Survival and Axonal Outgrowth of the Mauthner Cell Following Spinal Cord Crush Does Not Drive Post-injury Startle Responses.

A pair of Mauthner cells (M-cells) can be found in the hindbrain of most teleost fish, as well as amphibians and lamprey. The axons of these reticulospinal neurons cross the midline and synapse on interneurons and motoneurons as they descend the length of the spinal cord. The M-cell initiates fast C-type startle responses (fast C-starts) in goldfish and zebrafish triggered by abrupt acoustic/vibratory stimuli. Starting about 70 days after whole spinal cord crush, less robust startle responses with longer latencies manifest in adult goldfish, Carassius auratus. The morphological and electrophysiological identifiability of the M-cell provides a unique opportunity to study cellular responses to spinal cord injury and the relation of axonal regrowth to a defined behavior. After spinal cord crush at the spinomedullary junction about one-third of the damaged M-axons of adult goldfish send at least one sprout past the wound site between 56 and 85 days postoperatively. These caudally projecting sprouts follow a more lateral trajectory relative to their position in the fasciculus longitudinalis medialis of control fish. Other sprouts, some from the same axon, follow aberrant pathways that include rostral projections, reversal of direction, midline crossings, neuromas, and projection out the first ventral root. Stimulating M-axons in goldfish that had post-injury startle behavior between 198 and 468 days postoperatively resulted in no or minimal EMG activity in trunk and tail musculature as compared to control fish. Although M-cells can survive for at least 468 day (∼1.3 years) after spinal cord crush, maintain regrowth, and elicit putative trunk EMG responses, the cell does not appear to play a substantive role in the emergence of acoustic/vibratory-triggered responses. We speculate that aberrant pathway choice of this neuron may limit its role in the recovery of behavior and discuss structural and functional properties of alternative candidate neurons that may render them more supportive of post-injury startle behavior.

Invasion of microglia/macrophages and granulocytes into the Mauthner axon myelin sheath following spinal cord injury of the adult goldfish, Carassius auratus.

Rapid activation of resident glia occurs after spinal cord injury. Somewhat later, innate and adaptive immune responses occur with the invasion of peripheral immune cells into the wound site. The activation of resident and peripheral immune cells has been postulated to play harmful as well as beneficial roles in the regenerative process. Mauthner cells, large identifiable neurons located in the hindbrain of most fish and amphibians, provided the opportunity to study the morphological relationship between reactive cells and Mauthner axons (M-axons) severed by spinal cord crush or by selective axotomy. After crossing in the hindbrain, the M-axons of adult goldfish, Carassius auratus, extend the length of the spinal cord. Following injury, the M-axon undergoes retrograde degeneration within its myelin sheath creating an axon-free zone (proximal dieback zone). Reactive cells invade the wound site, enter the axon-free dieback zone and are observed in the vicinity of the retracted M-axon tip as early as 3 hr postinjury. Transmission electron microscopy allowed the detection of microglia/macrophages and granulocytes, some of which appear to be neutrophil-like, at each of these locations. We believe that this is the first report of the invasion of such cells within the myelin sheath of an identifiable axon in the vertebrate central nervous system (CNS). We speculate that microglia/macrophages and granulocytes that are attracted within a few hours to the damaged M-axon are part of an inflammatory response that allows phagocytosis of debris and plays a role in the regenerative process. Our results provide the baseline from which to utilize immunohistochemical and genetic approaches to elucidate the role of non-neuronal cells in the regenerative process of a single axon in the vertebrate CNS.

Two Forms of Electrical Transmission Between Neurons.

Electrical signaling is a cardinal feature of the nervous system and endows it with the capability of quickly reacting to changes in the environment. Although synaptic communication between nerve cells is perceived to be mainly chemically mediated, electrical synaptic interactions also occur. Two different strategies are responsible for electrical communication between neurons. One is the consequence of low resistance intercellular pathways, called "gap junctions", for the spread of electrical currents between the interior of two cells. The second occurs in the absence of cell-to-cell contacts and is a consequence of the extracellular electrical fields generated by the electrical activity of neurons. Here, we place present notions about electrical transmission in a historical perspective and contrast the contributions of the two different forms of electrical communication to brain function.

Rescue of homeostatic regulation of striatal excitability and locomotor activity in a mouse model of Huntington's disease.

We describe a fast activity-dependent homeostatic regulation of intrinsic excitability of identified neurons in mouse dorsal striatum, the striatal output neurons. It can be induced by brief bursts of activity, is expressed on a time scale of seconds, limits repetitive firing, and can convert regular firing patterns to irregular ones. We show it is due to progressive recruitment of the KCNQ2/3 channels that generate the M current. This homeostatic mechanism is significantly reduced in striatal output neurons of the R6/2 transgenic mouse model of Huntington's disease, at an age when the neurons are hyperactive in vivo and the mice begin to exhibit locomotor impairment. Furthermore, it can be rescued by bath perfusion with retigabine, a KCNQ channel activator, and chronic treatment improves locomotor performance. Thus, M-current dysfunction may contribute to the hyperactivity and network dysregulation characteristic of this neurodegenerative disease, and KCNQ2/3 channel regulation may be a target for therapeutic intervention.

Tonic mGluR5/CB1-dependent suppression of inhibition as a pathophysiological hallmark in the striatum of mice carrying a mutant form of huntingtin.

Changes in the activity of striatal output neurons (SONs) have been implicated in the pathogenesis of Huntington's disease (HD). In this inherited polyglutamine disorder, accumulation of intracellular toxins causes a variety of deficits, including synaptic dysfunction, but it is still unclear to what extent striatal GABA release is afflicted as well. Two murine HD models were used, a recently created knock-in mouse (Z_Q175_KI) and an established model of HD (R6/2). In sagittal slices with relatively well-preserved glutamatergic connections throughout the basal ganglia, we have characterized the following: (i) the excitability of SONs; (ii) their spontaneous action potential-dependent GABAergic synaptic activity; (iii) the capacity of exogenous GABA to inhibit spontaneous action potential generation; and (iv) the properties of GABAergic unitary evoked responses (eIPSCs) in response to intrastriatal minimal stimulation at low and high frequency. The HD SONs exhibited enhanced intrisic excitability and higher levels of GABAergic spontaneous activity without presenting evidence for homeostatic upregulation of endogenous or exogenous GABA actions. Unitary eIPSC amplitudes were reduced, with a clear deficit in the probability of release, as indicated by a higher paired-pulse ratio, failure rate and coefficient of variation. In conditions of high-frequency activation, GABAergic connections of HD SONs were prone to asynchronous release and delayed IPSC generation at the expense of synchronized release. Both in wild-type and in HD SONs, GABA was inhibitory. Our results support the conclusion that the enhanced spontaneous synaptic activity in the HD striatum reflects disinhibition. Pharmacological tests identified the HD-related tonic suppression of synaptic inhibition as a glutamate- and endocannabinoid-dependent process.

Role of the lateral line mechanosensory system in directionality of goldfish auditory evoked escape response.

Goldfish (Carassius auratus) escape responses to sudden auditory stimuli are mediated by a pair of reticulospinal neurons, the Mauthner (M-) cells, which integrate mechanosensory inputs from the inner ear and the lateral line (LL) to initiate a fast directional response away from the aversive stimulus. This behavior is context dependent; when near an obstruction the fish may rather turn towards the sound to avoid hitting the object. Mechanisms underlying this directionality remain unknown. Here we investigate the contribution of the LL system to auditory evoked escapes and provide behavioral evidence that it transmits stimulus - and environmental-dependent information that determines the initial response direction of the escape. We quantified escape latency, probability and directionality following abrupt sound stimuli before and after removal of the entire LL with 0.03 mmol l(-1) cobalt chloride (CoCl(2)), 0.002% gentamicin or selective posterior LL nerve (pLLn) transection. CoCl(2) significantly increased escape onset latency without affecting probability and reduced open field directionality from 77% to chance, 52%. This effect on directionality was also observed with gentamicin. Transection of the pLLn had no effect on directionality, indicating the anterior LL nerve (aLLn) afferents are more likely to transmit directional information to the M-cell. When the fish were near a wall, the error rate was quadrupled by both CoCl(2) and pLLn transection. Visual elimination had no influence on directionality unless combined with LL elimination.

Characteristics of the anterior lateral line nerve input to the Mauthner cell.

The goldfish Mauthner (M-) cells, a bilateral pair of reticulospinal neurons, initiate the auditory evoked escape behavior of teleosts. In an open field the fish reliably turns away from the sound source. This implies that the M-cells are capable of a decision-making process that requires the two cells to receive differential directional inputs. Recent studies have indicated that the lateral line (LL) system is necessary in the initial directionality of the escape. This information is thought to be transmitted to the M-cell by the anterior branch of the lateral line nerve (aLLn), which has a shorter conduction time then the posterior branch. This study is the first attempt to characterize the inputs from the aLLn to the M-cell. M-cell intracellular responses to aLLn stimulation indicate a fast monosynaptic input (0.80±0.03 ms) that has a small amplitude averaging 5.85±0.42 mV. This input is bilateral and has a significantly longer latency and smaller amplitude in the contralateral M-cell. Superimposed on the evoked excitatory postsynaptic potential (EPSP) is a shunting inhibition with a delay of 1 ms, which is characteristic of other sensory inputs to the M-cell. Pharmacological manipulation and 50 Hz stimulation reveal a component of the evoked EPSP that is electrotonic, a property favoring speed of transmission. In addition, this input is localized to the lateral dendrite proximal to the inputs from the inner ear. The short latency of these inputs and their proximity to the posterior eighth nerve afferents indicate a crucial role for the aLLn in influencing the excitability and directionality of the M-cell.

Atypical properties of release and short-term depression at a specialized nicotinic synapse in the Mauthner cell network.

Many synapses exhibit temporally complex forms of activity-dependent short-term synaptic plasticity. The diversity of these phenomena reflects the evolutionary specialization of synapses within networks. We examined the properties of transmission and plasticity, in vivo, at an identified, specialized axo-axonic nicotinic synapse between the goldfish Mauthner cell and one of its targets, the cranial relay neuron (CRN), using intracellular paired recordings and low frequency (0.33-2 Hz) train stimulations. Depression of successive excitatory postsynaptic potentials (EPSPs), which dominates short-term plasticity, had two components. A fast component reduced the amplitude of EPSP(2), to less than 50% of EPSP(1). A slow component produced an additional 10-30% of amplitude reduction and developed with a time constant of tens of seconds. The latencies of the later depressed responses were ∼0.1 ms longer than that of EPSP(1), suggesting a reduced release probability. The Ca(2+) chelators EGTA and BAPTA, injected presynaptically, reduced all EPSPs and slowed development of the second component of depression. Interestingly, spike broadening, produced by injecting K(+) channel blockers, reduced release, but accelerated the kinetics of the slow component. Finally, Ba(2+) in the external medium enhanced release, and reduced the first component and slowed the development of the second component of depression. Taken together, these last two results, which are in contrast to observations at other synapses, and the two-component depression suggest atypical release properties at the output synapses of the Mauthner cell, which triggers an escape behavior. We suggest that the second component of depression provides an additional safety factor to prevent repetitive firing of the CRN.

Fast synaptic transmission in the goldfish CNS mediated by multiple nicotinic receptors.

Usually nicotinic receptors in the central nervous system only influence the strength of a signal between neurons. At a few critical connections, for instance some of those involved in the flight response, nicotinic receptors not only modulate the signal, they actually determine whether a signal is conveyed or not. We show at one of the few such connections accessible for study, up to three different nicotinic receptor subtypes mediate the signal. The subtypes appear to be clustered in separate locations. Depending on the number and combination of the subtypes present the signal can range from short to long duration and from low to high amplitude. This provides a critical connection with a built-in plasticity and may enable it to adapt to a changing environment.

Field effects in the CNS play functional roles.

An endogenous electrical field effect, i.e., ephaptic transmission, occurs when an electric field associated with activity occurring in one neuron polarizes the membrane of another neuron. It is well established that field effects occur during pathological conditions, such as epilepsy, but less clear if they play a functional role in the healthy brain. Here, we describe the principles of field effect interactions, discuss identified field effects in diverse brain structures from the teleost Mauthner cell to the mammalian cortex, and speculate on the function of these interactions. Recent evidence supports that relatively weak endogenous and exogenous field effects in laminar structures reach significance because they are amplified by network interactions. Such interactions may be important in rhythmogenesis for the cortical slow wave and hippocampal sharp wave-ripple, and also during transcranial stimulation.

Phase encoding in the Mauthner system: implications in left-right sound source discrimination.

The paired teleost Mauthner (M)-cells and their associated network serve as an excellent system to study the biophysical basis of decision making. In teleosts, an abrupt sound evokes an M-spike, triggering a C-start escape that is usually directed away from a sound source. The response latency is minimized by electrical synapses between auditory afferents and the M-cell lateral dendrite. Here, we demonstrate that the electrical synapses also mediate phase encoding. Ramped sound pressure waves (150-250 Hz) evoked electrotonic postsynaptic potentials in the M-cell locked to two diametrically opposed phase angles that were frequency dependent but intensity independent. Phase encoding was also evident at the behavioral level underwater, because the stimuli evoked directional C-starts with an onset that was phase locked to the sound wave. In interneurons inhibitory to the M-cell, these same stimuli also evoked phase-locked electrotonic postsynaptic potentials and action potentials. The resulting chemical and electrical (i.e., field effect) inhibitions functioned tonically and phasically, respectively. Phase encoding could be important in underwater sound source localization, which is thought to require a neural computation involving a phase comparison between the pressure and the directional particle motion components of sound. This computation may be implemented by an interplay between phase-dependent afferent excitation and feedforward inhibition that activates the appropriate M-cell and directs the C-start away from the sound source.

Validation of a 2-day water maze protocol in mice.

We present a 2-day water maze protocol that addresses some of potential confounds present in the water maze when using the aged subjects typical of studies of neurodegenerative disorders, such as Alzheimer's disease. This protocol is based on an initial series of training trials with a visible platform, followed by a memory test with a hidden platform 24h later. We validated this procedure using aged (15-18m) mice expressing three Alzheimer's disease-related transgenes, PS1(M146 V), APP(Swe), and tau(P301L). We also tested these triple transgenic mice (3xTG) and age and sex-matched wild-type (WT) in a behavioral battery consisting of tests of motor coordination (balance beam), spatial memory (object displacement task) visual acuity (novel object recognition task) and locomotor activity (open field). 3xTG mice had significantly longer escape latencies in the memory trial of the 2-day water maze test than WT and than their own baseline performance in the last visible platform trial. In addition, this protocol had improved sensitivity compared to a typical probe trial, since no significant differences between genotypes were evident in a probe trial conducted 24h after the final training trial. The 2-day procedure also resulted in good reliability between cohorts, and controlled for non-cognitive factors that can confound water maze assessments of memory, such as the significantly lower locomotor activity evident in the 3xTG mice. A further benefit of this method is that large numbers of animals can be tested in a short time.

A role of electrical inhibition in sensorimotor integration.

Although it is accepted that extracellular fields generated by neuronal activity can influence the excitability of neighboring cells, whether this form of neurotransmission has a functional role remains open. In vivo field effects occur in the teleost Mauthner (M)-cell system, where a combination of structural features support the concept of inhibitory electrical synapses. A single spike in one M-cell evoked within as little as 2.2 ms of the onset of an abrupt sound, simulating a predatory strike, initiates a startle-escape behavior [Zottoli SJ (1977) J Exp Biol 66:243-254]. We show that such sounds produce synchronized action potentials in as many as 20 or more interneurons that mediate feed-forward electrical inhibition of the M-cell. The resulting action currents produce an electrical inhibition that coincides with the electrotonic excitatory drive to the M-cell; the amplitude of the peak of the inhibition is approximately 40% of that of the excitation. When electrical inhibition is neutralized with an extracellular cathodal current pulse, subthreshold auditory stimuli are converted into ones that produce an M-spike. Because the timing of electrical inhibition is often the same as the latency of M-cell firing in freely swimming fish, we conclude that electrical inhibition participates in regulating the threshold of the acoustic startle-escape behavior. Therefore, a field effect is likely to be essential to the normal functioning of the neural network.

Effects of temperature acclimation on a central neural circuit and its behavioral output.

In this study, we address the impact of temperature acclimation on neuronal properties in the Mauthner (M-) system, a brain stem network that initiates the startle-escape behavior in goldfish. The M-cell can be studied at cellular and behavioral levels, since it is uniquely identifiable physiologically within the intact vertebrate brain, and a single action potential in this neuron determines not only whether a startle response will occur but also the direction of the escape. Using animals acclimated to 15 degrees C as a control, 25 degrees C-acclimated fish showed a significant increase in escape probability and a decrease in the ability to discriminate escape directionality. Intracellular recordings demonstrated that M-cells in this population possessed decreased input resistance and reduced strength and duration of inhibitory inputs. In contrast, fish acclimated to 5 degrees C were behaviorally similar to 15 degrees C fish and had increased input resistance, increased strength of inhibitory transmission, and reduced excitatory transmission. We show here that alterations in the balance between excitatory and inhibitory synaptic transmission in the M-cell circuit underlie differences in behavioral responsiveness in acclimated populations. Specifically, during warm acclimation, synaptic inputs are weighted on the side of excitation and fish demonstrate hyperexcitability and reduced left-right discrimination during rapid escapes. In contrast, cold acclimation results in transmission weighted on the side of inhibition and these fish are less excitable and show improved directional discrimination.

Otolith endorgan input to the Mauthner neuron in the goldfish.

The Mauthner (M-) cell of the goldfish, Carassius auratus, triggers the rapid escape response of the fish in response to various stimuli, including visual and auditory. The large size and accessibility of the M-cell make it an ideal model system for the study of synaptic transmission, membrane properties, and sensory-motor gating. Although physiological recordings have suggested that afferents from all three of the inner ear endorgans (the saccule, lagena, and utricle) synapse directly on the ipsilateral M-cell, the specific contacts and anatomical distributions of these inputs along the M-cell lateral dendrite remain unknown. We traced specific branches of the auditory (VIIIth) nerve from the three otolith organs of the fish inner ear to the M-cell. The goldfish sacculus gives rise to the vast majority of inputs that contact a large portion of the M-cell lateral dendrite, and these inputs vary greatly in size. In contrast to the ubiquitous distribution of saccular inputs, those from the lagena are segregated to distal regions of the M-cell and synapse on the distal dorsal branch of the lateral dendrite. Similarly, inputs from the utricle are also segregated to distal regions, synapsing on the ventral branch of the lateral dendrite. These results demonstrate that nerves from all three endorgans contact the M-cell, with input-specific segregation of synapses along the M-cell lateral dendrite.

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

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.

Neural representation of object approach in a decision-making motor circuit.

Although behavior is ultimately guided by decision-making neurons and their associated networks, the mechanisms underlying neural decision-making in a behaviorally relevant context remain mostly elusive. To address this question, we analyzed goldfish escapes in response to distinct visual looming stimuli with high-speed video and compared them with electrophysiological responses of the Mauthner cell (M-cell), the threshold detector that initiates such behaviors. These looming stimuli evoke powerful and fast body-bend (C-start) escapes with response probabilities between 0.7 and 0.91 and mean latencies ranging from 142 to 716 ms. Chronic recordings showed that these C-starts are correlated with M-cell activity. Analysis of response latency as a function of the different optical parameters characterizing the stimuli suggests response threshold is closely correlated to a dynamically scaled function of angular retinal image size, (t), specifically kappa(t) = (t-delta x e(-beta(t-delta)), where the exponential term progressively reduces the weight of (t). Intracellular recordings show that looming stimuli typically evoked bursts of graded EPSPs with peak amplitudes up to 9 mV in the M-cell. The proposed scaling function kappa(t) predicts the slope of the depolarizing envelope of these EPSPs and the timing of the largest peak. An analysis of the firing rate of presynaptic inhibitory interneurons suggests the timing of the EPSP peak is shaped by an interaction of excitatory and inhibitory inputs to the M-cell and corresponds to the temporal window in which the probabilistic decision of whether or not to escape is reached.

The Mauthner cell half a century later: a neurobiological model for decision-making?

The Mauthner (M) cell is a critical element in a vital escape "reflex" triggered by abrupt or threatening events. Its properties at the molecular and synaptic levels, their various forms of plasticity, and the design of its networks, are all well adapted for this survival function. They guarantee that this behavior is appropriately unilateral, variable, and unpredictable. The M cell sets the behavioral threshold, and, acting in concert with other elements of the brainstem escape network, determines when, where, and how the escape is executed.

Transient electrical coupling delays the onset of chemical neurotransmission at developing synapses.

The formation and subsequent elimination of electrical coupling between neurons has been demonstrated in many developing vertebrate and invertebrate nervous systems. The relationship between the disappearance of electrical synaptic connectivity and the appearance of chemical neurotransmission is not well understood. We report here that identified motoneurons from the snail Helisoma formed transient electrical and chemical connections during regeneration both in vivo and in vitro. Electrical connections that formed in vivo were strongest by day 2 and no longer detectable by day 7. During elimination of this electrical connection, an inhibitory chemical connection from 110 onto 19 formed. This sequence of synaptic development was recapitulated in cell culture with a similar time course. The relationship between the appearance of transient electrical coupling and its possible effects on the subsequent chemical synaptogenesis were examined by reducing transient intercellular coupling. Trophic factor-deprived medium resulted in a 66% reduction in coupling coefficient. In these conditions, the unidirectional chemical connection formed readily; in contrast, chemical synaptogenesis was delayed in cell pairs exposed to trophic factors where transient electrical coupling was strong. Dye coupling and synaptic vesicle cycling studies supported electrophysiological results. Exposure to cholinergic antagonists, curare and hexamethonium bromide, which block chemical neurotransmission in these synapses, resulted in prolonged maintenance of the electrical connection. These studies demonstrated an inverse relationship between chemical and electrical connectivity at early stages of synaptic development and suggest a dynamic interaction between these forms of neuronal communication as adult neural networks are constructed or regenerated.

Central cellular mechanisms underlying temperature-dependent changes in the goldfish startle-escape behavior.

Activation of auditory afferents on the lateral dendrite of the Mauthner (M)-cell triggers an escape response (C-start) in goldfish. To study distinct behavioral changes and their physiological correlates on a cellular level we examined the effect of acute changes of temperature on M-cell membrane properties and intracellular responses to sound clicks and on C-start kinematics and behavior, focusing on threshold and initial escape direction, two properties determined on the M-cell level. Cooling slowed C-start motor performance, increasing response latency and decreasing peak velocity and peak acceleration, but increased the probability of triggering the escape. In addition, the likelihood of escapes in an inappropriate direction (e.g., responses toward the stimulus instead of away from it) increased at low temperatures. On a cellular level, cooling caused a distinct increase in input resistance of the M-cell and in the dendritic space constant for the auditory-evoked synaptic potentials. Moreover, cooling decreased the magnitude and delayed the onset of feedforward inhibition of the M-cell. These temperature-induced changes in the network and in the intrinsic M-cell properties combine to support behavioral hyperexcitability, but apparently also alter the directional decision-making process during an escape. More generally, our results illustrate that the balance between excitatory and inhibitory influences can determine the expression of a behavior and its modification and at the same time underline the significance of temperature for nervous system function and behavior.