Turning the heterogeneous into homogeneous: studies on selectively isolated GABAergic interneuron subsets

The amazing morphological and electrophysiological diversity of cortical GABAergic interneurons subserves the broad diversity of processes these cells modulate in neuronal networks. Until recently, interneuron development and functions have been extensively studied in heterogeneous in vitro and in vivo systems containing both excitatory and inhibitory components. However, mechanisms of interneuron specification during development, key signaling mechanisms controlling the establishment of particular inhibitory neuron subsets, and the spatial and temporal regulation of their integration in neuronal microcircuits remain poorly understood. Selective isolation of particular interneuron subsets may significantly extend our knowledge on the scenario of neurochemical and electrophysiological specification of developing interneurons, identification of signaling cues directing their axon growth, and principles of their anterograde and retrograde synaptic communication with other cell types. Here, we show that selective isolation of perisomatic inhibitory cells containing either parvalbumin or cholecystokinin reveals major differences in the temporal dynamics of their functional differentiation, and their dependence on target-derived signals like brain-derived neurotrophic factor and endocannabinoids. In addition, we discuss therapeutic prospects of modulating increased excitatory output in the hippocampus and subthalamic nucleus by re-adjusting the inhibitory control of principal cells.     

Digital holographic imaging of dynamic cytoskeleton changes

A variety of physical and biochemical cell properties depends on the function and integrity of the actin cytoskeleton. The actin cytoskeleton mediates crucial cellular functions as migration, intracellular transport, exocytosis, endocytosis, cell stiffness and force generation. Highly dynamic actin fibers are therefore targets for several drugs and toxins. However, the study of actin interfering processes by standard microscopy techniques fails in the detailed resolution of dynamic spatial alterations required for a deeper understanding of toxic or drug effects. Here we applied digital holographic microscopy in the online functional analysis of the actin cytoskeleton changes of a highly differentiated and a dedifferentiated pancreas tumor cell line induced by the marine toxin Latrunculin B. Scanning electron microscopy (SEM) and fluorescence microscopy showed rapid Latrunculin B induced alterations in cell morphology and actin fiber degradation in both pancreatic tumor cell lines. In contrast digital holographic in vivo analysis of the drug dependent dynamic cellular processes revealed unequal changes in cell morphology. While tumor cells with a low metastatic potential showed Latrunculin B induced cell collapse within 4 h the metastatic tumor cells were resistant to Latrunculin B treatment. Spatial resolution of morphological alterations by digital holography detected so far unknown differences in the actin cytoskeleton stability of highly differentiated and dedifferentiated pancreas tumor cell lines. These data demonstrate that marker-free, non-destructive online analysis of cellular morphology and dynamic spatial processes in living cells by digital holography offers new insights in actin dependent cellular mechanisms. Digital holographic microscopy was shown to be a versatile tool in the screening of toxic drug effects and cancer cell biology.     

Subcellular receptor redistribution and enhanced microspike formation by a Ret receptor preferentially recruiting Dok

Ret is a receptor tyrosine kinase for the GDNF family of ligands and plays important roles during nervous system development for cell proliferation, cell migration and neurite growth. Signaling initiated from intracellular tyrosine 1062, by recruitment of several different phosphotyrosine binding (PTB) proteins (i.e. Shc, Frs2 and Dok), is important for these biological effects. By a single amino acid substitution in the PTB domain binding sequence of Ret, we have rewired the receptor such that it preferentially recruits Dok (Ret^Dok+) with little or no remaining interactions with Shc and Frs2. Ret^Dok+ displays a sustained MAP kinase activation and a loss of Akt signaling compared to Ret^WT. We show that early events after ligand stimulation of Ret^Dok+ include massive formation of fine microspikes that are believed to be priming structures for neurite growth from the cell soma. The Ret^Dok+ receptors relocated in the membrane compartment into focal clusters at the tip of the microspikes, which was associated with Cdc42 activation. These results suggest that engagement of different adaptor proteins by Ret results in very different downstream signaling and functions within neurons and that Dok recruitment leads to a rapid receptor relocation and formation of microspikes.     

Die Kirschner-Draht-Spickung der Hüftkopfepiphyse bei Epiphyseolysis capitis femoris juvenilis

OBJECTIVE: To stabilize the unstable capital femoral epiphysis to stop further slippage. To prevent imminent epiphyseal dislocation with subsequent articulation disorders of the hip joint and joint degeneration. To prevent additional growth disorders of the proximal femur. INDICATIONS: Epiphyseolysis capitis femoris acuta.Epiphyseolysis capitis femoris lenta. Epiphyseolysis capitis femoris incipiens (incipient epiphyseolysis). Epiphyseolysis capitis femoris imminens (imminent epiphyseolysis of the so-called healthy contralateral side). CONTRAINDICATIONS: None. The diagnosis of slipped capital femoral epiphysis is an absolute indication for surgery unless there is an internal or pediatric disorder that dictates a conservative approach. SURGICAL TECHNIQUE: Three, possibly four, Kirschner wires, at least 2 mm thick, are inserted through a short subtrochanteric approach under image intensification into the femoral neck so that they are seen to be spread uniformly over the cross section of the femoral head when viewed in both planes. The opposite side is transfixed prophylactically. POSTOPERATIVE MANAGEMENT: 3 weeks partial loading at 10 kg, then increased loading after radiologic assessment. Radiologic assessment every 6 months until the end of the growth phase so that migration of the Kirschner wiresout of the femoral head can be identified early. If necessary, renewed Kirschner wire transfixation. Implants are removed after consolidation and closure of the growth plate. RESULTS: 59 therapeutic and 48 prophylactic transfixations of slipped capital femoral epiphysis were performed from 1970 to 1995. The average follow-up period was 4 years (0.1-17.3 years). The most frequent complications were incipient secondary joint degeneration arising in most cases from femoral head necrosis, and postoperative identification of Kirschner wire malalignment. At the time of follow-up 27 patients had an excellent result on the pathologic side according to Southwick's classification, 26 a good, three a moderate, and three a poor result. In particular, a poor result ensued subsequent to femoral head necrosis.     

A simple method for long-term storage of Acanthamoeba species

We present a novel and simple technique for storing live Acanthamoeba for long periods of time. The amoebae are maintained at refrigerator temperatures in a peptone–yeast extract–glucose (PYG) medium normally used for cultivation. Using this method, we obtained survival rates of at least 4 years for Acanthamoeba polyphaga and 3 years for Acanthamoeba castellanii and Acanthamoeba rhysodes. Advantages of this storage method are: (1) it is quick and simple, (2) inexpensive, (3) does not require encystment before storage, (4) resuscitation of cysts can be achieved within a week of culture in PYG medium at 27°C, and does not require co-culture with bacteria or any special equipment.     

Psychomotor performance in relation to acute oral administration of Δ9-tetrahydrocannabinol and standardized cannabis extract in healthy human subjects

Abnormalities in psychomotor performance are a consistent finding in schizophrenic patients as well as in chronic cannabis users. The high levels of central cannabinoid (CB₁) receptors in the basal ganglia, the cerebral cortex and the cerebellum indicate their implication in the regulation of motor activity. Based on the close relationship between cannabis use, the endogenous cannabinoid system and motor disturbances found in schizophrenia, we expected that administration of cannabinoids may change pattern of psychomotor activity like in schizophrenic patients. This prospective, double-blind, placebo-controlled cross-over study investigated the acute effects of cannabinoids on psychomotor performance in 24 healthy right-handed volunteers (age 27.9 ± 2.9 years, 12 male) by comparing Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and standardized cannabis extract containing Δ⁹-THC and cannabidiol. Psychomotor performance was assessed by using a finger tapping test series. Cannabis extract, but not Δ⁹-THC, revealed a significant reduction of right-hand tapping frequencies that was also found in schizophrenia. As to the pure Δ⁹-THC condition, left-hand tapping frequencies were correlated with the plasma concentrations of the Δ⁹-THC metabolite 11-OH-THC. These effects are thought to be related to cannabinoid actions on CB₁ receptors in the basal ganglia, the cerebral cortex and the cerebellum. Our data further demonstrate that acute CB₁ receptor activation under the cannabis extract condition may also affect intermanual coordination (IMC) as an index of interhemispheric transfer. AIR-Scale scores as a measure of subjective perception of intoxication were dose-dependently related to IMC which was shown by an inverted U-curve. This result may be due to functional changes involving GABAergic and glutamatergic neurotransmission within the corpus callosum. I. Gorynia and A.M. Stadelmann have equally contributed to this work.