Autoradiographic in Situ Hybridizations for Total G S Mrna (Both Endogenous and Transgenic

Gs mice show schizophrenia-related phenotypes

EXPERIMENTAL PROCEDURES

Electrophysiological experiments. Mice were sacrificed by cervical dislocation and hippocampi were rapidly dissected in iced oxygenated ACSF. 400µm thick transverse hippocampal slices were placed in an interface chamber and continuously perfused with

30 C oxygenated ACSF while they equilibrated for at least 1.5 hours before starting electrophysiological recordings (Abel et al., 1997). A bipolar stimulating electrode (A-M systems, Inc., Sequim, WA; 0.002” diameter nichrome wire) placed in the stratum radiatum was used to stimulate CA3 axons. An ACSF-filled glass microelectrode (A-M systems, Inc. Sequim, WA; 1.5mm x 0.85mm) with a resistance between 0.5 and 3 M placed in the stratum radiatum region of CA1 and was used to record the resulting excitatory postsynaptic field potentials (fEPSPs). Data were acquired using Clampex 8.2

(Axon Instruments, Inc. Union City, CA) and analyzed using Clampfit 8.2 (Axon

Instruments, Inc. Union City, CA). Peak fEPSP amplitude was required to be at least 3 mV, and stimulus intensity was set to produce 40% of the maximal response.

Stimulations occurred every minute. Baseline responses were recorded for 20 minutes, and LTP was then induced by applying four 100 Hz, 1 second duration trains of stimuli with 5 minutes between each train. Recordings continued for 2 hours after LTP induction. Initial fEPSP slopes were normalized against the average of the 20 baseline traces, and were expressed as percent. Input-output characteristics were also examined in separate hippocampal slices by recording fEPSPs in CA1 resulting from stimuli of increasing intensity. Initial fEPSP slopes were plotted against the corresponding presynaptic fiber volley amplitudes. Paired-pulse facilitation, a short-term form of synaptic plasticity, was also monitored. Pairs of stimuli were delivered with varying

1 Gs mice show schizophrenia-related phenotypes delays (300, 200, 100, 50, 25 ms) between the two stimuli, and the initial fEPSP slope from the second stimulus was plotted as a percentage of the slope from the first stimulus.

RESULTS

Adulthood overexpression in Gs mice is necessary to increase paired-pulse facilitation and long-term potentiation. To identify a potential cellular correlate of the cognitive deficits observed in the Gs bigenic mice, we measured basal electrophysiological properties (input/output curves), short-term synaptic plasticity (paired-pulse facilitation;

PPF), and long-lasting synaptic plasticity (long-term potentiation; LTP) in the CA1 region of hippocampus. Gs mice show normal input/output properties relative to control littermates, demonstrating that basal synaptic transmission is unchanged (Figure

6A). In contrast, Gs overexpression increases PPF (Figure 7B), with Gs mice showing a significant increase at the 50 msec interval (t(13) = 3.83, p = 0.002) and a trend towards an increase at the 200 msec interval (t(14) = 2.02, p = 0.063). Similarly, there is a significant effect of genotype on LTP induced by four 100 Hz trains (Figure 6D; effect of

genotype x trace: F(160, 1600) = 2.80, p < 0.001). Post hoc analyses shows that Gs mice exhibit significantly enhanced LTP from minutes 80-140 (60 minutes following

induction) (F(1,590) = 10.62, p = 0.009). Developmental overexpression of Gs is not sufficient to induce these electrophysiological alterations as Gsdev mice show normal

PPF (Figure 7C) and LTP (Figure 7E) relative to control littermates.

2 Gs mice show schizophrenia-related phenotypes

DISCUSSION

G  s mice exhibited increased PPF and LTP: It is quite possible the deficits in memory retrieval exhibited by Gs mice may be related to the enhanced PPF and LTP measured in these mice. These results contribute to a growing body of literature associating learning and memory deficits with increases in measures of synaptic plasticity (e.g.,

Janus et al., 2001; Jolas et al., 2002; Vaillend et al., 2004; Pineda et al., 2004;

Bourtchouladze et al., 2006). It is possible that with Gs overexpression, memories may be so easily laid down that individual traces become obscured—much like writing over and over on a piece of paper eventually leaves it unreadable.

Impaired vision unlikely to account for phenotypes observed in G  s transgenic mice:

Our transgene was initially injected into pronuclei of B6SJLF1/J, a strain heterozygous for the retinal degeneration-1 and albino mutation reported to impair vision when inherited in the homozygous state. We do not believe phenotypes exhibited by Gs mice are due to visual impairments for several reasons. First, we control for confounding effects introduced by genetic background by comparing sex-matched littermates (i.e., a male control mouse is compared to a littermate male transgenic), as noted in the

METHODS. Second, the C57BL/6J strain is one of the strains most resistant to age- related retinal degeneration (Danciger et al., 2007) and our tTA line is N20+ on the

C57BL/6J background. Thus, we believe it is a statistical improbability that offspring even of an N3 tetO-Gs (which, statistically, only retains 12.5% of the original genetic background) x N20 CaMKII-tTA mating (which, statistically, retains only 0.0001% of the original genetic background) would even be heterozygous, let alone homozygous for

3 Gs mice show schizophrenia-related phenotypes either mutation. Indeed all parents and offspring clearly exhibited pigmented eyes.

Further, although some characterization was conducted on offspring of N3-5 tetO-Gs mice (x N20 CaMKII-tTA mice), animals were tested on each of the three main behavioral paradigm (startle/PPI, CFC, water maze) after the tetO-Gs line had been further backcrossed N10+ (then, statistically, retaining only 0.1% of the original genetic background) and no change was seen in the pattern of deficits observed in our transgenic mice. These two facts together strongly suggest to us that deficits observed in Gs transgenic mice are not due to a confound of genetic background. Third, Gs transgenic mice perform normally relative to controls (even when born of N3-5 tetO-Gs mice) on rotarod (data not shown), visible watermaze , and zero maze—performance on all of which has been suggested to be influenced by visual abilities (e.g., Cook et al., 2001;

McFayden et al., 2003). Finally, it is important to note that retinal degeneration is unlikely to be the cause of the contextual fear conditioning deficit and sensorimotor gating impairments exhibited by Gs mice as the mutation does not affect these behaviors (Bolivar et al., 2001; Clapcote et al., 2003; Garcia et al., 2004).

Justification of sample sizes: With regard to the fact that some group sizes are < n = 10, we believe it is important to note that reports in the schizophrenia literature frequently employ group sizes ranging from n = 6-9 (e.g., Clapcote et al., 2007; Li et al., 2007;

Kellendock et al., 2006). We believe the striking p values of our comparisons reflect the power of our statistical analyses and the sufficiency of the sample sizes employed herein

(e.g., hyperactivity seen in Gasadult mice in Figure 3C with n = 6 per genotype, p = 0.002).

In addition, it is important to point out that many of the phenotypes described herein are

4 Gs mice show schizophrenia-related phenotypes assessed in separate experiments (separate groups of mice) under similar conditions, which lends confidence to conclusions regarding the presence or absence of a phenotype.

For example, PPI deficits in Gas mice are seen under basal conditions as well as under vehicle-treatment (Figure 2) and the reversal of the contextual fear conditioning deficit by dox is shown under two conditions as well (Gasdev and Gastraining, Figure 5). In conclusion, we suggest that the power of our statistical analyses, coupled with precedence in the literature, support the sample sizes chosen herein.

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FIGURES and LEGENDS

Figure S1. Overexpression of Gs during adulthood is necessary to cause increases in paired-pulse facilitation and long-term potentiation in adult Gs mice. A) Gs mice show no differences in input/output measures relative to control mice (n = 7-8 per genotype). B) In contrast, Gs mice do show significantly increased paired-pulse facilitation (PPF) relative to control mice (n = 8 per genotype). C) The increase in PPF is not simply due to developmental effects of the transgene because Gαsdev mice show normal PPF relative to control littermates (n = 8 per genotype). D) Similarly, Gs mice show significant increases in long-term potentiation (induced by four 100 Hz trains of stimulation) relative to control mice (n = 5-7 per genotype). E) This increase in LTP is not due to developmental effects of the transgene, either, because Gαsdev mice show normal LTP relative to control littermates (n = 8 per genotype). vs CT, *p < 0.01.