Neuroscience Vol. 44, No. I, pp. 205-214, 1991 Printed in Great Britain 0306-4522/91 $3.00+0.00 Pergamon Press plc 0 1991 IBRO LIMITED DISTRIBUTION OF PERTUSSIS TOXIN IN RAT BRAIN AFTER INJECTION INTO THE LATERAL CEREBRAL VENTRICLES* I. VAN DER Pmm,t$ A. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDC CINTRA,~ N. ALTIOK,~ P. AsKE~~F,/~ K. FUR@ and B. B. FREDHOLM$ SDepartment of Pharmacology and 4Department of Histology and Neurobiology, Karolinska Institute& s-10401 Stockhohn, Sweden [INational Bacteriological Laboratory, S-105 21 Stockholm, Sweden Almtrac-Zn uiuo administration of pertussis toxin is often used to study the involvement of guanine nucleotide binding proteins in signal transduction. Especially when it is administered in the brain the effect is often poor. This could be due to the fact that pertussis toxin does not reach the area of interest. To evaluate the extent to which pertussis toxin is distributed in rat brain after intraventricular injection, different techniques were used. Immunohistochemical studies with an antibody against pertussis toxin showed that immunoreactivity was limited to periventricular brain structures less than 0.5 mm from the lumen. The highest immunoreactivity was seen 16-24 h after injection. After 96 h the labeling was very weak. The proportion of guanine nucleotide binding proteins that were ADP-ribosylated by in oiuo injection of pertussis toxin into the ventricles as assessed by in vitro [32P]-back-ADP-ribosylation was very low 48 h after the injection, in all regions studied. Direct injection of pertussis toxin into the brain caused a marked ADP-ribosylation local&cd to the region injected that was maximal at 72 h after injection. At 96 h there were also effects after control injections, indicating non-specific effects. Synaptosomal membranes and other membranes were equally affected by pertussis toxin. The results suggest that in studies regarding the effect of pertussis toxin treatment on signal transduction, the toxin must be injected very close to the brain region of interest and, furthermore, that the rats should be killed 48-72 h after injection. In case of lack of effect on the response of interest one should examine whether the ADP-ribosylation of pertussis toxin-sensitive guanine nucleotide binding proteins in the area of concern has been affected. Pertussis toxin (PTX) is frequently used as a tool for studying signal transduction through guanine nucleo- tide binding proteins (G-proteins). PTX catalyses the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD) to a specific C-terminal cysteine present on several G-proteins (e.g. members of the Gi-family and GO) and thus prevents the activation of these G-proteins by receptor stimu- lation.‘* When a receptor-mediated response is blocked by prior PTX treatment the interpretation is therefore straightforward. By contrast, the lack of a G-protein-mediated response can be explained in at least two ways. One possibility is that the G-proteins that mediate the physiological response of interest are not sensitive to PTX. Another possibility is that the *Parts of this material have been presented at the XIth International Congress of Pharmatilogy, Amsterdam, l-6 July 1990, and at the 13th Annual Meeting of the European Neuroscience Association, Stockholm, 8-12 September 1990. tTo whom correspondence should be addressed. Abbreuiations: BSA, bovine serum albumin; CSF, cere- brospinal fluid; DTT, l&dithiothreitol; EDTA, ethylenediarninetetra-acetate; EGTA, ethyleneglycol- bis-(B-aminoethyl ether)-iV,N’-tetra-acetic acid; G- proteins, guanine nucleotide binding proteins; NAD, nicotinamide adenine dinucleotide; PAGE, polvacrvl- amide gel electrophoresis; PBS, phosphat&bbe&.l saline; PMSF, phenyhnethyl-sulfonyl fluoride; PTX, per- tussis toxin; SDS, sodium dodecyl sulfate, TCA, trichloroacetic acid. toxin does not reach the area of interest in sufficient amounts. In an earlier study we showed that in vivo FTX treatment led to inhibition of postsynaptic, but not of presynaptic, adenosine A, responses in slices of the rat hippocampus.’ This could have been due to the fact that the postsynaptic G-proteins that couple to the A, receptors are sensitive to PTX, while the presynaptic G-proteins coupling to A, receptors are PTX-insensitive. Another possibility is that PTX reached the presynaptic G-proteins poorly or not at all. We have addressed the uncertainty regarding the degree of penetration of PTX after intraventricular injection by studying the distribution of PTX in the rat brain using an immunohistochemical technique. The effect of in vivo administration of PTX on PTX-sensitive G-proteins was studied by in vitro [32P]ADP-ribosylation in different brain regions. To examine whether there was a differential effect on synaptic G-proteins we prepared cortical/ hippocampal synaptosomes from control and PTX- treated rats and compared the degree of inhibition of [32P]ADP-ribosylation with that obtained with hom- ogenates. EXPERIMENTAL PROCEDUaEs Materials PTX was obtained from List (Campbell, CA, U.S.A.) or from the National Bacteriological Laboratory (Sweden). 205