Regional distribution of muscarinic cholinergic receptors in rat brain

Brain Research, 154 (1978) 13-23 © Elsevier/North-Holland BiomedicalPress

13

REGIONAL DISTRIBUTION OF MUSCARINIC CHOLINERGIC RECEPTORS IN RAT BRAIN

R O N A L D M. KOBAYASHI, MIKLOS PALKOVITS, ROBERT E. HRUSKA, R I C H A R D ROTHSCHILD and H E N R Y I. Y A M A M U R A

Veterans Administration Hospital, 3350 La Jolla Village Drive, San Diego, Calif. 92161 and Department of Neurosciences, University o f California, San Diego, Calif. ( U. S..4.); (M.P.) First Department of Anatomy, Semmelweis Medical University, 1450 Budapest, Tuzolto-u 58 (Hungary) and (R.E.H. and H.I.Y.) Department of Pharmacology, College of Medicine, University of Arizona Health Sciences Center, Tucson, Ariz. 85724 (U.S.A.) (Accepted January 26th, 1978)

SUMMARY

Using the tritiated ligand [aH]quinuclidinyl benzilate ([3H]QNB), a specific muscarinic cholinergic antagonist, the distribution of muscarinic receptors was determined in 75 specific brain regions and nuclei removed by microdissection as well as in large brain areas. [3H]QNB binding was highest in the caudate-putamen; other regions with high binding included the nucleus accumbens, cerebral cortex and hippocampus, certain limbic and thalamic nuclei, superior colliculus, pontine nuclei and the hypoglossal nucleus. Muscarinic cholinergic receptors may be concentrated in these regions. [aH]QNB binding generally correlated well with acetylcholinesterase staining in terminals and is consistent with certain sites as particularly enriched in cholinergic synapses.

INTRODUCTION

A variety of biochemical and histochemical techniques have been utilized to investigate brain neuronal systems in which acetylcholine is the neurotransmitter. These biochemical markers include acetylcholine, (ACh)2, 6 choline acetyltransferase (CAT)l,2, 5,14 and high affinity choline uptakeT, s while acetylcholinesterase (ACHE) has been extensively investigated by histochemistrya,10,1a,16,17. Recently specific muscarinic cholinergic receptors in rat brain homogenates have been identified using [3H]quinuclidinyl benzilate ([aH]QNB), a potent muscarinic antagonist19, 20. In this study, the distribution of [aH]QNB binding was determined in 75 discrete nuclei and regions of the rat brain removed by microdissectionl,5,12,14.

14 MATERIAL AND METHODS Male adult Sprague-Dawley rats weighing 200-225 g were housed under diurnal lighting conditions and allowed free access to standard laboratory chow and water ad libitum. After decapitation, the brain was rapidly removed. In the initial study, the brain was divided into larger regions% The samples were placed in 0.05 M ice-cold sodium-potassium phosphate buffer (pH 7.4) in microtubes and homogenized with a cell disrupter (Kontes Instrument Co.). In subsequent microdissection studies, the brain was frozen on dry-ice and 300 #m coronal sections were cut in a microtomecryostat at --10 °C. Specific regions and nuclei were removed with special hollow needles or a microknife under a stereomicroscope as described 1,5,12,14. Samples were placed in 150 #1 of ice-cold 0.05 M sodium-potassium phosphate buffer, pH 7.4, and homogenized with a cell disrupter. Each sample consisted of similar regions and nuclei pooled from 3 rats. Muscarinic receptor binding was determined using the specific ligand [3H]QNB by a micromodification of the previously described method19, 2°. Twenty-five #1 of homogenate was incubated in a final volume of 200 #1 of the 0.05 M phosphate buffer, containing 1.35 n M [3H]QNB (13 Ci/mmole). A parallel set of tubes to determine non-specific binding contained the above plus 1 # M atropine. All samples were assayed in duplicate. After incubation at 37 °C for 1 h, the reaction was terminated by rapid filtration under vacuum through Whatman GF/B filters. The incubation tubes were twice washed with 5 ml of the ice-cold phosphate buffer. The filters were placed in vials containing 8 ml of Triton X-100-toluene phosphor for approximately 24 h and radioactivity measured by liquid scintillation spectrometry at 4 5 ~ efficiency. Specific [3H]QNB binding was calculated as total binding minus binding in the presence of atropine (non-specific binding). In the area showing the lowest specific binding, i.e. the cerebellar nuclei, the specific binding was 30 ~ of the non-specific binding. The caudate-putamen had the highest amount of specific binding which was 600 ~ of the non-specific binding. Therefore, the ratio of specific to nonTABLE I Distribution o f muscarinic receptors in rat brain

Data are means 4- S.E.M. (n) expressed as pmol [SH]QNBspecificallybound/g protein. Rank order Area

[3H] Q N B binding

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

603 4- 16 (23) 518 i 17 (24) 508 i 21 (23) 310 4- 22 (6) 269 4- 23 (3) 250 ± 27 (6) 248 4- 62 (3) 170 4- 20 (10) 157 4- 15 (3) 79 4- 4 (10) 51 4- 6(9)

Caudate-putamen Cerebral cortex Hippocampus Amygdala, posterior Thalamus Amygdala, anterior Pons Hypothalamus Medulla Cervical spinal cord Cerebellum

15 TABLE II

Distribution of muscarinic cholinergic receptors in rat brain Each value is the mean 4- S.E.M. (n) expressed as specific [aH]QNB bound (pmol/g protein).

Region

Punch No. (Figs. 1-5)

[3H] Q NB binding

No. of Samples

(1) Forebrain Nucleus accumbens Caudate-putamen Globus pallidus Cortex Cingulate cortex Hippocampus Parietal cortex Temporal cortex Entorhinal cortex Limbic Bulbus olfactorius Tuberculum olfactorium Tractus olfactorius lateralis Nucleus tractus diagonalis Nucleus interstitialis stria terminalis dorsalis Nucleus interstitialis stria terminalis ventralis Nucleus septi lateralis Nucleus septi medialis Nucleus habenularis medialis Nucleus habenularis lateralis Amygdaloid nuclei Nucleus amygdaloideus corticalis Nucleus amygdaloideus lateralis Nucleus amygdaloideus medialis Nucleus amygdaloideus basalis Nucleus amygdaloideus centralis Thalamus Nucleus ventralis thalami Nucleus paraventricularis thalami Nucleus anterior ventralis thalami Nucleus anterior medialis thalami Nucleus medialis thalami Nucleus lateralis thalami Nucleus posterior thalami Hypothalamus Nucleus preopticus medialis Nucleus suprachiasmatis Nucleus periventricularis Nucleus supraopticus Medial forebrain bundle (rostral) Nucleus hypothalamicus anterior Nucleus paraventricularis Median eminence Nucleus arcuatus Nucleus ventromedialis Nucleus dorsomedialis

(ac) (cp) (gp)

1 2 3

503 q- 37* 625 4- 43" 110 q- 15

6 6 6

(Ci) (Hi) (Par) (Temp) (Ent)

4 5 6 7 8

384 387 493 492 461

44444-

23" 19" 41 * 18" 18"

6 6 5 4

(TU) (TOL) (td)

9 10 11

416 484 133 181

44± 4-

17" 50* 19 18

9 6 6 6

(std)

12

259 4- 45

6

(stv) (sl) (sm) (hm) (hi)

13 14 15 16 17

104 210 199 87 160

4- 18 4- 14 ± 17 4- 10 -4- 10

6 6 6 6 6

(aco) (al) (am) (ab) (ace)

18 19 20 21 22

197 341 180 185 200

44444-

30 26* 6 28 8

6 6 6 6

(tv) (tpv) (tav) (tam) (tm) (tl) (tp)

23 24 25 26 27 28 29

196 272 316 267 279 320 300

4444444-

18 24 54* 18 8 28* 33

4 4 4 4 4 4 4

216 -4- 30 217 q- 54 180 4- 26 204 4- 28 247 4- 26 230 ± 22 168 ± 18 216 4- 55 158 4- 45 172 4- 38 260 4- 106

5 3 5 5 5 6 5 5 5 6 4

(pom) 30 (rise) 31 (ripe) 32 (nso) 33 (MFB) 34 (nha) 35 (npv) 36 (me) 37 (ha) 38 (nvm) 39 (ndm) 40

5

16 Region

Nucleus hypothalamicus posterior Mamillary nuclei Medial forebrain bundle (caudal)

Punch No. (Figs. 1 5)

/:~H)QNB binding

No. of Samples

(nhp) (ran) (MFB)

41 42 43

280 ± 60 221 ± 14 194 -_L 32

5 5 5

(cgm) (cgl) (rn) (sn) (VTA) (ipn) (CS) (CI) (scg)

44 45 46 47 48 49 50 51 52

232 171 139 116 130 160 384 210 201

8 12 8 7 12 12 17" 25 7

6 6 6 6 6 6 6 6 6

(po) (ntd) (lc)

53 54 55

490 5- 37* 186 5- 13 178 ± 8

6 6 6

(rd) (rm)

56 57

116 ± 11 118 5- 25

6 6

(co) (tb) (so) (11) (io)

58 59 60 61 62

58 90 61 84 110

± ± ± ± 5-

8 13 3 12 5

6 6 6 6 6

(nV) (nVII) (amb) (XII) (ntV) (nts) (vl)

63 64 65 66 67 68 69

241 274 131 365 159 113 65

5555± ± ±

16 19 11 27* 14 27 8

6 6 6 6 6 6 6

(rp) (rgi) (rmo)

70 71 72

139 5- 9 186 i 28 173 5- 23

6 6 6

(CC) (Cn)

73 74

55 ± 7 41 ~ 6

6 6

(11) Hindbrain Mesencephalon Corpus geniculatum mediale Corpus geniculatum laterale Nucleus ruber Substantia nigra Ventral tegemental area Nucleus interpeduncularis Colliculus superior Colliculus inferior Substantia grisea centralis Pons Nuclei pontis Nucleus tegmenti dorsalis Locus coeruleus Raphe nuclei Nucleus raphe dorsalis Nucleus raphe magnus Acoustic system Nuclei cochleares Trapezoid body Superior olive Nucleus lemnisci lateralis Inferior olive Cranial nerve nuclei Nucleus originis nervi trigemini (V) Nucleus orginis nervi facialis (VII) Nucleus ambiguus Nucleus motorius nervi hypoglossi (XII) Nucleus tractus spinalis nervi trigemini (V) Nucleus tractus solitarius Nucleus vestibularis lateralis Reticular formation Nucleus reticularis pontis Nucleus reticularis gigantocellularis Nucleus reticularis medulla oblongata Cerebellum Cerebellar cortex Cerebellar nuclei

± 555± ± 5± 5-

* Indicates high binding (greater than 50 ~ of binding observed in the highest region which was the caudate-putamen).

specific binding varied from 0.3:1.0 to 6.0:1.0. For each region, non-specific binding remained constant and only the specific binding varied. In most areas the ratio was at least 2.0:1.0. There was no regional variation of KD in 8 different brain regions examined, with a range of 50-100 pM. Protein in the homogenate was determined by a micromodification of the method of Lowry et al. 11. The results are expressed as picomoles of [aH]QNB specifically bound per g protein.

17

)

cA( \

CO

0

3 mm

Figs. 1-5. Schematic frontal sections of the rat brain. The left half identifies the structures. The circles on the right half indicate the location and diameter of the tissue sample; the number is the 'punch number' identified in Table II. Abbreviations are indicated in Table II. Additional abbreviations are: CA, commissura anterior; CAI, capsula interna; CC, crus cerebri; F, columna fornicis; FLM, fasciculus longitudinalis medialis; FMT, fasciculus mamillothalamicus; FR, fasciculus retroflexus; GCC, genu corporis callosi; LM, leminiscus medialis; P, tractus corticospinalis; PCI, pedunculuscerebellaris inferior; PCS, pedunculus cerebellaris superior; SM, stria medullaris thalami; TS, tractus solitarius; TSV, tractus spinalis nervi trigemini; co, chiasma opticum; vm, nucleus vestibularis medialis.

18 SM

m,p

I

0

I

I

I

3ram

Fig. 2. RESULTS Muscarinic cholinergic receptor distribution in larger regions of rat brain is shown in Table I. Highest binding occurred in the caudate-putamen, cerebral cortex and hippocampus, while lowest binding was observed in the cerebellum and cervical spinal cord. The range from lowest to highest binding was 12-fotd. [aH]QNB binding measured in 75 specific nuclei and regions of the rat removed

19

VTA (48)

P

0

5 mm

Fig. 3.

by microdissection is summarized in Table II and Figs. 1-5. The highest binding was again in the caudate-putamen while binding in the adjacent globus pallidus was 6-fold lower. Cerebral cortical regions were uniformly high, including cingulate, parietal, temporal and entorhinal cortices and hippocampus. As a group, limbic regions exhibited high density of receptors, especially the nucleus accumbens ~which was second only to the caudate-putamen), olfactory tubercle and olfactory bulb. Among thalamic nuclei, the lateral and anterior ventral nuclei were higher while the others were intermediate in binding. Hypothalamic nuclei were low to intermediate in binding. The medial habenular nucleus and the interpeduncular nucleus, considered to be an important cholinergic pathway, were both low. The lowest binding was observed in the cerebellar cortex and nuclei, which were less than 10 ~o of the caudate-putamen. Among brain stem regions, high binding was present in the superior colliculus, pontine nuclei and motor nucleus of the hypoglossal nerve.

20

p

.~

~.n

~

~

7

74

4

0

FLM

~"~1.~ /7/~'"

TSV nVll

\~If~,,\\ ~\

~>Jf--~\ ..~,_~./b{s4)~" "~~ ~ j ~ ~ p

TS

nVII

57

LM

nts

FLM

64

v ~ ~ LM P 0

rm 1571 3 mm

Fig. 4. DISCUSSION

By combining a micromodification of the radioreceptor assay for muscarinic receptors with a microdissection method, it was possible to determine the density of muscarinic receptors in specific nuclei and regions of the rat brain. Our results are similar to the regional distribution in the rhesus monkey which also measured [aH]QNB bindingis. Cholinergic muscarinic receptors examined by light microscopic

21 .68

"IS nts FLM

TSV

65

PC! arab

62 P

nts

Xll / - 6 8

,66

TSV

amb

io

p nts

62 Xll

68

66

TSV

ntV amb

/

,

~rmo

io

~

•

65

p

I

0

I

I

I

5 mm

Fig. 5.

radioautography after [aH]QNB injection9 also correspond directly with the present observations. Both techniques demonstrated high [aH]QNB binding in the hippocampus, caudate-putamen, nucleus accumbens and cerebral cortex and low to intermediate binding in the septum, interpeduncular nucleus, habenular nuclei and medial thalamus 9. Both ACh and muscarinic receptors are regarded as being concentrated in sites of cholinergic nerve terminals. Certain notable correlations exist for these two markers. Both are high in the nucleus accumbens and caudate-putamen and both are low in the globus pallidus2. The nucleus interpeduncularis is high in ACh 2 but, in contrast, is low in muscarinic receptors and this suggests the possibility that this nucleus may be predominately nicotinic. Since similar dissection techniques were employed previously to determine the

22 distribution of CAT 1,2,5,14, the regional distribution of [3H]QNB and CAT may be compared. Positive correlations, with high levels of both markers was present in the caudate-putamen2,5, lateral amygdaloid nucleus 14 and the hypoglossal nucleus~ while low levels of both were measured in the globus pallidus, cerebellar cortex and nuclei, substantia nigra and acoustic system2,5. In contrast, high [3H]QNB binding with low CAT activity has been found in cerebral cortical regions, hippocampus and nucleus accumbens 14. Furthermore, low [aH]QNB binding in the interpeduncular nucleus and medial habenular nucleus does not correspond to high CAT activity in these regions'~,14. This disparity is particularly striking for the interpeduncular nucleus, in which the highest brain levels of CAT have been identified z,14. While CAT is regarded as a valid marker of cholinergic neurons, it may more closely identify cholinergic cell bodies 2. There appears to be a strong correlation when [3H]QNB binding is compared to AChE-containing terminals (as contrasted to AChE cell bodies). High QNB binding obtained in the present studies and histochemically rich AChE in terminals has been reported for the caudate-putamen, nucleus accumbens, olfactory tubercle, thalamic anterior ventral nucleus, lateral amygdaloid nucleus, superior colliculus and the hypoglossal nucleus4,10,13,16,17. Cerebral cortical and hippocampal regions are rich in both muscarinic receptors and in AChE 16. In contrast to AChE-rich terminal sites, cell bodies rich in AChE 4,1a,16 exhibit only low to intermediate QNB binding. Interestingly, regions low to intermediate in QNB binding were also low to intermediate in AChE terminal staining. Notable exceptions to positive correlation are the substantia nigra, interpeduncular nucleus and ventral tegmental area which are rich in AChE terminals4,10,1a but are low in QNB binding. Cranial nerve motor nuclei contain high AChE staining in cells and terminals la and high CAT activity5 but only an intermediate density of muscarinic receptors, with the exception of the hypoglossal nucleus (Table !I). Of salient interest is that peripheral section of the hypoglossal nerve reduced by 50 ~o both binding of the muscarinic ligand [aH]propylbenzilylcholine mustard and the number of synapses visualized by electron microscopy in the hypoglossal nucleus 1'~. This lesion approach is consistent with cholinergic terminals being characterized by high muscarinic receptor density. Abundant [3H]QNB binding in certain regions correspond to intense AChE staining in terminals and is compatible with the cholinergic projection systems as previously suggested 4,1°,ta,~6AT. Thus, cholinergic synapses appear to be associated with both a high density of [3H]QNB binding and with intensity of AChE staining in terminals. ACKNOWLEDGEMENTS We greatly appreciate the technical assistance of Patricia Kraus and Steven Painter and the typing of the manuscript by Dea Pechnick. R. M. Kobayashi is supported by a Clinical Investigator Award of the Veterans Administration and MH 26072. H. I. Yamamura is the recipient of a Research Scientist Development Award MH 00095 and MH 27257. R. E. Hruska is supported by USPHS Post-Doctoral

23 Fellowship NS-05585. R. Rothschild was supported by the University of California President's Fellowship Program and is presently a student at Harvard Medical School.

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