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Cardiovascular responses to blockade of GABA synthesis in the hypothalamus of the spontaneously hypertensive rat
Cardio~~cular Responses to Blockade ofG4BA Synthesis in the Hypothalamus of the Spo~~neously Hype~ensi~e Rat C. A. SHONIS,
C. A. PEANO,
Received
G. H, DILLON
6 April 1992; Accepted
AND
T. G. WALDROP’
22 November
1992
SHONIS, C. A., C. A. PEANO, G. 11. DILLON ANDT. G. WALDROP. Cardiovascularresponrrf m bipeds ofGAB.4 synrhms in fhe ~~~~~~~~~~ #the ~~n~~~e~~~~ ~~~r~~~~~~ r& BRAIN RES BULL 31(S) 4934% 1~3.~Previous studies have suggested that a decreased inhibitor input onto neurons within the posterior h~tba~amus (PH), a known pressor area, may contribute to hy~~en~ou in the spontaneously hypertensive rat (SHR). Recent experiments from this laboratory have shown that neurons in the PH afthe SHR have an altered and elevated discharge frequency compared to those in the normotensive rat, fn addition, bi~hern~~ studies have reported that there is a decreased concentration of the inhibitory neurotra~smi~er, GABA, in the hy~th~amus of the SHR. The objective of the present study was to assess any variations in CABAergic modulation of cardiovascular activity in SHRs compared to normotensive Wistar-Kyoto (WKY) rats and Sprague-Dawley {SD) rats. Arterial pressure and heart rate responses to microinj~tio~s of -the GABA synthesis inhibitor 3-mer~ptopropionic acid I%-MP) into the posterior h~t~lamic area of anesthetized young (6-8 weeks) and mature (1 I-16 weeks) hypertensive and no~otensive rats were recorded. Microinjection of 3-MP elicited increases in arterial pressure of i 7.4 ri: 3.9 mmHg, 18.1 + 7.8 mmH8, 16.9 zt 6.4 mmHg, and 10.4 rt 3.5 mmHg in the mature WKY, mature SD, young WKY, and young SHR, respectively. In addition, heart rate was elevated bv 33.2 z&21.9 beats/min. 70.0 f 25.3 beats/min, 56.3 rt 15.0 beats/min and, 45.9 + 10 beats/min in the mature WKY, adult SD, young WKY, and’youig SHR groups, r&pctively. In contrast, microinjection of 3-MP. into the posterior hypot~lamus of adult SHRs produced no significant change in arterial pressure (-5.0 c I.8 mmHg) or heart rate (1-5.3 i- 6.1 ~ts~rn~n~. fn three of the adult SHRs, ~iov~uIar responses to electrical stimulation in the PH were compared to responses elicited by m~cro~n~ction of 3-MP into the same PH site. Efecttical stimu~tio~ produced Iarge increases in both arterial pressure and heart rate; however, m~~i~jec~on of 3-MP produced no ~gni~~nt changes in ~~~nva~~~ activity. These rest&s indicate that spontaneously h~~~ens~ve rats have a defziency in the tonic GABAergic input onto posterior b~~th~arnic neurons. This alteration may contribute to the maintenance ofthe elevated blood pressure in spontaneously hypertensive rats.
Arterial pressure
GABA
Heart rate
Hypertension
THE posterior hypothalamus has long been known to exert an excitatory influence upon the cardiovascular system. Numerous studies have shown that electrical stimulation in this area produces pronounced increases in arteriai pressure and heart rate (4,9,1 I, 17,2Sf. forever, recent studies have suggested that this ~~~~alarnic region receives tonic i~ibitiou by a ~A~Aer~c input. ~ic~injection of GABAA receptor antagonists into the posterior hy~~alamus of rats and cats evokes increases in arterial pressure and heart rate f 3,6,7,20,22-24). This cardiovascular response is known to be sym~thetic~ly mediated because gangiionic blockade prevents the increases in arterial pressure and heart rate (6). ~icro~nject~ons of compounds that inhibit GABA synthesis also produce increases in arterial pressure and heart rate in normotensive rats ( 1,6,15).
Posterior hypothalamus
SHR
Several studies have shown that spontaneously hypertensive rats (SHR) have altered GABAergic activity in the posterior hypothalamus. Reduced levels of both CABA and CABA receptors have been reported for the posterior hygothalamus in SHRs f&g), fn addition, it has been shown that t&e is a decreased activity of the GABA s~nthe~z~ng enzyme, glutamic acid decarboxylase, in the posterior hypothalamus of the SHR (5). Moreover, microinjection of the GABA agonist, muscimol, into the posterior hypothalamus results in larger depressor responses in SHRs compared to their normotensive controls (26). A defect in a GABAergic inhibitory mechanism in this pressor region of the ~ypothaIamus could contribute to the hypertension in the spontaneously hypertensive rat.
’ Requests for reprints should be addressed to Tony G. Waldrop, PhD, Department of Ph~ioi~
Goodwin Avenue, Urbana, IL 6 I 80 f .
493
& Bjo~h~~,
524 Burrill HaIt, 407 South
SWONJS ET AL. POST 3MP
A
200 ARTERlAL PRESSURE (mm Hd
HEART
RGTE
(beats/minf
B ARTERIAL (mm
PRESSURE Hr)
100
--
400 350
200 100
HEART RATE (beats/mix%)
HEART RATE (beat#/min)
D
3MP
150 ARTERIAL (mm
PRESSURE W
-4
75
REAR7
RATE (beata/min)
E ARTERLAL (mm
PRESSURE %t)
BEAR3 RATE (beate/min)
550 900 E-
,15 set,
FIG.I.Examplesof cardiovascularresponsesto unilateral m~croinject~onof 3-~ercaptopropion~c acid (3-MP) into the posterior hypothalamus ofmature(1 I -16week-old) rats; (A)WKY rat? (R) SD rat, {C)SHR rat. and young (&B-week-old) rats; (D) WKY rat, (E) SNR rat.
The purpose of the present study was to determine if inhibition of GABA synthesis in the posterior hypothalamus of the spontaneousiy hypertensive rat elicits different cardiovascular responses than those evoked in normotensive rats. The level of endogenous GABA in the posterior hypothalamus was reduced by m~croinje~ng a GABA synthesis inhibitor, 3-MP, into the area and moRito~~g arteriaI pressure and heart rate.
METHOD
Male rats weighing between 170 and 381 g were used for these experiments. Five groups of rats were studied: I. young (6-8 weeks) spontaneously hypertensive rats (SEW., n = II), _. > mature f I I- 16 weeks) SHRs (n = 1X),
HYPOTHALAMIC
GABA AND HYPERTENSION
495
additional anesthetic, A tracheotomy was performed and the rats were allowed to breathe spontaneously. Body temperature was monitored with a rectal probe and controlled between 36 and 38.5”C with a heating pad and radiant heat lamp. Fine insulated wires (0.127 mm diameter) with uninsulated tips were placed into the belly of the left and right gastrocnemius muscles to measure electromyographic (EMG) activity. The EMG activity was amplified with a high impedance probe and an AC preamplifier (Grass P5 11). Gastrocnemius EMG activity was recorded in order to rule out the possibility that any cardiovascular responses observed were due to feedback from contracting muscles (24). After each rat was placed in a stereotaxic apparatus (Smelting), a partial craniotomy was performed to permit placement of the tip of a micropipette or stimulating electrode (Rhodes, SNE 300) into the posterior hypothalamic area. The stereotaxic coordinates
I3 z
a
-10
A
i
B
t
I
a
-l-
-10 '
B FIG. 2. Mean responses k SEM for (A) arterial pressure and (B) heart
rate to microinjection of 3-MP in the mature SHR (n = 9), WKY (n = 22), and SD (n = 4) rats and in the young SHR (n = 11) and WKY (n = 8) rats. +Response is statistically different from preinjection control values. *Response is statistically different from other group responses @ < 0.05).
3. young Wistar-Kyoto rats (WKY, n = 8), 4. mature WKYs (n = 25), and 5. mature Sprague-Dawley rats (SD, n = 4).
All rats were anesthetized with a mixture of cr-chloralose (65 mg/kg) and urethane (800 mg/kg) given intraperitoneally. Additional anesthetic was given when necessary based upon a withdrawal reflex to pinching of a hindpaw. A common carotid artery was cannulated to measure arterial pressure with a pressure transducer. Heart rate was determined from the pressure tracing using a biotachometer. An external jugular vein was cannulated for administration of fluids and
-10 '
60pA
lOOpA
200pA
FIG. 3. Mean responses + SEM for arterial pressure (A) to electrical stimulation (B)(50, 100and 200 @A; 70 Hz; 1 ms duration) and injection of 3-MP (200 nl) into the PH of adult SHRs (n = 3). ‘Indicates response is statistically different from control baseline values 0, < 0.05).
SHONIS ET AL. TABLE
I
CHANGES IN MEAN ARTERIAL PRESSURE ANU HEART RATE IN RESPONSE TO MKROINJECTION OF 3-MP INTO THE POSTERIOR HYPOTHALAMUS
Mature WKY Mature SD Mature SHR Young WKY Young SHR
Change rn MAP (mmHg)
Percent Change in MAP
Change m HR (min-‘)
Percent Change in HR
+17 + 4t +18 F 8.f
27% 14%
+33 c 22t +70 + 2%
16%
-4%
-15k 6* +56* 1st +46 + lot
I% 15% 9%
-5 t 3* +17 2 ht
28%
t10*4t
10%
I I&
* Indicates significantly different from all other groups (p s 0.05). t Indicates significantly different from preinjection values (p 5 0.05).
used to locate this area in the mature group were AP -I .2, L 0.7, and V -8.5 relative to bregma with the incisor bar at +5 mm (16). It was found upon histological verification that these coordinates were not appropriate for the posterior h~~alamic area in the young groups of rats. Therefore, the stereotaxic coordinates were adjusted for the young rats as a result of these pilot experiments. Coordinates for the young groups were AP -3.0,L 0.7,and V -7.5relative to bregma in a flat skull orientation ( 14). Micropipettes were made by pulling fine tips on glass tubing ( 1.O mm diameter) with a pipette puller (Narishige 700C). The tips were broken back to a diameter of 20-50 pm. The pipettes were filled with the GABA synthesis inhibitor, 3-mercaptopropionic acid (3-MP, 1.2 g/nl). Fast green dye was placed in the top of each pipette separated from the 3-MP by a thin layer of mineral oil. Unilateral microinje~ions (200 nl) into the posterior hypothalamic area were made using a pressure injection device (General Valve Picospritzer If). A calibrated reticle in the eyepiece of a microscope was used to determine the movement of the oil meniscus in order to measure the amount injected. It has been estimated that injection of this volume into brain tissue results in a spread of less than 1.0 mm with less diffusion for higher molecular weight compounds ( 12). 3-MI?, as supplied by Sigma, is a very viscous solution; thus, it is less prone to spread outside the initial injection site. 3-MP is available only as a neat liquid; therefore, an equivalent volume of acidified (pH = 2.3) mineral oil was microinjected into the PH of three adult WKY rats in order to control for the acidity and mechanical effects of injecting 3-MP. All injection sites were marked at the end of the experiment by microinj~~ng fast gteen dye (200 nl). Each rat was allowed to recover from the surgical procedures for at least 30 min before resting levels for heart rate and arterial pressure were recorded on a Gould chart recorder. The cardiovascular and EMG responses to microinjection of 3-MP into the posterior hypothalamus were then recorded in all rats studied. In three adult SHRs, the posterior hypothalamus was stimulated electrically (50, 100,200 mA, 70 Hz, 1 ms pulse duration) before microinjection of 3-MP into the same site. After a rat was sacrificed at the end of the experiment, the brain was removed and placed in formalin for at least 7 days. A block of the brain containing the hypothalamus was frozen and sliced into 50 pm sections on a microtome. Alternate sections were stained with cresyl violet. Injection sites were marked on drawings using a microslide projector and microscope. Drawings
were compared to a stereotaxic atlas in order to verify that injection sites were located in the posterior hypothalamus ( 14). Means + SEM were calculated for arterial pressure and heart rate. The values given represent the peak responses to the microinjections. Latency of onset was expressed as the time in minutes from microinjection of 3-MP until a change in mean arterial pressure of at least 10 mmHg or a change in heart rate of 20 beats/min was seen. Differences among groups were analyzed with Student’s t-test and analysis of variance; p < 0.05 was accepted as significant. RESUL.TS
An initial resting blood pressure was recorded for each rat before being placed in the stereotaxic apparatus. The resting mean arterial pressure at this time was significantly higher in the mature SHR group (152.3 ? 4.9 mmHg) compared to the adult WKY group (94.0 + 4.8 mmHg) and the adult SD group ( 130 rt 7.2 mmHg). The resting mean arterial pressure was also significantly higher for the young SHR group (143.9 + 9.6 mmHg) compared to the young WKY group (109.3 f 10.0 mmHg). Representative examples of the cardiovascular responses to microinjection of 3-MP into the posterior hypothalamus of the mature WKY, SD, and SHR rats as well as young WKY and SHR rats are shown in Fig. 1. Increases in arterial pressure and heart rate occurred in all mature WKY rats studied. Latency of onset for changes in the mean arterial pressure and heart rate was 18.5 + 2.3 min. 3-MP microinjections also evoked increases in mean arterial pressure and heart rate in the mature SD rats. The mean latency for a response to 3-MP in the SD rat was 16.2 t 3.1 min. In contrast to the other mature rats, mature SHRs had no significant ~rdiovascuIar responses to microinjections of 3-MP. Figure 1C shows a typical example of the lack of arterial pressure and heart rate responses to 3-MP in an adult SHR. Young SHRs had a modest increase in mean arterial pressure that was not significantly different from age-matched WKYs, but it was significantly greater than the mature SHR’s response. The increase in heart rate in the young SHRs is similar to the heart rate response in mature WKYs, SDS, and young WKYs. Young WKYs responded to 3-MP microinjections with increases in mean arterial pressure and heart rate. Mean changes for arterial pressure and heart rate in response to 3-MP for all young and mature groups are shown in Fig. 2 and Table 1. Electrical stimulation ofthe PH in the SHR resulted in pressor and tachycardic responses in all three animals tested at every
HYPOTHALAMIC
GABA
AND
HYPERTENSION
497
-3.60 mm
FIG. 4. Summary of microinjection sites in the posterior hy~th~amus of (A) mature SHR, WKY, and SD rats and (B) young SHR and WKY rats. l = 3-MP injection into the PH of a WKY, A = 3-MP injection into the PH of a SHR, and El = 3-MP injection into the PH of a SD. A = 3-MP injection outside the PH of a SHR and 0 = 3-MP injection outside the PH of a WKY. * = electrical stimulation and 3-MP injection into the PH of a SHR. + = acidified mineral oil injection into the PH of a WKY. Drawings and abbreviations were adapted from Paxinos and Watson (14). The stereotaxic coordinates given in mm posterior to bregma are noted on the right side of each drawing. 3V = 3rd ventricle, Arc = arcuate hypothalamic nucleus, DM = dorsomedial hypothalamic nucleus, f = fomix, mtb = medial forebrain bundle, mt = mammillothalamic tract, PH = posterior hypothalamic area, PMD = premammilky nucleus, dorsal, PMV = premammillary nucleus, ventral, VMH = ventromedial hypothalamic nucleus.
level of stimulaltion. The magnitude of the cardiovascular responses were graded in relation to the level of current used. Mean responses rt SEM for arterial pressure and heart rate are shown in Fig. 3. In striking contrast, microinj~tion of 3-MP into the same site produced no increases in arterial pressure or heart rate in these three animals. No significant cardiovascular changes were observed with injections of 3-MP into sites surrounding the posterior hypothalamus (n = 18). Mean changes in mean arterial pressure and heart rate in response to 3-MP outside the PH for mature SHRs (n = 3), mature WKYs (n = 4) young SHRs (n = 7), and young WKYs (n = 4) were -2 + 2 mmHg and -3 f 3 beats/min, -3 + 3 mmHg and 0 +- 7 beats/min, -1 + 1 mmHg and -1 + 2 beats/min, and 1 f 10 mmHg and 3 + 6 beats/min, respectively. In addition, microinjection of acidified mineral oil into the PH
of three adult WKY rats had no significant effects on blood pressure (0 + 0 mmHg) or heart rate (2 f 11 beatslmin). Gastrocnemius EMG activity increased in two mature WKYs, two SHRs, one young WKY, five young SHRs, and one adult SD rat in response to microinjection of 3-MP into the posterior hypothalamus. Increased EMG activity, however, was not responsible for the cardiovascular effects seen because increases in arteriai pressure and heart rate were observed prior to any changes in EMG activity. in addition, some of the mature SHRs had increases in EMG activity in the absence of any cardiovascular response. In most cases, EMG activity was increased in the hindlimb contralateral to the hypothalamic injection site. Forelimb movement and vibrissae twitching were also observed in rats following 3-MP microinjections.
SHONE
roles this brain site may play in essential hypertension. This possibility has been examined in the s~ntaneousiy hypertensive rat developed by Okamoto and his co-workers in Japan (13). initial studies demonstrated that surgical separation of the connections between the mesen~phalon and the caudal hypothalamus evokes larger decreases in arterial pressure in the SHR than in the normotensive WKY rat (27). In addition. electrical stimulation produces larger increases in blood pressure and sympathetic nerve activity in the SHR and in the SHR maintained normotensive with pharmacological treatment than in the WKY rat (9). However, these latter results are difficult to interpret because electrical stimulation affects fibers of passage as well as ceD bodies. The exact disruption in the posterior hypothalamus of the spontaneously hypertensive rat which may be responsible for the hype~ension has not been determined; however. aherations in the GABAergic system have been observed. Endogenous GABA levels and GABA receptor numbers in the posterior hypothalamus are significantly lower in the SHR as compared to age-matched WKYs (8). In addition to reduced levels ofGABA, investigators have reported a decreased activity of the enzyme responsible for GABA synthesis, glutamic acid decarboxylase, in the posterior hypothalamus of the SHR (5). An indication that this alteration in GABAergic function might be important in cardiovascular regulation was provided wrhen responses to hypothalamic stimulation before and after intracerebral ventricular administration of GABA were examined ( IQ). The pressor and sympathetic nerve responses to electrical stimulation were initially greater in the SHR than in the WKY: however, similar responses were observed in both groups after ventricular administration of GABA. Even though this study indicated that an altered concentration of GABA might contribute to hypertension, the exact loci for the deficiency was not identified. Rccent evidence suggests, however, that the posterior hypothalamus may be the responsible brain site. Investigators have reported that microinjection of the GABA agonist, muscimol, into the posterior hypothalamus elicits greater decreases in blood pressure in the SHR than in the WKY (26). However, it was surprising that mi~roinjection of a GABA antagonist. bi~uc~illine. had simitar responses in both strains of rats. If one assumes that the synthesis inhibitor, 3-MP, equally retards the synthesis in both rat strains regardless of age, the findings of the present study indicate that a disturbance in GABA synthesis in the posterior hypothalamus may contribute to the high blood pressure present in spontaneously hypertensive rats. Microinjection of3-MP into the posterior hypothalamic area in the present study elicited large increases in arterial pressure and heart rate in mature and young no~otens~ve rats and in young spontaneously hypertensive rats. In contrast, no cardiovascular responses were evoked by 3-MP microinjections in mature spontaneously hypertensive rats. Small but significant increases in arterial pressure were evoked in young SHRs. The lack of cardiovascular response in the adult spontaneousIy hypertensive rat cannot be explained by the inability ofthis animal to increase arterial pressure above the already elevated levei. Electrical stimulation in the PH of adult SHRs in the present study evoked large increases in both arterial pressure and heart rate. Based on the findings from this study and previous evidence, one could speculate that as the SHR develops h~~e~sion there is a progressive loss of GABAergic inhibition onto the posterior hypothalamus. Loss of this inhibition would enable hypothalamic neurons to exert a potent effect upon brain stem neurons that regulate sympathetic discharge, resulting in an elevated arterial pressure. This hypothesis is supported by the different basal what possible
I
POSTERIOR HYPOT~~US
I f+>
It
BLOOD PRESSURE
I
I
FIG. 5. Proposed mechanism for the increased blood pressure in spontaneously hypertensive rats. Evidence suggests adult SHRs have a diminished GABAergic influence on the posterior hypothalamus. This would lead to an increased level of neuronal activity in this area which has been shown to have excitatory projections to brainstem cardiovascular neurons. Brain stem cardiovascular neurons would, in turn. increase sympathetic outflow and thereby increase blood pressure.
A summary of the rn~~roinj~~tion sites for mature SHR. WKY, and SD rats and for young SHR and WKY rats are shown in Fig. 4. Rata obtained onfy from m~croinj~t~on sites located within the posterior hypothalamic area or in very close proximity were used for comparisons between animal groups. DISCUSSION
Activation of neurons in the posterior hy~thalamus has long been known to exert a strong influence upon the cardiovascular system. Electrical stimulation of this region increases heart rate, arterial pressure, and myocardial contractility (4,l I, 17). In addition, h~thalamic stimulation produces a redist~bution of organ bfood flows with increased flow to the heart, diaphragm, and limb skeletal muscles concurrent with increased vascular resistance in the renal and mesenteric beds (2.5). Similar results have been obtained with chemical activation (d~s~nh~bition) of hypothalamic neurons (24). This cardiovascular activation is not surprising, considering the direct hypothalamic-autonomic connections that have been described f 18). In addition. the posterior hypothalamus has been shown to contain many neurons that have a basal discharge that is correlated temporally with inferior cardiac nerve activity andfor the catdiac cycle (2). Because the posterior hypothalamus has the ability to profoundly alter cardiovascular function, it is intriguing to consider
ET Al,.
HYPOTHALAMIC
GABA
AND
499
HYPERTENSION
arterial pressures and cardiovascular responses to 3-MP observed in the young compared to the mature SHR. One would expect that a loss of GABAergic input onto posterior hypothalamic neurons should lead to an elevated level of neuronal activity as a result of this disinhibition. Recent studies have provided experimental evidence in support of this hypothesis. Krukoff and Weigel used hexokinase histochemistry to compare the metabolic activity of various brain regions in spontaneously hypertensive rats and normotensive rats (10). The elevated activity in the posterior hypothalamus of the SHRs led these investigators to conclude that this region may participate in the origination of hypertension. It has also been shown that neurons in the posterior hypothalamus that have a discharge rate related to the cardiac cycle have an elevated discharge rate in the SHR as compared to the WKY rat (21). In addition, it was shown that posterior hypothalamic neurons studied in a brain slice preparation also possess a higher discharge rate in the SHR than in the WKY (21).
The combination of the results of the present study with findings from other laboratories lead to a hypothesis concerning the elevated blood pressure in the spontaneously hypertensive rat. A reduction in the synthesis of GABA in the posterior hypothalamus results in a disinhibition of posterior hypothalamic neurons. Elevated activity of neurons in this known pressor region results in an increased level of sympathetic discharge, thereby eliciting an increase in arterial pressure, Figure 5 shows this proposed mechanism. Future studies are needed to test this hypothesis and to determine if this deficiency contributes to hypertension in the SHR. ACKNOWLEDGEMENTS
This research was supported by NIH grant HL38276 and the American Heart Association-Illinois Affiliate. T.G.W. is an Established Investigator of the American Heart Association. C.A.S. and G.H.D. were supported by predoctoral fellowships from AHA-Illinois Affiliate.
REFERENCES 1. Abshire, V. M.; Hankins, K. D.; Roehr, K. E.; DiMicco. J. A. In-
2
3.
4.
5.
6.
7.
8. 9.
10. 11. 12. 13. 14.
jection of L-Allylglycine into the posterior hypothalamus in rats causes decreases in local GABA which correlate with increases in heart rate. Neuropharmacology 27: 117 l-l 177, 1988. Barman, S. Descending projections of hypothalamic neurons with sympathetic nerve-related activity. J. Neurophysiol. 64: 1019- 1032; 1990. Bauer, R. M.; Vela, M. B.; Simon, T.; Waldrop, T. G. A GABAergic mechanism in the posterior hypothalamus modulates baroreflex bradycardia. Brain Res. Bull. 20:633-641; 1988. Bunag, R. D.; Eferakeya. A. E.; Langdon, D. S. Enhancement of hypothalamic pressor responses in spontaneously hypertensive rats. Am. J. Phvsiol. 228:217-222: 1975. Czyzewska-Szafran, H.; Wutkiewicz, M.; Remiszewska, M.; Jastrazebski, Z.; Czamecki, A.; Danysz, A. Down-regulation of the GABAergic system in selected brain areas of spontaneously hypertensive rats (SHR). Pol. J. Pharrnacol. Pharm. 41:619-627; 1988. DiMicco, J. A.; Abshire, V. M. Evidence of GABAergic inhibition of a hypothalamic sympathoexcitatory mechanism in anesthetized rats. Brain Res. 402:1-10; 1987. DiMicco, J. A.; Abshire, V. M.; Hankins, K. D.; Sample, R. H. B.; Wible, J. H., Jr. Microinjections ofGABA antagonists into posterior hypothalamus elevates heart rate in anesthetized rats. Neuropharmacology 25:1063-1066; 1986. Hambley, J. W.; Johnston, G. A. R.; Shaw, J. Alterations in a hypothalamic GABA system in the spontaneously hypertensive rat. Neurochem. Int. 6:8 13-822; 1984. Juskevich, J. C.; Robinson, D. S.; Whitehorn, D. Effect of hypothalamic stimulation in spontaneously hypertensive and Wistar-Kyoto rats. Eur. J. Pharmacol. 5 1:429-439; 1978. Krukoff, T. L.; Weigel, M. A. Metabolic alterations in discrete regions of the rat brain during development of spontaneous hypertension. Brain Res. 499: l-6; 1989. Manning, J. W.; Peiss, C. N. Cardiovascular responses to electrical stimulation in the diencephalon. Am. J. Physiol. 198:366-370; 1960. Myers, R. D. Injection of solutions into cerebral tissue: Relation between volume and diffusion. Physiol. Behav. 1:17I- 174; 1966. Okamoto, K. Spontaneous hypertension in rats. Int. Rev. Exp. Pathol. 7~227-270: 1969. Paxinos, G.; Watson, C. The rat brain in stereotaxic coordinates. Orlando, FL: Academic Press; 1986.
15. Peano, C. A.; Shonis, C. A.; Dillon, G. H.; Waldrop, T. G. Hypothalamic GABAergic mechanism involved in the respiratory response to hypercapnia. Brain Res. Bull. 28: 107-l 13; 1991. 16. Pellegrino, L. J.; Pellegrino, A. S.; Cushman, A. J. A stereotaxic atlas of the rat brain. New York: Plenum Press; 1979. 17. Rosen, A. Augmented cardiac contraction, heart acceleration and skeletal muscle vasodilatation produced by hypothalamic stimulation in cats. Acta Physiol. Stand. 52:291-308; 1961. 18. Saper, C. B.; Loewy, A. D.; Swanson, L. W.; Cowan, W. M. Direct hypothalamo-autonomic connections. Brain Res. 117:305-3 12; 1976. 19. Sasaki, S.; Lee, L.; Iyota, 1.; Kambara, S.; Okajima, H.; Inoue, A.; Takahashi, H.; Takeda, K.; Youshimura, M.; Nakagawa, M.; Ijichi, H. Central GABA-ergic stimulation attenuates hypertension and hypothalamic hyperactivity in spontaneously hypertensive rats. J. Hypertens. 4:S171-S174; 1986. 20. Shekhar, A.; DiMicco, J. A. Defense reaction elicited by injection of GABA antagonists and synthesis inhibitors into the posterior hypothalamus inrats. Neuropharmacology 26:407-4 17; 1987. 21. Shonis. C. A.: Waldrop. T. G. Augmented neuronal activitv in the posterior hypothalamus of sponta\eously hypertensive rats. Brain Res. Bull. (in press). 22. Soltis, R. P.; DiMicco, J. A. GABAA and excitatory amino acid receptors in dorsomedial hypothalamus and heart rate in rats. Am. J. Physiol. 260:Rl3-R20; 1991. 23. Waldrop, T. G.; Bauer, R. M. Modulation of sympathetic discharge by a hypothalamic GABAergic mechanism. Neuropharmacology 28: 263-269; 1988. 24. Waldrop, T. G.; Bauer, R. M.; Iwamoto, G. A. Microinjection of GABA antagonists into the posterior hypothalamus elicits locomotor activity and a cardiorespiratory activation. Brain Res. 444:84-94; 1988. 25. Waldrop, T. G.; Henderson, M. C.; Iwamoto, G. A.; Mitchell, G. H. Regional blood flow responses to stimulation of the subthalamic locomotor region. Respir. Physiol. 64:93-102; 1986. 26. Wible, J. H., Jr.; DiMicco, J. A.; Luft, F. C. Hypothalamic GABA and sympathetic regulation in spontaneously hypertensive rats. Hypertension 14:623-628; 1989. 27. Yamori, Y.; Okamoto, D. Hypothalamic tonic regulation of blood pressure in spontaneously hypertensive rats. Jpn. Circ. J. 33:509519; 1969.
C. A. PEANO,
Received
G. H, DILLON
6 April 1992; Accepted
AND
T. G. WALDROP’
22 November
1992
SHONIS, C. A., C. A. PEANO, G. 11. DILLON ANDT. G. WALDROP. Cardiovascularresponrrf m bipeds ofGAB.4 synrhms in fhe ~~~~~~~~~~ #the ~~n~~~e~~~~ ~~~r~~~~~~ r& BRAIN RES BULL 31(S) 4934% 1~3.~Previous studies have suggested that a decreased inhibitor input onto neurons within the posterior h~tba~amus (PH), a known pressor area, may contribute to hy~~en~ou in the spontaneously hypertensive rat (SHR). Recent experiments from this laboratory have shown that neurons in the PH afthe SHR have an altered and elevated discharge frequency compared to those in the normotensive rat, fn addition, bi~hern~~ studies have reported that there is a decreased concentration of the inhibitory neurotra~smi~er, GABA, in the hy~th~amus of the SHR. The objective of the present study was to assess any variations in CABAergic modulation of cardiovascular activity in SHRs compared to normotensive Wistar-Kyoto (WKY) rats and Sprague-Dawley {SD) rats. Arterial pressure and heart rate responses to microinj~tio~s of -the GABA synthesis inhibitor 3-mer~ptopropionic acid I%-MP) into the posterior h~t~lamic area of anesthetized young (6-8 weeks) and mature (1 I-16 weeks) hypertensive and no~otensive rats were recorded. Microinjection of 3-MP elicited increases in arterial pressure of i 7.4 ri: 3.9 mmHg, 18.1 + 7.8 mmH8, 16.9 zt 6.4 mmHg, and 10.4 rt 3.5 mmHg in the mature WKY, mature SD, young WKY, and young SHR, respectively. In addition, heart rate was elevated bv 33.2 z&21.9 beats/min. 70.0 f 25.3 beats/min, 56.3 rt 15.0 beats/min and, 45.9 + 10 beats/min in the mature WKY, adult SD, young WKY, and’youig SHR groups, r&pctively. In contrast, microinjection of 3-MP. into the posterior hypot~lamus of adult SHRs produced no significant change in arterial pressure (-5.0 c I.8 mmHg) or heart rate (1-5.3 i- 6.1 ~ts~rn~n~. fn three of the adult SHRs, ~iov~uIar responses to electrical stimulation in the PH were compared to responses elicited by m~cro~n~ction of 3-MP into the same PH site. Efecttical stimu~tio~ produced Iarge increases in both arterial pressure and heart rate; however, m~~i~jec~on of 3-MP produced no ~gni~~nt changes in ~~~nva~~~ activity. These rest&s indicate that spontaneously h~~~ens~ve rats have a defziency in the tonic GABAergic input onto posterior b~~th~arnic neurons. This alteration may contribute to the maintenance ofthe elevated blood pressure in spontaneously hypertensive rats.
Arterial pressure
GABA
Heart rate
Hypertension
THE posterior hypothalamus has long been known to exert an excitatory influence upon the cardiovascular system. Numerous studies have shown that electrical stimulation in this area produces pronounced increases in arteriai pressure and heart rate (4,9,1 I, 17,2Sf. forever, recent studies have suggested that this ~~~~alarnic region receives tonic i~ibitiou by a ~A~Aer~c input. ~ic~injection of GABAA receptor antagonists into the posterior hy~~alamus of rats and cats evokes increases in arterial pressure and heart rate f 3,6,7,20,22-24). This cardiovascular response is known to be sym~thetic~ly mediated because gangiionic blockade prevents the increases in arterial pressure and heart rate (6). ~icro~nject~ons of compounds that inhibit GABA synthesis also produce increases in arterial pressure and heart rate in normotensive rats ( 1,6,15).
Posterior hypothalamus
SHR
Several studies have shown that spontaneously hypertensive rats (SHR) have altered GABAergic activity in the posterior hypothalamus. Reduced levels of both CABA and CABA receptors have been reported for the posterior hygothalamus in SHRs f&g), fn addition, it has been shown that t&e is a decreased activity of the GABA s~nthe~z~ng enzyme, glutamic acid decarboxylase, in the posterior hypothalamus of the SHR (5). Moreover, microinjection of the GABA agonist, muscimol, into the posterior hypothalamus results in larger depressor responses in SHRs compared to their normotensive controls (26). A defect in a GABAergic inhibitory mechanism in this pressor region of the ~ypothaIamus could contribute to the hypertension in the spontaneously hypertensive rat.
’ Requests for reprints should be addressed to Tony G. Waldrop, PhD, Department of Ph~ioi~
Goodwin Avenue, Urbana, IL 6 I 80 f .
493
& Bjo~h~~,
524 Burrill HaIt, 407 South
SWONJS ET AL. POST 3MP
A
200 ARTERlAL PRESSURE (mm Hd
HEART
RGTE
(beats/minf
B ARTERIAL (mm
PRESSURE Hr)
100
--
400 350
200 100
HEART RATE (beats/mix%)
HEART RATE (beat#/min)
D
3MP
150 ARTERIAL (mm
PRESSURE W
-4
75
REAR7
RATE (beata/min)
E ARTERLAL (mm
PRESSURE %t)
BEAR3 RATE (beate/min)
550 900 E-
,15 set,
FIG.I.Examplesof cardiovascularresponsesto unilateral m~croinject~onof 3-~ercaptopropion~c acid (3-MP) into the posterior hypothalamus ofmature(1 I -16week-old) rats; (A)WKY rat? (R) SD rat, {C)SHR rat. and young (&B-week-old) rats; (D) WKY rat, (E) SNR rat.
The purpose of the present study was to determine if inhibition of GABA synthesis in the posterior hypothalamus of the spontaneousiy hypertensive rat elicits different cardiovascular responses than those evoked in normotensive rats. The level of endogenous GABA in the posterior hypothalamus was reduced by m~croinje~ng a GABA synthesis inhibitor, 3-MP, into the area and moRito~~g arteriaI pressure and heart rate.
METHOD
Male rats weighing between 170 and 381 g were used for these experiments. Five groups of rats were studied: I. young (6-8 weeks) spontaneously hypertensive rats (SEW., n = II), _. > mature f I I- 16 weeks) SHRs (n = 1X),
HYPOTHALAMIC
GABA AND HYPERTENSION
495
additional anesthetic, A tracheotomy was performed and the rats were allowed to breathe spontaneously. Body temperature was monitored with a rectal probe and controlled between 36 and 38.5”C with a heating pad and radiant heat lamp. Fine insulated wires (0.127 mm diameter) with uninsulated tips were placed into the belly of the left and right gastrocnemius muscles to measure electromyographic (EMG) activity. The EMG activity was amplified with a high impedance probe and an AC preamplifier (Grass P5 11). Gastrocnemius EMG activity was recorded in order to rule out the possibility that any cardiovascular responses observed were due to feedback from contracting muscles (24). After each rat was placed in a stereotaxic apparatus (Smelting), a partial craniotomy was performed to permit placement of the tip of a micropipette or stimulating electrode (Rhodes, SNE 300) into the posterior hypothalamic area. The stereotaxic coordinates
I3 z
a
-10
A
i
B
t
I
a
-l-
-10 '
B FIG. 2. Mean responses k SEM for (A) arterial pressure and (B) heart
rate to microinjection of 3-MP in the mature SHR (n = 9), WKY (n = 22), and SD (n = 4) rats and in the young SHR (n = 11) and WKY (n = 8) rats. +Response is statistically different from preinjection control values. *Response is statistically different from other group responses @ < 0.05).
3. young Wistar-Kyoto rats (WKY, n = 8), 4. mature WKYs (n = 25), and 5. mature Sprague-Dawley rats (SD, n = 4).
All rats were anesthetized with a mixture of cr-chloralose (65 mg/kg) and urethane (800 mg/kg) given intraperitoneally. Additional anesthetic was given when necessary based upon a withdrawal reflex to pinching of a hindpaw. A common carotid artery was cannulated to measure arterial pressure with a pressure transducer. Heart rate was determined from the pressure tracing using a biotachometer. An external jugular vein was cannulated for administration of fluids and
-10 '
60pA
lOOpA
200pA
FIG. 3. Mean responses + SEM for arterial pressure (A) to electrical stimulation (B)(50, 100and 200 @A; 70 Hz; 1 ms duration) and injection of 3-MP (200 nl) into the PH of adult SHRs (n = 3). ‘Indicates response is statistically different from control baseline values 0, < 0.05).
SHONIS ET AL. TABLE
I
CHANGES IN MEAN ARTERIAL PRESSURE ANU HEART RATE IN RESPONSE TO MKROINJECTION OF 3-MP INTO THE POSTERIOR HYPOTHALAMUS
Mature WKY Mature SD Mature SHR Young WKY Young SHR
Change rn MAP (mmHg)
Percent Change in MAP
Change m HR (min-‘)
Percent Change in HR
+17 + 4t +18 F 8.f
27% 14%
+33 c 22t +70 + 2%
16%
-4%
-15k 6* +56* 1st +46 + lot
I% 15% 9%
-5 t 3* +17 2 ht
28%
t10*4t
10%
I I&
* Indicates significantly different from all other groups (p s 0.05). t Indicates significantly different from preinjection values (p 5 0.05).
used to locate this area in the mature group were AP -I .2, L 0.7, and V -8.5 relative to bregma with the incisor bar at +5 mm (16). It was found upon histological verification that these coordinates were not appropriate for the posterior h~~alamic area in the young groups of rats. Therefore, the stereotaxic coordinates were adjusted for the young rats as a result of these pilot experiments. Coordinates for the young groups were AP -3.0,L 0.7,and V -7.5relative to bregma in a flat skull orientation ( 14). Micropipettes were made by pulling fine tips on glass tubing ( 1.O mm diameter) with a pipette puller (Narishige 700C). The tips were broken back to a diameter of 20-50 pm. The pipettes were filled with the GABA synthesis inhibitor, 3-mercaptopropionic acid (3-MP, 1.2 g/nl). Fast green dye was placed in the top of each pipette separated from the 3-MP by a thin layer of mineral oil. Unilateral microinje~ions (200 nl) into the posterior hypothalamic area were made using a pressure injection device (General Valve Picospritzer If). A calibrated reticle in the eyepiece of a microscope was used to determine the movement of the oil meniscus in order to measure the amount injected. It has been estimated that injection of this volume into brain tissue results in a spread of less than 1.0 mm with less diffusion for higher molecular weight compounds ( 12). 3-MI?, as supplied by Sigma, is a very viscous solution; thus, it is less prone to spread outside the initial injection site. 3-MP is available only as a neat liquid; therefore, an equivalent volume of acidified (pH = 2.3) mineral oil was microinjected into the PH of three adult WKY rats in order to control for the acidity and mechanical effects of injecting 3-MP. All injection sites were marked at the end of the experiment by microinj~~ng fast gteen dye (200 nl). Each rat was allowed to recover from the surgical procedures for at least 30 min before resting levels for heart rate and arterial pressure were recorded on a Gould chart recorder. The cardiovascular and EMG responses to microinjection of 3-MP into the posterior hypothalamus were then recorded in all rats studied. In three adult SHRs, the posterior hypothalamus was stimulated electrically (50, 100,200 mA, 70 Hz, 1 ms pulse duration) before microinjection of 3-MP into the same site. After a rat was sacrificed at the end of the experiment, the brain was removed and placed in formalin for at least 7 days. A block of the brain containing the hypothalamus was frozen and sliced into 50 pm sections on a microtome. Alternate sections were stained with cresyl violet. Injection sites were marked on drawings using a microslide projector and microscope. Drawings
were compared to a stereotaxic atlas in order to verify that injection sites were located in the posterior hypothalamus ( 14). Means + SEM were calculated for arterial pressure and heart rate. The values given represent the peak responses to the microinjections. Latency of onset was expressed as the time in minutes from microinjection of 3-MP until a change in mean arterial pressure of at least 10 mmHg or a change in heart rate of 20 beats/min was seen. Differences among groups were analyzed with Student’s t-test and analysis of variance; p < 0.05 was accepted as significant. RESUL.TS
An initial resting blood pressure was recorded for each rat before being placed in the stereotaxic apparatus. The resting mean arterial pressure at this time was significantly higher in the mature SHR group (152.3 ? 4.9 mmHg) compared to the adult WKY group (94.0 + 4.8 mmHg) and the adult SD group ( 130 rt 7.2 mmHg). The resting mean arterial pressure was also significantly higher for the young SHR group (143.9 + 9.6 mmHg) compared to the young WKY group (109.3 f 10.0 mmHg). Representative examples of the cardiovascular responses to microinjection of 3-MP into the posterior hypothalamus of the mature WKY, SD, and SHR rats as well as young WKY and SHR rats are shown in Fig. 1. Increases in arterial pressure and heart rate occurred in all mature WKY rats studied. Latency of onset for changes in the mean arterial pressure and heart rate was 18.5 + 2.3 min. 3-MP microinjections also evoked increases in mean arterial pressure and heart rate in the mature SD rats. The mean latency for a response to 3-MP in the SD rat was 16.2 t 3.1 min. In contrast to the other mature rats, mature SHRs had no significant ~rdiovascuIar responses to microinjections of 3-MP. Figure 1C shows a typical example of the lack of arterial pressure and heart rate responses to 3-MP in an adult SHR. Young SHRs had a modest increase in mean arterial pressure that was not significantly different from age-matched WKYs, but it was significantly greater than the mature SHR’s response. The increase in heart rate in the young SHRs is similar to the heart rate response in mature WKYs, SDS, and young WKYs. Young WKYs responded to 3-MP microinjections with increases in mean arterial pressure and heart rate. Mean changes for arterial pressure and heart rate in response to 3-MP for all young and mature groups are shown in Fig. 2 and Table 1. Electrical stimulation ofthe PH in the SHR resulted in pressor and tachycardic responses in all three animals tested at every
HYPOTHALAMIC
GABA
AND
HYPERTENSION
497
-3.60 mm
FIG. 4. Summary of microinjection sites in the posterior hy~th~amus of (A) mature SHR, WKY, and SD rats and (B) young SHR and WKY rats. l = 3-MP injection into the PH of a WKY, A = 3-MP injection into the PH of a SHR, and El = 3-MP injection into the PH of a SD. A = 3-MP injection outside the PH of a SHR and 0 = 3-MP injection outside the PH of a WKY. * = electrical stimulation and 3-MP injection into the PH of a SHR. + = acidified mineral oil injection into the PH of a WKY. Drawings and abbreviations were adapted from Paxinos and Watson (14). The stereotaxic coordinates given in mm posterior to bregma are noted on the right side of each drawing. 3V = 3rd ventricle, Arc = arcuate hypothalamic nucleus, DM = dorsomedial hypothalamic nucleus, f = fomix, mtb = medial forebrain bundle, mt = mammillothalamic tract, PH = posterior hypothalamic area, PMD = premammilky nucleus, dorsal, PMV = premammillary nucleus, ventral, VMH = ventromedial hypothalamic nucleus.
level of stimulaltion. The magnitude of the cardiovascular responses were graded in relation to the level of current used. Mean responses rt SEM for arterial pressure and heart rate are shown in Fig. 3. In striking contrast, microinj~tion of 3-MP into the same site produced no increases in arterial pressure or heart rate in these three animals. No significant cardiovascular changes were observed with injections of 3-MP into sites surrounding the posterior hypothalamus (n = 18). Mean changes in mean arterial pressure and heart rate in response to 3-MP outside the PH for mature SHRs (n = 3), mature WKYs (n = 4) young SHRs (n = 7), and young WKYs (n = 4) were -2 + 2 mmHg and -3 f 3 beats/min, -3 + 3 mmHg and 0 +- 7 beats/min, -1 + 1 mmHg and -1 + 2 beats/min, and 1 f 10 mmHg and 3 + 6 beats/min, respectively. In addition, microinjection of acidified mineral oil into the PH
of three adult WKY rats had no significant effects on blood pressure (0 + 0 mmHg) or heart rate (2 f 11 beatslmin). Gastrocnemius EMG activity increased in two mature WKYs, two SHRs, one young WKY, five young SHRs, and one adult SD rat in response to microinjection of 3-MP into the posterior hypothalamus. Increased EMG activity, however, was not responsible for the cardiovascular effects seen because increases in arteriai pressure and heart rate were observed prior to any changes in EMG activity. in addition, some of the mature SHRs had increases in EMG activity in the absence of any cardiovascular response. In most cases, EMG activity was increased in the hindlimb contralateral to the hypothalamic injection site. Forelimb movement and vibrissae twitching were also observed in rats following 3-MP microinjections.
SHONE
roles this brain site may play in essential hypertension. This possibility has been examined in the s~ntaneousiy hypertensive rat developed by Okamoto and his co-workers in Japan (13). initial studies demonstrated that surgical separation of the connections between the mesen~phalon and the caudal hypothalamus evokes larger decreases in arterial pressure in the SHR than in the normotensive WKY rat (27). In addition. electrical stimulation produces larger increases in blood pressure and sympathetic nerve activity in the SHR and in the SHR maintained normotensive with pharmacological treatment than in the WKY rat (9). However, these latter results are difficult to interpret because electrical stimulation affects fibers of passage as well as ceD bodies. The exact disruption in the posterior hypothalamus of the spontaneously hypertensive rat which may be responsible for the hype~ension has not been determined; however. aherations in the GABAergic system have been observed. Endogenous GABA levels and GABA receptor numbers in the posterior hypothalamus are significantly lower in the SHR as compared to age-matched WKYs (8). In addition to reduced levels ofGABA, investigators have reported a decreased activity of the enzyme responsible for GABA synthesis, glutamic acid decarboxylase, in the posterior hypothalamus of the SHR (5). An indication that this alteration in GABAergic function might be important in cardiovascular regulation was provided wrhen responses to hypothalamic stimulation before and after intracerebral ventricular administration of GABA were examined ( IQ). The pressor and sympathetic nerve responses to electrical stimulation were initially greater in the SHR than in the WKY: however, similar responses were observed in both groups after ventricular administration of GABA. Even though this study indicated that an altered concentration of GABA might contribute to hypertension, the exact loci for the deficiency was not identified. Rccent evidence suggests, however, that the posterior hypothalamus may be the responsible brain site. Investigators have reported that microinjection of the GABA agonist, muscimol, into the posterior hypothalamus elicits greater decreases in blood pressure in the SHR than in the WKY (26). However, it was surprising that mi~roinjection of a GABA antagonist. bi~uc~illine. had simitar responses in both strains of rats. If one assumes that the synthesis inhibitor, 3-MP, equally retards the synthesis in both rat strains regardless of age, the findings of the present study indicate that a disturbance in GABA synthesis in the posterior hypothalamus may contribute to the high blood pressure present in spontaneously hypertensive rats. Microinjection of3-MP into the posterior hypothalamic area in the present study elicited large increases in arterial pressure and heart rate in mature and young no~otens~ve rats and in young spontaneously hypertensive rats. In contrast, no cardiovascular responses were evoked by 3-MP microinjections in mature spontaneously hypertensive rats. Small but significant increases in arterial pressure were evoked in young SHRs. The lack of cardiovascular response in the adult spontaneousIy hypertensive rat cannot be explained by the inability ofthis animal to increase arterial pressure above the already elevated levei. Electrical stimulation in the PH of adult SHRs in the present study evoked large increases in both arterial pressure and heart rate. Based on the findings from this study and previous evidence, one could speculate that as the SHR develops h~~e~sion there is a progressive loss of GABAergic inhibition onto the posterior hypothalamus. Loss of this inhibition would enable hypothalamic neurons to exert a potent effect upon brain stem neurons that regulate sympathetic discharge, resulting in an elevated arterial pressure. This hypothesis is supported by the different basal what possible
I
POSTERIOR HYPOT~~US
I f+>
It
BLOOD PRESSURE
I
I
FIG. 5. Proposed mechanism for the increased blood pressure in spontaneously hypertensive rats. Evidence suggests adult SHRs have a diminished GABAergic influence on the posterior hypothalamus. This would lead to an increased level of neuronal activity in this area which has been shown to have excitatory projections to brainstem cardiovascular neurons. Brain stem cardiovascular neurons would, in turn. increase sympathetic outflow and thereby increase blood pressure.
A summary of the rn~~roinj~~tion sites for mature SHR. WKY, and SD rats and for young SHR and WKY rats are shown in Fig. 4. Rata obtained onfy from m~croinj~t~on sites located within the posterior hypothalamic area or in very close proximity were used for comparisons between animal groups. DISCUSSION
Activation of neurons in the posterior hy~thalamus has long been known to exert a strong influence upon the cardiovascular system. Electrical stimulation of this region increases heart rate, arterial pressure, and myocardial contractility (4,l I, 17). In addition, h~thalamic stimulation produces a redist~bution of organ bfood flows with increased flow to the heart, diaphragm, and limb skeletal muscles concurrent with increased vascular resistance in the renal and mesenteric beds (2.5). Similar results have been obtained with chemical activation (d~s~nh~bition) of hypothalamic neurons (24). This cardiovascular activation is not surprising, considering the direct hypothalamic-autonomic connections that have been described f 18). In addition. the posterior hypothalamus has been shown to contain many neurons that have a basal discharge that is correlated temporally with inferior cardiac nerve activity andfor the catdiac cycle (2). Because the posterior hypothalamus has the ability to profoundly alter cardiovascular function, it is intriguing to consider
ET Al,.
HYPOTHALAMIC
GABA
AND
499
HYPERTENSION
arterial pressures and cardiovascular responses to 3-MP observed in the young compared to the mature SHR. One would expect that a loss of GABAergic input onto posterior hypothalamic neurons should lead to an elevated level of neuronal activity as a result of this disinhibition. Recent studies have provided experimental evidence in support of this hypothesis. Krukoff and Weigel used hexokinase histochemistry to compare the metabolic activity of various brain regions in spontaneously hypertensive rats and normotensive rats (10). The elevated activity in the posterior hypothalamus of the SHRs led these investigators to conclude that this region may participate in the origination of hypertension. It has also been shown that neurons in the posterior hypothalamus that have a discharge rate related to the cardiac cycle have an elevated discharge rate in the SHR as compared to the WKY rat (21). In addition, it was shown that posterior hypothalamic neurons studied in a brain slice preparation also possess a higher discharge rate in the SHR than in the WKY (21).
The combination of the results of the present study with findings from other laboratories lead to a hypothesis concerning the elevated blood pressure in the spontaneously hypertensive rat. A reduction in the synthesis of GABA in the posterior hypothalamus results in a disinhibition of posterior hypothalamic neurons. Elevated activity of neurons in this known pressor region results in an increased level of sympathetic discharge, thereby eliciting an increase in arterial pressure, Figure 5 shows this proposed mechanism. Future studies are needed to test this hypothesis and to determine if this deficiency contributes to hypertension in the SHR. ACKNOWLEDGEMENTS
This research was supported by NIH grant HL38276 and the American Heart Association-Illinois Affiliate. T.G.W. is an Established Investigator of the American Heart Association. C.A.S. and G.H.D. were supported by predoctoral fellowships from AHA-Illinois Affiliate.
REFERENCES 1. Abshire, V. M.; Hankins, K. D.; Roehr, K. E.; DiMicco. J. A. In-
2
3.
4.
5.
6.
7.
8. 9.
10. 11. 12. 13. 14.
jection of L-Allylglycine into the posterior hypothalamus in rats causes decreases in local GABA which correlate with increases in heart rate. Neuropharmacology 27: 117 l-l 177, 1988. Barman, S. Descending projections of hypothalamic neurons with sympathetic nerve-related activity. J. Neurophysiol. 64: 1019- 1032; 1990. Bauer, R. M.; Vela, M. B.; Simon, T.; Waldrop, T. G. A GABAergic mechanism in the posterior hypothalamus modulates baroreflex bradycardia. Brain Res. Bull. 20:633-641; 1988. Bunag, R. D.; Eferakeya. A. E.; Langdon, D. S. Enhancement of hypothalamic pressor responses in spontaneously hypertensive rats. Am. J. Phvsiol. 228:217-222: 1975. Czyzewska-Szafran, H.; Wutkiewicz, M.; Remiszewska, M.; Jastrazebski, Z.; Czamecki, A.; Danysz, A. Down-regulation of the GABAergic system in selected brain areas of spontaneously hypertensive rats (SHR). Pol. J. Pharrnacol. Pharm. 41:619-627; 1988. DiMicco, J. A.; Abshire, V. M. Evidence of GABAergic inhibition of a hypothalamic sympathoexcitatory mechanism in anesthetized rats. Brain Res. 402:1-10; 1987. DiMicco, J. A.; Abshire, V. M.; Hankins, K. D.; Sample, R. H. B.; Wible, J. H., Jr. Microinjections ofGABA antagonists into posterior hypothalamus elevates heart rate in anesthetized rats. Neuropharmacology 25:1063-1066; 1986. Hambley, J. W.; Johnston, G. A. R.; Shaw, J. Alterations in a hypothalamic GABA system in the spontaneously hypertensive rat. Neurochem. Int. 6:8 13-822; 1984. Juskevich, J. C.; Robinson, D. S.; Whitehorn, D. Effect of hypothalamic stimulation in spontaneously hypertensive and Wistar-Kyoto rats. Eur. J. Pharmacol. 5 1:429-439; 1978. Krukoff, T. L.; Weigel, M. A. Metabolic alterations in discrete regions of the rat brain during development of spontaneous hypertension. Brain Res. 499: l-6; 1989. Manning, J. W.; Peiss, C. N. Cardiovascular responses to electrical stimulation in the diencephalon. Am. J. Physiol. 198:366-370; 1960. Myers, R. D. Injection of solutions into cerebral tissue: Relation between volume and diffusion. Physiol. Behav. 1:17I- 174; 1966. Okamoto, K. Spontaneous hypertension in rats. Int. Rev. Exp. Pathol. 7~227-270: 1969. Paxinos, G.; Watson, C. The rat brain in stereotaxic coordinates. Orlando, FL: Academic Press; 1986.
15. Peano, C. A.; Shonis, C. A.; Dillon, G. H.; Waldrop, T. G. Hypothalamic GABAergic mechanism involved in the respiratory response to hypercapnia. Brain Res. Bull. 28: 107-l 13; 1991. 16. Pellegrino, L. J.; Pellegrino, A. S.; Cushman, A. J. A stereotaxic atlas of the rat brain. New York: Plenum Press; 1979. 17. Rosen, A. Augmented cardiac contraction, heart acceleration and skeletal muscle vasodilatation produced by hypothalamic stimulation in cats. Acta Physiol. Stand. 52:291-308; 1961. 18. Saper, C. B.; Loewy, A. D.; Swanson, L. W.; Cowan, W. M. Direct hypothalamo-autonomic connections. Brain Res. 117:305-3 12; 1976. 19. Sasaki, S.; Lee, L.; Iyota, 1.; Kambara, S.; Okajima, H.; Inoue, A.; Takahashi, H.; Takeda, K.; Youshimura, M.; Nakagawa, M.; Ijichi, H. Central GABA-ergic stimulation attenuates hypertension and hypothalamic hyperactivity in spontaneously hypertensive rats. J. Hypertens. 4:S171-S174; 1986. 20. Shekhar, A.; DiMicco, J. A. Defense reaction elicited by injection of GABA antagonists and synthesis inhibitors into the posterior hypothalamus inrats. Neuropharmacology 26:407-4 17; 1987. 21. Shonis. C. A.: Waldrop. T. G. Augmented neuronal activitv in the posterior hypothalamus of sponta\eously hypertensive rats. Brain Res. Bull. (in press). 22. Soltis, R. P.; DiMicco, J. A. GABAA and excitatory amino acid receptors in dorsomedial hypothalamus and heart rate in rats. Am. J. Physiol. 260:Rl3-R20; 1991. 23. Waldrop, T. G.; Bauer, R. M. Modulation of sympathetic discharge by a hypothalamic GABAergic mechanism. Neuropharmacology 28: 263-269; 1988. 24. Waldrop, T. G.; Bauer, R. M.; Iwamoto, G. A. Microinjection of GABA antagonists into the posterior hypothalamus elicits locomotor activity and a cardiorespiratory activation. Brain Res. 444:84-94; 1988. 25. Waldrop, T. G.; Henderson, M. C.; Iwamoto, G. A.; Mitchell, G. H. Regional blood flow responses to stimulation of the subthalamic locomotor region. Respir. Physiol. 64:93-102; 1986. 26. Wible, J. H., Jr.; DiMicco, J. A.; Luft, F. C. Hypothalamic GABA and sympathetic regulation in spontaneously hypertensive rats. Hypertension 14:623-628; 1989. 27. Yamori, Y.; Okamoto, D. Hypothalamic tonic regulation of blood pressure in spontaneously hypertensive rats. Jpn. Circ. J. 33:509519; 1969.