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Allan-Herndon-Dudley syndrome (AHDS) is a severe psychomotor retardation characterized by neurological impairment and abnormal thyroid hormone (TH) levels. Mutations in the TH transporter, monocarboxylate transporter 8 (MCT8), are associated with AHDS. MCT8 knock-out mice exhibit impaired TH levels; however, they lack neurological defects. Here, the zebrafish mct8 gene and promoter were isolated, and mct8 promoter-driven transgenic lines were used to show that, similar to humans, mct8 is primarily expressed in the nervous and vascular systems. Morpholino-based knockdown and rescue experiments revealed that MCT8 is strictly required for neural development in the brain and spinal cord. This study shows that MCT8 is a crucial regulator during embryonic development and establishes the first vertebrate model for MCT8 deficiency that exhibits a neurological phenotype.

Varicosities (VRs) are ubiquitous neuronal structures that are considered to serve as presynaptic structures. The mechanisms of their assembly are unknown. Using cultured Aplysia neurons, we found that in the absence of postsynaptic targets, VRs form at the leading edge of extending neurites when anterogradely transported organelles accumulate within the palm of the growth cone (GC) at a rate that exceeds their utilization by the GC machinery. The aggregation of excess organelles at the palm of the GC leads to slowdown of the GC's advance. As the size of the organelle clusters increases, the rate of organelle sequestration diminishes and the supply of building blocks to the GC resumes. The GCs' advance is re-initiated, “leaving behind” an organelle-loaded nascent VR. These mechanisms account for the recurrent “deposition” of almost equally spaced VRs by advancing GCs. Consistent with the view that VRs serve as “ready-to-go” presynaptic terminals, we found that a short train of action potentials leads to exocytosis of labeled vesicles within the varicosities. We propose that the formation and spacing of VRs by advancing GCs is the default outcome of the balance between the rate of supply of growth-supporting resources and the usage of these resources by the GC's machinery at the leading edges of specific neurites.

The use of the sensory–motor (SN-MN) synapse of the Aplysia gill withdrawal reflex has contributed immensely to the understanding of synaptic transmission, learning and memory acquisition processes. Whereas the majority of the studies focused on analysis of the presynaptic mechanisms, recent studies indicated that as in mammalian synapses, long term potentiation (LTP) formed by Aplysia SN-MN synapse depends on elevation of the postsynaptic free intracellular calcium concentration ([Ca 2+ ] i ). Consistently, injection of the fast calcium chelator BAPTA to the MN prevents the formation of serotonin-induced LTP. Nevertheless, currently there are no published reports that directly examine and document whether evoked synaptic transmission is associated with transient increase in the postsynaptic [Ca 2+ ] i . In the present study we imaged, for the first time, alterations in the postsynaptic [Ca 2+ ] i in response to presynaptic stimulation and analyzed the underlying mechanisms. Using live imaging of the postsynaptic [Ca 2+ ] i while monitoring the EPSP, we found that evoked transmitter release generates excitatory postsynaptic calcium concentration transients (EPSCaTs) by two mechanisms: (a) activation of DNQX-sensitive postsynaptic receptors-gated calcium influx and (b) calcium influx through nitrendipine-sensitive voltage-gated calcium channels (VGCCs). Concomitant confocal imaging of presynaptic boutons and EPSCaTs revealed that approximately 86% of the presynaptic boutons are associated with functional synapses.

The double C2 domain protein family (DOC2) is characterized by two calcium-binding domains (C2). Upon binding to calcium, the affinity of the protein to phospholipids is significantly increased, leading to translocation of the protein from the cytosol to the plasma membrane. These properties, and the binding domain of DOC2B to Munc13, suggested that DOC2B could play a role in augmentation and potentiation of synaptic release. Nevertheless, the level of the free intracellular calcium concentration ([Ca(2+)](i)) which triggers its translocation under in vivo conditions, is not known. Using cultured Aplysia neurons that express rat EGFP-DOC2B, we found that the [Ca(2+)](i) increment necessary to induce EGFP-DOC2B translocation is approximately 200 nM in the bulk of the cytoplasm. The rate of EGFP-DOC2B recruitment to the plasma membrane is slower than the [Ca(2+)](i) elevation rate, while the detachment of EGFP-DOC2B from it is faster than the calcium removal. The extent of EGFP-DOC2B translocation to the plasma membrane reflects local submembrane [Ca(2+)](i). Our observations are consistent with the view that DOC2B can participate in the regulation of neurotransmitter release. It should be noted that EGFP-DOC2B could be used as a tool to map sub-membrane calcium dynamics under physiological conditions.

Two-photon imaging of endogenous fluorescence can provide important physiological and metabolic information from intact tissues in a label-free and non-invasive way. However, imaging of multiple intrinsic fluorophores, such as NADH, FAD, retinoids and porphyrins in living systems is generally hampered by sequential multi-wavelength excitation resulting in long acquisition times and motion artifacts. We report an efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores with absorption spectra spanning the 700-1040nm range, using wavelength mixing. By using two synchronized pulse trains at two different wavelengths, an additional “virtual” two-photon excitation wavelength is generated, and simultaneous excitation of blue, green and red endogenous fluorophores is achieved. This method permits fast and reliable simultaneous imaging of the metabolic coenzymes NADH and FAD to being implemented, overcoming the difficulties associated with their difference in absorption spectra and disparity in concentration. We achieve efficient ratiometric redox imaging and simultaneous efficient two-photon fluorescence lifetime imaging (FLIM) of NADH and FAD in living tissues. Lifetime gradients of NADH and FAD associated with different cellular metabolic and differentiation states were measured in both reconstructed human skins and live C. elegans worms. Finally, we perform hyperspectral imaging of endogenous fluorophores during early zebrafish development.