Brm Rexurch Bullem. Vol. 32, pp. 285-291, 1993 0361-9230/93 $6.00 + .OO

Printed in the USA. All rights reserved. Copyright 0 1993 Pergamon Press Ltd.

Distribution of Catecholamines in the Central Nervous System of the Pig

R. K. AGARWAL,* V. K. CHANDNA,? L. R. ENGELKING,$ K. LIGHTBOWNg AND M. S. A. KUMAR*’

Departments of *Anatomy and Cellular Biology, fSurgery and fkfedicine, and the #Division of Laboratory Animal Medicine, Tufts University School of Veterinary Medicine, 200 Westboro Road, North Grafton, MA 015361895

Received 8 January 1993; Accepted 19 March 1993

AGARWAL, R. K., V. K. CHANDNA. L. R. ENGELKING. K. LIGHTBOWN AND M. S. A. KUMAR. Distribufion qf

catecholamrnes in [he central nervous system of’the pig. BRAIN RES BULL 32(3) 285-291, 1993.-The objective of this study was to document, through comprehensive means, normal distribution and concentration of catecholamines in various regions of the CNS of pigs, an increasingly popular animal model used for transgenic manipulation of neural genes. The effects of gonadal steroidal status on this distribution were also assessed by comparing CNS catecholamine concentrations among mature male pigs (boars), immature (gilts) and mature female pigs (sows), and adult male pigs castrated prepuberally (barrows). Dissected tissue samples from the CNS were extracted in 2 N acetic acid, filtered through a 0.2 micron filter, then quantitated by reverse-phase high performance liquid chromatography using a C- 18 reverse phase column with electrochemical detection. In both boars and sows the highest concentrations of norepinephrine (NE) were found in the diencephalic areas and brain stem. Gilts exhibited elevated concentrations of NE in the olfactory bulbs (OB), hypothalamus, pons, and corpus trapezoideum-locus ceruleus (LC) compared to lower concentrations in corresponding areas of sows. Prepuberal castration of the male was associated with significantly lower NE concentrations in the striatum, periaqueductal area (PAG), pons, LC, and spinal cord. The sow exhibited significantly lower NE concentrations in the mammillary area (Mam), PAG, pons, and spinal cord than those in corresponding areas of the boar. Dopamine concentrations appeared to be similar in all areas of the brain and spinal cord studied in the sow and boar. Results demonstrated that prepuberal castration of the male appears to significantly alter the DA content of the Mam and dorsal spinal cord, in contrast to gilts who possess significantly higher concentrations of DA. It is concluded from our studies that in general, catecholamine concentrations in various regions of the brain and spinal cord of sexually mature pigs parallel distributions of neuropeptides, substance P, and methionine enkephalin, as previously reported. In addition, significant association was found between gonadal activity and catecholamine concentrations in discrete areas of the pig brain.

Norepinephrine Dopamine Catecholamines CNS Pig Barrow Gilt Gonadal steroids

NUMEROUS studies have documented important physiological roles played by catecholaminergic neural networks in both central (CNS) and peripheral nervous systems. Mapping catecholamin- ergic systems in the CNS subsequently allowed investigators to study regulatory roles played by catecholaminergic neurons on the endocrine system, various behaviors, and autonomic nervous system control. For many laboratory animals, both basal con- centrations of catecholamines (17) and the effects of various gonadal steroid feedback effects on catecholaminergic systems have been well documented (1,5,9,11). For example, different doses of hormones can facilitate or inhibit catecholaminergic neurotransmission (5,8).

Many peptidergic neural systems are known to regulate CNS catecholaminergic systems (2 1). Although Erlander et al. (6) demonstrated the involvement of brain catecholamines (cortex, cerebellum, olfactory bulb, hypothalamus, thalamus, caudate nucleus, putamen, and substantia nigra) in porcine stress sus-

ceptibility, comprehensive studies have not been performed to document normal concentrations of catecholamines in various areas of the porcine CNS. Although the pig is becoming increas- ingly popular as an animal model for transgenic manipulation of neural genes, there are few studies reported on the concen- trations or distribution of neurotransmitters in the CNS of this species. We have, thus, undertaken a series of comprehensive studies to document basal concentrations of various neurotrans- mitters within the CNS of pigs. In our previous report, we de- scribed the concentrations of Substance P, GnRH, and methi- onine-enkephalin in the porcine CNS (14). As a followup in this study, we describe basal concentrations of catecholamines in similar areas of the CNS. In addition, we also report basal con- centrations of catecholamines in various regions of the CNS of long-term castrated male pigs (barrows), and immature female pigs (gilts) in an attempt to correlate the effects of gonadal steroid status on this distribution.

’ To whom requests for reprints should be addressed

285

286

METHOD

Twenty-three pigs were used in this study: adult males (boars; n = .5), adult females (sows; n = 6), castrated males (barrows; n = 6), and prep&era1 females (gilts; n = 6). All pigs were healthy and were maintained in indoor barns. Six male pigs were cas- trated under general anaesthesia at 3 months of age. A blood sample was collected from each pig before the animal was hu- manely euthanatized with T-6 1 (N-[2-methoxy-phenyll-2-ethyl- butyl-[ I ]-gamma-hydroxy butyramide, National Laboratories Corporation, Kansas City, MO). Gross morphology of the ovaries was recorded (gross size of ovaries, presence/absence of follicular activity, and size of follicles, if present). The heads of each animal were sectioned midsagittaly with a band saw, with the brains further dissected (Fig. 1). The cervical spinal cord was isolated and divided into dorsal (DSC) and ventral (VSC) halves as pre- viously described (9). Brain dissections were accomplished within 15 min after death, with tissue samples isolated from the fol- lowing areas: Olfactory bulb (OB), preoptic area (POA), supra- chiasmatic area (SCA), hypothalamus (Hypo), mammillary area (Mam), periaqueductal area (PAG), pons (PONS), corpus tra- pezoideum locus ceruleus (LC), and striatum (Str). Brain tissues were stored at -80°C until assayed.

Tissues were named in this study according to major ana- tomical areas dissected. Other nuclei and fiber tract systems contained in each tissue region are listed below along with the wet weights of each region (mg; mean + standard deviation): OB, includes anterior olfactory nucleus (14 I .4 -t 19.8); POA. includes medial preoptic area, diagonal band of Broca ( 1 14. I +- 14.6): SCA, includes the suprachiasmatic nucleus and part of the anterior hypothalamic nucleus ( 148.9 f 26.0); Hype, includes the median eminence, ventromedial nucleus of the hypothala-

mus, periventricular, and paraventricular nucleus (90.3 -I 12.2); Mam. includes both lateral and medial mammillary nuclei and the nucleus tuberomammillaris hypothalami ( 158.4 t 14.2): the PAG. also includes the oculomotor nucleus, red nucleus, inter- peduncular nucleus, and the substantia nigra ( 143.5 + 16.8): the Pons, also includes the corticospinal tract ( 146.3 -i: 18.2); the LC also includes the motor nucleus of the trigeminal. olivary and periolivary nucleus. the nucleus reticularis gigantocellularis. and part of the corticospinal tract (226.7 i 14.7). The dorsal spinal cord ( 132.7 t 14.8). ventral spinal cord ( 145. I i 19.2). and the striatum ( 146.6 F 9.0) are dissected based on gross land- marks.

Blood samples collected before euthanasia were spun for 10 min at 1000 X g, with the plasma separated and stored at -80°C. Approximately 2 weeks thereafter, samples were thawed for hor- monal assay. Plasma was extracted as previously described (19). Briefly, I ng ofdihydroxybenzylamine (DHBA: as internal stan- dard) and 30 mg of acid washed alumina (BAS) were added to I ml of serum with the pH adjusted to 8.2 +. 0. I with I .5 M Tris buffer. Tubes were vortexed for 4-5 min and the alumina was allowed to settle. Solutions were washed twice with distilled water (pH 8.0), then alumina-adsorbed catecholamines were eluted with 200 @I of 0. I M perchloric acid (2 K 100 ,uI) and filtered through a 0.2 micron filter at 1000 X .Y. Recovery effi- ciency for plasma catecholamines was 80%.

Tissue catecholamines were extracted with IO ~1 of 2 N acetic acid/mg of tissue, using 2 ng of DHBA/ml of 2 N acetic acid as internal standard. Tissues were quickly homogenized and cen- trifuged with the clear supernatant collected in a fresh tube. The tissue pellet was again homogenized and spun as before. Both supematants were mixed together and filtered two times through

D Str

I

FIG. I. A diagrammatic representation of the pig brain shown in a midsagittal view. The pig head was rapidly sectioned with a band saw and the brain and cervical spinal cord removed and dissected further to yield the following tissue samples: Olfactory bulb (OB); preoptic area (POA); suprachiasmatic area (SCA); hypothalamus (Hypo); mam- milky area (Mam); periaqueductal grey (PAG); pons; locus ceruleus area (LC); dorsal spinal cord (DSC) and ventral spinal cord (VSC).

PIG CNS CATECHOLAMINES 287

CE POA SCA Hype Mam PAG Pons LC DSC VSC Str

Braln Areas

FIG. 2. Catecholamine concentrations (pg/mg; Mean + SEM) in various areas of the boar and barrow brain and spinal cord (refer to Fig. 1 legend for explanation of the abbreviations). Tissues were extracted in 2 N acetic acid and cate- cholamines measured using an HPLC with electrochemical detection. The level of statistical significance is indicated on the corresponding pairs of bars.

0.2 grn microfilters (BAS). Recovery efficiency for CNS tissues was 90%. The filtrate was then stored at -80°C until assayed by HPLC. Extracted catecholamines from plasma and CNS tissue samples were injected onto an HPLC reversed phase column, using a 50 @I sample loop. All samples were injected two or three times, with mean peak heights taken for calculation of cate- cholamine concentrations.

Catecholamines were fractionated over a microsorb reverse phase C-18 column (Rainin) at a flow rate of 0.8 ml/min. The instrumentation was identical to that described by Pate1 and co- workers (19). The mobile phase composition was as follows:

0.01 M NaOH, 0.07 M citric acid (monohydrate), 1.6% (wt./ vol) disodium EDTA, 0.033% (wt./vol) SDS, 0.425% (v/v) di- ethanolamine, 12% (v/v) acetonitrile, with pH adjusted to 3.15. The electrochemical detector fitted with a glassy carbon electrode, was set at f0.75 volt vs. an Ag/AgCl reference electrode (16).

Statistical Analysis

Data were analyzed with the aid of a StatView 5 12 (Brain Power Inc., Calabasas, CA) program. Comparisons between groups of animals were performed by analysis of variance (AN-

1500

T n Gilt

CB POA SCA Hype Mam PAG KM u: DSC VSC Str

FIG. 3. Catecholamine concentrations in various regions of the sow and gilt brain and spinal cord. Refer to Fig. 1 and 2 for explanation of the abbreviations.

288 ACiARWAL El’ Al.

TABLE I

DIFFERENCES IN CAI~ECHOLAMINE CONCENTRATIONS (pg/mg OF I’ISX~E:

MEAN + SEM) BETWEEN THE BOAR AND SOW __~ -.

Boar sow

CNS Area NE DA NE DA

OB 105.1 * 14.4 12.1 t 6.1 96.1 + 13.5 25.4 +- 3. I POA 692.5 + 128.4 276.0 + 83.2 742. I i 152.8 916.8 + 390.3 SCA 579.1 * 105.5 99.3 + 18.1 323.5 + 85.2 109.5 + II.5 HYPE 908.9 + I 15.0 99.3 of- 18.1 599.5 + 126.9 171.6 + 13.8 Mam 484.9 _t 64.3 125.1 f 16.7 232.1 f 23.1* 218.5 -t 65.6 PAG 791.7 t 108.7 227.2 If- 2 1.4 403.1 + 99.2 314.8 + 38.3 Pons 334.6 I? 48.1 34.1 +- 4.3 102.0 t 13.1* 24.1 ?z 6.1 LC 327.8 + 19.2 30.6 + 2.9 114.1 + 21.8* 40.2 + 20.1 DSC 135.4 +- 21.5 39.7 + 7.0 44.7 f 5.8* 26.6 f 7.4 vsc 120.7 ? 23.3 41.6 t 11.5 40.3 .f 6.5* 12.4 * 1.7 str 282.4 t 10. I 36 15.4 ? 674.0 215.5 + 72.0 37 12.4 + 472.3

NE = Norepinephrine; DA = dopamine. * p < 0.05 vs. corresponding NE concentrations in the boar.

OVA), and paired comparisons (e.g., NE concentrations of Boars vs. barrows) were performed using two-tailed t-tests.

RESULTS

Sows in our study exhibited multiple follicles, while gilts had immature ovaries, a finding that corresponded well with serum estradiol and progesterone concentrations. Progesterone and es- tradiol concentrations of gilts were well below detection limits of the assay (progesterone less than 1 &ml plasma and estradiol less than 1 pg/ml plasma). In contrast, sows exhibited high con- centrations of progesterone (14.42 t 7.10 ng/ml, mean + SEM; range: 0.55-25.5 q/ml) and estradioi (85 f 56.28 pgml; mean + SEM; range: 0.7-354.6 pg/ml). Sows used in this study were found to be in different phases of both luteal and follicular stages. Testosterone concentrations of barrows (less than 0.5 ng/ml) were also significantly lower than testosterone concentrations of boars (12.19 f 7.7 &ml; mean + SEM). However, basal serum catecholamine concentrations were similar among the four groups studied (NE 578.71 + 56.87, and E 946.28 f 207.68 pg/ ml; mean f SEM).

Norepinephrine Distribution in the CNS of the Pig

Concentrations of NE were in general high, extending from the POA to the PAG area, and then declined to lower concen- trations in the spinal cord of all animals studied. The NE con- centrations in the olfactory bulb, hypothalamus, pons, and LC areas of the sow were significantly lower than corresponding concentrations in the gilt (Fig. 3). In the boar, NE concentrations were significantly higher in the PAG, pons, LC, spinal cord areas, and the striatum than those in corresponding areas of the barrow.

The NE concentrations were strikingly lower in the striatum of the barrow when compared with NE concentrations in the stria- turn of the boar (Fig. 2). Although concentrations of NE were similar in many areas of the central nervous system of the sow and boar, in certain areas of the central nervous system, NE concentrations of the boar were significantly (p < 0.05) higher than in the sow (i.e., mammillary area, pons, LC area, dorsal, and ventral spinal cords; Table 1).

Dopamine Distribution in the CNS of the Pig

The highest concentrations of dopamine were found, as ex- pected, in the striatum of all pigs examined (Figs. 4B and 5B). The POA, hypothalamus, mammillary area, and PAG also con- tained substantial amounts of DA (Figs. 4B and 5B). The spinal cord, olfactory bulbs, and LC areas contained low concentrations of DA (Figs. 4A and SA). Compared to barrows, DA concen- trations were significantly lower in the mammillary area of the boar (p < 0.05), and significantly higher in the dorsal spinal cord (p < 0.05; Figs. 4A and B). Only the striatum of the sow exhibited significantly lower concentrations of DA 0, < 0.05) compared to corresponding areas of the gilt (Fig. 5A and B). No significant differences in dopamine concentrations were noted between boars and sows across CNS areas examined.

DISCUSSION

We report here for the first time, a comprehensive study on both the distribution and concentration of catecholamines in various areas of the porcine central nervous system as well as the effects of gonadal steroidal status on this distribution. Al- though catecholamine turnover studies have been performed in rats using synthetic chemical blockers such as alpha-methyl par- atyrosine, we did not attempt to measure turnover in pigs because such studies are impractical in large animals.

It is interesting to note that both the distribution and con- centration gradient for catecholamines in the porcine CNS par- allel those of substance P and enkephalin (14). Also, the highest concentrations of dopamine, as expected, were found in the striatum of the pig, which is in agreement with a previously published report (6). The striatum has also been shown to contain the highest concentrations of enkephahns ( 14). Numerous studies indicate extensive interactions among catecholamines, substance P, and enkephalin neural systems in the brain [( 16), review (7)]. Parallel distribution of high concentrations of catecholamines in areas containing high concentrations of substance P and en- kephalins may indicate a mutually interactive catecholamine- peptidergic neural network in the porcine central nervous system.

Significant differences in regional CNS concentrations of NE among male and female pigs were documented for the first time in this study. Barrows exhibited significantly elevated NE con-

PIG CNS CATECHOLAMINES 289

n Barrow

E Boar 80

70

60

50

Pons

Brain Areas

1000

:B 900 -

, W Barrow

W Boar

600

500

400

300

200

100

0 POA SCA Hype Mam PAG Str

Brain Areas

FIG. 4. (A,B) Dopamine concentrations in various regions of the boar and barrow central nervous system. The boar and barrow differed with reference to dopamine concentrations in two areas of the central nervous system. *Dopamine concentration of the striatum is only expressed as pg/O. 1 mg tissue (wet wt.). Refer to Figs. 1 and 2 for explanation of abbreviations.

centrations in brain stem and spinal cord areas compared with ventricles has been associated with significant increases in the boars; whereas in females, gilts generally contained lower con- turnover rate of NE in the hypothalamus and cerebral cortex, centrations of NE in the olfactory bulbs, hypothalamus, and and decreases in dopamine turnover rate in the corpus striatum parts of the brain stem. It is well known that gonadal steroids (18). Chronic treatment with estrogen has also been shown to have profound effects on catecholaminergic neurons, and such inhibit the brain monoamine oxidase system ( 15). In addition, effects are seen in these studies in both hypothalamic and ex- chronic estradiol administration has also been shown to signif- trahypothalamic areas of the pig. Estrogens have been shown to icantly alter striatal dopamine receptor densities (3), and pro- regulate tyrosine hydroxylase activities in the hypothalamus of gesterone has been shown to increase striatal dopaminergic ac- rats (2,12,22), and continuous infusion of estradiol into cerebral tivity (4). Furthermore, immunocytochemical studies have

290 AGAKWAI. I- I‘ Al.

H Gilt

160 - n sow

80

POA HYP Mall PAG Str

Brain Ames

FIG. 5. (A,B) Dopamine concentrations in various regions of the sow and gilt brain and spinal cord. *Dopamine concentrations of striatum only is expressed as pg/O. 1 mg tissue (wet wt.). The dopamine concentrations in the striatum of the sow were significantly lower than those of the gilt. Refer to Figs. 1 and 2 for explanation of abbreviations.

demonstrated colocalization of progesterone receptors and cat- echolamines in the same neurons of the brain (13). Catechol- aminergic neurons may also have the selective ability to con- centrate gonadal steroids ( 10). Unfortunately, there are no reports on the relative distribution of estrogen and androgen receptors in the pig. A recent study indicates that in contrast to estrogen receptor mRNA, androgen receptor mRNA concentrations were high in the brain stem and spinal cord of male rats (20). It would be interesting to correlate the abundance of androgen receptors with catecholamine concentrations in the CNS of the boar. Per- haps the relative abundance of androgen receptors in the brain stem and spinal cord areas of the boar is partly responsible for

higher concentrations of NE observed in these areas (Table I). Generally, the NE concentrations of barrows tended to be higher in brain stem and spinal cord areas compared with corresponding concentrations in the boar. The reason for this remains unclear.

Compared to gilts, NE concentrations in sows were signifi- cantly higher in olfactory bulbs, hypothalamus, and brain stem areas containing the locus ceruleus. These areas have been im- plicated in sexual behaviour and regulation of gonadotropin se- cretion; therefore, differences observed in catacholamine con- centrations of these regions could be taken to indicate a dynamic status of neural control systems during the sexual cycle of the sow.

PIG CNS CATECHOLAMINES 291

Dopamine concentrations, as expected, are quite high in the striatum of male and female pigs. The lack of gonadal steroids seems to affect striatal dopamine concentrations more pro- foundly in the female than in the male pig. Striatal dopaminergic neurons play an important role in coordinating general as well as lordotic motor control, and dopamine turnover in the striatum appears to be sensitive to the regulatory effects of circulating gonadal steroids in mature females (4). In the male pig, long- term castration significantly altered dopamine concentrations only in mammillary and dorsal spinal cord areas (Fig. 4A and B). Because the majority of central nervous system structures

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with detectable androgen receptor populations are thought to be associated with sensory function (20), it is not surprising that the boar differed significantly in dopamine concentrations ob- served in the dorsal spinal cord, which contains sensory afferent fibers.

In summary, we have documented for the first time basal concentrations of catecholamines in various areas of the cen- tral nervous system of the adult male and female pig, and compared those concentrations with concentrations in the immature female, as well as in the long-term castrated adult male pig.

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