Brain slice technique

The suprachiasmatic nucleus controls synthesis and release of mdatonin, the hormone of the pineal gland. Melatonin itself feeds back to the suprachiasmatic nucleus (Cassone et al. 1987, Stehle et al. 1989, McArthur etal. 1991). Using brain slice technique and immunocytochemistry Kopp etal. (1997) demonstrated that pituitary adenylate cyclase-activating polypeptide induces the phosphorylation of the transcription factor cyclic AMP response element binding protein in the suprachiasmatic nucleus of the Wistar rat during the late subjective day and that melatonin inhibits this pituitary adenylate cyclase-activating polypeptide-induced phosphorylation. 

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... 

In the upper panel are shown hippocampal cerebral blood volume (CBV) maps generated in mice (right) and humans (left). In the lower panel an in vitro horizontal brain slice (left) at the same position where the MRI generated images (right). These types of preparations exemplified the techniques that can be used crosssspecies (upper panel) and techniques that complement each other (lower panel)... 

Fleck et al., 1993). This result was also obtained under conditions mimicking high neuronal activity (Szerb, 1988). Also in the intact striatum using the microdialysis technique, a selective increase in Asp release after chemical depolarization (with GABA antagonists or NMDA) of the neocortex has been demonstrated (Palmer et al., 1989). Such selectivity suggests that Asp is released independently of Glu both in brain slices and the intact brain. 

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. 

The sections that follow describe how to make the carbon fiber electrodes used as the working electrodes in FCV, how to set up an FCV amplifier, and how to test and calibrate the carbon fiber electrodes before use. These techniques are suitable for the detection and measurement of dopamine, noradrenaline, and serotonin (5-HT) in the whole bram or in brain slices. 

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