Metyrapone and fluoxetine suppress enduring behavioral but not cardiac effects of subchronic stress in rats

In humans, chronic stressors have long been recognized as potential causes for cardiac dysregulation. Despite this, the underlying mechanistic links responsible for this association are still poorly understood. The purpose of this study was to determine whether exposure to a paradigm of subchronic stress can provoke enduring changes on the heart rate of experimental rats and, if so, to reveal the autonomic and neural mechanisms that mediate these effects. The study was conducted on adult male Sprague-Dawley rats instrumented for telemetric recording of heart rate and locomotor activity. Animals were submitted to a subchronic stress protocol, consisting of a 1-h foot shock session on five consecutive days. Heart rate and locomotor activity were recorded continuously for 3 days before and for 6 days after the subchronic stress period. Subchronic foot shock produced significant and enduring reduction in heart rate both during the dark/active [Δ= −23 ± 3 beats per minute (bpm)] and light/inactive (Δ= −20 ± 3 bpm) phases of the circadian cycle, and a reduction in locomotor activity during the dark/active phase [Δ= −54 ± 6 counts per hour (cph)]. The bradycardic effect of subchronic stress was not related to a reduced locomotion. Selective sympathetic (atenolol) and vagal (methyl-scopolamine) blockades were performed to reveal which autonomic component was responsible for this effect. We found that the fall in heart rate persisted after subchronic stress in animals treated with atenolol (active phase Δ= −16 ± 3 bpm, inactive phase Δ= −19 ± 2 bpm), whereas vagal blockade with scopolamine transiently prevented this effect, suggesting that the bradycardia following subchronic stress was predominantly vagally mediated. Fluoxetine (selective serotonin reuptake inhibitor) and metyrapone (inhibitor of corticosterone synthesis) treatments did not affect heart rate changes but prevented the reduction in locomotion. We conclude that subchronic stress exposure in rats reduces heart rate via a rebound in vagal activation and that this effect is serotonin- and corticosterone-independent.

Acute Ethanol Exposure Elevates Muscarinic Tone in the Septohippocampal System

The septohippocampal system has been implicated in the cognitive deficits associated with ethanol consumption, but the cellular basis of ethanol action awaits full elucidation. In the medial septum/diagonal band of Broca (MS/DB), a muscarinic tone, reflective of firing activity of resident cholinergic neurons, regulates that of their noncholinergic, putatively GABAergic, counterparts. Here we tested the hypothesis that ethanol alters this muscarinic tone. The spontaneous firing activity of cholinergic and noncholinergic MS/DB neurons were monitored in acute MS/DB slices from C57Bl/6 mice. Exposing the entire slice to ethanol increased firing in both cholinergic and noncholinergic neurons. However, applying ethanol focally to individual MS/DB neurons increased firing only in cholinergic neurons. The differential outcome suggested different mechanisms of ethanol action on cholinergic and noncholinergic neurons. Indeed, with bath-perfused ethanol, the muscarinic antagonist methyl scopolamine prevented the increase in firing in noncholinergic, but not cholinergic, MS/DB neurons. Thus, the effect on noncholinergic neuronal firing was secondary to ethanol's direct action of acutely increasing muscarinic tone. We propose that the acute ethanol-induced elevation of muscarinic tone in the MS/DB contributes to the altered net flow of neuronal activity in the septohippocampal system that underlies compromised cognitive function.

Peripheral cholinergic pathway modulates hyperthermia induced by stress in rats exposed to open-field stress

Exposure to an open field is psychologically stressful and leads to an elevation in core temperature (Tc). Methyl scopolamine (MS), a muscarinic antagonist, and pyridostigmine (PYR), a carbamate that inhibits acetylcholinesterase, do not cross the blood-brain barrier and have little effect on Tc in resting, nonstressed animals. However, we have found that MS has an antipyretic effect on Tc that is caused by handling and cage-switch stress. PYR should act pharmacologically to reverse the effects of MS. To this end, we assessed the effects of MS and PYR on stress-induced hyperthermia. Male Sprague-Dawley rats at 90 days of age were housed individually at an ambient temperature of 22°C. Tc and motor activity were monitored by radiotelemetry in an open-field chamber. Rats were dosed intraperitoneally at 1200 with 1.0 mg/kg MS, 0.1 mg/kg PYR, a combination of MS and PYR, or saline and placed immediately inside the open-field chamber for 60 min. Stress-induced hyperthermia was suppressed immediately by MS and enhanced by PYR. Tc only increased by 0.3°C in the MS-treated animals. The hyperthermic response in the PYR group was nearly 0.6°C above that of rats dosed with saline. Coadministration of PYR and MS led to a stress-induced hyperthermia response nearly identical to that of rats injected with saline. Overall, open-field stress exacerbated the effects of MS and PYR on body Tc and provides support for a peripheral cholinergic mechanism that mediates stress-induced hyperthermia.

Actions of cholinergic drugs in the nematode Ascaris suum. Complex pharmacology of muscle and motorneurons.

The cholinergic agonists acetylcholine (ACh), nicotine, and pilocarpine produced depolarizations and contractions of muscle of the nematode Ascaris suum. Dose-dependent depolarization and contraction by ACh were suppressed by about two orders of magnitude by 100 microM d-tubocurarine (dTC), a nicotinic antagonist, but only about fivefold by 100 microM N-methyl-scopolamine (NMS), a muscarinic antagonist. NMS itself depolarized both normal and synaptically isolated muscle cells. The muscle depolarizing action of pilocarpine was not consistently antagonized by either NMS or dTC. ACh receptors were detected on motorneuron classes DE1, DE2, DI, and VI as ACh-induced reductions in input resistance. These input resistance changes were reversed by washing in drug-free saline or by application of dTC. NMS applied alone lowered input resistance in DE1, but not in DE2, DI, or VI motorneurons. In contrast to the effect of ACh, the action of NMS in DE1 was not reversed by dTC, suggesting that NMS-sensitive sites may not respond to ACh. Excitatory synaptic responses in muscle evoked by depolarizing current injections into DE1 and DE2 motorneurons were antagonized by dTC; however, NMS antagonized the synaptic output of only the DE1 and DE3 classes of motorneurons, an effect that was more likely to have been produced by motorneuron conduction failure than by pharmacological blockade of receptor. The concentration of NMS required to produce these changes in muscle polarization and contraction, ACh antagonism, input resistance reduction, and synaptic antagonism was 100 microM, or more than five orders of magnitude higher than the binding affinity for [3H]NMS in larval Ascaris homogenates and adult Caenorhabditis elegans (Segerberg, M. A. 1989. Ph.D. thesis. University of Wisconsin-Madison, Madison, WI). These results describe a nicotinic-like pharmacology, but muscle and motorneurons also have unusual responses to muscarinic agents.

M2 muscarinic ([3H]N-methyl scopolamine) binding in micropunches of rat ventricular myocardium: characterization and modification by progesterone

A new technique is outlined for the characterization and quantification of M2 muscarinic binding sites (receptors) in micro-punches (1 mm diam.), cut from slices (350 μm), of fresh cardiac tissue using the hydrophilic antagonist [3H]N-methyl scopolamine. The use of this water-soluble ligand allows us to label, and quantify, M2 receptors on the cell surface of intact cells contained within the micropunch. We believe that cardiac micropunches offer a simple but powerful approach to the investigation of membrane receptor regulation in tissue that largely retains the in vivo cytoarchitecture. Specific binding is reversible, stereospecific, saturable, of high affinity, and has the drug specificity typical of an M2 muscarinic receptor. In rat left ventricle, Bmax was 151.2 ± 10.3 fmol/mg protein while KD was 1.0 ± 0.1 nM. Nonspecific binding of the ligand was very low, varying from 2.8% (at 0.27 nM) to 7.7% (at 3.58 nM). This micropunch assay was used to determine that progesterone can compete with the muscarinic ligand for the M2 receptor in vitro (IC50 = 50 × 10−6 M). The steroids estradiol and testosterone, as well as ouabain, were without effect. Progesterone inhibited [3H]N-methyl scopolamine binding competitively (KD reduced from 1.9 to 4.3 nM) without affecting the rate of association of the ligand. However, progesterone induced a rapid dissociation of the ligand from its receptor. We conclude that the micropunch assay described here is suitable for the continued study of sex hormone effects on cardiac function.Key words: cardiac micropunches, muscarinic receptor, progesterone, [3H]N-methyl scopolamine.