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Small ocellar interneurons in the brain of the cabbage looper moth Trichoplusia ni (hubner) (Lepidoptera: Noctuidae)
Int. J. Insect Morphol. & Embryol. 7(4): 337-345. ©Persamon Press Ltd. 1978. Printed in Great Britain.
0020-7322/78/0801-0337502.00/0
SMALL OCELLAR INTERNEURONS IN THE BRAIN OF THE CABBAGE LOOPER MOTH T R I C H O P L U S I A N I (HUBNER) (LEPIDOPTERA: NOCTUIDAE)* JOHN L. EATON and LARRY G. PAPPAS Department of Entomology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, U.S.A.
(Accepted 13 February 1978) Abstract--The small ocellar interneurons in the brain of the adult cabbage looper moth, Trichoplusia ni, have been examined after cobalt infiltration and intensification by the Timm's sulfide-silver technique. Small interneuron tracts pass from the ocellar synaptic region to the central body and the ipsilateral corpora pedunculata, optic tubercle and Iobula. Additional ipsilateral and contralateral Iobula tracts arise from the ipsilateral large interneuron tract and pass to both Iobulas. Small interneurons also pass to the deutocerebrum and tritocerebrum via fibers in the anterior median tract and the antennal glomerular tracts. Index descriptor (in addition to those in title): Ocellar interneurons-cobalt infiltration.
INTRODUCTION ONE OF the key elements in understanding ocellar function in insects is the definition o f the supporting network of interneurons which convey information from the ocelli to the various integrative centers of the brain. The development and improvement o f the cobalt staining technique has made it possible to study pathways of interneurons in the brain (Goodman, 1974; Strausfeld and Obermayer, 1976). Most studies have concentrated on large relatively easy to see ocellar first order interneurons portraying a fairly simple version of ocellar connectivity (Goodman, 1974; G o o d m a n et al., 1975; Cooter, 1975; Bernard, 1976; Koontz, 1976; Pappas and Eaton, 1977). Recently, a more complex situation has been found in the locust, Schistocerca vaga, and honey bee, Apis mellifera, with the discovery of numerous connections between small ocellar interneurons and various integrative centers in the brain ( G o o d m a n and Williams, 1976; Heinzeller, 1976a). In the cabbage looper moth, T. ni, the receptor cell axons in the ocellar nerve synapse with first order interneurons in the dorsal protocerebrum (Dow and Eaton, 1976; Eaton and Pappas, 1977). This area is hereafter referred to as the synaptic region. Six to 7 large first order interneurons project from the synaptic region o f each ocellus to the ipsilateral and contralateral sides of the protocerebrum. In addition, a large number of small interneurons extend from the ocellar synaptic area to several areas of the brain of the cabbage looper moth (Pappas and Eaton, 1977). This report describes the results of studies o f the small ocellar interneurons of the cabbage looper moth and compares them to small interneurons of other insects. MATERIALS AND METHODS The cabbage looper moths used for this study came from a colony maintained at 25°C on an ambient * Supported by NSF Grant BMS 75-07645 and by the VPI & SU Research Division. 337
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photoperiod in the Department of Entomology at VPI and SU. The rearing methods have been described previously (Eaton, 1976). Cobalt chloride (250 mM) with BSA added was used to infiltrate ocelli (Strausfeld and Obermayer, 1976). The method of infiltration has been described elsewhere (Pappas and Eaton, 1977). Only one ocellus was infiltrated in each brain. After incubation for 4-6 hr the cobalt was precipitated with 0"570 ammonium sulfide in saline and the brains were fixed in alcoholic Bouin's, embedded in paraffin and sectioned at 15 wm. The cobalt in the sectioned brains was intensified by Timm's sulfide-silver method using freshly prepared solutions (Tyrer and Bell, 1974). Over 80 brains have been studied by this method. Photomontages prepared from photomicrographs of individual specimens have been used to reconstruct the interneuron tracts in the brain of tile cabbage looper moth. The terminology used to describe ocellar interneuron projections is that of Strausfeld (1976). Pearson's (1971) study of the sphinx moth also proved useful in the identification of brain areas. RESULTS Several filling methods and incubation times were tested in these studies of the cabbage looper moth ocellar interneurons. The best results, for whole mounts or for intensification by the Timm's sulfide-siWer method, have been obtained using cobalt solutions with added BSA and incubation times of 4-6 hr. Longer incubations appeared to diminish the intensity of the cobalt infiltrations. While not all preparations filled to the same degree, the fibers which did fill were consistent from preparation to preparation. Less complete infiltrations were useful for study of fibers near the fill site which would be obscured by more extensive fills. Correspondingly, the extensive infiltrations were advantageous for study of more distant fibers. In the more extensive infiltrations it is apparent that some second or third order interneurons were filled. However, most infiltrated cells are first order interneurons. Small ocellar interneurons connect the ocelli to several areas of the protocerebrum including the central body, corpora pedunculata and optic lobes. Other small ocellar interneurons join the ocellus and the deuto- and tritocerebrum. Accurate counts of fiber numbers were not possible because of the large number o f fibers present. The details of the pathways of these fibers follow.
Central body The interneurons to the central body arise from the ventral side of the ocellar synaptic region lateral to the anterior median tract (AM) (Figs. 1, 6). Cobalt fills o f one ocellus stain both the ipsilateral and contralateral central body tracts. The contralateral central body tract stains less intensely and cobalt presumably enters it through the fibers in the large ocellar interneuron tract extending to the contralateral ocellus (Pappas and Eaton, 1977). From the synaptic region the small interneuron tract passes anterolaterally to the anterior surface of the central body where fibers enter the dorsal edge of the fan-shaped body (fb) (Figs. 1, 2). The remaining fibers continue downward across the anterolateral surface of the superior arch where one group of fibers turns posteriorly to the ventral edge of the fanshaped body. The remaining fibers turn laterally to enter the lateral protuberance tract of the central body (Fig. 3). The fibers in these tracts are very small and while exact counts are FIo. 1. Reconstruction of small ocellar interneuron tracts in a 90-pm-thick saggital plane of brain adjacent to circumesophageal commissure and ipsilateral to infiltrated ocellus. Retouched. Antennal glomerular tract (AG), antennal lobe (AL), anterior median tract (AM), beta lobe (B), cell bodies (CB), central body (C), fan-shaped body (fb), superior arch (sa), subesophageal ganglion (SO), ocellar synaptic region (SR), tritocerebrum (T), trachea (Tr). x 223 FiG. 2. Small ocellar interneuron tracts in a 15-pm-thick saggital plane of the brain adjacent to circumesophageal commissure and contralateral to infiltrated ocellus. Antennal glomerular tract (AG), antennal lobe (AL), cell bodies (CB), central body (C), fan-shaped body (fb), synaptic region (SR), tritocerebrum (T). x 223
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not possible, we estimate that there are about 20 fibers in each tract. The cell bodies o f these fibers are located in the pars intercerebralis.
Corpora pedunculata Small first order interneurons enter the corpora pedunculata via separate tracts from the synaptic region. Fibers in one tract enter the calyx (Fig. 6) and fibers in other tracts enter the alpha and beta lobes at the base of the stalk (Figs. 1, 3, 6). The interneurons projecting to the calyx arise from the synaptic region lateral to those of the central body. They pass ventrolaterally to the apex of the stalk of the corpora pedunculata. These fibers surround the stalk but do not appear to enter it. Branches from these fibers enter the calyx from the ventral side. We were unable to estimate fiber numbers in these tracts, The cell bodies o f these fibers are located on the dorsal edge o f the calyx in the pars intercerebralis. Fibers in both calyces fill from infiltration of a single ocellus, but the ipsilaterai side fills most intensely. The contralateral side fills via fibers in the large ocellar interneuron tract extending to the contralateral ocellus. Small interneurons running from the synaptic region to the base of the stalk arise anterior and lateral to those to the central body and pass to the alpha lobe. Five tracts are present. One tract arises from the tract to the central body and terminates on the medial edge of the beta lobe (Fig. 1). The 2 dorsal tracts enter the dorsal area of the alpha lobe from the posterior side. The 2 ventral tracts enter the ventral edge of the alpha lobe (Fig. 3). Cell bodies of these interneurons are located in the protocerebral cell body layer dorsal and anterior to the alpha lobe.
Optic lobe A tract composed of 2 or 3 small interneurons, arising from the tract of the large contralateral interneurons and passing to the ipsilateral lobula, was observed by Pappas a~d Eaton (1977). It is now known that there are ipsilateral and contralateral tracts passing to each lobula. These lobula tracts are revealed by single ocellar fills. They arise from the tracts of the large contralateral interneurons (Figs. 4, 6). These ipsilateral and contralateral lobula tracts, consisting of 2 or 3 fibers, pass through the posterior protocerebral neuropile on the ventral side of the corpora pedunculata and terminate on the medial side of their respective lobulas. It has not been possible to identify the cell bodies of these fibers. Two additional diffuse tracts pass laterally from the ocellar synaptic region through the dorsal protocerebral neuropile to the dorsal area o f the lobula. These fibers are restricted to the ipsilateral side in fills of single ocelli.
FIG. 3. Reconstruction of small ocellar interneuron tracts to alpha lobe in a 45-1am-thicksaggital plane beginning 90 lain from midline and ipsilateral to infiltrated ocellus. Alpha lobe (A), antennal lobe (AL), cell bodies (CB), central body (C), synaptic region (SR). x 223 FIG. 4. Reconstruction of a 90-pm-cross-sectional plane of the brain showing posterior view ipsilateral (IL) and contralateral (CL) lobula tracts from fill of right oceilus. A part of ipsilateral (Li) large interneurons is removed to reveal the underlying IL tract. Two lines (arrows) mark removed area. Cell bodies (CB), circumesophageal commissure (CC), contralateral large interneurons (Lc), protocerebrum (P), synaptic region (SR). x 125 FIG. 5. Cross section through tritocerebrum (T) showing infiltrated interneurons and cell bodies (CB) lying between tritocerebrum and antennal lobe (AL). × 236
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Optic tubercle Interneurons projecting to the optic tubercle arise from the lateral edge of the ocellar synaptic region and pass anteriorly to the posterior surface of optic tubercle. They pass under and over the tubercle and turn laterally to follow the optic tubercle to the lobula (Pappas and Eaton, 1977) (Fig. 6). Numerous filled cell bodies surround the optic tubercle. The fibers from these cells terminate in the edge of the optic lobe.
Deutocerebrum and tritocerebrum Interneurons pass to the deutocerebrum and tritocerebrum via the antennal glomerular tract and the anterior median tract. In the case of the fibers in the antennal glomerular tract both ipsilateral and contralateral tracts are infiltrated only when the ipsilateral ocellus is filled with cobalt. As was the case above, the ipsilateral side fills more extensively (Figs. 1, 2, 6). Small fibers from the ocellar interneuron tracts of the posterior slope enter the antennal giomerular tract at the level of the protocerebral bridge. They pass through the tract to the posterior surface of the antennal glomerulus where some fibers terminate. Other branches turn downward parallel to the anterior median tract and terminate in the dorsal tritocerebral neuropile. The cell bodies of these cells are in the cell body lobes of the posterior protocerebrum. The fibers of the anterior median tract are the most anterior group of fibers leaving the synaptic region. These fibers arise from the anterior median edge of the synaptic region and pass in an anterior-ventral direction between the protocerebral neuropile and the cell bodies of the anterior surface of the protocerebrum (Figs. 1, 2). The anterior median tract fibers continue downward to the posterior surface of the antennal lobe where fibers from the antennal glomerular tract turn downward parallel to those of the anterior median tract. Some fibers apparently terminate on the posterior surface of the antennal glomerulus and the remainder terminate in the dorsal neuropile of the tritocerebral lobes. Occasionall~ fibers which appear to be axons of the medial neurosecretory cells are observed. These fibers are few in number and are not consistently filled. Cell bodies of these fibers are in the pars intercerebralis. Two fibers in each frontal nerve are often filled by cobalt infiltration of one ocellus (Fig. 5). There are 2 possible pathways for these fills. The most common is through synaptic contact with filled fibers of the antennal glomerular tract or the anterior median tract. Another pathway which has been observed begins at the terminations of the large first order interneurons of the posterior slope of the protocerebrum (Pappas and Eaton, 1977). Fibers arise from ~his area and pass anteriorly through the ventral protocerebral neuropile to the tritocerebral lobes. The fibers in the frontal nerve must be at least second order fibers. Their cell bodies are in the frontal ganglion. DISCUSSION
From the results presented here, it is clear that the ocelli of moths communicate visual information to many integrative areas of the brain via their small interneurons. Most of these contacts are believed to be through first order interneurons, but a few may be second or third order interneurons. The addition of BSA to cobalt chloride solutions has been shown to enhance the ability of cobalt to cross synapses of functionally contiguous units and to fill the finest branches of neurons (Strausfeld and Obermayer, 1976). This method also proved to be highly effective for the interneurons of the cabbage looper and is to a
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FIG. 6. Illustration of frontal view of brain of cabbage looper showing small interneuron tracts revealed by infiltration of right ocellus. Alpha lobe (ct), antennal glomerular tract (AG), antennal lobe (AL), anterior median tract (AM), beta lobe (13),central body (C), contralateral lobula tract (CL), corpora pedunculata (CP), ipsilateral lobula tract (IL), lobula (L), Ocellar nerve (OcN), Optic tubercle (OT), pars intercerebralis (PI), subesophageal ganglion (SG), synaptic region (SR), Tritocerebrum (T). Bar equals 100 ~tm. large degree responsible for the large number of neurons filled. Also important, as pointed out earlier, is the time allowed for infiltration. In our hands the best fills were obtained after 4-6 hr of incubation. Leaving the cobalt to infiltrate for periods of 16-20 hr produced fills which were much less intense. Accordingly, it seems there is an optimal filling time for cobalt infiltration. One possible criticism of this work is that some of the interneuron fills could have been produced by leakage of cobalt into the extracellular space of the neuropile via the ocellar nerve. As pointed out above, examination of the area near the site of infiltration in preparations incubated for short time intervals where leakage was not significant reveals a partial infiltration of interneuron tracts which were more completely filled by longer incubation intervals where some leakage did occur. Since these tracts are partially visible after short incubation intervals, we believe that extracellular cobalt has not contributed to the more extensive fills of these same tracts seen after longer incubations. This view is supported by Strausfeld and Obermayer's (1976) study of transneuronal migration o f cobalt in flies. We would further point out that our incubation times are among the shortest which have been used for studies o f this nature. In other work in which comparable methods have been used to study the small interneurons of insects, the degree of filling obtained seems to have been substantially less than we obtained with the looper preparations ( G o o d m a n and Williams, 1976; Pan and Goodman, 1977). There are several possible explanations for this including use of cobalt chloride with added BSA, incubation interval, differences in the number o f interneurons in different insects, and filling o f second or higher order neurons in the moth preparations. Because of these different possibilities, comparison o f the ocellar interneurons in the looper
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and other insects must be approached cautiously at this time. If one considers the central body, the corpora pedunculata and the lobula of the optic lobe, it is clear that ocellar interneurons apparently pass to these structures in both the cabbage looper and the locust. There are, however, apparent differences in the pathways and in degree of innervation of these structures in these insects. For example, the fibers to the central body in the locust and the honey bee apparently enter via the protocerebral bridge. In the case of the looper these fibers pass over the anterior surface of the central body and enter through that pathway. Similarly, in the case of the corpora pedunculata the fibers apparently enter the calyx from the lateral ocellar tract in the locust and in the looper they enter the calyx from the ventral side near the stalk. Fibers to the corpora pedunculata have not been reported in the honey bee. In the looper there is also innervation of the alpha and beta lobes which has not been described in the locust or honey bee (Goodman and Williams, 1976; Heinzeller, 1976a). The locust and the honey bee have ipsilateral fibers extending from both lateral and medial ocellar tracts through the posterior protocerebrum to the lobula (Goodman and Williams, 1976; Heinzeller, 1976a). There are apparently homologous fibers in the looper which extend ipsilaterally to the lobula. The moth has an additional fiber extending to the contralateral lobula as well as other fibers which extend laterally through the dorsal protocerebrum from the ocellar synaptic region to the ipsilateral lobula of the looper. Such fibers have not been described in other insects at this time. The posterior fibers to the Iobula were also observed in the cockroach and in the house fly (Bernard, 1976; Strausfeld, 1976). Fibers extending anteriorly from the ocellar synaptic region to the optic tubercle in the looper have not been described in other insects. The anterior median tract fibers from the ocelli of the looper also have not been seen in other insects. Heinzeller (1976a) does indicate that the ocellar interneurons end among the medial neurosecretory cells of the honey bee and both Heinzeller (1976b) and BrousseGaury (1970, 1971) have indicated that the ocelli may affect the neurosecretory activity of the medial neurosecretory cells. While the association of the ocellar interneurons with th~ neurosecretory cells is not clearly shown in the cabbage looper, there is some evidence that it may exist. Some of the fibers which fill in the anterior median tract may be neurosecretory cell fibers. Certainly some of them are not, for they extend to the deuto- and tritocerebrum rather than into the tract of corpora cardiaca nerve l (NCCI). We have seen some faintly stained fibers in NCCI, but no complete fills have been observed. In the case of the antennal glomerular tract fibers, Goodman and Williams (1976) observed ocellar fibers entering the antennal glomerular tract in the locust, but were unable to determine that they went any further. In the cabbage looper these fibers extend through the antermal glomerular tract to innervate deutocerebral neuropile on the posterior surface of the olfactory glomeruli. Fibers from the antennal glomerular tract also extend into the dorsal area of the tritocerebrum. The filling of the frontal nerve in some of our ocellar infiltrations must represent at least a second order and perhaps a third interneuron fill. In these preparations, filled cell bodies are observed around the edges of the tritocerebrum. A cell body has also been located in the frontal ganglion which apparently belongs to the filled interneuron. This could represent a third order fiber. We also regularly observed a fill of the tegumentary nerve when filling the ocellar receptor cells. Pan and Goodman (1977) observed similar fills when they filled cut setae on the head of the honey bee. We have not cut setae and these fills are produced by cobalt entering the oceilus. We know that the tegumentary nerve comes into very close proximity with the ocellar receptor cell area, and it is possible that the tegumentary nerve fills due to
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m e m b r a n e d a m a g e caused by o s m o t i c i m b a l a n c e s t h r o u g h the a p p l i c a t i o n o f c o b a l t to the ocellus. In s u m m a r y , c o b a l t infiltration o f the ocellus in the c a b b a g e l o o p e r p r o d u c e s a m u c h m o r e extensive fill o f i n t e r n e u r o n s t h a n has been observed i n o t h e r insects. W h i l e some of these m a y be second or third order i n t e r n e u r o n s , we believe m o s t o f t h e m to be first o r d e r i n t e r n e u r o n s . W e also believe t h a t future studies will reveal m o r e ocellar i n t e r n e u r o n s in o t h e r insects when this i m p r o v e d m e t h o d is a d o p t e d a n d used for those insects.
REFERENCES
BERNARD,A. 1976. Etude topographique des interneurones ocellaires et de quelques uns de leurs prolongemerits chez Periplaneta americana. J. Insect Physiol. 22: 569-77. BROUSSE-GAURY, P. 1970. Les stimuli ocellaires par les neuros6cr6tions contr61ent les corpora allata des Blattes. Ann. Sci. Nat. Zool. Biol. Anita. Paris. 12: 61~. BROUSSE-GAURV,P. 1971. Description d'arcs r6flexes neuro-endocriniens partant des ocelles chez quelque orthopt6res Bull. Biol. Fr. Belg. 105: 84-93. COOTER, R. J. 1975. Ocellus and ocellar nerves of Periplaneta americana L. (Orthoptera: Dictyoptera). Int. J. bisect Morphol. Embryol. 4: 273-88. Dow, M. A. and J. L. EATON.1976. Fine structure of the ocellus of the cabbage looper moth (Trichoplusia ni). Cell Tissue Res. 171: 523-33. EATON,J. L. 1976. Spectral sensitivity of the ocelli of the adult cabbage looper moth, Trichoplusia ni. J. Comp. Physiol. 109: 17-24. EATON, J. L. and L. G. PAPPAS. 1977. Synaptic organization of the cabbage looper moth ocellus. Cell Tissue Res. 183: 291-97. GOODMAN, C. 1974. Anatomy of locust ocellar interneurons constancy and variability. J. Comp. Physiol. 95: 185-201. GOODMAN, C. and J. L. D. WILLIAMS.1976. Anatomy of the ocellur interneurons of acridid grasshoppers. II. The small interneurons. Cell Tissue Res. 175: 203-26. GOODMAN,L. J., J. A. PATTERSONand P. G. MOSBS. 1975. The projection of ocellar neurons within the brain of the locust, Schistocerca gregaria. Cell Tissue Res. 157: 467-92. HEINZELLER,T. 1976a. Second-order ocellar neurons in the brain of the honey bee (Apis mellifera). Cell Tissue Res. 171: 91-9. HEINZELLER,T. 1976b. Circadiane Anderung im endokrinen System der Honigbiene, Apis mellifera, Effekt yon Haft und Ocellenblendung. J. bisect Physiol. 22: 315-21. KOONTZ, M. 1976. Neuronal pathways from the dorsal ocelli of the house cricket, Acheta domesticus. J. MorphoL 149: 105-20. PAN, K. C. and L. J. GOODMAN.1977. Ocellar projections within the central nervous system of the worker honey bee, Apis mellifera. Cell Tissue Res. 176: 505-27. PAPPAS, L. G. and J. L. EATON. 1977. Large ocellar interneurons in the brain of the cabbage looper moth, Trichoplusia ni (Lepidoptera). Zoomorphologie 87: 237-46. PEARSON,L. 1971. The corpora pedunculata of Sphinx ligustri L. and other Lepidoptera. Phil. Trans. R. Soc. Lond. B. 259: 477-516. STRAUSFELD,N. J. 1976. Atlas of an Insect Brain. Springer, Berlin. STRAUSFELD,N. J. and M. OBERMAVER.1976. Resolution of intraneuronal and transynaptic migration of cobalt in the insect visual and central nervous systems. J. Comp. Physiol. 110: 1-12. TYRER,N. M. and E. M. BELL. 1974. The intensification of cobalt filled neuron profiles using a modification of Timm's sulfide-silver method. Brain Res. 73: 151-55.
0020-7322/78/0801-0337502.00/0
SMALL OCELLAR INTERNEURONS IN THE BRAIN OF THE CABBAGE LOOPER MOTH T R I C H O P L U S I A N I (HUBNER) (LEPIDOPTERA: NOCTUIDAE)* JOHN L. EATON and LARRY G. PAPPAS Department of Entomology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, U.S.A.
(Accepted 13 February 1978) Abstract--The small ocellar interneurons in the brain of the adult cabbage looper moth, Trichoplusia ni, have been examined after cobalt infiltration and intensification by the Timm's sulfide-silver technique. Small interneuron tracts pass from the ocellar synaptic region to the central body and the ipsilateral corpora pedunculata, optic tubercle and Iobula. Additional ipsilateral and contralateral Iobula tracts arise from the ipsilateral large interneuron tract and pass to both Iobulas. Small interneurons also pass to the deutocerebrum and tritocerebrum via fibers in the anterior median tract and the antennal glomerular tracts. Index descriptor (in addition to those in title): Ocellar interneurons-cobalt infiltration.
INTRODUCTION ONE OF the key elements in understanding ocellar function in insects is the definition o f the supporting network of interneurons which convey information from the ocelli to the various integrative centers of the brain. The development and improvement o f the cobalt staining technique has made it possible to study pathways of interneurons in the brain (Goodman, 1974; Strausfeld and Obermayer, 1976). Most studies have concentrated on large relatively easy to see ocellar first order interneurons portraying a fairly simple version of ocellar connectivity (Goodman, 1974; G o o d m a n et al., 1975; Cooter, 1975; Bernard, 1976; Koontz, 1976; Pappas and Eaton, 1977). Recently, a more complex situation has been found in the locust, Schistocerca vaga, and honey bee, Apis mellifera, with the discovery of numerous connections between small ocellar interneurons and various integrative centers in the brain ( G o o d m a n and Williams, 1976; Heinzeller, 1976a). In the cabbage looper moth, T. ni, the receptor cell axons in the ocellar nerve synapse with first order interneurons in the dorsal protocerebrum (Dow and Eaton, 1976; Eaton and Pappas, 1977). This area is hereafter referred to as the synaptic region. Six to 7 large first order interneurons project from the synaptic region o f each ocellus to the ipsilateral and contralateral sides of the protocerebrum. In addition, a large number of small interneurons extend from the ocellar synaptic area to several areas of the brain of the cabbage looper moth (Pappas and Eaton, 1977). This report describes the results of studies o f the small ocellar interneurons of the cabbage looper moth and compares them to small interneurons of other insects. MATERIALS AND METHODS The cabbage looper moths used for this study came from a colony maintained at 25°C on an ambient * Supported by NSF Grant BMS 75-07645 and by the VPI & SU Research Division. 337
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JOHN L. EATONand I.kggv G. PAPPA$
photoperiod in the Department of Entomology at VPI and SU. The rearing methods have been described previously (Eaton, 1976). Cobalt chloride (250 mM) with BSA added was used to infiltrate ocelli (Strausfeld and Obermayer, 1976). The method of infiltration has been described elsewhere (Pappas and Eaton, 1977). Only one ocellus was infiltrated in each brain. After incubation for 4-6 hr the cobalt was precipitated with 0"570 ammonium sulfide in saline and the brains were fixed in alcoholic Bouin's, embedded in paraffin and sectioned at 15 wm. The cobalt in the sectioned brains was intensified by Timm's sulfide-silver method using freshly prepared solutions (Tyrer and Bell, 1974). Over 80 brains have been studied by this method. Photomontages prepared from photomicrographs of individual specimens have been used to reconstruct the interneuron tracts in the brain of tile cabbage looper moth. The terminology used to describe ocellar interneuron projections is that of Strausfeld (1976). Pearson's (1971) study of the sphinx moth also proved useful in the identification of brain areas. RESULTS Several filling methods and incubation times were tested in these studies of the cabbage looper moth ocellar interneurons. The best results, for whole mounts or for intensification by the Timm's sulfide-siWer method, have been obtained using cobalt solutions with added BSA and incubation times of 4-6 hr. Longer incubations appeared to diminish the intensity of the cobalt infiltrations. While not all preparations filled to the same degree, the fibers which did fill were consistent from preparation to preparation. Less complete infiltrations were useful for study of fibers near the fill site which would be obscured by more extensive fills. Correspondingly, the extensive infiltrations were advantageous for study of more distant fibers. In the more extensive infiltrations it is apparent that some second or third order interneurons were filled. However, most infiltrated cells are first order interneurons. Small ocellar interneurons connect the ocelli to several areas of the protocerebrum including the central body, corpora pedunculata and optic lobes. Other small ocellar interneurons join the ocellus and the deuto- and tritocerebrum. Accurate counts of fiber numbers were not possible because of the large number o f fibers present. The details of the pathways of these fibers follow.
Central body The interneurons to the central body arise from the ventral side of the ocellar synaptic region lateral to the anterior median tract (AM) (Figs. 1, 6). Cobalt fills o f one ocellus stain both the ipsilateral and contralateral central body tracts. The contralateral central body tract stains less intensely and cobalt presumably enters it through the fibers in the large ocellar interneuron tract extending to the contralateral ocellus (Pappas and Eaton, 1977). From the synaptic region the small interneuron tract passes anterolaterally to the anterior surface of the central body where fibers enter the dorsal edge of the fan-shaped body (fb) (Figs. 1, 2). The remaining fibers continue downward across the anterolateral surface of the superior arch where one group of fibers turns posteriorly to the ventral edge of the fanshaped body. The remaining fibers turn laterally to enter the lateral protuberance tract of the central body (Fig. 3). The fibers in these tracts are very small and while exact counts are FIo. 1. Reconstruction of small ocellar interneuron tracts in a 90-pm-thick saggital plane of brain adjacent to circumesophageal commissure and ipsilateral to infiltrated ocellus. Retouched. Antennal glomerular tract (AG), antennal lobe (AL), anterior median tract (AM), beta lobe (B), cell bodies (CB), central body (C), fan-shaped body (fb), superior arch (sa), subesophageal ganglion (SO), ocellar synaptic region (SR), tritocerebrum (T), trachea (Tr). x 223 FiG. 2. Small ocellar interneuron tracts in a 15-pm-thick saggital plane of the brain adjacent to circumesophageal commissure and contralateral to infiltrated ocellus. Antennal glomerular tract (AG), antennal lobe (AL), cell bodies (CB), central body (C), fan-shaped body (fb), synaptic region (SR), tritocerebrum (T). x 223
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JOHN L. EATONand LARRYG. PAPPAS
not possible, we estimate that there are about 20 fibers in each tract. The cell bodies o f these fibers are located in the pars intercerebralis.
Corpora pedunculata Small first order interneurons enter the corpora pedunculata via separate tracts from the synaptic region. Fibers in one tract enter the calyx (Fig. 6) and fibers in other tracts enter the alpha and beta lobes at the base of the stalk (Figs. 1, 3, 6). The interneurons projecting to the calyx arise from the synaptic region lateral to those of the central body. They pass ventrolaterally to the apex of the stalk of the corpora pedunculata. These fibers surround the stalk but do not appear to enter it. Branches from these fibers enter the calyx from the ventral side. We were unable to estimate fiber numbers in these tracts, The cell bodies o f these fibers are located on the dorsal edge o f the calyx in the pars intercerebralis. Fibers in both calyces fill from infiltration of a single ocellus, but the ipsilaterai side fills most intensely. The contralateral side fills via fibers in the large ocellar interneuron tract extending to the contralateral ocellus. Small interneurons running from the synaptic region to the base of the stalk arise anterior and lateral to those to the central body and pass to the alpha lobe. Five tracts are present. One tract arises from the tract to the central body and terminates on the medial edge of the beta lobe (Fig. 1). The 2 dorsal tracts enter the dorsal area of the alpha lobe from the posterior side. The 2 ventral tracts enter the ventral edge of the alpha lobe (Fig. 3). Cell bodies of these interneurons are located in the protocerebral cell body layer dorsal and anterior to the alpha lobe.
Optic lobe A tract composed of 2 or 3 small interneurons, arising from the tract of the large contralateral interneurons and passing to the ipsilateral lobula, was observed by Pappas a~d Eaton (1977). It is now known that there are ipsilateral and contralateral tracts passing to each lobula. These lobula tracts are revealed by single ocellar fills. They arise from the tracts of the large contralateral interneurons (Figs. 4, 6). These ipsilateral and contralateral lobula tracts, consisting of 2 or 3 fibers, pass through the posterior protocerebral neuropile on the ventral side of the corpora pedunculata and terminate on the medial side of their respective lobulas. It has not been possible to identify the cell bodies of these fibers. Two additional diffuse tracts pass laterally from the ocellar synaptic region through the dorsal protocerebral neuropile to the dorsal area o f the lobula. These fibers are restricted to the ipsilateral side in fills of single ocelli.
FIG. 3. Reconstruction of small ocellar interneuron tracts to alpha lobe in a 45-1am-thicksaggital plane beginning 90 lain from midline and ipsilateral to infiltrated ocellus. Alpha lobe (A), antennal lobe (AL), cell bodies (CB), central body (C), synaptic region (SR). x 223 FIG. 4. Reconstruction of a 90-pm-cross-sectional plane of the brain showing posterior view ipsilateral (IL) and contralateral (CL) lobula tracts from fill of right oceilus. A part of ipsilateral (Li) large interneurons is removed to reveal the underlying IL tract. Two lines (arrows) mark removed area. Cell bodies (CB), circumesophageal commissure (CC), contralateral large interneurons (Lc), protocerebrum (P), synaptic region (SR). x 125 FIG. 5. Cross section through tritocerebrum (T) showing infiltrated interneurons and cell bodies (CB) lying between tritocerebrum and antennal lobe (AL). × 236
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Optic tubercle Interneurons projecting to the optic tubercle arise from the lateral edge of the ocellar synaptic region and pass anteriorly to the posterior surface of optic tubercle. They pass under and over the tubercle and turn laterally to follow the optic tubercle to the lobula (Pappas and Eaton, 1977) (Fig. 6). Numerous filled cell bodies surround the optic tubercle. The fibers from these cells terminate in the edge of the optic lobe.
Deutocerebrum and tritocerebrum Interneurons pass to the deutocerebrum and tritocerebrum via the antennal glomerular tract and the anterior median tract. In the case of the fibers in the antennal glomerular tract both ipsilateral and contralateral tracts are infiltrated only when the ipsilateral ocellus is filled with cobalt. As was the case above, the ipsilateral side fills more extensively (Figs. 1, 2, 6). Small fibers from the ocellar interneuron tracts of the posterior slope enter the antennal giomerular tract at the level of the protocerebral bridge. They pass through the tract to the posterior surface of the antennal glomerulus where some fibers terminate. Other branches turn downward parallel to the anterior median tract and terminate in the dorsal tritocerebral neuropile. The cell bodies of these cells are in the cell body lobes of the posterior protocerebrum. The fibers of the anterior median tract are the most anterior group of fibers leaving the synaptic region. These fibers arise from the anterior median edge of the synaptic region and pass in an anterior-ventral direction between the protocerebral neuropile and the cell bodies of the anterior surface of the protocerebrum (Figs. 1, 2). The anterior median tract fibers continue downward to the posterior surface of the antennal lobe where fibers from the antennal glomerular tract turn downward parallel to those of the anterior median tract. Some fibers apparently terminate on the posterior surface of the antennal glomerulus and the remainder terminate in the dorsal neuropile of the tritocerebral lobes. Occasionall~ fibers which appear to be axons of the medial neurosecretory cells are observed. These fibers are few in number and are not consistently filled. Cell bodies of these fibers are in the pars intercerebralis. Two fibers in each frontal nerve are often filled by cobalt infiltration of one ocellus (Fig. 5). There are 2 possible pathways for these fills. The most common is through synaptic contact with filled fibers of the antennal glomerular tract or the anterior median tract. Another pathway which has been observed begins at the terminations of the large first order interneurons of the posterior slope of the protocerebrum (Pappas and Eaton, 1977). Fibers arise from ~his area and pass anteriorly through the ventral protocerebral neuropile to the tritocerebral lobes. The fibers in the frontal nerve must be at least second order fibers. Their cell bodies are in the frontal ganglion. DISCUSSION
From the results presented here, it is clear that the ocelli of moths communicate visual information to many integrative areas of the brain via their small interneurons. Most of these contacts are believed to be through first order interneurons, but a few may be second or third order interneurons. The addition of BSA to cobalt chloride solutions has been shown to enhance the ability of cobalt to cross synapses of functionally contiguous units and to fill the finest branches of neurons (Strausfeld and Obermayer, 1976). This method also proved to be highly effective for the interneurons of the cabbage looper and is to a
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FIG. 6. Illustration of frontal view of brain of cabbage looper showing small interneuron tracts revealed by infiltration of right ocellus. Alpha lobe (ct), antennal glomerular tract (AG), antennal lobe (AL), anterior median tract (AM), beta lobe (13),central body (C), contralateral lobula tract (CL), corpora pedunculata (CP), ipsilateral lobula tract (IL), lobula (L), Ocellar nerve (OcN), Optic tubercle (OT), pars intercerebralis (PI), subesophageal ganglion (SG), synaptic region (SR), Tritocerebrum (T). Bar equals 100 ~tm. large degree responsible for the large number of neurons filled. Also important, as pointed out earlier, is the time allowed for infiltration. In our hands the best fills were obtained after 4-6 hr of incubation. Leaving the cobalt to infiltrate for periods of 16-20 hr produced fills which were much less intense. Accordingly, it seems there is an optimal filling time for cobalt infiltration. One possible criticism of this work is that some of the interneuron fills could have been produced by leakage of cobalt into the extracellular space of the neuropile via the ocellar nerve. As pointed out above, examination of the area near the site of infiltration in preparations incubated for short time intervals where leakage was not significant reveals a partial infiltration of interneuron tracts which were more completely filled by longer incubation intervals where some leakage did occur. Since these tracts are partially visible after short incubation intervals, we believe that extracellular cobalt has not contributed to the more extensive fills of these same tracts seen after longer incubations. This view is supported by Strausfeld and Obermayer's (1976) study of transneuronal migration o f cobalt in flies. We would further point out that our incubation times are among the shortest which have been used for studies o f this nature. In other work in which comparable methods have been used to study the small interneurons of insects, the degree of filling obtained seems to have been substantially less than we obtained with the looper preparations ( G o o d m a n and Williams, 1976; Pan and Goodman, 1977). There are several possible explanations for this including use of cobalt chloride with added BSA, incubation interval, differences in the number o f interneurons in different insects, and filling o f second or higher order neurons in the moth preparations. Because of these different possibilities, comparison o f the ocellar interneurons in the looper
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and other insects must be approached cautiously at this time. If one considers the central body, the corpora pedunculata and the lobula of the optic lobe, it is clear that ocellar interneurons apparently pass to these structures in both the cabbage looper and the locust. There are, however, apparent differences in the pathways and in degree of innervation of these structures in these insects. For example, the fibers to the central body in the locust and the honey bee apparently enter via the protocerebral bridge. In the case of the looper these fibers pass over the anterior surface of the central body and enter through that pathway. Similarly, in the case of the corpora pedunculata the fibers apparently enter the calyx from the lateral ocellar tract in the locust and in the looper they enter the calyx from the ventral side near the stalk. Fibers to the corpora pedunculata have not been reported in the honey bee. In the looper there is also innervation of the alpha and beta lobes which has not been described in the locust or honey bee (Goodman and Williams, 1976; Heinzeller, 1976a). The locust and the honey bee have ipsilateral fibers extending from both lateral and medial ocellar tracts through the posterior protocerebrum to the lobula (Goodman and Williams, 1976; Heinzeller, 1976a). There are apparently homologous fibers in the looper which extend ipsilaterally to the lobula. The moth has an additional fiber extending to the contralateral lobula as well as other fibers which extend laterally through the dorsal protocerebrum from the ocellar synaptic region to the ipsilateral lobula of the looper. Such fibers have not been described in other insects at this time. The posterior fibers to the Iobula were also observed in the cockroach and in the house fly (Bernard, 1976; Strausfeld, 1976). Fibers extending anteriorly from the ocellar synaptic region to the optic tubercle in the looper have not been described in other insects. The anterior median tract fibers from the ocelli of the looper also have not been seen in other insects. Heinzeller (1976a) does indicate that the ocellar interneurons end among the medial neurosecretory cells of the honey bee and both Heinzeller (1976b) and BrousseGaury (1970, 1971) have indicated that the ocelli may affect the neurosecretory activity of the medial neurosecretory cells. While the association of the ocellar interneurons with th~ neurosecretory cells is not clearly shown in the cabbage looper, there is some evidence that it may exist. Some of the fibers which fill in the anterior median tract may be neurosecretory cell fibers. Certainly some of them are not, for they extend to the deuto- and tritocerebrum rather than into the tract of corpora cardiaca nerve l (NCCI). We have seen some faintly stained fibers in NCCI, but no complete fills have been observed. In the case of the antennal glomerular tract fibers, Goodman and Williams (1976) observed ocellar fibers entering the antennal glomerular tract in the locust, but were unable to determine that they went any further. In the cabbage looper these fibers extend through the antermal glomerular tract to innervate deutocerebral neuropile on the posterior surface of the olfactory glomeruli. Fibers from the antennal glomerular tract also extend into the dorsal area of the tritocerebrum. The filling of the frontal nerve in some of our ocellar infiltrations must represent at least a second order and perhaps a third interneuron fill. In these preparations, filled cell bodies are observed around the edges of the tritocerebrum. A cell body has also been located in the frontal ganglion which apparently belongs to the filled interneuron. This could represent a third order fiber. We also regularly observed a fill of the tegumentary nerve when filling the ocellar receptor cells. Pan and Goodman (1977) observed similar fills when they filled cut setae on the head of the honey bee. We have not cut setae and these fills are produced by cobalt entering the oceilus. We know that the tegumentary nerve comes into very close proximity with the ocellar receptor cell area, and it is possible that the tegumentary nerve fills due to
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m e m b r a n e d a m a g e caused by o s m o t i c i m b a l a n c e s t h r o u g h the a p p l i c a t i o n o f c o b a l t to the ocellus. In s u m m a r y , c o b a l t infiltration o f the ocellus in the c a b b a g e l o o p e r p r o d u c e s a m u c h m o r e extensive fill o f i n t e r n e u r o n s t h a n has been observed i n o t h e r insects. W h i l e some of these m a y be second or third order i n t e r n e u r o n s , we believe m o s t o f t h e m to be first o r d e r i n t e r n e u r o n s . W e also believe t h a t future studies will reveal m o r e ocellar i n t e r n e u r o n s in o t h e r insects when this i m p r o v e d m e t h o d is a d o p t e d a n d used for those insects.
REFERENCES
BERNARD,A. 1976. Etude topographique des interneurones ocellaires et de quelques uns de leurs prolongemerits chez Periplaneta americana. J. Insect Physiol. 22: 569-77. BROUSSE-GAURY, P. 1970. Les stimuli ocellaires par les neuros6cr6tions contr61ent les corpora allata des Blattes. Ann. Sci. Nat. Zool. Biol. Anita. Paris. 12: 61~. BROUSSE-GAURV,P. 1971. Description d'arcs r6flexes neuro-endocriniens partant des ocelles chez quelque orthopt6res Bull. Biol. Fr. Belg. 105: 84-93. COOTER, R. J. 1975. Ocellus and ocellar nerves of Periplaneta americana L. (Orthoptera: Dictyoptera). Int. J. bisect Morphol. Embryol. 4: 273-88. Dow, M. A. and J. L. EATON.1976. Fine structure of the ocellus of the cabbage looper moth (Trichoplusia ni). Cell Tissue Res. 171: 523-33. EATON,J. L. 1976. Spectral sensitivity of the ocelli of the adult cabbage looper moth, Trichoplusia ni. J. Comp. Physiol. 109: 17-24. EATON, J. L. and L. G. PAPPAS. 1977. Synaptic organization of the cabbage looper moth ocellus. Cell Tissue Res. 183: 291-97. GOODMAN, C. 1974. Anatomy of locust ocellar interneurons constancy and variability. J. Comp. Physiol. 95: 185-201. GOODMAN, C. and J. L. D. WILLIAMS.1976. Anatomy of the ocellur interneurons of acridid grasshoppers. II. The small interneurons. Cell Tissue Res. 175: 203-26. GOODMAN,L. J., J. A. PATTERSONand P. G. MOSBS. 1975. The projection of ocellar neurons within the brain of the locust, Schistocerca gregaria. Cell Tissue Res. 157: 467-92. HEINZELLER,T. 1976a. Second-order ocellar neurons in the brain of the honey bee (Apis mellifera). Cell Tissue Res. 171: 91-9. HEINZELLER,T. 1976b. Circadiane Anderung im endokrinen System der Honigbiene, Apis mellifera, Effekt yon Haft und Ocellenblendung. J. bisect Physiol. 22: 315-21. KOONTZ, M. 1976. Neuronal pathways from the dorsal ocelli of the house cricket, Acheta domesticus. J. MorphoL 149: 105-20. PAN, K. C. and L. J. GOODMAN.1977. Ocellar projections within the central nervous system of the worker honey bee, Apis mellifera. Cell Tissue Res. 176: 505-27. PAPPAS, L. G. and J. L. EATON. 1977. Large ocellar interneurons in the brain of the cabbage looper moth, Trichoplusia ni (Lepidoptera). Zoomorphologie 87: 237-46. PEARSON,L. 1971. The corpora pedunculata of Sphinx ligustri L. and other Lepidoptera. Phil. Trans. R. Soc. Lond. B. 259: 477-516. STRAUSFELD,N. J. 1976. Atlas of an Insect Brain. Springer, Berlin. STRAUSFELD,N. J. and M. OBERMAVER.1976. Resolution of intraneuronal and transynaptic migration of cobalt in the insect visual and central nervous systems. J. Comp. Physiol. 110: 1-12. TYRER,N. M. and E. M. BELL. 1974. The intensification of cobalt filled neuron profiles using a modification of Timm's sulfide-silver method. Brain Res. 73: 151-55.