Histochemistry (1992) 97 : 511-516

Histochemistry © Springer-Verlag 1992

Glutamate-like immunoreactivity in the leech central nervous system Peter D. Brodfuehrer 1,2 and Avis H. Cohen 1,3

1 Section of Neurobiology and Behavior, Seeley G. Mudd Hall, Cornell University, Ithaca, NY 14853, USA 2 Department of Biology, Bryn Mawr College, Bryn Mawr, PA 19010, USA 3 Department of Zoology, University of Maryland, College Park, MD 20742, USA Accepted April 28, 1992

Summary. Using a monoclonal antibody for glutamate the distribution was determined of glutamate-like immunoreactive neurons in the leech central nervous system (CNS). Glutamate-like immunoreactive neurons (GINs) were found to be localized to the anterior portion of the leech CNS: in the first segmental ganglion and in the subesophageal ganglion. Exactly five pairs of G I N s consistently reacted with the glutamate antibody. Two medial pairs of G I N s were located in the subesophageal ganglion and shared several morphological characteristics with two medial pairs of G I N s in the first segmental ganglion. An additional lateral pair of G I N s was also located in segmental ganglion 1. A pair of glutamate-like immunoreactive neurons, which are potential homologs of the lateral pair of G I N s in segmental ganglion 1, were occasionally observed in more posterior segmental ganglia along with a selective group of neuronal processes. Thus only a small, localized population of neurons in the leech CNS appears to use glutamate as their neurotransmitter.

Introduction

The amino acid L-glutamate is the predominant excitatory neurotransmitter in the vertebrate central nervous system (Collingridge and Singer 1990) and acts in a similar fashion in both the central and peripheral nervous systems of invertebrates (Takeuchi and Takeuchi 1964; Walker et al. 1980; Shinozaki 1988). Electrophysiological studies have indicated that neurons that release glutamate as their neurotransmitter function in a variety of neuronal processes, ranging from activation of rhythmic m o t o r patterns (Grillner and Matsushima 1991 ; M c C r i m m o n et al. 1989) to activity dependent synaptic plasticity, learning and m e m o r y (Malenka 1991; Collingridge and Singer 1990). In the medicinal leech, one possible function ascribed to the release of L-glutamate from central neurons is

Correspondence to: P.D. Brodfuehrer

the initiation of swimming activity, since pressure ejection of L-glutamate and several of its analogs onto a single segmental ganglion is sufficient to elicit the swim m o t o r p r o g r a m in the isolated ventral nerve cord (Brodfuehrer and Cohen 1990). However, no identified central neurons that are associated with the generation of swimming activity in the leech are known to contain glutamate, although a variety of putative neurotransmitters and neuromodulators have been identified in the leech central nervous system (CNS) (Hashemzadeh and Friesen 1989; Leake et al. 1986; Osborne et al. 1982; Muller et al. 1981). In this paper, the location of putative glutamatergic neurons in the leech CNS was determined using a monoclonal antibody for glutamate.

Materials and methods

Animals Adult leeches, Hirudo medicinalis, were purchased from Leeches USA and kept, for up to 2 months prior to use, at room temperature in an aquarium filled with artificial pond water. The leech CNS consists of a head and a tail ganglion connected by a ventral nerve cord, a chain of 21 segmental ganglia and their intersegmental connectives (Muller et al. 1981). The head ganglion is comprised of a supraesophageal ganglion (SupraEG) and a subesophageal ganglion (SubEG), which is further divided into four divisions referred to as rostral 1 through rostral 4 (R1-R4; see Fig. 1), anterior to posterior (E. Macagno, M. Shankland, D. Weisblat, K. Muller, and W. Kristan, Jr., personal communication). The segmental ganglia are numbered sequentially, from i to 21, beginning with the first ganglion posterior to the SubEG (Muller et al. 1981), and designated M1-M21. The neurons in each segmental ganglion are separated by septa into six groups or packets, with each packet surrounded by one glial cell (Muller et al. 1981).

Tissue preparation and immunocytochemical procedures Whole-mount preparations. Chains of 3-5 ganglia were surgically removed from the animal and pinned in clear Sylgard-coated dishes. The ganglia were fixed for 1 h using 4% paraformaldehyde and 0.2-0.5% glutaraldehyde in 0.1 M phosphate-buffered saline

512 (PBS), pH 7.2-7.4. Fresh glutaraldehyde was added to a stock solution of 4% paraformaldehyde just prior to use. Following fixation the tissue was rinsed in several changes of PBS for 30-60 min. To improve penetration of the primary antibody into the leech ganglia, two procedures were employed: a 2 h incubation at room temperature in 0.2 M ethanolamine diluted with PBS containing 0.4% Triton X (PBS-X) and then a 1 h incubation at room temperature in a collagenase/dispase (Boehringer Mannheim Biochemicals, Indianapolis, Ind., USA) solution (1 mg/ml of PBS-X), prepared immediately before use. Following each of the above procedures, the ganglia were rinsed thoroughly in several changes of PBS-X over 1 h. Prior to incubation in primary antiserum, the ganglia were blocked in PBS-X with 3 % normal goat serum (NGS) for 2-6 h and then incubated overnight in a solution of primary antibody diluted either 1:1000 or 1:2500 with PBS-X and 3% NGS. Visualization of the primary antibody was done using commercially available kits from Vectastain or Biomedia for avidinbiotinylated peroxidase complex second antibody, developed with 3,3'-diaminobenzidine according to the glucose oxidase method (Itoh et al. 1979 ; R. Booker, personal communication). The ganglia were then dehydrated in an alcohol series, cleared in xylene, and mounted onto glass slides with Permount. The glutamate antibody used was purchased from INCSTAR and was originally generated from mouse in response to gamma-Lglutamyl-L-glutamic acid, the product of L-glutamate reacting with fixative, and conjugated to keyhole limpet hemocyanin using glutaraldehyde-borohydride (Madl et al. 1986). In preabsorption controls, 500 gg of gamma-I~-glutamyl-L-glutamicacid (Sigma) per ml of antibody diluted 1 : 2500 was incubated with the antiserum for approximately 4-6 h at room temperature, spun for 15 min at approximately 14000 x g and the supernatant applied to the ganglia. Both the preabsorption controls (see Fig. 4) and the negative controls (no antibody) eliminated specific staining of neurons in the leech nerve cord. In some instances, glutamate-like immunoreactivity was also localized to the nucleus of connective tissue cells that surround the ventral nerve cord (see Fig. 3 A). This may be partially due to a slight cross-reactivity between this glutamate antibody and c~-tubutin (McDonald et al. 1989).

Cryostat sections. Following fixation of the ganglia and after several washes in PBS, the tissue was incubated in PBS containing 30% sucrose from periods of several hours to up to 3 days. Sections, 20 gm thick, were cut and recovered on gelatin-coaiLed glass slides. The sections were then processed for glutamate immunoreactivity (antibody dilution 1 : 1000) and visualized according to the procedures described above.

Results Distribution o f immunoreactive neurons in the head ganglion In w h o l e - m o u n t p r e p a r a t i o n s f o u r g l u t a m a t e - l i k e i m m u n o r e a c t i v e n e u r o n s ( G I N s ) were c o n s i s t e n t l y o b s e r v e d o n the v e n t r a l surface o f the m o s t p o s t e r i o r d i v i s i o n o f the S u b E G , R 4 (Figs. 1 a n d 2 A ) . These f o u r G I N s a p p e a r e d to r e p r e s e n t two p a i r s o f b i l a t e r a l n e u r o n s . T h e m o s t p o s t e r i o r p a i r o f G I N s (referred to as G I N 1 ) was a n a t o m i c a l l y different f r o m the m o r e a n t e r i o r p a i r o f G I N s (referred to as G I N 2 ) . Firstly, the G I N 1 cell p a i r was g e n e r a l l y slightly l a r g e r in d i a m e t e r a n d s e c o n d ly, was p o s i t i o n e d closer to the m i d l i n e o f R 4 t h a n the G I N 2 cell p a i r (Fig. 2 A ) . In a d d i t i o n , G I N 1 cells s h o w e d m o r e intense g l u t a m a t e - l i k e i m m u n o r e a c t i v i t y t h a n G I N 2 cells. G I N 1 a n d G I N 2 were similar in t h a t b o t h h a d a n t e r i o r l y p r o j e c t i n g a x o n s w h i c h e x t e n d e d ipsilater-

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Fig. 1. Diagram of the anterior portion of the leech central nervous system showing the relative position of glutamate-like immunoreactive neurons (GIN). Filled circles (GINI-GIN5) represent neurons that consistently reacted with the glutamate antiserum, while open circles in R2, R3 and in the supraesophageal ganglion indicate neurons that only occasionally showed glutamate-like immunoreactivity. SupraEG, supraesophageal ganglion; subEG, subesophageal ganglion; RI R4, four divisions of the SubEG; MI, first segmental ganglion

ally, with r e s p e c t to the p o s i t i o n o f their cell b o d i e s , in the S u b E G (Fig. 2B). C r y o s t a t sections t h r o u g h the h e a d g a n g l i o n r e v e a l e d t h a t the a x o n s o f G I N 1 a n d G I N 2 e x t e n d e d u p i n t o the c i r c u m e s o p h a g e a l c o n n e c tives, while g l u t a m a t e - l i k e i m m u n o r e a c t i v e processes o f G I N 1 a n d G I N 2 were d i s t r i b u t e d t h r o u g h o u t the S u b E G n e u r o p i l e (Fig. 2B). C r y o s t a t sections t h r o u g h the S u p r a E G n e u r o p i l e also r e v e a l e d m a n y g l u t a m a t e like i m m u n o r e a c t i v e processes (Fig. 2 C). T h e cell b o d i e s o f several o t h e r n e u r o n s in the h e a d g a n g l i o n o c c a s i o n a l ly s h o w e d g l u t a m a t e - l i k e i m m u n o r e a c t i v i t y in w h o l e m o u n t p r e p a r a t i o n s , i n c l u d i n g a p a i r o f cell b o d i e s l o c a t ed at the a p p r o x i m a t e m i d l i n e o f the S u p r a E G a n d a cell b o d y l o c a t e d a t the a n t e r i o r l a t e r a l edge o f e a c h h e m i - g a n g l i o n o f R 2 a n d R3 in the S u b E G (Fig. 1).

Distribution o f immunoreactive neurons in segmental ganglia In whole-mount preparations, glutamate-like immunorea c t i v i t y w a s o b s e r v e d c o n s i s t e n t l y o n l y in s e g m e n t a l gan-

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Fig. 2 A-C. Glutamate-like immunoreactive neurons in the head ganglion. A Whole-mount preparation. Glutamate-like immunoreactivity localized to two pairs of cell bodies, GIN1 and GIN2, in the most posterior division of the SubEG. B, C Glutamate-like immunoreactive processes distributed throughout the neuropile of both the SubEG and the SupraEG. B Cryostat section (20 txm) through the SubEG. GIN1 cell bodies (asterisks) and their anteriorly projecting axons show glutamate-like immunoreactivity. The axon from one GIN2 cell (arrowhead) is also evident. C Cryostat section (20 gin) through SupraEG. Scale bar = 100 gm

glion 1 (M1) (Figs. 1A and 3A). A total of six cell bodies, which appeared to represent three pairs of GINs, reacted with the glutamate antiserum; two pairs (referred to as GIN3 and GIN4) were located in the posterio-medial packet and one cell (GIN5) in each anteriolateral packet. With respect to their morphology and immunoreactive characteristics, GIN3 and G I N 4 were similar to GIN1 and GIN2, respectively: firstly, both GIN3 and G I N 4 had anteriorly projecting axons; secondly, the soma diameter of GIN3 was generally larger than G I N 4 ; and thirdly, the intensity of glutamate-like immunoreactivity was greater in GIN3 than in GIN4. Unlike G I N 1 - G I N 4 , glutamate-like immunoreactivity was observed in both the cell body and processes of GIN5 in whole-mount preparations. The processes of GIN5 arborized exclusively within the hemi-ganglion that contained the soma, and extended along the entire anterior-posterior length of the ganglion (cf. Fig. 3B and C). In the anterio-lateral packet of segmental ganglia posterior to M 1 a pair of glutamate-like immunoreactive neurons were periodically observed, particularly in M2

and M3 (Fig. 3B), and as far posterior as M l l (Fig. 4B). Based on cell body location and sometimes on cell morphology it appeared that these glutamate-like immunoreactive neurons in segmental ganglia posterior to MI may be homologs of GIN5. Furthermore, glutamate-like immunoreactive processes i n the general region of the arborization of GIN5 within M1 were observed in all segmental ganglia examined and were absent in preabsorption controls (Figs. 4 A and B).

Discussion Our results demonstrate the presence of glutamate-like immunoreactivity in the CNS of the medicinal leech. However the number of neuronal cell bodies that reacted with the glutamate antiserum used in this study was small. In total, only five pairs of neuronal cell bodies repeatedly showed glutamate-like immunoreactivity. Moreover, the distribution of these five pairs of GINs

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Fig. 3A-C. Glutamate-like immunoreactive neurons in segmental ganglia 1 and 2. A Whole-mount preparation. Three pairs of cell bodies, GIN3, GIN4 and GIN5, show glutamate-like immunoreactivity on the ventral surface of segmental ganglion I. Note that the GIN5 cell located in the right anterio-lateral packet is slightly out of the focal plane of the picture. B Pair of cells in the anteriolateral packet in segmental ganglion 2 that reacted with the glutamate antiserum. Note that this cell pair is in the same stereotypic position in segmental ganglion 2 as the GIN5 cell pair in segmental ganglion 1. C Camera lucida drawing of cell pair from B. Scale bars: A 60 gin, B 200 gin, C 100 gm

in the leech CNS was restricted primarily to the SubEG and MI. The small number and limited distribution of GINs in the leech CNS suggest that glutamate is not a widely used neurotransmitter in the leech CNS. The leech CNS is organized in a highly stereotypic manner. Cell counts performed on midbody (M8-M11) segmental ganglia indicate that the number of neurons per ganglion is constant (within 2%) along the ventral nerve cord, excluding the sex ganglia (Macagno 1980). Physiological examinations of many segmental ganglia also suggest that to the first approximation all segmental ganglia are organized similarly (Muller et al. 1981). Clearly, however, segmental specializations do exist. Not all identified neurons occur in each segmental ganglion. For example, swim-initiating interneurons, cells 204, are located only in MI0-M16 (Weeks and Kristan 1978) and heart interneurons are limited to the first seven segmental ganglia (Calabrese and Peterson 1983). Biochemical and histochemical studies on the identification of putative neurotransmitter substances in the leech CNS have documented other segmental specializations (Marsden and Kerkut 1969; Leake et al. 1986). The distribution of glutamate-like immunoreactive neurons along the ventral nerve cord also highlights the existence of segmental specializations in the leech CNS. Cells GIN1 and GIN2 and cells GIN3 and GIN4 are located exclusively in R4 and M1, respectively. It is not clear whether homologs of these GINs, with homology based on soma size and position within the ganglion, occur in other divisions of the SubEG and segmental ganglia, respectively. Visual examination of many segmental ganglia suggests that neurons occur, which are in the same position and have approximately the same soma size as GIN3 and GIN4 (P.D. Brodfuehrer, personal observation). Our immunocytochemical results indicate that if homologs of GIN3 and GIN4 are present in other segmental ganglia they either do not use glutamate as their transmitter, or the concentration of glutamate in these neurons is below the detection level of our immunocytochemical assay. Neurons similar to GIN1 and GIN2 in the other divisions of the SubEG were not clearly identifiable (P.D. Brodfuehrer, personal observation). Unlike GIN1 GIN4, however, GIN5 and its segmental homologs appear to be located in most, if not all, segmental ganglia. The motive for this study was to determine whether glutamate may be used as a neurotransmitter by neurons associated with the initiation of swimming activity. Specifically, since the manner in which cell Trl stimulation initiates swimming can be mimicked by pressure ejection of L-glutamate onto a segmental ganglion (Brodfuehrer and Friesen 1986a, b; Brodfuehrer and Cohen 1990), the neurotransmitter released by Trl could possibly be glutamate. However, neurons located in the region of Trl's cell body in R1 did not show glutamate-like immunoreactivity. The possibility cannot be ruled out that the long-term effect of Trl stimulation on swim initiation pathways is not direct, but occurs via an intermediate cell, possibly a GIN. The most likely candidates would be GIN5 and its putative segmental homologs since these GINs are located in several, if not all, segmental ganglia

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Fig. 4A, B. Glutamate-like immunoreactive cell body (arrowhead), a putative GIN5 homolog, and neuropile processes in segmental ganglion 11. A Preabsorption control; B Experimental. Both A and B are 20-~tm-thick cryostat sections of the approximate lateral half of segmental ganglion 11. Scale bar = 100 gm

w h e r e they w o u l d h a v e i n t r a g a n g l i o n i c access to the swim g e n e r a t i n g c i r c u i t r y ( F r i e s e n 1989). A l t e r n a t i v e l y , the l o c a l i z a t i o n o f G I N s to the a n t e r i o r n e r v o u s system o f the leech suggests t h a t G I N s c o u l d be a s s o c i a t e d with feeding b e h a v i o r , i n v o l v i n g p r e d o m i n a n t l y the a n t e r i o r p o r t i o n o f the a n i m a l ( L e n t a n d D i c k i n s o n 1984; D i c k i n s o n a n d L e n t 1984). Recently, G r o o m e a n d L e n t (1.991) have s h o w n t h a t c h e m i c a l stimu l a t i o n o f the p r o s t o m a l lip, w h i c h n o r m a l l y increases a c t i v i t y in Retzius cells, is b l o c k e d b y b a t h a p p l i c a t i o n o f the g l u t a m a t e a n t a g o n i s t k y n u r e n i c acid. Since Retzius cell activity is an i n t e g r a l c o m p o n e n t o f the p h y s i o logical m e c h a n i s m s i n d u c i n g feeding there is a s t r o n g p o s s i b i l i t y t h a t G I N s m a y f u n c t i o n in the p a t h w a y cont r o l l i n g feeding b e h a v i o r in the leech. Acknowledgements. We thank Dr. R. Booker, Dr. M. Grober, M.

Read, M. Marchaterre, and M. O'Neill for histological assistance, A. Burns for photographic assistance and M.S. Thorogood for critical comments on earlier versions of this manuscript. This work was supported by NIMH grant MH44809 to A.H.C. and a Whitehall Foundation grant and NIH grant NS29509 to P.D.B.

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