Summary The ability to chronically monitor neuronal activity in the living brain is essential for understanding the organization and function of the nervous system. The genetically encoded green fluorescent protein based calcium sensor GCaMP provides a powerful tool for detecting calcium transients in neuronal somata, processes, and synapses that are triggered by neuronal activities. Here we report the generation and characterization of transgenic mice that express improved GCaMPs in various neuronal subpopulations under the control of the Thy1 promoter. In vitro and in vivo studies show that calcium transients induced by spontaneous and stimulus-evoked neuronal activities can be readily detected at the level of individual cells and synapses in acute brain slices, as well as chronically in awake behaving animals. These GCaMP transgenic mice allow investigation of activity patterns in defined neuronal populations in the living brain, and will greatly facilitate dissecting complex structural and functional relationships of neural networks.

Neurogenesis in the dentate gyrus occurs throughout adult mammalian life and is essential for proper hippocampal function. Early in their development, adult-born neurons express homomeric ␣7-containing nicotinic acetylcholine receptors (␣7-nAChRs) and receive direct cholinergic innervation. We show here that functional ␣7-nAChRs are necessary for normal survival, maturation, and integration of adult-born neurons in the dentate gyrus. Stereotaxic retroviral injection into the dentate gyrus of wild-type and ␣7-knock-out (␣7KO) male and female mice was used to label and birthdate adult-born neurons for morphological and electrophysiological measures; BrdU (5-bromo-2-deoxyuridine) injections were used to quantify cell survival. In ␣7KO mice, we find that adult-born neurons develop with truncated, less complex dendritic arbors and display GABAergic postsynaptic currents with immature kinetics. The neurons also have a prolonged period of GABAergic depolarization characteristic of an immature state. In this condition, they receive fewer spontaneous synaptic currents and are more prone to die during the critical period when adult-born neurons are normally integrated into behaviorally relevant networks. Even those adult-born neurons that survive the critical period retain long-term dendritic abnormalities in ␣7KO mice. Interestingly, local infection with retroviral constructs to knockdown ␣7-mRNA mimics the ␣7KO phenotype, demonstrating that the relevant ␣7-nAChR signaling is cell autonomous. The results indicate a profound role for ␣7-nAChRs in adult neurogenesis and predict that ␣7-nAChR loss will cause progressive impairment in hippocampal circuitry and function over time as fewer neurons are added to the dentate gyrus and those that are added integrate less well.

Intracellular calcium (Ca(2+)) signals are essential for several aspects of muscle development, including myofibrillogenesis-the terminal differentiation of the sarcomeric lattice. Ryanodine receptor (RyR) Ca(2+) stores must be operative during this period and contribute to the production of spontaneous global Ca(2+) transients of long duration (LDTs; mean duration approximately 80 s). In this study, high-speed confocal imaging of intracellular Ca(2+) in embryonic myocytes reveals a novel class of spontaneous Ca(2+) transient. These short duration transients (SDTs; mean duration approximately 2 s) are blocked by ryanodine, independent of extracellular Ca(2+), insensitive to changes in membrane potential, and propagate in the subsarcolemmal space. SDTs arise from RyR stores localized to the subsarcolemmal space during myofibrillogenesis. While both LDTs and SDTs occur prior to myofibrillogenesis, LDT production ceases and only SDTs persist during a period of rapid sarcomere assembly. However, eliminating SDTs during this period results in only minor myofibril disruption. On the other hand, artificial extension of LDT production completely inhibits sarcomere assembly. In conjunction with earlier work, these results suggest that LDTs have at least two roles during myofibrillogenesis-activation of sarcoplasmic regulatory cascades and regulation of gene expression. The distinct spatiotemporal patterns of LDTs versus SDTs may be utilized for differential regulation of cytosolic cascades, control of nuclear gene expression, and localized activation of assembly events at the sarcolemma.

The hippocampus is one of only two regions in the adult brain where neurons are generated in significant numbers throughout the lifetime of the animal. Numerous studies have demonstrated that these adult-born neurons are essential for optimal cognitive function with unimpaired memory formation and retrieval. The extent to which adult-born neurons survive through an early “critical period” and become integrated into functional networks has been shown to depend on the richness of stimulation they receive during these formative stages. The dentate gyrus in the hippocampus – home of the adult-born neurons – receives extensive cholinergic innervation, and newly generated neurons in the adult hippocampus express substantial numbers of both major types of neuronal nicotinic acetylcholine receptors. Early studies indicated that nicotinic signaling may be important for the development of adult-born neurons: repeated exposure to nicotine impaired their long-term survival. Recent studies with mutant mice lacking either one of the two major nicotinic receptor subtypes demonstrate that receptor loss results in fewer adult-born neurons surviving the critical period and becoming integrated into neural networks. The key nicotinic receptor mediating the largest effects is one that has a high relative permeability to calcium. In view of this feature, it may not be surprising that excessive exposure to nicotine can have detrimental effects on survival and maturation of adult-born neurons in the dentate; these same receptors appear to be key. The results pose serious challenges for therapeutic strategies targeting an individual class of nicotinic receptors for global treatment in the recipient.