A role for the beta isoform of protein kinase C in fear conditioning

Abstract

The protein kinase C family of enzymes has been implicated in synaptic plasticity and memory in a wide range of animal species, but to date little information has been available concerning specific roles for individual isoforms of this category of kinases. To investigate the role of the beta isoform of PKC in mammalian learning, we characterized mice deficient in the PKC beta gene using anatomical, biochemical, physiological, and behavioral approaches. In our studies we observed that PKC beta was predominantly expressed in the neocortex, in area CA1 of the hippocampus, and in the basolateral nucleus of the amygdala. Mice deficient in PKC beta showed normal brain anatomy and normal hippocampal synaptic transmission, paired pulse facilitation, and long-term potentiation and normal sensory and motor responses. The PKC beta knock-out animals exhibited a loss of learning, however; they suffered deficits in both cued and contextual fear conditioning. The PKC expression pattern and behavioral phenotype in the PKC beta knock-out animals indicate a critical role for the beta isoform of PKC in learning-related signal transduction mechanisms, potentially in the basolateral nucleus of the amygdala.

Fig. 1.

PKCβ distribution in the brain. X-gal staining (A, B) shows prominent staining in CA1 of the hippocampus with mild staining in CA3. Moderate staining is seen in both the lateral and basolateral nuclei of the amygdala, striatum, somatosensory cortex, and cerebellar and entorhinal/perirhinal cortical areas. Immunohistochemistry using antibodies against the βI isoform of PKC (C, D) shows essentially no staining in the hippocampus (C) or amygdala (D). Immunohistochemistry using antibodies against the βII isoform (E, F) shows staining in the stratum oriens and stratum radiatum of CA1 of the hippocampus but not in the stratum pyramidali (E). The amygdala (F) shows staining of fibers in the basolateral nucleus. The structures represented in D–Fwere obtained from a single mouse and processed in parallel.

Fig. 2.

Electrophysiological responses at Schaffer collateral synapses in area CA1 of hippocampus. A, Loss of PKCβ had no effect on baseline synaptic transmission in stratum radiatum of the CA1 region of the hippocampus measured at 25°C in PKCβ-deficient mice (○) or wild-type mice (●).Inset, Representative traces (mean of 6 successive field EPSPs) of previous baseline synaptic transmission. Calibration: 2 mV, 4 msec. B, Paired pulse facilitation was also unaffected in PKCβ-deficient mice.

Fig. 3.

Hippocampal LTP. In the following experiments, hippocampal slices obtained from PKCβ-deficient mice (○) or wild-type mice (●) were given an LTP-inducing stimulus (arrows) delivered after stable baseline responses were recorded for 20 min. Each set of tetani consisted of two trains of 100 Hz stimulation for 1 sec, separated by 20 sec. A, Mutant hippocampal slices showed normal LTP compared with wild types after a modest LTP-inducing protocol consisting of a single set of tetani while maintaining slices at 25°C. B, The extent of depotentiation in PKCβ-deficient mice was equal to that of wild types determined from low-frequency 5 Hz stimulation (5 min) after a single set of tetani at 25°C. C, No differences in PKCβ mutant LTP were observed in experiments using a more robust LTP-inducing protocol consisting of three sets of 100 Hz tetani delivered 10 min apart while maintaining slices at an elevated temperature of 32°C.

Fig. 4.

Lack of compensatory changes in PKC in PKCβ-deficient mice. A, Top, Representative Western analysis of PKCβ, PKCα, and PKCγ protein from hippocampal homogenates of PKCβ knock-outs, control mice, or purified PKCβII protein. Bottom, The percent change in protein kinase C expression in PKCβ knock-out mice versus control mice is shown for each of the Ca²⁺-dependent protein kinase C isoforms. All PKC densitometric measurements were normalized to corresponding total protein amounts obtained from whole hippocampal homogenates. No statistically significant changes in PKC expression levels were observed for PKCα (n = 4) or PKCγ (n = 4). N/D, Not detectable. B, Phorbol ester-induced potentiation of synaptic transmission was reduced in PKCβ-deficient mice. Results shown are the percent increase in baseline synaptic transmission (relative to predrug application at time 0) produced by a 25 min application of 5 μm phorbol 12, 13 diacetate (PDA) in control (+/+; n = 4) and PKCβ-deficient (−/−; n = 7) hippocampal slices. The difference in control versus knock-out mice is statistically significant (p < 0.01) at all times from 25 min after drug application and is consistent with the biochemical data indicating a lack of other PKC isoform compensatory changes in PKCβ-deficient mice.

Fig. 5.

Fear conditioning. Left, Our initial findings comparing PKCβ knock-out mice (○) and littermate wild-type mice (●). Right, Replications of these experiments using a set of naïve PKCβ-deficient mice and age-matched C57BL/6 control mice. A, Freezing behavior on the day of training for PKCβ-deficient or wild-type mice. Wild-type mice displayed significantly higher freezing in response to the shock (p < 0.05) than did PKCβ knock-out mice in the initial results, but this difference was not significant when the experiment was replicated. The acoustic CS is presented for the two periods underlined. Foot shocks are presented at the arrowheads. B, In the initial results, PKCβ-deficient mice showed significantly less freezing in response to replacement in the training context (p < 0.01) compared with wild-type mice. This effect was replicated in the second experiment (p < 0.01). C, For these experiments the animals are in a different context than that in which they were trained. Acoustic CS presentation is indicated by theline. PKCβ knock-out mice were impaired in freezing in response to CS presentation 24 hr after training in both the initial experiment (p < 0.01) and the replication experiment (p < 0.001). For all graphs, freezing was scored every 5 sec and averaged over 1 min epochs.

Fig. 6.

A, Passive avoidance, hotplate, shock threshold, and prepulse inhibition. Passive avoidance was tested for step-through latencies from a lighted compartment to a dark compartment. This test uses the natural tendency for mice to retreat from a lighted area to darker area during the training session. On entering the dark area a mild foot shock was given, and learning was assessed as the avoidance of the dark area after the training session. Results are shown as step-through latency during trial sessions 1–3 d after training for PKCβ-deficient mice (■) or wild-type mice (▪); mean ± SEM. A hotplate test was used to compare mutant (■) versus wild-type (▪) sensitivity to a noxious stimuli. Thermal nociception was measured on a 55°C hotplate as the latency to hindpaw lick. As an additional control to the hotplate test, the shock threshold test was used to compare sensitivity to foot shock measured by the extent of flinching, jumping, or vocalization to increasing foot shock intensities. Mice normally exhibit a startle response to loud noise. Interestingly, if a modest noise is presented immediately preceding the loud noise, the startle response is significantly attenuated, a phenomenon referred to as prepulse inhibition. Prepulse inhibition is a very sensitive test to evaluate sensorimotor gating as well as hearing, because animals reliably give quantitatively different responses to prepulses varying by only a few decibels. The effect of a pretone (sound intensity given in decibels) to diminish the magnitude of acoustic startle is shown in the bottom graph. Results are given as percent diminution of the force of the subsequent 120 dB startle response for PKCβ-deficient mice (■) or wild- type mice (●). B, Open field behavior. As a test of general activity levels and to test for anxiety, animals were monitored using the open-field test. With this test general activity levels are evaluated by measurements of horizontal activity, vertical activity, and total distance traveled during a 10 min test session in an open box in a lighted room. The open field test also measures anxiety levels, as assessed by the center distance to total distance ratio. Results shown are for PKCβ-deficient mice (○) or wild-type mice (●); mean ± SEM.Top graph, Total distance traveled per minute for a 30 min period. Bottom graph. Ratio of center distance to total distance traveled for each minute over a 30 min period. There were no differences in the total distance traveled or the center to total distance ratio between groups. A small decrease in the general vertical activity of mutants was seen (data not shown).C, Rotarod behavior. We analyzed coordination and motor skill acquisition using the rotarod test. The amount of time an animal can stay on a rotating rod is an index of its general level of coordination. Mice also improve their performance with training, which is an indicator of motor learning. Results shown are total times the animals remain on the rotating rod per training period. Three training trials were given on a single day. The increase in time the animal remained on the rod is taken as an index of motor learning. Results shown are for PKCβ-deficient mice (○) or wild-type mice (●); mean ± SEM.

Fig. 7.

Retraining of PKCβ-deficient and control mice on day 3. A, Freezing behavior on retraining of PKCβ-deficient mice (○) or control mice (●). One day after cue and contextual testing (day 3), these mice were retrained with five pairings of acoustic CS (dark bar) and foot shock (gray arrowhead). Compared with control mice, PKCβ knock-out mice displayed significantly lower freezing during baseline measurements (minutes 1–2; p < 0.05). This freezing deficit disappeared with overtraining (minutes 3–7).B, Freezing in response to CS presentation 1–2 hr after training on day 3. There was no significant difference in freezing levels in response to the CS between PKCβ knock-out mice and controls. C, Freezing in response to representation of the training context on day 4. PKCβ knock-out mice were impaired in terms of freezing behavior in response to the training context (p < 0.05). D, Freezing in response to CS presentation on day 4. PKCβ-deficient mice also showed significantly less freezing in response to the CS than did controls (p < 0.01). In all experiments, freezing is scored every 5 sec and averaged over 1 min epochs.