Behavioral Consequences of a Combination of Gad1 Haplodeficiency and Adolescent Exposure to an NMDA Receptor Antagonist in Long-Evans Rats

Glutamate decarboxylase 67-kDa isoform (GAD67), which is encoded by the GAD1 gene, is one of the key enzymes that produce GABA. The reduced expression of GAD67 has been linked to the pathophysiology of schizophrenia. Additionally, the excitatory glutamatergic system plays an important role in the development of this disorder. Animal model studies have revealed that chronic blockade of NMDA-type glutamate receptors can cause GABAergic dysfunction and long-lasting behavioral abnormalities. Based on these findings, we speculated that Gad1 haplodeficiency combined with chronic NMDA receptor blockade would lead to larger behavioral consequences relevant to schizophrenia in a rat model. In this study, we administered an NMDAR antagonist, MK-801 (0.2 mg/kg), to CRISPR/Cas9-generated Gad1 +/− rats during adolescence to test this hypothesis. The MK-801 treated Gad1 +/− rats showed a shorter duration in each rearing episode in the open field test than the saline-treated Gad1 +/+ rats. In contrast, immobility in the forced swim test was increased and fear extinction was impaired in Gad1 +/− rats irrespective of MK-801 treatment. Interestingly, the time spent in the center region of the elevated plus-maze was significantly affected only in the saline-treated Gad1 +/− rats. Additionally, the MK-801-induced impairment of the social novelty preference was not observed in Gad1 +/− rats. These results suggest that the synergistic and additive effects of Gad1 haplodeficiency and NMDA receptor blockade during adolescence on the pathogenesis of schizophrenia may be more limited than expected. Findings from this study also imply that these two factors mainly affect negative or affective symptoms, rather than positive symptoms.

move freely in the maze for 10 min. The durations spent in the closed arms, open arms, and center area were measured with TimeEP software (O'Hara & Co., Ltd., Tokyo, Japan).

Open field test
A 90-cm × 90-cm open field apparatus (O'Hara, Tokyo, Japan) was used as described previously (Fujihara et al., 2020(Fujihara et al., , 2021. The center of the arena was illuminated at 50 lx. Each rat was allowed to move freely during a 5-min session (Miyata et al., 2019). The behavior of the rats was recorded with an attached CCD camera. The distance traveled during a session and the time spent in the central area (36% of the field) was assessed accordingly. Furthermore, we measured the rearing behaviors automatically with an infrared beam sensor. Data analysis was carried out using the automated software TimeOFCR1 (O'Hara & Co., Ltd., Tokyo, Japan).

Social interaction test
The social behaviors of the rats were tested as described previously (Fujihara et al., 2020). An open field apparatus was used for this test. Two identical wire cages were placed inside the arena diagonally. First, rats were allowed to move freely around the arena for 10 min for habituation. A stranger rat younger than the subjects was enclosed in one of the wire cages immediately after habituation. Then, the rat to be tested was put back in the arena (sociability test). The amount of time the rats spent around the cages was measured for 5 min. After a 5-min interval, we placed a different stranger rat in the empty wire cage, and again, the time spent around each cage was recorded for 5 min (social novelty preference test). The data were analyzed using TimeSSI (O'Hara & Co., Ltd., Tokyo, Japan).

Porsolt forced Swim test
The forced swim test was carried out according to a previously described protocol with minor modifications (Fujihara et al., 2020). Plexiglas cylinders (20 cm diameter × 45 cm height; O'Hara, Tokyo, Japan) were filled with water (25 cm depth), whose temperature was maintained at 22 ± 2°C.
On the first day, each rat was placed in water for 10 min. On the second day, each rat was put into water again, and its behavior was recorded for 6 min by a CCD camera. The data were analyzed using TimeFZ1 (O'Hara & Co., Ltd., Tokyo, Japan).

Cued fear conditioning test
The cued fear conditioning test was conducted in a box surrounded by a sound-attenuated chamber (O'Hara & Co., Ltd., Tokyo, Japan) as described previously (Fujihara et al., 2021). This test consisted of a conditioning trial (day 1) and 4-day cued test trials (day 2 to day 5) (Supplementary Figure S3).
The rats were placed in a clear acrylic conditioning chamber (33 cm × 25 cm × 28 cm) equipped with a stainless-steel grid floor connected to a shock generator. The brightness of the chamber was set at 120 lx. On day 1, the rats were habituated to the conditioning chamber for 180 s. Immediately after, they received five auditory tones (CS; 10 kHz, 65 dB, duration: 20 s) with an 80 s interval; each tone was delivered simultaneously with a foot shock at the end of it (US; 0.7 mA, duration: 1 s). One hundred seconds after the last foot shock, the rats were returned to their home cages. A cued test was performed in a chamber with different contexts. The test chamber differed from the conditioning chamber in brightness (30 lx), color (black), and shape (triangular prism, 33 cm on one side and 31.5 cm high). The schedule for the cue test was the same as that for the conditioning, except that no foot shock was administered in this case. The acquired cued fear was tested through five CS periods (without foot shock). From day 3 to day 5, we repeated the same cued test to assess extinction learning.
The percentage of time that the rats exhibited freezing responses was measured as an index of fear memory. The decrease in freezing level in each rat during the fear extinction test was expressed as a ratio to the freezing level of day 2 (extinction rate [%]). The data were analyzed using TimeFZ1