Molecular Brain Research, 8 (1990) 311-317 311 Elsevier

BRESM 70238

Cellular and subcellular localization of protein kinase C in cat visual cortex

Weiguo Jia 1, Clermont Beaulieu 1, Freesia L. Huang 2 and Max S. Cynader 1

1Department of Ophthalmology, University of British Columbia, Vancouver, B.C. (Canada) and 2Department of Health and Human Services, NIH, Bethesda, MD 20892 (U.S.A.)

(Accepted 8 May 1990)

Key words: Protein kinase C; Visual cortex; Cortical plasticity; Immunocytochemistry

Polyclonal antibodies against 3 protein kinase C (PKC) subtypes (I, II and III) were applied to localize the kinase in cat visual cortex. These antibodies exclusively stained neuronal cells. Both pyramidal and non-pyramidal cells exhibiting PKC-like immunoreactivity were concentrated in layers II, III, V and VI with relatively few cells in layer IV. Electron microscopic examination did not reveal any presynaptic localization of the kinase. PKC immunoreactivity remained normal in a zone of cortex surgically isolated from the rest of the brain by an undercut procedure. These results suggest that PKC is heterogeneously distributed in adult cat visual cortex; the kinase recognized by the polyclonal antibodies is localized postsynaptically in intracortical neurons of the superficial and deep cortical layers and the expression of the kinase is not regulated by extracortical input.

INTRODUCTION

Prote in kinase C (PKC) is a Ca2+/phospholipid -

dependen t kinase which is act ivated by diacylglycerol

when this second messenger is re leased by hydrolysis of

phosphat idyl inos i to l 4 ,5-bisphosphate (PIPE) 25'26. The

hydrolysis of PIP 2 results from the occupancy of a wide

var ie ty of neuro t ransmi t t e r and neu romodu la to r recep-

tors. Hence , PKC can be act ivated under a var ie ty of

c i rcumstances and play different roles in various cellular

funct ions 1'5. In the deve lopmen t of the nervous system,

P K C is requi red for nerve growth factor s t imulated

neurogenes is 7 and it phosphory la tes various develop-

men t - re l a t ed prote ins such as GAP43 ~8. In addi t ion,

recent work shows that the expression of PKC itself is deve lopmenta l ly regula ted 8'14'32'35 (W. Jia et al . , in

p repara t ion) . In the adul t , brain PKC has been shown to

be involved in several dist inct neuronal processes, such as

enhancemen t of neuro t ransmi t t e r release 19,23, and mod-

ula t ion of receptors and ion channels 4,22.

Ear l i e r s tudies with complemen ta ry D N A ( c D N A )

suggested that there were four subtypes of PKC, i.e. a ,

i l l , f12 and ~,2o, which co r responded to PKC III , PKC II

( i l l and f12), and PKC I isolated from rat brain with hydroxyapa t i t e columns 12. Later , Ono 27 and his col-

leagues ident i f ied 3 addi t ional m R N A s belonging to PKC

family in rat brain, named 6-, e-, and C-subtypes,

a l though these 3 new isozymes have not yet been

isolated. The P K C I or ), i sozyme is r epor t ed to be bra in

specific while the PKC II (fll/fl2) and I I I ( a ) are found in bra in and o ther tissues 2'35. Previous immunocy tochem-

ical studies using ant ibodies against P K C 1°'11A3'29 or PKC

isozymes 31'33 have revea led that P K C is he te rogeneous ly

dis t r ibuted in the bra in and is especial ly rich in hippo-

campus, cerebra l cor tex and cerebe l lum 11. However ,

de ta i led informat ion about cel lular and subceUular dis-

t r ibut ion of the kinase in sensory processing systems such

as cat visual cor tex has not been descr ibed. In this paper ,

polyclonal ant ibodies against three PKC isozymes (I, II

and 111) 15 was uti l ized to invest igate the dis t r ibut ion of

PKC in cat visual cortex.

MATERIALS AND METHODS

Animals Three adult cats were utilized in this experiment. In one cat, the

visual cortex on one side of the brain was surgically undercut two weeks before peffusion. This surgical procedure has been described previously 3°. Briefly, a portion of the visual cortex of one hemi- sphere was isolated by three 1-cm-deep scalpel cuts: two cuts extended from the midline to the lateral edge of the marginal gyms, the third cut run parallel to the midline, but was angled at a 45 ° angle from the lateral edge of the marginal gyrus to the medial bank. In this manner, the white matter underlying a portion of visual cortex was completely cut and this part of visual cortex was isolated from any input from subcortical and other cortical areas.

The cats were deeply anesthetized with euthanyl and perfused through the aorta with 500 ml of 0.1 M phosphate buffer (pH 7.2, PB) followed by two liters of 4% paraformaldehyde in PB at room temperature. For electron microscopy, 0.1% glutaraldehyde was

Correspondence: W. Jia, Eye Care Center, 2550 Willow Street, Vancouver, B.C. V5Z 3N9, Canada.

0169-328X/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)

312

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313

added. The brain was removed and postfixed in 4% paraformalde- hyde overnight at 4 °C. The visual cortex (areas 17 and 18) was dissected out and cut into 50/~m sections with a vibratome.

lmmunoreaction The sections were washed 3 x 10 min in 0.1 M PB and were

sometimes preincubated for 30 min at room temperature with 10% normal rabbit serum. The polyclonal antibodies against PKC used were those raised by Drs. K.-P. Huang and EL. Huang 17. Sections were incubated with primary antibodies for 24-72 h at 4 °C at a dilution of either 1:2000 or 1:8000. After washing for 3 x 10 min in PB, the sections were incubated for 2 h at room temperature with biotinylated rabbit-anti-goat IgG. The sections were then washed for 3 x 5 min in PB and followed by 1 h incubation with avidin-biotin-HRP reagent (ABC kit from Vector). Following another 3 x 5 min wash, the HRP was developed by reacting with 0.003% H20 2 and 0.05% DAB in 0.1 M Tris buffer (pH 7.2). Occasionally, 7% NiC12 was added to intensify the staining.

As controls, normal goat serum was substituted for the antisera in adjacent sections. No immunoreactivity was found in those sections, indicating specificity of immunostaining.

Sections used for light microscopic observations were washed in PB, dehydrated in ascending concentrations of ethanol and cover- slipped with Permount.

Sections for electron microscopic observations were washed in phosphate buffer and postfixed for 30--60 min in 1% osmium tetroxide dissolved in 0.1 M PB (pH 7.4). They were washed again in PB, then dehydrated in ethanol (1% uranyl acetate was included in the 70% ethanol stage for 40 min), immersed in propylene oxide (2 x 10 min) and finally embedded on glass slides in Durcupan ACM (Fluka) resin. Portions of interest were cut out from the slides and rembedded. Serial ultrathin sections were cut on an ultrami- crotome and viewed under a Philips 400 at 40 kV.

RESULTS

Light microscopic observations T h e i m m u n o r e a c t i o n of the po lyc lona l an t ibod ies

aga ins t P K C in cat v isua l cor tex is shown in Fig. 1. The

d e g r e e of cell s t a in ing var ies s t r ik ingly a m o n g cort ical

l a m i n a e . T h e u p p e r pa r t o f layer II a n d layer VI (Fig.

l b , c ) c o n t a i n e d the h ighes t p r o p o r t i o n of dense ly s ta ined

n e u r o n s t h r o u g h o u t the cor tex , g iving the a p p e a r a n c e of

the h ighes t i m m u n o r e a c t i o n . A n o t h e r p o p u l a t i o n of

n e u r o n s , m a i n l y loca ted in layers I I I a n d V, were also

i m m u n o p o s i t i v e b u t the s ta in ing was cons is ten t ly w e a k e r

t h a n tha t s h o w n in layers I I a n d VI . In layers I I I and V,

Fig. 2. Immunostaining of PKC in area 19 of cat visual cortex. Compared with Fig. la, many pyramidal cells in layer V present strong PKC immunoreactivity and the overall staining is also stronger in all cortical layers. Bar = 200/tm.

however , a few highly i m m u n o r e a c t i v e cells were found.

In add i t ion , neu rop i l in layer I was also s ta ined a l though

we could no t rule ou t the in f luence of an ' edge effect ' in

this layer. In cont ras t to these layers, layer IV con ta ined

very few i m m u n o r e a c t i v e n e u r o n s , giving the appearance

of the least immunoreac t i v i t y in the visual cortex. It is

in te res t ing to no t e that by increas ing the concen t ra t ion of

the p r imary an t ibod ies (up to 1/2000), the inc idence of

weakly s ta ined cells increased in layer IV while no

changes were observed in o the r layers. Cells con ta in ing

Fig. 1. Light micrographs of visual cortex labelled with polyclonal antibodies for PKC. a: laminar distribution of immunoreaction stained with the polyclonal antibody. PKC-like immunoreactivity was located in the superficial and deep layers of the visual cortex. The most densely stained cells were in layers II and VI while many less strongly stained cells were found in layers III and V. Neuropile in layer I and some cells in white matter also showed PKC immunoreaction. Layer IV showed the least staining. The somewhat heterogeneous staining in superficial layers, which can be observed in Fig. la) was not consistent from section to section and some of the staining in layer I may represent an edge artifact. Bar = 100/~m. b: higher-power photograph of layers I and II of the visual cortex shows PKC-positive neuropile and neurons. Neurons in upper layer II presented very strong immunoreactivity while cells in lower part of the layer showed less intense staining. The entire cell membrane was stained in those neurons with strong immunoreaction. In other PKC-positive cells, the immunoreactivity was present only in the cytoplasm. Most of the immunoreactive neurons in these layers were small pyramidal cells. Bar = 50 pm. c: higher-power photograph of lower layer V and upper layer VI in the visual cortex. A few large pyramidal cells in layer V shown in the top part of the picture were PKC positive. Many neurons including pyramidal (big arrowhead) and non-pyramidal (big arrow) cells in layer VI contained PKC immunoreactivity to various degrees. In most cases, nuclei were not stained. Numerous long processes presumed to be apical dendrites (small arrows) were stained. Sometimes, stained fibers bifurcated in upper layer VI (small arrowhead). Notice that similar fibers were not found in the superficial layers of cortex. Bar = 50 ~m. d: white matter of cat visual cortex stained with the polyclonal antibodies. The polyclonal antibodies stained a few cells in the white matter. Some of the cells appeared bipolar with ovoid-shaped cell bodies. Neither axons nor glial cells were stained. Bar = 20/~m.

314

Fig. 4. Electron micrographs of visual cortex tissue labelled with polyclonal antibodies for PKC. a: cytoplasm of a PKC-immunore- active cell. Note that the end-product can be found throughout the cytoplasm and that the membranes of mitochondria present some degree of immunoreactivity. Compare for example the membranes of the mitochondria marked with a curved arrow with those of this organelles present in the neuropil outside the cytoplasm. The membranes of the cytoplasm are densely stained (arrows) and appear as a thin rim around the cell body. cyt, cytoplasm; nu, nucleoplasm. Bar = 1/~m. b: positive dendrite. The end-product is principally seen on the microtubules (small arrows) and to a lesser extent on the plasma membranes (arrowheads). The postsynaptic side of the synaptic contacts (large arrows) is slightly darker in the immunopositive dendrite (+) than in the other immunonegative postsynaptic target (-). ter, synaptic terminal. Bar = 0.25/~m.

Fig. 3. Immunoreaction of PKC in the undercut visual cortex, a: a portion of visual cortex was isolated surgically (see Methods and Materials) and the white matter underlying the area was completely sectioned Bar = 1 mm. b and c show a control region and an operated region of the visual cortex, respectively. There was no significant difference in immunoreaction between these two areas. Bar = 100/~m.

PKC immunoreactivity were also localized in the white matter especially in the vicinity of layer VI (Fig. ld). Axons in the white matter were not stained by the antibodies.

Compared with adjacent sections stained by Cresyl violet, PKC-like immunoreactivity was present in about 70% of neurons in layers II, I!I, V and VI, respectively. Although both pyramidal and non-pyramidal cells were stained, pyramidal cells were more likely to be stained and stained more densely than other types of cells. Under the light microscope, the immunoreactivity appeared in both perikaryal and dendritic cytoplasm including the proximal portions of the dendrites. In some cells, nuclei were lightly stained. Neuropil staining was also present in the upper part of layer II but tended to be irregularly distributed (Fig. la,b). However, neither axons nor axonal terminals were found to be PKC-positive.

The staining patterns in areas 18 and 19 were similar to that of area 17. However, the intensity of the immunoreactivity varied between the three areas. It seemed that there were more PKC-positive pyramidal cells in layer V in area 18 and the most heavily stained neurons, especially pyramidal cells in layer V were found in area 19 (Fig. 2).

In the undercut cat, the lesion completely isolated the visual cortex (areas 17 and 18) from the subadjacent cortex and subcortical structures (Fig. 3). However, there was no difference in immunostaining between the under- cut part of the cortex and control areas in either the number of stained cells or in the pattern of the staining (Fig. 3b,c).

Electron microscopic observations Fig. 4 presents electron micrographs of visual cortex

tissue labelled with the polyclonal antibodies of PKC. As suggested by light microscopic observations, electron microscopy showed that no glial cells were labelled with the antibodies; only neurons were stained. These neurons could receive synaptic contacts from unlabelled termi- nals. At the level of the cell body, end-product could be found throughout the cytoplasm and to a lesser extent into the nucleoplasm. In addition, while the membranes of mitochondria, Golgi apparatus, or endoplasmic reti- culum presented some degree of immunoreactivity, no clear labelling was present within these cell organelles (Fig. 4a). It is also shown that the cytoplasmic mem- branes were densely stained (Fig. 4a) forming a thin rim delimiting the cytoplasm. Positive staining was also found in dendrites (Fig. 4b). In these structures, the end- product was principally seen on the microtubules (Fig. 4b) and to a lesser extent on the plasma membranes. In our preparations, the internal and external membranes of dendritic mitochondria did not present any clear label-

315

ling. At the level of the synaptic contact, the immuno- reaction became more marked at the postsynaptic side of the contact (Fig. 4b). This increased immunoreaction might be due to the presence of the postsynaptic opacity which is the characteristic feature of asymmetrical syn- apses. We could not decide whether this structure was especially highly immunoreactive for PKC or alterna- tively that the increased immunoreaction was due only to the summation of dark appearance of the postsynaptic opacity with PKC labelling of the plasma membrane.

Despite extensive searches, PKC immunoreactivity was never found in vesicle-containing terminals. Only postsynaptic elements (somata and dendrites) were la- belled. In some situations at the light microscopic level, some roundish immunoreactive profiles of different sizes can appear as possible terminals. Electron microscopic observations of serial ultrathin sections through some of these profiles revealed instead that they were dendrites cut transversally.

DISCUSSION

Our results showed that the polyclonal antibodies exclusively stained neurons. These results are consistent with those of F. Huang 12 with the antibodies in rat brain.

The polyclonal antibodies utilized in our experiments recognized PKC I, II, III isozymes in Western blot 11A2. As a comparison, we utilized a monoclonal antibody (MC5, Amersham) claimed to react with PKC II and III isozymes in western blot (Amersham, data sheet). Inter- estingly, the two different antibodies for PKC gave rise to different staining patterns. In contrast to the staining pattern of the polyclonal antibodies which stain neurons only, the MC5 monoclonal antibody heavily stained glial cells in the white matter (data not shown). Numerous glia as well as neurons in the grey matter showed fairly weak immunoreactivity of the MC5 antibody in the adult cat visual cortex. The distribution of MC5-positive cells were homogenous over all layers of the cortex. Our results with this monoclonal antibody are consistent with those of Stichel and Singer a2. It is possible that the polyclonal antibodies may not detect the low level of PKC III in fixed cat brain, since the antibodies titer against type III was lower than those of the other two subtypes in immunoblot analysis 17. The level of PKC in glial cells may be low 2 and if the PKC III is the only subtype of PKC expressed in gila of adult cat visual cortex, the polyclonal antibodies may not be able to label them. Further studies with well-defined oligonucleotide probes and in situ hybridization would be necessary to address these issues.

Neurons with the strongest immunoreactivity for the polyclonal antibodies of PKC are located in layers II and

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VI of the visual cortex while many cells in layers III and V also showed less marked PKC immunoreactivity. This laminar pattern of immunostaining for the polyclonal antibodies resembles that observed using a monoclonal antibody against the f12 isozyme in rat neocortex 33, suggesting that major isozyme detected by our antibodies in adult cat visual cortex is possible the f12 one. This pattern is also similar to the results obtained with [3H]PDBu autoradiography in cat visual cortex 24 and to

the results of in situ hybridization with PKC ~', i l l , /32 cDNA probes in rat caudal neocortex z. The expression of PKC in visual cortical neurons may, however, not be 'all-or-none' since cells in the middle layers showed weak but specific immunoreaction with higher concentration of the antibodies. The differential expression of PKC isozymes at various levels in adult visual cortex may relate to its functions in intracellular signal transduction. High levels of PKC immunoreaction in the neurons of layer II and VI suggest that the kinase may be important in maintenance of synaptic plasticity in these layers as has been suggested in the hippocampus 2s.

An important result of our electronmicroscopic exam- ination is that PKC immunoreactivity is found only at postsynaptic locations in adult cat visual cortex. These results support previous work by Wolf et al. 34 who

showed that PKC was concentrated at postsynaptic densities (PSDs) prepared from rat brain. Most recently, Tsujino et al. reported that no immunoreactivity of three subspecies (ill,/32 and y) of PKC was found in presyn- aptic terminals of rat neocortex detected by monoclonal antibodies 33. In contrast, PKC immunoreactivity has

been localized in presynapt ic terminals of rat cerebral cortex 6 and in cerebellar Purkinje cell terminals 2~ with different antibodies. It is well established that PKC has a wide spectrum of substrates in vitro 25. The diversity in subcellular localization of the kinase in different brain areas suggests that PKC may play different roles in different neurons depending on its location within cells and on the physiological substrates colocalized with the enzyme. In addition, the subcellular location of PKC

indicated by different antibodies may also reflect the distribution of various isoforms of the kinase. It is clear that PKC isozymes are subtly different in their activities. For example, PKC I and III are much less well activated by diacylglycerol in the presence of phosphatidylserine and are more dependent on calcium than PKC II while unlike PKC III, PKC II is poorly activated by free arachidonic acid 16'26.

No difference in the staining of the polyclonal antibody was found in the undercut area of the visual cortex. Two conclusions were drawn from the results indicating that PKC immunoreactivity did not change after this manip- ulation. Firstly, PKC is localized in intracortical neurons of the visual cortex but not in the terminals of subcortical inputs or association fibers. This conclusion is coincident with the observation that PKC was located in presynaptic elements by EM examination. Secondly, PKC-immuno- reactivity in adult visual cortex is independent of the activity of those presynaptic terminals from subcortical or other cortical areas, at least after the survival period (2 weeks) allowed in these experiments. Taken together, these results imply that protein kinase C mainly plays a role in regulation of intrinsic neuronal activity in adult visual cortex. Interestingly, in experiments performed in kitten visual cortex, the same manipulation resulted in an increase in PKC immunoreactivity in the isolated region (Jia et al., in preparation), suggesting that the level of PKC is use-dependent in the developing visual cortex but not in adulthood. In contrast, immunoreactivity for Ca2+/calmodulin kinase, another calcium-dependent pro- tein kinase, is strongly regulated by retinogeniculate afferents in adult monkey visual cortex 9. When one eye was removed or closed, the immunoreactivity of Ca2+/ calmodulin kinase increased in the ocular dominance columns originally driven by the removed or closed eye. Although both of the kinases are thought to be important in modulation of neuronal activities, the difference in response to the alteration of cortical inputs in adulthood suggests that the two kinases are regulated in very different ways.

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