It has long been apparent that neuropeptides can coexist with small molecule neurotransmitters. For example, Hökfelt and coworkers reported somatostatin-like immunoreactivity in noradrenergic neurons in principle ganglion cells of sympathetic ganglia in 1977 (Hökfelt et al., 1977). The demonstration of coexistence then led to the obvious question, do “co-existing” peptides function as neurotransmitters? (an alternative possibility would be that they simply act as trophic factors).

In the early 1980s the cotransmission question was addressed by Jan and Jan (1982) in experiments in the sympathetic nervous system of the bullfrog. There is general agreement that certain criteria have to be met for a substance to be classified as a neurotransmitter. Although there is some disagreement as to how many of these criteria there are, Jan and Jan (1982) were able to convincingly satisfy those that are most commonly considered crucial. For example, they demonstrated that the peptide they studied (LH-RH) is present in presynaptic terminals, and is released in a calcium dependent manner, Further, a late, slow EPSP was mimicked by application of exogenous LH-RH, and blocked by LH-RH antagonists (for a detailed discussion of this work see Nusbaum, 2017).

A further question that was subsequently raised was, under what circumstances does peptide release occur? Early experiments in the sympathetic nervous system of the pig used a radioimmunoassay (RIA) to quantify NPY-like immunoreactivity in the perfusate following low frequency (i.e., 2 Hz) nerve stimulation as compared to release induced by intermittent stimulation at a high frequency (20 Hz; Lundberg et al., 1986). Release was greater at the higher frequency. Data such as these led to the hypothesis that peptide release only occurs if neurons fire at high/excessive frequencies that are observed under pathological conditions (Hökfelt, 1991). This line of thinking led to the prediction that peptides would only be important for mediating responses to injury or stress (Hökfelt, 1991).

Subsequent invertebrate research clearly established that this is not the case. Many of these early studies were conducted in neuromuscular systems (O’Shea and Schaffer, 1985). An initial goal of this work was to verify peptide release by making direct biochemical measurements. Peptide release was induced either by raising the potassium concentration in the saline, or by stimulating motor neurons at relatively high frequencies. For example, Adams and O’Shea (1983) demonstrated proctolin release from a slow skeletal motoneuron (Ds) in the cockroach with stimulation at 50 Hz. Other lower frequencies were not tested when release was directly monitored, presumably because the method used to detect released peptides was not very sensitive. However, in other experiments in this study, peptide release was monitored indirectly, i.e., by monitoring a physiological response clearly not mediated by the release of the primary neurotransmitter (glutamate). Thus, Adams and O’Shea (1983) also demonstrated that when a burst of action potentials is triggered in Ds, a delayed slow increase in muscle tension is observed that is not associated with excitatory junctional potentials (EJPs). This delayed response was observed when Ds was stimulated at a frequency that was not specified but was clearly way below 50 Hz.

In another early invertebrate study, proctolin release was monitored in a neuromuscular preparation of the crayfish using a sensitive and quantifiable bioassay, i.e., samples were applied to a subset of muscle fibers from the main extensor muscle of the locust leg and changes in contraction frequency were noted (O’Shea and Bishop, 1982). In this situation, it was possible to detect release when motor neurons were stimulated at a fairly low frequency (e.g., 10 Hz; Bishop et al., 1987).

Other experiments were conducted in a molluscan (Aplysia) preparation that consists of a muscle utilized in feeding, the accessory radula closer (ARC) and its two cholinergic motor neurons (B15 and B16; Cohen et al., 1978). Initially, peptide release in this system was monitored indirectly. For example, investigators measured cAMP levels in the ARC muscle (Whim and Lloyd, 1989; Cropper et al., 1990b). In later studies, however, a sensitive RIA was developed that permitted direct detection of released material (Vilim et al., 1996a).

Research in the ARC neuromuscular system was unusual in its emphasis on mimicking naturally occurring patterns of motor neuron activity. Thus, extra junctional currents (EJCs) induced by B15 and B16 were recorded from the ARC muscle during normal feeding behavior in intact animals (Cropper et al., 1990a). Physiologically relevant patterns of neural activity were then simulated in subsequent release experiments that confirmed that the amount of peptide released depends on firing frequency in the ARC neuromuscular system (Vilim et al., 1996a, 2000) as it does in bullfrog sympathetic ganglia (e.g., Lundberg et al., 1986, 1989; Peng and Horn, 1991; Figure 1A). Importantly, however, release did occur at the low end of the physiological range (which is 7.5 Hz for B15 and 10 Hz for B16 (Cropper et al., 1990a; Vilim et al., 1996a,b, 2000). These data provide direct evidence for the release of peptides at physiologically relevant levels of activity, and obviously contradict the idea that peptide cotransmitters solely mediate responses to stress.

It has long been recognized that neuropeptides are generally packaged in dense core vesicles whereas small clear vesicles generally contain low molecular weight neurotransmitters. In a number of neurons, data suggest that exocytosis from the two types of vesicles occurs in different regions in the presynaptic terminal. In some cases, release from the small clear vesicles occurs in the active zone and release from peptidergic large dense core vesicles appears to occur elsewhere (e.g., Zhu et al., 1986; Vilim et al., 1996b; Lysakowski et al., 1999; Karhunen et al., 2001). Further, release from the two types of vesicles is often differentially sensitive to increases in the intracellular calcium concentration. Release from peptidergic large dense core vesicles occurs at lower [Ca²⁺]_i (Verhage et al., 1991; Peng and Zucker, 1993; Ohnuma et al., 2001). These data suggest that the patterning of neural activity could impact peptide cotransmitter release in a manner that differs from its impact on the release of a low molecular weight transmitter.

As described above, a number of investigators have demonstrated that more peptide is released when neurons fire at higher frequencies (Lundberg et al., 1986, 1989; Peng and Horn, 1991; Vilim et al., 1996a, 2000). Obviously as firing frequency increases, there is an increase in the number of action potentials triggered in a given period of time. One method that has been used to correct for this is to calculate the amount of peptide released per action potential. Even with this correction more release at higher firing frequencies has been demonstrated in the ARC neuromuscular system (if neurons are stimulated within a physiologically relevant range; Figure 1A; Vilim et al., 1996a, 2000).

An additional question that has been addressed is, are periods of rest necessary to maintain peptide cotransmitter release? That this could be the case had been suggested by experiments that studied peptide hormone release from the hypothalamus (Cazalis et al., 1985). Investigators working in the bullfrog sympathetic ganglia demonstrated that rest periods are not essential, i.e., LHRH-induced slow currents were recorded from postsynaptic neurons when presynaptic neurons were stimulated continuously (Peng and Horn, 1991).

Subsequently, research conducted in the ARC neuromuscular system elaborated on these findings (Vilim et al., 1996a, 2000). With excessive stimulation, depletion of peptide cotransmitters obviously occurs. Experiments were, however designed so that all parameters chosen were behaviorally relevant. For example, motor neurons were not fired at frequencies higher than those observed during normal behavior and burst durations and interburst intervals were all within physiological limits. Under these conditions, periods of rest were actually detrimental, i.e., there was a decrease in the amount of peptide released per action potential as the interburst interval was increased (Figure 1B). This result suggests that effects of modulatory neuropeptides will be manifested in a manner that is at least to some extent determined by how a behavior is executed. Namely, if it is executed rapidly, effects of peptides will be more pronounced.

Additionally, ARC investigators characterized the dynamics of peptide release when neural activity was maintained for a relatively long period of time (e.g., ~ an hour) with no change in either the motor neuron firing frequency or bursting pattern (Figure 1C; Vilim et al., 1996b, 2000; Brezina et al., 2000; Karhunen et al., 2001). In some of these experiments, motor neurons were stimulated at the high end of the physiological range, and pauses between bursts of activity were on the short side. Nevertheless, initially relatively little peptide was released. Over time however release facilitated and reached a peak (Figure 1C). Thereafter, it declined. These data suggest that even when behavior is constant, modulatory effects of peptide cotransmitters will be dynamically manifested. When a behavior is initiated, it may not be greatly impacted by peptide release. However, as it is repeated, peptidergic effects may become more pronounced (up to a point).

Taken together, these results indicate that neuropeptides are released during normal behavior. The amount of peptide released per action potential can vary greatly and be altered by the firing pattern of the neuron. Consequently, peptide release is likely to be determined by how behavior is executed (e.g., quickly or slowly). Additionally, even when patterns of neural activity do not change, peptide release may occur dynamically, e.g., effects of modulatory peptides may become more pronounced as a behavior progresses. Below we discuss potential functional consequences of these forms of plasticity in a specific situation—in the situation where peptide cotransmitters are intrinsic to a behavior generating circuit.