Molecular Brain Research, 19 (1993) 269-276 269 Elsevier Science Publishers B.V.

BRESM 70631 Research Reports

Quantitative distribution of protein kinase C a,/3, y, and e mRNAS in the hippocampus of control and nictitating membrane

conditioned rabbits

Ann Marie Craig *, James L. Olds, Bernard G. Schreurs, Andrew M. Scharenberg and Daniel L. Alkon

Laboratory of Adaptive Systems, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 (USA)

(Accepted 16 February 1993)

Key words: Protein kinase C; Gene expression; In situ hybridization; Hippocampus; Classical conditioning; Rabbit

We used oligonucleotide in situ hybridization and film autoradiography to quantitate the distributions of protein kinase C (PKt~) a,/3, 3', and mRNAs in subregions of rabbit hippocampus. Levels of each of the hippocampal PKC isozyme mRNAs and patterns of their regional distributions were remarkably invariant between individuals. Within stratum pyramidale, the highest levels of PKC a mRNA were in the CA2 region, while PKC/3 mRNA was maximally expressed in CA1, and PKC ~ mRNA in CA3; PKC y mRNA was abundantly expressed throughout Ammon's horn. Previous experiments employing quantitative autoradiography for [3H]PDBU (Olds et al., Science, 245 (1989) 866-869) revealed an increase in membrane-bound PKC in the CA1 region of rabbit hippocampus up to 3 days following classical conditioning of the nictitating membrane response. We report here that there were no differences in levels of PKC a, /3, y, or E mRNA between conditioned and control rabbits in any hippocampal region one day after training. These data are consistent with the hypothesis that PKC is post-translationally activated and translocated to the membrane during memory storage.

INTRODUCTION

Activation of the phospholipid-dependent enzyme

protein kinase C (PKC) is one of the critical events in

the transduction of many extracellular signals, includ- ing the response of neurons to synaptic stimulation 24.

Specific transmitter binding can bring about phospho-

lipid hydrolysis and transient intracellular increases in

the PKC activators diacylglycerol (DG), Ca 2+, and

arachidonic acid (AA). Activation of PKC is generally

accompanied by translocation from the cytosol to the membrane 7. PKC activation in neurons can lead to

changes in ion channel properties, transmitter release, and transmitter sensitivity t7. Activation of PKC or in-

troduction of activated PKC into Hermissenda pho-

toreceptors mimics the changes in protein phosphory- lation and reduction of voltage-dependent K + currents which occur during classical conditioning 2'3'13'23.

PKC is particularly abundant in the mammalian

hippocampus, a structure long known to be involved in memory formation 4°'44 and more recently the site of

studies of long-term potentiation (LTP) of synaptic efficacy18. PKC may be activated in hippocampal neu-

rons by multiple mechanisms including activation of

muscarinic cholinergic and excitatory amino acid re- ceptors 31'46. Injection of PKC into CA1 neurons poten-

tiates the synaptic response 15. PKC translocation and

activation are associated with and required for induc- tion of LTP in the perforant path and in the Schaffer collateral pathway 1,5,22. A role for PKC in hippocampal

memory processes was suggested by changes in the

distribution of membrane-bound PKC in the rabbit

hippocampus following classical conditioning of the nictitating membrane response (NMR) and in the rat hippocampus following discrimination learning 4'6'31'32'37.

This idea is further supported by the observation that

Correspondence: A.M. Craig, Present address: Department.of Neuroscience, University of Virginia School of Medicine, MR4 Annex, Room 5147, Charlottesville, VA 22908, USA. Fax: (1) (804) 982 4380.

270

both LTP and classical conditioning lead to a similar enhancement of phosphoinositide metabolism in rat dentate gyrus 2~. The possibility that transcriptionally based differences in PKC activity may affect memory consolidation was suggested by a correlation between spatial learning performance and hippocampal PKC activity in recombinant inbred strains of mice 48.

Molecular cloning has revealed the existence of at least 8 mammalian PKC isozymes, which differ in tis- sue and subcellular distribution and in kinetics of acti- vation by DG, Ca 2+, and AA 25'27. It is not known which

of the PKC isozymes are involved in LTP or memory processes. The first goal of this work was to extend previous in situ hybridization studies of PKC m4<4'~ to obtain quantitative relative distributions of PKC a, /3, 7, and e mRNAs in sub-regions of the hippocampus. The second goal was to test the hypothesis that an increase in PKC mRNA accompanies the increase seen in membrane-bound PKC in rabbit hippocampus dur- ing acquisition and consolidation of NMR condition- ing 31'37. We thus compared the levels of PKC c~, /3, 3,,

and e mRNA in hippocampal sub-regions of condi- tioned and control rabbits one day after training, the timepoint of maximal increase in phorbol ester bind- ing. While the possibility of changes in other isozymes or at other timepoints cannot be ruled out, our present observations tend to suggest that the change in distri- bution of membrane-bound PKC described previously occurs at the post-translational level.

M A T E R I A L S A N D M E T H O D S

Animal training Male New Zealand White rabbits (80-100 days old; = 2 kg) were

adapted to the training chambers for 80 min 1 day prior to training. Paired subjects received 80 presentat ions per day of a 400-ms, 1000-Hz, 82-decibel tone conditioned stimulus (CS) that cotermi- nated with a 100-ms, 50-Hz periorbital electrical pulse unconditioned stimulus (US). Paired presentations were delivered, on average, every 60 s. Unpaired subjects received 80 CS alone and 80 US alone presentations per day delivered, on average, every 30 s in an explic- itly unpaired manner. Naive subjects were restrained in the training chambers for the 80 min period corresponding to the duration of the paired and unpaired stimulus presentations. All animals received 3 days of training, at which time paired subjects exhibited > 90% conditioned responses as measured by a transducer attached to the nictitating membrane. Stimulus delivery and data collection were performed using a C o m p a q / A S Y S T system 39.

Probes and Northern blot analysis Synthetic 48-base oligodeoxyribonucleotide probes were prepared

on an Applied Biosystems DNA synthesizer (courtesy of Dr. J.C. Venter, NINDS). The sequences chosen were complementary to coding regions of published rabbit PKC cDNAs. The nomenclature used here is that of Ohno et al. 3°, which corresponds to that used for the rat isozymes. The PKC a probe ( C G G C T G C T T C C T G T C T T C - C G A G G G A C T G A T C A C C T T G T T G C C A G C G G G ) was comple- mentary to nt 934-981 of Ohno et al. 29. The PKC ~, probe ( G G A A G A A G A G G G G C C C A T C C G C A C C C G C T C A T A C A A C T C - C A G G G G G T A ) was complementary to nt 919-966 of Ohno et al. 3t~.

The PKC e probe ( G G T G G A T G A T G C T G C C C G G T G T T C C T C C - C C T C G G T T G T C A A A T G A C A ) was complementary to nt 1099- 1146 of Ohno et al. 2s. These probes all correspond to the D3 region between the regulatory and kinase domains. For PKC /3, probes were synthesized corresponding to both /31 ( G A G C T T A T C A G T - G G G A G T C A G T T C C A C A G G C T G T C T G G T G A A C T C T T T ) and /31I ( T T C C T G G T C G G G A G G T G T T A G G A C T G G T G G A T G G C G - G G T G A A A A A C C G ) ; these sequences are complementary to nt 1831 1878 and 1891-1938, respectively, in region D4 of Ohno el al. eg. Since differences in hybridization signal between the /3I and /3II probes were not apparent in preliminary experiments, these probes were pooled for the experiments reported in this paper. A random 48-mer ( C G C G C A G A T G T T A T A C T T C T G C T T T G T G C C - A T C C C A A T G T G G G G T T A ) was used as a control for specificity of hybridization.

RNA for Northern blot analysis was isolated from naive rabbit hippocampi either by the guan id in ium/ho t phenol method for total RNA 3s or by detergent-mediated lysis and extraction of cytoplasmic RNA 12. Poly(A) + and poly(A) RNA were separated by chromatog- raphy on oligo(dT)-cellulose. RNA was electrophoresed on a 1.1~ agarose gel containing formaldehyde and MOPS 3s and blotted onto GeneScreenPlus (NEN). Probes were labelled by tailing with termi- nal transferase (BRL) and [32P]~-dATP (NEN) to a specific activity of -~ 107 cpm/pmol . Prebyhridization and hybridization were per- formed as recommended by NEN. Wash conditions were exactly as for in situ hybridization (below), in order to test probe specificity under these conditions.

In situ hybridization One day after the third day of training, rabbits were anesthetized

with sodium pentobarbitol (5 m g / k g ) and decapitated. Brains were immediately frozen on dry ice and stored at - 8 0 ° C prior to prepara- tion of 12 # m sections cut 3 -4 mm posterior to the bregma. Sections were thaw-mounted on chrom-alum-coated slides and again stored at -80°C until use. Cresyl violet staining of a few sections per animal was performed to ensure corresponding rostral to caudal alignment of all sections used for in situ hybridization.

In situ hybridization was performed according to Young 5~ with the following modifications. Oligonucleotides were tailed with [3SS]a-dATP using terminal transferase to a specific activity of = 2 N 11) 7 cpm/pmol . Probe (6x 1W' cpm) was hybridized at 38.5°C overnight to 2 rabbit forebrain sections per slide in a volume of 95 ill. Sections were dipped in l × SSC, washed for 1.5 h in four changes of 2 × SSC/50% formamide at 43°C and then 1 h in two changes of II.5XSSC at room temperature, rinsed in water and then 70% ethanol, and air dried. These wash conditions were 15-20°C below the calculated melting temperatures3S'4S: addition of a 3'-tail has been found to change the melting temperature by only a few de- grees 47.

Sections were exposed to X-ray film (Kodak XAR-5). Autoradio- graphic standards (range 70-4900 nCi /g ) consisting of 12 # m sec- tions of [35S]dATP mixed with rabbit forebrain tissue were included for each piece of film. The hippocampal signal was well within the linear range of the film for all exposures used for quantitation. [This is not the case for the images shown in Fig. 2, where, to allow comparison by eye, a single exposure was used resulting in underex- posure (a ) or overexposure (/3 and y) of some images.] For higher resolution (Fig. 3), slides were exposed to Ilford K.5D emulsion and counterstained with Cresyl violet after development.

Quantitation of probe binding All image analysis was performed so that the experimenter was

blind to the experimental group to which each image belonged. Probe binding was measured from autoradiograms using the MCID image-analysis system (Imaging Research, St. Catharines, Ont.. Canada). Each autoradiographic image was digitized and each hip- pocampal image was then individually magnified by a factor of 2 and subjected to a low-frequency 3 × 3 convolution matrix filter opera- tion. Regions of interest were defined by manually tracing the outline of stratum pyrimidale or the granule cell layer with a line 3 pixels thick (which just covered the cell body layer). This thick line

A-A* 7

A-A* E

A-A*

271

means and SEMs were determined using NPlot (Dr. T. Nelson, NINDS) and plotted using CricketGraph.

6 - 5 - 4 - 3 -

2 -

Fig. 1. Northern blot analysis demonstrating specificity of PKC oligonucleotide probes. RNA was isolated from rabbit hippocampaus and electrophoresed in sets consisting of poly(A) + RNA (1.6 /~g cytoplasmic for a and E probes; and 5.6/,tg total for/3 and y probes) on the right and poly(A)- RNA (8 /.~g) on the left. Blots were probed with 32p-labelled oligonucleotides as indicated. Size markers

(BRL DNA ladder) are given in kilobases.

was subdivided into regions of interest as illustrated in Fig. 4A. Average probe binding values over all pixels within each region of interest were obtained by comparison with autoradiographic stan- dards. The values for each region of interest were averaged for both left and right hippocampi for all sections from each rabbit. Group

RESULTS

Probes and Northern blot analysis Antisense oligonucleotides specific for portions of

the coding regions of PKC a , /3 (I + II), y, and • were used for in situ hybridization of rabbit forebrain sec- tions. Probe specificity was demonstrated by Northern blot analysis using hippocampal RNA (Fig. 1). The sizes of mRNA species recognized by these probes were consistent with previous reports: a - 9.5 kb; /3 - 10 kb; 3' - 3.4 kb; and • - 7.5 kb 29'30"49. Smaller bands at 3.4-4 kb have also been reported to hybridize with a and /3 PKC probes in rabbit and other species, but with varying intensity, sometimes much less than the 9-10 kb bands t°'29.

Distribution of PKC isozyme mRNAs in rabbit hip- pocampus

Computerized autoradiographic images depicting oligonucleotide probe binding following in situ hy- bridization in the rabbit hippocampal formation are shown in Fig. 2. Distributions of the PKC isozyme

Fig. 2. Digitized autoradiographic images of rabbit hippocampus following in situ hybridization for specific PKC isozymes. These images were obtained from serial sections which were hybridized to the indicated probes and exposed on the same piece of film. [The section probed for PKC

a mRNA was underexposed, and the/3 and y sections overexposed to allow direct visual comparison.]

272

m R N A s in s t ra tum pyramidale were overlapping but

strikingly different. With in s t ra tum pyramidale, regions

of maximal expression differed be tween PKC isozyme

transcripts, with a maximal in CA2, /3 maximal in

CA1, and e maximal in CA3; PKC 3' m R N A was

a b u n d a n t th roughout the pyramidal cell layer. In the

denta te granule cell layer, levels of PKC • m R N A

were highest, a l though PKC /3, y and a transcripts

Fig. 3. Photomicrographs of rabbit hippocampus following in situ hybridization for specific PKC isozymes: (A) PKC a in the CA2 region; (B) PKC/3 in the CA1 region; (C) PKC 3, in the CA1 region; (D) PKC 3' in the CA3 region; (E) PKC ~ in the CA3 region; and (F) random control in the CA1 region. Following in situ hybridization, sections were processed for emulsion auroradiography and counterstained with Cresyl violet.

Bar = 10 ~tm.

were also present. While PKC /3, 3', and e mRNAs were most abundant within Ammon's horn, PKC ot mRNA was also very abundant in the fasciola cinereum.

Photomicrographs depicting hybridization signals at the cellular level are shown in Fig. 3. With all probes, the hybridization signal was greater over the large pyramidal cells than over the smaller interneurons.

Regional quantitative analysis of PKC mRNA levels Probe binding to PKC mRNAs was analysed by a

method similar to that used for quantitative autoradio- graphic [3H]phorbol ester binding to membrane-associ- ated PKC 31'32'37. Autoradiographic optical densities were measured for each of the regions of interest (shown schematically in Fig. 4) and converted to meas- ures of bound radioactive probe by comparison with [35S]tissue autoradiographic standards. The different isozyme probes were of identical length, 53-62% G + C, and were hybridized under identical conditions at 16-20°C below the calculated melting temperatures. Thus, after normalizing for small differences in specific activity of the probes, the different signal intensities are likely a reasonable estimate of the relative amounts

alpha

beta

~. gamma

~- epsilon 3000

2o00

~" 1000

4000

CA1 CA3 I~G

. . . . ' 0 ' 2 2 4 6 8 1 1

hippocampal region

2 it

I t

Fig. 4. PKC mRNA isozyme levels as measured by probe binding to hippocampal regions of naive rabbits. The diagram below the graph indicates the regions of interest for measurement of specific oligonu- cleotide probe binding. For each digitized autoradiographic image, a thick (3 pixel wide) line encompassing stratum pyramidale was di- vided into 10 regions of equal length for sampling. The outer and inner blades of the dentate gyrus were also sampled as individual regions. For each region of interest, data from left and right hip- pocampi were pooled. Hybridization of a random oligonucleotide resulted in less than 200 nCi /g control probe binding throughout the

regions of interest.

273

of mRNA present for each isozyme. Fig. 4B depicts PKC mRNA probe binding profiles (averaged from 5 naive rabbits).

Hippocampal PKC mRNA expression following NMR conditioning

Animals for training were randomly assigned to one of 3 groups: P (paired presentation of CS and US), UP (explicitly unpaired presentation of CS and US), and N (naive). After 3 days of training, all group P animals exhibited reliable conditioned extension of the nictitat- ing membrane (> 90% conditioned responses). One day after the 3 days of training, brains were processed to determine levels of hippocampal PKC mRNAs. The profiles in Fig. 5 were obtained by analysis of at least 3 sections per animal and either 7 (P, UP) or 5 (N) animals per group; data from the left and right hip- pocampal formations were pooled. There were no ap- parent differences in PKC a, /3, 3', or E mRNA levels between groups, either by visual inspection of original autoradiograms or by quantitative analysis. Statistical comparisons between the conditioned (P) and control (UP) groups indicated no significant differences (P > 0.2, t-test) in any of the PKC isozyme mRNA levels in any of the regions of interest. The sensitivity of the assay to detect possible between-group differences was indicated by the consistent demonstration of regional differences in probe distribution.

We also assayed mRNA levels in the hippocampus of conditioned and control rabbits immediately follow- ing day 1 of training, during acquisition of NMR condi- tioning. We previously reported a significant increase in phorbol ester binding in the CA3 stratum oriens in conditioned animals at this timepoint 37. However, no differences in PKC a, /3, y or E mRNA levels were apparent, although probe binding was rigorously quan- tified only for the CNS-specific isoform, PKC 3" (data not shown).

DISCUSSION

We used oligonucleotide in situ hybridization and film autoradiography to quantitate the distribution of PKC a, /3, 3' and E mRNAs within the rabbit hip- pocampus. The distribution is in general agreement with previous (non-quantitative) reports for the distri- bution of these mRNAs in rat hippocampus and for PKC a in rabbit hippocampus ~°'16'49. As suggested by Young 49 and Ito et al. 16, PKC a mRNA was indeed expressed at lower levels than the other isozyme tran- scripts and, as in rat, was particularly concentrated in the CA2 region of Ammon's horn and the fasciola cinereum. The striking expression of PKC /3 in the

0 uP

CA1 region was similar in rabbit (here) and r a t 10'34'49.

PKC/3 mRNA was not absent from CA2 in rabbit as it is in rat 10,49, and the ratio of PKC/3 mRNA expression

in CA1 to CA3 appeared to be higher in rabbit. A relatively high level of expression of PKC 3' mRNA across the whole pyramidal cell layer, as reported here in rabbit, has also been described for the rat 1°'49, and

the somewhat higher level in CA1 versus CA3 is con- sistent with protein levels in rat 19. The expression of

PKC • mRNA was the converse of PKC/3 and 7, with higher levels in CA3 than in CA1 in both rabbit and rat 49. Finally, the relative levels of PKC mRNAs we observed in rabbit dentate granule cells, with • highest and a lowest, also appears to be the case for rat 49.

An attempt to detect PKC/31 and PKC/311 mRNAs separately using probes against divergent sequences as reported by Ohno et al. 29 resulted in an essentially identical hybridization pattern with both probes. A similar phenomenon was also reported by Brandt et al. 1° using different probes for the rat mRNAs. The in situ hybridization results for PKC /3I mRNA are not consistent with the reported immunolocalization of PKC /31, which indicated that it was present at very low levels and mostly restricted to non-pyramidal cells in rat hippocampus 14'34, or with the specific activity

ratio of 43 : 1 for PKC/311 :/3I in the soluble fraction of rat hippocampus 25. The /3II subtype mRNA may be preferentially translated, or the /3I probe used here

N

1200 4000

1100

tooo ~ 3000

7OO

600 1000 i I | - I " I l

0 2 4 6 8 10 12

hippocampaJ region

274

t I I I • I • i

2 4 6 8 10 12

hippocampal region

4000 - 3000

E

~= ~; ~oo

1000 , = . . , , ~ 1000

2 6 8 10 12 0 hippocampal region hippocampal region

Fig. 5. PKC m R N A isozyme levels as measured by probe binding to hippocampal regions of nictitating membrane conditioned (P, paired) versus control (UP, unpaired; N, naive) rabbits. Animals were trained for 3 days, resulting in > 90% conditioned responses in the paired group, m R N A levels were assayed 24 h after the third day of training. The hippocampal regions sampled are as indicated in Fig. 4. The data were collected from

the left and right hippocampi of either 7 (P, UP) or 5 (N) animals per group.

may cross-react with the/311 form (although this is not predicted on the basis of published sequences).

The participation of PKC isoforms in LTP and memory processes may be regulated by their anatomi- cal localization and intracellular compartmentalization. The high level of PKC /3 in the CA1 region would be consistent with a function in consolidation of the NMR. PKC 3,, which is absent from presynaptic terminals but present in spines 19, may participate in postsynaptic consolidation events.

Although hippocampal lesions do not abolish simple delay conditioning of the NMR as performed here 38, the hippocampus is implicated in modulating higher order aspects of NMR conditioning. The hippocampus is essential for proper extinction, blocking, sensory preconditioning, latent inhibition, and appropriate stimulus generalization 33'38'41'42. The cholinergic blocker scopolamine retards NMR conditioning only when the hippocampus is intact 43. Furthermore, CA1 and CA3 pyramidal neurons show increases in within-trial firing rate during NMR acquisition 8'9.

Both biophysical and biochemical changes in a large proportion of cells in the CA1 region have been re- ported up to 3 days following conditioning. These include reductions in the afterhyperpolarization and the CaZ+-dependent K + current IAHP, enhanced synaptic responsiveness, and an increase in membrane- bound and decrease in soluble PKC activity 6a1'2°'36. Using a very sensitive assay, we did not detect any change in the levels of transcripts encoding PKC a,/3, 3' (Ca2+-dep endent) or • (CaZ+-independent) z7 at the 24 h post-conditioning timepoint. Although there must be a delay between a transcriptional change and a resultant change in enzyme activity, this delay need not be long as evidenced by the nearly parallel timecourse in changes in phorbol ester binding and PKC/3 mRNA during in vitro differentiation z6. We have also been unable to detect changes in PKC mRNA levels during acquisition of the conditioned response. Based on these results and those of Bank et al. 6, it is likely that the conditioning-specific changes in PKC activity during the early phases of conditioning and consolidation are due to post-transcriptional activation and subcellular translocation rather than overall changes in amount of PKC mRNA or protein. However, any final conclu- sions regarding the nature of such changes must de- pend upon the study of multiple timepoints during both acquisition and consolidation.

A striking observation was the invariance of PKC isozyme mRNA levels between individuals, in spite of large regional and inter-isozyme differences. This re- sult underscores the potential use of in situ hybridiza- tion combined with film autoradiography and imaging

275

in detecting subtle changes in specific mRNA levels in small, anatomically defined regions.

Acknowledgements. We are grateful to Dr. W.S. Young, III, and to D.L. McPhie for technical advice. A.M.C. was supported by a Medi- cal Research Council of Canada Fellowship, and A.M.S. was the recipient of a Howard Hughes Medical Institute-National Institutes of Health Research Scholars Fellowship.

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