An atlas and 3-D reconstruction of the antennal lobes in the worker honey bee, Apis mellifera L. (Hymenoptera : Apidae)

Int. J. InsectMorphol. & Embryol.. Vol. 18, No. 2/3, pp. 145-159, 1989 Printed in Great Britain

0020-7322/89 $3.00+ .130 Pergamon Press plc

A N ATLAS A N D 3-D RECONSTRUCTION OF THE A N T E N N A L LOBES IN THE WORKER H O N E Y BEE, APIS MELLIFERA L. ( H Y M E N O P T E R A • A P I D A E )

DANIEL FLANAGAN and ALISON R.

MERCER

Department of Zoology, University of Otago, Box 56, Dunedin, New Zealand

(Accepted28 February 1989)

model of the 3-dimensional structure of the antennal lobes in the brain of the worker honey bee, Apis mellifera L. (Hymenoptera : Apidae), has been constructed using retrograde cellular marking and computer reconstruction techniques. The model provides a pictorial representation of the spatial organization of structures within the antennal lobe neuropil. In addition, an atlas is provided to aid the identification of invariant morphological landmarks within the antennal lobe, some of which have been described previously. Readily identifiable invariant glomeruli are described in detail, and a hitherto unreported afferent tract of the antennal nerve, the tract "T2-2", is also identified. Precise descriptions of the arborizations patterns of several deutocerebral interneurones stained intracellularly with cobalt serve to illustrate the advantages of using the atlas and 3-dimensional representations of the antennal lobe represented in this paper. Abstract -- A

Index descriptors (in addition to those in title): Glomeruli, computer reconstruction, deutocerebral neurones.

INTRODUCTION THE ANTENNAL lobes, which lie in the deutocerebrum of the insect brain, represent the first point of interaction between incoming signals of antennal sensory neurones and the integrating processes of the central nervous system. The precise role of the antennal lobes in the processing of olfactory information remains unclear, although in recent years considerable progress has been made in determining the structure and physiology of neurones in the antennal lobes of insects, such as the sphinx moth, Manduca sexta ( M a t s u m o t o and Hildebrand, 1981; Christensen and Hildebrand, 1987), the cockroach, Periplaneta americana (Ernst and Boeckh, 1983; Burrows et al., 1982) and the honey bee, Apis mellifera ( H o m b e r g , 1984; Flanagan and Mercer, 1989). In the honey bee, antennal sensory neurones project into the antennal lobes in 4 m a j o r tracts, T1 to T4 respectively (Suzuki, 1975). The sensory neurones converge onto the dendrites of deutocerebral neurones, forming discrete spheres, or glomeruli, which surround a central feltwork of fibres at the core of each antennal lobe (see Pareto, 1972; Suzuki, 1975; Mobbs, 1982, 1984; Masson, 1982; Masson and Arnold, 1987). Arnold et al. (1985) have shown that m a n y of the glomeruli in the antennal lobes of the honey bee satisfy criteria used to identify glomerular invariance, and there is some evidence to suggest that glomeruli m a y represent functional units within the antennal lobes (Arnold et al., 1985; 145

146

D. FLANAGANand A. R. MERCE~

Chambille et al., 1980). Information on functional aspects of olfactory glomeruli relates principally to macroglomerular complexes (MGC) found in the antennal lobes of the males of some insect species (Jawlowski, 1948, 1954; Boeckh et al., 1977; Sanes et al., 1977; Boeckh and Boeckh, 1979; Chambille et al., 1980; Arnold et al., 1984; Koontz and Schneider, 1987). There is good evidence that MGCs are involved in processing signals from receptors sensitive to female pheromone (e.g. Boeckh and Boeckh, 1979; Matsumoto and Hildebrand, 1981; Burrows et al., 1982; Beockh and Ernst, 1987), but very little is known about the functional roles of glomeruli other than the sex-specific MGCs. An important prerequisite to determining the roles of non-MGC glomeruli in the antennal lobe neuropil is the identification of invariant glomeruli that can be easily recognized from one animal to the next of the same species. The large number of glomeruli in the antennal lobes of the honey bee (Arnold et al., 1985) makes visualization of the spatial organization of the antennal lobe and identification of specific giomeruli in this insect difficult. In order to overcome this problem, the present investigation has used computer reconstruction techniques to provide detailed representations of the 3dimensional structure of the antennal lobes of the worker honey bee and presents also a serial atlas of the antennal lobe that can be used in conjunction with the 3-dimensional reconstructions to locate invariant glomeruli that can be readily identified from one worker bee to the next. The arborization patterns of single cells within the antennal lobes of the worker bee are described in detail using the atlas and 3-dimensional representations presented in this paper.

MATERIALS

AND

METHODS

Cobalt impregnation Worker bees were captured at the entrance of a hive. The bees were fed ad libitum, chilled and mounted in metal tubes, their heads held in place with tape. The left antenna was cut at the level of the 5th segment of the flagellum and inserted into a fine glass tube filled with 0.2 M hexaminecobaltic chloride. A seal was made between the antenna and the tube with petroleum jelly. The bee was left at room temperature for between 16 and 24 hr. The head was then removed and a small window was cut in the frontal surface of the head capsule. The brain was placed in approximately 0.2% ammonium sulphide for 4 min to induce cobalt precipitation. After overnight fixation in alcoholic Bouin, the brain was removed from the head capsule, dehydrated and embedded in wax. Serial sections (10 p~m thick) of cobalt impregnated brains were cut in the 3 major planes (frontal, sagittal, and horizontal) and the sections intensified according to the Tyrer and Bell modification of the Timm's method (Tyrer and Bell, 1974). The best of the frontal sections were used for computer reconstruction purposes (see below). Computer acquisition and reconstruction o f serial sections Serial sections were photograped using a Zeiss Axiophot microscope. For the purposes of reconstruction, the antennal lobe was divided into 3 major components: the peripheral rind of cell bodies, the glomeruli, and the branches of the antennal nerve. Each component of the antennal lobe was traced individually from the photographs using a Hipad Digitizer drawing tablet and the information was fed into an Apple Macintosh Plus microcomputer. Sections were acquired serially, each section being carefully aligned with the section before it. This was repeated until the entire antennal lobe was digitalized. The programme used for 3-D reconstructions was written by Dr Yves Usson and kindly supplied to us for the purpose of 3-D reconstruction of the bee brain. The programme allows the computer to redraw the sections, or any given component of the sections, at any angle. In this way, a 2-dimensional representation of the 3-dimensional structure is produced. For the identification of individual glomeruli in the antennal lobes of the worker bee, each glomerulus is ranked according to its x, y, z coordinates and numbered accordingly, an approach similar to that used for the numbering of invariant glomeruli by Rospars (1983) in the moth, Mamestra brassicae and the butterfly, Pieris brassicae, and by Chambille et al. (1980), Chambille and Rospars (1981, 1985) and Rospars and Chambille (1986) in the cockroach, Blaberus craniifer Burm. Numbers that have already been assigned to invariant giomeruli in the worker honey bee (see Arnold et al., 1985) have, where possible, been retained in this paper.

Antennal Lobes of Apis mellifera Workers

147

In contrast to earlier reports, however, the glomeruli are named according to their position relative to the 4 major tracts of afferent fibres that project into the antennal lobe, namely T1, T2, T3, and T4 rather than on the basis of their general position within the antennal lobe neuropil (dorsal, ventral, intermediate, posterior; see Arnold et al., 1985). In the current investigation, all glomeruli that receive input from the T1 bundle, for example, are referred to here as T1 glomeruli. In some cases, the glomeruli are further classified according to the particular subdivisions of the major tract of sensory neurones from which they receive input. For example, there are 7 subdivisions of the T4 tract (T4-1 to T4-7 respectively). A glomerulus receiving input from the T4-1 subdivision of the T4 tract is named accordingly and numbered using the ranking system described above (e.g. T4-1(1)). It should be noted, however, that the methods used in this study do not exclude the possibility that some glomeruli may receive input from more than one tract.

Routine histology and staining of single cells Brains prepared for routine histology were fixed in alcoholic Bouin, dehydrated and embedded in wax. Serial sections (10 p~m thick) were stained using the Bodian protargol technique described by Gregory (1980). For examination of single cells within the antennal lobe, glass microelectrodes filled with 0.2 M hexaminecobaltic chloride were used to impale and stain the cells. Intracellular injection of cobalt was achieved by passing pulses of positive current (40 nA) through the cobalt filled electrodes for 4 min. The stain was developed in 0.2% ammonium sulphide and intensified according to the Tyrer and Bell (1974) modification of the Timm's method. Histological sections were prepared as described by Flanagan and Mercer (1989).

Single cell reconstruction Histological sections were viewed using phase-contrast optics which provided sufficient detail to identify glomerular profiles. Only cells closely associated with easily identifiable glomeruli or landmarks were reconstructed in this investigation. Single cells identified in this study were drawn into a 3-dimensional representation of the deutocerebrum. This was done either manually or with the assistance of the computer reconstruction programme. For computer reconstruction, serial photographs of the histological sections were traced onto the digitizing tablet connected to the Apple Macintosh Plus microcomputer. Cell profiles thus acquired can be rotated by the computer to provide a view from any angle.

RESULTS T h i r t y b e e s w e r e u s e d in t h e c o b a l t backfill p r e p a r a t i o n s a n d 3 o f the p r e p a r a t i o n s w e r e e x a m i n e d in specific d e t a i l in this study. T h e largest v a r i a t i o n in n u m b e r s of g l o m e r u l i was f o u n d in t h e T3 r e g i o n . T h r e e b r a i n s cut in the f r o n t a l p l a n e w e r e u s e d to e x a m i n e in d e t a i l i n v a r i a n c e in t h e d i s t r i b u t i o n o f g l o m e r u l i within t h e a n t e n n a l l o b e n e u r o p i l . G l o m e r u l i in t h e a n t e n n a l l o b e s of all 3 b r a i n s w e r e f o u n d to b e closely homologous.

Computer reconstruction o f the antennal lobe A c o m p u t e r r e c o n s t r u c t i o n of o n e o f t h e 3 c o b a l t backfill p r e p a r a t i o n s e x a m i n e d in d e t a i l is p r e s e n t e d in Fig. 1. F e a t u r e s h i g h l i g h t e d in t h e r e c o n s t r u c t i o n s can b e r e a d i l y i d e n t i f i e d in t h e a n t e n n a l l o b e s o f m o s t w o r k e r h o n e y bees. In Fig. 1, t h e left a n t e n n a l l o b e is v i e w e d l a t e r a l l y f r o m b o t h t h e r i g h t - h a n d side (Fig. l a ) to show the m e d i a l surface o f t h e l o b e , a n d f r o m t h e l e f t - h a n d side (Fig. l b ) to show t h e l a t e r a l surface o f t h e lobe. C o m p u t e r r e c o n s t r u c t i o n s of sections c o n t a i n i n g i m p o r t a n t l a n d m a r k s a n d r e a d i l y i d e n t i f i a b l e i n v a r i a n t g l o m e r u l i (Figs 2 - 4 ) p r o v i d e a serial atlas of the a n t e n n a l l o b e in the w o r k e r h o n e y b e e . T h e atlas aids t h e i d e n t i f i c a t i o n o f i n v a r i a n t g l o m e r u l i in serial s e c t i o n s , i r r e s p e c t i v e of t h e histological t e c h n i q u e u s e d to p r e p a r e t h e tissue. In using the atlas, h o w e v e r , a l l o w a n c e m u s t be m a d e for d i f f e r e n c e s in p l a n e of s e c t i o n b e t w e e n preparations.

Morphological landmarks in the antennal lobe I n t h e a n t e n n a l l o b e s o f t h e h o n e y b e e , c e r t a i n " l a n d m a r k s " , m o s t o f which h a v e b e e n

148

D. FLANAGANand A. R. MERCER

D

V

FIG. la, b. Computer reconstruction of left antennal lobe in brain of worker honey bee. Left antennal lobe is viewed anteriorly from (a) 60 ° to right and (b) 60 ° to left of a direct frontal aspect. Major divisions of deutocerebral neuropil are explained in key provided. Easily identifiable invariant glomeruli are numbered in the reconstructions. Whole-brain reconstructions above each figure indicate position of antennal lobes in brain of honey bee and can be used for orientation purposes. A = anterior; D = dorsal; L = left; l.p. = lateral passage; P = posterior; R = right; V = ventral. Scale = 100 ~m.

Antennal Lobes of Apis melliferaWorkers

149

i:i:i:

iii:

TI Glomeruli

•

T2 Glomeruli T3 Glomeruli T4 Glomeruli

I

I

50 ~ m

I0 pm

d

40 ~.m

70 ~m

FIGS 2-4. An atlas of antennal lobe. A series of computer reconstructions of frontal sections of antennal lobe taken at increasing depths beneath frontal surface of brain. Glomeruli are numbered according to their ranked position and an asterisk is used to indicate readily identifiable invariant glomeruli. Because of space limitations, the regional prefix (e.g. "T1"(33)) has been omitted from T1 and T3 glomeruli. 2a~l. Serial reconstructions of sections taken at depths of 10, 40 and 70 p,m from surface of antennal lobe. An indication of depth is given below computer reconstruction of each section. Region in which particular glomeruli are located is indicated by shading whose explanation is given in Fig. 2a.

d e s c r i b e d p r e v i o u s l y ( P a r e t o , 1972; Suzuki, 1975; M o b b s , 1982, 1984; A r n o l d et al., 1985), can b e u s e d to i d e n t i f y specific g l o m e r u l i .

Major regions of the antennal lobe. T h e a n t e n n a l l o b e can b e d i v i d e d i n t o 4 r e g i o n s b a s e d u p o n t h e divisions o f t h e a n t e n n a l n e r v e within t h e a n t e n n a l lobe. O n e o f t h e l a r g e r r e g i o n s is t h e v e n t r a l l y p l a c e d T 3 - r e g i o n receiving i n p u t f r o m t h e tract T3 (see Fig. l a , b). I n t h e p r e s e n t i n v e s t i g a t i o n , this r e g i o n was f o u n d to conthin b e t w e e n 72 a n d 77 g l o m e r u l i . T h e o t h e r large r e g i o n o f t h e a n t e n n a l l o b e is t h e d o r s a l l y l o c a t e d T l - r e g i o n

150

D. FLANAGANand A. R. MERCER II

•~i~/~ii- (

100 I.tm

160 pm

130 pm

170 lira

FIG. 3a-d. Serial reconstructions of sections taken at depths of 100, 130, 160, and 170 Ixm from surface of antennal lobe. Key and scale bar provided in Fig. 2a also applies to these sections.

innervated by the T1 tract. This region contained between 66 and 69 glomeruli among the bees examined in this study. The T2-region consists of a narrow band of glomeruli on the medial surface of the lobe (Fig. la) and the T4-region is located at rear of the lobe (Figs la, b; 7a). There are 7 invariant T2 glomeruli and 7 invariant T4 glomeruli in the antennal lobe of the worker bee. The T2- and T4-regions of the antennal lobe are innervated by the T2 and T4 divisions of the antennal nerve respectively.

The lateral passage. The antennal lobe has a peripheral rind of cell bodies, whose largest concentration is the lateral population located around the lateral surface of the

Antennal Lobes of Apis mellifera Workers

151

b

-~4)*

l.p. ~

~

•

220 ~m

190 pm

¢

d

J 2,30 Mnl

f

250 pm

FIG. 4a-d. Serial reconstructions of sections taken at depths of 190, 220, 230, and 250 ~m from

surface of antennal lobe. Key and scale bar provided in Fig. 2a also applies to these sections. lobe (Suzuki, 1975; Mobbs, 1982; Arnold et al., 1985). The majority of these cells send processes into the central neuropil of the lobe through a disjunction in the arrangement of the glomeruli, referred to here as the lateral passage (Fig. lb). The lateral passage is a narrow oval passage that runs along the lateral face of the lobe with a m a x i m u m dorsoventral width of about 50 ~ m and an anteroposterior length of about 120 I~m. It begins about 100 Izm f r o m the frontal surface of the lobe (Fig. 3a) and extends to the T4region, at a depth of about 220 ~m. In frontal sections, the lateral passage appears as a b r e a k in the circle of glomeruli (Figs 3a-d, 4a; 5b). It is a useful landmark, not only because it is obvious in all preparations regardless of the stain used, but also because it

152

D. FLANAGANand A. R. MERCER

4-a~

FIc. 5a-e, a. Frontal section of a cobalt filled antennal lobe at a depth of 230 p,m shows medial portion of a useful landmark, crescent tract (arrow). Cell bodies of deutocerebral neurones (dn) surround neuropil of antennal lobe. Five T4 glomeruli can be identified in this section and are n u m b e r e d as follows: 1 represents glomerulus T4-1(1); 2 represents glomerulus T4-2(1); represents

Antennal Lobes of Apis meUiferaWorkers

153

separates the T3-region from the Tl-region on the lateral side of the lobe (Fig. lb). The lateral passage is delineated by 4 T1 glomeruli and 3 T3 glomeruli. From front to back these are Tl(18), T I ( l l ) , Tl(7) and Tl(4) on the dorsal side and T3(4), T3(6) and T3(2) on the ventral surface of the lateral passage (Fig. lb). Sometimes an extra T3 glomerulus is observed near the rear of the lateral passage. The crescent tract. At the back of the antennal lobe is a network of fibres, the T4 network. Within this network is a cresent shaped tract that runs beneath most of the T4 glomeruli (Fig. 5a). The fibres in this tract are large (2-4 Ixm) and make the tract readily identifiable with a variety of stains. The crescent tract can be used to identify at least 2 of the T4 glomeruli because it originates between T4-2(1) and T4-3(1) (Fig. 7a). Identifiable glomeruli Within the antennal lobe, there are a number of glomeruli that are easy to distinguish because of their size, shape or location. It is possible to identify other less exceptional glomeruli by their position relative to these particular landmarks. Arnold et al. (1985) have already described some of these glomeruli, and we adhere to their numbering system where possible. The T1(44) glomerulus. This is the largest glomerulus in the worker brain, (Arnold et al., 1985; referred to as D44 in that study), and can be found at the most dorsal surface of the lobe at a depth of about 160 I~m (Fig. 3c). It is possible to identify other glomeruli by their position relative to this obvious landmark. Two comparatively large glomeruli are associated with T1(44). Immediately posterior to T1(44) is the glomerulus T1(32) (Fig. 4a) and lateral to T1(44) is T1(13) (Fig. 3d). The T2-1(2) glomerulus. In the T2 region, about 190 txm from the frontal surface of the brain, there is a relatively large, roughly spherical glomerulus (T2-1(2)) that is notable because it is displaced inwards from the ring of glomeruli (Fig. 4a). This glomerulus is one of the most obvious glomeruli to be invaded by the T2-1 subdivision of the T2 tract (Fig. 5c). Arnold et al. (1985) call this the I2 glomerulus (I = intermediate). T2-1(2) is invariant and provides a valuable landmark. Most of the remaining T2 glomeruli can be identified by their position relative to T2-1(2). They lie almost in a straight line beside T2-1(2) on the medial side of the lobe, with T2-1(2) situated in the

glomerulus T4-4(1) and 5 represents glomerulus T4-5(1). Scale = 100 I~m. b. Silver stained frontal section of antennal lobe showing main entry point, or lateral passage (lp), of deutocerebral neurones into feltwork of antennal lobe. Identifiable glomerulus T2-1(2) is indicated (arrow). Scale = 100 p.m.c. Frontal section of a cobalt filled antennal lobe at a depth of 160 p.m showing 3 major branches of antennal nerve, namely T1 (single arrowhead), T2-1 (double arrowhead) and T3 (arrow). T2-I tract can be seen to invade 3 T2-1 glomeruli which are numbered as follows: 2 representing glomerulus T2-1(2); 4 representing glomerulus T2-1(4) and 5 representing T2-1(5). Three other identifiable glomeruli are present in this section and are represented by 6 for glomerulus T3(6); 7 for glomerulus Tl(7) and 44 for glomerulus T1(44). Scale = 100 p.m.d. Frontal section of a cobalt filled antennal lobe at a depth of 110 p.m showing newly identified tract T2-2 (arrow). This tract appears to innervate a single glomerulus T2-2(1) (arrowhead). Scale = 100 i~m. e. A sagittal section through antennal lobe of worker bee stained using Bodian protargol method. T1 tract (arrow) and T3 tract (double arrowhead) are clearly visible. Group of small glomeruli called ventral lobule (vl), which is innervated by T3 tract, is also apparent. Scale = 100 ixm.

154

D. FLANAGANand A. R. MERCER

dl

ma

f"'~

FIG. 6a~d. a. Reconstruction of a neurone that intensely invades the identifiable glomerulus T2-1(6) (arrowhead). This cell has a heterogeneous distribution of arborizations within a number of glomeruli and its cell body is located in frontal portion of lateral population. Scale bar = 100 ixm. b. A partial reconstruction of an AGT neurone that has a concentration of processes in T2-1(3) glomerulus (arrowhead). Cell body of this neurone is located in medial population. Scale bar = 100 I~m. c. A partial reconstruction of an AGT neurone that invades the T2-1(5) glomerulus (arrowhead). Cell body of this neurone is also located within medial population. Scale bar = 100 i~m. d. A partial reconstruction of an AGT neurone that invades T4-1(1) glomerulus (arrowhead). Cell body of this neurone is located in lateral population. Scale bar = 100 ~xm. a p p r o x i m a t e m i d p o i n t o f this line (Fig. l a ) . T h e T2-1(5) g l o m e r u l u s lies slightly a n t e r i o r a n d m e d i a l to T2-1(2), w h e r e a s T2-1(4) is v e n t r a l to T2-1(5) a n d p l a c e d v e n t r o m e d i a l l y to T2-1(2) (Figs 3, 4). H a v i n g i d e n t i f i e d the T2 g l o m e r u l i t h e s e can be used to d i v i d e t h e l o b e r o u g h l y into t h e T1 r e g i o n , which lies d o r s a l to t h e T2 g l o m e r u l i , a n d t h e T3 r e g i o n w h i c h is s i t u a t e d v e n t r a l l y (Fig. l a ) .

The T2-2(1) glornerulus. T2-2(1) is an eUiptically s h a p e d g l o m e r u l u s just a n t e r i o r to t h e T2-1(2) g l o m e r u l u s d e s c r i b e d a b o v e . L i k e T2-1(2), the T2-2(1) g l o m e r u l u s is d i s p l a c e d slightly into t h e c e n t r e o f the a n t e n n a l l o b e n e u r o p i l (Fig. 3a, b). H o w e v e r , T22(1), which is a b o u t 120 Ixm d e e p , is n o t as large as T2-1(2), a n d T2-2(1), as its n a m e

Antennal Lobes of Apis mellifera Workers

155

I FIG. 7a, b. a. Computer reconstruction of left deutocerebrum in brain of worker bee viewed from a posterior aspect. T4 glomeruli are clearly shown in this reconstruction as are some of T2 glomeruli. Major divisions of lobe are explained in key provided for Fig. 1 and reconstruction of whole brain at top of figure is provided for orientation purposes. A = anterior; D = dorsal; Left = left; P = posterior; R = right; V = ventral. Scale bar = 100 v,m. b. A reconstruction of a neurone with a process in crescent tract (double arrowhead) at rear of antennal lobe. cb = cell body.

suggests, is innervated by the T2-2 subdivision of the T2 tract. T h e T2-2 tract, which has not b e e n r e p o r t e d previously, is a thin b r a n c h (2-5 ~ m in diameter) that projects about 20 Ixm anterior to the main T2 tract in the central feltwork of the antennal lobe (Fig. 5d). Lying dorsolateral to T2-2(1), is a n o t h e r identifiable glomerulus, T l ( 5 1 ) , which lies at the inner margin of the fan region of the T1 tract (Fig. 3b).

The T4 glomeruli. T h e 7 T4 glomeruli are, on average, the largest in v o l u m e in the antennal lobe and are supplied by 7 distinct branches of the T4 tract, T4-1 to T4-7 respectively. T h e y are situated at the back of the lobe (Fig. l b ; 4b--d). All but 2 of t h e m , T4-2(1) and T4-7(1)), lie a b o v e the crescent tract and the T4 network. T4-7(1) is the m o s t posterior glomerulus in the antennal lobe and lies behind the T3 glomeruli. T w o large T1 glomeruli can be located using the positions of the T4 glomeruli. T1(58) lies dorsal to T46(1) (Fig. 4b, c) and T l ( 4 1 ) is lateral to T1(58) and dorsal to T4-5(1) (Fig. 4b).

156

D. FLANAGANand A. R. MERCER

The ventral lobule. The ventral lobule (Arnold et al., 1985) is a group of between 10 and 20 small T3 glomeruli just ventral to the T1 bundle as it enters the lobe (Fig. 5e). The number of glomeruli within this region of the antennal lobe varies (Arnold et al., 1985; present investigation), therefore it is difficult to identify invariant glomeruli in this region of the antennal lobe. Single cell morphology Identification of single cells with characteristic arborization patterns within the antennal lobes offers the potential for examining the physiology of specific cell types within the antennal lobe neuropil and the functional roles of the glomeruli invaded by such cells. The serial atlas and 3-D reconstructions of the antennal lobes (Figs 1-4) can be used to identify and describe the morphology of single neurones in the deutocerebrum of the honey bee. The serial atlas has proved critical for the identification of invariant glomeruli invaded by cells stained intracellularly with cobalt (present investigation; Flanagan and Mercer, 1989). Figure 6 is a reconstruction of a neurone which has a heterogeneous distribution of arborizations that are restricted to the antennal lobe neuropil. The cell has a concentration of arborizations within the T2-1(6) glomerulus (Fig. 4a) and invades a number of other glomeruli less intensely. This cell can be classified as an AL(M)He(T2-1(6)) neurone according to the classification system suggested by Flanagan and Mercer (1989). The arborizations of 3 neurones that intensively invade single glomeruli and project to the calyces of the mushroom bodies in the protocerebrum of the brain via the inner antenno-glomerular tract (AGT), are reconstructed in Fig. 6b-d. The neurone reconstructed in Fig. 6b intensely invades the T2-1(3) glomerulus (see Fig. 4b) and is classified as an iAGT(T2-1(3)) neurone. Figure 6c shows the reconstruction of a neurone that intensely invades the T2-1(5) glomerulus (see Figs la; 3c) and is classified as an iAGT(T2-1(5)) neurone. The neurone reconstructed in Fig. 6d intensely invades the T41(1) glomerulus (see Fig. 4b) and is classified as an iAGT(T4-1(1)) neurone. Not all cells can be identified on the basis of their glomerular projections. However, some neurones can be categorized according to other distinctive morphological features. The neurone in Fig. 7, for example, is readily identifiable because of the location of one of its major branches in the crescent tract, described earlier. Neurones of this type, which have processes in both the dorsal lobe and the antennal lobe neuropil (D(M)Ho neurones; Flanagan and Mercer, 1989) as well as projections in the crescent tract are classified as D(M)Ho;c neurones. The D(M)Ho;c neurone reconstructed in Fig. 7b is viewed from a posterior aspect. A reconstruction of the antennal lobe is included for orientation purposes (Fig. 7a).

DISCUSSION The serial atlas and 3-D reconstructions of the antennal lobe presented in this paper provide important tools for detailed investigation of the processing of olfactory signals in the brain of the worker honey bee. The serial atlas, prepared from sections of a single brain, does not represent a general mathematical model of the antennal lobe such as those established for Mamestra brassicae (Rospars, 1983) and Blaberus craniifer (Chambille et al., 1980), but does provide a model from which generalizations can be made. Precise calculations of glomerular coordinates and the degree of rotation of the sectioning plane are not required in order to use effectively the atlas and 3-D representations presented in this paper. The atlas can be used to determine the precise

Antennal Lobesof Apis mellifera Workers

157

locations of invariant glomeruli in the antennal lobe of the worker bee, and can be used to describe the arborization patterns of single neurones identified in the antennal lobe neuropil. As shown in the present investigation, it is now possible to identify specific glomeruli invaded by single cells stained intracellularly using electrophysiological techniques (e.g. Figs 6; 7). Identification of invariant glomeruli is aided greatly by the structural consistency of the deutocerebral neuropil in insects. In Periplaneta americana and Locusta migratoria (Ernst et al., 1977), Polistes gallicus (Masson and Strambi, 1977), Camponotus vagus and Mesoponera caffraria (Masson, 1972) the branches of the root of the antennal sensory nerve provide invariant reference points that can be used for orientation purposes within the antennal lobe. The same is true in the honey bee. The nomenclature introduced by Suzuki (1975) to describe the major divisions of the antennal nerve in the honey bee (T1T6) is now widely adopted in the literature (e.g. Mobbs, 1982, 1984; Arnold et al., 1985; Masson, 1987). The minor amendments to this classification made by Arnold et al. (1985), relating in particular to the precise nature of tracts T2 and T4, are confirmed in the present study. The previously unreported T2-2 tract also identified in the present investigation provides another useful landmark for the localization of glomeruli in the T2 region of the lobe, in particular, the invariant glomerulus T2-2(1). Classification of glomeruli, where possible, according to the subdivisions of the major tracts of sensory neurones from which they receive input, ensures precise identification as well as a clear indication of the spatial organization of the glomeruli within the antennal lobe neuropil. Like Arnold et al. (1985), we have found little variation between individuals in the numbers of glomeruli innervated by each of the 4 tracts of sensory neurones that project into the antennal lobe of the worker honey bee. Arnold and his co-workers suggest that approximately one-third of the glomeruli are invariant. We have specifically identified 27 of the invariant glomeruli (Figs 1-4). Some variation in the number and distribution of glomeruli is apparent in the antennal lobes of the honey bee. In the present investigation 66-69 glomeruli were found in the T1 region of the antennal lobe of the worker honey bee, whereas Arnold et al., (1985) counted 71 glomeruli in this region. Similarly 72-77 glomeruli were identified in the T3 region of the antennal lobe in the present investigation, compared with between 82 and 90 glomeruli identified in that region by Arnold et al. (1985). However, Arnold et al. (1985) found that some variation in glomerular numbers occurs especially between individuals from different hives, which may explain the differences between our findings and those of Arnold and his co-workers for the T1 and T3 regions of the antennal lobes (dorsal-anterior and ventral regions respectively according to the nomenclature used by Arnold et al., 1985). Such differences lead to problems when glomeruli in these regions of the antennal lobe are numbered, especially if the numbering system is based on the ranking of glomeruli according to their x, y, z coordinates. We suggest that numbers that have already been used in the literature to describe invariant glomeruli be retained, and that they be used as "names" rather than specific indications of ranking. Numbers given to glomeruli that do not appear to be invariant can be adjusted accordingly. It has been suggested that glomeruli represent the functional subunits of the olfactory neuropil (Mobbs, 1982; Stocker et al., 1983; Arnold et al., 1985; Christensen and Hildebrande, 1987; Masson and Arnold, 1987). The identification of invariant glomeruli is the essential first step for investigating this question. There is good evidence that the MGCs identified in the antennal lobes of the males of many insect species are the

158

D. FLANAGANand A. R. MERCER

p r i m a r y site f o r p r o c e s s i n g o f i n f o r m a t i o n f r o m s e x - s p e c i f i c p h e r o m o n e s e n s i t i v e r e c e p t o r s , a n d t h a t M G C s a r e f u n c t i o n a l l y d i s t i n c t f r o m all o t h e r g l o m e r u l i in t h e a n t e n n a l l o b e s o f t h e s e i n s e c t s . It c a n n o t b e a s s u m e d , h o w e v e r , t h a t b e c a u s e t h e s e h i g h l y s p e c i a l i z e d s e x - s p e c i f i c g l o m e r u l i a p p e a r to f u n c t i o n as i n d e p e n d e n t units, t h a t this is t y p i c a l o f all g l o m e r u l i w i t h i n t h e a n t e n n a l l o b e s o f t h e i n s e c t b r a i n . D e t a i l e d examination of the physiology of neurones with projections into identifiable glomeruli, o t h e r t h a n M G C s , s h o u l d p r o v i d e insight i n t o t h e f u n c t i o n s o f i n v a r i a n t g l o m e r u l i in t h e a n t e n n a l l o b e s o f t h e w o r k e r h o n e y b e e . T h e atlas p r e s e n t e d in this p a p e r s h o u l d p r o v i d e a valuable tool for such studies.

Acknowledgements--We acknowledge the expert advice and help of Mr Gerald Stokes and Mr Douglas Sanderson.

REFERENCES ARNOLD, G., C. MASSONand S. BUDHARUGSA.1984. Demonstration of a sexual dimorphism in the olfactory pathways of the drones of Apis mellifica L. (Hymenoptera, Apidae). Experientia 40: 723-25. ARNOLD,G., C. MASSONand S. BUDHARUGSA.1985. Comparative study of the antennai lobes and their afferent pathway in the worker bee and the drone (Apis Mellifera). Cell Tissue Res. 242: 593-605. BOECrH, J. and V. BOECrH. 1979. Threshold and odor specificity of pheromone-sensitive neurones in the deutocerebrum of Antheraea pernyi and A. polyphemus (Saturnidae). J. Comp. Physiol. 132: 235-42. BOECKH, J. and K. D. ERNST. 1987. Contribution of single unit analysis in insects to an understanding of olfactory function. J. Comp. Physiol. A 161: 549-65. BOECKH, J., V. BOECKH,and A. KUHN. 1977. Further data on the topography and physiology of central olfactory neurones in insects, pp. 315-21. In J. LE MAGNENand P. MACLEOo(eds) Olfaction and Taste VI. I.R.L., London. BURROWS,M., J. BOECKHand J. ESSLEN. 1982. Physiological and morphological properties of interneurones in the deutocerebrum of male cockroaches which respond to female pheromone. J. Comp. Physiol. 145: 447-57. OtaMaIeLE, I. and J. P. ROSr'ARS. 1981. Deutocerebrum de la blatte Blaberus craniifer Burm. (Dictyoptera : Blaberidae): 6tude qualitative et identification morphologiques des glom6rules. Int. J. Insect Morphol. Embryol. 10: 141--65. CHAMaILLE, I. and J. P. ROSPARS. 1985. Neurons and identified glomeruli of antennal lobes during post embryonic development in the cockroach Blaberus craniifer Burm. (Dictyoptera : Blaberidae). Int. J. Insect Morphol. Embryol. 14: 203-26. CHAMalLLE, I., C. MASSONand J. P. ROSPARS. 1980. The deutocerebrum of the cockroach Blaberus craniifer Burm. Spatial organization of the sensory glomeruli. J. Neurobiol. 11: 135-57. CHRISTENSEN,T. A. and J. G. HILDEBI~ANt).1987. Male specific, sex pheromone-selective projection neurones in the antennal lobes of the moth Manduca sexta. J. Comp. Physiol. A 160: 553--69. ERNST, K. D. and J. BOECKr~. 1983. A neuroanatomical study on the organization of central antennal pathways in insects. III. Neuroanatomical characterization of physiologically defined response types of deutocerebral neurons in Periplaneta americana. Cell Tissue. Res. 229: 1-22. ERNST, K. D., J. BOECKrt and V. BOECKH. 1977. A neuroanatomical study on the organization of central antennal pathways in insects. II. Deutocerebral connections in Locusta migratoria and Periplaneta americana. Cell Tissue Res. 176: 295-308. FLANAGAN, D. and A. R. MERCER. 1989. Morphology and response characteristics of neurones in the deutocerebrum of the honeybee Apis mellifera. J. Comp. Physiol. A 164: 483-94. HOMBERG,U. 1984. Processing of antennal information in extrinsic mushroom body neurons of the bee brain. J. Comp. Physiol. A 154: 825-36. GREGORY,G. E. 1980. The Bodian protargal technique, pp. 75-95. In N. J. STRAtJSFELDand T. A. MILLER(eds) Neuroanatomical Techniques Insect Nervous System. Springer, New York. JAWLOWS~X,H. 1948. Studies on the insect's brain. Ann. Univ. Mariae Curie-Sklodowska (C) 3: 1-30. JAWLOWSKI,H. 1954. Uber die Struktur des Gehirnes bei Saltatoria. Ann. Univ. Mariae Curie-Sklodowska (C) 11: 403-34. KOONTZ, M. A. and D. SCHNEIDER.1987. Sexual dimorphism in neuronal projections from the antennae of silk moths ( Bombyx mori, Antheraea polyphemus) and the gypsy moth (Lymantria dispar.). Cell Tissue Res. 249: 39--50.

Antennal Lobes of Apis mellifera Workers

159

MASSON, C, 1972. Le systeme antennaire chez les fourmis. I. Histologie et ultrastructure du deutocerebron. Etude comparee chez Componotus vagus (Formicinae) et Mesoponera caffraria. Z. Zellforsch. 134: 31-64. MASSON, C. 1982. Basic mechanisms of sensory antennal information processing in insects, with special reference to social insects, pp. 380-84. In M. D. BREED, C. D. MrrCHENERand H. E. EVANS(eds) The Biology of Social Insects. Westview Press, Colorado. MASSON, C, and G. ARNOLD. 1987. Organization and plasticity in the olfactory system of the honeybee, Apis meUifera, pp. 280-95. In R. MENZELand A. MERCER(eds) Neurobiology and Behavior of Honeybees. Springer, Berlin. MASSON,C. and C. STRAMBI.1977. Sensory antennal organization in an ant and a wasp. J. Neurobiol. 8: 537-48. MATSUMOTO, S. G. and J. G. HILDEBRAND. 1981. Olfactory mechanisms in the moth Manduca sexta: response characteristics and morphology of the central neurones in the antennal lobes. Proc. R. Soc. Lond. B 213: 249-27. MOBBS, P. G. 1982. The brain of the honeybee, Apis mellifera. I. The connections and spatial organization of the mushroom bodies. Philos. Trans. R. Soc. Lond. B 298: 309-54. MOBBS, P. G. 1984. Neural networks in the mushroom bodies of the honeybee. J. Insect. Physiol. 30: 43--58. PARETO, A. 1972. Die zentrale Verteilung der Fuhlerafferenz bei Arbeiterinnen der Honigbiene, Apis mellifera L. Z. Zellforsch. 131: 109--40. ROSPARS,J. P. 1983. Invariance and sex-specific variations of the glomerular organization in the antennal lobes of a moth. Mamestra brassicae, and a butterfly, Pieris brassicae. J. Comp. Neurol. 220: 80-96. ROSPARS, J. P. and I. CHAMBILLE. 1986. Postembryonic growth of the antennal lobes and their identified glomeruli in the cockroach Blaberus craniifer Burm. (Dictyoptera : Blaberidae): a morphometric study. Int. J. Insect Morphol. Embryol. 15: 393-415. SANES,J. R., D. J. PRESCOTTand J. G. HILDEBRAND. 1977. Cholinergic neurochemical development of normal and deafferented antennal lobes during metamorphosis of the moth, Manduca sexta. Brain Res. 199: 389-402. STOCKER, R. F., R. N, SINGH, M, SCHORDERETand O. SIDDIQI.1983. Projection patterns of different types of antennal sensilla in the antennal glomeruli of Drosophila melanogaster. Cell Tissue Res. 232: 237-48. SUZUKI, H. 1975. Antennal movements induced by odour and central projection of antennal neurones in the honeybee. J. Insect Physiol. 21: 831-47. TYRER, N. M. and E. M. BELL. 1974. The intensification of cobalt-filled neurone profiles using a modification of Timm's sulphide-silver method. Brain Res. 73: 151-55.