DENDRITE2BEHAVIOR · From dendrite to behavior: fiberoptic Imaging and optogenetic manipulation of dendritic activity in behaving animals

From dendrite to behavior: fiberoptic Imaging and optogenetic manipulation of dendritic activity in behaving animals

Dendrites are branched extensions of a neural cell, or neuron. They receive electrical signals emitted from other neurons and transfer these signals to the cell body, or soma. Dendrites were believed to be passive "antennas" that only have the ability to propagate electrical signals, but now it is clear that dendrites have active properties that have a strong influence on the output of the cell, or somatic action potentials [1]. The goal of our project was to test the hypothesis that optically manipulating the activity in a specific region of dendrites should influence animal behavior. To achieve this goal we developed a 'micro-periscope' system that is able to deliver a light beam whose diameter is as small as 0.2 mm. Since the thickness of rodent cortex is 1 to 2 mm, the light beam may selectively stimulate a specific region of the cortex. The extreme lightweight of the system allowed us to measure or manipulate the activity of a specific region of dendrites in freely behaving animals. The development of this tiny device turned out to be a breakthrough that led us to produce highly significant results described below. We are currently preparing four journal articles, two of which are of exceptional importance and therefore will be submitted to the top scientific journals. 1. Dendritic calcium spikes greatly affect EEG signals The cerebral cortex is the outermost layer of the brain that generates our cognitive functions such as attention, memory, perception, and language. The cortex can be divided into six layers labeled 1 to 6. Dendrites of cortical layer 5 (L5) pyramidal neurons are known to have voltage-gated calcium channels that enable the dendrites to produce characteristic depolarizing plateau potentials (called "dendritic calcium spikes") [1]. They are large, long-lasting events that take place near the cortical surface. A recent study demonstrated the significant contribution of dendritic calcium spikes to animal behavior [2]. Despite their magnitude, functional significance, and proximity to the cortical surface, dendritic calcium spikes have not been considered as a major contributor to the electroencephalogram (EEG) signals, that instead have been thought to primarily reflect cooperative postsynaptic activity [3, 4]. Through combined use of electrophysiological and optogenetic techniques, we found for the first time that dendritic calcium spikes greatly affect the potential measured at the cortical surface; they are not only clearly detectable but their amplitude is as large as the amplitude of excitatory postsynaptic potentials. This finding was made possible by our establishment of a method to evoke dendritic calcium spikes in vivo. The micro-periscope system we developed played a crucial role in this method. The results we obtained could have a huge impact in the field of neuroscience and neurology because our data challenge the prevailing assumption that EEG signals primarily reflect the excitatory postsynaptic activity. Our data suggest that dendritic calcium spikes cannot be neglected, but rather significantly modulate EEG signals. As EEG method is so widely used all over the world in clinical and non-clinical applications, our data may have huge influence on the way in which EEG data is interpreted in healthy and diseased individuals. 2. Pyramidal neurons are extremely sensitive to coincidentally arriving inputs at different cortical layers Intelligent behavior depends on the ability to generate flexible, context-dependent actions. Psychological experiments consistently show that experience or expectation influences behavior, for instance in learned tasks or illusory perception. This influence has been shown to depend on top-down signaling from higher to lower cortical areas [5]. Sensory cortices receive a top-down signal from higher cortical areas (e.g., prefrontal cortex), which provides "executive control" and specifies the context or rules currently in effect [6]. However, little is known as to how this top-down signal influences the sensory, bottom-up signals, and thereby the behavior of the animal. This has long been a central question in neuroscience. Previous anatomical studies revealed that top-down signals tend to arrive at the outermost cortical layer 1 whereas the sensory or bottom-up signals are fed to cortical layer 4. Layer 5 pyramidal neurons are known to extend their dendrites into these layers and therefore are ideally suited to associating top-down signals with sensory, bottom-up signals. This issue was previously addressed in in vitro setup [7], but in vivo evidence was lacking for various technical reasons. Using two micro-periscope systems simultaneously, we have established the method for the first time to activate two separate cortical layers in vivo. The timing of two light pulses was controlled with millisecond precision. We obtained the first in vivo evidence that L5 pyramidal neurons are extremely sensitive to coincidentally arriving inputs at different cortical layers. Their activity significantly increased when dendrites in two separate layers 1 and 5 were stimulated within a very narrow time window. We further found that this sensitivity depends on the voltage-gated calcium channels in the pyramidal cell dendrites. The results we obtained could have a huge impact on the field of neuroscience because we have established the method to precisely activate different cortical layers in vivo, and because our data is the first in vivo evidence that pyramidal neurons are highly sensitive to coincidentally arriving inputs at different cortical layers. In summary, our project supported by Marie Curie International Incoming Fellowship produced highly important results that have huge potential to influence the fields of neuroscience and neurology. The manuscripts are currently in preparation and will be submitted to top scientific journals by the end of this year. References 1. Major, G., M.E. Larkum, and J. Schiller, Active properties of neocortical pyramidal neuron dendrites. Annu Rev Neurosci, 2013. 36: p. 1-24. 2. Xu, N.L., et al., Nonlinear dendritic integration of sensory and motor input during an active sensing task. Nature, 2012. 492(7428): p. 247-51. 3. Mitzdorf, U., Current source-density method and application in cat cerebral cortex: investigation of evoked potentials and EEG phenomena. Physiol Rev, 1985. 65(1): p. 37-100. 4. Buzsaki, G., C.A. Anastassiou, and C. Koch, The origin of extracellular fields and currents--EEG, ECoG, LFP and spikes. Nat Rev Neurosci, 2012. 13(6): p. 407-20. 5. Gilbert, C.D. and M. Sigman, Brain states: top-down influences in sensory processing. Neuron, 2007. 54(5): p. 677-96. 6. Miller, E.K. and J.D. Cohen, An integrative theory of prefrontal cortex function. Annu Rev Neurosci, 2001. 24: p. 167-202. 7. Larkum, M.E., J.J. Zhu, and B. Sakmann, A new cellular mechanism for coupling inputs arriving at different cortical layers. Nature, 1999. 398(6725): p. 338-41.

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