The Mauthner cell and other identified neurons of the brainstem escape network of fish.

This paper reviews the development of our research on the motor consequences of Mauthner cell function and related brainstem neurons. These cells activate fast-start responses such as seen in fishes escaping from predatory attacks. Our goal was to devise a neuroethological theory of fish escape that accurately reconciled the underlying neural function with a correct concept of the motor act. The identified neuron concept of invertebrates greatly influenced the initial studies. Horseradish peroxidase technology allowed us and other workers to identify principal neurons in the brainstem escape system. Digital imaging technology permitted adequate kinematic characterization of the behavior. Resulting experiments showed that Mauthner system demonstrates two general principles of motor organization: (1) the Mauthner cell is a command-like higher order neuron that serially outputs to a lower level central pattern generator; and (2) the Mauthner cell participates in a larger parallel, brainstem escape network. In this network, we showed that the spatio-temporal pattern of activity codes the timing and magnitude of agonist and antagonist trunk muscle contractions during the behavior. Because the approach angle of the stimulus determines these parameters, we were able to discover the overall sensorimotor relationship between stimulus angle and motor output. This relationship is given as a set of descriptive equations written in terms of stimulus angle, magnitude and timing variables of trunk muscle contractions, and resulting escape trajectory. The equations unify the apparent variability of C-start movement patterns into a single, quantitative theory. Recent studies by other workers show how this concept can make accurate predictions about the underlying neural processes, even at the level of the single, identified cell.

Mauthner and reticulospinal responses to the onset of acoustic pressure and acceleration stimuli.

We determined how the Mauthner cell and other large, fast-conducting reticulospinal neurons of the goldfish responded to acoustic stimuli likely to be important in coordinating body movements underlying escape. The goal was to learn about the neurophysiological responses to these stimuli and the underlying processes of sensorimotor integration. We compared the intracellularly recorded postsynaptic responses (PSPs) of 9 Mauthner cells and a population of 12 other reticulospinal neurons to acoustic pressure and acceleration stimuli. All recorded cells received both pressure and acceleration inputs and responded to stimuli regardless of initial polarity. Thus these cells receive acoustic components necessary to determine source direction. We observed that the Mauthner cell was broadly tuned to acoustic pressure from 100 to 2,000 Hz, with a Q(10dB) of 0.5-1.1 over the best frequency range, 400-800 Hz. This broad tuning is probably due to input from S1 afferents and is similar to tuning of the behavioral audiogram. Our data suggest that cells have relatively more sustained responses to acceleration than to pressure stimuli, to which they rapidly adapted. For a given cell, PSP latencies and amplitudes varied inversely with stimulus intensity. For the entire population of cells studied, minimum onset latencies (i.e., those at the highest intensities) ranged from 0.7 to 7.6 ms for acoustic pressure and 0.7 to 9.8 ms for acceleration. This distribution in minimum onset latencies is consistent with earlier EMG and kinematic findings and supports our previous hypothesis that escape trajectory angle is controlled, in part, by varying the activation time of neurons in the escape network. While the Mauthner cell latency did not differ to both onset polarities of pressure and acceleration, this was not true of all cells. Also, the Mauthner cell responses to pressure were approximately 0.6 ms faster than to acceleration; for the other cells, this difference was 1.1 ms with some cells having differences

A connectionist model of left-right sound discrimination by the Mauthner system.

Artificial neural networks were used to explore the auditory function of the Mauthner system, the brainstem circuit in teleost fishes that initiates fast-start escape responses. The artificial neural networks were trained with backpropagation to assign connectivity and receptive fields in an architecture consistent with the known anatomy of the Mauthner system. Our first goal was to develop neurally specific hypotheses for how the Mauthner system discriminates right from left in the onset of a sound. Our model was consistent with the phase model for directional hearing underwater, the prevalent theory for sound source localization by fishes. Our second goal was to demonstrate how the neural mechanisms that permit sound localization according to the phase model can coexist with the mechanisms that permit the Mauthner system to discriminate between stimuli based on amplitude. Our results indicate possible computational roles for elements of the Mauthner system, which has provided us a theoretical context within which to consider past and future experiments on the cellular physiology. Thus, these findings demonstrate the potential significance of this approach in generating experimentally testable hypotheses for small systems of identified cells.

Sex dimorphic alterations in postnatal brain catecholamines after gestational morphine.

The concentration of brain catecholamines was measured in the hypothalamus, preoptic area (POA), frontal cortex, cerebellum, and striatum of rats exposed in utero to morphine (5-10 mg/kg/twice daily) during gestation days 11-18. Prenatal morphine induced regionally specific, sexually dimorphic alterations in male and female norepinephrine (NE), and dopamine (DA) content at different postnatal ages. Prenatal morphine significantly increased NE content in the hypothalamus of both sexes at postnatal day (PND) 23. In the POA, on the other hand, morphine increased NE content in exposed males at PND 23 and in females at PND 33. In the cerebellum, the NE content of both sexes was significantly elevated at PND 45. In the striatum, NE content was increased by the prenatal morphine only in females at PND 16. The concentration of DA was also affected in a sexually dimorphic manner. At PND 16, prenatal morphine increased the levels of hypothalamic DA only in males, and it reduced the content of DA in female but not male POA. At PND 45, prenatal morphine increased DA in the hypothalamus of females and decreased it in males. In the cerebellum of 16-day-old morphine-exposed animals, DA levels were increased only in males; at PND 45, the levels of DA were still increased in males but had not changed in females. In the striatum, the DA content was reduced only in males at PND 16. Thus, prenatal morphine alters the development of both NE and DA neurotransmitter systems in the hypothalamus, POA, striatum, and cerebellum in a sexually dimorphic manner.

The octavolateralis system and Mauthner cell: interactions and questions.

This paper is an overview of some of the major points to arise in the accompanying contributions of this special symposium issue. The symposium papers arose out of discussions among investigators interested in the inner ear and Mauthner cell, with the focus on hydrodynamic components that activate the Mauthner cell through the octavolateralis system. The intention of the symposium was to investigate the possibility of using our knowledge of the Mauthner system to help understand acoustic processing by the ear, and of using our knowledge of fish hearing to better understand Mauthner cell function. This is the first attempt to take a broad look at both systems to see how they might function together. As such, these proceedings can serve as a mini-tutorial for investigators interested in one system or the other. In this summary paper we also identify some of the major uncertainties in our understanding of the ear-Mauthner connection. These include questions about: (1) the identity of the acoustic stimuli that are neuroethologically relevant to the Mauthner system; (2) the relative importance of the various octavolateralis inputs (acoustic, vestibular, or lateral line); (3) the contribution of the different various acoustic endorgans to the Mauthner system; (4) whether the Mauthner system can distinguish sound source location, and (5) whether Mauthner neurobiology is compatible with the prevailing model (the phase model) for determining sound source location by fishes. We believe these issues provide potentially useful avenues of future investigation that should give important insights in both acoustic processing by fish and the function of the mauthner system.

Left-right discrimination of sound onset by the Mauthner system.

We present a neural model for how the Mauthner system could compute the direction of a transient sound stimulus originating on either the left or right side of a fish. This computation results in an initial orientation of an escape response away from the side of the stimulus. Our idea is based on the phase model of underwater sound localization by fishes. If the phase model is applicable to the Mauthner system, then the problem of sound localization can be reduced to a logical operator, the EXCLUSIVE-NOR (or XNOR). We show how this can be solved by the Mauthner system using afferents that convey separate inputs of sound pressure transduced by the swimbladder (rarefaction and compression) and particle displacement (left and right) from the inner ear. In our model, both pressure components are responsible for bringing the Mauthner cell to threshold. Mauthner firing is gated by the inhibitory PHP neurons receiving specific combinations of pressure and displacement that implement the XNOR logic. We refer to this as the XNOR model. This model is experimentally verifiable and makes specific predictions about the expected acoustic response characteristics of the Mauthner and PHP neurons. Our model places a component of PHP function into a new neuroethological context and may provide insights into the central neurophysiological mechanisms of directional hearing in fishes. In particular, we show how the XNOR model can be applied to predict the activity of diverse neural elements involved in acoustic localization by fishes.

Beating the competition: the reliability hypothesis for Mauthner axon size.

The Mauthner cell has an axon that is among the largest in diameter of any vertebrate neuron. It is commonly thought that the large size is needed for short latency escape responses involving a major contraction of the trunk musculature. Previous work, however, has shown that there is nothing unique about the strength of the Mauthner initiated response, compared to responses initiated by other smaller cells, and it is debatable that there is any important improvement in response latency due to Mauthner axon size. In this paper we advance an alternative explanation: although the Mauthner cell has a powerful excitatory influence on motoneurons, the large size of the Mauthner axon is most important in rapidly spreading an inhibitory signal that turns off other competing motor commands. Such competing commands are likely to arise in the presence of ongoing swimming behavior or ambiguous stimuli that could activate a fast turn either toward or away from the stimulus. These stimuli include apparent food items, or lures, presented by predators (such as anglerfish) and escape eliciting sounds which, in the presence of background noise, may have 180 degrees directional ambiguity. Thus, large size of the axon contributes most to the reliable expression of the escape behavior. We base this reliability hypothesis on a retrospective analysis of previous neurophysiological data and new anatomical measurements of the diameters of the large spinal cord axons from which we calculated conduction velocities. Our calculations show that the Mauthner-derived inhibition is fast enough that it allows an escape response to occur even when a conflicting motor command enters the spinal cord at the same time as the Mauthner axon impulse. The rapid spread of inhibitory influence, along with excitation, may be a general feature of motor system cells with large axonal diameters.

Modulation of catecholamine turnover rate in brain regions of rats exposed prenatally to morphine.

The concentration and turnover rate of brain catecholamines were measured in the hypothalamus, preoptic area (POA), frontal cortex, striatum and cerebellum of adult male and female rats exposed in utero to morphine (5-10 mg/kg/twice a day) during gestation days 11-18. Norepinephrine (NE) and dopamine (DA) turnover rates were estimated following alpha-methylparatyrosine (AMPT) administration. Prenatal morphine altered NE content and turnover in male and female rats in a regionally specific, sexually dimorphic manner. Basal NE content increased approximately 60% in the hypothalamus of male rats, but it decreased about 30% in the hypothalamus of female rats. NE turnover in the hypothalamus of morphine-exposed rats increased 50% in males and decreased 50% in females. Prenatal morphine had no effects on NE turnover in the male POA, but in female rats NE turnover decreased approximately 60%. Alterations in the frontal cortex of morphine-exposed male and female rats resembled the pattern in the hypothalamus; however, the differences did not reach statistical significance. In addition, prenatal morphine had no effect on striatal or cerebellar NE or on basal levels or turnover of DA in any brain regions. These results demonstrate that prenatal morphine alters the content and turnover of NE in a sexually dimorphic manner in specific brain regions of male and female rats, suggesting alterations in the density of terminals and/or utilization of NE. These sexually dimorphic alterations in hypothalamic NE induced by prenatal morphine may be related to the changes observed in adult male and female sexual behavior in our previous work.

A D1 agonist in the MPOA facilitates copulation in male rats.

The classic dopamine agonist apomorphine, microinjected into the medial preoptic area (MPOA), enhances the copulatory behavior of male rats, while pharmacological blockade of endogenous dopamine inhibits sexual behavior. We now report that MPOA injections of 10 micrograms of the selective D1 agonist dihydroxyphenyl-tetrahydrothienopyridine (THP) significantly increased the number of ejaculations, while decreasing the latency to ejaculate in a 30-min test. These effects were not observed following coadministration of the selective D1 antagonist SCH-23390 with 10 micrograms THP. This enhancement may be related to a D1-stimulated facilitation of penile erections.

GABAergic innervation of the Mauthner cell and other reticulospinal neurons in the goldfish.

The Mauthner cells are pair of identifiable hindbrain neurons that participate in the escape response of fishes. Membrane excitability in these cells is regulated by inhibitory neurons that use glycine as a transmitter. We examined the possibility that the inhibitory transmitter gamma-amino butyric acid (GABA) may also act on the Mauthner cells. We used immunocytochemical methods involving an antibody against glutamic acid decarboxylase (GAD), the synthesizing enzyme for GABA. Our study revealed dense GAD immunoreactive terminals surrounding the Mauthner cells. Puncta counts showed that the distribution of GAD immunoreactivity was densest at the distal lateral dendrite of the Mauthner cells; the distribution of puncta tapers gradually in regions closer to the soma. The axon cap was devoid of GABAergic immunoreactivity. We also performed unilateral lesions of the octaval nuclei to evaluate the origin of the GAD immunoreactive terminals. Following the lesions, we found marked decreases in GAD immunoreactive terminals on the proximal lateral dendrite, soma, and proximal ventral dendrite of both Mauthner cells. These results suggest that the octaval region contributes to bilateral inhibition of the Mauthner cells. The distal lateral dendrite of the ipsilateral Mauthner cell also showed a reduction in GAD immunoreactive terminals. This suggests that GABA mediates remote dendritic inhibition of this cell. GAD immunoreactive puncta also surrounded other large reticulospinal neurons, some of which are serially reiterated along the anterior-posterior axis of the hindbrain. Thus, GABA may also exert an influence not only on the Mauthner cells, but also on other reticulospinal neurons.

The direction change concept for reticulospinal control of goldfish escape.

This is an analysis of whether biomechanical or kinematic variables are controlled by descending reticulospinal commands to the spinal cord during escape responses (C-starts) in the goldfish. We studied how the animal contracted its trunk musculature to orient an escape trajectory. We used trunk EMG recordings as a measure of the reticulospinal output to the musculature and we simultaneously gathered high-speed cinematic records of the resulting movements. We found that the escape trajectory is controlled by (1) the relative size of the agonist versus the antagonist muscle contractions on two sides of the body and (2) the timing between these contractions. We found no separate signal for forward propulsion (or force) apart from the initial stage 1 bending of the body. Rather, the neural specification of force is embedded in the commands to bend the body. Thus, our findings demonstrate the importance of the angular kinematic components, or direction changes, caused by the descending reticulospinal command. This new direction change concept is important for two reasons. First, it unifies the diversity of C-start movement patterns into a single and rather simple quantitative model. Second, the model is analogous to the systematic EMG and kinematic changes observed by others to underlie single joint movements of limbs in other vertebrates such as primates. As in these cases, the fish capitalizes on the mechanical properties of the muscle by setting the extent and timing of agonist and antagonist contractions. This, plus the fact that sensory feedback is likely to be minimal, may enable the animal to reduce the number of computational steps in its motor commands used to produce the escape response. Because horizontal body movements in fish are a fundamental vertebrate movement pattern produced by a highly conserved brainstem movement system, our findings may have general implications for understanding the neural basis of rapid movements of diverse body parts.

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.

Copulation increases dopamine activity in the medial preoptic area of male rats.

Dopamine (DA) metabolites in microdialysates from the medial preoptic area (MPOA) of male rats increased during copulation. These increases were not observed during eating of a highly palatable food, or if the animal failed to copulate, or if the microdialysis probe was anterior or dorsal to the MPOA. The only two animals with measurable serotonin (5-HT) levels while the female was present were also the only two that either failed to copulate or copulated but failed to ejaculate. These data are consistent with previous evidence for a facilitative role of MPOA DA in the control of male sexual behavior; however, 5-HT activity in the MPOA may impair copulation.

Opposite influence of medial preoptic D1 and D2 receptors on genital reflexes: implications for copulation.

Dopamine D1 and D2 receptors may synergize with or oppose each other's effects. We suggest that stimulation of D1 and D2 receptors in the medial preoptic area (MPOA) of male rats have opposing effects on genital reflexes. In Experiment 1 a D1 agonist injected into the MPOA increased the number of ex copula erections but decreased the number of seminal emissions. In Experiment 2 a D1 antagonist had the opposite effects (decreased erections and increased seminal emissions), as had a D2 agonist previously. We also suggest that D1 and D2 mechanisms in the MPOA have different thresholds of activation. In Experiment 3 a low dose of the mixed D1/D2 agonist apomorphine increased erections and anteroflexions, an effect blocked by the D1 antagonist. In Experiments 3 and 4 a high dose of apomorphine increased seminal emissions, an effect blocked by the D2 antagonist. Thus, low levels of dopaminergic stimulation may facilitate erections and anteroflexions (controlled by the parasympathetic system and striated muscles) via D1 receptors; higher or more prolonged stimulation may shift to seminal emission (controlled by the sympathetic system) via D2 receptors. This may explain the progression from erectile to ejaculatory mechanisms during copulation.

How stimulus direction determines the trajectory of the Mauthner-initiated escape response in a teleost fish.

Fishes use the Mauthner-initiated C-start for short-latency evasion of predators. C-starts consist of a sudden turn (stage 1) and a rapid acceleration (stage 2). We analyzed high-speed ciné films of goldfish C-starts elicited by dropping a ball into the water. It was previously thought that stage 1 angle does not vary concomitantly with the angle of the threatening stimulus relative to the position of the fish. We found, however, a significant inverse relationship between the direction of the impact of the ball and the angle turned by the end of stage 1. When starting near a wall, or when its usual trajectory was blocked by a wall, the fish used an escape route that was not predictable from the stimulus angle. The fish did not appear to correct its trajectory if it began to turn towards the ball. This behavioral evidence supports the previous notion that the underlying neural command is ballistic and does not use sensory information from the stimulus once the movement begins. If this is so, the fish probably utilizes information on obstacle location in the interval leading up to the trigger stimulus.

Dopamine receptors in the ventral tegmental area affect motor, but not motivational or reflexive, components of copulation in male rats.

Microinjection of apomorphine into the ventral tegmental area (VTA) of male rats was previously shown to delay the onset of copulation and slow its rate, presumably by stimulating impulse-regulating autoreceptors on cell bodies of the A10 mesocorticolimbic dopamine tract. Such stimulation would be expected to slow the firing rate of these neurons and, thereby, to impair locomotion and/or motivational processes. The present experiments tested whether the delayed onset and slowed rate of copulation were related to deficits in motor performance, sexual motivation, and/or genital reflexes. In X-maze tests the speed of running to all 4 goal boxes was slowed; however, the percentage of trials on which the male chose the female's goal box was not decreased. Examination of videotaped copulation tests revealed that the male showed fewer complete copulatory behaviors (mounts, intromissions, and ejaculations), but more misdirected or incomplete copulatory attempts after apomorphine in the VTA. There were also fewer scores of active, as opposed to inactive, behaviors, and the onset and rate of copulation were slowed. The total number of female directed behaviors was not different in apomorphine tests, compared to vehicle. Finally, tests of ex copula genital reflexes revealed no significant effects of apomorphine in the VTA on erections, penile movements, or seminal emissions. These data suggest a role of the VTA in the motor aspects and/or sensorimotor integration of copulation. Sexual motivation and ex copula genital reflexes appeared to be unaffected by apomorphine in the VTA.

D2 receptors in the paraventricular nucleus regulate genital responses and copulation in male rats.

The D2 dopamine receptor agonist quinelorane (LY-163502), microinjected into the paraventricular nucleus (PVN), affected genital response of restrained supine male rats in a biphasic dose-dependent fashion. A moderate dose (1 microgram) facilitated penile responses (intense erections and penile movements), and decreased the latency to the first response. A high dose of quinelorane (10 micrograms) facilitated seminal emission while inhibiting penile responses. The addition of the D1 antagonist SCH-23390 to the 1 microgram dose of quinelorane potentiated quinelorane's increase in seminal emission. We suggest that D1 receptors in the PVN may be antagonistic to D2 receptor-mediated seminal emission, and possibly also penile responses. In copulation tests 1 microgram quinelorane decreased mount latency, whereas 10 micrograms quinelorane increased mount and intromission latencies and slowed copulatory rate. Both 1 and 10 micrograms quinelorane, and also 1 and 10 micrograms of the mixed D1 and D2 agonist apomorphine, decreased the number of intromissions preceding ejaculation.

Identifiable reticulospinal neurons of the adult zebrafish, Brachydanio rerio.

Reticulospinal neurons of the larval zebrafish Brachydanio rerio have been categorized into 27 different types (Kimmel et al.: Journal of Comparative Neurology 205:112-127, 1982; Metcalfe et al.: Journal of Comparative Neurology 251:147-159, 1986). Nineteen of these occur as bilateral pairs which are individually identifiable. Since considerable remolding of brain structures (e.g., cell death and modifications of neuronal architecture) occurs during development, we ask if these cells are preserved in the adult zebrafish and the extent to which neuronal morphology of the larva is conserved during ontogeny. In our analysis, we studied reticular neurons from 84 brains retrogradely labelled from the spinal cord with HRP. We show that all reticulospinal types of the larva are retained without considerable change in morphology in the adult. Many neurons, including the Mauthner cell and two of its serial homologues, MiD2cm and MiD3cm, can be individually and unambiguously identified. In addition, the appearance of later developing (tertiary) neurons leads to an increase in the numbers of some neuron types. Although tertiary neurons are often isomorphic with neighboring cells, they can have unique morphologies of their own and, therefore, are also individually identifiable. We suggest that the appearance of tertiary neurons may serve to extend the behavioural repertoire of the embryo. Moreover, morphological repetitions in adjacent segments of the otic region (level of VIIIth nerve entry) may represent the replication of a functional motif, perhaps involving the C-type escape response which is known to involve the Mauthner cell.

Microinjection of the dopamine antagonist cis-flupenthixol into the MPOA impairs copulation, penile reflexes and sexual motivation in male rats.

Microinjection of the dopamine antagonist cis-flupenthixol into the medial preoptic area was previously shown to impair male rat copulatory behavior. The present experiments provide further evidence of cis-flupenthixol's inhibitory effects on male sexual behavior. Following microinjections of moderate to high doses of cis-flupenthixol, males exhibited slower copulatory rates and fewer ejaculations in copula, fewer ex copula erections and penile movements, and reduced sexual motivation in an X-maze. Locomotion in the X-maze was not significantly affected. Microinjections of the inactive isomer trans-flupenthixol produced no change in any behavioral measure, indicating that cis-flupenthixol's effects were receptor mediated. We suggest that dopamine receptors in the MPOA influence copulation primarily by regulating reflexive and motivational factors, but not locomotion.