Probes for PARVALBUMIN

Spinal cord dorsal horn (DH) inhibition is critical to the processing of sensory inputs, and its impairment leads to mechanical allodynia. How this decreased inhibition occurs and whether its restoration alleviates allodynic pain is poorly understood. Here, we show that the calcium (Ca2+)-binding protein, parvalbumin (PV), controls the activity of inhibitory PV-expressing neurons (PVNs) by enabling them to sustain high-frequency tonic firing patterns. Upon nerve injury, PVNs transition to adaptive firing and decrease their PV expression. Interestingly, decreased PV is necessary and sufficient to the development of mechanical allodynia and the transition of PVNs to adaptive firing. This transition of firing pattern is due to the recruitment of calcium-activated potassium (SK) channels and blocking them during chronic pain restores normal tonic firing. Our findings indicate that PV is essential to the firing activity of PVNs and in preventing allodynia, these observations may lead to novel strategies for chronic pain relief.

Memory retrieval refers to the fundamental ability of organisms to make use of acquired, sometimes inconsistent, information about the world. Although memory acquisition has been studied extensively, the neurobiological mechanisms underlying memory retrieval remain largely unknown. Conditioned taste aversion (CTA) is a robust associative paradigm, through which animals can be trained to express aversion toward innately appetitive tastants. The anterior insula (aIC) is indispensable in the ability of mammals to retrieve associative information regarding tastants that have been previously linked with gastric malaise. Here, we show that CTA memory retrieval promotes cell-type-specific activation in the aIC. Using chemogenetic tools in the aIC, we found that CTA memory acquisition requires activation of excitatory neurons and inhibition of inhibitory neurons, whereas retrieval necessitates activation of both excitatory and inhibitory aIC circuits. CTA memory retrieval at the aIC activates parvalbumin (PV) interneurons and increases synaptic inhibition onto activated pyramidal neurons projecting to the basolateral amygdala (aIC-BLA). Unlike innately appetitive taste memory retrieval, CTA retrieval increases synaptic inhibition onto aIC-BLA-projecting neurons that is dependent on activity in aIC PV interneurons. PV aIC interneurons coordinate CTA memory retrieval and are necessary for its dominance when conflicting internal representations are encountered over time. The reinstatement of CTA memories following extinction is also dependent on activation of aIC PV interneurons, which increase the frequency of inhibition onto aIC-BLA-projecting neurons. This newly described interaction of PV and a subset of excitatory neurons can explain the coherency of aversive memory retrieval, an evolutionary pre-requisite for animal survival.

Chronic stress represents a major contributor for the development of mental illness. This study aimed to investigate how animals exposed to chronic mild stress (CMS) responded to an acute stress (AS), as a vulnerability's challenge, and to establish the potential effects of the antipsychotic drug lurasidone on such mechanisms. Adult male Wistar rats were exposed or not (controls) to a CMS paradigm for 7 weeks. Starting from the end of week 2, animals were randomized to receive vehicle or lurasidone for 5 weeks. Sucrose intake was used to measure anhedonia. At the end, half of the animals were exposed to an acute stress before sacrifice. Exposure to CMS produced a significant reduction in sucrose consumption, whereas lurasidone progressively normalized such alteration. We found that exposure to AS produced an upregulation of Brain derived neurotrophic factor (Bdnf) in the prefrontal cortex of controls animals. This response was impaired in CMS rats and restored by lurasidone treatment. While in control animals, AS-induced increase of Bdnf mRNA levels was specific for Parvalbumin cells, CMS rats treated with lurasidone show a significant upregulation of Bdnf in pyramidal cells. Furthermore, when investigating the activation of different brain regions, CMS rats showed an impairment in the global response to the acute stressor, that was largely restored by lurasidone treatment. Our results suggest that lurasidone treatment in CMS rats may regulate specific circuits and mechanisms, which will ultimately contribute to boost resilience under stressful challenges.

Several neurodevelopmental disabilities are strongly associated with alterations in GABAergic transmission, and therapies to stimulate its normal development are lacking. Erythropoietin (EPO) is clinically used in neonatology to mitigate acute brain injury, and to stimulate neuronal maturation. Yet it remains unclear whether EPO can stimulate maturation of the GABAergic system. Here, with the use of a transgenic mouse line that constitutively overexpresses neuronal EPO (Tg21), we show that EPO stimulates postnatal GABAergic maturation in the hippocampus. We show an increase in hippocampal GABA-immunoreactive neurons, and postnatal elevation of interneurons expressing parvalbumin (PV), somatostatin (SST) and neuropeptide Y (NPY). Analysis of perineuronal net formation and innervation of glutamatergic terminals onto PV+ cells, shows to be enhanced early in postnatal development. Additionally, an increase in GABAAergic synapse density and inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal cells from Tg21 mice is observed. Detection of erythropoietin receptor (EPOR) mRNA was observed to be restricted to glutamatergic pyramidal cells and increased in Tg21 mice at postnatal day 7, along with reduced apoptosis. Our findings show that EPO can stimulate postnatal GABAergic maturation in the hippocampus, by increasing neuronal survival, modulating critical plasticity periods, and increasing synaptic transmission. Our data supports EPO's clinical use to balance GABAergic dysfunction.Significance Statement Using a mouse model that overexpresses recombinant human EPO in the CNS, we observed stimulation of the postnatal maturation of GABAergic transmission in the hippocampus, notably accelerated maturation of PV+ interneurons, enhanced glutamatergic inputs onto these interneurons, and enhanced inhibitory postsynaptic currents (IPSCs) onto pyramidal cells. We show that EPORs are expressed on pyramidal cells, therefore the impact of EPO on GABAergic maturation is likely to be indirect. Our data show that EPO can modulate hippocampal network maturation and support ongoing trials of the use of EPO in clinical neonatology to stimulate neuronal maturation after perinatal brain injury.

Background: The Psychiatric Ratings using Intermediate Stratified Markers (PRISM) project focuses on understanding the biological background behind social deficits, specifically social withdrawal irrespective of diagnosis. Reduced connectional integrity in fiber tracts such as Forceps minor has been indicated in low social individuals as a part of the PRISM 1 project. These fiber tracts are also involved in the Default Mode Network (DMN) and the Social network and they share a common region, the Orbitofrontal Cortex (OFC).This study aims to back-translate the clinical data to preclinical studies and associate social dysfunction in rodents with DMN and particularly OFC. Parvalbumin interneurons are targeted based on their fundamental role in maintaining Excitatory Inhibitory (E/I) balance in brain circuits. Numerous studies indicate behavioral impairment in rodents by increasing excitability of PV+ interneurons. Methods: As an initial step, we characterized the population of projection neurons within OFCs by combining Cholera Toxin subunit B (CTB) as a retrograde tracer and In situ hybridization (ISH) technique (RNAscope). We identified the expression of mRNAs marking glutamatergic (vesicular glutamate transporter [VGLUT]) and GABAergic (vesicular GABA transporter [VGAT]) by using Slc17a7 and Slc32a1 probes. CTB was injected unilaterally in the left OFC (AP=2.68, ML=-0.8, DV=2.2). after 10 days mice were perfused and RNAscope assay was performed using RNAscope™ Multiplex Fluorescent kit (ACDBio™).For inducing hypoactivation of OFC, we introduced an excitatory DREADD (designer receptors exclusively activated by designer drugs) to PV+ interneurons by using a PV-Cre mouse line. Mice were injected either AAV-hSyn-DIO-hM3D(Gq)-mCherry virus (n=12) or AAV-hSyn-DIO-mCherry (n=12) as control virus. As a novel behavioral tool, Radiofrequency identification (RFID)-assisted SocialScan combined with video tracking has been used, which provides a long-term observation of social behaviors. Monitoring the behavior in groups of four was performed for 7 days in total. After two pre-application days, Clozapine-N-oxide (CNO) was injected three times on consecutive days intraperitoneally (5mg/kg) as an activator of hM3D. application days were followed by two post-application days. Mice were perfused and RNAscope was performed to visualize c-fos mRNA expression as neuronal activity marker, and PV expression to validate our virus and mouse line efficacy. Results: ISH results indicated VGLUT1 has the highest expression within projection neurons (81%). 6% are VGAT+ and only 3% are both VGLUT1/VGAT positive neurons. Despite demonstrating the GABAergic projection neurons as a minority, their crucial role as local interneurons to moderate the excitatory neurons is indisputable.In in vivo study, CNO administration induced social dysregulation in DREAAD mice, demonstrated by a reduction in different social parameters (approach, fight, etc.) in terms of duration. During post-application days, DREAAD mice showed significantly higher social interaction in all definedparameters (Social Approach: p=0.0009, unpaired T-test) and locomotion as a non-social parameter (p= 0.0207).Results from ISH support our hypothesis that DREADD activation of PV+ interneurons is followed by high expression of neuronal activity markers in these targeted interneurons. Conclusion: This study indicates that manipulation of PV+ interneurons using artificially engineered activating protein receptors, generates in effect activation of these interneurons, and this manipulation particularly in OFC could cause social dysfunction in mice.