Ultrastructure of the Mauthner neurons in goldfish fry after their adaptation to vestibular stimulation

The ultrastructure of the Mauthner cells (M-cells) of goldfish fries was investigated under four different functional states: a) intact (native fishes), b) fatigue (intact fishes subjected to a prolonged vestibular stimulation), c) adapted (intact fishes after a prolonged training session of the daily short vestibular stimuli), d) excited (adapted fishes subjected to a prolonged vestibular stimulation). It has been first found that the fatigue of the M-cells may result in destructive changes of their cytoskeleton. Besides, in the afferent synapses of both adapted and excited M-cells numerous dense-cored vesicles were revealed near the active zones. The data show the neuronal cytoskeleton to be the central target susceptible to damage upon stimulation. The training leads presumably to stabilization of the cytoskeleton ultrastructure. The dense-cored vesicles were suggested to play an active role in the process.     

The structure of the reticulum of Mauthner's neurons in tadpoles of the clawed toad grown under increased gravitational force]

The ultrastructure of the Mauthner cells (M-cells) and the behaviour of Xenopus laevis tadpoles, reared from eggs under increased gravity (2.9 g) which changes the activity of an afferent vestibular input, were investigated. Besides, a study was made of tadpoles after the hindbrain ablation at earlier embryonal stages which significantly altered the microenvironment of M-cells. It is shown that experimental treatments enhance the proliferation of endoplasmic reticulum and its derivatives, so called subsurface cisterns, in the subsynaptic areas. Some structural changes of the synaptic active zones and the cytoskeleton of M-cells were also noticed. It is assumed that the development of the endoplasmic reticulum promotes an intense removal of calcium ions from subsynaptic areas. The plasticity of the endoplasmic reticulum together with other ultrastructural changes apparently stipulate the adaptation of neurons to changed conditions of functioning.     

Ultrastructural localization of calcium pyroantimonate in Mauthner neurons of goldfish fry adapted to natural stimulation

The localization of Ca2+ in control and adapted goldfish fry Mauthner cells (M-cells) revealed by sedimentation with potassium pyroantimonate technique was investigated. It has been shown the following. 1. In the control M-cells electron dense precipitates are present in the extracellular space, commonly within the active zone clefts of chemical synapses, throughout the whole apposition of the mixed synapses and in the synaptoplasm of both type afferent boutons. No precipitates were seen in the cytoplasm of M-cells. 2. After long term natural (vestibular) stimulation (LTNS), resulting in a strong functional suppression of M-cells, precipitates disappeared entirely from active zones but remained numerous in the cytoplasm of M-cells. The distribution of precipitates within the cytoplasm was non-uniform, the highest density was observed on the surfaces of intracellular organelles and elements of the cytoskeleton. 3. In fatigued M-cells after LTNS and after a subsequent one day rest the distribution of precipitates was less intensive, while in the whole it resembled that of fatigued M-cells. 4. In adapted M-cells the distribution of precipitates was similar to that observed in control. M-cells after LTNS, but the amount and size of the precipitated grains were noticeably increased. 5. The most numerous precipitates were seen in adapted M-cells after LTNS. They were localized throughout the postsynaptic cytoplasm and in a lesser order in the presynaptic cytoplasm. 6. After one day rehabilitation the intensitivity of cytochemical reaction of Ca2+ ion precipitation restored to the initial stage characteristic of adapted M-cells before LTVS. The results obtained suggest that the total concentration of Ca2+ ions in adapted M-cells and the dynamics of their exchanges between cytosole and intracellular depots, such as the smooth endoplasmic reticulum, may increase to keep a normal or even increased functional activity of M-cells, both before and after the LTNS.     

Stabilization of motor asymmetry in the goldfish under the influence of optokinetic stimulation

Adaptation as a memory model appears, at the cellular level, as an increase in the resistivity of neurons to fatigue under the influence of repetitive natural training stimulation. Selective induction of adaptational changes in separate compartments of one and the same neuron can also serve as an important instrument for identification of the roles of these compartments in the integrative function of the individual neuron. Mauthner neurons (MNs) of fishes (the goldfish in particular) possess a clearly differentiated soma and two dendrites, lateral and ventral ones. The soma and lateral dendrite of each MN receive afferentation from the ipsilateral vestibular apparatus; at present, the functional and morphological aspects of selective adaptational modifications induced in these compartments by adequate vestibular stimulation have been examined in detail. As to the ventral MN dendrite receiving visual afferentation from the contralateral eye via the ipsilateral tectum, it remained impossible until now to realize the respective approach. We found that training sessions of visual optokinetic stimulation performed in certain modes provide selective activation of one MN through its ventral dendrite and increase the resistivity of this cell to fatiguing stimulation. Therefore, we first demonstrated the possibility of adaptational changes in an individual ventral dendrite of the MN. If fishes were preliminarily adapted with respect to vestibular stimulation, and the resistivity of the soma and lateral dendrite was selectively increased, the resistivity to fatiguing visual test stimulation also increased. On the other hand, if fishes were preliminarily adapted with respect to visual stimulation, the resistivity to fatiguing vestibular stimulation also increased. The observed increase in the resistivity of MNs of fishes adapted due to sensory stimulation of one afferent input with respect to sensory stimulation of other sensory input, as well as an increase in the resistivity to sensory stimulation of one modality, probably show that the mechanism of increase in the resistivity is the same in both cases. Neirofiziologiya/Neurophysiology, Vol. 40, No. 3, pp. 211–220, May–June, 2008.     

Eliminating afferent impulse activity does not alter the dendritic branching of the amphibian Mauthner cell

In the developing amphibian, the formation of extra vestibular contacts on the Mauthner cell (M-cell) enhances dendritic branching, while deprivation reduces it (Goodman and Model, 1988a). The mechanism underlying the interaction between afferent fibers and developing dendritic branches is not known; neural activity may be an essential component of the stimulating effect. We examined the role of afferent impulse activity in the regulation of M-cell dendritic branching in the axolotl (Ambystoma mexicanum) embryo. M-cells occur as a pair of large, uniquely identifiable neurons in the axolotl medulla. Synapses from the ipsilateral vestibular nerve (nVIII) are restricted to a highly branched region of the M-cell lateral dendrite. We varied the amount of nVIII innervation and eliminated neural activity. First, unilateral transplantation of a vestibular primordium deprived some M-cells of nVIII innervation and superinnervated others. Second, surgical fusion of axolotls to TTX-harboring California newt (Taricha torosa) embryos paralyzed the Ambystoma twin: voltage-sensitive Na+ channel blockade by TTX eliminated action potential propagation. Reconstruction of M-cells in 18 mm larvae revealed that dendritic growth was influenced by in-growing axons even in the absence of incoming impulses: impulse blockade had no effect on the stimulation of dendritic growth by the afferent fibers.     

Developmental interactions in the growth and branching of the lateral dendrite of Mauthner's cell (Ambystoma mexicanum)

The axolotl Mauthner (M) cell receives synapses from the vestibular and lateral line nerves on highly branched regions located ventrally and dorsally, respectively, of its lateral dendrite. One of the pair of M-cells was deprived of all ipsilateral vestibular supply and of some lateral line supply by unilateral ablation of the otic vesicle at stage 34, before nerve outgrowth and M-cell differentiation. Histological reconstruction of such deprived M-cells at stages 42 and 45, following M-cell differentiation, revealed that the deprived dendrite was poorly developed, being thinner and much less ramified than in controls. This effect was specific; no changes were consistently observed in the shape or size of the M-cell body, the medial dendrite, or the axon. Electron microscopic examination of deprived M-cells showed that morphologically normal synapses were present on the lateral dendrite; however, synaptic knobs identifiable as being of vestibular origin were absent. We suggest that patterned growth and branching of the M-lateral dendrite during differentiation is regulated through interactions with afferent axons.     

Features of morphofunctional rehabilitation of Mauthner neurons after fatigue

Structural reorganization of smooth endoplasmic reticulum (SER) in relation to changes in functional state of neurons has been investigated using fatigue and subsequent rhabilitation of the goldfish Mauthner (M-) cells as experimental approach. The recovery of original structure of SER in distal parts of dendrities after its significant proliferation, caused by a 3 h natural stimulation, markedly retarded, as compared with quickly normalized functional activity of M-cells. At the same time in somata and proximal parts of dendrites the structural recovery of SER coincided with restoration of the initial function of M-cells. The results suggest that within a single neuron SER with its obvious structural plastisity the neuron functional activoty is supported and restored through regulating the extent of proliferation angmenting Ca(2+)-accumulation in its compartments. Nevertheless SER posseses certain autonomy in structural recovery within somata and dendrites. Such differences of SER plasticity in different parts of the same neuron presumable reflect differences in interaction of its individual compartments with the cytoskeleton and adjacent cytoplasm, or may be caused by different activity of synapses situated on the soma and dendrites.     

Flight-correlated activity changes in neurons of the lateral accessory lobes in the brain of the locust Schistocerca gregaria

The study investigates activity changes in neurons of the lateral accessory lobes in the brain of the locust Schistocerca gregaria during wind-elicited tethered flight. Neurons with ascending projections from the ventral nerve cord to the lateral accessory lobes showed flight-associated excitations which were modulated in the flight motor rhythm. Descending neurons with ramifications in the lateral accessory lobes were tonically excited corresponding to flight duration. The onset of wind-elicited responses in the descending neurons preceded the onset of flight motor activity by 22–60 milliseconds. Neurons connecting the lateral accessory lobes with the central body, the anterior optic tubercles, or other brain areas showed a variety of responses including activity changes during flight initiation and flight termination. Activity in many of these neurons was less tightly coupled to the flight situation and often returned to background levels before flight was terminated. Most of the recorded neurons responded, in addition, to stationary visual stimuli. The results suggest that the lateral accessory lobes in the locust brain are integrative links between the central body, visual pathways, and the ventral nerve cord. The possible involvement of these brain areas in flight control is discussed.     

Ultrastructural changes in the MP3 neuron of the mollusk Lymnaea stagnalis after cryopreservation of the isolated brain

Investigation of a possibility of long-term storage of frozen (-196 degrees C) viable neurons and nervous tissue is one of the central present day problems. In this study ultrastructural changes in neurons of frozen-thawed snail brain were examined as a function of time. We studied the influence of cryopreservation, cryoprotectant (Me2SO), cooling to 4-6 degrees C, and a prolonged incubation in physiological solution at 4-6 degrees C on dictyosomes of Golgi apparatus, endoplasmic reticulum (ER) cisternae and mitochondria. It has been found that responses of these intracellular structures of cryopreserved neurons to the above influences are similar: dissociation of Golgi dictyosomes, swelling of endoplasmic reticulum cisternae and mitochondrial cristae. Both freezing-thawing and cryoprotectant were seen to cause an increase in the number of lysosomes, liposomes, myelin-like structures, and to form large vacuoles. The structural changes in molluscan neurons caused by cryopreservation with Me2SO (2 M) were reversible.     

Bilateral otolith contribution to spatial coding in the vestibular system

Recent work on the coding of spatial information in central otolith neurons has significantly advanced our knowledge of signal transformation from head-fixed otolith coordinates to space-centered coordinates during motion. In this review, emphasis is placed on the neural mechanisms by which signals generated at the bilateral labyrinths are recognized as gravity-dependent spatial information and in turn as substrate for otolithic reflexes. We first focus on the spatiotemporal neuronal response patterns (i.e. one- and two-dimensional neurons) to pure otolith stimulation, as assessed by single unit recording from the vestibular nucleus in labyrinth-intact animals. These spatiotemporal features are also analyzed in association with other electrophysiological properties to evaluate their role in the central construction of a spatial frame of reference in the otolith system. Data derived from animals with elimination of inputs from one labyrinth then provide evidence that during vestibular stimulation signals arising from a single utricle are operative at the level of both the ipsilateral and contralateral vestibular nuclei. Hemilabyrinthectomy also revealed neural asymmetries in spontaneous activity, response dynamics and spatial coding behavior between neuronal subpopulations on the two sides and as a result suggested a segregation of otolith signals reaching the ipsilateral and contralateral vestibular nuclei. Recent studies have confirmed and extended previous observations that the recovery of resting activity within the vestibular nuclear complex during vestibular compensation is related to changes in both intrinsic membrane properties and capacities to respond to extracellular factors. The bilateral imbalance provides the basis for deranged spatial coding and motor deficits accompanying hemilabyrinthectomy. Taken together, these experimental findings indicate that in the normal state converging inputs from bilateral vestibular labyrinths are essential to spatiotemporal signal transformation at the central otolith neurons during low-frequency head movements.     

Eye movements and brainstem neuronal responses evoked by cerebellar and vestibular stimulation in chicks

Summary The vestibulo-ocular reflex undergoes adaptive changes that require inputs from the cerebellar flocculus onto brainstem vestibular neurons. As a step toward developing an in vitro preparation in chicks for studying the synaptic basis of those changes, we have elucidated the organization of the pathways through which the flocculus influences vestibulo-ocular movements. Electrical stimulation of the vestibular ampulla evoked brief, contralaterally directed movements in both eyes. Although single current pulses to the flocculus elicited no response, conjunctive stimulation of the flocculus and the vestibular apparatus significantly reduced the vestibularly-evoked movement. Trains of current pulses applied to the flocculus and ampulla evoked eye movements directed toward and away from the side of stimulation, respectively. Recordings from the brainstem revealed neurons that were activated by ipsilateral vestibular stimulation and inhibited by ipsilateral floccular stimulation. Our sample included neurons in the lateral vestibular nucleus, the ventrolateral portion of the medial vestibular nucleus, and the superior vestibular nucleus. Similarities between these findings and those of similar studies in mammals indicate that the chick will provide a good model system for cellular studies of adaptive changes in the vestibulo-ocular reflex.Abbreviations FTN flocculus target neuron - VOR vestibuloocular reflex     

Experimental testing of the role of cytoskeleton in the solution by neurons of problems facing the brain

Investigation of the influence of cAMP on neuronal electric activity suggests that nerve cells can solve problems using an intraneuronal calculating medium based on the cytoskeleton. When a new problem is posed, this structure has to be disassembled and assembled by the neuronal molecular computer according to the program recorded in DNA. If DNA lacks an appropriate program, the cytoskeleton will not be assembled. In our experiments, fishes which were rotated simultaneously around two mutually perpendicular axes lost their swimming ability, and some dramatic changes were observed in the cytoskeleton of their Mauthner neurons. These changes disappeared after a long-term rest: the cytoskeleton was restored simultaneously with the ability for normal swimming.     

Aspects of Mauthner Cell Differentiation in the Axolotl, Ambystoma mexicanum

SYNOPSIS. In premetamorphic amphibians, the Mauthner cells (M-cells),a single pair of large neurons, are present in the medulla.M-cells differentiate early, are easily recognized morphologically,and in the axolotl embryo, may be approached experimentally:This system is a unique one for the study of neuronal development. The withdrawal of a neuron from the cell division cycle is anearly event in its differentiation. Gastrulae, neurulae andtailbud embryos were each given a single injection of ³H-thymidine.Radioautographs of larvae showed label over M-cell nuclei wheninjections were made before the end of gastrulation, but notwhen injections were made at later stages. Thus, the cells thatgive rise to M-cells cease DNA synthesis during late gastrulation. Unilateral rotations of prospective hindbrain through 180°were performed to see if M-cell axes are specified during neurulation.Rigid axial polarization of the M-cell does not appear to occurin the neurula: The rotated cell regulates and develops normallywith respect to its axes. A major source of input to the M-cell is from the ipsilateralvestibular system. To study the interaction of the M-cell withingrowing axons, unilateral implants of otic vesicles were madeanterior to the otic vesicle in host midtailbud embryos. Preliminarydata suggests a mechanism for the formation of specific neuronalconnections not dependent upon position-time relationships:The ectopic vestibular axons enter the medulla and course caudadto terminate in the region of the ipsilateral M-cell. Whetherthese axons actually form synapses on the M-cell remains tobe established.     

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals¹. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact^2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)^5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.     

The effects of hypergravity and substrate vibration on vestibular function in developing chickens

We used linear vestibular evoked potentials (VsEPs) to characterize peripheral and central vestibular function in birds following embryogenesis at 2G centrifugation or at elevated levels of vibration (+20dB re: background levels). Additionally, we characterized peripheral and central vestibular adaptation to 2G centrifugation in early post-hatch birds. Linear VsEP response peak latencies, amplitudes, thresholds and input/output functions were quantified and compared between experimental and control animals. Birds vibrated throughout embryogenesis and up to one-week post-hatch revealed no changes in linear VsEP response components compared to control siblings. Birds centrifuged at 2G throughout embryogenesis also evidenced no changes in the linear VsEP measured at hatch (P0). Significant changes were seen, however, for linear VsEPs of post-hatch birds placed at 2G for 7 days beginning on post-hatch day 5. Linear VsEPs for these animals displayed significant reductions in response amplitudes associated with peaks P2, N2 and P3, response peaks generated by central neural relays of gravity receptors. The earliest response components, generated by the peripheral vestibular nerve (i.e., P1, N1), were not significantly altered with the 7-day exposure to 2G. Thus, there was no evidence of generalized changes in peripheral gravity receptor excitability or in the rate of maturation in developing animals under increased levels of gravity or vibration. If gravity level plays a critical role in shaping peripheral vestibular ontogeny at magnitudes between 1 and 2G, then it may serve to stabilize function under changing G-fields or it may operate on physiological features that can not be resolved by the VsEP. In contrast, exposure to elevated gravity during post-hatch periods does alter central vestibular function thus providing direct evidence for central vestibular adaptation to the gravitational environment. The fact that central functional change was observed in hatchlings and not embryos, raises the possibility that the first 2-weeks post-hatch may be a critical period of "heightened developmental sensitivity" to hypergravity.     

Efferent vestibular neurons in the guinea pig by horseradish peroxidase and fluorochrome labeling techniques

Neurons with projections into the vestibular receptor apparatus (efferent vestibular neurons) were identified in different medullary regions by retrograde labeling with horseradish peroxidase and transport-specific fluorochromes in the guinea pig. Two groups of efferent vestibular neurons could be distinguished, located dorsally and ventrally to the facial nerve fiber pathway. The dorsal group of efferent vestbular neurons consisted of small cells located close to the genu and the root of the facial nerve and the subependymal granular layer of the 4th ventricle floor. The ventral group was primarily composed of medium-sized cells, usually with only slight tracer accumulation; these were scattered over an extensive area of the lateral tegmental field within nucleus reticularis lateralis parvocellularis. The question of whether the test cells belong to the system of true vestibular efferents and satellite cells is discussed in the light of findings on cell location, morphology, and pattern of tracer accumulation.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 6, pp. 738–747, November–December, 1986.     

Modeling postural control in the lamprey

 A phenomenological model of the mechanism of stabilization of the body orientation during locomotion (dorsal side up) in the lamprey is presented. The mathematical modeling is based on experimental results obtained during investigations of postural control in lampreys using a combined in vivo and robotics approach. The dynamics of the model agree qualitatively with the experimental data. It is shown by computer simulations that postural correction commands from reticulospinal neurons provide information sufficient to stabilize body orientation in the lamprey. The model is based on differences between the effects exerted by the vestibular apparatus on the left and the right side. Received: 16 February 2000 / Accepted in revised form: 29 September 2000     

Contribution of REM sleep to Fos and FRA expression in the vestibular nuclei of rat leading to vestibular adaptation during the STS-90 Neurolab Mission

1. Electrophysical studies performed in ground-based experiments have shown that VN neurons respond to labyrinthine signals following stimulation of macular gravity receptors. Additional evidence indicates that VN neurons may also respond to extralabyrinthine signals of pontine origin, which occur during the PGO waves typical of REM sleep (Bizzi et al., 1964a, b; cf. also Pompeiano, 1967, 1970, 1974 for ref.). 2. In a previous study (Pompeiano et al., 2002) changes in Fos and FRA expression were used to identify the short-term (Fos) and the long-term (FRA) molecular changes which affect the VN neurons at different time points of the space flight. In particular, while Fos protein persists in the brain tissue only for a few hours (6-8 hrs) after its induction, FRA proteins, which can also be induced in the same experimental conditions, persist in the brain tissue for longer periods of time (i.e. from 12/24 hrs to days). 3. In order to relate the changes in gene expression which occurred in the VN during the space flight either to gravity changes or to REM sleep, we investigated in a recent study (Centini et al, 2006) the changes in Fos and FRA expression which occurred in different phases of the sleep-waking cycle, thus being indicative of the animal state. We could then compare the results obtained during the space lab Mission with those previously observed either in ground-based experiments during the physiological state of waking and slow-wave (SWS) or during neurochemically induced episodes of PS, as obtained after microinjection of appropriate agents in dorsal pontine structures of rats. 4. Our findings indicated that a waking state possibly associated with episodes of SWS, occurred at FD2 and FD14, i.e. at launch and after exposure of the animal to microgravity. It appeared also that at the reentry (R + 1) rather than at launch (FD2), an increase in Fos and FRA expression affected the noradrenergic LC neurons, as well as several related structures. These findings probably resulted from the acceleration stress, or immobilization stress as shown by the appearance of a starle reaction (or arrest reaction) which occurred after landing. This condition of stress was followed after landing by an increase in Fos and FRA expression which affected ventromedial medullary reticular structures, whose descending projections are involved in the suppression of postural activity during PS. Moreover, their ascending projections were likely to increase the FRA expression in the neocortex as well as in several regions of the limbic system, such as the dentate gyrus and the hippocampus, which lead to EEG desynchronization and the theta activity during PS. FRA expression affected also at the reentry pontine and diencephalic structures, such as the lateral parabrachial nucleus and the central nucleus of the amygdala, which are known to contribute to the occurrence of pontine waves and the related bursts of REM. 5. Observations made on the various components of the vestibular complex indicated that no Fos and FRA expression occurred in the LVN at the four different mission time points. However, an increase in Fos and FRA expression occurred particularly in the medial (MVN) and spinal vestibular nuclei (SpVN) at FD2 and at R + 1, i.e. 1 day after launch and 12-24 hours after landing, respectively. The pattern of FRA expression observed in the VN during the space flight was generally similar to that of Fos, except at the reentry, when FRA positive cells were observed throughout the whole SpVN, but not the MVN, which showed only a few labeled cells in its rostral part. In contrast to this finding, a prominent Fos expression was found not only in the SpVN, but also throughout the entire MVN. In this case the Fos labeling affected not only the caudal but also the rostral part of this structure, including the dorsal (MVePc) rather than the ventral aspect (MVeMc). Grounded on their different time of persistence, both Fos and FRA expression which occurred in the SpVe could be attributed to the increase in gravity force experienced during take-off and landing, while the Fos pattern which affected particularly the MVN soon after the reentry could additionally be attributed to the rebound episode of PS following the forced period of waking which occurred after landing and after the prolonged (12 days) exposure to microgravity. 6. The results of the present experiments provide the first molecular evidence that pontine activity sources producing rhythmic discharges of vestibulo-ocular neurons during REM sleep may substitute for labyrinthine signals after prolonged (12 days) exposure to microgravity, thus contributing to activity-related plastic changes in the VN leading to readaptation of the vestibular system to 1 G.     

Virus-neuron interactions in the mouse brain infected with Japanese encephalitis virus

The virus-host interactions between Japanese encephalitis (JE) virus and mouse brain neurons were analyzed by electron microscopy. JE virus replicated exclusively in the rough endoplasmic reticulum (RER) of neurons. In the early phase of infection, the perikaryon of infected neurons had relatively normal-looking lamellar RER whose cisternae showed focal dilations containing progeny virions and characteristic endoplasmic reticulum (ER) vesicles. The reticular RER, consisted of rows of ribosomes surrounding irregular-shaped, membrane-unbounded cisternae and resembled that observed in JE-virus-infected PC12 cells, were also seen adjacent to the lamellar RER. The appearance of the reticular RER indicated that RER morphogenesis occurred in infected neurons in association with the viral replication. The fine network of Golgi apparatus was extensively obliterated by fragmentation and dissolution of the Golgi membranes and their replacement by the electron-lucent material. As the infection progressed, the lamellar RER was increasingly replaced by the hypertrophic RER which had diffusely dilated cisternae containing multiple progeny virions and ER vesicles. The Golgi apparatus, at this stage, was seen as coarse, localized Golgi complexes near the hypertrophic RER. In the later phase of infection, RER of infected neurons showed a degenerative change, with the cystically dilated cisternae being filled with ER vesicles and virions. Small, localized Golgi complexes frequently showed vesiculation, vacuolation, and dispersion. The present study, therefore, indicated that during the viral replication the normal lamellar RER which synthesized neuronal secretory and membrane proteins was replaced by the hypertrophic RER which synthesized the viral proteins. The hypertrophic RER eventually degenerated into cystic RER whose cisternae were filled with viral products. The constant degenerative change which occurred in the Golgi apparatus during the viral replication suggested that some of the viral proteins transported from RER to the Golgi apparatus were harmful to the Golgi apparatus and that increasing damage to the Golgi apparatus during the viral replication played the principal role in the pathogenesis of JE-virus-infected neurons in the central nervous system.