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Gene: 936	Name: Gabra3	Family: Channel	Subfamily: GABA Receptor	Accession: M86568	GI: 193390

Gene 936
Summary of Phenotypic Analysis

Changes related to genotype:

Home cage observations:

Hyperactivity in a 68 day hemizygous mutant male.

Behavior :

Hemizygous mutants displayed increased total distance traveled scores on the open field test.
Hemizygous mutants had significantly lower pre pulse inhibition scores on the Startle/PPI test.

ES cells derived from the 129/OlaHsd mouse substrain were used to generate chimeric mice. F1 mice were generated by breeding with C57BL/6 females. The resultant F1N0 heterozygotes and hemizygotes were backcrossed to C57BL/6 mice to generate F1N1 heterozygotes and hemizygotes. F2N1 mutant mice were produced by intercrossing F1N1 heterozygous females and hemizygous males.

As this gene is X-linked, the cohort consists of homozygous mutant females, hemizygous mutant males (-/0), heterozygous control females, and wild-type control males (+/0). No wild-type females or heterozygous males were analyzed for this target, as such mice cannot be generated from a male -/0 x female -/+ mating.

Female homozygous mutant mice, male hemizygous mutant mice, female heterozygous control mice, and male wild-type control mice were evaluated by the following examinations or tests:

Physical examination
Necropsy, including body length, body weight, and organ weight measurements
Histological examination of tissues and organs
Bone marrow section evaluation
Complete blood counts and differential cell counts
Clinical chemistry panels
Densitometry
Behavioral tests
Fertility
Aging studies

When compared to age- and gender-matched wild-type control mice, hemizygous mutant(s) were hyperactive, as observed in a 68 day hemizygous mutant male during home cage observation, and at behavior in that they explored the open field significantly more.

Hemizygous mutants were significantly impaired in the Startle/PPI task, demonstrating lower prepulse inhibition scores when the prepulse was 90dB preceeding a 110dB startle stimulus.

Certain differences between mice, including lower triglyceride levels, were present that may have occurred spontaneously in this age group. When compared to age- and gender-matched contemporaneous wild-type control mice, such differences were present in four of five 49 day cohort hemizygous mutant males and two 300 day cohort hemizygous mutant males. We have not presented these findings as phenotypic changes, but we have presented them here for your consideration.

Gene 936
Behavior

Changes related to genotype:

Hemizygous mutants displayed increased total distance traveled scores on the open field test.
Hemizygous mutants had significantly lower pre pulse inhibition scores on the Startle/PPI test.

Hemizygous mutant and wild-type control mice were evaluated for phenotypic changes by testing on seven behavioral tasks: Open field test, Tail suspension test, Rotarod test, Hot plate test, Startle/PPI, Tail flick test, and Metrazol test.

Mouse ID numbers are as follows:

11 hemizygous mutant males (192789, 181100, 192802, 188938, 192795, 181101, 192800, 188939, 185970, 185972, 181106)
10 wild-type control males (181104, 192797, 192801, 192793, 192803, 185971, 181105, 188936, 181107, 192787)

ES cells derived from the 129/OlaHsd mouse substrain were used to generate chimeric mice. F1 mice were generated by breeding with C57BL/6 females. The resultant F1N0 heterozygotes were backcrossed to C57BL/6 mice to generate F1N1 heterozygotes. F2N1 homozygous mutant mice were produced by intercrossing F1N1 heterozygous males and females.

Behavior Findings:
When compared to age- and gender-matched wild-type control mice, hemizygous mutants were hyperactive, in that they explored the open field significantly more. This finding correlates with hyperactivity observed during home cage observations.

Hemizygous mutants were significantly impaired in the Startle/PPI task, demonstrating lower prepulse inhibition scores when the prepulse was 90dB preceeding a 110dB startle stimulus. This effect on stimulus processing is similar to that seen in schizophrenic patients. Furthermore, hemizygous mutants displayed an increased startle response to several sound stimulus types including single bursts at 100db.

There were no other genotype-related differences noted between homozygous mutant and wild-type control mice for any other parameters evaluated during behavior testing.

Gene 936
Fertility

Both hemizygous mutant males and homozygous mutant females were fertile. Their progeny were viable until weaning.

Three homozygous mutant female mice and three hemizygous male mice were set up in a fertility mating one on one with each other at seven to ten weeks of age. The number of pups born from three litters was recorded. Three weeks later, the live pups were counted and weaned.

Mouse ID numbers are as follows:

3 hemizygous mutant males (202910, 195498, 195509)

3 homozygous mutant females (202906, 195503, 195504)

Gene 936
Expression Summary

RT-PCR Summary:
The highest levels of RNA transcripts are detectable in brainstem and olfactory bulb.

Lower levels of RNA transcripts are also detectable in all other tissues analyzed: brain, cortex, subcortical region, cerebellum, spinal cord, eye, Harderian glands, heart, lung, liver, pancreas, kidney, spleen, thymus, lymph nodes, bone marrow, skin, gallbladder, urinary bladder, pituitary gland, adrenal gland, salivary gland, skeletal muscle, tongue, stomach, small intestine, large intestine, cecum, testis, epididymis, seminal vesicle, coagulating gland, prostate gland, ovaries, uterus and white fat.

LacZ Summary:
LacZ (beta-galactosidase) expression was detected in brain, dorsal horns and dorsal root ganglia of the spinal cord and lens of the eye. Expression was also detectable in aorta, a few cells of the heart and kidney, some blood vessels of the lung and liver and tunica albuginea of testis.

In wholemount staining of the brain, strong lacZ expression was detectable in olfactory bulb, thalamus, cortex and hypothalamus. Weaker staining was also detectable in small focal regions of cerebellum and brainstem. On coronal sections of brain weak to moderate expression was detectable in amygdala, cortex, hippocampus and hypothalamus.

LacZ expression was not detected in sciatic nerve, thymus, spleen, lymph nodes, bone marrow, pancreas, urinary bladder, thyroid gland, pituitary gland, adrenal glands, skeletal muscle, skin, prostate gland, ovary and uterus.

Gene 936
Densitometry

There were no significant differences detected in the female homozygous and male hemizygous mutant mice when compared with age- and gender-matched heterozygous and wild-type control mice.

The following mice were evaluated by dual-energy x-ray absorptiometry.

Densitometric Findings:

Certain densitometric differences between mice, including decreased fat %, are present that occasionally occur spontaneously in this age group. In this target, such differences are present in two 300 day cohort hemizygous mutant males. We are not reporting this finding as a phenotypic change, but we present it here for your consideration.

Other incidental densitometric differences were present between some mice. These findings were considered to represent background differences occasionally seen in this strain of mice, differences due to spontaneous disease, age-related differences, and/or differences of a nonspecific etiology. They were not considered to be genotype related.

Gene 936
Histopathology

There were no significant differences detected in the female homozygous and male hemizygous mutant mice when compared with age- and gender-matched heterozygous and wild-type control mice.

Tissues from the following mice were evaluated histologically.

The following tissues were examined and considered to have no genotype-related abnormality: brain, pituitary gland, ears, nasal cavity, salivary glands, oral cavity, lymph nodes, aorta, lungs, gallbladder, pancreas, spleen, kidneys, urinary bladder, stomach, small and large intestines, larynx, esophagus, trachea, thyroid gland, thymus gland, tongue, skeletal muscle, sciatic nerve, mammary glands, vertebrae, spinal cord, bone (skull, sternum, femur, tibia, and stifle joint), reproductive tract (including gonads), eyes, Harderian glands, integumentary system (skin and either clitoral or preputial glands), and bone marrow.

Bone marrow was examined in sections of sternum, vertebrae, and/or femur and tibia. Marrow cellularity, myeloid:erythroid (M:E) ratio, myeloid and erythroid maturation sequences, and numbers of megakaryocytes were evaluated.

Incidental lesions, such as spermatic granulomas in one hemizygous mutant male (173477) and one wild-type control male (173471), are present in some tissues. These findings are considered to represent background lesions occasionally seen in this strain of mice, lesions due to spontaneous disease, age-related lesions, lesions due to procedural artifacts, and/or lesions of a nonspecific etiology. They are not considered to be genotype related.

Gene 936
Necropsy

There were no significant differences detected in the female homozygous and male hemizygous mutant mice when compared with age- and gender-matched heterozygous and wild-type control mice.

The following mice were necropsied. Body weight, body length, and organ weights were obtained, and gross pathological findings were recorded.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (173482, 173483, 173485)
4 heterozygous mutant females (192805, 192809, 200379, 200381)
5 hemizygous mutant males (173473, 173474, 173477, 200367, 200368)
2 wild-type control males (173470, 173471)

300 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (188945, 192807, 192810)
2 heterozygous mutant females (192774, 192811)
2 hemizygous mutant males (183362, 183364)
2 wild-type control males (183371, 183373)

Mice were examined for the following observables: adrenal glands, body length, body weight, bone marrow, bone - cranium, bone - femur, bone - sternum, bone - stifle joint, bone - vertebral column, brain, cecum, colon, duodenum, epididymis - seminal vesicle, esophagus, eyes, gallbladder, general appearance, Harderian glands, heart, heart weight, ileum, jejunum, kidney weight, kidneys, liver, liver weight, lungs, lymph nodes, mesentery, ovaries, pancreas, penis, salivary glands, sciatic nerve, scrotum, skeletal muscle, skin, skinned mouse, spleen, spleen weight, stomach, testes, testes - epididymis weight, thymus, thymus weight, tongue, trachea, urinary bladder, urine, uterus, and vagina. (Gender-specific observables apply to the appropriate gender.)

Necropsy Findings:

There were no genotype-related or biologically significant differences noted between mutant and control mice for any of the parameters evaluated at necropsy. Incidental lesions may have been present in some tissues. These findings were considered to represent background lesions occasionally seen in this strain of mice, lesions due to spontaneous disease, age-related lesions, lesions due to procedural artifacts, and/or lesions of a nonspecific etiology. They were not considered to be related to genotype.

Body and Organ Weight Findings:

When compared to contemporaneous but not historical age- and gender-matched wild-type control mice, 300 day cohort hemizygous mutant males have a lower body weight. We are not reporting this finding as a phenotypic change but are reporting it for your consideration.

Differences in body length, body weight, organ weights, and/or organ weight to body weight ratios were present between individual mice. The variability between mice usually fell within our historical reference ranges and was not correlated with genotype.

Gene 936
Clinical Chemistry

There were no significant differences in the female homozygous and male hemizygous mutant mice when compared with age- and gender-matched heterozygous and wild-type control mice.

Serum samples from the following mice were evaluated by a clinical chemistry panel.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (173482, 173483, 192780)
4 heterozygous mutant females (192805, 192809, 195492, 200379)
5 hemizygous mutant males (173473, 173474, 173477, 200367, 200368)
2 wild-type control males (173470, 173471)

90 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (188944, 188945, 192807, 192810)
4 heterozygous mutant females (192774, 192776, 192777, 192811)
4 hemizygous mutant males (183361, 183362, 183364, 183365)
4 wild-type control males (183363, 183371, 183372, 183373)

180 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (188944, 188945, 192807, 192810)
4 heterozygous mutant females (192774, 192777, 192811, 202896)
4 hemizygous mutant males (183361, 183362, 183364, 183365)
4 wild-type control males (183363, 183371, 183372, 183373)

300 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (188945, 192807, 192810)
2 heterozygous mutant females (192774, 192811)
2 hemizygous mutant males (183362, 183364)
2 wild-type control males (183371, 183373)

Certain differences between mice, including lower triglyceride levels, were present that may have occurred spontaneously in this age group. When compared to age- and gender-matched contemporaneous wild-type control mice, such differences were present in four of five 49 day cohort hemizygous mutant males (173473, 173474, 200367, 200368) and two 300 day cohort hemizygous mutant males (183362, 183364). We have not presented these findings as phenotypic changes, but we have presented them here for your consideration.

Values for the various other analytes evaluated were generally similar between the female homozygous mutant, male hemizygous mutant, female heterozygous control, and male wild-type control mice. Although variations in clinical chemistry values were present in some mice, they were not related to genotype and, thus, were not considered phenotypically relevant.

Gene 936
Hematology

There were no significant differences in the female homozygous and male hemizygous mutant mice when compared with age- and gender-matched heterozygous and wild-type control mice.

Blood samples from the following mice were evaluated by a complete blood count and differential cell count.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (173483, 173485, 192780)
4 heterozygous mutant females (192809, 195492, 200379, 200381)
5 hemizygous mutant males (173473, 173474, 195497, 200368, 202899)
2 wild-type control males (173470, 173471)

90 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (188944, 188945, 192807, 192810)
4 heterozygous mutant females (192774, 192811, 202893, 202894)
4 hemizygous mutant males (183361, 183362, 183364, 183365)
3 wild-type control males (183363, 183372, 183373)

180 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (188944, 188945, 192807, 192810)
4 heterozygous mutant females (192774, 192776, 192777, 192811)
4 hemizygous mutant males (183361, 183362, 183364, 183365)
3 wild-type control males (183363, 183371, 183373)

300 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (188945, 192807, 192810)
2 heterozygous mutant females (192774, 192811)
2 hemizygous mutant males (183362, 183364)
2 wild-type control males (183371, 183373)

Although minor variations of hematological values were present in some mice, these changes were not related to genotype and, thus, were not considered phenotypically relevant.

Gene 936
Physical Examination

There were no significant differences detected in the female homozygous and male hemizygous mutant mice when compared with age- and gender-matched heterozygous and wild-type control mice.

The following mice were evaluated by physical examination.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (173482, 173483, 173485)
4 heterozygous mutant females (192805, 192809, 200379, 200381)
5 hemizygous mutant males (173473, 173474, 173477, 200367, 200368)
2 wild-type control males (173470, 173471)

300 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (188945, 192807, 192810)
2 heterozygous mutant females (192774, 192811)
2 hemizygous mutant males (183362, 183364)
2 wild-type control males (183371, 183373)

Mice were examined in detail as follows: anus, behavior, body shape, claws, coat - fur, coat color - back, coat color - belly, ear - left, ear - right, eye - left, eye - right, eye color - left, eye color - right, feces, forelimb - left, forelimb - right, forelimb number of amputated digits - left, forelimb number of amputated digits - right, forelimb number of digits - left, forelimb number of digits - right, general appearance, genitals - female, genitals - male, hair type, head shape, hindlimb - left, hindlimb - right, hindlimb number of amputated digits - left, hindlimb number of amputated digits - right, hindlimb number of digits - left, hindlimb number of digits - right, injuries, lesions, limb shape, locomotor, lumps - masses, mammary glands, mice in cage, respiration, skin appearance, snout, swelling - joints, tail, teeth color, teeth length, urine, and whiskers. (Gender-specific observables apply to the appropriate gender.)

Individual female homozygous and male hemizygous mutant mice had only occasional minor differences in observed physical features compared to female heterozygous and male wild-type control mice. These findings are considered to represent individual variability, background features occasionally seen in this strain of mice, findings due to spontaneous disease, age-related findings, procedural artifacts, and/or findings of a nonspecific etiology. However, none of these differences was regarded as biologically significant or genotype related.

Gene 936
Aging Metrics

There were no significant differences detected in the homozygous mutant mice when compared with age- and gender-matched wild-type control mice.

Body weights and body lengths were measured for mice at 49, 90, 180, and 300 days of age.

49 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (188944, 188945, 192807, 192810)
5 heterozygous mutant females (192774, 192776, 192777, 192811, 195499)
6 hemizygous mutant males (183361, 183362, 183364, 183365, 202899, 202910)
4 wild-type control males (183363, 183371, 183372, 183373)

90 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (188944, 188945, 192807, 192810)
4 heterozygous mutant females (192774, 192776, 192777, 192811)
4 hemizygous mutant males (183361, 183362, 183364, 183365)
4 wild-type control males (183363, 183371, 183372, 183373)

180 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (188944, 188945, 192807, 192810)
4 heterozygous mutant females (192774, 192776, 192777, 192811)
4 hemizygous mutant males (183361, 183362, 183364, 183365)
4 wild-type control males (183363, 183371, 183372, 183373)

300 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (185976, 192807, 192810)
5 heterozygous mutant females (192774, 192811, 200375, 202896, 202897)
4 hemizygous mutant males (183361, 183362, 183364, 183365)
3 wild-type control males (183363, 183371, 183373)

Body Weight and Length Findings:

When compared to contemporaneous but not historical age- and gender-matched wild-type control mice, 300 day cohort hemizygous mutant males had a lower total tissue mass. We are not reporting this finding as a phenotypic change but are presenting it for your consideration.

Other differences in body length and body weight were present between individual mice. The variability between mice usually fell within our historical reference ranges and was not correlated with genotype.

Gene 936
Home Cage Observations

Changes related to genotype:

Movement : Hyperactivity.

Home cage observations are collected by animal care personnel over the lifetime of the animals. The observables are similar to those found in the physical examination.

The following mouse had home cage observations:

68 Day Mouse ID number is as follows:
1 hemizygous mutant male (188938)

A hemizygous mutant male (188938) was reported to be hyperactive. This finding correlates with the findings of behavioral studies.