Steven J. Zottoli

Localization of optic tectal input to the ventral dendrite of the goldfish Mauthner cell

Although visually evoked Mauthner cell (M-cell) startle responses occur in the goldfish, the afferent projections underlying these reactions have not been previously studied. We have recorded from the M-cell while stimulating the left optic nerve and/or right optic tectum and have traced projections of the optic nerve and restricted areas of the optic tectum using HRP histochemistry and autoradiography. Tectal stimulation elicits similar postsynaptic potentials (PSPs) in both M-cells. The responses recorded in the right (ipsilateral) cell were localized to its ventral dendrite. The existence of uncrossed tectal projections to the ventral dendrite was confirmed morphologically following application of horseradish peroxidase (HRP) to the optic tectum. The PSPs contained both inhibitory and excitatory components, but with adequate stimulus strength, excitation of either M-cell dominated. Thus, this pathway is probably sufficient to trigger visually evoked startle responses mediated by the M-cell. Stimulation of the left optic nerve also evoked PSPs capable of bringing both M-cells to threshold. The blockage of this response by conditioning stimulation of the right tectum suggests that the visual information is relayed to the M-cells through this structure. In support of these findings, no label was found near any portion of the M-cell after either intraocular injection of tritiated proline or application of HRP to the cut end of the optic nerve. In summary, visual input to the M-cell is mediated via projections from the tectum, is segregated onto the ventral dendrite, and is capable of bringing this neuron to threshold. This pathway presumably accounts for the demonstrated behavioral efficacy of visual stimuli in evoking a startle response.

■ Review : The Mauthner Cell: What Has it Taught us?

The Mauthner cell (M-cell) is one of the few identifiable neurons in the vertebrate central nervous system. The ability to locate the M-cell, along with its inputs and outputs, has resulted in important findings in diverse areas of neurobiology including the molecular biology of neurons, synaptic and systems physiology, behavior, development, and neuroethology. The review provides a brief overview of the M-cell and then focuses on recent studies applying state-of-the-art techniques to address new issues and revisit old ones. One advantage of this preparation is the ability to conduct multidisciplinary studies from the subcellular to behavioral levels. For example, studies of activity-dependent changes in the strength of mixed electrotonic and chemical synapses on the M-cells lateral dendrite in vivo have been correlated with changes in the probability of eliciting a fast startle response initiated by the M-cell and its associated circuits. Similarly, it is now possible to image the activity of the M-cell and its homologs while observing motor behavior in zebrafish larvae. These approaches will provide direct tests of the functional properties of complex neural networks. Moreover, molecular mechanisms that underlie neuronal development can be tested directly with this neuron and its segmental homologs, because these cells occur in singular pairs at defined locations. Finally, after spinal cord injury, the M-cells axon regenerates, but does not follow its original course, and the startle response gradually recovers. The accessibility of the M-cell system offers the promise that strategies employed in restoring the function of a neural network will be revealed. Thus, we anticipate that the M-cell system will become a favored preparation for multidisciplinary studies on the neuronal basis of behavior and the recovery of behavior after injury. NEUROSCIENTIST 6:26-38, 2000

Correlation of C-start behaviors with neural activity recorded from the hindbrain in free-swimming goldfish (Carassius auratus).

SUMMARY Startle behaviors in teleost fishes are well suited for investigations of mechanisms of sensorimotor integration because the behavior is quantifiable and much of the underlying circuitry has been identified. The teleost C-start is triggered by an action potential in one of the two Mauthner (M) cells. To correlate C-start behavior with electrophysiology, extracellular recordings were obtained from the surface of the medulla oblongata in the hindbrain, close to the M-axons, in freely swimming goldfish monitored using high-speed video. The recordings included action potentials generated by the two M-axons, as well as neighboring axons in the dorsal medial longitudinal fasciculus. Axonal backfills indicated that the latter originate from identifiable reticulospinal somata in rhombomeres 2-8 and local interneurons. Diverse auditory and visual stimuli evoked behaviors with kinematics characteristic of the C-start, and the amplitude of the first component of the hindbrain field potential correlated with the C-start direction. The onset of the field potential preceded that of the simultaneously recorded trunk EMG and movement initiation by 1.08±0.04 and 8.13±0.17 ms, respectively. A subsequent longer latency field potential was predictive of a counterturn. These results indicate that characteristic features of the C-start can be extracted from the neural activity of the M-cell and a population of other reticulospinal neurons in free-swimming goldfish.

Comparative Studies on the Mauthner Cell of Teleost Fish in Relation to Sensory Input

Most physiological and behavioral studies of the Mauthner cells have used the goldfish and a few other fish from the superorder Ostariophysi, series Otophysi (= otophysans). We first provide some background and recent findings on the Mauthner cells of otophysan fish and then compare this information to that known about the Mauthner cells in certain non-otophysan fish. These comparisons are meant to provide the impetus for a comparative approach to understanding the role of the Mauthner cells in behavior.

Comparison of Acetylcholinesterase and Choline Acetyltransferase Staining Patterns in the Optic Tectum of the Goldfish Carassius auratus

Although the optic tectum of nonmammalian vertebrates has been extensively studied anatomically, there is little information about the identification of neurotransmitters and the enzymes critical to their synthesis. Choline acetyltransferase (ChAT), the enzyme responsible for acetylcholine synthesis, is presently regarded as the most reliable marker for cholinergic neurons, and its localization within putative cholinergic neurons has been made possible by the development of antibodies specific to ChAT. We have compared the immunocytochemical localization of ChAT to the histochemical staining of acetylcholinesterase (AChE) in the goldfish optic tectum. Goldfish brains reacted with the monoclonal antibody AB8 to ChAT have revealed that: (1) type XIV neurons are the only ChAT-positive cells in the tectum, and there are approximately 15,000 such cells per tectal hemisphere; (2) these neurons and other ChAT-containing afferent fibers form bands of label which correspond to those seen after AChE staining, and (3) many AChE-stained neurons do not contain ChAT. The immunohistochemical localization of ChAT has provided a direct method for determining the localization and organization of putative cholinergic structures in the optic tectum of goldfish. Future studies may elucidate the relationship of these cholinergic systems to the retinotectal projections, as there is close correspondence between AChE and ChAT location and the retinotectal termination patterns.

Chapter 18 Spinal cord regeneration in adult goldfish: implications for functional recovery in vertebrates

Publisher Summary Many central nervous system (CNS) neurons damaged by spinomedullary level (SML) crush in adult goldfish extend sprouts that choose a peripheral nervous system (PNS) pathway. This chapter gives suggestion that this inappropriate choice delays, impairs, or prevents recovery of behaviors, including equilibrium and C-starts. If this pathway choice is responsible for the limited regenerative capacity, it may also explain limited behavioral recovery in nonmammalian and mammalian vertebrates alike. The goldfish PNS may present a more permissive environment to regenerating fibers than the CNS after SML crush. Such a pathway preference demonstrates a parallel to mammalian systems and makes the goldfish more valuable as a model system for regeneration. While advances have been made in techniques that allow mammalian CNS neurons to regenerate over distances longer than 1 mm, such as peripheral nerve implants, conditioned media, and antibodies that neutralize inhibitory substances, there is no guarantee that regenerating sprouts will make appropriate pathway choices, that behavioral recovery will result in the return of function or that neurons normally involved in a behavior will contribute equally to the recovery. The adult goldfish nervous system provides an exciting model to address these issues.

Comparison of Fast Startle Responses between Two Elongate Bony Fish with an Anguilliform Type of Locomotion, the African Lungfish, Protopterus annectens, and the American Eel, Anguilla rostrata, and the Implications for the Underlying Neuronal Basis of Escape Behavior

A comparative anatomical and behavioral approach was used to elucidate the role of medullary networks associated with Mauthner cells (M-cells) in determining the type of fast startle responses of elongate bony fish with an anguilliform type of locomotion. The M-cell initiates fast startle responses in goldfish and is thought to initiate such responses in other fish as well [Wilson, 1959; Eaton et al., 1977; Currie and Carlsen, 1987; Currie, 1991]. Goldfish M-cells have a specialized structure, termed an axon cap, that surrounds their axon hillock-initial segment region and is critical for the control of M-cell excitability. The M-cell axon cap is present in some elongate fish such as the African lungfish (Protopterus, Lepidosireniformes) and absent in others such as the American eel (Anguilla, Anguilliformes). The lungfish startle response is characterized by a rotation of the head and upper trunk, similar to the initial phase of the goldfish (Carassius, Cypriniformes) startle response. The American eel, on the other hand, displays a withdrawal-type startle response. We hypothesize that the withdrawal-type startle response of the American eel results from the absence of inhibitory networks associated with the M-cell axon cap. The correlation of the absence of an axon cap and a withdrawal-type startle response may be a general feature of all Anguilliformes and elongate ray-finned fish with an anguilliform type of locomotion.

Localization of choline acetyltransferase to somata of posterior lateral line efferents in the goldfish

The somata of posterior lateral line efferents in goldfish have been identified by retrograde transport of horseradish peroxidase. Co-localization of retrogradely transported horseradish peroxidase and choline acetyltransferase, detected by immunohistochemical staining with the monoclonal antibody AB8, supports the view that some lateral line efferent neurons in the goldfish are cholinergic.

Choline acetyltransferase immunohistochemical staining in the goldfish (Carassius auratus) brain: Evidence that the Mauthner cell does not contain choline acetyltransferase

In the hatchetfish, the Mauthner cell (M-cell) is thought to be cholinergic based on electrophysiological studies using cholinergic agents and on the localization of acetylcholinesterase (AChE) and alpha-bungarotoxin to M-cell-giant fiber synapses. Immunocytochemical studies have shown that mammalian and non-mammalian cholinergic neurons stain positive for choline acetyltransferase (ChAT), the enzyme responsible for synthesizing acetylcholine. We processed tissue from the goldfish (Carassius auratus) for the immunohistochemical detection of ChAT using the monoclonal antibody AB8 and the peroxidase-antiperoxidase procedure. ChAT immunoreactivity was found in selected areas of the goldfish brain including the cranial nerve nuclei and the ventral horn motoneurons of the spinal cord. Interestingly, the M-cell soma which stains positive for AChE was ChAT negative. This immunohistochemical evidence does not support cholinergic functioning of the Mauthner cell.

Evolution of the Mauthner axon cap.

Studies of vertebrate brain evolution have focused primarily on patterns of gene expression or changes in size and organization of major brain regions. The Mauthner cell, an important reticulospinal neuron that functions in the startle response of many species, provides an opportunity for evolutionary comparisons at the cellular level. Despite broad interspecific similarities in Mauthner cell morphology, the motor patterns and startle behaviors it initiates vary markedly. Response diversity has been hypothesized to result, in part, from differences in the structure and function of the Mauthner cell-associated axon cap. We used light microscopy techniques to compare axon cap morphology across a wide range of species, including all four extant basal actinopterygian orders, representatives of a variety of teleost lineages and lungfishes, and we combined our data with published descriptions of axon cap structure. The ‘composite’ axon cap, observed in teleosts, is an organized conglomeration of glia and fibers of inhibitory and excitatory interneurons. Lungfish, amphibian tadpoles and several basal actinopterygian fishes have ‘simple’ axon caps that appear to lack glia and include few fibers. Several other basal actinopterygian fishes have ‘simple-dense’ caps that include greater numbers of fibers than simple caps, but lack the additional elements and organization of composite caps. Phylogenetic mapping shows that through evolution there are discrete transitions in axon cap morphology occurring at the base of gnathostomes, within basal actinopterygians, and at the base of the teleost radiation. Comparing axon cap evolution to the evolution of startle behavior and motor pattern provides insight into the relationship between Mauthner cell-associated structures and their functions in behavior.