Proc. Nail. Acad. Sci. USA Vol. 85, pp. 3208-3212, May 1988 Neurobiology Phospholipase C I and II brain isozymes: Immunohistochemical localization in neuronal systems in rat brain (immunohistochemistry/second-messenger systems) CHARLES R. GERFEN*t, WON CHUL CHOIt, PANN GHILL SUHt, AND SUE Goo RHEEf *Laboratory of Cell Biology, National Institute of Mental Health, and tLaboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892 Communicated by E. R. Stadtman, December 17, 1987

ABSTRACT Two distinct inositol phospholipid-specific dylinositol-4,5-bisphosphate (PtdInsP2) in the presence of phospholipase C (PLC; phosphatidylcholMe phosphatidohy- calcium. At low calcium concentrations PtdInsP2 is the drolase, EC 3.1.4.3) isozymes, PLC-I and PLC-il, have been preferred substrate for both isozymes. However, PLC-I has purified and characterized from bovine brain. Monoclonal a greater specificity for PtdInsP2 than does PLC-II. The antibodies that distinguish between these isozymes are used in activities of the two isozymes appear to be differentially the present study to map isozyme distribution in the rat brain affected by nucleotides in the presence or absence of Ca2 . with immunohistothemical techniques. Both isozymes are For example, PLC-I activity is enhanced by both GTP and localized in neurons, and, whereas PLC-il is rather ubiqui- ATP but to a much greater extent in the absence of Ca2`, tous-being expressed in most neurons, PLC-I is restricted in whereas PLC-II activity appears unaffected by nucleotides its distribution. The strongest immunoreactive labeling for regardless of Ca2+ concentration. Furthermore, the intracel- PLC-I is in the neurons of the , which provide inputs lular distribution of the two forms of PLC may be different. to the and substantia nigra, where terminals PLC-I is present in nearly equal amounts in both particulate are also densely labeled. The neuronal targets of these termi- and cytosolic fractions of brain homogenates, whereas nals in the globus pallidus and substantia nigra do not express nearly 90% of PLC-II is present in cytosolic fractions (7). PLC-I immunoreactivityj but they do display PLC-il immu- The existence of at least two forms of PLC in the brain noreactivity. PLC-I immunoreactivity is also particularly well justified an examination of their immunohistochemical dis- pronounced in the pyramidal cells of the and, to tribution to determine whether they are expressed in differ- a lesser extent, in the granule cells of the . In the ent neuronal systems. , PLC-I is localized to neurons in the reticular thalamic nucleus, in the medial subdivision of the mediodorsal MATERIALS AND METHODS thalamic nucleus, and in the anteromedial thalamic nucleus. Other areas displaying PLC-I immunoreactive neurons in- Adult Sprague-Dawley rats were used in this study. Under clude the dorsal lateral septal nucleus and the basolateral chloral hydrate anesthesia rats were perfused transcardially . The expression of at least one or more forms of with 0.9% saline followed by 500 ml of a fixative solution PLC in most neurons of the brain suggests that this enzyme containing 2% (wt/vol) formaldehyde (from paraformalde- may be part of a common system of signal transduction used hyde), 1.5% lysine, and 0.2% sodium periodate in sodium universally by all neurons. However, the differential expres- phosphate buffer, pH 7.4 (8). Following perfusion the brains sion of PLC isozymes suggests further that certain neurotrans- were removed and placed in fixative for an additional 4-48 mitter and receptor interactions may differ in the forms of the hr at room temperature and were then transferred to 0.1 M PLC enzyme used for signal transduction. potassium phosphate-buffered saline (KPBS, pH 7.4) for 12 hr at 40C. The brains were then sectioned on a Vibratome One of the mechanisms whereby receptor- (TPI, Saint Louis) at 30 ,um and collected in KPBS. Sections mediated signal transduction produces second messengers is were then incubated in dilutions of mAbs directed against by the activation, via as yet unspecified guanine nucleotide- either PLC-I (5 tug/ml) or PLC-II (10 ,g/ml) in KPBS plus binding proteins, of inositol phospholipid-specific phospho- 2% normal goat serum (NGS) and 0.5% Triton X-100 at 40C lipase C -(PLC; phosphatidylcholine phosphatidohydrolase, for 24-48 hr. Sections were rinsed twice in KPBS for 5 min EC 3.1.4.3; for reviews, see refs. 1-4). This enzyme hydro- each time and treated for immunoperoxidase localization lyzes inositolphospholipids to generate two second-messen- using the avidin-biotin-peroxidase method (9). Briefly, sec- ger molecules, diacylglycerol (acyl2Gro) and inositol 1,4,5- tions were incubated in a dilution of biotin-labeled goat triphosphate. Recently, two distinct PLC isozymes have anti-mouse IgG (2.5 mg/ml) in KPBS/2% NGS/0.5% Triton been purified and characterized from bovine brain, PLC-I X-100 for 2 hr at room temperature, rinsed in KPBS, and and -II (5, 6). Monoclonal antibodies (mAbs) have been incubated in streptavidin-conjugated peroxidase (2.5 ttg/ml) raised that distinguish between these two forms of PLC and in KPBS for 1 hr. Sections were then rinsed three times for do not show crossreactivity for the heterologous isozyme (5, 5 min each in 0.1 M sodium acetate buffer, pH 8.0, and then 6). Under denaturing conditions the molecular weights ofthe incubated in a solution of 10 ml of 0.1 M sodium acetate two forms differ, being 150,000 for PLC-I and 145,000 for buffer, pH 6.0, containing 5 mg of 3,3' diaminobenzidine PLC-Il. Moreover, PLC-I appears under nondenaturing (Sigma), 20 mM dextrose, 7.5 mM NH4Cl, and 5-30 units of conditions to be present mainly as a dimer, whereas PLC-II glucose oxidase for 30 min at room temperature (10). Sec- is present as a monomer. Both forms exhibit similar sub- strate specificity-catalyzing the hydrolysis of phosphatidyl- Abbreviations: PLC, phospholipase C; PLC-I and PLC-I, PLC inositol, phosphatidylinositol4-phosphate, and phosphati- isozyme I and isozyme II; mAb, monoclonal antibody; acyl2Gro, diacylglycerol; PKC, protein kinase C; P(BtO)2, phorbol 12,13- dibutyrate. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Laboratory of payment. This article must therefore be hereby marked "advertisement" Cell Biology, National Institute of Mental Health, Building 36, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Room 2D-10, Bethesda, MD 20892. 3208 Neurobiology: Gerfen et A Proc. Natl. Acad. Sci. USA 85 (1988) 3209 tions were then placed in sodium acetate buffer containing 10% (vol/vol) formalin for 5 min, rinsed in KPBS, and PLC-1 PLC-2 mounted onto gelatin-coated slides, air dried, defatted, counterstained with ethidium bromide, and dehydrated be- fore coverslips were added. mot ~ ~ ,, c tx

RESULTS OX General Characteristics of Immunohistochemical Labeling. The mAbs directed against PLC-I and PLC-I1 have been characterized for specific binding to the appropriate antigens 2n in previous studies and show minimal crossreactivity for the Al Ac heterologous isozyme by immunoblot analysis (6). Specific- ity for immunohistochemical staining was assessed by pread- sorbing dilutions of the antibodies (5 gg/ml for PLC-I and PLC-I1 mAbs) with either the homologous or heterologous antigen at 50 pug/ml. Antibodies preadsorbed with the ho- mologous antigen did not produce immunohistochemical ss staining in sections adjacent to those analyzed for specific OtX immunohistochemical staining patterns, whereas preadsorp- tion with the heterologous antigen had no effect on the staining. Furthermore, immunohistochemical labeling de- creased with increasing dilutions of mAb. Both PLC-I and PLC-I mAb produced specific and wide- -bla spread patterns of labeling in the rat brain (Fig. 1). In terms of distinguishing the cellular localization of immunoreactive labeling, neuron perikarya in many brain areas show the B' B2 clearest labeling. Additionally, there is labeling of specific fiber-projection systems and terminal fields and some non- )ftS- L distinct labeling of brain neuropil. In general, for equal -;Pi concentrations of antibodies, labeling of PLC-I-positive sys- tems is denser than that of PLC-II-positive systems. As will 'p be described below, labeling with PLC-Il-specific mAb is somewhat dependent on fixation conditions, whereas PLC-I mAb-specific labeling is less affected by such variations. The patterns of labeling with the two antibodies appear in some areas to be similar and in other areas to be distinct. Fig. 1 shows labeling obtained with mAb to PLC-I and PLC-II in adjacent coronal sections at three different brain levels- Cl through the rostral forebrain at A, a level containing the striatum and ; B, a rostral thalamic level; and C, a caudal thalamic level that includes the hippocampus. A more FIG. 1. Bright-field photomicrographs (in which labeling is dark) thorough comparison of the distribution of PLC-I and PLC-Il of coronal sections at three different brain levels from rostral to immunoreactivity in different systems is presented below. caudal (A to C) showing immunohistochemical localization of PLC-I Basal Ganglia. Both PLC-I and PLC-Il immunoreactivity (Left) and PLC-II (Right). PLC-I and -II immunoreactivity is most is clearly evident in the cell bodies of the majority, and dense in the caudate and nuclei (cp, all levels) with PLC-I a staining more pronounced than PLC-I staining. The basolateral perhaps all, of cells in the striatum as determined by amygdala (bla in B) also contains both PLC-I- and -I-immunoreactive careful analysis of labeling in sections counterstained with neurons, but PLC-I staining is considerably darker. At rostral levels ethidium bromide (Fig. 2 A' and A2). With comparable (A) PLC-I immunoreactivity is dense in the lateral septum and absent dilutions of antibodies PLC-I labeling is denser per cell than in the medial septum and . PLC-II immunoreactive that obtained for PLC-II. Labeled cells are evenly distrib- neurons are present in all areas. In the thalamus (B and C) PLC-I uted in the striatum; thus, no apparent differences exist in immunoreactivity is densest in neurons of the reticular nucleus (rt, B labeling of the striatal "patch" and "matrix" compartment and C) and shows differential patterns of neuropil staining in other systems of medium-sized striatal projection neurons (11). nuclei; the mediodorsal and anteromedial nuclei contain both stained Labeling with both antibodies is also evident in the projec- neurons and dense neuropil staining, the posterior nucleus shows neuropil staining, while the ventral posterior nuclei and habenular tion systems of the labeled striatal neurons, with immuno- nuclei (h) are relatively devoid of labeling. PLC-I immunoreactivity reactivity present in the terminal fields in the globus pallidus is homogenously distributed in all thalamic nuclei. In the hypothala- (Fig. 2 B' and B2), entopeduncular nucleus, and substantia mus (B) the paraventricular nucleus (pvn) stands out as being devoid nigra (Fig. 2 C' and C2). PLC-II immunoreactivity also labels of PLC-I immunoreactivity while displaying PLC-II-immunoreactive neurons that are the targets of the striatopallidal and stria- neurons. There is dense labeling of PLC-I in the hippocampus (hipp, tonigral systems (Fig. 2 B2 and C2). In contrast, PLC-I C). The dentate gyrus granule cells are labeled, but their efferent immunoreactivity is absent in these neurons, which appear mossy fibers are less so. PLC-II labeling is homogenously distributed as perikaryal ghosts embedded in immunoreactivity present in the hippocampus. mot ctx, Motor cortex; ss ctx, somatosensory in the neuropil surrounding them (Fig. 2 B' and C'). cortex; s, septum; thal, thalamus. Hippocampus and Cortex. Next to striatum, the hippo- campus shows the densest immunoreactive labeling for the ability to label neurons in these areas depends, to some PLC-I. The hippocampal pyramidal neurons are densely extent, on fixation, in that shorter fixation times (6- to 10-hr labeled, with the dentate granule cells showing less-dense postfixation) are necessary to reveal labeling of all neurons labeling. PLC-II immunoreactivity was expressed in all in these areas; with longer fixation times (24- to 48-hr neurons in the hippocampus and dentate gyrus. However, postfixation) a very restricted population of neurons show 3210 Neurobiology: Gerfen et A Proc. Natl. Acad. Sci. USA 85 (1988)

ta 1 e; FIG. 2. PLC-I (A', B', and C') and PLC-II (A2, B2, and C2) labeling in the basal ganglia. In the caudate putamen 90 ...IR-Alp B1 (CP), medium-sized neurons display dense immunoreac- tive labeling for both PLC-I (A') and PLC-I (A2). These neurons provide efferent projections to the globus pallidus (GP) and substantia nigra (SN), and these projections contain both isozymes as evidenced by neuropil staining in both nuclei. In both the globus pallidus (B') and substantia nigra (C'), PLC-I labels the neuropil but does not label the recipient neurons that are identified as perikaryal ghosts (arrows). On the other hand, in both these nuclei (B2 and C2), PLC-II labels both the neuropil and the neurons in these areas (arrows). PLC-I immunoreactivity (Fig. 3). In these cases all neurons in the are labeled (Fig. 3 A and C), whereas in : x ::PLC2.: both the hippocampus (Fig. 3 A and D) and dentate gyrus (Fig. 3A), longer fixation regimens result in labeling of only restricted populations of neurons, which appear possibly to ...... 2. be interneurons. PLC-I immunoreactivity is distributed in the neuropil of most of the cerebral cortex, where the expression in the CAl:Iden entorhinal and olfactory cortical areas is considerably lower. PLC-I immunoreactive neurons are also seen scattered throughout the cortex, being most prevalent in motor and A CA4 sensorimotor cortical areas. PLC-I-immunoreactive neurons are most often pyramidal neurons in layers IV-VI. PLC-II immunoreactivity is, as is the case with labeling in the hippocampus, dependent on postfixation times. Again, with shorter postfixation nearly all cortical neurons show PLC-II immunoreactivity, whereas with longer postfixation times, a slightly more restricted labeling pattern occurs, which is not as marked as in the hippocampus. With postfixation regi- mens of 24-48 hr, groups of cortical pyramidal cells, which appear to have fasciculated apical dendrites, stand out with more intense labeling than neighboring neurons (Fig. 3B). Thalamus. Within the thalamus, PLC-II immunoreactivity appears in all neurons (Fig. 4B2), whereas PLC-I immuno- reactivity is restricted to a distinct subset of neurons (Fig. ICAI CA4 4B'). PLC-I immunoreactivity is present in the majority of - neurons in the thalamic reticular nucleus and is also present in some neurons in the medial part of the mediodorsal thalamic nucleus and neurons in the paratenial thalamic nucleus (Fig. 4B'). PLC-I immunoreactivity also shows a differential expression in the neuropil of thalamic nuclei, showing mod- erate staining in the mediodorsal, lateral dorsal, ventrolateral, ventromedial, reuniens, and posterior thalamic nuclei, but being very weak in the anterior dorsal, anterior ventral, intralaminar, and ventral posterior thalamic nuclei. One ofthe clearest examples of the differences in the thalamic distribu- tion of the two PLC-immunoreactive patterns exists in the medial , which is devoid of PLC-I immunoreactivity FIG. 3. PLC-II immunoreactivity in the cortex and hippocampus (Fig. SB'), but which contains PLC-II immunoreactive neu- in which differential labeling of specific neurons resulted from a long rons (Fig. SB2). Additionally, the target of the projections of postfixation regimen (see text). (A) Low-magnification photomicro- the medial habenula, the in the graph of area of somatosensory cortex (ctx), subiculum (sub), hippo- (12), shows PLC-Il labeling of the neuropil (Fig. campus (CA1), and hippocampal C4 hilar region (CA4) labeled for 5C2) but not `PLC-I immunoreactivity (Fig. SC'). PLC-II immunoreactivity. den, Dentate gyrms. (B) In cortex many . Compared with labeling in the rest of the neurons in all laminae are labeled, but in layer 5 clusters ofpyramidal brain, PLC-I immunoreactivity is relatively sparse in the cells with fasciculated dendrites are more densely labeled than are neighboring neurons. (C) In the subiculum most neurons are densely hypothalamus, although there is a weak neuropil labeling. As labeled. (D) In the CA1 region ofthe hippocampus nonpyramidal cells for labeling in the hippocampus, PLC-II immunoreactivity in are labeled in the pyramidal layer and just subjacent to the white the hypothalamus depends on fixation, and short postfixa- matter. (E) In the CA4 hilar region, between the blades ofthe dentate tion regimens were required to reveal labeling of nearly all gyrus, nonpyramidal cells are labeled. Neurons labeled in the hippo- neurons. Longer fixation regimens abolished hypothalamic campus are structurally of the interneuron type. Neurobiology: Gerfen et al. Proc. Natl. Acad. Sci. USA 85 (1988) 3211 PLC.] PLC 2 4. pvn pvn Isi Msd Ms~~~~s Al A2 ms A2 ms Al eamh ad mh 4 w f, _i l., md lh

B1.., Bl --FIt: B2 .... ~avBl~~~~~~. av $A vXm / ,so ... osvg I rt pt Cl ipn C ipn VI am FIG. 5. PLC-I (A', B', and C') and PLC-2 (A2, B2, and C2) B2 re immunoreactivity in the paraventricular nucleus of the hypothala- mus (A) and in the habenulo-interpeduncular nucleus system (B and C). The hypothalamus, in general, and the paraventricular nucleus FIG. 4. PLC-I- (A' and B') and PLC-Il- (A2 and B2) immuno- (pvn), in particular, shows low levels of PLC-I immunoreactivity reactive labeling in the septal area (A) and thalamus (B). (A) In the (A') in the neuropil, whereas PLC-I1 immunoreactivity (A2) labels septal area PLC-I immunoreactivity (A') labels many neurons and most paraventricular neurons. In the habenula (B) PLC-I immuno- densely labels the neuropil in the dorsal lateral septal nucleus (lsd), reactivity (B') is weak in the neuropil of the lateral habenula (lh) and labels scattered neurons and lightly labels the neuropil in the almost absent in the medial habenula (mh), and little labeling occurs intermediate lateral septal nucleus (lsi), and is absent in the medial in the interpeduncular nucleus (ipn, C'). PLC-I immunoreactivity septal nucleus (ms). PLC-I immunoreactivity (A2) labels most (B2 and C2) labels most neurons in the lateral and medial habenula neurons in all septal nuclei. (B) In the thalamus PLC-I immunoreac- (B2) and labels the neuropil in the interpeduncular nucleus (C2), tivity (B') labels most neurons in the reticular thalamic nucleus (rt) where projections of the medial habenula terminate. and labels scattered neurons in the paratenial (pt) and mediodorsal thalamic nucleus (md). Also, moderate labeling of the neuropil both of occurs in the ventrolateral (vl), mediodorsal (md), paratenial (pt), these isozymes share certain biochemical properties, and reuniens (re) thalamic nuclei, and there is a paucity of neuropil primarily in having similar substrate specificities, they differ labeling in the anterodorsal (ad) and anteroventral (av) thalamic in their relative abundance in the brain and other tissues and nuclei. PLC-I immunoreactivity (B2) is expressed in most neurons in their localization within cells (6, 7). PLC-I is approximately in all thalamic nuclei. am, Anteromedial. 4 times more abundant in brain than is PLC-Il. PLC-I appears to be relatively brain specific, whereas PLC-I is, at least labeling with PLC-II antibodies, except in the paraventricu- immunologically, similar to isozymes present in most other lar nucleus. Labeling in this nucleus is notable in that PLC-I tissues immunoreactivity is absent, such that the nucleus stands out including seminal vesicles, heart, kidney, spleen, and against the weak labeling in the rest of the hypothalamus lung (S.G.R., unpublished work). Biochemical purification (Fig. 5A'). By contrast, PLC-Il immunoreactivity is present data suggest that PLC-I is equally distributed between mem- in most paraventricular neurons (Fig. 5A2). brane and cytosolic fractions, whereas >90%o of PLC-II is Other Areas. Two other areas are notable for localization purified from the cytosolic fraction (7). Using the highly of PLC-I-immunoreactive neurons. One is the basolateral purified mAbs that had been used to biochemically distinguish nucleus of the amygdala, whose neurons are intensely la- PLC-I and PLC-Il (6), we demonstrate differential distribu- beled with PLC-I immunoreactivity. The other area is the tion of these isozymes in the rat brain with immunohisto- septal area; in this area, PLC-I immunoreactive neurons are chemical techniques. Both PLC-I and PLC-II immunoreac- restricted to the dorsal segment of the lateral septal nucleus, tivity appears to be localized to neurons, and, at least at the whereas all areas of the lateral septal area show immuno- light-microscopic level, there is little suggestion of glial reactive neuropil, but the medial septal area, including and expression. PLC-Il immunoreactivity is rather ubiquitous, extending into the basal forebrain nuclei that contain the such that it is localized in nearly all neurons in all brain areas major cholinergic cell groups, are distinct in their absence of examined. However, the relative density of immunoreactiv- any PLC-I immunoreactivity. PLC-II immunoreactivity is ity, particularly at the cellular level, suggests that PLC-I is homogenously localized in neurons of the lateral septal more abundant than is PLC-II. Although such qualitative complex, as well as in the and the observations must be viewed with caution, they are consis- basal forebrain nuclei. tent with biochemical data, which shows that PLC-I is -4 times more abundant than PLC-Il in the brain (S.G.R., unpublished work). Additionally, the patterns of immuno- DISCUSSION reactive labeling for PLC-Il are variable, being somewhat At least two isozymes of PLC have been purified and char- dependent on fixation properties, whereas PLC-I immuno- acterized from the brain, PLC-I and PLC-II (5, 6). Although reactivity is consistently stronger in cells in which it is 3212 Neurobiology: Gerfen et al. Proc. Natl. Acad. Sci. USA 85 (1988) localized. This may reflect the differential distribution of the A simple correspondence between isozymes of PLC and two isozymes within cells suggested by biochemical data, PKC may not occur, but rather their relative distributions in which show that PLC-II is not only less abundant, but is individual neurons may be determined by multiple constitu- confined principally to the cytosol. It is important to stress ents of the metabolic pathways regulating signal transduc- that the variability in immunolabeling for PLC-Il, which tion. The striatonigral system may be a useful system for appears dependent on fixation conditions, precludes a defin- unraveling such complex interactions. This system shows itive description of the relative abundance of these isozymes the highest concentration of PLC-I immunoreactivity. Fur- in different brain areas or neuronal cell types. thermore, other markers of second-messenger systems, such The present immunohistochemical data suggest that PLC-I1 as some isozymes of PKC (15), P(BtO)2 (13, 14), forskolin is rather ubiquitously expressed by most neurons in the brain, binding to mark adenylate cyclase-binding sites (13), and although PLC-Il may be present in higher concentrations in immunoreactivity for the G protein Go (18) are each localized some neurons than in others. On the other hand, PLC-I in the striatonigral system. Although markers for PKC and appears to have a much more restricted distribution. PLC-I Go are present in the striatum at levels comparable to that in immunoreactivity is particularly dense in the neurons of the cortex, forskolin binding is similar to PLC-I immunoreactiv- striatum and in their efferent projections to the globus pal- ity in being present in considerably higher levels in the lidus, entopeduncular nucleus, and substantia nigra. Notably, striatum. Adenylate cyclase (19), which is thought to be PLC-I immunoreactivity is absent in the target neurons of the marked by forskolin binding (13), and DARPP-32 (20) are striatal projections, whereas PLC-II immunoreactivity does linked to D1 dopamine receptors (21), which are present on label both the projections to and the target neurons in the striatonigral neurons. Various peptides in these neurons, globus pallidus and substantia nigra. The hippocampus also which are displays a particularly high level of PLC-I immunoreactivity; those same neurons that display high concentra- both pyramidal cells of the hippocampus and granule cells in tions of PLC-I immunoreactivity, have been shown to be the dentate gyrus are densely labeled. PLC-II immunoreac- regulated by dopamine (22). Cyclic AMP has been suggested tivity is also present in these neurons but may be more to inhibit the activation of PLC (23). It is thus of interest to concentrated in the interneurons of the hippocampus than in explore the possible interactions between the various con- the pyramidal cells. The septal nuclei and the thalamus are stituents of second-messenger systems in the regulation of also examples of brain areas that display a restricted distri- peptide expression by dopaminergic receptors. bution of PLC-I immunoreactivity as compared with a more 1. Berridge, M. J. & Irvine, R. F. (1984) Nature (London) 312, ubiquitous distribution of PLC-II immunoreactivity. 315-321. PLC functions in hormonal or transmitter transduction by 2. Nishizuka, Y. (1984) Science 225, 1365-1370. producing two second messengers, acyl2Gro and inositol 3. Majerus, P. W., Connolly, T. M., Deckmyn, H., Ross, T. S., phosphates, after being stimulated via a receptor linked G Bross, T. E., Ishii, H., Bansal, V. S. & Wilson, D. V. (1986) protein (1-4). The functional significance of the localization Science 234, 1519-1526. of PLC in a particular neuron will be clarified by determining 4. Worley, P. F., Baraban, J. M. & Snyder, S. H. (1987) Ann. the specific receptor-linked G protein that regulates its Neurol. 21, 217-229. stimulation and the targets of the second messengers that it 5. Ryu, S. H., Cho, K. S., Lee, K.-Y., Suh, P.-G. & Rhee, S. G. generates. The widespread distribution of suggests (1986) Biochem. Biophys. Res. Commun. 141, 137-144. PLC-II 6. Ryu, S. H., Cho, K. S., Lee, K.-Y., Suh, P.-G. & Rhee, S. G. that it may function with multiple neurotransmitter/hormo- (1987) J. Biol. Chem., 262, 12511-12518. nal receptor transducing complexes. On the other hand, 7. Lee, K.-Y., Ryu, S.-H., Suh, P.-G., Choi, W. C. & Rhee, because of its more restricted distribution, a comparison of S. G. (1987) Proc. Nati. Acad. Sci. USA 84, 5540-5544. the distribution of PLC-I with the distributions of specific 8. McLean, I. W. & Nakane, P. K. (1974) J. Histochem. Cyto- receptors, G proteins, and targets of acyl2Gro may be more chem. 22, 1077-1083. informative. For example, one product of PLC hydrolysis is 9. Hsu, S. M., Raine, L. & Fanger, H. (1981) J. Histochem. acyl2Gro, which has a stimulatory effect on protein kinase C Cytochem. 29, 577-580. (PKC), and recent studies have mapped the distribution of 10. Itoh, K., Konishi, A., Nomura, S., Mizuno, N., Nakamura, Y. this family of enzymes (13-16). The binding of & Sugimoto, T. (1979) Brain Res. 175, 341-346. distribution 11. Gerfen, C. R. (1984) Nature (London) 311, 461-464. the phorbol ester phorbol 12,13-dibutyrate [P(BtO)2, refs. 12. Herkenham, M. H. & Nauta, W. J. H. (1979) J. Comp. Neu- 13, 14], whose binding site is thought identical to that of PKC rol. 187, 19-48. (13, 14, 17), matches in many respects the pattern of PLC-I 13. Worley, P. F., Baraban, J. M., De Souza, E. B. & Snyder, immunoreactivity. Both P(BtO)2 binding sites and PLC-I S. H. (1986) Proc. Nati. Acad. Sci. USA 83, 4053-4057. immunoreactivity are present in the cortex, striatum, hippo- 14. Worley, P. F., Baraban, J. M. & Snyder, S. H. (1986) J. campus, and thalamus, although levels in the hypothalamus, Neurosci. 6, 199-207. particularly in the paraventricular nucleus, are low. The 15. Brandt, S. J., Niedel, J. E., Bell, R. M. & Young, W. S. relative distribution of these two markers are similar in some (1987) Cell 49, 57-63. areas; for instance, specific patterns of thalamic labeling 16. Mochly-Rosen, D., Basbaum, A. I. & Koshland, D. E., Jr. (1987) Proc. Nati. Acad. Sci. USA 84, 4660-4664. roughly correlate. However, in other areas marked differ- 17. Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, ences exist; for example, in the cortex P(BtO)2 labeling is U. & Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847-7851. comparable with that in the striatum. Although P(BtO)2 18. Worley, P. F., Baraban, J. M., Van Dop, C., Neer, E. J. & binding may provide a sum of PKC distribution, recent Snyder, S. H. (1986) Proc. Nati. Acad. Sci. USA 83, 4561- immunohistochemical (16) and in situ hybridization studies 4565. (15) have revealed differential patterns of distribution for the 19. Kebabian, J. W. & Calne, D. B. (1979) Nature (London) 277, various PKC isozymes. However, none of these isozyme 93-%. maps matches exactly with that for PLC-I. In some cases 20. Ouimet, C. C., Miler, P. E., Hemmings, H. C., Jr., Walaas, there appeared to be a complementary distribution of an S. I. & Greengard, P. (1984) J. Neurosci. 4, 111-124. 21. Dawson, T. M., Gehlert, D. R., Yamamura, H. I., Barnett, A. isozyme of PKC with PLC-I. For example, in the study by & Walmsley, J. K. (1985) Eur. J. Pharmacol. 108, 323-325. Mochly-Rosen et al. (16), mAb CK 1.4 did not label the 22. Young, W. S., III, Bonner, T. I. & Brann, M. R. (1986) Proc. cortex, striatum, or hippocampus, and in the thalamus did Nati. Acad. Sci. USA 83, 9827-9831. not label the reticular thalamic nucleus-areas in which 23. Watson, S. P., McConnell, R. T. & Lapetina, E. G. (1984) J. PLC-I immunoreactivity was highly concentrated. Biol. Chem. 259, 13199-13203.