Hippocampal c-Jun-N-terminal kinases serve as negative regulators of associative learning

In the adult mouse, signaling through c-Jun N-terminal kinases (JNKs) links exposure to acute stress to various physiological responses. Inflammatory cytokines, brain injury and ischemic insult, or exposure to psychological acute stressors induce activation of hippocampal JNKs. Here we report that exposure to acute stress caused activation of JNKs in the hippocampal CA1 and CA3 subfields, and impaired contextual fear conditioning. Conversely, intrahippocampal injection of JNKs inhibitors sp600125 (30 mum) or D-JNKI1 (8 mum) reduced activity of hippocampal JNKs and rescued stress-induced deficits in contextual fear. In addition, intrahippocampal administration of anisomycin (100 mug/mul), a potent JNKs activator, mimicked memory-impairing effects of stress on contextual fear. This anisomycin-induced amnesia was abolished after cotreatment with JNKs selective inhibitor sp600125 without affecting anisomycin\u27s ability to effectively inhibit protein synthesis as measured by c-Fos immunoreactivity. We also demonstrated milder and transient activation of the JNKs pathway in the CA1 subfield of the hippocampus during contextual fear conditioning and an enhancement of contextual fear after pharmacological inhibition of JNKs under baseline conditions. Finally, using combined biochemical and transgenic approaches with mutant mice lacking different members of the JNK family (Jnk1, Jnk2, and Jnk3), we provided evidence that JNK2 and JNK3 are critically involved in stress-induced deficit of contextual fear, while JNK1 mainly regulates baseline learning in this behavioral task. Together, these results support the possibility that hippocampal JNKs serve as a critical molecular regulator in the formation of contextual fear

JIP1-Mediated JNK Activation Negatively Regulates Synaptic Plasticity and Spatial Memory

The c-Jun N-terminal kinase (JNK) signal transduction pathway is implicated in learning and memory. Here, we examined the role of JNK activation mediated by the JIP1 scaffold protein. We compared male wild-type mice with a mouse model harboring a point mutation in the Jip1 gene that selectively blocks JIP1-mediated JNK activation. These male mutant mice exhibited increased NMDA receptor currents, increased NMDA receptor-mediated gene expression, and a lower threshold for induction of hippocampal long-term potentiation. The JIP1 mutant mice also displayed improved hippocampus-dependent spatial memory and enhanced associative fear conditioning. These results were confirmed using a second JIP1 mutant mouse model that suppresses JNK activity. Together, these observations establish that JIP1-mediated JNK activation contributes to the regulation of hippocampus-dependent, NMDA receptor-mediated synaptic plasticity and learning. SIGNIFICANCE STATEMENT: The results of this study demonstrate that JNK activation induced by the JIP1 scaffold protein negatively regulates the threshold for induction of long-term synaptic plasticity through the NMDA-type glutamate receptor. This change in plasticity threshold influences learning. Indeed, mice with defects in JIP1-mediated JNK activation display enhanced memory in hippocampus-dependent tasks, such as contextual fear conditioning and Morris water maze, indicating that JIP1-JNK constrains spatial memory. This study reports the identification of JIP1-mediated JNK activation as a novel molecular pathway that negatively regulates NMDA receptor-dependent synaptic plasticity and memory

Association of Cholecystokinin-2 and corticotropin-releasing factor receptor with mood and anxiety disorders

Ph.DDOCTOR OF PHILOSOPH

Polymorphisms at the DRD2 locus in early-onset alcohol dependence in the Indian population

The susceptibility to alcohol dependence is probably of polygenic origin. Association studies have attempted to identify possible candidate genes that may contribute to the risk to developing dependence. Severe forms of the alcoholism phenotype have been associated with an increased frequency of the Taq A1 allele at the DRD2 locus. Ethnic stratification and non-comparable phenotype may have contributed to the contradictory results in previous studies. We identified probands, using the Schedules of Assessment of Neuropsychiatry (SCAN) schedule, who had onset of alcohol dependence (ICD-10) before 25 years of age. Family members were interviewed using the Family Interview for Genetic Studies (FIGS) schedule to identify patients who had two first-degree relatives with alcohol dependence. Fifty subjects who fulfilled the criteria were selected for the study. These were compared to a normal population from a similar background. The allele frequencies did not differ between the two groups. The Taq1a polymorphism does not seem to be associated with alcoholism in this group of severely affected, young age of onset probands in the southern Indian population

Small-conductance Ca2+-activated potassium type 2 channels regulate the formation of contextual fear memory.

Small-conductance, Ca2+ activated K+ channels (SK channels) are expressed at high levels in brain regions responsible for learning and memory. In the current study we characterized the contribution of SK2 channels to synaptic plasticity and to different phases of hippocampal memory formation. Selective SK2 antisense-treatment facilitated basal synaptic transmission and theta-burst induced LTP in hippocampal brain slices. Using the selective SK2 antagonist Lei-Dab7 or SK2 antisense probes, we found that hippocampal SK2 channels are critical during two different time windows: 1) blockade of SK2 channels before the training impaired fear memory, whereas, 2) blockade of SK2 channels immediately after the training enhanced contextual fear memory. We provided the evidence that the post-training cleavage of the SK2 channels was responsible for the observed bidirectional effect of SK2 channel blockade on memory consolidation. Thus, Lei-Dab7-injection before training impaired the C-terminal cleavage of SK2 channels, while Lei-Dab7 given immediately after training facilitated the C-terminal cleavage. Application of the synthetic peptide comprising a leucine-zipper domain of the C-terminal fragment to Jurkat cells impaired SK2 channel-mediated currents, indicating that the endogenously cleaved fragment might exert its effects on memory formation by blocking SK2 channel-mediated currents. Our present findings suggest that SK2 channel proteins contribute to synaptic plasticity and memory not only as ion channels but also by additionally generating a SK2 C-terminal fragment, involved in both processes. The modulation of fear memory by down-regulating SK2 C-terminal cleavage might have applicability in the treatment of anxiety disorders in which fear conditioning is enhanced

Inhibition of SK2 channel function impaired contextual fear memory.

<p>(A, B) Mice injected intrahippocampally (i.h.) or intracerebroventricularly (i.c.v.) with antisense-SK2 ODNs showed reduced freezing when compared to naive animals. No significant difference in freezing scores was seen in mice injected with vehicle or control ODNs (n = 9–11/ group). (C) Intrahippocampal injection of the selective SK2 antagonist Lei-Dab⁷ impaired freezing when compared to non-injected animals, whereas vehicle-injection had no effect (n = 8–9/group). (D) When Lei-Dab⁷ was injected intracortically (i.c.), freezing was not different from non-injected animals. Similarly, vehicle-injected mice showed no significant difference in freezing when compared to non-injected mice (n = 6/ group). Percentage of freezing during pre-shock and post-shock was not significantly different in any treatment group from percentage of freezing of non-injected animals. Freezing was measured in the memory test 24 h after training. Data presented are the mean ± SEM. Statistics was performed by repeated measures two-way ANOVA with Bonferroni multiple comparisons test (*<i>p</i> < 0.05).</p

Antisense-SK2 treatment reduced expression of hippocampal SK2 channels.

<p>(A) RT-PCR analysis was performed on RNA extracted from a single hippocampus of naïve mice or from a single hippocampus of mice that were pre-treated with vehicle, control oligonucleotides (ODNs) or SK2 antisense ODNs. Each reaction mixture contained a set of primers specific for the cDNA of hypoxantine-phosphoribosyl-transferase (HPRT), used as internal control. (B) Bar graphs show the relative band intensities on the basis of densitometric analysis as ratios of SK2 and HPRT mRNA after 27 cycles of co-amplification from 7–8 mRNA samples. Representative western blots showing the analysis of SK2 (C), SK3 (D) and SK1 (G) protein homogenates from single hippocampi of mice pre-treated with vehicle, control oligonucleotides (ODNs) or SK2 antisense ODNs. Bars represent mean western blot band intensities ± SEM for SK2 (D), SK3 (F) and SK1 (H) proteins from hippocampal homogenates (n = 9) (Bonferroni multiple comparisons test: * <i>p</i> < 0.05).</p

Cleavage of SK2 channel protein after contextual fear conditioning training.

<p>Western blot analysis of SK2 protein levels in (A) hippocampal tissue obtained from naïve mice or 1 h and 3 h after training (context+shock). In the context group, mice were exposed to the training context without receiving a foot-shock. In the shock group, mice received a foot-shock immediately after they were exposed to the training context and were removed immediately after the foot-shock. Experiment was performed twice. (B) Representative immunoblot of SK2 protein levels in hippocampal tissue from naïve, trained and trained vehicle- or Lei-Dab⁷-injected mice. Mice were injected either 0.5 h before or 0 h after training as indicated. Hippocampal tissue was removed 1 h after training. The SK2_(538–555) antibody recognized the 64-kDa SK2 protein and a 10-kDa SK2 C-terminal fragment (top). (C) SK2 protein levels in hippocampal tissue from naïve non-injected, Lei-Dab⁷-injected naïve, or vehicle-injected trained mice. Hippocampal tissue was removed 90 or 210 min after injection (1h and/or 3h after training). The number of individual samples per treatment was five. Data presented are the mean ± SEM. Statistically significant differences: *<i>p</i> < 0.05 versus naive, ^#<i>p</i> < 0.005 versus 3 hours context and conditioning groups, ^a<i>p</i><0.05 versus training + vehicle.</p

SK2 leucine zipper (SK2-LZ) domain peptide enhances fear conditioning and TBS-LTP by inhibiting SK2 current.

<p>(A) Mice intrahippocampally (i.h.) injected at 30 before or immediately after training with either the SK2-LZ peptide _(488–526), a random sequenced control peptide or vehicle showed no difference in freezing when compared to non-injected animals. Injection of the selective SK2 antagonist Lei-Dab⁷ 0.5 h before training impaired freezing when compared to non-injected animals. This impairment was rescued if mice were injected with the SK2-LZ peptide _(488–526) immediately after training. I.h. injection of SK2-LZ peptide _(488–526) alone, either 30 min before training or immediately following training did not affect contextual fear. Freezing was measured in the memory test 24 h after training. n = 7–10 mice/group. Statistics were performed by one-way ANOVA with Bonferroni multiple comparisons test (*<i>p</i> < 0.05). (B) TBS-LTP elicited in slices from mice that were pre-treated with SK2-LZ peptide _(488–526) was significantly enhanced when compared to LTP induced in slices from naive mice. There was no statistical difference between control peptide-injected mice and naïve mice. Statistics were performed by two-way ANOVA with Bonferroni multiple comparisons test (*<i>p</i> < 0.05). Insets: Responses shown are fEPSPs recorded during baseline (upper row) and 55–60 min (bottom row) after the induction of LTP (post-induction). Traces are averages of five consecutive responses. (C) Average whole-cell currents were recorded in Jurkat cells endogenously expressing SK2 channels. Voltage ramps were from -100 mV to 100 mV for 100 ms delivered at 2 s intervals. SK2 current was measured at 80 mV and normalized to cell size (in pF) to show current density over time. After 200 seconds external solutions containing 100 μM SK2-LZ peptide (filled circles, n = 6) was applied to the cells for 150 seconds or no external solution application (control; open circles, n = 4). Arrows at 196 s and 348 s indicate the time points at which the current-voltage (I-V) curves in D were obtained (D) Representative I-V curves for endogenous SK2 in Jurkat cells taken after 196 s, right before application (dotted line) and at the end of the experiment at 348 s (solid line) in unexposed control cells (left panel) or when exposed to 100 μM SK2-LZ peptide. Data presented are the mean ± SEM.</p

Enhanced basal synaptic transmission and LTP in hippocampal slices from mice pre-treated with SK2 antisense ODNs.

<p>The distance between the stimulating and recording electrodes was kept constant between slices. (A) Input-output curve of fEPSP slope (mV/ms) versus stimulus (V) at the SC-CA1 pyramidal cell synapse in naive mice and mice pre-treated with vehicle, antisense ODNs against SK2 channels and control ODNs. The maximal fEPSP slopes were significantly larger in the SK2 antisense-treated mice than those in the naive, vehicle and control ODNs-treated mice. Data are presented as mean ± s.e.m. (B) Relationship between the slope of the evoked fEPSPs from panel A and the corresponding fiber volley amplitude. SK2 antisense-treated mice exhibit a greater postsynaptic response than control groups to similar presynaptic depolarization. Data are presented as mean ± s.e.m. (C) Comparison of PPF in naive mice and mice pre-treated with vehicle, antisense ODNs against SK2 channels and control ODNs. No differences were found between these four groups of mice. Data presented are the mean ± SEM of the facilitation of the second response relative to the first response. Insets: Field EPSPs recorded in response to paired-pulse stimulation at an interstimulus interval of 50 ms in slices from all four treatment groups as indicated. (D) TBS-LTP elicited in slices from mice that were pre-treated with SK2 antisense ODNs was significantly enhanced when compared to LTP induced in slices from naive mice. There was no statistical difference between control ODNs-injected mice and mice that were pre-injected with vehicle. Insets: Responses shown are fEPSPs recorded during baseline (upper row) and 55–60 min (bottom row) after the induction of LTP (post-induction). Traces are averages of five consecutive responses. Statistical significance was determined by two-way ANOVA followed by Bonferroni multiple comparisons test (*<i>p</i> < 0.05).</p