A simple dual pressure-ejection system and calibration method for brief local applications of drugs and modified salines

We report a minimal method for dual pressure ejection through a single micropipette, which uses standard theta-type glass tubing and a connection through thinned polythene catheters without any sealing. We also describe a simple calibration method using a standard electrometer. The linearity of the ejected volume with respect to pulse duration and to applied pressure was maintained, as measured by the resistivity of ultrapure water after ejection of a saline solution. The method was used to analyse the inhibitory effect of glutamate on crayfish motoneurones with local and reversible applications of low-chloride saline and picrotoxin.     

A simple and rapid method to isolate rat retinal cells for biochemical analysis

We have developed a rapid method to enzymatically disperse rat retinae and to separate large quantities of various cell types by centrifugation through a metrizamide density gradient. Seven distinct bands of cells were detected in the gradient (I–VII). Cells from layers III and V were morphologically identified as red blood cells and photoreceptor cells, respectively. [¹²⁵I]Horseradish peroxidase injected into the lateral geniculate bodies labels the ganglion cells via retrograde transport. This technique allowed us to demonstrate the presence of ganglion cells in layer I of the metrizamide gradient. This method will be useful for biochemical and pharmacological characterization of several types of retinal cells.     

A simple method for the Schwann cell preparation from newborn rat sciatic nerves

We have developed a simple and relatively rapid method for obtaining a sufficient number of Schwann cells with a favorable purification ratio from newborn rat sciatic nerves. Perineurium-free nerves were torn into small fascicles of approximately 150-200 microns in diameter and explanted twice on type I collagen gel every 2 days of the culture period in order to reduce the number of contaminant fibroblasts. The last explanted tissues were fed with Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and 100 micrograms/ml bovine pituitary extract for 10 days. More than 10 X 10(4) Schwann cells (greater than 95% purity) were obtained from newborn rat sciatic nerves.     

A method for the intracranial delivery of reagents to voltammetric recording sites

Carbon fiber microelectrodes are widely used for electrochemical monitoring in the intact brain. The local delivery of reagents to the recording site is often desirable. The approach of co-implanting a micropipette near the microelectrode presents some limitations that are overcome by the use of double-barreled devices. One barrel supports the carbon fiber and the other barrel serves as a pipet for local reagent delivery. Some studies have used iontophoretic delivery but here we consider the alternative approach of pressure ejection. However, placing the pipet so close to the electrode raises the risk that reagent can leak into the recording site. This problem is easily solved. We filled the tip of the pipet with vehicle solution, the barrel with a reagent solution, and separated the two solutions with an air gap to prevent their mixing. With this approach, reagent is delivered only after 'priming' pressure pulses: we show in two examples that unintended reagent delivery (leakage) prior to the priming pulses is non-detectable.     

A simple and economical method for studying protein phosphorylation in vivo in the rat brain

A simple and economical procedure, capable of routine application, is described for the labelling of cerebral phosphoproteins in vivo. [32P]Orthophosphate, in high concentration, was infused into selected brain areas of anaesthetised rats under stereotaxic control. The animals were frozen with liquid N2 and the labelled tissue punched out of frozen thick sections. [32P]Polypeptides were analysed by high-resolution two-dimensional gel electrophoresis. Several phosphoproteins on the gels were provisionally identified, including synapsin I, MAP-2 and an 82-87 kdalton substrate of protein kinase 'C'.     

A simple method to study cellular migration

We describe here a simple and fast method for the characterisation of cell motion. By projecting on a single plane different positions of the cell a ribbon is generated, whose characteristics can be related to the type of motion. The proposed method allows both to determine, very quickly, the motility of a population of cells and to investigate and characterise properties of a single cell's motion. The methodology presented here can be applied to a large range of cell movement and also adapted and extended to other problems involving biological motion.     

Rapid and simple determination of delivery after iontophoretic and pressure injections of radiolabeled tracer substances

A fluorographic method is described using X-ray film analysis for the determination of delivery of radiolabeled tracer substances both in Agar plates and in tissue sections. This method is most useful in neuroanatomical autoradiographic studies for providing rapid identification of delivery, placement, and extent of an injection site after iontophoresis or pressure injections of radiolabeled axonal tracer substances.     

A simple method for the construction of electrode arrays

A simple method is described for the construction of electrode arrays consisting of insulated metal wires (33 microns diameter) spaced at small, equal distances (0.1 mm). No specialized instrumentation and techniques are needed, as only simple mechanical tools are sufficient. The electrode arrays are used for field potential recording from in vitro brain slice preparations.     

A method for combining field potential recording with local perfusion in the hippocampus of the anaesthetized rat

A method is described for combining local perfusion of discrete brain areas and simultaneous recording of field potentials using a push-pull cannula with attached electrodes.     

A simple sleep stage detector for the rat.

A simple sleep stage detector for the rat is described, which uses as its sources of input the signal from a single hippocampal recording electrode and the signal generated by a motion indicator. The device is suitable for use in sleep studies and in experiments on REM sleep deprivation.     

A method for intracochlear drug delivery in the mouse

The confluence of two rapidly emerging research arenas - development of mouse models of human deafness and inner ear drug therapy for treatment and prevention of hearing loss - provides an opportunity for unprecedented approaches to study and treat deafness. Toward such goals, we have developed a method for intracochlear drug delivery in the mouse. The bulla was exposed using a ventral approach and the stapedial artery cauterized. An opening made into the inferior-medial aspect of the bulla, where the basal cochlear wall fuses with tympanic bulla, provided direct access to the scala tympani without separately opening the bulla or elevating auditory response thresholds. Cochlear responses, assayed by frequency-specific effects on ABRs and DPOAEs, were stable with infusion (1 microl/h) of an artificial perilymph solution (80 min). The glutamate receptor antagonist, CNQX (100 microM; 175 min), reduced ABR responses without affecting DPOAEs. Salicylate (5mM; 165 min) altered both. Both drugs had greatest effects at high frequencies, but distributed throughout the cochlea and were reversible. The safe delivery of drugs into the cochlea by this approach has immediate application in the study and treatment of various forms of human hearing loss that can be modeled in the mouse.     

A simple, sensitive method for detecting early peripheral nerve dysfunction in the rat following acrylamide treatment

The "ocular zingerone test" was employed to detect early alterations in peripheral nerve function which were associated with acrylamide intoxication in the rat. Acrylamide treatment resulted in a dose-related prolongation of the behavioral response to ocular zingerone. Significant alterations in the zingerone response occurred prior to detectable alterations in peripheral sensory and motor nerve function. Acrylamide-induced prolongation of the zingerone response appears to be the result of functional alterations in cholinergic neurotransmission in the autonomic nervous system. It is concluded that the "ocular zingerone test" is a simple, sensitive technique with which the degree of acrylamide intoxication may be quantified in the rat.     

A simple pressure microinjecting system for delivery of small substance volumes to the brain: application to the developmental study of thalamo-cortical projections in foetal and neonatal rats

We describe a reliable and inexpensive method for placing injections of anatomical tracers into the brain of lower mammals. The pressure microinjecting system we developed is specifically designed to deliver very small amount of substances. The injecting portion of the system is relatively easy to assemble and can be repeatedly used for multiple experimental sessions. The system has been validated with experiments of multiple fluorescent retrograde tracing. In these experiments the populations of thalamo-cortical neurons were consistently labeled by the tracers injected bilaterally and symmetrically in the cortex of foetal and neonatal rats.Themes: Development and regenerationTopics: Pattern formation, compartments, and boundaries     

A simple, sensitive method for the determination of extracellular catecholamines in the rat hypothalamus using in vivo dialysis

The design, construction and characterisation of a dialysis probe suitable for perfusing any deep brain structures is described. Using high-performance liquid chromatography with electrochemical detection (HPLC-ECD) the effects of flow rate, concentration, dialysate composition and temperature on the recovery within the dialysate of authentic catecholamines are detailed. The dialysis probe was used to collect endogenous catecholamines from the anterior hypothalamus of urethane-anaesthetised rats. Following organic phase extraction of the in vivo samples, a small basal release of noradrenaline (NA) of 37 +/- 4 pg/30 min sample was found. Potassium stimulation markedly elevated the release of NA and dopamine from the anterior hypothalamus in a calcium ion and dose dependent manner. It appears therefore that the dialysis probe described here, in conjunction with HPLC-ECD, can be used to follow changes in neuronally released catecholamines within the anterior hypothalamus, providing a valuable tool to study the role of these neurotransmitters in physiological and pharmacological function.     

Virus vectors for gene delivery to the nervous system

We have recently shown that optic nerve transection is followed by a delayed appearance (7-14 days after lesion) of putative guidance activities for retinal axons in the superior colliculus (SC) of adult rats. In the present study we analysed with immunohistological methods whether there is a correlation between localized reactive changes in the cellular compartment of the SC after deafferentation and the reappearance of putative guidance activities. The optic nerve of adult female Sprague-Dawley rats was transected. After 1 day to 1 year the contralateral SC was sectioned sagitally and examined with antibodies directed against Vimentin and glial fibrillary acidic protein (GFAP) to label astrocytes, myelin basic protein (MBP) to detect oligodendrocytes, OX-42 to stain microglia and SMI-31 to determine degeneration of afferent retinal ganglion cell axons. These stainings were performed in order to detect differences in the cellular composition of macro- and microglial cells between the anterior and posterior regions of the SC. All cell populations we examined in the SC showed reactive changes after optic nerve transection. Microglia activation occurred as soon as 2 days after optic nerve transection in the anterior part of the SC. After 4 days, a homogeneous distribution of activated microglia was seen in the whole SC. GFAP upregulation occurred without a specific regional distribution. Only Vimentin-expressing astrocytes appeared with a delay of 1-2 weeks after deafferentation and were strongly restricted to the afferent optic tract and the very anterior region of the SC. In conclusion, a temporo-spatial correlation between the re-expression of guidance activities and changes in the cellular compartment of the SC exists in the delayed re-expression of Vimentin in astrocytes and their strong spatial restriction. Activated microglia might be involved in triggering regional changes in an anterior-posterior graded fashion in the deafferented SC.     

A methoxyflurane delivery system for stereotaxic surgery

Methoxyflurane (2,2-dichloro-1,1-difluro-ethyl methyl ether; Metofane) is a potent general inhalation anesthetic that is well-suited for small animal surgery. Methoxyflurane is particularly attractive as an anesthetic agent in neurological stereotaxic surgery, because methoxyflurane does not markedly attenuate the rate of anterograde or retrograde transport of horseradish peroxidase, or reduce the consistency and/or extent of excitatory neurotoxin damage. Methoxyflurane also is non-flammable when mixed with O2 or air at anesthetic concentrations. The use of methoxyflurane anesthesia in stereotaxic surgery has been limited because methoxyflurane must be delivered via a vaporizer system that will easily interface with standard stereotaxic headholders. The present report describes a simple, reliable and inexpensive methoxyflurane delivery system for stereotaxic surgery.