In Vivo Functional Bioluminescence Brain Imaging Highlights Learning and Memory Process
Material below summarizes the article PKA and cAMP/CNG Channels Independently Regulate the Cholinergic Ca2+-Response of Drosophila Mushroom Body Neurons, published on April 8, 2015, in eNeuro and authored by Pierre Pavot, Elena Carbognin, and Jean-René Martin.
As neuroscientists, we try to understand how billions of neurons work together to perform either simple daily tasks, such as coordinated walking, or more complex ones, such as learning and memory. How are the multiple integrated functions — like playing the piano, which uses learning and memory — encoded in the brain? And how does that compare to the multiple integrated functions, such as coordinated locomotor activity, or more sophisticated motor-based functions, such as playing piano — which refers in part to learning and memory — or yet to encoding other tasks, such as the memory of odor or even the learning of more intellectual and abstract things, such as poetry? In other words, where in the brain and at which levels (genetic, molecular, cellular, and/or network) do these given integrated functions take place?
The ultimate goal is to understand the functioning of our brain, but its high complexity and impossibility to perform experimental research (particularly its genetic dimension) prompt us to develop animal models. Since biologically and fundamentally speaking, the functioning of a neuron is similar between different mammals, and even between vertebrates and invertebrates, the fruit fly Drosophila melanogaster is used to address these questions.
About a century ago, neuroanatomists and entomologists showed that the mushroom bodies (MBs), composed of about 2,500 neurons, are the most prominent structures in the adult Drosophila brain. About 50 years later, neurobiologists successfully showed that the MBs are involved in several crucial functions, such as olfactory learning and memory (L&M), locomotor activity, and more recently, in sleep and even in decision-making. Thus, the Drosophila, which is a genetically tractable and an excellent amenable experimental system, is well-suited to study the neural bases of such sophisticated functions, and particularly at all different system levels, from genetic, molecular, cellular, and neuronal networks, and even up to whole animal behavior.
In this continuity, the first and thus historical L&M genes, such as dunce and rutabaga, were identified more than 40 years ago, in Drosophila. These two genes have been shown to be preferentially expressed and are required in the MBs to perform their function. In addition, they regulate the level of the cAMP, a major intracellular second messenger involved in several crucial cellular function and notably in L&M. Though combined genetic and behavioral approaches have brought substantial amount of results highlighting the L&M process, curiously, the in vivo functional effects of these two genes, on the cellular and physiological mechanisms in the intrinsic neurons of the MBs and particularly on the Ca2+-responses, still remain largely unknown. Indeed Ca2+-activity, which is an excellent indicator of neuronal activity, is involved in the vast majority of intracellular processes, including L&M.
In the second half of the last century, several techniques such as electrophysiology have been developed to investigate neuronal activity or, in other words, how a neuron functions. Although these approaches have been informative, they have been mainly dedicated to the resolution of single cell level activity. Therefore, they are limited in describing whole brain activity and remained relatively poor when attempting to assign a specific role to a defined structure or group of neurons (structure/function relationship).
To temptingly complement them in the last two decades several different optical imaging techniques have been developed to study neuronal activity with the aim to map and decipher the neural code underlying major neurophysiologic functions, such as odor perception, learning and memory, locomotor activity, and sleep. The first generation of these techniques was principally based on detecting either transmembrane voltage or calcium activity using fluorescent dye markers. The development of genetically encoded calcium indicators (GECIs), for which the most representative candidate is the GCaMP, has extended our limits, increasing the accessibility of deeper structures. More importantly, GECIs have allowed investigators to precisely label and identify the desired neurons. However, as with other fluorescent Ca2+-sensitive dyes, these indicators/probes require light excitation. As a consequence, these experimental techniques are hampered by autofluorescence, photobleaching, and phototoxicity, which limit the duration of recording and prevent the recording of deep structures, and consequently, of the whole brain. Consequently, they could not be used continuously over a long time period and made them very limited in detecting unpredicted spontaneous activity.
More recently, a new bioluminescence approach based on the fusion protein between the GFP and the aequorin, a Ca2+-sensitive protein (both extracted from the jellyfish), has been successfully developed, allowing recording of the neuronal and Ca2+-activity. Bioluminescence is light produced by an enzymatic reaction occurring between the aequorin and its cofactor coelenterazine. In the presence of calcium, aequorin is activated and catalyzes the coelenterazine into coelenteramide, and consequently a photon is emitted. Since light is produced by a chemical reaction, light excitation is not required. Although the light signal produced by bioluminescence is generally weak, the signal-to-noise ratio is excellent because the background is more or less nil. Therefore, the light emission could be monitored continuously, over long time periods of time —hours and even days — making this genetically encoded bioluminescent probe suitable to detect both induced or the spontaneous Ca2+-activity.
Here, in our work performed in Drosophila, we took advantage of this new in vivo bioluminescence imaging approach, which allowed real-time monitoring of the entire strangely shaped structure of the MBs: the calyx (the dendritic bush), the cell-bodies, and the axonal projections (the lobes). We imaged neuronal Ca2+-activity continuously, over a long time period, and characterized the nicotine-evoked Ca2+-response. Since the olfactory circuitry (neurons) uses the acetylcholine as neurotransmitters to carry the odor information up to the MBs, nicotine (an agonist of the acetylcholine receptor) mimics an olfactory input and per extension simulate a learning and memory process.
Using both genetic and pharmacological approaches to interfere with the multiple components of the cAMP signaling pathway, we show that the Ca2+-response in the MB is proportional to the levels of cAMP. Then, we reveal that an acute change in cAMP levels is sufficient to trigger a Ca2+-response similar to the nicotine-triggered one (OK-JRM). This observation opens the door to speculate that the intrinsic neurons of the MBs could trigger, by themselves, a response that could support a reminiscence process. We also found a genetic manipulation of protein kinase A (PKA), a direct effector of cAMP, suggests that cAMP has both PKA-dependent and (OK-JRM) PKA-independent effects through the cyclic nucleotide-gated Ca2+-channel (CNG). Finally, the disruption of calmodulin, one of the main regulators of the rutabaga adenylate cyclase (AC) (which synthetizes the cAMP), yields different effects in the calyx/cell-bodies and in the lobes, suggesting a differential and regionalized regulation of AC.
Our results provide new insight into the complex Ca2+-response in the MBs, leading to the conclusion that cAMP modulates the Ca2+-responses through both PKA-dependent and independent mechanisms, the latter through CNG-channels. In perspective, these results may serve as a foundation for disentangling the complex roles of cAMP in memory formation, as well as guiding new behavioral experiments that focus on CNG-channels and calmodulin.
PKA and cAMP/CNG Channels Independently Regulate the Cholinergic Ca2+-Response of Drosophila Mushroom Body Neurons. Pierre Pavot, Elena Carbognin, Jean-René Martin. eNeuro Feb 2015, 2 (2). DOI: 10.1523/ENEURO.0054-14.2015