Acetylcholine Mediates Excitatory Input to Chromatophore Motoneurons in the Squid, Loligo pealeii

All cephalopods save the Nautilidae possess the ability to rapidly modify their body coloration (1). Cephalopod color patterning probably serves foremost as camouflage, yet many also use species-specific color patterns for communication (1–3). Color patterning in cephalopods is achieved by the synchronized neural manipulation of many thousands of small chromatophore organs distributed throughout the skin (4). These chromatophore organs consist of centrally located, pigment-containing cells surrounded by 6–30 radially-arranged muscle fibers (4, 5). Stimulation of chromatophore nerves causes contraction of muscle fibers and thereby chromatophore expansion (6, 7). Motoneurons project uninterrupted to the chromatophores from brain structures known as the chromatophore lobes (8). Anterior chromatophore lobe motoneurons project to chromatophores on the head, arms, and tentacles, while the posterior chromatophore lobe (PCL) motoneurons innervate ipsilateral chromatophores on the fins and mantle. The chromatophore lobes receive their principal inputs from the lateral basal lobes (LBL) and from the contralateral chromatophore lobes (8, 9). Although the physiological role of the LBL in the control of chromatophore activity has been demonstrated (8), neither the pharmacology nor the complexity of the synaptic inputs to the chromatophore lobes has been investigated. The goal of this project was to identify neurotransmitters that can effectively stimulate chromatophore motoneurons located in the PCL of the squid, Loligo pealeii. Pharmacological agents were applied to cell bodies located peripherally in the right or left PCL of intact, unanesthetized male and female squids (8–10 cm mantle length). All experiments were performed in a 500-ml tank of cold (12–15 °C) flowing seawater with a white background. Under these conditions, a squid remains pale in color. The head was restrained against a plexiglass plate with a small hole positioned over the skull, and a small incision was made just above the site where the esophagus exits from the skull. The esophagus and salivary glands were gently displaced, and care was taken not to damage the cephalic aorta. The PCLs could be localized under a dissecting microscope, allowing micropipettes (2–5 m tips) to be lowered into the PCL under visual control. A video camera was positioned to record chromatophore activity on the dorsal side of the mantle and fins. Several transmitters suspected of being active in the PCL (2) were tested. Drugs were applied under pressure (Picospritzer II, General Valve Corp.) in concentrations ranging from 10 6 to 10 3 M (10–50 picoliters). Most drugs were dissolved in artificial seawater (ASW), but neither ASW nor distilled water elicited chromatophore expansion when applied to the PCL. In some experiments, to confirm the injection site, the vital dye methylene blue was included in the carrier solution. Of all the drugs tested, only L-glutamate and acetylcholine (ACh) were observed to cause rapid, localized and reproducible chromatophore expansion (Table 1). However, the effects of glutamate were irregular and were often followed by prolonged deactivation of chromatophores over large areas of the mantle. Because the effects of ACh were more consistent and reproducible, they were explored in greater detail. When a pipette containing 10 6 M ACh was pushed through the outer capsule of the PCL and into the cellular layer, it was common to see a flickering of chromatophore activity at a localized position somewhere on the ipsilateral mantle. Then, when ACh was applied, many chromatophores around that site would rapidly expand. In every case, chromatophores of multiple colors were observed to expand in response to the injection. Effects typically lasted 30–60 s. The response location, the total area of the mantle to exhibit chromatophore expansion, the duration of the response, and the spread of the response over time were assessed digitally after each experiment and were found to vary with the quantity of solution delivered and the concentration of the drug. ACh injections repeated at the same pipette position elicited similar responses from the same group of chromatophores. Injections of ACh into the neuropil of the PCL or more caudally into the base of the pallial nerve did not elicit chromatophore activity. The ACh agonist nicotine was also tested and was found to be equally effective in generating chromatophore expansion. ACh antagonists have not yet been investigated. These experiments suggest that ACh may be an important excitatory input to the chromatophore motoneurons in the squid. This conclusion is supported by histochemical evidence that acetylcholinesterase is present in the PCL (10). Whether nicotinic ACh receptors are present on the motoneurons, and whether ACh represents input derived from the LBL or elsewhere in the squid brain, remains to be determined. This work was supported by a Grass Fellowship.