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Psychostimulants, including ritalin (methylphenidate), were used as antidepressives in the '50s but were then replaced by tricyclics and MAO inhibitors. Treatment of depression with psychostimulants is still controversial. Several anecdotal reports in the past decade approved the use of tricyclic antidepressants (TCA) together with methylphenidate in apathetic and withdrawal states in medically ill and in elderly patients. Ritalin elevates mood by releasing catecholamines and blocking their re-uptake, and also increases serum TCA levels. 5 men and 5 women between the ages of 65 and 79 were diagnosed as suffering from major depressive disorders, either single or recurrent, based on the Revised Diagnostic and Statistical Manual for Mental Disorders (DSM-III-R). They had been treated with TCA for up to several months with no response. Following addition of methylphenidate, 5-15 mg/d for 2 weeks, 4 men and 3 women improved rapidly, 2 of them within 24 hours.

6 patients came to our psychiatric emergency room during the first 2 weeks of the Gulf War presenting the clinical picture of acute delusional paranoid psychosis (4 women and 2 men between the ages of 30-77). 4 were without previously known psychopathology, while the other 2 were known to have had some nonpsychotic cognitive impairment. The first 4 recovered completely within a short time while the other 2 continued to have psychopathological symptoms. We discuss psychogenic or reactive psychosis, and the concept of reactivation when cumulative trauma exceed the individual's personal threshold, as may occur during a war. Different phenomenological syndromes may follow.

We describe a temperature-jump device that permits time-resolved studies of thin cryo-transmission electron microscopy specimens. The specimen is rapidly heated to induce a change in microstructure just prior to cryo-fixation. The apparatus consists of a xenon arc lamp equipped with a shutter controlled by timing circuitry, used in conjunction with an environmental specimen preparation chamber. The specimen is heated by exposure to focused light from the lamp, and then plunged into cryogen. Using a thermocouple constructed from an electron microscope grid, we show that temperature jumps of 30-60 K are achieved with exposure times of 150-450 milliseconds. Micrographs of dimyristoyl phosphatidylcholine (DMPC) vesicles and n-docosane films, subjected to these exposures, show that the specimens are still at least 20-30 K above their initial temperature when they contact the cryogen. This method could be applied to a variety of biological and chemical systems which undergo structural changes activated by a rise in temperature.

The vesicle-micelle transition of egg phosphatidylcholine (PC) and sodium cholate was described by comparing cryo-transmission electron microscopic (cryo-TEM) images of the structures formed to the associated turbidity changes. These experiments were designed to identify the morphology of the intermediates between vesicles and small spheroidal mixed micelles. With increasing cholate concentration, the vesicular structures changed size and more multilamellar vesicles were seen. Between the apparent upper and lower phase boundaries, three structures were observed: open vesicles, large bilayer sheets (twenty to several hundred nanometers in diameter), and long (150-300 nm) flexible cylindrical micelles. The cylindrical micelles evolved from the edges of the bilayer sheets. At higher relative cholate concentration, the phase boundary was sharply defined by optical clarification of the egg PC-cholate mixtures. Cryo-TEM revealed only small spheroidal mixed micelles at this transition. These results provide the first direct evidence of the structural pathway or of molecular intermediates between a lamellar and a micellar state. Understanding these specific intermediates and the transitions between them is essential to developing reconstitution protocols and properly analyzing either activity or structural data obtained from cholate-dispersed membrane proteins.

Fat emulsions containing soy triacylglycerols (100-300 g/l) and egg-yolk phospholipids (12 g/l) are often used for intravenous feeding. Previous studies have shown that these emulsions contain chylomicron-like emulsion particles of diameters of 300-400 nm and excess phospholipids aggregated as vesicles (liposomes), which remain in the infranatant upon floatation of the emulsion particles by ultracentrifugation. This work is devoted to the characterization of the commercial lipid emulsions commonly denoted Intralipids, with special emphasis on the presently ill-defined liposomes. The lipid particles composing commercial lipid emulsions (10%, 20% and 30% Intralipids, Kabivitrum Nutrition) were characterized by the combined use of physical and chemical methods. Each of the emulsions was fractionated by ultracentrifugation in saline into a 'cream' layer which floats to the top of the dispersion upon ultracentrifugation and a relatively transparent infranatant. The cream layer contains large emulsion particles of diameters ranging from 300 to 400 nm, in agreement with theoretical considerations based on their chemical composition as determined by chemical analysis. The infranatants contain about 1 g/l triacylglycerols in addition to phospholipids (from 7.2 g/l in 10% Intralipid to 2.4 g/l in 30% Intralipid) in the form of smaller particles of 70-100 nm diameter. Cryo-transmission electron microscopy shows that the infranatants contain vesicles (mostly unilamellar) at the side of residual small emulsion particles. This conclusion is also consistent with the distribution of phospholipids between outer and inner lamellae, as determined by 31P-NMR.

We describe a new technique, time-resolved cryotransmission electron microscopy (TRC-TEM), that can be used to study changes in microstructure occurring during dynamic processes such as phase transitions and chemical reactions. The sample is prepared on an electron microscope grid maintained at a fixed temperature in a controlled atmosphere. The dynamic process is induced on the grid by a change in pH, salt, or reactant concentration by rapid mixing with appropriate solutions. Alternatively, induction is by rapid change of specimen temperature, or by controlled evaporation of a volatile component. We call such procedures on-the-grid processing. The dynamic process is permitted to run for a defined time and then the thin-film specimen is thermally fixed by plunging into liquid ethane at its freezing point, producing a cryotransmission electron microscopy specimen. By repeating this procedure with varying delays between induction and sample fixation, we can observe transient microstructures. We demonstrate the use of TRC-TEM to study the intermediate structures that form during the transitions between L alpha, III, and HII liquid crystalline phases in phospholipid systems. We also identify several other possible applications of the technique.

The least artifact-laden fixation technique for examining colloidal suspensions, microemulsions, and other microstructured liquids in the electron microscope appears to be thermal fixation, i.e., ultrafast freezing of the liquid specimen. For rapid-enough cooling and for observation in TEM/STEM a thin sample is needed. The need is met by trapping a thin layer ( approximately 100 nm) of liquid between two polyimide films ( approximately 40 nm thickness) mounted on copper grids and immersing the resulting sandwich in liquid nitrogen at its melting point. For liquids containing water, polyimides films are used since this polymer is far less susceptible to the electron beam damage observed for the commonly used polymer films such as Formvar and collodion in contact with ice. Transfer of the frozen sample into the microscope column without deleterious frost deposition and warming is accomplished with a new transfer module for the cooling stage of the JEOL JEM-100CX microscope, which makes a true cold stage out of a device originally intended for cooling specimens inside the column. Sample results obtained with the new fast-freeze, cold-stage microscopy system are given.

A guillotine-type quick freezing device and a bio-hazard containment box have been designed, constructed, and used to prepare vitrified-hydrated specimens of viruses in their native environment. Special design considerations include the preservation of the specimen in its natural state in vitrified ice, prevention of virus aerosols escape, and control of the potentially explosive air-coolant vapor mixtures.

Dialkyldimethylammonium hydroxide surfactants are highly soluble in water and form spontaneous stable vesicles. These vesicles can be grown to size with added acid, and appear to provide an ideal membrane mimetic system for the study of fusion and ion transport. These phenomena are a consequence of strong hydration forces that are not necessarily limited to the hydroxide ions. The forces can be used to design a variety of model systems whose behavior differs from that of systems in which double-chained surfactants form insoluble liquid crystalline phases in water and unstable vesicle suspensions on prolonged sonication.

The controlled environment vitrification system (CEVS) permits cryofixation of hydrated biological and colloidal dispersions and aggregates from a temperature- and saturation-controlled environment. Otherwise, specimens prepared in an uncontrolled laboratory atmosphere are subject to evaporation and heat transfer, which may introduce artifacts caused by concentration, pH, ionic strength, and temperature changes. Moreover, it is difficult to fix and examine the microstructure of systems at temperatures other than ambient (e.g., biological systems at in vivo conditions and colloidal systems above room temperature). A system has been developed that ensures that a liquid or partially liquid specimen is maintained in its original state while it is being prepared before vitrification and, once prepared, is vitrified with little alteration of its microstructure. A controlled environment is provided within a chamber where temperature and chemical activity of volatile components can be controlled while the specimen is being prepared. The specimen grid is mounted on a plunger, and a synchronous shutter is opened almost simultaneously with the release of the plunger, so that the specimen is propelled abruptly through the shutter opening into a cryogenic bath. We describe the system and its use and illustrate the value of the technique with TEM micrographs of surfactant microstructures in which specimen preparation artifacts were avoided. We also discuss applications to other instruments like SEM, to other techniques like freeze-fracture, and to novel "on the grid" experiments that make it possible to freeze successive instants of dynamic processes such as membrane fusion, chemical reactions, and phase transitions.

A fast-freeze, cold-stage transmission electron microscopy technique which can incorporate in situ freeze-drying of the sample is described. Its use in elucidating structure in unstained and stained, hydrated and freeze-dried, aqueous vesicular dispersions of biological and chemical interest is demonstrated with vesicles of L-alpha-phosphatidylcholine (bovine phosphatidylcholine) and of the synthetic surfactant sodium 4-(1'-heptylnonyl)benzenesulfonate (SHBS). The contrast features observed in transmission electron microscope images of frozen, hydrated samples are identified and explained with the dynamical theory of electron diffraction. Radiolysis by the electron beam is shown to increase contrast in vesicle images and to change their structure and size. Micrographs illustrate the freeze-drying of a dispersion in the microscope; the process causes vesicles to shrink and collapse.