Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.     

An Ibotenate-Selective Metabotropic Glutamate Receptor: Mediates Protein Phosphorylation in Cultured Hippocampal Pyramidal Neurons

Abstract: Previous results showed that within 30 s after glutamate stimulation of cultured rat hippocampal pyramidal neurons there occurred an elevation of Ca²⁺ and diacylglycerol, and the phosphorylation of three acidic protein kinase C substrates, i.e., an 87-kDa protein known as myristoylated alanine-rich C kinase substrate and a 120-and a 48-kDa protein. In addition, it was suggested that a metabotropic-type glutamate receptor might be responsible for the phosphorylation observed. This work examines the ability of metabotropic and ionotropic glutamate receptor agonists to quickly activate phospholipases in 1.26 mM versus 50 nM extracellular Ca²⁺ by measuring the generation of inositol phosphates. NMDA, quisqualate, and trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid did not stimulate the generation of inositol phosphates in the presence of normal or low extracellular Ca²⁺ in pyramidal neurons. Kainate stimulated the production of inositol phosphates in the presence of 1.26 mM extracellular Ca²⁺ but not in 50 nM extracellular Ca²⁺. Other than glutamate, only ibotenate was able to stimulate the generation of inositol phosphates in both normal and low extracellular Ca²⁺. The maximal response to ibotenate was approximately equal to that of glutamate, when pyramidal neurons were stimulated in 50 nM extracellular Ca²⁺. The generation of inositol phosphates by glutamate and ibotenate could be partially blocked (50–60% reduction) by pretreatment of neurons with pertussis toxin (250 ng/ml),-suggesting that a GTP-binding protein might be involved. In addition, ibotenate stimulated the immediate phosphorylation of the same three protein kinase C substrates as glutamate. The NMDA receptor blocker MK-801 had no effect on this phosphorylation. These results suggest that the stimulation of phosphorylation in pyramidal neurons by glutamate occurs predominantly through the activation of an ibotenate-selective metabotropic glutamate receptor.     

Translocation and Activation of Protein Kinase C in Striatal Neurons in Primary Culture: Relationship to Phorbol Dibutyrate Actions on the Inositol Phosphate Generating System and Neurotransmitter Release

The actions of the tumor-promoting phorbol ester phorbol dibutyrate were examined, under identical physiological conditions, on three distinct cellular processes in striatal neurons: the distribution of protein kinase C, the carbachol-stimulated generation of [3H]inositol monophosphate, and the KCl-evoked release of gamma-[3H]aminobutyric acid ([3H]GABA). Phorbol dibutyrate induced a rapid (complete in 5 min), dose-dependent, entirely reversible (t0.5 = 15 min) translocation of protein kinase C from cytosol to membrane. On longer exposure to phorbol dibutyrate, membrane-associated protein kinase C returned toward the control level, and total cellular enzyme activity declined markedly. Phorbol dibutyrate also induced the dose-dependent attenuation of carbachol-stimulated [3H]inositol monophosphate production and potentiation of KCl-evoked release of [3H]GABA. The translocation of protein kinase C and the potentiation of KCl-evoked [3H]GABA release were both rapidly reversed following washout of phorbol dibutyrate. In addition, for both processes, the effect of a 1-h exposure to phorbol dibutyrate was markedly less than that observed following a 5-min exposure to the agent. In direct contrast, inhibition of carbachol-stimulated [3H]inositol monophosphate production was not rapidly reversed following washout of phorbol dibutyrate and was actually more pronounced following a 1-h exposure, compared with a 5-min exposure. These findings indicate that some, but not all, of the actions of phorbol dibutyrate are closely associated with the translocation of protein kinase C in striatal neurons in primary culture.