Application Of Bicuculline Research Articles

Perinatal High Fat Diet Exposure Reduces KCC2 Expression and Alters GABAergic Signaling in the Dorsal Motor Nucleus of the Vagus

Perinatal high fat diet (pHFD) exposure alters the development of vagal neurocircuits controlling gastrointestinal (GI) functions resulting in gastric motor dysfunction. GABAergic synaptic inhibition of vagal efferent motoneurons within the dorsal motor nucleus of the vagus (DMV) is critical in the brain’s ability to modulate gastric motility. This inhibition is maintained by KCC2, a Cl− transporter, who determines intracellular chloride concentration. The current study was designed to test the hypothesis that pHFD reduces KCC2 levels in DMV neurons, subsequently decreasing GABAergic inhibition and delaying gastric emptying rates. Pregnant Sprague-Dawley rats were fed either a control or high fat (14% vs 60% kcal from fat, respectively) diet from embryonic day 13, and offspring were used starting postnatal day 28. In vitro electrophysiological recordings were made from adult DMV neurons in brainstem slices. To assess the GABA-mediated inhibition, cell-attached recordings were made to preserve the internal Cl− concentration, and firing rate was quantified before and after application of bicuculline (BIC; 30μM), a GABAA antagonist. To determine the Cl− reversal potential, perforated patch clamp recordings were performed and the response to picospritz application of the GABAA agonist muscimol (100μM) was measured. KCC2 expression was quantified using pharmacology in the in vitro slice preparation, as well as immunofluorescence in brainstem cross-sections. Gastric emptying rates were quantified using the in vivo13C octanoic acid breath test technique. KCC2 transfection and induced expression in the DMV of pHFD rats was achieved using an adeno-associated viral vector. BIC increased firing rate in control DMV neurons (143.1% of baseline), but failed to increase DMV firing rate in DMV neurons from pHFD rats (96.27% of baseline; p=0.0103 using unpaired t-test). When control DMV neurons were treated with VU0240551 (VU; 10μM), a KCC2 blocker, BIC application led to a decrease in the firing rate (3.118 Hz to 1.606 Hz; p=0.0375 via paired t-test), demonstrating that antagonizing KCC2 attenuates GABAergic inhibition at this synapse. In control DMV neurons, the Cl− reversal potential was calculated to be -64.6mV. There was a depolarizing shift in Cl− reversal potential observed in pHFD neurons (-43.0mV; p=0.0007 using unpaired t-test). Application of VU shifted the chloride reversal from -64.6mV to -57.3mV in control neurons (p=0.0416 using a paired t-test). VU application had a reduced impact on pHFD neurons, shifting the reversal from -46.7mV to -45.0mV (p=0.1689 using a paired t-test), suggesting KCC2 downregulation. In fact, compared to control rats, there was a significant reduction in KCC2 expression in the pHFD DMV (2.237% area fluorescence in pHFD vs. 5.335%; p=0.0147 using unpaired t-test). When KCC2 expression was virally induced in young adult pHFD rats, GABA became inhibitory (163.7% of baseline firing rate after BIC application vs. 93.25% in pHFD + empty vector (EV); p=0.0019 using unpaired t-test), the Cl− reversal potential was normalized (-63.0mV vs. -49.7mV in pHFD + EV; p=0.0012 using an unpaired t-test), and gastric emptying rates were accelerated (58.7min vs 72.1min in pHFD + EV). Taken together, these results suggest that the loss of KCC2 function is a driver of the gastric pathophysiology seen in the pHFD rats, and that normalizing KCC2 levels can rescue the cellular and gastric phenotype. DK 111667. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Decreased GABAergic Neurotransmission Promotes Breathing Upon Emergence From Hibernation

The respiratory network must produce reliable output for an animal to survive. Although respiratory motor plasticity is well appreciated, how plasticity mechanisms work to promote robustness following environmental perturbations that disrupt breathing is less clear. During underwater hibernation, respiratory neurons of bullfrogs remain inactive for months, triggering a suite of compensatory mechanisms that aim to maintain respiratory function. We hypothesized that GABAergic inhibition would decrease following hibernation. In control animals, respiratory frequency was under control of GABAAR signaling. Specifically, block of GABAARs dose-dependently decreased respiratory burst frequency in the in vitro brainstem preparation (n=5 preparations). After hibernation, GABAA receptor block had little to no impact on respiratory burst frequency, suggesting the network was no longer reliant on GABAergic inhibition to maintain normal frequency (n=5 preparations). The loss of inhibition was specific to the respiratory network because non-respiratory motor activity and large seizure-like bursts were similarly triggered by GABAA receptor blockade in intact brainstem preparations from controls and hibernators. We next used a semi-intact preparation that maintains functional respiratory-synaptic input onto rhythmic motor neurons to identify compensatory changes in GABAergic tone at the level of the motor neuron. In controls, the firing rate of respiratory motoneurons was constrained by a phasic GABAAR tone because focal application of bicuculline, a GABAAR antagonist, led to an increase in phasic firing frequency (n=12 cells). After hibernation, this bicuculline-mediated increase in phasic firing frequency was attenuated suggesting phasic inhibition onto the motor neuron had decreased (n=9 cells). Brain slice recordings of identified vagal motor neurons revealed that postsynaptic GABAA receptor strength was unchanged following hibernation (n=16 control cells; n=16 hibernation cells). Taken together, our results suggest that GABAergic activity is decreased in premotor networks following hibernation. Despite this dramatic loss of GABAergic function, respiratory output from control preparations was indistinguishable from hibernators. To elucidate the functional impacts of loss of GABAergic activity, we tested the network’s ability to produce output at colder temperatures. In controls, respiratory output slowed as temperature decreased, with most networks nearly silent at 8 degrees Celsius (n=5 preparations). Interestingly, in controls low dose block of GABAA receptors promoted respiratory activity at cold temperatures, with frequency not statistically different than baseline at any temperature tested (n=4 preparations). This suggests that the loss of premotor GABAergic activity we observed could work to promote motor output at colder temperatures, which works adaptively for the animal when emerging from hibernation. NIH R01NS114514 (JS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Activation of 5-HT5A receptor in the ventrolateral orbital cortex produces antinociceptive effects in rat models of neuropathic and inflammatory pain

The ventrolateral orbital cortex (VLO) is identified as an integral component of the endogenous analgesic system comprising a spinal cord - thalamic nucleus submedius - VLO - periaqueductal gray (PAG) - spinal cord loop. The present study investigates the effects of 5-HT5A receptor activation in the VLO on allodynia induced by spared nerve injury and formalin-evoked flinching behavior and spinal c-Fos expression in male SD rats, and further examines whether GABAergic modulation is involved in the effects evoked by VLO 5-HT5A receptor activation. We found an upregulation of 5-HT5A receptor expression in the VLO during neuropathic and inflammatory pain states. Microinjection of the non-selective 5-HT5A receptor agonist 5-CT into the VLO dose dependently alleviated allodynia, and flinching behavior and spinal c-Fos expression, which were blocked by the selective 5-HT5A receptor antagonist SB-699551. Moreover, application of the GABAA receptor antagonist bicuculline in the VLO augmented the analgesic effects induced by 5-CT in neuropathic and inflammatory pain states, whereas the GABAA receptor agonist muscimol attenuated these analgesic effects. Additionally, the 5-HT5A receptors were found to be colocalized with GABAergic neurons in the VLO. These results provide new evidence for the involvement of central 5-HT5A receptors in the VLO in modulation of neuropathic and inflammatory pain and support the hypothesis that activation of 5-HT5A receptors may inhibit the inhibitory effect of GABAergic interneurons on output neurons projecting to the PAG (GABAergic disinhibitory mechanisms), consequently activating the brainstem descending inhibitory system that depresses nociceptive transmission at the spinal cord level.

Gray and white matter astrocytes differ in basal metabolism but respond similarly to neuronal activity.

Astrocytes are a heterogeneous population of glial cells in the brain, which adapt their properties to the requirements of the local environment. Two major groups of astrocytes are protoplasmic astrocytes residing in gray matter as well as fibrous astrocytes of white matter. Here, we compared the energy metabolism of astrocytes in the cortex and corpus callosum as representative gray matter and white matter regions, in acute brain slices taking advantage of genetically encoded fluorescent nanosensors for the NADH/NAD+ redox ratio and for ATP. Astrocytes of the corpus callosum presented a more reduced basal NADH/NAD+ redox ratio, and a lower cytosolic concentration of ATP compared to cortical astrocytes. In cortical astrocytes, the neurotransmitter glutamate and increased extracellular concentrations of K+ , typical correlates of neuronal activity, induced a more reduced NADH/NAD+ redox ratio. While application of glutamate decreased [ATP], K+ as well as the combination of glutamate and K+ resulted in an increase of ATP levels. Strikingly, a very similar regulation of metabolism by K+ and glutamate was observed in astrocytes in the corpus callosum. Finally, strong intrinsic neuronal activity provoked by application of bicuculline and withdrawal of Mg2+ caused a shift of the NADH/NAD+ redox ratio to a more reduced state as well as a slight reduction of [ATP] in gray and white matter astrocytes. In summary, the metabolism of astrocytes in cortex and corpus callosum shows distinct basal properties, but qualitatively similar responses to neuronal activity, probably reflecting the different environment and requirements of these brain regions.

GABAergic CaMKIIα+ Amygdala Output Attenuates Pain and Modulates Emotional-Motivational Behavior via Parabrachial Inhibition.

Pain and emotion are strongly regulated by neurons in the central nucleus of the amygdala (CeA), a major output of the limbic system; yet, the neuronal signaling pathways underlying this modulation are incompletely understood. Here, we characterized a subpopulation of CeA neurons that express the CaMKIIα gene (CeACAM neurons) and project to the lateral parabrachial nucleus (LPBN), a brainstem region known for its critical role in distributing nociceptive and other aversive signals throughout the brain. In male Sprague Dawley rats, we show that CeACAM-LPBN neurons are GABAergic and mostly express somatostatin. In anaesthetized rats, optogenetic stimulation of CeACAM-LPBN projections inhibited responses of LPBN neurons evoked by electrical activation of Aδ- and C-fiber primary afferents; this inhibition could be blocked by intra-LPBN application of the GABAA receptor antagonist bicuculline. CeACAM-LPBN stimulation also dampened LPBN responses to noxious mechanical, thermal, and chemical stimuli. In behaving rats, optogenetic stimulation of CeACAM-LPBN projections attenuated nocifensive responses to mechanical pressure and radiant heat, disrupted the ability of a noxious shock to drive aversive learning, reduced the defensive behaviors of thigmotaxis and freezing, induced place preference, and promoted food consumption in sated rats. Thus, we suggest that CeACAM-LPBN projections mediate a form of analgesia that is accompanied by a shift toward the positive-appetitive pole of the emotional-motivational continuum. Since the affective state of pain patients strongly influences their prognosis, we envision that recruitment of this pathway in a clinical setting could potentially promote pain resilience and recovery.SIGNIFICANCE STATEMENT Pain and emotion interact on multiple levels of the nervous system. Both positive and negative emotion may have analgesic effects. However, while the neuronal mechanisms underlying "stress-induced analgesia" have been the focus of many studies, the neuronal substrates underlying analgesia accompanied by appetitive emotional-motivational states have received far less attention. The current study focuses on a subpopulation of amygdala neurons that form inhibitory synapses within the brainstem lateral parabrachial nucleus (LPBN). We show that activation of these amygdalo-parabrachial projections inhibits pain processing, while also reducing behaviors related to negative affect and enhancing behaviors related to positive affect. We propose that recruitment of this pathway would benefit pain patients, many of whom suffer from psychological comorbidities such as anxiety and depression.

A brainstem circuit for nausea suppression.

SUMMARYNausea is a discomforting sensation of gut malaise that remains a major clinical challenge. Several visceral poisons induce nausea through the area postrema, a sensory circumventricular organ that detects blood-borne factors. Here, we use genetic approaches based on an area postrema cell atlas to reveal inhibitory neurons that counteract nausea-associated poison responses. The gut hormone glucose insulinotropic peptide (GIP) activates area postrema inhibitory neurons that project locally and elicit inhibitory currents in nausea-promoting excitatory neurons through γ-aminobutyric acid (GABA) receptors. Moreover, GIP blocks behavioral responses to poisons in wild-type mice, with protection eliminated by targeted area postrema neuron ablation. These findings provide insights into the basic organization of nausea-associated brainstem circuits and reveal that area postrema inhibitory neurons are an effective pharmacological target for nausea intervention.

Thyroid hormones during the perinatal period are necessary to respiratory network development of newborn rats

Thyroid hormones (THs) are essential for foetal brain development. Because the gestating mother is the main source of THs to the foetus, maternal hypothyroidism and/or premature birth compromise neurological outcomes in the offspring. Respiratory instability and recurrent apneas due to immaturity of the respiratory control network are major causes of morbidity in infants. Inadequate TH supply may be sufficient to delay perinatal maturation of the respiratory control system; however, this hypothesis remains untested. To address this issue, maternal hypothyroidism was induced by adding methimazole (MMI; 0.02% w/v) to the drinking water of pregnant dams from conception to postpartum day 4 (P4). The effect of TH supplementation on respiratory function was tested by injecting levothyroxine (L-T4) in newborns at P1. Respiratory function was assessed by plethysmography (in vivo) and recording of phrenic output from medullary preparations (in vitro). By comparison with controls, TH deficiency increased the frequency of apneas and decreased basal ventilation in vivo and prevented the age-dependent increase in phrenic burst frequency normally observed in vitro. The effects of TH deficiency on GABAergic modulation of respiratory activity were measured by bath application of muscimol (GABAA agonist) or bicuculline (GABAA antagonist). The phrenic burst frequency responses to GABAergic agents were consistently greater in preparations from TH deficient pups. L-T4 supplementation reversed part of the respiratory anomalies related to MMI treatment in vitro. We conclude that TH deficiency during the perinatal period is sufficient to delay maturation of the respiratory control network development. Excessive GABAergic inhibition may contribute to this effect.

The effect of GABAergic neurotransmission on the seizure-related activity of the laterodorsal thalamic nuclei and the somatosensory cortex in a genetic model of absence epilepsy

The present study aimed to investigate the alterations of the GABAergic system in the laterodorsal nucleus (LDN) of the thalamus and the somatosensory cortex (SC) in an experimental model of absence seizure. The effects of pharmacological manipulation of both GABAA and GABAB receptor subunits in the LDN on the generation of spike-wave discharges (SWD) were evaluated. The experiments were carried out in four groups of both WAG/Rij and Wistar rats with 2 and 6 months of age. The expressions of various GABA receptor subunits were studied in the LDN and SC. Furthermore, recordings of unit activity from the LDN and electrocorticography were simultaneously monitored before, during, and after the application of GABAA and GABAB antagonists in the LDN. The generation of SWD in the older WAG/Rij rats was associated with significant alterations in the expression of GABAARα1, GABAARβ3, and GABABR2 subunits in the LDN as well as GABAARα1, GABAARβ3, GABAARγ2, and GABABR2 subunits in the SC. Furthermore, the occurrence of SWD was associated with a significant reduction of gene expression of GABAARα1 and increase of GABAARβ3 in the LDN as well as reduction of GABAARα1, GABAARβ3, GABAARγ2, and GABABR2 in the SC. The microionthophoretic application of the GABAA antagonist bicuculline resulted in a significant increase in the population firing rate of LDN neurons as well as the mean number and duration of SWD. The application of the GABAB antagonist CGP35348 significantly increased the population firing rate of LDN neurons but decreased the mean number of SWD. Our data indicate the regulatory effect of the GABAergic system of the LDN and SC in absence seizures.

Dual synaptic inhibitions of brainstem neurons by GABA and glycine with impact on Rett syndrome.

Rett syndrome (RTT) is a neurodevelopmental disease caused mostly by mutations in the MECP2 gene. People with RTT show breathing dysfunction attributable to the high rate of sudden death. Previous studies have shown that insufficient GABA synaptic inhibition contributes to the breathing abnormalities in mouse models of RTT, while it remains elusive how the glycine system is affected. We found that optogenetic stimulation of GAD-expressing neurons in mice produced GABAergic and glycinergic postsynaptic inhibitions of neurons in the hypoglossal nucleus (XII) and the dorsal motor nucleus of vagus (DMNV). By sequential applications of bicuculline and strychnine, such inhibition appeared approximately 44% GABAA ergic and 52% glycinergic in XII neurons, and approximately 49% GABAA ergic and 46% glycinergic in DMNV neurons. Miniature inhibitory postsynaptic potentials (mIPSCs) in these neurons were approximately 47% GABAA ergic and 49% glycinergic in XII neurons, and approximately 48% versus 50% in DMNV neurons, respectively. Consistent with the data, our single-cell polymerase chain reaction studies indicated that transcripts of GABAA receptor γ2 subunit (GABAA Rγ2) and glycine receptor β subunit (GlyRβ) were simultaneously expressed in these cells. In MeCP2R168X mice, proportions of GABAA ergic and glycinergic mIPSCs became approximately 28% versus 69% in XII neurons, and approximately 31% versus 66% in DMNV cells. In comparison with control mice, the GABAA ergic and glycinergic mIPSCs decreased significantly in the XII and DMNV neurons from the MeCP2R168X mice, so did the transcripts of GABAA Rγ2 and GlyRβ. These results suggest that XII and DMNV neurons adopt dual GABAA ergic and glycinergic synaptic inhibitions, and with Mecp2 disruption these neurons rely more on glycinergic synaptic inhibition.

Seizures Alter Cortical Representations for Complex Movements

Alterations in the functional organization of motor cortex and interictal motor deficits are observed in people with epilepsy. While seizures in the rat lead to more cortical area devoted to simple cortical forelimb movement representations (motor maps) assessed using short-duration intracortical microstimulation (ICMS), the effect of seizures on complex movements derived with long-duration ICMS is unknown. Further, the relationship between motor map expression and motor impairment is not well understood. We used long-duration ICMS in the rat to test the hypothesis that repeated seizure activity (cortical kindling) increases the extent of overlapping cortical representation where multiple forelimb movements are evoked to stimulation. Cortical kindling (n = 7) significantly expanded (100%) forelimb motor maps characterized by a proportional increase in both complex and simple movement representation areas, and significantly increased (285%) overlapping forelimb representation compared to sham-kindled controls (n = 5). In a second experiment, motor maps were derived with long-duration ICMS under acute cortical application of bicuculline (n = 6) to reduce intracortical inhibition or saline control (n = 10). Bicuculline also significantly expanded forelimb motor maps (108%) but without increasing representational overlap. Moreover, expanded map areas in bicuculline rats evoked qualitatively distinct forelimb movements to long-duration, but not short-duration (n = 5), ICMS that were truncated. Our evidence indicates that motor map expansion following repeated experimental seizures is associated with reduced segregation between cortical movement representations that is not entirely due to reduced cortical inhibition but may contribute to interictal motor deficits in some individuals with epilepsy.

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings.

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

Proteomic analysis involved with synaptic plasticity improvement by GABAA receptor blockade in hippocampus of a mouse model of Alzheimer’s disease

GABAergic system plays a part in synaptic plasticity in the hippocampus. We had reported a long-term potentiation (LTP)-like facilitation in vivo, known as synaptic plasticity, through GABAA receptor blockade by bicuculline and the expression of proteins involved with this synaptic plasticity in mouse hippocampus. In the present study, we aimed to show improvement of impaired synaptic plasticity through GABAA receptor blockade and to clarify the molecular mechanisms involved with this improvement in the hippocampus of mice overexpressing human amyloid precursor protein with the E693Δ mutation (APPOSK-Tg) as an Alzheimer’s disease model showing impaired synaptic plasticity. Electrophysiological study showed that the LTP-like facilitation expressed with application of bicuculline in vivo was significantly greater than impaired tetanic LTP in APPOSK-Tg mice, which was improved by bicuculline. Proteomic analysis showed that the expression of 11 proteins in the hippocampus was significantly changed 8 h after bicuculline application to APPOSK-Tg mice. The identified proteins could be functionally classified as chaperone, cytoskeletal protein, energy metabolism, metabolism, neuronal development, and synaptic component. Additionally, western blotting validated the changes in four proteins. We therefore propose that the improvement of impaired synaptic plasticity through GABAA receptor blockade could be mediated by the changed expression of these proteins.

Co‐release of inhibitory neurotransmitters in the RVLM

Sympathetic nerve activity (SNA) and blood pressure (BP) are largely determined by the level of excitability of presympathetic neurons (C1 neurons) in the rostral ventrolateral medulla (RVLM). GABAergic inhibition in the RVLM is the primary mechanism for depressor and sympathoinhibitory responses; however, the role of glycinergic inhibition in the control of RVLM excitability is unknown. There is evidence that both, GABA and glycine efficiently inhibit RVLM neurons, but functional evidence for their co‐release or the mechanism of co‐inhibition is lacking. In our study we used both male and female glycine transporter 2 Cre (GlyT2‐Cre) mice. Glycinergic neurons sending projections to the RVLM were identified with viral labeling. Targeted expression of channelrhodopsin 2 (ChR2) in GlyT2‐expressing neurons and whole‐cell recordings from presympathetic RVLM neurons were used to study the mechanism of glycine release. We found that under steady‐state conditions spontaneous inhibitory postsynaptic current (sIPSC) are primarily mediated by GABA. Photostimulation of glycinergic fibers expressing GlyT2 in the RVLM resulted in evoked IPSCs with both glycinergic and GABAergic components. The time course of synaptic signaling were identical for the two transmitters. Bath application of GABAA receptors antagonist bicuculline (10 μM) or glycine receptor antagonist strychnine (1 μM) revealed that ~40% of the evoked inhibitory currents are GABAergic. High frequency photostimulation (20 Hz) of co‐releasing inhibitory fibers produced a short‐term synaptic depression. Blockade of GlyT2 prevented the glycinergic component of the light evoked IPSCs. In this study we showed that GABA and glycine are co‐released in the RVLM. Our findings suggest that the two neurotransmitters may be functionally interchangeable or complement each other, and thus provide diverse mechanisms for maintaining inhibitory homeostasis.

Co‐modulation of the Na+/K+ pump and hyperpolarization‐activated currents as a mechanism for robust neuromodulation

Specialized oscillatory circuits, central pattern generators (CPGs), control rhythmic motor behaviors such as locomotion and breathing. To accommodate a motor pattern to environmental changes and behavioral goals, neuromodulators adjust the dynamics of CPGs by orchestrating changes in various ionic currents in a wide range of their biophysical parameters to expand temporal characteristics of the functional pattern. Recent studies provide evidence that the Na+/K+ pump contributes to the dynamics of CPGs and is controlled by neuromodulation [1– 4]. In the leech heartbeat CPG, the neuropeptide myomodulin reduces the period of bursting activity by increasing the hyperpolarization‐activated (h)‐current and decreasing the Na+/K+ pump current [4]. The application of myomodulin reduces the period of oscillatory activity by 17%. Application of Cs+, which is an h‐current blocker, increases the period of bursting by 24% relative to control. The application of myomodulin along with Cs+ decreases the period by 12% relative to treatment with Cs+ [4]. Here we investigate how the period of a bursting can be controlled in a wide range while functional bursting is maintained with a focus on the role of the Na+/K+ pump. We also investigate the risk associated with multistability in neuronal dynamics.We optimized a model of the leech heart interneuron (HN), which includes the Na+/K+ pump current and intracellular Na+ dynamics [1] and investigated the activity regimes of pairs of mutually inhibitory coupled HNs forming half‐center oscillators (HCOs). HCOs form the kernel of the leech heartbeat CPG. We investigated eight model variants representing combinations of three experimental treatments: the blockade of chemical synapses representing the application of bicuculline, the blockade of h‐current representing the application of Cs+, and the enhancement of the h‐current and inhibition of the Na+/K+ pump current representing the application of myomodulin. The model captures the qualitative trends in change of cycle period observed in experiments with myomodulin and Cs+. We found ranges of parameters where neurons showed functional bursting. The coordinated changes of maximal conductance of h‐current (Gh) and maximal pump activity (IPumpMax) increases the range of period of functional bursting as well as the range of parameters Gh and IPumpMax. We hypothesize that myomodulin co‐modulates h‐ and pump currents to expand the domain of the functional activity.Support or Funding InformationSupported by NINDS 1 R01 NS085006 to RLC and 1 R21 NS111355 to RLC and GSC.

L-type Ca2+ channel-mediated Ca2+ influx adjusts neuronal mitochondrial function to physiological and pathophysiological conditions.

L-type voltage-gated Ca2+ channels (LTCCs) are implicated in neurodegenerative processes and cell death. Accordingly, LTCC antagonists have been proposed to be neuroprotective, although this view is disputed, because intentional LTCC activation can also have beneficial effects. LTCC-mediated Ca2+ influx influences mitochondrial function, which plays a crucial role in the regulation of cell viability. Hence, we investigated the effect of modulating LTCC-mediated Ca2+ influx on mitochondrial function in cultured hippocampal neurons. To activate LTCCs, neuronal activity was stimulated by increasing extracellular K+ or by application of the GABAA receptor antagonist bicuculline. The activity of LTCCs was altered by application of an agonistic (Bay K8644) or an antagonistic (isradipine) dihydropyridine. Our results demonstrated that activation of LTCC-mediated Ca2+ influx affected mitochondrial function in a bimodal manner. At moderate stimulation strength, ATP synthase activity was enhanced, an effect that involved Ca2+-induced Ca2+ release from intracellular stores. In contrast, high LTCC-mediated Ca2+ loads led to a switch in ATP synthase activity to reverse-mode operation. This effect, which required nitric oxide, helped to prevent mitochondrial depolarization and sustained increases in mitochondrial Ca2+ Our findings indicate a complex role of LTCC-mediated Ca2+ influx in the tuning and maintenance of mitochondrial function. Therefore, the use of LTCC inhibitors to protect neurons from neurodegeneration should be reconsidered carefully.

Burst and Tonic Spinal Cord Stimulation Both Activate Spinal GABAergic Mechanisms to Attenuate Pain in a Rat Model of Chronic Neuropathic Pain.

BackgroundExperimental and clinical studies have shown that tonic spinal cord stimulation (SCS) releases gamma‐aminobutyric acid (GABA) in the spinal dorsal horn. Recently, it was suggested that burst SCS does not act via spinal GABAergic mechanisms. Therefore, we studied spinal GABA release during burst and tonic SCS, both anatomically and pharmacologically, in a well‐established chronic neuropathic pain model.MethodsAnimals underwent partial sciatic nerve ligation (PSNL). Quantitative immunohistochemical (IHC) analysis of intracellular GABA levels in the lumbar L4 to L6 dorsal spinal cord was performed after 60 minutes of burst, tonic, or sham SCS in rats that had undergone PSNL (n = 16). In a second pharmacological experiment, the effects of intrathecal administration of the GABAA antagonist bicuculline (5 μg) and the GABAB antagonist phaclofen (5 μg) were assessed. Paw withdrawal thresholds to von Frey filaments of rats that had undergone PSNL (n = 20) were tested during 60 minutes of burst and tonic SCS 30 minutes after intrathecal administration of the drugs.ResultsQuantitative IHC analysis of GABA immunoreactivity in spinal dorsal horn sections of animals that had received burst SCS (n = 5) showed significantly lower intracellular GABA levels when compared to sham SCS sections (n = 4; P = 0.0201) and tonic SCS sections (n = 7; P = 0.0077). Intrathecal application of the GABAA antagonist bicuculline (5 μg; n = 10) or the GABAB antagonist phaclofen (5 μg; n = 10) resulted in ablation of the analgesic effect for both burst SCS and tonic SCS.ConclusionsIn conclusion, our anatomical and pharmacological data demonstrate that, in this well‐established chronic neuropathic animal model, the analgesic effects of both burst SCS and tonic SCS are mediated via spinal GABAergic mechanisms.

Focal electrical stimulation of dorsal nucleus of the lateral lemniscus modulates auditory response properties of inferior collicular neurons in the albino mouse

The inferior colliculus (IC) receives and integrates excitatory and inhibitory inputs from many bilateral lower auditory nuclei, intrinsic projections within IC, contralateral IC through the commissure of IC and from the auditory cortex (AC). These excitatory and inhibitory inputs from both ascending and descending auditory pathways contribute significantly to auditory response properties and temporal signal processing in IC. The present study examines the contribution of gamma-aminobutyric acid-ergic (GABAergic) inhibition of dorsal nucleus of the lateral lemniscus (DNLL) in influencing the response properties and amplitude sensitivity of contralateral IC neurons using focal electrical stimulation of contralateral DNLL and by the application of bicuculline to the recording site of modulated IC neurons. Focal electrical stimulation of contralateral DNLL produces inhibition (78.1%), facilitation (7.1%) or no effect (14.8%) in the number of spikes, firing duration and the first-spike latency of modulated IC neurons. The degree of modulation is inversely correlated to the difference in best frequency (BF) between electrically stimulated DNLL neurons and modulated IC neurons (p < 0.01). The application of bicuculline to the recording site of modulated IC neurons abolishes the inhibitory effect of focal electrical stimulation of DNLL neurons. DNLL inhibition also modulates the amplitude sensitivity of IC neurons by changing the dynamic range (DR) and the slope of rate-amplitude function (RAF) of modulated IC neurons. Possible biological significance of these findings in relation to auditory signal processing is discussed.

Behavioral and electrophysiological changes in female mice overexpressing ORAI1 in neurons

Orai proteins form highly selective Ca2+ release-activated channels (CRACs). They play a critical role in store-operated Ca2+ entry (SOCE; i.e., the influx of external Ca2+ that is induced by the depletion of endoplasmic reticulum Ca2+ stores). Of the three Orai homologs that are present in mammals (Orai1–3), the physiological function of Orai1 is the best described. CRACs are formed by both homomeric assemblies and heteromultimers of Orais. Orai1 and Orai2 can form heteromeric channels that differ in conductivity during SOCE, depending on their Orai1-to-Orai2 ratio. The present study explored the potential consequences of ORAI1 overexpression in neurons where the dominant isoform is Orai2. We established the Tg(ORAI1)Ibd transgenic mouse line that overexpresses ORAI1 in brain neurons. We observed seizure-like symptoms in aged (≥15-month-old) female mice but not in males of the same age. The application of kainic acid and bicuculline to slices that were isolated from 8-month-old (±1 month) female Tg(ORAI1)Ibd mice revealed a significantly lower frequency of interictal bursts compared with samples that were isolated from wildtype mice. No differences were observed in male mice of a similar age. A battery of behavioral tests showed that context recognition decreased only in female transgenic mice. The phenotype that was observed in female mice suggests that ORAI1 overexpression may affect neuronal activity in a sex-dependent manner.This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Spatial coherence resonance and spatial pattern transition induced by the decrease of inhibitory effect in a neuronal network

Spiral waves were observed in the biological experiment on rat brain cortex with the application of carbachol and bicuculline which can block inhibitory coupling from interneurons to pyramidal neurons. To simulate the experimental spiral waves, a two-dimensional neuronal network composed of pyramidal neurons and inhibitory interneurons was built. By decreasing the percentage of active inhibitory interneurons, the random-like spatial patterns change to spiral waves and to random-like spatial patterns or nearly synchronous behaviors. The spiral waves appear at a low percentage of inhibitory interneurons, which matches the experimental condition that inhibitory couplings of the interneurons were blocked. The spiral waves exhibit a higher order or signal-to-noise ratio (SNR) characterized by spatial structure function than both random-like spatial patterns and nearly synchronous behaviors, which shows that changes of the percentage of active inhibitory interneurons can induce spatial coherence resonance-like behaviors. In addition, the relationship between the coherence degree and the spatial structures of the spiral waves is identified. The results not only present a possible and reasonable interpretation to the spiral waves observed in the biological experiment on the brain cortex with disinhibition, but also reveal that the spiral waves exhibit more ordered degree in spatial patterns.

Evidence for Tonic Control by the GABAA Receptor of Extracellular D-Serine Concentrations in the Medial Prefrontal Cortex of Rodents.

Endogenous D-serine is a putative dominant co-agonist for the N-methyl-D-aspartate glutamate receptor (NMDAR) in the mammalian forebrain. Although the NMDAR regulates the higher order brain functions by interacting with various neurotransmitter systems, the possible interactions between D-serine and an extra-glutamatergic system largely remain elusive. For the first time, we show in the rat and mouse using an in vivo microdialysis technique that the extracellular D-serine concentrations are under tonic increasing control by a major inhibitory transmitter, GABA, via the GABAA (GABAAR) in the medial prefrontal cortex (mPFC). Thus, an intra-mPFC infusion of a selective GABAAR antagonist, bicuculline (BIC), caused a concentration-dependent and reversible decrease in the extracellular levels of D-serine in the rat mPFC without affecting those of another intrinsic NMDAR coagonist, glycine and an NMDAR agonist, L-glutamate. The decreasing effects of BIC were eliminated by co-infusion of a selective GABAA agonist, muscimol (MUS) and were mimicked by a GABAA antagonist, gabazine (GBZ). In contrast, selective blockade of the GABAB or homomeric ρGABAA (formerly GABAC) receptor by saclofen or (1,2,5,6-tetrahydropyridin-4-yl)-methylphosphinic acid (TPMPA), respectively, failed to downregulate the prefrontal extracellular D-serine levels. Moreover, the local BIC application attenuated the ability of NMDA given to the mPFC to increase the cortical extracellular concentrations of taurine, indicating the hypofunction of the NMDAR. Finally, in the mouse mPFC, the reduction of the extracellular D-serine levels by a local injection of BIC into the prefrontal portion was replicated, and was precluded by inhibition of the neuronal or glial activity by co-local injection with tetrodotoxin (TTX) or fluorocitrate (Fluo), respectively. These findings suggest that the GABAAR-mediated regulation of the D-serine signaling may exert fine-tuning of the NMDAR function and require both neuronal and glial activities in the mammalian mPFC.