Improved Force Spectroscopy Using Focused-Ion-Beam-Modified Cantilevers.

Atomic force microscopy (AFM) is widely used in biophysics, including force-spectroscopy studies of protein folding and protein-ligand interactions. The precision of such studies increases with improvements in the underlying quality of the data. Currently, data quality is limited by the mechanical properties of the cantilever when using a modern commercial AFM. The key tradeoff is force stability vs short-term force precision and temporal resolution. Here, we present a method that avoids this compromise: efficient focused-ion-beam (FIB) modification of commercially available cantilevers. Force precision is improved by reducing the cantilever's hydrodynamic drag, and force stability is improved by reducing the cantilever stiffness and by retaining a cantilever's gold coating only at its free end. When applied to a commonly used short cantilever (L=40µm), we achieved sub-pN force precision over 5 decades of bandwidth (0.01-1000Hz) without significantly sacrificing temporal resolution (~75µs). Extending FIB modification to an ultrashort cantilever (L=9µm) also improved force precision and stability, while maintaining 1-µs-scale temporal resolution. Moreover, modifying ultrashort cantilevers also eliminated their inherent underdamped high-frequency motion and thereby avoided applying a rapidly oscillating force across the stretched molecule. Importantly, fabrication of FIB-modified cantilevers is accessible after an initial investment in training. Indeed, undergraduate researchers routinely modify 2-4 cantilevers per hour with the protocol detailed here. Furthermore, this protocol offers the individual user the ability to optimize a cantilever for a particular application. Hence, we expect FIB-modified cantilevers to improve AFM-based studies over broad areas of biophysical research.

Target-derived astroglia regulate axonal outgrowth in a region-specific manner.

The potential neuroanatomical specificity of astrocyte influence on neurite outgrowth was studied using an in vitro coculture system in which neurons from embryonic rat spinal cord or hippocampus were grown for 4 days in the presence of, but not in direct contact with, astrocytes derived either from the same region (homotopic coculture) or from different regions (heterotopic coculture) of the rat central nervous system. The results showed that axonal outgrowth was greatly enhanced in heterotopic cocultures in which spinal cord or hippocampal neurons were grown with astrocytes derived from their appropriate CNS target regions. This effect was remarkably specific, because the astroglia harvested from spinal or hippocampal target regions were not effective in promoting axon growth of nonafferent neuronal populations. Dendritic outgrowth was similar under all coculture conditions. These data suggest that diffusible signals, produced by astrocytes, can regulate neurite extension in vitro in a neuroanatomically specific manner and that axons are more sensitive than dendrites to the regional astrocyte environment.

Immunocytochemical identification of GABA in astrocytes located in white matter after inhibition of GABA-transaminase with gamma-acetylenic GABA.

Several lines of evidence suggest that astrocytes contribute to the uptake and degradation of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Recent immunohistochemical studies have shown that GABA-like immunoreactivity can be demonstrated in astrocytes in the grey matter of the rat's brainstem. The present study investigates whether GABA is also present in astrocytes located in the white matter. Adult rats were given gamma-acetylenic GABA (GAG), which inhibits the GABA-degratory enzyme GABA-alpha-ketoglutaric acid aminotransferase, and tissue sections from the cerebral cortex and brainstem were processed for GABA immunohistochemistry using an antiserum to GABA. Light microscopic examination of the sections showed numerous small GABA-immunoreactive cells in fibre tracts as well as in nuclear regions. Electron microscopic examination of the immunoreactive cells showed that they were fibrous astrocytes. The results provide evidence that the large increase in GABA in fibre tracts found in biochemical studies of rats injected with GAG is due to an increase in astrocytic GABA and suggest that fibrous astrocytes regulate GABA levels in the extracellular space of white matter.

Cerebellar projections to the somatic pretectum in the cat.

Neurons in the somatic pretectum receive input from the dorsal column nuclei (DCN) and project to a comparable "somatic" portion of the dorsal accessory nucleus of the inferior olive (DAO). This somatic DAO is reciprocally connected with the anterior interpositus nucleus of the cerebellum. One question that arises is whether this circuitry is further controlled by an output specifically from the anterior interpositus nucleus to the somatic pretectum. Wheatgerm agglutinin conjugated to horseradish peroxidase was injected into various parts of the cat pretectum. Injection sites were interpreted as including the somatic pretectum if neurons in the DCN were retrogradely labeled and if anterograde terminal labeling occurred in somatic DAO. The locations of retrogradely labeled neurons within the deep cerebellar nuclei were then compared in cases in which the injection sites included or excluded the somatic pretectum. In all cases in which the injection site included the somatic pretectum, retrogradely labeled neurons were observed in the anterior interpositus nucleus as well as in the lateral cerebellar nuclei. In some of these cases, neurons in the posterior interpositus and medial nuclei were also labeled. In contrast, in cases in which the pretectal injection site was located outside or at the border of the somatic pretectum, retrogradely labeled neurons were observed only in the lateral, posterior interpositus, and medial nuclei. Thus, the somatic pretectum appears to receive input primarily from neurons in the anterior interpositus nucleus, along with some input from neurons in the lateral nucleus. These results provide additional evidence for a pathway through the DCN in which sequentially processed somatic information has access to and is modulated by cerebellar circuitry. The existence of such a pathway supports the conclusion that neurons in the DCN convey somatic information important not only for cutaneous, kinesthestic, and other bodily sensations, but also for the control of movement.

Convergent inputs to the inferior olive from the dorsal column nuclei and pretectum in the cat.

The dorsal column nuclei (DCN) consist of an anatomically heterogeneous population of neurons, some of which project to the inferior olive and pretectum. Recent anatomical experiments on cats have shown that neurons in the parts of the pretectum which receive input from DCN also project to the inferior olive. Thus, DCN neurons provide an input to the inferior olive via both a direct DCN-olivary pathway and an indirect pathway through the pretectum. This connective situation provides a mechanism by which incoming somatic sensory information that is processed at different levels of the brainstem (i.e. DCN and pretectum) has access to the cerebellum by way of the inferior olive. It is of interest whether the two sets of differently processed information are conveyed to the same group of inferior olive neurons. Although DCN and pretectal projections to the inferior olive have been generally described, the relationship between the DCN targets in the inferior olive and those specifically from the DCN-recipient parts of the pretectum have not. To address this question, this study used single and double anterograde labeling strategies with a variety of tracers to compare the two targets in the inferior olive of cats. It was found that projections to the inferior olive from the DCN-recipient parts of the pretectum were located predominantly in the dorsal accessory portion of the inferior olive where they overlapped extensively with projections directly from DCN. These results provide evidence for a pathway by which sequentially processed somatic sensory information, first in the DCN and then in the pretectum, has access to the cerebellum by way of the same group of inferior olive neurons.

Simultaneous determination of fenbendazole, its sulphoxide and sulphone metabolites with the corresponding metabolites of triclabendazole in the plasma of sheep and cattle by high-performance liquid chromatography.

Pharmacokinetic studies following the simultaneous treatment of sheep or cattle with the anthelmintics triclabendazole and fenbendazole required an assay for determining the analytes in the plasma. An extraction and clean-up procedure is described which uses a C(18) Sep Pak cartridge, involves no evaporation steps and produces a clean extract from sheep and cattle plasma in which triclabendazole, fenbendazole and their respective oxidation products may be determined by reversed-phase high-performance liquid chromatography. Results from application of the method to a pharmacokinetic study of sheep are presented.

Differences in the neurons that project from the dorsal column nuclei to the diencephalon, pretectum, and tectum in the cat.

The dorsal column nuclei (DCN) project to a number of targets in the nervous system besides the ventroposterolateral nucleus (VPL) of the thalamus. Recent evidence obtained using double-labeling techniques indicates that DCN's diencephalic-projecting neurons differ in their location and morphology from those that project to some of its other targets, such as the cerebellum and tectum. The purpose of the present study was to characterize anatomically the DCN neurons that project another of DCN's targets, the pretectum, and to determine if any of these neurons have collateral projections to the tectum or diencephalon. The projections were studied using two double-labeling methods. One method made use of either tritiated inactivated horseradish peroxidase ([3H]apoHRP) or tritiated N-acetyl wheatgerm agglutinin ([3H]WGA) as a marker and HRP or WGA conjugated to HRP. The other method made use of the dyes Fast Blue and Nuclear Yellow. In each cat, one marker was injected into the DCN-recipient portions of the pretectum, tectum, or diencephalon, and the other marker was injected into another of these three targets. Neurons labeled by pretectal or tectal injections were of all sizes, fusiform and multipolar in shape, and similarly located. They were scattered through the rostral zone of DCN, but were distributed at the periphery of and at the junction between the gracile and cuneate nuclei in DCN's middle and caudal zones. In contrast to the pretectal- and tectal-labeled neurons, neurons labeled by diencephalic injections were round and large. They were found throughout the DCN complex, but were concentrated in DCN's middle and caudal zones. When both the pretectum and diencephalon were injected in the same cat, the two groups of neurons occupied similar locations in the rostral zone, but were distinct in the middle and caudal zones, with the pretectal-projecting neurons surrounding the clusters of diencephalic-projecting neurons. Very few neurons were double-labeled. These results demonstrate that the projections to the pretectum, tectum, and diencephalon originate from different populations of neurons within specific domains in DCN. When these results are compared with the results of electrophysiological and other anatomical studies, it appears that the pretectal- and tectal-projecting neurons may be part of a previously unrecognized system originating in DCN.(ABSTRACT TRUNCATED AT 400 WORDS)