Cellular neurochemical characterization and subcellular localization of phospholipase C ß1 in rat brain.

The present study describes a complete and detailed neuroanatomical distribution map of the phospholipase C beta1 (PLCß1) isoform along the adult rat neuraxis, and defines the phenotype of cells expressing PLCß1, along with its subcellular localization in cortical neurons as assessed by double-immunofluorescence staining and confocal laser scanning. Immunohistochemical labeling revealed a considerable morphological heterogeneity among PLCß1-positive cells in the cortex, even though there was a marked predominance of pyramidal morphologies. As an exception to the general non-matching distribution of GFAP and PLCß1, a high degree of co-expression was observed in radial glia-like processes of the spinal cord white matter. In the somatosensory cortex, the proportion of GABAergic neurons co-stained with PLCß1 was similar (around 2/3) in layers I, II-III, IV and VI, and considerably lower in layer V (around 2/5). Double immunofluorescence against PLCß1 and nuclear speckle markers SC-35 and NeuN/Fox3 in isolated nuclei from the rat cortex showed a high overlap of both markers with PLCß1 within the nuclear matrix. In contrast, there was no apparent co-localization with markers of the nuclear envelope and lamina. Finally, to assess whether the subcellular expression pattern of PLCß1 involved specifically one of the two splice variants of PLCß1, we carried out Western blot experiments in cortical subcellular fractions. Notably, PLCß1a/1b ratios were statistically higher in the cytoplasm than in the nuclear and plasma membrane fractions. These results provide a deeper knowledge of the cellular distribution of the PLCß1 isoform in different cell subtypes of the rat brain, and of its presence in the neuronal nuclear compartment.

Immunohistochemical localization of CB1 cannabinoid receptors in frontal cortex and related limbic areas in obese Zucker rats: effects of chronic fluoxetine treatment.

In the present study, we report on the application of two specific polyclonal antibodies to different intracellular domains of the CB1 cannabinoid receptor to define the expression of the neural CB1 cannabinoid receptor at the histochemical level in frontal cortex and related limbic areas of the obese Zucker rats. Higher levels of CB1 receptor expression in frontal, cingulated and piriform cortex, without differences in temporal, parietal and occipital cortex, were observed in obese Zucker rats, with respect to their lean littermates. CB1 phosphorylated receptor (CB1-P) levels were also higher in frontal, temporal, parietal and occipital cortex in obese rats with respect to lean controls. Potential involvement of brain cortical CB1 cannabinoid receptors in the long-term effects of fluoxetine was studied. Experimental animals were administered with fluoxetine (10 mg/kg, i.p.) daily for 3 weeks, whereas the control group was given 0.9% NaCl solution. In obese Zucker rats, a significant decrease in CB1 receptor levels, measured by western blot, was observed in brain cortex after fluoxetine treatment. Immunostaining for CB1 receptor expression was also carried out, showing a significant decrease in the density of neural cells positive for CB1 receptor in frontal, cingulate and piriform cortex, without changes in parietal, temporal and occipital regions. Regional prosencephalic immunostaining for CB1-P receptor level showed a significant decrease in the density of stained neural cells in frontal, temporal and parietal cortex, without changes in cingulated, piriform and occipital cortex. These results suggest the involvement of endocannabinoid system in the chronic effects of fluoxetine, especially in the frontal cortex.

Depresión y sistemas de generación de segundos mensajeros / Depression and seconf messenger systems

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Transmembrane signaling through phospholipase C in cortical and hippocampal membranes of psychogenetically selected rat lines.

RATIONALE: One of the major pathways for neurotransmitter signaling involves phosphoinositide-specific and G-protein-dependent phospholipase C-beta (PLC-beta), which stimulates the formation of inositol 1,4,5-trisphosphate and diacylglycerol. Serotonergic and muscarinic-cholinergic signals in the brain are largely mediated through the hydrolysis of phosphoinositides by PLC. OBJECTIVES: The aim of the experiments reported here was to explore the potential differences in neurotransmitter receptor coupling to PLC in Roman high-avoidance (RHA)/Verh and Roman low-avoidance (RLA)/Verh rats, by examining the changes in agonist (carbachol, 5-methyltryptamine)-stimulated phosphoinositide hydrolysis in hippocampal and cortical membranes derived from the two rat lines. METHODS: To investigate changes in receptor and G-protein coupling to PLC in the brains of these two psychogenetically selected rat lines, which differ in their emotional profiles/learning abilities, we examined GTPgammaS-, agonist (carbachol, 5-methyltryptamine)-, and calcium-stimulated phosphoinositide hydrolysis in cortical and hippocampal membranes of RHA/Verh and RLA/Verh rats. RESULTS: The results indicated that calcium-induced increase in PLC activity was larger in the cortex and hippocampus of RHA/Ver rats, as compared to their RLA/Verh counterparts. Conversely, GTPgammaS- and agonist-induced PLC activity was less pronounced in the hippocampus of RHA/Verh with respect to RLA/Verh rats. Western blot analysis showed no significant differences in the relative values of the G-proteins alphaq/11 and betagamma subunits between both groups of rats in any brain region. However, the levels of PLC-beta1, PLC-beta3, and PLC-beta4 were significantly lower in the hippocampus of RHA/Verh than in RLA/Verh rats. CONCLUSIONS: It is concluded that the hippocampus of RHA/Verh rats has severe deficiencies in PLC activity stimulated by guanine nucleotides and agonists, which are specifically related to a lower level of expression of the PLC-beta type isozymes, a fact that may account for the differential behavioral phenotype observed in these psychogenetically selected rat lines.

Regulation of phospholipase Cbeta activity by muscarinic acetylcholine and 5-HT(2) receptors in crude and synaptosomal membranes from human cerebral cortex.

Stimulation of phospholipase Cbeta by receptor agonists and G proteins has been characterized in crude cerebral membrane preparations, but little is known about their presynaptic localizations and little information is currently available for human brain tissue. The characteristics of phosphoplipase C transmembrane signaling were studied in crude and synaptosomal plasma membranes from postmortem human prefrontal cortex by measuring the hydrolysis of exogenous [(3)H]phosphatidylinositol4,5bisphosphate(PIP(2)) and the immunoreactive levels of phospholipase C (PLC) and G(alphaq/11) proteins. Regulation of PLC activity by Ca(2+) and the 5-HT(2) receptor agonist 5-methyltryptamine, but not by guanosine 5'-O-[3-thiotriphosphate] and the muscarinic acetylcholine receptor agonist carbachol were different between crude and synaptosomal membranes. KCl (20 mM) stimulation was absent in both preparations. Levels of G(alphaq/11)-protein subunits differed between preparations. The functional inhibition carried out with pirenzepine in crude membranes in order to reverse the carbachol-induced PLC stimulation indicates the existence of a component (53%) of the response that is activated by the M(1) muscarinic acetylcholine receptor subtype, and another component (47%) probably mediated by the M(3) muscarinic acetylcholine receptor subtype. In synaptosomal plasma membranes an increased inhibition of carbachol-induced PLC activation through M(1) was found. The PLC activation by 5-methyltryptamine (ketanserin-sensitive in crude membranes) was absent in synaptosomal plasma membranes suggesting the lack of activity mediated by 5-HT(2)-serotonin receptors.