Faculty Bibliography

High-throughput screening (HTS) of chemical libraries has become a critical tool in basic biology and drug discovery. However, its implementation and the adaptation of high-content assays to human embryonic stem cells (hESCs) have been hampered by multiple technical challenges. Here we present a strategy to adapt hESCs to HTS conditions, resulting in an assay suitable for the discovery of small molecules that drive hESC self-renewal or differentiation. Use of this new assay has led to the identification of several marketed drugs and natural compounds promoting short-term hESC maintenance and compounds directing early lineage choice during differentiation. Global gene expression analysis upon drug treatment defines known and novel pathways correlated to hESC self-renewal and differentiation. Our results demonstrate feasibility of hESC-based HTS and enhance the repertoire of chemical compounds for manipulating hESC fate. The availability of high-content assays should accelerate progress in basic and translational hESC biology

OBJECTIVE: Intraoperative identification of cerebrospinal fluid (CSF) leakage is critical in successful closure after endoscopic cranial base surgery. Intrathecal injection of fluorescein is quite useful in identifying CSF leaks. However, complications have been reported with various doses and the technique has fallen out of favor. We explored the safety of low-dose intrathecal fluorescein administered to patients undergoing endoscopic cranial base surgery. METHODS: A retrospective chart review and postoperative patient survey were performed. The nature and incidence of complications and subjective complaints were recorded in 54 patients who underwent endoscopic, endonasal approaches to the anterior cranial base and received intrathecal fluorescein after premedication with dexamethasone and diphenhydramine. RESULTS: Intraoperative CSF leak was identified with fluorescein in 46.3% of the patients and helped determine the reconstruction technique. Postoperative CSF leak occurred in 9.3% of the patients and resolved with lumbar drainage. There were no seizures. Most side effects were nonspecific, transient, and likely not caused by fluorescein including malaise (57.4%), headache (51.9%), dizziness (31.5%), or nausea/vomiting (24.1%). Three patients (5.6%) experienced persistent subjective lower extremity weakness (n = 2) and numbness (n = 2) postoperatively; however, two of them had undergone lumbar drainage. CONCLUSION: Low-dose injection of intrathecal fluorescein after premedication with steroid and antihistamine agents is generally safe. Most symptoms are nonspecific and transient, likely caused by the surgery or lumbar drainage. However, fluorescein should be administered with some caution because it may be responsible for occasional lower extremity weakness and numbness

OBJECTIVES: Reconstruction following endoscopic skull base surgery requires a high degree of success to avoid the morbidity of postoperative cerebrospinal fluid (CSF) leak. The impact on outcomes of CSF visualization with intrathecal fluorescein, however, is unknown. STUDY DESIGN: A retrospective review of patients undergoing endoscopic skull base surgery with intrathecal fluorescein. A possible correlation between intraoperative fluorescein identification and postoperative CSF leak was analyzed. RESULTS: 61 patients underwent surgery for a variety of lesions including pituitary adenoma (55.7%), encephalocele (14.8%), and meningioma (9.8%). Seven (19.4%) of the 37 patients with intraoperative fluorescein leak experienced postoperative CSF leak compared to 0 of the 24 patients who did not have intraoperative fluorescein leak (P = 0.02). All cases of CSF leak resolved with lumbar drainage alone. CONCLUSIONS: The lack of intraoperative fluorescein leakage correlates strongly with a low risk for postoperative CSF leak. This can be used to stratify the extent of skull base reconstruction required during endoscopic skull base surgeries

Do continuous subthreshold oscillations in membrane potential within an electrically coupled network depend on gap junctional coupling? For the inferior olive (IO), modeling and developmental studies suggested that the answer is yes, although physiological studies of connexin36 knock-out mice lacking electrical coupling suggested that the answer is no. Here we addressed the question differently by using a lentivirus-based vector to express, in the IO of adult rats, a single amino acid mutation of connexin36 that disrupts the intracellular trafficking of wild-type connexin36 and blocks gap junctional coupling. Confocal microscopy of green fluorescence protein-labeled dendrites revealed that the mutant connexin36 prevented wild-type connexin36 from being expressed in dendritic spines of IO neurons. Intracellular recordings from lentivirally transduced IO networks revealed that robust and continuous subthreshold oscillations require gap junctional coupling of IO neuron somata within 40 microm of one another. Topological studies indicated that the minimal coupled network for supporting such oscillations may be confined to the dendritic arbor of a single IO neuron. Occasionally, genetically uncoupled IO neurons showed transient oscillations; however, these were not sustained longer than 3 s and were 69% slower and 71% smaller than the oscillations of normal IO neurons, a finding replicated with carbenoxolone, a pharmacological antagonist of gap junctions. The experiments provided the first direct evidence that gap junctional coupling between neurons, specifically mediated by connexin36, allows a continuous network oscillation to emerge from a population of weak and episodic single-cell oscillators. The findings are discussed in the context of the importance of gap junctions for cerebellar rhythms involved in movement

Intradiploic cerebrospinal fluid (CSF) collections represent a rare complication of either head trauma or neurosurgical procedures. Their formation is thought to depend partly on violation of the meninges and the inner cortical bone abutting the neuraxis. The authors present two pediatric cases involving diploic CSF collections following neurosurgical interventions. In the first case, a CSF fistula was found within the occipital bone and the petrous portion of the temporal bone 9 years after a suboccipital craniectomy. The second case features the extremely rare occurrence of a CSF fistula within a thoracic vertebra, 9 years after a laminectomy. Both patients underwent successful surgical procedures for repair of the fistulas

The hypothesis is presented that a disruption in brain synchronization contributes to autism by destroying the coherence of brain rhythms and slowing overall cognitive processing speed. Particular focus is on the inferior olive, a precerebellar structure that is reliably disrupted in autism and which normally generates a coherent 5-13 Hz rhythmic output. New electrophysiological data reveal that the continuity of the rhythmical oscillation in membrane potential generated by inferior olive neurons requires the formation of neuronal assemblies by the connexin36 protein that mediates electrical synapses and promotes neuronal synchrony. An experiment with classical eyeblink conditioning is presented to demonstrate that the inferior olive is necessary to learn about sequences of stimuli presented at intervals in the range of 250-500 ms, but not at 700 ms, revealing that a disruption of the inferior olive slows stimulus processing speed on the time scale that is lost in autistic children. A model is presented in which the voltage oscillation generated by populations of electrically synchronized inferior olivary neurons permits the utilization of sequences of stimuli given at, or faster than, 2 per second. It is expected that the disturbance in inferior olive structure in autism disrupts the ability of inferior olive neurons to become electrically synchronized and to generate coherent rhythmic output, thereby impairing the ability to use rapid sequences of cues for the development of normal language skill. Future directions to test the hypothesis are presented

Inferior olive (IO) neurons are electrically coupled by cytosolic pores formed by the neuron-specific connexin 36 (Cx36). Electrical coupling in the IO figures prominently in current views about brain control of movement. However, a role for Cx36 in movement has been questioned and not definitively demonstrated. Previous reports have shown that embryonic deletion of the Cx36 gene resulted in almost complete loss of cytosolic and electrical coupling in the IO without an obvious deficit in movement, possibly due to developmental compensations in ionic conductances that can confound the approach of embryonic gene deletion. We used a replication-incompetent lentiviral vector to stably express a dominant-negative Cx36 mutant in the IO of adult rats. We show that interneuronal cytosolic coupling is severely reduced by the mutant Cx36, without effect on neuron morphology or electrical properties. Multisite electromyography revealed that blocking Cx36 in the IO impaired the coherence of muscle firing during harmaline tremor without affecting its rhythm. The data demonstrate that gap junction coupling within the IO mediated by Cx36 adds 10-20 ms of precision to the fine temporal coordination of muscle firing during movement

Rat connexin 36 (Cx36) was mutated by substituting serine for cysteine at residue 231 (C231S) and the mutant's effect on the subcellular localization of wild-type Cx36 and the intercellular permeability that it confers was determined in human HeLa and rat PC12 cells. Cells transfected with the mutant or wild-type Cx36 cDNA expressed the expected 36 kDa protein and Cx36 immunoreactivity. Co-immunoprecipitation experiments with monkey COS-7 cells transiently transfected with both mutant and wild-type Cx36 cDNAs demonstrated that the mutant protein bound to the wild-type. Double immunofluorescence microscopy of stably transfected HeLa cells demonstrated that mutant Cx36 blocked the transport of the wild-type Cx36 to the cell membrane, primarily by trapping it in the endoplasmic reticulum around the nucleus. Coexpression of the mutant Cx36 with the wild-type protein abolished the ability of the latter to permit dye transfer in both HeLa and PC12 cells. The findings are the first demonstration of a mutation of Cx36 that inhibits wild-type Cx36 function in mammalian cells