Final Report Summary - VISUALDENDRITE (In vitro and in vivo examination of the spatial and temporal distribution of synaptic inputs and synaptic integration in layer 2/3 visual cortical neurons)
The brain constantly processes information arising from internal sources and the outside world, a process that is crucial for controlling behaviour and navigating in the outside world. This ability relies on the power of neuronal networks, which are capable of encoding of sensory and proprioceptive information into electrical signals that are sensed, transmitted and transformed by neurons within neuronal microcircuits. Deciphering the mechanisms by which single neurons combine or integrate signals arising from multiple sources is therefore crucial for furthering our understanding of how the brain functions. Decades of past experimental and modeling work have led to the consensus that neuronal dendrites can support non-linear integration of synaptic inputs, and that these non-linearities are critical for neuronal computations. There is still much unknown the mechanisms and role of these transformations, shaped by the precise spatial and temporal distribution of incoming synaptic inputs, and the morphological and electrical properties of the integrating neuron.
We use the cerebellum as a model, an anatomically well-defined structure with a small number of different cell types, where multiple sensory afferences converge and are integrated to guide motor learning and behavior. We focused on the integrative properties of stellate cells (SCs), an interneuron that receives synaptic inputs from thousands of parallel fibers, and provides feed-forward inhibition onto the dendritic tree of Purkinje cell, the principle cell and sole output of the cerebellum. Abrahamsson et al (2012) have demonstrated that SCs summate neighbouring synaptic inputs following a sublinear summation rule. Building on this result we have tried to investigate further the mechanisms underlying this rather unusual integrative behaviour, and to explore its functional consequences. In order to tackle the complex question of how stellate cells transform and integrate their inputs, using as signals the transformations of their membrane voltage and intracellular Ca2+, we have used a combination of state-of-the art electrophysiological and imaging techniques.
Results
Sublinear integration in thin dendrites of SCs has been hypothesized to be due to the large local depolarization upon synaptic conductance activation. Unfortunately these extremely thin (0.4 um) dendrites are therefore not accessible to electrode based recording methods, which can be used on structures of at least 1µm. We therefore used an optical tool to record the dendritic voltage during synaptic activity. The two-component voltage reporter described by Bradley et al (2009) consists of DiO, a neuronal tracer dye as a FRET donor and DPA as a non-fluorescent acceptor that quenches DiO fluorescence. This dye presents the advantage of a great sensitivity, allowing measurements of small amplitude depolarization, and excellent temporal resolution. We calibrated the dye in SC dendrites and have been able to measure the amplitude and time course of local synaptic depolarization in SC dendrites.
Using Ca2+ imaging and glutamate uncaging, we have been able to characterize the input-output functions for Ca2+ and voltage responses during synaptic stimulation, and their responses to stimulation at different frequencies.
Conclusions
Thin dendrites of SCs support extraordinarily large amplitude depolarizations, which allow sublinear summation of concomitant, neighbouring synaptic inputs. The direct consequence of this integration property is that spatially clustered synaptic inputs are less likely to produce a depolarization sufficient to trigger action potential firing.
Surprisingly we observed a different summation mode for dendritic Ca2+, which is linear or supralinear. To date, supralinear synaptic input-output relationships described in cortical or hippocampal pyramidal neurons result from voltage-dependent activation of NMDA Rs and Ca2+ channels (Branco and Häusser, 2011; Losonczy and Magee, 2006; Schiller et al., 2000), thus producing coordinate transformations of synaptic activity into dendritic voltage and [Ca2+]. Therefore we show for the first time that dendritic [Ca2+] signaling can summate linearly or supra-linearly, while simultaneously synaptic voltage summates according to a sublinear rule. Linear and supralinear Ca2+ integration modes are involved in triggering, respectively, long and short-term synaptic plasticity.
Therefore, in stark contrast with pyramidal neurons, where supralinear integration promotes the detection of clustered inputs and has promoted the idea that dendritic branch can act as an independent integrative unit and the elementary brick of neuronal computation, the firing of SC is preferentially activated by scattered inputs, therefore requiring the knowledge of synaptic activity impinging on its dendritic tree on a global scale in order to define the neuronal computation.
Outcomes
Our findings regarding the integration properties of SCs provide a critical knowledge to build upon in order to understand the information flow in the cerebellar microcircuit. Specifically, the cerebellum is known to be a site of convergence of multiple sources of sensory information. A recent study has shown that cerebellar granule cells, the neuron that provide presynaptic innervation to SC, supply a first step in this process by integrating inputs from multiple sensory sources (Chabrol et al., 2015). How their output is further integrated and transformed by SCs will therefore be essential in understanding how multi-sensory information is performed in the cerebellum. Such future research might have important consequences as synaptic properties and multi-sensory integration are altered in the cerebellum in neurological and psychiatric disorders, notably in some cases of autism spectrum disorders (ASD) (Wang et al. 2014). The prevalence of ASD is of about 1/160 in the population (source: World Health Organization, http://www.who.int/features/qa/85/en/) and the better understanding of their biological bases will provide a high socio-economic impact and benefits for public health. Additionally, studying synaptic integration in canonical microcircuits provides information for the functioning of neuronal circuits known to be involved in higher cognitive. This knowledge is also important in contributing to the understanding of other severe psychiatric disorders such as schizophrenia and neurodegenerative disorders.
References
Abrahamsson, T., Cathala, L., Matsui, K., Shigemoto, R., and Digregorio, DA. (2012). Thin dendrites of cerebellar interneurons confer sublinear synaptic integration and a gradient of short-term plasticity. Neuron 73:1159-1172.
Bradley J., Luo R., Otis TS., DiGregorio DA. (2009) Submillisecond Optical Reporting of Membrane Potential In Situ Using a Neuronal Tracer Dye. The Journal of Neuroscience 29(29):9197-9209.
Branco T., Häusser M. (2011). Synaptic integration gradients in single cortical pyramidal cell dendrites. Neuron 69(5):885-92.
Chabrol, FP., Arenz, A., Wiechert, MT., Margrie, TW., DiGregorio, DA. (2015). Synaptic diversity enables temporal coding of coincident multisensory inputs in single neurons. Nature Neuroscience 18:728-727.
Losonczy A., Magee JC. (2006). Integrative properties of radial oblique dendrites in hippocampal CA1 pyramidal neurons. Neuron 50(2):291-307.
Schiller J., Major G., Koester HJ., Schiller Y. (2000). NMDA spikes in basal dendrites of cortical pyramidal neurons. Nature 404: 285–289.
Wang SS-H, Kloth AD, and Badura A (2014) The cerebellum, sensitive periods, and autism (Perspective). Neuron, 83:518-532.
We use the cerebellum as a model, an anatomically well-defined structure with a small number of different cell types, where multiple sensory afferences converge and are integrated to guide motor learning and behavior. We focused on the integrative properties of stellate cells (SCs), an interneuron that receives synaptic inputs from thousands of parallel fibers, and provides feed-forward inhibition onto the dendritic tree of Purkinje cell, the principle cell and sole output of the cerebellum. Abrahamsson et al (2012) have demonstrated that SCs summate neighbouring synaptic inputs following a sublinear summation rule. Building on this result we have tried to investigate further the mechanisms underlying this rather unusual integrative behaviour, and to explore its functional consequences. In order to tackle the complex question of how stellate cells transform and integrate their inputs, using as signals the transformations of their membrane voltage and intracellular Ca2+, we have used a combination of state-of-the art electrophysiological and imaging techniques.
Results
Sublinear integration in thin dendrites of SCs has been hypothesized to be due to the large local depolarization upon synaptic conductance activation. Unfortunately these extremely thin (0.4 um) dendrites are therefore not accessible to electrode based recording methods, which can be used on structures of at least 1µm. We therefore used an optical tool to record the dendritic voltage during synaptic activity. The two-component voltage reporter described by Bradley et al (2009) consists of DiO, a neuronal tracer dye as a FRET donor and DPA as a non-fluorescent acceptor that quenches DiO fluorescence. This dye presents the advantage of a great sensitivity, allowing measurements of small amplitude depolarization, and excellent temporal resolution. We calibrated the dye in SC dendrites and have been able to measure the amplitude and time course of local synaptic depolarization in SC dendrites.
Using Ca2+ imaging and glutamate uncaging, we have been able to characterize the input-output functions for Ca2+ and voltage responses during synaptic stimulation, and their responses to stimulation at different frequencies.
Conclusions
Thin dendrites of SCs support extraordinarily large amplitude depolarizations, which allow sublinear summation of concomitant, neighbouring synaptic inputs. The direct consequence of this integration property is that spatially clustered synaptic inputs are less likely to produce a depolarization sufficient to trigger action potential firing.
Surprisingly we observed a different summation mode for dendritic Ca2+, which is linear or supralinear. To date, supralinear synaptic input-output relationships described in cortical or hippocampal pyramidal neurons result from voltage-dependent activation of NMDA Rs and Ca2+ channels (Branco and Häusser, 2011; Losonczy and Magee, 2006; Schiller et al., 2000), thus producing coordinate transformations of synaptic activity into dendritic voltage and [Ca2+]. Therefore we show for the first time that dendritic [Ca2+] signaling can summate linearly or supra-linearly, while simultaneously synaptic voltage summates according to a sublinear rule. Linear and supralinear Ca2+ integration modes are involved in triggering, respectively, long and short-term synaptic plasticity.
Therefore, in stark contrast with pyramidal neurons, where supralinear integration promotes the detection of clustered inputs and has promoted the idea that dendritic branch can act as an independent integrative unit and the elementary brick of neuronal computation, the firing of SC is preferentially activated by scattered inputs, therefore requiring the knowledge of synaptic activity impinging on its dendritic tree on a global scale in order to define the neuronal computation.
Outcomes
Our findings regarding the integration properties of SCs provide a critical knowledge to build upon in order to understand the information flow in the cerebellar microcircuit. Specifically, the cerebellum is known to be a site of convergence of multiple sources of sensory information. A recent study has shown that cerebellar granule cells, the neuron that provide presynaptic innervation to SC, supply a first step in this process by integrating inputs from multiple sensory sources (Chabrol et al., 2015). How their output is further integrated and transformed by SCs will therefore be essential in understanding how multi-sensory information is performed in the cerebellum. Such future research might have important consequences as synaptic properties and multi-sensory integration are altered in the cerebellum in neurological and psychiatric disorders, notably in some cases of autism spectrum disorders (ASD) (Wang et al. 2014). The prevalence of ASD is of about 1/160 in the population (source: World Health Organization, http://www.who.int/features/qa/85/en/) and the better understanding of their biological bases will provide a high socio-economic impact and benefits for public health. Additionally, studying synaptic integration in canonical microcircuits provides information for the functioning of neuronal circuits known to be involved in higher cognitive. This knowledge is also important in contributing to the understanding of other severe psychiatric disorders such as schizophrenia and neurodegenerative disorders.
References
Abrahamsson, T., Cathala, L., Matsui, K., Shigemoto, R., and Digregorio, DA. (2012). Thin dendrites of cerebellar interneurons confer sublinear synaptic integration and a gradient of short-term plasticity. Neuron 73:1159-1172.
Bradley J., Luo R., Otis TS., DiGregorio DA. (2009) Submillisecond Optical Reporting of Membrane Potential In Situ Using a Neuronal Tracer Dye. The Journal of Neuroscience 29(29):9197-9209.
Branco T., Häusser M. (2011). Synaptic integration gradients in single cortical pyramidal cell dendrites. Neuron 69(5):885-92.
Chabrol, FP., Arenz, A., Wiechert, MT., Margrie, TW., DiGregorio, DA. (2015). Synaptic diversity enables temporal coding of coincident multisensory inputs in single neurons. Nature Neuroscience 18:728-727.
Losonczy A., Magee JC. (2006). Integrative properties of radial oblique dendrites in hippocampal CA1 pyramidal neurons. Neuron 50(2):291-307.
Schiller J., Major G., Koester HJ., Schiller Y. (2000). NMDA spikes in basal dendrites of cortical pyramidal neurons. Nature 404: 285–289.
Wang SS-H, Kloth AD, and Badura A (2014) The cerebellum, sensitive periods, and autism (Perspective). Neuron, 83:518-532.