Recent success in identifying gene regulatory elements in the context of recombinant adeno-associated virus vectors have enabled cell type-restricted gene expression. However, within the cerebral cortex these tools are presently limited to broad classes of neurons. To overcome this limitation, we developed a strategy that led to the identification of multiple novel enhancers to target functionally distinct neuronal subtypes. By investigating the regulatory landscape of the disease gene Scn1a, we identified enhancers that target the breadth of its expression, including two that are selective for parvalbumin and vasoactive intestinal peptide cortical interneurons. Demonstrating the functional utility of these elements, we found that the PV-specific enhancer allowed for the selective targeting and manipulation of these neurons across species, from mice to humans. Finally, we demonstrate that our selection method is generalizable to other genes and characterize four additional PV-specific enhancers with exquisite specificity for distinct regions of the brain. Altogether, these viral tools can be used for cell-type specific circuit manipulation and hold considerable promise for use in therapeutic interventions.Large-scale transcriptomic studies are rapidly revealing where and when genes associated with neuropsychiatric disease are expressed within specific cell types (1-4). Approaches for understanding and treating these disorders will require methods for targeting and manipulating specific neuronal subtypes. Thus, gaining access to these populations in non-human primates and humans has become paramount. AAVs are the method of choice for gene delivery in the nervous system but have a limited genomic payload and are not intrinsically selective for particular neuronal populations (5). We and others have identified short regulatory elements capable of restricting viral expression to broad neuronal classes. In addition, systematic enhancer discovery has been accelerated by the recent development of technologies allowing for transcriptomic and epigenetic studies at single-cell resolution (6-12). Despite these advances, the search space for enhancer selection remains enormous and to date success has been limited. To focus our enhancer selection, we chose to specifically examine the regulatory landscape of Scn1a, a gene expressed in distinct neuronal populations and whose disruption is associated with severe epilepsy (13).Combining single-cell ATAC-seq data with sequence conservation across species, we nominated ten candidate regulatory sequences in the vicinity of this gene. By thoroughly investigating each of these elements for their ability to direct viral expression, we identified three enhancers that collectively target the breadth of neuronal populations expressing Scn1a. Among these, one particular short regulatory sequence was capable of restricting viral expression to parvalbumin-expressing cortical interneurons (PV cINs). To fully assess the utility of this element beyond reporter expression, we validated it in a v...

Sound localization critically depends on detection of differences in arrival time of sounds at the two ears (acoustic delay). The fundamental mechanisms are debated, but all proposals include a process of coincidence detection and a separate source of internal delay which offsets the acoustic delay and determines neural tuning. We obtained in vivo patch clamp recordings of binaural neurons in the Mongolian gerbil, combined with pharmacological manipulations, to directly compare neuronal input to output and to separate excitation from inhibition. The results cannot be accounted for by existing models and reveal that coincidence detection is not an instantaneous process but is shaped by the interaction of intrinsic conductances with preceding synaptic activity. This interaction generates an internal delay as an intrinsic part of the process of coincidence detection. The multiplication and time-shifting stages thought to extract synchronous activity in many brain areas can thus be combined in a single operation.

The brainstem’s lateral superior olive (LSO) is thought to be crucial for localizing high-frequency sounds by coding interaural sound level differences (ILD). Its neurons weigh contralateral inhibition against ipsilateral excitation, making their firing rate a function of the azimuthal position of a sound source. Since the very first in vivo recordings, LSO principal neurons have been reported to give sustained and temporally integrating ‘chopper’ responses to sustained sounds. Neurons with transient responses were observed but largely ignored and even considered a sign of pathology. Using the Mongolian gerbil as a model system, we have obtained the first in vivo patch clamp recordings from labeled LSO neurons and find that principal LSO neurons, the most numerous projection neurons of this nucleus, only respond at sound onset and show fast membrane features suggesting an importance for timing. These results provide a new framework to interpret previously puzzling features of this circuit.

To understand a visual scene, the brain segregates figures from background by assigning borders to foreground objects. Neurons in primate visual cortex encode which object owns a border (border ownership), but the underlying circuitry is not understood. Here, we used multielectrode probes to record from border ownership-selective units in different layers in macaque visual area V4 to study the laminar organization and timing of border ownership selectivity. We find that border ownership selectivity occurs first in deep layer units, in contrast to spike latency for small stimuli in the classical receptive field. Units on the same penetration typically share the preferred side of border ownership, also across layers, similar to orientation preference. Units are often border ownership-selective for a range of border orientations, where the preferred sides of border ownership are systematically organized in visual space. Together our data reveal a columnar organization of border ownership in V4 where the earliest border ownership signals are not simply inherited from upstream areas, but computed by neurons in deep layers, and may thus be part of signals fed back to upstream cortical areas or the oculomotor system early after stimulus onset. The finding that preferred border ownership is clustered and can cover a wide range of spatially contiguous locations suggests that the asymmetric context integrated by these neurons is provided in a systematically clustered manner, possibly through corticocortical feedback and horizontal connections.

Coincidence detection by binaural neurons in the medial superior olive underlies sensitivity to interaural time difference (ITD) and interaural correlation (ρ). It is unclear whether this process is akin to a counting of individual coinciding spikes, or rather to a correlation of membrane potential waveforms resulting from converging inputs from each side. We analyzed spike trains of axons of the cat trapezoid body (TB) and auditory nerve (AN) in a binaural coincidence scheme. ITD was studied by delaying “ipsi-” vs. “contralateral” inputs; ρ was studied by using responses to different noises. We varied the number of inputs; the monaural and binaural threshold and the coincidence window duration. We examined physiological plausibility of output “spike trains” by comparing their rate and tuning to ITD and ρ to those of binaural cells. We found that multiple inputs are required to obtain a plausible output spike rate. In contrast to previous suggestions, monaural threshold almost invariably needed to exceed binaural threshold. Elevation of the binaural threshold to values larger than 2 spikes caused a drastic decrease in rate for a short coincidence window. Longer coincidence windows allowed a lower number of inputs and higher binaural thresholds, but decreased the depth of modulation. Compared to AN fibers, TB fibers allowed higher output spike rates for a low number of inputs, but also generated more monaural coincidences. We conclude that, within the parameter space explored, the temporal patterns of monaural fibers require convergence of multiple inputs to achieve physiological binaural spike rates; that monaural coincidences have to be suppressed relative to binaural ones; and that the neuron has to be sensitive to single binaural coincidences of spikes, for a number of excitatory inputs per side of 10 or less. These findings suggest that the fundamental operation in the mammalian binaural circuit is coincidence counting of single binaural input spikes.