Corticotropin-releasing factor receptors couple to multiple G-proteins to activate diverse intracellular signaling pathways in mouse hippocampus: role in neuronal excitability and associative learning

Abstract

Corticotropin-releasing factor (CRF) exerts a key neuroregulatory control on stress responses in various regions of the mammalian brain, including the hippocampus. Using hippocampal slices, extracts, and whole animals, we investigated the effects of human/rat CRF (h/rCRF) on hippocampal neuronal excitability and hippocampus-dependent learning in two mouse inbred strains, BALB/c and C57BL/6N. Intracellular recordings from slices revealed that application of h/rCRF increased the neuronal activity in both mouse inbred strains. Inhibition of protein kinase C (PKC) by bisindolylmaleimide I (BIS-I) prevented the h/rCRF effect only in hippocampal slices from BALB/c mice but not in slices from C57BL/6N mice. Inhibition of cAMP-dependent protein kinase (PKA) by H-89 abolished the h/rCRF effect in slices from C57BL/6N mice, with no effect in slices from BALB/c mice. Accordingly, h/rCRF elevated PKA activity in hippocampal slices from C57BL/6N mice but increased only PKC activity in the hippocampus of BALB/c mice. These differences in h/rCRF signal transduction were also observed in hippocampal membrane suspensions from both mouse strains. In BALB/c mice, hippocampal CRF receptors coupled to G(q/11) during stimulation by h/rCRF, whereas they coupled to G(s), G(q/11), and G(i) in C57BL/6N mice. As expected on the basis of the slice experiments, h/rCRF improved context-dependent fear conditioning of BALB/c mice in behavioral experiments, and BIS-I prevented this effect. However, although h/rCRF increased neuronal spiking in slices from C57BL/6N mice, it did not enhance conditioned fear. These results indicate that the CRF system activates different intracellular signaling pathways in mouse hippocampus and may have distinct effects on associative learning depending on the mouse strain investigated.

Fig. 1.

A, Representative intracellular recordings from CA1 pyramidal neurons in hippocampal slices from C57BL/6N mice and BALB/c mice showing responses to 600 msec depolarizing current pulses. B, Number of spikes elicited in 100 msec fragments during a single depolarizing (depol.) current pulse (600 msec, 1 nA).C, Plot of the number of spikes elicited by a 600 msec depolarizing pulse versus stimulus (stim.) intensity.

Fig. 2.

Effect of h/rCRF on neuronal spiking of BALB/c mouse CA1 pyramidal cells elicited by 600 msec depolarizing current pulses. A, Traces were sampled before, during, and 30 and 90 min after h/rCRF (250 nm, 10 min) application.B, Recordings were made before and 20 min after coapplication of h/rCRF (250 nm, 10 min) and [Glu^11,16] astressin (1 μm) over a period of 10 min. Pulse intensity was kept constant during each experiment; holding potential, −65 mV.

Fig. 3.

Effect of the PKC inhibitor BIS-I and of the PKA inhibitor H-89 on h/rCRF-mediated modulation of excitability. Representative recordings in CA1 pyramidal cells from C57BL/6N (A, C) and BALB/c (B, D) mice showing the effect of 250 nm h/rCRF applied over a period of 20 min after preincubation with BIS-I (1.2 μm, 1 hr) or H-89 (10 μm, 3 hr). E, Spiking behavior of CA1 pyramidal cells from C57BL/6N before and during bath application of PDBu (100 nm). Pulse intensity was kept constant during each experiment.

Fig. 4.

PKA and PKC activity in hippocampal slices of C57BL/6N and BALB/c mice. Hippocampal slices were incubated in either 250 nm h/rCRF (30 min) or aCSF (30 min, as control). Partially purified homogenates of these slices (n = 11) from six animals were tested for the ability to phosphorylate a PKA-specific (L-R-R-A-S-L-G; Kemptide) (A) or a PKC-specific (P-L-S-R-T-L-S-V-A-A-K) (B) peptidic substrate in a nonradioactive assay. Identical amounts of protein were used for each sample.

Fig. 5.

h/rCRF-induced activation of G_s-, G_I-, and G_q/11-proteins. A, Basal levels of G_s, G_i, and G_q/11 in hippocampal membrane fractions from C57BL/6N and BALB/c mice. The bar graph summarizes Western blot data (mean ± SEM) of three independent experiments each with five animals per mouse strain. B, Autoradiograph of h/rCRF-induced photolabeling of Gα subunit subtypes from hippocampal membranes of C57BL/6N (n = 30) and BALB/c (n= 30) mice. Membranes were incubated with ³²P-GTP-AA in the presence and absence of h/rCRF (100 nm), followed by UV cross-linking and immunoprecipitation of the Gα subunit subtypes using specific antibodies. Proteins were resolved by SDS-PAGE, followed by autoradiographic visualization. C, Bar graph summarizing autoradiograph data. *p < 0.05 indicates statistically significant differences.

Fig. 6.

Effect of h/rCRF on context-dependent fear conditioning of BALB/c (A) and C57BL/6N (B) mice injected with aCSF, h/rCRF, [Glu^11,16] astressin, PDBu, or 4α-phorbol 2 hr before the training as indicated. For combined treatment, [Glu^11,16] astressin and BIS-I were given 15 min before h/rCRF application. Freezing was measured in the retention test performed 24 hr after training. Injections were performed intracerebroventricularly (i.c.v.) or intrahippocampally (i.h.) as indicated. *p < 0.05 indicates statistically significant differences versus vehicle-injected animals and naive animals.