Peptide Cotransmitters as Dynamic, Intrinsic Modulators of Network Activity

Neurons can contain both neuropeptides and “classic” small molecule transmitters. Much progress has been made in studies designed to determine the functional significance of this arrangement in experiments conducted in invertebrates and in the vertebrate autonomic nervous system. In this review article, we describe some of this research. In particular, we review early studies that related peptide release to physiological firing patterns of neurons. Additionally, we discuss more recent experiments informed by this early work that have sought to determine the functional significance of peptide cotransmission in the situation where peptides are released from neurons that are part of (i.e., are intrinsic to) a behavior generating circuit in the CNS. In this situation, peptide release will presumably be tightly coupled to the manner in which a network is activated. For example, data obtained in early studies suggest that peptide release will be potentiated when behavior is executed rapidly and intervals between periods of neural activity are relatively short. Further, early studies demonstrated that when neural activity is maintained, there are progressive changes (e.g., increases) in the amount of peptide that is released (even in the absence of a change in neural activity). This suggests that intrinsic peptidergic modulators in the CNS are likely to exert effects that are manifested dynamically in an activity-dependent manner. This type of modulation is likely to differ markedly from the modulation that occurs when a peptide hormone is present at a relatively fixed concentration in the blood

Urotensin II in invertebrates: from structure to function in Aplysia californica.

Neuropeptides are ancient signaling molecules that are involved in many aspects of organism homeostasis and function. Urotensin II (UII), a peptide with a range of hormonal functions, previously has been reported exclusively in vertebrates. Here, we provide the first direct evidence that UII-like peptides are also present in an invertebrate, specifically, the marine mollusk Aplysia californica. The presence of UII in the central nervous system (CNS) of Aplysia implies a more ancient gene lineage than vertebrates. Using representational difference analysis, we identified an mRNA of a protein precursor that encodes a predicted neuropeptide, we named Aplysia urotensin II (apUII), with a sequence and structural similarity to vertebrate UII. With in-situ hybridization and immunohistochemistry, we mapped the expression of apUII mRNA and its prohormone in the CNS and localized apUII-like immunoreactivity to buccal sensory neurons and cerebral A-cluster neurons. Mass spectrometry performed on individual isolated neurons, and tandem mass spectrometry on fractionated peptide extracts, allowed us to define the posttranslational processing of the apUII neuropeptide precursor and confirm the highly conserved cyclic nature of the mature neuropeptide apUII. Electrophysiological analysis of the central effects of a synthetic apUII suggests it plays a role in satiety and/or aversive signaling in feeding behaviors. Finding the homologue of vertebrate UII in the numerically small CNS of an invertebrate animal model is important for gaining insights into the molecular mechanisms and pathways mediating the bioactivity of UII in the higher metazoan

Composite Modulatory Feedforward Loop Contributes to the Establishment of a Network State

Feedforward loops (FFLs) are one of many network motifs identified in a variety of complex networks, but their functional role in neural networks is not well understood. We provide evidence that combinatorial actions of multiple modulators may be organized as FFLs to promote a specific network state in the Aplysia feeding motor network. The Aplysia feeding central pattern generator (CPG) receives two distinct inputs—a higher-order interneuron cerebral-buccal interneuron-2 (CBI-2) and the esophageal nerve (EN)—that promote ingestive and egestive motor programs, respectively. EN stimulation elicits a persistent egestive network state, which enables the network to temporarily express egestive programs following a switch of input from the EN to CBI-2. Previous work showed that a modulatory CPG element, B65, is specifically activated by the EN and participates in establishing the egestive state by enhancing activity of egestion-promoting B20 interneurons while suppressing activity and synaptic outputs of ingestion-promoting B40 interneurons. Here a peptidergic contribution is mediated by small cardioactive peptide (SCP). Immunostaining and mass spectrometry show that SCP is present in the EN and is released on EN stimulation. Importantly, SCP directly enhances activity and synaptic outputs of B20 and suppresses activity and synaptic outputs of B40. Moreover, SCP promotes B65 activity. Thus the direct and indirect (through B65) pathways to B20 and B40 from SCPergic neurons constitute two FFLs with one functioning to promote egestive output and the other to suppress ingestive output. This composite FFL consisting of the two combined FFLs appears to be an effective means to co-regulate activity of two competing elements that do not inhibit each other, thereby contributing to establish specific network states

apUII inhibits the generation of spontaneously occurring programs.

<p>(<b>A</b>) In control condition, motor programs occurred spontaneously. (<b>B</b>) Perfusion of 10⁻⁵ M apUII reduced the frequency of motor programs. (<b>C</b>) Upon washout of the peptide, motor programs occurred more frequently. Each cycle of motor programs consists of a protraction (open bar) and retraction (filled bar) sequence.</p

apUII actions on motor programs elicited by a command-like interneuron CBI-2.

<p>(<b>A</b>) Representative examples. CBI-2 was stimulated throughout the protraction phase (open bar) to elicit single cycle motor programs; the inter-trial interval is 1 min. In control condition (<b>A1</b>), B8 is predominantly active during the retraction phase (filled bar), while B61 is only active during protraction. Perfusion of 10⁻⁶ M (<b>A2</b>) and 10⁻⁵ M (<b>A3</b>) apUII’ reduced the firing frequency of both B8 and B61, but had a lesser effect on the firing frequency of B4/5. Protraction is monitored via activity of I2, retraction via activity of BN2. (<b>B</b>) Group data showing the suppressive effects of 10⁻⁶ M and 10⁻⁵ M apUII’ on B8 firing frequency during protraction (<b>B1</b>), or during retraction (<b>B2</b>), and B61/62 firing frequency during protraction (<b>B3</b>). (<b>C</b>) Group data showing the lengthening of the latency to protraction initiation, measured as the time that elapsed from the onset of CBI-2 stimulation to the onset of I2 nerve activity (<b>C1</b>). apUII’ also reduced protraction duration (<b>C2</b>) and retraction duration (<b>C3</b>). *, p<0.05; **, p<0.01; ***, p<0.001; error bars represent SEM.</p