The Authoring of Observational Cinema: Conversations with Colin Young

Based on a series of conversations with Colin Young that have taken place over more than thirty years, this article explores how a certain set of practical and institutional circumstances, in combination with a series of philosophical and aesthetic ideas about the nature of cinema, first led to the emergence over the late 1960s and early 1970s of the approach to ethnographic filmmaking that would become known as “Observational Cinema.” Although it was those whom Colin Young trained, inspired or simply influenced who worked out the practical filmmaking applications of his ideas, it was he who initially formulated the foundational concepts underpinning this approach to ethnographic filmmaking. As such, although he has been a “filmmaker-maker” rather than a filmmaker himself, Colin Young has a rightful claim to be considered, in the sense defined by Roland Barthes, as the original “author” of Observational Cinema.     

Primary Neuron Culture for Nerve Growth and Axon Guidance Studies in Zebrafish (Danio rerio)

Zebrafish (Danio rerio) is a widely used model organism in genetics and developmental biology research. Genetic screens have proven useful for studying embryonic development of the nervous system in vivo, but in vitro studies utilizing zebrafish have been limited. Here, we introduce a robust zebrafish primary neuron culture system for functional nerve growth and guidance assays. Distinct classes of central nervous system neurons from the spinal cord, hindbrain, forebrain, and retina from wild type zebrafish, and fluorescent motor neurons from transgenic reporter zebrafish lines, were dissociated and plated onto various biological and synthetic substrates to optimize conditions for axon outgrowth. Time-lapse microscopy revealed dynamically moving growth cones at the tips of extending axons. The mean rate of axon extension in vitro was 21.4±1.2 µm hr⁻¹ s.e.m. for spinal cord neurons, which corresponds to the typical ∼0.5 mm day⁻¹ growth rate of nerves in vivo. Fluorescence labeling and confocal microscopy demonstrated that bundled microtubules project along axons to the growth cone central domain, with filamentous actin enriched in the growth cone peripheral domain. Importantly, the growth cone surface membrane expresses receptors for chemotropic factors, as detected by immunofluorescence microscopy. Live-cell functional assays of axon extension and directional guidance demonstrated mammalian brain-derived neurotrophic factor (BDNF)-dependent stimulation of outgrowth and growth cone chemoattraction, whereas mammalian myelin-associated glycoprotein inhibited outgrowth. High-resolution live-cell Ca²⁺-imaging revealed local elevation of cytoplasmic Ca²⁺ concentration in the growth cone induced by BDNF application. Moreover, BDNF-induced axon outgrowth, but not basal outgrowth, was blocked by treatments to suppress cytoplasmic Ca²⁺ signals. Thus, this primary neuron culture model system may be useful for studies of neuronal development, chemotropic axon guidance, and mechanisms underlying inhibition of neural regeneration in vitro, and complement observations made in vivo.     

Graphical determination of mean activation energy and standard deviation in a microheterogeneity model of enzyme deactivation

Traditionally, enzyme populations have been treated as if they were either homogenous, or heterogeneous with distinct and separable subpopulations. The microheterogeneity model, however, assumes that there is a continuous distribution of properties in the population. In the area of enzyme deactivation kinetics, this model describes the heterogeneous population as having a continuous distribution of activation energy of deactivation. This distribution is characterized by mean activation energy, and a standard deviation of activation energy. The microheterogeneity model contains two parameters, (0) and sigma. Parameter (0) is the mean value of for a heterogeneous enzyme population; is the activation energy divided by absolute temperature and the ideal gas constant. Parameter sigma is the standard deviation of the Gaussian distribution of values in the population. If the population is homogeneous, then = (0) for all enzyme molecules and sigma = 0. There are certain ratios which are independent of (0) and dependent upon sigma. Two important ratios are t(1/4)/t(1/2) and t(1/2)/t(1/2) ('), where t(1/2) (') represents t(1/2) for a homogeneous enzyme population with the same mean ((0)), as the heterogeneous population. If there is experimental deactivation data for the heterogeneous population which is well behaved, the first ratio, t(1/4)/t(1/2), can be determined by estimating the time in minutes at which the enzyme has lost 25% of its activity (t(1/4)), and the time in minutes at which the enzyme has lost 50% of its activity (t(1/2)), and then taking the ratio t(1/4)/t(1/2). The corresponding value of sigma can be estimated from a graph. The ratio t(1/2)/t(1/2) (') can be found directly as a function of t(1/4)/t(1/2), and can be estimated from another graph. The value of (0) can then be calculated from the formulasgiven in the article.     

Enantioselective hydroxylation of 4-alkylphenols by vanillyl alcohol oxidase

Vanillyl alcohol oxidase (VAO) from Penicillium simplicissimum catalyzes the enantioselective hydroxylation of 4-ethylphenol, 4-propylphenol, and 2-methoxy-4-propylphenol into 1-(4'-hydroxyphenyl)ethanol, 1-(4'-hydroxyphenyl)propanol, and 1-(4'-hydroxy-3'-methoxyphenyl)propanol, respectively, with an ee of 94% for the R enantiomer. The stereochemical outcome of the reactions was established by comparing the chiral GC retention times of the products to those of chiral alcohols obtained by the action of the lipases from Candida antarctica and Pseudomonas cepacia. Isotope labeling experiments revealed that the oxygen atom incorporated into the alcoholic products is derived from water. During the VAO-mediated conversion of 4-ethylphenol/4-propylphenol, 4-vinylphenol/4-propenylphenol are formed as side products. With 2-methoxy-4-propylphenol as a substrate, this competing side reaction is nearly abolished, resulting in less than 1% of the vinylic product, isoeugenol. The VAO-mediated conversion of 4-alkylphenols also results in small amounts of phenolic ketones indicative for a consecutive oxidation step. Copyright 1998 John Wiley & Sons, Inc.     

Effect of thiobencarb on growth and photosynthesis of the soil alga Protosiphon botryoides (Chlorophyta)

The effects of the herbicide thiobencarb (Saturn) were tested on the growth and physiology of the chlorophyte Protosiphon botryoides isolated from an Egyptian paddy. Assays were conducted using 16-day batch cultures. Chlorophyll and dry weight biomass yields were significantly reduced at 2–3 mg L^-1 thiobencarb, and dark respiration increased and protein decreased significantly at 3 mg L^-1. Reductions in exponential specific growth rate (μ) were generally small, but in some cases significant. Thiobencarb also slightly, but significantly, reduced the 77 K fluorescence parameter Fv/Fm, an indicator of maximum photosynthetic efficiency. No consistent dose-dependent changes occurred in chlorophyll per unit dry weight, total carbohydrate or gross photosynthetic capacity. Whereas half of the added thiobencarb was recovered from control (uninoculated) medium, it was largely absent from cells and culture medium after sixteen days, indicating biodegradation by the alga or associated bacteria. P. botryoides recovered fully within sixteen days following subculture in thiobencarb-free medium. Independently varying phosphate and nitrate nine-fold had no clear effect on the sensitivity of P. botryoides to thiobencarb. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ethnographic film: Technology,practice and anthropological theory

Despite the enthusiasm of the pioneer generation of anthropologists for the camera as a means of ethnographic research, filmmaking remained marginal to the anthropological project for most of the course of the last century. However a combination of technological developments and recent theoretical paradigm shifts within anthropology generally now offers the possibility of greater integration of filmmaking into ethnographic research. This article1 seeks to identify the basis for this theoretical incorporation and discusses some of the practical ways in which film can now be used as a means of generating ethnographic understanding,     

Drug-primed reinstatement of cocaine seeking in mice: increased excitability of medium-sized spiny neurons in the nucleus accumbens

To examine the mechanisms of drug relapse, we first established a model for cocaine IVSA (intravenous self-administration) in mice, and subsequently examined electrophysiological alterations of MSNs (medium-sized spiny neurons) in the NAc (nucleus accumbens) before and after acute application of cocaine in slices. Three groups were included: master mice trained by AL (active lever) pressings followed by IV (intravenous) cocaine delivery, yoked mice that received passive IV cocaine administration initiated by paired master mice, and saline controls. MSNs recorded in the NAc shell in master mice exhibited higher membrane input resistances but lower frequencies and smaller amplitudes of sEPSCs (spontaneous excitatory postsynaptic currents) compared with neurons recorded from saline control mice, whereas cells in the NAc core had higher sEPSCs frequencies and larger amplitudes. Furthermore, sEPSCs in MSNs of the shell compartment displayed longer decay times, suggesting that both pre- and postsynaptic mechanisms were involved. After acute re-exposure to a low-dose of cocaine in vitro, an AP (action potential)-dependent, persistent increase in sEPSC frequency was observed in both NAc shell and core MSNs from master, but not yoked or saline control mice. Furthermore, re-exposure to cocaine induced membrane hyperpolarization, but concomitantly increased excitability of MSNs from master mice, as evidenced by increased membrane input resistance, decreased depolarizing current to generate APs, and a more negative Thr (threshold) for firing. These data demonstrate functional differences in NAc MSNs after chronic contingent versus non-contingent IV cocaine administration in mice, as well as synaptic adaptations of MSNs before and after acute re-exposure to cocaine. Reversing these functional alterations in NAc could represent a rational target for the treatment of some reward-related behaviors, including drug addiction.     

Dynamin-dependent Membrane Drift Recruits AMPA Receptors to Dendritic Spines

The surface expression and localization of AMPA receptors (AMPARs) at dendritic spines are tightly controlled to regulate synaptic transmission. Here we show that de novo exocytosis of the GluR2 AMPAR subunit occurs at the dendritic shaft and that new AMPARs diffuse into spines by lateral diffusion in the membrane. However, membrane topology restricts this lateral diffusion. We therefore investigated which mechanisms recruit AMPARs to spines from the shaft and demonstrated that inhibition of dynamin GTPase activity reduced lateral diffusion of membrane-anchored green fluorescent protein and super-ecliptic pHluorin (SEP)-GluR2 into spines. In addition, the activation of synaptic N-methyl-d-aspartate (NMDA) receptors enhanced lateral diffusion of SEP-GluR2 and increased the number of endogenous AMPARs in spines. The NMDA-invoked effects were prevented by dynamin inhibition, suggesting that activity-dependent dynamin-mediated endocytosis within spines generates a net inward membrane drift that overrides lateral diffusion barriers to enhance membrane protein delivery into spines. These results provide a novel mechanistic explanation of how AMPARs and other membrane proteins are recruited to spines by synaptic activity.AMPA³ receptors (AMPARs) are of fundamental importance because they mediate the majority of fast excitatory synaptic transmission in the mammalian central nervous system (1). Most excitatory synapses are characterized morphologically by dendritic spines that contain an electron-dense postsynaptic density (PSD) at their head (2, 3). PSD is highly enriched in AMPARs and associated proteins equired for synaptic transmission and signal transduction (4-6). Activity-evoked changes in functional postsynaptic AMPARs mediate the two main forms of synaptic plasticity believed to underlie learning and memory in the hippocampus (7). Long term potentiation involves the activity-dependent recruitment of AMPARs to the postsynaptic membrane and a concurrent increase in AMPA-mediated transmission, whereas long term depression is a decrease in synaptic AMPAR function (8).The number and subunit composition of synaptic AMPARs are stringently regulated, but despite intense investigation, the processes by which AMPARs are delivered to and retained at the PSD remain controversial. Using photoreactive antagonists and electrophysiology, it has been proposed that AMPARs are only inserted in the plasma membrane at the cell body and laterally diffuse long distances to synapses (9). In direct contrast, approaches using real-time imaging have suggested that AMPARs are inserted in the plasma membrane of the dendritic shaft close to, but not at, dendritic spines (10). It has also been suggested that AMPARs could be inserted directly into the plasma membrane of the PSD (11).Independent of the route of delivery for new AMPARs to synapses, it is well established that lateral diffusion in the plasma membrane allows the exchange of receptors in and out of the PSD (12-14). Using palmitoylated membrane-anchored GFP (mGFP), which partitions to the inner leaflet of the plasma membrane, it has also been reported that diffusion is significantly retarded within spines compared with the shaft and that AMPAR activation increases the rates of mGFP diffusion in spines (15). In addition, we have shown previously that membrane protein movement into and out of spines is slow compared with lateral diffusion on non-spiny membrane (16), and modeling studies have predicted that spine length is a major determinant of the time a protein takes to reach the PSD (17). More recently, it has been proposed that endocytosis at specialized endocytic zones close to the PSD within spines is required to maintain the steady state complement of synaptic AMPARs (18).Taken together these findings suggest that endocytosis and exocytosis as well as lateral diffusion and membrane topology may all play important roles in regulating membrane protein mobility in spines. The interrelationships between these processes, however, remain unclear. Here we used FRAP (fluorescence recovery after photobleaching) and multisite FLIP (fluorescence loss in photobleaching) to visualize super-ecliptic pHluorin-tagged GluR2 surface expression and AMPAR movement in real time. We examined how lateral diffusion is regulated in spines both by blocking dynamin GTPase activity and stimulating NMDARs. Combined with Monte Carlo simulations on lattices fitting theoretical spines, our data indicate that the membrane topology of spines alone is sufficient to constrain lateral diffusion. NMDAR activation facilitates AMPAR recruitment to spines by a process that involves the recruitment of plasma membrane, together with the constituent membrane proteins, from adjacent regions of the dendritic shaft being drawn into the spine to replace membrane that is internalized during endocytosis. In other words, our results suggest a mode of lateral diffusion that is neither free nor anomalous. Rather, we show the directional diffusion of membrane-embedded proteins toward the postsynapse driven by the endocytosis within the spine. These results provide a new mechanistic explanation of how synaptic activity can overcome topology-induced diffusion barriers to recruit new membrane proteins to the spine.     

Differential responsiveness to immunoablative therapy in refractory rheumatoid arthritis is associated with level and avidity of anti-cyclic citrullinated protein autoantibodies: a case study

In order to identify pathogenic correlates of refractory rheumatoid arthritis (RA), antibodies against anti-cyclic citrullinated protein (ACPAs) were investigated in RA patients in whom the dysregulated immune system had been ablated by high-dose chemotherapy (HDC) and autologous haematopoietic stem cell transplantation (HSCT). Six patients with refractory RA were extensively characterized in terms of levels of total immunoglobulins, RA-specific autoantibodies (ACPAs and rheumatoid factor) and antibodies against rubella, tetanus toxoid (TT) and phosphorylcholine before and after HDC plus HSCT. Additionally, the avidity of ACPAs was measured before and after treatment and compared with the avidity of TT antibodies following repeated immunizations. Synovial biopsies were obtained by arthroscopy before HDC plus HSCT, and analyzed by immunohistochemistry. In the three patients with clinically long-lasting responses to HDC plus HSCT (median 423 days), significant reductions in ACPA-IgG levels after therapy were observed (median level dropped from 215 to 34 arbitrary units/ml; P = 0.05). In contrast, stable ACPA-IgG levels were observed in three patients who relapsed shortly after HDC plus HSCT (median of 67 days). Clinical responders had ACPA-IgG of lower avidity (r = 0.75; P = 0.08) and higher degree of inflammation histologically (r = 0.73; P = 0.09). Relapse (after 38 to 530 days) in all patients was preceded by rising levels of low avidity ACPA-IgG (after 30 to 388 days), in contrast to the stable titres of high avidity TT antibodies. In conclusion, humoral autoimmune responses were differentially modulated by immunoablative therapy in patients with synovial inflammation and low avidity ACPA-IgG autoantibodies as compared with patients with high levels of high avidity ACPA-IgG. The distinct clinical disease course after immunoablative therapy based on levels and avidity of ACPA-IgG indicates that refractory RA is not a single disease entity.     

PHOTOACCLIMATION AND GROWTH RATE RESPONSES OF ULVA ROTUNDATA (CHLOROPHYTA) TO INTRADAY VARIATIONS IN GROWTH IRRADIANCE1

A vegetative clone of the chlorophyte macroalga Ulva rotundata was maintained in an outdoor continuous flow system under nutrient sufficient conditions and various light regimes. Step changes between 9 and 100% incident irradiance (I^o) were employed to simulate cloud passage. Temporary (1–4 h) midday (I^o)perturbations evoked net changes in growth rate (μ) and chlorophyll (chl) content. Under I_o alternating at various periodicities from 15 min to 7 h, net μ was the average of the μ under steady state 9 and 100% I^o, regardless of periodicity. However, the μ in alternating light was considerably less than μ under steady state 55% I^o(? 9%+ 100%/2), as expected based on the nonlinear shape of the μ-I relationship. Unlike μ, chl content depended primarily on the total daily irradiance, probably clue to the slower response of chl compared to photosynthetic rate. On time scales ≥ one day, chl was linearly correlated with light-regulated daily μ under both steady state and intraday fluctuating irradiance, consistent with photosynthetic feedback regulation of chl concentration.