Jacqueline Blundell

Neuroligin-1 Deletion Results in Impaired Spatial Memory and Increased Repetitive Behavior

Neuroligins (NLs) are a family of neural cell-adhesion molecules that are involved in excitatory/inhibitory synapse specification. Multiple members of the NL family (including NL1) and their binding partners have been linked to cases of human autism and mental retardation. We have now characterized NL1-deficient mice in autism- and mental retardation-relevant behavioral tasks. NL1 knock-out (KO) mice display deficits in spatial learning and memory that correlate with impaired hippocampal long-term potentiation. In addition, NL1 KO mice exhibit a dramatic increase in repetitive, stereotyped grooming behavior, a potential autism-relevant abnormality. This repetitive grooming abnormality in NL1 KO mice is associated with a reduced NMDA/AMPA ratio at corticostriatal synapses. Interestingly, we further demonstrate that the increased repetitive grooming phenotype can be rescued in adult mice by administration of the NMDA receptor partial coagonist d-cycloserine. Broadly, these data are consistent with a role of synaptic cell-adhesion molecules in general, and NL1 in particular, in autism and implicate reduced excitatory synaptic transmission as a potential mechanism and treatment target for repetitive behavioral abnormalities.

Pharmacological Inhibition of mTORC1 Suppresses Anatomical, Cellular, and Behavioral Abnormalities in Neural-Specific Pten Knock-Out Mice

PTEN (phosphatase and tensin homolog deleted on chromosome ten) is a lipid phosphatase that counteracts the function of phosphatidylinositol-3 kinase (PI3K). Loss of function of PTEN results in constitutive activation of AKT and downstream effectors and correlates with many human cancers, as well as various brain disorders, including macrocephaly, seizures, Lhermitte–Duclos disease, and autism. We previously generated a conditional Pten knock-out mouse line with Pten loss in limited postmitotic neurons in the cortex and hippocampus. Pten-null neurons developed neuronal hypertrophy and loss of neuronal polarity. The mutant mice exhibited macrocephaly and behavioral abnormalities reminiscent of certain features of human autism. Here, we report that rapamycin, a specific inhibitor of mammalian target of rapamycin complex 1 (mTORC1), can prevent and reverse neuronal hypertrophy, resulting in the amelioration of a subset of PTEN-associated abnormal behaviors, providing evidence that the mTORC1 pathway downstream of PTEN is critical for this complex phenotype.

Postreactivation glucocorticoids impair recall of established fear memory.

Pavlovian fear conditioning provides one of the best rodent models of acquired anxiety disorders, including posttraumatic stress disorder. Injection of a variety of drugs after training in fear-conditioning paradigms can impair consolidation of fear memories. Indeed, early clinical trials suggest that immediate administration of such drugs after a traumatic event may decrease the risk of developing posttraumatic stress disorder in humans (Pitman et al., 2002; Vaiva et al., 2003). The use of such a treatment is limited by the difficulty of treating every patient at risk and by the difficulty in predicting which patients will experience chronic adverse consequences. Recent clinical trials suggest that administration of glucocorticoids may have a beneficial effect on established posttraumatic stress disorder (Aerni et al., 2004) and specific phobia (Soravia et al., 2006). Conversely, glucocorticoid administration after training is known to enhance memory consolidation (McGaugh and Roozendaal, 2002; Roozendaal, 2002). From a clinical perspective, enhancement of a fear memory or a reactivated fear memory would not be desirable. We report here that when glucocorticoids are administered immediately after reactivation of a contextual fear memory, subsequent recall is significantly diminished. Additional experiments support the interpretation that glucocorticoids not only decrease fear memory retrieval but, in addition, augment consolidation of fear memory extinction rather than decreasing reconsolidation. These findings provide a rodent model for a potential treatment of established acquired anxiety disorders in humans, as suggested by others (Aerni et al., 2004; Schelling et al., 2004), based on a mechanism of enhanced extinction.

Increased Anxiety-like Behavior in Mice Lacking the Inhibitory Synapse Cell Adhesion Molecule Neuroligin 2

Neuroligins (NL) are postsynaptic cell adhesion molecules that are thought to specify synapse properties. Previous studies showed that mutant mice carrying an autism‐associated point mutation in NL3 exhibit social interaction deficits, enhanced inhibitory synaptic function and increased staining of inhibitory synaptic puncta without changes in overall inhibitory synapse numbers. In contrast, mutant mice lacking NL2 displayed decreased inhibitory synaptic function. These studies raised two relevant questions. First, does NL2 deletion impair inhibitory synaptic function by altering the number of inhibitory synapses, or by changing their efficacy? Second, does this effect of NL2 deletion on inhibition produce behavioral changes? We now show that although NL2‐deficient mice exhibit an apparent decrease in number of inhibitory synaptic puncta, the number of symmetric synapses as determined by electron microscopy is unaltered, suggesting that NL2 deletion impairs the function of inhibitory synapses without decreasing their numbers. This decrease in inhibitory synaptic function in NL2‐deficient mice correlates with a discrete behavioral phenotype that includes a marked increase in anxiety‐like behavior, a decrease in pain sensitivity and a slight decrease in motor co‐ordination. This work confirms that NL2 modulates inhibitory synaptic function and is the first demonstration that global deletion of NL2 can lead to a selective behavioral phenotype.

Lasting anxiogenic effects of feline predator stress in mice: Sex differences in vulnerability to stress and predicting severity of anxiogenic response from the stress experience

Previous work in male Swiss Webster (CFW) mice demonstrated a long lasting effect of predator stress on risk assessment in the elevated plus maze (EPM). Most severe effects (increases in risk assessment) were seen following a brief unprotected exposure to a cat. Lesser effects were produced by a brief exposure of mice to the cat exposure room without a cat in the room (room stress). This graded response is analogous to the covariation of symptom severity and severity of the precipitating stressor in posttraumatic stress disorder (PTSD). The present study extended these findings to another strain of mice, C57/BL6, and a broader range of tests of anxiety-like behavior, including EPM, acoustic startle response and light/dark box test. Sex was introduced as a variable to investigate if females might be more susceptible to the effects of stressors than males, as has been suggested in human PTSD. Graded and lasting (7 days) effects of a 10 min exposure to a cat (predator stress) or to the cat exposure room only (room stress) were observed on lighted chamber avoidance in the light/dark box. Room stress was without effect on startle responses, but predator stress enhanced peak startle amplitudes measured in the light or in the dark. There was no evidence of light-enhancement of startle in C57 mice. Female mice were more susceptible to the effects of predator and room stress, depending on the measure. Females only responded to cat exposure with a lasting increase in average startle amplitude. This was due to an increased and more prolonged multipeak response to startle after the first and maximal peak startle response. In addition, in females, room and predator stress were equally anxiogenic in measures of open arm avoidance in the EPM. In contrast, room stress was without effect on open arm avoidance in males, but cat exposure was as anxiogenic in males as it was in females. These findings suggest EPM anxiety in females is affected more by the milder stress of room exposure. Severity of effects of predator stress on anxiety-like behaviors in EPM and startle were well predicted (60% of the variance) by measures of cat behavior and probability of mouse defensive response to particular cat behaviors during the cat exposure. Finally, factor analysis indicated that different tests of anxiety-like behavior may be measuring different and independent aspects of mouse affect. Moreover, stressors had no lasting effects on sugar solution consumption. Implications of these findings for modeling PTSD and using transgenic strains of mice to study lasting effects of stress on affect are discussed.

Systemic Inhibition of Mammalian Target of Rapamycin Inhibits Fear Memory Reconsolidation

BACKGROUND Established traumatic memories have a selective vulnerability to pharmacologic interventions following their reactivation that can decrease subsequent memory recall. This vulnerable period following memory reactivation is termed reconsolidation. The pharmacology of traumatic memory reconsolidation has not been fully characterized despite its potential as a therapeutic target for established, acquired anxiety disorders including posttraumatic stress disorder (PTSD). The mammalian target of rapamycin (mTOR) kinase is a critical regulator of mRNA translation and is known to be involved in various forms of synaptic plasticity and memory consolidation. We have examined the role of mTOR in traumatic memory reconsolidation. METHODS Male C57BL/6 mice were injected systemically with the mTOR inhibitor rapamycin (1-40mg/kg), at various time points relative to contextual fear conditioning training or fear memory retrieval, and compared to vehicle or anisomycin-treated groups (N=10-12 in each group). RESULTS Inhibition of mTOR via systemic administration of rapamycin blocks reconsolidation of an established fear memory in a lasting manner. This effect is specific to reconsolidation as a series of additional experiments make an effect on memory extinction unlikely. CONCLUSIONS Systemic rapamycin, in conjunction with therapeutic traumatic memory reactivation, can decrease the emotional strength of an established traumatic memory. This finding not only establishes mTOR regulation of protein translation in the reconsolidation phase of traumatic memory, but also implicates a novel, FDA-approved drug treatment for patients suffering from acquired anxiety disorders such as PTSD and specific phobia.

Neural circuit changes mediating lasting brain and behavioral response to predator stress.

This paper reviews recent work which points to critical neural circuitry involved in lasting changes in anxiety like behavior following unprotected exposure of rats to cats (predator stress). Predator stress may increase anxiety like behavior in a variety of behavioral tests including: elevated plus maze, light dark box, acoustic startle, and social interaction. Studies of neural transmission in two limbic pathways, combined with path and covariance analysis relating physiology to behavior, suggest long term potentiation like changes in one or both of these pathways in the right hemisphere accounts for stress induced changes in all behaviors changed by predator stress except light dark box and social interaction. Findings will be discussed within the context of what is known about neural substrates activated by predator odor.

Vulnerability to mild predator stress in serotonin transporter knockout mice

Effect of predator stress on rat and mouse anxiety-like behavior may model aspects of post traumatic stress disorder (PTSD). A single cat exposure of wild type (C57, CFW) mice can produce lasting anxiety-like effects in the elevated plus maze, light/dark box tests and startle. In addition, female but not male C57 mice are made more anxious in the plus maze by exposure to predator odors alone, suggesting differential vulnerability to predator stressors of differing intensity. There is a link between genetic variation in the serotonin (5-HT) transporter (SERT) and anxiety in humans. This prompted the generation of SERT knockout mice [see Holmes A, Murphy DL, Crawley, JN. Biol Psychiatry 2003;54(10):953-9]. Present work used these mice to determine if there was a link between vulnerability to the anxiogenic effects of predator odors and abnormalities of 5-HT transmission induced by a life long reduction in 5-HT reuptake. Wild type (WT, C57 background), heterozygous (SERT +/-, HET) mice and homozygous knockout (SERT -/-, KO) were assigned to handled control groups or groups exposed for 10 min to a large testing room rich in cat odor. One week after handling or room exposure, anxiety testing took place in the dark phase of the light/dark cycle, in red light. Predator odor exposure was selectively anxiogenic in the plus maze and light/dark box tests in SERT -/- mice. Exposure to predator odor did not potentiate startle. Findings suggest a role for abnormalities in 5-HT transmission in vulnerability to some of the lasting anxiogenic effects of species relevant stressors and possibly in vulnerability to PTSD.

Relationship of the predatory attack experience to neural plasticity, pCREB expression and neuroendocrine response.

Aggression takes at least two, an attacker and a target. This paper will address the lasting consequences of being a target of aggression. We review the lasting impact of predatory attack on brain and behavior in rodents. A single brief unprotected exposure of a rat to a cat lastingly alters affective responses of rats in a variety of contexts. Alterations of these behaviors resembles both generalized anxiety comorbid with post traumatic stress disorder (PTSD), and the hyper arousal expressed in enhanced startle in PTSD. Examination of neural transmission and neural plasticity in limbic circuits implicates changes in transmission in two connecting pathways in many but not all of the behavioral changes. Quantification of the predator encounter reveals that both the behavior of the predator and the reaction of the rat to attack are highly predictive of the effects of predatory attack on molecular biological (pCREB expression) and electrophysiological measures of limbic neuroplastic change. Moreover, a case will be made that the pattern of change of corticosteroid level over three hours after the predator encounter, in interaction with the predatory experience, plays an important part in initiation of lasting changes in brain and behavior.

RIM1α phosphorylation at serine-413 by protein kinase A is not required for presynaptic long-term plasticity or learning

Activation of presynaptic cAMP-dependent protein kinase A (PKA) triggers presynaptic long-term plasticity in synapses such as cerebellar parallel fiber and hippocampal mossy fiber synapses. RIM1α, a large multidomain protein that forms a scaffold at the presynaptic active zone, is essential for presynaptic long-term plasticity in these synapses and is phosphorylated by PKA at serine-413. Previous studies suggested that phosphorylation of RIM1α at serine-413 is required for presynaptic long-term potentiation in parallel fiber synapses formed in vitro by cultured cerebellar neurons and that this type of presynaptic long-term potentiation is mediated by binding of 14-3-3 proteins to phosphorylated serine-413. To test the role of serine-413 phosphorylation in vivo, we have now produced knockin mice in which serine-413 is mutated to alanine. Surprisingly, we find that in these mutant mice, three different forms of presynaptic PKA-dependent long-term plasticity are normal. Furthermore, we observed that in contrast to RIM1α KO mice, RIM1 knockin mice containing the serine-413 substitution exhibit normal learning capabilities. The lack of an effect of the serine-413 mutation of RIM1α is not due to compensation by RIM2α because mice carrying both the serine-413 substitution and a RIM2α deletion still exhibited normal long-term presynaptic plasticity. Thus, phosphorylation of serine-413 of RIM1α is not essential for PKA-dependent long-term presynaptic plasticity in vivo, suggesting that PKA operates by a different mechanism despite the dependence of long-term presynaptic plasticity on RIM1α.