of modeling and experiment, we characterize in detail a cerebellar circuit in weakly electric fish, showing how it can carry out this computation. We use a model incorporating the wide range of experimentally estimated parallel fiber feedback delays and a burst-induced LTD rule derived from in vitro experiments to explain the precise cancellation of redundant signals observed in vivo. Our model demonstrates how the backpropagation-dependent burst dynamics adjusts the temporal pairing width of the plasticity mechanism to precisely match the frequency of the redundant signal. The model also makes the prediction that this cerebellar feedback pathway must be composed of frequency-tuned channels; this prediction is subsequently verified in vivo, highlighting a novel and general capability of cerebellar circuitry.

Long-term recognition memory of individual conspecifics is associated with telencephalic expression of Egr-1 in the electric fish Apteronotus leptorhynchus.

Primates and songbirds can learn to recognize individual conspecifics based on complex sensory cues; this requires a large, highly differentiated dorsal telencephalon. Here we show that the electric fish Apteronotus leptorhynchus can learn to recognize individual conspecifics based on a simple cue, the beat frequency of their summed sinusoidal electric organ discharges (EOD). Male fish produce transient communication signals (chirps) in response to mimic EODs. The chirp response habituates over repeated stimulus presentations within one experimental session, continues to habituate over successive daily sessions and is nearly extinguished after 5-7 days. Habituation of the chirp response was specific to the presented beat frequency. The conversion of short- to long-term habituation could be disrupted by cooling the head 30 minutes after the daily habituation trials. Consolidation of long-term memory in mammals is thought to involve induced expression of an immediate early gene, Egr-1. We cloned the Apteronotid homolog of the Egr-1 gene and found that chirp-evoking stimuli induced strong expression of its mRNA within the dorsal (Dd), central (DC), and lateral (DL) subdivisions of the dorsal telencephalon. Interestingly, the dorsolateral region is hypothesized to be homologous to the amniote hippocampal formation. We conclude that A. leptorhynchus can learn to identify individual conspecifics based on their EOD frequency and can remember these frequencies for several days. We hypothesize that this form of learning, as in primates and songbirds, requires a subset of dorsal telencephalic areas and involves a consolidation-like process that includes the expression of the transcription factor AptEgr-1.

Burst-induced anti-Hebbian depression acts through short-term synaptic dynamics to cancel redundant sensory signals.

Weakly electric fish can enhance the detection and localization of important signals such as those of prey in part by cancellation of redundant spatially diffuse electric signals due to, e.g., their tail bending. The cancellation mechanism is based on descending input, conveyed by parallel fibers emanating from cerebellar granule cells, that produces a negative image of the global low-frequency signals in pyramidal cells within the first-order electrosensory region, the electrosensory lateral line lobe (ELL). Here we demonstrate that the parallel fiber synaptic input to ELL pyramidal cell undergoes long-term depression (LTD) whenever both parallel fiber afferents and their target cells are stimulated to produce paired burst discharges. Paired large bursts (4-4) induce robust LTD over pre-post delays of up to +/-50 ms, whereas smaller bursts (2-2) induce weaker LTD. Single spikes (either presynaptic or postsynaptic) paired with bursts did not induce LTD. Tetanic presynaptic stimulation was also ineffective in inducing LTD. Thus, we have demonstrated a form of anti-Hebbian LTD that depends on the temporal correlation of burst discharge. We then demonstrated that the burst-induced LTD is postsynaptic and requires the NR2B subunit of the NMDA receptor, elevation of postsynaptic Ca(2+), and activation of CaMKIIbeta. A model incorporating local inhibitory circuitry and previously identified short-term presynaptic potentiation of the parallel fiber synapses further suggests that the combination of burst-induced LTD, presynaptic potentiation, and local inhibition may be sufficient to explain the generation of the negative image and cancellation of redundant sensory input by ELL pyramidal cells.

Regulated expression of N-methyl-D-aspartate receptors and associated proteins in teleost electrosensory system and telencephalon.

Several types of N-methyl-D-aspartate (NMDA) receptor-dependent synaptic plasticity are characterized by differences in polarity, induction parameters, and duration, which depend on the interactions of NMDARs with intracellular synaptic and signaling proteins. Here, we examine the NMDAR signaling components in the brain of the weakly electric fish Apteronotus leptorhynchus. Compared with mammalian orthologs, high levels of sequence conservation for known functional sites in both NMDAR subunits (NR1, NR2A-C) and signaling proteins (fyn tyrosine kinase, RasGRF-1 and -2) were found. In situ hybridization analysis demonstrated that, similar to the case in the adult mammal brain, NR2A and NR2B are expressed at moderate levels in most brain regions and at very high levels in the dorsal telencephalon. RasGRF-1 and fyn have a similar distribution and appear to be coexpressed with NR2B in telencephalic regions known to support learning and long-term memory. Both NR2A and NR2B are highly expressed in pyramidal cells of the electrosensory lateral line lobe (ELL) known to exhibit the short-term synaptic plasticity that underlies adaptive feedback cancellation of redundant sensory input. In contrast, nonplastic pyramidal cells expressed only the NR2A subunit. Furthermore, field recordings show that ifenprodil-sensitive NR2B-containing NMDARs predominate for the plastic feedback input to ELL pyramidal cells. However, RasGRF-1 and fyn are expressed only at low levels in a subset of these pyramidal cells. Our data suggest that NMDAR functions are highly conserved between fish and mammals and that synaptic plasticity dynamics in different brain regions are related to the expression patterns of the synaptic signaling proteins interacting with NMDARs.

SK channels provide a novel mechanism for the control of frequency tuning in electrosensory neurons.

One important characteristic of sensory input is frequency, with sensory neurons often tuned to narrow stimulus frequency ranges. Although vital for many neural computations, the cellular basis of such frequency tuning remains mostly unknown. In the electrosensory system of Apteronotus leptorhynchus, the primary processing of important environmental and communication signals occurs in pyramidal neurons of the electrosensory lateral line lobe. Spike trains transmitted by these cells can encode low-frequency prey stimuli with bursts of spikes and high-frequency communication signals with single spikes. Here, we demonstrate that the selective expression of SK2 channels in a subset of pyramidal neurons reduces their response to low-frequency stimuli by opposing their burst responses. Apamin block of the SK2 current in this subset of cells induced bursting and increased their response to low-frequency inputs. SK channel expression thus provides an intrinsic mechanism that predisposes a neuron to respond to higher frequencies and thus specific, behaviorally relevant stimuli.

Excitatory amino acid receptors of the electrosensory system: the NR1/NR2B N-methyl-D-aspartate receptor.

The amino acid sequence of the N-methyl-D-aspartate (NMDA) receptor subunit NR2B from the brown ghost knife fish Apteronotus leptorhynchus has been determined and compared with the sequence of the murine NR2B. This comparison revealed high levels of sequence conservation throughout the ligand binding and membrane spanning segments. The functional properties of the NR1 and NR2B receptor complex were examined by coexpression in HEK cells. The recombinant AptNR1/NR2B receptors produced robust currents after stimulation with glutamate or NMDA in the presence of glycine. Measurements of the concentration dependencies for these agonists indicated that the agonist binding sites on the apteronotid receptor are highly conserved, with nearly identical agonist affinities to those of the murine NR1/NR2B receptor. The kinetic responses of the fish receptor were also highly conserved, with deactivation rates for the AptNR2B receptor matching those of the murine NR2B containing receptor. Evidently, most of the unique functional properties that reside in the NR2B receptor subunit have been well conserved in teleost NMDA receptors. On the other hand, the apteronitid receptor displayed a lowered sensitivity to voltage-dependent Mg(2+) block and a reduced affinity for the NR2B-specific noncompetitive antagonist ifenprodil. We conclude that the functional properties that result from the incorporation of the NR2B receptor in the NMDA receptor complex have been maintained since the evolutionary divergence of teleost and mammalian organisms.