Ion channel drug discovery and research: the automated Nano-Patch-Clamp technology.

Unlike the genomics revolution, which was largely enabled by a single technological advance (high throughput sequencing), rapid advancement in proteomics will require a broader effort to increase the throughput of a number of key tools for functional analysis of different types of proteins. In the case of ion channels -a class of (membrane) proteins of great physiological importance and potential as drug targets- the lack of adequate assay technologies is felt particularly strongly. The available, indirect, high throughput screening methods for ion channels clearly generate insufficient information. The best technology to study ion channel function and screen for compound interaction is the patch clamp technique, but patch clamping suffers from low throughput, which is not acceptable for drug screening. A first step towards a solution is presented here. The nano patch clamp technology, which is based on a planar, microstructured glass chip, enables automatic whole cell patch clamp measurements. The Port-a-Patch is an automated electrophysiology workstation, which uses planar patch clamp chips. This approach enables high quality and high content ion channel and compound evaluation on a one-cell-at-a-time basis. The presented automation of the patch process and its scalability to an array format are the prerequisites for any higher throughput electrophysiology instruments.

High quality ion channel analysis on a chip with the NPC technology.

In evaluating ion channel function, electrophysiology, e.g., patch clamping, provides the highest information content. For the analysis of ion channel-modulating compounds, one variant of the patch-clamp technique, the whole-cell configuration, is particularly useful. We present here patch-clamp recordings in the whole-cell configuration and single channel recordings performed with planar patch-clamp chips, which are microstructured from borosilicate glass substrate. The chips are used in the Port-a-Patch, an ion channel research/screening instrument that enables automated patch-clamp experiments on a single cell. A software runs the experiment by executing user-determined protocols for cell positioning, as well as for electrical stimulation and current readout. In various electrophysiological experiments, the high quality of recordings and the versatility of the perfusion of the recorded cells are demonstrated. Quantitative pharmacological experiments are performed on sodium channels expressed in HEK cells using solution volumes in the low microliter range. The exceptionally low volume consumption in the experiments make the system attractive for work on rare or expensive compounds. Due to the low volumes necessary, a rapid solution exchange is facilitated, which is shown on RBL cells. The patch-clamp chip enables a rapid and precise perfusion, allowing sophisticated investigations on ion channel function with the Port-a-Patch.

Reduced number of functional glutamatergic synapses in hippocampal neurons overexpressing full-length TrkB receptors.

Brain-derived neurotrophic factor (BDNF) acutely modulates the efficacy of central glutamatergic synapses via activation of the receptor tyrosine kinase TrkB. On a longer time scale, recent evidence suggests an additional role of TrkB signaling in the formation of excitatory synaptic connections. Here, we have overexpressed full-length TrkB receptors (fl-TrkB) in hippocampal neurons, to investigate the contribution of BDNF signaling to the maturation of glutamatergic synapses. Using patch clamp recordings, we show a three-fold reduction in glutamatergic excitatory autaptic and synaptic current amplitudes in neurons overexpressing fl-TrkB, and application of saturating concentrations of BDNF and NT-4/5 completely reverses this effect. Compatible with these overexpression data, in untransfected neurons, scavenging of endogenous BDNF and NT-4/5 by TrkB-IgGs reduces excitatory autaptic current (EAC) amplitudes. By overexpression of truncated TrkB receptors (TrkB.T1, TrkB.T2) and a chimeric receptor containing only the intracellular domain of fl-TrkB, we show that intra- and extracellular domains of fl-TrkB are necessary to observe the EAC reduction. Labeling of presynaptic terminals with FM 4-64 revealed, that the reduced EAC amplitudes in fl-TrkB overexpressing neurons are accompanied by a two-fold reduction in synapse number. These results suggest, that ligand-independent signaling through fl-TrkB receptors can decrease glutamatergic synaptic strength, if sufficient amounts of BDNF or NT-4/5 are not available.

Continuing medical education for AIDS: an organizational response.

The Management of the Southern California Kaiser-Permanente Medical Group mandated a day-long program of continuing medical education on AIDS for all staff physicians at one facility. Presentations dealt with cognitive (transmission/diagnosis), skills (risk factors, history taking), and affective (discomfort/attitudes) domains. All physicians participated in 2 small-group workshops on sexual history taking and ethical issues. An evaluation compared primary care physicians' attitudes and practices before and after the program at the experimental site (Medical Center), a comparison site (written materials only), and a "no-treatment" facility. A sample of new patients (N = 2,689) seen by study physicians (N = 253) before and after the educational intervention were telephoned to determine if sexual history questions were asked by their physicians. There was a significant increase in the frequency of sexual history questions asked by internists at the experimental site. Over 95% of all patients were accepting of sexual history questions. This program has since been repeated at 11 medical centers involving over 3,500 Kaiser-Permanente physicians throughout Southern California.

Metastatic squamous cell carcinoma in the neck invading the facial canal--a case report.

The results of histopathologic examination of the temporal bone of a 71-year-old woman with squamous cell carcinoma of the tonsil and ipsilateral facial palsy are presented. The right temporal bone was directly involved by metastatic spread of the primary lesion to the right upper cervical lymph nodes. Tumor cells had invaded the canal of the facial nerve, the chorda tympani nerve, and the stapedius muscle, as well as the air cells in the mastoid region. However, although tumor cells had infiltrated the facial canal to a considerable distance from the metastatic tumor mass, the facial nerve had not been infiltrated. Slight degeneration of the facial nerve, however, was observed and appeared to have been caused by compression by the tumor.