The endoplasmic reticulum kinase PERK interacts with the oxidoreductase ERO1 to metabolically adapt mitochondria.

Endoplasmic reticulum (ER) homeostasis requires molecular regulators that tailor mitochondrial bioenergetics to the needs of protein folding. For instance, calnexin maintains mitochondria metabolism and mitochondria-ER contacts (MERCs) through reactive oxygen species (ROS) from NADPH oxidase 4 (NOX4). However, induction of ER stress requires a quick molecular rewiring of mitochondria to adapt to new energy needs. This machinery is not characterized. We now show that the oxidoreductase ERO1⍺ covalently interacts with protein kinase RNA-like ER kinase (PERK) upon treatment with tunicamycin. The PERK-ERO1⍺ interaction requires the C-terminal active site of ERO1⍺ and cysteine 216 of PERK. Moreover, we show that the PERK-ERO1⍺ complex promotes oxidization of MERC proteins and controls mitochondrial dynamics. Using proteinaceous probes, we determined that these functions improve ER-mitochondria Ca2+ flux to maintain bioenergetics in both organelles, while limiting oxidative stress. Therefore, the PERK-ERO1⍺ complex is a key molecular machinery that allows quick metabolic adaptation to ER stress.

Astrocytes show increased levels of Ero1α in multiple sclerosis and its experimental autoimmune encephalomyelitis animal model.

Multiple sclerosis (MS) and its animal models are characterized by cellular inflammation within the central nervous system (CNS). The sources and consequences of this inflammation are currently not completely understood. Critical signs and mediators of CNS inflammation are reactive oxygen species (ROS) that promote inflammation. ROS originate from a variety of redox-reactive enzymes, one class of which catalyses oxidative protein folding within the endoplasmic reticulum (ER). Here, the unfolded protein response and other signalling mechanisms maintain a balance between ROS producers such as ER oxidoreductin 1α (Ero1α) and antioxidants such as glutathione peroxidase 8 (GPx8). The role of ROS production within the ER has so far not been examined in the context of MS. In this manuscript, we examined how components of the ER redox network change upon MS and experimental autoimmune encephalomyelitis (EAE). We found that unlike GPx8, Ero1α increases within both MS and EAE astrocytes, in parallel with an imbalance of other oxidases such of GPx7, and that no change was observed within neurons. This imbalance of ER redox enzymes can reduce the lifespan of astrocytes, while neurons are not affected. Therefore, Ero1α induction makes astrocytes vulnerable to oxidative stress in the MS and EAE pathologies.

A sensitive and specific genetically-encoded potassium ion biosensor for in vivo applications across the tree of life.

Potassium ion (K+) plays a critical role as an essential electrolyte in all biological systems. Genetically-encoded fluorescent K+ biosensors are promising tools to further improve our understanding of K+-dependent processes under normal and pathological conditions. Here, we report the crystal structure of a previously reported genetically-encoded fluorescent K+ biosensor, GINKO1, in the K+-bound state. Using structure-guided optimization and directed evolution, we have engineered an improved K+ biosensor, designated GINKO2, with higher sensitivity and specificity. We have demonstrated the utility of GINKO2 for in vivo detection and imaging of K+ dynamics in multiple model organisms, including bacteria, plants, and mice.

Mediation of Sinusoidal Network Oscillations in the Locus Coeruleus of Newborn Rat Slices by Pharmacologically Distinct AMPA and KA Receptors.

Brain control by locus coeruleus (LC) neurons involves afferent glutamate (Glu) inputs. In newborns, LC Glu receptors and responses may be sparse due to immaturity of the brain circuits providing such input. However, we reported, using newborn rat brain slices, that Glu and its ionotropic receptor (iGluR) agonist NMDA transform spontaneous local field potential (LFP) rhythm. Here, we studied whether α-amino-3-hydroxy-5-methyl-4-isoxazole propionic-acid (AMPA) and kainate (KA) iGluR subtypes also transform the LFP pattern. AMPA (0.25-0.5 µM) and KA (0.5-2.5 µM) merged ~0.2 s-lasting bell-shaped LFP events occurring at ~1 Hz into ~40% shorter and ~4-fold faster spindle-shaped and more regular sinusoidal oscillations. The AMPA/KA effects were associated with a 3.1/4.3-fold accelerated phase-locked single neuron spiking due to 4.0/4.2 mV depolarization while spike jitter decreased to 64/42% of the control, respectively. Raising extracellular K+ from 3 to 9 mM increased the LFP rate 1.4-fold or elicited slower multipeak events. A blockade of Cl--mediated inhibition with gabazine (5 µM) plus strychnine (10 µM) affected neither the control rhythm nor AMPA/KA oscillations. GYKI-53655 (25 µM) blocked AMPA (but not KA) oscillations whereas UBP-302 (25 µM) blocked KA (but not AMPA) oscillations. Our findings revealed that AMPA and KA evoke a similar novel neural network discharge pattern transformation type by acting on pharmacologically distinct AMPAR and KA receptors. This shows that already the neonatal LC can generate oscillatory network behaviors that may be important, for example, for responses to opioids.

NMDA Enhances and Glutamate Attenuates Synchrony of Spontaneous Phase-Locked Locus Coeruleus Network Rhythm in Newborn Rat Brain Slices.

Locus coeruleus (LC) neurons are controlled by glutamatergic inputs. Here, we studied in brain slices of neonatal rats NMDA and glutamate effects on phase-locked LC neuron spiking at ~1 Hz summating to ~0.2 s-lasting bell-shaped local field potential (LFP). NMDA: 10 µM accelerated LFP 1.7-fold, whereas 25 and 50 µM, respectively, increased its rate 3.2- and 4.6-fold while merging discrete events into 43 and 56% shorter oscillations. After 4-6 min, LFP oscillations stopped every 6 s for 1 s, resulting in 'oscillation trains'. A dose of 32 µM depolarized neurons by 8.4 mV to cause 7.2-fold accelerated spiking at reduced jitter and enhanced synchrony with the LFP, as evident from cross-correlation. Glutamate: 25-50 µM made rhythm more irregular and the LFP pattern could transform into 2.7-fold longer-lasting multipeak discharge. In 100 µM, LFP amplitude and duration declined. In 25-50 µM, neurons depolarized by 5 mV to cause 3.7-fold acceleration of spiking that was less synchronized with LFP. Both agents: evoked 'post-agonist depression' of LFP that correlated with the amplitude and kinetics of Vm hyperpolarization. The findings show that accelerated spiking during NMDA and glutamate is associated with enhanced or attenuated LC synchrony, respectively, causing distinct LFP pattern transformations. Shaping of LC population discharge dynamics by ionotropic glutamate receptors potentially fine-tunes its influence on brain functions.

Autocrine Neuromodulation and Network Activity Patterns in the Locus Coeruleus of Newborn Rat Slices.

Already in newborns, the locus coeruleus (LC) controls multiple brain functions and may have a complex organization as in adults. Our findings in newborn rat brain slices indicate that LC neurons (i) generate at ~1 Hz a ~0.3 s-lasting local field potential (LFP) comprising summated phase-locked single spike discharge, (ii) express intrinsic 'pacemaker' or 'burster' properties and (iii) receive solely excitatory or initially excitatory−secondary inhibitory inputs. µ-opioid or ɑ2 noradrenaline receptor agonists block LFP rhythm at 100−250 nM whereas slightly lower doses transform its bell-shaped pattern into slower crescendo-shaped multipeak bursts. GABAA and glycine receptors hyperpolarize LC neurons to abolish rhythm which remains though unaffected by blocking them. Rhythm persists also during ionotropic glutamate receptor (iGluR) inhibition whereas <10 mV depolarization during iGluR agonists accelerates spiking to cause subtype-specific fast (spindle-shaped) LFP oscillations. Similar modest neuronal depolarization causing a cytosolic Ca2+ rise occurs (without effect on neighboring astrocytes) during LFP acceleration by CNQX activating a TARP-AMPA-type iGluR complex. In contrast, noradrenaline lowers neuronal Ca2+ baseline via ɑ2 receptors, but evokes an ɑ1 receptor-mediated 'concentric' astrocytic Ca2+ wave. In summary, the neonatal LC has a complex (possibly modular) organization to enable discharge pattern transformations that might facilitate discrete actions on target circuits.

Expiratory abdominal muscle nerve is active at flexor phase, while inspiratory phrenic nerve is not active during locomotion evoked by 5-HT and NMDA in the neonatal rat.

Abdominal muscles are involved in respiration and locomotion. In the isolated pons-spinal cord-rib attached preparation from neonatal rat, the phrenic nerve and abdominal muscles show inspiratory and expiratory activity, respectively. Using this preparation, we investigated whether the bath application of NMDA and 5-HT could evoke locomotor activities in the fourth cervical ventral root (C4VR), phrenic nerve, and abdominal muscle nerve (ilioinguinal nerve, IIG-n). We also observed rib and abdominal muscle movements visually. The phrenic nerve and C4VR showed inspiratory activity consistently under the control conditions, whereas IIG-n showed expiratory activity only at the beginning of the experiment. During the chemically-induced locomotion, both C4VR and IIG-n showed locomotor activity, and IIG-n in particular showed flexor activity. During the flexor activity, lateral bending of the rib cage to the recording site was observed. The phrenic nerve showed weak or no apparent locomotor activity. We concluded that the central pattern generator (CPG) for locomotion provides stronger excitatory synaptic inputs to C4 motoneurons innervating neck and shoulder muscles than the inputs to the phrenic motoneurons. Thus, the locomotor CPG provides a suitable amount of inputs to the functionally proper motoneurons. This preparation will be useful to explore how the respiratory and locomotor CPGs select proper motoneurons to give synaptic inputs and are coordinated with each other.

Mapping the Dynamic Recruitment of Spinal Neurons during Fictive Locomotion.

The basic rhythmic activity that underlies stepping is generated by a neural network, situated in the spinal cord, known as the locomotor central pattern generator (CPG). While a series of lesion experiments have demonstrated that the mammalian locomotor CPG is distributed throughout the ventral portion of the caudal spinal cord, the specific transverse distribution of this neural network is unclear. Here we evoke fictive locomotor activity of various frequencies in upright spinal cords prepared from male and female neonatal mice. This preparation enables us to use an imaging approach to identify locomotor-related cells across the transverse plane of the spinal cord. Results indicate that there is a clear shift in the recruitment of cells toward the ventromedial, and away from the ventrolateral, spinal cord as the frequency of fictive locomotion increases. Surprisingly, the analysis of multiple frequencies of fictive locomotion in the same spinal cord indicates that few neurons are involved in locomotor outputs across multiple speeds. Collectively, these experiments allow us to map the transverse distribution of the locomotor CPG and highlight the pattern of dynamic recruitment that occurs within this neural circuit as the frequency is altered. Our findings are consistent with data indicating that there is a speed-dependent recruitment of interneuronal populations during locomotion and suggest that the locomotor CPG is not a static network, but rather the specific cells recruited vary extensively based on demand.SIGNIFICANCE STATEMENT In this article, we use an imaging approach to identify all those cells that are rhythmically active at the same frequency as fictive locomotion recorded from the ventral roots of the isolated spinal cord. These experiments allow us to map the distribution of locomotor-related cells across the transverse plane of the spinal cord and identify the recruitment pattern of these cells as the frequency of locomotor outputs is altered. Our results indicate that there are drastic changes in the specific neurons activated at different frequencies and provide support for the concept that the locomotor central pattern generator is a modular network with speed-dependent recruitment of interneuronal components.

The ER chaperone calnexin controls mitochondrial positioning and respiration.

Chaperones in the endoplasmic reticulum (ER) control the flux of Ca2+ ions into mitochondria, thereby increasing or decreasing the energetic output of the oxidative phosphorylation pathway. An example is the abundant ER lectin calnexin, which interacts with sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). We found that calnexin stimulated the ATPase activity of SERCA by maintaining its redox state. This function enabled calnexin to control how much ER Ca2+ was available for mitochondria, a key determinant for mitochondrial bioenergetics. Calnexin-deficient cells compensated for the loss of this function by partially shifting energy generation to the glycolytic pathway. These cells also showed closer apposition between the ER and mitochondria. Calnexin therefore controls the cellular energy balance between oxidative phosphorylation and glycolysis.

Endoplasmic reticulum stress in the dorsal root ganglia regulates large-conductance potassium channels and contributes to pain in a model of multiple sclerosis.

Neuropathic pain is a common symptom of multiple sclerosis (MS) and current treatment options are ineffective. In this study, we investigated whether endoplasmic reticulum (ER) stress in dorsal root ganglia (DRG) contributes to pain hypersensitivity in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Inflammatory cells and increased levels of ER stress markers are evident in post-mortem DRGs from MS patients. Similarly, we observed ER stress in the DRG of mice with EAE and relieving ER stress with a chemical chaperone, 4-phenylbutyric acid (4-PBA), reduced pain hypersensitivity. In vitro, 4-PBA and the selective PERK inhibitor, AMG44, normalize cytosolic Ca2+ transients in putative DRG nociceptors. We went on to assess disease-mediated changes in the functional properties of Ca2+ -sensitive BK-type K+ channels in DRG neurons. We found that the conductance-voltage (GV) relationship of BK channels was shifted to a more positive voltage, together with a more depolarized resting membrane potential in EAE cells. Our results suggest that ER stress in sensory neurons of MS patients and mice with EAE is a source of pain and that ER stress modulators can effectively counteract this phenotype.

Correction to: A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein eqFP578.

In the online version of the article [ 1 ], Figure S1 was mistakenly replaced with Figure 1.

Using an upright preparation to identify and characterize locomotor related neurons across the transverse plane of the neonatal mouse spinal cord.

BACKGROUND: The basic rhythmicity underlying stepping in mammals is generated by a neural network, situated in the spinal cord, known as the locomotor central pattern generator (CPG). While a molecular approach has provided information regarding neuronal populations that participate in locomotor activity and their specific function, the distributed nature of the locomotor CPG has made it difficult to identify and characterize the specific neurons belonging to each population that are rhythmically-active during stepping. NEW METHOD: We describe a preparation in which we isolate the spinal cord from a neonatal mouse, section it at a lumbar segment, situate it in an upright orientation under the objective lens of a 2- photon microscope, and evoke fictive locomotion. RESULTS: This preparation allows us to image rhythmic Ca2+ oscillations in spinal neurons, and visually identify those that are involved in fictive locomotor activity. We can then characterize unique features of these neurons. COMPARISON WITH EXISTING METHODS: This builds on existing fictive locomotor preparations and is the first which allows for the visual identification of locomotor related neurons spanning the transverse plane of the spinal cord, facilitating their electrophysiological and anatomical characterization CONCLUSIONS: This approach promises to provide new information regarding the distribution of the locomotor CPG in the transverse plane, the characteristics of its component interneurons, as well as the cellular mechanisms and network properties which underlie rhythm generation. By altering the location of Ca2+ indicator application it can also be used to identify and characterize neurons involved in other facets of sensorimotor processing.

Receptor dependence of BDNF actions in superficial dorsal horn: relation to central sensitization and actions of macrophage colony stimulating factor 1.

Peripheral nerve injury elicits an enduring increase in the excitability of the spinal dorsal horn. This change, which contributes to the development of neuropathic pain, is a consequence of release and prolonged exposure of dorsal horn neurons to various neurotrophins and cytokines. We have shown in rats that nerve injury increases excitatory synaptic drive to excitatory neurons but decreases drive to inhibitory neurons. Both effects, which contribute to an increase in dorsal horn excitability, appear to be mediated by microglia-derived BDNF. We have used multiphoton Ca2+ imaging and whole cell recording of spontaneous excitatory postsynaptic currents in defined-medium organotypic cultures of GAD67-GFP+ mice spinal cord to determine the receptor dependence of these opposing actions of BDNF. In mice, as in rats, BDNF enhances excitatory transmission onto excitatory neurons. This is mediated via presynaptic TrkB and p75 neurotrophin receptors and exclusively by postsynaptic TrkB. By contrast with findings from rats, in mice BDNF does not decrease excitation of inhibitory neurons. The cytokine macrophage colony-stimulating factor 1 (CSF-1) has also been implicated in the onset of neuropathic pain. Nerve injury provokes its de novo synthesis in primary afferents, its release in spinal cord, and activation of microglia. We now show that CSF-1 increases excitatory drive to excitatory neurons via a BDNF-dependent mechanism and decreases excitatory drive to inhibitory neurons via BDNF-independent processes. Our findings complete missing steps in the cascade of events whereby peripheral nerve injury instigates increased dorsal horn excitability in the context of central sensitization and the onset of neuropathic pain. NEW & NOTEWORTHY Nerve injury provokes synthesis of macrophage colony-stimulating factor 1 (CSF-1) in primary afferents and its release in the dorsal horn. We show that CSF-1 increases excitatory drive to excitatory dorsal horn neurons via BDNF activation of postsynaptic TrkB and presynaptic TrkB and p75 neurotrophin receptors. CSF-1 decreases excitatory drive to inhibitory neurons via a BDNF-independent processes. This completes missing steps in understanding how peripheral injury instigates central sensitization and the onset of neuropathic pain.

TARP mediation of accelerated and more regular locus coeruleus network bursting in neonatal rat brain slices.

Transmembrane AMPA receptor (AMPAR) regulatory proteins (TARP) increase neuronal excitability. However, it is unknown how TARP affect rhythmic neural network activity. Here we studied TARP effects on local field potential (LFP) bursting, membrane potential and cytosolic Ca2+ (Cai) in locus coeruleus neurons of newborn rat brain slices. LFP bursting was not affected by the unselective competitive ionotropic glutamate receptor antagonist kynurenic acid (2.5 mM). TARP-AMPAR complex activation with 25 µM CNQX accelerated LFP rhythm 2.2-fold and decreased its irregularity score from 63 to 9. Neuronal spiking was correspondingly 2.3-fold accelerated in association with a 2-5 mV depolarization and a modest Cai rise whereas Cai was unchanged in neighboring astrocytes. After blocking rhythmic activities with tetrodotoxin (1 µM), CNQX caused a 5-8 mV depolarization and also the Cai rise persisted. In tetrodotoxin, both responses were abolished by the non-competitive AMPAR antagonist GYKI 53655 (25 µM) which also reversed stimulatory CNQX effects in control solution. The CNQX-evoked Cai rise was blocked by the L-type voltage-activated Ca2+ channel inhibitor nifedipine (100 µM). The findings show that ionotropic glutamate receptor-independent neonatal locus coeruleus network bursting is accelerated and becomes more regular by activating a TARP-AMPAR complex. The associated depolarization-evoked L-type Ca2+ channel-mediated neuronal Cai rise may be pivotal to regulate locus coeruleus activity in cooperation with SK-type K+ channels. In summary, this is the first demonstration of TARP-mediated stimulation of neural network bursting. We hypothesize that TARP-AMPAR stimulation of rhythmic locus coeruleus output serves to fine-tune its control of multiple brain functions thus comprising a target for drug discovery.

Genetically encoded fluorescent indicators for imaging intracellular potassium ion concentration.

Potassium ion (K+) homeostasis and dynamics play critical roles in biological activities. Here we describe three genetically encoded K+ indicators. KIRIN1 (potassium (K) ion ratiometric indicator) and KIRIN1-GR are Förster resonance energy transfer (FRET)-based indicators with a bacterial K+ binding protein (Kbp) inserting between the fluorescent protein FRET pairs mCerulean3/cp173Venus and Clover/mRuby2, respectively. GINKO1 (green indicator of K+ for optical imaging) is a single fluorescent protein-based K+ indicator constructed by insertion of Kbp into enhanced green fluorescent protein (EGFP). These indicators are suitable for detecting K+ at physiologically relevant concentrations in vitro and in cells. KIRIN1 enabled imaging of cytosolic K+ depletion in live cells and K+ efflux and reuptake in cultured neurons. GINKO1, in conjunction with red fluorescent Ca2+ indicator, enable dual-color imaging of K+ and Ca2+ dynamics in neurons and glial cells. These results demonstrate that KIRIN1 and GINKO1 are useful tools for imaging intracellular K+ dynamics.

A Bioluminescent Ca2+ Indicator Based on a Topological Variant of GCaMP6s.

Fluorescent genetically encoded calcium ion indicators (GECIs) enable Ca2+ dynamics to be monitored in a diverse array of cell types and tissues. One drawback of green fluorescent GECIs, such as the widely used GCaMP6, is that the blue wavelengths of light used to excite the GECI also activate optogenetic actuators such as channelrhodopsins. Accordingly, it is particularly challenging simultaneously to use both optogenetic actuators and GECIs to both control and image cell signaling. Bioluminescence is an alternative imaging modality that circumvents this problem by avoiding the need for illumination for fluorescence excitation. Here, we report the development of a bioluminescent GECI, designated LUCI-GECO1, based on efficient bioluminescent resonance energy transfer (BRET) between the NanoLuc luciferase and a topological variant of GCaMP6s. LUCI-GECO1 is a sensitive ratiometric GECI that retains the highly optimized properties of GCaMP6s, as we demonstrate by imaging of chemically and optogenetically induced Ca2+ concentration changes in cultured cells and neurons.

Voluntary wheel running reveals sex-specific nociceptive factors in murine experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an inflammatory, neurodegenerative autoimmune disease associated with sensory and motor dysfunction. Although estimates vary, ∼50% of patients with MS experience pain during their disease. The mechanisms underlying the development of pain are not fully understood, and no effective treatment for MS-related pain is available. Previous work from our laboratory demonstrated that voluntary exercise (wheel running) can reduce nociceptive behaviours at the disease onset in female mice with experimental autoimmune encephalomyelitis (EAE), an animal model used to study the immunopathogenesis of MS. However, given the established sex differences in the underlying mechanisms of chronic pain and MS, we wanted to investigate whether wheel running would also be effective at preventing nociceptive behaviours in male mice with EAE. C57BL/6 mice of both sexes were given access to running wheels for 1 hour/day until the disease onset, when nociceptive behaviour was assessed using von Frey hairs. Daily running effectively reduced nociceptive behaviour in female mice, but not in male mice. We explored the potential biological mechanisms for these effects and found that the reduction in nociceptive behaviour in female mice was associated with reduced levels of inflammatory cytokines from myelin-reactive T cells as well as reduced dorsal root ganglia excitability as seen by decreased calcium responses. These changes were not seen in male mice. Instead, running increased the levels of inflammatory cytokines and potentiated Ca responses in dorsal root ganglia cells. Our results show that voluntary wheel running has sex-dependent effects on nociceptive behaviour and inflammatory responses in male and female mice with EAE.

WT1-Expressing Interneurons Regulate Left-Right Alternation during Mammalian Locomotor Activity.

The basic pattern of activity underlying stepping in mammals is generated by a neural network located in the caudal spinal cord. Within this network, the specific circuitry coordinating left-right alternation has been shown to involve several groups of molecularly defined interneurons. Here we characterize a population of spinal neurons that express the Wilms' tumor 1 (WT1) gene and investigate their role during locomotor activity in mice of both sexes. We demonstrate that WT1-expressing cells are located in the ventromedial region of the spinal cord of mice and are also present in the human spinal cord. In the mouse, these cells are inhibitory, project axons to the contralateral spinal cord, terminate in close proximity to other commissural interneuron subtypes, and are essential for appropriate left-right alternation during locomotion. In addition to identifying WT1-expressing interneurons as a key component of the locomotor circuitry, this study provides insight into the manner in which several populations of molecularly defined interneurons are interconnected to generate coordinated motor activity on either side of the body during stepping.SIGNIFICANCE STATEMENT In this study, we characterize WT1-expressing spinal interneurons in mice and demonstrate that they are commissurally projecting and inhibitory. Silencing of this neuronal population during a locomotor task results in a complete breakdown of left-right alternation, whereas flexor-extensor alternation was not significantly affected. Axons of WT1 neurons are shown to terminate nearby commissural interneurons, which coordinate motoneuron activity during locomotion, and presumably regulate their activity. Finally, the WT1 gene is shown to be present in the spinal cord of humans, raising the possibility of functional homology between these species. This study not only identifies a key component of the locomotor circuitry but also begins to unravel the connectivity among the growing number of molecularly defined interneurons that comprise this neural network.

Characterization of the Nile Grass Rat as a Unique Model for Type 2 Diabetic Polyneuropathy.

Type 2 diabetes (T2D) has reached pandemic proportions worldwide. Almost half of T2D patients suffer from polyneuropathy that can present as paresthesia, hyperalgesia, allodynia, or hypoesthesia. Therapeutic treatment options are largely incomplete, suggesting new avenues of research are needed. Herein, we introduce the African Nile Grass rat (NGR), which develops T2D solely by diet manipulation, as a novel T2D polyneuropathy model. The purpose of this study was to first characterize T2D-induced polyneuropathy in the NGRs before highlighting their strength as a potential prediabetic model of T2D. NGRs with long-term T2D exhibit hallmark features of polyneuropathy such as decreased motor nerve conduction velocity, intraepidermal denervation, and hyposensitivity to noxious mechanical and thermal stimulation. At the dorsal root ganglia, T2D neurons have altered sodium channel expression, specifically increased Nav1.7 and Nav1.9, and their surrounding satellite glial cells express glial fibrillary acidic protein. Now that these T2D NGRs have been characterized and shown to have a similar presentation to human and other animal models of T2D, the strength of this diet-induced model can be exploited. The prediabetic changes can be observed over their long progression to develop T2D which may allow for a therapeutic window to prevent T2D before permanent damage occurs.

Suction electrode recording in locus coeruleus of newborn rat brain slices reveals network bursting comprising summated non-synchronous spiking.

The brainstem locus coeruleus (LC) controling behaviors like arousal, sleep, breathing, pain or opioid withdrawal is an established model for spontaneous action potential synchronization. Such synchronous 'spiking' might produce an extracellular field potential (FP) which is a crucial tool for neural network analyses. We found using ≥10 µm tip diameter suction electrodes in newborn rat brainstem slices that the LC generates at ∼1 Hz a robust rhythmic FP (rFP). During distinct rFP phases, LC neurons discharge with a jitter of ±33 ms single spikes that summate to a ∼200 ms-lasting population burst. The rFP is abolished by blocking voltage-gated Na+ channels with tetrodotoxin (TTX, 50 nM) or gap junctions with mefloquine (100 µM) and activating µ-opioid receptors with [D-Ala2,N-Me-Phe4,Gly5-ol]-Enkephalin (DAMGO, 1 µM). Raising superfusate K+ from 3 to 7 mM either increases rFP rate or transforms its pattern to slower and longer multipeak bursts similar to those during early recovery from DAMGO. The results show that electrical coupling of neonatal LC neurons does not synchronize their spiking as previously proposed. They also indicate that both increased excitability (by elevated K+) and recovery from inhibition (by opioids) can enhance spike desynchronization to transform the population burst pattern. Both observations show that this gap junction-coupled neural network has a more complex connectivity than currently assumed. These new findings along with the inhibitory drug effects that are in line with previous reports based on single neuron recording point out that field potential analysis is pivotal to further the understanding of this brain circuit.