Distinct startle responses are associated with neuroanatomical differences in pufferfishes.

Despite the key function of the Mauthner cells (M-cells) in initiating escape responses and thereby promoting survival, there are multiple examples of M-cell loss across the teleost phylogeny. Only a few studies have directly considered the behavioral consequences of naturally occurring M-cell variation across species. We chose to examine this issue in pufferfishes, as previous research suggested that there might be variability in M-cell anatomy in this group of fish. We characterized the M-cell anatomy and fast-start responses of two pufferfish species, Tetraodon nigroviridis and Diodon holocanthus. T. nigroviridis showed robust fast-starts to both tactile and acoustic startling stimuli. These fast-starts occurred with a latency typical of M-cell initiation in other fish, and retrograde labeling of spinal-projection neurons revealed that T. nigroviridis does have M-cells. By contrast, D. holocanthus only rarely exhibited fast-start-like behavior, and these responses were at a substantially longer latency and were much less extensive than those of T. nigroviridis. Using three complementary anatomical techniques we were unable to identify obvious M-cell candidates in D. holocanthus. These results provide a clear correlation between M-cell presence or absence and dramatic differences in fast-start behavior. The rich diversity within the pufferfish clade should allow future studies investigating the factors that contribute to this correlated anatomical and behavioral variation.

Comparison of Mauthner cell size in teleosts.

The Mauthner cells (M-cells) are a pair of neurons found in the medulla oblongata of most fish and amphibians. This neuron is not the same size in all teleosts. Based on the hypothesis that differences in M-cell size might be related to fish family and possibly fish habitat, M-cell and nuclear size were compared between fish families. In order to minimize effects of fish length on cell or nuclear size, statistical treatment of the data was made for four separate length classes. The results indicate at least three groupings by cell or nuclear size. The cell is large in fish from the Salmonidae, Catostomidae and Cyprinidae, small in the Stichaeidae, Cottidae and Pleuronectidae and absent in Batrachoididae, Lophiidae, Ogcocephalidae and Cylopteridae. In general a correlation exists between fish that lack or have small M-cells and a demersal habitat. Since these fish may rely on camouflage for protection against predation, the M-cells may have altered excitability or function as compared to more active fish in which the M-cell is known to function in initiating the startle response (larval zebrafish and goldfish). Differences in input to M-cells of different sizes in conjunction with known electrophysiological properties of the goldfish M-cell are discussed in relation to the function of this neuron.

Posterior lateral line afferent and efferent pathways within the central nervous system of the goldfish with special reference to the Mauthner cell.

The goldfish posterior lateral line nerve consists of a dorsal and a ventral branch, each of which is associated with a ramus of the sensory branch of the VIIth nerve (ramus recurrens facialis). The afferent and efferent pathways of these nerves within the central nervous system were studied by using horseradish peroxidase (HRP) histochemistry. The afferent fibers of the ramus recurrens facialis travel in the ventral portion of the VIIth nerve as it enters the brain and project predominantly to the ipsilateral half of the facial lobe. The afferent fibers of either the dorsal or ventral branch of the posterior lateral line nerve split into two bundles as they enter the brain. The caudally projecting fascicle terminates predominantly in the nucleus medialis. The fibers of the rostrally projecting bundle terminate predominantly in nucleus medialis and nucleus magnocellularis and in the eminentia granularis. The posterior lateral line efferent somata were located in the diencephalon as well as in the medulla oblongata. The medullary efferent neurons formed two distinct groups, a rostral and a caudal nucleus. The cell bodies of the latter were more numerous and larger than those of the former. The axons of the efferent neurons exit from the brain by one of two routes. The first is at the level of the rostral efferent nucleus and the second at the level of the Mauthner cell. Previous reports have described input of posterior lateral line afferent fibers to the Mauthner cell soma and proximal lateral dendrite of the goldfish. This electrophysiological input was bilateral and was interpreted as monosynaptic. The afferent input described in this study was ipsilateral and ended in the vicinity of the distal lateral dendrite. These differences are discussed in the context of the neuronal circuitry that may be present.

Segmental arrangement of reticulospinal neurons in the goldfish hindbrain.

The hindbrain is evolutionarily conserved among diverse vertebrate phyla. In vertebrate embryos, the hindbrain is segmentally organized as a series of overt swellings known as rhombomeres. In the larval zebrafish Brachydanio rerio, conspicuous and identifiable reticulospinal neurons are positioned in the center of rhombomeres. Segmentally homologous reticulospinal neurons that share a range of morphological, developmental, and biochemical features occupy adjacent rhombomeres. We have recently shown that reticulospinal neurons of the zebrafish survive ontogeny without considerable morphological modification and we suggested that homologous neurons may share similar functions at different stages of development (Lee and Eaton: Journal of Comparative Neurology 304:34-52, 1991). The goldfish Carassius auratus, a related cyprinid, is especially suited for neurophysiological and behavioral studies. However, it is not yet known if the various reticulospinal neurons of zebrafish are generalizable to other species such as the goldfish. Therefore, we sought to examine the extent to which reticulospinal neurons of the zebrafish are also present in the adult goldfish. Analysis of 45 brains retrogradely labeled with horseradish peroxidase (HRP) from the spinal cord showed that reticulospinal neurons are arranged as a series of seven segments within the hindbrain; a regular interval of approximately 200 microns separates adjacent segments. Although the goldfish reticulospinal system has more neurons than the zebrafish, many reticulospinal neuron types continue to be identifiable. Moreover, comparisons of dendritic arborizations and axon paths between the two species showed that the morphology between various neuron types is virtually identical. The cross-taxonomic similarities between the reticulospinal systems of these related cyprinids make it possible to pursue functional considerations of segmentally homologous neurons in the goldfish hindbrain.

Origin and function of spiral fibers projecting to the goldfish Mauthner cell.

Two neuron types contact the Mauthner cell (M cell) in the axon cap, a specialized region of high electrical resistance surrounding the initial segment of the M cell axon. One type produces a mixed electrical and chemical inhibition of the M cell. The second sends axons into the central core of the axon cap, where they spiral around the initial segment making both conventional synapses and gap junction contacts. The origin and synaptic effects of these spiral fibers have not been studied previously. When goldfish M cells were filled with Lucifer yellow, presynaptic spiral fibers were seen in the axon cap. These fibers could be traced back through the medial longitudinal fasciculus to their somata, near the contralateral fifth nerve motor nucleus. The same somata were labeled by horseradish peroxidase injected extracellularly into the axon cap. Recordings were made in the axon cap and the M cell after stimulation of hindbrain areas near the spiral fiber somata and axons. Extracellularly, a negative potential was observed close to the termination of the spiral fibers and termed the spiral fiber potential (SFP). Intracellularly, a graded, short latency depolarization of the M cell corresponded to the SFP and could cause the M cell to spike. This depolarization did not shunt the membrane, indicating that it may be produced through gap junctions. Intracellular responses to hindbrain stimulation also had a chloride-dependent, second component that shunted the membrane during paired-pulse testing. This inhibitory second component was probably evoked by cells other than the spiral fiber cells themselves.

The axon reaction of the goldfish mauthner cell and factors that influence its morphological variability.

The axon reaction of the goldfish Mauthner cell, elicited by spinal cord transection, included somatic swelling, nuclear eccentricity, chromatolysis, nuclear infolding, and a perinuclear buildup of basophilic material. The latter three changes were found most consistently and showed gradations which were ranked quantitatively. The time of onset of chromatolysis and nucleus-associated changes depended upon the distance of the wound from the Mauthner cell soma. Specifically, for Mauthner axons cut at 5, 10.5, and 20 mm distal to their somata, the approximate postoperative times of onset were 10, 20, and 40 days, respectively. Mauthner cells axotomized 42 mm distally did not display a consistent axon reaction. Cell atrophy and death were not found in cells axotomized 10.5, 20, or 42 mm from their somata up to 285 postoperative days, but were observed at the longer postoperative intervals (421 days) in neurons cut 5 mm distally and were consistently found in neurons axotomized less than 1.6 mm from their somata. The axon reactions of Mauthner cells within a pair were frequently different. This variability cannot be explained by the influence of cut site or postoperative interval and is hypothesized to result from different metabolic conditions of the individual cells.

Morphological and physiological survival of goldfish Mauthner axons isolated from their somata by spinal cord crush.

Axon segments isolated from their somata degenerate within days or months depending on species and neuronal type. To better understand the time course of morphological and physiological changes associated with degeneration of axon segments of vertebrate central neurons, we have studied the goldfish Mauthner axon (M-axon) when it has been separated from its soma by spinal cord crush. M-axon segments survive morphologically for at least 77 days at 14 degrees C. Cross-sectional areas of isolated M-axon segments (measured 25-30 mm caudal to the wound site at postoperative days 64 and 77) were greater than those of control axons at the same level. Sheath areas did not change. Electron microscopic observations at the same spinal cord location indicated no clear changes in the configuration or number of neurofilaments or any other organelle. M-axon segments studied morphologically after 87 postoperative days had all degenerated. Mauthner axon segments were capable of conducting action potentials and eliciting ipsilateral EMG responses. Repetitive firing of the M-axon segments elicited EMG responses that fatigued more easily and remained fatigued over a longer interval than did those of control axons. The long duration of M-axon segment survival is unusual in a vertebrate and may be due to the low temperature at which the experiments were conducted (14 degrees C) and/or temperature-independent factors. The increased susceptibility to synaptic depression, which has not reported previously, may represent an early sign of the degenerative process.

Putative cholinergic projections from the nucleus isthmi and the nucleus reticularis mesencephali to the optic tectum in the goldfish (Carassius auratus).

The nucleus isthmi of fish and amphibians has reciprocal connections with the optic tectum, and biochemical studies suggested that it may provide a major cholinergic input to the tectum. In goldfish, we have combined immunohistochemical staining for choline acetyltransferase with retrograde labeling of nucleus isthmi neurons after tectal injections of horseradish peroxidase. Seven fish received tectal horseradish peroxidase injections, and brain tissue from these animals was subsequently processed for the simultaneous visualization of horseradish peroxidase and choline acetyltransferase. In many nucleus isthmi neurons the dense horseradish peroxidase label obscured the choline acetyltransferase reaction product but horseradish peroxidase and choline acetyltransferase were colocalized in 54 cells from nine nuclei isthmi. The somata of nucleus reticularis mesencephali neurons stained so intensely for choline acetyltransferase that we could not determine whether they were labelled also with horseradish peroxidase. However, the large choline acetyltransferase-immunoreactive axons of nucleus reticularis mesencephali neurons stained intensely enough for us to follow them rostrally; the axons are clustered together until the level of the rostral tectum where two groupings form: one travels into the tectum and the other travels rostroventrally to cross the midline and enter the contralateral diencephalic preoptic area. We conclude therefore that cholinergic neurons project to the optic tectum from the nucleus isthmi as well as nucleus reticularis mesencephali in goldfish.

Central nervous system lesion triggers inappropriate pathway choice in adult vertebrate system.

Damaged neurons within the CNS of the goldfish are able to regrow to appropriate target areas with resultant recovery of swimming behavior. However, after a whole spino-medullary level crush, many adult goldfish do not recover all behavior. Brain neurons regenerating past a crush wound at this level have a choice between the spinal cord and the first ventral root. Many CNS neurons faced with this decision do not make the same pathway choice as they made during development but rather project axons into the first ventral root, away from their normal target areas in the spinal cord. In fact, more regenerating fibers, including those of reticulospinal and vestibulospinal neurons, choose the peripheral nervous system (PNS) over the CNS, which may limit behavioral recovery. The goldfish PNS may present a more permissive environment to regenerating fibers than the CNS, as is the case in mammals. We suggest that the goldfish is a better model for mammalian regeneration than previously thought.

Axotomy-induced changes in cell structure and membrane excitability are sustained in a vertebrate central neuron.

The retrograde reactions of the goldfish Mauthner neuron to axotomy 8-10 mm caudal to its soma are detectable within a few weeks and persist for more than 200 days. Morphological changes include chromatolysis, reflecting a redistribution of cytoplasmic ribosomes, and infolding of the nuclear membrane. At the time, the normally inexcitable soma-dendritic membrane becomes capable of impulse initiation; this induced excitability also persists for at least 200 days.

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.

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.

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.

Mauthner axon diameter and impulse conduction velocity decrease with growth of goldfish.

Conduction velocities of antidromically evoked impulses along the goldfish Mauthner axon were found to be inversely correlated with body length. To test the hypothesis that such a relation is accompanied by a reduction in axonal diameter with increasing fish size, Mauthner axon diameters were measured. A parabolic relationship with respect to body length was obtained, axonal diameter being maximal in 9.5 cm fish. To our knowledge, this is the first report of a decrease in conduction velocity and axonal size during growth of an organism were functioning of the cell is maintained.

A hindbrain segmental scaffold specifying neuronal location in the adult goldfish, Carassius auratus.

The vertebrate hindbrain develops as a series of well-defined neuroepithelial segments or rhombomeres. While rhombomeres are visible in all vertebrate embryos, generally there is not any visible segmental anatomy in the brains of adults. Teleost fish are exceptional in retaining a rhombomeric pattern of reticulospinal neurons through embryonic, larval, and adult periods. We use this feature to map more precisely the segmental imprint in the reticular and motor basal hindbrain of adult goldfish. Analysis of serial sections cut in three planes and computer reconstructions of retrogradely labeled reticulospinal neurons yielded a segmental framework compatible with previous reports and more amenable to correlation with surrounding neuronal features. Cranial nerve motoneurons and octavolateral efferent neurons were aligned to the reticulospinal scaffold by mapping neurons immunopositive for choline acetyltransferase or retrogradely labeled from cranial nerve roots. The mapping corresponded well with the known ontogeny of these neurons and helps confirm the segmental territories defined by reticulospinal anatomy. Because both the reticulospinal and the motoneuronal segmental patterns persist in the hindbrain of adult goldfish, we hypothesize that a permanent "hindbrain framework" may be a general property that is retained in adult vertebrates. The establishment of a relationship between individual segments and neuronal phenotypes provides a convenient method for future studies that combine form, physiology, and function in adult vertebrates.