New dancers to the tunes of the rap “ GABA”
One of the cardinal questions in the field of neurosciences is to understand how brain receives, process, consolidates and retrieve information. Work from many laboratories for many decades reached a consensus that synaptic plasticity, in other words the plasticity displayed by the synapses.
(https://en.wikipedia.org/wiki/Chemical_synapse#Volume_transmission) in the brain encodes the behavioural plasticity. The dogma of memory suggests that multiple mechanisms are required to process information when every new information is perceived and old information is recalled. The current view on the field is that the brain networks that sub serve both function and plasticity within the networks is mediated by extra cellular and intracellular molecular signaling. Therefore it is important to understand their functional organization, connectivity, neurochemical integration, protein signaling molecules and basic organization of these networks that are involved in long-term plasticity.
In this context understanding the synaptic structure, function, synaptic receptor trafficking, modulation of receptors, contribution of receptor trafficking to synaptic plasticity in vivo, compared with pre-synaptic changes and protein synthesis-dependent mechanisms are fundamental to the appreciation of plasticity at the molecular level. However, before understanding the molecular machineries responsible for inducing plasticity that enables the neuronal circuit to learn, it is pivotal to understand how neurons form its circuits during the developmental stages such as how the axons find its targets, what the guidance cues are led the neurons to finds its partner and how pruning in dendrites and axons establishes connectivity independent and dependent of activity. Molecular neuroscience emphasize to work on the mechanisms and molecules involved in synaptic plasticity, which in turn is tightly associated with modification of synaptic proteins and dynamic rearrangement of synaptic structure, which in turn offers answer to really exciting questions such as on how synapses are pruned or eliminated during development?
For answering the exciting questions of how the phenomena of multiple memory phases, parallel processing of information and anatomic transfer all be explained by biochemical changes in synaptic strength, neuroscientist use model organisms such as mouse, C.elegans, fruit flies, which in turn provides the behavior – genetic tools to unravel the anatomical and biochemical complexities that translate synaptic plasticity in to experience dependent behavior responses. In this issue of journal “Neuron”, two groups (1, 2) have elegantly shown that a tripartite trans membrane complex of MADD4, Neurexin (NRX-1) and Neuroligin (NLG-1) regulates the clustering of post-synaptic GABA receptors (involved in inhibitory synaptic transmission) which are juxtaposed to the pre-synaptic GABAergic boutons in C.elegans. Both of the studies corroborate very clearly the need of the formation of the tripartite complex for the clustering of the GABA receptors in C.elegans, interestingly it opens a few question to test and answer.
- How does the binding of NLG-1 directly and MADD4 indirectly through the recruitment of UNC-40/DCC (Netrin receptor) affect the kinetics and dynamics of the turn over of the GABA receptors at the synapses? In other words what is the time constant for the turn over of those receptors in the synapses in wild type and in the mutant background?
By measuring the mean displacement of the GABA receptors on the synapses in cultured mammalian neurons using single molecule imaging might answer how the tripartite complex is regulating the clustering.
- Does NLG-1 and MADD4 complex acts as an anchor to capture the laterally diffusing GABA receptors on the synapses to stabilize them?
- Given C.elegans synapses are refractory to changes upon activity, It will be interesting to test in mammalian system whether the recruitment of GABA receptors can be regulated by activity? In other words, will there be a concomitant increase in the number of NLG-1 as well as UNC-40 to hold the GABA receptors during long-term potentiation and depression as well as during homeostatic plasticity? FRAP (Fluorescent recovery after photo bleaching) and single molecule imaging might provide the answer if this interactions hold true in specific mammalian inhibitory synapses. On the same line of thought, it will be interesting to test whether activity regulates the remodeling of extracellular matrix (ECM), where MADD4 is present.
- It is tempting to speculate whether those axons secretes netrin (UNC-6) and MADD4 (either concentration dependent or the intensity of the signaling which I am surmising) operates the phenomenon of “ Winner takes all “ in wading the competition from other axons to stabilize the synapses in the neuromuscular junction (NMJ’s).
- What are the downstream signaling pathways operates through UNC-40 when MADD4 binds to it for clustering the GABA receptors? Does the signaling cascade different from the canonical pathway of netrin binding to its cognate receptor, UNC-40?
Along the same line, a study published in the issue of journal “Nature “ (3) also throws light on the organization of the inhibitory circuits in the worms. They have shown a single transmembrane protein, OIG1, which is under the control of a transcription factor UNC-30 in concert with other factors regulate the input as well as the output of the inhibitory circuit in a spatiotemporal pattern during the developmental stages of the worm. Hereby in this work, they demonstrates a single perisynaptic organizer plays a pivotal role in restricting the neural connection at L1 stage (as C.elegans undergoes L1 to L4 stage before they become adults) and during later stages it is involved in regulating the neural re-wiring of the input and output connections. As the authors pointed out that the ventrally located OIG1 regulates the dorsal organization of the synapses opens up a gamut of questions, which will shed light on how inhibitory circuits assembles in this microcircuit A couple of questions, which ignite my interest, are enumerated below:
- What are the receptors for OIG1 in DD and VD motor neurons? Do they share the same receptor or different?
- How do OIG1 acts as a non-permissive signal to restrict the DD motor neurons not to form ectopic synapses at the dorsal muscle during the L1 stage, while later it acts as a permissive signal in the ventral part to remodel the input and output from the VD motor neurons? What are the different signaling cascades regulated by OIG1 in two different context in different developmental stages?
By using laser micro dissection techniques, one will be able to pull out the genetically fluorescently labeled DD neurons at L1 stage and VD neurons after L1 stage. Alternatively, the approach will be dissociating the worms at above mentioned stages and FACS sorting the individual neurons. Once this neurons are isolated or sorted one can perform RNA sequencing of the DD and VD neurons at different stages of development in wild type, olig1-/-, unc-30 -/-, unc55-/- backgrounds or in combinations to unravel the operating signaling pathway. This may offers clues of how OIG1 orchestrates the assembly of inhibitory synapses in motor neurons in the nerve cord.
Given the seminal importance played by transmembrane receptors or adhesive molecular match codes flagging the juxtaposed pre and post-synaptic partners in assembling the excitatory and inhibitory circuits, I put a meager effort to barcode them by visualizing their expression from public databases such as Allen brain atlas map, which in turn provides a plethora of information of expression patterns of genes across different regions of the brain during development as well as in adult stages. In essence, I obtained a list of transmembrane domain from mouse proteins from Ensembl Biomart (Ensembl Genes 75). A list of 11368 transcripts/proteins (many were isoforms) was obtained, which refers to the subset of the Mouse proteome (genome version GRcm38.P2 & Ensembl annotation release 75). The transmembrane receptors representing ion channels, solute carriers, G-protein coupled receptors and their isoforms were omitted by manual curating. The rest of them were verified against the expression pattern in the Allen brain atlas and color coded according to their expression in the different layers in the developing brain. Potentially, one can color code the expression pattern of any genes in the different layers in the developing as well as adult brain comparing with the insitu expression pattern found in the public database, Allen brain atlas (http://www.brain-map.org) for easing the visualisation. ( If you need the data files of the 11368 transcripts as well as the colour coded visualisation, please contact me through my e-mail).
What roads are ahead?
Unlike a simple nervous system with a total of 302 neurons in worms, mammalian nervous system possess a gamut of inhibitory interneurons differs in their morphological and physiological properties with specialized functions and distinct localization to execute gain modulation not only in microcircuits but also in shaping the oscillations in the brain. (4). Moreover, the development of nervous system can be divided into multiple phases 1. Path finding 2. Synapse formation 3. Synapse stabilization 4. Synapse maintenance. This poses multiple questions: What are the molecules that regulate the formation and maintenance of GABAergic synapses for distinct class of interneurons? How similar and different they are? For instance, Martinotti cells, one of the subtypes of inhibitory interneurons exclusively localized in the layer 5, can send their axonal projections from layer 5 to layer 1, while neuroglia form cells are found in all layers of the cortex. Distinctly, the latter can make both chemical and electrical synapses with pyramidal neurons and other interneuron subtypes respectively thereby connecting with multiple networks of interneurons to shape synchronised activity. Besides, they possess dense local axonal projection in contrast to Martinotti cells and display unique synaptic properties, which includes a biphasic response in post-synaptic cells called “ Volume transmission”. An emerging question would be which molecules are in common and which differ in regulating the synapse formation in different types of interneurons with their different targets? Experimental approaches to unravel the complexity behind this biological phenomenon will keep the field busy.
With the advent of advanced tools such as high-resolution imaging, RNA sequencing technology, proteomics, we are now able to isolate and probe distinct classes of interneurons. Using specific CRE lines, it is possible now to isolate specific classes of interneurons and combining with the discovery of new molecular makers, we will be in a position to isolate neurons in early and in late stages of development. Other alternative methods for isolating the subtypes of neurons will be performing translating ribosome affinity purification (TRAP) in tandem with the reporter line (5, 6). Along the same line, another exciting prospect will be profiling activated ribosomes from neurons in those regions of the brain which are active for a particular function there by capturing the activity dependent neuron type transcripts (7). Molecular profiling of neurons depends on their connectivity (8) is an another powerful tool given the electrical and chemical synapses formed by distinct classes of interneurons across different regions with specific cell types in modulating the output of a microcircuit. Another recent method I believe worth mentioning is the recent “INTACT” method developed to delineate the epigenomic signature in two classes of interneurons (PV and VIP) as well as in excitatory pyramidal neurons (9). I believe this technique will help us to explore new frontiers in synapse biology, enables us in understanding what synaptic changes happens in neurodevelopmental diseases such as autism, schizophrenia. Using this method, one can understand what kind of epiegentic changes are happening in different neuronal subtypes during path finding, synapse formation, synapse stabilization, synapse maturation and also later in development how activity remodels this synapses such as strengthening or weakening. Combining this epigenetic modifications with RNA sequencing of this sorted cell types and further profiling of ribosomes on this cells one can find the causality of those epigenetic modifications. This also offers profound implications where one can compare the changes in those neuronal subtypes in developmental disorders to wild type.
It seems inevitable that with the army of new molecular as well as technological tools will play a major share in dissecting new molecular performers, who are obliged as well as recruited to facilitate to dance to the tunes of the new rap word in the lexicon “ GABA
- MADD-4/Punctin and Neurexin Organize C elegans GABAergic Postsynapses through Neuroligin, Neuron, 2015
- C.elegans Punctin Clusters GABAA Receptors via Neuroligin Binding and UNC-40/DCC Recruitment, Neuron, 2015
- Spatiotemporal control of a novel synaptic organizer molecule. Nature, 2015.
- New insights into the classification and nomenclature of cortical GABAergic interneurons. Nat. Rev. Neuroscience, 2013
- A Translational Profiling Approach for the molecular characterization of CNS Cell Types. Cell 2008.
- Application of a Translational Profiling Approach for the Comparative Analysis of CNS Cell Types. Cell 2008.
- Molecular profiling of activated neurons by phosphorylated ribosome capture. Cell 2012.
- Molecular profiling of neurons based on connectivity, Cell 2014.
- Epigenomic Signatures of Neuronal Diversity in the Mammalian Brain. Neuron, 2015.