Identification of pressor regions activated by central cholinergic stimulation in rat brain.

The acetylcholinesterase inhibitor neostigmine (2 microg) was microinjected into the lateral cerebral ventricle (i.c.v.) of unanesthetized rats to activate central cholinergic receptors. Changes in arterial blood pressure were correlated with changes in Fos-like immunoreactivity in the hypothalamus and forebrain following cholinergic stimulation. Neostigmine increased mean arterial pressure by 39 +/- 3 mmHg at peak (P < 0.05) from a pretreatment level of 104 +/- 4 mmHg. Blood pressure remained elevated for more than 30 min. Distinct Fos-like immunoreactivity was found in the posterior hypothalamic nucleus, the paraventricular nucleus and the supraoptic nucleus of the hypothalamus, the ventral premamillary nucleus, the central nucleus of amygdala, the lateral septum and the medial preoptic area. In contrast, only a very small amount of Fos-like immunoreactivity was scattered in those regions in a control group injected i.c.v. with saline. Pretreatment with the muscarinic receptor antagonist methylatropine (i.c.v., 0.5 microg) prevented the pressor response to neostigmine and evoked a reduced Fos-like immunoreactivity compared to animals given neostigmine without methylatropine. The pressor response to neostigmine was blocked after pretreatment with phenoxybenzamine, however, this did not prevent the development of Fos-like immunoreactivity. These results indicate that the pressor response induced by central cholinergic stimulation may result from muscarinic receptor activation in specific regions of the hypothalamus and the forebrain that are implicated in regulating cardiovascular activity.

Effect of hemicholinium-3 on the hypothalamic concentration of a cytochemically detectable glucose-6-phosphate dehydrogenase-stimulating substance.

Hypothalamus and plasma of salt-loaded rats, spontaneously hypertensive rats (SHR), and hypertensive reduced renal mass rats (RRM), and the plasma of patients with essential hypertension and of Milan hypertensive rats contain an increased concentration of a cytochemically detectable glucose-6-phosphate dehydrogenase (G6PD)-stimulating substance that has properties similar to that of a possible choline derivative di-methyl methylene immonium ion. Intracerebroventricular (i.c.v.) administration of hemicholinium-3 (HC-3) selectively blocks high-affinity neuronal choline uptake, inhibits brain acetylcholine (ACh) synthesis, and decreases arterial pressure in SHR through an inhibiting effect on hypothalamic cholinergic function. The experiments were performed to study the effect of centrally administered HC-3 on the content of the cytochemically detectable cholinelike substance in hypothalamus and plasma of SHR. HC-3 or saline was infused into the lateral cerebral ventricle for 6 days with a minipump in 14 SHR. On day 7, the hypothalamic and plasma concentration of the cytochemically detectable substance was significantly reduced in rats that received HC-3. The hypothalamic concentration was 225 +/- 95.6 x 10(8) G6PD U per hypothalamus (range 38.2-775) in SHR that received saline and 1.037 +/- 0.45 x 10(8) G6PD U (range 0.112-3.61) (p < 0.05) in SHR that received HC-3. The respective plasma concentrations were 284.9 +/- 26 U/ml (range 192-374) and 72.7 +/- 14.7 U/ml (range 24-119) (p < 0.05). The findings are consistent with the physicochemical evidence, which suggests that the cytochemically detectable substance is a choline derivative.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of repeated administration of soman on schedule-controlled behavior and brain in the rat.

The effects of repeated SC administration of soman on schedule-controlled performance and brain pathology were studied in the rat. Soman suppressed response rates in both components of a multiple fixed interval 50-sec fixed-ratio 25 (mult. FI 50-sec FR 25) schedule of reinforcement, although all animals revealed marked tolerance to repeated drug administration. Response rates generally recovered to baseline levels within 1-3 sessions. Three of the six animals studied, however, demonstrated marked deterioration of steady state schedule performance, particularly during the FI 50-sec component of the multiple schedule. Compared to untreated controls, all soman-treated animals exhibited pathological changes in brain. The most salient finding was glial cell proliferation in layer 4 and deep parts of layer 3 of the cerebral cortex. Glial cell proliferation was most marked in animals that exhibited deterioration of steady state schedule performance.

Acetylcholine in the posterior hypothalamic nucleus is involved in the elevated blood pressure in the spontaneously hypertensive rat.

Intravenous injection of physostigmine, 40 and 80 ug/kg, in unanesthetized normotensive rats increased systolic blood pressure (SBP) by 21 +/- 3 and 42 +/- 7 mmHg. This pressor response was 80% inhibited by intracerebroventricular (icv) injection of hemicholinium-3 (HC-3), 20 ug. Simultaneous icv injection of HC-3 and choline (365 ug) prevented the inhibition of the pressor response by HC-3. In spontaneously hypertensive rats, injection of HC-3 either icv (20 ug) or bilaterally into the posterior hypothalamic nuclei (1 ug) decreased SBP by about 40 mmHg. The effect of intrahypothalamic HC-3 was completely blocked by simultaneous injection of choline (24.3 ug) into the same site. The hypotensive effect of icv HC-3 was completely blocked by icv choline (243 ug) and was inhibited up to 60% by injections of choline (24.3 ug) into the posterior hypothalamic nuclei.

The role of M2 muscarinic receptors in the posterior hypothalamus in the pressor response to intracerebroventricularly-injected neostigmine.

Injection of neostigmine into the lateral cerebral ventricle of urethane-anesthetized rats increases arterial blood pressure. Prior injection of atropine or the muscarinic M2 antagonist 4-DAMP into the posterior hypothalamic nuclei inhibited the pressor response to neostigmine by up to approximately 56%. The same maximum degree of inhibition was elicited by bilateral electrical lesions of the posterior hypothalamic nuclei. The response was not modified by intrahypothalamic injection of pirenzepine or intraventricular injection of hexamethonium, but was prevented by intraventricular injection of 4-DAMP. The results indicate that about half of the pressor response to intraventricular injection of neostigmine was mediated through M2 muscarinic receptors in the posterior hypothalamic nuclei, and the remainder through M2 muscarinic receptors in other regions of the brain.

Suppression of hypertension during chronic reduction of brain acetylcholine in spontaneously hypertensive rats.

Experiments were conducted to determine the effects of chronic depletion of brain acetylcholine (ACh) on the development and maintenance of hypertension in spontaneously hypertensive rats (SHR). Synthesis of brain ACh was inhibited by chronic infusion of hemicholinium-3 (HC-3) into the cerebral ventricles, and systolic blood pressure was monitored by tail cuff occlusion. In 5-week-old SHR, infusion of HC-3 (0.25 micrograms/h) suppressed development of hypertension when compared to saline-infused control SHR during the 21 days of infusion (140 versus 190 mmHg on day 21). Hypothalamic and brain-stem ACh during this period was reduced by 50% and by 60-75%, respectively. In 18-week-old SHR with established hypertension, HC-3 (0.25 and 0.5 micrograms/h) reduced systolic blood pressure by 35-40 mmHg for 8 days, after which pressures returned to control hypertensive levels (191 mmHg) by day 14. The increase in blood pressure was accompanied by recovery of hypothalamic ACh levels to 75% of control. The specificity and physiological effectiveness of HC-3 was shown by its ability to inhibit the centrally mediated pressor response to physostigmine but not to oxotremorine. Infusion of HC-3 did not affect body growth, water consumption, body temperature or gross behavior. From this study, it can be concluded that brain cholinergic neurons are an important component in the development and the maintenance of hypertension in the SHR.

Blockade of brain M2 muscarinic receptors lowers blood pressure in spontaneously hypertensive rats.

Injections of the M2 muscarinic receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; 1.5-40 micrograms) into the cerebral ventricles of urethane-anesthetized rats caused a dose-related inhibition of the pressor response to intravenously injected physostigmine. A similar reduction was obtained with 1/80th the dose of methylatropine, but not with the selective M1 antagonist pirenzepine. Intraventricular injection of 4-DAMP (6.25-25 micrograms) caused a dose-related reduction in blood pressure in unanesthetized spontaneously hypertensive rats (SHR), but not in normotensive controls. Systolic pressure fell 42 +/- 6 mm Hg at the 25-micrograms dose. Pirenzepine did not lower blood pressure in SHR and inhibited the antihypertensive effect of 4-DAMP.

A comparison of the central and peripheral antimuscarinic effects of atropine and methylatropine injected systemically and into the cerebral ventricles.

We compared the relative abilities of atropine sulfate and methylatropine, injected i.v. and into the cerebral ventricles (icv), to block pharmacological responses mediated through central and peripheral muscarinic receptors. The hypotensive response to i.v. injection of acetylcholine (peripheral muscarinic receptors) was inhibited 50% by i.v. injection of 14.3 nmol (5.5 micrograms)/kg methylatropine and 147.8n molar equivalents (50 micrograms)/kg atropine sulfate. A similar degree of inhibition followed icv injection of 49.4 nmol/kg methylatropine and 384.2 nmol equivalents/kg atropine sulfate, indicating significant leakage out of the ventricular space. The pressor response to icv injection of neostigmine (central muscarinic receptors) also was inhibited more effectively by icv methylatropine than by atropine sulfate. Methylatropine was not effective in blocking central muscarinic receptors when injected i.v.

Protection by physostigmine against the pressor effect of soman in the rat.

Intravenous injection of soman, 30 ug/kg, increased mean arterial blood pressure by 57 mmHg in urethane-anesthetized rats. The response declined slightly after a few minutes and then remained stable at about 39 mmHg for the next 20 minutes. The increase in pressure was accompanied by marked inhibition of brain acetylcholinesterase (AChE). In rats pretreated with a threshold pressor dose of physostigmine (50 ug/kg, i.v.), the peak pressor response to soman rose to the same level as that in the control group, but showed a more rapid recovery, reaching 17 mmHg at 20 minutes. The recovery of blood pressure was accompanied by partial recovery of brain AChE.

Increased central cholinergic activity in rat models of hypertension.

The role of brain acetylcholine (ACh) in the development and maintenance of experimental hypertension was evaluated. Single, acute injections of hemicholinium-3 (HC-3), 20 micrograms, were made into the lateral cerebral ventricle of unanesthetized rats during development of hypertension in the spontaneously hypertensive rat (SHR) or following induction of deoxycorticosterone-salt (DOCA), aortic coarctation (AoCo), and Grollman hypertension. HC-3 caused nonsignificant reductions in blood pressure (BP) when injected in SHR and Wistar-Kyoto (WKY) controls at 5 and 8 weeks of age. At 12 weeks in SHR, depressor responsiveness increased concomitantly with the development of hypertension and reached maximal stable effect during established hypertension (18-60 weeks). Intravenous (i.v.) infusion of sodium nitroprusside (NaNP) normalized BP of adult SHR, yet did not prevent the hypotensive response to HC-3. In DOCA hypertension, the HC-3 hypotensive effect (1 week postinduction) preceded establishment of significantly elevated BP (3 weeks), but then remained constant despite a continual rise in BP (2-6 weeks). In the AoCo model, onset of severe hypertension (day 2 postinduction) preceded the appearance of significant depressor responsiveness to HC-3 (day 10). Although BP remained uniformly elevated from 10 to 40 days, the magnitude of the depressor response was stable between 10 and 28 days, then increased further at day 40. At the only time studied, 4 weeks after induction of hypertension, HC-3 also lowered BP of Grollman rats. Marked bradycardia occurred in all models and controls. The results suggest that enhancement of central cholinergic neurotransmission is a common feature of experimental hypertension regardless of its etiology. Increased brain cholinergic function appears to be involved in the development (but not the initiation) and maintenance of elevated BP in the SHR, whereas it may play a role in the initiation of DOCA hypertension and in the maintenance of AoCo hypertension.

Cerebral cholinergic control of rat arterial blood pressure in streptozotocin-induced diabetes.

Rats were made diabetic with a single intravenous injection of streptozotocin (STZ; 40 mg/Kg). Buffer treated animals were used as controls. Experiments were performed 7 and 14 days thereafter. One week diabetic rats (plasma glucose = 3.34 +/- 0.58 mg/ml), compared with control animals (plasma glucose = 0.94 +/- 0.33 mg/ml), showed higher (P less than 0.05), more prolonged and dose-dependent pressor and bradycardic responses to intracerebroventricular (icv) injection of carbachol (125, 250 and 500 ng), together with a significantly lower bradycardia after icv injection of physostigmine (1.25, 2.5 and 5 mcg). The pressor response to icv injection of physostigmine (1.25 mcg) was significantly reduced in diabetic rats. Pressor and bradycardic responses induced by angiotensin II (100 and 200 ng, icv) did not show any differences between control and diabetic animals, thus ruling out an impairment of peripheral nerve conduction. Diabetic rats exhibited higher content of acetylcholine (Ach) in the striatum (123.8 +/- 3.09 nmoles/g) and in the hypothalamus (45.7 +/- 1.31 nmoles/g). Three weeks diabetic animals (plasma glucose = 2.76 +/- 0.23 mg/ml) had neither different cardiovascular responsiveness to icv injection of muscarinic agonists nor changes in hypothalamus and striatum Ach content. Data strongly suggest that STZ-induced diabetes temporarily alters cerebral acetylcholine control of cardiovascular apparatus.

Effect of soman on schedule-controlled behavior and brain acetylcholinesterase in rats.

Rats were trained to press a lever under a multiple fixed-ratio 25 fixed-interval 50-second (FR25 FI50-sec) schedule of food reinforcement. Soman, 70-90 micrograms/kg, s.c., suppressed response rates in both components, with a slightly greater effect in the FI schedule. The pattern of responding under the FI schedule, however, was maintained until lever-pressing was nearly completely suppressed. At the highest doses, soman occasionally caused tremors or mild tonic seizures with hindlimb abduction. The suppression of response rate was correlated with inhibition of acetylcholinesterase (AChE) in all brain regions examined: cortex, striatum, hippocampus, hypothalamus and brainstem. Cortical AChE was inhibited to the highest degree, while striatal AChE was most resistant to inhibition by soman.

Inhibition of acetylcholinesterase in the gut inhibits schedule-controlled behavior in the rat.

Rats were trained to press a lever under a multiple Fixed-Ratio 25 Fixed-Interval 50-second schedule of food reinforcement. Subcutaneous injection of soman, 80 micrograms/kg, suppressed responding under both schedules and inhibited acetylcholinesterase (AChE) in the brain. AChE activity in the gastrointestinal tract was not significantly inhibited. In contrast, i.p. injection of either soman (10-40 micrograms/kg), neostigmine (75 micrograms/kg) or DFP (350 micrograms/kg) caused marked suppression of behavior and AChE activity of the gut, without affecting brain AChE. These doses caused marked increases in peristaltic activity and likely caused gastrointestinal spasm. Injection of DFP, 500 micrograms/kg, s.c., inhibited AChE in both the brain and gut. The results indicate that inhibition of AChE in the gastrointestinal tract by certain anticholinesterase agents may be involved in the behavioral effects attributed to these drugs.

Effect of repeated intraperitoneal injections of soman on schedule-controlled behavior in the rat.

Intraperitoneal (IP) administration of the acetylcholinesterase inhibitor, soman (10-40 micrograms/kg), suppressed in a dose-related manner response rates in rats maintained under a multiple fixed-interval 50-s fixed-ratio 25 schedule of food delivery. Chronic administration of soman at weekly intervals resulted in tolerance to the response. When soman administration was separated by 2-5 weeks in individual rats, the suppressive effects of the agent again became apparent. Analysis of acetylcholinesterase activity revealed that enzyme inhibition was limited to gastrointestinal areas near the site of injection. There was no significant effect on brain acetylcholinesterase even following IP injection of doses which completely suppressed responding. The IP route may be useful for studying tolerance and other chronic effects of soman without producing generalized toxicity.

The hypertensive response to soman and its relation to brain acetylcholinesterase inhibition.

Intravenous injection of soman in the rat produced a rapid and dose related increase in blood pressure. The dose response curve was very steep, threshold responses occurring after intravenous injection of 10 micrograms/kg, and maximum increases of about 50 mmHg occurring after 40 micrograms/kg. Heart rate also generally increased. An increase in blood pressure also followed injection of soman subcutaneously, intramuscularly, intraperitoneally and into the cerebral ventricles, although the onset was slower and higher doses were required. The magnitude of the pressor response was correlated with the degree of AChE activity in the cortex, hypothalamus and brain stem, but not in the striatum. The pressor response was aborted or prevented by atropine, but not by methylatropine. It also was prevented by phenoxybenzamine. Atropine increased survival following an LD50 dose of soman; phenoxybenzamine prevented the pressor response but did not alter the survival rate.

Effects of selective central muscarinic blockade on schedule-controlled behavior and on the rate-decreasing effects of physostigmine.

N-(4-diethylamino-2-butynyl)-succinimide, or DKJ-21, is a muscarinic receptor antagonist with a high degree of selectivity for the central nervous system. In the present study of 6 rats maintained under a fixed-interval 50-sec schedule of food reinforcement, atropine and methylatropine reduced responding in a dose dependent manner, while DKJ-21 had little or no effect. Our findings suggest that the suppression caused by atropine and methylatropine may be the result of the dry mouth induced by these agents. Doses of DKJ-21 which had no effect on schedule performance antagonized the rate-lowering effects of physostigmine in all of the animals. Neither atropine nor methylatropine consistently antagonized the inhibitory effects of physostigmine. Some antagonism may be inferred, however, from the findings that response rates were suppressed less by combinations of atropine and physostigmine than by either drug alone.

Cardiovascular regulation by brain acetylcholine.

Evidence is presented that implicates brain acetylcholine (ACh) in the control of blood pressure (BP) and in hypertension. Central cholinergic stimulation by muscarinic agonists or inhibitors of acetylcholinesterase (AChE) evokes a hypertensive response in several animal species, including humans. The elevation in BP after injection of AChE inhibitors is mediated centrally by ACh acting on muscarinic receptors and peripherally through increased sympathetic nerve activity. The pressor response is accompanied by inhibition of reflex tachycardia and potentiation of both reflex bradycardia and the pressor reflex to carotid artery occlusion. Intracerebroventricular injection of hemicholinium 3 in doses that deplete brain ACh lowers BP in the spontaneously hypertensive and the deoxycorticosterone acetate-salt hypertensive rat. Little or no reduction occurs in the normotensive rat or in animals made hypertensive by aortic coarctation. In addition, atropine and the selective central muscarinic receptor antagonist N-(4-diethylamino-2-butynyl)-succinimide lower BP in the spontaneously hypertensive rat but not in the normotensive rat.

Potentiation of the carotid artery occlusion reflex by a cholinergic system in the posterior hypothalamic nucleus.

Bilateral occlusion of the common carotid arteries of urethane-anesthetized rats evoked a pressor response of 14 +/- 1 mm Hg. Injection into the lateral cerebral ventricle of neostigmine (0.2-1.0 microgram) or physostigmine (10-15 microgram) caused a dose-dependent increase in basal blood pressure and in the magnitude of the carotid artery occlusion (CAO) pressor reflex. Neostigmine (1 microgram) and physostigmine (15 microgram) caused nearly maximal and approximately equal degrees of cholinesterase inhibition in several brain regions. The recovery of the cardiovascular parameters and of brain cholinesterase activity was significantly faster following physostigmine compared to neostigmine. Prior intracerebroventricular injection of atropine (0.3 microgram) or hemicholinium-3 (20 microgram) prevented the increases in basal pressure and the CAO pressor response. Potentiation of the CAO reflex also followed injection of physostigmine or neostigmine into the posterior hypothalamic nucleus and of injection of physostigmine intravenously. Injection of atropine bilaterally into ther posterior hypothalamic nucleus prior to intravenous injection of physostigmine prevented the potentiation of the CAO reflex but not the increase in basal blood pressure. These results indicate that acetylcholine in the posterior hypothalamic nucleus serves as a neurotransmitter in a pathway which can potentiate the pressor response to carotid artery occlusion and thus modulate baroreceptor reflexes.