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Distribution of GABA and glycine in the lamb nucleus of the solitary tract
BRAIN RESEARCH ELS1{VIER
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Distribution ol-GABA am.1glycine in the lamb nucleus of the sol itary tract Rd-rerl D. Swc:imy
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.——— Abstract illt) ilcids y-i IIII[ntohutyric ,,lcid (G A13/\ ) :Iud ~!ly~il]c :I[c iIIY{~1vcd in several IILIC]CUSof’ the solitary Irx[ The inhibitory :1111 {Ii’IIICWalrin(}~ICIdS in the NS’I’ O( the ]amb. J ~pccics (’T-CtINCII[ly used in investiglticms of (NS’1’)-illL:[ii~ltedtnnctions. The distrib(l[i~~rl NST media(cd bchaiitw~. h~i~11{)1 Ixxn descrIhed. Therefore. (hiscIne l]llllll]llol-c:ic.[i~c L“cII!:LIKIpunc(u WCI-Cunevenly distribultxi in [ht: lamb NST. ‘1’hchi@sL density of tiAJ3A irrlrrl[lr){)t,::l~[il~:ccl 1~W;ISL)und In the in[ernmdiatt> zone t~f’lhc NST, Ilwdi;]l to the solitary h-ac[ (ST). High [{) Ilitxlcmte levels ot puncwI I:thc]ing w[:rc (Jw:[;cd {It]t]uglrout the .!4S’1’.particularly arxwnd Ihc ST in in[crmmrhte and caudal ztmc’s. M(xlcmIc II] I(JW Icvels of gl}cinc illlr~l~lrl(}r~’:lt[ ivi[] ii L:IL:[dx+med, with most .glycinc imrnunt~rc~lc[ivc CCIISund prmcta found in the caud!Il [wo-[hi[.ds ~)1’the NS’I’II) t17emcdid. LLIIIr(Il:IILI:IliIIId d~~ls{~lncdiolNS’I’. Only :L[ew g[y~inc irrlllll~r]orczlctive cells and punctti were f(xmd in the rmtml zone ol’ [he NST. ‘1’lM wide.sprcxt distribution ol’ GABA and glycinc imrnunoreactivity in intcrmedialc and cauLIal zones of’ the NST SLI,ggCStSthat [hcsc ir~hihilclr) wnino acids plu) an imporlant role in modukrting NST-mediated functions like swallowing, respiration and c:lr-diok:tsc~]l~ltrcgulntion in the lwnb. The much higher density of GABA immunorcactivity it> in (he u)str:d zone of the NST suggests tlml GABA, but not glycine, is an important cornpwcd tu glycine immunol-mc[il ncurotransrnitter in the proccssin: of’ [:lste il]l’[)lrll:l.ior] h} dw Itunb NST.
1. Introduction Inhibitory amino acids, particularly y-aminobutyric acid (GABA), are distributed hOLl@OLll the vertebrae centt-d nervous system anci serve as important netrrottansmilters in neural pathways that under-lie numerous diverse beha\ iors. Anatomical and biochemical studies conducted in cat and rat have demonstrated that the inhibitory amino acids GABA and glycinc. and their receptors. arc widespread throughout the brainstcm. inc]udin: lhe nLlcleLls of the solitary ttxt (NST) [7, 10,12.Is.24,32.36..37.42,431. The NST is the primary central nertous system rcla}) for sensory information transmitted \ia the facial. glossoph:Lryngeal and vagos nerves [X).26.27] aIId is an importan[ component in the neural pathway~ that underlie :Ltstatt)r}, cardiovascular, respiratory. and alilnentary tract functions. Studies conducted in many laboratoric~ indicate a role of’ inhibitory amino acids in the man) functions of’the NST.
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Wan: and Bicger [53] found that GABA agonists inhibit the buccopharyngetil phase of’swallowing and esophageal peristalsis, Physiological studies conducted in vivo and in vitro have irnplicatcd both GABA and glycine as neurotransrnitlers influencing NST control of cardiovascular and respiratory function [ 19,31,4I,48,49,5 1]. Similar lines of evidence suggest that GABA serves as an important inhibitor-yncurotransrnitter in the rostral gustatory portion of the NST [34,44,52]. Although several studies have described the distribution 01”inhibitory amino acids in the NST of the rat, cat and rabbit, eqtrivalen( information for lambs is currently lacking. Lambs have been used extensively for investigation of’ the physiology and development of taste, deglutition, upper airway rei’lexes, cardiovascular function and respiration. functions in which the NST plays an important role [S.6.2S.39,47,S4]. A description of the distribution of inhibitory alnino acids GABA and gl}cine in the lamb NST would contribute information important to understanding the significance of’ these neurotransmitters in the various functions controlled by this nucleus. Therefore, the goal of
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this study was todcscribe the distribution ot’GABA and glycinein the lamb NSTusing immunohistochemistry on thick and semi-thin tissue sectit)ns.
2. Materials and methods Experiments were performed using 151Suffolk lambs aged 30-60 days, weighing 9– 16 kg. An overdose ot sodium pentobarbita] was given by jugultir puncture and the animals perfused through the carotid arteries. Blood was flushed from the vascular system using 1.5 1 01 phosphate buffered saline (PBS) (pH 7.4). followed by I 1 of a solution of 3–4% paraformaldehyde and ().1–().5% ,gIutaraldehyde in ().I M phosphate buffer (pH 7.3–7.4) for the 9 animals evaluated for GABA il~~mlll~ore:lctivit}, or 1 1 of a solution of I% par:ifotl~l:ildehyde and 2.25% glw taraldehyde in 0.1 M phosphate buffer (pH 7.3–7.4) for the 6 animals evaluated for glycinc illllnLln(>re~lcti~i[). After perfusion the brainstern was removed frotn the skull and stored overnight in 0.1 M phosphate buffer at 4°C. Five serial (io-Lm transverse sectiom of the lamb brainstem were collected every 1 mm throughout the rostrll caudal extent of’the NST. Following collection of [he t’if’th 60-pm section. a 150-~m section was taken for subsequent post-embedding illllllLlllt)cytocllclllistry as nt)[ed be. low. All tissue seclions were cut using a vibralomc. ‘“h free-floating 60-WIT]sections were processed for the ~isuaization of GABA or glycine according to the protocol of’ Helfert et al. [22]. Bricily, sections were first pre-incubated in a blocking solution containing 5Ycnormal goat serum in PBS. Sections were then incubated ovcrn i,ghtat 4(]C with gentle agitation in a solution containing one of the following affinity-purified, polyclonal rabbit antisera diluted in blocking serum: GABA 1:1500-I :2000” [56]: glycine I: 1000-I: 1400 [55]. The preparation. purification und irnmunostaining patterns of these two antibodies have been described in detail previously [55,56]. Following incubation in the primary atltisera the tissue was processed according to the avidin–hiotin method of’ Hsu et al. [23] using the Vectas[ain ABC kit (Vector Labs). The peroxidasc activity was ~isualized by exposing the sections to 0.05YGdiaminobenzidinc and 0.()()1YChydrogen peroxide in PBS for 5–lo rnin [22]. Tissue was then rinsed in distilled water, mounted on chron-alum subbed slides and coverslipped with Krystallon mounting medium (Harleco) or Permount. With only slight modification. the 150-Km thick sections were processed for post-embedding immunocytw chemistry according the technique of Helfert et al. [22]. Briefly, the sections were post-fixed in ().1% OSOJ in O.I M phosphate buffer for 2 h, dehydrated through a graded series of’ethanol followed by acetone, and flat-crnbedded in Epon resin. The embedded sections were then trimmed to include only the region around the NST and thin 1–2 Km sections through the NST were cut on an ultrarnicrm
tome and mounted on glass slides. To increase penetration of the antiserum, sections were etched in a solution of sodium ethoxide/ethanol and osmium was bleached from the sections using 1Y. sodium rr-periodate followed by distilled water rinses. Sections were then placed in s~. normal goal serum in PBS, followed by incubation in GABA or g]ycine antiserum diluted in blocking serum ( 1:400) in a humidified chamber at 4°C for 48 h. The tissue sections were then processed as noted above except that the Vectastain reagents were prepared at twice the concentration recommended by the manufacturer [22]. Controls for both thick and selni-thin sections included incubating sornc sections in the blocking serum minus the primary antisera and incubating sections in primary antisera preadsorbed with the appropriate amino acid. Both of these control conditions resulted in an absence of immunoIabeling, An additional control employed was taking sections through the cochlear nuclei to permit comparison witb previous studies of these areas [22,55,56]. In the lamb, both GABA and g]ycine immunoreactive neurons were observed in the lamb cochlcar nuclei and these immuuoreactive neurons were distributed in a pattern comparable to (hat observed previously. Data analysis WJSconducted in a manner similar to that reported earlier [46]. Briefly, the distribution of GABA and glycirl~in]nlLm~l.eaC[iVC cell bodies iind puncta was mapped onlo standard drawings of’the Itimb NST using a cameralucida. The density of immunoreactive labeling was subjectively evaluated for each animal separately, and rated on a four point scale of high to none. For immunoreactive cells in the NST these rankings roughly corresponded to the following: high, 8 or more immunoreactive cells/ 15 000 ~ m?; moderate, 3–7 immunoreactive cells/ I5 000”~m2; low, 1–2 immunoreactive cells/15 000 ~mz. For imrnunoreactive puncta, the subjective evaluations were: high, approximately 6 or more immunoreactive puncta/100 Lrnz: rnodcrate, 3–5 immunoreactive puncta\ 100”Lm?; low, 1-2 immunoreactive puncta/100 ~mz. Subjective evaluations were combined across subjects to produce the distribution maps shown in Figs. 1 and 2. For clarity, the NST was divided into caudal, intermediate and rostral zones. The caudal NST zone included all sections caudal to ().5 mm anterior to obex. The intermediate zone of the NST was from 0.5 mm to 3.5 mm rostral to obex und the rostral zone of the NST included all of the NST rostral to 3.5 mm. When appropriate, the nomenclature of Kalia and Mesulam [26] is used to describe homologous subnuclei of the lamb NST.
3. Results Both GABA immunoreactivity and glycine immunoreactivity were differentially distributed in the lamb NST. In general, the density of GABA immunoreactive (GABAi) CCIIand puncta staining was more intense and more widely
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3.1. GABA it?lttll{ilo~.e(~(:tiii~) h [he lumb NST GABAi cells were differentially distributed throughout the lamb NST and, regardless of the zone of the NST
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Fig. I. I)istribution of GABA illltllunc)r”~~(~li~rity in th~ lamb NS’i’. Ttle left column of schematic dr:iwings show’sthe ctist]ihutiorrof in)munnrcacti~c cells and the right column shows the distribution of”immunc)reactihrc puucta. The numbers adjaccrrl to the right column indicate the distance In mm of each section from the obex. The clensityof GAEJAif~lrnLtnc)lczic(i\it) is coded according tu the key at the upper right of the fi,gurc. AP.area PH. pl”epOSiLLl~ lhY’postrcmti: MLF, medial Inngitudimd f’2iSCiCLllLlS: puglossi; ST. solitary tmct: Vu, [rigeminui nucleus: X. dorsal Illt)tol” nucleus of the vagus: Xl1. bypoglossal UUCICUS. throughout the rostral to caudal extent of the Iamb NST than the density of glycine immunoreactivi~y (Figs. 1 and 2). This difference was espcciall} pronounced in the rostral zone of’the NST where much lc~wcrlevels of’ glycine immunoreactivity were obset-vcxl.Although GABAi structures were more numerous thtin glycine :lmmunoreactive (GLYi) structures in the ros[ral zone of the NST. it was found that the densities of both GABA immunoreactivity and glycine immunoreact ivity were ;grealerin the
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Fig. 2. Distributiuil Of glycine immunoreactivity in the lamb NST.The left column of schematic drawings shnws the distribution of immunoreacti\e CCIISand the right column shows [he distribution of immunoreactive puuctl. The uumbers adjucent to the right column indicate the distance in mm t)f’each scctiun from the obex. The density ot’ glycine immunoIreactivityis cudcd accurding to the key at the upper right of the figure. AP. area postremu; MLF, medid longitudimd fasciculus; PH. prepositus hypoglossi: ST. sulitary tract; Vn, trigeminal nucleus; X, dorsal motor UUCICUS of tbc hagus: XII. hypoglossal nucleus.
~i:, j, ~lloto)nicro:r~,l)t]s 01”G,\13A i!))ll](ltq,)]ciictil i[:, in dit”l”crcm[ trcgi,]]]it,I’[l], lamb NSI”. /1: (;ABAi cells (art”ow;heads)in mcdid regions Of the rostral pole nf the NST. “I”hcdensil} of”it)lt]l[[ll(]!”~~]L[i\t’ CCIISit) (his alea w:ts cI:I.. II’IccIits Iiyht. B: pllt)[(~!llicr(lgrtlpil from a thick section showing a GABAi )ncarthe Ievcl 01”OIXXshowing the region nt’the NST [lLllldU1n(ilrro~’h~ild)ildjlt~er)t(();111(]11 IIllllllll]oteL( L’i \L’C~!lill ti]L r(}stri~lN’s’I’.(‘: pll[~t[)!llicj-ogt-~~1~11 dol-~orme
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(Figs. I and 3A). The greatest numbers of GABAi cells in the rostral zone of the NST were observed ventral and medial to the solitary tract (ST) at posterior aspee[s of this NST zone. Near the rostral pole ol’ the NST more intense GABA immunoreactivity was observed laterally (Fig. I). The greatest density of GABAi cells was observed in areas of the intermediate and caudal NST zones, at or just rostral to the level of’ obex (Fig. 1). At these levels, the highest density of GABAi cells was found in the dorsolateral and medial subnuclei (Figs. 1 and 3C,D). Moderate to low numbers of GABAi cells were observed in the ventral and ventrolateral subnuclei (Fig. I). Moderate numbers of GABAi cells were also observed in the spaces between the bundles of fibers that make up the lamb ST, an area corresponding to the interstitial subnucleus described in cat and rat [26,27]. Moderate to low levels of GABAi neurons were observed in the commissural nucleus at the most caudal levels of the NST examined, and medial to the ST at the more rostra] levels of the intermediate zone of the NST (Fig. 1). Regardless of the level of’the NST examined, GABAi neurons were rarely observed in the subnucleus substantial gelatinous (Fig. 1). Like GABAi cells, GABAi puncta were observed in all zones of the NST. These puncta were observed on both GABAi and non-immunoreactive neurons, b~t in general puncta were more frequently found adjacent to non-immunot-eactive cells in the NST (Fig. 3B and 4A,B). Many of these non-immunoreactive cells exhibited fusiform- or multipolar-shaped cell bodies (Fig. 4A,B). In the caudal zone of-the NST at the level of obcx, high levels of ptmcta labeling were observed in the dorsolatet-al, intermediate and ventral subnuclei while at the most caudd NST levels examined a high level of puncta labeling was observed in the ventromedial and ventrolateral NST. Moderate to low levels of puncta labeling were seen in other regions of the caudal NST. The highest density of’ GABAi puncta labeling was obsetwed in the intermediate zone of the NST’.High levels of’ immunoreactive puncta were observed in the dorsolateral, ventrolateral and ventral subnuclei (Fig. 1, panels 1.5 and 2.7 mm rostral to obex; Fig. 3D). The density of immunoreactive labeling was moderate in th~ medial and interstitial subnuclei and was low in the subnucleus substantial gelatinous as one moves rostrally through the intermediate zone of the NST (Fig. l). In the rostral zone of the NST a high density of GABAi puncta labeling was seen medial to the ST (Fig. 1, panels 3.9 and 5.0 mm rostral to obex). Moderate punctu labeling occurred in dorsomedial and ventral regions of the NST
279
while only low numbers of immunoreactive puncta were observed lateral to the ST or in the interstitial subnucleus. Near the rostral pole of the NST a moderate density of immunorcactive puncta was evenly distributed throughout the nucleus (Fig. 1, panel 6.5 mm rostral to obex). 3.2. Glycine immunorcwctiui(y in the lamb NST It was noted above that the overall density of GLYi cells in the lamb NST was lower than that of GABAi cells in all NST zones. In fact, concentrations of GLYi cells that could be classified as high were never encountered in the lamb NST and moderate levels of GLYi cells were restricted to very small regions of the nucleus. The lack of GLYi neurons was especially striking at the most rostral locations of the NST where almost no GLYi cells were observed in medial regions of the nucleus and only a few immunoreactive neurons were scattered in lateral areas (Fig. 2). Like GABAi neurons, GLYi somata found in the rostral NST were small ( 10–20 p,m) and round or ovoid (Fig. 5A). In the intermediate zone of the NST, moderate numbers of GLYi somata were restricted to an area of the nucleus corresponding to the dorsolateral NST (Fig. 2). Throughout much of the rest of the intermediate zone of the NST only low numbers of GLYi somata were observed, and no GLYi cells were observed in the subnucleus substantial gelatinous. Most GLYi somata in the intermediate zone of the NST were small and round or ovoid (Fig. 5B), but an occasional fusiform-shaped soma was observed in the interstitial subnucleus (Fig. 5C). In the caudal zone of the NST near the level of the obex moderate numbers of GLYi somata were observed in the dorsolateral and ventrolateral NST (Fig. 2). Like more rostra] regions of the NST, in the caudal zone of the NST most GLYi neurons were characterized by small round or ovoid somata. However, some GLYi somata in the ventroIateral NST were larger in size and fusiform or multipolar in shape (Fig. 4D). Throughout the medial aspects of the caudal NST, including the commissural subnucleus, the density of GLYi cells was generally low and the immunoreactive cells widely scattered. Throughout the NST the density of GLYi puncta was greater than that of GLYi cells, but the density of GLYi puncta staining was usually lower than the GABAi puncta labeling at equivalent levels of the NST. GLYi puncta in the lamb NST were generally more numerous in the caudal and intermediate zones of the NST than in its more rostral aspect, but this difference was not as pronounced as that
Fig. 4.GABA and glycine immullc>histocllcrnistry’ in semi-thin sectioas ut’the lamb NST. A: GAt3Ai punct~ (arrowheads) adjacent to a non-immunoreactive cell located in an area 01’ the intcrmedi~tc N5T ventrola[eral to the ST. B: GAEtAi cell (arrowhead) and puncta (arrows) adjacent to a nun-immunoreactive soma in the medial NST. C: GL.Yi puncla (arr[lw’heads)adjacent to a non-immurmreactive cell in the medial NST at the level of the t”e:iclivecell that is embedded in the ST at the level of the caudal NST. Scale intermediate NST.D: GLYi puncta (arrom,hcads)adjacent to a ncJn-inlillun(> bal-: A, B and D, 17 ~m; C, 45 p.m.
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In [he rostrtil zone of the NST moderate levels of GLYi Puncta w’el”eobserved in the medial aspect of the nucleus Iw(wccn 3.5 and 5.() rnrn rostml to obex (Fig. 2). Moderate
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levels of’imrnunorcactivc punc[a were also obserted vem tral and ventromeclial to the ST at the more posterior levels of’the rostral NST. The density of’GLYi puncta was low’in
281
other mcas of the rostral NST and no immunoreactivity was observed in the interstitial subnucleus. The relative density of GLYi puncta in the intermediate
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zone of the NST was general Iy greater than that seen at more rostra] levels, but there were fcw differcnccs in the overall distribution pattern of imtnunoreactive puncta (Fig. ~). Within the in[crmediale Z.CJIW of the NST, a moderate density of GLYi pttncta was observed in the medial, intermediate, ventral and ventrolateral subnuciei. Similar to the distribution of GLYi somata. GL,Yi puncta were a{Tenerallynot observed in the subnucleus :substaotia gelatinosus. In contrast to more rostral NST locations where no GLYi puncta were observed in the intersl.itial subnucleus, low levels of GLYi puncta were observed in the intcrstiti~il subnucleus of the intermediate zone of the NST. The distribution of’GLYi puncta in the caudal zone of the NST showed some similarity to the distribution of GABAi puncta. For example. at the level of otwx one region with a greater relative density of (ILY i puncta labeling was the dorsolateral NST (Figs. 2 and SD). This region also contuined a relatively high level of CJABAi pLlncta labeling. However, other areas at this level of the NST that contained high densities of GABA immunc)reactivity, such as the intertnediate subnucleus. contained only low levels of GLYi puncta. At the most caudal levels of the NST examined, modcra[e numbers of GLYi puncta were evenly distributed throughout the nucleus.
4.
Discussion
GABAi, and to a lesser extent GLYi, cells and pttncta were widely distributed throuyhou{ the jamb NST. The results of the present study show”(hat (“;A.BA and glycinc immunoreactivity
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4.1. GABA
Several studies utilizing biochemical or irnrnunohistm chemical techniques have reported that moderate w higb levels of GABA, or the GABA-synthesizing enzyme glLltamic acid dectirboxylase (GAD), arc widely distributed throughout the NST [2, 10,12,24,32,36–38,43]. Other studies have shown that GABA binding sites are present in the NST as is a high-affinity GABA uptake mechanism [16,42,43]. The distribution of GABAi neurons in the lamb NST was comparable to the distribution of GABA ond
GAD immunoreactive neurons in other species [2,10,32,36,37]. For example, in the cat the greatest numbers of GABA or GAD immunoreactive cell bodies are observed medial to the ST at the level of the area postrema [24,36]. The corresponding region of the lamb NST con[ained some of the highes[ numbers of GABAi neurons (Fig. I). Likewise, in the lamb NST near the level of obex, there was a dense aggregation of immunoreactive cells located in the dorsolateral NST (Fig. 1). A similar aggregation of GAD immunoreactive neurons is observed in this region of the rabbit and rat [2,37]. The distribution of GABAi neurons in the rostral zone of the lamb NST resembled that previously seen in hamster [10] and rat [32]. Larger numbers of GABAi neurons were found in the posterior aspects of the rostral lamb NST than near the rostral pole and most of these neurons were located in the medial NST. However, near the rostral pole of the lamb NST grea(er numbers of GABAi somata were found in the lateral aspects of the NST, a finding opposite that observed in the rat and hamster where larger numbers of GABAi neurons were found in the medial NST [10,32]. Another similarity between the findings of the present study and previous immunohistochemical investigations conducted in other species [10,24,32,36] is that the distribution of GABAi puncta was widespread and this distribution was relatively homogeneous throughout the lamb NST. The large numbers of GABAi neurons in the lamb NST suggest that much of the observed puncta labeling reflects intrinsic GABAi projections. However, it is probable that extrinsic sources also contribute GABAergic inputs to the lamb NST since extrinsic neuronal inputs to the cat ventroIatcral NST have been previously identified [35]. The rclative]y high levels of GABA immunoreactivity observed in the intermediate and caudal zones of the lamb NST suggest an important role for this neurotransmitter in controlling the many functions subserved by this region. Neurons within the caudal two-thirds of the NST are part of neural networks controlling respiration, swallowing, cardiovascular and gastrointestinal functions, and several physiological studies have implicated GABA as a neurotransmitter in these networks. In the intermediate zone of the lamb NST, large numbers of GABAi puncta were found in regions ventromedial to the ST, areas associated with the control of swallowing in this species [6,25]. This observation suggests a role for this amino acid in the control of swallowing. Functional studies conducted in the rat by Wang and Bieger [53] found that applications of the GABA agonist muscimol to the surface of the NST inhib-
Fig. 5. Photomicrographs nf’glycinc illl]]lc~r]t)[-c~ict ii’i[:)’in diffcrcnl legions ol’the lamb NST. A: G1,Yi cell (arrowhcw-1)ventrmnedial to the ST in the rostra] NST. GI.Yi neurons were irarelyencountered at this Ic\cl ol’the NST. hut ]puncta(ar]-ows)were scuttercd Lhmughmrtthe neuropil. B: small round GLYi CCII(arrowhead) in [he intcrnwdiale NS”l_Iatc!mlttl the ST. [’: I’Llsil’t>rtll-\l)it[?ccl (;LYi cell (arrowhead) ernbcddcxlin the ST near the level of the obex. D: [~tlc,tc)illicr(>graph from a senli-thin scc[it,n shoving (;LYi CCIIS(open arr(~ws).CCIIprocesses (arrowheads) and puncta (arrows) located in the caudal NST kentrolatcml to ST. GLYi cells in thih region (II’the n~lclcuswere ftcqucn(l> I’usil’ormor Imul[ipolmin shape. Scale bar: A,60 km; B and C, 40 p,m; D, 70 &m.
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ited Jr] evoked swaJlow, while injecl ioJIs [)1’ LIK GABA antagonist bicucullint iii deglutiti vc loci in the NST produced buccopharyngea] and cst~phagcal components ()[ swallowing. CMm-areas ot’ [he intcmwdiatc and caudal ~rmcs t)] Ihc lamb NS’I’that contained large numhet-s of GA13Ai new rons ami punc(u wurc the k’entrola(eral N!j’1’,ao :U.CtI associaltd wilh the control of respirti(ion [29]. and mcdial and dorsomeclial 10 Ibe ST. alcus ossociatcri wi[h cardim vascular cun[lml 1{)].Numerous shldics IMVCshown that LGABAis involved in the control of”respir~lio(l and cal-cliovascular reflexes SUCIIw tk 13aroreceptorw flex (foI review see [1] and [451) and the high Ievcl; of GAliA immunoreactivity irr (hesc irrcas of the IalmbN,ST suggesl a similar role fo[-GABA in the lamb. The rostral 7one of (hc NST is the prilmary relay folgustatory inf(mnatioo carried in IIlt facial aNLlglossrrph:lryngcal ncmcs, and is often rcl’c]medto as the gustalory NST to reflect its imporkm[ role in processing gustatory information [20,57]. Examination of GABA or GAD inmunoreactivity in the rat and hamster rostra] NST found that GABA was localized in a subpopulation of rostra] NST neurons having small round {o ovoid somata, and in numerous puncta distributed throughout the mstral NST 110.32]. Both of’ these findings wc similar to those observed for GABAi neurom and puncta in the lamb NST. Using a combinti(ion O( GAPIA itllllllJnollist~)cllcL1listty and re(rogradc tracing techniques. Lasitcr and IKachele [32] determined thfii. (; A13Ai ncmrons in [bc rtil Iostral NST were mos( Iikcly intmneur(~rls auLI hypothesized {hal GABA-mediated inhibition may underlie some taste response decrements observed in clectrc]physic~logicalstudies of gustatory NST neurons. Physiol(Jgical studies conducted in vivo and in vitro support this hypothesis Microinjections of GABA into the hamster NST in vi~o inhibi[ the taste-evoked responses of a subpoprrlation of’rostral NST neurons and this cffec( was blocked by the CJABA,i receptor antagonist bicuculline [44]. In~estigtitions utilizing extracellular and whole cell recordings in an in vitro SIice preparation of the rat or hamster rostral NST found that over half of the stimpled NSI” neurons were inhibited by GABA or GABA agonists and this ell’ect was blocked by GABA antagonists [34,52]. Applications of GABA produce a reduction in input resistance and hypf~rpc:llalizu[iorl in many rostra] NST neurons, and these effects are mediated by either GABA 1 or GAB A,] receptors [34.52]. The widespread distribution and physiological actions of GABA in the rodent rostral NST suggest an important role for this transmitter in mediating [LLSW processirqg. The GABAi neurons and puncta observed at similar Icvels of’the lamb NST indicate that GABA probabl~’plays a similar role in gustatory processing in the lamb. 4.2. Gl?rine Although glycinc immunoreactivity is no( as widespread or as densely distributed in the lamb NST as GAf3A
283
illlllltlllol-e~ic[ivily,convc~-ginsevidence supports a role for glycine as a llclrl’otl”:lrlslllitter in lbe NS’I’.Biochcmical tind ilrlrlluilollis tocllclllictll studies, including the present siudy, h;ive shown dlat glyciue is present in the NST [7,14,38]. There are nurntm~usglycinc receptors distributed throughout Ihe NS’I’ [7, 1f] and there is a high affinity uptake system ii)r glycinc in the NST [42]. In iln in vitro brain SIice prepartilion applications of glycine reduced the I-cspuuscs of some NST neurons [o ST stimulation [13], while ir)~.rcasingpcrtilsate potassium corwcntl-ations in an in vitro prep:iration ot”the intermediate NSrl’ results in H release of glycine tha[ is in part CaJ+-dependent [38]. 1+’urthernlorc, clectl-opllysiological studies using LIissuciated NST neurons IIJVCLlernonsll-atccla strychnine-sensitive, glycine-induux-l cument dependent upon Cl- [40]. In contrast to the findings of biochemical studies that snowed cquivalcllt or higher basal levels of’ glycinc relative [o CTABAin the NST [30,38,421, the current sludy found that the density of glycine immunoreactivity was LIsudly less than the density of’GABA immunoreactivity. This was particularly true for GLYi neurons in that most NST regions contained only a few scattered GLYi neurons. Like GABA, the density of GLYi reactive cells and puncta varied depending upon the level and region of the lamb NST examined. In general the greatest numbers of GLYi cells and puncta were located in the intermediate and caudal zones of the NST. An imrnunohistochernical investigation of the distribution of GLYi terminals in the I-at NST found similar une(lual dish-ibutious of glycine imIllunorcactivity and higher ICVCISof imm~Jnor-eactivityat caudal and intermediate levels [7]. Densities of ,gIycine immunoreactivity classified as moderate were rare in the lamb NST. One area where rnoder-atelevels of GLYi cells and puncta were observed was in regions of the intermediate and caudal zones of the lamb NST associated with cardiovascular, respiratory and alimentary tract functions, suggesting a role for this transtnittcr in controlling these functions in lamb. Several studies have indicated that .glycine plays a role in respiratory and cardiovascular functions in other species [21,41]. Ion~ophoretic applications of glycine or its agonists into the NST depress the activity of NST respiratory neurons and NST-evoked respiratory activity, while glycine antagonists increase the firing rate [8,11,18,28]. Glycine is released from regions of the NST involved in cardiovascular function and has been localized in these areas immunohistochemical]y [7,30,38]. Glycine injected into the NST can either increase or decrease heart rate and blood pressure depending upon the site of injection [30,49]. Talman et al. [48.50] found that glycine microinjected into the medial NST decreased arterial pressure and heart rate in a manner similar to tha[ observed following injections of glutamate or acetylcholine. Further investigations revealed that this effect was not due to a direct effect of glycine on cardiovascular neurons, but rather the result of glycine-modulated release of acetylcholine in the cardiovascular NST
[48,50]. Thus it appears (hat gl}cine acts in the NST viti a number of direct and indirect tnechanisms. When compared to GABA. tbc uncvctl distribution of glycine immtrnoreac(ivity in [he roslroca Ll&L~axis Of the lamb NST was especially pronounced, GLYi cells were sparsely distributed at the most rostral NST Icvcls and no immurmreactive cells were (observed in the very medial regions of the NST at this level. The density of GLYi puncta was also tow near the rt)stral pole of lhe NST, btr[ increased at more posterior levels of the rostra] NST. [Little information exists concerning the distribl~tion of glycinc immunoreactivity in the ros[ral. gLis{atoryregioms of the NST because most s(ucliestha( have examined the distribw [ion of glycine in this nucleus have t’ocuscdtheir investig:ltions on more caudal NST localions. In a study relelant m the present investigation, ~’assell et al. [7] examined the distribution of g]ycine-containing terminals in the rat dolsal vagal complex using a prepar-a[ion aIndglycinc antibody similar to those used in the crrrrcnt study. Tlresc investigators divided the NST into ros(ral, intermediate and cwrdal levels. Their rostral Ie\el roughly corrcsp~)rlds[() Iamb sections bctwccn those Iabe]ed 3.9 wrd S.() mm rostral to obex in Fig. 2 above (c(’.F-ig.3 of Cassel I ct al. [7]). [n the rat rostra] NST the greatest concentrations of GLYi tel”minals were obser-vcd rncdial to the ST in [he medial and intei”rnediate subnuclei. Although anaiogous subnuclear boundaries of the NST have not been described in lamb, the positions of thcw areas of the rat r-ostralNST correspond to the rc,gions ot’ the lamb rostral NST corltaining the highest le~els of GLYi puncta. Sin:.ilar 10the ]LLmb,
the rostra] NST may be that it acts as a postsynaptic inhibitory transmitter. Another potential role for glycine in the rostral NST would be participation in the NMDA rcccptor cornplcx for which glycine is a modulator [3]. Expcrirncnts conducted in vitro and in vivo have demonstrated thut the majority of’taste neurons in the rostral NST are responsive to glutamate and NMDA agonists [4,33]. In summary, both GABAi and GLYi neurons and puncta were found throughout the Iamb NST and were distributed in a manner similar to that observed in other species. The density of GABAi neurons, and GLYi neurons and puucta, was generally higher in the caudal two-thirds of the nucleus, but GABAi puncta were more evenly distributed across the different zones of the NST. In most areas of the NST tbe density of GABA immunoreactivity was greater than glycinc irnmunoreactivity. This was especially true mzrr the rostra] pole of the NST where only a few scattered GLYi neurons and low levels of GLYi puncta were observed. The relatively dense GABA and glycine immunoreactivity observed at more catrdal levels of the NST associated with the control of respiration, cardiovascular functions and swallowing suggests a role for these inhibitory, amino acids in the control of visceral functions in the lamb. The abundance of GABA in the rostral NST suggests an important role for this amino acid in gustatory processing:, but the low levels of glycine immunoreactivity imply thot this rleurotransrnittcr is not an integral part of neural circuitry underlying taste processing in the NST.
ventrolateral
Acknowledgements
sLlbdivisiorls
of
(he
rat NST
contzLirmd
only
modera(e levels of GLYi terminals [7]. In addition. small numbers of fiber-s and terminals were observed within the rat rostral ST. bomcver. in the lamb. glycirw imrnunoreactivity was not observed in this ama at t“ostra] levels. In the present study the paucity of GLY’ineurons in the rostral NST suggests tha[ most GLYi punf;ta in the rmtra] NST represent [be terminations of efferent fibers from outside this NST level rather thtin tcrmin:dions from neurons making up an inlr-insic modulatory nelwor-k as has been hypothesized for GABA. Unt’ortunutely.information regarding the source of glycil~e ter-rninals in the rostral NST is not currently available. [t is possible that some of the GLYi puncta observed in the lamb rostral NST represent the terminations of oral al’ferent f’ibcr.s01 the facial and glossopharyngeal nerves. However. studies of the v:lgus nerve, which terminates xt lnore caudal NST locations. suggest that this is unlikely. tlo(h rwur”ochern ical und phal”rnacological sludies indicate that glycinc i!; not a trarlsmi[ter for va,galaff’ercnlfiber terminations in the NST [17,38]. Although a function for-glycirte in the rostra] NST has not been elucidated, the 10WJIcvels ot’ glycine immunw reactivity in the rostral NST suggest that this transmitter does not play a nui,jorrole in NST processing of gustator-y information. One possible function of this amino acid in lmv to
1 would like to thank Dr. R.A. Altschuler and Dr. R.J. Wcnthold for-providing the antisera used in this study. I would also like to thank J.A. Cook and E. Denman for rechnictil and photographic assistance. This research was supported by grant number DCO0735 from the National Institute of Deafness and Other Communication Disorders, National Institutes of Health.
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R{,\..439(l9h’8) 1[)5210. :l]l(ll{(~h~,r[<(~rl. S.t’.,~,l),.it)(, [W]’ 1’ahn,au. W.’l’.. ~’(>11111~,~.-kll[~. lnicl.(JlnjcclL;[l intt~nll(lt,lls Ir:ltllls.{llilnl iit}l’li]lluls ihl~~llyl]cll{)lill c~rgi(.tnc:c.h:lrlistl)\.A)}I..IP/I\.\i{,/.H4{,// (“i/{.l’/l\\if,/..2(lo (l’.)[)l) II 1326–H 13.;1. [49] ‘Tahmm.W.’l’.:tnd l{t,bcrlwrl. S.(.. Gl)ci,l~ lik~ :,IuII,,,,Ic. lllicl{)illIecred Into the nuc’ieu\ Wlcllls \illil:lrii ill’ tr:ll [icctcIIsc\ ;Ittciul J77 ( I98’)) ‘7–I 3. pressLnY and hem r:itc, /;r(/rll /cin~ elicits rclcaw of”:~cc?tjlcholinc l’rc)mthe nucleus (ruc(us solitarii in tr:(~.Bfcfitl R~.\.. 050”( 1994) 253–259. [51] Van Ciiersbcrgcm 1>. L.M., Pillk(\,I\. M. MCIdc Jong. W’.. ln\)l\cmcnl 01’neLlr(ltt:ltlstllillcrsin d]c Il(lclcu\ (IKILI: solit:wii i)] cmdi(]vascular lrcgul:ltion. P/~\\i~~/.R~/.. 72 ( I992) 789-824. R.M.. Iilf’1,,.tlce01”GABA on neuron. ot’ Iilc [52] Wang. L, ~lnciBI:KIle~.
~L15(il(c,1), ~11’(IW M II ULIeU\ (i tile soi it~t-y tract, Rmin Res., (rI6 ( I993) I-M 153. II {1 W’:jng Y.’i’.:Ind ijicgcr. r~., R(~lcof kt~litwid CJABAclgic mcchutlisll)~ iii c[)tlltt}l t)l’ swallowing, A/ii. J. l’hysiol. Rc,qul. lnrc>,qr (_’u/?I/~. f’/i\.}i{)/,,261 ( 1991) 1<63!)R64. A A. :md D<,vcnporl,P.W., VagiIlly i,diukxl [>4] Webb. Lt.. Ilmchism modulalitm [~finspirat(lr’ydumtilln in Ihc Inc(molul ~’ollllll~-.sio/,. 76 ( l!)!)~)397-402. [55] Wcnd,{lld, R.J., llLlie. D., Altschuler, R.A.;md Recks, K.A.. Glycinc iljlljlLLl){llc~~cti\it], Iocohzed m rile coch[e:ir nucleus and s~(pcrifw {]li\ol-yc<)mplex, Nc~f//.Tci<,ffcc,c,.22 ( 1987) 897–9 12. [i(l] WendKIILI. R.J.. zempel, J. M., Parakkal, M.H.,Reeks, K.A. and Altschu[er-.K.A.. IrllInlltl(>c)lt>chclnicaiIoculization of’GA13Ain the cocillc~ir InLIcleus of the guinea pig, Btai}t Ra., 380 ( i986) 7– 18. [571 Whi[chc:,d. M.(’., Functional connections of the rostra] nucleus of tile soiilm> tract in \isccr(hwnsory intcgratimr of ingestion reflexes. in: 1.R.A. i3mImco (ELI.) Nu[/efM of’the S[)/i/ar} Tract. CRC Press, inc.. Boca l<~~ton.1994. pp. 105-i 18. LL)LIL.
Bmll] i
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Distribution ol-GABA am.1glycine in the lamb nucleus of the sol itary tract Rd-rerl D. Swc:imy
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.——— Abstract illt) ilcids y-i IIII[ntohutyric ,,lcid (G A13/\ ) :Iud ~!ly~il]c :I[c iIIY{~1vcd in several IILIC]CUSof’ the solitary Irx[ The inhibitory :1111 {Ii’IIICWalrin(}~ICIdS in the NS’I’ O( the ]amb. J ~pccics (’T-CtINCII[ly used in investiglticms of (NS’1’)-illL:[ii~ltedtnnctions. The distrib(l[i~~rl NST media(cd bchaiitw~. h~i~11{)1 Ixxn descrIhed. Therefore. (his
1. Introduction Inhibitory amino acids, particularly y-aminobutyric acid (GABA), are distributed hOLl@OLll the vertebrae centt-d nervous system anci serve as important netrrottansmilters in neural pathways that under-lie numerous diverse beha\ iors. Anatomical and biochemical studies conducted in cat and rat have demonstrated that the inhibitory amino acids GABA and glycinc. and their receptors. arc widespread throughout the brainstcm. inc]udin: lhe nLlcleLls of the solitary ttxt (NST) [7, 10,12.Is.24,32.36..37.42,431. The NST is the primary central nertous system rcla}) for sensory information transmitted \ia the facial. glossoph:Lryngeal and vagos nerves [X).26.27] aIId is an importan[ component in the neural pathway~ that underlie :Ltstatt)r}, cardiovascular, respiratory. and alilnentary tract functions. Studies conducted in many laboratoric~ indicate a role of’ inhibitory amino acids in the man) functions of’the NST.
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Wan: and Bicger [53] found that GABA agonists inhibit the buccopharyngetil phase of’swallowing and esophageal peristalsis, Physiological studies conducted in vivo and in vitro have irnplicatcd both GABA and glycine as neurotransrnitlers influencing NST control of cardiovascular and respiratory function [ 19,31,4I,48,49,5 1]. Similar lines of evidence suggest that GABA serves as an important inhibitor-yncurotransrnitter in the rostral gustatory portion of the NST [34,44,52]. Although several studies have described the distribution 01”inhibitory amino acids in the NST of the rat, cat and rabbit, eqtrivalen( information for lambs is currently lacking. Lambs have been used extensively for investigation of’ the physiology and development of taste, deglutition, upper airway rei’lexes, cardiovascular function and respiration. functions in which the NST plays an important role [S.6.2S.39,47,S4]. A description of the distribution of inhibitory alnino acids GABA and gl}cine in the lamb NST would contribute information important to understanding the significance of’ these neurotransmitters in the various functions controlled by this nucleus. Therefore, the goal of
llwi i~; Scieilcc I!.V. ,\l I IrIglll\]rescncd
this study was todcscribe the distribution ot’GABA and glycinein the lamb NSTusing immunohistochemistry on thick and semi-thin tissue sectit)ns.
2. Materials and methods Experiments were performed using 151Suffolk lambs aged 30-60 days, weighing 9– 16 kg. An overdose ot sodium pentobarbita] was given by jugultir puncture and the animals perfused through the carotid arteries. Blood was flushed from the vascular system using 1.5 1 01 phosphate buffered saline (PBS) (pH 7.4). followed by I 1 of a solution of 3–4% paraformaldehyde and ().1–().5% ,gIutaraldehyde in ().I M phosphate buffer (pH 7.3–7.4) for the 9 animals evaluated for GABA il~~mlll~ore:lctivit}, or 1 1 of a solution of I% par:ifotl~l:ildehyde and 2.25% glw taraldehyde in 0.1 M phosphate buffer (pH 7.3–7.4) for the 6 animals evaluated for glycinc illllnLln(>re~lcti~i[). After perfusion the brainstern was removed frotn the skull and stored overnight in 0.1 M phosphate buffer at 4°C. Five serial (io-Lm transverse sectiom of the lamb brainstem were collected every 1 mm throughout the rostrll caudal extent of’the NST. Following collection of [he t’if’th 60-pm section. a 150-~m section was taken for subsequent post-embedding illllllLlllt)cytocllclllistry as nt)[ed be. low. All tissue seclions were cut using a vibralomc. ‘“h free-floating 60-WIT]sections were processed for the ~isuaization of GABA or glycine according to the protocol of’ Helfert et al. [22]. Bricily, sections were first pre-incubated in a blocking solution containing 5Ycnormal goat serum in PBS. Sections were then incubated ovcrn i,ghtat 4(]C with gentle agitation in a solution containing one of the following affinity-purified, polyclonal rabbit antisera diluted in blocking serum: GABA 1:1500-I :2000” [56]: glycine I: 1000-I: 1400 [55]. The preparation. purification und irnmunostaining patterns of these two antibodies have been described in detail previously [55,56]. Following incubation in the primary atltisera the tissue was processed according to the avidin–hiotin method of’ Hsu et al. [23] using the Vectas[ain ABC kit (Vector Labs). The peroxidasc activity was ~isualized by exposing the sections to 0.05YGdiaminobenzidinc and 0.()()1YChydrogen peroxide in PBS for 5–lo rnin [22]. Tissue was then rinsed in distilled water, mounted on chron-alum subbed slides and coverslipped with Krystallon mounting medium (Harleco) or Permount. With only slight modification. the 150-Km thick sections were processed for post-embedding immunocytw chemistry according the technique of Helfert et al. [22]. Briefly, the sections were post-fixed in ().1% OSOJ in O.I M phosphate buffer for 2 h, dehydrated through a graded series of’ethanol followed by acetone, and flat-crnbedded in Epon resin. The embedded sections were then trimmed to include only the region around the NST and thin 1–2 Km sections through the NST were cut on an ultrarnicrm
tome and mounted on glass slides. To increase penetration of the antiserum, sections were etched in a solution of sodium ethoxide/ethanol and osmium was bleached from the sections using 1Y. sodium rr-periodate followed by distilled water rinses. Sections were then placed in s~. normal goal serum in PBS, followed by incubation in GABA or g]ycine antiserum diluted in blocking serum ( 1:400) in a humidified chamber at 4°C for 48 h. The tissue sections were then processed as noted above except that the Vectastain reagents were prepared at twice the concentration recommended by the manufacturer [22]. Controls for both thick and selni-thin sections included incubating sornc sections in the blocking serum minus the primary antisera and incubating sections in primary antisera preadsorbed with the appropriate amino acid. Both of these control conditions resulted in an absence of immunoIabeling, An additional control employed was taking sections through the cochlear nuclei to permit comparison witb previous studies of these areas [22,55,56]. In the lamb, both GABA and g]ycine immunoreactive neurons were observed in the lamb cochlcar nuclei and these immuuoreactive neurons were distributed in a pattern comparable to (hat observed previously. Data analysis WJSconducted in a manner similar to that reported earlier [46]. Briefly, the distribution of GABA and glycirl~in]nlLm~l.eaC[iVC cell bodies iind puncta was mapped onlo standard drawings of’the Itimb NST using a cameralucida. The density of immunoreactive labeling was subjectively evaluated for each animal separately, and rated on a four point scale of high to none. For immunoreactive cells in the NST these rankings roughly corresponded to the following: high, 8 or more immunoreactive cells/ 15 000 ~ m?; moderate, 3–7 immunoreactive cells/ I5 000”~m2; low, 1–2 immunoreactive cells/15 000 ~mz. For imrnunoreactive puncta, the subjective evaluations were: high, approximately 6 or more immunoreactive puncta/100 Lrnz: rnodcrate, 3–5 immunoreactive puncta\ 100”Lm?; low, 1-2 immunoreactive puncta/100 ~mz. Subjective evaluations were combined across subjects to produce the distribution maps shown in Figs. 1 and 2. For clarity, the NST was divided into caudal, intermediate and rostral zones. The caudal NST zone included all sections caudal to ().5 mm anterior to obex. The intermediate zone of the NST was from 0.5 mm to 3.5 mm rostral to obex und the rostral zone of the NST included all of the NST rostral to 3.5 mm. When appropriate, the nomenclature of Kalia and Mesulam [26] is used to describe homologous subnuclei of the lamb NST.
3. Results Both GABA immunoreactivity and glycine immunoreactivity were differentially distributed in the lamb NST. In general, the density of GABA immunoreactive (GABAi) CCIIand puncta staining was more intense and more widely
Puncta
cells
El Jmmunorractivc Density [~ None [g Low iY#Jll htodcratc I-
T-’\
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caLIdal [wo-lhirds ()(” (1w NST corresponding to the intermediutc and caudal zones as compared to the roslra] zone of the lamb NST (Figs. I and 2).
3.1. GABA it?lttll{ilo~.e(~(:tiii~) h [he lumb NST GABAi cells were differentially distributed throughout the lamb NST and, regardless of the zone of the NST
MLF
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Fig. I. I)istribution of GABA illltllunc)r”~~(~li~rity in th~ lamb NS’i’. Ttle left column of schematic dr:iwings show’sthe ctist]ihutiorrof in)munnrcacti~c cells and the right column shows the distribution of”immunc)reactihrc puucta. The numbers adjaccrrl to the right column indicate the distance In mm of each section from the obex. The clensityof GAEJAif~lrnLtnc)lczic(i\it) is coded according tu the key at the upper right of the fi,gurc. AP.area PH. pl”epOSiLLl~ lhY’postrcmti: MLF, medial Inngitudimd f’2iSCiCLllLlS: puglossi; ST. solitary tmct: Vu, [rigeminui nucleus: X. dorsal Illt)tol” nucleus of the vagus: Xl1. bypoglossal UUCICUS. throughout the rostral to caudal extent of the Iamb NST than the density of glycine immunoreactivi~y (Figs. 1 and 2). This difference was espcciall} pronounced in the rostral zone of’the NST where much lc~wcrlevels of’ glycine immunoreactivity were obset-vcxl.Although GABAi structures were more numerous thtin glycine :lmmunoreactive (GLYi) structures in the ros[ral zone of the NST. it was found that the densities of both GABA immunoreactivity and glycine immunoreact ivity were ;grealerin the
p.,. ~,,,,!, ,:,,’
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-1.0
distributed
Fig. 2. Distributiuil Of glycine immunoreactivity in the lamb NST.The left column of schematic drawings shnws the distribution of immunoreacti\e CCIISand the right column shows [he distribution of immunoreactive puuctl. The uumbers adjucent to the right column indicate the distance in mm t)f’each scctiun from the obex. The density ot’ glycine immunoIreactivityis cudcd accurding to the key at the upper right of the figure. AP. area postremu; MLF, medid longitudimd fasciculus; PH. prepositus hypoglossi: ST. sulitary tract; Vn, trigeminal nucleus; X, dorsal motor UUCICUS of tbc hagus: XII. hypoglossal nucleus.
~i:, j, ~lloto)nicro:r~,l)t]s 01”G,\13A i!))ll](ltq,)]ciictil i[:, in dit”l”crcm[ trcgi,]]]it,I’[l], lamb NSI”. /1: (;ABAi cells (art”ow;heads)in mcdid regions Of the rostral pole nf the NST. “I”hcdensil} of”it)lt]l[[ll(]!”~~]L[i\t’ CCIISit) (his alea w:ts cI:I.. II’IccIits Iiyht. B: pllt)[(~!llicr(lgrtlpil from a thick section showing a GABAi )ncarthe Ievcl 01”OIXXshowing the region nt’the NST [lLllldU1n(ilrro~’h~ild)ildjlt~er)t(();111(]11 IIllllllll]oteL( L’i \L’C~!lill ti]L r(}stri~lN’s’I’.(‘: pll[~t[)!llicj-ogt-~~1~11 dol-~orme
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(Figs. I and 3A). The greatest numbers of GABAi cells in the rostral zone of the NST were observed ventral and medial to the solitary tract (ST) at posterior aspee[s of this NST zone. Near the rostral pole ol’ the NST more intense GABA immunoreactivity was observed laterally (Fig. I). The greatest density of GABAi cells was observed in areas of the intermediate and caudal NST zones, at or just rostral to the level of’ obex (Fig. 1). At these levels, the highest density of GABAi cells was found in the dorsolateral and medial subnuclei (Figs. 1 and 3C,D). Moderate to low numbers of GABAi cells were observed in the ventral and ventrolateral subnuclei (Fig. I). Moderate numbers of GABAi cells were also observed in the spaces between the bundles of fibers that make up the lamb ST, an area corresponding to the interstitial subnucleus described in cat and rat [26,27]. Moderate to low levels of GABAi neurons were observed in the commissural nucleus at the most caudal levels of the NST examined, and medial to the ST at the more rostra] levels of the intermediate zone of the NST (Fig. 1). Regardless of the level of’the NST examined, GABAi neurons were rarely observed in the subnucleus substantial gelatinous (Fig. 1). Like GABAi cells, GABAi puncta were observed in all zones of the NST. These puncta were observed on both GABAi and non-immunoreactive neurons, b~t in general puncta were more frequently found adjacent to non-immunot-eactive cells in the NST (Fig. 3B and 4A,B). Many of these non-immunoreactive cells exhibited fusiform- or multipolar-shaped cell bodies (Fig. 4A,B). In the caudal zone of-the NST at the level of obcx, high levels of ptmcta labeling were observed in the dorsolatet-al, intermediate and ventral subnuclei while at the most caudd NST levels examined a high level of puncta labeling was observed in the ventromedial and ventrolateral NST. Moderate to low levels of puncta labeling were seen in other regions of the caudal NST. The highest density of’ GABAi puncta labeling was obsetwed in the intermediate zone of the NST’.High levels of’ immunoreactive puncta were observed in the dorsolateral, ventrolateral and ventral subnuclei (Fig. 1, panels 1.5 and 2.7 mm rostral to obex; Fig. 3D). The density of immunoreactive labeling was moderate in th~ medial and interstitial subnuclei and was low in the subnucleus substantial gelatinous as one moves rostrally through the intermediate zone of the NST (Fig. l). In the rostral zone of the NST a high density of GABAi puncta labeling was seen medial to the ST (Fig. 1, panels 3.9 and 5.0 mm rostral to obex). Moderate punctu labeling occurred in dorsomedial and ventral regions of the NST
279
while only low numbers of immunoreactive puncta were observed lateral to the ST or in the interstitial subnucleus. Near the rostral pole of the NST a moderate density of immunorcactive puncta was evenly distributed throughout the nucleus (Fig. 1, panel 6.5 mm rostral to obex). 3.2. Glycine immunorcwctiui(y in the lamb NST It was noted above that the overall density of GLYi cells in the lamb NST was lower than that of GABAi cells in all NST zones. In fact, concentrations of GLYi cells that could be classified as high were never encountered in the lamb NST and moderate levels of GLYi cells were restricted to very small regions of the nucleus. The lack of GLYi neurons was especially striking at the most rostral locations of the NST where almost no GLYi cells were observed in medial regions of the nucleus and only a few immunoreactive neurons were scattered in lateral areas (Fig. 2). Like GABAi neurons, GLYi somata found in the rostral NST were small ( 10–20 p,m) and round or ovoid (Fig. 5A). In the intermediate zone of the NST, moderate numbers of GLYi somata were restricted to an area of the nucleus corresponding to the dorsolateral NST (Fig. 2). Throughout much of the rest of the intermediate zone of the NST only low numbers of GLYi somata were observed, and no GLYi cells were observed in the subnucleus substantial gelatinous. Most GLYi somata in the intermediate zone of the NST were small and round or ovoid (Fig. 5B), but an occasional fusiform-shaped soma was observed in the interstitial subnucleus (Fig. 5C). In the caudal zone of the NST near the level of the obex moderate numbers of GLYi somata were observed in the dorsolateral and ventrolateral NST (Fig. 2). Like more rostra] regions of the NST, in the caudal zone of the NST most GLYi neurons were characterized by small round or ovoid somata. However, some GLYi somata in the ventroIateral NST were larger in size and fusiform or multipolar in shape (Fig. 4D). Throughout the medial aspects of the caudal NST, including the commissural subnucleus, the density of GLYi cells was generally low and the immunoreactive cells widely scattered. Throughout the NST the density of GLYi puncta was greater than that of GLYi cells, but the density of GLYi puncta staining was usually lower than the GABAi puncta labeling at equivalent levels of the NST. GLYi puncta in the lamb NST were generally more numerous in the caudal and intermediate zones of the NST than in its more rostral aspect, but this difference was not as pronounced as that
Fig. 4.GABA and glycine immullc>histocllcrnistry’ in semi-thin sectioas ut’the lamb NST. A: GAt3Ai punct~ (arrowheads) adjacent to a non-immunoreactive cell located in an area 01’ the intcrmedi~tc N5T ventrola[eral to the ST. B: GAEtAi cell (arrowhead) and puncta (arrows) adjacent to a nun-immunoreactive soma in the medial NST. C: GL.Yi puncla (arr[lw’heads)adjacent to a non-immurmreactive cell in the medial NST at the level of the t”e:iclivecell that is embedded in the ST at the level of the caudal NST. Scale intermediate NST.D: GLYi puncta (arrom,hcads)adjacent to a ncJn-inlillun(> bal-: A, B and D, 17 ~m; C, 45 p.m.
280
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In [he rostrtil zone of the NST moderate levels of GLYi Puncta w’el”eobserved in the medial aspect of the nucleus Iw(wccn 3.5 and 5.() rnrn rostml to obex (Fig. 2). Moderate
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levels of’imrnunorcactivc punc[a were also obserted vem tral and ventromeclial to the ST at the more posterior levels of’the rostral NST. The density of’GLYi puncta was low’in
281
other mcas of the rostral NST and no immunoreactivity was observed in the interstitial subnucleus. The relative density of GLYi puncta in the intermediate
~g~
1<.1.) ,SII(,(IZI /IIIII;IZ Rc\c({I(/I 7.77 (1996) 275–286
zone of the NST was general Iy greater than that seen at more rostra] levels, but there were fcw differcnccs in the overall distribution pattern of imtnunoreactive puncta (Fig. ~). Within the in[crmediale Z.CJIW of the NST, a moderate density of GLYi pttncta was observed in the medial, intermediate, ventral and ventrolateral subnuciei. Similar to the distribution of GLYi somata. GL,Yi puncta were a{Tenerallynot observed in the subnucleus :substaotia gelatinosus. In contrast to more rostral NST locations where no GLYi puncta were observed in the intersl.itial subnucleus, low levels of GLYi puncta were observed in the intcrstiti~il subnucleus of the intermediate zone of the NST. The distribution of’GLYi puncta in the caudal zone of the NST showed some similarity to the distribution of GABAi puncta. For example. at the level of otwx one region with a greater relative density of (ILY i puncta labeling was the dorsolateral NST (Figs. 2 and SD). This region also contuined a relatively high level of CJABAi pLlncta labeling. However, other areas at this level of the NST that contained high densities of GABA immunc)reactivity, such as the intertnediate subnucleus. contained only low levels of GLYi puncta. At the most caudal levels of the NST examined, modcra[e numbers of GLYi puncta were evenly distributed throughout the nucleus.
4.
Discussion
GABAi, and to a lesser extent GLYi, cells and pttncta were widely distributed throuyhou{ the jamb NST. The results of the present study show”(hat (“;A.BA and glycinc immunoreactivity
were
differentially
distributed
across
the
NST and within the different zones of the NST. Similar results have been reported in the rat, hams[cr. rabbit and cat NST [2,7, 10,24,32>36,37]. rostral
to caudal
axis
of’ [he
lamb
4.1. GABA
Several studies utilizing biochemical or irnrnunohistm chemical techniques have reported that moderate w higb levels of GABA, or the GABA-synthesizing enzyme glLltamic acid dectirboxylase (GAD), arc widely distributed throughout the NST [2, 10,12,24,32,36–38,43]. Other studies have shown that GABA binding sites are present in the NST as is a high-affinity GABA uptake mechanism [16,42,43]. The distribution of GABAi neurons in the lamb NST was comparable to the distribution of GABA ond
GAD immunoreactive neurons in other species [2,10,32,36,37]. For example, in the cat the greatest numbers of GABA or GAD immunoreactive cell bodies are observed medial to the ST at the level of the area postrema [24,36]. The corresponding region of the lamb NST con[ained some of the highes[ numbers of GABAi neurons (Fig. I). Likewise, in the lamb NST near the level of obex, there was a dense aggregation of immunoreactive cells located in the dorsolateral NST (Fig. 1). A similar aggregation of GAD immunoreactive neurons is observed in this region of the rabbit and rat [2,37]. The distribution of GABAi neurons in the rostral zone of the lamb NST resembled that previously seen in hamster [10] and rat [32]. Larger numbers of GABAi neurons were found in the posterior aspects of the rostral lamb NST than near the rostral pole and most of these neurons were located in the medial NST. However, near the rostral pole of the lamb NST grea(er numbers of GABAi somata were found in the lateral aspects of the NST, a finding opposite that observed in the rat and hamster where larger numbers of GABAi neurons were found in the medial NST [10,32]. Another similarity between the findings of the present study and previous immunohistochemical investigations conducted in other species [10,24,32,36] is that the distribution of GABAi puncta was widespread and this distribution was relatively homogeneous throughout the lamb NST. The large numbers of GABAi neurons in the lamb NST suggest that much of the observed puncta labeling reflects intrinsic GABAi projections. However, it is probable that extrinsic sources also contribute GABAergic inputs to the lamb NST since extrinsic neuronal inputs to the cat ventroIatcral NST have been previously identified [35]. The rclative]y high levels of GABA immunoreactivity observed in the intermediate and caudal zones of the lamb NST suggest an important role for this neurotransmitter in controlling the many functions subserved by this region. Neurons within the caudal two-thirds of the NST are part of neural networks controlling respiration, swallowing, cardiovascular and gastrointestinal functions, and several physiological studies have implicated GABA as a neurotransmitter in these networks. In the intermediate zone of the lamb NST, large numbers of GABAi puncta were found in regions ventromedial to the ST, areas associated with the control of swallowing in this species [6,25]. This observation suggests a role for this amino acid in the control of swallowing. Functional studies conducted in the rat by Wang and Bieger [53] found that applications of the GABA agonist muscimol to the surface of the NST inhib-
Fig. 5. Photomicrographs nf’glycinc illl]]lc~r]t)[-c~ict ii’i[:)’in diffcrcnl legions ol’the lamb NST. A: G1,Yi cell (arrowhcw-1)ventrmnedial to the ST in the rostra] NST. GI.Yi neurons were irarelyencountered at this Ic\cl ol’the NST. hut ]puncta(ar]-ows)were scuttercd Lhmughmrtthe neuropil. B: small round GLYi CCII(arrowhead) in [he intcrnwdiale NS”l_Iatc!mlttl the ST. [’: I’Llsil’t>rtll-\l)it[?ccl (;LYi cell (arrowhead) ernbcddcxlin the ST near the level of the obex. D: [~tlc,tc)illicr(>graph from a senli-thin scc[it,n shoving (;LYi CCIIS(open arr(~ws).CCIIprocesses (arrowheads) and puncta (arrows) located in the caudal NST kentrolatcml to ST. GLYi cells in thih region (II’the n~lclcuswere ftcqucn(l> I’usil’ormor Imul[ipolmin shape. Scale bar: A,60 km; B and C, 40 p,m; D, 70 &m.
R.D, s’1’lw.7\/li,l(;fl /<,,$((/)(/7 7.+7( /<996)275--2<$’6
ited Jr] evoked swaJlow, while injecl ioJIs [)1’ LIK GABA antagonist bicucullint iii deglutiti vc loci in the NST produced buccopharyngea] and cst~phagcal components ()[ swallowing. CMm-areas ot’ [he intcmwdiatc and caudal ~rmcs t)] Ihc lamb NS’I’that contained large numhet-s of GA13Ai new rons ami punc(u wurc the k’entrola(eral N!j’1’,ao :U.CtI associaltd wilh the control of respirti(ion [29]. and mcdial and dorsomeclial 10 Ibe ST. alcus ossociatcri wi[h cardim vascular cun[lml 1{)].Numerous shldics IMVCshown that LGABAis involved in the control of”respir~lio(l and cal-cliovascular reflexes SUCIIw tk 13aroreceptorw flex (foI review see [1] and [451) and the high Ievcl; of GAliA immunoreactivity irr (hesc irrcas of the IalmbN,ST suggesl a similar role fo[-GABA in the lamb. The rostral 7one of (hc NST is the prilmary relay folgustatory inf(mnatioo carried in IIlt facial aNLlglossrrph:lryngcal ncmcs, and is often rcl’c]medto as the gustalory NST to reflect its imporkm[ role in processing gustatory information [20,57]. Examination of GABA or GAD inmunoreactivity in the rat and hamster rostra] NST found that GABA was localized in a subpopulation of rostra] NST neurons having small round {o ovoid somata, and in numerous puncta distributed throughout the mstral NST 110.32]. Both of’ these findings wc similar to those observed for GABAi neurom and puncta in the lamb NST. Using a combinti(ion O( GAPIA itllllllJnollist~)cllcL1listty and re(rogradc tracing techniques. Lasitcr and IKachele [32] determined thfii. (; A13Ai ncmrons in [bc rtil Iostral NST were mos( Iikcly intmneur(~rls auLI hypothesized {hal GABA-mediated inhibition may underlie some taste response decrements observed in clectrc]physic~logicalstudies of gustatory NST neurons. Physiol(Jgical studies conducted in vivo and in vitro support this hypothesis Microinjections of GABA into the hamster NST in vi~o inhibi[ the taste-evoked responses of a subpoprrlation of’rostral NST neurons and this cffec( was blocked by the CJABA,i receptor antagonist bicuculline [44]. In~estigtitions utilizing extracellular and whole cell recordings in an in vitro SIice preparation of the rat or hamster rostral NST found that over half of the stimpled NSI” neurons were inhibited by GABA or GABA agonists and this ell’ect was blocked by GABA antagonists [34,52]. Applications of GABA produce a reduction in input resistance and hypf~rpc:llalizu[iorl in many rostra] NST neurons, and these effects are mediated by either GABA 1 or GAB A,] receptors [34.52]. The widespread distribution and physiological actions of GABA in the rodent rostral NST suggest an important role for this transmitter in mediating [LLSW processirqg. The GABAi neurons and puncta observed at similar Icvels of’the lamb NST indicate that GABA probabl~’plays a similar role in gustatory processing in the lamb. 4.2. Gl?rine Although glycinc immunoreactivity is no( as widespread or as densely distributed in the lamb NST as GAf3A
283
illlllltlllol-e~ic[ivily,convc~-ginsevidence supports a role for glycine as a llclrl’otl”:lrlslllitter in lbe NS’I’.Biochcmical tind ilrlrlluilollis tocllclllictll studies, including the present siudy, h;ive shown dlat glyciue is present in the NST [7,14,38]. There are nurntm~usglycinc receptors distributed throughout Ihe NS’I’ [7, 1f] and there is a high affinity uptake system ii)r glycinc in the NST [42]. In iln in vitro brain SIice prepartilion applications of glycine reduced the I-cspuuscs of some NST neurons [o ST stimulation [13], while ir)~.rcasingpcrtilsate potassium corwcntl-ations in an in vitro prep:iration ot”the intermediate NSrl’ results in H release of glycine tha[ is in part CaJ+-dependent [38]. 1+’urthernlorc, clectl-opllysiological studies using LIissuciated NST neurons IIJVCLlernonsll-atccla strychnine-sensitive, glycine-induux-l cument dependent upon Cl- [40]. In contrast to the findings of biochemical studies that snowed cquivalcllt or higher basal levels of’ glycinc relative [o CTABAin the NST [30,38,421, the current sludy found that the density of glycine immunoreactivity was LIsudly less than the density of’GABA immunoreactivity. This was particularly true for GLYi neurons in that most NST regions contained only a few scattered GLYi neurons. Like GABA, the density of GLYi reactive cells and puncta varied depending upon the level and region of the lamb NST examined. In general the greatest numbers of GLYi cells and puncta were located in the intermediate and caudal zones of the NST. An imrnunohistochernical investigation of the distribution of GLYi terminals in the I-at NST found similar une(lual dish-ibutious of glycine imIllunorcactivity and higher ICVCISof imm~Jnor-eactivityat caudal and intermediate levels [7]. Densities of ,gIycine immunoreactivity classified as moderate were rare in the lamb NST. One area where rnoder-atelevels of GLYi cells and puncta were observed was in regions of the intermediate and caudal zones of the lamb NST associated with cardiovascular, respiratory and alimentary tract functions, suggesting a role for this transtnittcr in controlling these functions in lamb. Several studies have indicated that .glycine plays a role in respiratory and cardiovascular functions in other species [21,41]. Ion~ophoretic applications of glycine or its agonists into the NST depress the activity of NST respiratory neurons and NST-evoked respiratory activity, while glycine antagonists increase the firing rate [8,11,18,28]. Glycine is released from regions of the NST involved in cardiovascular function and has been localized in these areas immunohistochemical]y [7,30,38]. Glycine injected into the NST can either increase or decrease heart rate and blood pressure depending upon the site of injection [30,49]. Talman et al. [48.50] found that glycine microinjected into the medial NST decreased arterial pressure and heart rate in a manner similar to tha[ observed following injections of glutamate or acetylcholine. Further investigations revealed that this effect was not due to a direct effect of glycine on cardiovascular neurons, but rather the result of glycine-modulated release of acetylcholine in the cardiovascular NST
[48,50]. Thus it appears (hat gl}cine acts in the NST viti a number of direct and indirect tnechanisms. When compared to GABA. tbc uncvctl distribution of glycine immtrnoreac(ivity in [he roslroca Ll&L~axis Of the lamb NST was especially pronounced, GLYi cells were sparsely distributed at the most rostral NST Icvcls and no immurmreactive cells were (observed in the very medial regions of the NST at this level. The density of GLYi puncta was also tow near the rt)stral pole of lhe NST, btr[ increased at more posterior levels of the rostra] NST. [Little information exists concerning the distribl~tion of glycinc immunoreactivity in the ros[ral. gLis{atoryregioms of the NST because most s(ucliestha( have examined the distribw [ion of glycine in this nucleus have t’ocuscdtheir investig:ltions on more caudal NST localions. In a study relelant m the present investigation, ~’assell et al. [7] examined the distribution of g]ycine-containing terminals in the rat dolsal vagal complex using a prepar-a[ion aIndglycinc antibody similar to those used in the crrrrcnt study. Tlresc investigators divided the NST into ros(ral, intermediate and cwrdal levels. Their rostral Ie\el roughly corrcsp~)rlds[() Iamb sections bctwccn those Iabe]ed 3.9 wrd S.() mm rostral to obex in Fig. 2 above (c(’.F-ig.3 of Cassel I ct al. [7]). [n the rat rostra] NST the greatest concentrations of GLYi tel”minals were obser-vcd rncdial to the ST in [he medial and intei”rnediate subnuclei. Although anaiogous subnuclear boundaries of the NST have not been described in lamb, the positions of thcw areas of the rat r-ostralNST correspond to the rc,gions ot’ the lamb rostral NST corltaining the highest le~els of GLYi puncta. Sin:.ilar 10the ]LLmb,
the rostra] NST may be that it acts as a postsynaptic inhibitory transmitter. Another potential role for glycine in the rostral NST would be participation in the NMDA rcccptor cornplcx for which glycine is a modulator [3]. Expcrirncnts conducted in vitro and in vivo have demonstrated thut the majority of’taste neurons in the rostral NST are responsive to glutamate and NMDA agonists [4,33]. In summary, both GABAi and GLYi neurons and puncta were found throughout the Iamb NST and were distributed in a manner similar to that observed in other species. The density of GABAi neurons, and GLYi neurons and puucta, was generally higher in the caudal two-thirds of the nucleus, but GABAi puncta were more evenly distributed across the different zones of the NST. In most areas of the NST tbe density of GABA immunoreactivity was greater than glycinc irnmunoreactivity. This was especially true mzrr the rostra] pole of the NST where only a few scattered GLYi neurons and low levels of GLYi puncta were observed. The relatively dense GABA and glycine immunoreactivity observed at more catrdal levels of the NST associated with the control of respiration, cardiovascular functions and swallowing suggests a role for these inhibitory, amino acids in the control of visceral functions in the lamb. The abundance of GABA in the rostral NST suggests an important role for this amino acid in gustatory processing:, but the low levels of glycine immunoreactivity imply thot this rleurotransrnittcr is not an integral part of neural circuitry underlying taste processing in the NST.
ventrolateral
Acknowledgements
sLlbdivisiorls
of
(he
rat NST
contzLirmd
only
modera(e levels of GLYi terminals [7]. In addition. small numbers of fiber-s and terminals were observed within the rat rostral ST. bomcver. in the lamb. glycirw imrnunoreactivity was not observed in this ama at t“ostra] levels. In the present study the paucity of GLY’ineurons in the rostral NST suggests tha[ most GLYi punf;ta in the rmtra] NST represent [be terminations of efferent fibers from outside this NST level rather thtin tcrmin:dions from neurons making up an inlr-insic modulatory nelwor-k as has been hypothesized for GABA. Unt’ortunutely.information regarding the source of glycil~e ter-rninals in the rostral NST is not currently available. [t is possible that some of the GLYi puncta observed in the lamb rostral NST represent the terminations of oral al’ferent f’ibcr.s01 the facial and glossopharyngeal nerves. However. studies of the v:lgus nerve, which terminates xt lnore caudal NST locations. suggest that this is unlikely. tlo(h rwur”ochern ical und phal”rnacological sludies indicate that glycinc i!; not a trarlsmi[ter for va,galaff’ercnlfiber terminations in the NST [17,38]. Although a function for-glycirte in the rostra] NST has not been elucidated, the 10WJIcvels ot’ glycine immunw reactivity in the rostral NST suggest that this transmitter does not play a nui,jorrole in NST processing of gustator-y information. One possible function of this amino acid in lmv to
1 would like to thank Dr. R.A. Altschuler and Dr. R.J. Wcnthold for-providing the antisera used in this study. I would also like to thank J.A. Cook and E. Denman for rechnictil and photographic assistance. This research was supported by grant number DCO0735 from the National Institute of Deafness and Other Communication Disorders, National Institutes of Health.
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[~71’hve;17cy.] {.i). srrlitwyrrm
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~L15(il(c,1), ~11’(IW M II ULIeU\ (i tile soi it~t-y tract, Rmin Res., (rI6 ( I993) I-M 153. II {1 W’:jng Y.’i’.:Ind ijicgcr. r~., R(~lcof kt~litwid CJABAclgic mcchutlisll)~ iii c[)tlltt}l t)l’ swallowing, A/ii. J. l’hysiol. Rc,qul. lnrc>,qr (_’u/?I/~. f’/i\.}i{)/,,261 ( 1991) 1<63!)R64. A A. :md D<,vcnporl,P.W., VagiIlly i,diukxl [>4] Webb. Lt.. Ilmchism modulalitm [~finspirat(lr’ydumtilln in Ihc Inc(molul ~’ollllll~-