Contextual fear response is modulated by M-type K+ channels and is associated with subtle structural changes of the axon initial segment in hippocampal GABAergic neurons

Background: In the fear memory network, the hippocampus modulates contextual aspects of fear learning while mutual connections between the amygdala and the medial prefrontal cortex are widely involved in fear extinction. G-protein-coupled receptors (GPCRs) are involved in the regulation of fear and anxiety, so the regulation of GPCRs in fear signaling pathways can modulate the mechanisms of fear memory acquisition, consolidation and extinction. Various studies suggested a role of M-type K+ channels in modulating fear expression and extinction, although conflicting data prevented drawing of clear conclusions. In the present work, we examined the impact of M-type K+ channel blockade or activation on contextual fear acquisition and extinction. In addition, regarding the pivotal role of the hippocampus in contextual fear conditioning (CFC) and the involvement of the axon initial segment (AIS) in neuronal plasticity, we investigated whether structural alterations of the AIS in hippocampal neurons occurred during contextual fear memory acquisition and short-time extinction in mice in a behaviorally relevant context. Results: When a single systemic injection of the M-channel blocker XE991 (2 mg/kg, IP) was carried out 15 minutes before the foot shock session, fear expression was significantly reduced. Expression of c-Fos was increased following CFC, mostly in GABAergic neurons at day 1 and day 2 post-fear training in CA1 and dentate gyrus hippocampal regions. A significantly longer AIS segment was observed in GABAergic neurons of the CA1 hippocampal region at day 2. Conclusions: Our results underscore the role of M-type K + channels in CFC and the importance of hippocampal GABAergic neurons in fear expression

Incretin mimetics as pharmacological tools to elucidate and as a new drug strategy to treat traumatic brain injury

Traumatic brain injury (TBI), either as an isolated injury or in conjunction with other injuries, is an increasingly common occurring event. An estimated 1.7 million injuries occur within the US each year and 10 million people are affected annually worldwide. Indeed, some one-third (30.5%) of all injury-related deaths in the U.S. are associated with TBI, which will soon outstrip many common diseases as the major cause of death and disability. Associated with a high morbidity and mortality, and no specific therapeutic treatment, TBI has become a pressing public health and medical problem. The highest incidence of TBI occurs among young adults (15 to 24 years age) as well as in the elderly (75 years and older) who are particularly vulnerable as injury, often associated with falls, carries an increased mortality and worse functional outcome following lower initial injury severity. Added to this, a new and growing form of TBI, blast injury, associated with the detonation of improvised explosive devices in the war theaters of Iraq and Afghanistan, are inflicting a wave of unique casualties of immediate impact to both military personnel and civilians, for which long-term consequences remain unknown and may potentially be catastrophic. The neuropathology underpinning head injury is becoming increasingly better understood. Depending on severity, TBI induces immediate neuropathological effects that for the mildest form may be transient but with increasing severity cause cumulative neural damage and degeneration. Even with mild TBI, which represents the majority of cases, a broad spectrum of neurological deficits, including cognitive impairments, can manifest that may significantly influence quality of life. In addition, TBI can act as a conduit to longer-term neurodegenerative disorders. Prior studies of glucagon-like peptide-1 (GLP-1) and long-acting GLP-1 receptor agonists have demonstrated neurotrophic/neuroprotective activities across a broad spectrum of cellular and animal models of chronic neurodegenerative (Alzheimer's and Parkinson's diseases) and acute cerebrovascular (stroke) disorders. In line with the commonality in mechanisms underpinning these disorders as well as TBI, the current article reviews this literature and recent studies assessing GLP-1 receptor agonists as a potential treatment strategy for mild to moderate TBI

Time line of mouse studies.

<p>Anesthetized male ICR mice were subjected to either mTBI (a single 30 g weight drop) or a sham procedure (without weight drop) and 1 hr. later were administered either PFT-α (2 mg/kg, i.p.) or vehicle (0.2% DMSO/saline mixture, i.p.). Three parallel series of animals were then maintained for (i) 72 hr. and were prepared for immunohistochemical analyses of their brain tissue for quantification of degenerating neurons assessed by FJB and NeuN, (ii) 7 days and were behaviorally evaluated by novel object recognition and Y-maze paradigms, and (iii) 30 days and underwent similar behavior evaluation. The structure of PFT-α is shown as its synthesized HBr salt.</p

p53 inhibition by PFT-α/analog inhibits glutamate-induced excitotoxicity and oxidative stress mediated loss of cell viability in neuronal cultures.

<p>Human SH-SY5Y cells were subjected to p53 inactivation (PFT-α analog 1 to 10 μM) and then challenged with (<b>A</b>) glutamate (100 mM) excitotoxicity or (<b>B</b>) oxidative stress (H₂O₂: 500 μM). These insults alone significantly reduced cellular viability (* p<0.05 vs. control, Dunnetts t-test), which was mitigated by p53 inactivation (# p<0.05 vs. glutamate alone, Dunnetts t-test). (<b>C</b>) Rat primary cortical neuron cultures undergo time-dependent degeneration [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079837#B44" target="_blank">44</a>] that was mitigated by the addition of PFT-α (2 nM to 1 μM; * p<0.05, ** p<0.01, *** p<0.001 vs. untreated controls that are expressed as 100% (Dunnett’s t-test). A 10 μM PFT-α concentration proved to be toxic to primary neurons (*** p<0.001 vs. untreated controls; Dunnett’s t-test). (<b>D</b>) In an alike manner to SH-SY5Y cells, exposure of primary cortical neurons to glutamate (100 μM) resulted in reduced survival (* p<0.05 vs. control, Dunnetts t-test),) and pre-treatment with 2 to 100 nM PFT-α ameliorated this (NS not significantly different from untreated controls, Dunnetts t-test). Analysis of viable neurons was undertaken by MTS assay at 24 hr.</p

PFT-α mitigates mTBI-induced degeneration of neurons in the dentate gyrus.

<div><p>(<b>A</b>) Representative images of Fluoro Jade B (FJB) (green) and NeuN (red) positive neurons in the dentate gyrus 72 hr. after mTBI. Scale bar= 100µm.</p> <p>(<b>B</b>) The field in the box indicates the hilus of the dentate gyrus, which is represented in a higher magnification. (<b>C</b>) Bar graph shows the quantification of neuronal degeneration in the dentate gyrus as a ratio of number of neurons positively stained with FJB (degenerating neurons) divided by neurons positively stained with anti-NeuN in sham control, mTBI and mTBI PFT-α groups. (**P<0.01; Bonferroni <i>post </i><i>hoc</i> [F_(2,19)= 9.219, <i>p</i>=0.002). Values are mean ± SEM, of n= 6 - 10 mouse brains.</p></div

PFT-α inhibits mTBI-induce deficits in Y maze.

<p>(<b>A</b>) PFT-α administration 1 hr. post trauma improved mTBI spatial memory deficits. mTBI mice had a significantly lower visual memory compared with all other groups, a deficit that was corrected with the administration of PFT-α 7 days post trauma (**p<0.01; Fisher’s LSD <i>post </i><i>hoc</i> [F_(3,72)=4.155, <i>p</i>=0.009]). (<b>B</b>) 30 days post trauma the differences between mTBI and PFT-α mice reached statistical significance (#p<0.01; Bonferroni <i>post </i><i>hoc</i> [F_(3,45)= 4.337, <i>p</i>=0.009]). Performance of mice was quantitatively assessed as a preference index, calculated as (time at the new arm - time at the old arm)/(time at the new arm + time at the old arm). Values are mean ± SEM, of n= 10 - 20.</p