Mar 21, 1991 - of phorbol 12,13-dibutyrate (PBt2) in brain of overtrained rabbits sacrificed 72 hr after completion of training showed. PKC redistribution within ...
Proc. Natl. Acad. Sci. USA Vol. 88, pp. 6637-6641, August 1991
Neurobiology
Protein kinase C redistribution within CA3 stratum oriens during acquisition of nictitating membrane conditioning in the rabbit (leaming/memory/hippocampus)
ANDREW M. SCHARENBERG, JAMES L. OLDS, BERNARD G. SCHREURS, ANNE M. CRAIG, AND DANIEL L. ALKON* Section on Neural Systems, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, MD 20892
Communicated by James L. McGaugh, March 21, 1991 (received for review June 8, 1990)
This manuscript describes experiments deABSTRACT signed to investigate protein kinase C redistribution occurring during acquisition of the rabbit nictitating membrane (NM) conditioned response (CR). The first experiment defined the acquisition phase of the NM response for our laboratory. A group of rabbits (n = 6) was given 2 days of paired NM training; a second group (n = 6) was given 2 days of unpaired NM training. The data document a variable level of responding on day 1 for rabbits given paired training (mean ± SEM, 21 ± 11% CRs) but show that on day 2 most rabbits reached the behavioral asymptote (five of six rabbits responding with >85% CRs). Rabbits responding at the behavioral asymptote were defined as having acquired the NM conditioned response. These data were interpreted to indicate that 1 day of training initiated processes necessary for behavioral acquisition (i.e., responding at the behavioral asymptote). A quantitative finm autoradiographic study of [3H]phorbol 12,13-dibutyrate binding was then used to determine the distribution of hippocampal protein kinase C in rabbits sacrificed after receiving either 1 day of paired stimuli (n = 10), 1 day of unpaired stimuli (n = 6), or no stimuli (n = 6). Autoradiograms were analyzed by measuring binding in strictly defined regions of interest and from transept profiles. A significant increase in binding of the phorbol ester was found in the CA3 stratum oriens in the paired group relative to unpaired and naive controls. No other significant differences were found.
The nictitating membrane (NM) conditioned response (CR) is an associative learning model in which rabbits learn to associate a tone (conditioned stimulus, CS) with a shock (unconditioned stimulus, US) to the periorbital region (1, 2). Retraction of the NM in response to the tone (a CR) is used to index the progression of learning. The hippocampus has been shown to be necessary for both proper consolidation of the NM response in undertrained rabbits and proper extinction of the response in overtrained rabbits (3, 4). Firing patterns for CA1 and CA3 pyramidal cells formed a striking "neural model" of the CR during paired NM training (5).
Furthermore, changes in neuronal firing within the CA1 and CA3 areas of the hippocampus occurred within the first 10 paired trials and increased throughout the training period (5, 6). Extensive studies of hippocampal neurons have been performed with rabbits that were overtrained using this paradigm and sacrificed 24 hr later. In such rabbits, postsynaptic biophysical changes in CA1 neurons were demonstrated, including increased input resistance (7), decreased after hyperpolarizations (8, 9), and increased summation of postsynaptic potentials (10). Accompanying the biophysical changes at this time point were biochemical changes (11-13), with two studies localizing an increase in membrane-bound
protein kinase C (mPKC) to the CAl region (11, 12). Binding of phorbol 12,13-dibutyrate (PBt2) in brain of overtrained rabbits sacrificed 72 hr after completion of training showed PKC redistribution within CAl, although qualitatively different than that seen at 24 hr. The PKC redistribution seen within CA1 in overtrained rabbits sacrificed 24 or 72 hr after their training ended implied a role for PKC in encoding retention-related changes in the CA1 neurons. The overall goal of the present study was to begin to elucidate how PKC redistribution develops during NM conditioning. We started by asking whether PKC redistribution occurs during acquisition of the response. Addressing this question required defining a time point during the NM training protocol that would allow examination of acquisition-related biochemical processes. Our first experiment documented NM response development and illustrated the suitability of animals sacrificed at the end of day 1 for analysis of acquisition-related biochemical processes. Our second experiment used quantitative [3H]PBt2 autoradiography to measure the distribution of mPKC within the hippocampus at that time point.
METHODS Behavior. Thirty-four male rabbits (New Zealand White, 80-100 days old, -2.0 kg, maintained in a standard manner) were used. The rabbits were subjected to 1 day of adaptation and received their behavioral experience starting 24 hr later. Paired subjects received 80 paired trials per day consisting of presentations of a 400-msec, 1000-Hz, 82-decibel tone CS that coterminated with a 100-msec, 50-Hz electrical pulse US. Paired trials were delivered, on average, every 60 sec (range, 50-70 sec). Unpaired subjects received 80 CS-alone and 80 US-alone presentations per day, delivered, on average, every 30 sec in an explicitly unpaired manner. Naive subjects were restrained and placed in the training chambers for the 80-min-period that corresponded to the duration of the paired and unpaired stimulus presentations. Experiment 1 consisted of two groups of rabbits given 2 days of training with paired (paired group no. 1, n = 6) or unpaired (unpaired group no. 1, n = 6) stimuli. A mixed repeated-measures analysis of variance (ANOVA) was used to assess the effects by day of each stimulus paradigm on conditioned responding. Experiment 2 consisted of three groups of rabbits, which were each given 1 day of their respective training and then processed for [3H]PBt2 autoradiography. These groups inAbbreviations: NM, nictitating membrane; CR, conditioned response; CS, conditioned stimulus; US, unconditioned stimulus; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; ROI, region of interest; DG, dentate gyrus; PKC, protein kinase C; mPKC, membrane-bound PKC; PBt2, phorbol 12,13-dibutyrate; ANOVA, analysis of variance. *To whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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cluded paired group no. 2, (n = 10), unpaired group no. 2 (n = 6), and a naive group (n = 6). A one-way ANOVA was used to make between-groups comparisons of changes in conditioned responding. Stimulus delivery and data collection were accomplished using a Compaq/ASYST system (14). Statistical analyses were performed using the Systat statistical analysis package (Systat, La Jolla, CA). Quantitative Autoradiography. Immediately after training on day 1, the rabbits in experiment 2 were anesthetized with sodium pentobarbital (5 mg/kg) and decapitated. Their brains were rapidly removed and processed (11) into 12-gm slidemounted frozen sections. Eighteen sequential sections 3-4 mm posterior to the bregma (15) were selected for each rabbit, processed for [3H]PBt2 autoradiography (16, 17) using 2.5 nM [3H]PBt2 (specific activity, 18.0 Ci/mmol; New England Nuclear; 1 Ci = 37 GBq), and apposed to 3Hsensitive film (LKB Ultrofilm). Nonspecific binding was assessed for each rabbit by incubating two sections per animal with 2.5 nM [3H]PBt2 plus 2.5 tiM unlabeled PBt2. Specific and nonspecific binding were measured from autoradiograms using the MCID image-analysis system (Imaging Research, St. Catherines, ON, Canada). All image analysis was performed in a manner that did not allow the experimenter to know the experimental group to which the autoradiogram belonged. Two methods were used to sample data from the autoradiograms: regions of interest (ROIs) and transept profiles. For both methods, left and right sides were pooled. ROI sampling analysis required digitization of each autoradiographic section and manual delineation of ROIs. Global section binding was sampled by outlining the raw digitized image. Each hippocampal image was then individually magnified by a factor of 2 and subjected to a low-frequency 3 x 3 convolution-matrix filter operation. Global hippocampal [3H]PBt2 binding was sampled by outlining the hippocampus. Fig. 1A illustrates the method used to sample the DG, CA1, and CA3 subregions within the hippocampus. The MCID system produced average binding values over all pixels within each ROI and recorded these in a "data log" file. The values for each region were averaged for all sections from each rabbit and these average values were then compared between groups with one-way ANOVAs. Transept profiles were obtained from each image as illustrated in Fig. 1B. All binding values along each transept profile were recorded in a data log file and imported into a spreadsheet (Lotus 123, Lotus Development Corporation). Transept profiles from each section were aligned by the pixel corresponding to their exterior (SO/alveus), and border and pixel-by-pixel average transept profiles were constructed for each rabbit. Subregion layer widths [SO, stratum pyramidale (SP), and stratum radiatum (SR)] were measured from the two sections immediately preceding the sections used in the binding assays. These sections were stained with cresyl violet, images of each section were digitized, and cell-layer widths along CA1 and CA3 transept profiles were measured in pixels with the MCID system. The SO was measured as the distance from the alveus/SO border to the exterior border of the cell layer staining. The SP was measured as the width of the cell layer staining. The SR was taken as two-thirds of the distance (to the nearest pixel) from the interior border of the cell layer staining to the hippocampal fissure. The pixel width values for each layer were then used to make an average binding value for those pixels in each rabbits's average transept profile corresponding to the respective subregion layer (data are shown in Fig. 4). The values generated for each subregion layer were compared between groups using one-way ANOVAs. Nonspecific binding was measured as the difference between the binding in global-section ROIs sampled from
Proc. Natl. Acad. Sci. USA 88 (1991)
A
B
CAl Transept Profile
CA3 Transept Profile
FIG. 1. (A) Three subregions were sampled from each side of the hippocampus for each section. Representative areas encompassed by RO1s 1-3 are illustrated. ROT 1 included the region of Ammon's horn determined medially by a line connecting the alveus at the external border of the hippocampus to the medial extent of the dentate gyrus (DG), and laterally by a line perpendicular to a tangent line at the CA1/CA3 border. This allowed it to include all of CA1 in all of the sections; in some cases it contained a small amount of prosubiculum as well. Starting from the lateral border of ROI 1 and ending in the genu of the DG, ROT 2 encompassed the rest of Ammon's horn. ROT 3 encompassed all of the DG, including both buried and exposed blades, in every section. (B) CA1 and CA3 transept profiles were drawn on each side of the hippocampus for each section. CAl transept profiles were placed at the midpoint of a region of Ammon's horn defined medially by a line perpendicular to the tangent line at the dorsal border of the genu of the CAl region, and laterally by a perpendicular to a tangent line at the border of CA1/CA3. CA3 transept profiles were drawn as the angle bisector of an angle formed by vertical and horizontal lines extending from the tip of the DG to the stratum oriens (SO)/alveus border. "Vertical" and "horizontal" were defined relative to the whole brain section. Each transept profile was drawn so as to encompass the entire dendritic arborization of the hippocampal pyramidal cells, extending from the SO/alveus border to the interior border of the stratum moleculare at the hippocampal fissure.
images of nonspecific binding sections and the background density of the film in a nearby area.
RESULTS Behavior. Experiment 1 documented the time course of development of the CR during paired NM training in our laboratory. On day 1 three rabbits in the paired group showed very little responding (50% CRs in 80 paired trials), and 1 rabbit showed an intermediate level of responding (Fig. 2A). On day 2 in the paired group all but one rabbit were responding immediately at high levels (Fig. 2B), including two of the three rabbits who had shown 0.10 in each case, one-way ANOVA, data not shown). Additionally, comparisons of DG/CA3/CA1 ROT binding values revealed no significant between-groups differences (data not shown). Autoradiograms of areas CA3a and CA3b (Fig. 3) suggested the presence of increased binding in the CA3 SO specific to the paired group. This was borne out by the transept profile data (Fig. 4). No significant betweengroups differences were present in any of the CA1 cell layers or in the CA3 SP or SR. The CA3 SO, however, showed a highly significant increase in binding relative to unpaired and naive controls (=18% greater than controls, P < 0.01,
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FIG. 4. [3H]PBt2 binding for the SO, SP, and SR for CA1 (A) and CA3 (B). Data were sampled from each individual animal's average transept profile. Solid bars, paired group; hatched bars, unpaired group; stippled bars, naive group. One-way ANOVAs were used to compare the values in each subregion between groups. The only significant between-groups difference found was in CA3 SO [group effect, F(2,19) = 6.2, P < 0.01]. Planned orthogonal contrasts showed this to be due to an increased binding in the paired group relative to unpaired and naive controls (orthogonal contrast, P < 0.01, indicated by two stars). There was no difference between controls (orthogonal contrast, P > 0.20).
planned orthogonal contrast). Between-rabbit variability in transept profiles is illustrated in Fig. 5, which shows pixelby-pixel group-average transept profiles, constructed as de-
scribed in the figure legend. Only the section of the CA3 profiles corresponding to SO diverges signif-
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FIG. 3. Representative images of [3H]PBt2 autoradiograms of the right hippocampus from one rabbit in each group. The images were digitized from autoradiograms from the same piece of film. Each image was subjected to a 50% reduction, and then approximately the onefourth of the hippocampus including CA3a and CA3b was subjected to a x2 magnification to produce the images presented. A calibrated color bar is shown in the lower left for quantitative comparison.
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Proc. Natl. Acad. Sci. USA 88
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icantly between groups. The transept profiles within groups highly reproducible as evidenced by the standard error bars on each plotted point. Nonspecific binding values were very close to the background film density values. No measured nonspecific binding values was >7% of the binding in the corresponding ROIs of the same rabbit, and most were
