Changes in central cholinergic neurons in the spontaneously hypertensive rat

Brain Research, 188 (1980) 425-436 © Elsevier/North-Holland Biomedical Press

425

C H A N G E S IN C E N T R A L C H O L I N E R G I C N E U R O N S IN T H E S P O N T A N E O U S L Y HYPERTENSIVE RAT

CINDA J. HELKE, ERIC A. MUTH and DAVID M. JACOBOWITZ Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Md. 20205 (U.S.A.)

(Accepted September 27th, 1979) Key words: acetylcholine- - choline acetyltransferase- - central cholinergic neurons - - spontaneously

hypertensive rats - - hypertension

SUMMARY The activity of choline acetyltransferase (CHAT) was measured in discrete areas of the brain in 4-, 8- and 12-week-old spontaneously hypertensive rats (SH rats) and age matched Wistar Kyoto (WKY rats) controls. The concentration of acetylcholine (ACh) was also measured in certain hindbrain nuclei of 12 week SH and W K Y rats. An increase in the ChAT activity and ACh concentration in the locus coeruleus was detected in 12-week-old SH rats. Decreases in the ChAT activity were found in several hypothalamic nuclei of SH rats, specifically in the paraventricular nucleus of 4week-old rats, in the dorsomedial nucleus at 8 and 12 weeks and in the posterior hypothalamic nucleus at 12 weeks. Changes in ChAT activity were also detected in 4and 8-week-old SH rats in the anterior ventral thalamus and in the nucleus gigantocellularis. These results suggest that cholinergic nerve activity in certain rat brain areas, several of which play a role in cardiovascular control, is altered in spontaneously hypertensive rats.

INTRODUCTION In recent years the role of the sympathetic nervous system in the development of hypertension in spontaneously hypertensive rats (SH rats) has been well established lz, 19,~z,z4. Likewise, evidence has accumulated that the central nervous system is responsible for the increased sympathetic activity seen in SH ratslt,16, 4°. However, with the exception of the catecholamine systems~O,16,3a,4a,46, the participation of CNS neurotransmitter systems in the elevated blood pressure of SH rats has not been thoroughly investigated.

426 Recent pharmacological studies demonstrate that CNS cholinergic neurons and receptors may be involved in blood pressure control. Intracerebroventricular injections of acetylcholine (ACh), cholinergic agonists and cholinesterase inhibitors in rats result in an elevation of blood pressure3,4,t7, 2n which is in part mediated by the sympathetic nervous system 26. Endogenous acetylcholine may also be important since the ability of cholinesterase inhibitors to raise blood pressure is dependent upon intact acetylcholine stores4, ~. Other data also suggest that central cholinergic mechanisms may be altered in SH rats. The pressor response to centrally administered carbachol is greater in these animals than in their normotensive controlslS, 50. In addition, Yamori4a, 50 reported elevated levels of choline acetyltransferase (CHAT) and acetylcholinesterase in the hindbrain of SH rats. These findings lead Yamori 49 to speculate that the central pressor mechanism in SH rats is cholinergic. The purpose of this study was to investigate whether biochemical differences in cholinergic neurons of discrete brain nuclei exist between 4-, 8- and 12-week-old SH rats and their age-matched normotensive controls. This was done by measuring the activity of the ACh synthesizing enzyme, choline acetyltransferase, and in a few nuclei, the ACh concentration. METHODS Male spontaneously hypertensive Okamoto-Aoki strain rats (SH rats) and corresponding age-matched normotensive Wistar-Kyoto controls (WKY rats) were obtained from Taconic Farms (Germantown, N.Y.). The rats (4, 8 and 12 weeks old) were caged in groups of 6 and kept in a 12 h light/dark schedule. Food and water were available ad libitum. The animals were housed in the laboratory for 6 days before sacrifice. Systolic blood pressure measurements (SBP) were made by the indirect tail-cuff method on conscious rats on two consecutive days. The animals were placed in a heated restraining device (Narco Bio Systems, Houston, Texas) for approximately 10 min prior to SBP determination. The arterial pulse was detected by a pneumatic pulsetransducer (Narco Bio Systems) and displayed on a Harvard recorder. Five pressure measurements were recorded for each rat and the median of these readings taken as the rat's SBP. Heart rate was obtained from the pulse tracings. All animals were killed between 09.00 and 12.00 h. Tissue to be assayed for ChAT activity was obtained from rats killed by decapitation. Tissue to be analyzed for ACh content was obtained from rats killed by focused microwave irradiation (2.0-2.5 sec) using a Gerling Moore Unit with a metabostat controller (Model 4094) and with the microwave power source set in the high power mode (3.5 kW). Tissue sectioning and microdissection Both fresh and microwave-fixed brains were frozen on dry-ice and sectioned (300 #m) in a cryostat at about --8 °C. Alternate 60 # m sections were stained with thionine for reference. Brain regions were dissected from the 300 # m frozen coronal sections using the

427

Fig. 1. Coronal sections of rat brain from atlas of Jacobowitz and Palkovits 23and Palkovits and Jacobowitz42. The approximate location of regions dissected and cannula sizes are shown by the black circles. Abbreviations are defined in Table I.

micropunch method described by Palkovits 41. Cannulae of 1, 0.75, 0.5 and 0.3 m m internal diameter were used to remove brain regions at the approximate coordinates shown in Fig. 1. C h A T assay Tissue was homogenized by sonication in 25 #1 of 10 m M E D T A (pH 7.4) containing 0.2 ~ Triton X-100. Aliquots of 5 or 10 pl were taken for protein analysis by the method of Lowry et al. zl. ChAT activity was then assayed by the method of Fonnum 13. Samples were incubated for 10 min (hypoglossal nucleus, interpeduncular nucleus, caudate-putamen) or 30 min (all other samples) at 37 °C and p H 7.4. Acetyl [1-14C]coenzyme A was obtained from New England Nuclear (Boston, Mass.). This assay has a sensitivity of approximately 15 pmol/h. ACh assay Because of the difficulties and inconsistencies we encountered in our attempts to obtain an adequate microwave fixation of the hindbrain, the inactivation of acetylcholinesterase (ACHE) in these tissues was confirmed prior to ACh analysis. To do this, AChE activity was assayed as previously described 20 in the hypoglossal and facial nuclei of all the microwave-fixed brain. Tissues from those animals which exhibited

428 TABLE [ ChAT activity in discrete nuclei o f S H and W K Y rats

Data are expressed as the mean i S.E.M. of individual values obtained from the number of rats shown Area

N. accumbens N. caudatus-putamen N. sept± N. tractus diagonalis N. interstitialis striae terminalis (pars ventral±s) Cingulate cortex Dorsal hippocampus N. preopticus medialis N. periventricularis hypothalamis N. hypothalamicus anterior N. supraopticus N. paraventricularis N. ventromedialis N. arcuatus Median eminence N. dorsomedialis N. hypothalamicus posterior N. perifornicalis N. mamillaris medialis N. premamillaris dorsalis N. anterior ventralis thalami N. habenulae N. amygdaloideus lateralis Area tegmentalis ventralis Tsai N. interpeduncularis N. cuneiformis N. tegmenti dorsalis lateralis N. tegmenti dorsalis Gudden Locus coeruleus N. prepositus hypoglossi N. reticularis parvocellularis N. reticularis gigantocellularis N. reticularis lateralis N. reticularis paramedianus N. cuneatuslateralis N. ambiguus - - rostra l N. ambiguus - - medial N. ambiguus - - caudal N. originis dorsalis vagi N. originis nervi hypoglossi N. tractus solitarii - - medial N. tractus solitarii - - caudal N. intercalatus and N. commisuralis * Abbreviations are those used in Fig. 1. ** P < 0.05.

Abbreviations*

AC CP S TD ST CC DH PM PE AH SO PVN VM AR ME DM PH PF MM PMD TAV H AL TV IP C TDL NTD LC PH RPC RG RL NRP CL NA-r NA-m NA-c X X! I TS-r TS-c IC

Number punches/ rat

2 1 2 2 2 1 1 1 2 2 2 2 2 4 3 2 2 4 l 2 2 1 2 2 2 2 2 2 4 2 2 2 2 2 2 2 4 4 8 2 8 8 1

ChAT activity 4-week-old W K Y rats

187.4&28.2 (7) 426.2 dz 10.6 (9) 50.5&4.7 (9) 317.7 & 27.4 (9) 3 6 . 7 i 3 . 8 (7) 82.5±3.3 (9) 76.0~4.5 (9) 27.1 dz2.3 (7) 25.0±2.3 (9) 29.3±3.1 (9) 160.0~ 16.9 (8) 27.3 ~3.9 (9) 28.8 ±5.0 (9) 30.7±2.8 (9) 100.2±4.1 (7) 32.0+2.9 (9) 44.9-J 5.9 (9) 45.2±3.5 (9) 22.9:~1.3 (8) 16.8~1.5 (8) 129.2 ± 13.1 (9) 375.5±24.8 (9) 244.0 ± 22.6 (9) 83.1 ± 12.4 (7) 1708.8~62.3 (9) 141.1 ± 22.8 (9) 444.4i28.6 (8) 91.3 ± 13.1 (9) 87.8±9.9 (10) 74.7±5.0 (8) 87.8 ±7.5 (9) 93.1 ±6.6 (9) 91.9±4.9 (9) 148.1 ± 15.2 (9) 65.5±5.1 (10) 316.8 ± 46.5 (9) 230.2 ± 27.0 (9) -794.2±85.5 (9) 1022.3 ± 114.3 (9) 204.0 ± 18.7 (8) 221.6 ± 24.3 (8) 186.1 ± 19.1 (9)

429

in parentheses. (pmolAChformed/#gprotein/h) 8-week-old SHrats

152.5±24.1 (8) 450.0± 18.6 (9) 51.14-4.8 (9) 326.94-24.2 (8) 31.54-3.1 (8) 93.4±6.1 (9) 85.44-7.6 (9) 28.2+4.2 (8) 22.62_1.5 (9) 29.34-2.4 (7) 206.6t34.8(9) 17.6±1.2"*(8) 25.84-2.7 (9) 29.74-2.2 (8) 112.8±5.5 (8) 32.12_2.4 (9) 47.64-3.8 (9) 41.54-6.2 (9) 24.82_1.8 (9) 20.54-1.7 (8) 207.54-21.0"* (9) 373.1 ±12.0 (9) 242.3 4-29.7 (9) 79.7±16.5 (8) 1494.24-89.9 (8) 175.5±31.7 (9) 410.2± 17.2 (10) 94.65_9.8 (8) 109.94-8.5 ( 1 2 ) 80.94-6.0 (9) 86.0±-6.4 (8) 119.44-8.8"* (9) 117.04-12.4(8) 140.64-15.1 (9) 73.54-2.1 ( 1 0 ) 352.72_53.6 (9) 217.22_21.2 (8) 759.84-45.4 (8) 115.05_100.2 (9) 298.0 2_50.0 (9) 278.54-39.2 (9) 191.2±14.9 (7)

W K Y rats

111.04-7.3 (9) 28.14-2.7 (8)

49.24-1.6 (9) 50.54-2.0 ( 1 0 ) 31.64-3.1 ( 1 0 ) 25.04-2.5 ( 1 0 ) 234.8±14.3 (9)

340.84-29.9 (9) 77.64-5.6 ( 1 0 ) 118.54-3.5 (9) 54.14-4.3 (8)

12-week-oM SHrats

W K Y rats

S H rats

186.04-21.9 (5) 175.94-28.0 (6) 420.22_17.2(8) 436.02_9.2 (7) 59.7±6.0 (6) 50.5±5.8 (7) 334.44-41.2 (5) 339.5±29.7 (5) 33.6tl.8 (8) 36.0±3.3 (6) 80.1-I-2.4 (7) 84.4±2.0 (8) 79.02-5.9 (8) 78.32-3.9 (8) 23.9±2.1 (8) 27.24-2.1 (7) 18.64-1.5 (8) 17.34-3.2 (7) 37.04-3.0 (8) 35.52-3.4 (9) 111.04-5.8 (8) 92.04-19.3 (8) 81.34-10.8 (7) 26.44-1.8 (9) 32.64-3.8 (15) 24.84-2.8 (16) 19.24-4.8 (8) 21.64-2.1 (9) 33.04-2.9 (8) 32.34-6.10(9) 95.64-7.8 (7) 94.44-14.3 (9) 39.72_1.8"*(9) 43.54-3.2 (16) 33.64-2.3** (18) 44.8±2.7 (10) 75.02-4.5 (8) 57.64-3.0** (9) 46.64-5.3 (8) 45.24-7.0 (8) 32.42_2.6 (11) 37.42_2.2 (7) 35.24-3.0 (7) 22.92_2.0 (11) 19.05-0.9 (8) 17.34-0.8 (9) 308.34-11.9"* (9) 177.94-14.8 (8) 206.84-23.1(9) 313.8±15.1 (8) 288.34-17.7 (9) 357.44-28.9 (8) 345.72_24.2 (8) 180.42_28.3 (9) 183.65-23.7 (9) 1489.34-76.6 (8) 1234.2±91.7 (9) 134.24-14.6(7) 108.1±8.8 (9) 335.34-21.9 (9) 301.74-21.7 (9) 262.74-20.4 (8) 94.34-14.8 (9) 95.1 4-16.2 (9) 79.2±8.5 (11) 52.04-4.9 (9) 93.1 4-12.4"* (9) 68.04-2.7 (9) 74.6±8.5 (9) 85.94-4.1 (9) 84.14-2.5 (9) 97.64-4.8** (9) 88.22-5.8 (9) 90.84-8.1 (9) 85.84-5.4 (9) 78.74-6.9 (9) 138.2±9.9 (9) 127.54-13.0 (8) 59.44-3.1 (10) 44.1±4.7 (9) 47.14-2.0 (9) 239.64-24.6 (9) 249.24-13.9 (9) 144.14-11.2(9) 163.22_7.1 (9) 117.04-6.6 (9) 115.2±6.1 (9) 530.52_32.0 (9) 606.04-26.7 (9) 810.04-55.6 (9) 856.64-54.4 (9) 145.24-17.2 (9) 183.4--13.8 (8) 129.14-14.4(9) 160.14-17.6(9) 239.4±31.3 (9) 227.5±23.2 (9)

430

180

w

160

,=

140 ¢:) E E

oo

120

,.%

100

• * SH Rats • ...... WKY Rats

500

•.~

~

450

v

w

",

400

x

~ \,

350

300

4

8

12

AGE (weeks)

Fig. 2. Systolic blood pressure and heart rate data in 4-, 8- and 12-week-old SH and W K Y rats. *, P < 0.05 compared to value in S H rat o f same age.

AChE activity greater than twice blank in these areas were not included in the ACh assay. Analysis of ACh in brain micropunches was performed as previously described by Hoover et al. 21. Micropunches of tissue were homogenized by sonication in 50 #l cold 0.1 N HC1. Protein was assayed in 5 or l0/zl aliquots. 25 ,ul aliquots were taken for the assay of ACh by the method of Goldberg and McCaman 14, including the tetraphenylboron extraction procedure. [7-32P]ATP (1 #Ci/tube) was obtained from New England Nuclear (Boston, Mass.). Following barium acetate precipitation, the [32P]phosphorylcholine reaction product was separated by the modified anionexchange chromatography described by McCaman and Stetzler32. This method has a sensitivity of 0.5-1 pmol.

Statistical analysis Data were analyzed using two-tailed Student's t-test for grouped data. The criterion for statistical significance was P ~ 0.05. All values are reported as mean ± S.E. of the mean.

431

200

]

4 Week

]

8 Week

~]

12 Week

o be

,k

I00

:':"

!ii!i! z

i

50

TAV

PVN

DM

PH

LC

1 RG

Fig. 3. ChAT activity in those areas of rat brain which demonstrated significant differences between SH and age matched W K Y rats. Expressed as per cent of age matched WKY value. Abbreviations are explained and baseline values are listed in Table I. *, ChAT activity in SI-I rats was significantly different (P < 0.05) than the value in W K Y rats at that age.

RESULTS

Cardiovascular data The systolic blood pressure (SBP) and heart rate (HR) data for 4-, 8- and 12week-old SH rats and their age-matched W K Y controls are presented in Fig. 2. At all ages, the SBP of the SH rats was significantly higher than that of the W K Y rats. While the 4-week-old SH rats are considered prehypertensive, there is a marked increase in SBP of the SH rats at 8 and 12 weeks of age. In contrast, the H R tended to decrease with age in both groups. However, at 8 and 12 weeks the SH rats exhibited a significantly higher H R than their age-matched controls. ChAT activity Table I presents the activity of ChAT in various discrete nuclei for 4-, 8- and 12week-old SH rats and their controls. Fig. 2 contains histograms of all those areas that showed significant differences in ChAT activity between SH and W K Y rats in at least one age group. Changes in ChAT activity of several nuclei were observed in all 3 age groups of SH rats. While there were no alterations in ChAT activity detected in any of the telencephalic regions investigated (Table I), several diencephalic and hindbrain nuclei exhibited changes. A significant activation of ChAT was detected in the anterior ventral thalamus of 4- and 8-week SH rats but a decline to control levels was seen in 12-week SH rats (Fig. 2 and Table I). In contrast, other diencephalic regions in which ChAT activity was altered in SH rats showed decreased activity of the enzyme. The

432 TABLE II ACh values in 12-week-old S H and W K Y rats

Data are expressed as the mean ± S.E.M. of individual values obtained from the number of rats shown in parentheses. Area

Number punches/ rat * *

Approximate ACh values (pmol//zg protein) coordinates* * * W K Y rats S H rats

Locus coeruleus N. reticularis lateralis N. ambiguus N. originis dorsalis vagi N. tractus solitarii - rostral N. tractus solitarii - caudal

6 4 4 8

P2800, P2300 P7000, P6500 P6000, P5500 P6500, P6000

8

P6500, P6000 0.389 ± 0.029 (7)

0.454 ± 0.080 (10)

8

P7000, P7400

0.378 ~ 0.046 (9)

0.304 ~: 0.022 (9) 0.258 ± 0.057 (7) 0.301 -- 0.029 (9) 0.586 ± 0.078 (6)

0.437 i 0.034 (7)

0.399 0.276 0.342 0.444

~: 0.038* (9) i 0.043 (9) ± 0.021 (9) i 0.079 (10)

* P < 0.05. ** Size of punches used was the same as indicated in Fig. 1. * * * Based on Palkovits and Jacobowitz4L

C h A T in the p a r a v e n t r i c u l a r nucleus o f the 4-week-old S H rats h a d 36 ~ less activity t h a n t h a t o f W K Y rats, while the 8- a n d 12-week values were n o t different from their controls. Conversely, in the d o r s o m e d i a l h y p o t h a l a m i c nucleus the decrease in C h A T activity was detected in the 8- a n d 12-week S H rats but n o t in the 4-week rats, while the p o s t e r i o r h y p o t h a l a m i c nuclei o f S H rats h a d significantly less C h A T activity t h a n controls only at 12 weeks o f age. The m o s t striking change in C h A T activity a m o n g the brain nuclei measured was found in the locus coeruleus, where a 79~o increase was detected in 12-week-old S H rats. R a t h e r inconsistent results were observed in the nucleus gigantocellularis, where C h A T activity in the S H rats was increased at 4 weeks, decreased at 8 weeks, a n d u n c h a n g e d at 12 weeks o f age c o m p a r e d to a g e - m a t c h e d controls. A Ch c o n t e n t

The A C h c o n c e n t r a t i o n s o f 6 h i n d b r a i n regions o f 12-week-old S H rats a n d W K Y rats are presented in Table II. In the locus coeruleus, A C h content was 31 ~i greater in the S H rats while no differences were evident in the o t h e r regions assayed. DISCUSSION In these experiments we have investigated the differences in C h A T activity a n d A C h content in discrete areas o f the brain which a c c o m p a n y genetically d e t e r m i n e d hypertension. The finding t h a t the b l o o d pressure in S H rats increased m a r k e d l y between 4 a n d 12 weeks o f age confirms previous studies a4,~5. A n i m a l s older t h a n 12 weeks were n o t examined in this study since the b l o o d pressure reaches a plateau at

433 this age 34,35. Furthermore, the role of the nervous system in the maintenance of hypertension may be of diminishing importance after this time11, 27,49. The results of these studies demonstrate that cholinergic neurons in certain nuclei of the brains of SH rats differ from those of the W K Y controls. This is shown by changes in the activity of CHAT, a specific marker for the presence of cholinergic nerves 45. Increases and decreases of ChAT activity have been correlated with increases and decreases of impulse flow in cholinergic neurons 9. The role of central cholinergic nerves in either the development or the maintenance of the elevated blood pressure in SH rats cannot be ascertained from these studies. However, certain of these nuclei have been demonstrated to be involved in central neural and endocrine control of cardiovascular function. Of particular interest is the finding of elevated ChAT activity in the locus coeruleus in the 12-weekold but not in the 4- and 8-week-old rats. The noradrenergic cell bodies in the locus coeruleus have been implicated in blood pressure regulation 39,47,48. The presence of ACh and ChAT in the locus coeruleus suggests the presence of a cholinergic innervation 1,7 although the origin is unknown. Furthermore, the presence of a high concentration of AChE in the locus coeruleus cell bodies 4~ suggests that this nucleus is highly cholinoceptive. ACh has an excitatory influence on the locus coeruleus15, 29. The finding of elevated dopamine-fl-hydroxylase in the locus coeruleus of SH rats 36 may be a reflection of cholinergic activation. The lack of differences in ChAT activity in the prehypertensive (4 week) and developmental (8 week) stages of hypertension suggest a secondary cholinergic influence on the locus coeruleus in the animals with establishcd hypertension (12 week). This is also reflected in an increased concentration of ACh in the locus coeruleus of the 12-week-old SH rats. This might occur as a result of changes in baroreceptor function 38, chronic alterations in blood flow resulting from vascular damage 30 and/or a variety of homeostatic endocrine changes 37 in the 12-week-old SH rats. Alterations in the neurochemistry of the locus coeruleus in adult SH rats are consistent with the findings of Kawamura et al. 25 who demonstrated altered cardiovascular responses to electrical stimulation of the locus coeruleus in these rats. In 4-week-old SH rats only, a change in ChAT activity was detected in the paraventricular nucleus, which is a site of oxytocin and vasopressin cell bodies. A cholinergic input here may be involved in triggering the endocrine imbalances found in SH rats. Crofton et al. s have shown increased plasma, pituitary and urinary vasopressin concentrations in SH rats, and it is known that there is a cholinergic modulation of vasopressin release2S, 44. Hoffman et al. 18 have recently proposed that the ability of centrally administered cholinergic drugs to produce enhanced pressor responses in SH rats is due in part to their ability to cause vasopressin release. Because it has been well established that hypertensive response can be elicited from the posteromedial region of the hypothalamus and that it modulates cardiovascular reflexes6, the differences in ChAT activity of the dorsomedial nucleus and posterior nucleus of the hypothalamus between SH rats and age-matched W K Y rats may also be related to cardiovascular differences. Brezenoff and colleagues z,5 have shown that the caudal hypothalamic nuclei contain cholinergic mechanisms capable of

434 affecting arterial blood pressure. Injections of carbachol or anti-cholinesterase agents into the posterior hypothalamus of rats result in hypertension~, 5 while carbachol evokes a hypotensive response when injected into the dorsomedial hypothalamus 2. A decrease in ChAT activity in the dorsomedial nucleus in the 8- and 12-week-old SH rats may be a reflection of reduced cholinergic activity in this nucleus which results in the development of hypertension. The role of the anterior ventral nucleus of the thalamus in cardiovascular control is not known. Thus it is difficult to speculate on the meaning of the elevated ChAT activity in the 4- and 8-week-old SH rats. Likewise, although the nucleus gigantocellularis appears to be involved in blood pressure control 6 the fluctuating changes in ChAT in this nucleus in the 3 ages of animals are difficult to interpret. It is of interest that areas of the medullary reticular formation known to be involved in cardiovascular control such as the nucleus tractus solitarius, paramedian reticular nucleus, lateral reticular nucleus, etc. 6, failed to exhibit alterations of C h A T activity or ACh content in the SH rats. However, recent studies report alterations in biochemical indices of noradrenergic36, 46 and adrenergic36, 43,46 function in certain of these nuclei. These findings indicate that the primary alteration at least in the nuclei comprising input to the hindbrain baroreceptor pathway 6 in SH rats may involve catecholaminergic rather than cholinergic neurons. In summary, these studies demonstrate that central cholinergic neurons of specific brain nuclei are biochemically altered in SH rats. The greatest alteration was found in the ChAT activity (a 79 ~ increase) of the locus coeruleus of 12-week-old SH rats. Lesser changes were demonstrated in SH rats in the paraventricular nucleus at 4 weeks, the dorsomedial nucleus at 8 and 12 weeks, the posterior hypothalamus at 12 weeks and the anterior ventral thalamus and nucleus gigantocellularis at 4 and 8 weeks of age. ACKNOWLEDGEMENTS The expert technical assistance of Mr. Robert P. McDevitt and Ms. Lisa Boswell and the excellent secretarial work of Ms. Judy G. Blumenthal and Ms. Doreen E. White are gratefully acknowledged. C.J.H. is a Research Associate in Pharmacology-Toxicology, National Institute of General Medical Science, National Institutes of Health, Bethesda, Md. 20205, U.S.A.

REFERENCES Amaral, D. G. and Sinnamon, H. M., The locus coeruleus: neurobiology of a central noradrenergic nucleus, Progr. Neurobiol., 9 (1977) 147-196. 2 Brezenoff,H. E., Cardiovascular responses to intrahypothalamic injections ofcarbachol and certain cholinesterase inhibitors, Neuropharmacology, 11 (1972)637-644. 3 Brezenoff, H. E., Centrally induced pressor responses to intravenous and intraventricular physostigmine evoked via different pathways, Europ. J. Pharmacol., 23 (1973) 290-292. 4 Brezenoff, I-t. E. and Rusin, J., Brain acetylcholine mediates the hypertensive response to physostigmine in the rat, Europ. J. Pharmacol., 29 (1974) 262-266. 1

435 5 Buccafusco, J. J. and Brezenoff, H. E., The hypertensive response to injectionof physostigmine into the hypothalamus of the unanesthetized rat, Clin. exp. Hypertension, 1 (1978) 219-227. 6 Calaresu, F. R., Faiers, A. A. and Mogenson, G. J., Central neural regulation of heart and blood vessels in mammals, Progr. NeurobioL, 5 (1975) 1-35. 7 Cheney, D. L., LeFevre, H. F. and Racagni, G., Choline acetyltransferase activity and mass fragmentographic measurement of acetylcholine in specific nuclei and tracts of rat brain, Neuropharmacology, 14 (1975) 801-809. 8 Crofton, J. T., Share, L., Shade, R. E., Allen, C. and Tarnowski, D., Vasopressin in the rat with spontaneous hypertension, Amer. J. Physiol., 235 (1978) H361-H366. 9 Ekstr6m, J., Acetylcholine synthesis and its dependence on nervous activity, Experientia (Basel), 34 (1978) 1247-1253. 10 Finch, L., Cohen, M. and Horst, W. D., Effects of 6-hydroxydopamine at birth on the development of hypertension in the rat, Life Sci., 13 (1973) 1403-1410. 11 Folkow, B. U. G. and Hallback, M. I. L., Physiopathology of spontaneous hypertension in rats. In J. Genest, E. Koiw and O. Kuchel (Eds.), Hypertension, McGraw-Hill, New York, 1977, pp. 507-528. 12 Folkow, B., Hal lb~ick, M., Lundgren, Y., and Weiss L., The effects of immunosympathectomy on blood pressure and vascular reactivity in normal and spontaneously hypertensive rats, Acta physiol, scand., 84 (1972) 512-523. 13 Fonnum, F., A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407-409. 14 Goldberg, A. M. and McCaman, R. E.,The determination of picomole amounts of acetylcholine in mammalian brain, J. Neurochem., 20 (1973) 1-8. 15 Guyenet, P. G. and Aghajanian, G. K., ACh, substance P and met-enkephalin in the locus coeruleus: pharmacological evidence for independent sites of action, Europ. J. Pharmacol., 53 (1979) 319-328. 16 Hauseler, G., Finch, L. and Thoenen, H., Central adrenergic neurones and the initiation and development of experimental hypertension, Experientia (Basel), 28 (1972) 1200--1203. 17 Hoffman, W. E. and Phillips, M. I., A pressor response to intraventricularinjections of carbachol, Brain Research, 105 (1976) 157-162. 18 Hoffman, W. E., Schmid, P. G. and Phillips, M. I., Central cholinergic and noradrenergic stimulation in spontaneously hypertensive rats, J. Pharmacol. exp. Ther., 206 (1978) 644-651. 19 Honda, K., Maekawa, S., Tamura, T., Uchiyama, S., Suzuki, K., Yashima, O., Ozawa, K., Takahashi, E. and Kimura, T., The role of peripheral sympathetic nerves and the adrenal medulla in maintainingblood pressure of essential hypertension, Jap. Circulat. J., 39 (1975) 591-594. 20 Hoover, D. B., Kosa, J., Colasanti, B. K. and Craig, C. R., A modified assay for cholinesterase, Microchem. J., 21 (1976) 267-271. 21 Hoover, D. B., Muth, E. A. and Jacobowitz, D. M., A mapping of the distribution of acetylcholine, choline acetyltransferase and acetylcholinesterase in discrete areas of rat brain, Brain Research, 153 (1978) 295-306. 22 lriuchijima, J., Sympathetic discharge rate in SHR. In S. Julius and M. D. Esler (Eds.), The Nervous System in Arterial Hypertension, C. C. Thomas, Springfield, I11., 1976, pp. 51-57. 23 Jacobowitz, D. M. andPalkovits, M.,Topographicatlasofcatecholamineandacetylcholinesterasecontaining neurons in the rat brain. I. Forebrain, J. comp. NeuroL, 157 (1974) 13-28. 24 Judy, W. V., Murphy, W. R., Watanabe, A. M., Besch, H. R., Henry, D. P. and Yu, P. L., Sympathetic nerve activity in spontaneously hypertensive rats. In Spontaneous Hypertension: its Pathogenesis and Complications. U.S. Government Printing Office, Washington, D.C., 1977, pp. 223-236. 25 Kawamura, H., Gunn, C. G. and Frohlich, E. D., Cardiovascular alteration by nucleus locus coeruleus in spontaneously hypertensive rat, Brain Research, 140 (1978) pp. 137-147. 26 Krstic, M. K. and Djurkovic, D., Cardiovascular response to intracerebroventricularadministration of acetylcholine in rats, Neuropharmacology, 17 (1978) 341-347. 27 Kubo, T. and Hashimoto, M., Effects of intraventricular and intraspinal 6-hydroxydopamine on blood pressure of spontaneously hypertensive rats, Arch. int. Pharmacodyn. Ther., 232 (1978) 166176. 28 Kuhn, E. R.,Cholinergicandadrenergicreleasemecbanismfor vasopressin in the malerat: astudy with injections of neurotransmitters and blocking agents into the third ventricle, Neuroendocrinology, 16 (1974) 255-264. 29 Lewander, T., Job, T. H. and Reis, D. J., Prolonged activation of tyrosine hydroxylase in nora-

436 drenergic neurons of rat brain by cholinergic stimulation, Nature (Lond.), 258 (1975) 440-441. 30 Lovenberg, W., Yamabe, H., Nakada, T. and Yamori, Y., Vascular protein synthesis in the spontaneously hypertensive rat. In Spontaneous Hypertension - - its Pathogenesis and Complications, U.S. Government Printing Office, 1977, pp. 71-75. 31 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 32 McCaman, R. E. and Stetzler, J., Radiochemical assay for ACh: modifications for sub-picomole measurements, J. Neurochem., 28 (1977) 669-671. 33 Nagatsu, T., Ikuta, K., Numata, Y., Kato, T., Sano, M., Nagutsu, 1. and Takeuchi, T., Vascular and brain dopamine fl-hydroxylase activity in young spontaneously hypertensive rats, Science, 191 (1976) 290-291. 34 Nagatsu, T., Mizutani, K., Nagatsu, I., Umezawa, H., Matsuzaki, M. and Takeuchi, T., Enzymes of catecholamine biosynthesis and metabolism in spontaneously hypertensive rats and hypotensive effects of the specific inhibitors from microbiol origin. In K. Okamoto (Eds.), Spontaneous Hypert e n s i o n - its Pathogenesis and Complications, Springer-Verlag, New York, 1972, pp. 31-36. 35 Nakamura, K., Gerold, M. and Thoenen, H., DOCA-salt and spontaneously hypertensive rats: comparative studies on norepinephrine turnover in central and peripheral adrenergic neurons. In K. Okamoto (Ed.), Spontaneous Hypertension - - its Pathogenesis and Complications, SpringerVerlag, New York, 1972, pp. 51-58. 36 Nakamura, K. and Nakamura, K., Role of brainstem and spinal noradrenergic and adrenergic neurons in the development and maintenance of hypertension in spontaneously hypertensive rats, Naunyn-Schmiedeberg 's exp. Path. Pharmak., 305 (1978) 127-133. 37 Nakamura, K., Nakamura, K. and Suzuki, T., Reciprocal changes in adrenomedullary and cortical functions in spontaneously hypertensive rats. In Spontaneous Hypertension - - its Pathogenesis and Complications, U.S. Government Printing Office, 1977, pp. 149-158. 38 Nosaka, S. and Wang, S. C., Baroreceptor reflex functions in the spontaneously hypertensive rat. In K. Okamoto (Ed.), Spontaneous Hypertenstion - - its Pathogenesis and Complications, SpringerVerlag, New York, 1972, pp. 79-82. 39 Ogawa, M., Fujita, Y., Niwa, M., Takami, N. and Ozaki, M., Role on blood pressure regulation of noradrenergic neurons originating from the locus coeruleus in the Wistar-Kyoto rat, Jap. Heart J., 18 (1977) 586--587. 40 Okamoto, K., Nosaka, S., Yamori, Y. and Matsumoto, M., Participation of neural factors in the pathogenesis of hypertension in the spontaneously hypertensive rats, Jap. Heart J., 8 (1967) 168-180. 41 Palkovits, M., Isolated removal of hypothalamic or other brain nuclei of the rat, Brain Research, 59 (1973) 449-450. 42 Palkovits, M. and Jacobotwitz, D. M., Topographic atlas of catecholamine and acetylcholinesterase containing neurons in the brain. I1. Hindbrain, J. comp. NeuroL, 157 (1974) 29-42. 43 Saavedra, J. M., Grobecker, H. and Axelrod, J., Changes in central catecholaminergic neurons in the spontaneously (genetic) hypertensive rat, Circulat. Res., 42 (1978) 531-534. 44 Sladek, C. D. and Knigge, K. M., Cholinergic stimulation of vasopressin release from the rat hypothalamic-neurohypophyseal system in organ culture, Endocrinology, 101 (1977) 411-420. 45 Storm-Mathisen, J., Localization oftransmitter candidates in the brain : the hippocampal formation as a model, Progr. Neurobiol., 8 (1977) 119-181. 46 Versteeg, D. H. G., Palkovits, M., Van der Gugten, J., Wijnen, H. L. J. M., Smeets, G. W. M. and DeJong, W., Catecholamine content of individual brain regions of spontaneously hypertensive rats (SH-rats), Brain Research, 112 (1976) 429--434. 47 Ward, D. G. and Gunn, C. G., Locus coeruleus complex: elicitation of a pressor response and a brain stem region necessary for its occurrence, Brain Research, 107 (1976) 401-406. 48 Ward, D. G. and Gunn, C. G., Locus coeruleus complex: differential modulation of depressor mechanisms, Brain Research, 107 (1976) 407-411. 49 Yamori, Y., Interaction of neural and nonneural factors in the pathogenesis of spontaneous hypertension. In S. Julius and M. D. Esler (Eds.), The Nervous System in Arterial Hypertension, C. C. Thomas, Springfield, I11., 1976, pp. 17-43. 50 Yamori, Y., Ooshima, A., Nosaka, S. and Okamoto, K., Metabolic basis for central blood pressure regulation in spontaneously hypertensive rats. In K. Okamoto (Ed.), Spontaneous Hypertension - its Pathogenesis and Complications, Springer-Verlag, New York, 1972, pp. 73-78.