Complement 3a Receptor in Dorsal Horn Microglia Mediates Pronociceptive Neuropeptide Signaling

The complement 3a receptor (C3aR1) participates in microglial signaling under pathological conditions and was recently shown to be activated by the neuropeptide TLQP‐21. We previously demonstrated that TLQP‐21 elicits hyperalgesia and contributes to nerve injury‐induced hypersensitivity through an unknown mechanism in the spinal cord. Here we determined that this mechanism requires C3aR1 and that microglia are the cellular target for TLQP‐21. We propose a novel neuroimmune signaling pathway involving TLQP‐21‐induced activation of microglial C3aR1 that then contributes to spinal neuroplasticity and neuropathic pain. This unique dual‐ligand activation of C3aR1 by a neuropeptide (TLQP‐21) and an immune mediator (C3a) represents a potential broad‐spectrum mechanism throughout the CNS for integration of neuroimmune crosstalk at the molecular level

Recommendations, guidelines, and best practice for the use of human induced pluripotent stem cells for neuropharmacological studies of neuropsychiatric disorders

The number of individuals suffering from neuropsychiatric disorders (NPDs) has increased worldwide, with 3 million disability-adjusted life-years calculated in 2019. Though research using various approaches including genetics, imaging, clinical and animal models has advanced our knowledge regarding NPDs, we still lack basic knowledge regarding the underlying pathophysiological mechanisms. Moreover, there is an urgent need for highly effective therapeutics for NPDs. Human induced pluripotent stem cells (hiPSCs) generated from somatic cells enabled scientists to create brain cells in a patient-specific manner. However, there are challenges to the use of hiPSCs that need to be addressed. In the current paper, consideration of best practices for neuropharmacological and neuropsychiatric research using hiPSCs will be discussed. Specifically, we provide recommendations for best practice in patient recruitment, including collecting demographic, clinical, medical (before and after treatment and response), diagnostic (including scales) and genetic data from the donors. We highlight considerations regarding donor genetics and sex, in addition to discussing biological and technical replicates. Furthermore, we present our views on selecting control groups/lines, experimental designs, and considerations for conducting neuropharmacological studies using hiPSC-based models in the context of NPDs. In doing so, we explore key issues in the field concerning reproducibility, statistical analysis, and how to translate in vitro studies into clinically relevant observations. The aim of this article is to provide a key resource for hiPSC researchers to perform robust and reproducible neuropharmacological studies, with the ultimate aim of improving identification and clinical translation of novel therapeutic drugs for NPDs

Neuropathic Pain Phenotype Does Not Involve the NLRP3 Inflammasome and Its End Product Interleukin-1β in the Mice Spared Nerve Injury Model.

The NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome is one of the main sources of interleukin-1β (IL-1β) and is involved in several inflammatory-related pathologies. To date, its relationship with pain has not been studied in depth. The aim of our study was to elucidate the role of NLRP3 inflammasome and IL-1β production on neuropathic pain. Results showed that basal pain sensitivity is unaltered in NLRP3-/- mice as well as responses to formalin test. Spared nerve injury (SNI) surgery induced the development of mechanical allodynia and thermal hyperalgesia in a similar way in both genotypes and did not modify mRNA levels of the NLRP3 inflammasome components in the spinal cord. Intrathecal lipopolysaccharide (LPS) injection increases apoptosis-associated speck like protein (ASC), caspase-1 and IL-1β expression in both wildtype and NLRP3-/- mice. Those data suggest that NLRP3 is not involved in neuropathic pain and also that other sources of IL-1β are implicated in neuroinflammatory responses induced by LPS

Recommendations, guidelines, and best practice for the use of human induced pluripotent stem cells for neuropharmacological studies of neuropsychiatric disorders

The number of individuals suffering from neuropsychiatric disorders (NPDs) has increased worldwide, with 3 million disability-adjusted life-years calculated in 2019. Though research using various approaches including genetics, imaging, clinical and animal models has advanced our knowledge regarding NPDs, we still lack basic knowledge regarding the underlying pathophysiological mechanisms. Moreover, there is an urgent need for highly effective therapeutics for NPDs i. Human induced pluripotent stem cells (hiPSCs) generated from somatic cells enabled scientists to create brain cells in a patient-specific manner. However, there are challenges to the use of hiPSCs that need to be addressed. In the current paper, consideration of best practices for neuropharmacological and neuropsychiatric research using hiPSCs will be discussed. Specifically, we provide recommendations for best practice in patient recruitment, including collecting demographic, clinical, medical (before and after treatment and response), diagnostic (incl. scales) and genetic data from the donors. We highlight considerations regarding donor genetics and sex, in addition to discussing biological and technical replicates. Furthermore, we present our views on selecting control groups/lines, experimental designs, and considerations for conducting neuropharmacological studies using hiPSC-based models in the context of NPDs. In doing so, we explore key issues in the field concerning reproducibility, statistical analysis, and how to translate in vitro studies into clinically relevant observations. The aim of this article is to provide a key resource for hiPSC researchers to perform robust and reproducible neuropharmacological studies, with the ultimate aim of improving identification and clinical translation of novel therapeutic drugs for NPDs

Selective activation of microglia facilitates synaptic strength

Synaptic plasticity is thought to be initiated by neurons only, with the prevailing view assigning glial cells mere specify supportive functions for synaptic transmission and plasticity. We now demonstrate that glial cells can control synaptic strength independent of neuronal activity. Here we show that selective activation of microglia in the rat is sufficient to rapidly facilitate synaptic strength between primary afferent C-fibers and lamina I neurons, the first synaptic relay in the nociceptive pathway. Specifically, the activation of the CX3CR1 receptor by fractalkine induces the release of interleukin-1β from microglia, which modulates NMDA signaling in postsynaptic neurons, leading to the release of an eicosanoid messenger, which ultimately enhances presynaptic neurotransmitter release. In contrast to the conventional view, this form of plasticity does not require enhanced neuronal activity to trigger the events leading to synaptic facilitation. Augmentation of synaptic strength in nociceptive pathways represents a cellular model of pain amplification. The present data thus suggest that, under chronic pain states, CX3CR1-mediated activation of microglia drives the facilitation of excitatory synaptic transmission in the dorsal horn, which contributes to pain hypersensitivity in chronic pain states

Impaired excitatory drive to spinal GABAergic neurons of neuropathic mice.

Adequate pain sensitivity requires a delicate balance between excitation and inhibition in the dorsal horn of the spinal cord. This balance is severely impaired in neuropathy leading to enhanced pain sensations (hyperalgesia). The underlying mechanisms remain elusive. Here we explored the hypothesis that the excitatory drive to spinal GABAergic neurons might be impaired in neuropathic animals. Transgenic adult mice expressing EGFP under the promoter for GAD67 underwent either chronic constriction injury of the sciatic nerve or sham surgery. In transverse slices from lumbar spinal cord we performed whole-cell patch-clamp recordings from identified GABAergic neurons in lamina II. In neuropathic animals rates of mEPSC were reduced indicating diminished global excitatory input. This downregulation of excitatory drive required a rise in postsynaptic Ca(2+). Neither the density and morphology of dendritic spines on GABAergic neurons nor the number of excitatory synapses contacting GABAergic neurons were affected by neuropathy. In contrast, paired-pulse ratio of Aδ- or C-fiber-evoked monosynaptic EPSCs following dorsal root stimulation was increased in neuropathic animals suggesting reduced neurotransmitter release from primary afferents. Our data indicate that peripheral neuropathy triggers Ca(2+)-dependent signaling pathways in spinal GABAergic neurons. This leads to a global downregulation of the excitatory drive to GABAergic neurons. The downregulation involves a presynaptic mechanism and also applies to the excitation of GABAergic neurons by presumably nociceptive Aδ- and C-fibers. This then leads to an inadequately low recruitment of inhibitory interneurons during nociception. We suggest that this previously unrecognized mechanism of impaired spinal inhibition contributes to hyperalgesia in neuropathy

Density and morphological parameters of dendritic spines on GABAergic neurons.

<p>Neither density nor morphological parameters of dendritic spines on GABAergic neurons were significantly different between sham-treated (n = 12) and CCI (n = 13) animals (Mann-Whitney rank sum test, for all groups: P>0.1). Data: mean ± s.e.m.</p

Time course of thermal (A) and mechanical (B) withdrawal thresholds after CCI.

<p>Withdrawal thresholds in CCI (n = 19) and sham-operated (n = 17) animals for both ipsi- and contralateral hindpaws. Tests were performed on days 2 and 1 before operation and on days 1, 3, 5, 7 and 9 after surgery. (<b>A</b>) Thermal hyperalgesia is indicated by a significant reduction in the paw withdrawal latency. (<b>B</b>) Mechanical hyperalgesia is indicated by a significant reduction of the 50% paw withdrawal threshold. Data are expressed as mean ± s.e.m. (* P < 0.05; ** P < 0.01, two-way ANOVA).</p

Paired-pulse ratio recorded from EGFP-labeled, GABAergic neurons following dorsal root stimulation is increased after CCI treatment.

<p>(<b>A</b>) Overlaid monosynaptically evoked Aδ-fiber EPSCs during 10 Hz stimulation (scale bars: 10 ms, 50 pA). (<b>B</b>) Averaged traces of paired-pulse recordings following Aδ-fiber stimulation at 50 ms interstimulus interval (scale bars: 20 ms, 100 pA). (<b>C</b>) Paired-pulse ratio following Aδ-fiber stimulation was increased in CCI treated mice at 50 ms interstimulus interval (sham n = 19, CCI n = 20, ** P < 0.01, two-way ANOVA) and at 300 ms interstimulus interval (sham n = 21, CCI n = 20, * P < 0.05, two-way ANOVA). Data: mean ± s.e.m. (<b>D</b>) Overlaid monosynaptically evoked C-fiber EPSCs during 1 Hz stimulation (scale bars: 10 ms, 50 pA). (<b>E</b>) Averaged traces of paired-pulse recordings following C-fiber stimulation at 300 ms interstimulus interval (scale bars: 100 ms, 100 pA). (<b>F</b>) Paired-pulse ratio following C-fiber stimulation was increased in CCI treated mice at 300 ms interstimulus interval (sham n = 13, CCI n = 13, ** P < 0.01, two-way ANOVA) and at 500 ms interstimulus interval (sham n = 16, CCI n = 16, * P < 0.05, two-way ANOVA). Data: mean ± s.e.m.</p

c-Fos expression in GABAergic neurons.

<p>Upper row: c-Fos expression after noxious heat stimulus in a sham-treated mouse colocalizes with EGFP-labeled GABAergic neurons. Lower row: In a CCI-operated mouse c-Fos can be detected in fewer EGFP-labeled neurons as compared to a sham-treated mouse. (scale bar: 50 µm, left  =  lateral, right  =  medial) Border between dorsal horn and white matter is indicated by a solid line, lamina borders by a dashed line. Lamina II is indicated by L II.</p