GABA-receptive microglia selectively sculpt developing inhibitory circuits.

Microglia, the resident immune cells of the brain, have emerged as crucial regulators of synaptic refinement and brain wiring. However, whether the remodeling of distinct synapse types during development is mediated by specialized microglia is unknown. Here, we show that GABA-receptive microglia selectively interact with inhibitory cortical synapses during a critical window of mouse postnatal development. GABA initiates a transcriptional synapse remodeling program within these specialized microglia, which in turn sculpt inhibitory connectivity without impacting excitatory synapses. Ablation of GABAB receptors within microglia impairs this process and leads to behavioral abnormalities. These findings demonstrate that brain wiring relies on the selective communication between matched neuronal and glial cell types.

Sparse Labeling and Neural Tracing in Brain Circuits by STARS Strategy: Revealing Morphological Development of Type II Spiral Ganglion Neurons.

Elucidating axonal and dendritic projection patterns of individual neurons is a key for understanding the cytoarchitecture of neural circuits in the brain. This requires genetic approaches to achieve Golgi-like sparse labeling of desired types of neurons. Here, we explored a novel strategy of stochastic gene activation with regulated sparseness (STARS), in which the stochastic choice between 2 competing Cre-lox recombination events is controlled by varying the lox efficiency and cassette length. In a created STARS transgenic mouse crossed with various Cre driver lines, sparse neuronal labeling with a relatively uniform level of sparseness was achieved across different brain regions and cell types in both central and peripheral nervous systems. Tracing of individual type II peripheral auditory fibers revealed for the first time that they undergo experience-dependent developmental refinement, which is impaired by attenuating external sound input. Our results suggest that STARS strategy can be applied for circuit mapping and sparse gene manipulation.

Differential Receptive Field Properties of Parvalbumin and Somatostatin Inhibitory Neurons in Mouse Auditory Cortex.

Cortical inhibitory circuits play important roles in shaping sensory processing. In auditory cortex, however, functional properties of genetically identified inhibitory neurons are poorly characterized. By two-photon imaging-guided recordings, we specifically targeted 2 major types of cortical inhibitory neuron, parvalbumin (PV) and somatostatin (SOM) expressing neurons, in superficial layers of mouse auditory cortex. We found that PV cells exhibited broader tonal receptive fields with lower intensity thresholds and stronger tone-evoked spike responses compared with SOM neurons. The latter exhibited similar frequency selectivity as excitatory neurons. The broader/weaker frequency tuning of PV neurons was attributed to a broader range of synaptic inputs and stronger subthreshold responses elicited, which resulted in a higher efficiency in the conversion of input to output. In addition, onsets of both the input and spike responses of SOM neurons were significantly delayed compared with PV and excitatory cells. Our results suggest that PV and SOM neurons engage in auditory cortical circuits in different manners: while PV neurons may provide broadly tuned feedforward inhibition for a rapid control of ascending inputs to excitatory neurons, the delayed and more selective inhibition from SOM neurons may provide a specific modulation of feedback inputs on their distal dendrites.

Synaptic mechanisms underlying functional dichotomy between intrinsic-bursting and regular-spiking neurons in auditory cortical layer 5.

Corticofugal projections from the primary auditory cortex (A1) have been shown to play a role in modulating subcortical processing. However, functional properties of the corticofugal neurons and their synaptic circuitry mechanisms remain unclear. In this study, we performed in vivo whole-cell recordings from layer 5 (L5) pyramidal neurons in the rat A1 and found two distinct neuronal classes according to their functional properties. Intrinsic-bursting (IB) neurons, the L5 corticofugal neurons, exhibited early and rather unselective spike responses to a wide range of frequencies. The exceptionally broad spectral tuning of IB neurons was attributable to their broad excitatory inputs with long temporal durations and inhibitory inputs being more narrowly tuned than excitatory inputs. This uncommon pattern of excitatory-inhibitory interplay was attributed initially to a broad thalamocortical convergence onto IB neurons, which also receive temporally prolonged intracortical excitatory input as well as feedforward inhibitory input at least partially from more narrowly tuned fast-spiking inhibitory neurons. In contrast, regular-spiking neurons, which are mainly corticocortical, exhibited sharp frequency tuning similar to L4 pyramidal cells, underlying which are well-matched purely intracortical excitation and inhibition. The functional dichotomy among L5 pyramidal neurons suggests two distinct processing streams. The spectrally and temporally broad synaptic integration in IB neurons may ensure robust feedback signals to facilitate subcortical function and plasticity in a general manner.

Slit/Robo signaling mediates spatial positioning of spiral ganglion neurons during development of cochlear innervation.

During the development of periphery auditory circuits, spiral ganglion neurons (SGNs) extend their neurites to innervate cochlear hair cells (HCs) with their soma aggregated into a cluster spatially segregated from the cochlear sensory epithelium. The molecular mechanisms underlying this spatial patterning remain unclear. In this study, in situ hybridization in the mouse cochlea suggests that Slit2 and its receptor, Robo1/2, exhibit apparently complementary expression patterns in the spiral ganglion and its nearby region, the spiral limbus. In Slit2 and Robo1/2 mutants, the spatial restriction of SGNs was disrupted. Mispositioned SGNs were found to scatter in the space between the cochlear epithelium and the main body of spiral ganglion, and the neurites of mispositioned SGNs were misrouted and failed to innervate HCs. Furthermore, in Robo1/2 mutants, SGNs were displaced toward the cochlear epithelium as an entirety. Examination of different embryonic stages in the mutants revealed that the mispositioning of SGNs was due to a progressive displacement to ectopic locations after their initial normal settlement at an earlier stage. Our results suggest that Slit/Robo signaling imposes a restriction force on SGNs to ensure their precise positioning for correct SGN-HC innervations.

Broadening of inhibitory tuning underlies contrast-dependent sharpening of orientation selectivity in mouse visual cortex.

Orientation selectivity (OS) in the visual cortex has been found to be invariant to increases in stimulus contrast, a finding that cannot be accounted for by the original, purely excitatory Hubel and Wiesel model. This property of OS may be important for preserving the quality of perceived stimulus across a range of stimulus intensity. The synaptic mechanisms that can prevent a broadening of OS caused by contrast-dependent strengthening of excitatory inputs to cortical neurons remain unknown. Using in vivo loose-patch recordings, we found in excitatory neurons in layer 4 of mouse primary visual cortex (V1) that the spike response to the preferred orientation was elevated as contrast increased while that to the orthogonal orientation remained unchanged, resulting in an overall sharpening rather than a weakening of OS. Whole-cell voltage-clamp recordings further revealed that contrast increases resulted in a scaling up of excitatory conductance at all stimulus orientations. Inhibitory conductance was enhanced at a similar level as excitation for the preferred orientation, but at a significantly higher level for the orthogonal orientation. Modeling revealed that the resulting broadening of inhibitory tuning is critical for maintaining and sharpening OS at high contrast. Finally, two-photon imaging guided recordings from parvalbumin-positive (PV) inhibitory neurons revealed that the broadening of inhibition can be attributed to a contrast-dependent broadening of spike-response tuning of PV neurons. Together our results suggest that modulation of synaptic inhibition in the mouse V1 cortical circuit preserves the sharpness of response selectivity during changes of stimulus strength.

Mining the jewels of the cortex's crowning mystery.

Neocortical Layer 1 consists of a dense mesh of excitatory and inhibitory axons, dendrites of pyramidal neurons, as well as neuromodulatory inputs from diverse brain regions. Layer 1 also consists of a sparse population of inhibitory interneurons, which are appropriately positioned to receive and integrate the information from these regions of the brain and modulate cortical processing. Despite being among the sparsest neuronal population in the cortex, Layer 1 interneurons perform powerful computations and have elaborate morphologies. Here we review recent studies characterizing their origin, morphology, physiology, and molecular profiles, as well as their connectivity and in vivo response properties.

Viral manipulation of functionally distinct interneurons in mice, non-human primates and humans.

Recent success in identifying gene-regulatory elements in the context of recombinant adeno-associated virus vectors has enabled cell-type-restricted gene expression. However, within the cerebral cortex these tools are largely limited to broad classes of neurons. To overcome this limitation, we developed a strategy that led to the identification of multiple new enhancers to target functionally distinct neuronal subtypes. By investigating the regulatory landscape of the disease gene Scn1a, we discovered enhancers selective for parvalbumin (PV) and vasoactive intestinal peptide-expressing interneurons. Demonstrating the functional utility of these elements, we show that the PV-specific enhancer allowed for the selective targeting and manipulation of these neurons across vertebrate species, including humans. Finally, we demonstrate that our selection method is generalizable and characterizes additional PV-specific enhancers with exquisite specificity within distinct brain regions. Altogether, these viral tools can be used for cell-type-specific circuit manipulation and hold considerable promise for use in therapeutic interventions.

Cross-Modality Sharpening of Visual Cortical Processing through Layer-1-Mediated Inhibition and Disinhibition.

Cross-modality interaction in sensory perception is advantageous for animals' survival. How cortical sensory processing is cross-modally modulated and what are the underlying neural circuits remain poorly understood. In mouse primary visual cortex (V1), we discovered that orientation selectivity of layer (L)2/3, but not L4, excitatory neurons was sharpened in the presence of sound or optogenetic activation of projections from primary auditory cortex (A1) to V1. The effect was manifested by decreased average visual responses yet increased responses at the preferred orientation. It was more pronounced at lower visual contrast and was diminished by suppressing L1 activity. L1 neurons were strongly innervated by A1-V1 axons and excited by sound, while visual responses of L2/L3 vasoactive intestinal peptide (VIP) neurons were suppressed by sound, both preferentially at the cell's preferred orientation. These results suggest that the cross-modality modulation is achieved primarily through L1 neuron- and L2/L3 VIP-cell-mediated inhibitory and disinhibitory circuits.

EphA7 regulates spiral ganglion innervation of cochlear hair cells.

During the development of periphery auditory circuitry, spiral ganglion neurons (SGNs) form a spatially precise pattern of innervation of cochlear hair cells (HCs), which is an essential structural foundation for central auditory processing. However, molecular mechanisms underlying the developmental formation of this precise innervation pattern remain not well understood. Here, we specifically examined the involvement of Eph family members in cochlear development. By performing RNA-sequencing for different types of cochlear cell, in situ hybridization, and immunohistochemistry, we found that EphA7 was strongly expressed in a large subset of SGNs. In EphA7 deletion mice, there was a reduction in the number of inner radial bundles originating from SGNs and projecting to HCs as well as in the number of ribbon synapses on inner hair cells (IHCs), as compared with wild-type or heterozygous mutant mice, attributable to fewer type I afferent fibers. The overall activity of the auditory nerve in EphA7 deletion mice was also reduced, although there was no significant change in the hearing intensity threshold. In vitro analysis further suggested that the reduced innervation of HCs by SGNs could be attributed to a role of EphA7 in regulating outgrowth of SGN neurites as knocking down EphA7 in SGNs resulted in diminished SGN fibers. In addition, suppressing the activity of ERK1/2, a potential downstream target of EphA7 signaling, either with specific inhibitors in cultured explants or by knocking out Prkg1, also resulted in reduced SGN fibers. Together, our results suggest that EphA7 plays an important role in the developmental formation of cochlear innervation pattern through controlling SGN fiber ontogeny. Such regulation may contribute to the salience level of auditory signals presented to the central auditory system.

Functional response properties of VIP-expressing inhibitory neurons in mouse visual and auditory cortex.

Despite accounting for about 20% of all the layer 2/3 inhibitory interneurons, the vasoactive intestinal polypeptide (VIP) expressing neurons remain the least thoroughly studied of the major inhibitory subtypes. In recent studies, VIP neurons have been shown to be activated by a variety of cortico-cortical and neuromodulatory inputs, but their basic sensory response properties remain poorly characterized. We set out to explore the functional properties of layer 2/3 VIP neurons in the primary visual (V1) and primary auditory cortex (A1), using two-photon imaging guided patch recordings. We found that in the V1, VIP neurons were generally broadly tuned, with their sensory response properties resembling those of parvalbumin (PV) expressing neurons. With the exception of response latency, they did not exhibit a significant difference from PV neurons across any of the properties tested, including overlap index, response modulation, orientation selectivity, and direction selectivity. In the A1, on the other hand, VIP neurons had a strong tendency to be intensity selective, which is a property associated with a subset of putative pyramidal cells and virtually absent in PV neurons. VIP neurons had a best intensity that was significantly lower than that of PV and putative pyramidal neurons. Finally, sensory evoked spike responses of VIP neurons were delayed relative to pyramidal and PV neurons in both the V1 and A1. Combined, these results demonstrate that the sensory response properties of VIP neurons do not fit a simple model of being either PV-like broadly tuned or pyramidal-like narrowly tuned. Instead, the selectivity pattern varies with sensory area and can even be, as in the case of low sound intensity responsiveness, distinct from both PV and pyramidal neurons.

Auditory cortex controls sound-driven innate defense behaviour through corticofugal projections to inferior colliculus.

Defense against environmental threats is essential for animal survival. However, the neural circuits responsible for transforming unconditioned sensory stimuli and generating defensive behaviours remain largely unclear. Here, we show that corticofugal neurons in the auditory cortex (ACx) targeting the inferior colliculus (IC) mediate an innate, sound-induced flight behaviour. Optogenetic activation of these neurons, or their projection terminals in the IC, is sufficient for initiating flight responses, while the inhibition of these projections reduces sound-induced flight responses. Corticocollicular axons monosynaptically innervate neurons in the cortex of the IC (ICx), and optogenetic activation of the projections from the ICx to the dorsal periaqueductal gray is sufficient for provoking flight behaviours. Our results suggest that ACx can both amplify innate acoustic-motor responses and directly drive flight behaviours in the absence of sound input through corticocollicular projections to ICx. Such corticofugal control may be a general feature of innate defense circuits across sensory modalities.

Linear transformation of thalamocortical input by intracortical excitation.

Neurons in thalamorecipient layers of sensory cortices integrate thalamocortical and intracortical inputs. Although we know that their functional properties can arise from the convergence of thalamic inputs, intracortical circuits could also be involved in thalamocortical transformations of sensory information. We silenced intracortical excitatory circuits with optogenetic activation of parvalbumin-positive inhibitory neurons in mouse primary visual cortex and compared visually evoked thalamocortical input with total excitation in the same layer 4 pyramidal neurons. We found that intracortical excitatory circuits preserved the orientation and direction tuning of thalamocortical excitation, with a linear amplification of thalamocortical signals of about threefold. The spatial receptive field of thalamocortical input was slightly elongated and was expanded by intracortical excitation in an approximately proportional manner. Thus, intracortical excitatory circuits faithfully reinforce the representation of thalamocortical information and may influence the size of the receptive field by recruiting additional inputs.