Behavioral analysis of Ste20 kinase SPAK knockout mice.

SPAK/STK39 is a mammalian protein kinase involved in the regulation of inorganic ion transport mechanisms known to modulate GABAergic neurotransmission in the both central and the peripheral nervous systems. We have previously shown that disruption of the gene encoding SPAK by homologous recombination in mouse embryonic stem cells results in viable mice that lack expression of the kinase. With the exception of reduced fertility, these mice do not exhibit an overt adverse phenotype. In the present study, we examine the neurological phenotype of these mice by subjecting them to an array of behavioral tests. We show that SPAK knockout mice displayed a higher nociceptive threshold than their wild-type counterparts on the hot plate and tail flick assays. SPAK knockout mice also exhibited a strong locomotor phenotype evidenced by significant deficits on the rotarod and decreased activity in open-field tests. In contrast, balance and proprioception was not affected. Finally, they demonstrated an increased anxiety-like phenotype, spending significantly longer periods of time in the dark area of the light/dark box and increased thigmotaxis in the open-field chamber. These results suggest that the kinase plays an important role in CNS function, consistent with SPAK regulating ion transport mechanisms directly involved in inhibitory neurotransmission.

Sensorimotor gating in neurotensin-1 receptor null mice.

BACKGROUND: Converging evidence has implicated endogenous neurotensin (NT) in the pathophysiology of brain processes relevant to schizophrenia. Prepulse inhibition of the startle reflex (PPI) is a measure of sensorimotor gating and considered to be of strong relevance to neuropsychiatric disorders associated with psychosis and cognitive dysfunction. Mice genetically engineered to not express NT display deficits in PPI that model the PPI deficits seen in schizophrenia patients. NT1 receptors have been most strongly implicated in mediating the psychosis relevant effects of NT such as attenuating PPI deficits. To investigate the role of NT1 receptors in the regulation of PPI, we measured baseline PPI in wildtype (WT) and NT1 knockout (KO) mice. We also tested the effects of amphetamine and dizocilpine, a dopamine agonist and NMDA antagonist, respectively, that reduce PPI as well as the NT1 selective receptor agonist PD149163, known to increase PPI in rats. METHODS: Baseline PPI and acoustic startle response were measured in WT and NT1 KO mice. After baseline testing, mice were tested again after receiving intraperatoneal (IP) saline or one of three doses of amphetamine (1.0, 3.0 and 10.0 mg/kg), dizocilpine (0.3, 1.0 and 3.0 mg/kg) and PD149163 (0.5, 2.0 and 6.0 mg/kg) on separate test days. RESULTS: Baseline PPI and acoustic startle response in NT1 KO mice were not significantly different from NT1 WT mice. WT and KO mice exhibited similar responses to the PPI-disrupting effects of dizocilpine and amphetamine. PD149163 significantly facilitated PPI (P < 0.004) and decreased the acoustic startle response (P < 0.001) in WT but not NT1 KO mice. CONCLUSIONS: The data does not support the regulation of baseline PPI or the PPI disruptive effects of amphetamine or dizocilpine by endogenous NT acting at the NT1 receptor, although they support the antipsychotic potential of pharmacological activation of NT1 receptors by NT1 agonists.

Unveiling the molecular mechanisms behind selenium-related diseases through knockout mouse studies.

Selenium (Se), in the form of the 21st amino acid selenocysteine, is an integral part of selenoproteins and essential for mammals. While a large number of health claims for Se has been proposed in a diverse set of diseases, little is known about the precise molecular mechanisms and the physiological roles of selenoproteins. With the recent and rigorous application of reverse genetics in the mouse, great strides have been made to address this on a more molecular level. In this review, we focus on results obtained from the application of mouse molecular genetics in mouse physiology and discuss these insights into the physiological actions of selenoproteins in light of evidence from human genetics.

Elevated anxiety-like and depressive behavior in Desert hedgehog knockout male mice.

To investigate the functional role of Desert hedgehog (Dhh) gene in the nervous system, we examined motor, sensory, learning and memory functions as well as mood in Dhh knockout (KO) mice. Dhh KO male mice exhibited prolonged immobility time compared with wild-type male mice in the forced swimming test, and showed enhanced inhibition in the Vogel's conflict model. These findings suggest that Dhh KO male mice exhibited enhanced anxiety and depressive behavior compared with wild-type male mice. In contrast, Dhh KO female mice did not show any significant difference compared to wild-type female mice. These behavioral abnormalities of Dhh KO male mice may be due to lower testosterone levels with abnormal development of the testes caused by Dhh-null mutation.

Behavioral and biochemical characterization of a mutant mouse strain lacking D-amino acid oxidase activity and its implications for schizophrenia.

D-amino acid oxidase (DAO) degrades D-serine, a co-agonist at the NMDA receptor (NMDAR). Hypofunction of the NMDAR has been suggested to contribute to the pathophysiology of schizophrenia. Intriguingly, DAO has been recently identified as a risk factor for schizophrenia through genetic association studies. A naturally occurring mouse strain (ddY/DAO-) has been identified which lacks DAO activity. We have characterized this strain both behaviorally and biochemically to evaluate DAO as a target for schizophrenia. We have confirmed that this strain lacks DAO activity and shown for the first time it has increased occupancy of the NMDAR glycine site due to elevated extracellular D-serine levels and has enhanced NMDAR function in vivo. Furthermore, the ddY/DAO- strain displays behaviors which suggest that it will be a useful tool for evaluation of the clinical benefit of DAO inhibition in schizophrenia.

Neural cell adhesion molecule-null mice are not deficient in prepulse inhibition of the startle response.

Mice constitutively deficient in the neural cell adhesion molecule have morphological changes in the brain, which are hallmarks of schizophrenia. Schizophrenic patients are impaired in sensorimotor processing indicated by a deficit in prepulse inhibition of the acoustic startle response. Here we tested whether prepulse inhibition and prepulse facilitation are changed in neural cell adhesion molecule-deficient mice compared with their wild-type littermates. Neither prepulse inhibition nor prepulse facilitation (which occurred only at the lowest prepulse intensity used and was weak) was altered. This result is discussed in the light of the 'two-hit' hypothesis of schizophrenia, suggesting that in neural cell adhesion molecule-deficient mice, a prepulse inhibition deficit may become apparent only after treatment with a 'second hit' (such as increased stress).

Involvement of serotonin in the hypoglycemic response to 2 Hz electroacupuncture of zusanli acupoint (ST36) in rats.

In our previous studies, an insulin-dependent hypoglycemic effect produced by electroacupuncture (EA) was shown to be mediated by endogenous opioid peptides (EOP). In the present study, we applied 2 Hz EA to both zusanli acupoints (ST36) in the test group for 30 min, and to a nonacupoint area in the control group for 30 min to compare the acupoint specific character in the hypoglycemic effect of EA. Assays of plasma beta-endorphin and insulin levels were performed by ELISA kits. The insulin-dependent mechanism of the hypoglycemic effect was also investigated in streptozotocin (STZ)-induced diabetic rats. The mediation of EOP and the role of mu-opioid receptor were examined by naloxone and mu-opioid receptor knockout mice (MOR-KOM). The serotonin depletion was carried out by injecting (i.p.) p-chlorophenylalanine (PCPA); two low doses of serotonin were also injected (i.v.) to analyze the direct effect on plasma glucose levels. The hypoglycemic effect of EA was much greater in rats stimulated at ST36 than in rats receiving the same stimulation at the nonacupoint area. The plasma levels of insulin and beta-endorphin were also significantly elevated after stimulation of both zusanli acupoints, but remained unchanged following stimulation at the nonacupoint area. There was no sharp hypoglycemic response to 2 Hz EA at zusanli acupoint of STZ-induced diabetic rats. However, the hypoglycemic effect of this EA was not totally blocked by the sufficient dose of naloxone (1 mg/kg, i.v.). Additionally, 2 Hz EA at ST36 also showed a sharp decrease in plasma glucose levels of MOR-KOM. Pretreatment with PCPA did not reproduce hypoglycemic response to 2 Hz EA in naloxone-treated rats and MOR-KOM mice. Furthermore, injection of serotonin decreased the plasma glucose levels significantly. Therefore, we suggest that serotonin also involved in the hypoglycemic action of 2 Hz EA at both zusanli acupoints of normal rats.

Type IX collagen knock-out mouse shows progressive hearing loss.

Type IX collagen is one of the important components, together with type II, V, and XI collagens, in the tectorial membrane of the organ of Corti. To confirm the significance of type IX collagen for normal hearing, we assessed the detailed morphological and electrophysiological features of type IX collagen knock-out mice, which have recently been reported as a deafness model. Through assessment by auditory brainstem response (ABR), knock-out mice were shown to have progressive hearing loss. At the light microscopic level, the tectorial membrane of knock-out mice was found to be abnormal in shape. These morphological changes started in the basal turn and were progressive toward the apical turn. Electron microscopy confirmed disturbance of organization of the collagen fibrils. These results suggest that mutations in type IX collagen genes may lead to abnormal integrity of collagen fibers in the tectorial membrane.

Behavioural characterization of vitamin D receptor knockout mice.

Vitamin D (calcitriol) is a nuclear transcription regulator acting via a nuclear hormone receptor (VDR). In addition to its role in the regulation of calcium and phosphate homeostasis and in bone formation, Vitamin D is also thought to be involved in brain function. The aim of this study was to behaviourally phenotype VDR knockout mice. We characterized the behaviour of VDR null mutant mice and wildtype littermate controls by subjecting them to a range of tests including a primary behavioural screen (using the SHIRPA protocol), rotarod, gait analysis, Y-maze, marble burying test, bedding test, holeboard test, elevated plus maze, open field test and prepulse inhibition of the acoustic startle response. There were no effects of genotype on most of the scores from the SHIRPA protocol except that VDR -/- mice had alopecia, were shorter and weighed less than VDR +/+ mice. VDR -/- mice had a shorter gait as well as impairments on the rotarod, in the bedding test and impaired habituation in both the open field and on the acoustic startle response. The VDR -/- mice had normal acoustic startle responses but had impaired PPI at long (256 ms) but not short (64 ms) prepulse to pulse intervals. The VDR -/- mice were less active in the open field and buried fewer marbles in the marble burying test. However, there were no differences in the time spent on the open arms of the elevated plus maze or in working memory as assessed by repeat arm entries on the Y-maze. Therefore, it appears that VDR -/- mice have muscular and motor impairments that significantly affects locomotor behaviour but seemingly no impairments in cognition as indicated by exploration, working memory or anxiety.

Characteristics of behavioral abnormalities in alpha1d-adrenoceptors deficient mice.

To investigate the functional role of alpha1d-adrenergic receptor (alpha1d-AR) in the CNS, we have generated mutant mice lacking the alpha1d-AR using a gene targeting approach and examined in detail the effects of alpha1d-AR knockout mice on motor function, sensory function, and learning and memory. alpha1d-AR knockout mice showed better motor coordination at the highest rotating speed of the rotarod performance and stronger muscle tone using the traction meter, but their locomotor activity and swimming ability in the water maze were not affected. In the water maze requiring reference memory, alpha1d-AR knockout mice showed normal spatial learning. In the Y-maze task requiring working memory or attention, alpha1d-AR knockout mice displayed an impaired spontaneous alternation performance. The alpha1d-AR knockout mice tended to display lower levels of acoustic startle responses than the wild-type group at lower pulse intensities, although the acoustic prepulse inhibition was not impaired in the alpha1d-AR knockout mice. Furthermore, the NMDA receptor antagonist, MK-801-induced deficits of acoustic prepulse inhibition were not observed in the alpha1d-AR knockout mice. These results clearly demonstrate that the alpha1d-AR receptor plays an important role in the process of auditory sensory function, attention or working memory rather than reference memory, and the sensorimotor gating deficits induced by the NMDA receptor antagonist.

Learning deficits in forebrain-restricted brain-derived neurotrophic factor mutant mice.

Brain-derived neurotrophic factor (BDNF) participates in synaptic plasticity and the adaptive changes in the strength of communication between neurons thought to underlie aspects of behavioral adaptation. By selectively deleting BDNF from the forebrain of mice using the Cre site-specific DNA recombinase, we were able to study the requirements for BDNF in behaviors such as learning and anxiety. Early-onset forebrain-restricted BDNF mutant mice (Emx-BDNF(KO)) that develop in the absence of BDNF in the dorsal cortex, hippocampus, and parts of the ventral cortex and amygdala failed to learn the Morris Water Maze task, a hippocampal-dependent visuo-spatial learning task. Freezing during all phases of cued-contextual fear conditioning, a behavioral task designed to study hippocampal-dependent associative learning, was enhanced. These mice learned a brightness discrimination task well but were impaired in a more difficult pattern discrimination task. Emx-BDNF(KO) mice did not exhibit altered sensory processing and gating, as measured by the acoustic startle response or prepulse inhibition of the startle response. Although they were less active in an open-field arena, they did not show alterations in anxiety, as measured in the elevated-plus maze, black-white chamber or mirrored chamber tasks. Combined, these data indicate that although an absence of forebrain BDNF does not disrupt acoustic sensory processing or alter baseline anxiety, specific forms of learning are severely impaired.

Prostaglandin E2 inhibits the potassium current in sensory neurons from hyperalgesic Kv1.1 knockout mice.

Prostaglandin E(2) (PGE(2)) enhances the sensitivity of sensory neurons to various forms of noxious stimulation. This occurs, in part, by the suppression of a delayed rectifier-like potassium current in these neurons. However, the molecular identity of this current remains unclear. Recent studies demonstrated that a mutant mouse lacking a delayed rectifier potassium channel gene, Kv1.1, displayed lowered thresholds to thermal stimulation in behavioral assays of pain perception, i.e. the Kcna1-null mice were hyperalgesic. Here we examined whether PGE(2) can alter the sensitivity of Kcna1-null mice to noxious stimulation and examine the capability of PGE(2) to inhibit the potassium current in these knockout mice. Behavioral assays were used to assess the effect of PGE(2) on either thermal hyperalgesia or mechanical sensitivities. In addition, the whole-cell patch-clamp technique was used to study the effects of PGE(2) on the total potassium current recorded from isolated mouse sensory neurons. Even with a reduced threshold to thermal stimulation, PGE(2) could still sensitize the response of Kcna1-null mice to thermal and mechanical stimulation by amounts that were similar to that in wild type mice. The activation properties of the potassium current were similar for both the wild type and the Kcna1-null mice, whereas the inactivation properties were different in cells exhibiting large amounts of steady-state inactivation (>50%) measured at +20 mV. PGE(2) suppressed the total potassium current in both groups of mice by 40-50% without altering the voltage dependence of activation. In addition, PGE(2) produced similar amounts of suppression in both groups of mice when currents were examined with the steady-state inactivation protocol. Based on these results, it is unlikely that Kv1.1 is the molecular identity of the potassium channel(s) modulated by PGE(2) to sensitize nociceptive sensory neurons. Also, the enhanced thermal sensitivity as observed in the Kcna1-null mice might be due to more central neurons of the pain sensing pathway.

Loss of cortical and thalamic neuronal tenascin-C expression in a transgenic mouse expressing exon 1 of the human Huntington disease gene.

A transgenic mouse containing the first exon of the human Huntington's disease (HD) gene has revealed a variety of behavioral and pathophysiological anomalies reminiscent of certain aspects of human Huntington's disease (HD). The present study has found that expression of the extracellular matrix glycoprotein tenascin-C appears to be unaffected in astroglial cells in wild-type and R6/2 transgenic mice that express the mutant huntingtin protein but that it is conspicuously absent in two neuronal populations within the cerebral cortex and thalamus of the R6/2 mice. Loss of tenascin-C expression begins between the fourth and eighth postnatal weeks, coincidental with the onset of abnormal behavioral phenotype and the appearance of intranuclear inclusion bodies and neuropil aggregates. By 12 weeks, R6/2 mice exhibit a complete absence of tenascin-C neuronal immunolabeling, a disappearance of cRNA probe-positive neurons across discrete cytoarchitectonic regions of the dorsal thalamus (e.g., the ventromedial, parafascicular, lateral posterior, and posterior thalamic groups) and frontal cortex, and an accompanying thalamic astrogliosis. The loss of neuronal tenascin-C expression includes structures that are known to send converging excitatory axonal projections to the caudate-putamen, the structure that is most at risk for neurodegeneration in HD. Altered neuronal expression of tenascin-C in R6/2 mice implicates altered transcriptional activities of the mutant huntingtin protein. The abnormal biochemistry and possibly abnormal activity of thalamostriate and corticostriate projection neurons may also affect abnormal neuronal activities in their primary connectional target, the neostriatum, which is severely compromised in HD.

Startle responses, heart rate, and temperature in 5-HT1B receptor knockout mice.

Relative to wildtype mice, mice lacking 5-HT1B receptors (5-HT1B KO) exhibit exaggerated heart rate and body temperature responses to environmental stimuli. In contrast, acoustic startle reactivity is reduced in 5-HT1B KO mice. We combined heart rate and temperature measurement with startle response paradigms in order to elucidate this apparent contradiction. Habituation and footshock-induced sensitization paradigms modulate startle reactivity. Reduced startle reactivity and unaltered habituation in 5-HT1B KO mice were replicated. Heart rate and temperature were unaffected by startle stimuli, but increased markedly in response to transportation and handling procedures. Footshocks caused a mild startle-sensitization and tachycardia in both genotypes. The physiological hyper-reactivity in 5-HT1B KO mice is a subtle phenotypic difference that contrasts with the phenotypic decrease in startle reactivity.

Natural melatonin 'knockdown' in C57BL/6J mice: rare mechanism truncates serotonin N-acetyltransferase.

Pineal melatonin synthesis (serotonin --> N-acetylserotonin --> melatonin) is severely compromised in most inbred strains of mice, in many cases because serotonin is not acetylated by serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT). We have found that in the C57BL/6J strain, AANAT mRNA encodes a severely truncated AANAT protein, because a pseudo-exon containing a stop codon is spliced in. This is the first identification of a natural mutation which knocks down melatonin synthesis. The decrease in melatonin signaling may have been a selective factor in the development of laboratory strains of mice because melatonin can inhibit reproduction and modify circadian rhythmicity.

Mechanism of increased lung injury after acute pancreatitis in IL-10 knockout mice.

BACKGROUND: IL-10 is a potent anti-inflammatory cytokine which inhibits inflammatory cytokine release from all tissue sites. Hepatic cytokine release from Kupffer cells (KC) is one important source of inflammatory cytokines and may be the main one causing lung damage in acute pancreatitis (AP). Here we studied the KC contribution to lung injury in IL-10 knockout (KO) mice, in which tissue inflammatory cytokine release from all sites is unrestrained, and AP is more severe. METHODS: Three- to 4-week-old C57BL/6J mice and KO mice on a C57BL/6J background were used. Control mice received regular chow and gadolinium chloride (GD; 1 mg/100 g iv), to inhibit KC activity, or saline. Pancreatitis mice received a choline-deficient, ethionine-supplemented diet for 66 h to induce AP and saline or GD injections iv. After 66 h, lung tissue was assessed for edema, myeloperoxidase (MPO), and superoxide dismutase (SOD). Histology was scored in a blinded fashion. RESULTS: In pancreatitis KO mice, KC blockade had no effect on the degree of lung edema, lung neutrophil infiltration, and lung histology score. As expected, each of these parameters was more severe in the KO mice than in the normal mice: lung wet/dry ratio 5.3 +/- 0.2 versus 4.3 +/- 0.13; lung MPO (U/g) 1.9 +/- 0.2 versus 1.1 +/- 0.08; histology score 7.1 +/- 0.8 versus 5.3 +/- 0.5. CONCLUSION: Endogenous IL-10 is important in reducing the lung injury in this model of AP. KC-derived cytokines were of minor importance, compared to those derived from all other tissue sites.

L-arginine prevents xanthoma development and inhibits atherosclerosis in LDL receptor knockout mice.

BACKGROUND: The potential antiatherosclerotic actions of NO were investigated in four groups of mice (n = 10 per group) lacking functional LDL receptor genes, an animal model of familial hypercholesterolemia. Group 1 was fed a regular chow diet. Groups 2 through 4 were fed a 1.25% high-cholesterol diet. In addition, group 3 received supplemental L-arginine and group 4 received L-arginine and N omega-nitro-L-arginine (L-NA), an inhibitor of NO synthase (NOS). METHODS AND RESULTS: Animals were killed at 6 months; aortas were stained with oil red O for planimetry and with antibodies against constitutive and inducible NOSs. Plasma cholesterol was markedly increased in the animals receiving the high-cholesterol diet. Xanthomas appeared in all mice fed the high-cholesterol diet alone but not in those receiving L-arginine. Aortic atherosclerosis was present in all mice on the high-cholesterol diet. The mean atherosclerotic lesion area was reduced significantly (P < .01) in the cholesterol-fed mice given L-arginine compared with those receiving the high-cholesterol diet alone. The mean atherosclerotic lesion area was significantly larger (P < .01) in cholesterol-fed mice receiving L-arginine + L-NA than in those on the high-cholesterol diet alone. Within the atherosclerotic plaques, endothelial cells immunoreacted for endothelial cell NOS; macrophages, foam cells, and smooth muscle cells immunostained strongly for inducible NOS and nitrotyrosine residues. CONCLUSIONS: The data indicate that L-arginine prevents xanthoma formation and reduces atherosclerosis in LDL receptor knockout mice fed a high-cholesterol diet. The abrogation of the beneficial effects of L-arginine by L-NA suggests that the antiatherosclerotic actions of L-arginine are mediated by NOS. The data suggest that L-arginine may be beneficial in familial hypercholesterolemia.