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Multiple innervation by substance P-containing fibers in the parabrachial area of the rat
Neuroscienee Letters, 33 (1982) 2 7 1 - 2 7 4
271
Elsevier Scientific Put-lishers ~eland Ltd. :;
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INNERVA]rION~BYSUI~TANCE P-CONTAINING FI-KERS
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SAKAN.~(A, KANJI U M E G A K I
SENBA. and
Department of Neuroanatomy, Institute of Higher Nervous Acti~,ity, Osaka University Medical School, 4-3-57 Nakanoshima, Kitaku, Osaka (Japan) (Received July 13th, 1982; Revised version received September :~th, 1982.; Accepted September 29th, 1982)
Key words: fiber cor~nection - substance P - parabrachial area - immurtohistochemistr) - rat
The present study demonsirates that a large number of substartce P (SP) l~bers in the parabrachial area (PB) of the .--atodgilr.ate from at least three sources. The majarlty of SP fibers in the lateral surface of the lateral l~arabrachial area (PBLI) and medial parabrachial erea originate from SP neurons located caudal to the PB, Some of the SP fibers in the PBLI originate from SP neurons located rostral to the PB. SP fibers in the ventral part of the lateral parabraehial area originate from SP cells from the pens at the level of the PB.
It has been previously reported theft the parabrachial area (PB) is involved in various functior~s such as taste cond~:ction and respiration, etc. [3, 6]. In addition, recent immunohistochemical studies have shown that large amounts of substance P (SP)-containing fibers exist in this area [2, 4, 5, 8], suggesting that SP mey play some rolein the function of the PB. Howc~,e.r, little is known as to the origin of these fibers. The present study describes how these fibers may originate from at least three types of SP-containing cells. SP antiserum was produced after immunization of a rabbit with SP--bovine serum albumin conjugate. Specificity of the SP antiserum was determined by radioimmunoassay. The S P •antiserum was shown to cross-react less than 0.1°70 with physalemin: and eledoisin, There was no cross-reaction with other peptides such as enkephalins, endorphins, neurotensin, somatostatin, ACTH, TRH, CCK-8, etc Absorption of antiserum by excessive SP (0.08 p:g/m!) completely eli.minatec. immunostaining, so the structures stained with SP antiserum a~'e considered specific, These:structures should correctly be described as SPqike imn~.unoreactive, and the simplerterm SP! is used in this s~udy. A total of 37 male rats (body weight 30-40 g) we~'c used in ~.[s study. Experimen0304-3940/82/0000-0000/$ 02.75 © 1982 El,;cvicrScientific Publishers )roland Lzd.
272
tal animals were divided into 4 groups. The first group of rats (5 rats) was used for observation of SPI structures in the normal brain. In the other groups o f rats, various operations were conducted under sodium l~ntobarbitone (Nembutal, 40 mg/kg, i.p.) anesthesia. In groups two (14 rats) and three (11 rats), herai- or total transections of the brain just rostralto the PB were made stereotaxically.:In the fourth group of rats (7 rats), total transections of the brain were made simultaneously rostral arid caudal to the PB. All the animals were kept alive for 5-7 days after the operations. All the animals were perfused transcardially with 50 ml of ice-cold saline followed by 300 ml of Zamboni's fixative [11] under sodium pentobarbitone anesthesia (40 mg/kg, i.p.). The brain was removed stereotaxically with a stereotaxic apparatus, immersed in the same fixative for 12 h, and then rinsed in phosphate buffer containing 30e/o sucrose. Serial frontal, sagittal or horizontal sections were cut in a cryostat with a section thickness of 10/an, with careful attention paid to the stereotaxic levels of the sections. After sectioning, the sections were immediately processed for the indirect immunofluorescent technique of Coons [1]. Terminology was based upon the atlases of Liungdahi et al [4], Palkovits and Jacobowitz [7], and Taber [9]. As shown in Figs. IA, B (r:" ]t half) and 2B, numerous SPl Hbers were seen in the PB, particularly in the lateral parabrachial area (PBL). This dense fiber plexus could be further subdivided into two components: one located in the lateral surface of the PBL (PBLI), and the other in the ventral part of the PBL (PBLv) (see Figs. I and 2). SPI fibers located ~n the PBLI extended medially to occupy the medial parabrachial area (PBM) and ventrally to fuse with the SPl fiber meshwork in the PBLv (see Figs. I and 2). Hemitransection of the brain caudal to the PB caused a marked reduction in the number of SPI fibers in the PBL! (Figs. 1A and 2A) and ~BM (Fig. 1A) on the operated side, but failed to dec:ease the SPI fibers in the PBLv (Figs. 1A and 2A, B). The char~ge in the number of SPl fibers in the PBLI and PBM subsequent to hemitransection of the brain caudal to the PB was identified throughout its rostrocaudal extent (Fig. 1A, left halo (from the level of P 15 to P 2.3 mm of the atlas of P,alkovits and Jacobowitz [7]). Total transection of the brain caudal to the PB also caused a similar reduction in bilateral PBLI and PBM compared with a normal rat. These findings strongly suggest that the majority of SPI fibers in the PBL1 and PBM originate from SPI cells caudal to PB. Hemitranseetion of the brain just rostral to the PB resulted in a slight decrease of SPI fibers in the ipsilaterat PBLI, but failed to decrease t h o ~ in the PBM and PBLv (Fig. IB). Furthermore, it should be stressed that the changes detected in the PBLi were confined to the rostral part of the PBL! (Fig. IB) (from the level of Pl.5 to P2.0 mm of tee ~ame atlas described above [7]). Total transection at a similar level also caused a similar reduction of SPI fibers in the bilateral PBLI compared to that ,~een subsequent to the hemitransection of the brain at a similar level. These findings strongly suggest that some of the SPI fibers at the rostral part of the PBLl may originate from SPI cells located rostral to the PB.
273
.y.:.. B v
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CAUDAL-SECTION
A
ROSTRAL-SECTION
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Fig. 1. Schematic drawing showing the~changes of the SPI fibers in the parabrachial area (PB) following hemitransection of the brain just caudal to the PB (A) (case P 7), and rostral to the PB (B) (case P19). Frontal planes. Left half is the operated side, and right the normal side. Hemitran~ection of the brain caudal to the PB resulted in a marked decrease in the number of SPI fibers in the medial PB (PBM) and lateral surface (PBLI) of the lateral PB (PBL) throughout their rostrocaudal extent (A). Hemitransecfion of the brain rostral to the PB resulted in a slight decrease in the number of SP1 fibers in the rostral part of the PBLI (B). Note that the SPI fibers in the ventral part of the PBL (PBLv) remain intact after these sections. Abbreviations: i¢, locus coeruleus; ntm, mesencephalic trigeminal nucleus; nV, trigeminat motor nucleus; PCS, superior eerebellar peduncle.
Fig. 2. Fluorescent photomicrographs showing the chal~ges of the SPI fibers in the PBLI after hemitranseetion of the brain caudal to the PB. Case P 7. Frontal sections. Note a remarkable decrease in the number of SP! fibers in the PBL1 on the operated side (A). B: control side. x 60. Note that SPI fibers in the PBLv remain intact on the operated side. Explanation of the abbreviations see Fig. I.
274
Total transection of the brain simultaneously rustral and caudal to tan PB resulted in a marked decrease of SPl l i a r s in the PBLI and PBM, bt~t failed to decrease the number of SPI fiber.q in the PBLv. It should be noted that the reductioa of SP[ fibers in the PBLI seen, i u ~ ::c~es was m u c h : m ~ : ~ : : ~ i i ~ in ~ cases, such as henri-or total t r ~ Of the brM~ ~ t O ithePR, T h e ! f ~ obtainedfrom simultaneous}:nifeincisionsr ~ and caudalto thePB su~,;~ SPI fiberslocatedin the PBL,, may originatefrom SPI cellsinthe arealocatedbetween these two sections, namely mstral ports at thele~elof PB, ::~;~ Little is known about the f:mctions of PB. However,: ~veralauthors have suggested that PBLv may be relatedto respiratoryfunction [3], wt,~le others ~ a relationship to the taste pathways [6]. The present' study has shown that the SPI fibers located in the PB were supplied by heterogen0us origins ~o that the'majority of SPI fibers in the PBLI and PBM originate from SPI cells located caudai to the PB. Some of the SP'I fibers in the rostral PBL! originate from t h ~ locatedrostral to the PB, and the 3Pl fibers in the PBLv originate mostly from those located at the level of the PB. It is, therefore, most likely that the, SPl fibers locat~ in the PBLv, which is innervated by SPI cells in the pons at the level of the PB, may relate to respiratory function, and the majority of SPl fibers in the PBLI and PBM to the t~ste pathways. It is certain, though, that in order to explore ~he functions of SP in the PB, further careful anatomical, physiological and pharnmcologicai analyses are needed. I Coons, A.H., Fluoresc~t antibody methods. In J.F. Danielli fed.), Genera~.Cytochemical Methods, Academic Press, New Vork, 1958, pp. 399-422. 2 Cuello, A.C. and Kanazawa, !., The distribution of substance P immunoreactive fibers in the rat central nervous system, J. comp. Neurol., 178 (19:18) 129-156. 3 Denavit-Saibi~, M. and Riche, D., Descending input from the pneumotaxic system to the lateral t~piratory nucleus of the medulla. An anatomical study with the horseradish peroxidase technique, l~eurosci. Lett., 6 (1977) 121-126. 4 i.jungdahl, .~., H6kfelt, T., Nilsson, G., Di:stribution of substance P-Uke immunoresctivity in the c~ntral nervous system of the rat-l. Cell bodies and nerve terminals, Neuroscience, 3 (1978) 861-943. 5 Mat,~uzaki~T~, Shiosakt, S,, lnag~.ki, S , i S a k ~ t t i M~, T a k ~ ; : K ~ , S e n ~ E ~ : i : ~ : Y , ami Tohytma, M , , ~ b u t i o n Of 6 Norsten, R., Ttste pathW~to ihypothalatnus a n ~ i ~ l l ~ j ~
M.,
:S.,
~i~!~ol.:,i:i~
O9~6)~ 1 7 ~
-,
mu'~ohistechem[cel analysis - If, Lower br~nstem.Neur~iT(1982) l~!126i": 9 Taber, E., The cytoarchitectureof the brain stem o:~the cat - I. Brain stem nucleiof the cat, ~ cc.~o2.
Neurol., 116 (1961) 27--70. . . . . . 10 Von Euler, U.S. and Pernow, B., Substance P, Nol~elSymposium 37, Ravers P~ess, New Yc~.~, 1975. 11 Zamboni, L. and De Marti:no, C., Buffered picric.acidfor~mldehyd¢: ~t newrapid fixative I~r ¢lec:tron microscopy, I. Celd Biol., 35 (1967) l~.gA.
271
Elsevier Scientific Put-lishers ~eland Ltd. :;
i
.,
" ~!
.
.:
~ ~ L E IN T
mo
'
-
'
i
INNERVA]rION~BYSUI~TANCE P-CONTAINING FI-KERS
tF. :P
S ,
vtJ
" ~ "
r
CH ,i.
" :. :
L XREA .
'
• .
.
.
.
"
SmNOnU.IN^GAZI,SAD.
K E N ! C H I a ' A K A T S U K !, M A S A H I R O MAS v rORrA A
,THE lok: r .
O smos
x.
SAKAN.~(A, KANJI U M E G A K I
SENBA. and
Department of Neuroanatomy, Institute of Higher Nervous Acti~,ity, Osaka University Medical School, 4-3-57 Nakanoshima, Kitaku, Osaka (Japan) (Received July 13th, 1982; Revised version received September :~th, 1982.; Accepted September 29th, 1982)
Key words: fiber cor~nection - substance P - parabrachial area - immurtohistochemistr) - rat
The present study demonsirates that a large number of substartce P (SP) l~bers in the parabrachial area (PB) of the .--atodgilr.ate from at least three sources. The majarlty of SP fibers in the lateral surface of the lateral l~arabrachial area (PBLI) and medial parabrachial erea originate from SP neurons located caudal to the PB, Some of the SP fibers in the PBLI originate from SP neurons located rostral to the PB. SP fibers in the ventral part of the lateral parabraehial area originate from SP cells from the pens at the level of the PB.
It has been previously reported theft the parabrachial area (PB) is involved in various functior~s such as taste cond~:ction and respiration, etc. [3, 6]. In addition, recent immunohistochemical studies have shown that large amounts of substance P (SP)-containing fibers exist in this area [2, 4, 5, 8], suggesting that SP mey play some rolein the function of the PB. Howc~,e.r, little is known as to the origin of these fibers. The present study describes how these fibers may originate from at least three types of SP-containing cells. SP antiserum was produced after immunization of a rabbit with SP--bovine serum albumin conjugate. Specificity of the SP antiserum was determined by radioimmunoassay. The S P •antiserum was shown to cross-react less than 0.1°70 with physalemin: and eledoisin, There was no cross-reaction with other peptides such as enkephalins, endorphins, neurotensin, somatostatin, ACTH, TRH, CCK-8, etc Absorption of antiserum by excessive SP (0.08 p:g/m!) completely eli.minatec. immunostaining, so the structures stained with SP antiserum a~'e considered specific, These:structures should correctly be described as SPqike imn~.unoreactive, and the simplerterm SP! is used in this s~udy. A total of 37 male rats (body weight 30-40 g) we~'c used in ~.[s study. Experimen0304-3940/82/0000-0000/$ 02.75 © 1982 El,;cvicrScientific Publishers )roland Lzd.
272
tal animals were divided into 4 groups. The first group of rats (5 rats) was used for observation of SPI structures in the normal brain. In the other groups o f rats, various operations were conducted under sodium l~ntobarbitone (Nembutal, 40 mg/kg, i.p.) anesthesia. In groups two (14 rats) and three (11 rats), herai- or total transections of the brain just rostralto the PB were made stereotaxically.:In the fourth group of rats (7 rats), total transections of the brain were made simultaneously rostral arid caudal to the PB. All the animals were kept alive for 5-7 days after the operations. All the animals were perfused transcardially with 50 ml of ice-cold saline followed by 300 ml of Zamboni's fixative [11] under sodium pentobarbitone anesthesia (40 mg/kg, i.p.). The brain was removed stereotaxically with a stereotaxic apparatus, immersed in the same fixative for 12 h, and then rinsed in phosphate buffer containing 30e/o sucrose. Serial frontal, sagittal or horizontal sections were cut in a cryostat with a section thickness of 10/an, with careful attention paid to the stereotaxic levels of the sections. After sectioning, the sections were immediately processed for the indirect immunofluorescent technique of Coons [1]. Terminology was based upon the atlases of Liungdahi et al [4], Palkovits and Jacobowitz [7], and Taber [9]. As shown in Figs. IA, B (r:" ]t half) and 2B, numerous SPl Hbers were seen in the PB, particularly in the lateral parabrachial area (PBL). This dense fiber plexus could be further subdivided into two components: one located in the lateral surface of the PBL (PBLI), and the other in the ventral part of the PBL (PBLv) (see Figs. I and 2). SPI fibers located ~n the PBLI extended medially to occupy the medial parabrachial area (PBM) and ventrally to fuse with the SPl fiber meshwork in the PBLv (see Figs. I and 2). Hemitransection of the brain caudal to the PB caused a marked reduction in the number of SPI fibers in the PBL! (Figs. 1A and 2A) and ~BM (Fig. 1A) on the operated side, but failed to dec:ease the SPI fibers in the PBLv (Figs. 1A and 2A, B). The char~ge in the number of SPl fibers in the PBLI and PBM subsequent to hemitransection of the brain caudal to the PB was identified throughout its rostrocaudal extent (Fig. 1A, left halo (from the level of P 15 to P 2.3 mm of the atlas of P,alkovits and Jacobowitz [7]). Total transection of the brain caudal to the PB also caused a similar reduction in bilateral PBLI and PBM compared with a normal rat. These findings strongly suggest that the majority of SPI fibers in the PBL1 and PBM originate from SPI cells caudal to PB. Hemitranseetion of the brain just rostral to the PB resulted in a slight decrease of SPI fibers in the ipsilaterat PBLI, but failed to decrease t h o ~ in the PBM and PBLv (Fig. IB). Furthermore, it should be stressed that the changes detected in the PBLi were confined to the rostral part of the PBL! (Fig. IB) (from the level of Pl.5 to P2.0 mm of tee ~ame atlas described above [7]). Total transection at a similar level also caused a similar reduction of SPI fibers in the bilateral PBLI compared to that ,~een subsequent to the hemitransection of the brain at a similar level. These findings strongly suggest that some of the SPI fibers at the rostral part of the PBLl may originate from SPI cells located rostral to the PB.
273
.y.:.. B v
"~ .. =~';.~.
.t.
I¢
CAUDAL-SECTION
A
ROSTRAL-SECTION
B
Fig. 1. Schematic drawing showing the~changes of the SPI fibers in the parabrachial area (PB) following hemitransection of the brain just caudal to the PB (A) (case P 7), and rostral to the PB (B) (case P19). Frontal planes. Left half is the operated side, and right the normal side. Hemitran~ection of the brain caudal to the PB resulted in a marked decrease in the number of SPI fibers in the medial PB (PBM) and lateral surface (PBLI) of the lateral PB (PBL) throughout their rostrocaudal extent (A). Hemitransecfion of the brain rostral to the PB resulted in a slight decrease in the number of SP1 fibers in the rostral part of the PBLI (B). Note that the SPI fibers in the ventral part of the PBL (PBLv) remain intact after these sections. Abbreviations: i¢, locus coeruleus; ntm, mesencephalic trigeminal nucleus; nV, trigeminat motor nucleus; PCS, superior eerebellar peduncle.
Fig. 2. Fluorescent photomicrographs showing the chal~ges of the SPI fibers in the PBLI after hemitranseetion of the brain caudal to the PB. Case P 7. Frontal sections. Note a remarkable decrease in the number of SP! fibers in the PBL1 on the operated side (A). B: control side. x 60. Note that SPI fibers in the PBLv remain intact on the operated side. Explanation of the abbreviations see Fig. I.
274
Total transection of the brain simultaneously rustral and caudal to tan PB resulted in a marked decrease of SPl l i a r s in the PBLI and PBM, bt~t failed to decrease the number of SPI fiber.q in the PBLv. It should be noted that the reductioa of SP[ fibers in the PBLI seen, i u ~ ::c~es was m u c h : m ~ : ~ : : ~ i i ~ in ~ cases, such as henri-or total t r ~ Of the brM~ ~ t O ithePR, T h e ! f ~ obtainedfrom simultaneous}:nifeincisionsr ~ and caudalto thePB su~,;~ SPI fiberslocatedin the PBL,, may originatefrom SPI cellsinthe arealocatedbetween these two sections, namely mstral ports at thele~elof PB, ::~;~ Little is known about the f:mctions of PB. However,: ~veralauthors have suggested that PBLv may be relatedto respiratoryfunction [3], wt,~le others ~ a relationship to the taste pathways [6]. The present' study has shown that the SPI fibers located in the PB were supplied by heterogen0us origins ~o that the'majority of SPI fibers in the PBLI and PBM originate from SPI cells located caudai to the PB. Some of the SP'I fibers in the rostral PBL! originate from t h ~ locatedrostral to the PB, and the 3Pl fibers in the PBLv originate mostly from those located at the level of the PB. It is, therefore, most likely that the, SPl fibers locat~ in the PBLv, which is innervated by SPI cells in the pons at the level of the PB, may relate to respiratory function, and the majority of SPl fibers in the PBLI and PBM to the t~ste pathways. It is certain, though, that in order to explore ~he functions of SP in the PB, further careful anatomical, physiological and pharnmcologicai analyses are needed. I Coons, A.H., Fluoresc~t antibody methods. In J.F. Danielli fed.), Genera~.Cytochemical Methods, Academic Press, New Vork, 1958, pp. 399-422. 2 Cuello, A.C. and Kanazawa, !., The distribution of substance P immunoreactive fibers in the rat central nervous system, J. comp. Neurol., 178 (19:18) 129-156. 3 Denavit-Saibi~, M. and Riche, D., Descending input from the pneumotaxic system to the lateral t~piratory nucleus of the medulla. An anatomical study with the horseradish peroxidase technique, l~eurosci. Lett., 6 (1977) 121-126. 4 i.jungdahl, .~., H6kfelt, T., Nilsson, G., Di:stribution of substance P-Uke immunoresctivity in the c~ntral nervous system of the rat-l. Cell bodies and nerve terminals, Neuroscience, 3 (1978) 861-943. 5 Mat,~uzaki~T~, Shiosakt, S,, lnag~.ki, S , i S a k ~ t t i M~, T a k ~ ; : K ~ , S e n ~ E ~ : i : ~ : Y , ami Tohytma, M , , ~ b u t i o n Of 6 Norsten, R., Ttste pathW~to ihypothalatnus a n ~ i ~ l l ~ j ~
M.,
:S.,
~i~!~ol.:,i:i~
O9~6)~ 1 7 ~
-,
mu'~ohistechem[cel analysis - If, Lower br~nstem.Neur~iT(1982) l~!126i": 9 Taber, E., The cytoarchitectureof the brain stem o:~the cat - I. Brain stem nucleiof the cat, ~ cc.~o2.
Neurol., 116 (1961) 27--70. . . . . . 10 Von Euler, U.S. and Pernow, B., Substance P, Nol~elSymposium 37, Ravers P~ess, New Yc~.~, 1975. 11 Zamboni, L. and De Marti:no, C., Buffered picric.acidfor~mldehyd¢: ~t newrapid fixative I~r ¢lec:tron microscopy, I. Celd Biol., 35 (1967) l~.gA.