Pharmacological characterization of the response of the leech pharynx to acetylcholine.

In this study we document the sensitivity of the leech pharynx to acetylcholine and begin to characterize the acetylcholine receptor mediating this response by examining the effects of selective cholinergic agonists and antagonists on the contractile behavior of the pharynx. The order of potency derived from the EC50 of each agonist was (+/-)epibatidine > acetylcholine (in the presence of physostigmine) >> McN A-343 >> carbachol > nicotine. However, when response amplitude was considered, the order of potency to the tested agonists was (+/-)epibatidine >> nicotine >> McN A-343 >> carbachol > acetylcholine. Acetylcholine-induced contractions of the pharynx were antagonized by d-tubocurarine, but not by alpha-bungarotoxin, alpha-conotoxin M1, or mecamylamine. Application of high concentrations of hexamethonium (1 mM) augmented the acetylcholine-induced contractions. However, this augmentation was apparently due to inhibition of acetylcholinesterase by hexamethonium. The muscarinic antagonist atropine produced complex actions and apparently acted as a mixed agonist/antagonist. Atropine by itself produced an increase in basal tonus and increased the frequency and amplitude of phasic contractions. Atropine increased the peak tension of the acetylcholine-induced response; however, it reduced the amplitude of both the acetylcholine-induced increase in basal tonus and integrated area. Based on the pharmacological profile of the pharyngeal acetylcholine response, we conclude that the acetylcholine receptor mediating the response is a nicotinic receptor. However, the responsiveness of the pharynx to muscarinic agents diverges from that of a classical nicotinic receptor.

Serotonin induces four pharmacologically separable contractile responses in the pharynx of the leech Hirudo medicinalis.

Stimulation of the serotoninergic innervation of the leech pharynx or application of serotonin to the isolated pharynx induced four distinct types of contractile activity: an increase in basal tonus, large phasic contractions of 10-15 s in duration, smaller phasic contractions occurring at approximately 1 Hz, and a relaxation after washout of serotonin. Application to the isolated pharynx of the selective serotonin agonists (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin, N-(3-trifluoromethylphenyl)piperazine, 1-(m-chlorophenyl)-piperazine, (+/-)-2,5-dimethoxy-4-iodoamphetamine, 2-methyl-5-hydroxytrypamine, alpha-methyl-5-hydroxytryptamine, and 5-methoxytryptamine induced distinct types of pharyngeal contractile activity. The results of this study suggest that the leech pharynx possesses more than one type of serotonin receptor.

Regulation of pharyngeal motility by FMRFamide and related peptides in the medicinal leech, Hirudo medicinalis.

The medicinal leech possesses FMRFamide-like immunoreactivity in neural processes and somata associated with the pharynx and pharyngeal ganglia. The pharynx possessed about 25 immunoreactive somata; about half of the approximately 20 neurons of the pharyngeal ganglia were immunoreactive. We provide brief descriptions of several neurons located in the first neuromere of the subesophageal ganglion involved in controlling pharyngeal motility. Double-labeling experiments indicate that one of these cells, named Swallow neuron 1 (SW1), contains a FMRFamide-like peptide. Stimulation of SW1 caused the mouth to open and the pharynx to dilate. Upon termination of SW1 stimulation, the mouth closed, and a peristaltic wave progressed from the mouth down the length of the pharynx. Stimulation of SW1 did not produce 1:1 postsynaptic potentials in pharyngeal muscle cells. Thus, SW1 is apparently not a motor neuron. The pharynx responded to application of FMRFamide and related peptides by producing a series of 20- to 35-s phasic contractions superimposed upon an increase in basal tonus. The peptide-induced response was quantified by measuring increases in basal tonus, peak tension, and integrated area. Although there were some differences in the order of potency depending upon which parameter was considered, the approximate order of potency of RFamide peptides tested was: pQDPFLRFamide > or = FMRFamide approximately YGGFMRFamide > or = YMRFamide approximately FLRFamide approximately GGKYMRFamide approximately YLRFamide > leucomyosuppressin approximately perisulfakinin. Except for differences in potency, each of the RFamide peptides produced similar contractile waveforms. FMRFamide-induced responses were reduced by the protein kinase C inhibitor bisindolylmaleimide I (10 microM), the nonspecific protein kinase inhibitor H-7 (50 microM), and were increased by the protein phosphatase inhibitor okadaic acid (1 microM). However, the FMRFamide-induced response was unaffected by the protein kinase A inhibitor H-89 (1 microM), the phosphodiesterase inhibitor theophylline (1 mM), the phospholipase A(2) inhibitor OBAA (0.1 microM) or the cation channel blocker amiloride (100 microM).

Termination of leech swimming activity by a previously identified swim trigger neuron.

Cell Tr2 is a neuron in the subesophageal ganglion of the leech that can trigger swim episodes. In this report, we describe the ability of Tr2 to terminate ongoing swim episodes as well as to trigger swimming. Stimulation of Tr2 terminated ongoing swim episodes in nearly every preparation tested, while Tr2 stimulation triggered swim episodes in only a minority of the preparations. We suggest that the primary role of Tr2 is in the termination rather than the initiation of swimming activity. The swim trigger neuron Tr3 and a swim-gating neuron, cell 21, hyperpolarized during Tr2-induced swim termination. Another swim-gating neuron, cell 204 was sometimes slightly excited, but more often, hyperpolarized during Tr2-induced swim termination. In contrast to these cells, Tr2 stimulation excited another swim-gating neuron, cell 61. The responses of the swim-gating cells were variable in amplitude and sometimes not evident during Tr2-induced swim termination. Hence, the effects of Tr2 stimulation on swim-gating neurons seem unlikely to be the direct cause of swim termination. Oscillator cells examined during Tr2-induced swim termination include: 27, 28, 33, 60, 115, and 208. The largest effect seen in an oscillator neuron was in cell 208, which was repolarized by up to 10 mV during Tr2 stimulation. Tr2 stimulation did not produce any obvious synaptic effects in motor neurons DI-1, VI-1, and DE-3. Our findings indicate that other, yet undiscovered, connections are likely to be important in Tr2-induced swim termination. Therefore, we propose that cell Tr2 is probably a member of a distributed neural network involved in swim termination.

Neuronal control of leech swimming.

Leech swimming is produced by the antiphasic contractions of dorsal and ventral longitudinal muscles that travel rearward along the animal and propel it forward. Research over the past three decades has focused on identifying the underlying neuronal circuit and mechanisms that produce and control this coordinated movement pattern. Investigations have also tested whether leech swimming is modifiable, both by experience and by neuromodulators. One outcome has been the identification of several functional classes of neurons associated with swimming. Systematic analysis of the interactions between these neurons had led to the elucidation of a neuronal circuit that adequately accounts for the generation of the swim motor program cord. The swim motor program appears to be produced by a chain of coupled segmental oscillators whose intrinsic properties and intersegmental connections ensure the coordinated expression of swimming along the nerve cord. In addition, neurons identified in the head ganglion comprise two parallel, but opposite-acting, systems that control the initiation of swimming in response to sensory input. Also, the pathway by which body wall stimulation initiates swimming shows a simple form of learning, that is habituation. Repeatedly stroking the leech body wall decreases both the probability of initiating swimming and the length of elicited swim episodes. Finally, the biogenic amine serotonin, which is found in the nerve cord, affects leech swimming in a number of ways. Serotonin's modulation of swimming is due, in part, to its effect of the membrane properties of swim-initiating interneurons and several swim motor neurons.

Modification of leech behavior patterns by reserpine-induced amine depletion.

A single injection of 100 micrograms reserpine into the crop of the medicinal leech, Hirudo medicinalis, reduced CNS serotonin and dopamine levels to less than 1% of control values within 3 d. High-pressure liquid chromotography- (HPLC) determined CNS serotonin and dopamine levels remained maximally depressed for approximately 1 month following reserpine injection. Subsequently, amine levels recovered slowly, but remained depressed 6 months after reserpine injection. Following reserpine treatment, glyoxylic acid-induced fluorescence or neutral red staining closely mirrored the HPLC-determined time course of amine depletion and recovery. Acute exposure of isolated ganglia to 10 microM reserpine for periods up to 6 hr produced a 20-30% reduction of serotonin and dopamine content. The threshold concentration of reserpine necessary to produce amine depletion was approximately 1 microM. We found that reserpine treatment eliminated biting behavior within 4 d following injection. Biting behavior remained depressed below control levels for approximately 4 months, but returned to control values while CNS serotonin and dopamine levels remained significantly depressed at this time. Unexpectedly, reserpine treatment increased rather than reduced the duration of stimulus-evoked swimming activity. This behavioral change was evident within 3 d and persisted for approximately 3.5 months. To rapidly restore amine levels in reserpine-treated animals, we bathed intact leeches in pond water containing serotonin, dopamine, or octopamine. We found that biting behavior was restored following reserpine treatment by bathing intact leeches in pond water containing serotonin or dopamine, but not octopamine. Also contrary to expectations, the increase in swim duration was not reversed by bath exposure to serotonin, dopamine, octopamine, or histamine. However, all swimming activity in reserpine-treated leeches was eliminated by the amine antagonist cyproheptadine. We propose that the presence of low levels of amines is critical for the expression of both biting and swimming activity in leeches. However, the minimal levels of amines necessary for the expression of these behaviors are lower for swimming than for biting.