Dopamine release during reward-driven behaviors influences synaptic plasticity. However, dopamine innervation and release in the hippocampus and its role during aversive behaviors are controversial. Here we show that in vivo hippocampal synaptic plasticity in the CA3-CA1 circuit underlies contextual learning during inhibitory avoidance (IA) training. Immunohistochemistry and molecular techniques verified sparse dopaminergic innervation of the hippocampus from the midbrain. The long-term synaptic potentiation (LTP) underlying the learning of IA was assessed with a D1-like dopamine receptor agonist or antagonist in ex vivo hippocampal slices and in vivo in freely-moving mice. Inhibition of D1-like dopamine receptors impaired memory of the IA task and prevented the training-induced enhancement of both ex vivo and in vivo LTP induction. The results indicate that dopamine-receptor signaling during an aversive contextual task regulates aversive memory retention and regulates associated synaptic mechanisms in the hippocampus that likely underlie learning.

Channelrhodopsin-2 (ChR2) has quickly gained popularity as a powerful tool for eliciting genetically targeted neuronal activation. However, little has been reported on the response kinetics of optogenetic stimulation across different neuronal subtypes. With excess stimulation, neurons can be driven into depolarization block, a state where they cease to fire action potentials. Herein, we demonstrate that light-induced depolarization block in neurons expressing ChR2 poses experimental challenges for stable activation of specific cell types and may confound interpretation of experiments when ‘activated’ neurons are in fact being functionally silenced. We show both ex vivo and in vivo that certain neuronal subtypes targeted for ChR2 expression become increasingly susceptible to depolarization block as the duration of light pulses are increased. We find that interneuron populations have a greater susceptibility to this effect than principal excitatory neurons, which are more resistant to light-induced depolarization block. Our results highlight the need to empirically determine the photo-response properties of targeted neurons when using ChR2, particularly in studies designed to elicit complex circuit responses in vivo where neuronal activity will not be recorded simultaneous to light stimulation.DOI: http://dx.doi.org/10.7554/eLife.01481.001

Proper brain function relies on exquisite balance between excitation and inhibition, where inhibitory circuits play fundamental roles toward sculpting principle neuron output and information processing. In prominent models of olfactory bulb circuitry, inhibition of mitral cells by local interneurons sharpens odor tuning and provides contrast enhancement. Mitral cell inhibition occurs at both mitral cell apical dendrites and deep-layer dendrodendritic synapses between granule cells, the most abundant population of inhibitory interneurons in the olfactory bulb. However, it remains unclear whether other local interneurons make inhibitory connections onto mitral cells. Here, we report a novel circuitry with strong and reciprocal connectivity between a subpopulation of previously uncharacterized Corticotropin-Releasing Hormone (CRH)-expressing interneurons located in the external plexiform layer (EPL), and mitral cells. Using cell type-specific genetic manipulations, imaging, optogenetic stimulation, and electrophysiological recordings, we reveal that CRH-expressing EPL interneurons strongly inhibit mitral cell firing, and that they are reciprocally excited by fast glutamatergic mitral cell input. These findings functionally identify a novel subpopulation of olfactory bulb interneurons that show reciprocal connectivity with mitral cells, uncovering a previously unknown, and potentially critical player in olfactory bulb circuitry that may influence lateral interactions and/or facilitate odor processing.

Elucidating patterns of functional synaptic connectivity and deciphering mechanisms of how plasticity influences such connectivity is essential toward understanding brain function. In the mouse olfactory bulb (OB), principal neurons (mitral/tufted cells) make reciprocal connections with local inhibitory interneurons, including granule cells (GCs) and external plexiform layer (EPL) interneurons. Our current understanding of the functional connectivity between these cell types, as well as their experience-dependent plasticity, remains incomplete. By combining acousto-optic deflector-based scanning microscopy and genetically targeted expression of Channelrhodopsin-2, we mapped connections in a cell-type-specific manner between mitral cells (MCs) and GCs or between MCs and EPL interneurons. We found that EPL interneurons form broad patterns of connectivity with MCs, whereas GCs make more restricted connections with MCs. Using an olfactory associative learning paradigm, we found that these circuits displayed differential features of experience-dependent plasticity. Whereas reciprocal connectivity between MCs and EPL interneurons was nonplastic, the connections between GCs and MCs were dynamic and adaptive. Interestingly, experience-dependent plasticity of GCs occurred only in certain stages of neuronal maturation. We show that different interneuron subtypes form distinct connectivity maps and modes of experiencedependent plasticity in the OB, which may reflect their unique functional roles in information processing.

Summary Neural activity either enhances or impairs de novo synaptogenesis and circuit integration of neurons, but how this activity is mechanistically relayed in the adult brain is largely unknown. Neuropeptide-expressing interneurons are widespread throughout the brain and are key candidates for conveying neural activity downstream via neuromodulatory pathways that are distinct from classical neurotransmission. With the goal of identifying signaling mechanisms that underlie neuronal circuit integration in the adult brain, we have virally traced local Corticotropin-releasing hormone (CRH)-expressing inhibitory interneurons with extensive presynaptic inputs onto new neurons that are continuously integrated into the adult rodent olfactory bulb. Local CRH signaling onto adult-born neurons promotes and/or stabilizes chemical synapses in the olfactory bulb, revealing a neuromodulatory mechanism for continued circuit plasticity, synapse formation, and integration of new neurons in the adult brain.

Neuropeptides play important physiological functions during distinct behaviors such as arousal, learning, memory, and reproduction. However, the role of local, extrahypothalamic neuropeptide signaling in shaping synapse formation and neuronal plasticity in the brain is not well understood. Here, we characterize the spatiotemporal expression profile of the neuropeptide CRH and its receptor CRHR1 in in the mouse OB throughout development. We found that CRH-expressing interneurons are enriched in the external plexiform layer, that its cognate receptor is expressed by granule cells, and show that both CRH and CRHR1 expression enriches in the postnatal period when olfaction becomes important towards olfactory related behaviors. Further, we provide electrophysiological evidence that CRHR1-expressing granule cells functionally respond to CRH ligand, and that the physiological circuitry of CRHR1 knockout mice is abnormal, leading to impaired olfactory behaviors. Together, these data suggest a physiologically relevant role for local CRH signaling towards shaping the neuronal circuitry within the mouse OB.

Transsynaptic circuit tracing using genetically modified Rabies virus (RV) is an emerging technology for identifying synaptic connections between neurons. Complementing this methodology, it has become possible to assay the basic molecular and cellular properties of neuronal lineages derived from embryonic stem (ES) cells in vitro, and these properties are under intense investigation towards devising cell replacement therapies. Here, we report the generation of a novel mouse ES cell (mESC) line that harbors the genetic elements to allow RV-mediated transsynaptic circuit tracing in ES cell-derived neurons and their synaptic networks. To facilitate transsynaptic tracing, we have engineered a new reporter allele by introducing cDNA encoding tdTomato, the Rabies-G glycoprotein, and the avian TVA receptor into the ROSA26 locus by gene targeting. We demonstrate high-efficiency differentiation of these novel mESCs into functional neurons, show their capacity to functionally connect with primary neuronal cultures as evidenced by immunohistochemistry and electrophysiological recordings, and show their ability to act as source cells for presynaptic tracing of neuronal networks in vitro and in vivo. Together, our data highlight the potential for using genetically engineered stem cells to investigate fundamental mechanisms of synapse and circuit formation with unambiguous identification of presynaptic inputs onto neuronal populations of interest.

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