Repeated daily administration of increasing doses of lipopolysaccharide provides a model of sustained inflammation-induced depressive-like behaviour in mice that is independent of the NLRP3 inflammasome

Mounting preclinical evidence has implicated the NLRP3 inflammasome in depression-related behaviours elicited by chronic stress or acute lipopolysaccharide (LPS) challenge. However, the relevance of acute LPS as a model of depression has been questioned and behavioural time-courses of its effects can be inconsistent. The aims of this study were (1) to develop a novel protocol for repeated daily LPS administration and (2) to use this model to assess the involvement of NLRP3 inflammasome signalling in sustained inflammation-induced depressive-like behaviour in adult C57BL/6J mice deficient in NLRP3.Acute LPS (0.83 mg/kg; i.p.) induced sickness behaviour evident as hypolocomotor activity. However, there was no significant increase in depressive-like behaviour in the forced swim test 24 h post-administration. Interestingly, depressive-like behaviours were observed in the female urine sniffing test and in the sucrose preference test at 24 h, but not 48 h, post-administration of acute LPS. To mimic a period of sustained inflammation, 3-day repeated increasing LPS doses (0.1, 0.42 and 0.83 mg/kg; i.p.) was compared to constant LPS doses (0.83 mg/kg; i.p.). Sickness behaviour was seen in response to increasing doses, but tolerance developed to repeated constant doses of LPS. Furthermore, 3-day increasing doses of LPS resulted in a significant increase in immobility time in the forced swim test, consistent with depressive-like behaviour. When NLRP3−/− mice received this 3-day increasing dose regimen of LPS, sickness behaviours were attenuated compared to wild-type mice. The behaviour in the forced swim test was not significantly altered in NLRP3−/− mice.We propose that this increasing repeated dosing LPS model of inflammation-induced depressive-like behaviour may better model the sustained inflammation observed in depression and may provide a more translationally relevant paradigm to study the inflammatory mechanisms that contribute to depression

Panic results in unique molecular and network changes in the amygdala that facilitate fear responses

Recurrent panic attacks (PAs) are a common feature of panic disorder (PD) and post-traumatic stress disorder (PTSD). Several distinct brain regions are involved in the regulation of panic responses, such as perifornical hypothalamus (PeF), periaqueductal grey, amygdala and frontal cortex. We have previously shown that inhibition of GABA synthesis in the PeF produces panic-vulnerable rats. Here, we investigate the mechanisms by which a panic-vulnerable state could lead to persistent fear. We first show that optogenetic activation of glutamatergic terminals from the PeF to the basolateral amygdala (BLA) enhanced the acquisition, delayed the extinction and induced the persistence of fear responses 3 weeks later, confirming a functional PeF-amygdala pathway involved in fear learning. Similar to optogenetic activation of PeF, panic-prone rats also exhibited delayed extinction. Next, we demonstrate that panic-prone rats had altered inhibitory and enhanced excitatory synaptic transmission of the principal neurons, and reduced protein levels of metabotropic glutamate type 2 receptor (mGluR2) in the BLA. Application of an mGluR2 positive allosteric modulator (PAM) reduced glutamate neurotransmission in the BLA slices from panic-prone rats. Treating panic-prone rats with mGluR2 PAM blocked sodium lactate (NaLac)-induced panic responses and normalized fear extinction deficits. Finally, in a subset of patients with comorbid PD, treatment with mGluR2 PAM resulted in complete remission of panic symptoms. These data demonstrate that a panic-prone state leads to specific reduction in mGluR2 function within the amygdala network and facilitates fear, and mGluR2 PAMs could be a targeted treatment for panic symptoms in PD and PTSD patients

Selective inhibition of intestinal guanosine 3,5-cyclic monophosphate signaling by small-molecule protein kinase inhibitors

The guanosine 3,5-cyclic monophosphate (cGMP)-dependent protein kinase II (cGKII) serine/threonine kinase relays signaling through guanylyl cyclase C (GCC) to control intestinal fluid homeostasis. Here, we report the discovery of small-molecule inhibitors of cGKII. These inhibitors were imidazole-aminopyrimidines, which blocked recombinant human cGKII at submicromolar concentrations but exhibited comparatively little activity toward the phylogenetically related protein kinases cGKI and cAMP-dependent protein kinase (PKA). Whereas aminopyrimidyl motifs are common in protein kinase inhibitors, molecular modeling of these imidazole-aminopyrimidines in the ATP-binding pocket of cGKII indicated an unconventional binding mode that directs their amine substituent into a narrow pocket delineated by hydrophobic residues of the hinge and the C-helix. Crucially, this set of residues included the Leu-530 gatekeeper, which is not conserved in cGKI and PKA. In intestinal organoids, these compounds blocked cGKII-dependent phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). In mouse small intestinal tissue, cGKII inhibition significantly attenuated the anion secretory response provoked by the GCC-activating bacterial heat-stable toxin (STa), a frequent cause of infectious secretory diarrhea. In contrast, both PKA-dependent VASP phosphorylation and intestinal anion secretion were unaffected by treatment with these compounds, whereas experiments with T84 cells indicated that they weakly inhibit the activity of cAMP-hydrolyzing phosphodiesterases. As these protein kinase inhibitors are the first to display selective inhibition of cGKII, they may expedite research on cGMP signaling and may aid future development of therapeutics for managing diarrheal disease and other pathogenic syndromes that involve cGKII

Repeated daily administration of increasing doses of lipopolysaccharide provides a model of sustained inflammation-induced depressive-like behaviour in mice that is independent of the NLRP3 inflammasome

Mounting preclinical evidence has implicated the NLRP3 inflammasome in depression-related behaviours elicited by chronic stress or acute lipopolysaccharide (LPS) challenge. However, the relevance of acute LPS as a model of depression has been questioned and behavioural time-courses of its effects can be inconsistent. The aims of this study were (1) to develop a novel protocol for repeated daily LPS administration and (2) to use this model to assess the involvement of NLRP3 inflammasome signalling in sustained inflammation-induced depressive-like behaviour in adult C57BL/6J mice deficient in NLRP3.Acute LPS (0.83 mg/kg; i.p.) induced sickness behaviour evident as hypolocomotor activity. However, there was no significant increase in depressive-like behaviour in the forced swim test 24 h post-administration. Interestingly, depressive-like behaviours were observed in the female urine sniffing test and in the sucrose preference test at 24 h, but not 48 h, post-administration of acute LPS. To mimic a period of sustained inflammation, 3-day repeated increasing LPS doses (0.1, 0.42 and 0.83 mg/kg; i.p.) was compared to constant LPS doses (0.83 mg/kg; i.p.). Sickness behaviour was seen in response to increasing doses, but tolerance developed to repeated constant doses of LPS. Furthermore, 3-day increasing doses of LPS resulted in a significant increase in immobility time in the forced swim test, consistent with depressive-like behaviour. When NLRP3−/− mice received this 3-day increasing dose regimen of LPS, sickness behaviours were attenuated compared to wild-type mice. The behaviour in the forced swim test was not significantly altered in NLRP3−/− mice.We propose that this increasing repeated dosing LPS model of inflammation-induced depressive-like behaviour may better model the sustained inflammation observed in depression and may provide a more translationally relevant paradigm to study the inflammatory mechanisms that contribute to depression