Chemogenetic activation of noradrenergic A5 neurons increases blood pressure and visceral sympathetic activity in adult rats.

In mammals, the pontine noradrenergic system influences nearly every aspect of central nervous system function. A subpopulation of pontine noradrenergic neurons, called A5, are thought to be important in the cardiovascular response to physical stressors, yet their function is poorly defined. We hypothesized that activation of A5 neurons drives a sympathetically mediated increase in blood pressure (BP). To test this hypothesis, we conducted a comprehensive assessment of the cardiovascular effects of chemogenetic stimulation of A5 neurons in male and female adult rats using intersectional genetic and anatomical targeting approaches. Chemogenetic stimulation of A5 neurons in freely behaving rats elevated BP by 15 mmHg and increased cardiac baroreflex sensitivity with a negligible effect on resting HR. Importantly, A5 stimulation had no detectable effect on locomotor activity, metabolic rate, or respiration. Under anesthesia, stimulation of A5 neurons produced a marked elevation in visceral sympathetic nerve activity (SNA) and no change in skeletal muscle SNA, showing that A5 neurons preferentially stimulate visceral SNA. Interestingly, projection mapping indicates that A5 neurons target sympathetic preganglionic neurons throughout the spinal cord and parasympathetic preganglionic neurons throughout in the brainstem, as well as the nucleus of the solitary tract, and ventrolateral medulla. Moreover, in situ hybridization and immunohistochemistry indicate that a subpopulation of A5 neurons coreleases glutamate and monoamines. Collectively, this study suggests A5 neurons are a central modulator of autonomic function with a potentially important role in sympathetically driven redistribution of blood flow from the visceral circulation to critical organs and skeletal muscle.

Calpain-Dependent Degradation of Nucleoporins Contributes to Motor Neuron Death in a Mouse Model of Chronic Excitotoxicity.

Glutamate-mediated excitotoxicity induces neuronal death by altering various intracellular signaling pathways and is implicated as a common pathogenic pathway in many neurodegenerative diseases. In the case of motor neuron disease, there is significant evidence to suggest that the overactivation of AMPA receptors due to deficiencies in the expression and function of glial glutamate transporters GLT1 and GLAST plays an important role in the mechanisms of neuronal death. However, a causal role for glial glutamate transporter dysfunction in motor neuron death remains unknown. Here, we developed a new animal model of excitotoxicity by conditionally deleting astroglial glutamate transporters GLT1 and GLAST in the spinal cords of mice (GLAST+/-/GLT1-cKO). GLAST+/-/GLT1-cKO mice (both sexes) exhibited nuclear irregularity and calpain-mediated degradation of nuclear pore complexes (NPCs), which are responsible for nucleocytoplasmic transport. These abnormalities were associated with progressive motor neuron loss, severe paralysis, and shortened lifespan. The nuclear export inhibitor KPT-350 slowed but did not prevent motor neuron death, whereas long-term treatment of the AMPA receptor antagonist perampanel and the calpain inhibitor SNJ-1945 had more persistent beneficial effects. Thus, NPC degradation contributes to AMPA receptor-mediated excitotoxic motor neuronal death, and preventing NPC degradation has robust protective effects. Normalization of NPC function could be a novel therapeutic strategy for neurodegenerative disorders in which AMPA receptor-mediated excitotoxicity is a contributory factor.SIGNIFICANCE STATEMENT Despite glial glutamate transporter dysfunction leading to excitotoxicity has been documented in many neurological diseases, it remains unclear whether its dysfunction is a primary cause or secondary outcome of neuronal death at disease state. Here we show the combined loss of glial glutamate transporters GLT1 and GLAST in spinal cord caused motor neuronal death and hindlimb paralysis. Further, our novel mutant exhibits the nuclear irregularities and calpain-mediated progressive nuclear pore complex degradation. Our study reveals that glial glutamate transporter dysfunction is sufficient to cause motor neuronal death in vivo.

Multiomic profiling of the acute stress response in the mouse hippocampus.

The acute stress response mobilizes energy to meet situational demands and re-establish homeostasis. However, the underlying molecular cascades are unclear. Here, we use a brief swim exposure to trigger an acute stress response in mice, which transiently increases anxiety, without leading to lasting maladaptive changes. Using multiomic profiling, such as proteomics, phospho-proteomics, bulk mRNA-, single-nuclei mRNA-, small RNA-, and TRAP-sequencing, we characterize the acute stress-induced molecular events in the mouse hippocampus over time. Our results show the complexity and specificity of the response to acute stress, highlighting both the widespread changes in protein phosphorylation and gene transcription, and tightly regulated protein translation. The observed molecular events resolve efficiently within four hours after initiation of stress. We include an interactive app to explore the data, providing a molecular resource that can help us understand how acute stress impacts brain function in response to stress.

Presence of ethanol-sensitive and ethanol-insensitive glycine receptors in the ventral tegmental area and prefrontal cortex in mice.

BACKGROUND AND PURPOSE: Glycine receptors composed of α1 and ß subunits are primarily found in the spinal cord and brainstem and are potentiated by ethanol (10-100 mM). However, much less is known about the presence, composition and ethanol sensitivity of these receptors in higher CNS regions. Here, we examined two regions of the brain reward system, the ventral tegmental area (VTA) and the prefrontal cortex (PFC), to determine their glycine receptor subunit composition and sensitivity to ethanol. EXPERIMENTAL APPROACH: We used Western blot, immunohistochemistry and electrophysiological techniques in three different models: wild-type C57BL/6, glycine receptor subunit α1 knock-in and glycine receptor subunit α2 knockout mice. KEY RESULTS: Similar levels of α and ß receptor subunits were detected in both brain regions, and electrophysiological recordings demonstrated the presence of glycine-activated currents in both areas. Sensitivity of glycine receptors to glycine was lower in the PFC compared with VTA. Picrotoxin only partly blocked the glycine-activated current in the PFC and VTA, indicating that both regions express heteromeric αß receptors. Glycine receptors in VTA neurons, but not in PFC neurons, were potentiated by ethanol. CONCLUSION AND IMPLICATIONS: Glycine receptors in VTA neurons from WT and α2 KO mice were potentiated by ethanol, but not in neurons from the α1 KI mice, supporting the conclusion that α1 glycine receptors are predominantly expressed in the VTA. By contrast, glycine receptors in PFC neurons were not potentiated in any of the mouse models studied, suggesting the presence of α2/α3/α4, rather than α1 glycine receptor subunits.

Modulation of glycine receptor single-channel conductance by intracellular phosphorylation.

Glycine receptors (GlyRs) are anion-permeable pentameric ligand-gated ion channels (pLGICs). The GlyR activation is critical for the control of key neurophysiological functions, such as motor coordination, respiratory control, muscle tone and pain processing. The relevance of the GlyR function is further highlighted by the presence of abnormal glycinergic inhibition in many pathophysiological states, such as hyperekplexia, epilepsy, autism and chronic pain. In this context, previous studies have shown that the functional inhibition of  GlyRs containing the α3 subunit is a pivotal mechanism of pain hypersensitivity. This pathway involves the activation of EP2 receptors and the subsequent PKA-dependent phosphorylation of α3GlyRs within the intracellular domain (ICD), which decrease the GlyR-associated currents and enhance neuronal excitability. Despite the importance of this mechanism of glycinergic dis-inhibition associated with dysfunctional α3GlyRs, our current understanding of the molecular events involved is limited. Here, we report that the activation of PKA signaling pathway decreases the unitary conductance of α3GlyRs. We show in addition that the substitution of the PKA-targeted serine with a negatively charged residue within the ICD of α3GlyRs and of chimeric receptors combining bacterial GLIC and α3GlyR was sufficient to generate receptors with reduced conductance. Thus, our findings reveal a potential biophysical mechanism of glycinergic dis-inhibition and suggest that post-translational modifications of the ICD, such as phosphorylation, may shape the conductance of other pLGICs.

PGE(2) selectively blocks inhibitory glycinergic neurotransmission onto rat superficial dorsal horn neurons.

Despite the crucial role that prostaglandins (PGs) have in the sensitization of the central nervous system to pain, their cellular and molecular targets leading to increased pain perception have remained elusive. Here we investigated the effects of PGE(2) on fast synaptic transmission onto neurons in the rat spinal cord dorsal horn, the first site of synaptic integration in the pain pathway. We identified the inhibitory (strychnine-sensitive) glycine receptor as a specific target of PGE(2). PGE(2), but not PGF(2 alpha), PGD(2) or PGI(2), reduced inhibitory glycinergic synaptic transmission in low nanomolar concentrations, whereas GABAA, AMPA and NMDA receptor-mediated transmission remained unaffected. Inhibition of glycine receptors occurred via a postsynaptic mechanism involving the activation of EP2 receptors, cholera-toxin-sensitive G-proteins and cAMP-dependent protein kinase. Via this mechanism, PGE(2) may facilitate the transmission of nociceptive input through the spinal cord dorsal horn to higher brain areas where pain becomes conscious.

Linking altered central pain processing and genetic polymorphism to drug efficacy in chronic low back pain.

BACKGROUND: Inability to predict the therapeutic effect of a drug in individual pain patients prolongs the process of drug and dose finding until satisfactory pharmacotherapy can be achieved. Many chronic pain conditions are associated with hypersensitivity of the nervous system or impaired endogenous pain modulation. Pharmacotherapy often aims at influencing these disturbed nociceptive processes. Its effect might therefore depend on the extent to which they are altered. Quantitative sensory testing (QST) can evaluate various aspects of pain processing and might therefore be able to predict the analgesic efficacy of a given drug. In the present study three drugs commonly used in the pharmacological management of chronic low back pain are investigated. The primary objective is to examine the ability of QST to predict pain reduction. As a secondary objective, the analgesic effects of these drugs and their effect on QST are evaluated. METHODS/DESIGN: In this randomized, double blinded, placebo controlled cross-over study, patients with chronic low back pain are randomly assigned to imipramine, oxycodone or clobazam versus active placebo. QST is assessed at baseline, 1 and 2 h after drug administration. Pain intensity, side effects and patients' global impression of change are assessed in intervals of 30 min up to two hours after drug intake. Baseline QST is used as explanatory variable to predict drug effect. The change in QST over time is analyzed to describe the pharmacodynamic effects of each drug on experimental pain modalities. Genetic polymorphisms are analyzed as co-variables. DISCUSSION: Pharmacotherapy is a mainstay in chronic pain treatment. Antidepressants, anticonvulsants and opioids are frequently prescribed in a "trial and error" fashion, without knowledge however, which drug suits best which patient. The present study addresses the important need to translate recent advances in pain research to clinical practice. Assessing the predictive value of central hypersensitivity and endogenous pain modulation could allow for the implementation of a mechanism-based treatment strategy in individual patients. TRIAL REGISTRATION: Clinicaltrials.gov, NCT01179828.

Diversity of glycine receptors in the mouse retina: localization of the alpha2 subunit.

Gamma-aminobutyric acid (GABA) and glycine are the major inhibitory neurotransmitters in the retina, glycine being produced in approximately half of all amacrine cells. Whereas retinal cell types expressing the glycine receptor (GlyR) alpha1 and alpha3 subunits have been mapped, the role of the alpha2 subunit in retinal circuitry remains unclear. By using immunocytochemistry, we localized the alpha2 subunit in the inner plexiform layer (IPL) in brightly fluorescent puncta, which represent postsynaptically clustered GlyRs. This was shown by doubly labeling sections for GlyR alpha2 and bassoon (a presynaptic marker) or gephyrin (a postsynaptic marker). Synapses containing GlyR alpha2 were rarely found on ganglion cell dendrites but were observed on bipolar cell axon terminals and on amacrine cell processes. Recently, an amacrine cell type has been described that is immunopositive for glycine and for the vesicular glutamate transporter vGluT3. The processes of this cell type were presynaptic to GlyR alpha2 puncta, suggesting that vGluT3 amacrine cells release glycine. Double labeling of sections for GlyR alpha1 and GlyR alpha2 subunits showed that they are clustered at different synapses. In sections doubly labeled for GlyR alpha2 and GlyR alpha3, approximately one-third of the puncta were colocalized. The most abundant GlyR subtype in retina contains alpha3 subunits, followed by those containing GlyR alpha2 and GlyR alpha1 subunits.

Hyperalgesia by low doses of the local anesthetic lidocaine involves cannabinoid signaling: an fMRI study in mice.

Lidocaine is clinically widely used as a local anesthetic inhibiting propagation of action potentials in peripheral nerve fibers. Correspondingly, the functional magnetic resonance imaging (fMRI) response in mouse brain to peripheral noxious input is largely suppressed by local lidocaine administered at doses used in a clinical setting. We observed, however, that local administration of lidocaine at doses 100 × lower than that used clinically led to a significantly increased sensitivity of mice to noxious forepaw stimulation as revealed by fMRI. This hyperalgesic response could be confirmed by behavioral readouts using the von Frey filament test. The increased sensitivity was found to involve a type 1 cannabinoid (CB(1)) receptor-dependent pathway as global CB(1) knockout mice, as well as wild-type mice pretreated systemically with the CB(1) receptor blocker rimonabant, did not display any hyperalgesic effects after low-dose lidocaine. Additional experiments with nociceptor-specific CB(1) receptor knockout mice indicated an involvement of the CB(1) receptors located on the nociceptors. We conclude that low concentrations of lidocaine leads to a sensitization of the nociceptors through a CB(1) receptor-dependent process. This lidocaine-induced sensitization might contribute to postoperative hyperalgesia.