Role of local premotor nonspiking interneurons in walking pattern generation of the stick insect Carausius morosus

In the course of this thesis, neural mechanisms underlying the generation of single leg stepping in the stick insect Carausius morosus were investigated at the premotor level. Local nonspiking interneurons (NSIs) are important premotor elements within the leg muscle control system of insects, which integrate sensory signals from different sources and provide synaptic drive onto motoneurons (MNs). The single middle leg preparation used allows intracellular recordings from identified NSIs while the active animal performs stepping movements on a treadmill. For identification, NSIs were stained following physiological characterization by iontophoretical dye injection and viewed with a confocal laser scanning microscope. The alternating activity of flexor and extensor tibiae MNs during single middle leg stepping, which characterizes stance and swing phase, respectively, was monitored by extracellular recordings. In the first part of the thesis, the activity pattern of NSIs driving tibial MNs during single leg stepping was studied and their contribution to the generation of stepping motor output was revealed. With the initiation of stepping, modulations of membrane potential were generated in all NSIs that were closely related to the step cycle. The activity pattern comprised distinct excitatory or inhibitory phasic input, during at least one phase of the step cycle. Most NSI types showed an inversion of membrane potential polarization from one phase of the step cycle to the other. It was shown that the activity pattern of the individual NSIs during stepping was not predictable from the synaptic drive, i.e., excitatory or inhibitory, they provide onto MNs in the resting animal. Artificial alterations of membrane potential and measurements of local input resistance for individual NSIs revealed that phasic excitatory and inhibitory modulations of membrane potential during stepping results from true excitatory and inhibitory synaptic input. Current injections into NSI I1 immediately terminated stepping sequences, indicating an important role of I1 in the control of motor output for stepping. The amplitude of phasic membrane potential modulation of NSIs during stepping varied markedly. The maximum peak-to-peak amplitude of membrane potential modulation during stepping amounted to 16.9 ± 6.0 mV on average for all NSIs presented in this study and ranged from 5 to 34 mV for individual recordings. The time of peak and trough potential occurrence within a step cycle appears to contribute substantially to the patterning of motor output, since the extensor MN activity was closely correlated with the membrane potential of individual NSIs, e.g., E2/3, E4, E8 and I2. For the first time, it could be shown that the activity of NSIs during stepping can largely be explained by the state dependency of their inputs from the femoral chordotonal organ, one of the main leg sensors. Hence, the results presented here strongly support the notion that the motor response during the �active reaction� represents a part of the control regime for the generation of single leg stepping. In the second part of the thesis, the interest was to investigate neural mechanisms underlying adaptivity in locomotor systems. Therefore, it was examined which parameters contribute to alterations in stepping velocity. An important finding was that stepping velocity varies with membrane potential alterations of NSIs activated during stance phase, but not with NSIs activated during swing phase. Furthermore, the results suggest that the stance part of the locomotor network is stronger activated during fast stepping velocities and that the swing part is simultaneously inhibited to the same extent. However, investigation of extensor MN activity failed to show a correlation with stepping velocity. This finding implies that swing phase activity is independent of stepping velocity and, hence, corroborates the notion that the swing part of the premotor network does not contribute to alterations in stepping velocity. Finally, it was investigated whether there is a correlation between swing phase activation and stance phase velocity during single leg stepping. The results indicate that there is no influence between stance and swing phase activation in the single middle leg preparation, at least, not in the way that activation strength of stance would influence the subsequent activation of swing phase. The insights gained on premotor NSIs within the femur-tibia joint control system of the stick insect raise the assumption of a premotor network organized into functionally different and partly overlapping pools of NSIs. In the single middle leg preparation, individual NSI types appear to control the actual magnitude of stepping motor output (e.g., E2/3, E8, I2) or the stepping velocity (e.g., E1, I1, I2), while others seem to control step phase transitions (e.g., E2/3, E4, I4) or phase duration (e.g., I8, I1, E1)

ADPβS evokes microglia activation in the rabbit retina in vivo

To investigate whether stimulation of purinergic P2Y1 receptors modulates the activation of microglial and Müller glial cells in the rabbit retina in vivo, adenosine 5-O-(2-thiodiphosphate) (ADPβS; 2 mM in 100 μl saline), a non-hydrolyzable ADP analogue, was intravitreadly applied into control eyes or onto retinas that were experimentally detached from the pigment epithelium. Both retinal detachment and application of ADPßS onto control retinas induced phenotype alterations of the microglial cells (decrease of soma size, retraction of cell processes) and had no influence on microglial cell density. ADPßS application onto detached retinas accelerated the process retraction and resulted in a strongly decreased density of microglial cells. The effects of ADPßS on microglia density and phenotype in detached retinas were partially reversed by co-application of the selective inhibitor of P2Y1 receptors, MRS-2317 (3 mM in 100 μl saline). ADPßS apparently did not influence Müller cell gliosis, as determined by electrophysiological and calcium imaging records. It is concluded that rabbit retinal microglial cells express functional P2Y1 receptors in vivo, and that activation of these receptors stimulates phenotype alterations that are characteristical for microglia activation

Radiation enhancement and "temperature" in the collapse regime of gravitational scattering

We generalize the semiclassical treatment of graviton radiation to gravitational scattering at very large energies $\sqrt{s}\gg m_P$ and finite scattering angles $\Theta_s$, so as to approach the collapse regime of impact parameters $b \simeq b_c \sim R\equiv 2G\sqrt{s}$. Our basic tool is the extension of the recently proposed, unified form of radiation to the ACV reduced-action model and to its resummed-eikonal exchange. By superimposing that radiation all-over eikonal scattering, we are able to derive the corresponding (unitary) coherent-state operator. The resulting graviton spectrum, tuned on the gravitational radius $R$, fully agrees with previous calculations for small angles $\Theta_s\ll 1$ but, for sizeable angles $\Theta_s(b)\leq \Theta_c = O(1)$ acquires an exponential cutoff of the large $\omega R$ region, due to energy conservation, so as to emit a finite fraction of the total energy. In the approach-to-collapse regime of $b\to b_c^+$ we find a radiation enhancement due to large tidal forces, so that the whole energy is radiated off, with a large multiplicity $\langle N \rangle\sim Gs \gg 1$ and a well-defined frequency cutoff of order $R^{-1}$. The latter corresponds to the Hawking temperature for a black hole of mass notably smaller than $\sqrt{s}$.Comment: 5 pages, 2 figures, talk presented at the European Physical Society Conference on High Energy Physics, 5-12 July, Venice, Ital

A rigid coherent anti-Stokes Raman scattering endoscope with high resolution and a large field of view

Nonlinear optical endoscopy is an attractive technique for biomedical imaging since it promises to give access to high resolution imaging in vivo. Among the various techniques used for endoscopic contrast generation, coherent anti-Stokes Raman scattering (CARS) is especially interesting. CARS endoscopy allows molecule specific imaging of unlabeled samples. In this contribution, we describe the design, implementation, and experimental characterization of a rigid, compact CARS endoscope with a spatial resolution of 750 nm over a field of view of roughly 250 μm. Omission of the relay optics and use of a gradient index lens specifically designed for this application allow one to realize these specifications in an endoscopic unit which is 2.2 mm wide over a length of 187 mm, making clinical applications during surgical interventions possible. Multimodal use of the endoscope is demonstrated with images of samples with neurosurgical relevance.Nonlinear optical endoscopy is an attractive technique for biomedical imaging since it promises to give access to high resolution imaging in vivo. Among the various techniques used for endoscopic contrast generation, coherent anti-Stokes Raman scattering (CARS) is especially interesting. CARS endoscopy allows molecule specific imaging of unlabeled samples. In this contribution, we describe the design, implementation, and experimental characterization of a rigid, compact CARS endoscope with a spatial resolution of 750 nm over a field of view of roughly 250 μm. Omission of the relay optics and use of a gradient index lens specifically designed for this application allow one to realize these specifications in an endoscopic unit which is 2.2 mm wide over a length of 187 mm, making clinical applications during surgical interventions possible. Multimodal use of the endoscope is demonstrated with images of samples with neurosurgical relevance

Ectonucleotidases in Müller glial cells of the rodent retina: Involvement in inhibition of osmotic cell swelling

Extracellular nucleotides mediate glia-to-neuron signalling in the retina and are implicated in the volume regulation of retinal glial (Müller) cells under osmotic stress conditions. We investigated the expression and functional role of ectonucleotidases in Müller cells of the rodent retina by cell-swelling experiments, calcium imaging, and immuno- and enzyme histochemistry. The swelling of Müller cells under hypoosmotic stress was inhibited by activation of an autocrine purinergic signalling cascade. This cascade is initiated by exogenous glutamate and involves the consecutive activation of P2Y1 and adenosine A1 receptors, the action of ectoadenosine 5′-triphosphate (ATP)ases, and a nucleoside-transporter-mediated release of adenosine. Inhibition of ectoapyrases increased the ATP-evoked calcium responses in Müller cell endfeet. Müller cells were immunoreactive for nucleoside triphosphate diphosphohydrolases (NTPDase)2 (but not NTPDase1), ecto-5′-nucleotidase, P2Y1, and A1 receptors. Enzyme histochemistry revealed that ATP but not adenosine 5′-diphosphate (ADP) is extracellularly metabolised in retinal slices of NTPDase1 knockout mice. NTPDase1 activity and protein is restricted to blood vessels, whereas activity of alkaline phosphatase is essentially absent at physiological pH. The data suggest that NTPDase2 is the major ATP-degrading ectonucleotidase of the retinal parenchyma. NTPDase2 expressed by Müller cells can be implicated in the regulation of purinergic calcium responses and cellular volume

Receptor regulation of osmolyte homeostasis in neural cells

The capacity of cells to correct their volume in response to hyposmotic stress via the efflux of inorganic and organic osmolytes is well documented. However, the ability of cell-surface receptors, in particular G-protein-coupled receptors (GPCRs), to regulate this homeostatic mechanism has received much less attention. Mechanisms that underlie the regulation of cell volume are of particular importance to cells in the central nervous system because of the physical restrictions of the skull and the adverse impact that even small increases in cell volume can have on their function. Increases in brain volume are seen in hyponatraemia, which can arise from a variety of aetiologies and is the most frequently diagnosed electrolyte disorder in clinical practice. In this review we summarize recent evidence that the activation of GPCRs facilitates the volume-dependent efflux of osmolytes from neural cells and permits them to more efficiently respond to small, physiologically relevant, reductions in osmolarity. The characteristics of receptor-regulated osmolyte efflux, the signalling pathways involved and the physiological significance of receptor activation are discussed. In addition, we propose that GPCRs may also regulate the re-uptake of osmolytes into neural cells, but that the influx of organic and inorganic osmolytes is differentially regulated. The ability of neural cells to closely regulate osmolyte homeostasis through receptor-mediated alterations in both efflux and influx mechanisms may explain, in part at least, why the brain selectively retains its complement of inorganic osmolytes during chronic hyponatraemia, whereas its organic osmolytes are depleted.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79149/1/jphysiol.2010.190777.pd

Social Class

Discussion of class structure in fifth-century Athens, historical constitution of theater audiences, and the changes in the comic representation of class antagonism from Aristophanes to Menander

Relationship between the magnitude of intraocular pressure during an episode of acute elevation and retinal damage four weeks later in rats

PURPOSE: To determine relationship between the magnitude of intraocular pressure (IOP) during a fixed-duration episode of acute elevation and the loss of retinal function and structure 4 weeks later in rats. METHODS: Unilateral elevation of IOP (105 minutes) was achieved manometrically in adult Brown Norway rats (9 groups; n = 4 to 8 each, 10-100 mm Hg and sham control). Full-field ERGs were recorded simultaneously from treated and control eyes 4 weeks after IOP elevation. Scotopic ERG stimuli were white flashes (-6.04 to 2.72 log cd.s.m(-2)). Photopic ERGs were recorded (1.22 to 2.72 log cd.s.m(-2)) after 15 min of light adaptation (150 cd/m(2)). Relative amplitude (treated/control, %) of ERG components versus IOP was described with a cummulative normal function. Retinal ganglion cell (RGC) layer density was determined post mortem by histology. RESULTS: All ERG components failed to recover completely normal amplitudes by 4 weeks after the insult if IOP was 70 mmHg or greater during the episode. There was no ERG recovery at all if IOP was 100 mmHg. Outer retinal (photoreceptor) function demonstrated the least sensitivity to prior acute IOP elevation. ERG components reflecting inner retinal function were correlated with post mortem RGC layer density. CONCLUSIONS: Retinal function recovers after IOP normalization, such that it requires a level of acute IOP elevation approximately 10 mmHg higher to cause a pattern of permanent dysfunction similar to that observed during the acute event. There is a 'threshold' for permanent retinal functional loss in the rat at an IOP between 60 and 70 mmHg if sustained for 105 minutes or more

Infrared Spectroscopic Studies of Cells and Tissues: Triple Helix Proteins as a Potential Biomarker for Tumors

In this work, the infrared (IR) spectra of living neural cells in suspension, native brain tissue, and native brain tumor tissue were investigated. Methods were developed to overcome the strong IR signal of liquid water so that the signal from the cellular biochemicals could be seen. Measurements could be performed during surgeries, within minutes after resection. Comparison between normal tissue, different cell lineages in suspension, and tumors allowed preliminary assignments of IR bands to be made. The most dramatic difference between tissues and cells was found to be in weaker IR absorbances usually assigned to the triple helix of collagens. Triple helix domains are common in larger structural proteins, and are typically found in the extracellular matrix (ECM) of tissues. An algorithm to correct offsets and calculate the band heights and positions of these bands was developed, so the variance between identical measurements could be assessed. The initial results indicate the triple helix signal is surprisingly consistent between different individuals, and is altered in tumor tissues. Taken together, these preliminary investigations indicate this triple helix signal may be a reliable biomarker for a tumor-like microenvironment. Thus, this signal has potential to aid in the intra-operational delineation of brain tumor borders. © 2013 Stelling et al