JPhysiology (1993) 87, 141 -151 141 © Elsevier, Paris Physiology and biochemistry of peptidergic cotransmission in Aplysia KR Weiss a, V Brezina a, EC Cropper a, J Heierhorst a, SL Hooper a, WC Probst a, SC Rosen b, FS Vilim a, I Kupfermann b aDepartment of Physiology and Biophysics, Mt Sinai School of Medicine, 1 Gustave Levy Place, New York, NY 10029; hCenter for Neurobiology and Behavior, College of Physicians and Surgeons, Columbia University, 722 West 168rh Street, New York, NY 10032, USA Summary - The marine mollusc Aplysia. whose simple nervous system facilitates study of the neural basis of behavior, was used to investigate the role of peptidergic cotransmission in feeding behavior. Several novel modulatory neuropeptides were purified and localized to identified cholinergic motorneurons. Physiological and biochemical studies demonstrated that these peptides are released when the motorneurons fire at frequencies that occur during normal behavior, and that the peptides modify the relationship between muscle contraction amplitude and relaxation rate so as to maintain optimal motor output when the intensity and frequency of feeding behavior change. Aplysia / peptidergic cotransmission / feeding behavior Introduction Prior to the 1970s it was generally accepted that each neuron contains a single neurotransmitter. Also, it was felt that the number of transmitter substances was very limited. More recent dis- coveries have led to the revision of these tradi- tional views of synaptic transmission. For example, in the last two decades it has become apparent that numerous small bioactive peptides are present in the nervous system (H6kfelt et al, 1984). Hundreds of these peptides are likely to act as neurotransmitters (Snyder, 1980). Often neuropeptides are present in neurons that contain either additional neuropeptides or classical trans- mitters. Thus, it now appears that individual neu- rons typically contain various combinations of cotransmitters, at least some of them neuropep- tides (for reviews see Bartfai et al, 1986; H6kfelt et al, 1984; Kupfermann, 1991). This very prob- ably contributes to the complexity of signaling in the brain. Although it is now widely accepted that neu- rons contain cotransmitters, technical difficulties have limited attempts to characterize the role that cotransmitters play in the nervous system under physiological conditions (Campbell, 1987; Kupfermann, 1991). For instance, cotransmitters are usually released in smaller quantities than classical transmitters. Consequently, in most sys- tems direct evidence for cotransmitter release has been impossible to obtain. Moreover, in order to establish that a substance functions as a cotransmitter it is necessary to demonstrate not only that it is released, but that it is released in quantities that are sufficient to activate receptors and have physiological consequences. Since the biological actions of putative cotransmitters are often subtle, it has been even more difficult to demonstrate physiological consequences of cotransmitter release than release itself. Indeed, in a recently published exhaustive review of cotransmission, Kupfermann (1991) concluded that while the cumulative evidence suggested that cotransmission may occur under some circum- stances, at the time of writing there was as yet "no conclusive evidence that indicates that cotransmission occurs under normal operation of the nervous system". Many of the technical difficulties encountered in studying cotransmission can be avoided by using relatively simple invertebrate preparations. Many invertebrate neurons are large and reiden- tifiable. Consequently, they can be individually stimulated in physiological studies and then bio- chemically characterized. There is no need for