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The distribution of glycine receptors in the human brain. A light microscopic autoradiographic study using [3H]strychnine
Neuroscience Vol. 17, No. I, PP. 11-3.5,1986 Printed in Great Britain
0306-4522/86$3.00+ 0.00 Pergamon Press Ltd IBRO
THE DISTRIBUTION OF GLYCINE RECEPTORS IN THE HUMAN BRAIN. A LIGHT MICROSCOPIC AUTORADIOGRAPHIC STUDY USING [3H]STRYCHNINE A. F%OBST,* R. CORTES~ and J. M. PALACIOS*$ *Preclinical Research, Sandoz Ltd, CH-4002 Basle, Switzerland and tDepartment of Pathology, Division of Neuropathology, University of Basle, Schiinbeinstrasse 40, CH-4003 Basle, Switzerland Abstract--Glycine receptors were localized autoradiographically in postmortem human brain material using [3H]strychinine as a ligand. Slide mounted tissue sections were labeled in vitro by incubation with [rH]strychnine and autoradiograms obtained using [3H]Ultrofilm. Receptor densities were quantified by computer assisted microdensitometry. No specific binding of [)H]strychnine was observed in any of the forebrain areas studied. Low densities were seen in the midbrain except for dorsal and lateral parts of the periaqueductal grey matter and the oculomotor nuclei. In pons, medulla oblongata and upper cervical cord high densities of [‘HIstrychnine
binding sites were associated with some nuclei including the motor and sensory trigeminal nuclei, the facial and the hypoglossal nuclei. The highest densities of grains were associated with the substantia gelatinosa of the trigeminal nucleus in the medulla oblongata. A peculiar spotty distribution of [3H]strychnine binding sites were found in the gracilis and cuneatus nuclei. The distribution of glycine receptors in the human brain is comparable to that seen in the rat brain, although densities are much higher in the rat. The distribution of glycine receptors in the human brain provides an anatomical substrate for the understanding
of the effects of drugs acting in these receptors, particularly strychnine.
human brain chemistry and present characteristics The amino acid glycine appears to play an important similar to those found in studies in experimental role as an inhibitory neurotransmitter in the brain of animals. mammals and other animals.1~2~3~4~12*13~37~43~“~45 GlycinIn an attempt to expand the knowledge of the ergic synapses are mainly concentrated in the phylodistribution of GR in the human CNS we have genetically oldest parts of the brain, such as the spinal now examined the whole CNS of the neurologically cord and medulla,3~‘0~s’their relative number decreas“normal” human brain using quantitative autoradioing in a rostra1 progression of the neuraxis. Receptors for glycine have been characterized mainly by graphic techniques. In this paper we report a detailed mapping of these sites at the light microscopic electrophysiological methods and the ionic and resolution level. pharmacological properties of these sites are well known.5~i0*“~L7~22~23*24@‘~4’ More recently the use of EXPERIMENTAL PROCEDURES radioligand binding techniques has permitted a more direct biochemical study of these receptors allowHuman brains from a total of eight patients (four males, mean age 51 years, and four females, mean age 70 years) ing for detailed studies of structure activity relationwere obtained at autopsy after an average postmortem delay ships, ion requirements and will eventually lead to of 6 h. None of the cases studied showed clinical or neurothe molecular characterization of the glycine receppathological evidence of a brain disease. Individual tor.2’*2g*30,35,39,48*49,50 The ‘H-labeled glycine antagonist characteristics of these subjects, including sex, age, interval [‘HIstrychnine is a useful tool for the study of these between death and brain freezing, premortem condition and cause of death have been reported elsewhere.’ After removreceptors.48*49*so Recently Zarbin et ~1.~’have used this ing the brain, one cerebral and one cerebellar hemisphere, ligand for the autoradiographic mapping of glycine as well as the brainstem and the upper cervical cord, were receptors (GR) in the rat CNS. GR in the human stored at - 8o”C, to be later cut into 4-6 mm thick blocks. CNS have only been studied to a limited extent.‘4,26 The remainder of the CNS was immersed in formaldehyde Particularly changes in GR in the spinal cord of (4%) and further processed for routine neuropathological examination. Frozen blocks were then brought to -20°C patients dying from amyotrophic lateral sclerosis have been examined by membrane binding assays20 and mounted onto microtome chucks. Ten micrometerthick sections were cut using a cryostat (Leitz, 1720; from and by autoradiography.& These studies have L&z, Wetzlar, Federal Republic of Germany) and mounted revealed, that GR, like other neurotransmitter onto gelatin coated glass slides. GR were labeled as previously described5’ by incubating tissue sections with 4 nM receptors,‘*“*” survive postmortem modifications in $To whom correspondence should be addressed. Abbreviations: GR, glycine receptors; 3H-SBS, [‘Hlstrychnine binding sites. NSC. 1711-a
[‘HIstrychnine (23 Ci mmol-‘) in 0.05 M sodium potassium phosphate buffer, pH 7.4, at room temperature. Nonspecific binding was determined with the addition of 1O-2 M glycine. To generate autoradiograms, [“HlUltrofilm (LKB, Sweden) was exposed to the labeled slides as described by 11
A. Paoesl
I’
Unnerstall PI ol.“’ during three months. Grey and white brain matter standards containing varying amounts of tritium were exposed along with the incubated tissue sections. Quantification of autoradiograms was performed using a computer assisted image processing system. as previously described.’ Brain areas and nuclei were identified using atlases of the human brain.“.‘? [‘HIStrychnine was obtained from New England Nuclear (Dreieich F.R.G.). Other chemicals and reagents were 01 commercial origin. RESULTS
Distribution of [‘HJstrychnine binding sites in the brainstem and upper cervical cord
Autoradiographic grains were found to be highly localized within grey matter structures and nuclei of the brainstem and spinal cord. The non-specific binding was homogeneous and low (below 10 fmolmg protein-‘) in all the brain regions studied with the exception of the locus coeruleus and substantia
(‘I
(il.
nigra where high levels of non-specific binding were apparently associated with the neuronal cell bodies. Amounts of binding over the white matter did not differ from background
levels.
The density of glycine receptors was greater In the caudal parts of the brainstem and decreased in more rostra1 regions. The highest densities of [‘HIstrychnine binding sites (‘H-SBS) were found in the substantia gelatinosa of the trigeminal nucleus. in the hypoglossal nucleus and in the motor and principal sensory nuclei of the trigeminal nerve. Negligible densities of ‘H-SBS were observed in forebrain structures. such as putamen (I. I + 0.6 fmol mg protein ‘), globus pallidus (3.7 k 2.2 fmol mg protein ‘) and neocortex (3.4 + I .7 fmol mg protein I), as well as diencephalon (I 8.7 k 5.4 fmol mg protein ‘) and cerebellum (5.4 + 3.2 fmol mg protein ‘). In order to simplify the description we arbitrarily designated those areas and nuclei of the brainstem with values above 300 fmol mg protein ’
Abbreviationsused in figures
ATV BCI CM
area tegmentalis ventralis brachium colliculi inferioris cellulae motoriae colliculus inferior colliculus superior :J Cl-r central tegmental tract dc dorsal cortex of the inferior colliculus dm dorsomedial nucleus of the inferior colliculus decussatio pyramidum DP fasciculus cuneatus FC fasciculus gracilis FG formatio reticularis lateralis FRL griseum centrale mescncqhali GCM GcMet griseum centrale metencephali g&urn centrale pontis GCP HY hypothalamus locus coeruleus LC LL lemniscus lateralis medial lemniscus ML MLF medial longitudinal faxiculus N III nucleus nervi oculomotorii N IV nucleus nervi trochkaris nucleus nervi abducentis N VI N VII nucleus nervi facialis nucleus nervi hypoglossi N XII NcN.V nervus trigeminus Nerv.VII nervus facialis NA nucleus ambiguus nuclei arcuati NAr NC nucleus cuneiformis nucleus cochlearis dorsalis NCD NCM nucleus cuneatus medialis nucleus dorsalis nervi vagi NDX NFA nucleus funicuIi anterioris NFL nucleus funiculi later& (syn.: n. reticulari lateralis) NG nucleus gracilis NGC nucleus reticularis gigantoccIIuIaris NI nucleus interudatus NIP nucleus intcrpeduncularis NLL nuckw lcmniaci Iate&is NM V nucku.9 maeaaqkkm nervi wi NMOC nucleus medullae oblongatae centralis
NMot V nucleus motorius nervi trigemini NOAD nucleus olivaris accessorius dorsalis NOAM nucleus olivaris accessorius medialis NO1 nucleus olivaris inferior nuclei pontis NP NPBL nucleus parabrachialis lateralis NPBM nucleus parabrachialis medialis NPH nucleus praepositus hypoglossi NPL nucleus paralemniscalis nucleus paranigralis NPN NPro nucleus proprius NPro V nucleus proprius nervi trigemini NR nucleus ruber NRC nucleus rapht centralis NRC p.a. nucleus raphi centralis, pars annularis nucleus raphi dorsalis NRD NRD D.s1nucleus raphe dorsalis. pars supratrochlearis NRL ’ nucleus raphC linearis NRM nucleus raphC magnus NRO nucleus raphC obscurus NRP nucleus raphC pontis NRPC nucleus reticularis pontis caudalis nucleus reticularis pontis oralis NRPO NRTP nucleus reticulotegmentalis pontis nucleus solitarius NS NS V nucleus sensorius principalis nervi trigemini NSp V nucleus spinalis nervi trigemini nucleus supraspinalis NSS NTPP nucleus tegmentalis pedunculopontinus NV1 nucleus vestibularis inferior nucleus vestibularis medialis zs” nucleus vestibularis superior pedunculus arebdlaris superior PCS PR processus reticularis PT pyramidal tract substantia gelatinosa SG substantia gelatinosa nervi trigemini SG V substantia intermedia SI substantia nigra SN TPL tractus pyramidalis lateralis trachls solitarius TS tractus spinalis nervi trigemini TSp V
Figs l-7. Bright-field photomicrographs of autoradiograms generated by apposing tissue labeled with (‘H]strychnine to [‘H]Ultrofilm. The images show the distribution of autoradiographic grains in the human brainstem, dark regions representing areas with high densities of receptor sites. Abbreviations used in photomicrographs are listed opposite in the text. Fig. 1. Bright-field photomicrograph demonstrating the distribution of autoradiographic grains over the upper midbrain. The highest densities of 3H-SBS are found over lateral and dorsal aspects of periaqueductal grey matter @CM) and in the oculomotor nucleus (N III). Negligible amounts of silver grains are seen over the white matter tracts. Detail of the anatomical structures is shown in the corresponding schematic of the atlas (Fig. 8). See text for details. Bar = 0.5 cm.
13
Fig. 2. Photomicrograph of glycine receptors in lower midbrain tcgmentum and tectum. Note high densities of 3H-SBS over the dorsolateral parts of the periaq~ctal grey matter @CM). Low densities of glycine receptors are found over the nuclei paranigralis (NPM on both sides of the nudeus interpeduncularis (NIP) and the nucleus interpadwn~ukis it&f, as well as over the inferior coliiculus, the nucleus cuneiformis (NC), the nucleus te+mentab @eduncuiop@nus (NTPP) and the substantia nigra (SN). Low concentrations of grains are also observed over the tipbe nuclei. See schkmatic representation in Fig. 9 for anatomical &tails. Bar = 0.5 cm.
14
Fig. 3. Photomicrograph of glycine receptors at the midpontine level. The most prominently labeled structures are the motor trigeminal nucleus (NMot V) and the principal trigeminal sensory nucleus (NS V). Note the presence of intermediate concentrations of glycine receptors in the formation reticularis lateralis (FRL) and low concentrations in the raphe nuclei, the nucleus reticularis pontis caudalis (NRPC), and the griseum centrale pontis (GCP). Very low concentrations of glycine receptors are seen over the nuclei pontis (NP). See corresponding schematic diagram in Fig. 11 for details of the anatomical structures. Bar = 0.5 cm.
15
Fig. 4. Photania@ of the g&&e reaptors at the transirioar~ peas and Note the high density of grains over the spinai nudeus of the &igemW iterve (MuI the nucleus nervi facialis. Intermediate density of g&ine receptors are ais observed over the fomatio reticularis lateralis, auclens prqmbls hypu@otBi . only low concentrations of stry&nine kbding sites are rwe nuclei and the inferior olivary m&i. See schematic drawing in Fig. 13. l&w= 0.5 cm.
16
Fig. 5. Bright-tield photomicrograph of glycine receptors at the upper level of the medulla oblongata. Note very high density of receptors in the nuckus nervi hypoglossi (N XII) and in the spinal trigeminal nucleus (pars interpolaris) (NSp V). Intermediate densities of glycine receptors are observed over the nucleus solitarius (NS) and low densities over the formatio reticularis of the medulla oblongata and dorsal aRerent nucleus of the vagus. See corresponding schematic diagram in Fig. 14 for details of anatomical structures. Bar = 0.5 cm.
17
I’. .:- ’ Fig. 6. Phatomicrograph of &eke ra#ptws at the ieve1 of the decwaatio of the lam&& kc+& very k&b ~f&he tweptors in the substantia gelatinosa of the tfisctminai nubus @G V). Note also patches of very high receptor densities in the -us (BiCM). Some small patches of high receptor density are obaerwd in the nsdeus grac&s (NO). Wxmediate to high receptor Sties ant ‘&aerwd in the nucleus tractus solitarius and hypoglossal nucleus at this level. Intermediate concentrations of gtycine receptors are present over the n’uckws ‘funiculi anterioris. See corresponding schematic representation in Fig. 15 for anatomical details. Bar = 0.3 cm.
Fig. 7. Bright-field photomicrograph of an autoradiogram from a section through the cervica1 cord. The highest glycine receptor densities are observed in the substantia gelatinosa (SG). High levels of binding are associated with the motor cells (CM). The other structures at this level contain intermediate concentrations of GIL See Fig. 17 of the schematics for anatomical details. Bar = 0.3 cm.
19
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Figs 8-17. Schematic illustrations of the distribution of 3W-SB!Jin transverse sections of the buman brainstem and upper cervical cord. Density scale indicates the relative concentration of %4&&J with assigned values ranging from low or very low (1oDfmol mgprotein-’ or teas), medium (100~200fmoI mg protein-‘), high @OO-3OOfmolmgprotein-‘) and very high (more than 300 fmol mg protein-‘). Abbreviations used in schematics are listed separately in the text. Fig. 8. Rostra1 midbrain. 20
dc
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.
Fig. 9. Caudal midbrain. 21
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.
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Fig. 10. Rostra1 pons.
Fig. 11. Mid-caudal pons, at the level of the motor and sensory trigeminal nuclei.
23
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.
Fig. 12. Caudal pons at the level of the facial nucleus.
24
Fig. 13. Pons-medulla
transition.
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.
,
.
-. +.
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,
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Fig. 14. Rostra1 medulla oblongata.
26
*
.
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,
.
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Fig. 15. Medulla oblongata at the level of the decussatio of the lemnisci.
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*-...
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Fig. 16. Caudal medulla oblongata at the level of the pyramidal decussation.
28
Fig. 17. Cervical spinal cord.
Fig. 18. Sagittal section of the rat brain. (a) Photomicrograph of an autoradiogram showing the distribution of ‘H-SBS. Note the lack of binding in the forebrain and a rostrocaudal incmtsing gradient of GR. Very high densities are obsrved in the substantia gelatinosa and nucleus nervi hypogiossi. (b) Acetylcholinesterase staining of the same section showed in (a). The abbreviations used are listed sparately. Bar = 0.3 cm.
30
Glycine receptor autoradiography
in human brain Table l-continued
Table 1. Specific [)H]strychnine binding in the human brain Brain area Midbrain Griseum centrale mesencephali Griseum centrale mesencephali laterodorsal part Nucleus cuneiformis Nucleus accessorius nervi oculomotorii (Edinger-Westphal) Nucleus nervi oculomotorii Nucleus tegmentalis pedunculopontinus pars compacta Nucleus interpeduncularis Nucleus paranigralis Substantia nigra, pars compacta Substantia nigra, pars reticulata Area tegmentalis ventralis Colliculus inferior nucleus centralis lamina superficialis Nucleus intercollicularis Colliculus superior Nucleus ruber, pars magnocellularis pars parvocellularis Pons Nucleus nervi facialis Nucleus nervi abducentis Nucleus sensorius principalis nervi trigemini Nucleus sensorius principalis nervi
trigemini (spots) Nucleus motorius nervi trigemini Nucleus mesencephalicus nervi trigemini Nucleus reticularis gigantocellularis Formatio reticularis lateralis Nucleus reticularis pontis caudalis Nucleus reticularis pontis oralis Nucleus reticularis tegrnenti pontis Nuclei pontis Nucleus raphes magnus Nucleus raphes pontis Nucleus raphes centralis Nucleus lemnisci lateralis Nucleus parabrachialis medialis Nucleus parabrachialis lateralis Corpus parabigeminum Locus coeruleus Medulla oblongata Nucleus gracilis Nucleus cuneatus medialis Nucleus cuneatus medialis (spots) Nucleus cuneatus lateralis Nucleus supraspinalis Nucleus medullae oblongatae centralis Nucleus funiculi lateralis Nucleus funiculi anterioris Nucleus olivaris inferior Nucleus olivaris accessorius medialis Nucleus olivaris accessorius dorsalis Nucleus spinalis nervi trigemini pars caudalis: substantia gelatinosa nucleus proprius pars interpolaris pars oralis Nucleus nervi hypoglossi Nucleus intercalatus Nucleus dorsalis nervi vagi Nucleus solitarius
fmolmgprotein-’ k SEM (n) 79.2 + 7.0 (8) 242.3 + 41.1 (3) 69.4 f 8.9 (5) 269.5 &-8.3 (2) 68.7 + 11.1 (3) 28.2 f 6.2 (5) 35.1 Jt 13.3 (4) 26.3 + 7.9 (5) 44.1 k 10.6 (5) 41.9 f 5.5 (3) 22.0 & 1.8 (2) 42.0 f 8.4 (5) 98.6 5 11.7 (5) 42.5 f 7.2 (3) 52.1 + 2.9 (2) 8.5 & 1.4(2) 17.4 + 3.0 (2) 279.3 f 39.7 (4) 62.4 + 1.5 (2) 97.1 + 16.3 (4) 310.2 k 29.8 (3) 359.2 + 75.8 (4) 26.5 + 2.9 (8) 87.9 + 11.7 (7) 158.7 & 17.6 (5) 52.8 f 9.7 (3) 14.5 + 2.4 (8) 12.7 k 1.7 (8) 10.4 f 1.7 (8) 15.7 + 5.4 (5) 32.7 f 8.1 (2) 43.9 * 11.2 (8) 10.3 f 4.1 (5) 15.4 k 2.5 (7) 28.3 k 4.4 (8) 26.7 k 5.9 (5) 89.3 * 14.0 (7)
57.9 + 6.5 (6) 88.9 + 9.7 (6) 344.5 f 15.1 (6) 83.3 k 9.7 (6) 186.8 f 16.3 (5) 69.9 f 5.8 (8) 57.7 + 7.1 (8) 159.1 + 10.8 (6) 45.6 + 5.5 (8) 54.0 & 8.7 (8) 25.4 f 4.2 (6) 457.6 & 37.3 (7) 186.9 k 10.9 (7) 312.5 + 26.6 (6) 246.6 + 34.9 (7) 383.9 + 26.2 (7) 88.4 f 9.0 (8) 44.3 f 6.2 (8) 200.2 f 23.8 (7)
31
Brain area Medulla Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus
oblongata ambiguus vestibularis inferior vestibularis medialis cochlearis dorsalis praeopositus hypoglossi raphes obscurus
Cervical cord Substantia gelatinosa Nucleus proprius Processus reticularis Substantia intermedia Cellulae motoriae
fmolmgprotein-’ f SEM (n) 226.5 &-17.4 (5) 28.8 & 5.6 (8) 111.5 f 12.2 (5) 136.3 + 25.5 (5) 150.1 f 22.2 (5) ND 389.7 + 47.0 (2) 185.3 k 23.6 (2) 146.3 f 31.6(2) 125.8 + 18.5 (2) 233.5&11.8(2)
(n); Number of cases measured. ND; Not detectable.
as structures with very high density; high densities were between 200 and 300 fmol mg protein-‘; intermediate densities were between 100 and 200 fmol mg protein-’ and low to very low densities were below 100 fmol mg protein-‘. Table 1 summarizes the quantitative results obtained in the eight brains analyzed in this study. Each value represents the average amount of receptor sites computed from several brains for each particular region. The reader is referred to the photographic atlas (Figs l-7) and to the corresponding representations of these areas in the schematics (Figs 8-17). In the upper half of the midbrain (Figs 1 and 8) high concentrations of ‘H-SBS were associated with the lateral and the dorsal parts of the pxiaqueductal grey matter. Lower densities of grains were found just laterally from the midline, dorsally from the aqueduct and in the periaqueductal grey situated ventrally from the aqueduct. High concentrations of binding sites were observed over the oculomotor nucleus. Low concentrations of 3H-SBS characterized the nucleus of Edinger-Westphal, the nucleus cuneiformis and the superior colliculus. Slightly higher ‘H-SBS concentrations were found in the stratum griseum superficial than in the stratum griseum intermediale and profundum. Very low amounts of grains were present over the nucleus ruber (pars magnocellularis and pars parvocellularis), area tegmentalis ventralis and the corpus geniculatum mediale. The substantia nigra was characterized by low densities of binding sites with a slightly heterogeneous distribution of grains over the pars compacta and reticulata. Relatively high levels of non-specific [‘HIstrychnine binding were observed over the cell bodies of the substantia nigra compacta. The medial longitudinal fasciculus and the central tegmental tract were associated with 3H-SBS densities not different from the background. In the caudal midbrain (Figs 2 and 9) intermediate to low concentrations of silver grains were computed in the periaqueductal grey matter including the area occupied by the nucleus raphe dorsalis pars
31
.4. P~oesr (‘I al.
supratrochlearis and the nucleus trochlearis. On the contrary, high concentrations of grains were found over the peripheral aspects of the dorsal and lateral periaqueductal grey matter. although lower concentrations of grains were found just around the aqueduct. Low to very low concentrations of silver grains were noted over other grey structures of the midbrain tegmentum including the nucleus paranigralis, nucleus interpeduncularis, nucleus tegmcntalis pedunculopontinus (pars dissipata and compacta), nucleus cuneiformis and nucleus raphe centralis. Similar concentrations of ‘H-SBS were found over the inferior colliculus, however. higher densities were measured over its dorsal cortex. The most prominently labeled nuclei in the pons (Figs 3, 4. IO, I I and 12) were the motor nucleus of the trigeminal nerve and the nucleus of the facial nerve. Intermediate concentrations of ‘H-SBS were found in the sensory trigeminal nucleus (principal nucleus). However, high to very high concentrations of binding sites were found in irregularly shaped spots distributed over this nucleus. Intermediate concentrations of binding sites characterized the lateral reticular formation. Low to very low degrees of binding were found over the remaining pontine grey matter including the nucleus reticularis pontis caudalis and oralis, nucleus reticularis tegmenti pontis, raphe and parabrachial nuclei. Only negligible amounts of grains were found over the nuclei pontis and nucleus lemnisci lateralis. Low densities of ‘H-SBS characterized the locus coeruleus, which contained high levels of non-specific binding. As in the pons. the distribution of autoradiographic grains in the medulla oblongata (Figs 4, 5, 6, 13, 14, I5 and 16) was highly localized. High to very high concentrations of ‘H-SBS were found over the spinal trigeminal nucleus. This nucleus showed a definite increase of receptor sites from its rostra1 to its caudal portions. Thus, its pars oralis contained high concentrations whereas very high concentrations of silver grains were observed in the pars interpolaris and caudalis of this nucleus. The substantia gelatinosa of the pars caudalis contained the highest degree of receptor labeling found in the human brainstem and upper spinal cord, presenting an irregular distribution with patches of a higher density of silver grains. The nucleus proprius was associated with lower densities, however, very high concentrations of receptor sites were probably present in its outer parts. Also very high levels of (‘HIstrychnine binding were observed in the nucleus of the XIIth nerve. Spots of very high silver grain density were also seen in the nucleus cuneatus medialis at the level of the pyramidal decussation. These spots were very irregular in shape and are likely to correspond to the known clustering of neurons in this nucleus.32 Only medium to low concentration of receptor sites were found between these high density spots. A somewhat
spotty distribution of ‘H-SBS was also obserlcd 111 the nucleus gracilis. However, grain concentrations in this nucleus were lower than in the cuncate nucleus. High concentrations of grains characterized the nucleus ambiguus and the nucleus solitarius. The distribution of silver grains in the nucleus solitarius was irregular with highest concentrations found surrounding the tractus solitarius. Intermediate levels of ‘H-SBS were found in the dorsal cochlcar nuclei. nucleus praepositus hypoglossi and nucleus vcstibularis medialis. Medium to low grain densities wcrc associated to the nucleus cuneatus lateralis, nucleus medullae oblongatae ccntralis, the funicular nuclei and the nucleus intercalatus. The inferior olivary complex and nucleus vcstibularis inferior showed very low levels of receptor binding. In the upper cervical cord (Fig. 17) very high densities of ‘H-SBS were observed in the substantia gelatinosa. However. the levels of binding in this region were markedly lower than those measured more rostrally in the substantia gelatinosa of the trigeminal nucleus. In the ventral horn high densities of GR were found in the motor neurons. Intermediate levels of specific binding were associated with other structures including nucleus proprius. processus reticularis and substantia intermedia. The rat brain was examined in parallel to human tissues. The distribution of ‘H-SBS in the rat brain is illustrated in Fig. I8 and quantitative values are given in Table 2 for the sake of comparison with values obtained in human brain samples, Densities in the rat brain were much higher than those found in humans. with the highest levels of ‘H-SBS being observed in the substantia gelatinosa of the cervical cord, spinal nucleus of the trigeminal nerve (substantia gelatinosa and nucleus proprius) and nucleus nervi hypoglossi. Intermediate densities were seen in the medial vestibular nucleus. The reticular formation, central grey and superficial grey layer of the superior COIliculus contained rather low densities of GR. Finally, very low densities were detected over the inferior colliculus, pontine nuclei and hypothalamus. Table 2. Specific [‘HIstrychnine binding in the rat brain Brain area Colliculus superior stratum griseum superficiale stratum griseum intermediale Colliculus inferior Grkeum centrale mesenceohali
Nuclei pontis N UC Ieus reticularis pontis oralis Nucleus reticularis pontis caudalis Nucleus reticularis gigantocellularis Nucleus vestibularis medialis Nucleus nervi hypoglossi Nucleus spinalis nervi trigemini substantia gclatinosa nucleus proprius Cervical cord, substantia gelatinosa
fmolmgprotein + SEM 240.9 f 16.7 138.1 + 16.3 63.8 f 4.2 279.3 t 29.8
72.2 + 1.7 250.6 2 15.4 319.0 * 31.5
267.0 f 5.2 450.2 + 15.7 955.7 + 37.6 988.9 + 12.5 922.1 + 28.6 825.1 f 17.6
Data were obtained from two different animals
’
Glycine receptor autoradiography in human brain DISCUSSION
This report presents an atlas of the distribution of high affinity glycine binding sites labeled with the antagonist [3H]strychnine in the brain of individuals without evidence of neurological or psychiatric disorders. High concentrations of ‘H-SBS were present in several nuclei and areas of the brainstem including motor and sensory trigeminal nuclei, nucleus cuneatus, as well as the facial and hypoglossal nuclei. The highest concentrations of 3H-SBS were found in the grey matter of the upper cervical cord and medulla oblongata where the most prominently labeled structure was the substantia gelatinosa of the trigeminal nucleus. The overall distribution of glycine receptors observed in this study is in agreement with previous results obtained in human and experimental animal CNS by microdissection techniques26*48and in the rat and mouse by light microscopic autoradiographic techniques.38s5’ Like in these studies, we observed that the distribution of ‘H-SBS was highly localized and a decrease of receptor concentration was found from the spinal cord level to the upper brainstem and forebrain. Grey structures of the forebrain, including the thalamus, the striatum and the cerebral cortex, were almost devoid of receptor sites. This caudalrostra1 gradient of GR concentrations is in agreement with previous studies showing decrement in a rostra1 progression of the relative number of glycinergic synapses as defined neurophysiologically,5~8~11,15,16,17~18,23,24,28~4~~47 by differential
distribution
or a high affinity glycine uptake mechof glycine,2*3*6 anism 21.25,27,36,39 Differences were seen between the rat and the human brain. In identical incubation conditions the densities of 3H-SBS were two to three-fold higher in the rat than in the human brain, These data are in good agreement with the results obtained by De Montis et ~1.‘~ using classical membrane binding assays. These authors reported maximal densities of more than 2000 fmol mg protein-’ in the rat spinal cord, while the total number of ‘H-SBS in the human spinal cord was about 600 fmol mg protein-‘. In addition, the rank order of densities of ‘H-SBS in different nuclei of the rat and human brains was not completely identical. For instance we found a different distribution of 3H-SBS in the midbrain, the highest concentrations being observed in the periaqueductal grey matter and the oculomotor nuclei, whereas in the rat highest concentrations have been noted in the nucleus cuneiformis5’ In the human pons, the relative concentrations of 3H-SBS were significantly higher than those noted in the rat. Higher concentrations of binding sites were also noted in the human tractus solitarius and in the nucleus ambiguus. Detailed studies of the distribution of GR in the human brain have limited themselves to the spinal cord because of the relevance of glycinergic mech-
33
anisms in spinal motoneuron functions in human diseases, particularly in amyotrophic lateral sclerosis and spasticity.igJO” High concentrations of GR have been found by membrane binding studies in the grey matter of human control spinal cord with values slightly higher in the ventral than in the dorsal half of the grey matter. 20In a recent study by Whitehouse et al.& high concentrations of 3H-SBS were found in the grey matter of the cervical spinal cord of control cases by light microscopic autoradiographic techniques. Like these authors we found higher receptor concentrations in laminae II (substantia gelatinosa) and III of Rexed and in ventral horn in layer IX, which contains motoneurons. This distribution of 3H-SBS in human anterior horn appears to be different from that of the rat spinal cords1 where the highest concentrations of grains were found in lamina VII of Rexed, whereas receptor concentrations were much lower in the lamina IX. Glycine has been shown to play a major role in postsynaptic inhibition in the spinal cord where it is released mainly by segmental intemeurons, including the Renshaw cells, responsible for recurrent inhibition of motor neurons and I-a afferent interneurons.2,4,5*11,24Furthermore, putative glycinergic nerve terminals have been mapped in the ventral horn of the rat by localizing glycine uptake sites.27*36 Uptake sites were noted in the neuropil and around the cell bodies of motor neurons in a distribution very similar to that of GR in the ventral horn of rat5’ and human.46 In the report of Whitehouse et al.,& decrement of muscarinic, benzodiazepine and glycine receptors was shown to occur in spinal grey matter of patients with amyotrophic lateral sclerosis. The major loss of GR was observed in lamina IX and was highly correlated with the degree of neuronal loss in the same region, suggesting that the majority of the receptors are localized on motor neurons. There is only scanty information on the distribution of GR in supraspinal grey structures in human. In one study, 26 3H-SBS distribution has been compared with the distribution of [3H]y-aminobutyrate binding sites in macroscopically dissected brain regions. Whereas y-aminobutyrate receptors were found to be predominant in the forebrain and upper brainstem, GR were more localized in the lower brainstem and in the spinal cord. However, both receptors were colocalized in some structures like the substantia nigra and the superior colliculus. Surprisingly, significant, although low amounts of [3H]strychnine binding were found in the cerebral cortex, a result which contrasts with the absence of significant amounts of 3H-SBS in autoradiographic studies in animal? and in human (results in this paper). In a recent study using membrane preparations of the brain of non-neurological patients, high and low affinity sites for [‘HIstrychnine were found in the spinal cord and pars compacta of the substantia nigra.i4 High levels of [3H]strychnine binding with
34
A.
PRORST CI cd
similar values were found in both regions. [‘HIStrychnine binding was significantly reduced in the substantia nigra of Parkinson’s disease patients, both in pars compacta and reticulata.“’ In our studies.
basis of high ‘H-SBS concentrations in loci of the brainstem and spinal cord of the rat can now hc explained in the basis of GR localization in the human brain. For instance, visual or acoustic stimuh
where only high affinity binding sites are visualized. we have detected low densities of ‘H-SBS in the substantia nigra, which are of the same order of magnitude as those reported by DeMontis et ~1.‘~ The distribution of GR as shown by in rirro autoradiography in the rat and in human parallels that observed for the neurophysiological responses after local application of glycine.8,“,‘5 ‘8242840.47 Correlations between loci of high ‘H-SBS density and electrophysiologic data as well as physiologic functions associated with GR have been extensively dis-
induced convulsions in humans suffering from strychnine toxicity have been tentatively explained by high SBS densities in the superior colliculus. In this structure we found ‘H-SBS in amounts comparable to those found in the rat. Also the very high levels 01 SBS in the sensory and motor portions of the trigeminal nuclei and in the facial nuclei in human suggest these arcas as a likely anatomical locus for strychnine induced hyperreflexia, muscle stiffness and spasm in masticatory and facial muscles.
cussed by Zarbin et al.” In particular, some clinical effects of strychnine which were explained on t&
Acknowledgemenr-- We are grateful to Dr R. Foote for critical reading of the manuscript.
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Glycine receptor autoradiography
in human brain
35
25. Logan W. L. and Snyder S. H. (1972) High affinity uptake system for glycine glutamic and aspartic acids in synaptosomes of rat central nervous tissues. Bruin Res. 42, 41-31. 26. Lloyd K. G., DeMontis G., Broekkamp C. L., Thuret F. and Worms P. (1983) Neurochemical and neuropharmacological indications of the involvement of GABA and glycine receptors in neuropsychiatric disorders. In CNS Receptors -From Molecular Pharmacology to Behauior (eds Mandel P. and DeFeudis F. V.), pp. 137-148. Raven Press, New York. 27. Matus A. I. and Dennison, M. E. (1971) Autoradiographic localization of tritiated glycine at “flat-vesicle” synapses in spinal cord. Brain Res. 32, 195-197. 28. Morimoto T., Takata M. and Kawamura Y. (1968) Effect of lingual nerve stimulation on hypoglossal motoneurons. Exp. Neurol. 22, 174190. 29. Miiller W. E. and Snyder S. H. (1978) Glycine high affinity uptake and strychnine binding associated with glycine receptors in the frog central nervous system. Brain Res. 143, 487498. 30. Miiller W. E. and Snyder S. H. (1978) Strychnine binding associated with synaptic glycine receptors in rat spinal cord membranes: ionic influences. Bruin Res. 147, 107-l 16. 31. Nieuwenhuis R., Voogd J. and Van Huijzen C. (1978) The Human Central Nervous System. A Synopsis and Atlas. Springer, Berlin. 32. Olszewski J. and Baxter D. (1982) Cytoarchitecture of the Human Brainstem, 2nd Edn. Karger, Basel. 33. Perry E. K. and Perry R. H. (1983) Human brain neurochemistry-Some postmortem problems. Life Sci. 33, 1733-1743. 34. Perry E. K., Perry R. H. and Tomlinson B. E. (1982) The influence of agonal status on some neurochemical activities of postmortem human brain tissue. Neurosci. Lett. 29, 303-307. 35. Pfeiffer F., Graham D. and Betz H. (1982) Purification by affinity chromatography of the glycine receptor of rat spinal cord. J. biol. Chem. 257, 93899393. 36. Price D. L., Stocks A., Griffin J. W., Young A. and Peck K. (1976) Glycine specific synapses in rat spinal cord: identification by electron microscopic autoradiography. J. cell. Biol. 68,’ 384-395. 37. Pycock C. J. and Kerwin R. W. (1981) The status of glycine as a supraspinal neurotransmitter. Life Sci. 28,26792686. 38. Rotter A., Schultz C. H. and Frostholm A. (1984) Regulation of glycine receptor binding in the mouse hypoglossal nucleus in response to axotomy. Brain Res. Bull. 13, 487492. 39. Snyder S. H. (1975) The glycine receptor in the mammalian central nervous system. Br. J. Pharmac. 53, 473-484. 40. Tebecis A. K. (1973) Transmitters and reticulospinal neurons. Exp. Neural. 40, 297-308. 41 Tebecis A. K. and DiMaria A. (1972) Strychnine-sensitive inhibition in the medullary reticular formation: evidence for glycine as an inhibitory transmitter: B&n Res. 40, 373-383. 42. Unnerstall J. R., Niehoff D. L., Kuhar M. J. and Palacios J. M. (1982) Quantitative receptor autoradiography using ‘H-Ultrofilm: application to multiple benzodiazepine receptors. J. Neurosci. Meth. 6, 59-73. 43. Werman R., Davidoff R. A. and Aprison M. H. (1966) Glycine and postsynaptic inhibition in the cat spinal cord. Physiologist 9, 318. 44. Werman R., Davidoff R. A. and Aprison M. H. (1967) Inhibition of motoneurons by iontophoresis of glycine. Nature 214, 681683. 45. Werman R., Davidoff R. A. and Aprison M. H. (1968) Inhibitory action of glycine on spinal neurons in the cat. J. Neurophysiol. 31, 81-95. 46. Whitehouse P. J., Wamsley J. K., Zarbin M. A., Price D. L., Tourtellote W. W. and Kuhar M. J. (1983) Amyotrophic lateral sclerosis: alterations in neurotransmitter receptors. Ann. Neural. 14, 8-16. 47. Yokota T., Nishikawa N. and Nishikawa Y. (1979) Effects of strychnine upon different classes of trigeminal subnucleus caudalis neurons. Brain Res. 168, 430-434. 48. Young A. B. and Snyder S. H. (1973) Strychnine binding associated with glycine receptors of the central nervous system. Proc. natn. Acad. Sci. U.S.A. 79 2832-2836. 49. Young A. B. and Snyder S. H. (1974) The glycine synaptic receptor: evidence that strychnine binding is associated with the ionic conductance mechanism. Proc. natn. Acad. Sci. U.S.A. 71, 40024005. 50. Young A. B. and Snyder S. H. (1974) Strychnine binding in rat spinal cord membranes associated with the synaptic _ _ glycine receptor: cooperativity of glycine interactions. Mblec. Phirmac. 10, 79&809. 51. Zarbin M. A., Wamsley J. K. and Kuhar M. J. (1981) Glycine receptor: light microscopic autoradiographic localization with ‘H-strychnine. J. Neurosci. 1, 532-547. (Accepted 14 August 1985)
0306-4522/86$3.00+ 0.00 Pergamon Press Ltd IBRO
THE DISTRIBUTION OF GLYCINE RECEPTORS IN THE HUMAN BRAIN. A LIGHT MICROSCOPIC AUTORADIOGRAPHIC STUDY USING [3H]STRYCHNINE A. F%OBST,* R. CORTES~ and J. M. PALACIOS*$ *Preclinical Research, Sandoz Ltd, CH-4002 Basle, Switzerland and tDepartment of Pathology, Division of Neuropathology, University of Basle, Schiinbeinstrasse 40, CH-4003 Basle, Switzerland Abstract--Glycine receptors were localized autoradiographically in postmortem human brain material using [3H]strychinine as a ligand. Slide mounted tissue sections were labeled in vitro by incubation with [rH]strychnine and autoradiograms obtained using [3H]Ultrofilm. Receptor densities were quantified by computer assisted microdensitometry. No specific binding of [)H]strychnine was observed in any of the forebrain areas studied. Low densities were seen in the midbrain except for dorsal and lateral parts of the periaqueductal grey matter and the oculomotor nuclei. In pons, medulla oblongata and upper cervical cord high densities of [‘HIstrychnine
binding sites were associated with some nuclei including the motor and sensory trigeminal nuclei, the facial and the hypoglossal nuclei. The highest densities of grains were associated with the substantia gelatinosa of the trigeminal nucleus in the medulla oblongata. A peculiar spotty distribution of [3H]strychnine binding sites were found in the gracilis and cuneatus nuclei. The distribution of glycine receptors in the human brain is comparable to that seen in the rat brain, although densities are much higher in the rat. The distribution of glycine receptors in the human brain provides an anatomical substrate for the understanding
of the effects of drugs acting in these receptors, particularly strychnine.
human brain chemistry and present characteristics The amino acid glycine appears to play an important similar to those found in studies in experimental role as an inhibitory neurotransmitter in the brain of animals. mammals and other animals.1~2~3~4~12*13~37~43~“~45 GlycinIn an attempt to expand the knowledge of the ergic synapses are mainly concentrated in the phylodistribution of GR in the human CNS we have genetically oldest parts of the brain, such as the spinal now examined the whole CNS of the neurologically cord and medulla,3~‘0~s’their relative number decreas“normal” human brain using quantitative autoradioing in a rostra1 progression of the neuraxis. Receptors for glycine have been characterized mainly by graphic techniques. In this paper we report a detailed mapping of these sites at the light microscopic electrophysiological methods and the ionic and resolution level. pharmacological properties of these sites are well known.5~i0*“~L7~22~23*24@‘~4’ More recently the use of EXPERIMENTAL PROCEDURES radioligand binding techniques has permitted a more direct biochemical study of these receptors allowHuman brains from a total of eight patients (four males, mean age 51 years, and four females, mean age 70 years) ing for detailed studies of structure activity relationwere obtained at autopsy after an average postmortem delay ships, ion requirements and will eventually lead to of 6 h. None of the cases studied showed clinical or neurothe molecular characterization of the glycine receppathological evidence of a brain disease. Individual tor.2’*2g*30,35,39,48*49,50 The ‘H-labeled glycine antagonist characteristics of these subjects, including sex, age, interval [‘HIstrychnine is a useful tool for the study of these between death and brain freezing, premortem condition and cause of death have been reported elsewhere.’ After removreceptors.48*49*so Recently Zarbin et ~1.~’have used this ing the brain, one cerebral and one cerebellar hemisphere, ligand for the autoradiographic mapping of glycine as well as the brainstem and the upper cervical cord, were receptors (GR) in the rat CNS. GR in the human stored at - 8o”C, to be later cut into 4-6 mm thick blocks. CNS have only been studied to a limited extent.‘4,26 The remainder of the CNS was immersed in formaldehyde Particularly changes in GR in the spinal cord of (4%) and further processed for routine neuropathological examination. Frozen blocks were then brought to -20°C patients dying from amyotrophic lateral sclerosis have been examined by membrane binding assays20 and mounted onto microtome chucks. Ten micrometerthick sections were cut using a cryostat (Leitz, 1720; from and by autoradiography.& These studies have L&z, Wetzlar, Federal Republic of Germany) and mounted revealed, that GR, like other neurotransmitter onto gelatin coated glass slides. GR were labeled as previously described5’ by incubating tissue sections with 4 nM receptors,‘*“*” survive postmortem modifications in $To whom correspondence should be addressed. Abbreviations: GR, glycine receptors; 3H-SBS, [‘Hlstrychnine binding sites. NSC. 1711-a
[‘HIstrychnine (23 Ci mmol-‘) in 0.05 M sodium potassium phosphate buffer, pH 7.4, at room temperature. Nonspecific binding was determined with the addition of 1O-2 M glycine. To generate autoradiograms, [“HlUltrofilm (LKB, Sweden) was exposed to the labeled slides as described by 11
A. Paoesl
I’
Unnerstall PI ol.“’ during three months. Grey and white brain matter standards containing varying amounts of tritium were exposed along with the incubated tissue sections. Quantification of autoradiograms was performed using a computer assisted image processing system. as previously described.’ Brain areas and nuclei were identified using atlases of the human brain.“.‘? [‘HIStrychnine was obtained from New England Nuclear (Dreieich F.R.G.). Other chemicals and reagents were 01 commercial origin. RESULTS
Distribution of [‘HJstrychnine binding sites in the brainstem and upper cervical cord
Autoradiographic grains were found to be highly localized within grey matter structures and nuclei of the brainstem and spinal cord. The non-specific binding was homogeneous and low (below 10 fmolmg protein-‘) in all the brain regions studied with the exception of the locus coeruleus and substantia
(‘I
(il.
nigra where high levels of non-specific binding were apparently associated with the neuronal cell bodies. Amounts of binding over the white matter did not differ from background
levels.
The density of glycine receptors was greater In the caudal parts of the brainstem and decreased in more rostra1 regions. The highest densities of [‘HIstrychnine binding sites (‘H-SBS) were found in the substantia gelatinosa of the trigeminal nucleus. in the hypoglossal nucleus and in the motor and principal sensory nuclei of the trigeminal nerve. Negligible densities of ‘H-SBS were observed in forebrain structures. such as putamen (I. I + 0.6 fmol mg protein ‘), globus pallidus (3.7 k 2.2 fmol mg protein ‘) and neocortex (3.4 + I .7 fmol mg protein I), as well as diencephalon (I 8.7 k 5.4 fmol mg protein ‘) and cerebellum (5.4 + 3.2 fmol mg protein ‘). In order to simplify the description we arbitrarily designated those areas and nuclei of the brainstem with values above 300 fmol mg protein ’
Abbreviationsused in figures
ATV BCI CM
area tegmentalis ventralis brachium colliculi inferioris cellulae motoriae colliculus inferior colliculus superior :J Cl-r central tegmental tract dc dorsal cortex of the inferior colliculus dm dorsomedial nucleus of the inferior colliculus decussatio pyramidum DP fasciculus cuneatus FC fasciculus gracilis FG formatio reticularis lateralis FRL griseum centrale mescncqhali GCM GcMet griseum centrale metencephali g&urn centrale pontis GCP HY hypothalamus locus coeruleus LC LL lemniscus lateralis medial lemniscus ML MLF medial longitudinal faxiculus N III nucleus nervi oculomotorii N IV nucleus nervi trochkaris nucleus nervi abducentis N VI N VII nucleus nervi facialis nucleus nervi hypoglossi N XII NcN.V nervus trigeminus Nerv.VII nervus facialis NA nucleus ambiguus nuclei arcuati NAr NC nucleus cuneiformis nucleus cochlearis dorsalis NCD NCM nucleus cuneatus medialis nucleus dorsalis nervi vagi NDX NFA nucleus funicuIi anterioris NFL nucleus funiculi later& (syn.: n. reticulari lateralis) NG nucleus gracilis NGC nucleus reticularis gigantoccIIuIaris NI nucleus interudatus NIP nucleus intcrpeduncularis NLL nuckw lcmniaci Iate&is NM V nucku.9 maeaaqkkm nervi wi NMOC nucleus medullae oblongatae centralis
NMot V nucleus motorius nervi trigemini NOAD nucleus olivaris accessorius dorsalis NOAM nucleus olivaris accessorius medialis NO1 nucleus olivaris inferior nuclei pontis NP NPBL nucleus parabrachialis lateralis NPBM nucleus parabrachialis medialis NPH nucleus praepositus hypoglossi NPL nucleus paralemniscalis nucleus paranigralis NPN NPro nucleus proprius NPro V nucleus proprius nervi trigemini NR nucleus ruber NRC nucleus rapht centralis NRC p.a. nucleus raphi centralis, pars annularis nucleus raphi dorsalis NRD NRD D.s1nucleus raphe dorsalis. pars supratrochlearis NRL ’ nucleus raphC linearis NRM nucleus raphC magnus NRO nucleus raphC obscurus NRP nucleus raphC pontis NRPC nucleus reticularis pontis caudalis nucleus reticularis pontis oralis NRPO NRTP nucleus reticulotegmentalis pontis nucleus solitarius NS NS V nucleus sensorius principalis nervi trigemini NSp V nucleus spinalis nervi trigemini nucleus supraspinalis NSS NTPP nucleus tegmentalis pedunculopontinus NV1 nucleus vestibularis inferior nucleus vestibularis medialis zs” nucleus vestibularis superior pedunculus arebdlaris superior PCS PR processus reticularis PT pyramidal tract substantia gelatinosa SG substantia gelatinosa nervi trigemini SG V substantia intermedia SI substantia nigra SN TPL tractus pyramidalis lateralis trachls solitarius TS tractus spinalis nervi trigemini TSp V
Figs l-7. Bright-field photomicrographs of autoradiograms generated by apposing tissue labeled with (‘H]strychnine to [‘H]Ultrofilm. The images show the distribution of autoradiographic grains in the human brainstem, dark regions representing areas with high densities of receptor sites. Abbreviations used in photomicrographs are listed opposite in the text. Fig. 1. Bright-field photomicrograph demonstrating the distribution of autoradiographic grains over the upper midbrain. The highest densities of 3H-SBS are found over lateral and dorsal aspects of periaqueductal grey matter @CM) and in the oculomotor nucleus (N III). Negligible amounts of silver grains are seen over the white matter tracts. Detail of the anatomical structures is shown in the corresponding schematic of the atlas (Fig. 8). See text for details. Bar = 0.5 cm.
13
Fig. 2. Photomicrograph of glycine receptors in lower midbrain tcgmentum and tectum. Note high densities of 3H-SBS over the dorsolateral parts of the periaq~ctal grey matter @CM). Low densities of glycine receptors are found over the nuclei paranigralis (NPM on both sides of the nudeus interpeduncularis (NIP) and the nucleus interpadwn~ukis it&f, as well as over the inferior coliiculus, the nucleus cuneiformis (NC), the nucleus te+mentab @eduncuiop@nus (NTPP) and the substantia nigra (SN). Low concentrations of grains are also observed over the tipbe nuclei. See schkmatic representation in Fig. 9 for anatomical &tails. Bar = 0.5 cm.
14
Fig. 3. Photomicrograph of glycine receptors at the midpontine level. The most prominently labeled structures are the motor trigeminal nucleus (NMot V) and the principal trigeminal sensory nucleus (NS V). Note the presence of intermediate concentrations of glycine receptors in the formation reticularis lateralis (FRL) and low concentrations in the raphe nuclei, the nucleus reticularis pontis caudalis (NRPC), and the griseum centrale pontis (GCP). Very low concentrations of glycine receptors are seen over the nuclei pontis (NP). See corresponding schematic diagram in Fig. 11 for details of the anatomical structures. Bar = 0.5 cm.
15
Fig. 4. Photania@ of the g&&e reaptors at the transirioar~ peas and Note the high density of grains over the spinai nudeus of the &igemW iterve (MuI the nucleus nervi facialis. Intermediate density of g&ine receptors are ais observed over the fomatio reticularis lateralis, auclens prqmbls hypu@otBi . only low concentrations of stry&nine kbding sites are rwe nuclei and the inferior olivary m&i. See schematic drawing in Fig. 13. l&w= 0.5 cm.
16
Fig. 5. Bright-tield photomicrograph of glycine receptors at the upper level of the medulla oblongata. Note very high density of receptors in the nuckus nervi hypoglossi (N XII) and in the spinal trigeminal nucleus (pars interpolaris) (NSp V). Intermediate densities of glycine receptors are observed over the nucleus solitarius (NS) and low densities over the formatio reticularis of the medulla oblongata and dorsal aRerent nucleus of the vagus. See corresponding schematic diagram in Fig. 14 for details of anatomical structures. Bar = 0.5 cm.
17
I’. .:- ’ Fig. 6. Phatomicrograph of &eke ra#ptws at the ieve1 of the decwaatio of the lam&& kc+& very k&b ~f&he tweptors in the substantia gelatinosa of the tfisctminai nubus @G V). Note also patches of very high receptor densities in the -us (BiCM). Some small patches of high receptor density are obaerwd in the nsdeus grac&s (NO). Wxmediate to high receptor Sties ant ‘&aerwd in the nucleus tractus solitarius and hypoglossal nucleus at this level. Intermediate concentrations of gtycine receptors are present over the n’uckws ‘funiculi anterioris. See corresponding schematic representation in Fig. 15 for anatomical details. Bar = 0.3 cm.
Fig. 7. Bright-field photomicrograph of an autoradiogram from a section through the cervica1 cord. The highest glycine receptor densities are observed in the substantia gelatinosa (SG). High levels of binding are associated with the motor cells (CM). The other structures at this level contain intermediate concentrations of GIL See Fig. 17 of the schematics for anatomical details. Bar = 0.3 cm.
19
PT
Figs 8-17. Schematic illustrations of the distribution of 3W-SB!Jin transverse sections of the buman brainstem and upper cervical cord. Density scale indicates the relative concentration of %4&&J with assigned values ranging from low or very low (1oDfmol mgprotein-’ or teas), medium (100~200fmoI mg protein-‘), high @OO-3OOfmolmgprotein-‘) and very high (more than 300 fmol mg protein-‘). Abbreviations used in schematics are listed separately in the text. Fig. 8. Rostra1 midbrain. 20
dc
NPNtNiP . “
.
Fig. 9. Caudal midbrain. 21
PHITP
.
. .
J
Fig. 10. Rostra1 pons.
Fig. 11. Mid-caudal pons, at the level of the motor and sensory trigeminal nuclei.
23
‘,.
.
Fig. 12. Caudal pons at the level of the facial nucleus.
24
Fig. 13. Pons-medulla
transition.
* .‘.
. . . :.
.
,
.
-. +.
I,
**
,
_- .,.
* :
Fig. 14. Rostra1 medulla oblongata.
26
*
.
*
,
.
* *
Fig. 15. Medulla oblongata at the level of the decussatio of the lemnisci.
..
*-...
. * - -. . . .
I-
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. .
.
.
._.
.
.
..
,; . *; . ‘.
.
.
.
. . ..-
.
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Fig. 16. Caudal medulla oblongata at the level of the pyramidal decussation.
28
Fig. 17. Cervical spinal cord.
Fig. 18. Sagittal section of the rat brain. (a) Photomicrograph of an autoradiogram showing the distribution of ‘H-SBS. Note the lack of binding in the forebrain and a rostrocaudal incmtsing gradient of GR. Very high densities are obsrved in the substantia gelatinosa and nucleus nervi hypogiossi. (b) Acetylcholinesterase staining of the same section showed in (a). The abbreviations used are listed sparately. Bar = 0.3 cm.
30
Glycine receptor autoradiography
in human brain Table l-continued
Table 1. Specific [)H]strychnine binding in the human brain Brain area Midbrain Griseum centrale mesencephali Griseum centrale mesencephali laterodorsal part Nucleus cuneiformis Nucleus accessorius nervi oculomotorii (Edinger-Westphal) Nucleus nervi oculomotorii Nucleus tegmentalis pedunculopontinus pars compacta Nucleus interpeduncularis Nucleus paranigralis Substantia nigra, pars compacta Substantia nigra, pars reticulata Area tegmentalis ventralis Colliculus inferior nucleus centralis lamina superficialis Nucleus intercollicularis Colliculus superior Nucleus ruber, pars magnocellularis pars parvocellularis Pons Nucleus nervi facialis Nucleus nervi abducentis Nucleus sensorius principalis nervi trigemini Nucleus sensorius principalis nervi
trigemini (spots) Nucleus motorius nervi trigemini Nucleus mesencephalicus nervi trigemini Nucleus reticularis gigantocellularis Formatio reticularis lateralis Nucleus reticularis pontis caudalis Nucleus reticularis pontis oralis Nucleus reticularis tegrnenti pontis Nuclei pontis Nucleus raphes magnus Nucleus raphes pontis Nucleus raphes centralis Nucleus lemnisci lateralis Nucleus parabrachialis medialis Nucleus parabrachialis lateralis Corpus parabigeminum Locus coeruleus Medulla oblongata Nucleus gracilis Nucleus cuneatus medialis Nucleus cuneatus medialis (spots) Nucleus cuneatus lateralis Nucleus supraspinalis Nucleus medullae oblongatae centralis Nucleus funiculi lateralis Nucleus funiculi anterioris Nucleus olivaris inferior Nucleus olivaris accessorius medialis Nucleus olivaris accessorius dorsalis Nucleus spinalis nervi trigemini pars caudalis: substantia gelatinosa nucleus proprius pars interpolaris pars oralis Nucleus nervi hypoglossi Nucleus intercalatus Nucleus dorsalis nervi vagi Nucleus solitarius
fmolmgprotein-’ k SEM (n) 79.2 + 7.0 (8) 242.3 + 41.1 (3) 69.4 f 8.9 (5) 269.5 &-8.3 (2) 68.7 + 11.1 (3) 28.2 f 6.2 (5) 35.1 Jt 13.3 (4) 26.3 + 7.9 (5) 44.1 k 10.6 (5) 41.9 f 5.5 (3) 22.0 & 1.8 (2) 42.0 f 8.4 (5) 98.6 5 11.7 (5) 42.5 f 7.2 (3) 52.1 + 2.9 (2) 8.5 & 1.4(2) 17.4 + 3.0 (2) 279.3 f 39.7 (4) 62.4 + 1.5 (2) 97.1 + 16.3 (4) 310.2 k 29.8 (3) 359.2 + 75.8 (4) 26.5 + 2.9 (8) 87.9 + 11.7 (7) 158.7 & 17.6 (5) 52.8 f 9.7 (3) 14.5 + 2.4 (8) 12.7 k 1.7 (8) 10.4 f 1.7 (8) 15.7 + 5.4 (5) 32.7 f 8.1 (2) 43.9 * 11.2 (8) 10.3 f 4.1 (5) 15.4 k 2.5 (7) 28.3 k 4.4 (8) 26.7 k 5.9 (5) 89.3 * 14.0 (7)
57.9 + 6.5 (6) 88.9 + 9.7 (6) 344.5 f 15.1 (6) 83.3 k 9.7 (6) 186.8 f 16.3 (5) 69.9 f 5.8 (8) 57.7 + 7.1 (8) 159.1 + 10.8 (6) 45.6 + 5.5 (8) 54.0 & 8.7 (8) 25.4 f 4.2 (6) 457.6 & 37.3 (7) 186.9 k 10.9 (7) 312.5 + 26.6 (6) 246.6 + 34.9 (7) 383.9 + 26.2 (7) 88.4 f 9.0 (8) 44.3 f 6.2 (8) 200.2 f 23.8 (7)
31
Brain area Medulla Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus
oblongata ambiguus vestibularis inferior vestibularis medialis cochlearis dorsalis praeopositus hypoglossi raphes obscurus
Cervical cord Substantia gelatinosa Nucleus proprius Processus reticularis Substantia intermedia Cellulae motoriae
fmolmgprotein-’ f SEM (n) 226.5 &-17.4 (5) 28.8 & 5.6 (8) 111.5 f 12.2 (5) 136.3 + 25.5 (5) 150.1 f 22.2 (5) ND 389.7 + 47.0 (2) 185.3 k 23.6 (2) 146.3 f 31.6(2) 125.8 + 18.5 (2) 233.5&11.8(2)
(n); Number of cases measured. ND; Not detectable.
as structures with very high density; high densities were between 200 and 300 fmol mg protein-‘; intermediate densities were between 100 and 200 fmol mg protein-’ and low to very low densities were below 100 fmol mg protein-‘. Table 1 summarizes the quantitative results obtained in the eight brains analyzed in this study. Each value represents the average amount of receptor sites computed from several brains for each particular region. The reader is referred to the photographic atlas (Figs l-7) and to the corresponding representations of these areas in the schematics (Figs 8-17). In the upper half of the midbrain (Figs 1 and 8) high concentrations of ‘H-SBS were associated with the lateral and the dorsal parts of the pxiaqueductal grey matter. Lower densities of grains were found just laterally from the midline, dorsally from the aqueduct and in the periaqueductal grey situated ventrally from the aqueduct. High concentrations of binding sites were observed over the oculomotor nucleus. Low concentrations of 3H-SBS characterized the nucleus of Edinger-Westphal, the nucleus cuneiformis and the superior colliculus. Slightly higher ‘H-SBS concentrations were found in the stratum griseum superficial than in the stratum griseum intermediale and profundum. Very low amounts of grains were present over the nucleus ruber (pars magnocellularis and pars parvocellularis), area tegmentalis ventralis and the corpus geniculatum mediale. The substantia nigra was characterized by low densities of binding sites with a slightly heterogeneous distribution of grains over the pars compacta and reticulata. Relatively high levels of non-specific [‘HIstrychnine binding were observed over the cell bodies of the substantia nigra compacta. The medial longitudinal fasciculus and the central tegmental tract were associated with 3H-SBS densities not different from the background. In the caudal midbrain (Figs 2 and 9) intermediate to low concentrations of silver grains were computed in the periaqueductal grey matter including the area occupied by the nucleus raphe dorsalis pars
31
.4. P~oesr (‘I al.
supratrochlearis and the nucleus trochlearis. On the contrary, high concentrations of grains were found over the peripheral aspects of the dorsal and lateral periaqueductal grey matter. although lower concentrations of grains were found just around the aqueduct. Low to very low concentrations of silver grains were noted over other grey structures of the midbrain tegmentum including the nucleus paranigralis, nucleus interpeduncularis, nucleus tegmcntalis pedunculopontinus (pars dissipata and compacta), nucleus cuneiformis and nucleus raphe centralis. Similar concentrations of ‘H-SBS were found over the inferior colliculus, however. higher densities were measured over its dorsal cortex. The most prominently labeled nuclei in the pons (Figs 3, 4. IO, I I and 12) were the motor nucleus of the trigeminal nerve and the nucleus of the facial nerve. Intermediate concentrations of ‘H-SBS were found in the sensory trigeminal nucleus (principal nucleus). However, high to very high concentrations of binding sites were found in irregularly shaped spots distributed over this nucleus. Intermediate concentrations of binding sites characterized the lateral reticular formation. Low to very low degrees of binding were found over the remaining pontine grey matter including the nucleus reticularis pontis caudalis and oralis, nucleus reticularis tegmenti pontis, raphe and parabrachial nuclei. Only negligible amounts of grains were found over the nuclei pontis and nucleus lemnisci lateralis. Low densities of ‘H-SBS characterized the locus coeruleus, which contained high levels of non-specific binding. As in the pons. the distribution of autoradiographic grains in the medulla oblongata (Figs 4, 5, 6, 13, 14, I5 and 16) was highly localized. High to very high concentrations of ‘H-SBS were found over the spinal trigeminal nucleus. This nucleus showed a definite increase of receptor sites from its rostra1 to its caudal portions. Thus, its pars oralis contained high concentrations whereas very high concentrations of silver grains were observed in the pars interpolaris and caudalis of this nucleus. The substantia gelatinosa of the pars caudalis contained the highest degree of receptor labeling found in the human brainstem and upper spinal cord, presenting an irregular distribution with patches of a higher density of silver grains. The nucleus proprius was associated with lower densities, however, very high concentrations of receptor sites were probably present in its outer parts. Also very high levels of (‘HIstrychnine binding were observed in the nucleus of the XIIth nerve. Spots of very high silver grain density were also seen in the nucleus cuneatus medialis at the level of the pyramidal decussation. These spots were very irregular in shape and are likely to correspond to the known clustering of neurons in this nucleus.32 Only medium to low concentration of receptor sites were found between these high density spots. A somewhat
spotty distribution of ‘H-SBS was also obserlcd 111 the nucleus gracilis. However, grain concentrations in this nucleus were lower than in the cuncate nucleus. High concentrations of grains characterized the nucleus ambiguus and the nucleus solitarius. The distribution of silver grains in the nucleus solitarius was irregular with highest concentrations found surrounding the tractus solitarius. Intermediate levels of ‘H-SBS were found in the dorsal cochlcar nuclei. nucleus praepositus hypoglossi and nucleus vcstibularis medialis. Medium to low grain densities wcrc associated to the nucleus cuneatus lateralis, nucleus medullae oblongatae ccntralis, the funicular nuclei and the nucleus intercalatus. The inferior olivary complex and nucleus vcstibularis inferior showed very low levels of receptor binding. In the upper cervical cord (Fig. 17) very high densities of ‘H-SBS were observed in the substantia gelatinosa. However. the levels of binding in this region were markedly lower than those measured more rostrally in the substantia gelatinosa of the trigeminal nucleus. In the ventral horn high densities of GR were found in the motor neurons. Intermediate levels of specific binding were associated with other structures including nucleus proprius. processus reticularis and substantia intermedia. The rat brain was examined in parallel to human tissues. The distribution of ‘H-SBS in the rat brain is illustrated in Fig. I8 and quantitative values are given in Table 2 for the sake of comparison with values obtained in human brain samples, Densities in the rat brain were much higher than those found in humans. with the highest levels of ‘H-SBS being observed in the substantia gelatinosa of the cervical cord, spinal nucleus of the trigeminal nerve (substantia gelatinosa and nucleus proprius) and nucleus nervi hypoglossi. Intermediate densities were seen in the medial vestibular nucleus. The reticular formation, central grey and superficial grey layer of the superior COIliculus contained rather low densities of GR. Finally, very low densities were detected over the inferior colliculus, pontine nuclei and hypothalamus. Table 2. Specific [‘HIstrychnine binding in the rat brain Brain area Colliculus superior stratum griseum superficiale stratum griseum intermediale Colliculus inferior Grkeum centrale mesenceohali
Nuclei pontis N UC Ieus reticularis pontis oralis Nucleus reticularis pontis caudalis Nucleus reticularis gigantocellularis Nucleus vestibularis medialis Nucleus nervi hypoglossi Nucleus spinalis nervi trigemini substantia gclatinosa nucleus proprius Cervical cord, substantia gelatinosa
fmolmgprotein + SEM 240.9 f 16.7 138.1 + 16.3 63.8 f 4.2 279.3 t 29.8
72.2 + 1.7 250.6 2 15.4 319.0 * 31.5
267.0 f 5.2 450.2 + 15.7 955.7 + 37.6 988.9 + 12.5 922.1 + 28.6 825.1 f 17.6
Data were obtained from two different animals
’
Glycine receptor autoradiography in human brain DISCUSSION
This report presents an atlas of the distribution of high affinity glycine binding sites labeled with the antagonist [3H]strychnine in the brain of individuals without evidence of neurological or psychiatric disorders. High concentrations of ‘H-SBS were present in several nuclei and areas of the brainstem including motor and sensory trigeminal nuclei, nucleus cuneatus, as well as the facial and hypoglossal nuclei. The highest concentrations of 3H-SBS were found in the grey matter of the upper cervical cord and medulla oblongata where the most prominently labeled structure was the substantia gelatinosa of the trigeminal nucleus. The overall distribution of glycine receptors observed in this study is in agreement with previous results obtained in human and experimental animal CNS by microdissection techniques26*48and in the rat and mouse by light microscopic autoradiographic techniques.38s5’ Like in these studies, we observed that the distribution of ‘H-SBS was highly localized and a decrease of receptor concentration was found from the spinal cord level to the upper brainstem and forebrain. Grey structures of the forebrain, including the thalamus, the striatum and the cerebral cortex, were almost devoid of receptor sites. This caudalrostra1 gradient of GR concentrations is in agreement with previous studies showing decrement in a rostra1 progression of the relative number of glycinergic synapses as defined neurophysiologically,5~8~11,15,16,17~18,23,24,28~4~~47 by differential
distribution
or a high affinity glycine uptake mechof glycine,2*3*6 anism 21.25,27,36,39 Differences were seen between the rat and the human brain. In identical incubation conditions the densities of 3H-SBS were two to three-fold higher in the rat than in the human brain, These data are in good agreement with the results obtained by De Montis et ~1.‘~ using classical membrane binding assays. These authors reported maximal densities of more than 2000 fmol mg protein-’ in the rat spinal cord, while the total number of ‘H-SBS in the human spinal cord was about 600 fmol mg protein-‘. In addition, the rank order of densities of ‘H-SBS in different nuclei of the rat and human brains was not completely identical. For instance we found a different distribution of 3H-SBS in the midbrain, the highest concentrations being observed in the periaqueductal grey matter and the oculomotor nuclei, whereas in the rat highest concentrations have been noted in the nucleus cuneiformis5’ In the human pons, the relative concentrations of 3H-SBS were significantly higher than those noted in the rat. Higher concentrations of binding sites were also noted in the human tractus solitarius and in the nucleus ambiguus. Detailed studies of the distribution of GR in the human brain have limited themselves to the spinal cord because of the relevance of glycinergic mech-
33
anisms in spinal motoneuron functions in human diseases, particularly in amyotrophic lateral sclerosis and spasticity.igJO” High concentrations of GR have been found by membrane binding studies in the grey matter of human control spinal cord with values slightly higher in the ventral than in the dorsal half of the grey matter. 20In a recent study by Whitehouse et al.& high concentrations of 3H-SBS were found in the grey matter of the cervical spinal cord of control cases by light microscopic autoradiographic techniques. Like these authors we found higher receptor concentrations in laminae II (substantia gelatinosa) and III of Rexed and in ventral horn in layer IX, which contains motoneurons. This distribution of 3H-SBS in human anterior horn appears to be different from that of the rat spinal cords1 where the highest concentrations of grains were found in lamina VII of Rexed, whereas receptor concentrations were much lower in the lamina IX. Glycine has been shown to play a major role in postsynaptic inhibition in the spinal cord where it is released mainly by segmental intemeurons, including the Renshaw cells, responsible for recurrent inhibition of motor neurons and I-a afferent interneurons.2,4,5*11,24Furthermore, putative glycinergic nerve terminals have been mapped in the ventral horn of the rat by localizing glycine uptake sites.27*36 Uptake sites were noted in the neuropil and around the cell bodies of motor neurons in a distribution very similar to that of GR in the ventral horn of rat5’ and human.46 In the report of Whitehouse et al.,& decrement of muscarinic, benzodiazepine and glycine receptors was shown to occur in spinal grey matter of patients with amyotrophic lateral sclerosis. The major loss of GR was observed in lamina IX and was highly correlated with the degree of neuronal loss in the same region, suggesting that the majority of the receptors are localized on motor neurons. There is only scanty information on the distribution of GR in supraspinal grey structures in human. In one study, 26 3H-SBS distribution has been compared with the distribution of [3H]y-aminobutyrate binding sites in macroscopically dissected brain regions. Whereas y-aminobutyrate receptors were found to be predominant in the forebrain and upper brainstem, GR were more localized in the lower brainstem and in the spinal cord. However, both receptors were colocalized in some structures like the substantia nigra and the superior colliculus. Surprisingly, significant, although low amounts of [3H]strychnine binding were found in the cerebral cortex, a result which contrasts with the absence of significant amounts of 3H-SBS in autoradiographic studies in animal? and in human (results in this paper). In a recent study using membrane preparations of the brain of non-neurological patients, high and low affinity sites for [‘HIstrychnine were found in the spinal cord and pars compacta of the substantia nigra.i4 High levels of [3H]strychnine binding with
34
A.
PRORST CI cd
similar values were found in both regions. [‘HIStrychnine binding was significantly reduced in the substantia nigra of Parkinson’s disease patients, both in pars compacta and reticulata.“’ In our studies.
basis of high ‘H-SBS concentrations in loci of the brainstem and spinal cord of the rat can now hc explained in the basis of GR localization in the human brain. For instance, visual or acoustic stimuh
where only high affinity binding sites are visualized. we have detected low densities of ‘H-SBS in the substantia nigra, which are of the same order of magnitude as those reported by DeMontis et ~1.‘~ The distribution of GR as shown by in rirro autoradiography in the rat and in human parallels that observed for the neurophysiological responses after local application of glycine.8,“,‘5 ‘8242840.47 Correlations between loci of high ‘H-SBS density and electrophysiologic data as well as physiologic functions associated with GR have been extensively dis-
induced convulsions in humans suffering from strychnine toxicity have been tentatively explained by high SBS densities in the superior colliculus. In this structure we found ‘H-SBS in amounts comparable to those found in the rat. Also the very high levels 01 SBS in the sensory and motor portions of the trigeminal nuclei and in the facial nuclei in human suggest these arcas as a likely anatomical locus for strychnine induced hyperreflexia, muscle stiffness and spasm in masticatory and facial muscles.
cussed by Zarbin et al.” In particular, some clinical effects of strychnine which were explained on t&
Acknowledgemenr-- We are grateful to Dr R. Foote for critical reading of the manuscript.
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