The mammalian brain is an enormously complex set of circuits composed of interconnected neuronal cell types. The analysis of central neural circuits will be greatly served by the ability to turn off specific neuronal cell types while recording from others in intact brains. Because drug delivery cannot be restricted to specific cell types, this can only be achieved by putting "silencer" transgenes under the control of neuron-specific promoters. Towards this end we have created a line of transgenic mice putting the Drosophila allatostatin (AL) neuropeptide receptor (AlstR) under the control of the tetO element, thus enabling its inducible expression when crossed to tet-transactivator lines. Mammals have no endogenous AL or AlstR, but activation of exogenously expressed AlstR in mammalian neurons leads to membrane hyperpolarization via endogenous G-protein-coupled inward rectifier K(+) channels, making the neurons much less likely to fire action potentials. Here we show that this tetO/AlstR line is capable of broadly expressing AlstR mRNA in principal neurons throughout the forebrain when crossed to a commercially-available transactivator line. We electrophysiologically characterize this cross in hippocampal slices, demonstrating that bath application of AL leads to hyperpolarization of CA1 pyramidal neurons, making them refractory to the induction of action potentials by injected current. Finally, we demonstrate the ability of AL application to silence the sound-evoked spiking responses of auditory cortical neurons in intact brains of AlstR/tetO transgenic mice. When crossed to other transactivator lines expressing in defined neuronal cell types, this AlstR/tetO line should prove a very useful tool for the analysis of intact central neural circuits.

In vitro studies suggest that the intracellular C-terminus of Neuroligin1 (NL1) could play a central role in the maturation of excitatory synapses. However, it is unknown how this activity affects synapses in vivo, and whether it may impact the development of complex behaviors. To determine how NL1 influences the state of glutamatergic synapses in vivo, we compared the synaptic and behavioral phenotypes of mice overexpressing a full length version of NL1 (NL1FL) with mice overexpressing a version missing part of the intracellular domain (NL1ΔC). We show that overexpression of full length NL1 yielded an increase in the proportion of synapses with mature characteristics and impaired learning and flexibility. In contrast, the overexpression of NL1ΔC increased the number of excitatory postsynaptic structures and led to enhanced flexibility in mnemonic and social behaviors. Transient overexpression of NL1FL revealed that elevated levels are not necessary to maintain synaptic and behavioral states altered earlier in development. In contrast, overexpression of NL1FL in the fully mature adult was able to impair normal learning behavior after one month of expression. These results provide the first evidence that NL1 significantly impacts key developmental processes that permanently shape circuit function and behavior, as well as the function of fully developed neural circuits. Overall, these manipulations of NL1 function illuminate the significance of NL1 intracellular signaling in vivo, and enhance our understanding of the factors that gate the maturation of glutamatergic synapses and complex behavior. This has significant implications for our ability to address disorders such as ASD.

The craniotomy is a commonly performed procedure to expose the brain for in vivo experiments. In mouse research, most labs utilize a small craniotomy, typically 3 mm x 3 mm. This protocol introduces a method for creating a substantially larger 7 mm x 6 mm cranial window exposing most of a cerebral hemisphere over the mouse temporal and parietal cortices (e.g., bregma 2.5 - 4.5 mm, lateral 0 - 6 mm). To perform this surgery, the head must be tilted approximately 30° and much of the temporal muscle must be retracted. Due to the large amount of bone removal, this procedure is intended only for acute experiments with the animal anesthetized throughout the surgery and experiment. The main advantage of this innovative large lateral cranial window is to provide simultaneous access to both medial and lateral areas of the cortex. This large unilateral cranial window can be used to study the neural dynamics between cells, as well as between different cortical areas by combining multi-electrode electrophysiological recordings, imaging of neuronal activity (e.g., intrinsic or extrinsic imaging), and optogenetic stimulation. Additionally, this large craniotomy also exposes a large area of cortical blood vessels, allowing for direct manipulation of the lateral cortical vasculature.

The interaural level difference (ILD) is a sound localization cue that is extensively processed in the auditory brain stem and midbrain and is also represented in the auditory cortex. Here, we asked whether neurons in the auditory cortex passively inherit their ILD tuning from subcortical sources or whether their spiking preferences were actively shaped by local inhibition. If inherited, the ILD selectivity of spiking output should match that of excitatory synaptic input. If shaped by local inhibition, by contrast, excitation should be more broadly tuned than spiking output with inhibition suppressing spiking for nonpreferred stimuli. To distinguish between these two processing strategies, we compared spiking responses with excitation and inhibition in the same neurons across a range of ILDs and average binaural sound levels. We found that cells preferring contralateral ILDs (often called EI cells) followed the inheritance strategy. In contrast, cells that were unresponsive to monaural sounds but responded predominantly to near-zero ILDs (PB cells) instead showed evidence of the local processing strategy. These PB cells received excitatory inputs that were similar to those received by the EI cells. However, contralateral monaural sounds and ILDs >0 dB elicited strong inhibition, quenching the spiking output. These results suggest that in the rat auditory cortex, EI cells do not utilize inhibition to shape ILD sensitivity, whereas PB cells do. We conclude that an auditory cortical circuit computes sensitivity for near-zero ILDs.

How does the brain accomplish sound localization with invariance to total sound level? Sensitivity to interaural level differences (ILDs) is first computed at the lateral superior olive (LSO) and is observed at multiple levels of the auditory pathway, including the central nucleus of inferior colliculus (ICC) and auditory cortex. In LSO, this ILD sensitivity is level-dependent, such that ILD response functions shift toward the ipsilateral (excitatory) ear with increasing sound level. Thus early in the processing pathway changes in firing rate could indicate changes in sound location, sound level, or both. In ICC, while ILD responses can shift toward either ear in individual neurons, there is no net ILD response shift at the population level. In behavioral studies of human sound localization acuity, ILD sensitivity is invariant to increasing sound levels. Level-invariant sound localization would suggest transformation in level sensitivity between LSO and perception of sound sources. Whether this transformation is completed at the level of the ICC or continued at higher levels remains unclear. It also remains unknown whether perceptual sound localization is level-invariant in rats, as it is in humans. We asked whether ILD sensitivity is level-invariant in rat auditory cortex. We performed single-unit and whole cell recordings in rat auditory cortex under ketamine anesthesia and measured responses to white noise bursts presented through sealed earphones at a range of ILDs. Surprisingly, we found that with increasing sound levels ILD responses shifted toward the ipsilateral ear (which is typically inhibitory), regardless of whether cells preferred ipsilateral, contralateral, or binaural stimuli. Voltage-clamp recordings suggest that synaptic inhibition does not contribute substantially to this transformation in level sensitivity. We conclude that the level invariance of ILD sensitivity seen in behavioral studies is not present in rat auditory cortex.