Adrenergic signaling is involved in the regulation of synaptic plasticity39,40
. However, the precise mechanisms of this regulation are poorly understood. We show that octopamine regulates behavioral and synaptic plasticity through an autoregulatory mechanism that promotes the growth of type II innervation and in turn the expansion of excitatory glutamatergic arbors. This process seems to be associated with physiological stimuli that lead to increased locomotion. We propose that food deprivation elicits the release of octopamine by type II terminals. Octopamine binds to Octβ2R receptors and thereby increases cAMP, which activates CREB-dependent regulation of transcription and leads to new type II synaptic growth (Supplementary Fig. 8a
). This autoregulatory mechanism might control the amount of octopamine released by type II arbors. In turn, octopamine release stimulates the growth of type I arbors through Octβ2R at type I motor neurons. This mechanism would regulate, in a global fashion, excitatory transmission at the NMJ (Supplementary Fig. 8a
Increases in larval locomotion, type II motor neuron activity or exogenous octopamine resulted in the extension of synaptopods. With the demonstration that synaptopod extension constitutes a mechanism for the formation of type II boutons, these results suggest that the above events control the growth of octopaminergic endings in an acute manner. Analysis of mutations in octopamine receptors and components of the cAMP cascade revealed an autoregulatory mechanism that controls this growth. First, expression of Octβ2R in type II motor neurons was required for type II synaptic growth. Second, altering cAMP levels by mutations in dnc
modified this response in a manner consistent with positive regulation by cAMP. This regulation was cell autonomous in octopaminergic motor neurons, as the defects in synaptopod formation and type II synaptic growth were also elicited or rescued by transgene expression in octopaminergic motor neurons alone, in a chronic or acute manner. The presence of auto-octopamine receptors had been suggested in locusts41
, although the identity of the proposed autoreceptor was not known. However, it was proposed that the locust octopamine autoreceptors served to inhibit octopamine release. By contrast, our experiments are consistent with a positive feedback mechanism that enhances synaptic growth. Autoregulatory mechanisms that control the amount of neuromodulator release have been previously demonstrated for neuromodulators such as dopamine42
As in other forms of synaptic plasticity, including late LTP and long-term memory35
, the autoregulatory mechanism required the function of CREB and new protein synthesis. This finding underscores the universality of mechanisms by which the nervous system modifies the efficacy of connections in a long-lasting manner. Octopamine receptor activation leading to CREB signaling has also been demonstrated in Caenorhabditis elegans10
Our studies showed that this pathway regulated the structure of octopaminergic arbors in an autoregulatory fashion, and that this influenced the growth of type I excitatory arbors. The presence of a positive feedback that controls the growth of modulatory inputs in an acute manner provides a mechanism by which animal experience can modify circuitry and thus by which animals can adapt to a changing environment.
Activity was absolutely required for innervation of body-wall muscles by type II arbors, as reduced activity perturbed type II synaptogenesis. This is in contrast to the widely held view that although activity is important for the refinement of connections, it is not required for initial synaptogenesis43
. Part of this view arises from the examination of arbors that mediate classical neurotransmission43
. By contrast, the dependence of modulatory terminal growth on activity has been less studied. Our studies provide compelling evidence that octopamine has an influence on bouton outgrowth in octopaminergic type II and type I motor neurons. Studies of type I bouton outgrowth have identified local factors that influence the development of pre- or postsynaptic compartments, including Wnts and BMPs15,16
. We suggest that octopamine release by type II arbors might mediate a more global regulation of outgrowth.
At the Drosophila
larval NMJ, glutamatergic type Ib motor neurons innervate each muscle in an approximately 1:1 manner44
(Supplementary Fig. 8b
, type Ib). In addition, two glutamatergic type Is motor neurons innervate the entire ventral or dorsal muscle field within each hemisegment44,43
(Supplementary Fig. 8b
, type Is). By contrast, the three octopaminergic neurons per segment innervate most of the body-wall muscles in a bilateral fashion13
(Supplementary Fig. 8b
, type II). The layout of this innervation suggests that type II synapses might establish global regulation of the plasticity of type I arbors. This might serve as a mechanism for setting excitability levels in the entire body wall, and thereby keep synaptic function in a dynamic range. Similarly, studies in mammalian systems have shown that adrenergic signaling can affect plasticity at glutamatergic synapses, either through changes in ionotropic GluR localization2
or through regulation of metabotropic GluR, which affects the ability of a synapse to become potentiated depending on its history3
. Octopamine might regulate the ability of type I NMJs to trigger muscle contraction by long-term regulation of type I synaptic growth.
Two previous studies at the Drosophila
larval NMJ have shown that octopamine enhances synaptic transmission46,47
. However, another study reported that octopamine might inhibit glutamatergic transmission in first-inslar larvae48
. Our studies suggest that blocking activity or interfering with octopamine signaling in type II neurons leads to a decrease in type I synaptic outgrowth, consistent with the idea that octopamine release is a positive regulator of type I transmission. We suggest that in the short term, octopamine enhances synaptic strength, as observed in our electrophysiology experiments, leading to the observed increase in crawling behavior after starvation. This would be consistent with studies showing that increases in locomotor speed induced by food deprivation led to an enhancement of synaptic efficacy17
Octopamine is a potent modulator of invertebrate behavior and is secreted during starvation in invertebrates10,22
. Nevertheless, its function at the synaptic level is poorly understood. Our study shows that octopamine can influence synapses at the structural level through the activation of Octβ2R autoreceptors in octopamine neurons and through the presence of these receptors in type I motor neurons.
An important question is whether octopamine is simply involved in locomotion and the lack of starvation response in mutants that cannot synthesize octopamine is an indirect effect of defective locomotion. It is not possible to answer this question in tbh mutants, as basal locomotion was reduced in these mutants. However, our experiments revealed conditions in which changes in basal activity could be genetically separated from changes in the starvation response. One such case is rut mutants, which have normal locomotion but lack the starvation response. This effect seemed to be due to the function of Rut in octopamine neurons, as the defective starvation response was completely rescued by expressing a Rut transgene in octopamine neurons. A second, albeit less clear observation regards octβ2R mutants. Although baseline locomotion was much less altered in these mutants than in tbh mutants, these animals still could not mount a starvation response (). Thus, it is likely that octopamine neurons are involved not only in locomotion, but also in the response to starvation.
Octopamine is also required for appetitive memory in adult fruit flies7
. Notably, the appetitive memory procedure requires starvation before the assay, and tbh
mutants cannot learn in this procedure. Octopamine has been proposed to mediate the reinforcing effects of sugar in appetitive memory formation5,7
. Our studies raise the possibility that this mechanism might involve structural changes at synaptic sites.
Although our studies focused on structural changes at type II NMJs, many of our manipulations affected all octopamine neurons, as Tdc2-Gal4 drives Gal4 in all octopaminergic neurons. Thus, our studies cannot rule out an influence from other octopaminergic neurons, besides motor neurons, in the changes observed and in the behavior. However, the finding that the manipulations resulted in specific changes in type II NMJ terminals and that octopamine modulates synaptic strength at the NMJ argues that at least some of the effects are likely to be due to the peripheral octopamine innervation.
In summary, our studies reveal important mechanisms by which activity regulates the ability of motor neurons to scale the release of regulatory signals, which is important for the adaptation of the organism to the environment. In addition, they show a mechanism by which excitatory synapses are regulated in a global manner, presumably to maintain synaptic plasticity in a dynamic range.