Generation of secretin receptor-deficient mice
To analyze the function of the secretin receptor in vivo, we generated secretin receptor-deficient mice using embryonic stem cell technology. A targeting vector was constructed which was designed to replace exon 1 of the secretin receptor with the lacZ reporter and a PGKneobpA selection marker (). ES cells with a targeted allele were obtained at a rate of 10% following electroporation of the targeting vector in the AB2.2 ES cell line. This allele, termed sctrm1Brd (m1), was established in mice using standard procedures. Intercross of m1/+ mice generated m1/m1 homozygous mutants at the expected 25% frequency (). Secretin receptor-deficient mice were fully viable and fertile and had normal body weight. RT-PCR analysis of tissues which normally express secretin receptor mRNA (cerebrum, cerebellum, kidney, muscle, stomach and pancreas) revealed no expression of secretin receptor in m1/m1 mice, confirming that the m1 allele was null (). We examined the kidney, stomach and pancreas of the mutant mice histologically. All pancreata had multiple variably sized islets. Acinar cells had typical zymogen granules and the ducts contained secretory material. No cell death, degeneration or inflammation was seen. The stomachs had a normal fundic epithelium, with normal-appearing chief, parietal and foveolar cells. The kidneys were unexceptional in appearance (data not shown).
Figure 1 Generation of secretin receptor-deficient mice. (A) The targeting vector (Sctr-TV1) was designed to replace exon 1 of the secretin receptor with the lacZ reporter and a PGKneobpA selection marker. (B) Southern blot analysis distinguishes the secm1Brd (more ...)
Neuropathological examination and LacZ reporter expression
We examined sections of the brain in secretin receptor-deficient adult mice for neuroanatomical changes with standard hematoxylin-eosin stains and immuno-histochemistry using antibodies to neurofilament, glial fibrillary acidic protein (GFAP), MAP2, tryptophan hydroxylase and tyrosine hydroxylase. Hematoxylin-eosin stains revealed no obvious malformation or degeneration in secretin receptor-deficient brain sections. Changes in structural proteins of the pyramidal neurons were examined using antibodies to MAP2 and alterations in neurotransmitters were analyzed using antibodies to tyrosine hydroxylase and tryptophan hydroxylase. The staining patterns of these markers were indistinguishable between the secretin receptor-deficient mice and their wild-type littermates (). There was no evidence of a glial reaction as determined using antibodies to GFAP (). These results suggest that, using these techniques, the neuroanatomy in the mutants and wild-type littermates is similar. However, analysis of the dendritic spines in the CA1 region of the hippocampus does reveal an abnormality in the secretin receptor-deficient mice.
Figure 2 Neuronal and glial marker analysis in adult brain of secretin receptor-deficient mice. Similar expression patterns are observed in both secretin receptor-deficient and wild-type littermates detected by immunohistochemistry using antibodies to (A and (more ...)
To analyze the expression of the secretin receptor, we stained the brains of secretin receptor m1/+ and m1/m1 mice with X-Gal. We found strong expression of the secretin receptor in the hippocampal CA1 region, the lower layer of cerebral cortex, the anterior olfactory nuclei, the anterior ventrolateral thalamus, the lateral region of hypothalamus, substantia nigra, tegmental area and central nucleus of the inferior colliculus, the ventral supramamillary nucleus and the cerebellum ().
Figure 3 Secretin receptor expression in adult brain revealed by staining of the lacZ reporter. The expression pattern was not different between secretin receptor m1/+ and m1/m1 mice. Expressions in (A) anterior olfactory neuron, (B) hippocampal CA1 region and (more ...)
Impaired synaptic transmission and long-term potentiation in secretin receptor mutant mice
To better understand the role of secretin in the hippocampus, the well-defined Schaffer collateral synapses were examined for altered synaptic function. Synaptic transmission was determined from population excitatory postsynaptic potentials (pEPSPs) of field recordings from the hippocampal CA1 synapses. These were evaluated by determining the amplitude of the evoked fiber volley versus the slope of the field EPSP at increasing stimulus intensities (). Loss of the secretin receptor appears to have a deleterious effect on overall synaptic transmission at Schaffer collateral synapses. shows a significant difference when an F-test is performed on the best fit curves (F[2,16] = 4.095, P = 0.0366; m1/m1, n = 21; wild-type, n = 11). Interestingly, the reduction in synaptic transmission for m1/m1 mice was due to a decrease in the slope of the pEPSP at increasing stimulus intensities and not due to a change in the fiber volley amplitude (data not shown). Since the amplitude of the fiber volley is a measurement of presynaptic firing and the slope of the pEPSP is a measurement of the postsynaptic response to stimulation, these results suggest that the reduced synaptic transmission is a result of miscommunication across the Schaffer collateral synapse, which is not due to a deficit in presynaptic terminal firing.
Figure 4 Impaired synaptic transmission and LTP of hippocampal CA1 slices. (A) Synaptic transmission was determined from pEPSPs of field recordings from the hippocampal CA1 synapses. Loss of the secretin receptor appears to have a deleterious effect on overall (more ...)
In light of the deficit in synaptic transmission, we sought to determine whether there was a change in the short-term facilitation in the CA1 area by examining paired-pulse facilitation (PPF). PPF is the facilitation of neurotransmitter release likely due to residual calcium present in the presynaptic terminal following depolarization (28
) and is likely to be involved in some forms of learning and memory (29
). Since PPF varies with the stimulus intensity and the size of the pEPSP of the first response, the stimulus intensity was adjusted so that the slopes of the pEPSP from the first stimulus for both secretin receptor-deficient and wild-type mice were the same (data not shown). Normalizing the slopes of the first response was particularly important in light of the difference in input/output in secretin receptor-deficient mice. The percent of PPF was determined at interpulse intervals of 20, 50, 100, 200 or 300 ms. m1/m1
= 20) showed no significant deficits in PPF compared with +/+ mice (n
= 12) at any of the interpulse intervals tested (). This suggests that the mechanisms controlling PPF in secretin receptor-deficient mice is intact and supports the hypothesis that presynaptic function is intact in secretin receptor-deficient mice.
Hippocampal long-term potentiation (LTP) is an use-dependent increase in synaptic efficacy believed to be similar to the processes underlying the long-term memory formation in mammals. Given that the absence of the secretin receptor appears to be responsible for the deficits in synaptic transmission, we sought to examine the possibility that this would also affect long-lasting synaptic plasticity. Hippocampal LTP was induced by stimulation of the Schaffer collateral pathway in the CA1 area with a modest protocol consisting of two trains of tetani (2 × 1 s, 100 Hz, separated by 20 s.). We observed a significant reduction in overall LTP induction in secretin receptor-deficient mice immediately following high-frequency stimulation and throughout the induction and maintenance phases of LTP (two-way ANOVA: genotype: F[1,481] = 83.97, P < 0.0001; time: F[25,481] = 1.90, P < 0.0001, m1/m1, n = 13; +/+, n = 11) (). These results demonstrate that secretin receptors are necessary for normal LTP induction in the CA1 area of the hippocampus.
Reduced dendritic spine number in secretin receptor-deficient mice
One possible cause of reduced synaptic transmission is an alteration in dendritic morphology or spine density. Therefore, we compared the structure of the dendritic tree and the dendritic spine density between secretin receptor-deficient mice and their wild-type littermates. Sholl analysis revealed that the average number of dendritic branches in CA1 pyramidal neurons in the mutant mice was similar to the number in the wild-type mice for both apical and basal dendrites (P-values >0.05) (). However, there were significantly fewer spines on CA1 apical dendrites in secretin receptor mutant mice (). The average dendritic spine number for the wild-type animals was 58.6 spines per 100 μm of dendrite and for the secretin receptor-deficient mice was 43.9 spines (unpaired t-test, n = 30 per group, P < 0.0001) (). These results suggest that the reduced ‘postsynaptic’ synaptic transmission displayed by the mutant mice correlates with a reduction in spine density.
Figure 5 Reduced dendritic spine number in secretin receptor-deficient mice. The average number of apical (A) and basilar (B) dendritic branches of CA1 pyramidal neurons in mutant mice was similar to the number in wild-type mice. (C) Higher magnification images (more ...)
Reversal water maze behavior abnormalities in secretin receptor mutant mice
To examine behavioral consequences of reduced dendritic spine number and the defect in synaptic plasticity in the hippocampus, secretin receptor-deficient mice were tested in the Morris water maze, which is a hippocampal-dependent spatial memory test. In this hidden platform test, the mice learn the spatial relationships between objects in the room and the position of the platform they can use to escape from the water. Both the secretin receptor-deficient mice and the control mice showed significant reductions in latency (search time) over the eight blocks of training. There were no significant differences between the mutants and the wild-type mice (P-values >0.05) (). However, in the reversal platform test, which tests the ability to learn the position of the hidden platform that has been moved to the opposite quadrant of its original location, secretin receptordeficient mice did not show a decrease in latency over the four blocks of the test compared with the wild-type mice (two-way ANOVA: genotype: F[1,43] = 8.656, P < 0.005; time: F[3,129] = 3.828, P < 0.02) ().
Figure 6 Reversal water maze behavior abnormalities in secretin receptor mutant mice. (A) Hidden platform test (latency). Both secretin receptor-deficient mice (m1/m1) and wild-type littermates (+/+) showed significant reductions in latency (search time) over (more ...)
Social behavior abnormalities in secretin receptor mutant mice
As an initial screen for normal social behavior, mice were tested in a tube test for social dominance (30
). We have previously found this simple test to be useful in predicting impairments in social interaction (32
). In this test, one mutant mouse and one wild-type mouse from different home cages were placed at opposite ends of a tube. A mouse was declared a ‘winner’ when the opposing mouse backed out first. We performed 39 matches with 13 mutant and 13 wild-type mice. Secretin receptor-deficient mice ‘won’ significantly more (29/36 or 81%) of their matches against wild-type littermates than expected by chance (χ2
= 13.4, P
< 0.001) (). Thus, secretin receptor-deficient mice showed abnormal social behavior. To study this further, we evaluated their responses in the partition test for social interest and recognition. The advantage of utilizing the partition test for social behavior analysis comes from the ability to assess sociability without direct physical contact (34
). The time spent near the partition during the baseline assessment with the original partner was not significantly different between secretin receptor-deficient and wild-type mice (P
>0.05) (). For the wild-type mice, the time spent at the partition when paired with an unfamiliar partner was 136% more than the time spent when paired with the original partner. In contrast, the secretin receptor-deficient mice only increased their time at the partition by 38% in the presence of an unfamiliar partner. Thus, the unfamiliar recognition ratio was significantly lower (P
< 0.03) in secretin receptor-deficient mice compared with wild-type mice (). In contrast, when the initial familiar partner was returned into the cage, the recognition ratio was similar between the two genotypes (P
>0.05). These findings indicate that secretin receptor-deficient mice have impaired social recognition behavior in the partition test.
Figure 7 Secretin receptor-deficient mice showed abnormal social behavior. (A) Tube test of social dominance. Secretin receptor-deficient mice won more matches in the tube test against wild-type littermates than expected by chance (P < 0.001). (B) Partition (more ...)
Locomotor activity and motor skills of secretin receptor-deficient mice
Locomotor activity of secretin receptor-deficient mice was evaluated by open-field test. Secretin receptor-deficient mice moved significantly faster than wild-type mice in the open-field test (), suggesting hyperactivity. Other open-field measurements such as distance traveled, time spent moving and rearing responses were not significantly different between m1/m1 and wild-type mice (P-values >0.05). Motor coordination and skill learning were assessed on the accelerating rotarod. Wild-type and m1/m1 mice performed similarly on the first day of testing; however, the secretin receptor-deficient mice performed significantly worse than wild-type mice during the last trials () on the second day of training (trial X genotype interaction, P = 0.045; trials 1-6, P-values >0.05; trials 7-8, P-values <0.05).
Normal conditional fear in secretin receptor-deficient mice
Mice were tested for their ability to learn and remember an association between a training context and a footshock, which served as unconditioned stimulus, and between an auditory conditioned stimulus (CS) and the footshock. Freezing behavior was assessed during training and during the context and auditory CS tests 24 h and 2 weeks after training. Secretin receptor-deficient mice showed significantly more freezing than wild-type littermates during the first 2 min in the conditioning chamber prior to the first footshock (m1/m1 freezing, 4.5%; wild-type freezing, 0.0%; P = 0.006). However, there were no differences (P-values >0.05) between the mutant mice and their wild-type littermates in the levels of freezing during the context tests or the auditory CS tests either 24 h or 2 weeks after training ().