- Email: [email protected]
Evidence for the existence of a neurotensin-containing pathway from the endopiriform nucleus and the adjacent prepiriform cortex to the anterior olfactory nucleus and nucleus of diagonal band (Broca) of the rat
EVIDENCE FOR THE EXISTENCE OF A NEUROTENSIN-CONTAINING PATHWAY FROM THE ENDOPIRIFORM NUCLEUS AND THE ADJACENT PREPIRIFORM CORTEX TO THE ANTERIOR OLFACTORY NUCLEUS AND NUCLEUS OF DIAGONAL BAND ~BROCA~ OF THE RAT S. INAGAKI, K. SHINODA, Y. KUBOTA, S. SHIOSAE;A,T. MATSUZAKI and M. T~HYAMA Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, 4-3-57 Nakanoshima, Kitaku. Osaka 530. Japan
Abstract-The origin of neurotensin-iike immunoreactive fibers to the anterior olfactory nucleus and nucleus of the diagonal band of Broca of the rat were elucidated experimentally using the indirect immunofluorescence method. Neurotensin-like immunoreactive fibers located in these areas decreased remarkably in numbers on the operated side after the destruction of the ventral part of the endopiriform nucleus and the adjacent prepiriform cortex where numerous cells with neurotensin-like immunoreactivity were detected. This strongly suggests that such cells located in the endopiriform nucleus and the adjacent prepiriform cortex send a neurotensin-Ike projection ipsi~atera~~yto the anterior olfactory nucleus and to the nucleus of the diagonal band of Broca.
Neurotensin
is a basic polypeptide which was isolated
from the hypothalamus and characterized by Carraway & Leeman. 2*3 subsequent radioimmunoassay and immunohistochemjcal studies have suggested that neurotensin is distributed unevenly in the central nervous system, is concentrated in the synaptosomal and microsomal fraction, and binds to a specific receptor. 7,8,9,10,11,16,21,22,24.25,27 However, knowledge of neurotensin-containing neuron tracts in the central nervous system is incomplete, with such tracts having been described only in reports on the amygdaloid neurotensin systemz2824 and cortical neurotensin system. 21 The present study was carried out to see whether the numerous neurotensin fibers located in the anterior olfactory nucleus (OA) and nucleus of the diagonal band of Broca (DB) are supplied by neurotensin-containing neurons in the ventral part of the endopiriform nucleus @NV) and adjacent prepiriform cortex (PF). EXPERIMENTAL
PROCEDURES
Seventy-one young male rats (body weight 30_5Og, about 2-3 weeks old) were used in this study. lmmunohistochemical
study
Experimental animals and preparation of tissue. A total of 42 male rats were used in a immunohistochemical study. Abbreviations: DB, nucleus of the diagonal band of Broca; DB-EN, area between DB and EN; EN, endopiriform nucleus; ENv, ventral part of EN; HRP, horseradish peroxidase; PBS, phosphate buffer saline; PF, prepiriform Cortex; OA, anterior olfactory nucleus.
Three animals were used for the observation of the normal distribution of neurotensin-like immunoreactive structures. The remanning animals were used for the ~xperimentai immunohistochem~cal studies. In 6 animals, hem~transection of the brain just caudal to the DB (at the level of A 5.3 mm of the atlas of Sherwood & Timiras23) were made stereotaxically with a fine knife. In 9 animals, hemi- or total transection of the brain just caudal to the ENv (at the level of A 2.9 mm of the same atlasz3) were also performed in the same way. These knife-cut studies and the horseradish peroxidase (HRP) study (see below) suggested that the origins of neurotensin-like immunoreactive fibers in the OA and OB may be localized in the ENv and PF. Accordingly, in 24 animals, electrolytic lesions (passing an AC current of 1mA for I s through a monopolar electrode) were made stereotaxically in the ENv and PF (A 4.7-3.5 mm, L 3.8-4.8 mm, H 1.7 mm).23 The animals were kept alive for 3-7 days after the operations. All the animals were perfused via the ascending aorta with 50 ml ice-cold saline followed by 300ml of Zamboni’s fixative.” The brains were removed stereotaxically to obtain the same area on both the operated side and the control side. The brains were immersed in the same fixative for 24-48 h and then rinsed for 24-72 h in 0.1 M phosphate buffer pH 7.4 containing 30”/, sucrose. Frontal, sag&al and horizontat serial sections with a section thickness of 1Opm were cut on a cryostat. The slides were pretreated with chrom-alum gelatin to prevent detachment of the sections during incubation. Immune serum. Antiserum against neurotensin was raised in a rabbit following injections of neurotensin-hemocyanin conjugates linked with (l-ethyl-B, 3-dimethylaminopropylfcarbodiimide. The specificity of this antiserum was tested in a radioimmunoassay system and less than 0.01% crossreactivity was found to occur to related peptides such as substance P, enkephalins, endorphins, somatostatin, gluca-
bradykinin
&on.
specificity
and
cholecystokinin
of the antiserum
tion
test at the morphological
The
neurotensin-antiserum
treated
in
stained brain
with
cresyl
regions.
chemistry
The
The
indirect
was
prc-
Half of the sections
were
sections
immunoreactivity
the
the
at IO C wlfh
Triton
rinsed three times Priton
fluorescein
the sections
(15min
Incubated sorbed
in buffer
antiserum
immuno-
with
prro.\-irlasr
Fig. I. A-G. anterior
atmosphere
each) with
absorption
test
olfactory
RESULTS
(0.X jig ml). These
above.
Ittitnut2oki.~tolhrtt~icul
whether or
of the ventral
nucleus
and
part of endopiriform
nucleus
of
study
The specificity of the immunoreaction was checked by comparing sections stained with test.
nucleus
and the adjacent
neurotensin-like
diagonal
band
prepiriform
immunoreactivity
of
Broca.
A-G
Control
side (A). operated
in the horizontal
Control limbs
of the DB.
fibers in the vertical side (The midline
immunoreactive
show
by an arrow).
the electrolytic
Frontal
sections.
Abbreviations B
basal amygdaloid
c
central
CA
commissure
CL
claustrum
DB
nucleus
DB-EN
the area between
ENv
ventral
0 OA
nucleus of the olfactory tract anterior olfactory nucleus
L
lateral
amygdaloid
nucleus
M
medial
amygdaloid
nucleus
NT
neurotensin
PF POM
prepiriform medial
side (E), operated
in these areas after
showing
by arrows).
Control
Note
destruction lesion
Case ENv
of Ihe ENv
of the ENv
used in figures
nucleus
amygdaloid
nucleus
anterior
of the diagonal
band (Broca)
the DB and endopiriform
part of the endopiriform
cortex
preoptic
area
nucleus
nucleus
change
ipsilateral
the change decrease
the adjacent
and the adjacent
x 80, H.
fibers in
side and the right and
x 19
in
in immunoreactive
side (F). G shons
a remarkable
21. A-G.
the
fibers in the OA
in immunoreactlve
side (DI. E and F show the change
limb of the DB. The left side is the control
is indicated
fibers
photomicrograph
side (B). C and D show the change
side (C), operated
cortex
in the ipsilateral
fibers in the OA and DB. A and B show the change in immunoreactive
immunoreactive
Light-field
M~S ah-
decrease of fibers displaying
the OA pars medialis.
number
first
not the ENv and the :idjaccnt PF‘ project to the OA and DB. and whether the distribution of HR P-labeled cells in the ENv and the adjacent PF after ()A or DR injection is similar to the distribution of neurotcnslnlike immunorractive cells obtained bj immunohistochemical analysis. A total of 29 animals were used in the HRP study. A W’,, solution of HRP (Sigma type VI) in ;I ghs micropipette was connected to ;I high-voltage-current source. which provided a pulsed positive current (7s on. alternating with 7 s off ). A single injection of HRP was made stereotaxically using :I positive current 01’ 2.5 ,uA for I5 min. into the OA and DB. Followmg a survival time of 24 h. the brain was pcrfuscd with 1.?I”,, glutaraldehyde 4.W,, paraformaldehl;de in 0.1 M phosphate buffer (pH 7.4). The brains were removed, immersed in the same ti.xativo at 4 (‘ overnight and then rinsed for 2472 h :It 4 c’ in 0.1 M phosphate buffer containing Jo”,, sucrose. Serial frontal sections with a section thickness of 30 /lrn \vcre cut on ;I cryostat. The sections Mere then reacted with 3.3.‘5,5’-tetra-methyl benzidine. ’ a The terminology used was that in the atlases of Kijnig & Kiippel,‘” Krettek & Price’4,” and Sherwood & Timirss.”
Ahsorptiott
Destruction
the operated
(SO”,,)
were
which
neurotensin described
at
PBS at 4 C
.stztrlj~
immunoreactive
fibers
with
(Miles
m a PBS glycerine
the
in a marked
pars dorsalis.
then
Incubated
antlbodies
The HRP method was used to ascertain
resulted
PBS conhere
PBS contaimng
to neurotensin as
were processed
test The
atmosphere
They
and
(I 5 mm
mounted
by excess synthetic
Horsrutrdi.sl~
diluted
1:2000.
for I h in 21 humid
for
groups:
immunoserum.
conjugated
Mere finally with
the
each) in ice cold
rinsed
sections
two
neurotensin-like
at
three rinses
The
into
of neurotensin-like
antiserum
Lid) at I :50 dilution,
mixture.
of Coons’
for 23 h in a humid
isothiocyanate
37 C. Follorting
saline
half for the absorption of
X-100
X-100.
technique
subdivided
specificity
neurotensin
(~.I”,,
buffer
used for the immuno-
demonstration
were incubated
taining
immunohisto-
phosphate
The sections
used for demonstrating
reactivity
for
were of the
10 staining.
and the other
checking
sections
identification
intended
In cold
were further
was used for
sections
exact
immunofluorescence
histochemistry
sections
for
bvere incubated
was used in this study.
Co.
stud!
and alternate
violet
(PBS) for IO min prior
O.l”,,
this
by hemocyamin.’
fttirrlitrtolristo~~~~ttii~~u~ pr~tcduru.
for
The
by the absorp-
level (for results, see beloir). used
used for immunohistochemistry
one
octapeptide.
was also tested
PF
m
side is in the PF.
H:
(indicated
Fig. 1
489
Fig. 2. A-C. Light-field photomicrographs showing an injection site of horseradish peroxidase into the ante) rior olfactory nucleus pars medialis (indicated by an arrow) where the highest density of fi\,ers displ aying neurotensin-like immunoreactivity was seen (A), and HRP-labeled cells in the ventral par t of cortex (B, C) subsequent to HRP injection into the endc bpiriform nucleus and the adjacent prepiriform cells found in the ree ;ion OA pars medialis. Frontal sections. Case ENv 14. In Fig. 2C, HRP-labeled (HRP-labeled cells are shown by arrows). A. x 34. outli ned in Fig. 2B are shown in greater magnification photomicrograph showing neurotensin-immunoreactive cells in the B. x 130, C. x 320. D: Fluorescence ENv and the adjacent PF. Frontal sections. Case ENv 6. x 200. 490
Neurotensin afferents to anterior olfactory nucleus neurotensin-antiserum and absorption control. Since the structures stained with neurotensin-antiserum were not seen in the control sections, the former were considered specific, These materials should correctly be described as showing neurotensin-like immunoreactivity. The specificity of the neurotensin-antiserum obtained by radioimmunoassay was discussed under Experimental Procedures. Effects of knife cuts. As shown in Figs lA, C, E, G, numerous neurotensin-like immunoreactive fibers were found in the OA, particularly in the pars dorsalis (Fig. IA), pars medialis (Fig. 1C) and pars posterior, and also in the DB both in the horizontal (Fig. 1E) and vertical limbs (Fig. 1G). The number of these fibers decreased remarkably on the operated side after unilateral transection of the brain just caudal to the DB (at the level of A 5.3 mm of the atlas of Sherwood & Timirasz3). On the other hand, hemi- or total transection of the brain just caudal to the ENv and the adjacent PF in which numerous neurotensin-like immunoreactive cells exist (Fig. 2D) at the level of A 2.9mm of the same atlas) failed to decrease the number of immunoreactive fibers in these areas. These results strongly suggest that the major source of neurotensin-like immunoreactive fibers in the OA and DB is localized in the forebrain and diencephalic areas between these two levels (DB and ENv) (from the level of A 2.9-5.3 mm). We call this the DB-EN area hereafter. In the DB-EN area, several groups of cells showing neurotensin-like immunoreactivity were found, e.g. numerous cells in the ENv and the adjacent PF, some in the medial preoptic area, bed nucleus of the stria terminalis, hypothalamic paraventricular nucleus and lateral hypothalamus (Fig. 3, right half). Furthermore, some of the immunoreactive cells located in the ENv and the adjacent PF extended rostrally to the lateral part of the anterior amygdaloid area and medially to the amygdaloid complex. The location of immunoreactive cells in the DB-EN area is shown in Fig. 3 (right half).
491
general similar to that seen after HRP injection into OA pars medialis. In addition, in cases in which HRP was injected into the DB (Case ENv 21, 23), a wider distribution of HRP-labeled cells than that seen after HRP injection into OA was identified in the DB-EN area. For example, in addition to HRP labeled groups found subsequent to HRP injection into OA (Fig. 3, left side), the group of central thalamic nuclei was found to be labeled. A comparison of the locations of immunoreactive cells and HRP-labeled cells after injection of HRP into OA (Fig. 3) or DB (Figs 2B-D, 3), suggests that neurotensin-immunoreactive cells located in the ENv and the adjacent PF may project to the OA and DB.
Horseradish peroxidase study
In order to examine afferents to the anterior olfactory nucleus (OA) (Fig. 2A) and nucleus of the diagonal band of Broca (DB), HRP was injected into these two areas, and the locations of HRP labeled cells identified in the DB-EN area were compared with those of cells displaying neurotensin-like immunoreactivity in the same area (Figs 2B-D, 3). In Fig. 3 (left half), the location of HRP-labeled cells is shown in the ENv and the adjacent PF following HRP injection into OA, pars medialis (Fig. 2A) where the highest density of immunoreactive fibers were seen (Fig. 1). In other cases in which HRP was injected into OA pars dorsalis (Case ENv 15, 17) or OA pars posterior (Case ENv 19, 20), or in other OA injected cases (Case ENv 13, 16), the location of HRP-labeled cells found in the DEEN area was in
Fig. 3. Schematic representation of the distribution of cells showing neurotensin (NT)-like immunoreactivity (right side, shown by closed circles) in the DB-EN area and horseradish peroxidase-labeled cells (left side, shown by closed triangles) in the same area after injection of horseradish peroxidase into the anterior olfactory nucleus pars medialis (Case ENv 14) outlined on a stereotaxic atlas of the rat brain. The injection site and HRP-labeled cells in the ENv and the adjacent PF is shown in Figs 2A, B and C. Note that the ENv and the adjacent PF contained both immunoreactive cells and HRP-labeled cells.
iZffe~‘tsof’ the destruction of the uentral part of endopiri,fbrm mcleus und the ad¢ prepiriforvn cortex In cases in which electrolytic destruction included the compIete ENv and the adjacent PF area which contains numerous ~mmunor~~~f~ve cells, changes in the numbers of immunoreactive fibers were striking (Figs IA-H): in other cases where the lesion partly included the ENv and the adjacent PF area, changes in the immunoreactive fibers were more variable. Following the destruction of the ENv and the adjacent PF (Case ENv 22. 25, 28, 31, 37). a remarkable decrease in immunoreactive fibers was found in the ipsilateral OA, particularly in the pars dorsalis, medialis (Figs IA-D) and posterior. and DB both in the horizontal and vertical limbs (Figs 1E-G).
IXXUSSION
Regarding the fiber connections of the neurotensinlike neuron system, there is good evidence for an amygdaloid neurotensin neuron system”2*2s and cortical neurotensin neuron system.2’ LittIe so far is known, however, about other systems containing neurotensin-iike immunoreactive material. The present study clearly demonstrated that cells displaying neurotensin like immunoreactivity, which are located in the ENv and the adjacent PF area, send their axons to the OA and DB. Prev~ous)y, the ascending tract from this area to the olfactory bulb and nucleus of the diagonal band of Broca had been demonstrated by the HRP method
and by the autoradiographic method.” ’ ‘.’ ’ ’ . .I bus. it is probable that one of the main components of thi* ascending system is the neurotensin-contairllng one. It should be noted that f<>llowing transection of the brain just caudal to the anterior olfactory nucleus (OA) or nucleus diagonal band of Brocn if>B). a significant number of immunoreactive fibers was stilt found in the OA and DB. This fact suggests that some of the neurotensin-immunoreactive fibers in the OA and DB are intrinsic, because both in OA and DB. ;\ nt~mber of ~mmunoreactive cells were found.In addition. our previous olltogeneti~~~~ studies’ concerning the neurotensin-containing system have shown that the initial differentiation and development of the neurotensin-containing neuron system in the rat brain occurs during the fetal period before the establishment of normal synaptic transmission. suggesting that the neurotensin-~~~nt~~il~ing neuron system might have some functional roie in the development of the brain in addition to its neurontransmitter or neuromodulator functions. Based upon the present and previous findings. ENv and the adjacent PF is one of the major sources of neurotensin in the anterior olfactory nucleus (OA) and the nucleus of the diagonal band of Broca (DB) and might play an important role in regulation of these olfactory or limbic functions, beside its well known effects. hypothermia. inhibition to nociception etc.‘~4~r’~“‘~‘0~‘s~z~fn addition, the facts that OA and DB are innervated by more than one NT neuron system suggest complex functions of ~euro~e~~jrl in the OA and DB.
REFERENCES Bissette G., Nemeroff C.,B., Soosen P. T., Prange A. J. Jr & Lipton M. A. (1976) Hypothermia and intolerance to cold induced by intracisternal administration of the hypothalamic peptide neurotensin. Nature, Land. 262, 607-609. Carraway R. & Leeman S. E. (1973) The isolation of a new hypothalamic peptide, neurotensin. from bovine hypothalami. .i. hiol. Chem. 248, 6854-6861. Carraway R. & Leeman S. E. (1976) Characterization of radioimmunoassayable neurotensin in the rat. J. hioi. Chern. 25t, 70567052. Clineschmidt B. V. & McGrifiin J. C. (1977) Neurotensin administrated intracisternaily inhibits responsiveness of mice to noxious stimuli. Eur. J. Pharmac. 46, 3955396. Coons A. H. (1953) Fluorescent antibody methods, In General Cytochemicul Methods (ed. Danielli J. F.) pp. 399 -422. Academic Press, New York. De Olmos J.. Hardy I-i. & Heimer L. (1978) The afferent connections of the main and the accessory olfactory bulb formations in the rat: an experimental HRP-study. J. camp. Neural. 181, 213-244. Ham Y.. Shiosaka S., Senba E., Sakanaka M., Inagaki S., Takagi H., Kawai Y., Takatsuki K.. Matsuzaki T. & Tohyama M. (1982) Ontogeny of the neurofensjn-containing neuron system of the rat: immunohistochemical analysis-1. Forebrain and Diencephalon. J. camp. Neural. 208, 177-195. Kahn D., Abram G. M., Zimmerman E. A. & Leeman S. E. (1980) Neurotensin neurons in the rat hypothalamus: an immunocytochemicaf study. Endocrinology 107,47-54. Kataoka K. and Frohman L. A. (1979) Regional distribution of immunoreactive neurotensin in monkey brain. Brfrir~ Res. Bull. 4, 57-60. Kitabgi P., Carraway R., Van Rietschoten J., Granier C., Morgat J. L.. Menez A., Leeman S. & Freychet P. (1977f Neurotensin: specific binding to synaptic membranes from rat brain. Proc. ~atn. Acad. Sci. U.S.A. 74, f846l850. Kobayashi R. M., Brown M. and Vale W. (1977) Regional distribution of neurotensin and somatostatin in the rat brain. Brain Res. 126, 584-588. Kiinig J. F. R. & Klippel R. A. (1963) The Rat Brain. A stereotaxic Atlas of the Forebruin and Lower pcrrts qf’the Bruin Stem. Williams & Wilkins, Baltimore.
Neurotensin afferents to anterior olfactory nucleus
493
13. Krettek J. E. & Price J. L. (1977) Projections from the amygdaloid complex to the cerebral cortex and thahmus in the rat and cat. J. camp. Neural. 172, 687-722. 14. Krettek J. E. & Price J. L. (1978) Amygdaloid projection to subcortical structures within the basal forebrain and brainstem in the rat and cat. J. camp. Neurol. 178, 225-254. 15. Krettek .I. E. & Price J. L. (1978) A description of the amygdaloid complex in the rat and cat with observations on intra-amygdaloid complex in the rat and cat with observations on intra-amygdalod axonal connections. J. camp. Neurol. 178, 255-280. 16. Lazarus L., Brown M. & Perrin M. (1977) Distribution, localization, and characteristics of neurotensin binding sites in the rat brain. Neuropharmacology 16, 625-629. 17. Maeda K. & Frohman L. A. (1978) Dissociation of systemic and central effects of neurotensin on the secretion of growth hormone, prolactin and thyrotropin. Endocrinology 103, 1903. 18. Mesulam M.-M. (1978) Tetramethylbenzidine for horseradish peroxidase neurohistochemistry : a noncarcinogenic blue reaction product with superior sensitivity for visualizing neuronal afferents and efferents. J. Histochem. Cytothem. 26, 106-l 17. 19. Nemeroff C. B., Osbahr A. J., III Manberg P. J., Ervin G. N. & Prange A. J. Jr (1979) Alterations in nociception and
body temperature after intracisternal administration of neurotensin, betaendorphine, other endogenous peptides, and morphine. Proc. natn. Acad. Sci. U.S.A. 76, 5368-5371. 20. Prange A. 3. Jr, Nemeroff C. B. & Loosen P. T. (1978) Behavioral effects of hypothalamic peptides. In Centrally Acting Peptides (ed. Danielli J. F.) pp. 99-l 18. The Macmillan Press, London. 21. Roberts G. W., Crow T. J. & Polak J. M. (1981) Neurotensin: first report of a cortical pathway. Peptides Suppl. 1, 3744. 22. Roberts G. Woodhams P. L., Polak J. M. & Crow T. J. (1982) Distribution of neuropeptides in the limbic system of the rat: the amygdaloid complex. Neuroscience 7, 99-l 3 1. 23. Sherwood N. M. & Timiras P. S. (1970) A Stereotaxic Atlas of the Developing Raf Brain. University of California Press, Berkeley. 24. Uhl G. R., Goodman R. R. & Snyder S. H. (1979) Neurotensin-containing cell bodies, fibers and nerve terminals in the brain stem of the rat; immunohistochemical mapping. Brain Res. 167, 77-91. 25. Uhl G. R. & Snider S. H. (1979) Neurotensin: a neuronal pathway projecting from amygdala through stria terminalis. Brain Res. 161, 522-526. 26. Vijayan E. & McCann S. M. (1980) Effects of substance P and neurotensin release in uioo and in uifro. Life Sci. 26, 321-327.
on growth hormone and thyrotropin
27. Young W. S. III., Ubl G. R. & Kuhar M. J. (1978) Iontophoresis of neurotensin in the area of the locus coeruleus. Brain Res. 150,431-435. 28. Zamboni L. KcDe Martin0 C. (1967) Buffered picric-acid formaldehyde: a new rapid fixative for electron-microscopy. J. Cell Biol. 35, 148A.
(Accepted
16 September 1982)
Abstract-The origin of neurotensin-iike immunoreactive fibers to the anterior olfactory nucleus and nucleus of the diagonal band of Broca of the rat were elucidated experimentally using the indirect immunofluorescence method. Neurotensin-like immunoreactive fibers located in these areas decreased remarkably in numbers on the operated side after the destruction of the ventral part of the endopiriform nucleus and the adjacent prepiriform cortex where numerous cells with neurotensin-like immunoreactivity were detected. This strongly suggests that such cells located in the endopiriform nucleus and the adjacent prepiriform cortex send a neurotensin-Ike projection ipsi~atera~~yto the anterior olfactory nucleus and to the nucleus of the diagonal band of Broca.
Neurotensin
is a basic polypeptide which was isolated
from the hypothalamus and characterized by Carraway & Leeman. 2*3 subsequent radioimmunoassay and immunohistochemjcal studies have suggested that neurotensin is distributed unevenly in the central nervous system, is concentrated in the synaptosomal and microsomal fraction, and binds to a specific receptor. 7,8,9,10,11,16,21,22,24.25,27 However, knowledge of neurotensin-containing neuron tracts in the central nervous system is incomplete, with such tracts having been described only in reports on the amygdaloid neurotensin systemz2824 and cortical neurotensin system. 21 The present study was carried out to see whether the numerous neurotensin fibers located in the anterior olfactory nucleus (OA) and nucleus of the diagonal band of Broca (DB) are supplied by neurotensin-containing neurons in the ventral part of the endopiriform nucleus @NV) and adjacent prepiriform cortex (PF). EXPERIMENTAL
PROCEDURES
Seventy-one young male rats (body weight 30_5Og, about 2-3 weeks old) were used in this study. lmmunohistochemical
study
Experimental animals and preparation of tissue. A total of 42 male rats were used in a immunohistochemical study. Abbreviations: DB, nucleus of the diagonal band of Broca; DB-EN, area between DB and EN; EN, endopiriform nucleus; ENv, ventral part of EN; HRP, horseradish peroxidase; PBS, phosphate buffer saline; PF, prepiriform Cortex; OA, anterior olfactory nucleus.
Three animals were used for the observation of the normal distribution of neurotensin-like immunoreactive structures. The remanning animals were used for the ~xperimentai immunohistochem~cal studies. In 6 animals, hem~transection of the brain just caudal to the DB (at the level of A 5.3 mm of the atlas of Sherwood & Timiras23) were made stereotaxically with a fine knife. In 9 animals, hemi- or total transection of the brain just caudal to the ENv (at the level of A 2.9 mm of the same atlasz3) were also performed in the same way. These knife-cut studies and the horseradish peroxidase (HRP) study (see below) suggested that the origins of neurotensin-like immunoreactive fibers in the OA and OB may be localized in the ENv and PF. Accordingly, in 24 animals, electrolytic lesions (passing an AC current of 1mA for I s through a monopolar electrode) were made stereotaxically in the ENv and PF (A 4.7-3.5 mm, L 3.8-4.8 mm, H 1.7 mm).23 The animals were kept alive for 3-7 days after the operations. All the animals were perfused via the ascending aorta with 50 ml ice-cold saline followed by 300ml of Zamboni’s fixative.” The brains were removed stereotaxically to obtain the same area on both the operated side and the control side. The brains were immersed in the same fixative for 24-48 h and then rinsed for 24-72 h in 0.1 M phosphate buffer pH 7.4 containing 30”/, sucrose. Frontal, sag&al and horizontat serial sections with a section thickness of 1Opm were cut on a cryostat. The slides were pretreated with chrom-alum gelatin to prevent detachment of the sections during incubation. Immune serum. Antiserum against neurotensin was raised in a rabbit following injections of neurotensin-hemocyanin conjugates linked with (l-ethyl-B, 3-dimethylaminopropylfcarbodiimide. The specificity of this antiserum was tested in a radioimmunoassay system and less than 0.01% crossreactivity was found to occur to related peptides such as substance P, enkephalins, endorphins, somatostatin, gluca-
bradykinin
&on.
specificity
and
cholecystokinin
of the antiserum
tion
test at the morphological
The
neurotensin-antiserum
treated
in
stained brain
with
cresyl
regions.
chemistry
The
The
indirect
was
prc-
Half of the sections
were
sections
immunoreactivity
the
the
at IO C wlfh
Triton
rinsed three times Priton
fluorescein
the sections
(15min
Incubated sorbed
in buffer
antiserum
immuno-
with
prro.\-irlasr
Fig. I. A-G. anterior
atmosphere
each) with
absorption
test
olfactory
RESULTS
(0.X jig ml). These
above.
Ittitnut2oki.~tolhrtt~icul
whether or
of the ventral
nucleus
and
part of endopiriform
nucleus
of
study
The specificity of the immunoreaction was checked by comparing sections stained with test.
nucleus
and the adjacent
neurotensin-like
diagonal
band
prepiriform
immunoreactivity
of
Broca.
A-G
Control
side (A). operated
in the horizontal
Control limbs
of the DB.
fibers in the vertical side (The midline
immunoreactive
show
by an arrow).
the electrolytic
Frontal
sections.
Abbreviations B
basal amygdaloid
c
central
CA
commissure
CL
claustrum
DB
nucleus
DB-EN
the area between
ENv
ventral
0 OA
nucleus of the olfactory tract anterior olfactory nucleus
L
lateral
amygdaloid
nucleus
M
medial
amygdaloid
nucleus
NT
neurotensin
PF POM
prepiriform medial
side (E), operated
in these areas after
showing
by arrows).
Control
Note
destruction lesion
Case ENv
of Ihe ENv
of the ENv
used in figures
nucleus
amygdaloid
nucleus
anterior
of the diagonal
band (Broca)
the DB and endopiriform
part of the endopiriform
cortex
preoptic
area
nucleus
nucleus
change
ipsilateral
the change decrease
the adjacent
and the adjacent
x 80, H.
fibers in
side and the right and
x 19
in
in immunoreactive
side (F). G shons
a remarkable
21. A-G.
the
fibers in the OA
in immunoreactlve
side (DI. E and F show the change
limb of the DB. The left side is the control
is indicated
fibers
photomicrograph
side (B). C and D show the change
side (C), operated
cortex
in the ipsilateral
fibers in the OA and DB. A and B show the change in immunoreactive
immunoreactive
Light-field
M~S ah-
decrease of fibers displaying
the OA pars medialis.
number
first
not the ENv and the :idjaccnt PF‘ project to the OA and DB. and whether the distribution of HR P-labeled cells in the ENv and the adjacent PF after ()A or DR injection is similar to the distribution of neurotcnslnlike immunorractive cells obtained bj immunohistochemical analysis. A total of 29 animals were used in the HRP study. A W’,, solution of HRP (Sigma type VI) in ;I ghs micropipette was connected to ;I high-voltage-current source. which provided a pulsed positive current (7s on. alternating with 7 s off ). A single injection of HRP was made stereotaxically using :I positive current 01’ 2.5 ,uA for I5 min. into the OA and DB. Followmg a survival time of 24 h. the brain was pcrfuscd with 1.?I”,, glutaraldehyde 4.W,, paraformaldehl;de in 0.1 M phosphate buffer (pH 7.4). The brains were removed, immersed in the same ti.xativo at 4 (‘ overnight and then rinsed for 2472 h :It 4 c’ in 0.1 M phosphate buffer containing Jo”,, sucrose. Serial frontal sections with a section thickness of 30 /lrn \vcre cut on ;I cryostat. The sections Mere then reacted with 3.3.‘5,5’-tetra-methyl benzidine. ’ a The terminology used was that in the atlases of Kijnig & Kiippel,‘” Krettek & Price’4,” and Sherwood & Timirss.”
Ahsorptiott
Destruction
the operated
(SO”,,)
were
which
neurotensin described
at
PBS at 4 C
.stztrlj~
immunoreactive
fibers
with
(Miles
m a PBS glycerine
the
in a marked
pars dorsalis.
then
Incubated
antlbodies
The HRP method was used to ascertain
resulted
PBS conhere
PBS contaimng
to neurotensin as
were processed
test The
atmosphere
They
and
(I 5 mm
mounted
by excess synthetic
Horsrutrdi.sl~
diluted
1:2000.
for I h in 21 humid
for
groups:
immunoserum.
conjugated
Mere finally with
the
each) in ice cold
rinsed
sections
two
neurotensin-like
at
three rinses
The
into
of neurotensin-like
antiserum
Lid) at I :50 dilution,
mixture.
of Coons’
for 23 h in a humid
isothiocyanate
37 C. Follorting
saline
half for the absorption of
X-100
X-100.
technique
subdivided
specificity
neurotensin
(~.I”,,
buffer
used for the immuno-
demonstration
were incubated
taining
immunohisto-
phosphate
The sections
used for demonstrating
reactivity
for
were of the
10 staining.
and the other
checking
sections
identification
intended
In cold
were further
was used for
sections
exact
immunofluorescence
histochemistry
sections
for
bvere incubated
was used in this study.
Co.
stud!
and alternate
violet
(PBS) for IO min prior
O.l”,,
this
by hemocyamin.’
fttirrlitrtolristo~~~~ttii~~u~ pr~tcduru.
for
The
by the absorp-
level (for results, see beloir). used
used for immunohistochemistry
one
octapeptide.
was also tested
PF
m
side is in the PF.
H:
(indicated
Fig. 1
489
Fig. 2. A-C. Light-field photomicrographs showing an injection site of horseradish peroxidase into the ante) rior olfactory nucleus pars medialis (indicated by an arrow) where the highest density of fi\,ers displ aying neurotensin-like immunoreactivity was seen (A), and HRP-labeled cells in the ventral par t of cortex (B, C) subsequent to HRP injection into the endc bpiriform nucleus and the adjacent prepiriform cells found in the ree ;ion OA pars medialis. Frontal sections. Case ENv 14. In Fig. 2C, HRP-labeled (HRP-labeled cells are shown by arrows). A. x 34. outli ned in Fig. 2B are shown in greater magnification photomicrograph showing neurotensin-immunoreactive cells in the B. x 130, C. x 320. D: Fluorescence ENv and the adjacent PF. Frontal sections. Case ENv 6. x 200. 490
Neurotensin afferents to anterior olfactory nucleus neurotensin-antiserum and absorption control. Since the structures stained with neurotensin-antiserum were not seen in the control sections, the former were considered specific, These materials should correctly be described as showing neurotensin-like immunoreactivity. The specificity of the neurotensin-antiserum obtained by radioimmunoassay was discussed under Experimental Procedures. Effects of knife cuts. As shown in Figs lA, C, E, G, numerous neurotensin-like immunoreactive fibers were found in the OA, particularly in the pars dorsalis (Fig. IA), pars medialis (Fig. 1C) and pars posterior, and also in the DB both in the horizontal (Fig. 1E) and vertical limbs (Fig. 1G). The number of these fibers decreased remarkably on the operated side after unilateral transection of the brain just caudal to the DB (at the level of A 5.3 mm of the atlas of Sherwood & Timirasz3). On the other hand, hemi- or total transection of the brain just caudal to the ENv and the adjacent PF in which numerous neurotensin-like immunoreactive cells exist (Fig. 2D) at the level of A 2.9mm of the same atlas) failed to decrease the number of immunoreactive fibers in these areas. These results strongly suggest that the major source of neurotensin-like immunoreactive fibers in the OA and DB is localized in the forebrain and diencephalic areas between these two levels (DB and ENv) (from the level of A 2.9-5.3 mm). We call this the DB-EN area hereafter. In the DB-EN area, several groups of cells showing neurotensin-like immunoreactivity were found, e.g. numerous cells in the ENv and the adjacent PF, some in the medial preoptic area, bed nucleus of the stria terminalis, hypothalamic paraventricular nucleus and lateral hypothalamus (Fig. 3, right half). Furthermore, some of the immunoreactive cells located in the ENv and the adjacent PF extended rostrally to the lateral part of the anterior amygdaloid area and medially to the amygdaloid complex. The location of immunoreactive cells in the DB-EN area is shown in Fig. 3 (right half).
491
general similar to that seen after HRP injection into OA pars medialis. In addition, in cases in which HRP was injected into the DB (Case ENv 21, 23), a wider distribution of HRP-labeled cells than that seen after HRP injection into OA was identified in the DB-EN area. For example, in addition to HRP labeled groups found subsequent to HRP injection into OA (Fig. 3, left side), the group of central thalamic nuclei was found to be labeled. A comparison of the locations of immunoreactive cells and HRP-labeled cells after injection of HRP into OA (Fig. 3) or DB (Figs 2B-D, 3), suggests that neurotensin-immunoreactive cells located in the ENv and the adjacent PF may project to the OA and DB.
Horseradish peroxidase study
In order to examine afferents to the anterior olfactory nucleus (OA) (Fig. 2A) and nucleus of the diagonal band of Broca (DB), HRP was injected into these two areas, and the locations of HRP labeled cells identified in the DB-EN area were compared with those of cells displaying neurotensin-like immunoreactivity in the same area (Figs 2B-D, 3). In Fig. 3 (left half), the location of HRP-labeled cells is shown in the ENv and the adjacent PF following HRP injection into OA, pars medialis (Fig. 2A) where the highest density of immunoreactive fibers were seen (Fig. 1). In other cases in which HRP was injected into OA pars dorsalis (Case ENv 15, 17) or OA pars posterior (Case ENv 19, 20), or in other OA injected cases (Case ENv 13, 16), the location of HRP-labeled cells found in the DEEN area was in
Fig. 3. Schematic representation of the distribution of cells showing neurotensin (NT)-like immunoreactivity (right side, shown by closed circles) in the DB-EN area and horseradish peroxidase-labeled cells (left side, shown by closed triangles) in the same area after injection of horseradish peroxidase into the anterior olfactory nucleus pars medialis (Case ENv 14) outlined on a stereotaxic atlas of the rat brain. The injection site and HRP-labeled cells in the ENv and the adjacent PF is shown in Figs 2A, B and C. Note that the ENv and the adjacent PF contained both immunoreactive cells and HRP-labeled cells.
iZffe~‘tsof’ the destruction of the uentral part of endopiri,fbrm mcleus und the ad¢ prepiriforvn cortex In cases in which electrolytic destruction included the compIete ENv and the adjacent PF area which contains numerous ~mmunor~~~f~ve cells, changes in the numbers of immunoreactive fibers were striking (Figs IA-H): in other cases where the lesion partly included the ENv and the adjacent PF area, changes in the immunoreactive fibers were more variable. Following the destruction of the ENv and the adjacent PF (Case ENv 22. 25, 28, 31, 37). a remarkable decrease in immunoreactive fibers was found in the ipsilateral OA, particularly in the pars dorsalis, medialis (Figs IA-D) and posterior. and DB both in the horizontal and vertical limbs (Figs 1E-G).
IXXUSSION
Regarding the fiber connections of the neurotensinlike neuron system, there is good evidence for an amygdaloid neurotensin neuron system”2*2s and cortical neurotensin neuron system.2’ LittIe so far is known, however, about other systems containing neurotensin-iike immunoreactive material. The present study clearly demonstrated that cells displaying neurotensin like immunoreactivity, which are located in the ENv and the adjacent PF area, send their axons to the OA and DB. Prev~ous)y, the ascending tract from this area to the olfactory bulb and nucleus of the diagonal band of Broca had been demonstrated by the HRP method
and by the autoradiographic method.” ’ ‘.’ ’ ’ . .I bus. it is probable that one of the main components of thi* ascending system is the neurotensin-contairllng one. It should be noted that f<>llowing transection of the brain just caudal to the anterior olfactory nucleus (OA) or nucleus diagonal band of Brocn if>B). a significant number of immunoreactive fibers was stilt found in the OA and DB. This fact suggests that some of the neurotensin-immunoreactive fibers in the OA and DB are intrinsic, because both in OA and DB. ;\ nt~mber of ~mmunoreactive cells were found.In addition. our previous olltogeneti~~~~ studies’ concerning the neurotensin-containing system have shown that the initial differentiation and development of the neurotensin-containing neuron system in the rat brain occurs during the fetal period before the establishment of normal synaptic transmission. suggesting that the neurotensin-~~~nt~~il~ing neuron system might have some functional roie in the development of the brain in addition to its neurontransmitter or neuromodulator functions. Based upon the present and previous findings. ENv and the adjacent PF is one of the major sources of neurotensin in the anterior olfactory nucleus (OA) and the nucleus of the diagonal band of Broca (DB) and might play an important role in regulation of these olfactory or limbic functions, beside its well known effects. hypothermia. inhibition to nociception etc.‘~4~r’~“‘~‘0~‘s~z~fn addition, the facts that OA and DB are innervated by more than one NT neuron system suggest complex functions of ~euro~e~~jrl in the OA and DB.
REFERENCES Bissette G., Nemeroff C.,B., Soosen P. T., Prange A. J. Jr & Lipton M. A. (1976) Hypothermia and intolerance to cold induced by intracisternal administration of the hypothalamic peptide neurotensin. Nature, Land. 262, 607-609. Carraway R. & Leeman S. E. (1973) The isolation of a new hypothalamic peptide, neurotensin. from bovine hypothalami. .i. hiol. Chem. 248, 6854-6861. Carraway R. & Leeman S. E. (1976) Characterization of radioimmunoassayable neurotensin in the rat. J. hioi. Chern. 25t, 70567052. Clineschmidt B. V. & McGrifiin J. C. (1977) Neurotensin administrated intracisternaily inhibits responsiveness of mice to noxious stimuli. Eur. J. Pharmac. 46, 3955396. Coons A. H. (1953) Fluorescent antibody methods, In General Cytochemicul Methods (ed. Danielli J. F.) pp. 399 -422. Academic Press, New York. De Olmos J.. Hardy I-i. & Heimer L. (1978) The afferent connections of the main and the accessory olfactory bulb formations in the rat: an experimental HRP-study. J. camp. Neural. 181, 213-244. Ham Y.. Shiosaka S., Senba E., Sakanaka M., Inagaki S., Takagi H., Kawai Y., Takatsuki K.. Matsuzaki T. & Tohyama M. (1982) Ontogeny of the neurofensjn-containing neuron system of the rat: immunohistochemical analysis-1. Forebrain and Diencephalon. J. camp. Neural. 208, 177-195. Kahn D., Abram G. M., Zimmerman E. A. & Leeman S. E. (1980) Neurotensin neurons in the rat hypothalamus: an immunocytochemicaf study. Endocrinology 107,47-54. Kataoka K. and Frohman L. A. (1979) Regional distribution of immunoreactive neurotensin in monkey brain. Brfrir~ Res. Bull. 4, 57-60. Kitabgi P., Carraway R., Van Rietschoten J., Granier C., Morgat J. L.. Menez A., Leeman S. & Freychet P. (1977f Neurotensin: specific binding to synaptic membranes from rat brain. Proc. ~atn. Acad. Sci. U.S.A. 74, f846l850. Kobayashi R. M., Brown M. and Vale W. (1977) Regional distribution of neurotensin and somatostatin in the rat brain. Brain Res. 126, 584-588. Kiinig J. F. R. & Klippel R. A. (1963) The Rat Brain. A stereotaxic Atlas of the Forebruin and Lower pcrrts qf’the Bruin Stem. Williams & Wilkins, Baltimore.
Neurotensin afferents to anterior olfactory nucleus
493
13. Krettek J. E. & Price J. L. (1977) Projections from the amygdaloid complex to the cerebral cortex and thahmus in the rat and cat. J. camp. Neural. 172, 687-722. 14. Krettek J. E. & Price J. L. (1978) Amygdaloid projection to subcortical structures within the basal forebrain and brainstem in the rat and cat. J. camp. Neurol. 178, 225-254. 15. Krettek .I. E. & Price J. L. (1978) A description of the amygdaloid complex in the rat and cat with observations on intra-amygdaloid complex in the rat and cat with observations on intra-amygdalod axonal connections. J. camp. Neurol. 178, 255-280. 16. Lazarus L., Brown M. & Perrin M. (1977) Distribution, localization, and characteristics of neurotensin binding sites in the rat brain. Neuropharmacology 16, 625-629. 17. Maeda K. & Frohman L. A. (1978) Dissociation of systemic and central effects of neurotensin on the secretion of growth hormone, prolactin and thyrotropin. Endocrinology 103, 1903. 18. Mesulam M.-M. (1978) Tetramethylbenzidine for horseradish peroxidase neurohistochemistry : a noncarcinogenic blue reaction product with superior sensitivity for visualizing neuronal afferents and efferents. J. Histochem. Cytothem. 26, 106-l 17. 19. Nemeroff C. B., Osbahr A. J., III Manberg P. J., Ervin G. N. & Prange A. J. Jr (1979) Alterations in nociception and
body temperature after intracisternal administration of neurotensin, betaendorphine, other endogenous peptides, and morphine. Proc. natn. Acad. Sci. U.S.A. 76, 5368-5371. 20. Prange A. 3. Jr, Nemeroff C. B. & Loosen P. T. (1978) Behavioral effects of hypothalamic peptides. In Centrally Acting Peptides (ed. Danielli J. F.) pp. 99-l 18. The Macmillan Press, London. 21. Roberts G. W., Crow T. J. & Polak J. M. (1981) Neurotensin: first report of a cortical pathway. Peptides Suppl. 1, 3744. 22. Roberts G. Woodhams P. L., Polak J. M. & Crow T. J. (1982) Distribution of neuropeptides in the limbic system of the rat: the amygdaloid complex. Neuroscience 7, 99-l 3 1. 23. Sherwood N. M. & Timiras P. S. (1970) A Stereotaxic Atlas of the Developing Raf Brain. University of California Press, Berkeley. 24. Uhl G. R., Goodman R. R. & Snyder S. H. (1979) Neurotensin-containing cell bodies, fibers and nerve terminals in the brain stem of the rat; immunohistochemical mapping. Brain Res. 167, 77-91. 25. Uhl G. R. & Snider S. H. (1979) Neurotensin: a neuronal pathway projecting from amygdala through stria terminalis. Brain Res. 161, 522-526. 26. Vijayan E. & McCann S. M. (1980) Effects of substance P and neurotensin release in uioo and in uifro. Life Sci. 26, 321-327.
on growth hormone and thyrotropin
27. Young W. S. III., Ubl G. R. & Kuhar M. J. (1978) Iontophoresis of neurotensin in the area of the locus coeruleus. Brain Res. 150,431-435. 28. Zamboni L. KcDe Martin0 C. (1967) Buffered picric-acid formaldehyde: a new rapid fixative for electron-microscopy. J. Cell Biol. 35, 148A.
(Accepted
16 September 1982)