Mechanisms of the Cardiovascular Response to Posterior Hypothalamic Nucleus Administration of Carbachol

Abstract

Summary: 

Unilateral microinjection of the cholinergic agonist carbachol (CCh) into the posterior hypothalamic nucleus (PHN) of conscious rats evoked a dose-dependent increase in mean arterial pressure (MAP). The pressor response was accompanied by tachycardia at all doses of CCh used (0.8-13.2 nmol), although the tachycardia was followed by a secondary bradycardia after the two highest doses (5.5 and 13.2 nmol). To determine the involvement of the autonomic nervous system and arginine vasopressin (AVP) in these cardiovascular changes, we administered selective receptor antagonists intravenously (i.v.) before microinjection of CCh into the PHN. The pressor response evoked by 3.3 nmol CCh could be attenuated by prazosin (an α₁-adrenoceptor blocker) or yohimbine (an α₂-adrenoceptor blocker) and completely blocked by the combination of prazosin and yohimbine. In contrast, the increase in MAP evoked by 5.5 and 13.2 nmol CCh could be attenuated by prazosin, yohimbine, or D[(CH₂)₅Tyr(Me)]AVP (AVPX, a V₁-vasopressin receptor blocker), and completely blocked by the combination of prazosin and AVPX. The tachycardia evoked by the 3.3-, 5.5-, and 13.2-nmol doses of CCh could be attenuated by propranolol (a β-adrenoceptor blocker), and the secondary bradycardia evoked by 5.5 and 13.2 nmol CCh could be attenuated by either methylatropine (a muscarinic receptor blocker) or AVPX. These results suggest that administration of CCh into the PHN increases sympathetic nervous system activity, which increases MAP and heart rate (HR). The increase in MAP activates a baroreflex-mediated bradycardia by increasing vagal tone. This bradycardia is potentiated by an increase in circulating levels of AVP, which also contributes to the increased blood pressure (BP).

A hypertensive event can be evoked by the application of certain chemicals to the posterior hypothalamic nucleus (PHN) of rats ^(1-3). Some of these chemicals interact with the neurotransmitter acetylcholine (ACh), which is synthesized and released from neurons in this brain region ^(4,5). Therefore, a hypertensive response can be evoked by the direct administration into the PHN of acetylcholinesterase inhibitors or the cholinergic receptor agonist carbachol (CCh) ^(1,3,6-9). These pressor responses are due to activation of muscarinic receptors since they can be blocked by prior administration of muscarinic receptor antagonists into this brain region ^(3,10). Furthermore, the muscarinic receptor subtype involved in the pressor response evoked by microinjection of CCh into the posterior hypothalamic nucleus is the M₃ muscarinic receptor ⁽¹⁰⁾.

Although the central muscarinic receptor in the PHN involved in the pressor response to CCh has been characterized, the peripheral receptors and thus the peripheral neurotransmitters and neurohormones responsible for mediating this pressor response have not been well characterized. An increase in blood pressure (BP) evoked by intracere-broventricular administration of CCh has been attributed to an increase in sympathetic nervous system activity ⁽¹¹⁾ as well as to simultaneous increases in both sympathetic nerve activity and circulating levels of vasopressin ⁽¹²⁾. Pretreatment of rats with phentolamine, a nonselective α-adrenoceptor antagonist, partially blocked the pressor response induced by microinjection of CCh into the PHN of intact and hypophysectomized rats ⁽⁸⁾. Because hypophysectomized rats lack arginine vasopressin (AVP), the remaining increase in BP was suggested to be due to an increase in cardiac output (CO). However, another study showed that CO did not change during the pressor response induced by microinjection of CCh into the PHN ⁽⁹⁾.

The results of these earlier studies are equivocal and do not adequately characterize the peripheral neurotransmitters and neurohormones involved in the pressor response evoked by the administration of CCh directly into the PHN. Instead, these studies imply that the pressor response may be due to an increase in (a) sympathetic nervous system activity, (b) sympathetic nervous system activity and plasma levels of vasopressin, and/or (c) CO. Therefore, we wished to characterize, by use of selective receptor antagonists, the neurotransmitters and neurohormones responsible for the changes in arterial pressure and HR evoked by CCh microinjection into the PHN. Our results show that the pressor response evoked by direct administration of CCh into the PHN of conscious rats involves increases in sympathetic nervous system activity and plasma levels of vasopressin. The changes in HR that occur are due to an increase in sympathetic nerve activity that evokes tachycardia and to an increase in vagal activity and plasma levels of vasopressin that can produce a secondary bradycardia.

METHODS

Animals and preparation

Sprague-Dawley rats (Hilltop, Scottdale, PA, U.S.A.) weighing 270-330 g were used in all experiments. The rats were housed for 6-7 days before surgical preparation, at which time they were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneally, i.p.). The left femoral artery and vein were isolated and catheterized with medical grade polyvinyl chloride (PVC) tubing (0.2 mm ID × 0.5 mm OD) connected to S-54-HL PVC tubing (0.5 mm ID × 1.5 mm OD) for direct arterial pressure measurement and intravenous administration of receptor antagonists. The entire length (3.0-3.5 cm) of the smaller diameter tubing was slid into and anchored to the blood vessel with suture. The larger diameter catheter was tunneled subcutaneously to the nape of the neck, exteriorized, and loosely anchored with suture to the skeletal muscle underlying the skin.

After catheter implantation, the rats were placed in a stereotaxic apparatus (Stoelting, Wood Dale, IL, U.S.A.). The scalp was incised, exposing the skull. On the basis of the atlas of Paxinos and Watson ⁽¹³⁾, the coordinates A-3.8 mm and L-0.5 mm with respect to bregma on a flat skull were used in making a burr hole, which exposed the sagittal sinus, in the skull overlying the PHN. The dura mater to the left of and adjacent to the superior sagittal sinus was cut with the point of a 27-gauge hypodermic needle so that a 23-gauge stainless-steel guide cannula (0.33 mm ID), previously melded to a plastic pedestal (Plastics One, Roanoke, VA, U.S.A.), could be lowered into the brain tissue. The tip of the guide cannula was lowered to a level 5.7 mm below the surface of the skull at an angle of 0 °. The guide cannula at these coordinates (A-3.8 mm, L-0.5 mm, H-5.7 mm, θ = 0 °) directed a 30-gauge stainless-steel injection cannula (0.30 mm OD) to the PHN, consistent with methods used in earlier studies ^(3,9,14). The guide cannula was anchored to the skull by three mounting screws and dental caulk.

Each rat was used for the first experiment, which consisted of CCh microinjection into the PHN 2-3 days post-operatively. After the first experiment, rats were used for subsequent experiments every 2-3 days. These subsequent experiments consisted of intravenous administration of receptor antagonists alone or in combination before the microinjection of 0.9% saline or CCh. Each rat was used for no more than four experiments. Previously, similar pressor responses were shown to result from four consecutive microinjections of CCh when 2-3 days were allowed to elapse between consecutive administrations ⁽¹⁰⁾. Each experiment was performed during the light cycle at an ambient temperature of 21 ° ± 1 °C. Rats were not allowed access to food or water during the experiments, but were left in their home cage. Arterial and venous catheters were kept patent by flushing with 0.2 ml 200 U/ml heparin in 0.9% saline 1 day postoperatively and then at the end of each experiment.

Experimental protocol

On the day of an experiment, the arterial catheter was connected to a pressure transducer (COBE, Denver, CO, U.S.A.), and the venous catheter was connected to a 1-ml syringe. Each rat was then left alone so that BP could stabilize. Once BP had remained stable for 10 min, each rat received 0.9% saline (0.3 ml) or receptor antagonists intravenously, followed by microinjection of CCh or 0.9% saline into the PHN. The intravenous injections were given through the venous catheter, beginning 20-30 min and ending 10 min before microinjection. Microinjections were given by lowering the 30-gauge stainless-steel injection cannula through the 23-gauge guide cannula. The injection cannula extended 2 mm beyond the tip of the guide cannula so that microinjections were given at a level of approximately H-7.7 mm. The injection cannula was connected to a 5.0-μl Hamilton syringe (model 7005) with polyurethane tubing (0.305 mm ID × 0.635 mm OD). Both the Hamilton syringe and connecting tubing were filled with water. The Hamilton syringe was placed in a manual microinjector (David Kopf, Tujunga, CA, U.S.A.), and air was drawn into the injection cannula, followed by a 50-nl volume of injectate, which ensured that no rat was microinjected with a volume >50 nl. The injection cannula was lowered into the brain through the guide cannula during the minute immediately preceding microinjection and was removed 11 min after microinjection. Arterial BP and HR were monitored with a BP analyzer (Micro Med, Inc., Louisville, KY, U.S.A.) connected either to a printer for a digital printout of BP and HR or to a Grass polygraph (model 7D) equipped with a tachograph. Arterial BP and HR were recorded during the stabilization period and for 62 min after microinjection.

Histology

Each rat was anesthetized with urethane (1.6 g/kg i.p.) after completion of the experiments and immediately killed by transcardial perfusion with 10% phosphate-buffered formalin. The brains were removed and stored in formalin for at least 1 week, at which time each brain was transferred to a 30% sucrose solution in formalin. Each brain was stored in this second solution until prepared histologically. Frozen brain sections (75 μm thick) were cut through the PHN region with a freezing microtome, mounted onto slides, and stained with cresyl violet.

Statistical methods

Baseline BP was determined by averaging the mean arterial BP (MAP) obtained each minute during the 10-min period immediately preceding microinjection. Changes in MAP were determined by subtracting the baseline MAP from the MAP observed at each minute for the first 10 min after microinjection and then at the end of each 5-min interval thereafter. This generated a time-dependent curve which showed that the maximum increase in MAP occurred during the 5- to 8-min time period after CCh microinjection. The maximum change in MAP for each rat was then determined by averaging the changes in MAP obtained at each minute from 5 to 8 min after microinjection. Averaging the increase in MAP over this time period decreased the influence of transient increases in MAP caused by rearing, grooming, or other motor movements of the rats. These changes are presented as the maximum increase in MAP.

Baseline HR and changes in HR were determined in the same manner as that described for BP determination. Because the two higher doses of CCh evoked a biphasic change in HR (an initial tachycardia followed by a bradycardia), two changes in HR were determined. The initial tachycardia was determined by averaging the greatest increase in HR observed for each rat during the first 7 min after microinjection and is reported as the maximum increase in HR. The peak bradycardic response occurred 15 min after CCh microinjection. Therefore, the peak bradycardic response is reported together with the change in MAP at 15 min after CCh administration.

We determined the attenuation of the CCh-evoked pressor response provided by selective and combined receptor blockade by calculating the percent of the mean maximum increase in MAP evoked by microinjection of CCh into rats pretreated with these antagonists. We made this calculation by first correcting for artifacts of microinjection by determining a control response to each dose of CCh. This control response was obtained by subtracting the mean change in MAP evoked by microinjection of 0.9% saline into rats pretreated intravenously with 0.9% saline from the increase in MAP evoked by CCh microinjection into each rat pretreated intravenously with 0.9% saline. The average of these control responses was then calculated to determine a “mean maximum response” for each dose of CCh. We then calculated a “test” response for each rat by subtracting the mean change in MAP evoked by microinjection of 0.9% saline into rats pretreated intravenously with antagonists from the maximum change in MAP evoked by CCh microinjection into each rat pretreated intravenously with the same antagonists. We calculated a percent of the mean maximum response for each rat using the formula: percent of mean maximum response = 100-[((mean maximum response-test response)/mean maximum response) × 100]. These percent changes were averaged and are reported as percent of mean maximum response.

Changes in MAP, HR, and percent of mean maximum response are reported as the mean ± SEM. Differences between treatment groups were tested for statistical significance by analysis of variance, followed by Newman-Keul's multiple-range test for multicomparison analysis (p < 0.05 was considered significant). We determined dose dependency of the increase in MAP evoked by CCh by calculating coefficients of correlation and dose dependency of the change in HR by contingency table analysis.

Drugs

Carbamylcholine chloride (CCh, Sigma Chemical Co., St. Louis, MO, U.S.A.); atropine methylbromide (methylatropine, Sigma); D,L-propranolol hydrochloride (Sigma); yohimbine hydrochloride (Sigma); [D(CH₂)₅Tyr(Me)]arginine vasopressin (AVPX, Peninsula Laboratories, Belmont, CA, U.S.A.); (-)-arterenol [norepinephrine (NE), Sigma]; L-phenylephrine hydrochloride (Sigma); AVP acetate salt (Sigma); and (+)-isoproterenol hydrochloride (Sigma) were dissolved in 0.9% saline. Prazosin hydrochloride (Sigma) was dissolved in 50% propylene glycol solution. CCh or 0.9% saline was microinjected in a volume of 50 nl for 30 s directly into brain tissue. Prazosin, yohimbine, AVPX, methylatropine, and propranolol were administered intravenously in a volume of 1 ml/kg. NE, phenylephrine, AVP, and isoproterenol were administered in a volume of 0.1 ml per rat. All dosages, except for AVPX and (-)-NE, are expressed as the salt.

RESULTS

Cardiovascular responses to microinjection of CCh into the PHN

Unilateral microinjection of CCh in a volume of 50 nl directly into the PHN of conscious, freely-moving rats in doses ranging from 0.8 to 13.2 nmol evoked a dose-dependent increase (r² = 0.970, p < 0.01) in MAP (Fig. 1A). All five doses of CCh evoked an initial tachycardia (Fig. 1A); and 5.5-and 13.2-nmol doses of CCh evoked a secondary bradycardia that peaked 15 min after CCh administration (Fig. 1B). The increase in MAP was still dose-dependent (r² = 0.956, p < 0.01) 15 min after microinjection of CCh into the PHN (Fig. 1B).

Microscopic examination of tissue slices through the PHN region showed cannula tracts ending in or near the border of the PHN of rats that responded to CCh (Fig. 2A). The increase in MAP evoked by microinjection of 3.3 and 5.5 nmol CCh into the PHN did not differ, as shown in the three-dimensional graphs of Fig. 2C and D, across the four sections shown in Fig. 2A. Additional rats were prepared so that the guide cannula directed the injection cannula 0.5 mm lateral or caudal to the border of the PHN (Fig. 2A). Microinjections of 0.9% saline or of 3.3 or 5.5 nmol CCh into these areas failed to evoke an increase in MAP (Fig. 2B-D). Neither did microinjection of 0.9% saline directly into the PHN evoke a change in MAP (Fig. 2B). The baseline MAP of all rats averaged 108 ± 1 mm Hg; baseline HR rate averaged 387 ± 2 beats/min (n = 493) before drug administration.

To evaluate the effect of the dose of CCh and site of microinjection on HR, we placed the rats into one of several groups based on change in HR, site of microinjection, and dose of CCh (Table 1). Contingency table analysis of these data showed that the change in HR was best predicted by the dose of CCh administered rather than by site of microinjection (Table 1). The changes in HR evoked by microinjection of 3.3 or 5.5 nmol CCh lateral or caudal to the PHN did not differ from the changes induced by microinjection of 0.9% saline into these same areas or directly into the PHN (data not shown).

Selectivity of receptor antagonists

To evaluate the selectivity of the receptor antagonists, we used four vasoactive agents to challenge the receptor blockade provided by the antagonists. The data in Table 2 show that the antagonists, at the doses used, blocked only the receptors they were intended to block. Prazosin blocked the pressor response to the selective α₁-adrenoceptor agonist phenylephrine, AVPX blocked the pressor response induced by AVP, and propranolol blocked the depressor response to the β-adrenoceptor agonist isoproterenol. Yohimbine was selective for the α₂-adrenoceptor, as illustrated by its lack of effect on the pressor response to the selective α₁-adrenoceptor agonist phenylephrine but its attenuation of the nonselective α-adrenoceptor agonist NE, and by its complete blockade of the NE-evoked pressor response when combined with prazosin.

Attenuation of pressor responses to CCh by selective receptor blockade

To evaluate the contribution of α-adrenoceptors and V₁-vasopressin receptors to the pressor response evoked by CCh microinjection into the PHN, we pretreated rats intravenously with prazosin, yohimbine, AVPX, or a combination of these receptor antagonists before microinjection of CCh. The selective α₁-adrenoceptor antagonist prazosin (0.2 mg/kg) attenuated the maximum increase in MAP evoked by microinjection of 3.3 nmol CCh (62 ± 10% inhibition, n = 9), 5.5 nmol CCh (73 ± 7% inhibition, n = 9), and 13.2 nmol CCh (69 ± 5% inhibition, n = 8) into the PHN (Fig. 3). Blockade of α₂-adrenoceptors with yohimbine (0.3 mg/kg) attenuated the increase in MAP evoked by 3.3 nmol CCh (37 ± 13% inhibition, n = 8), 5.5 nmol CCh (43 ± 10% inhibition, n = 9), and 13.2 nmol CCh (45 ± 3% inhibition, n = 8) (Fig. 3). The combination of yohimbine and prazosin completely inhibited (102 ± 6%, n = 10) the increase in MAP evoked by 3.3 nmol CCh (Fig. 3). In contrast, this combination of antagonists did not attenuate the increase in MAP evoked by 5.5 nmol CCh (75 ± 8% inhibition, n = 10) or 13.2 nmol CCh (72 ± 9% inhibition, n = 8) any better than did prazosin alone (Fig. 3). Yohimbine had little effect on baseline arterial BP, whereas prazosin caused a slight decrease in baseline arterial BP (Table 3). The combination of prazosin and yohimbine caused a greater decrease in baseline arterial BP than did prazosin alone (Table 3). The selective V₁-vasopressin receptor antagonist AVPX (20 μg/kg) attenuated the increase in MAP evoked by 5.5 nmol CCh (23 ± 5% inhibition, n = 9) and 13.2 nmol CCh (47 ± 2% inhibition, n = 8), but not the increase induced by 3.3 nmol CCh (10 ± 12% inhibition, n = 8) (Fig. 3). The combination of prazosin and AVPX completely inhibited the increase in MAP evoked by 5.5 nmol CCh (92 ± 5% inhibition, n = 12) and 13.2 nmol CCh (96 ± 6% inhibition, n = 8). Prazosin and AVPX combined caused inhibition of the increase induced by 3.3 nmol CCh (74 ± 3% inhibition, n = 10) that was similar to that induced by prazosin alone (Fig. 3). AVPX had no effect on baseline BP nor did it affect, when combined with prazosin, the decrease in baseline BP caused by prazosin (Table 3).

The pressor response to 3.3, 5.5, and 13.2 nmol CCh was completely inhibited by the combination of α₁-adrenoceptor, α₂-adrenoceptor, and V₁-vasopressin receptor blockade. Addition of AVPX to prazosin and yohimbine inhibited the increase in MAP evoked by 3.3 nmol CCh (n = 10) to the same degree as prazosin and yohimbine, whereas addition of yohimbine to prazosin and AVPX inhibited the increase in MAP evoked by 5.5 nmol (n = 11) and 13.2 nmol (n = 8) CCh to the same degree as prazosin and AVPX (Fig. 3). The combination of these three antagonists caused a modest decrease in baseline arterial BP that was approximately the same as that caused by the combination of prazosin and yohimbine (Table 3).

Attenuation of the initial tachycardic response to CCh by propranolol

Pretreatment of rats with the β-adrenoceptor antagonist propranolol (1 mg/kg) did not affect the maximum increase in MAP evoked by 3.3, 5.5, or 13.2 nmol CCh (Fig. 4A). Propranolol pretreatment did inhibit the tachycardia that occurred after these three doses of CCh (Fig. 4A), yet had no effect on the increase in MAP or the bradycardia that occurred 15 min after the 5.5- and 13.2-nmol doses of CCh (data not shown). Propranolol inhibited the increase in HR evoked by intravenous administration of 2 μg isoproterenol by 55 ± 9% and caused a significant decrease in baseline HR without affecting baseline arterial BP (Table 3).

Attenuation of the secondary bradycardic response to CCh by methylatropine

Pretreatment of rats with the muscarinic receptor antagonist methylatropine (1 mg/kg) attenuated the secondary bradycardia evoked by 5.5 and 13.2 nmol CCh (Fig. 5B). Methylatropine pretreatment did not affect the initial tachycardia evoked by these doses of CCh (data not shown), despite an increase in baseline HR of ≈50 beats/min (Table 3). Methylatropine decreased the magnitude of the maximum increase in MAP induced by 13.2, but not that induced by 5.5 nmol CCh (data not shown), while attenuating the increase in MAP observed 15 min after both doses of CCh (Fig. 5A).

Alteration of the CCh-induced changes in HR by AVPX

The secondary bradycardia evoked by microinjection of 5.5 and 13.2 nmol CCh was also attenuated by the presence of AVPX, which actually reversed the bradycardia to a tachycardia (Fig. 6B). AVPX also enhanced the tachycardia evoked by the 5.5- and 13.2-nmol doses of CCh (Fig. 6A), while significantly attenuating the mean maximum increase (Fig. 3) and the increase in MAP observed 15 min after these doses of CCh (data not shown). Pretreatment with AVPX alone did not alter baseline HR (Table 3).

DISCUSSION

Microinjection of the nonselective cholinergic agonist CCh in doses ranging from 0.8 to 13.2 nmol directly into the PHN of conscious, freely moving rats evoked a dose-dependent increase in MAP. Administration of 3.3 or 5.5 nmol CCh in a volume of 50 nl into areas ≈0.5 mm lateral or caudal to the PHN failed to evoke changes in BP or HR different from those evoked by microinjection of 50 nl 0.9% saline directly into the PHN. The failure of CCh to elicit a response when administered in a volume of 50 nl lateral or caudal to the PHN suggests that the diffusion of this volume through brain tissue is restricted to a sphere with a radius of ≈0.5 mm. These results are similar to those obtained for the diffusion of 50 nl [³H]bicuculline around its site of administration into brain tissue, which was through a sphere with a radius of 0.6 ± 0.1 mm ⁽¹⁵⁾. Therefore, our present findings demonstrate that the increase in arterial BP and changes in HR which occur after microinjection of 50 nl CCh into the PHN is the result of activation of cholinergic receptors in the PHN and not in nearby hypothalamic structures such as the fields of Forel which, when electrically stimulated, increase BP ⁽¹⁶⁾. The cholinergic receptor involved in this CCh-induced increase in MAP was previously shown to be the M₃ muscarinic receptor ⁽¹⁰⁾ which is consistent with the demonstration of M₃ muscarinic receptors in this region by autoradiographic techniques ⁽¹⁷⁾.

To determine the mechanisms responsible for the pressor response evoked by administration of CCh into the PHN, we used antagonists of the receptors involved in the sympathetic nervous system to assess the contribution of these receptors to the CCh-induced changes in BP. Pretreatment of rats with the selective α₁-adrenoceptor antagonist prazosin resulted in 62-73% inhibition of the increase in MAP evoked by 3.3, 5.5, and 13.2 nmol CCh. Similarly, prazosin inhibited an increase in MAP evoked by intravenous administration of the nonselective α-adrenergic agonist NE by 71%. The similarity in the level of inhibition provided by prazosin of the pressor response to centrally administered CCh and peripherally administered NE suggests that the attenuation of the CCh-evoked pressor response was due to blockade of peripheral α₁-adrenoceptors and not to blockade of central α₁-adrenoceptors. These results demonstrate that a major portion of the increase in MAP induced by CCh microinjection into the PHN results from stimulation of α₁-adrenoceptors.

Pretreatment of rats with the selective α₂-adrenoceptor antagonist yohimbine, used at a dose previously reported not to have significant central effects in normotensive Wistar rats ⁽¹⁸⁾, attenuated the pressor response evoked by 3.3, 5.5, and 13.2 nmol CCh 37-45%, which was similar to the 37% inhibition induced by yohimbine of the increase in MAP evoked by intravenous injection of NE. These results indicate that a portion of the increase in MAP induced by CCh results from stimulation of α₂-adrenoceptors.

Pretreatment of rats with the combination of prazosin and yohimbine completely inhibited the increase in MAP evoked by the 3.3-nmol dose of CCh, indicating that both α-adrenoceptors are involved in the pressor response to this dose of CCh. In contrast, this combination of antagonists did not inhibit the increase in MAP induced by 5.5 or 13.2 nmol CCh to any greater degree than did prazosin alone. These results imply that the increase in MAP evoked by 5.5 and 13.2 nmol CCh is largely the result of activation of α₁-adrenoceptors and receptors for at least one other endogenous pressor substance.

Several endogenous substances could have been responsible for mediating the remaining pressor response to the higher doses of CCh, including AVP, angiotensin II, and endothelin. To assess the possible involvement of AVP, we administered AVPX, which selectively blocked the pressor response to AVP, intravenously before CCh microinjection. Blockade of vasopressin receptors did not affect the pressor response evoked by 3.3-nmol CCh, but did significantly attenuate the pressor response to the 5.5- and 13.2-nmol doses. These results indicate an involvement of AVP in the hypertensive response to the two higher doses of CCh. In addition, the combination of prazosin and AVPX completely blocked the pressor response to the two higher doses while failing to inhibit the increase in MAP evoked by 3.3 nmol CCh to any greater degree than prazosin alone. These results further suggest that AVP is involved in the pressor response to the higher doses of CCh. Why BP did not increase after the two higher doses of CCh in the presence of prazosin and AVPX due to stimulation of α₂-adrenoceptors is not known. However, the combination of prazosin and AVPX did not affect the tachycardia evoked by the two higher doses of CCh (J. R. Martin, unpublished observations) which would be expected to increase BP. Therefore, the higher doses of CCh may lead to greater stimulation of the adrenal medullae than does the 3.3-nmol dose of CCh. The released epinephrine acting on α₂-, β₁-, and β₂-adrenoceptors may result in opposing effects on BP, resulting in no overall change in MAP. Because other plausible explanations exist for this observation, the exact mechanism involved in this phenomenon deserves further study.

All the doses of CCh used in this study evoked tachycardia that was significantly attenuated by pretreatment with the β-adrenoceptor antagonist propranolol. These results indicate that the increase in HR, like the increase in BP, is due primarily to an increase in sympathetic nerve activity. An increase in sympathetic nerve activity is consistent with an increase in renal sympathetic nerve activity previously observed in urethane-anesthetized rats ⁽¹⁴⁾. The tachycardia evoked by the 5.5- and 13.2-nmol doses of CCh was followed by a secondary bradycardia that was not affected by the presence of propranolol but was attenuated by pretreatment with methylatropine or AVPX. These results indicate that an increase in vagal tone, probably caused by activation of the baroreceptor reflex by the increased BP, is partly responsible for the bradycardia. This bradycardia may be potentiated by an increase in plasma levels of vasopressin. Because AVP enhances bradycardic responses resulting from baroreceptor reflex activation ^(19-21), blockade of vasopressin receptors would be expected to inhibit the secondary bradycardia evoked by the higher doses of CCh. These were exactly the results obtained in the present study. Because AVPX selectively blocks the V₁-vasopressin receptor ⁽²²⁾, the present results suggest that the vasopressin receptor involved in the bradycardia resulting from the CCh-evoked increase in MAP is the V₁-vasopressin receptor. Recent studies suggest that these V₁-vasopressin receptors might be located in the area postrema ⁽²³⁾.

The increase in plasma levels of AVP may have resulted from diffusion of CCh from the PHN to the paraventricular nucleus of the hypothalamus, where activation of muscarinic receptors stimulates the release of AVP into the circulation ⁽²⁴⁾. Because a 50-nl volume of CCh diffuses from the tip of the injection cannula through a sphere with a radius of ≈0.5 mm and the paraventricular nucleus lies at least 1 mm rostral to the sites of CCh microinjection, it is unlikely that the CCh diffused from the PHN to the paraventricular nucleus. Alternatively, CCh may have diffused into the cerebrospinal fluid (CSF) of the third ventricle and was then carried to the paraventricular nucleus through the CSF. However, microinjection of 5.5 nmol CCh into the PHN of urethane-anesthetized rats results in an increase in MAP that is accompanied only by tachycardia despite the use of an injection volume of 1 μl (J. R. Martin, unpublished observations). Therefore, if CCh had diffused to the paraventricular nucleus, there should have been a secondary bradycardia in the anesthetized rats similar to that which occurred in the conscious rats. In addition, it is unlikely that vasopressin was released as a result of baroreceptor reflex activation since reflex release of vasopressin is stimulated by hypotension accompanied by hypovolemia. Therefore, the vasopressin released into the circulation after CCh microinjection into the PHN was most likely due to activation of a neuronal connection between the PHN and the paraventricular nucleus.

The present results further localize a CCh-induced increase in MAP accompanied by tachycardia to the PHN ⁽¹⁴⁾. The mechanisms underlying these cardiovascular changes, as determined by the use of selective receptor antagonists, are an increase in the activity of the sympathetic nervous system which results in an increase in total peripheral resistance ⁽⁹⁾, MAP, and HR. The increase in total peripheral resistance and MAP evokes a baroreceptor-mediated increase in vagal activity which then predominates over the action of the sympathetic nervous system on the heart, thereby resulting in bradycardia. In addition, the action of CCh in the PHN results in release of AVP into the circulation, especially at higher doses of CCh, which mediates a portion of the increase in MAP. Furthermore, the circulating AVP potentiates the baroreceptor reflex somewhere along the baroreceptor reflex arc (possibly the area postrema), resulting in an enhancement of the baroreceptor-mediated bradycardia. These conclusions are consistent with present knowledge about the effects of the autonomic nervous system and AVP on the cardiovascular system, including the baroreceptor reflex.

Acknowledgment: This work was supported by National Institutes of Health Grant No. HL-44531 and by a grant-in-aid from the Missouri Affiliate of the American Heart Association. I thank Ruth Chronister for expert secretarial help during the preparation of the manuscript; Hector Salas, Akram Farr, and Barbara Redel for technical help in conducting experiments and in histological preparation of rat brains; and Dr. Robert Baer for helpful discussions of the data.

FIG. 1: . Microinjection of carbachol (CCh) directly into the posterior hypothalamic nucleus in increasing doses from 0 to 13.2 nmol resulted in a dose-dependent maximum increase in mean arterial pressure (MAP) (left y-axis) and an initial tachycardia (HR, heart rate) (right y-axis) as shown in A. The dose-dependent increase in MAP persisted 15 min after microinjection of CCh (B). The initial tachycardia became bradycardia 15 min after microinjection of 5.5 and 13.2 nmol CCh (B). ^aSignificantly different from rats microinjected with 50 nl 0.9% saline (0-nmol dose of CCh); ^bsignificantly different from rats microinjected with 0.8 nmol CCh; ^csignificantly different from rats microinjected with 0.8, 1.6, or 3.3 nmol CCh. Numbers in parentheses are numbers of rats injected with that particular dose of CCh.

FIG. 2: . Histological verification of injection sites. A: The tissue sections show the sites of injection cannula tips: microinjections into the posterior hypothalamic nucleus PHN (open circles); microinjections lateral and caudal to the PHN (open squares). The number to the left of each tissue section indicates how far (in mm) the section is caudal to bregma; the number below those numbers represents the number of rats in which microinjection sites were localized to the tissue section shown. B-D: Three-dimensional representation of the change in MAP (z-axis) based on the location of the microinjection with respect to bregma (x-axis), midline (y-axis), and the dose of CCh (0 nmol in B, 3.3 nmol in C, 5.5 nmol in D). Zl, zona incerta; STh, subthalamic nucleus; SN, substantia nigra; LH, lateral hypothalamic area; mt, mammillothalamic tract; f, fornix; Arc, arcuate nucleus; DM, dorsomedial nucleus of hypothalamus; VM, ventromedial nucleus of hypothalamus; PMV, premammillary nucleus, ventral; PMD, premammillary nucleus, dorsal; VTA, ventral tegmental area; MM, medial mammillary nucleus.

FIG. 3: . Pretreatment of rats with various antagonists attenuated to differing degrees the mean maximum increase in mean arterial pressure (MAP) evoked by microinjection of 3.3, 5.5, or 13.2 nmol carbachol (CCh) into the posterior hypothalamic nucleus (PHN). ^aSignificantly different from the percent of mean maximum response of rats pretreated with 0.9% saline (SAL); ^bsignificantly different from the percent of mean maximum response of rats pretreated with D[(CH₂)₅Tyr(Me)]arginine vasopressin (AVPX) or yohimbine (YOH); ^csignificantly different from the percent of mean maximum response of rats pretreated with SAL, AVPX, YOH, prazosin (PRAZ), or PRAZ and YOH; ^dsignificantly different from the percent of maximum response of rats pretreated with SAL, AVPX, YOH, PRAZ, or PRAZ and AVPX.

FIG. 4. A:: Propranolol (PRO) pretreatment had no effect on the increase in MAP evoked by microinjection of 3.3, 5.5, or 13.2 nmol CCh into the posterior hypothalamic nucleus (PHN). B: PRO pretreatment significantly attenuated the increase in heart rate (HR, beats/min) evoked by microinjection of 3.3, 5.5, or 13.2 nmol CCh into the PHN. ^*Significantly different from the change in MAP or HR evoked by microinjection of 0.9% saline (0-nmol dose of CCh) into the PHN nucleus of rats pretreated with 0.9% saline (SAL) or PRO, and ^**significantly different from the change in HR evoked by microinjection of CCh into the PHN of rats pretreated with SAL.

FIG. 5. A:: Methylatropine (MeATR) pretreatment attenuated the increase in mean arterial pressure (MAP) evoked by 5.5 or 13.2 nmol CCh 15 min after microinjection. B: MeATR pretreatment significantly attenuated the bradycardia observed 15 min after microinjection of 5.5 or 13.2 nmol CCh into the posterior hypothalamic nucleus (PHN). ^*Significantly different from the change in MAP or HR evoked by microinjection of 0.9% saline (0-nmol dose of CCh) into the PHN, and ^**significantly different from the change in MAP or HR evoked by microinjection of CCh into the PHN of rats pretreated with 0.9% saline (SAL).

FIG. 6. A:: Pretreatment of rats with D[(CH₂)₅Tyr(ME)]arginine vasopressin (AVPX) enhanced the tachycardia observed after the microinjection of 5.5 or 13.2 nmol CCh into the posterior hypothalamic nucleus (PHN). ^*Significantly different from the change in heart rate (HR, beats/min) observed after the microinjection of 3.3, 5.5, or 13.2 nmol CCh into the PHN, and ^**significantly different from the change in HR observed in rats pretreated with 0.9% saline (SAL) and microinjected with 5.5 nmol CCh. B: AVPX pretreatment attenuated the bradycardia observed after the 5.5- and 13.2-nmol doses of CCh so that a tachycardia was present 15 min after microinjection of these two doses of CCh. ^*Significantly different from the change in HR of rats microinjected with 0.9% saline (0-nmol dose of CCh), and ^**significantly different from the change in HR observed in rats pretreated with AVPX microinjected with 5.5 or 13.2 nmol CCh.