Henry E. Brezenoff

A comparison of the central and peripheral antimuscarinic effects of atropine and methylatropine injected systematically 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.

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.

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.

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.

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.

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.

Brain neurotransmitters and the development and maintenance of experimental hypertension

Animal models of human hypertensive disease have been available since 1933, when Goldblatt and co-workers [1] described persistent hypertension in dogs following controlled constriction of the renal arteries. Of the several species utilized, the rat has provided the most diverse number of experimental approaches. In recent years, the most widely employed rat model of essential hypertension has been the spontaneously hypertensive (SH) rat. Introduced in 1962, following selective breeding of the Wistar Kyoto strain [2], the SH rat is heralded as closely resembling the human disease. Hypertension develops in these animals during maturation and persists throughout life without surgical, pharmacological or dietary intervention. In both SH rats and human essential hypertension, the increased blood pressure initially is associated with increased cardiac output, and later with increased vascular resistance and normal cardiac output.

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

Intraperitoneal (IP) administration of the acetylcholinesterase inhibitor, soman (10–40 μg/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.

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.

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.