Application Of Bicuculline Research Articles

Study on the mechanisms of seizure-like events suppression effect by electrical stimulation using a microelectrode array.

In this paper, we studied the mechanisms underlying the suppression of seizure-like events (SLEs) by electrical stimulation. We conducted an in-vitro experiment using entorhinal cortex combined hippocampal slices and two convulsant drugs, bicuculline and 4-aminopyridine, to induce spontaneous SLEs. We used a microelectrode array to observe network dynamics over the entire hippocampal area simultaneously, including regions far from the stimulation site. We stimulated the entorhinal cortex region, which has been determined to be a focus of SLEs by Granger causality analysis of multichannel time series data, by an external electrode. In bicuculline application, electrical stimulation showed a marked suppression effect, even though the sizes of the effective region differed. In 4-aminopyridine application, however, stimulation under the same conditions did not suppress the activities in ∼80% of SLEs. The suppression effect was more remarkable in the areas surrounding the stimulation site in both cases. Our experimental results could support the neuronal depolarization blockade mechanism by accumulation of extracellular potassium ions, which is one of the most convincing mechanisms to understand seizure suppression phenomena because of electrical stimulation. Computer simulation using a neuronal network model also confirmed the mechanism.

Isolated bullfrog rostral brainstems exhibit a DAMGO‐insensitive lung‐like episodic rhythm in the presence of bicuculline

Respiratory behavior in terrestrial animals is primarily controlled via neural circuits in the medulla. A site in the amphibian rostral medulla at the level of the abducens nerve (CN VI) is believed to be homologous to the pre‐Bötzinger complex in mammals. The μ‐opioid agonist DAMGO has a suppressive effect, and the GABAA receptor antagonist bicuculline has an excitatory effect on lung activity in mammals and amphibians. However, the locations of action in amphibians are unknown. Therefore, we examined the effect of DAMGO and bicuculline on isolated rostral brainstem tissue.We isolated brainstems from bullfrog tadpoles, placed them in a recording chamber, and allowed them to recover for 1 hour perfused with artificial cerebral spinal fluid (aCSF) equilibrated with 98.5% O2 and 1.5% CO2 (pH 7.8). During this time, neural activity from the facial (CN VII) and hypoglossal (CN XII) cranial nerves was recorded. Following recovery, brainstems were transected just rostral to the trigeminal (CN V) nerve, and at the level of the glossopharyngeal nerve (CN IX), removing the caudal medulla. Then activity from the trigeminal (CN V) and facial (CN VII) nerves was recorded. A cocktail containing either DAMGO (120 nM) and bicuculline methochloride (5 μM) or bicuculline alone was subsequently bath applied for 30 minutes.The transection alone resulted in a loss of discernable respiratory rhythm. However, bicuculline application caused a lung‐like episodic rhythm to reappear. Contrary to the suppressive effect DAMGO has on lung activity in other preparations (including intact isolated amphibian brainstems), this rhythm was not affected by the addition of DAMGO in our transected preparation. This work informs the comparison of amphibian and mammalian respiratory oscillators and the consideration of their homology and differences.Support or Funding InformationNIH under Award Numbers UL1GM118991, TL4GM118992, or RL5GM118990.

Early-Onset Network Hyperexcitability in Presymptomatic Alzheimer's Disease Transgenic Mice Is Suppressed by Passive Immunization with Anti-Human APP/Aβ Antibody and by mGluR5 Blockade.

Cortical and hippocampal network hyperexcitability appears to be an early event in Alzheimer’s disease (AD) pathogenesis, and may contribute to memory impairment. It remains unclear if network hyperexcitability precedes memory impairment in mouse models of AD and what are the underlying cellular mechanisms. We thus evaluated seizure susceptibility and hippocampal network hyperexcitability at ~3 weeks of age [prior to amyloid beta (Aβ) plaque deposition, neurofibrillary pathology, and cognitive impairment] in a triple transgenic mouse model of familial AD (3xTg-AD mouse) that harbors mutated human Aβ precursor protein (APP), tau and presenilin 1 (PS1) genes. Audiogenic seizures were elicited in a higher proportion of 3xTg-AD mice compared with wild type (WT) controls. Seizure susceptibility in 3xTg-AD mice was attenuated either by passive immunization with anti-human APP/Aβ antibody (6E10) or by blockade of metabotropic glutamate receptor 5 (mGluR5) with the selective antagonist, 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP). In in vitro hippocampal slices, suppression of synaptic inhibition with the GABAA receptor antagonist, bicuculline, induced prolonged epileptiform (>1.5 s in duration) ictal-like discharges in the CA3 neuronal network in the majority of the slices from 3xTg-AD mice. In contrast, only short epileptiform (<1.5 s in duration) interictal-like discharges were observed following bicuculline application in the CA3 region of WT slices. The ictal-like activity in CA3 region of the hippocampus was significantly reduced in the 6E10-immunized compared to the saline-treated 3xTg-AD mice. MPEP acutely suppressed the ictal-like discharges in 3xTg-AD slices. Remarkably, epileptiform discharge duration positively correlated with intraneuronal human (transgenic) APP/Aβ expression in the CA3 region of the hippocampus. Our data suggest that in a mouse model of familial AD, hypersynchronous network activity underlying seizure susceptibility precedes Aβ plaque pathology and memory impairment. This early-onset network hyperexcitability can be suppressed by passive immunization with an anti-human APP/Aβ antibody and by mGluR5 blockade in 3xTg-AD mice.

Methylmercury induces an initial increase in GABA-evoked currents in Xenopus oocytes expressing α1 and α6 subunit-containing GABAA receptors

Early onset effects of methylmercury (MeHg) on recombinant α1β2γ2S or α6β2γ2S subunit-containing GABAA receptors were examined. These are two of the most prevalent receptor types found in cerebellum–a consistent target of MeHg-induced neurotoxicity. Heterologously expressed receptors were used in order to: (1) isolate receptor-mediated events from extraneous effects of MeHg due to stimulation of the receptor secondary to increased release of GABA seen with MeHg in neurons in situ and (2) limit the phenotypes of GABAA receptors present at one time. Initial changes in IGABA in Xenopus laevis oocytes expressing either α1β2γ2S or α6β2γ2S receptors were compared during continuous bath application of MeHg. A time-dependent increase in IGABA mediated by both receptor subtypes occurred following the first 25–30min of MeHg (5μM) exposure. In α6β2γ2S containing receptors, the MeHg-induced increase in IGABA was less pronounced compared to that mediated by α1β2γ2S containing receptors, although the pattern of effects was generally similar. Washing with MeHg-free solution reversed the increase in current amplitude. Application of bicuculline at the time of peak potentiation of IGABA rapidly and completely reversed the MeHg-induced currents. Therefore these MeHg-increased inward currents are mediated specifically by the two subtypes of GABAA receptors and appear to entail direct actions of MeHg on the receptor. However bicuculline did not affect stimulation by MeHg of oocyte endogenous Cl− -mediated current, which presumably results from increased [Ca2+]i. Thus, MeHg initially potentiates IGABA in oocytes expressing either α1β2γ2S or α6β2γ2S receptors prior to its more defined later effects, suggesting that MeHg may initially interact directly with GABAA receptors in a reversible manner to cause this potentiation.

Effects of bicuculline application on the somatosensory responses of secondary vestibular neurons

Limb somatosensory signals modify the discharge of vestibular neurons and elicit postural reflexes, which stabilize the body position. The aim of this study was to investigate the contribution of the γ-amino-butyric-acid (GABA) to the responsiveness of vestibular neurons to somatosensory inputs. The activity of 128 vestibular units was recorded in anesthetized rats in resting conditions and during sinusoidal foreleg rotation around the elbow or shoulder joints (0.026–0.625Hz, 45° peak amplitude). None of the recorded units was influenced by elbow rotation, while 40% of them responded to shoulder rotation. The selective GABAA antagonist receptor, bicuculline methiodine (BIC), was applied by microiontophoresis on single vestibular neurons and the changes in their activity at rest and during somatosensory stimulation was studied. In about half of cells the resting activity increased after the BIC application: 75% of these neurons showed also an increased response to somatosensory inputs whereas 17% exhibited a decrease. Changes in responsiveness in both directions were detected also in the units whose resting activity was not influenced by BIC. These data suggest that the responses of vestibular neurons to somatosensory inputs are modulated by GABA through a tonic release, which modifies the membrane response to the synaptic current. It is also possible that a phasic release of GABA occurs during foreleg rotation, shaping the stimulus-elicited current passing through the membrane. If this is the case, the changes in the relative position of body segments would modify the GABA release inducing changes in the vestibular reflexes and in learning processes that modify their spatio-temporal development.

CNQX facilitates inhibitory synaptic transmission in rat hypoglossal nucleus

6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) is a most commonly used antagonist of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor in the central nervous system. During the past two decades, studies had demonstrated that CNQX could partially activate AMPA receptors that are located on the hippocampal and cerebellar interneurons, thus subsequently leading to the facilitation of inhibitory transmission. However, whether CNQX could enhance inhibitory synaptic transmission in the hypoglossal nucleus remains elusive. Here, using whole-cell patch-clamp recording in the brainstem slice, we showed that CNQX greatly increased both frequency and amplitude of spontaneous inhibitory postsynaptic currents in the hypoglossal motoneurons, whereas D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5), N-methyl-D-aspartate (NMDA) receptor antagonist, had no effect on inhibitory synaptic transmission. Application of bicuculline and strychnine further identified that CNQX not only increased GABAergic sIPSCs but also glycinergic one in these motoneurons. Similar enhancement of inhibitory transmission was observed with application of 6, 7-dinitroquinoxaline-2, 3-dione (DNQX), a quinoxaline derivative of CNQX, but not with application of GYKI 53655, a non-competitive antagonist of AMPA receptor. In the presence of tetradotoxin, the effect of CNQX on sIPSCs was abolished, suggesting that an increase in presynaptic interneuron spike firing rate induced by CNQX was responsible for the facilitation of sIPSCs. Taken together, these results demonstrated that the excitatory effect of CNQX on presynaptic interneurons triggered enhancement of both GABAergic and glycinergic synaptic transmission within the rat hypoglossal nucleus.

Role of GABAA-Mediated Inhibition and Functional Assortment of Synapses onto Individual Layer 4 Neurons in Regulating Plasticity Expression in Visual Cortex.

Layer 4 (L4) of primary visual cortex (V1) is the main recipient of thalamocortical fibers from the dorsal lateral geniculate nucleus (LGNd). Thus, it is considered the main entry point of visual information into the neocortex and the first anatomical opportunity for intracortical visual processing before information leaves L4 and reaches supra- and infragranular cortical layers. The strength of monosynaptic connections from individual L4 excitatory cells onto adjacent L4 cells (unitary connections) is highly malleable, demonstrating that the initial stage of intracortical synaptic transmission of thalamocortical information can be altered by previous activity. However, the inhibitory network within L4 of V1 may act as an internal gate for induction of excitatory synaptic plasticity, thus providing either high fidelity throughput to supragranular layers or transmittal of a modified signal subject to recent activity-dependent plasticity. To evaluate this possibility, we compared the induction of synaptic plasticity using classical extracellular stimulation protocols that recruit a combination of excitatory and inhibitory synapses with stimulation of a single excitatory neuron onto a L4 cell. In order to induce plasticity, we paired pre- and postsynaptic activity (with the onset of postsynaptic spiking leading the presynaptic activation by 10ms) using extracellular stimulation (ECS) in acute slices of primary visual cortex and comparing the outcomes with our previously published results in which an identical protocol was used to induce synaptic plasticity between individual pre- and postsynaptic L4 excitatory neurons. Our results indicate that pairing of ECS with spiking in a L4 neuron fails to induce plasticity in L4-L4 connections if synaptic inhibition is intact. However, application of a similar pairing protocol under GABAARs inhibition by bath application of 2μM bicuculline does induce robust synaptic plasticity, long term potentiation (LTP) or long term depression (LTD), similar to our results with pairing of pre- and postsynaptic activation between individual excitatory L4 neurons in which inhibitory connections are not activated. These results are consistent with the well-established observation that inhibition limits the capacity for induction of plasticity at excitatory synapses and that pre- and postsynaptic activation at a fixed time interval can result in a variable range of plasticity outcomes. However, in the current study by virtue of having two sets of experimental data, we have provided a new insight into these processes. By randomly mixing the assorting of individual L4 neurons according to the frequency distribution of the experimentally determined plasticity outcome distribution based on the calculated convergence of multiple individual L4 neurons onto a single postsynaptic L4 neuron, we were able to compare then actual ECS plasticity outcomes to those predicted by randomly mixing individual pairs of neurons. Interestingly, the observed plasticity profiles with ECS cannot account for the random assortment of plasticity behaviors of synaptic connections between individual cell pairs. These results suggest that connections impinging onto a single postsynaptic cell may be grouped according to plasticity states.

Coregulation of the Na/K pump and the h-current as a mechanism for robust neuromodulation

To achieve the behavioral flexibility necessary for survival in a variable environment, neuronal circuits have to produce activity over a broad range of functional characteristics. Central pattern generators (CPGs), rhythmically active neuronal networks that control motor functions such as breathing, swimming, and walking, are ideal systems for addressing questions about how neuromodulation efficiently adjusts network output to meet environmental demands. Neuromodulators modify the dynamics of CPGs by orchestrating changes in multiple ionic channels and the electrogenic pump. The Na+/K+ pump has been implicated as critical in the dynamics of CPGs [1,2]. In the leech heartbeat CPG, the Na+/K+ pump is a target for neuromodulation. The neuropeptide myomodulin decreases the burst period; it increases the h-current and decreases the Na+/K+ pump current. We developed a model of the leech heart interneuron (HN) including the Na+/K+ pump current and intracellular Na+ dynamics. We separately considered the HN and pairs of HNs coupled through mutual inhibition that form half-center oscillators (HCOs). HCOs form the kernel of the leech heartbeat CPG. To investigate the role of the h-current and the Na+/K+ pump current, we changed corresponding biophysical parameters of these currents. We investigated eight model instantiations representing combinations of three experimental treatments: the blockade of chemical synapses representing application of bicuculline, the blockade of h-current representing application of Cs+, and the enhancement of the h-current and inhibition of the Na+/K+ pump current representing the application of myomodulin. Experiments with Cs+ and myomodulin showed the separate effects of myomodulin on the h-current and the Na+/K+ pump current [1]. Experimental application of myomodulin decreases the period of oscillatory activity by 17%. Application of Cs+ increases the period of bursting by 24% relative to control. The application of myomodulin in addition to Cs+ decreases the period of bursting by 12% relative to treatment with Cs+. The model captures the qualitative trends in change of cycle period observed in experiments with myomodulin and Cs+. Since the application of myomodulin decreases the period of activity when the h-current is present or absent, the Na+/K+ pump current plays a significant role in the dynamics of the HCO. We investigated activity of the model with different values of the maximal conductance of the h-current, g ¯h and the maximal pump current, IPumpMax, for isolated neurons and HCOs. We found ranges of parameters where neurons showed bursting activity. In the HCO, we identified the region of the parameter space that supported functional bursting activity. Joint changes of IPumpMax and g ¯h allows neurons to preserve functional activity over a larger span of IPumpMax and g ¯h and to produce functional activity with a larger range of period.

Modulation of synchronous sympathetic firing behaviors by endogenous GABAA and glycine receptor-mediated activities in the neonatal rat spinal cord in vitro

Delivering effective commands in the nervous systems require a temporal integration of neural activities such as synchronous firing. Although sympathetic nerve discharges are characterized by synchronous firing, its temporal structures and how it is modulated are largely unknown. This study used a collagenase-dissociated splanchnic sympathetic nerve–thoracic spinal cord preparation of neonatal rats in vitro as an experimental model. Several single-fiber activities were recorded simultaneously and verified by rigorous computational algorithms. Among 3763 fiber pairs that had spontaneous fiber activities, 382 fiber pairs had firing positively correlated. Their temporal relationship was quantitatively evaluated by cross-correlogram. On average, correlated firing in a fiber pair occurred in scales of ∼40ms lasting for ∼11ms. The relative frequency distribution curves of correlogram parametrical values pertinent to the temporal features were best described by trimodal Gaussians, suggesting a correlated firing originated from three or less sources. Applications of bicuculline or gabazine (noncompetitive or competitive GABAA receptor antagonist) and/or strychnine (noncompetitive glycine receptor antagonist) increased, decreased, or did not change individual fiber activities. Antagonist-induced enhancement and attenuation of correlated firing were demonstrated by a respective increase and decrease of the peak probability of the cross-correlograms. Heterogeneity in antagonistic responses suggests that the inhibitory neurotransmission mediated by GABAA and glycine receptors is not essential for but can serve as a neural substrate to modulate synchronous firing behaviors. Plausible neural mechanisms were proposed to explain the temporal structures of correlated firing between sympathetic fibers.

Duration sensitivity of neurons in the primary auditory cortex of albino mouse

Many neurons in the central auditory system of a number of species have been found to be sensitive to the duration of sound stimuli. While previous studies have shown that γ-aminobutyric acid (GABA)-ergic inhibitory input is important for duration sensitivity in the inferior colliculus (IC), it is still unknown whether (GABA)-ergic inhibitory input plays an important role in generating duration sensitivity in the cortex. Using free-field sound stimulation and in vivo extracellular recording, we investigated duration sensitivity in primary auditory cortical (AI) neurons of the Nembutal anesthetized albino mouse (Mus musculus, Km) and examined the effect of the GABAA receptor antagonist bicuculline on AI neuron duration sensitivity. A total of 63 duration tuning curves were measured in AI neurons. Of these, 44% (28/63) exhibited duration sensitive responses, while 43% (27/63) lacked duration sensitivity. The remaining 13% (8/63) exhibited long-pass properties likely reflecting both duration sensitive and insensitive features. We found that duration sensitive neurons had shorter first spike latency (FSL) and longer firing duration (FD) when stimulated with best duration (p < 0.05), while duration insensitive neurons had invariable FSL and FD at different sound durations (p>0.05). Furthermore, 60% (6/10) of duration sensitive neurons and 75% (3/4) long-pass neurons lost duration sensitivity following bicuculline application. Taken together, our results show that cortical neurons in the albino mouse are sensitive to sound duration, and that GABAergic inhibition may play an important role in the formation of de novo duration sensitivity in AI. The possible mechanism and behavioral significance of duration sensitivity in AI neurons is discussed.

Emergence of dominant initiation sites for interictal spikes in rat neocortex.

Neuronal populations with unbalanced inhibition can generate interictal spikes (ISs), where each IS starts from a small initiation site and then spreads activation across a larger area. We used in vivo voltage-sensitive dye imaging to map the initiation site of ISs in rat visual cortex disinhibited by epidural application of bicuculline methiodide. Immediately after the application of bicuculline, the IS initiation sites were widely distributed over the entire disinhibited area. After ∼ 10 min, a small number of sites became "dominant" and initiated the majority of the ISs throughout the course of imaging. Such domination also occurred in cortical slices, which lack long-range connections between the cortex and subcortical structures. This domination of IS initiation sites may allow timing-related plasticity mechanisms to provide a spatial organization where connections projecting outward from the dominant initiation site become strengthened. Understanding the spatiotemporal organization of IS initiation sites may contribute to our understanding of epileptogenesis in its very early stages, because a dominant IS initiation site with strengthened outward connectivity may ultimately develop into a seizure focus.

Stress-induced inhibition of LH pulses in female rats: role of GABA in arcuate nucleus.

Stress exerts profound inhibitory effects on reproductive function by suppression of the pulsatile release of GnRH and therefore LH. Besides the corticotrophin-releasing factor (CRF), this effect also might be mediated via GABAergic signaling within the arcuate nucleus (ARC) since its inhibitory effects on LH pulses and increased activity during stress. In the present study, we investigated the role of endogenous GABAergic signaling within the ARC in stress-induced suppression of LH pulses. Ovariectomised oestradiol-replaced rats were implanted with bilateral and unilateral cannulae targeting toward the ARC and lateral cerebral ventricle respectively. Blood samples (25 μl) were taken via chronically implanted cardiac catheters every 5 min for 6 h for measurement of LH pulses. Intra-ARC infusion of GABAA receptor antagonist, bicuculline (0.2 pmol in 200 nl artificial cerebrospinal fluid (aCSF) each side, three times at 20-min intervals) markedly attenuated the inhibitory effect of lipopolysaccharide (LPS; 25 μg/kg i.v.) but not restraint (1 h) stress on pulsatile LH secretion. In contrast, restraint but not LPS stress-induced suppression of LH pulse frequency was reversed by intra-ARC administration of GABABR antagonist, CGP-35348 (1.5 nmol in 200 nl aCSF each side, three times at 20-min intervals). Moreover, intra-ARC application of either bicuculline or CGP-35348 attenuated the inhibitory effect of CRF (1 nmol in 4 μl aCSF, i.c.v.) on the LH pulses. These data indicate a pivotal and differential role of endogenous GABAA and GABAB signaling mechanisms in the ARC with respect to mediating immunological and psychological stress-induced suppression of the GnRH pulse generator respectively.

Mice deficient in carbonic anhydrase type 8 exhibit motor dysfunctions and abnormal calcium dynamics in the somatic region of cerebellar granule cells

The waddles (wdl) mouse is characterized by a namesake “side-to-side” waddling gait due to a homozygous mutation of the Car8 gene. This mutation results in non-functional copies of the protein carbonic anhydrase type 8. Rota-rod testing was conducted to characterize the wdl mutations’ effect on motor output. Results indicated that younger homozygotes outperformed their older cohorts, an effect not seen in previous studies. Heterozygotes, which were thought to be free of motor impairment, displayed motor learning deficiencies when compared with wild type performance. Acute cerebellar slices were then utilized for fluorescent calcium imaging experiments, which revealed significant alterations in cerebellar granule cell somatic calcium signaling when exposed to glutamate. The contribution of GABAergic signaling to these alterations was also verified using bath application of bicuculline. Changes in somatic calcium signals were found to be applicable to an in vivo scenario by comparing group responses to electrical stimulation of afferent mossy fiber projections. Finally, intracellular calcium store function was also found to be altered by the wdl mutation when slices were treated with thapsigargin. These findings, taken together with previous work on the wdl mouse, indicate a widespread disruption in cerebellar circuitry hampering proper neuronal communication.

Activity-Dependent Ubiquitination of GluA1 and GluA2 Regulates AMPA Receptor Intracellular Sorting and Degradation

SUMMARYAMPA receptors (AMPARs) have recently been shown to undergo post-translational ubiquitination in mammalian neurons. However, the underlying molecular mechanisms are poorly understood and remain controversial. Here, we report that all four AMPAR subunits (GluA1–4) are rapidly ubiquitinated upon brief application of AMPA or bicuculline in cultured neurons. This process is Ca2+ dependent and requires the activity of L-type voltage-gated Ca2+ channels and Ca2+/calmodulin-dependent kinase II. The ubiquitination of all subunits occurs exclusively on AMPARs located on the plasma membrane post-endocytosis. The sites of ubiquitination were mapped to Lys-868 in GluA1 and Lys-870/Lys-882 in GluA2 C-terminals. Mutation of these lysines did not affect basal surface expression or AMPA-induced internalization of GluA1 and GluA2 subunits. Instead, it reduced the intracellular trafficking of AMPARs to the late endosomes and thus protein degradation. These data indicate that ubiquitination is an important regulatory signal for controlling AMPAR function, which may be crucial for synaptic plasticity.

Influence of GABAergic pathway on retinal adaptation-related response changes

Retinal ganglion cells (RGCs) exhibit adaptive changes in response to sustained light stimulation, which include decrease in firing rate, tendency to shrink in receptive field (RF) size and reduction in synchronized activities. Gamma-aminobutyric acid-ergic (GABAergic) pathway is an important inhibitory pathway in retina. In the present study, the effects of GABAergic pathway on the contrast adaptation process of bullfrog RGCs were studied using multi-electrode recording technique. It was found that the application of bicuculline (BIC), a gamma-aminobutyric acid A (GABAA) receptor antagonist, caused a number of changes in the RGCs’ response characteristics, including attenuation in adaptation-dependent firing rate decrease and the adaptation-dependent weakening in synchronized activities between adjacent neuron-pairs, whereas intensified the adaptation-dependent RF size shrinkage. These results suggest that GABAA receptors are involved in the modulation of the firing activity and synchronized activities in contrast adaptation process of the RGCs, whereas the adaptation-related RF property changes involve more complicated mechanisms.

Co-activation of synaptic and extrasynaptic NMDA receptors by neuronal insults determines cell death in acute brain slice

Overactivation of NMDA receptors is linked to cell death during neuronal insults. However the precise role of synaptic and extrasynaptic NMDA receptors remains to be further determined. In this study, we used the acute brain slice to examine the contributions of synaptic and extrasynaptic NMDA receptors to neuronal death. By activation of synaptic NMDA receptors with bath application of 100μM bicuculline in acute brain slices, we observed a significant up-regulation in activation of neuronal survival-related signaling (p-CREB, p-ERK1/2 and p-AKT), without an obvious increase of LDH release and neuronal death. Interestingly, activation of extrasynaptic NMDA receptors alone by high dose of glutamate (200μM) following blockade of synaptic NMDA receptors with co-application of 20μM MK801 and 100μM bicuculline, we failed to observe inhibition of neuronal survival signaling and neuronal damage. In contrast, co-activation of synaptic and extrasynaptic NMDA receptors by applying 200μM glutamate or oxygen–glucose deprivation (OGD) to acute brain slices for 30min, we observed a significant inhibition of CREB, ERK1/2 and AKT activation, an increase of LDH release and neuronal condensation. Together, co-activation of synaptic and extrasynaptic NMDA receptors by neuronal insults contributes to cell death in acute brain slice.

Propagation of cortical spreading depression into the hippocampus: The role of the entorhinal cortex.

Propagation of cortical spreading depression (CSD) to the subcortical structures could be the underlying mechanism of some neurological deficits in migraine with aura. The entorhinal cortex (EC) as a gray matter bridge between the neocortex and subcortical regions plays an important role in this propagation. In vitro combined neocortex-hippocampus brain slices were used to study the propagation pattern of CSD between the neocortex and the hippocampus. The effects of different compounds as well as tetanic electrical stimulations in the EC on propagation of CSD to the hippocampus were investigated. Repetitive induction of CSD by KCl injection in the somatosensory cortex enhanced the probability of CSD entrance to the hippocampus via EC. Local application of AMPA receptor blocker CNQX and cannabinoid receptor agonist WIN 55212-2 in EC facilitated the propagation of CSD to the hippocampus, whereas application of NMDA receptor blocker APV and GABAA receptor blocker bicuculline in this region reduced the probability of CSD penetration to the hippocampus. Application of tetanic stimulation in EC also facilitated the propagation of CSD entrance to the hippocampus. Our data suggest the importance of synaptic plasticity of EC in filtering the propagation of CSD into subcortical structures and possibly the occurrence of concomitant neurological deficits. Synapse 68:574-584, 2014. © 2014 Wiley Periodicals, Inc.

Corticofugal projections induce long-lasting effects on somatosensory responses in the trigeminal complex of the rat

The sensory information flow at subcortical relay stations is controlled by the action of topographic connections from the neocortex. To determinate the functional properties of the somatosensory corticofugal projections to the principal (Pr5) and caudal spinal (Sp5C) trigeminal nuclei, we performed unitary recordings in anesthetized rats. To examine the effect of these cortical projections we used tactile stimulation of the whisker and electrical stimulation of somatosensory cortices. Corticofugal anatomical projections to Pr5 and Sp5C nuclei were detected by using retrograde fluorescent tracers. Neurons projecting exclusively to Pr5 were located in the cingulate cortex while neurons projecting to both Sp5C and Pr5 nuclei were located in the somatosensory and insular cortices (>75% of neurons). Physiological results indicated that primary somatosensory cortex produced a short-lasting facilitating or inhibiting effects (<5 min) of tactile responses in Pr5 nucleus through activation of NMDA glutamatergic or GABAA receptors since effects were blocked by iontophoretically application of APV and bicuculline, respectively. In contrast, stimulation of secondary somatosensory cortex did not affect most of the Pr5 neurons; however both cortices inhibited the nociceptive responses in the Sp5C nucleus through activation of glycinergic or GABAA receptors because effects were blocked by iontophoretically application of strychnine and bicuculline, respectively. These and anatomical results demonstrated that the somatosensory cortices projects to Pr5 nucleus to modulate tactile responses by excitatory and inhibitory actions, while projections to the Sp5C nucleus control nociceptive sensory transmission by only inhibitory effects. Thus, somatosensory cortices may modulate innocuous and noxious inputs simultaneously, contributing to the perception of specifically tactile or painful sensations.

Tonic GABAergic inhibition of RVLM neurons: a novel target for sympathetic control (1172.3)

The rostral ventrolateral medulla (RVLM) is a critical component of the sympathetic nervous system regulating homeostatic functions including arterial blood pressure (ABP). GABAA receptors mediate the majority of inhibition in the brain and GABAergic inhibition of preautonomic neurons in the RVLM is well accepted. Decreased inhibition of RVLM neurons increases sympathetic nervous system activity and thereby elevates ABP. Despite our increasing understanding of the functional significance of GABAA receptors in the regulation of neuronal excitability, the mechanisms underlining synaptic and extrasynaptic control of preautonomic neurons in the RVLM is unknown. Using the transsynaptic retrograde viral tracer, PRV‐152, we identified preautonomic neurons in the RVLM. We found that RVLM neurons displayed persistent, tonic current to GABA, via GABAA receptors, in the absence of large‐scale phasic neurotransmission using whole‐cell patch‐clamp recordings. Application of the GABAA receptor blocker bicuculline (30 µM), revealed a robust somatic tonic inhibitory current mediated by extrasynaptic GABAA receptors with an average amplitude of 196.3 ± 39.3 pA. Our data suggest that extrasynaptic tonic current mediated by GABAA receptors plays a major role in determining the resting membrane potential and firing activity of preautonomic neurons in the RVLM.Grant Funding Source: NIH P30GM103337

Adaptive changes in the motor cortex during and after longterm forelimb immobilization in adult rats

Experimental and clinical studies have attempted to evaluate the changes in cortical activity seen after immobilization-induced longterm sensorimotor restriction, although results remain controversial. We used intracortical microstimulation (ICMS), which provides topographic movement representations of the motor areas in both hemispheres with optimal spatial characterization, combined with behavioural testing to unravel the effects of limb immobilization on movement representations in the rat primary motor cortex (M1). Unilateral forelimb immobilization in rats was achieved by casting the entire limb and leaving the cast in place for 15 or 30 days. Changes in M1 were bilateral and specific for the forelimb area, but were stronger in the contralateral-to-cast hemisphere. The threshold current required to evoke forelimb movement increased progressively over the period in cast, whereas the forelimb area size decreased and the non-excitable area size increased. Casting resulted in a redistribution of proximal/distal movement representations: proximal forelimb representation increased, whereas distal representation decreased in size. ICMS after cast removal showed a reversal of changes, which remained partial at 15 days. Local application of the GABAA-antagonist bicuculline revealed the impairment of cortical synaptic connectivity in the forelimb area during the period of cast and for up to 15 days after cast removal. Six days of rehabilitation using a rotarod performance protocol after cast removal did not advance map size normalization in the contralateral-to-cast M1 and enabled the cortical output towards the distal forelimb only in sites that had maintained their excitability. These results are relevant to our understanding of adult M1 plasticity during and after sensorimotor deprivation, and to new approaches to conditions that require longterm limb immobilization.