Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of the glutamate sensor iGluSnFR that are functionally brighter, detect sub-micromolar to millimolar glutamate, and have blue, cyan, green, or yellow emission profiles. These variants allow
in vivo
imaging where original iGluSnFR was too dim, can resolve glutamate transients in dendritic spines and axonal boutons, and permit kilohertz imaging.
The medial prefrontal cortex (mPFC) plays a critical role in the control of cognition and emotion. Reciprocal circuits between the mPFC and basolateral amygdala (BLA) are particularly important for emotional control. However, the neurons and synapses that link these brain regions remain largely unknown. Here we examine long-range connections between the mouse mPFC and BLA, using whole-cell recordings, optogenetics, and two-photon microscopy. We first identify two non-overlapping populations of layer 2 pyramidal neurons that directly project to either the BLA or contralateral mPFC. We then show that pyramidal neurons projecting to the BLA receive much stronger excitatory inputs from this same brain region. We next assess the contributions of both presynaptic and postsynaptic mechanisms to this cell-type and input-specific connectivity. We use two-photon mapping to reveal differences in both the synaptic density and subcellular targeting of BLA inputs. Finally, we simulate and experimentally validate how the number, volume, and location of active spines all contribute to preferential synaptic drive. Together, our findings reveal a novel and strong reciprocal circuit that is likely to be important for how the mPFC controls cognition and emotion.
In vivo imaging at high spatiotemporal resolution holds the key to the fundamental understanding of complex biological systems. Integrating an optical phase-locked ultrasound lens into a conventional two-photon fluorescence microscope, we achieved microsecond scale axial scanning, which enabled high-speed volumetric imaging. We applied this system to multicolor volumetric imaging of fast processes, including calcium dynamics in the cerebral cortex of behaving mice, and transient morphology changes and trafficking of immune cells.
Pyramidal neurons in the prefrontal cortex (PFC) are important for the control of cognitive and emotional behavior. The medial PFC (mPFC) receives diverse long-range excitatory inputs from the midline thalamus, contralateral mPFC, basolateral amygdala and ventral hippocampus. While axons from these different regions have distinct distributions in the mPFC, their functional connections at the cellular and subcellular levels remain unknown. Here, we use optogenetics to show that layer 2 pyramidal neurons in acute slices of the mouse mPFC receive excitatory inputs from each of these regions. Using a combination of optogenetics and two-photon microscopy, we then determine the subcellular properties of these inputs. We find that different types of inputs make selective contacts at the levels of both dendrites and spines. Using two-photon uncaging, we show that this subcellular targeting strongly influences synaptic efficacy in these neurons. Together, our results show that functional connectivity is finely tuned, with important implications for signal processing in the mPFC.
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