Glutamate receptor 5/6/7-like and glutamate transporter-1-like immunoreactivity in the leech central nervous system

Glutamate Receptor 5/6/7-Likeand Glutamate Transporter-1-Like

Immunoreactivity in the LeechCentral Nervous System

MARIA STELLA E. THOROGOOD, VANIA W. ALMEIDA,

AND PETER D. BRODFUEHRER*Biology Department, Bryn Mawr College, Bryn Mawr, Pennsylvania 19010

ABSTRACTPrevious physiological and pharmacological evidence has suggested a neurotransmitter

role for the excitatory amino acid glutamate in the leech central nervous system (CNS). In thepresent study, we sought to localize glutamate receptor (GluR) subunits (GluR 5/6/7, GluR 2/3and N-methyl-D-aspartate receptor 1 [NMDAR 1]) and a glutamate transporter subtype[GLT-1] within the leech CNS using mono- and polyclonal antibodies. In whole-mountedtissue, small cells of the outer capsule and putative microglia labeled with both GluR 5/6/7 andGluR 2/3 but not NMDAR 1 subunit antisera. In general, GluR 5/6/7-like immunofluorescencewas both more intense and more widespread than GluR 2/3-like immunolabeling. Cryostat-sectioned tissue revealed extensive GluR 5/6/7-like immunoreactivity throughout the neuropilas well as labeling within a few neuronal somata. GLT-1-like immunoreactivity localized tothe inner capsule, which is the interface between neuronal somata and the neuropil and isdeeply invested by processes of neuropil glia. These results complement previous physiologi-cal and pharmacological findings indicating that the leech CNS possesses the cellularmachinery to respond to glutamate and to transport glutamate from extracellular spaces.Together, they provide further evidence for glutamate’s role as a neurotransmitter within theleech CNS. J. Comp. Neurol. 405:334–344, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: Hirudo medicinalis; GluR 5/6/7; GLT-1

Understanding the circuitry underlying behavior hasbeen an important endeavor in neurobiology. In addition toidentifying the synaptic connections and interactions com-prising the circuit, it is important to determine the molecu-lar and chemical components giving rise to the characteris-tics of each individual neuron, such as membrane receptorphenotype. As the molecular and chemical traits of indi-vidual neurons are established, one begins to understandthe synaptic interactions that give rise to the circuit, andthen ultimately the interactions of different circuits result-ing in specific behaviors.

The classification of the amino acid L-glutamate as anexcitatory neurotransmitter in the vertebrate central ner-vous system (CNS) has been well documented (Colling-ridge and Lester, 1989). Evidence for glutamate’s role as aneurotransmitter in the invertebrate CNS is rapidly accu-mulating and is based primarily on physiological andpharmacological data. For example, several connectionsbetween identified neurons involved in the leech swimcircuitry appear to be glutamatergic. Swim trigger cellTr1, swim gating interneuron cell 204, and swim oscillator

cell 208 depolarize following focal application of L-glutamate or one of its agonists (Brodfuehrer and Cohen,1990; Thorogood et al., 1996). Non-N-methyl-D-aspartate(NMDA) antagonists also block synaptic input to each ofthese cells, as well as to two additional members of theswim oscillator—cells 115 and 28 (Thorogood and Brodfue-hrer, 1995; Thorogood et al., 1996). In addition, exposure toglutamate agonists directly induces membrane depolariza-tion of leech DE-3 and L motor neurons, Retzius andeffector AP and AE cells, and neuropil glia (Mat Jais et al.,1984; Brodfuehrer and Cohen, 1990; Deitmer and Munsch,1992; Dorner et al., 1994; Thorogood et al., 1995, 1996;Dierkes et al., 1996). Agonist-induced excitation of neuro-pil glia and DE-3 are also blocked by non-NMDA antago-

Grant sponsor: NSF; Grant number: 9514617.*Correspondence to: Peter D. Brodfuehrer, Biology Department, Bryn

Mawr College, Bryn Mawr, PA 19010. E-mail: [email protected] 27 April 1998; Revised 28 September 1998; Accepted 9 October

1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 405:334–344 (1999)

r 1999 WILEY-LISS, INC.

nists (Deitmer and Munsch, 1992; Thorogood et al., 1996).Recently, Deitmer and Schneider (1997) have demon-strated that antagonists of glutamate transporter activitypotentiate glutamate agonist-induced pH changes in neu-ropil glia. Taken together, these findings suggest a neuro-transmitter role for glutamate in the leech CNS: mediat-ing synaptic connections within the leech swim circuitryand from mechanosensory cells to their postsynaptic tar-gets. In the present study, three antisera raised againstvertebrate sequences of GluR 5/6/7, GluR 2/3, and NMDAR1 subunits (Huntley et al., 1993; Wenthold et al., 1992;Siegel et al., 1994, respectively) were employed to localizeGluR-like immunoreactivity in leech ganglia. Preliminaryresults from this work have been previously published(Thorogood et al., 1997).

MATERIALS AND METHODS

Organization of the leech CNS

The leech CNS is composed of an anterior head ganglionand a posterior tail ganglion, which are connected by theventral nerve cord, a chain of 21 midbody segmentalganglia (designated M1–M21, anterior to posterior, begin-ning with the first ganglion posterior to the subesophagealganglion) and their intersegmental connectives. The headganglion (H) includes the supraesophageal ganglion andfour fused ganglia, which comprise the subesophagealganglion. Seven fused ganglia constitute the tail ganglion.Intersegmental connectives consist of a small mediannerve bundle, Faivre’s nerve, which is flanked by two largelateral nerve bundles, and join together adjacent ganglia.A pair of nerve roots issues bilaterally from each ganglion;these nerves carry neuronal signals both to and from theperiphery.

Each ganglion consists of a cortex, which includesapproximately 400 unipolar neurons, and a core (i.e.,neuropil) where a majority of synapses between neuronsare confined (Fernandez and Fernandez, 1974; Fernandez,1978). Each ganglion is encapsulated within a sheath orouter capsule, beneath which exists a monolayer of neuro-nal somata (Fig. 1). Although ultrastructural studies haveindicated that neuronal somata are free of synapses(Coggeshall and Fawcett, 1964), a more recent physiologi-cal study has suggested the presence of extrasynapticreceptors on the cell bodies themselves (Sargent et al.,1977). Neurons are separated into six smaller groups orpackets, one anteriomedial, one posteriomedial, two ante-rior lateral, and two posterior lateral packets. The medialpackets are located ventrally, whereas the other fourpackets wrap around the lateral edges and thus containboth ventrally and dorsally situated somata. Each of thesesix packets includes a single large glial cell, the packetglia, whose processes fill the interstitial spaces betweenthe cell bodies within each packet and envelope theneuronal somata as well as their apical processes (Fernan-dez, 1978). The periphery of the neuropil is termed theinner capsule of the ganglion (Fig. 1). Each neuron insertsits main process through the inner capsule and into theneuropil. Neuronal processes branch extensively uponentering the neuropil and form synapses with intragangli-onic as well as interganglionic processes. In addition, twolarge stellate cells are found internal to the inner capsuleand are located close to the ventral aspect of the eachganglion. These neuropil glia cells extend their processes

throughout the neuropil and line the surface of the innercapsule (Coggeshall and Fawcett, 1964).

Two other types of glial cells have been identified in theleech CNS. Microglia are very small cells (, 3–6 µm),located within neuronal packets near the inner and outercapsule, lodged in invaginations of the neuronal cellsurface, and surrounding the neuropil septa. Microglia aremigratory and have been observed to migrate to sites ofnerve lesion (Morgese et al., 1983; McGlade-McCulloh etal., 1989). A pair of glial cells is also located approximatelymidway between successive ganglia within the bundle ofeach lateral connective (Coggeshall and Fawcett, 1964;Muller et al., 1981). In addition to surrounding eitherindividual or groups of axons coursing through the lateralnerves, processes of these connective glial cells also wraparound the axons of Faivre’s nerve.

Animal dissection

Hirudo medicinalis were obtained from Leeches USA(Westbury, NY). Adult leeches were maintained in aquaria

Fig. 1. Sectional view of a leech segmental ganglion. The illustra-tion depicts the plane of section through a segmental ganglion (a) andidentifies relevant anatomical structures (b). Note that the outercapsule or sheath, surrounds the neuronal somata, whereas the innercapsule is the interface between neuronal somata and the neuropil.Top of figure corresponds with anterior of animal in b.

GluR 5/6/7- AND GLT-1-LIKE IMMUNOREACTIVITY 335

filled with artificial pond water (NaCl [28 mg/L], KCl[0.0005 mg/L], MgSO4[0.0008 mg/L], Ca[NO3]2

.4H2O[0.0003 mg/L], Tris buffer [0.0056 mg/L]) at room tempera-ture for up to 6 months before use. Prior to and duringdissection, leeches were anesthetized by immersion incold, normal physiological saline (115 mM NaCl, 4.0 mMKCl, 1.8 mM CaCl2

.2H2O, 2.0 mM MgCl2.6H2O, 10 mM

HEPES [or Tris] buffer, and 10 mM D-glucose; adjusted topH 7.4 with 2 M NaOH). Ganglia and their associatedroots and connectives were isolated as described by Mulleret al. (1981). Immunohistochemical experiments wereperformed on isolated head and segmental ganglia, inwhich the outer capsule had been removed (i.e., desheathed)with fine scissors.

Immunohistochemistry

Whole-mount tissue. Chains of H-M2 or of midbodyganglia were dissected from the animal and pinned inSylgard dishes. The tissue was fixed for 20 minutes in 4%paraformaldehyde and 0.1% glutaraldehyde in Dulbecco’sphosphate-buffered saline (PBS; Sigma), pH 7.4, followedby a 6 hour incubation in 4% paraformaldehyde and 0.2%picric acid in PBS. Afterward, the ganglia were rinsed in atleast three changes of PBS for 30–60 minutes. Threeprotocols were employed to improve penetration of theprimary antibody and the avidin-biotinylated peroxidasesecondary antibody: 1) dehydration to 95% ethanol (EtOH),followed by rehydration back to PBS, 2) incubation in 0.2M ethanolamine (Sigma) diluted in PBS-X (PBS contain-ing 0.4% Triton X-100) for 2 hours, and lastly 3) incubationin collagenase/dispase (Boehringer Mannheim, Indianapo-lis, IN) diluted in PBS-X (1 mg/mL) for 1 hour. When usingimmunofluorescence to visualize the primary antibody, thetissue was only subjected to dehydration/rehydration toimprove penetration. The above procedure(s) was per-formed at room temperature; following each step, thetissue was thoroughly rinsed in several changes of PBS-Xfor 1 hour. The ganglia were subsequently incubated inblocking solution (PBS-X with 3–10% normal goat serum[NGS]) for 2–6 hours and then incubated overnight at 4°Cin the primary antibody diluted in blocking solution. Thetissue was then rinsed in several changes of PBS for 1 hourand the primary antibody visualized using an avidin-biotinylated peroxidase complex secondary antibody (Bio-meda), developed with 3,38-diaminobenzidine according tothe glucose oxidase method (Itoh et al., 1979). Subse-quently, ganglia were dehydrated in an alcohol series,cleared in either xylene or methyl salicylate, and mountedwith Permount (Fisher, Norcross, GA).

Alternatively, a rhodamine-conjugated secondary anti-body was used to detect the primary antibody. In suchcases, dissected ganglia were fixed for 30 minutes in 4%paraformaldehyde in Dulbecco’s PBS (pH 7.4), rinsed withPBS, dehydrated to 95% EtOH, and then rehydrated toPBS prior to applying the primary antibody. Followingrinsing of the primary antibody, the tissue was incubatedwith the rhodamine-tagged secondary antibody for 4–24hours and then rinsed again with PBS. Subsequently, thestained ganglia were pinned out on Sylgard dishes, dehy-drated in an alcohol series, and cleared and mounted ineither xylene or methyl salicylate.

Sectioned tissue. Chains of H-M2 or of midbodyganglia were dissected from the leech and pinned inSylgard dishes. In some cases, identified cells were filled

with Lucifer Yellow for double-labeling morphological stud-ies. Ganglia were fixed for 30 minutes in 4% paraformalde-hyde in Dulbecco’s PBS (Sigma), pH 7.4 and subsequentlyrinsed in at least three changes of PBS, followed byincubation overnight in 30% sucrose in Dulbecco’s PBS.Individual ganglia were then mounted in TBS TissueFreezing Mediumy (Fisher), frozen on dry ice, and cryostat-sectioned with an IEC cryostat. Horizontal sections (10µm) were collected in series on either HistoGrip-coated(Zymed, South San Francisco, CA) or TESTA-coated slides(Rentrop et al., 1986). The sectioned tissue was subse-quently blocked and then incubated overnight in theprimary antibody diluted in blocking solution at 4°C. Thetissue was rinsed in several changes of PBS-X for 1 hour,incubated with a fluorescent-tagged secondary antibodyfor 2 hours at room temperature, and then rinsed inseveral changes of PBS. Cover slips were mounted byusing Gel/Mount (Biomeda).

Microscopy and analysis. Specimens were viewedusing an Olympus BH2-RFCA microscope equipped forepifluorescence and photographed with a Nikon cameraloaded with Ektachrome 320T (Kodak). Slide images werethen digitally scanned, and figures were prepared andconverted to black and white with Adobe Photoshop.

Antibody characteristics

Antisera to three different glutamate receptor (GluR)subunits (GluR 5/6/7, GluR 2/3 and NMDAR 1 [Phar-Mingen, San Diego, CA; Chemicon, Temecula, CA; andPharMingen, respectively]) were employed in this study. Amonoclonal antibody (mAb) 4F5 was prepared to a se-quence of the putative extracellular domain of the GluR 5receptor subunit (amino acid residues 233–518); it recog-nizes kainate receptor subtypes GluR 5, GluR 6, and GluR7 subunits (GluR 5/6/7; Huntley et al., 1993). The poly-clonal antiserum Ab25 was raised against a C-terminalportion of an (6)-a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subtype (amino acid resi-dues 850–862); it selectively labels both GluR 2 and GluR 3subunits (GluR 2/3; Wenthold et al., 1992). Finally, amonoclonal antibody (mAb 54.1) was generated againstNMDAR 1 amino acid residues 660–811, which representsthe intracellular loop between transmembrane regions IIIand IV; this antibody recognized only NMDAR 1 subunits(Siegel et al., 1994). The preparation and preadsorptioncontrols for the specificity of these antibodies have beendescribed in detail elsewhere (Petralia and Wenthold,1992; Wenthold et al., 1992; Petralia et al., 1994; Huntleyet al., 1993). Briefly, preadsorption of mouse a-GluR 5/6/7antiserum with GluR 5 resulted in the absence of specificstaining in monkey brain (Huntley et al., 1993). Similarly,preadsorption controls for rabbit a-GluR 2/3, as well asmouse a-NMDA R1, antisera remained unstained in ratbrain (Petralia and Wenthold, 1992; Petralia et al., 1994).

Two other antisera were tested in this study. A poly-clonal antibody, generated against the C-terminal 18 aminoacids (NGKSADCSVEEEPWKREK) of the rat glutamatetransporter GLT-1 subtype (Affinity BioReagents, Golden,CO; R. S. Roginski, personal communication), was em-ployed. A monoclonal antibody (mAb 39; gift from Dr. K. J.Muller), which recognizes a sequence homologous to thesquid neurofilament protein, was used to identify putativeleech glia (Luthi et al., 1993); mAb 39 also labels smallcells of the outer capsule. Luthi et al. (1993) referred to

336 M.S.E. THOROGOOD ET AL.

specific staining by the mAb 39 antibody as G39 immuno-reactivity.

Fluorescent-tagged secondary antibodies (AccurateChemical & Scientific, Westbury, NY) were used to visual-ize the binding of the primary antibody to specific antigensthroughout the leech CNS. Rhodamine-conjugated (Rh-)goat a-mouse IgG, Rh-goat a-rabbit IgG, and Rh-goata-mouse IgM were used to detect NMDAR 1, GluR 2/3, andGluR 5/6/7 antisera, respectively. GLT-1-like immunoreac-tivity was visualized by means of either Rh-goat a-rabbitIgG or a kit (Biomeda) for avidin-biotinylated peroxidasecomplex second antibody, which was developed with 3,3’-diaminobenzidine according to the glucose oxidase method.G39 immunoreactivity was detected with a fluorescein-tagged goat a-mouse IgG.

Several whole-mounted ganglia or ganglion sections ofeach experiment were reserved for controls. In all immuno-histochemical experiments, omission of the primary antise-rum resulted in loss of immunostaining where stainingoccurred in experimental sections.

RESULTS

Specificity of immunostaining

Control tissue was subjected to the same procedure asexperimental tissue, with the exception of exposure to theprimary antibody; each immunohistochemical experimentincluded a set of controls. In all experiments, controltissues were devoid of any obvious immunofluorescence(Figs. 2b,d, 3c, 4b, 5c,e, 6e,f), demonstrating that anystaining detected in experimental tissue was attributableto interactions between the primary antibody (specific for aparticular GluR subunit(s) or for GLT-1) and the epitopepresent within the leech CNS.

Distribution of glutamate receptor-likeimmunoreactivity in whole-mounted ganglia

When compared with control tissue, immunoreactivitywas detected in both head and midbody ganglia exposed tothe GluR 5/6/7 subunit antibody (Fig. 2) and to GluR 2/3subunit antisera (not shown). The former yielded stronger(more intense) and more extensive labeling overall. Incontrast, whole-mounted tissue exposed to NMDAR 1antiserum did not differ from controls (not shown), indicat-ing that the antibody did not detect the NMDAR 1 epitopein the leech CNS. Thus, non-NMDA receptor-like but notNMDA receptor-like immunoreactivity was present inleech ganglia, which is consistent with previous physiologi-cal and pharmacological studies (Ballanyi et al., 1989;Brodfuehrer and Cohen, 1990; Dorner et al., 1990, 1994;Deitmer and Munsch, 1992; Thorogood and Brodfuehrer,1995; Dierkes et al., 1996).

GluR 5/6/7-like immunoreactivity was observed in bothhead and midbody ganglia; identical staining patternswere obtained when visualizing the epitope with either therhodamine-conjugated secondary antibody (Fig. 2) or theavidin-biotinylated peroxidase complex secondary anti-body (not shown). Discrete staining was most noticeable inspecific regions of the outer capsule on both the dorsal andventral aspects of the ganglion. In particular, small immu-noreactive cells, of similar size and shape, occurred atregularly spaced intervals and extended throughout theouter capsule (Fig. 2a). Labeling with the GluR 5/6/7

antiserum was confined to the cytoplasm of these cells;nuclei remained unlabeled. Immunoreactive cells of compa-rable size, location, and distribution were also evidentalong the intersegmental connectives (not shown). Immu-nolabeling of whole-mounted ganglia with mAb 39, anantiserum that recognizes a sequence homologous to squidneurofilament protein (Luthi et al., 1993), also stainedsmall cells (Fig. 2e), which were of similar size anddistribution to those labeled with the GluR 5/6/7 subunitantibody. Whereas G39 immunoreactive cells include both1) cells of the outer capsule or perineurial sheath and 2)amoebocytes or putative microglia in the leech (Luthi etal., 1993), the identity of GluR 5/6/7-like immunoreactivecells is unknown. Since GluR 5/6/7-like immunofluores-cence was more intense than that seen using the GluR 2/3subunit antibody, we focused on GluR 5/6/7-like immunore-activity within leech ganglia.

Immunohistochemical localization of GluR5/6/7 in cryostat-sectioned ganglia

Bright immunofluorescence present within the outercapsule of the ganglion made it difficult to discern discretestaining within the neuronal packets and neuropil inwhole-mount tissue. Thus, individual ganglia were horizon-tally sectioned to visualize GluR-like immunoreactivitybetter within the ganglion. Although there was someautofluorescence of the tissue itself, positive staining wasof much greater intensity than that of background fluores-cence. Brilliant punctate staining was present in theneuropil of 10-µm sections of experimental but not controltissue (Fig. 3). In addition, GluR 5/6/7-like immunoreactiv-ity was consistently located within the same region ofadjacent sections (Fig. 3a,b,d). Thus the positive stainingobserved in experimental tissue can be attributed torecognition of the GluR 5/6/7-like antigen by the primaryantibody, and not to randomly distributed fluorescent-labeled aggregates of the secondary antibody.

Positive immunofluorescence was detected in the regionof the outer capsule of the glial packet. GluR 5/6/7-likeimmunoreactivity in this area tended to be punctate innature (Fig. 3) or appeared as elliptically shaped fluores-cence (not shown), and may be associated with the labeledmicroglia observed in whole-mounted tissue. Occasionally,fine punctate staining was detected within neuronal pack-ets of the ganglion; this staining was not obviously associ-ated with neuronal cell membranes. Bright GluR 5/6/7-likeimmunofluorescence was also evident within the cyto-plasm of several neuronal somata in cryostat-sectionedtissue (Fig. 3e,f). Regions of the inner capsule, representedby the periphery of the neuropil in sectioned tissue (Fig. 1),also exhibited sparsely distributed punctate GluR 5/6/7-like immunoreactivity (Fig. 3).

Specific staining consisting of both fine punctate stain-ing and larger aggregates of immunofluorescence werescattered throughout the neuropil. GluR 5/6/7-like immu-noreactivity was also present around the periphery of thetwo neuropilar septa, appearing as discrete ellipticallyshaped fluorescence (not shown). No immunofluorescencewas detected in axon tracts that exited the ganglia viaeither peripheral nerves or the intersegmental connectives(not shown). Thus, in addition to labeling putative microg-lia in whole-mounted ganglia, GluR 5/6/7-like immunoreac-tivity localizes to neuronal cell bodies and to the neuropil


of sectioned leech ganglia. These data suggest the presenceof GluR 5/6/7-like epitopes within these regions.

Immunolabeling of glutamate transporterGLT-1 subunit in the leech CNS

Having detected glutamate receptor-like immunoreactiv-ity within the leech nervous system, we sought to examinethe distribution of a glutamate transporter subtype, GLT-1(Kanai et al., 1993). Immunohistochemical localization of aglutamate transporter-like antigen within the leech CNSwould provide further evidence in support of glutamate’sclassification as a neurotransmitter in the leech.

In whole-mounted ganglia, GLT-1-like immunoreactiv-ity appeared to localize to the same set of cells that labeledwith antibodies raised against GluR 5/6/7 subunit (Fig. 2),GluR 2/3 subunit, and glia (Fig. 2). Like the G39 immuno-reactive cells described by Luthi et al. (1993), the GLT-1antiserum labeled two types of small cells: cells that wereuniformly scattered at the level of the outer capsule (Fig.

4) and cells that accumulated close to the ends of cut ordamaged peripheral nerves (not shown). Unlike GluR-likeimmunoreactivity of whole-mounted ganglia (Fig. 2), GLT-1-like immunoreactivity occurred around the periphery(i.e., surface) of these immunoreactive cells; neither cyto-plasmic nor nuclear staining was obvious.

GLT-1-like immunoreactivity also differed from GluR5/6/7-like immunoreactivity within the neuropil of leechganglion. Cryostat-sectioned tissue revealed extensive andintense GLT-1-like immunoreactivity throughout the in-ner capsule (Figs. 5a, 6a,b). Punctate staining of GLT-1-like immunofluorescence was observed within the packetlayer of the ganglia; however staining was sparse withinthe neuropil itself (Fig. 5a). Occasionally, a cluster ofpunctate immunoreactivity was detected in the neuropil ofcryostat-sectioned ganglia (Fig. 6b). Although the locationof the cluster, if present, was consistent from section tosection within an individual ganglion, the location ofclusters differed in different ganglia. Omission of theGLT-1 subtype antibody prevented staining of the inner

Fig. 2. Immunolabeling of whole-mounted leech ganglia withGluR 5/6/7 or mAb 39 antiserum. GluR 5/6/7-like immunoreactivitylocalizes to small cells (arrows) that are regularly distributed through-out the glial sheath midbody ganglia (a). Similar staining patternswere observed in tissue exposed to the mAb 39 antibody (c). G39immunoreactivity also highlights neuronal somata (c) and has beenattributed to immunolabeling of the packet glia (Luthi et al., 1993; seealso Fig. 6), which is closely associated with neuronal cell membranes.Control sections were processed as above but in the absence of primaryantisera—GluR 5/6/7 subunit antibody (b). Note the lack of staining

throughout the ganglion; ventral aspect of the ganglion is shown here.Tissue exposed to different antisera were from separate experiments.Top of figure corresponds to the anterior of the animal in a and b; left offigure corresponds to the posterior of the animal in c. Note theperipheral nerves issuing from the right-hand side of the dorsal aspectof the ganglion in a; the intersegmental connectives emerge from theanterior of the ventral aspect of the ganglion in b and the posterior ofthe ventral aspect of the ganglion in c. Scale bars 5 50 µm in a and b,20 µm in c.


Fig. 3. Distribution of GluR 5/6/7-like im-munoreactivity in cryostat-sectioned tissue. Ex-posure of cryostat-sectioned tissue to mousea-GluR 5/6/7 antiserum resulted in extensivestaining throughout the neuropil (a, b, and d).Note the presence of GluR 5/6/7-like immunore-activity within the same region of adjacent10-µm ganglion sections (a, b, and d). Noimmunoreactivity was detected in the absenceof primary antibody (c). Occasional cytoplas-mic staining was detected within neuronalsomata in the cell body layer of the ganglion (eand f ). Scale bars 5 50 µm in a–d, 20 µm in eand f ).


Fig. 4. Distribution of GLT-1-like immunoreactivity within whole-mounted leech ganglia. Immunoreactive cells labeled with GLT-1subtype antibody were regularly distributed throughout the outercapsule of the ganglion (a and b). Discrete staining appeared to beconfined to the cell membranes (arrows) of these small cells. Thus,GLT-1-like immunoreactivity exhibits a similar staining pattern to

that achieved when using GluR 5/6/7, GluR 2/3, or mAb 39 (but notNMDAR 1) antisera. When ganglia were not exposed to a-GLT-1, thesesmall immunoreactive cells remained unlabeled (c). Top of figurecorresponds to the anterior of the animal in each panel. Note theperipheral nerve(s) issuing to the left in a and the medial tractcoursing through the ganglion in b. Scale bar 5 50 µm.

Fig. 5. Immunohistochemical localization of the GLT-1 subtype insectioned leech ganglia. Cryostat-sectioned ganglia were labeled witha GLT-1 antibody and visualized by using the ABC kit/peroxidasemethod (a). Note the extensive immunostaining along the inner

capsule of the ganglion (arrows). In the absence of the GLT-1 antibody,the inner capsule remained unlabeled (b). Top of figure corresponds tothe anterior of the animal in both panels. Scale bar 5 50 µm.


capsule of the ganglion as well as punctate staining withinthe cell body layer or neuronal packets (Figs. 5b, 6e).

Since GLT-1 transporters have been localized to glia inthe vertebrate CNS (Kanai et al., 1998), we sought todetermine whether GLT-1-like immunoreactivity was simi-larly associated with leech glia in cryostat-sectioned tis-sue. We used mAb 39, an antiserum that labels putativeglial cells within the leech CNS (Luthi et al., 1993). Aspreviously shown (Luthi et al., 1993), brilliant G39 immu-nofluorescence surrounded neuronal somata and theirapical processes as they plunged into the inner capsule(Figs. 5b, 6d); Luthi et al. (1993) previously attributed thestaining within the neuronal packets to packet glia. Thecell bodies of neuropil glia as well as their processes thatline the inner capsule also labeled with mAb 39 (Figs. 5b,6d). In double staining experiments of cryostat-sectionedtissue, GLT-1-like immunoreactivity overlapped with G39immunostaining of the inner capsule but not with theintense G39 immunoreactivity of either the packet gliathat envelope neuronal somata or neuropil glial cell bodies(Fig. 6). Some punctate immunofluorescence of GLT-1 wasscattered throughout the neuropil but could not be attrib-uted specifically to either neuronal or glial origin. Theseresults demonstrate the presence of GLT-1-like immunore-activity and suggest the presence of GLT-1-like epitopes inthe leech CNS. Furthermore, the GLT-1 antigen is ex-pressed on the cell surface of putative microglia and in theneuropil glial processes that line the inner capsule of leechganglia.

DISCUSSION

Our results complement previous physiological and phar-macological evidence for glutamate’s role as a neurotrans-mitter in the leech CNS. GluR 5/6/7-like immunofluores-cence was detected within putative microglia and punctateimmunolabeling within the neuropil of leech ganglia.GLT-1-like immunoreactivity also exhibited a discretestaining pattern, primarily confined to the inner capsule ofleech ganglia. Staining of putative glia also localize to theinner capsule, suggesting that neuropil glial processesbordering neuronal packets express a GLT-1-like epitope;scattered, punctate staining was also observed within theneuropil and perhaps localized to neuropil glia. Immunohis-tochemical detection of GluR 5/6/7-like and GLT-1-likeantigens in leech ganglia indicates that leech neurons maypossess 1) receptors that physiologically respond to endog-enously released glutamate and 2) transporters that re-move glutamate from extracellular spaces. Furthermore,our findings together with previous physiological evidencestrongly support glutamate’s role as a neurotransmitter inthe leech swim pathway and bring us closer to understand-ing the circuitry underlying swimming in the medicinalleech.

The data presented here are the first to describe immu-nohistochemical detection of the vertebrate GluR 5/6/7-like and GLT-1-like epitopes in an invertebrate CNS. Webelieve that the antisera raised against vertebrate recep-tors are applicable in this invertebrate system for tworeasons. First, a number of glutamate receptor subunitshave been cloned in invertebrates (Darlison, 1992; Maricqet al., 1995; Hart et al., 1995) and exhibit up to 48% aminoacid sequence identities with various vertebrate ionotropicglutamate receptor subunits. Furthermore, antagonists to

Fig. 6. Double-labeling with antisera to GLT-1 subtype and puta-tive leech glia. Cryostat-sectioned tissue was double-labeled withGLT-1 and glial (mAb 39) antisera. A rhodamine-conjugated secondaryantibody was used to detect GLT-1, and a fluorescein-conjugatedantibody was used for G39 visualization. GLT-1-like immunoreactivitylocalized primarily to the inner capsule, although some punctateimmunofluorescence was also detected within the neuropil and cellbody layer (a and c). In addition, G39 immunofluorescence labeled thepacket glia, which highlight individual neuronal cell bodies and apicalprocesses issuing from the soma (b and d). More importantly, G39immunoreactivity colocalized with GLT-1-like immunoreactivity aroundthe inner capsule (long arrows) and the lining of neuropil septae (shortarrows). The same field of view is presented for a and b and for c and d.In the absence of the primary antibodies, no staining was detectedwith either rhodamine (e) or fluorescein (f) in control sections. Top offigure corresponds to the anterior of the animal in each panel. Scalebar 5 50 µm.


glutamate are effective in both vertebrate and inverte-brate systems (Honore et al., 1988; Collingridge andLester, 1989; Quinlan et al., 1995; Dierkes et al., 1996;Stuhmer et al., 1996), suggesting that the two systems usesimilar mechanisms to block agonist-induced responses.The similarities in sequence and pharmacology of bothvertebrate and invertebrate glutamate receptors thusjustifies our use of GluR antibodies raised against verte-brate sequences in an invertebrate model. The samereasoning may be applied to the case of using antisera tovertebrate glutamate transporters in an invertebrate sys-tem, since vertebrate reuptake blockers have been found tobe effective in the leech (Deitmer and Schneider, 1997).Thus far, sequence homology of vertebrate and inverte-brate glutamate transporters has not been investigated.Certainly isolating, cloning, and sequencing leech gluta-mate receptors and glutamate transporters are necessaryto identify the similarities and differences with vertebratemolecules.

Distribution of GluR 5/6/7-likeimmunoreactivity

The present investigation is the first to examine GluR5/6/7-like immunoreactivity in the leech. Previous studieshave employed the GluR 5/6/7 antibody to localize immuno-histochemically glutamate receptor subunits in the verte-brate CNS (Huntley et al., 1993; Vickers et al., 1993; Broseet al., 1994; Siegel et al., 1995). Although direct compari-sons of regional GluR 5/6/7 distribution between verte-brate and leech nervous systems cannot be made, manysimilarities exist in the type of GluR 5/6/7-like immuno-reactivity and the cellular localization of the GluR 5/6/7epitope within the leech and vertebrate CNS.

Not surprisingly, GluR subunit immunoreactivity isdiscretely expressed throughout all of the neuronal tissuesexamined in vertebrates. In the monkey neocortex, aggre-gates of GluR 5/6/7-like immunoreactivity were diffuselyscattered throughout the cytoplasm of both neuronal cellbodies and dendrites (Huntley et al., 1993). Some of theneuronal somata were lightly stained whereas others wereintensely immunofluorescent throughout the cytoplasmand around the nuclear membrane. In the leech, a fewneuronal somata labeled with the GluR 5/6/7 antibody.Immunofluorescence within those cell bodies appearedintermittent throughout the cytoplasm; unlike the observa-tions of Huntley et al. (1993), cytoplasmic staining did notobviously delineate a nuclear membrane within leechneuronal cell bodies. In both systems, cytoplasmic stainingmay be attributed to various stages of synthesis, trans-port, assembly, and degradation of the glutamate receptorsubunits. Whereas neuroglial cells remained unstained inmonkey neocortex (Huntley et al., 1993), the cytoplasm ofputative leech microglial cells exhibited GluR 5/6/7-likeimmunoreactivity; microglial nuclei remained devoid ofstaining.

At the ultrastructural level, some but not all somata,asymmetrical synapses, dendritic shafts, or spines labeledwith the GluR 5/6/7 subunit antibody in the vertebrateCNS. Huntley et al. (1993) observed many prominentlylabeled apical dendrites as well as an occasional basilardendritic process in the neocortex. Vickers et al. (1993)noted intense GluR 5/6/7-like immunoreactivity in neuro-nal cell bodies and in long apical dendrites throughoutlayers II–VI of the Macaque prefrontal cortex; in addition,

shorter apical dendrites exhibited small punctate concen-trations of immunofluorescence. Siegel et al. (1995) alsodetected GluR 5/6/7-like immunolabeling of postsynapticdensities on distal dendritic segments within the stratumlucidum and strata radiatum and moleculare of the mon-key hippocampus. Whereas postsynaptic densities wereintensely immunofluorescent along lengths of stained den-drites in monkey neocortex, GluR 5/6/7-like immunoreac-tivity was absent from synaptic clefts and presynapticaxon terminals and axons (Huntley et al., 1993). Together,these studies suggest that GluR 5/6/7-like subunits areexpressed exclusively on postsynaptic cells.

In the present study, most GluR 5/6/7-like immunoreac-tivity occurred as brilliant punctate staining within theneuropil, but was not detected within the intersegmentalconnectives or peripheral nerves of sectioned tissue. Someof the staining appeared to overlap with processes ofpreviously identified glutamate-responsive leech neurons(e.g., cells Tr1, 204, 208 and the dorsal exciter motorneuron [DE-3]; M. S. E. Thorogood, personal observations).Since GluR 5/6/7-like epitopes have been localized todendritic processes and neuronal somata of vertebrates(Huntley et al., 1993; Vickers et al., 1993), the GluR 5/6/7receptor subunit may serve a similar postsynaptic role inthe leech CNS. However, one cannot definitively attributeneuropil staining to a particular type of cell, much less anidentified neuron, since the leech neuropil is packed withupward of thousands of neuronal and glial processes(Fernandez, 1978). Ample physiological and pharmacologi-cal evidence exists for the ability of vertebrate macroglia torespond to glutamate (Hertz, 1979; Teichberg, 1991; Stein-hauser and Gallo, 1996). In the leech, physiological andpharmacological studies have demonstrated that neuropilglia respond to exogenously applied glutamate and itsagonists; these glia are also susceptible to non-NMDAreceptor antagonism (Ballanyi et al., 1989; Ballanyi andSchlue, 1990; Dorner et al., 1990, 1994; Deitmer andMunsch, 1992; Hochstrate and Schlue, 1994; Munsch etal., 1994; Rose et al., 1995; Dierkes et al., 1996; Deitmerand Schneider, 1997; Munsch and Deitmer, 1997). Hence,GluR 5/6/7-like immunoreactivity within the neuropil mayalso be associated with the processes of neuropil glia.Without extensive ultrastructural analysis of ultrathinsections of leech ganglia, the identity of the elements (e.g.,neuronal or glial, pre- or postsynaptic) expressing theGluR 5/6/7-like epitope in the leech remains unknown.

Unlike the vertebrate models studied thus far with theGluR 5/6/7 subunit antibody, putative leech microgliaappear to express the GluR 5/6/7-like epitope. Previousstudies have noted autofluorescence of microglia in someimmunohistochemical preparations (Cammermeyer, 1970;Bowman et al., 1992). Autofluorescence of leech microgliain our experiments seems highly unlikely for severalreasons. First, omission of primary antibody from thestaining protocol resulted in the absence of microglialimmunofluorescence with GLT-1 subtype- and GluR 5/6/7and GluR 2/3 subunit antisera. Second, immunofluores-cent labeling of leech microglia was not detected with theNMDAR 1 subunit antibody. Finally, GLT-1-like immuno-reactivity of whole-mounted ganglia exhibited identicalstaining patterns when using either a rhodamine-conju-gated secondary antibody or an avidin-biotinylated peroxi-dase complex second antibody. Although the significance ofthe expression of GluR 5/6/7- and GluR 2/3-like epitopes in


putative leech microglial cells is presently unknown, someparallels between vertebrate and leech microglia havebeen noted. Following CNS damage, leech microglia tendto accumulate at sites of nerve injury (Morgese et al., 1983;McGlade-McCulloh et al., 1989), as does laminin (Masuda-Nakagawa et al., 1990)—an extracellular matrix glycopro-tein associated with axonal sprouting in both leech andvertebrates, and possibly expressed by microglia. (Masuda-Nakagawa et al., 1994). In the squid CNS, activation ofglutamate receptors on Schwann cells initiates a cascadeof events leading to increased cAMP (Evans et al., 1985,1992; Lieberman et al., 1989), which in turn may enhancelaminin gene expression (Yamamoto et al., 1994). Gluta-mate receptor activation on microglia may similarly in-duce laminin expression and ultimately repair damagedtissue in the medicinal leech.

Distribution of GLT-1 transporter-likeimmunoreactivity

Until now, the immunohistochemical distribution ofglutamate transporters in an invertebrate CNS has notbeen explored. Previous studies, however, have employedvarious antisera to localize immunohistochemically gluta-mate transporter subtypes in the vertebrate CNS. GLT-1transporters, in particular, are expressed predominantlywithin plasma membranes and processes of macroglialcells (Danbolt et al., 1992; Levy et al., 1993; Rothstein etal., 1994; Lehre et al., 1995; Chaudhry et al., 1995).Specifically astrocytes and Bergmann glia, but not oligoden-drocytes, are labeled with GLT-1 antisera. Chaudhry et al.(1995) recently suggested that GLT-1 might be present onsynaptic membranes of some excitatory glutamatergicsynapses, at levels 5–10% of that found on astrocytes.Ultrastructural analysis subsequently detected the GLT-1-immunoreactive protein in both astroglial processes andcell bodies (Danbolt et al., 1992; Rothstein et al., 1994),which may reflect synthesis, transport, assembly, or degra-dation of the glutamate transporter. These earlier observa-tions are consistent with our findings: GLT-1-like immuno-reactivity was confined to processes of neuropil glia liningthe inner capsule and to the cell surfaces of microglia ofleech ganglia. To a much lesser extent punctate stainingwas also detected within the neuropil where it might beassociated with plasma membranes of neuropil glia and/orneurons. GLT-1-like immunoreactive processes formeddiscrete sheaths around neuronal perikarya in the verte-brate CNS (Rothstein et al., 1994). In contrast, processes ofleech packet glia surrounding neuronal somata exhibitedonly scattered punctate immunoreactivity. This suggeststhat GLT-1 transporters act primarily at the interfacebetween the neuronal packets and the neuropil, perhaps toprotect synapses within the neuropil from glutamate’spotential excitotoxic effects (Rothstein et al., 1996).

ACKNOWLEDGMENTS

We thank Dr. Margaret Hollyday for valuable discus-sion, Dr. Karen F. Greif for critical reading of the manu-script, and Dr. Lauren Sweeney for the Figure 1 illustra-tion. This work is part of a dissertation by M.S.E.T. andwas supported by NSF grant 9514617 to P.D.B. and by aPatricia Roberts Harris Fellowship to M.S.E.T.

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Glutamate receptor 5/6/7-like and glutamate transporter-1-like immunoreactivity in the leech central nervous system