Search tips
Search criteria

Results 1-8 (8)

Clipboard (0)

Select a Filter Below

more »
Year of Publication
Document Types
1.  Persistence of excitatory shaft synapses adjacent to newly emerged dendritic protrusions 
In the early postnatal hippocampus, the first synapses to appear on excitatory pyramidal neurons are formed directly on dendritic shafts. Very few dendritic spines are present at this time. By adulthood, however, the overwhelming majority of synapses are located at the tips of dendritic spines. Several models have been proposed to account for the transition from mostly shaft to mostly spinous synapses but none have been demonstrated conclusively. To investigate the cellular mechanism underlying the shaft-to-spinous synapse transition, we designed live imaging experiments to directly observe the dynamics of shaft and spinous synapses on developing dendrites. Immunofluorescent synaptic labeling of GFP-filled neurons showed that the shaft-to-spinous synapse transition in dissociated culture mirrors that in vivo. Along with electron microscopy, the fluorescent labeling also showed that veritable shaft synapses are abundant in dissociated culture and that shaft synapses are frequently adjacent to spines or other dendritic protrusions, a configuration previously observed in vivo by others. We used live long term time lapse confocal microscopy of GFP-filled dendrites and VAMP2-DsRed-labeled boutons to record the fate of shaft synapses and associated dendritic protrusions and boutons with images taken hourly for up to 31 continuous hours. Inspection of the time lapse imaging series revealed that shaft synapses can persist adjacent to either existing or newly grown dendritic protrusions. Alternatively, a shaft synapse bouton can redistribute to contact an adjacent dendritic protrusion. However, we never observed shaft synapses transforming themselves into spines or any type of dendritic protrusions. We conclude that repeated iterations of dendritic protrusion or spine outgrowth adjacent to shaft synapses is very likely to be a critical component of the shaft-to-spinous synapse transition during CNS development.
PMCID: PMC3171181  PMID: 21784157
synaptogenesis; spinogenesis; synaptic plasticity; correlative light and electron microscopy (CLEM); laser scanning confocal microscopy (LSCM)
2.  A variable cytoplasmic domain segment is necessary for γ-protocadherin trafficking and tubulation in the endosome/lysosome pathway 
Molecular Biology of the Cell  2011;22(22):4362-4372.
The variable portion of the γ-protocadherin (Pcdh-γ) cytoplasmic domain (VCD) controls Pcdh-γ trafficking and organelle tubulation in the endolysosome system. Active VCD segments are conserved in Pcdh-γA and Pcdh-γB subfamilies.
Clustered protocadherins (Pcdhs) are arranged in gene clusters (α, β, and γ) with variable and constant exons. Variable exons encode cadherin and transmembrane domains and ∼90 cytoplasmic residues. The 14 Pcdh-αs and 22 Pcdh-γs are spliced to constant exons, which, for Pcdh-γs, encode ∼120 residues of an identical cytoplasmic moiety. Pcdh-γs participate in cell–cell interactions but are prominently intracellular in vivo, and mice with disrupted Pcdh-γ genes exhibit increased neuronal cell death, suggesting nonconventional roles. Most attention in terms of Pcdh-γ intracellular interactions has focused on the constant domain. We show that the variable cytoplasmic domain (VCD) is required for trafficking and organelle tubulation in the endolysosome system. Deletion of the constant cytoplasmic domain preserved the late endosomal/lysosomal trafficking and organelle tubulation observed for the intact molecule, whereas deletion or excision of the VCD or replacement of the Pcdh-γA3 cytoplasmic domain with that from Pcdh-α1 or N-cadherin dramatically altered trafficking. Truncations or internal deletions within the VCD defined a 26–amino acid segment required for trafficking and tubulation in the endolysosomal pathway. This active VCD segment contains residues that are conserved in Pcdh-γA and Pcdh-γB subfamilies. Thus the VCDs of Pcdh-γs mediate interactions critical for Pcdh-γ trafficking.
PMCID: PMC3216661  PMID: 21917590
3.  Streamlined embedding of cell monolayers on gridded glass-bottom imaging dishes for correlative light and electron microscopy 
Correlative light and electron microscopy (CLEM) has facilitated study of intracellular trafficking. Routine application of CLEM would be advantageous for many laboratories but previously described techniques are particularly demanding, even for those with access to laser scanning confocal microscopy (LSCM) and transmission electron microscopy (TEM). We describe streamlined methods for TEM of GFP-labeled organelles after imaging by LSCM using gridded glass bottom imaging dishes. GFP-MAP 1A/1B LC3 (GFP-LC3) transfected cells were treated with rapamycin, fixed and imaged by LSCM. Confocal image stacks were acquired enabling full visualization of each GFP-LC3 labeled organelle. After LSCM, cells were embedded for TEM using a simplified two step method that stabilizes the glass bottom such that the block can be separated from the glass by mild heating. All imaging and TEM processing are performed in the same dish. The LSCM imaged cells were relocated on the block and serial sectioned. Correlation of LSCM, DIC and TEM images was facilitated by cellular landmarks. All GFP labeled structures were successfully reidentified and imaged by serial section TEM. This method could make CLEM more accessible to non-specialized laboratories with basic EM expertise and could be used routinely to confirm organelle localization of fluorescent puncta.
PMCID: PMC2995264  PMID: 20961484
organelle; green fluorescent protein; live imaging; autophagosome; vesicle; trafficking
4.  Characterization of MSB Synapses in Dissociated Hippocampal Culture with Simultaneous Pre- and Postsynaptic Live Microscopy 
PLoS ONE  2011;6(10):e26478.
Multisynaptic boutons (MSBs) are presynaptic boutons in contact with multiple postsynaptic partners. Although MSB synapses have been studied with static imaging techniques such as electron microscopy (EM), the dynamics of individual MSB synapses have not been directly evaluated. It is known that the number of MSB synapses increases with synaptogenesis and plasticity but the formation, behavior, and fate of individual MSB synapses remains largely unknown. To address this, we developed a means of live imaging MSB synapses to observe them directly over time. With time lapse confocal microscopy of GFP-filled dendrites in contact with VAMP2-DsRed-labeled boutons, we recorded both MSBs and their contacting spines hourly over 15 or more hours. Our live microscopy showed that, compared to spines contacting single synaptic boutons (SSBs), MSB-contacting spines exhibit elevated dynamic behavior. These results are consistent with the idea that MSBs serve as intermediates in synaptic development and plasticity.
PMCID: PMC3197663  PMID: 22028887
5.  The Effects of Nitrous Oxide Administration in the Healthy Elderly: N2O Elimination and Alveolar CO2 
Anesthesia Progress  1983;30(6):187-192.
Healthy young and elderly males were administered sedative concentrations of nitrous oxide/oxygen (N2O/O2) under a protocol designed to mimic that used in a dental operatory. Samples of end-tidal expired gas were taken at the end of 30-minutes inhalation of, and periodically for 70 minutes after withdrawal from, nitrous oxide/oxygen. Samples were analyzed to monitor the decline of alveolar nitrous oxide levels and any changes in alveolar carbon dioxide levels, to determine if there were any age-related differences. The fall in alveolar N2O following cessation of administration was rapid, and in a double-exponental manner as was expected. No age-related difference in N2O decline was observed. Alveolar carbon dioxide (CO2) levels were lower and more variable in the elderly group. Both groups exhibited elevated CO2 levels at the end of the N2O period, and an unexplained rise in CO2 at approximately 30 min post N2O.
PMCID: PMC2235781  PMID: 6424516
6.  The Effect of Nitrous Oxide and Age on Psychological and Psychomotor Performance 
Anesthesia Progress  1984;31(2):64-69.
Healthy older and younger males were compared on several psychomotor and cognitive measures before, during, and after N2O inhalation. Age did not appear to be a significant factor in determining response to N2O though on several measures younger subjects are superior to older under all conditions. N2O had a significant effect only on reaction time and facial recognition tasks. The relation of these findings to previous work is discussed.
PMCID: PMC2515534  PMID: 6597685
7.  Effect of Age on the Digit Blood Flow Response to Sedative Concentrations of Nitrous Oxide 
Anesthesia Progress  1984;31(1):17-22.
Twenty healthy male subjects [11 young, x̄ = 25.4 ± 0.8 (SEM) years old; 9 elderly, x̄ = 64.5 ± 0.7 years] volunteered for a study designed to investigate the effect of age on several cardiovascular parameters to inhaled N2O-O2. The protocol was designed to mimic the administration of N2O-O2 for sedation in the dental office, although no dental treatment was performed. Clinical criteria were used to judge the appropriate sedative level for each subject; no attempt was made to establish doseresponse relationships. Digit blood flow was measured by strain-gauge plethysmography, and heart rate, arterial blood pressure, respiratory rate, and skin temperature were monitored and recorded. N2O and CO2 levels were monitored in end-tidal gas samples by gas chromatography; machine gauge readings were calibrated against known gas mixtures by the same technique.
Under the conditions of this experiment both healthy young and healthy elderly subjects experienced a marked (200-300%) increase in digit blood flow during N2O inhalation, compared to that during air and 100% O2 inhalation. There was no significant difference in the degree of flow increase between young and elderly subjects. Also, there were no significant differences in the response of these healthy young and healthy elderly subjects to sedative concentrations of N2O with regard to heart rate, arterial blood pressure, respiratory rate, skin temperature, or mean end-tidal CO2 levels. The data indicate that N2O, in the concentrations routinely administered in the dental office for sedation, does not have a differential effect on the measured parameters in healthy elderly and healthy young males.
PMCID: PMC2235798  PMID: 6587798
8.  Monitoring Microvascular Reactivity: (II) - Short-Term Effect of Nitrous-Oxide on the Peripheral Microcirculation in Humans 
Anesthesia Progress  1980;27(4):125-130.
A study was carried out to monitor directly the short-term effects of N2O/O2 inhalation-sedation on the peripheral microcirculation in healthy human subjects in a simulated dental setting. The inhalation of 40% N2O and 60% O2 led to a small but significant mean percent decrease (-6.89 ± 1.2 SEM) in relative vessel diameter in the nailfold capillaries, for the entire N2O-breathing period. Individual vessels showed a marked constriction (-31.7% ± 4.4 SEM, mean maximal decrease); the time to this maximum constriction varied with the individual vessels. This constriction could not be attributed to changes in oxygen or carbon dioxide levels in the alveolar gas, or to changes in heart rate, systolic blood pressure, respiratory rate or temperature. Further studies are indicated to determine whether this effect is sustained with long term administration, and whether it is clinically significant, especially in those patients with acquired cardiac disease or those taking vasoactive drugs.
PMCID: PMC2235718  PMID: 19598579

Results 1-8 (8)