Ultrastructural localization of the binding fragment of tetanus toxin in putative ?-aminobutyric acidergic terminals in the intermediolateral cell column: A potential basis for sympathetic

Ultrastructural Localization of theBinding Fragment of Tetanus Toxin in

Putative g-Aminobutyric AcidergicTerminals in the Intermediolateral

Cell Column: A Potential Basisfor Sympathetic Dysfunction in

Generalized Tetanus

MICHAEL A. LIGORIO, WENDY AKMENTIN, FRANCES GALLERY,

AND JOHN B. CABOT*

Department of Neurobiology and Behavior, State University of New York at Stony Brook,Stony Brook, New York 11794-5230

ABSTRACTTetanus toxin (TeTx) causes sympathetic hyperactivity, a major cause of mortality in gen-

eralized tetanus, apparently by obstructing the inhibition of sympathetic preganglionic neurons(SPNs). Neuroanatomic tracing and immunohistochemistry were used to investigate whetheraxon terminals in the intermediolateral cell column (IML) that synapse on SPNs and use theinhibitory neurotransmitter g-aminobutyric acid (GABA) may be infected transsynaptically withTeTx. The binding fragment of TeTx (TTC; an atoxic surrogate of TeTx) and the cholera toxin Bsubunit (CTB; a retrograde tracer) were injected into the rat superior cervical ganglion and, over16–48 hours, were transported to the ipsilateral IML in the caudal half of the last cervical andfirst three thoracic spinal cord segments. With light microscopy, diffuse CTB immunolabelingextended throughout SPN perikarya and dendrites. Punctate TTC and GABA immunolabelingwere accumulated densely in the neuropil between and surrounding SPN processes. Withelectron microscopy, 54% of the axon terminals in the IML (n 5 1,337 terminals) were TTCimmunolabeled (TTC1), and 25% contained putative neurotransmitter levels of GABA immuno-labeling (GABA1). On average, GABA1 terminals had a 76% chance of also being TTC1 and a62% greater chance of being TTC1 than GABA2 terminals (P , 0.000001). Axon terminals werejust as likely to be TTC1 and/or GABA1 regardless of whether the dendrites they synapsed onwere large (.1 mM) or small in cross-sectional area or were labeled retrogradely. Sympathetichyperactivity in tetanus may involve 1) retrograde and transsynaptic transport of TeTx by SPNsand 2) at least in part, an infection of GABAergic terminals in the IML. J. Comp. Neurol. 419:471–484, 2000. © 2000 Wiley-Liss, Inc.

Indexing terms: sympathetic preganglionic neuron; inhibition; spinal cord inhibitory

neurotransmitter; transsynaptic transport; axon terminal

Tetanus is a life-threatening, infectious disease of both thesomatomotor and the autonomic nervous systems. The well-described symptoms of somatomotor pathology are painfulspasms of the face, neck, limbs, and back (Bleck, 1989).Generally, symptoms of autonomic pathology are manifesta-tions of sympathetic hyperactivity, which include tachycar-dia, hypertension, and an elevated metabolic rate (Hortnagland Brucke, 1979; Bleck, 1989; Wright et al., 1989).

At the onset of infection, the anaerobic bacterium Clos-tridium tetani enters the body through a wound and even-

tually releases tetanus toxin (TeTx) into systemic circula-tion (Bleck, 1989). Numerous physiologic and anatomic

Grant sponsor: NHLBI; Grant number: HL24103.*Correspondence to: John B. Cabot, Department of Neurobiology and

Behavior, State University of New York at Stony Brook, Stony Brook, NY11794-5230. E-mail: [email protected]

Received 13 April 1999; Revised 14 December 1999; Accepted 14 Decem-ber 1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 419:471–484 (2000)

© 2000 WILEY-LISS, INC.

studies using TeTx or its atoxic binding fragment (TTC)have suggested that TeTx infects the somatomotor and thesympathetic nervous systems along the following path-way: 1) TeTx is endocytosed by a-motoneuron (a-MN) andsympathetic preganglionic neuron (SPN) axon terminals(Schwab and Thoenen, 1977; Parton et al., 1987; Meckleret al., 1990); 2) TeTx is transported intraaxonally bya-MNs and SPNs retrogradely to their cell bodies in thebrainstem and spinal cord (Price et al., 1975; Schwab andThoenen, 1976; Fishman and Carrigan, 1987, Fishmanand Carrigan, 1988; Cabot et al., 1991); 3) TeTx is trans-ported transsynaptically to axon terminals that are pre-synaptic to a-MN and SPN cell bodies and dendrites(Schwab and Thoenen, 1976; Fishman and Savitt, 1989;Cabot et al., 1991); and 4) TeTx obstructs the inhibition ofa-MNs and SPNs (Brooks et al., 1957; Paar and Well-honer, 1973; Kanda and Takano, 1983). The pathophysi-ologic consequence of disinhibition is a sustained increasein a-MN and SPN discharge activity, leading to somato-motor and sympathetic hyperactivity.

Although the above-described steps of infection are ac-knowledged widely, one group of investigators (Takano etal., 1989; Takano, 1993) disputed whether generalizedtetanus truly is caused by a specific effect on spinal inhi-bition. Indeed, there is ample physiologic and pharmaco-logic evidence that TeTx acts presynaptically to block therelease of inhibitory neurotransmitters, such asg-aminobutyric acid (GABA; Carrea and Lanari, 1962;Curtis and De Groat, 1968; Curtis et al., 1973; Davies andTongroach, 1979; Collingridge et al., 1981; Collingridgeand Davies, 1982; Empson and Jefferys, 1993; Empson etal., 1993). However, although ultrastructural studies havelocalized either TeTx or TTC to axon terminals that syn-apse on a-MNs and SPNs (Schwab and Thoenen, 1976;Fishman and Savitt, 1989; Ligorio, 1996), they have notshown that any of these axon terminals contain inhibitoryneurotransmitters.

Immunohistochemical studies suggest that many axonterminals in the intermediolateral cell column (IML) thatsynapse on SPN perikarya and dendrites are GABAergic(Bacon and Smith, 1988; Bogan et al., 1989; Chiba andSemba, 1991; Cabot et al., 1995). Furthermore, recentphysiologic studies suggest that GABA may decreasemean arterial pressure and/or heart rate by hyperpolariz-ing SPNs and inhibiting their discharge activity (Yo-shimura and Nishi, 1982; Backman and Henry, 1983;Clendening and Hume, 1990; Hassessian et al., 1991; Ino-kuchi et al., 1992; Wu and Dun, 1992; Madorin and Ca-laresu, 1994; Lewis and Coote, 1995). Therefore, GABAer-gic terminals synapsing on SPNs may be affected by TeTx.

To test this hypothesis, we have employed retrogradelabeling with the binding fragment of cholera toxin (CTB)and immunoelectron microscopic colocalization of TTCand GABA in axon terminals in the IML. In light ofcumulative physiologic and pharmacologic data, the ana-tomic observations below suggest that one cause of sym-pathetic hyperactivity during generalized tetanus is ablockade of GABAergic input to SPNs.

MATERIALS AND METHODS

Experimental animals and surgicalprocedures

Experiments were performed on five male SpragueDawley rats (Taconic Farms) weighing 125–250 g. All

surgical procedures were performed under aseptic condi-tions. Immediately after a subcutaneous injection of atro-pine sulfate (0.2–0.4 mg/kg), rats were anesthetized witha mixture of xylazine (10 mg/kg) and ketamine (90 mg/kg).The right superior cervical ganglion (SCG) was ap-proached ventrolaterally following a midline, anterior cu-taneous incision extending from approximately the secondcervical vertebrae (C2) to C6.

By using 10-ml Hamilton syringes (Hamilton, Reno, NV)affixed with glass micropipette tips, 1–2 ml of TTC (10%,50 mM sodium bicarbonate, pH 9–10; Roche MolecularBiochemicals) and/or CTB (1.0%; 0.05 M Tris; 0.2 M NaCl,pH 7.5; List Biological Laboratories, Campbell, CA) wereinjected into the right SCG. TTC only was injected intotwo rats, CTB only was injected into one rat, and both TTCand CTB were injected into two rats.

The survival time was 48 hours postinjection in fourrats and 16 hours in one rat in which only TTC wasinjected, at which time rats were given a lethal dose ofsodium pentobarbital (200–400 mg/kg). When unrespon-sive to nociceptive stimuli, rats were injected intracardi-ally with heparin (0.1 ml, 1,000 USP/ml) and perfusedtranscardially with saline (100–200 ml, 0.9% NaCl) im-mediately followed by 1 liter of cold fixative [4% parafor-maldehyde; 0.15% glutaraldehyde; 0.1 M sodium phos-phate buffer (PB), pH 7.4]. These procedures conformed toNational Institutes of Health guidelines and were ap-proved by the Animal Care and Use Committee of TheState University of New York at Stony Brook. The spinalcord was removed and immersed in fixative for 1–2 hoursat 4°C.

Immunohistochemistry

Light microscopy. The caudal half of C8 (cC8) andthe first three thoracic spinal cord segments (T1–T3) werefrozen sectioned in the transverse plane at 40 mm with asliding microtome. All immunoreagents were diluted in0.1 M PB, pH 7.4 containing 0.3% Triton X-100 unlessotherwise indicated. Sections were blocked for 30 minutesin 2.5% normal serum that came from the host species ofthe secondary antiserum to be used in step 2 (see below).The same normal serum (1.0%) also was included with theprimary antibody to be used in step 1 (see below).

After a brief rinse in PB, immunoperoxidase labelingproceeded as follows: step 1) overnight incubation at 4°Cin either mouse anti-TTC (1:2,000; Roche Molecular Bio-chemicals), goat anti-CTB (1:12,500; List Biological Labo-ratories), or rabbit anti-GABA (1:2,000; Sigma, St. Louis,MO) followed by six 10-minute rinses in PB; step 2) 1-hourincubation in either rat preadsorbed horse anti-mouseimmunoglobulin (IgG), rabbit anti-goat IgG, or goat anti-rabbit IgG biotinylated secondary antisera (1:200; VectorLaboratories, Burlingame, CA) followed by three 10-minute rinses in PB; step 3) 1-hour incubation in horse-radish peroxidase-conjugated avidin-biotin complex (ABC)reagent (Elite Vectastain; Vector Laboratories) followedby two 10-minute rinses in PB and two 10-minute rinsesin 0.1 M sodium acetate buffer (AB), pH 6.0; and step 4)3-minute peroxidase reaction [0.05% 3,39-diaminobenzidine(DAB; Sigma), 0.6% nickel-ammonium sulfate, 0.01% hy-drogen peroxide, and AB] followed by three 5-minuterinses in AB.

After immunoperoxidase labeling, sections were trans-ferred to 0.05 M PB, mounted on gelatin-coated slides, andair dried overnight. Slides were dehydrated through an

472 M.A. LIGORIO ET AL.

ascending series of ethanols, cleared through two changesof xylene, and coverslipped with Permount (Fisher Scien-tific, Fair Lawn, NJ).

Electron microscopy. Spinal cord segments cC8through T3 were sectioned on a Vibratome in the trans-verse plane at 50 mm in PB at 4°C. To enhance penetrationof the immunoreagents, tissue sections were snap frozenand thawed in 15% sucrose (PB) as described by Smileyand Goldman-Rakic (1993). Sections were rinsed briefly inPB, immersed for 20 minutes in 0.3% Triton X-100 (PB),and rinsed thoroughly with several changes of PB.

Preembedding immunoperoxidase labeling of TTC andCTB proceeded as outlined above for light microscopy withthe following differences: 1) Triton X-100 was omittedfrom the diluent of all immunoreagents, 2) primary anti-sera concentrations were doubled and their incubationperiods were lengthened to 2–3 days, and 3) secondaryantisera and ABC reagent incubation periods were ex-tended to 2 hours. When TTC and CTB were to be immu-noperoxidase labeled in the same section, the immunore-agents were applied in the following order: 1) mouse anti-TTC serum, 2) rat preadsorbed biotinylated horse anti-mouse IgG serum, 3) goat anti-CTB serum, 4) biotinylatedrabbit anti-goat IgG serum, 5) ABC reagent, and 6) im-munoperoxidase reaction.

After immunoperoxidase labeling, sections were fixedfor 30–60 minutes with osmium tetroxide (1–2%, 0.1 MPB), dehydrated through an ascending series of ethanols,and embedded in Durcupan epoxy resin (Fluka, Milwau-kee, WI). Sections were sandwiched between sheets ofACLAR (Ted Pella, Redding, CA) and cured at 60°C for atleast 24 hours. Blocks of tissue containing the IML weresectioned at 60–70 nm with a Reichert-Jung Ultracut Eultramicrotome (Nussloch, Germany). Ultrathin sectionswere mounted either on 300/400 extra-fine, nickel-meshgrids (Ted Pella) with the aid of Coat-Quick “G” adhesive(Electron Microscopy Sciences, Fort Washington, PA) oron Formvar-coated, nickel-slot grids.

Sections were postembedding immunogold labeled forGABA within 24 hours of sectioning by using a modifica-tion of the protocol of Phend et al. (1992). Mesh grids wereimmersed completely section-side-up in drops of a buffercontaining 0.05 M Tris buffer, pH 7.6, with 0.9% NaCl and0.1% Triton X-100 (TBST buffer) for 5 minutes. Formvar-coated slot grids were floated section-side-down on drop-lets. Immunogold labeling proceeded as follows: 1) over-night incubation in rabbit anti-GABA (dilution, 1:1,000–2,000; TBST, pH 7.6; Sigma) followed by two 5-minuterinses and one 30-minute rinse in TBST, pH 7.6, and one5-minute rinse in TBST, pH 8.2; and 2) 1-hour incubationin 15 nm gold-conjugated goat anti-rabbit IgG (dilution1:25; TBST, pH 8.2; Amersham Life Science, ArlingtonHeights, IL) followed by two 5-minute rinses in TBST, pH7.6, and several rinses in dH20. Grids were air dried,stained with uranyl acetate and lead citrate, and exam-ined at 60/80 kV with a JEOL 1200 EX electron micro-scope (JEOL, Peabody, MA).

Electron microscopic analysis ofimmunolabeled neuronal elements

in the IML

Sampling the IML neuropil. All electron microscopicanalyses were performed in animals that were killed 48hours after the SCG injection. At this time, TTC immuno-

peroxidase labeling was prevalent in the IML neuropil.Only the IML of thoracic spinal cord segment T2, whichhas the greatest number of SPNs that project to the SCG(Strack et al., 1988), was examined systematically. In twoanimals, CTB was used to label retrogradely SPNperikarya and dendrites. For each analysis, a single ultra-thin section from a different block of T2 was selected. Ineach analyzed section, an area within the IML was pho-tographed as one or more nonoverlapping montage(s) fromwhich 222–446 axon terminals and 3–5 SPN perikaryawere sampled. This sampling method avoided countingthe same neuronal elements twice (Cabot et al., 1995).

Identification of TTC-positive terminals and retro-

gradely labeled SPN perikarya and dendrites. Anelectron-dense, preembedding immunoperoxidase reac-tion product was used to label TTC and CTB. There wasvariation in the intensity of this reaction product, partic-ularly in axon terminals, due at least in part to limitedpenetration of the immunoreagents. To help control forthis variation, in each electron photomicrograph, one axonterminal and/or dendrite that clearly was immunolabeledand one matching neuronal element that clearly was un-labeled both were used as standards with which all otherneuronal elements in the field of view were compared.Axon terminals that were not unambiguously TTC-positive (TCC1) were classified as TTC-negative (TTC2).

Identification of GABA1 terminals. Postembeddingimmunogold histochemistry was used to label GABA inaxon terminals. Although most axon terminals exhibitedimmunogold labeling, they probably did not all use GABAas a neurotransmitter. Some background labeling mayresult either from low metabolic levels of GABA or fromnonspecific binding of anti-GABA serum to substancesother than GABA (Hodgson et al., 1985; see Controls forantibody specificity, below). To be considered GABA1,axon terminals were required to have less than a 0.5%chance of containing only background levels of immuno-gold labeling.

Immunogold labeling was quantified in axon terminalsas follows: 1) axon terminals were outlined on electronphotomicrographs (magnification, 38,000–10,000) by us-ing interpolation where membranes were not completelyvisible; 2) cross-sectional areas of these tracings weremeasured with the Bioscan Optimas image analysis sys-tem equipped with a SummaSketch II Plus pad (Summa-graphics); 3) gold particles within each outlined area, ex-cept those particles overlying mitochondria (presumably anyGABA in these organelles was not part of the readily releas-able neurotransmitter pool), were counted; and 4) the levelof GABA in each terminal was expressed as an immuno-gold density, i.e., the number of gold particles/mm2.

Background immunogold labeling was estimated bysampling terminal-sized areas (TSAs) within SPNperikarya (Cabot et al., 1995), which are non-GABAergic.For each analysis, the outline of an axon terminal with across-sectional area near the mean of all axon terminalareas in the sample was chosen as the TSA. All TSAimmunogold densities were collected in SPN perikarya, asillustrated schematically in Figure 1. For each IMLsample, 120 TSA immunogold densities were accumu-lated from among 3–5 SPN perikarya, with equal num-bers of TSA densities being measured in each perikarya.The total variance of all the TSA immunogold densitieswas the sum of 1) the variance of TSA immunogolddensities between SPN perikarya and 2) the variance of

473TETANUS TOXIN AND GABAERGIC TERMINALS IN THE IML

TSA immunogold densities within SPN perikarya. Be-cause the variance of immunogold densities is inverselyproportional to the area over which the individual mea-surements are made, and SPN perikarya are muchgreater in size than axon terminals, the use of TSAmeasurements avoids a sampling bias that otherwisewould result in an underestimation of the variance ofbackground immunogold labeling in axon terminals inthe IML (Cabot et al., 1995). An axon terminal wasdesignated as GABA1 if its immunogold density was2.62 TSA standard deviations above the mean TSA im-munogold density of SPN perikarya.

Controls for antibody specificity

The specificity of rabbit anti-GABA serum (lot021h48903; Sigma) was confirmed by using dot blots andpostembedding immunogold histochemistry. On dot blots,serial dilutions of a glutaraldehyde-condensed conjugateof GABA and bovine serum albumin (GABA-BSA) andglycine-BSA (Seguela et al., 1984) were spotted onto dry

nitrocellulose paper. Blots were dried for 10–15 minutesand then blocked for 30–60 minutes in 2% BSA. Primaryincubations were carried out overnight at 4°C (with gentleagitation) in rabbit anti-GABA serum diluted 1:10,000 inTris phosphate-buffered saline (TPBS; 10 mM Tris, 10mM sodium phosphate, 150 mM sodium chloride, and 5.4mM potassium chloride, pH 7.4) containing 2% normalgoat serum. Blots were rinsed in TPBS after this and eachsubsequent step. Secondary incubations in biotinylatedgoat anti-rabbit serum (1:200 in TPBS) and tertiary incu-bations in Elite ABC reagent were each 1 hour. Ensuingperoxidase reactions (50 mM Tris buffer, pH 7.6, contain-ing 0.01% DAB and 0.01% hydrogen peroxide) were car-ried out until spots clearly had developed on the nitrocel-lulose paper ('1 minute). Only GABA-BSA wasimmunoreactive; glycine-BSA was not. In other dot blots,when the primary antiserum contained 1% GABA-BSA,dot-blot immunoreactivity was suppressed completely,whereas 1% glycine-BSA had no such effect. In postem-bedding immunogold assays, primary incubations weredone on a series of three serial sections with the additionof 10 mM free GABA, 10 mM free glycine, or without anyfree amino acid added to the anti-GABA serum (i.e., nor-mal condition). All primary solutions were prepared inadvance and preadsorbed overnight at 4°C before use.Preadsorption of the primary antiserum with free GABAvirtually eliminated immunogold labeling (Fig. 2),whereas preadsorption with free glycine did not have astatistically significant effect on the percentage of GABA1

terminals detected compared with the normal section. Inanalogous experiments, preadsorbing the primary anti-serum with GABA-BSA suppressed immunogold labelingcompletely, but preadsorbing with glycine-BSA did not.

To control for any nonspecific binding of TTC- and CTB-specific antisera, the left IML of cC8–T3 was examinedroutinely, because SPN innervation of the SCG is ipsilat-eral, and injections were made exclusively into the rightSCG. By using light and electron microscopy, TTC and/orCTB immunoperoxidase labeling was observed in neuro-nal perikarya, neuronal processes, and axon terminalsonly in the right IML. In one exceptional case, relativelylow levels of TTC immunolabeling appeared in the leftIML, suggesting that there may be a minor contralateralprojection from the IML to the SCG in some animals. Nospecific immunolabeling associated with TTC, CTB, orGABA was observed when primary antisera were omittedfrom the immunohistochemical protocols.

RESULTS

Broad distribution of CTB, TTC, and GABAimmunolabeling in the IML

Light microscopy. Two rats with postinjection sur-vival times of 16 hours and 48 hours, respectively, wereused for this part of the study. After injection into theright SCG, both CTB and TTC were transported retro-gradely to the ipsilateral IML (Fig. 3) and to other SPNsubnuclei that have been identified previously in spinalcord segments cC8–T3 of the rat (Cabot, 1990; Hosoya etal., 1991; Cabot et al., 1994). Intracellular CTB immuno-peroxidase labeling extended throughout SPN perikaryaand dendritic processes, whereas TTC immunoperoxidaselabeling was accumulated densely in the neuropil betweenand surrounding SPN perikarya and dendrites. At shorter

Fig. 1. A schematic representation of a sympathetic preganglionicneuron (SPN) cell body illustrating how immunogold densities aresampled in terminal sized areas (TSAs) within the perikaryon. Foreach measurement, 1) a TSA template (small, numbered, shadedellipses) was placed arbitrarily somewhere in an SPN perikaryon (thecytoplasmic area excluding the nucleus), 2) all gold particles (smallblack dots) within the TSA were counted except those overlying mi-tochondria (not illustrated), and 3) the number of gold particlestherein were divided by the area of the TSA template. For example,given a unit TSA of 0.5 mm2, the immunogold density of TSA 1 wouldbe 6 gold particles/mm2. Between measurements, the same TSA tem-plate was shifted in a semirandom fashion to nonoverlapping areas inthe SPN perikaryon. The schematic illustrates a series of nine uniqueTSA immunogold density measurements (moving down and to theright) collected from a single SPN perikaryon.


survival times, TTC immunoperoxidase labeling was moreprevalent within SPN perikarya (e.g., 5–8 hours postin-jection; Ligorio, 1996). This time-dependent change inTTC localization is consistent with previous light micro-scopic observations in pigeon spinal cord (Cabot et al.,1991) and suggests that, after retrograde transport, TTCis transported transsynaptically to axon terminals thatare presynaptic to SPNs in the IML. Also consistent withprevious light microscopic studies (Bogan et al., 1989;Cabot et al., 1995), punctate GABA immunoperoxidase

labeling was accumulated densely in the IML (Fig. 3,bottom). Unlike CTB and TTC immunolabeling, GABAimmunolabeling was observed bilaterally throughout thegray and white matter (not shown).

Electron microscopy. For this part of the study, TTCwas injected into the SCG of three rats with postinjectionsurvival times of 48 hours. In two of these rats (animals 2and 3; Tables 1, 2), CTB was used to label SPN perikaryaand dendrites retrogradely. In IML sample 4 (animal 3),for example, 46% of the neurons (n 5 11 neurons) and 42%of the dendritic processes (n 5 180 dendritic processes)were labeled retrogradely.

A preembedding immunoperoxidase reaction was usedto label both CTB and TTC (Figs. 2, 4), and postembeddingimmunogold labeling was used to label GABA (Figs. 2,5–7). In SPN perikarya, CTB immunolabeling could bedistinguished from TTC by its localization to the Golgiendoplasmic reticulum network (Llewellyn-Smith et al.,1992; Ligorio, 1996). However, in dendrites, CTB and TTCboth were localized to neurofilaments and appeared thesame on electron photomicrographs. Because intracellularTTC labeling is more prevalent at earlier survival times(Ligorio, 1996), and because, in IML samples 1 and 2(animal 1), very few dendrites exhibited TTC labeling, thedendritic immunolabeling observed in IML samples 3 and4 (animals 2 and 3) most likely was due to CTB labeling.Only TTC labeling was present in axon terminals, becauseCTB is not transported transsynaptically.

The percentage of axon terminals that were TTC1 ineach IML sample ranged from 49% to 59% and averaged54% (Table 1), whereas the percentage of axon terminalsthat were GABA1 ranged from 15% to 33% and averaged25% (Table 1). In IML samples 1 and 2, axon terminalswere divided into two groups: those terminals synapsingon large-caliber dendrites (.1 mm in cross-sectional area)and those terminals synapsing on small-caliber dendrites(#1 mm). In both of these samples, axon terminals syn-apsing on small-caliber dendrites were just as likely to beTTC1 and/or GABA1 as axon terminals synapsing onlarge-caliber dendrites. Likewise, axon terminals synaps-ing on retrogradely labeled SPN processes were just aslikely to be TTC1 and/or GABA1 as other terminals in theIML (compare Table 1 with Table 2).

It is worth noting that, if axon terminals acquire TTCtranssynaptically from SPNs, then it may be expected thataxon terminals synapsing on retrogradely labeled SPNprocesses would be more likely to be TTC1 than axonterminals synapsing on unlabeled processes. Because our

Fig. 2. Electron photomicrographs showing an axon terminal thatwas immunoperoxidase labeled for the binding fragment of tetanustoxin (TTC) and immunogold labeled for the neurotransmitterg-aminobutyric acid (GABA) in three serial sections. The double-labeled terminal is synapsing on an unlabeled dendrite (ud) in theintermediolateral cell column (IML) of the second thoracic spinal cordsegment (T2). a: When the normal immunogold-labeling protocol wasused, the primary antiserum was not preadsorbed with any hapten,and the axon terminal was GABA-positive (GABA1), that is, it con-tained putative neurotransmitter levels of GABA. b: When the pri-mary antiserum was preadsorbed with 10 mM free GABA, immuno-gold labeling was virtually eliminated. c: When the primaryantiserum was preadsorbed with 10 mM glycine, the axon terminalremained GABA1. Arrowheads point to regions of synaptic special-ization. Scale bar 5 0.25 mm in b (also applies to a and c).


results did not confirm this expectancy, either CTB doesnot label all SPNs retrogradely that project to the SCG, orTTC may reach axon terminals through pathways other

than retrograde and transsynaptic transport. For a dis-cussion of the possibility that TTC may diffuse radiallythroughout the extracellular space, see Ligorio (1996).

Electron microscopic colocalization of TTCand GABA in axon terminals

A combination of preembedding immunoperoxidase la-beling and postembedding immunogold labeling was usedto colocalize TTC and GABA in axon terminals in the IML(Figs. 2, 7). Across all four samples, 66–84% (average,76%) of all GABA1 terminals also were TTC1, comparedwith only 43–53% (average, 47%) of all GABA2 terminals(Fig. 8). This difference in TTC1 percentages was statis-tically significant in every sample, including subsamplesof axon terminals that were presynaptic to retrogradelylabeled SPN processes (Fig. 9).

The results also were compared among animals. Datafrom samples 1 and 2 (both of which were collected fromanimal 1; see Table 1) were found to be similar by using achi-square test of homogeneity. These data were pooledand compared with the data from animals 2 and 3. Byusing a logistic model that measured the association of TTCand GABA in axon terminals as an odds ratio (Fleiss, 1981),the difference in the TTC1 percentages of GABA1 andGABA2 terminals among all animals was highly significant(P , 0.000001). On average, GABA1 terminals had a 63%greater chance of being TTC1 than GABA2 terminals.

TABLE 1. Percentages of g-Aminobutyric Acid-Positive and TetanusToxin Atoxic Binding Fragment-Positive Terminals in the

Intermediolateral Cell Column

Animal Sample No.1 %GABA1 %TTC1

1 1 386 15 491 2 446 24 592 3 222 32 503 4 283 33 56Average — 334 25 54

1The number of terminals in each intermediolateral cell column sample. GABA,g-aminobutyric acid; TTC, tetanus toxin atoxic binding fragment; 1, positive.

TABLE 2. Percentages of g-Aminobutyric Acid-Positive and Tetanus ToxinAtoxic Binding Fragment-Positive Terminals Synapsing on Retrogradely

Labeled Sympathetic Preganglionic Neurons

Animal Sample1 No.2 %GABA1 %TTC1

2 1 90 30 473 2 107 34 59Average — 99 32 53

1Cholera toxin B subunit-labeled samples 1 and 2 are subsamples of intermediolateralcell column samples 3 and 4 from Table 1.2The number of terminals in each sample.

Fig. 3. Light photomicrographs from transverse sections of T2comparing the localization of the binding fragment of cholera toxin(CTB), TTC, and GABA in the right IML. Diffuse CTB immunoper-oxidase labeling can be observed in a group of SPN perikarya and inSPN processes extending medially (to the left) and dorsolaterally (tothe upper right). Punctate TTC immunoperoxidase labeling is appar-ent in the neuropil between and surrounding SPN perikarya anddendrites at 16 hours postinjection. Diffuse TTC immunoperoxidaselabeling can be seen in a few SPN perikarya. Punctate GABA immu-noperoxidase labeling is especially dense in the IML and along SPNdendrites and also can be observed throughout the gray and whitematter. Scale bars 5 50 mm.


DISCUSSION

Reliability of GABA and TTCimmunolabeling

When GABA and TTC were colocalized, the preembed-ding immunoperoxidase reaction used to label TTC mayhave compromised postembedding immunogold labelingfor GABA either by neutralizing antigenic determinantsor simply by obscuring gold particle visualization. To as-sess the significance of any such error, the GABA1 termi-

nal percentages of the four IML samples in this studywere compared with those of a previous study in whichimmunogold labeling was quantified in the absence of anyperoxidase labeling in axon terminals. In the previousstudy, the average percentage of GABA1 terminals in theIML was 37% (Cabot et al., 1995), significantly higherthan the percentage of GABA1 terminals in IML sample 1but only marginally higher than the GABA1 terminalpercentages observed in samples 3 and 4 (Table 1). There-fore, under ideal circumstances, gold counts inperoxidase-labeled terminals can be fairly reliable. Al-

Fig. 4. Electron photomicrograph showing TTC-positive (TTC1)terminals and a retrogradely labeled SPN dendritic process (SPNd) inthe IML neuropil of T2. Axon terminal 1 is TTC1 and is presynapticto both the SPNd and an unlabeled dendrite (ud). Axon terminal 2 alsois TTC1 and is presynaptic to the same unlabeled dendrite as termi-

nal 1. Axon terminals 3 and 4 are both TTC-negative (TTC2). Axonterminal 3 is presynaptic to an unlabeled dendrite, and axon terminal4 is closely opposed to the SPNd. Arrowheads point to regions ofsynaptic specialization. These identifications were confirmed on serialsections. Scale bar 5 0.2 mm.


though it may have been more reliable to have usedpostembedding immunogold labeling for TTC, efforts todo so were not successful.

Errors in TTC labeling also were made. For example,some axon terminals that were identified on serial sec-tions were TTC1 on one section but not on others. Thisinconsistency may have been a consequence of limitedpenetration of the immunoreagents in the tissue, whichsuggests that the percentage of TTC1 terminals in theIML may have been underestimated. Presumably, thisTTC labeling error is equally as likely to occur in GABA1

terminals as in GABA2 terminals. Therefore, it does notchange the result that GABA1 terminals are more likelyto be TTC1 than GABA2 terminals.

Although the experimental conditions were not nec-essarily optimal for GABA and/or TTC immunolabeling,compromises were necessary to colocalize them underthe same experimental conditions. To our knowledge, noprevious anatomic studies have localized either TTC orTeTx to axon terminals with GABA or any other inhib-itory neurotransmitter in the central nervous system(CNS).

Sympathetic hyperactivity duringgeneralized tetanus may be due in part toan effect on GABAergic terminals in the

IML

The colocalization data are meaningful in a pathophys-iologic context, in that GABA1 terminals are GABAergicand TTC1 terminals may sequester and be blocked byTeTx. With regard to the neurotransmitter content ofGABA1 terminals, in addition to our own controls of an-tiserum specificity (see Materials and Methods), previousstudies have established that antisera raised againstGABA-BSA glutaraldehyde conjugates specifically labelGABAergic neurons and axon terminals in the CNS withminimal cross reactivity to other amino acid neurotrans-mitters, such as glutamate and glycine (Storm-Mathisenet al., 1983; Seguela et al., 1984; Hodgson et al., 1985;Somogyi and Hodgson, 1985; Somogyi et al., 1985; Spre-afico et al., 1994; Todd et al., 1995). With regard to thesusceptibility of TTC1 terminals to blockade by TeTx,TeTx is a heterodimer composed of a 50-kD light chain anda 100-kD heavy chain. The light chain is a zinc metallo-

Fig. 5. Electron photomicrographs of GABA1 terminals in the IML neuropil of T2 contacting anunlabeled dendritic process (ud; a) and synapsing on a retrogradely labeled SPNd (b). Arrows point toregions of synaptic specialization. Scale bars 5 0.2 mm.


protease with a substrate (synaptobrevin; a membraneprotein of synaptic vesicles) that is involved in exocytosis(Montecucco and Schiavo, 1994; Poulain, 1994). The heavychain can be cleaved in vitro into two moieties; its 50-kDcarboxyl terminus, C fragment or TTC, specifically com-petes with the holotoxin for binding sites on neuronal

membranes (Morris et al., 1980; Goldberg et al., 1981;Montecucco, 1986), and, like TeTx, TTC is transportedretrogradely and transsynaptically (Weller et al., 1986;Bizzini, 1989; Cabot et al., 1991; Halpern and Neale,1995). After transsynaptic transport, TTC has been local-ized to synaptic vesicles (Fishman and Savitt, 1989; Ligo-

Fig. 6. Histograms comparing background immunogold labeling in SPN perikarya withneurotransmitter-specific immunogold labeling in axon terminals. Frequency distributions from corre-sponding IML samples 1–4 (Table 1) are paired left to right.


rio, 1996), whereas, in vitro, TeTx can bind to synaptoso-mal proteins (Schengrund et al., 1992, 1996). Finally,although TTC itself is atoxic, it is an essential moiety thatallows the toxic light chain to be internalized by neuronalcells and obstruct synaptic transmission (Simpson, 1984).

Given these conditions, the colocalization data in thisstudy suggest that GABAergic terminals synapsing onSPNs can be blocked by TeTx. Because GABA appears toinhibit SPNs (Yoshimura and Nishi, 1982; Backman andHenry, 1983; Clendening and Hume, 1990; Hassessian etal.; 1991; Inokuchi et al., 1992; Wu and Dun, 1992; Ma-dorin and Calaresu, 1994; Lewis and Coote, 1995) andbecause TeTx abolishes the inhibition of SPNs duringlocal tetanus (Paar and Wellhoner, 1973), one cause ofsympathetic hyperactivity during generalized tetanusmay be a blockade of GABAergic transmission to SPNs.

Uptake of TeTx by GABAergic terminals

TeTx blocks synaptic transmission by impeding the exo-cytosis of neurotransmitters (Montecucco and Schiavo,1994; Poulain, 1994). Because all axon terminals (regard-less of their neurotransmitter contents) probably share acommon release mechanism, it is not clear why axon ter-minals with inhibitory neurotransmitters appear to bemore susceptible to blockade than those with excitatoryneurotransmitters during tetanus. Some experimental ev-idence suggests that differences in the ability of neuronsand secretory cells to bind and internalize TeTx determine

which are more sensitive to blockade (Dreyer, 1989;Marxen and Bigalke, 1989; Poulain et al., 1990). If this istrue, then the specific binding of TeTx to axon terminalswith inhibitory neurotransmitters also may explain thesusceptibility of inhibitory synaptic transmission to TeTxblockade in the mammalian CNS. In the ventral horn, lowdoses of TeTx block the inhibition of a-MNs before block-ing their excitation (Kanda and Takano, 1983); this time-and dose-dependent effect of TeTx appears to be criticallydependent on TTC (Takano et al., 1989). Furthermore, ithas long been established that TeTx blocks GABAergicsynaptic transmission (Carrea and Lanari, 1962; Curtisand De Groat, 1968; Curtis et al., 1973; Davies and Ton-groach, 1979; Collingridge et al., 1981; Collingridge andDavies, 1982; Empson and Jefferys, 1993; Empson et al.,1993). Our colocalization data suggest that GABAergicterminals are more likely to sequester TTC than otheraxon terminals.

Although the basis for this more selective uptake isunknown, one plausible explanation is that GABAergicterminals are closer to the source of TTC than non-GABAergic terminals. Evidence for this hypothesis areprevious light microscopic studies suggesting that TTC istransported transsynaptically, initially to axon terminalssynapsing on a-MNs and SPN perikarya and proximaldendrites (Fishman and Carrigan, 1987; Cabot et al. 1991;Ligorio, 1996), and that GABAergic synapses aggregate inrelatively high density on the cell bodies and proximal

Fig. 7. Electron photomicrograph showing two axon terminals (At1 and At2) contacting the sameretrogradely labeled SPNd in the IML neuropil of T2. At1 is both GABA1 and TTC1, whereas At2 isGABA2 and TTC2. Arrows point to a region of synaptic specialization. Scale bar 5 0.2 mm.


dendrites of a-MNs (Ramırez-Leon and Ulfhake, 1993). Inthe IML, however, GABA immunolabeling clearly wasevident along distal dendrites that extended far into thelateral funiculus (Fig. 3). Furthermore, axon terminalssynapsing on large-caliber (proximal) dendrites were notmore likely to be GABA1 than axon terminals synapsing

on small-caliber (distal) dendrites, and, by 48 hourspostinjection, the same was true for TTC1 terminals.

Because there is little anatomic evidence that eitherGABA1 or TTC1 terminals are closer to SPN cell bodiesthan other terminals in the IML, an alternative explana-tion for why GABA1 terminals were more likely to be

Fig. 8. Bar graph comparing the percentages of GABA1 andGABA2 terminals that were TTC1 (6S.E. 5 SQRT[(%TTC1

terminals)(%TTC2 terminals)/n]) in each of the four IML samplesshown in Table 1. The number of axon terminals in each group is

shown at the base of each column. For one-tailed chi-square tests, asingle asterisk indicates P , 0.005, double asterisks indicate P ,0.001, and triple asterisks indicate P , 0.000001. SQRT, square root.

Fig. 9. Bar graph illustrating the likelihood that axon terminalsthat synapse on retrogradely labeled SPN processes in the IML areTTC1. The paired bars to the left of the vertical dashed line comparethe TTC1 percentages (6 S.E. 5 SQRT[(%TTC1 terminals)(%TTC2

terminals)/n]) of all the GABA1 and GABA2 terminals pooled fromsamples 3 and 4 (Table 1). On the right, each pool was divided into twogroups: axon terminals synapsing on retrogradely labeled processes

(Table 2) and axon terminals synapsing on unlabeled processes. Thenumber of axon terminals in each group is shown at the base of theirdesignated column. In the whole IML and in both subdivisions,GABA1 terminals are more likely to be TTC1 than GABA2 terminals.Axon terminals synapsing on retrogradely labeled processes are notmore likely to be TTC1 than other axon terminals in the IML. Aster-isks indicate P , 0.001 (one-tailed chi-square test).


TTC1 than GABA2 terminals is that GABAergic termi-nals have an intrinsically higher affinity for TTC. Thishypothesis would explain why, even though both axonterminals in Figure 7 appear equally close to the sameretrogradely labeled SPN process (a presumptive source ofTTC), only the GABA1 terminal is TTC1. Because axonterminals that sequester TTC may be susceptible to block-ade by TeTx, this hypothesis also would explain why TeTxblocks GABAergic transmission in vivo (Curtis et al.,1973) and GABA release in vitro (Collingridge et al., 1981;Collingridge and Davies, 1982) when retrograde andtranssynaptic transport are by-passed by applying TeTxexogenously: It follows that GABAergic terminals mayexpress a high-affinity receptor for TTC and/or TeTx.

TeTx may affect axon terminals withneurotransmitters other than GABA

Thus far, this paper has discussed only the possibilitythat TeTx may obstruct GABAergic transmission. How-ever, there is long-standing evidence that glycinergictransmission is susceptible to TeTx blockade (Sher-rington, 1905; Osborne and Bradford, 1973; Bigalke et al.,1981; Williamson et al., 1992). Physiologic studies haveshown that glycine inhibits SPNs through strychnine-sensitive glycine receptors (Backman and Henry, 1983;Inokuchi et al., 1992). Because a blockade of glycinergicsynaptic transmission to SPNs can affect cardiac output(Madorin and Calaresu, 1994; Lewis and Coote, 1995), it ispossible that some symptoms of sympathetic dysfunctionin tetanus may be due to an effect of TeTx on glycinergictransmission in the IML. A speculative idea is that glycineand GABA are coreleased from the same axon terminals;thus, TeTx may block the release of both of these neuro-transmitters simultaneously.

Physiologic and anatomic studies conducted in otherareas of the spinal cord suggest that glycine and GABAmay be coreleased from axon terminals that synapse onmotoneurons (Ornung et al., 1996, 1998; Jonas et al.,1998; Nicoll and Malenka, 1998). There also is evidencethat glycine and GABA receptors may be colocalized onpostsynaptic membranes through their mutual associa-tion with gephyrin, a cytoplasmic peripheral membraneprotein (Kirsch et al., 1993; Betz, 1998; Essrich et al.,1998). Accordingly, recent immunoelectron microscopicstudies in lumbar spinal cord suggest that GABA andglycine and their respective receptors all may localize tothe same synapses (Todd et al., 1996).

In the IML, GABA has been localized in terminals syn-apsing on SPN dendrites that express gephyrin on theirpostsynaptic membranes (Chiba and Semba, 1991; Cabotet al., 1995). Because some TTC1 terminals also synapseon dendrites expressing gephyrin (unpublished observa-tions), some of the axon terminals in our samples thatwere both GABA1 and TTC1 may also have been glycin-ergic. However, it has been estimated that only 8.3% of theaxon terminals in the IML are glycinergic (Cabot et al.1992), which does not account for all of the TTC1 and/orGABA1 terminals in our samples. Moreover, GABA2 ter-minals had a 47% chance of being TTC1, and the absolutenumber of GABA2 terminals that were TTC1 was greaterthan the number of GABA1 terminals that were TTC1 inall but one sample. Because axon terminals in the IMLmay use a variety of different neurotransmitters (Cabot,1990), TeTx may affect axon terminals other than thosethat are glycinergic and/or GABAergic.

Elsewhere in the CNS, TeTx has been shown to blockthe release of serotonin (5-HT), dopamine (Britton et al.,1995), and met-enkephalin (Janicki and Habermann,1983; McMahon et al., 1992). It is not clear what func-tion(s) 5-HT and dopamine have in the IML (Coote, 1988).However, the neuropeptide met-enkephalin appears tohave an inhibitory action (Dun et al., 1993), and morphinehas been reported to control autonomic hyperactivity ingeneralized tetanus (Rie and Wilson, 1978). Finally, withprolonged exposure and/or at relatively high concentra-tions, TeTx can also block excitatory synaptic transmis-sion (Kanda and Takano, 1983; Bergey et al., 1987; Wil-liamson et al., 1992). Because the amount of TTC injectedinto the SCG was much greater than the minimum lethaldose of TeTx for mice and the survival time was 48 hours,some of the TTC1 terminals in the IML may have con-tained excitatory neurotransmitters.

In summary, by using immunoelectron microscopy, wehave provided anatomic evidence that GABAergic termi-nals in the IML sequester the binding fragment of tetanustoxin. In light of prior physiologic, pharmacologic, andmolecular biologic studies, this evidence suggests that ablockade of GABAergic synaptic transmission may be animportant basis for the disinhibition of SPNs during tet-anus. However, it seems likely that axon terminals in theIML with neurotransmitters other than GABA also maysequester TTC. The identification of these other neuro-transmitters, the synaptic release of which also may besusceptible to TeTx intoxication, will require further stud-ies.

ACKNOWLEDGMENTS

The authors thank Dr. Nancy Role Mendell, Professor ofApplied Mathematics and Statistics, for offering invalu-able insight with regard to statistical analysis of the data.The majority of this work was submitted by M.A.L. aspartial fulfillment of doctoral degree requirements to theDepartment of Neurobiology and Behavior, College of Artsand Sciences, State University of New York at StonyBrook. This work was supported by a MERIT award fromNHLBI to J.B.C. (HL24103).

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Ultrastructural localization of the binding fragment of tetanus toxin in putative ?-aminobutyric acidergic terminals in the intermediolateral cell column: A potential basis for sympathetic