The anuran mauthner cell and its synaptic bed

A’euroscwnc~ Vol 5. pp 1629 to 1646 Pergamon Press Ltd 1980 Prmted in Great 0

Bntan

IBRO

THE ANURAN MAUTHNER CELL AND ITS SYNAPTIC BED S. L. COCHRAN, J. T. HACKETT and D. L. BROWN Department of Physiology, University of Virginia School of Medicine, Charlottesville, Virginia, U.S.A.

Abstract-We have performed a general study of the premetamorphic anuran Mauthner cell using anatomical techniques. Anatomical findings confirm that, as in other amphibia, the tadpole Mauthner

cell possesses a large soma, a principle lateral dendrite, a secondary medial dendrite, and a large decussating, spinally directed axon. Afferent terminals completely cover the surface of the unmyelinated portion of the cell. An unusually dense neuropile of spiral fibers surrounds the initial axon segment. Six general types of afferent contact are found: (1) club endings which establish mixed junctions with the distal lateral dendrite; (2) Gray’s Type 1 junctions; (3) Gray’s Type 2 junctions; (4) mixed junctions; (5) spiral fiber endings; and (6) a previously unknown type of junction found upon the medial dendrite. Type 1, Type 2. and mixed junctions are dispersed upon the whole receptive surface of the cell excluding the axon hillock and initial segment. where only spiral fiber endings are found. The spiral fibers synapse with the initial segment and with each other. Some of the latter contacts are symmetrical synapses forming presynaptic-to-presynaptic profiles. This Mauthner cell is therefore quite similar to that of the teleost. the major difference being the absence of unmyelinated club endings associated with the axon cap. Electrophysiologlcal recordings reflect this difference in anatomy.

auditory stimulation has been shown 1978). Bartelmez’s hypothesis has thus been substanto provoke a specific behavior which has come to be tiated by the results of many researchers. These results have uncovered additional afferents in known as the startle reflex, consisting of a rapid flick of the tail to the side opposite the stimulus (see EATON fish, as at least four other types of terminals are found & BOMBARDIERI, 1978). This action propels the fish in addition to the club ending (BODIAN, 1937; forward and away from the direction of the stimulus. NAKAJIMA& KOHNO, 1978). A peculiar neuropile Bartelmez (1915) proposed that this reflex was maniengulfing the initial segment is invaded by the terfested by a three neuron circuit consisting of afferent minal of a population of cells, which are able to inauditory fibers, efferent motoneurons, and a single hibit the fish Mauthner cell both chemically and elecinterneuron-the Mauthner cell-which ‘short cir- trically (FABER & KORN, 1978; KORN. TR:LLER & FABER,1978; NAKAJIMA,1974). Type 1 contacts upon cuited’ the brainstem nuclei. Because of its readily identifiable nature, the the fish Mauthner cell would appear to be responsible Mauthner cell of fish has been the subject of many for chemical excitation, while Type 2 contacts act to investigations. Anatomists have found that the largest provoke chemical inhibition of the Mauthner cell (see axons in the VIIIth crania1 nerve terminate on the GRAY, 1969). A fifth type of afferent-the ‘spiral lateral dendrite of the fish Mauthner cell as club endfiber’*oils around the initial segment in the central ings (BARTELMEZ,1915; BODIAN, 1937; NAKAJIMA, zone of the fish axon cap (BARTELMEZ, 1915 ; BODIAN, 1974; NAKAJIMA& KOHNO,1978; ROBERTSON, BODEN- 1937), forming axeaxonic synapses with the initial HEIMER& STAGE, 1963; SZABO,RAVAILLE& LIBOU- segment and with other spiral fibers (KOHNO, 1970; BAN, 1978). These club endings exhibit ‘mixed’ synapNAKAJIMA,1974; NAKAJIMA& KOHNO, 1978). The tic junctions in that both gap and Type 1 (see GRAY, effect of impulses in this afferent is unknown. By vir1969) junctions are present in the same terminal contue of all these contacts, many afferents are capable of tact (NAKAJIMA,1974; SZABOet al., 1978). The fish affecting action potential generation by the fish Mauthner axon projects the length of the contraMauthner cell. lateral spinal cord and establishes contacts with denIn the fish, then, the Mauthner cell is a part of a drites of the tail motoneurones (CELIO, GRAY & highly developed neuronal network and has a rich YASARGIL,1979; DIAMOND,1971). Physiologists have afferent input and a highly integrative nature. Many demonstrated that stimulation of the VIIIth nerve aquatic vertebrates with tails, besides teleosts, have results in both electrical and chemical excitation of Mauthner cells which appear to play the same behavthe fish Mauthner cell (FABER& KORN, 1978; FURSH- ioral role (EATON & BOMBARDIERI, 1978; HACKETT, PAN, 1964), and that fish Mauthner cell activity is capC~CHRAN& BROWN,1979; KIMMEL& EATON, 1976; able of provoking motoneuron discharge and tail ROCK, 1978, 1980). However, terrestrial vertebrates movement (DIAMOND,1971; EATON& BOMBARDIERI, lack Mauthner cells (ZOTTOLI,1978). An interesting intersection of these two kinds of vertebrates is found Abbreviation. EPSP, excitatory postsynaptic potential. in the amphibian, particularly the anuran, whose

IN FISH. strong

1629

I630

S. L. COWKAN. J. 7 HAC‘KITT and D. L. BKO!V\

early life is aquatic and fishlike, and whose later extstence is terrestrial. Interestingly, as the anuran makes this metamorphic transition. the Mauthner cell atrophies and may eventually die (STEFANELLI, 1951). Of particular interest are the cellular alterations of the Mauthner cell and the mode of inductlon of these developmental alterations (see JACOBSON, 1978), To understand the nature of these changes. one must first understand the structural and functional features of this cell as it exists prior to metamorphosis. Only with this basic understanding can a proper assessment of metamorphic alteration be ac~omplished. In a previous electronm~~ros~opic study of tadpole Mauthner cell synapses, the spiral fibers were found to impinge on the axon initial segment (NAKAJIMA& KOHNO, 1978). In extending Kohno’s investigation, we have attempted to define the relevant features of this Mauthner cell as It exists in the anuran tadpole by performing a general survey utilizing both anatomical and physiological techniques (HACKETTet al.. 1979). Thus. the present study provides a general knowledge of this cell and its afferents, and a reference for investigations of its metamorphic alteration. A preliminary account of these investigations has been reported (COCHRAN,HACKETT,Hou & BROWN, 1978). EXPERIMENTAL PROCEDURES For the purpose of this analysis over one hundred tadbrains (Runa catesbeiana, GOSNER (1960) stage

pole

32-26-‘legless’) have been fixed and prepared for electronmicroscopy. Because excellent, uniform preservation of this tissue has been difficult for us to achieve, many fixation parameters have been varied. Included in these variations are: (1) the concentrations of substances within the fixatives such as aldehydes, ions, buffers and sugars; (2) the means of delivery of the fixative-i.e. immersion versus perfusion; (3) the concentrations of substances in postfixation buffers prior to osmication: (4) primary versus secondary osmication; and (5) fixation time and temperature. Mauthncr cells have been examined in over thirty such preparations which were adequately fixed. Brains were sectioned in horizontal, coronal and sagittal planes. A detailed analysis of two well-fixed cells forms the core of our results. Reference to the observations from these two cells IS representative of our general findings from the others we have sectloned. One brain was fixed in the following fixative: 4.0”; glutaraldehyde, 3.07: paraformaldehyde. 0.5”; OsO.,, 0.5”: sucrose. and 0.5’:; glucose in bicarbonate-buffered Ringer’s solution (pH 7.4) at 4°C for 20 h. The fixative was delivered by perfuston, which was imperfect, and by subsequent immersion. Although generally well preserved. the cellular elements exhibited occasional swelling and vacuolation. Figures 1, 3, 4 and 7 are made from coronal sections of this brain. Another brain was fixed in the following fixative: 5.07; paraformaldehyde, OS?; OsO&, OS‘; sucrose, and 0.5:~; glucose m bicarbonate-buffered Ringer’s solution at 4°C for 9 h. This brain was fixed by immersion. It, too, possessed a wellpreserved neurophile, although disrupted mitochondria were often observed Figures 6, 8. 9 and 10 are made from

saglttal sections of thts brain. A \mali quantity of h~cl~r,gun peroxide was added to both of thcsc fixatives (Pt I(.U c tn 1

& MITTLTR, 1972). Standard eleutronmlcroscopic techniques ha\c bee11 employed (PALAY A? CHAN-PAL AY, 1974). All bl-‘unh WC~C

dehydrated m a graded series of cold methanols. stamed cw bloc with uranyl acetate and then were mfiltratcd with Epon 812. Serial thick sectlons II~ Sham) were obtamcd using glass knives with an LKB III Ultratome. Serml thtn sectmns were obtained at given areas of tnterest such as Ihc distal lateral dendrite and axon cap regions RESULTS

The ventral funiculus of the tadpole spinal cord is populated by hundreds of large, myelinated, caudally coursing axons. There are a pair of axons whose diameters are greater than any other (approx 20--25 pm). These are the axons of the Mauthner cells. These axons project into the distal extreme of the spinal cord, ending as large diameter. finger-like processes, devoid of myelin: Moving rostrally, one finds a gradual dorsal displacement of these axons. At the levei of the entrance of the VIIIth nerve. just under the floor of the fourth ventricle, these axons abruptly decussate and course in the coronal plane to their parent somata. There is thus one Mauthner cell for each side of the brain. As shown in Fig. 1, this cell is spindIe-shape and has four basic parts. (1) a lateral dendrite; (2) a medial dendrite; (3) an axon; and (4) a large soma, which is located ventral to the sulcus limitans. A partial reconstruction of the cell in Fig. 1 is shown in Fig. 2. A more complete reconstruction has been possible with intracellular dye marking (HAGWT et aL. 1979; ROCK, 1980). Nevertheless, this simplified diagram illustrates the major Mauthner cell components. The lateral dendrite projects dorsolaterally to the lateral margin of the brain. At its distal extent, fine processes emerge from the maJor trunk of the dendrite. Similarly fine processes proceed from the ventral surface of the cell throughout its extent, becoming more numerous medially. Arising from the medial aspect of the perikaryon are two major processes. A ventral extension of the cell, the medial dendrite, sends its processes into the neuropile of the medial longitudinal fasciculus and has a profusion of ventrally directed branches. A stout axon hillock proceeds dorsomedially from the soma, tapers into a fine initial segment and then enlarges as the axon becomes myel~nated. Along the surface of the cell (Fig. 1) is a dense neurophile, which completely ensheathes the cell and its unmyelinated processes. This neuropile is the ‘synaptic bed’ (ROBERTSON et al.. 1963): which is composed primarily of afferents and their terminal boutons. These a&rents are particularly prornjn~t upon the axon hillock and initial segment where boutons appear to cluster on top of each other. This region is named the ‘axon cap‘ (BARTELWZ, 1915: BODIAN.

FE. 1. Light micrograph of the tadpole Mauthner cell. The lateral dendrite, soma, medial dendrite and myelioated axon can be seen in this 1 pm coronal section. A dense neurophile ensheathes the plasma membrane of the cell. Dorsal is up, and lateral is to the right.

AXON

I

DENDRITE

loopm FIG. 2. Diagram of the cell shown in Fig. 1 with the same orientation, drawn with camera lucida from serial 5 and 1 gm coronal sections. Abbreviations: IS, initial segment; AH, axon hillock. Bar represents 100fim.

1631

FIG. 3. A club ending makes contact with a branch of the distal lateral dendrite. The ending in this section establishes both gap (filled arrow) and Type 1 (open arrow) junctions with the Mauthner cell. Bar represents 1 pm.

FIG. 4. A gap junction

between

a club ending

and the distal lateral

1632

dendrite.

Bar represents

0.1 pm

.

FIG. 5. Afferents to the proximal lateral dendrite establishing Type 1 and Type 2 contacts (arrows). This brain was fixed in 1% par~ormaldehyde and 1.25% glutaraldehyde, buffered with sodium cacodylate and then post-osmicated. Bar represents 0.5 pm.

1633

_.-. FIG. 6. Synaptic contacts upon the medial dendrite. Note the dense presynaptic substance in one terminal (X, arrow). A type 2 junction is also shown (2, arrow). Bar represents 0.5 pm.

1634

FIG. 7. Coronal section through the axon cap (enlargement of section shown in Fig. mass themselves along the axon hillock (AH) and initial segment (IS). These endings initial segment approaches the myelinated axon (M). A less dense synaptic bed covers medial dendrite. Peripheral to these endings and to the axon cap are many large and axons. Bar represents 10 pm.

1635

1). Small boutons are sparse as the the surface of the small myelinated

--

FIG. 8. A sagittal section through the axon cap. Numerous spiral fiber terminals impinge upon the initial segment. These terminals are very dense in synaptic vesicles and form contiguous beads (A and B). These endings multiply-contact both the initial segment and each other. Fine processes (arrows) are synaptically contacted and were traced to the initial segment (IS) in adjacent sections. The initial segment, its fine processes, and two spiral fiber terminals are outlined in black.

1636

FIG. 9. Spiral fiber contacts on the initial segment (sagittal section). The top bouton is seen in close apposition (arrow) with the Mauthner cell. The lower bouton contacts the initial segment in the more conventional fashion of the spiral fiber. Bar represents 0.5 pm. FIG. 10. A ‘symmetrical’ synapse between spiral fiber endings. These contacts are not uncommon, but are less frequently found than asymmetrical junctions between spiral fibers. Fine processes between terminals (M) most probably arise from the initial segment. Bar represents 0.5 pm.

1637

The anuran Mauthner cell and its synaptic bed

1639

sections established mixed junctions of this sort. High magnification of a gap junction between a club ending and lateral dendrite is shown in Fig. 4. Gap junctions Ultrastructural features of the Mauthner cell with the lateral dendrite of amphibians have not been previously reported possibly because distal regions Within the soma of the Mauthner cell are many were not examined (see KIMMEL& SCHABTACH, 1974). cytoplasmic organelles commonly found within neuronal perikarya, including a prominent nucleus and Other types of afferents to the lateral dendrite outnumber by far the club endings. These other endings nucleolus surrounded by patches of rough endoplasform three general types of synaptic complex. Gray’s mic reticulum and free polyribosomes, which appear Type 1 contacts (with round presynaptic vesicles and intimately associated with large dense granular postsynaptic thickenings) and Type 2 contacts (with bodies, Golgi cisternae, and mitochondria. These flattened presynaptic vesicles and symmetrical synappatches are heavily concentrated around the nucleus tic thickenings) are common along the entire extent of and are less evident in the distal extents of the dendrites. Within all parts of these dendrites are the lateral dendrite (Fig. 5). In one instance a fine process of the lateral dendrite, distal to the region numerous mitochondria, occasional multi-vesicular contacted by the club endings, invaded the dorsobodies and dense-cored vesicles. Perhaps the most characteristic feature of the cytoplasm of the cell is lateral region of the brainstem and was found to be the incredibly dense array of neurofilaments which enveloped with terminals forming Type 2 contacts. For the most part, however, there is no discernible seem to provide the cell with a cytoplasmic skeleton segregation of these two kinds of terminals as to type throughout its extent. along the extent of the lateral dendrite. Equally disUltrastructural features of the synaptic bed persed are terminals which establish mixed junctions with the lateral dendrite. Thus terminals forming gap As stated above, the entire surface of the unmyelijunctions are usually found in adjacent sections to nated portion of the cell is coated with the terminals have Type 1 junctions as well. These terminals arise of impinging afferents. The majority of these terminals from both myelinated and unmyelinated processes. appear to be endbulbs less than 1 pm in diameter. Although these three types of junctions are readily Upon one cell, when the brain had only been fixed distinguishable from each other, particular terminals with 0s04, 390 terminals were counted along 113 pm do not always lend themselves to such easy distincof the proximal lateral dendrite, revealing that the tion. In addition, these afferents arborize in many difdensity of these terminals was approx 3.5 terminals per micron of the dendritic perimeter. This value im- ferent fashions which can only be ascertained by serial reconstructions or sections cut in a fortunate plane. plies that a cell such as that in Figs 1 and 2 has approx 200,000 contacts upon its surface. It would be Because of their undiscernible segregation along the length of the lateral dendrite, and because of the diffinearly impossible to characterize all of these contacts which, as Bodian states for the goldfish, ‘represent a culty in assessing the topography of the afferent impingements, it is difficult to detect the relative distrimore or less continuous series with many transitions of size and form. . .’ (BODIAN,1937; see also BODIAN, bution of particular afferent populations along given regions of the lateral dendrite. The club endings of the 1972). Certain contacts, however, are distinct. distal lateral dendrite form an exception to this diffiFurthermore, some of these contacts restrict their terculty by virtue of their morphological distinctiveness. minal fields to specific regions of the cell, while others Cell body. Afferents to this region are similar to are found over most of the dendritic expanse. those described for the proximal portion of the lateral Lateral dendrite. The extreme distal extension of the lateral dendrite receives a specific type of afferent dendrite. All three types of endfeet (Type 1, Type 2 and mixed) are found dispersed along the entire surknown as the club ending. Such a terminal, which may arise from the VIIIth nerve, is characterized by face of the cell body. Terminals appear somewhat more sparse on the dorsal surface of the soma, but in an abrupt ending of a myelinated axon with little no region are they absent (cf. KIMMEL& SCHABTACH, change in axonal diameter prior to its contact with 1974). These afferents are found in quite dense contact the lateral dendrite (BODIAN,1937; ROBERTSON et al., on the finer ventral dendrite projections of the cell. 1963). These endings, which contact other dendrites No particular distribution of these contacts is apparand somata as well, restrict their contact zone to the ent. most distal extent of the lateral dendrite. Club endings are both large and small by previous definitions Medial dendrite. This dendrite and its branches are (BODIAN,1937; NAKAJIMA& KOHNO, 1978), but two also completely covered with afferents. Terminals distinct populations are not easily discerned, possibly forming Type 1, Type 2 and mixed junctions are quite due to a reduced number of such contacts as comcommonly found intermingled along the length of the pared with the goldfish. dendrite, as found upon the lateral dendrite and Individual club endings form both gap junctions soma. In addition some terminals, resembling those of (see BENNETT,1977) and Gray’s Type 1 synaptic interthe spiral fibers (see below) are found impinging upon faces (GRAY, 1969) with the lateral dendrite (Fig. 3). portions of the adjacent medial dendrite. A most Seven club endings examined through adjacent serial remarkable synapse on the medial dendrite consists of 1937; KIMMEL & SCHABTACH,1974; NAKAJIMA& KOHNO,1978).

1640

S.

L. COCHRAN.J. T HACKETTand D. L. BROWK

a terminal type having round vesicles with an extremely dense presynaptic substance (Fig. 6, arrow-x). As many as five of these synaptic profiles have been seen in a single thin section. No attempt has been made to quantify their frequency of occurrence, although they appear quite frequently. Such terminals are found only uncommonly on the soma (and then only on the ventral soma adjacent to the medial dendrite) and not at all on the lateral dendrite. The axon cap. A striking feature of the afferent network comprising the synaptic bed is the axon cap. This extremely dense neurophlle appears through the light microscope to consist of a ‘stacked-up’ clustering of boutons (Fig. 7). These terminals comprise a conical surface which completely ensheathes the axon hillock and initial segment. Terminals are several levels thick in the region of the axon htllock, but their numbers decrease as the initial segment tapers toward its myelination. At the extreme end of the initial segment, afferent boutons are scarce-more scarce than on any other region of the cell prior to its myelination. Unlike the goldfish axon cap, which is roughly spherical and has a central zone of afferents and a peripheral zone of afferent. glial and dendritic elements, the tadpole axon cap has a zone of afferents which correspond to the central core of the fish axon cap (NAKAJIMA& KOHNO,1978). Bordering this zone, instead of the pronounced ‘glial wall’ (ROBERTSON et al., 1963) found in the goldfish, is a ring of small myelinated axons which can be seen sparsely to surround the central neuropile. Also lacking in the tadpole are the unmyelinated club endings, which enter the peripheral zone of the goldfish axon cap, as well as the associated cap dendrites projecting from the tr~sitional zone between the soma and the axon hillock. The absence of all these components commonly found in the goldfish axon cap suggested that the spiral fiber was the only afferent to the initial segment of the tadpole Mauthner cell. We therefore endeavored to cut serial sections of the entire cap in sagittal section to ascertain if other components existed. Over 500 thin serial sections were cut and examined under the electron microscope. Intervening 1 pm sections were cut as well and examined with the light microscope. Sections were traced from a point prior to the bifurcation of the axon hillock and media1 dendrite to a point past the myelinated portion of the axon in order to posttively identify the axon initial segment. The initial segment does not have typical ultrastructural features found in fish Maunther cells (PALAY& CHAN-PALAY, 1974). Fascicles of microtubules are seen in sagittal sections through the axon cap (Fig. 9) but these transverse profiles do not demonstrate well other initial segment features. This endeavor has confirmed many features regarding homologous afferents in fish (NAKAJIMA & KOHNO, 1978). We find that small myelinated fibers spiral around the cap, lose their myelin and enter the

dense neuropile. There. these axons continue to curve around themselves and contact the initial segment. Within the cap, these unmyelinated processes take thl: form of finely interconnected. beaded termmals (Fig. 8). Unique to these terminals are the kind of junction they form, these being characterized by accumulations of round vesicles (resistant to aldehydeinduced tlattening) and dense presynaptic thickenings (Figs 9 and 10). After examining the termmals throughout the axon cap in the tadpole, one terminal (Fig. 9, arrow) was found in closer apposition to the initial segment, but it did not appear to be a true gap junction (cf. Figure 16 in NAKAXMA.1974). The boutons of spiral fibers also establish the same type of contact with each other as with the Mauthner cell. These contacts are occasionally found opposite each other forming what KOHNO (1970) has called a ‘symmetrical’ synapse (Fig. 10). Perhaps most significantly, it is evident that the only type of terminal occupying the tadpole axon cap is that of the spiral fiber. No other type of afferent ending is observed within this region or peripherally. Fine glial processes are often seen interposed between these terminals. No ‘glial wall’ or ‘cap cells’ (NAKAJIMA & KOWNO,1978; ROBERTSON et cl/., 1963) were observed and, although a few fine cap dendrites appear to arise from the transitional zone of soma to axon hillock. these dendrites project for very short distances (5 m at most) and are occupied primarily by terminals resembling those of the spiral fibers. In fish, a dense amorphous substance is often observed in the extracellular space of the cap (NAKAYIMA & KOHNO,1978) and more prominently along the rest of the synaptic bed of the Mauthner cell (ROBERTSON rr u1., 1963) and other fish vestibular neurons (KORW. SOTELO& BENNETT,1977). No such substance was found occupying the extracellular space around the tadpole Mauthner cell or other large. neighboring neurons. Electrophysiologica/ studies. The anatomical differences between the goldfish and the tadpole axon cap might be expected to be reflected in el~trophysiolog~cal responses. This is especially so because in the goldfish antidromic stimulation of the contralateral spinal cord elicits a characteristic extracellular field potential which is largest in the vicinity of the axon cap (FURSHPAN& FURUKAWA,1972). In five tadpoles. antidromic field potentials were recorded from Mauthner cells using previously reported techniques (HACKETTet al., 1979). In each animal the antidromic field potential was recorded at its maximum amplitude near the Mauthner cell in several tracks through the brainstem. The tracks were spaced about 50 pm apart. Representative records of antidromic activation of two Mauthner cells are shown in Fig. 11. A and B, respectively. A short latency positive-negative response (peak negativity: 1.2 ms in A, 1.6 ms in B) was recorded in regions of the axon (Al) and cell body (50 pm from the axon in A3 and 50 pm more lateral in A2). In the region of the cell body, a second. later

The amran Mauthner cell and its synaptic bed

SC FIG. Ii. Extracelluiar field potentials elicited by antidromic stimulation of the Mauntker ceil in two tadpoles (A and B). The recording rni~r~f~tr~~ was positioned near the Mautkner axon (Al), the Mauthner cell body (A2Xand the cetf body near the axon cap (A3). Electrical stimulation of the spinal cord (SC, arrow head marks stimulus artifact) was straddling the threshold for Mautkner axon activation. After the recording in A3, the ceif was penetrated and an action patentiat was recorded (A4), Note that the peak of the action potential corresponds to the first negative peak recorded extrace&tlatly. The second negative peak (A3. arrow), is associated with repolarization of the action potential (dotted line). (B) Antidromic extracellular tieId potentiaf recorded in another experiment. One baseline sweep was recorded with a subthreskold stimulus. (Ccl)Tracing of antidromic response from B. Stippled area is the second negative component of the antidromic response. $2) Tracing of an antidromic response recorded from a goldfish axon cap. The stippled area is positive component of the response to antidromic stimulation.

1641

S.

I642

CLUB ENDING

+

k-

L. COTHRAN.

J

TYPE 1

TYPE 2

DISTAL

LATERAL DENDRITE

PROXIMAL LATERAL

T.

HACKETT

and D. L BROWN

SPIRAL FIBER

MIXED

_

I

DENDRITE AND SOMA-

MEDIAL DENDRITE

I

I

41

TIAL SEGMENTt

I

*I

FIG.

12. A schematic representation of the types of terminals found on the surface of the tadpole Mauthner cell, their relative distribution. and their likely actions. Club endings are restricted to the

distal lateral dendrite and are thought to provoke both electrical (via gap junctions, filled arrow) and chemical (via Type 1 junctions, open arrow) excitation (+). Type 1. Type 2 and mixed junctions are found upon the entire receptive surface of the cell (excluding the initial segment) and probably mediate respectively chemical excitation (open arrow. +), chemical inhibition (open arrow, -), and combined electrical (filled arrow. +) and chemical (open arrow, +) excitation. Spiral fibers form contacts with the medial dendrite and initial segment and with each other. This terminal contact IS characterized by a presynaptic density associated with accumulations of vesicles. A sixth type of afferent (X) found contacting the medial dendrite has an extremely dense presynaptic substance. The function of this afferent ts unknown.

component (peak: 2ms in A, 2.1 ms in B) was recorded and appeared to reach maximum amplitude near the axon cap (A3). In three experiments once the maximum amplitude of this second negative component was obtained, the cell bodies were penetrated with less than an additional 2 pm movement of the microelectrode tip. In all cases, the peak of the intracellularly recorded action potential (A4) corresponded to the first negative component recorded extracellularly. The peak of the second later component (arrow in A3) was reached during the repolarization after the cell body was invaded by the action potentials. It is likely that this response was generated by inward current at a site distant from the cell body, perhaps in the axon cap or in the medial dendrite. In Cl, a tracing of the response shown in B was drawn to coincide in time with a tracing (C2) of the antidromic response from a goldfish. Mauthner cell (FURSHPAN & FURUKAWA, 1962). In Cl the amplitude of the initial positive-negative spike potential is smaller than that of C2, but these responses have similar time courses. In the tadpole the late component is of negative polarity and begins slightly before the positive component in C2. Individual variation

Due to intraspecies polymorphism, each Mauthner cell has its own particular shape, size and dendritic

ramifications, but within a broad range, all could be identified by their large somata. their prominent lateral and medial dendrites and largeqaxon, and by their fairly consistent, bilateral location at the level of the entrance of the VIIIth cranial nerve (Zorro~r, 1978). There are often many cells comparable in size to the Mauthner cell, whose perikarya are also located in this brainstem region, just as there are many caudally projecting axons in the ventral funiculus of the spinal cord whose diameters approach that of the Mauthner cell. Even so, the particular geometry of the Mauthner cell clearly distinguishes it from its neighbors. In a few tadpoles, however, no Mauthner cells could be found. Occasionally, we have observed Mauthner cells which appear dark and shrunken both at the light and electron-microscopic level. In one brain, we observed a Mauthner cell having a normally pale cytoplasm, while the other Mauthner cell processes were quite dark and at the electron-microscopic level appeared as if in an advanced state of degeneration (CA~~~~~ERMEYER, 1962; COHEN & PAPPAS, 1972). The afferent synapses on each of these cells exhibited a normal light-appearing cytoplasm. We have also observed, in some instances, myelinated aBerents to the lateral dendrite which also had a dense cytoplasm. These afferents formed mixed junctions with the Mauthner cell and other cells. Dark debris of an unre-

The anuran Mauthner cell and its synaptic bed

1643

nerve (see ZOTTOLI, 1978). Other sources for these contacts have not been discovered. Ultrastructural features of the junctions formed by the club endings with the Mauthner cell are virtually identical in both species. Although some variability exists in the actual geometry of these afferents. all such junctions are mixed. Individual club endings in fish and in tadpoles thus form gap and Type 1 junctions with the Mauthner cell. Such junctions in the vertebrate central nervous system have been implicated in mediating both electrical (via a low resistance shunt at the gap junction) and chemical (via the Type 1 junction) excitation (BENNETT,1972; 1977; GRAY, DISCUSSION 1969; NAKAJIMA& KOHNO, 1978; PAPPAS & WAXMAN, 1973; ROBERTSON et al., 1963). Supporting this The present study was undertaken to obtain a general assessment of the structural features of the contention for the club endings have been electrophysiological studies in the goldfish which showed a dual Mauthner cell and its afferents in the tadpole. Little progress has been made, as yet, to uncover the source excitatory postsynaptic potential (EPSP) evident in the Mauthner cell following stimulation of the posof particular afferents or to directly correlate an individual afferent’s activity with its morphology. The terior branch of the VIIIth nerve, which presumably activated fibers from the sacculus (FURSHPAN,1964). results herein reported, however, provoke the generThis EPSP consisted of a fast and a slow component ation of tentative hypotheses and reasonable specusimilar to those which we have found following sacculations regarding the functioning and synaptic modular nerve stimulation in the tadpole (HACKETTet al., lation of the tadpole Mauthner cell. These efforts are greatly facilitated by the rich experimentation which 1979). The fast component has a very short latency implying electrical coupling between afferents and the has been conducted on the kindred cell in other aquatic vertebrates (EATON& BOMBARDIERI. 1978; FABER Mauthner cell. while the slow component is most & KORN, 1978; KIMMEL& SCHABTACH.1974; NAKA- likely chemically mediated. It is not clear that the same junction is capable of both electrical and chemiJIMA& KOHNO,1978 ; ZOITOLI, 1978). cal transmission as other activated afferents are likely There are, in fact, many similarities between these cells in anurans and teleosts. In common to these cells to contribute to this slower response. It is clear that more rigorous physiological investigation of individare their location, relative size, shape, and, apparently, their axonal distribution. The tadpole Mauthner cell ual afferents and their transmission properties are is smaller and somewhat more spindle-shaped than in needed before definitive conclusions are drawn. Our findings, however, are in good agreement with similar the teleost, and instead of having a large, ventrally directed dendrite, the tadpole’s cell has a medially investigations of the vestibular nuclei of fish (KORN extended dendrite. et al., 1977) frogs (PRECHT, RICHTER, OZAWA & The similarity of these two cells is reflected in their SHIMAZU,1974) and lamprey (ROVAINEN,1979). Type 2 junctions in a few instances seem to coat the afferents. Not only are afferent types alike in both species, but their segregation along regions of these distal tip of the lateral dendrite as it invades the dorcells is equivalent as well. Six types of afferent have solateral brainstem. These terminals have been imphbeen reported in the goldfish (NAKAJIMA& KOHNO, cated in chemically mediating inhibition (ECCLES, 1978). We have found five similar types in the tadpole 1964; GRAY, 1969). It is most likely, as has been sugand possibly a sixth type which has not been reported gested for other systems, that the categories of Type 1 in previous Mauthner cell studies (Fig. 12). It may be and 2 represent extremes of a distribution of morphoinferred from our physiological studies that the morlogical types (BODIAN,1972). A pronounced increase in conductance at the tip of this dendrite would prophological similarities reftect similar functions for most of these afferents in both species (HACKETTet al., vide a very effective means of shunting distally the 1979). EPSP produced by the club endings, thereby lessening any depolarizing perturbation of the soma and The distal lateral dendrite axon hillock, normally consequent from club ending The distal extreme of the lateral dendrite is conactivity (DIAMOND,1968). tained in a dense zone by large and small club endThe proximal lateral dendrite and somu ings. A great number of these afferents contact a large extent of the lateral dendrite in the goldfish (BODIAN, A similar potential for modulation of the transmembrane electrical activity is evident by virtue of the 1937; NAKAJIMA,1974), while in the tadpole a very vast number of afferents comprising the synaptic bed compact region is densely populated by a smaller number of these afferents. A quantitative assessment of the proximal lateral dendrite and soma. Afferents of this difference has not been attempted. Studies on forming Type 1, Type 2, and mixed junctions can be fish indicate that club endings arise from the saccular distinguished impinging upon this region of the cell.

cognizable nature can sometimes be observed in small clusters within the axon cap neuropil. In addition, from tadpole to tadpole, many other cells with a darkened and shrunken appearance are apparently randomly dispersed throughout the nervous system. Such appearances could be the result of fixation osmolarity effects upon these cells, post-mortem changes in these cells, or might reflect degenerative phenomena normal to the developing tadpole’s nervous system. It is difficult from these few observations to discern the relative contributions of each of these influences.

1644

S.

L. COCHHAN.J. T. HACKETTand D L BKOW~

These afferents correspond, respectively, to the large vesicle boutons, the small vesicle boutons. and possibly to the small club endings which Nakajima has found innervating the same region of the goldfish Mauthner cell (NAKAJIMA.1974; NAKAJIMA & KOHNO. 1978). A clear correspondence between the mixed junctions we find and the small club endings of the goldfish is not evident as these terminals in the tadpole are smaller than the small club endings of the goldfish and do not always seem to arise from myelinated fibers as in the goldfish. Type 1 junctions were most probably responsible for the late component of the EPSP evoked by saccular nerve stimulation. and Type 2 contacts most probably mediate inhibition (HACKETTet al., 1979). The medial dendrite

Similar contacts are found upon the medial dendrite. In addition, terminals of the spiral fibers are often observed along the surface of the medial dendrite as it leaves the soma. The function of these contacts is unknown. Another type of contact was found to possess an unusually dense presynaptic substance. These terminals are otherwise similar to the terminals of the spiral fibers, which also have a dense presynaptic substance. It may be that these terminals are extremes (morphologically and functionally) of the population of spiral fibers in a manner similar to that proposed for the Type 1 and Type 2 junctions above. However, such contacts may represent a type distinct from that of the spiral fiber. The medial dendrite is situated at such a point on the cell as to critically modulate its excitability. Thus depolarizing influences from active aRerents on the soma and lateral dendrite can be supplemented by similar activity of the medial dendrite. Alternatively, large conductance changes on this dendrite could effectively channel depolarizing current into it and block spike initiation by the initial segment. In addition, this dendrite could operate somewhat independently of the lateral dendrite as conductance changes distal to the interposed soma would have less of an effect upon depolarizing influences of the medial dendrite. Axon cap

The ultrastructure of the axon cap in Figs 8, 9 and 10 resembles previous descriptions of this region in urodeles (KIMMEL& SCHABTACH, 1974;NAKAJIMA& KOHNO, 1978) and anurans (NAKAJIMA & KOHNO, 1978). In general, the amphibian axon cap is a zone of afferents which ensheathes the axon hillock and initial segment (Fig. 12). These afferents spiral around the axon cap where they lose their myelin, curve around themselves, and synapse with the initial segment and with each other. Thus, spiral fiber is an appropriate designation. We agree with Kohno’s conclusion that the spiral fiber is the only neuronal element in the anuran’s axon cap: this contention is supported by similar ob-

servation in newts (NAKAJIMA & KOHNO.19781 Hoaever, KIMMEL& SCHABTACH (1974) suggest that tao types of terminals may be present m :tuolotls. Although there may be several classes or a contmuum of terminal characteristics. the identification of the origin of the axon terminals and the demonstration of their function will provide a firmer basis for understanding this neuropile. Kohno claims that there are no symmetrical synapses between spiral fibers in amphibians (NAKAJIMA & KOHNO, 1978) analogous to those he discovered in fish (KO~INO.1970). However. symmetrical synapses are clearly present in Fig. 10. These unusual junctions are also obvious in axolotls (KIMMEI.& SCHABTACH, 1974, see junction between two terminal profiles in upper right of their Fig. 9). In the axon cap of goldfish, electrical inhibition. which is signaled by a positive extracellular field potential (FURSHPAN& FURUKAU~A,1962) is thought to be mediated by fibers giving rise to the unmyelinated club ending (KORN et rtl.. 1978) Significantly. both electrical inhibition (HACKETTrt d., 1979) and the unmyelinated club endings are lacking in the tadpole. In fact, none of the large extracellular fields characteristic of the teleost axon cap, including the Mauthner cell spike Itself. are found in the tadpore (Fig. 11). These observations may correlate with the absence of the ‘glial wall’ delimiting the teleost cap and of the amorphous extracellular substance seen in the teleost. Both of these features have been suggested to contribute to the high cap resistivity (FURSHPAN& FURUKAWA,1962; FABER& KORN, 1978: NAKAJIMA & KOHNO, 1978) considered necessary for the generation of large fields. Consequently, the anuran Mauthner cell extracellular response may be concomitantly smaller in amplitude even if the action currents within the axon cap are of a similar magnitude. Another interesting structure in the amphibian axon cap is the finger-like evaginations issuing from the initial segment. The size of these processes is at the limits of resolution of light microscopes, but they are clearly seen under the electron microscope in material from axolotls (KIMMEL& SCHABTACH, 1974) and anurans (Fig. 8). Few papers report the presence of appendages arising from the axon initial segment. A recent communication describes an even more complex process protruding from the initial segment of a neuron in the olfactory bulb of goldfish (KOSAKA & HAMA,1979).

CONCLUSION The features of the tadpole Mauthner cell revealed by this study lead to the conclusion that this cell is receptive to a great number of afferents and is capable of complex integration of this afferent information. We calculated 200,000 afferent contacts on the cell surface shown in Fig. 2. However, a much more elaborate dendritic tree is seen when the cell is stained

The anuran Mauthner ceil and its synaptic bed with horseradish peroxidase reaction product (HACK1979). Thus, the Mauthner celi impulse is probably the result of the activity of a large population of neurons. These neurons influence the Mauthner cell in different manners and to different degrees depending upon their own activity, their afferent topography, and their synaptic action and efficacy. To an extent, the Mauthner cell is, as described by BARTELMEZ (1913, a ‘nucleus’ in itself, as it is capable of sensing a variety of information about the organism and of deciding whether or not to initiate movement.

ETT et al.,

1645

While it lives, the anuran Mauthner cell seems to be sensitive and responsive to nervous system activity as its counterpart is in fish, and thereby provides the larval anuran with a similar behavior. As the tadpole loses its tail and moves to land, the use for this behavior becomes obsolete and the once needed Mauthner cefl decays.

Aeknowledge~enr-his research was supported in part by RSDA X02 DA 00009 from NIDA and NSF grant BNS 77-15.5271.

REFERENCES BARTELMEZ C. W. (1915) Mauthner’s cell and the nucleus motorius tegmenti. J. camp. Neural. 25,87-128. BENNETTM. V. L. (1972) A comparison of electrically and chemically mediated transmission. In Structure and Function of Synapses (ed. PAPPASG. D. & PURPURAD. P.), pp. 221-256. Raven Press, New York. BENNETTM. V. L. (1977) Electrical transmission: a functional analysis and comparison to chemical transmission. In ~~~lu~arBiology of Neurons, Part f (ed. KANDELE. R.), Handbook of Physiology, section 1: The Nervous System, Vol. 1, pp. 357416. Amer. Physiol. Sot., Bethesda. BODIAND. (1937) The structure of the vertebrate synapse. A study of the axon endings on Mauthner’s cell and neighboring centers in the goldfish. J. camp. Neural. 68, 117-159. BODIAND. (1972) Synaptic diversity and characterization by electron microscopy. In Sfrucrure and Fa~ctjo~ of Synapses (ed. PAPPASG. D. & PURPURAD. P.), pp. 221-256. Raven Press, New York. CAMMERMEYER J. (1962) An evaluation of the significance of the ‘dark’ neuron. Ergebn. Anat. Entw-Gesch. 36, l-61. CELIOM. R., GRAYE. G. & YASARGIL G. M. (1979) Ultrastructure of the Mauthner axon collateral and its synapses in the goldfish spinal cord. .l. Neurocytof. 8, 19-29. COCHRANS. L., HACKETTJ. T., Hou S. M. & BROWND. L. (f978) The Mauthner cell of the premetamorph~c anuran. Neuroscience Abs 4, 388. COHEN E. B. & PAPPASG. D. (1972) Dark profiles in the apparently-normal

central nervous system: A problem in the electron microscopic identification of early anterograde axonal degeneration. J. camp. Nemo/. 136, 375-396. DIAMONDJ. (1968) The activation and distribution of GABA and L-glutamate receptors on goldfish Mauthner neurones: An analysis of dendritic remote inhibition. J. P~~s~o~.Land. 194, 669-723. DIAMONDJ. (1971) The Mauthner cell. In Fish Physiology (ed. HOAR W. S. & RANDALLD. J.), pp. 265-346. Academic Press, New York. EATON R. C. & BOMBARDIERI R. A. (1978) Behavioral functions of the Mauthner neuron. In Neurobiology of the Mauthner Cell (ed FABERD. S. & KORN H.), pp. 221-244. Raven Press, New York. ECCI~ES J. C. (1964) The Physiology of Synapses. Springer-Verlag, Berlin. FABERD. S. & KORN H. (1978) Electrophysiology of the Mauthner cell: Basic properties, synaptic mechanisms, and associated networks. In Neurobiology ofthe Mauthner Cell (ed. FABERD. S. & KORNH.), pp. 47-131. Raven Press. New York. FURSHPANE. J. (1964) EIectrical transmission at an excitatory synapse in a vertebrate brain. Science, N.Y. 144, 878-880. FURSHPANE. J. & FURLJKAWA T. (1962) Intracellular and extracellular responses of the several regions of the Mauthner cell of the goldfish. J. Neurophysiol. 25, 732-771. GOSNERK. L. (1960) A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologicu 16, 183-190.

GRAY E. G. (1969) Electron microscopy of excitatory and inhibitory synapses: a brief review. In ~ec~~n~sms of Synaptic Trunsmission. Vol. 31, Prog. Brain Res. (ed. ALKERTK. & WASSERP. 8.). pp. 141.-.155.Elsevier, Amsterdam. HACKETTJ. T.. COCHRANS. L. & BROWND. L. (1979) Functional properties of afferents which synapse on the Mauthner neuron in the amphibian tadpole. Brain Res. 176, 148-152. JACOBXINM. (1978) ~el~elopmental ~eurohiology. Plenum, New York, KIMMELC. B. & EATONR. C. (1976) Development of the Mauthner cell. In Simple Networks and Bekauior (ed. FENTRESS J, C.), pp. 186-202. Sinauer Associates, Sunderland, Mass. KIMMELC. B. Jc SCHABTACH E. (1974) Patterning in synaptic knobs which connect with Mauthner’s cell (Ambystoma mexicanurn. J. camp. Neural. 156, 49-80. KOHNOK. (1970) Symmetrical axo-axonic synapses in the axon cap of the goldfish Mauthner cell. Brain Res. 23,255-258. KORN H., SOTELOC. & BENNETTM. V. L. (1977) The lateral vestibular nucleus of the toadfish Opsamrs tau: Ultrastructural and electrophysiological observations with special reference to electrotonic transmission. Neuroscience 2, 851-884. KORN H.. TRILLERA. & FABERD. S. (1978) Structural correlates of recurrent collateral interneurons producing both electrical and chemical Inhibitions of the Mauthner cell. Proc. R. Sot. B, 202, 533-539. KOSAKAT. & HAMAK. (1979) A new type of neuron with a distinctive axon initial segment. Bruin Res. 163, 151-155. NAKAJ~MAY. (1974) Fine structure of the synaptic endings on the Mauthner cell of the goldfish. J. camp. Neural. 156,

375402.

1646

S. L. COCHRAN.J T HACKETTand D. L. BROWN

NAKAJIMAY. & KOHNOK. (1978) Fine structure of the Mauthner cell. Synaptic topography and comparatlvc study III Neurobiology of the Mauthner Cell (ed. FABERD. S. & KORN H.). pp. 133-166. Raven Press. New York. PALAYS L. & CHAN-PALAYV. (1974) Cerehellnr Cortex. Cytology and Orguni~ation. Springer-Verlag. Berhn. PAPPASG. D. & WAXMANS. G. (1972) Synaptic fine structure~~morphological correlates of chemical Jnd elcctrotonlc transmission. In Structure and Funcrion of Synapses (ed. PAPPASG. D.. & PURPURAD. P.). pp. l-43 Raven Press. N~M York. PERACCHIA C. & MITTLERB. S. (1972) Fixation by means of glutaraldehyde-hydrogen peroxide reactlon products. .I Cull. B~ol. 53. 234238. PRECHTW.. RICHTERA., OZAWAS. & SHIMAZUH. (1974) Intracellular study of frog‘s vestibular neurons m relation to the labyrmth and spinal cord. Expl Brain Res. 19, 377-393 ROBERTSON J. D.. BODENHEIMER T. S. & STAGED. E. (1963) The ultrastructure of Mauthner cell synapses and nodes m goldfish brains. J. Cell. Brol. 19, 159-199. ROCK M K. (1978) Mauthner cell of the bullfrog tadpole mediates rapid activation of contralateral tall musculature Neurosci. Ahs 4, 614. ROCKM. K. (1980) Functional properties of the Mauthner cell in the tadpole Rana catesbeiana. J. Neurophyslob (in press). ROVAINEN C. M. (1979) Electrophysiology of vestibulospinal and vestibulo-reticulospinal systems in lampreys. J. Neurophysiol. 42, 745-766.

STEFANELLI A. (1951) The Mautherian apparatus in ichthypsida: Its nature and function and correlated problem of neurolnstogenesis. Q. Reu. Biol. 26, 17-34. SZABOT., RAVAILLEM. & LIBOUBANS. (1978) Club endings of primary aflerent fibers identified by anterograde horseradish peroxidase labelling. An EM study. Neuroscience Letters 9, 7-15. ZOTTOLIS. J. (1978) Comparative morphology of the Mauthner cell m fish and amphibians. In Neurobiology of thr Mauthner Cell (ed. FABERD. S. & KORN H.), pp. 1345. Raven Press, New York.

(Accepted 25 March 1980)