Background: Calcium-binding proteins (CBPs) are instrumental in the control of Ca2+ signaling. They are the fastest players within the Ca2+ toolkit responding within microseconds to [Ca2+] changes. The CBPs compete for Ca2+ which plays a direct role in modulating Ca2+ transients and the resulting biochemical message. The kinetic properties of the CBPs have to be known to have a good understanding of Ca2+ signaling. Scope of review: Most techniques used to measure binding kinetics are too slow to accurately determine the fast kinetics of most CBP. Furthermore, many CBPs bind Ca2+ in a cooperative way, which should be incorporated in the kinetic modeling. Here we will review a new ultra-fast in vitro technique for measuring Ca2+ binding properties of CBPs following flash photolysis of caged Ca2+. Compartmental modeling is used to resolve the kinetics of fast cooperative Ca2+ binding to CBPs. Major conclusions: Currently this technique has only been used to quantify the kinetics of three CBPs (calbindin, calretinin and calmodulin), but has already provided remarkable insights into the specific role that these kinetics in Ca2+ signaling. General significance: The potential to gain novel insights into Ca2+ signaling by quantifying kinetics of other CBPs using this technique is very promising. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.

Many forms of signal transduction occur when Ca2+ enters the cytoplasm of a cell. It has been generally thought that there is a fast buffer that rapidly reduces the free Ca2+ level and that it is this buffered level of Ca2+ that triggers downstream biochemical processes, notably the activation of calmodulin (CaM) and the resulting activation of CaM-dependent enzymes. Given the importance of these transduction processes, it is crucial to understand exactly how Ca2+ activates CaM. We have determined the rate at which Ca2+ binds to CaM and found that Ca2+ binds more rapidly to CaM than to other Ca2+-binding proteins. This property of CaM and its high concentration support a new view of signal transduction: CaM directly intercepts incoming Ca2+ and sets the free Ca2+ level (that is, it strongly contributes to fast Ca2+ buffering) rather than responding to the lower Ca2+ level set by other buffers. This property is crucial for making CaM an efficient transducer. Our results also suggest that other Ca2+ binding proteins have a previously undescribed role in regulating the lifetime of Ca2+ bound to CaM and thereby setting the gain of signal transduction.

The endoribonuclease, Dicer, is indispensable for generating the majority of mature microRNAs (miRNAs), which are posttranscrip- tional regulators of gene expression involved in a wide range of developmental and pathological processes in the mammalian CNS. Although functions of Dicer-dependent miRNA pathways in neurons and oligodendrocytes have been extensively investigated, little is known about the role of Dicer in astrocytes. Here, we report the effect of Cre-loxP-mediated conditional deletion of Dicer selectively from postnatal astroglia on brain development. Dicer-deficient mice exhibited normal motor development and neurological morphology before postnatal week 5. Thereafter, mutant mice invariably developed a rapidly fulminant neurological decline characterized by ataxia, severe progressive cerebellar degeneration, seizures, uncontrollable movements, and premature death by postnatal week 9 –10. Inte- grated transcription profiling, histological, and functional analyses of cerebella showed that deletion of Dicer in cerebellar astrocytes altered the transcriptome of astrocytes to be more similar to an immature or reactive-like state before the onset of neurological symptoms or morphological changes. As a result, critical and mature astrocytic functions including glutamate uptake and antioxidant pathways were substantially impaired, leading to massive apoptosis of cerebellar granule cells and degeneration of Purkinje cells. Collectively, our study demonstrates the critical involvement of Dicer in normal astrocyte maturation and maintenance. Our findings also reveal non-cell- autonomous roles of astrocytic Dicer-dependent pathways in regulating proper neuronal functions and implicate that loss of or dysregu- lation of astrocytic Dicer-dependent pathways may be involved in neurodegeneration and other neurological disorders.

The nucleus accumbens (NAc) is positioned to integrate signals originating from limbic and cortical areas and to modulate reward-related motor output of various goal-directed behaviours. The major target of the NAc GABAergic output neurons is the ventral pallidum (VP). VP is part of the reward circuit and controls the ascending mesolimbic dopamine system, as well as the motor output structures and the brainstem. The excitatory inputs gov- erning this system converge in the NAc from the prefrontal cortex (PFC), ventral hippocampus (vHC), midline and intralaminar thalamus (TH) and basolateral nucleus of the amygdala (BLA). It is unclear which if any of these affer- ents innervate the medium spiny neurons of the NAc, that project to the VP. To identify the source of glutamatergic afferents that innervate neurons projecting to the VP, a dual- labelling method was used: Phaseolus vulgaris leucoag- glutinin for anterograde and EGFP-encoded adenovirus for retrograde tract-tracing. Within the NAc, anterogradely labelled BLA terminals formed asymmetric synapses on dendritic spines that belonged to medium spiny neurons retrogradely labelled from the VP. TH terminals also formed synapses on dendritic spines of NAc neurons pro- jecting to the VP. However, dendrites and dendritic spines retrogradely labelled from VP received no direct synaptic contacts from afferents originating from mPFC and vHC in the present material, despite the large number of fibres labelled by the anterograde tracer injections. These findings represent the first experimental evidence for a selective glutamatergic innervation of NAc neurons projecting to the VP. The glutamatergic inputs of different origin (i.e. mPFC, vHC, BLA, TH) to the NAc might thus convey different types of reward-related information during goal-directed behaviour, and thereby contribute to the complex regulation of nucleus accumbens functions.

The hippocampal formation is one of the brain regions most sensitive to ischemic damage. However, there are no studies about changes in hippocampal neuronal activity during and after a selective unilateral hippocampal ischemia. We developed a novel unilateral cerebro- vascular ischemia model in mice that selectively shuts down blood supply to the ipsilateral hippocampal formation. Using a modified version of the photothrombotic method, we stereotaxically targeted the initial ascending part of the longitudinal hippocampal artery in urethane anesthetized and rose bengal-injected mice. To block blood flow in the targeted artery, we photoactivated the rose bengal by illuminating the longitudinal hippocampal artery through an optical fiber inserted into the brain. In vivo field potential recordings in the CA1 region of the hippocampus before, during and after the induction of ischemia demonstrated a high-frequency discharge (HFD) reaching frequencies of 􏰶300 Hz and lasting 7–24 s during the illumination consistent with a massive synchronous neuronal activity. The HFD was invariably followed by a DC voltage shift and a decreased activity at both low (30 –57 Hz)- and high (63–119 Hz)-gamma frequencies. This decrease in gamma activity lasted for the entire duration of the recordings (􏰷160 min) following ischemia. The contralateral hippocampus displayed HFDs but with different frequency spectra and without DC voltage shifts or long-lasting decreases in gamma oscillations. Our findings reveal for the first time the acute effects of unilateral hippocampal ischemia on ensemble hippocam- pal neuronal activities.

The Rbfox family of RNA binding proteins regulates alternative splicing of many important neuronal transcripts, but its role in neuronal physiology is not clear1. We show here that central nervous system–specific deletion of the gene encoding Rbfox1 results in heightened susceptibility to spontaneous and kainic acid–induced seizures. Electrophysiological recording revealed a corresponding increase in neuronal excitability in the dentate gyrus of the knockout mice. Whole-transcriptome analyses identified multiple splicing changes in the Rbfox1−/− brain with few changes in overall transcript abundance. These splicing changes alter proteins that mediate synaptic transmission and membrane excitation. Thus, Rbfox1 directs a genetic program required in the prevention of neuronal hyperexcitation and seizures. The Rbfox1 knockout mice provide a new model to study the post-transcriptional regulation of synaptic function.