- Email: [email protected]
Distribution of neurotensin receptors in the primate hippocampal region: a quantitative autoradiographic study in the monkey and the postmortem human brain
\ezlt'os4'icll, ~' l,vllvr.s, 76 (1987) 145 150 Else\ier Scientific Publishers Ireland Lid.
145
NSL I)455S
Distribution of neurotensin receptors in the primate hippocampal region: a quantitative autoradiographic study in the monkey and the postmortem human brain Christer K6hler 1, Ann-Cathrin Rades~iter t and Victoria C han-Palay" ' I)~'pIIFIIH~'III
l*l
.\~'ltVOl)/ltlrDl~l~'O/Ok" ', ,4.~lrll ,l[~d, ,.I B, ,57;d~'rl~'ilj~' , .S'u~'d~,n ; a~ l :Nez rolo~,. (niw'r~ity
' hl,~'ll'lttl~',
tt,~s'pila/, Z z i r i c h f' , S ' w i l z c r h m d ;
(l~.eceixcd 27 ()ctobel- 1987; Revised \ersion rcceixed 16 December 1986: Accepted 2(I January, 1987) K e y ir+.'d~
t lippocampus: Entorhinal :.ll'e,:i; Neurotcnsila; Receptor; a.utorad ograph}; IVhmkc); t lu man; Brain
The distribution of [~H]neurolensin (plI]NT) binding sites in the morlkey and the postmortem human brain ~as studied b~ using quantitati,,c in xitro receptor auloradiography. Biochemical expel'in,thiS car-
fled out on lisstlc sections of the monkey hippocampus sho,aed that the binding of [~H]NT ~as stltur:.lblc. rc\crsible and of high spccilici D. The hippocampal ptt]NT binding was displaced by fragmenl NT, t: but not fragment NT~ . of the pcptide. The anatomical analysis showed a highly heterogeneous dislribution of [ flINT binding sites within both the monkey and the htmlan hippocampal region. In both species the highest densitx of [~It]NT binding sites ~r~ts found in the presubiculum (rank order of binding density: laNer 2 > 6 > I > 3. 4. 5 m both monkey and man) and Ihc cntorhina[ area (monkeN: ]a~er 4 > 6 > 5 :> I > 2 > 3: htlnltln: layer I - 2 > 5 > 3). The subiculum and Allllnon's horl] \~/el*Crclatixcly poor in [:II]NT binding sites in both species. In lhc area dentata the highest density o f l ~ l l ] N l binding sites x~;ts fotmd in lhe hilar region.
Neuroanatomical findings have provided evidence that different neuropeptides are located in cells and afferent projections innervating the hippocampal region [13, 17], and, thus. suggest that several of these substances may participate in the regulation of ncurotransmission along the intrinsic hippocampal pathways. Neurotensin (NT), a 13-amino acid peptide isolated from bovine hypothalamic extracts [2 4, 15], has been identilicd within the hippocampal region of the rodent and primate brain [4 N, 14]. -['he hippocampus contains binding sites t\w [~H]NT which show pharmacological characteristics of a biologically' relevant receptor [10, I1]. By application of in vitro receptor autoradiography, the NT receptors have been shown to be heterogeneously distributed within the rat hippocarnpal region [11, 16]. While the anatomical ('~,';'vspomh';tce. C. K6hler, Department of Neuropharmacolog.v. Astra Alab AB, S-151 85 S,3dertiilie. S~cdcn. 0304 3940 87 S 03.50 (c) 1987 Elsevier Scientific Publishers Ireland Ltd.
146
localization of NT receptors has been studied in detail in the rat brain [l I, 16] Icss is known about the localization of NT binding sites in the primate brain (lk)r the human brain sec refs. II and 18). In the present study we have analyzed thc anatomicai distribution of [-~H]NT binding sites within the hippocampal region o1 the monke\ and the postmortem human brain. The brains from two male Cynomologus monkeys (SBL, Stockholm, Sweden: weight approx. 4 kg) and 5 temporal lobes obtained at autopsy (postmortem interwd: 8 21 h) from patients who died without any diagnosed neurological illness. The brains were cut frozen in a cryostat ( - 15 C) and the sections (20 ira1 thick) were incubated with [~H]NT (New England Nuclear, spec. act. 67.6 Ci.mmol i) in 0.17 M Tris-HCl buffer containing 0.05% boiled bovine serum albumin (Sigma, St. Louis, U.S.A.) and 0.1 mM bacitracin (Sigma, St. Louis, U.S.A.) as previously described [11]. The sections were rinsed, dried in a stream of cold air and put in contact with -~H-sensitive fihn (Ultrofilm, LKB, Sweden). The autoradiograms were analyzed by using an IBAS 2000 image analyzer (Zeiss Kontron, F.R.G.) equipped with a program which converted optical densities to t)nol.mg i tissue using calibrated plastic standards (Amersham) co-exposed with the tissue sections. In a separate series o1" experiments the binding of different concentrations of [~H]NT to sections of the monkey hippocampal region was analyzed by scintillation spectroscopy after the sections had been scraped off'the glass slides [I I], The biochemical studies showed that [~H]NT bound in a saturable manner to sections of the monkey hippocampal region. The non-specific binding contributed less than 20% of the total binding at concentrations around the Kd value (I.5 nM). The [3HINT binding was dose dependently blocked by fragment 8 13 of the peptide (1C50,-~ 70 nM) while fragment I-8 was inactive at concentrations up to I0/zM (Fig. 1A.B).
A
B
1000
100
o
o
o
o :5 t~
®
100
l _ ~ l J J l [ l l l l 1
5
t 10
Con¢.3H-Neutoten~n(nM)
J
i
I
15
I
I
-9
-
18
-
17
I
-6
-
15
- l o g displacer (M)
Vig. I. Saturation analysis o f t t HINT binding to tissue sections (20 l*m thick) of the monkey hippocampal region. The tissue sections were obtained at a level corresponding to that shown in Fig. 2B. As can be seen the binding is saturable and it shows a low degree of non-specific binding throughout the (lose response range tested. The dose-dependent blockade by fragment 8 13 of the NT molecule is shown in the right panel. Fragment NT~ ~ is inactive in displacing [3H]NT binding to sections of the monkey hippocampus up to 10 ltM. Similar results were obtained in lwo separate experiments.
147
Fig. 2. Photon~icrogral~hs el" auloradiograms showing the distribution of ptt]NT (4 nMI binding sitcx m the m~mkc> I \,B.(') and human [D.E) hippocampal region. A and (" urc dark-licld pholtm'dcrogiaphs (v, hilc ille:t> i'el')lcscnl [~II]NT binding-sitcsL Photomicrographs of compulcr-gencralcd images of the ]:II]NT autoradiogt-ums arc sho~n in IL D. und [!. The urrows in A delimit an at'ca I[l:tl ellcotllpgtsses the prcsubicuhnl~ and p~.|l'I C)I"the hippocampal-am,,gdaloid-transition area. l)oublc arrowheads sh(~\~ the border bcl,,~ccn lhc subiculum and rcgio superior. Single arrowhead marks the laleral border of lilt onto rhiual :lrea. l~hotomicrograph in B is a high per, or ~icw of the photomicrograph in (" shm~mg the exact latmin:lr distribution of the pH]NT binding sites. P,indmg silos l\tr [zlI]NT in Ihe human hippocampal region arc sh¢~x\n in D and E. Thc laminar distribution o1" the cnlorhinal [StlINT binding sites is sho~n in I{. ,,\trox~s mark Ihe borders bclx,,cen individual sublields ~1" the hippocampal region, ah. nn,.g,uklr bundle: I!A. eiHorhinal area: l l. hilus; inl, molccular layer" PRI{. prcsubiculum: PS. parusubiculum: RI. regio inferior: P,S, regio superior: SUB. subiculum: SN<. subslantia nigra, pars compacla: SN,. subst~lntia nigra, pars Ieticu]aln. The layers of lhc prcsubiculum and el" EA are labelled I through (~
Tile autoradiographic analysis revealed a heterogeneous distribution of the [-~H]NT binding sites within both the monkey and the human hippocampal region. Thus~ in both species most of tile [3HINT binding is present in the retrohippocampal
148 TABLE I T H E SPECIFIC B I N D I N G ( f m o l . m g *) OF [3HINT {4 nM) IN D I F F E R E N T A R E A S A N D LAM1NA OF T H E M O N K E Y A N D T H E P O S T M O R T E M H U M A N H I P P O C A M P A L R E G I O N AS DETERM I N E D BY Q U A N T I T A T I V E R E C E P T O R A U T O R A D I O G R A P H Y Specilic binding was defined by 1 I~M N ] l i~. The values are the mean ( ± S . E . M . ) of 6 determinations in each monkey and h u m a n brain. While the pH]NT binding showed the same pattern in the hippocampi from all 5 cases, quantitative analyses were made onl> in the 3 cases most similar with regard lo age and postmortem intervals. The vahlcs were not corrected for quenching. N.D., not determined. Anatomical region
Entorhinal area I_ayer I 3 4 5
Parasubiculmn Presubiculum Layer I 2 35 6
[~H]Neurotensin specitically bound (finol.mg 'l Monke',
Human
13.88± 2.84 10.62+ 2.43 7.74_+ 0.75 43.06 _+ 12.00 20.0427 3.97 34.98_+ 5.3[
243.73± 244.2{)± 46.53+_ N.D. 54.10__+
3.01 6.47 1.84 2.80
N,D.
8.54± 0.19
243.86 _+3.06
9.72::+ (t.20 76.12 + 2.50
40.06± 6.11 146.63 ± 22.15 24.90± 1.95 N.D.
8.19_+ 0.42 22.86±
1.49
Subiculum Stratum pyramidale
30.32+ 8.65
Stratum moleculare
13.98± 3.57
Regio superior Stratmn oriens Stratum pyramidalc Stratum radiatum Slrattlnl molecular¢
8.08+ 0.86 7.74,: 0.70 8.02:2 0.69 8.5857 I.'47
Regio inlierior Stratum or±otis Stratum pyramidale Sl rat Ulll radiatum
17.66_+ 0.43 14.16± 0,52 14.80# t).94
9.{/0_+ 0.80 13.50+ 2.1¢1 14.43+ 1,61
15.10+ 0.20
35.764 12.73+ 29.96+__ 8.50+
Area dentala Hilus Granular cell layer Subgranular zone Molecular layer Angular bundle
10.93± 0.43 35.63+ 1,06 9.03± 0.24 4.02__+ 0.80
19.66± 1.18 23.7(t+_ 2.77
13.06+ I4.43:> 12.73Z 15.9347
1.82 [ 5 ~' 14~
2.0 ~)
0.96 3.26 0.'-18 2,13
6.10+- 1.10
structures, while the Ammon's horn and area dentata are relatively poor in [3HJNT binding sites (Fig. 2: Table I). There exist, however, some differences in the laminar distribution of [3HINT binding sites between the monkey and the human hippocam-
149
pal region. In tile monkey brain, most of the [~H]NT binding is present in layers 4 and 6 of the entorhinal area (EA) and in layers 2 and 6 of the presubiculunl (Fig. 2: Table I). The [3H]NT binding density showed the following rank-order within the EA: layer 4 > 6 > 5 > 1 > 2 > 3 and in the presubiculum: layer 2 > 6 > 1 >3, 4, 5. 111 the hunmn brain on the other hand, layer 2 of the EA harbours far more [3H]NT binding sites than any of the other layers, and the total number of binding sites in this la~,cr far cxceeds that 1\rand in tile corresponding layers in the monkey EA. In layer 2 of the human brain, the [~H]NT binding is discontinuous with islands of dense binding separated by zones of low or no binding (Fig. 2E) while in the monkey such arrangements are not seen in the EA. The binding in the para- and presubiculum show a similar distribution pattern in the hul'nan and the monkey brain (Fig. 2: Table II. In the subiculum, the regio superior and the regio inferior the [~H]NT binding is Io,a in all lamina, both in the monkey and the human brain (Fig. 2: Table 1). In the area dentata, the hilus is very rich m [3H]NT binding sites in both species (Fig. 2). Whilc in the human brain these sites occur throughout most of the hilar region, the\ arc more enriched in the subgranular zone in the monkey hippocampus (Fig. 2). In thc monkey as well as in the human hippocampus, the pattern of [3H]NT binding rcinains constant throughout the rostrocaudal axis of the structure but the number of [~H]NT binding sites increases within each subtield lit successively more rostral lcxels. Prcvious biochemical [9, 14, 20] and autoradiographic [11, 16, 18] studics in thc rat have shown that the hippocampus contains [3HINT binding sites which fulfill several pharmacological criteria for the delinition of a receptor and. furthermorc, that these binding sites are heterogeneously distributed within the hippocampal region. The present study has extended these earlier observations to the primate brain by providing a detailed anatomical description of the localization of [~H]NT recognition sites in the monkey and the postmortem human brain. Our present tindings that certain sublields of the primate hippocanlpus contain high density of [~H]NT receptors confirm previous obserwltions in both the monkey [I I] and human [1 I, IS] brain. While in all species examined, the highest densities of [3HINT binding sites are found in the EA. it should be pointed out that distinct differences in the relative binding density among individual layers exist between the species. Thus, in the rat [I 1] as wcll as the hnlllan brain (present study) the highest density of [~H]NT binding sites occurs in layer 2 of the EA, while in the Cynomologus monkey most of the [~H]NT binding sites are present in layers 4 through 6. The reason(s) for this discrepancy is not clear tit prcsent, but it could reflect the existence of species differences with regard to the wiring patterns of NT containing afferent projections in the EA. The high densit\ of presumed NT receptors in layer 2 of the EA is interesting since the cells of this laycr givc rise to the perforant path, which innervates and drives the granular cells of area dcntata [19]. The organization of the (apparently sparse)innervation [5, 6, 1 I, 17] of the hippocampus by NT is poorly documented. However. pyramidal cells in the subiculum have been shown to contain NT immunoreactivity [6, 17]. Since the subiculunl innervates the entorhinal area in the rat [12] as well as the monkey [1] brain, it is possiblc that NT released from some of these nerve terminals acts on NT receptors in the EA. The high density of [-~H]NT binding sites in layer 2 of thc presubiculum, in layers 2, 4 and 6 of the EA and in the hilus of the area dentata suggest
150 t h a t in t h e p r i m a t e , i n c l u d i n g t h e h u m a n
brain, NT may participate m the regulation
oF n e u r a l a c t i v i t y at s e v e r a l c r i t i c a l p o s i t i o n s a l o n g t h e i n t r a h i p p o c a m p a l neurophysiological
circuit. The
action(s) of NT at these sites remain(s) to be elucidated.
This work was supported
in p a r t b y R e s e a r c h G r a n t s A F 0 5 R
82-0328, USPHS
NS
14740; N S 0 8 5 3 9 : N S 19010, I Anmral, .1., Insausti, R. and ('owan, M.W., The commissural connections of the monkey hippocampal Ik~rmation, ,I. ('omp. Neurol., 224 (I 984) 307 336. 2 Carraway, R. and Lecman, S.E., The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami, J. Biol. ('hem., 248 (1973) 6854 6861. 3 ('arraway, R. and Lceman. S.E., The amino acid sequence, chemical synthesis and radioimmunoassa> oi'ncurotcnsin, Fed. Proc. Fed. Am. Soc. Exp. Biol., 33 (1974) 548. 4 ('arrawa), R. and Lceman, S.t!., ('haracterizalion of radioimmunoassayable neurotensin in the rat. Its distribution in the cenlral nervous system, small intestine and stomach, J. Biol. Chem., 251 (1976) 7045 7052. 5 Emson, P.(.. (ioedcrt, M.. l|orslield. P.. Rioux, E, and St. Pierre, S., The regional distribution and chromatographic characterization of ncurotcnsin-like immunoreaclivity in the rat central nervous systcm,.I. Ncurochcm., 38 (1982) 992 999. 6 l lara, Y.. Shiosaka, S., Seraba, E., Sakanaka, M., lnagaki, S., Takagi, H., Kawai, Y., Takatsuki, K., Matsuzaki, T. and Tohyama, M., Onlogcny of the neurotensin containing neuron system of the rat: hnmunohistochcmical analysis. I. Forebrain and diencephalon, J. Comp. Neurol.. 208 (1982) 177 195. 7 Jcnnes. L.. StumpF, W.E. and Kalivas, P.W,, Neurotensin: topographical distribution in rat brain by imnmnohistochemistry, ,I. Comp. Ncurol.. 210 (1982) 211 224. 8 Kataoka, K., mizuno. N. and Frohman, L.A.. Regional distribution of immunoreactive neurotensin in monkey brain. Brain Rcs. Bull.. 4 (1979) 57 60. g Kitabgi, P.. Carraway. P.. ~an Ritschoten, J., (iranier, C., Morgat, J.L., Mcnez, A., Leeman, S.E. and Frcychet, P.. Neurotcnsin: specilic binding to synaptic membranes from rat brain. Proc. Natl. Acad. Sci. USA, 74(I977) 1846 1856. l0 Kanbu. K.S.. Kanba, S.. Okazaki, tl. and Richelson, E., Binding of pH]neurotensin in human brain: properties and distribution. J. Ncurochem., 46 (1986) 946 952. I I Kiihler, ('.. Radcs~iler, A.-('.. Hall, 11. and Winblad, B., Autoradiographic localization of [~H]neurotensm binding sites m the hippocampal region of the rat and primate brain, Neuroscience, 16 (1985) 577 587. 12 Ki:,hlei-, ('., Intrinsic projections of Ihc retrohippocampal region in the rat brain. I. The subicular complex, ,1. ('onap. Ncurol., 234(1985) 504 522. 13 K~hlcr, ('., Chemical architecture of the entorhinal area. In R. Schwarez and Y. Ben-Art (Eds.), Excitatory Amino Acids and Epilepsy, Plenum, New York. 1986, pp. 83 98. 14 Manbcrg. P.J.. Youngblood, W.W.. Nemeroff. C.B., Rossor, M., lverscn, L.L., Prange, Jr., A.J. and Kizer. J.S., Regional distribution of ncurotensin in human brain, J. Neurochem., 38 (1982) 1777 1780. 15 Ncmcroff. C. and Prangc. Jr., A.J. (FMs.), Neurotensin, a brain gastrointestinal peptidc, Ann. N.Y. Acad. Sci., 400 (1982). 16 Quirion, R.. Gaudreau, P., St. Pierre, S., Rioux, F. and Pert, C.B., Autoradiographic distribution o1" [~tt]neurolcnsin receptors m rat brain: visualization by tritium sensitive [tin1, Pep(ides, 3 (1982) 757 767. 17 Roberts, G.W., Woodhams, P i . , Polak, J.M. and Crow, T.J., Distribution of neuropeptides in the limbic system of the rat: the hippocampus, Neuroscience, II ([984) 35 77. 18 Sarrieau, A., Ja~oy-Agid, F., Kitabgi, P., Dussaillant, M.. Vial, M.. Vincent, J.O., Agid, Y. and Roslone, W.H.. ('haracterization and autoradiographic distribution of neurotensin binding siles in the human brain. Brain Rcs., 348 (1985) 375 380. 19 Stcv, ard. O. and Scovillc, S.A., Cells of origin of enlorhinal cortical afferents lo the hippocampus and Fascia dcntata ol'the rat, J ('omp. Neurol.. 169 (1976) 347 370. 2() Uhl, G.R., Bennett, J.P. and Snyder, S.H., Neurotensin, a central nervous system peptide: apparent reccplorbindmgin brain membranes. Brain Res., 13(1(1977) 299 313.
145
NSL I)455S
Distribution of neurotensin receptors in the primate hippocampal region: a quantitative autoradiographic study in the monkey and the postmortem human brain Christer K6hler 1, Ann-Cathrin Rades~iter t and Victoria C han-Palay" ' I)~'pIIFIIH~'III
l*l
.\~'ltVOl)/ltlrDl~l~'O/Ok" ', ,4.~lrll ,l[~d, ,.I B, ,57;d~'rl~'ilj~' , .S'u~'d~,n ; a~ l :Nez rolo~,. (niw'r~ity
' hl,~'ll'lttl~',
tt,~s'pila/, Z z i r i c h f' , S ' w i l z c r h m d ;
(l~.eceixcd 27 ()ctobel- 1987; Revised \ersion rcceixed 16 December 1986: Accepted 2(I January, 1987) K e y ir+.'d~
t lippocampus: Entorhinal :.ll'e,:i; Neurotcnsila; Receptor; a.utorad ograph}; IVhmkc); t lu man; Brain
The distribution of [~H]neurolensin (plI]NT) binding sites in the morlkey and the postmortem human brain ~as studied b~ using quantitati,,c in xitro receptor auloradiography. Biochemical expel'in,thiS car-
fled out on lisstlc sections of the monkey hippocampus sho,aed that the binding of [~H]NT ~as stltur:.lblc. rc\crsible and of high spccilici D. The hippocampal ptt]NT binding was displaced by fragmenl NT, t: but not fragment NT~ . of the pcptide. The anatomical analysis showed a highly heterogeneous dislribution of [ flINT binding sites within both the monkey and the htmlan hippocampal region. In both species the highest densitx of [~It]NT binding sites ~r~ts found in the presubiculum (rank order of binding density: laNer 2 > 6 > I > 3. 4. 5 m both monkey and man) and Ihc cntorhina[ area (monkeN: ]a~er 4 > 6 > 5 :> I > 2 > 3: htlnltln: layer I - 2 > 5 > 3). The subiculum and Allllnon's horl] \~/el*Crclatixcly poor in [:II]NT binding sites in both species. In lhc area dentata the highest density o f l ~ l l ] N l binding sites x~;ts fotmd in lhe hilar region.
Neuroanatomical findings have provided evidence that different neuropeptides are located in cells and afferent projections innervating the hippocampal region [13, 17], and, thus. suggest that several of these substances may participate in the regulation of ncurotransmission along the intrinsic hippocampal pathways. Neurotensin (NT), a 13-amino acid peptide isolated from bovine hypothalamic extracts [2 4, 15], has been identilicd within the hippocampal region of the rodent and primate brain [4 N, 14]. -['he hippocampus contains binding sites t\w [~H]NT which show pharmacological characteristics of a biologically' relevant receptor [10, I1]. By application of in vitro receptor autoradiography, the NT receptors have been shown to be heterogeneously distributed within the rat hippocarnpal region [11, 16]. While the anatomical ('~,';'vspomh';tce. C. K6hler, Department of Neuropharmacolog.v. Astra Alab AB, S-151 85 S,3dertiilie. S~cdcn. 0304 3940 87 S 03.50 (c) 1987 Elsevier Scientific Publishers Ireland Ltd.
146
localization of NT receptors has been studied in detail in the rat brain [l I, 16] Icss is known about the localization of NT binding sites in the primate brain (lk)r the human brain sec refs. II and 18). In the present study we have analyzed thc anatomicai distribution of [-~H]NT binding sites within the hippocampal region o1 the monke\ and the postmortem human brain. The brains from two male Cynomologus monkeys (SBL, Stockholm, Sweden: weight approx. 4 kg) and 5 temporal lobes obtained at autopsy (postmortem interwd: 8 21 h) from patients who died without any diagnosed neurological illness. The brains were cut frozen in a cryostat ( - 15 C) and the sections (20 ira1 thick) were incubated with [~H]NT (New England Nuclear, spec. act. 67.6 Ci.mmol i) in 0.17 M Tris-HCl buffer containing 0.05% boiled bovine serum albumin (Sigma, St. Louis, U.S.A.) and 0.1 mM bacitracin (Sigma, St. Louis, U.S.A.) as previously described [11]. The sections were rinsed, dried in a stream of cold air and put in contact with -~H-sensitive fihn (Ultrofilm, LKB, Sweden). The autoradiograms were analyzed by using an IBAS 2000 image analyzer (Zeiss Kontron, F.R.G.) equipped with a program which converted optical densities to t)nol.mg i tissue using calibrated plastic standards (Amersham) co-exposed with the tissue sections. In a separate series o1" experiments the binding of different concentrations of [~H]NT to sections of the monkey hippocampal region was analyzed by scintillation spectroscopy after the sections had been scraped off'the glass slides [I I], The biochemical studies showed that [~H]NT bound in a saturable manner to sections of the monkey hippocampal region. The non-specific binding contributed less than 20% of the total binding at concentrations around the Kd value (I.5 nM). The [3HINT binding was dose dependently blocked by fragment 8 13 of the peptide (1C50,-~ 70 nM) while fragment I-8 was inactive at concentrations up to I0/zM (Fig. 1A.B).
A
B
1000
100
o
o
o
o :5 t~
®
100
l _ ~ l J J l [ l l l l 1
5
t 10
Con¢.3H-Neutoten~n(nM)
J
i
I
15
I
I
-9
-
18
-
17
I
-6
-
15
- l o g displacer (M)
Vig. I. Saturation analysis o f t t HINT binding to tissue sections (20 l*m thick) of the monkey hippocampal region. The tissue sections were obtained at a level corresponding to that shown in Fig. 2B. As can be seen the binding is saturable and it shows a low degree of non-specific binding throughout the (lose response range tested. The dose-dependent blockade by fragment 8 13 of the NT molecule is shown in the right panel. Fragment NT~ ~ is inactive in displacing [3H]NT binding to sections of the monkey hippocampus up to 10 ltM. Similar results were obtained in lwo separate experiments.
147
Fig. 2. Photon~icrogral~hs el" auloradiograms showing the distribution of ptt]NT (4 nMI binding sitcx m the m~mkc> I \,B.(') and human [D.E) hippocampal region. A and (" urc dark-licld pholtm'dcrogiaphs (v, hilc ille:t> i'el')lcscnl [~II]NT binding-sitcsL Photomicrographs of compulcr-gencralcd images of the ]:II]NT autoradiogt-ums arc sho~n in IL D. und [!. The urrows in A delimit an at'ca I[l:tl ellcotllpgtsses the prcsubicuhnl~ and p~.|l'I C)I"the hippocampal-am,,gdaloid-transition area. l)oublc arrowheads sh(~\~ the border bcl,,~ccn lhc subiculum and rcgio superior. Single arrowhead marks the laleral border of lilt onto rhiual :lrea. l~hotomicrograph in B is a high per, or ~icw of the photomicrograph in (" shm~mg the exact latmin:lr distribution of the pH]NT binding sites. P,indmg silos l\tr [zlI]NT in Ihe human hippocampal region arc sh¢~x\n in D and E. Thc laminar distribution o1" the cnlorhinal [StlINT binding sites is sho~n in I{. ,,\trox~s mark Ihe borders bclx,,cen individual sublields ~1" the hippocampal region, ah. nn,.g,uklr bundle: I!A. eiHorhinal area: l l. hilus; inl, molccular layer" PRI{. prcsubiculum: PS. parusubiculum: RI. regio inferior: P,S, regio superior: SUB. subiculum: SN<. subslantia nigra, pars compacla: SN,. subst~lntia nigra, pars Ieticu]aln. The layers of lhc prcsubiculum and el" EA are labelled I through (~
Tile autoradiographic analysis revealed a heterogeneous distribution of the [-~H]NT binding sites within both the monkey and the human hippocampal region. Thus~ in both species most of tile [3HINT binding is present in the retrohippocampal
148 TABLE I T H E SPECIFIC B I N D I N G ( f m o l . m g *) OF [3HINT {4 nM) IN D I F F E R E N T A R E A S A N D LAM1NA OF T H E M O N K E Y A N D T H E P O S T M O R T E M H U M A N H I P P O C A M P A L R E G I O N AS DETERM I N E D BY Q U A N T I T A T I V E R E C E P T O R A U T O R A D I O G R A P H Y Specilic binding was defined by 1 I~M N ] l i~. The values are the mean ( ± S . E . M . ) of 6 determinations in each monkey and h u m a n brain. While the pH]NT binding showed the same pattern in the hippocampi from all 5 cases, quantitative analyses were made onl> in the 3 cases most similar with regard lo age and postmortem intervals. The vahlcs were not corrected for quenching. N.D., not determined. Anatomical region
Entorhinal area I_ayer I 3 4 5
Parasubiculmn Presubiculum Layer I 2 35 6
[~H]Neurotensin specitically bound (finol.mg 'l Monke',
Human
13.88± 2.84 10.62+ 2.43 7.74_+ 0.75 43.06 _+ 12.00 20.0427 3.97 34.98_+ 5.3[
243.73± 244.2{)± 46.53+_ N.D. 54.10__+
3.01 6.47 1.84 2.80
N,D.
8.54± 0.19
243.86 _+3.06
9.72::+ (t.20 76.12 + 2.50
40.06± 6.11 146.63 ± 22.15 24.90± 1.95 N.D.
8.19_+ 0.42 22.86±
1.49
Subiculum Stratum pyramidale
30.32+ 8.65
Stratum moleculare
13.98± 3.57
Regio superior Stratmn oriens Stratum pyramidalc Stratum radiatum Slrattlnl molecular¢
8.08+ 0.86 7.74,: 0.70 8.02:2 0.69 8.5857 I.'47
Regio inlierior Stratum or±otis Stratum pyramidale Sl rat Ulll radiatum
17.66_+ 0.43 14.16± 0,52 14.80# t).94
9.{/0_+ 0.80 13.50+ 2.1¢1 14.43+ 1,61
15.10+ 0.20
35.764 12.73+ 29.96+__ 8.50+
Area dentala Hilus Granular cell layer Subgranular zone Molecular layer Angular bundle
10.93± 0.43 35.63+ 1,06 9.03± 0.24 4.02__+ 0.80
19.66± 1.18 23.7(t+_ 2.77
13.06+ I4.43:> 12.73Z 15.9347
1.82 [ 5 ~' 14~
2.0 ~)
0.96 3.26 0.'-18 2,13
6.10+- 1.10
structures, while the Ammon's horn and area dentata are relatively poor in [3HJNT binding sites (Fig. 2: Table I). There exist, however, some differences in the laminar distribution of [3HINT binding sites between the monkey and the human hippocam-
149
pal region. In tile monkey brain, most of the [~H]NT binding is present in layers 4 and 6 of the entorhinal area (EA) and in layers 2 and 6 of the presubiculunl (Fig. 2: Table I). The [3H]NT binding density showed the following rank-order within the EA: layer 4 > 6 > 5 > 1 > 2 > 3 and in the presubiculum: layer 2 > 6 > 1 >3, 4, 5. 111 the hunmn brain on the other hand, layer 2 of the EA harbours far more [3H]NT binding sites than any of the other layers, and the total number of binding sites in this la~,cr far cxceeds that 1\rand in tile corresponding layers in the monkey EA. In layer 2 of the human brain, the [~H]NT binding is discontinuous with islands of dense binding separated by zones of low or no binding (Fig. 2E) while in the monkey such arrangements are not seen in the EA. The binding in the para- and presubiculum show a similar distribution pattern in the hul'nan and the monkey brain (Fig. 2: Table II. In the subiculum, the regio superior and the regio inferior the [~H]NT binding is Io,a in all lamina, both in the monkey and the human brain (Fig. 2: Table 1). In the area dentata, the hilus is very rich m [3H]NT binding sites in both species (Fig. 2). Whilc in the human brain these sites occur throughout most of the hilar region, the\ arc more enriched in the subgranular zone in the monkey hippocampus (Fig. 2). In thc monkey as well as in the human hippocampus, the pattern of [3H]NT binding rcinains constant throughout the rostrocaudal axis of the structure but the number of [~H]NT binding sites increases within each subtield lit successively more rostral lcxels. Prcvious biochemical [9, 14, 20] and autoradiographic [11, 16, 18] studics in thc rat have shown that the hippocampus contains [3HINT binding sites which fulfill several pharmacological criteria for the delinition of a receptor and. furthermorc, that these binding sites are heterogeneously distributed within the hippocampal region. The present study has extended these earlier observations to the primate brain by providing a detailed anatomical description of the localization of [~H]NT recognition sites in the monkey and the postmortem human brain. Our present tindings that certain sublields of the primate hippocanlpus contain high density of [~H]NT receptors confirm previous obserwltions in both the monkey [I I] and human [1 I, IS] brain. While in all species examined, the highest densities of [3HINT binding sites are found in the EA. it should be pointed out that distinct differences in the relative binding density among individual layers exist between the species. Thus, in the rat [I 1] as wcll as the hnlllan brain (present study) the highest density of [~H]NT binding sites occurs in layer 2 of the EA, while in the Cynomologus monkey most of the [~H]NT binding sites are present in layers 4 through 6. The reason(s) for this discrepancy is not clear tit prcsent, but it could reflect the existence of species differences with regard to the wiring patterns of NT containing afferent projections in the EA. The high densit\ of presumed NT receptors in layer 2 of the EA is interesting since the cells of this laycr givc rise to the perforant path, which innervates and drives the granular cells of area dcntata [19]. The organization of the (apparently sparse)innervation [5, 6, 1 I, 17] of the hippocampus by NT is poorly documented. However. pyramidal cells in the subiculum have been shown to contain NT immunoreactivity [6, 17]. Since the subiculunl innervates the entorhinal area in the rat [12] as well as the monkey [1] brain, it is possiblc that NT released from some of these nerve terminals acts on NT receptors in the EA. The high density of [-~H]NT binding sites in layer 2 of thc presubiculum, in layers 2, 4 and 6 of the EA and in the hilus of the area dentata suggest
150 t h a t in t h e p r i m a t e , i n c l u d i n g t h e h u m a n
brain, NT may participate m the regulation
oF n e u r a l a c t i v i t y at s e v e r a l c r i t i c a l p o s i t i o n s a l o n g t h e i n t r a h i p p o c a m p a l neurophysiological
circuit. The
action(s) of NT at these sites remain(s) to be elucidated.
This work was supported
in p a r t b y R e s e a r c h G r a n t s A F 0 5 R
82-0328, USPHS
NS
14740; N S 0 8 5 3 9 : N S 19010, I Anmral, .1., Insausti, R. and ('owan, M.W., The commissural connections of the monkey hippocampal Ik~rmation, ,I. ('omp. Neurol., 224 (I 984) 307 336. 2 Carraway, R. and Lecman, S.E., The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami, J. Biol. ('hem., 248 (1973) 6854 6861. 3 ('arraway, R. and Lceman. S.E., The amino acid sequence, chemical synthesis and radioimmunoassa> oi'ncurotcnsin, Fed. Proc. Fed. Am. Soc. Exp. Biol., 33 (1974) 548. 4 ('arrawa), R. and Lceman, S.t!., ('haracterizalion of radioimmunoassayable neurotensin in the rat. Its distribution in the cenlral nervous system, small intestine and stomach, J. Biol. Chem., 251 (1976) 7045 7052. 5 Emson, P.(.. (ioedcrt, M.. l|orslield. P.. Rioux, E, and St. Pierre, S., The regional distribution and chromatographic characterization of ncurotcnsin-like immunoreaclivity in the rat central nervous systcm,.I. Ncurochcm., 38 (1982) 992 999. 6 l lara, Y.. Shiosaka, S., Seraba, E., Sakanaka, M., lnagaki, S., Takagi, H., Kawai, Y., Takatsuki, K., Matsuzaki, T. and Tohyama, M., Onlogcny of the neurotensin containing neuron system of the rat: hnmunohistochcmical analysis. I. Forebrain and diencephalon, J. Comp. Neurol.. 208 (1982) 177 195. 7 Jcnnes. L.. StumpF, W.E. and Kalivas, P.W,, Neurotensin: topographical distribution in rat brain by imnmnohistochemistry, ,I. Comp. Ncurol.. 210 (1982) 211 224. 8 Kataoka, K., mizuno. N. and Frohman, L.A.. Regional distribution of immunoreactive neurotensin in monkey brain. Brain Rcs. Bull.. 4 (1979) 57 60. g Kitabgi, P.. Carraway. P.. ~an Ritschoten, J., (iranier, C., Morgat, J.L., Mcnez, A., Leeman, S.E. and Frcychet, P.. Neurotcnsin: specilic binding to synaptic membranes from rat brain. Proc. Natl. Acad. Sci. USA, 74(I977) 1846 1856. l0 Kanbu. K.S.. Kanba, S.. Okazaki, tl. and Richelson, E., Binding of pH]neurotensin in human brain: properties and distribution. J. Ncurochem., 46 (1986) 946 952. I I Kiihler, ('.. Radcs~iler, A.-('.. Hall, 11. and Winblad, B., Autoradiographic localization of [~H]neurotensm binding sites m the hippocampal region of the rat and primate brain, Neuroscience, 16 (1985) 577 587. 12 Ki:,hlei-, ('., Intrinsic projections of Ihc retrohippocampal region in the rat brain. I. The subicular complex, ,1. ('onap. Ncurol., 234(1985) 504 522. 13 K~hlcr, ('., Chemical architecture of the entorhinal area. In R. Schwarez and Y. Ben-Art (Eds.), Excitatory Amino Acids and Epilepsy, Plenum, New York. 1986, pp. 83 98. 14 Manbcrg. P.J.. Youngblood, W.W.. Nemeroff. C.B., Rossor, M., lverscn, L.L., Prange, Jr., A.J. and Kizer. J.S., Regional distribution of ncurotensin in human brain, J. Neurochem., 38 (1982) 1777 1780. 15 Ncmcroff. C. and Prangc. Jr., A.J. (FMs.), Neurotensin, a brain gastrointestinal peptidc, Ann. N.Y. Acad. Sci., 400 (1982). 16 Quirion, R.. Gaudreau, P., St. Pierre, S., Rioux, F. and Pert, C.B., Autoradiographic distribution o1" [~tt]neurolcnsin receptors m rat brain: visualization by tritium sensitive [tin1, Pep(ides, 3 (1982) 757 767. 17 Roberts, G.W., Woodhams, P i . , Polak, J.M. and Crow, T.J., Distribution of neuropeptides in the limbic system of the rat: the hippocampus, Neuroscience, II ([984) 35 77. 18 Sarrieau, A., Ja~oy-Agid, F., Kitabgi, P., Dussaillant, M.. Vial, M.. Vincent, J.O., Agid, Y. and Roslone, W.H.. ('haracterization and autoradiographic distribution of neurotensin binding siles in the human brain. Brain Rcs., 348 (1985) 375 380. 19 Stcv, ard. O. and Scovillc, S.A., Cells of origin of enlorhinal cortical afferents lo the hippocampus and Fascia dcntata ol'the rat, J ('omp. Neurol.. 169 (1976) 347 370. 2() Uhl, G.R., Bennett, J.P. and Snyder, S.H., Neurotensin, a central nervous system peptide: apparent reccplorbindmgin brain membranes. Brain Res., 13(1(1977) 299 313.