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Absence of adrenaline neurons in the guinea pig brain: A combined immunohistochemical and high-performance liquid chromatography study
Neuroscience Letters, 63 (1986) 125-130 Elsevier Scientific Publishers Ireland Ltd.
125
NSL 03716
A B S E N C E OF A D R E N A L I N E N E U R O N S IN T H E G U I N E A PIG BRAIN: A COMBINED IMMUNOHISTOCHEM1CAL AND HIGH-PERFORMANCE LIQUID C H R O M A T O G R A P H Y S T U D Y
P. CUMMING, M. VON KROSIGK, P.B. REINER, E.G. McGEER and S.R. VINCENT* Division qf Neurological Sciences, Department q[' Psychiato', The University 0[" British ('olumhia. Vancouver, B.C. ['67" IW5 (Canada) (Received October 2nd, 1985: Accepted October 8th, 1985)
Key words:
adrenaline adrenaline neuron guinea pig immunohistochemistry hypothalamus
high-performance liquid chromatography
The distributions of catecholamine (CA) neurons in the medulla oblongata of the guinea pig and rat were compared using immunohistochemistry with rabbit antisera against the CA synthesizing enzymes, tyrosine hydroxylase (TH), dopamine-fl-hydroxylase (DflH) and phenylethanolamine-N-methyltransferase (PNMT). TH and DflH distributions were similar in the two species. In contrast, the central nerwms systems of both normal and colchicine-treated guinea pigs failed to demonstrate immunoreactivity for PNMT, the synthetic enzyme for adrenaline. The concentrations of biogenic amines in the hypothalamus and medulla were determined in guinea pig and rat tissues by high-performance liquid chromatography with electrochemical detection. No adrenaline was detected in the guinea pig brain. Thus it appears that guinea pigs lack central neurons capable of synthesizing adrenaline.
Since the first observation of phenylethanolamine-N-methyltransferase (PNMT) activity in mammalian brain [13], the presence of adrenaline neurons has been well documented in the rat [1, 5, 6, 10, 16]. Immunohistochemistry has revealed two distinct groups of PNMT-containing cells in the medulla of the rat. These cell groups, designated C~ and C2 [5], are contiguous with the A~ and A2 noradrenergic groups of Dahlstr6m and Fuxe [3]. The C~ adrenergic cell group is located in the ventrolateral medulla between the lateral reticular nucleus and the inferior olivary complex. C2, a smaller cell group, is found in the dorsomedial medulla in the area postrema, the nucleus of the solitary tract and the dorsal motor nucleus of the vagus. Some cells are also present in the medial longitudinal fasiculus (group C3 of Howe et al. [6]). PNMT-immunoreactive cells have also been observed in the nucleus of the solitary tract in the human brain [11]. In addition, a group of neurons containing P N M T but not tyrosine hydroxylase (TH) immunoreactivity has been observed in the lateral hypothalamus of colchicine-treated rats [17]. In an immunohistochemical study of the distribution of catecholamine (CA) syn-
*Author for correspondence. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
126
thetic enzymes in the central nervous system of the guinea pig (Cavm porcellus), a notable finding was the complete absence of P N M T immunoreactivity in the brain. Moreover, in contrast to results in the rat, no adrenaline could be detected in the guinea pig hypothalamus or medulla oblongata by high-performance liquid chromatography (HPLC) with electrochemical detection (ECD). This paper summarizes our immunohistochemical and biochemical evidence indicating a lack of adrenaline neurons in the guinea pig brain. Young adult male albino guinea pigs (n = 5) weighing about 325 g and albino rats (n = 2) were deeply anesthetized with pentobarbital and perfused with 50 ml heparinized saline followed by either 4~; paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) or buffered picric acid-formaldehyde [20]. One guinea pig received an i.c.v, injection of 80/~g colchicine in 20 Fd normal saline 24 h before perfusion with the aldehyde picric fixative. The brains and adrenal glands were removed and postfixed overnight at 4 C before being placed in 2~"~,,, sucrose with 10°o glycerol for 48 h and then sectioned at 30 ltm on a freezing microtome. The antisera were raised in rabbits against TH, dopamine-fl-hydroxylase (DflH) and P N M T purified from bovine adrenal gland (Eugene Tech Intl., Allendale, N J, U.S.A.). Sections were incubated in primary antisera, diluted !:50 in 0.05 M Trisbuffered saline (TBS) with 0.3% Triton X-100, 2% normal goat serum for 48 h at 4 C with agitation and then carefully washed in TBS before being processed by the avidin-biotin peroxidase method [7] using a commercial kit (Vector Labs., Burlingame, CA, U.S.A.). Rat tissues were processed in parallel with guinea pig sections. For HPLC analysis, rats (n = 3) and guinea pigs (n = 6) were sacrificed by cervical dislocation and the brains rapidly removed and chilled on ice. The hypothalami were dissected by the method of lversen and Glowinski [8]. Brainstems were placed on a microtome and frozen with carbon dioxide. In rats, a 1 mm thick slice was taken just rostral to the obex, while in guinea pigs, a 1.5-mm section was taken to compensate for greater brain size. The dorsomedial portion of each section, containing the region defined as C2, and a ventrolateral portion containing the Ct area were dissected out. The right adrenal glands were also removed. Samples were weighed, rapidly frozen and stored at 70 C. Aromatic amines and metabolites were simultaneously analysed by H P L C - E C D (Millipore; Waters Assoc., Miilford, MA, U.S.A.). Samples were homogenized in 10 vols. of cold 0.1 M H C I O 4 containing 100 ng/ml of the internal standard 3,4-dihydroxybenzylamine. Biogenic amines were separated on a 300 × 3.9 mm #Bondapak reverse-phase Cts column maintained at 2 6 C with a flow-gradient modification of ). Fig. 1. Micrographs of TH, DflH and P N M T immunoreactivity. Guinea pig dorsomedial medulla oblongata at the level of the area postrema (AP) is shown in A, C and E, and the ventrolateral medulla oblongata at the level of the lateral reticular nucleus (LRN) is shown in B, D and F illustrating TH (A and B). DflH (C and D) and P N M T (E and F) immunoreactivities. Note the absence of PNMT-positive perikarya in both regions of the guinea pig brain. PNMT-positive chromaffin cells are present in guinea pig adrenal medulla (G), and PNMT-immunoreactivc neurons are seen in the ventrolateral medulla oblongata of the rat (H). Bars indicate 500 l~m for A, (', E and (;, and 200 ~m for B, D, F, and H. Large arrowhead points dorsally, smaller arrow points medially in F
127
the method described by Cheng and Wooten [2]. Analysis of detector noise and response suggested a minimum detection limit for adrenaline of 7 ng/g wet tissue in the hypothalamus and 5 ng/g in the medulla oblongata. "
B
o
J
E
-
F
AP
LRN
G
H • w* ¸
~¢~
128
In both guinea pig and rat, TH- and D/~H-immunoreactive cells were localized in the area postrema, the nucleus of the solitary tract and the dorsal motor nucleus of the vagus (Fig. IA, C) and in the ventrolateral medulla (Fig. 1B, D). Deeply stained PNMT-immunoreactive perikarya were observed in the ventrolateral (C,) (Fig. I Hi and dorsomedial (C2, C3) medulla of the rat. However, PNMT immunoreactivity was not detected in the corresponding regions of either normal (Fig. 1E, F) or colchicinetreated guinea pigs. Chromaffin cells in the guinea pig adrenal medulla were intensely PNMT-immunoreactive (Fig. 1G). In the three rat brain areas examined adrenaline was readily detectable (Table I). However, the guinea pig brain samples contained no measurable adrenaline. Adrenal glands from the two species contained similar levels of adrenaline, Noradrenaline, dopamine and serotonin levels were similar in the medulla oblongata samples from both species; however, the hypothalamic amine levels were lower in the guinea pig than in the rat (Table I). The PNMT antibody used in this study shows good cross-reactivity with PNMT from a variety of species: human, rat, mouse, cow, chick (Eugene Tech Intl.). In our laboratory, positive staining has been demonstrated in rat, cat, duck and human brain with this antiserum. The presence of strongly immunoreactive chromaffin cells TABLE 1 C O N C E N T R A T I O N S OF A R O M A T I C AMINES IN RAT A N D G U I N E A PIG TISSUES Values are mean+S.E.M., N.D. = a t or below detector limit of 7 ng/g in hypothalamus and 5 ng/g in medulla, n = 6 for guinea pig and 3 for rat tissues. DA, dopamine; NA, noradrenaline; A, adrenaline: 5HT, serotonin. * P < 0.05; **P < 0.01; *** P < 0.001; Student's t-test for differences between species. Tissue weight (rag) Hypothalamus Guinea pig Rat
42-t-4 40 _+7
24+- 3 19+3
5-1tT
246_+28* 371 + 2 2
1588+ 68* 2105+227
N.D. 52+2
690+32** 1044+87
57+_5 54+-4
907_+104 746+ 37
N.D. 14+-1
650_+82 771_+22
1148+130 1092±2,12
N.D. 22+5
609+ 39 755+ 157
25+1"** 84±9
379±21 359+23
-
ng/g wet wt. 12+-4 8+-0
97+22 85±21 /tg/g wet wt.
Adrenal gland Guinea pig Rat
A
ng/g wet wt.
Medulla (C2) Guinea pig Rat
NA
ng/g wet wt.
Medulla (C0 Guinea pig Rat
DA
36_+0.4 29 +- 2
2.0_+0.1 1.9_+0.2
129
in the guinea pig adrenal medulla indicates that guinea pig P N M T does cross-react with this antibody. It might still be argued that P N M T in the guinea pig brain has different antigenic properties than the peripheral enzyme. However, the lack of adrenaline in the guinea pig brain, as demonstrated by HPLC analysis, is consistent with the absence of central PNMT. The levels of adrenaline found in the rat brain are in good agreement with other studies [12, 14]. Central CA neurons are thought to participate in a number of neuroendocrine and autonomic functions. Depletion of hypothalamic adrenaline in the rat is associated with decreased secretion of growth hormone and luteinizing hormone from the anterior pituitary [19] and with increased corticotropin-releasing factor immunoreactivity in the paraventricular nucleus [15]. In the rat medulla oblongata, adrenaline appears to be involved in the control of blood pressure [4, 18]. Immunohistochemical and biochemical studies have demonstrated the absence of both PNMT and its synthetic product adrenaline from guinea pig brain. We conclude that the guinea pig lacks the C~ and C2 adrenergic innervation of the hypothalamus and medulla oblongata present in the rat, therefore illustrating that fundamental neurochemical differences can exist between members of the order Rodentia. Given the important roles which have been ascribed to adrenaline in the rat brain, the present observation points to the danger of over-generalization between species. Supported by grants from the Medical Research Council and the British Columbia Health Care Research Foundation. P.B.R. is a Fellow, and S.R.V. a Scholar of the M.R.C. I Armstrong, D.M., Ross, C.A., Pickel, V.M., Joh, T.H. and Reis, D.J., Distribution ofdopamine, nor-
2 3
4
5 6
7
8 9
adrenaline and adrenaline containing celt bodies in rat medulla oblongata: demonstration by immum~cytochemical localization of catecholamine biosynthetic enzymes, J. Comp. Neurol., 212 (1982) 173 187. Cheng, C.H. and Wooten, G.F., Dopamine turnover estimated by simultaneous LCELC assay of, dopamine and dopamine metabolites, J. Pharmacol. Meth., 8 (1982) 123 134. Dahlslr6m, A. and Fuxe, K., Evidence for the existence ofmonoamine-containing neurones in the central nervous system. I. Demonstration of monoamines in the cell bodies of brainstem neurones, Acta Physiol. Stand., 62, Suppl. 232 (1964) 13 15. Feuerstein, G., Zerbe, R.L., Ben-Ishay, D., Kopin, I. and Jacobowitz, D.M., Catecholamines and vasopressin in hindbrain nuclei of hypertensive prone and resistant rats, Brain Res., 251 (1982) 169 173. H6kfelt, T., Fuxe, K., Goldstein, M. and Johansson, O., lmmunohistochemical evidence for the existence of adrenaline neurones in the rat brain, Brain Res., 66 (1974) 235 251. Howe, P.R.C., Costa, M., Furness, J.B. and Chalmers, J.P., Simultaneous demonstration of phenylethanolamine-N-methyltransferase immunofluorescent and catecholamine fluorescent nerve cell bodies in ral medulla oblongata, Neuroscience, 5 (1980) 2229-2238. Hsu, S.M., Rainc, L. and Fanger, H., The use of avidin biotin peroxidase complex (A.B.C.) immunoperoxidase techniques. A comparison between A.B.C. and unlabeled antibody (PAP) procedures. J. ttislochem. Cytochem., 29 (1981) 577-580. lversen, L.L. and Glowinski, J., Regional studies of calecholamines in various brain regions, J. Neurochem., 13 (1966) 671-682. Kalia, M., Fuxe, K. and Goldstein, M., Rat medulla oblongata. II. Dopaminergic, noradrenergic (AI and A2) and adrenergic neurones, nerve fibres and presumptive terminal processes, J. Comp. Neurol., 233 (1985) 308-332.
130
10 Kalia. M.. Fuxe. K. and Goldstein, nerve libres and presumptive
I I Kitahama.
K., Pearson,
in human
brain
methyltrdnsferase
M.. Rat medulla
terminal
J., Denoroy,
demonstrated (PNMT):
processes.
oblongata,
L., Kopp.
N., Ulrich,
by immunohistochemistry discovery
III. Adrenergic
J. Comp. Neurol.,
(Cl and C2) neurones.
233 (1985) 333- 349.
J. and Maeda,
with antibodies
T., Adrenergic
neurones
to phenylcthanolamine-N-
of a new group in the nucleus tractus
solitariux.
Neurosci.
Lett
53 (1985) 303_~308. I3 Koslow.
S.H. and Schlumpf.
mentography,
13 McGcer.
Nature
M.. Quantitation
(London),
P.L. and McGecr.
Commun.. 14 Mefford.
17(1964)
E.G..
following
Formation
of adrenaline
J.D., Changes
acute administration
by brain
tissue, Biochem.
in rat paravcntricular
in rat hypothalamic
ofa PNMT
15 Mezay. E.. Kiss. J.Z.. Skirboll. C.R.. Goldstein, factor staining
in rat brain nuclei and areas by mass fragBiophys.
Rcs.
502~507.
I.. Roth, K.A. and Barchas.
concentrations
ofadrenaline
I (1974) 630-63 I
25
inhibitor,
M. and Axelrod,
neurones
by depletion
and brainstem
Neurosci.
catecholamme
Lett.. 20 (1980) 67 71.
J., Increase of corticotropin
of hypothalamic
adrenaline,
releasmg
Nature
(Lon-
don), 310 (1984) 140~~141. I6 Ross, C.A.. Armstrong,
D.M.,
Ruggierdo.
neurones
in the rostra1 ventrolateral
chemical
and rctrogrdde
I7 Ross, C.A.. neurones
Ruggierdo.
transport D.A.,
D.A., Pickel, V.M.. Joh, T.H. and Reis, D.J.. Adrenaline
medulla
innervate
demonstration,
Meely, M.P.. Park,
in hypothalamus
contain
PNMT
J.H.. Brainstem
adrenergic
the thoracic
Ncurosci.
cord: a combined
immunohisto-
Lett., 25 (1981) 257~~262.
D.H., Joh. T.H. and Reis, D.J.. A new group
but not tyrosine
hydroxylase,
ot
Brain Res.. 306 (1984) 349
353. IX Saavedra,
(Eds.), Central gamon
Adrenaline
Press. New York.
19 Terry. L.C., Crowley, in regulation 20 Zamboni, microscopy,
Neurones.
neurones.
B. Hokfelt
and T. liiikfelt Function,
Pcr-
1980. pp. I9 47.
W.R.. Lynch. C.. Longserre,
of anterior
In K. Fuxe, M. Goldstein,
Basic Aspects and Their Role in Cardiovascular
pituitary
L. and DeMartino,
hormone
C., Buffered
J. (‘ell Biol., 35 (1967) 14Xa.
C. and Johnson,
secretion,
Peptides,
M.D., Role of central epinephrine
3 (1982) 31 l-318.
picric acid-formaldehyde:
a new rapid fixative for electron
125
NSL 03716
A B S E N C E OF A D R E N A L I N E N E U R O N S IN T H E G U I N E A PIG BRAIN: A COMBINED IMMUNOHISTOCHEM1CAL AND HIGH-PERFORMANCE LIQUID C H R O M A T O G R A P H Y S T U D Y
P. CUMMING, M. VON KROSIGK, P.B. REINER, E.G. McGEER and S.R. VINCENT* Division qf Neurological Sciences, Department q[' Psychiato', The University 0[" British ('olumhia. Vancouver, B.C. ['67" IW5 (Canada) (Received October 2nd, 1985: Accepted October 8th, 1985)
Key words:
adrenaline adrenaline neuron guinea pig immunohistochemistry hypothalamus
high-performance liquid chromatography
The distributions of catecholamine (CA) neurons in the medulla oblongata of the guinea pig and rat were compared using immunohistochemistry with rabbit antisera against the CA synthesizing enzymes, tyrosine hydroxylase (TH), dopamine-fl-hydroxylase (DflH) and phenylethanolamine-N-methyltransferase (PNMT). TH and DflH distributions were similar in the two species. In contrast, the central nerwms systems of both normal and colchicine-treated guinea pigs failed to demonstrate immunoreactivity for PNMT, the synthetic enzyme for adrenaline. The concentrations of biogenic amines in the hypothalamus and medulla were determined in guinea pig and rat tissues by high-performance liquid chromatography with electrochemical detection. No adrenaline was detected in the guinea pig brain. Thus it appears that guinea pigs lack central neurons capable of synthesizing adrenaline.
Since the first observation of phenylethanolamine-N-methyltransferase (PNMT) activity in mammalian brain [13], the presence of adrenaline neurons has been well documented in the rat [1, 5, 6, 10, 16]. Immunohistochemistry has revealed two distinct groups of PNMT-containing cells in the medulla of the rat. These cell groups, designated C~ and C2 [5], are contiguous with the A~ and A2 noradrenergic groups of Dahlstr6m and Fuxe [3]. The C~ adrenergic cell group is located in the ventrolateral medulla between the lateral reticular nucleus and the inferior olivary complex. C2, a smaller cell group, is found in the dorsomedial medulla in the area postrema, the nucleus of the solitary tract and the dorsal motor nucleus of the vagus. Some cells are also present in the medial longitudinal fasiculus (group C3 of Howe et al. [6]). PNMT-immunoreactive cells have also been observed in the nucleus of the solitary tract in the human brain [11]. In addition, a group of neurons containing P N M T but not tyrosine hydroxylase (TH) immunoreactivity has been observed in the lateral hypothalamus of colchicine-treated rats [17]. In an immunohistochemical study of the distribution of catecholamine (CA) syn-
*Author for correspondence. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
126
thetic enzymes in the central nervous system of the guinea pig (Cavm porcellus), a notable finding was the complete absence of P N M T immunoreactivity in the brain. Moreover, in contrast to results in the rat, no adrenaline could be detected in the guinea pig hypothalamus or medulla oblongata by high-performance liquid chromatography (HPLC) with electrochemical detection (ECD). This paper summarizes our immunohistochemical and biochemical evidence indicating a lack of adrenaline neurons in the guinea pig brain. Young adult male albino guinea pigs (n = 5) weighing about 325 g and albino rats (n = 2) were deeply anesthetized with pentobarbital and perfused with 50 ml heparinized saline followed by either 4~; paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) or buffered picric acid-formaldehyde [20]. One guinea pig received an i.c.v, injection of 80/~g colchicine in 20 Fd normal saline 24 h before perfusion with the aldehyde picric fixative. The brains and adrenal glands were removed and postfixed overnight at 4 C before being placed in 2~"~,,, sucrose with 10°o glycerol for 48 h and then sectioned at 30 ltm on a freezing microtome. The antisera were raised in rabbits against TH, dopamine-fl-hydroxylase (DflH) and P N M T purified from bovine adrenal gland (Eugene Tech Intl., Allendale, N J, U.S.A.). Sections were incubated in primary antisera, diluted !:50 in 0.05 M Trisbuffered saline (TBS) with 0.3% Triton X-100, 2% normal goat serum for 48 h at 4 C with agitation and then carefully washed in TBS before being processed by the avidin-biotin peroxidase method [7] using a commercial kit (Vector Labs., Burlingame, CA, U.S.A.). Rat tissues were processed in parallel with guinea pig sections. For HPLC analysis, rats (n = 3) and guinea pigs (n = 6) were sacrificed by cervical dislocation and the brains rapidly removed and chilled on ice. The hypothalami were dissected by the method of lversen and Glowinski [8]. Brainstems were placed on a microtome and frozen with carbon dioxide. In rats, a 1 mm thick slice was taken just rostral to the obex, while in guinea pigs, a 1.5-mm section was taken to compensate for greater brain size. The dorsomedial portion of each section, containing the region defined as C2, and a ventrolateral portion containing the Ct area were dissected out. The right adrenal glands were also removed. Samples were weighed, rapidly frozen and stored at 70 C. Aromatic amines and metabolites were simultaneously analysed by H P L C - E C D (Millipore; Waters Assoc., Miilford, MA, U.S.A.). Samples were homogenized in 10 vols. of cold 0.1 M H C I O 4 containing 100 ng/ml of the internal standard 3,4-dihydroxybenzylamine. Biogenic amines were separated on a 300 × 3.9 mm #Bondapak reverse-phase Cts column maintained at 2 6 C with a flow-gradient modification of ). Fig. 1. Micrographs of TH, DflH and P N M T immunoreactivity. Guinea pig dorsomedial medulla oblongata at the level of the area postrema (AP) is shown in A, C and E, and the ventrolateral medulla oblongata at the level of the lateral reticular nucleus (LRN) is shown in B, D and F illustrating TH (A and B). DflH (C and D) and P N M T (E and F) immunoreactivities. Note the absence of PNMT-positive perikarya in both regions of the guinea pig brain. PNMT-positive chromaffin cells are present in guinea pig adrenal medulla (G), and PNMT-immunoreactivc neurons are seen in the ventrolateral medulla oblongata of the rat (H). Bars indicate 500 l~m for A, (', E and (;, and 200 ~m for B, D, F, and H. Large arrowhead points dorsally, smaller arrow points medially in F
127
the method described by Cheng and Wooten [2]. Analysis of detector noise and response suggested a minimum detection limit for adrenaline of 7 ng/g wet tissue in the hypothalamus and 5 ng/g in the medulla oblongata. "
B
o
J
E
-
F
AP
LRN
G
H • w* ¸
~¢~
128
In both guinea pig and rat, TH- and D/~H-immunoreactive cells were localized in the area postrema, the nucleus of the solitary tract and the dorsal motor nucleus of the vagus (Fig. IA, C) and in the ventrolateral medulla (Fig. 1B, D). Deeply stained PNMT-immunoreactive perikarya were observed in the ventrolateral (C,) (Fig. I Hi and dorsomedial (C2, C3) medulla of the rat. However, PNMT immunoreactivity was not detected in the corresponding regions of either normal (Fig. 1E, F) or colchicinetreated guinea pigs. Chromaffin cells in the guinea pig adrenal medulla were intensely PNMT-immunoreactive (Fig. 1G). In the three rat brain areas examined adrenaline was readily detectable (Table I). However, the guinea pig brain samples contained no measurable adrenaline. Adrenal glands from the two species contained similar levels of adrenaline, Noradrenaline, dopamine and serotonin levels were similar in the medulla oblongata samples from both species; however, the hypothalamic amine levels were lower in the guinea pig than in the rat (Table I). The PNMT antibody used in this study shows good cross-reactivity with PNMT from a variety of species: human, rat, mouse, cow, chick (Eugene Tech Intl.). In our laboratory, positive staining has been demonstrated in rat, cat, duck and human brain with this antiserum. The presence of strongly immunoreactive chromaffin cells TABLE 1 C O N C E N T R A T I O N S OF A R O M A T I C AMINES IN RAT A N D G U I N E A PIG TISSUES Values are mean+S.E.M., N.D. = a t or below detector limit of 7 ng/g in hypothalamus and 5 ng/g in medulla, n = 6 for guinea pig and 3 for rat tissues. DA, dopamine; NA, noradrenaline; A, adrenaline: 5HT, serotonin. * P < 0.05; **P < 0.01; *** P < 0.001; Student's t-test for differences between species. Tissue weight (rag) Hypothalamus Guinea pig Rat
42-t-4 40 _+7
24+- 3 19+3
5-1tT
246_+28* 371 + 2 2
1588+ 68* 2105+227
N.D. 52+2
690+32** 1044+87
57+_5 54+-4
907_+104 746+ 37
N.D. 14+-1
650_+82 771_+22
1148+130 1092±2,12
N.D. 22+5
609+ 39 755+ 157
25+1"** 84±9
379±21 359+23
-
ng/g wet wt. 12+-4 8+-0
97+22 85±21 /tg/g wet wt.
Adrenal gland Guinea pig Rat
A
ng/g wet wt.
Medulla (C2) Guinea pig Rat
NA
ng/g wet wt.
Medulla (C0 Guinea pig Rat
DA
36_+0.4 29 +- 2
2.0_+0.1 1.9_+0.2
129
in the guinea pig adrenal medulla indicates that guinea pig P N M T does cross-react with this antibody. It might still be argued that P N M T in the guinea pig brain has different antigenic properties than the peripheral enzyme. However, the lack of adrenaline in the guinea pig brain, as demonstrated by HPLC analysis, is consistent with the absence of central PNMT. The levels of adrenaline found in the rat brain are in good agreement with other studies [12, 14]. Central CA neurons are thought to participate in a number of neuroendocrine and autonomic functions. Depletion of hypothalamic adrenaline in the rat is associated with decreased secretion of growth hormone and luteinizing hormone from the anterior pituitary [19] and with increased corticotropin-releasing factor immunoreactivity in the paraventricular nucleus [15]. In the rat medulla oblongata, adrenaline appears to be involved in the control of blood pressure [4, 18]. Immunohistochemical and biochemical studies have demonstrated the absence of both PNMT and its synthetic product adrenaline from guinea pig brain. We conclude that the guinea pig lacks the C~ and C2 adrenergic innervation of the hypothalamus and medulla oblongata present in the rat, therefore illustrating that fundamental neurochemical differences can exist between members of the order Rodentia. Given the important roles which have been ascribed to adrenaline in the rat brain, the present observation points to the danger of over-generalization between species. Supported by grants from the Medical Research Council and the British Columbia Health Care Research Foundation. P.B.R. is a Fellow, and S.R.V. a Scholar of the M.R.C. I Armstrong, D.M., Ross, C.A., Pickel, V.M., Joh, T.H. and Reis, D.J., Distribution ofdopamine, nor-
2 3
4
5 6
7
8 9
adrenaline and adrenaline containing celt bodies in rat medulla oblongata: demonstration by immum~cytochemical localization of catecholamine biosynthetic enzymes, J. Comp. Neurol., 212 (1982) 173 187. Cheng, C.H. and Wooten, G.F., Dopamine turnover estimated by simultaneous LCELC assay of, dopamine and dopamine metabolites, J. Pharmacol. Meth., 8 (1982) 123 134. Dahlslr6m, A. and Fuxe, K., Evidence for the existence ofmonoamine-containing neurones in the central nervous system. I. Demonstration of monoamines in the cell bodies of brainstem neurones, Acta Physiol. Stand., 62, Suppl. 232 (1964) 13 15. Feuerstein, G., Zerbe, R.L., Ben-Ishay, D., Kopin, I. and Jacobowitz, D.M., Catecholamines and vasopressin in hindbrain nuclei of hypertensive prone and resistant rats, Brain Res., 251 (1982) 169 173. H6kfelt, T., Fuxe, K., Goldstein, M. and Johansson, O., lmmunohistochemical evidence for the existence of adrenaline neurones in the rat brain, Brain Res., 66 (1974) 235 251. Howe, P.R.C., Costa, M., Furness, J.B. and Chalmers, J.P., Simultaneous demonstration of phenylethanolamine-N-methyltransferase immunofluorescent and catecholamine fluorescent nerve cell bodies in ral medulla oblongata, Neuroscience, 5 (1980) 2229-2238. Hsu, S.M., Rainc, L. and Fanger, H., The use of avidin biotin peroxidase complex (A.B.C.) immunoperoxidase techniques. A comparison between A.B.C. and unlabeled antibody (PAP) procedures. J. ttislochem. Cytochem., 29 (1981) 577-580. lversen, L.L. and Glowinski, J., Regional studies of calecholamines in various brain regions, J. Neurochem., 13 (1966) 671-682. Kalia, M., Fuxe, K. and Goldstein, M., Rat medulla oblongata. II. Dopaminergic, noradrenergic (AI and A2) and adrenergic neurones, nerve fibres and presumptive terminal processes, J. Comp. Neurol., 233 (1985) 308-332.
130
10 Kalia. M.. Fuxe. K. and Goldstein, nerve libres and presumptive
I I Kitahama.
K., Pearson,
in human
brain
methyltrdnsferase
M.. Rat medulla
terminal
J., Denoroy,
demonstrated (PNMT):
processes.
oblongata,
L., Kopp.
N., Ulrich,
by immunohistochemistry discovery
III. Adrenergic
J. Comp. Neurol.,
(Cl and C2) neurones.
233 (1985) 333- 349.
J. and Maeda,
with antibodies
T., Adrenergic
neurones
to phenylcthanolamine-N-
of a new group in the nucleus tractus
solitariux.
Neurosci.
Lett
53 (1985) 303_~308. I3 Koslow.
S.H. and Schlumpf.
mentography,
13 McGcer.
Nature
M.. Quantitation
(London),
P.L. and McGecr.
Commun.. 14 Mefford.
17(1964)
E.G..
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