Tori L. Schaefer

Deficiency in Na,K-ATPase α Isoform Genes Alters Spatial Learning, Motor Activity, and Anxiety in Mice

Several disorders have been associated with mutations in Na,K-ATPase α isoforms (rapid-onset dystonia parkinsonism, familial hemiplegic migraine type-2), as well as reduction in Na,K-ATPase content (depression and Alzheimers disease), thereby raising the issue of whether haploinsufficiency or altered enzymatic function contribute to disease etiology. Three isoforms are expressed in the brain: the α1 isoform is found in many cell types, the α2 isoform is predominantly expressed in astrocytes, and the α3 isoform is exclusively expressed in neurons. Here we show that mice heterozygous for the α2 isoform display increased anxiety-related behavior, reduced locomotor activity, and impaired spatial learning in the Morris water maze. Mice heterozygous for the α3 isoform displayed spatial learning and memory deficits unrelated to differences in cued learning in the Morris maze, increased locomotor activity, an increased locomotor response to methamphetamine, and a 40% reduction in hippocampal NMDA receptor expression. In contrast, heterozygous α1 isoform mice showed increased locomotor response to methamphetamine and increased basal and stimulated corticosterone in plasma. The learning and memory deficits observed in the α2 and α3 heterozygous mice reveal the Na,K-ATPase to be an important factor in the functioning of pathways associated with spatial learning. The neurobehavioral changes seen in heterozygous mice suggest that these mouse models may be useful in future investigations of the associated human CNS disorders.

Abnormal neurodevelopment, neurosignaling and behaviour in Npas3-deficient mice

Npas3 is a member of the bHLH‐PAS superfamily of transcription factors that is expressed broadly in the developing neuroepithelium. To study the function of this gene, mice deficient in Npas3 were generated and characterized. Npas3–/– mice were growth‐retarded and exhibited developmental brain abnormalities that included a reduction in size of the anterior hippocampus, hypoplasia of the corpus callosum and enlargement of the ventricles. A number of behavioural abnormalities were identified in Npas3–/– mice including locomotor hyperactivity, subtle gait defects, impairment of prepulse inhibition of acoustic startle, deficit in recognition memory and altered anxiety‐related responses. Characterization of neurosignaling pathways using several pharmacological agents revealed dysfunctional glutamate, dopamine and serotonin neurotransmitter signaling. Consistent with these findings, we identified a significant alteration in cortical PSD‐95 expression, a PDZ‐containing protein that has been shown to be involved in postsynaptic signal transduction. Together, our observations indicate an important role for Npas3 in controlling normal brain development and neurosignaling pathways.

Effects of neonatal (+)-methamphetamine on path integration and spatial learning in rats: effects of dose and rearing conditions

Postnatal day (P)11–20 (+)‐methamphetamine (MA) treatment impairs spatial learning and reference memory in the Morris water maze, but has marginal effects on learning in a labyrinthine maze. A subsequent experiment showed that MA treatment on P11–15, but not P16–20, is sufficient to induce Morris maze deficits. Here we tested the effects of P11–15 MA treatment under two different rearing conditions on Morris maze performance and path integration learning in the Cincinnati water maze in which distal cues were unavailable by using infrared illumination. Littermates were treated with 0, 10, 15, 20, or 25 mg/kg MA × 4/day (2 h intervals). Half the litters were reared under standard housing conditions and half under partial enrichment by adding stainless steel enclosures. All MA groups showed impaired Cincinnati water maze performance with no significant effects of rearing condition. In the Morris maze, the MA‐25 group showed impaired spatial acquisition, reversal, and small platform learning. Enrichment significantly improved Morris maze acquisition in all groups but did not interact with treatment. The male MA‐25 group was also impaired on probe trial performance after acquisition and on small platform trials. A narrow window of MA treatment (P11–15) induces impaired path integration learning irrespective of dose within the range tested but impairments in spatial learning are dependent on dose. The results demonstrate that a narrower exposure window (5 days) changes the long‐term effects of MA treatment compared to longer exposures (10 days).

Creatine Transporter (CrT; Slc6a8) Knockout Mice as a Model of Human CrT Deficiency

Mutations in the creatine (Cr) transporter (CrT; Slc6a8) gene lead to absence of brain Cr and intellectual disabilities, loss of speech, and behavioral abnormalities. To date, no mouse model of CrT deficiency exists in which to understand and develop treatments for this condition. The purpose of this study was to generate a mouse model of human CrT deficiency. We created mice with exons 2–4 of Slc6a8 flanked by loxP sites and crossed these to Cre:CMV mice to create a line of ubiquitous CrT knockout expressing mice. Mice were tested for learning and memory deficits and assayed for Cr and neurotransmitter levels. Male CrT−/y (affected) mice lack Cr in the brain and muscle with significant reductions of Cr in other tissues including heart and testes. CrT−/y mice showed increased path length during acquisition and reversal learning in the Morris water maze. During probe trials, CrT−/y mice showed increased average distance from the platform site. CrT−/y mice showed reduced novel object recognition and conditioned fear memory compared to CrT+/y. CrT−/y mice had increased serotonin and 5-hydroxyindole acetic acid in the hippocampus and prefrontal cortex. Ubiquitous CrT knockout mice have learning and memory deficits resembling human CrT deficiency and this model should be useful in understanding this disorder.

Mouse plasmacytoma-expressed transcript 1 knock out induced 5-HT disruption results in a lack of cognitive deficits and an anxiety phenotype complicated by hypoactivity and defensiveness.

Serotonin (5-HT) is involved in many developmental processes and influences behaviors including anxiety, aggression, and cognition. Disruption of the serotonergic system has been implicated in human disorders including autism, depression, schizophrenia, and ADHD. Although pharmacological, neurotoxin, and dietary manipulation of 5-HT and tryptophan hydroxylase has added to our understanding of the serotonergic system, the results are complicated by multiple factors. A newly identified ETS domain transcription factor, Pet-1, has direct control of major aspects of 5-HT neuronal development. Pet-1 is the only known factor that is restricted in the brain to 5-HT neurons during development and adulthood and exerts dominant control over 5-HT neuronal phenotype. Disruption of Pet-1 produces an approximately 80% loss of 5-HT neurons and content and results in increased aggression in male Pet-1(-/-) mice [Hendricks TJ, Fyodorov DV, Wegman LJ, Lelutiu NB, Pehek EA, Yamamoto B, Silver J, Weeber EJ, Sweatt JD, Deneris ES (2003) Neuron 37:233-247]. We hypothesized that Pet-1(-/-) mice would also exhibit changes in anxiety and cognition. Pet-1(-/-) mice were hypoactive which may have affected the observed lack of anxious behavior in the elevated zero maze and light-dark test. Pet-1(-/-) mice, however, were more defensive during marble burying and showed acoustic startle hyper-reactivity. No deficits in spatial, egocentric, or novel object recognition learning were found in Pet-1(-/-) mice. These findings were unexpected given that 5-HT depleting drugs given to adult or developing animals result in learning deficits [Mazer C, Muneyyirci J, Taheny K, Raio N, Borella A, Whitaker-Azmitia P (1997) Brain Res 760:68-73; Morford LL, Inman-Wood SL, Gudelsky GA, Williams MT, Vorhees CV (2002) Eur J Neurosci 16:491-500; Vorhees CV, Schaefer TL, Williams MT (2007) Synapse 61:488-499]. Lack of differences may be the result of compensatory mechanisms in reaction to a constitutive knock out of Pet-1 or 5-HT may not be as important in learning and memory as previously suspected.

Short‐ and long‐term effects of (+)‐methamphetamine and (±)‐3,4‐methylenedioxymethamphetamine on monoamine and corticosterone levels in the neonatal rat following multiple days of treatment

J. Neurochem. (2008) 104, 1674–1685.

Phosphodiesterase 1B differentially modulates the effects of methamphetamine on locomotor activity and spatial learning through DARPP32‐dependent pathways: evidence from PDE1B‐DARPP32 double‐knockout mice

Mice lacking phosphodiesterase 1B (PDE1B) exhibit an exaggerated locomotor response to d‐methamphetamine and increased in vitro phosphorylation of DARPP32 (dopamine‐ and cAMP‐regulated phosphoprotein, Mr 32 kDa) at Thr34 in striatal brain slices treated with the D1 receptor agonist, SKF81297. These results indicated a possible regulatory role for PDE1B in pathways involving DARPP32. Here, we generated PDE1B × DARPP32 double‐knockout (double‐KO) mice to test the role of PDE1B in DARPP32‐dependent pathways in vivo. Analysis of the response to d‐methamphetamine on locomotor activity showed that the hyperactivity experienced by PDE1B mutant mice was blocked in PDE1B–/–x DARPP32–/– double‐KO mice, consistent with participation of PDE1B and DARPP32 in the same pathway. Further behavioral testing in the elevated zero‐maze revealed that DARPP32–/– mice showed a less anxious phenotype that was nullified in double‐mutant mice. In contrast, in the Morris water maze, double‐KO mice showed deficits in spatial reversal learning not observed in either single mutant compared with wild‐type mice. The data suggest a role for PDE1B in locomotor responses to psychostimulants through modulation of DARPP32‐dependent pathways; however, this modulation does not necessarily impact other behaviors, such as anxiety or learning. Instead, the phenotype of double‐KOs observed in these latter tasks may be mediated through independent pathways.

Prenatal immune challenge in rats: Altered responses to dopaminergic and glutamatergic agents, prepulse inhibition of acoustic startle, and reduced route-based learning as a function of maternal body weight gain after prenatal exposure to Poly IC

Prenatal maternal immune activation has been used to test the neurodevelopmental hypothesis of schizophrenia. Most of the data are in mouse models; far less is available for rats. We previously showed that maternal weight change in response to the immune activator polyinosinic‐polycytidylic acid (Poly IC) in rats differentially affects offspring. Therefore, we treated gravid Harlan Sprague‐Dawley rats i.p. on embryonic day 14 with 8 mg/kg of Poly IC or Saline. The Poly IC group was divided into those that lost or gained the least weight, Poly IC (L), versus those that gained the most weight, Poly IC (H), following treatment. The study design controlled for litter size, litter sampling, sex distribution, and test experience. We found no effects of Poly IC on elevated zero maze, open‐field activity, object burying, light–dark test, straight channel swimming, Morris water maze spatial acquisition, reversal, or shift navigation or spatial working or reference memory, or conditioned contextual or cued fear or latent inhibition. The Poly IC (H) group showed a significant decrease in the rate of route‐based learning when visible cues were unavailable in the Cincinnati water maze and reduced prepulse inhibition of acoustic startle in females, but not males. The Poly IC (L) group exhibited altered responses to acute pharmacological challenges: exaggerated hyperactivity in response to (+)‐amphetamine and an attenuated hyperactivity in response to MK‐801. This model did not exhibit the cognitive, or latent inhibition deficits reported in Poly IC‐treated rats but showed changes in response to drugs acting on neurotransmitter systems implicated in the pathophysiology of schizophrenia (dopaminergic hyperfunction and glutamatergic hypofunction). Synapse 2012.

Effects of (+)-methamphetamine on path integration and spatial learning, but not locomotor activity or acoustic startle, align with the stress hyporesponsive period in rats

Rats treated with (+)‐methamphetamine (MA) on postnatal days (P) 11–20 exhibit long‐term spatial and path integration (Morris water maze (MWM) and Cincinnati water maze (CWM)) learning deficits whereas those treated on P1–10 do not. MA treatment increases corticosterone release in an age‐dependent U‐shaped pattern that corresponds to the stress hyporesponsive period (SHRP; P4–15). Here we tested the hypothesis that the cognitive effects induced by MA are associated with treatment that begins within the SHRP. Three treatment regimens were compared, P1–10, P6–15, and P11–20. One male/female pair/litter received 0, 10, or 25 mg/kg MA/dose (four doses/day at 2 h intervals given s.c. with 19–21 litters/regimen). Locomotor activity and acoustic startle were tested as behaviors not predicted to be associated with the SHRP. Cincinnati and Morris water maze findings were consistent with the hypothesis in that MA‐treated animals exposed from P6–15 or P11–20 showed impaired learning compared to those exposed from P1–10; however, on probe trials in the Morris water maze, MA‐induced memory impairments were not regimen‐specific and were contributed to by all treatment regimens. All MA treatment regimens induced reductions in locomotor activity and acoustic startle facilitation as expected. No differential effect on prepulse trials was seen suggesting no impairment in sensory gating. Cognitive deficits from neonatal MA treatment are associated with the SHRP and may be the product of hypothalamic–pituitary–adrenal (HPA) axis dysregulation during critical periods of brain development.

Alterations in Body Temperature, Corticosterone, and Behavior Following the Administration of 5-Methoxy-Diisopropyltryptamine (‘Foxy’) to Adult Rats: a New Drug of Abuse

Many drugs are used or abused in social contexts without understanding the ramifications of their use. In this study, we examined the effects of a newly popular drug, 5-methoxy-diisopropyltryptamine (5-MEO-DIPT; ‘foxy’ or ‘foxy-methoxy’). Two experiments were performed. In the first, 5-MEO-DIPT (0, 10, or 20 mg/kg) was administered to rats four times on a single day and animals were examined 3 days later. The animals that received 5-MEO-DIPT demonstrated hypothermia during the period of drug administration and delayed mild hyperthermic rebound for at least 48 h. Corticosterone levels in plasma were elevated in a dose-dependent manner compared to saline-treated animals with minor changes in 5-HT turnover and no changes in monoamine levels. In experiment 2, rats were examined in behavioral tasks following either 0 or 20 mg/kg of 5-MEO-DIPT. The animals treated with 5-MEO-DIPT showed hypoactivity and an attenuated response to (+)-methamphetamine-induced stimulation (1 mg/kg). In a test of path integration (Cincinnati water maze), 5-MEO-DIPT-treated animals displayed deficits in performance compared to the saline-treated animals. No differences were noted in the ability of the animals to perform in the Morris water maze or on tests of novel object or place recognition. The data demonstrate that 5-MEO-DIPT alters the ability of an animal to perform certain cognitive tasks, while leaving others intact and disrupts the endocrine system. 5-MEO-DIPT may have the potential to induce untoward effects in humans.