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GABAergic activity is important in neocortical development and plasticity. Because the maturation of GABAergic interneurons is regulated by neural activity, the source of excitatory inputs to GABAergic interneurons plays a key role in development. We show, by laser-scanning photostimulation, that layer 4 and layer 5 GABAergic interneurons in the auditory cortex in neonatal mice (

Autism spectrum disorder (ASD) involves deficits in speech and sound processing. Cortical circuit changes during early development likely contribute to such deficits. Subplate neurons (SPNs) form the earliest cortical microcircuits and are required for normal development of thalamocortical and intracortical circuits. Prenatal valproic acid (VPA) increases ASD risk, especially when present during a critical time window coinciding with SPN genesis. Using optical circuit mapping in mouse auditory cortex, we find that VPA exposure on E12 altered the functional excitatory and inhibitory connectivity of SPNs. Circuit changes manifested as “patches” of mostly increased connection probability or strength in the first postnatal week and as general hyper-connectivity after P10, shortly after ear opening. These results suggest that prenatal VPA exposure severely affects the developmental trajectory of cortical circuits and that sensory-driven activity may exacerbate earlier, subtle connectivity deficits. Our findings identify the subplate as a possible common pathophysiological substrate of deficits in ASD.

Lithium carbonate, a drug for the treatment of bipolar disorder, provides mood stability to mitigate recurrent episodes of mania and/or depression. Despite its long-term and widespread use, the mechanism by which lithium acts to elicit these psychological changes has remained unknown. Using nuclear magnetic resonance (NMR) methods, in this study we characterized the association of lithium with adenosine triphosphate (ATP) and identified a bimetallic (Mg·Li) ATP complex. Lithium’s affinity to form this complex was found to be relatively high (Kd∼1.6 mM) compared with other monovalent cations and relevant, considering lithium dosing and physiological concentrations of Mg2+and ATP. The ATP·Mg·Li complex reveals, for the first time, to the best of our knowledge, that lithium can associate with magnesium-bound phosphate sites and then act to modulate purine receptor activity in neuronal cells, suggesting a molecular mode for in vivo lithium action.

Loss of a sensory modality can lead to functional enhancement of the remaining senses. For example, short-term visual deprivations, or dark exposure (DE), can enhance neuronal responses in the auditory cortex to sounds. These enhancements encompass increased spiking rates and frequency selectivity as well as increased spiking reliability. Although we previously demonstrated enhanced thalamocortical transmission after DE, increased synaptic strength cannot account for increased frequency selectivity or reliability. We thus investigated whether other changes in the underlying circuitry contributed to improved neuronal responses. We show that DE can lead to refinement of intra- and inter-laminar connections in the mouse auditory cortex. Moreover, we use a computational model to show that the combination of increased transmission and circuit refinement can lead to increased firing reliability. Thus cross-modal influences can alter the spectral and temporal processing of sensory stimuli by refinement of thalamocortical and intracortical circuits.

Existing theory suggests that mitochondria act as significant, dynamic buffers of cytosolic calcium ([Ca2+]i) in heart. These buffers can remove up to one-third of the Ca2+that enters the cytosol during the [Ca2+]itransients that underlie contractions. However, few quantitative experiments have been presented to test this hypothesis. Here, we investigate the influence of Ca2+movement across the inner mitochondrial membrane during both subcellular and global cellular cytosolic Ca2+signals (i.e., Ca2+sparks and [Ca2+]itransients, respectively) in isolated rat cardiomyocytes. By rapidly turning off the mitochondria using depolarization of the inner mitochondrial membrane potential (ΔΨm), the role of the mitochondria in buffering cytosolic Ca2+signals was investigated. We show here that rapid loss of ΔΨmleads to no significant changes in cytosolic Ca2+signals. Second, we make direct measurements of mitochondrial [Ca2+] ([Ca2+]m) using a mitochondrially targeted Ca2+probe (MityCam) and these data suggest that [Ca2+]mis near the [Ca2+]ilevel (∼100 nM) under quiescent conditions. These two findings indicate that although the mitochondrial matrix is fully buffer-capable under quiescent conditions, it does not function as a significant dynamic buffer during physiological Ca2+signaling. Finally, quantitative analysis using a computational model of mitochondrial Ca2+cycling suggests that mitochondrial Ca2+uptake would need to be at least ∼100-fold greater than the current estimates of Ca2+influx for mitochondria to influence measurably cytosolic [Ca2+] signals under physiological conditions. Combined, these experiments and computational investigations show that mitochondrial Ca2+uptake does not significantly alter cytosolic Ca2+signals under normal conditions and indicates that mitochondria do not act as important dynamic buffers of [Ca2+]iunder physiological conditions in heart.

We report the synthesis and characterization of O[o-nitromandelyloxycarbonyl]-2,5-di(tert-butyl)hydroquinone (Nmoc-DBHQ), a new “caged” reagent for photoreleasing DBHQ, a membrane-permeant, reversible inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ATPase (SERCA). The Nmoc group is a new caging group developed for the current application. Photolysis of Nmoc-DBHQ proceeds with t1/2 = 126 ± 2 μs, and t1/2 for subsequent release of DBHQ is estimated to be ∼5 ms. Nmoc-DBHQ thus allows rapid and reversible modulation of SERCA activity in living cells. Through its acetoxymethyl ester, Nmoc-DBHQ can be loaded into cells easily by incubation. We demonstrate the use of Nmoc-DBHQ for photomodulating SERCA activity in fibroblasts and vagal sensory neurons. We further demonstrate the utility of pulsed DBHQ photorelease for probing and manipulating dynamic phenomena such as [Ca2+] oscillations in fibroblasts.

We report the synthesis, the physicochemical characterization, and biological evaluation of a new caged glutamate,N-(o-nitromandelyl)oxycarbonyl-l-glutamic acid (Nmoc-Glu), that liberates free glutamate on photolysis. The low affinity of certain glutamate receptors and their rapid entry into desensitization have effectively prevented the creation of an ideal caged glutamate. In the absence of an ideal compound, Nmoc-Glu was designed to resist spontaneous hydrolysis while maintaining reasonable photorelease yield and kinetics. Chemical and physiological analyses reveal that Nmoc-Glu, indeed, has exceptionally low residual activity and high chemical stability. The quantum yield of Nmoc-Glu is 0.11. Photolytic uncaging and release of free glutamate occur in two steps, consisting of an initial light-induced cleavage that proceeds on the sub-millisecond time scale, and a subsequent light-independent, pH-dependent decarboxylation step that proceeds on the millisecond time scale. The low residual activity and high chemical stability of Nmoc-Glu are important advantages in applications where pre-photolysis Glu receptor activation and desensitization must be minimized.

A caged molecule is an inert but photosensitive molecule that is transformed by photolysis into a biologically active molecule at high speed (typically 1 msec). The process is referred to as photorelease. The spatial resolution of photorelease is limited by the properties of light; submicrometer resolution is potentially achievable. Therefore, focal photorelease of caged molecules enables one to control biological processes with high spatio‐temporal precision. The principles underlying caged molecules as well as practical considerations for their use are discussed in this unit.