Targeted pruning of a neuron's dendritic tree via femtosecond laser dendrotomy.

Neurons are classified according to action potential firing in response to current injection. While such firing patterns are shaped by the composition and distribution of ion channels, modelling studies suggest that the geometry of dendritic branches also influences temporal firing patterns. Verifying this link is crucial to understanding how neurons transform their inputs to output but has so far been technically challenging. Here, we investigate branching-dependent firing by pruning the dendritic tree of pyramidal neurons. We use a focused ultrafast laser to achieve highly localized and minimally invasive cutting of dendrites, thus keeping the rest of the dendritic tree intact and the neuron functional. We verify successful dendrotomy via two-photon uncaging of neurotransmitters before and after dendrotomy at sites around the cut region and via biocytin staining. Our results show that significantly altering the dendritic arborisation, such as by severing the apical trunk, enhances excitability in layer V cortical pyramidal neurons as predicted by simulations. This method may be applied to the analysis of specific relationships between dendritic structure and neuronal function. The capacity to dynamically manipulate dendritic topology or isolate inputs from various dendritic domains can provide a fresh perspective on the roles they play in shaping neuronal output.

Four-dimensional multi-site photolysis of caged neurotransmitters.

Neurons receive thousands of synaptic inputs that are distributed in space and time. The systematic study of how neurons process these inputs requires a technique to stimulate multiple yet highly targeted points of interest along the neuron's dendritic tree. Three-dimensional multi-focal patterns produced via holographic projection combined with two-photon photolysis of caged compounds can provide for highly localized release of neurotransmitters within each diffraction-limited focus, and in this way emulate simultaneous synaptic inputs to the neuron. However, this technique so far cannot achieve time-dependent stimulation patterns due to fundamental limitations of the hologram-encoding device and other factors that affect the consistency of controlled synaptic stimulation. Here, we report an advanced technique that enables the design and application of arbitrary spatio-temporal photostimulation patterns that resemble physiological synaptic inputs. By combining holographic projection with a programmable high-speed light-switching array, we have overcome temporal limitations with holographic projection, allowing us to mimic distributed activation of synaptic inputs leading to action potential generation. Our experiments uniquely demonstrate multi-site two-photon glutamate uncaging in three dimensions with submillisecond temporal resolution. Implementing this approach opens up new prospects for studying neuronal synaptic integration in four dimensions.

Demonstration of on-sky calibration of astronomical spectra using a 25 GHz near-IR laser frequency comb.

We describe and characterize a 25 GHz laser frequency comb based on a cavity-filtered erbium fiber mode-locked laser. The comb provides a uniform array of optical frequencies spanning 1450 nm to 1700 nm, and is stabilized by use of a global positioning system referenced atomic clock. This comb was deployed at the 9.2 m Hobby-Eberly telescope at the McDonald Observatory where it was used as a radial velocity calibration source for the fiber-fed Pathfinder near-infrared spectrograph. Stellar targets were observed in three echelle orders over four nights, and radial velocity precision of ∼10 m/s (∼6 MHz) was achieved from the comb-calibrated spectra.

Simultaneous multi-site two-photon photostimulation in three dimensions.

We demonstrate simultaneous multi-site two-photon photolysis of caged neurotransmitters with close to diffraction-limited resolution in all three dimensions (3D). We use holographic projection of multiple focal spots, which allows full control over the 3D positions of uncaging sites with a high degree of localized excitation. Our system incorporates a two-photon imaging setup to visualize the 3D morphology of the neurons in order to accurately determine the photostimulation sites. We show its application to studies of synaptic integration by performing simultaneous and controlled glutamate delivery at multiple locations on dendritic trees.

Effect of growth hormone on body composition and visceral adiposity in middle-aged men with visceral obesity.

RATIONALE: GH replacement in GH-deficient adults results in an improvement in metabolic status. GH might also decrease visceral adiposity in obese adults that are not GH deficient. OBJECTIVE: Our objective was to determine the effects of supraphysiological GH therapy on the metabolic syndrome and visceral adiposity in men with low blood levels of IGF-I and the durability of these effects after stopping GH therapy. DESIGN: The study was a double-blind, placebo-controlled 6-month intervention trial followed by a blinded follow-up period of 6 months. SUBJECTS: Thirty nondiabetic middle-aged men with central adiposity (body mass index > 27 kg/m(2); waist circumference > 102 cm) participated. RESULTS: After 6 months of GH therapy, we observed an increase in weight and lean body mass (2.5 +/- 0.6 kg, P < 0.05 compared with baseline and placebo) and 8.8% reduction in visceral adiposity. GH increased resting energy expenditure by 172.5 +/- 41.6 kcal/24 h after 6 months of therapy. Fasting insulin, glucose, and the quantitative insulin sensitivity check index for insulin resistance increased during GH therapy. The effects of GH on fatness and visceral adiposity disappeared shortly after GH withdrawal, but weight remained increased over baseline and when compared with the placebo group (P < 0.05). CONCLUSION: These data suggest that GH therapy is associated with small but statistically significant decreases in visceral adiposity and an increase in lean mass and body weight. In viscerally obese subjects, supraphysiological GH administration is not an effective treatment; however, additional studies are needed to evaluate the effects of low-dose, physiological GH treatment.

Cortical efferent control of subcortical sensory neurons by synaptic disinhibition.

A long-standing hypothesis predicts that pyramidal neurons of the cerebral cortex control the influx of sensory information at the level of primary sensory representations areas. Yet little is known about the cellular mechanisms governing selective attention to behaviorally relevant objects in space. Neurons in the superficial layers of the superior colliculus are notably involved in this process, and they are directly targeted by retinal and cortical afferents. To study long-term and short-term effects of the visual cortex (VC) on subcortical visual neurons we established an in vitro model of the developing cortico-tectal projection. To this end, cortical explants expressing Green Fluorescent Protein were allowed to form connections with non-labeled dissociated tectal neurons. The presence of VC explants led to an enhancement of tectal activity by 2 mechanisms. First, glutamatergic input was increased. Second, intrinsic GABAergic inhibition was suppressed. The latter effect was shown to be acute and mediated through postsynaptic metabotropic glutamate receptor activation, G-protein acitivity, and endocannabinoid receptor activation. The VC-induced disinhibition was readily reversed by application of an mGluR antagonist. However, high-frequency activation of the glutamatergic cortico-tectal input turned the labile disinhibition into a persistent suppression of inhibition.

Spatial segregation of neuronal calcium signals encodes different forms of LTP in rat hippocampus.

Calcium regulates numerous processes in the brain. How one signal can coordinate so many diverse actions, even within the same neurone, is the subject of intense investigation. Here we have used two-photon calcium imaging to determine the mechanism that enables calcium to selectively and appropriately induce different forms of long-term potentiation (LTP) in rat hippocampus. Short-lasting LTP (LTP 1) required activation of ryanodine receptors (RyRs), which selectively increased calcium in synaptic spines. LTP of intermediate duration (LTP 2) was dependent on activation of inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) and subsequent calcium release specifically in dendrites. Long-lasting LTP (LTP 3) was selectively dependent on L-type voltage-dependent calcium channels (L-VDCCs), which generated somatic calcium influx. Activation of NMDA receptors was necessary, but not sufficient, for the generation of appropriate calcium signals in spines and dendrites, and the induction of LTP 1 and LTP 2. These results suggest that the selective induction of different forms of LTP is achieved via spatial segregation of functionally distinct calcium signals.

Spatiotemporal patterns of dorsal root-evoked network activity in the neonatal rat spinal cord: optical and intracellular recordings.

Spatiotemporal patterns of dorsal root-evoked potentials were studied in transverse slices of the rat spinal cord by monitoring optical signals from a voltage-sensitive dye with multiple-photodiode optic camera. Typically, dorsal root stimulation generated two basic waveforms of voltage images: dual-component images consisting of fast, spike-like signal followed by a slow signal in the dorsal horn, and small, slow signals in the ventral horn. To qualitatively relate the optical signals to membrane potentials, whole cell recordings were combined with measurements of light absorption in the area around the soma. The slow optical signals correlated closely with subthreshold postsynaptic potentials in all regions of the cord. The spike-like component was not associated with postsynaptic action potentials, suggesting that the fast signal was generated by presynaptic action potentials. Firing in a single neuron could not be detected optically, implying that local voltage images originated from synchronously activated neuronal ensembles. Blocking glutamatergic synaptic transmission inhibited excitatory postsynaptic potentials (EPSPs) and significantly reduced the slow optical signals, indicating that they were mediated by glutamatergic synapses. Suppressing glycine-mediated inhibition increased the amplitude of both optical signals and EPSPs, while blocking GABA(A) receptor-mediated synapses, increased the amplitude and time course of EPSPs and prolonged the duration of voltage images in larger areas of the slice. The close correlation between evoked EPSPs and their respective local voltage images shows the advantage of the high temporal resolution optical system in measuring both the spatiotemporal dynamics of segmental network excitation and integrated potentials of neuronal ensembles at identified sites.

Quantal analysis based on density estimation.

When direct measurements of the quantal parameters for a synapse cannot be made, these parameters can be extracted from an analysis of the fluctuations in the evoked response at that synapse. In this article, a decision tree is described in which the ability of the data to match simple models of quantal transmission is rigorously compared with its ability to fit progressively more complex models. The Wilks statistic is the basis for this comparison. The procedure commences with optimal transformation of peak amplitude measurements into a probability density function (PDF). It then examines this PDF against various models of transmission, commencing with a multi-modal but non-quantal distribution, then to a multi-modal distribution with quantal peak separation with and without quantal variability, and, finally, the constraints of uniform and non-uniform release probabilities are imposed. These procedures are illustrated by example. A comparison is made between the relative sensitivities of the Wilks statistic and the chi2 goodness-of-fit criteria in rejecting inappropriate models at all stages in these procedures.

Calcium dynamics, buffering, and buffer saturation in the boutons of dentate granule-cell axons in the hilus.

The axons of dentate gyrus granule cells form synapses in the hilus. Ca(2+) signaling was investigated in the boutons of these axons using confocal fluorescence imaging. Boutons were loaded with various concentrations of the Ca(2+) indicator Oregon Green BAPTA-1 by patch-clamping the cell bodies and allowing the dye to diffuse into the axon. Resting free [Ca(2+)] started at 74 nm, rose to approximately 1 microm immediately after an action potential, and then decayed to rest with a time constant of 43 msec (all extrapolated to a dye concentration of zero). Action potential-induced [Ca(2+)] rises were smaller in larger boutons, consistent with a size-independent Ca(2+) channel density of 45/microm(2). Action potential-induced [Ca(2+)] changes varied with dye concentration in a manner consistent with kappa(E) approximately 20 for the ratio of endogenous buffer-bound Ca(2+) to free Ca(2+). During trains of action potentials, [Ca(2+)] increments summed supralinearly by more than that expected from dye saturation. The amount of endogenous Ca(2+) buffering declined as [Ca(2+)] rose, and this saturation indicated a buffer with a dissociation constant of approximately 500 nm and a concentration of approximately 130 microm. This is similar to the dissociation constant of calbindin-D28K, a Ca(2+)-binding protein that is abundant in dentate granule cells. Thus, calbindin-D28K is a good candidate for the Ca(2+) buffer revealed by these experiments. The saturation of endogenous buffer can generate short-term facilitation by amplifying [Ca(2+)] changes during repetitive activity. Buffer saturation may also be relevant to the presynaptic induction of long-term potentiation at synapses formed by dentate granule cells.

Different calcium sources are narrowly tuned to the induction of different forms of LTP.

The essential role of calcium in the induction of long-term potentiation (LTP) has been well established. In particular, calcium influx via the N-methyl-D-aspartate (NMDA) receptor (NMDAR) is important for LTP induction in many pathways. However, the specific roles of other calcium sources in hippocampal LTP are less clear. The aim of the present study was to determine the appropriate conditions and extent to which non-NMDAR Ca(2+) sources contribute to the induction of different forms of LTP in area CA1 of hippocampal slices. Increasing numbers of theta-burst trains (1, 4, and 8 TBS) induced LTP of increasing magnitude and persistence. Inhibition of ryanodine receptors caused inhibition of weak LTP induced by 1 TBS, but had no effect on more robust forms of LTP. Inhibition of IP3 receptors inhibited moderate LTP induced by 4 TBS, but had no effect when 1 TBS or 8 TBS were used. Inhibition of L-type voltage-dependent Ca(2+) channels inhibited strong LTP induced by 8 TBS, but had no effect on weaker forms of LTP. These results show that different Ca(2+) sources have different thresholds for activation by TBS trains. Furthermore, each Ca(2+) source appears to be tuned to the induction of a different form of LTP. Such tuning could reflect an important link between different LTP induction and maintenance mechanisms.

Synaptic transmission on the Barrier Reef.

The IUPS Satellite Symposium on Synaptic Transmission in the Central Nervous System was held on Heron Island, Australia from 3-6 September 2001.