Brain slice technique studies

Since these studies utilized autoradiographic techniques, it was important to determine the chemical nature of the material measured using radiochemical procedures. Using punch biopsies of the brain slices which were measured autoradiographically, it was shown using thin-layer chromatography that about 30% of the radioactivity was associated with unchanged BCNU (14). It was therefore concluded that the measurements described above accurately reflect brain concentrations of BCNU. 

These approaches to receptor identification and classification were, of course, pioneered by studies with peripheral systems and isolated tissues. They are more difficult to apply to the CNS, especially in in vivo experiments, where responses depend on a complex set of interacting systems and the actual drug concentration at the receptors of interest is rarely known. However, the development of in vitro preparations (acute brain slices, organotypic brain slice cultures, tissue-cultured neurons and acutely dissociated neuronal and glial cell preparations) has allowed more quantitative pharmacological techniques to be applied to the action of drugs at neurotransmitter receptors while the development of new recording methods such as patch-clamp... 

This can be carried out in vitro (in brain slices, cultured cell preparations) or in vivo and involves penetrating the experimental tissue with a carbon-fibre electrode of 5-30 pm in diameter (Fig. 4.9). This serves as an oxidising electrode and the Faradaic current generated by the oxidation of solutes on the surface of the electrode is proportional to their concentration. Obviously, only neurotransmitters which can be oxidised can be measured in this way so the technique is mainly limited to the study of monoamines and their metabolites. The amplitude of each peak on the ensuing voltammogram is a measure of solute concentration and individual peaks can be identified because different... 

Studies on the effects of OPs/CMs have been performed with various preparations, including brain slices, cultured neurons, acutely isolated neurons, iieuroreceptors expressed in a host cell, and neuronal cell lines. The electrophysiologi-cal techniques utilized are extracellular recording, intracellular recording, and patch clamp. Similar to the studies of neuromuscular functions, various compounds were tested. Thus, it i.s extremely difficult to obtain a unified picture regarding the mechani,sm of action of OPs/CMs on. synaptic transmission. 

The multi-slice technique of H MRI was used to study regional effects of amphetamine on rat brain [49], a two-coil system enabling three-dimensional perfusion imaging. Amphetamine caused a significant increase in perfusion in many areas of the brain, including the cortex, cingulate and caudate putamen. 

The development of in vitro brain slice and isolated neuron techniques has greatly facilitated detailed studies of the electrophysiology of a wide range of neuronal types in the adult and neonatal vertebrate central nervous system (CNS). Particularly advantageous are the greater mechanical stability that these preparations provide over in vivo models and the control allowed over the composition of the extracellular environment. In addition, the development of the patch-clamp technique has opened up the possibility of direct access to the intracellular environment via internal patch pipet solutions. In combination, these approaches have enabled detailed investigations of neuronal membrane properties, the cellular actions of neurotransmitters, and synaptic mechanisms. 

SPT permits to study the influence of lateral diffusion in the organization of a large number of membrane proteins. The technique has been limited until lately to observations in cell culture. Only a recent paper showed SPT in brain slices, using spinning disk microscopy to obtain the required high temporal and spatial resolution [25]. Still it has to be kept in mind that the antibody-QD complex has the size of tens of nanometers resulting in steric hindrance and thereby in artificial diffusion barriers, limiting the potential use of conventional QDs in brain tissue. 

Almost half a century thereafter, the new frontier of immunocytochemistry applied to the study of the brain lies in its successful combination with other neural tracing techniques and its application to different animal models, not only in vivo but also in other experimental contexts such as those offered by slice studies. 

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