Bicuculline Methiodide Research Articles

Acquisition of Contextual Discrimination Involves the Appearance of a RAS-GRF1/p38 Mitogen-activated Protein (MAP) Kinase-mediated Signaling Pathway That Promotes Long Term Potentiation (LTP)

RAS-GRF1 is a guanine nucleotide exchange factor with the ability to activate RAS and RAC GTPases in response to elevated calcium levels. We previously showed that beginning at 1 month of age, RAS-GRF1 mediates NMDA-type glutamate receptor (NMDAR)-induction of long term depression in the CA1 region of the hippocampus of mice. Here we show that beginning at 2 months of age, when mice first acquire the ability to discriminate between closely related contexts, RAS-GRF1 begins to contribute to the induction of long term potentiation (LTP) in the CA1 hippocampus by mediating the action of calcium-permeable, AMPA-type glutamate receptors (CP-AMPARs). Surprisingly, LTP induction by CP-AMPARs through RAS-GRF1 occurs via activation of p38 MAP kinase rather than ERK MAP kinase, which has more frequently been linked to LTP. Moreover, contextual discrimination is blocked by knockdown of Ras-Grf1 expression specifically in the CA1 hippocampus, infusion of a p38 MAP kinase inhibitor into the CA1 hippocampus, or the injection of an inhibitor of CP-AMPARs. These findings implicate the CA1 hippocampus in the developmentally dependent capacity to distinguish closely related contexts through the appearance of a novel LTP-supporting signaling pathway.

Intrathecal Apelin-13 Produced Different Actions in Formalin Test and Tail-flick Test in Mice

Apelin was identified as natural ligand for APJ, a G protein-coupled receptor. APJ is expressed in spinal cord and dorsal root ganglion. This study was designed to investigate the effects and mechanisms of intrathecally (i.t.) administered apelin-13 on nociceptive response in formalin test and tail-flick test. In formalin test, i.t. injection of apelin-13 (0.3-3 nmol/mouse) had no effect on the nociceptive response in either acute phase (0-10 min) or interphase (10-20 min), but significantly produced hyperalgesic effect in late phase (20-30 min) at the dose of 3 nmol/mouse. The APJ receptor antagonist apelin-13(F13A) and GABAA receptor antagonist bicuculline methiodide, but not opioid receptor antagonist naloxone, significantly blocked the hyperalgesia caused by apelin-13 in late phase, indicating that i.t. apelin-13- induced hyperalgesia was mediated by APJ and GABAA receptor, rather than opioid receptor. However, in tail-flick test, i.t. injected apelin-13 (1 and 3 nmol/mouse) induced a significant antinociceptive effect, which was significantly antagonized by apelin-13(F13A) and naloxone, suggesting APJ and opioid receptor were involved in the antinociception of spinal apelin-13.

Posterior hypothalamic modulation of light-evoked trigeminal neural activity and lacrimation

Enhanced light sensitivity is a common feature of many neuro-ophthalmic conditions and some chronic headaches. Previously we reported that the bright light-evoked increases in trigeminal brainstem neural activity and lacrimation depended on a neurovascular link within the eye (Okamoto et al., 2012). However, the supraspinal pathways necessary for these light-evoked responses are not well defined. To assess the contribution of the posterior hypothalamic area (PH), a brain region closely associated with control of autonomic outflow, we injected bicuculline methiodide (BMI), a GABAa receptor antagonist, into the PH and determined its effect on the encoding properties of ocular neurons at the ventrolateral trigeminal interpolaris/caudalis transition (Vi/Vc) and caudalis/upper cervical cord junction (Vc/C1) regions and on reflex lacrimation in male rats under isoflurane anesthesia. BMI markedly reduced light-evoked (>80%) responses of Vi/Vc and Vc/C1 neurons at 10min with partial recovery by 50min after injection. BMI also reduced (>35%) the convergent cutaneous receptive field area of Vi/Vc and Vc/C1 ocular neurons indicating that both intra-ocular and periorbital cutaneous inputs were affected by changes in PH outflow. Light-evoked lacrimation was reduced by >35% at 10min after BMI, while resting mean arterial pressure increased promptly and remained elevated (>20mmHg) throughout the 50-min post-injection period. These results suggested that PH stimulation, acting in part through increased sympathetic activity, significantly inhibited light- and facial skin-evoked activity of ocular neurons at the Vi/Vc and Vc/C1 region. These data provide further support for the hypothesis that autonomic outflow plays a critical role in mediating light-evoked trigeminal brainstem neural activity and reflex lacrimation.

GABAergic transmission facilitates ictogenesis and synchrony between CA3, hilus, and dentate gyrus in slices from epileptic rats

The impact of regional hippocampal interactions and GABAergic transmission on ictogenesis remain unclear. Cortico-hippocampal slices from pilocarpine-treated epileptic rats were compared with controls to investigate associations between seizurelike events (SLE), GABAergic transmission, and neuronal synchrony within and between cortico-hippocampal regions. Multielectrode array recordings revealed more prevalent hippocampal SLE in epileptic tissue when excitatory transmission was enhanced and GABAergic transmission was intact [removal of Mg(2+) (0Mg)] than when GABAergic transmission was blocked [removal of Mg(2+) + bicuculline methiodide (0Mg+BMI)]. When activity within individual regions was analyzed, spectral and temporal slow oscillation/SLE correlations and cross-correlations were highest within the hilus of epileptic tissue during SLE but were similar in 0Mg and 0Mg+BMI. GABAergic facilitation of spectral "slow" oscillation and ripple correlations was most prominent within CA3 of epileptic tissue during SLE. When activity between regions was analyzed, slow oscillation and ripple coherence was highest between the hilus and dentate gyrus as well as between the hilus and CA3 of epileptic tissue during SLE and was significantly higher in 0Mg than 0Mg+BMI. High 0Mg-induced SLE cross-correlations between the hilus and dentate gyrus as well as between the hilus and CA3 were reduced or abolished in 0Mg+BMI. SLE cross-correlation lag measurements provided evidence for a monosynaptic connection from the hilus to the dentate gyrus during SLE. Findings implicate the hilus as an oscillation generator, whose impact on other cortico-hippocampal regions is mediated by GABAergic transmission. Data also suggest that GABAA receptor-mediated transmission facilitates back-propagation from CA3/hilus to the dentate gyrus and that this back-propagation augments SLE in epileptic hippocampus.

Intracerebroventricular injection of liposome‐entrapped GABA attenuates the renal sympathetic nerve activity response evoked by central administration of bicuculline in spontaneously hypertensive rats

Liposomes are lipid vesicles that allow a sustained release of entrapped substances. GABA is the most prevalent inhibitory neurotransmitter in the central nervous system. In this study we tested the efficacy of liposome‐entrapped GABA (GL) in attenuating the cardiovascular response evoked by intracerebroventricular (icv) administration of GABAA antagonist bicuculline methiodide (BMI). Liposomes were prepared by the freeze‐thawing method followed by extrusion (200‐nm final vesicle diameter). Under tribromoethanol anesthesia (250 mg/Kg i.p.), guide cannula was implanted into the lateral ventricle of spontaneously hypertensive rats (SHR). In two groups of animals (n=5–6), GL or empty liposomes (EL, 0.9% NaCl) were injected icv 48 hours before the GABAA antagonist (BMI, 0.5 nmol/2μL). The icv injection of BMI increased the renal sympathetic nerve activity in EL group, this effect was greatly attenuated in GL group (22 ± 6 vs. 0.8 ± 6 %, P = 0.032). Conversely, BMI injection did not change HR and MAP in both groups. Current data extends our previous findings suggesting that liposome‐entrapped GABA is a promising tool for long‐term intervention in central nervous system. Support: INCT NanoBiofar/CNPq/FAPEMIG

Perinatal high fat diet dysregulates GABAergic signaling to vagal efferent motoneurons regulating gastric motility

Parasympathetic innervation to the stomach is supplied by dorsal motor nucleus of the vagus (DMV) neurons. Studies have demonstrated that ongoing GABAergic inputs to DMV neurons play a major role in regulating vagal efferent output. The aim of this study was to investigate whether perinatal HFD affects the GABAergic regulation of vagal efferent output to the stomach. Male Sprague Dawley rats were fed a HFD (60%kcal from fat) from E13 until experimentation at 12 weeks of age. Miniaturized strain gauges sutured to the ventral surface of the gastric corpus were used to record gastric motility. The GABAA receptor antagonist, bicuculline methiodide (BMI; 60nl) was microinjected into the dorsal vagal complex and the resulting increase in gastric motility was measured at 3 time points after injection (1.5, 4 and 6.5min) and averaged. Responses were expressed relative to the maximal increase in motility in response to i.v. injection of the non‐selective muscarinic agonist, bethanecol (353pmol; 1ml), which itself was unaffected by diet (9.08±1.20g in control vs 9.27±1.03g in HFD; P>;0.05).In control rats, BMI induced a dose‐dependent increase in gastric motility (3.1±0.6%, 4.8±1.1% and 5.7±1.7% at 25, 50 and 100pmol, n=8,8,4, respectively, P<0.05 for each). In contrast, following HFD, the BMI‐induced increase in gastric motility was significantly attenuated (0.004±0.0001%, 1.2±0.3% and 1.25±0.3% at 25, 50 and 100pmol, n= 4,5,4, respectively, P<0.05 vs control). These data suggest that exposure to a HFD during the perinatal period alters GABAergic activity within brainstem neural circuits controlling gastric functions, which may contribute to the dysregulation of homeostatic reflexes, including appetite and metabolic regulation.Supported by NSF IOS 1148978

Activation of both phasic and tonic GABAA¬ receptors modulates vagal efferent outflow regulating gastric motor functions

The dorsal motor nucleus of the vagus (DMV) provides the parasympathetic innervation to the stomach. DMV neurons display both synaptic and extrasynaptic GABAA receptors that are activated by phasic and tonic GABA release, respectively. Studies have demonstrated that ongoing activation of GABAA receptors is critical in determining vagal efferent outflow to the stomach. The present study was designed to investigate whether phasic and/or tonic GABAA receptors are involved in the regulation of gastric tone.Miniaturized strain gauges were sutured to the ventral surface of the gastric corpus in male Sprague‐Dawley rats (175–300g). The GABAA receptor antagonists, gabazine (0.78–6.25pmol) and/or bicuculline methiodide (BMI; 25–100pmol), were microinjected into the dorsal vagal complex and the increase in gastric tone expressed relative to the maximal increase induced following i.v. application of the non‐selective muscarinic agonist, bethanecol (352pmol; 1ml). At low doses, gabazine acts selectively on extrasynaptic receptors, whereas BMI acts on both synaptic and extrasynaptic receptors.Gabazine increased gastric tone 5.1±1.7% at 0.78pmol, n=10, increasing to 15.3±1.74% and 19.1±5.94% in response to 3.125 and 6.25pmol, n = 5,6, respectively (P<0.05 vs 0.78pmol for each). At lower doses, BMI induced similar increases in gastric tone (11.0±4.7% and 14.9±5.5% at 25 and 50pmol, n = 8,8, respectively) whereas the response was significantly greater at 100pmol (35.7±17.8%, n=4, P<0.05). These data suggest that the activity of DMV neurons, hence vagal efferent outflow to the stomach, is regulated by both synaptic and extrasynaptic GABAA receptors implying a more complex synaptic modulation than previously considered.Supported by NSF IOS 11489364

Iberiotoxin-sensitive and -insensitive BK currents in Purkinje neuron somata

Purkinje cells have specialized intrinsic ionic conductances that generate high-frequency action potentials. Disruptions of their Ca or Ca-activated K (KCa) currents correlate with altered firing patterns in vitro and impaired motor behavior in vivo. To examine the properties of somatic KCa currents, we recorded voltage-clamped KCa currents in Purkinje cell bodies isolated from postnatal day 17-21 mouse cerebellum. Currents were evoked by endogenous Ca influx with approximately physiological Ca buffering. Purkinje somata expressed voltage-activated, Cd-sensitive KCa currents with iberiotoxin (IBTX)-sensitive (>100 nS) and IBTX-insensitive (>75 nS) components. IBTX-sensitive currents activated and partially inactivated within milliseconds. Rapid, incomplete macroscopic inactivation was also evident during 50- or 100-Hz trains of 1-ms depolarizations. In contrast, IBTX-insensitive currents activated more slowly and did not inactivate. These currents were insensitive to the small- and intermediate-conductance KCa channel blockers apamin, scyllatoxin, UCL1684, bicuculline methiodide, and TRAM-34, but were largely blocked by 1 mM tetraethylammonium. The underlying channels had single-channel conductances of ∼150 pS, suggesting that the currents are carried by IBTX-resistant (β4-containing) large-conductance KCa (BK) channels. IBTX-insensitive currents were nevertheless increased by small-conductance KCa channel agonists EBIO, chlorzoxazone, and CyPPA. During trains of brief depolarizations, IBTX-insensitive currents flowed during interstep intervals, and the accumulation of interstep outward current was enhanced by EBIO. In current clamp, EBIO slowed spiking, especially during depolarizing current injections. The two components of BK current in Purkinje somata likely contribute differently to spike repolarization and firing rate. Moreover, augmentation of BK current may partially underlie the action of EBIO and chlorzoxazone to alleviate disrupted Purkinje cell firing associated with genetic ataxias.

Asymmetry in the control of cardiac performance by dorsomedial hypothalamus

Dorsomedial hypothalamus (DMH) plays a key role in integrating cardiovascular responses to stress. We have recently reported greater heart rate responses following disinhibition of the right side of the DMH (R-DMH) in anesthetized rats and greater suppression of stress-induced tachycardia following inhibition of the R-DMH in conscious rats [both compared with similar intervention in the left DMH (L-DMH)], suggesting existence of right/left side asymmetry in controlling cardiac chronotropic responses by the DMH. The aim of the present study was to determine whether similar asymmetry is present for controlling cardiac contractility. In anesthetized rats, microinjections of the GABAA antagonist bicuculline methiodide (BMI; 40 pmol/100 nl) into the DMH-evoked increases in heart rate (HR), left ventricular pressure (LVP), myocardial contractility (LVdP/dt), arterial pressure, and respiratory rate. DMH disinhibition also precipitated multiple ventricular and supraventricular ectopic beats. DMH-induced increases in HR, LVP, LVdP/dt, and in the number of ectopic beats dependent on the side of stimulation, with R-DMH provoking larger responses. In contrast, pressor and respiratory responses did not depend on the side of stimulation. Newly described DMH-induced inotropic responses were rate-, preload- and (largely) afterload-independent; they were mediated by sympathetic cardiac pathway, as revealed by their sensitivity to β-adrenergic blockade. We conclude that recruitment of DMH neurons causes sympathetically mediated positive chronotropic and inotropic effects, and that there is an asymmetry, at the level of the DMH, in the potency to elicit these effects, with R-DMH > L-DMH.

Role of GABAergic inhibition in shaping the spatial frequency tuning of neurons and its contrast dependency in the dorsal lateral geniculate nucleus of cat

To understand how GABAergic inhibition contributes to the elaboration of spatial frequency (SF) tuning of neurons in the lateral geniculate nucleus, we examined the effects of the microiontophoretic administration of bicuculline methiodide (BIC) and gabazine, both antagonists of GABAA receptors, on visual responses to grating stimuli with high or low contrasts. BIC administration changed the shape of the SF tuning curve of the spike response from band-pass to low-pass. We took the tuning curve obtained under the BIC condition as an estimated excitatory contribution to the control tuning curve and then estimated the difference between tuning curves recorded with and without BIC as the tuning curve of the estimated GABAergic inhibitory contribution. The SF tuning profile of estimated inhibition (Estimated-Inh) varied widely from cell to cell, as did estimated excitation (Estimated-Ex). Nonetheless, the relationship that Estimated-Inh exhibited more low-pass tuning than did Estimated-Ex was well conserved in the majority of cells, and the relationship refined the SF tuning of Estimated-Ex toward the band-pass tuning of the geniculate output. Lowering the stimulus contrast decreased the response magnitude, but did not change the degree of band-pass tuning. The GABAergic refinement of the SF tuning was also observed at low stimulus contrast, but was weaker than at high contrast, suggesting that GABAergic inhibition is regulated in coordination with excitatory inputs to keep the degree of the band-pass tuning constant. We therefore concluded that the degree of band-pass tuning is conserved contrast invariantly in the lateral geniculate nucleus on the basis of the dynamic regulatory action of GABAergic inhibition.

Defense-Like Behaviors Evoked by Pharmacological Disinhibition of the Superior Colliculus in the Primate

Stimulation of the intermediate and deep layers of superior colliculus (DLSC) in rodents evokes both orienting/pursuit (approach) and avoidance/flight (defense) responses (Dean et al., 1989). These two classes of response are subserved by distinct output projections associated with lateral (approach) and medial (defense) DLSC (Comoli et al., 2012). In non-human primates, DLSC has been examined only with respect to orienting/approach behaviors, especially eye movements, and defense-like behaviors have not been reported. Here we examined the profile of behavioral responses evoked by activation of DLSC by unilateral intracerebral infusions of the GABA(A) receptor antagonist, bicuculline methiodide (BIC), in nine freely moving macaques. Across animals, the most consistently evoked behavior was cowering (all animals), followed by increased vocalization and escape-like behaviors (seven animals), and attack of objects (three animals). The effects of BIC were dose-dependent within the range 2.5-14 nmol (threshold dose of 4.6 nmol). The behaviors and their latencies to onset did not vary across different infusion sites within DLSC. Cowering and escape-like behaviors resembled the defense-like responses reported after DLSC stimulation in rats, but in the macaques these responses were evoked from both medial and lateral sites within DLSC. Our findings are unexpected in the context of an earlier theoretical perspective (Dean et al., 1989) that emphasized a preferential role of the primate DLSC for approach rather than defensive responses. Our data provide the first evidence for induction of defense-like behaviors by activation of DLSC in monkeys, suggesting that the role of DLSC in responding to threats is conserved across species.

Blunted endogenous GABA-mediated inhibition in the hypothalamic paraventricular nucleus of rats with streptozotocin-induced diabetes

The hypothalamic paraventricular nucleus (PVN) is involved in the regulation of sympathetic outflow and particularly affects the heart. This study sets out to determine the role of GABA of the paraventricular nucleus (PVN) in cardiovascular regulation in streptozotocin-induced diabetic rats. Pharmacological stimulation of glutamatergic receptors with DL-Homocysteic acid (200 mM in 100 nL) in the PVN region showed a significant depression in both mean arterial pressure (MAP) and heart rate (HR) of diabetic rats (Diabetic vs. non-diabetic: MAP 15.0 ± 1.5 vs. 35.8 ± 2.8 mmHg; HR 3.0 ± 2.0 vs. 30.0 ± 6.0 bpm, P < 0.05). Microinjection of bicuculline methiodide (1 mM in 100 nL), a GABAA receptor antagonist, produced an increase in baseline MAP and HR in both non-diabetic and diabetic rats. In the diabetic rats, bicuculline injection into the PVN reduced the pressor and HR responses (Diabetic vs. non-diabetic: MAP 6.2 ± 0.8 vs. 25.1 ± 2.2 mmHg; HR 1.8 ± 1.1 vs. 25.4 ± 6.2 bpm, P < 0.05). A microinjection of muscimol (2 mM in 100 nL), which is a GABAA receptor agonist, in the PVN elicited decreases in MAP and HR in both groups. The diabetic group showed a significantly blunted reduction in HR, but not MAP (Diabetic vs. non-diabetic: MAP -15.7 ± 4.0 vs. -25.0 ± 3.8 mmHg; HR -5.2 ± 2.1 vs. -39.1 ± 7.9 bpm). The blunted vasopressor and tachycardic responses to bicuculline microinjection in the diabetic rats are likely to result from decreased GABAergic inputs, attenuated release of endogenous GABA or alterations in GABAA receptors within the PVN.

Significance of horizontal propagation of synchronized activities in human epileptic neocortex investigated by optical imaging and immunohistological study

To characterize the physiological condition of human epileptic neocortex, we employed flavoprotein fluorescence imaging (FFI), an optical imaging method which detects intrinsic signals accompanying neural activation, and immunohistologically studied human cortical specimens. The experimented materials were cortical tissues surrounding various intracerebral lesions obtained from 5 patients with epilepsy (epileptic patients: EPs) and 5 without epilepsy (non-epileptic patients: NEPs). These tissues were immersed in oxygenated artificial cerebrospinal fluid immediately after removal in the operating room. Signal changes of FFI in the cortical layers subjected to electrical stimulation were observed under bicuculline methiodide perfusion. Immunohistological staining for parvalbumin (PV), calbindin, and calretinin were performed on the same specimens to evaluate expressions of calcium-binding protein positive cells. The FFI study showed the characteristic cortical propagation pattern of elicited activities horizontally along the cortical layers in EPs but not in NEPs. The propagated area with more than 0.5% signal changes was significantly larger in EPs than in NEPs (p=0.008). Only the expression of PV positive neurons was significantly lower in EPs than in NEPs (p=0.006). The propagated area on FFI and the decrease in PV positive neurons correlated significantly (R=-0.78, p=0.04). The present study visualized the unique horizontal propagation of signal changes on FFI and demonstrated a correlation of this propagation with immunohistological decreases in PV positive neurons in human epileptic cortex. Further investigations may elucidate the mechanism of hyper-excitability and hyper-synchronization in epileptic cortical tissue itself.

Abstract 412: Electrophysiological Properties of Kidney-related Neurons in the Rostral Ventrolateral Medulla

Presympathetic neurons in the rostral ventrolateral medulla (RVLM) play an important integrative role in the neuronal network mediating cardiovascular regulation. In this study, we employed a retrograde, transsynaptic pseudorabies viral label (PRV-152), a PRV construct which expresses enhanced green fluorescent protein (EGFP), to identify kidney-related neurons in the RVLM. Whole-cell, patch-clamp recordings were made from 86 kidney-related neurons in the RVLM in transverse brainstem slices from 4-7-week-old rats. Under control conditions, we identified two populations of PRV-labeled RVLM neurons based on their electrophysiological properties. The resting membrane potential of large kidney-related neurons was -48 ± 2 mV and the input resistance was 205 ± 28 MΩ. The resting membrane potential of small kidney-related neurons was -51 ± 2 mV and the input resistance was 430 ± 36 MΩ. Spontaneous firing was observed in 2 out of 15 large cells with frequency of firing 0.008 and 0.05 Hz and 7 out of 16 small cells with average of firing 0.4 ± 0.2 Hz. Both spontaneous and miniature inhibitory and excitatory postsynaptic currents (i.e., sEPSCs, sIPSCs and mEPSCs, mIPSCs) were observed in small and large neurons. Tonic inhibitory and excitatory currents were identified in large kidney-related neurons of the RVLM. Application of the type A γ-aminobutyric acid (GABA A ) receptor-linked Cl - channel blocker, bicuculline methiodide (30 μM), blocked sIPSCs and revealed a robust tonic inhibitory current with an average amplitude of 145.3 ± 30 pA. The cocktail of potent N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA)/kainate ionotropic glutamate receptor antagonist, AP-5 (50 μM) and CNQX (10 μM), blocked sEPSCs and revealed a two-component tonic excitatory current mediated by NMDA and AMPA/kainate ionotropic glutamate receptors with overall amplitude of 65.2 ± 11.3 pA. These data demonstrate the synaptic complexity involved in the regulation of kidney-related RVLM neurons.

An Antagonist of GABA-B Receptors Potentiates Activity of Cortical Epileptic Foci

Cortical epileptic foci elicited by local application of bicuculline methiodide represent a model of interictal epileptic activity with a transition into ictal phases. We studied a role of GABA-B receptors in this model using GABA-B receptor antagonist CGP35348 in adult rats with implanted cortical electrodes and cannula. CGP35348 (100 or 200 mg/kg i.p.) did not affect interictal discharges but it augmented ictal activity. Latency to the first ictal episode was decreased by the lower dose of CGP35348, duration of episodes was increased by the higher dose. GABA-B receptor antagonist did not influence purely cortical epileptic phenomenon but it is proconvulsant in ictal activity generated with participation of subcortical structures.

A new signalling pathway for parallel fibre presynaptic type 4 metabotropic glutamate receptors (mGluR4) in the rat cerebellar cortex

In the rodent cerebellum, pharmacological activation of mGluR4 acutely depresses excitatory synaptic transmission at parallel fibre–Purkinje cell synapses. This depression involves the inhibition of presynaptic calcium (Ca2+) influx that ultimately controls glutamate release. In this study, we investigate the molecular basis of mGluR4-mediated inhibition of presynaptic Ca2+ transients. Our results demonstrate that the mGluR4 effect does not depend on selective inhibition of a specific type of presynaptic voltage-gated Ca2+ channel, but rather involves modulation of all classes of Ca2+ channels present in the presynaptic terminals. In addition, this inhibitory effect does not involve the activation of G protein-activated inwardly rectifying potassium channels, TEA-sensitive potassium channels or two-pore-domain potassium channels. Furthermore, this inhibition does not require pertussis toxin-sensitive G proteins, and is independent of any effect on adenylyl cyclases, protein kinase A, mitogen-activated protein kinases or phosphoinositol-3 kinase activity. Interestingly we found that mGluR4 inhibition of presynaptic Ca2+ influx employs a newly defined signalling pathway, notably that involving the activation of phospholipase C and ultimately protein kinase C.

Comparison of the cardiovascular effects produced by interference with GABA transmission in the left and right sides of dorsomedial hypothalamus in conscious rats

In anesthetized rats activation of neurons in the right DMH (RDMH) evoked by bicuculline methiodide (BMI) caused greater tachycardia when compared to left DMH (LDMH) [Xavier et al, Neuroscience 2009]. In this study, we evaluated whether the same results can be obtained in awake animals. Experiments were approved (Protocol 160/2011) and performed using male Wistar rats (290–330g) submitted to implantation of unilateral guide cannula targeted to DMH. Five days after, the femoral artery was cannulated for recording of cardiovascular parameters. After one day to recovery, these animals were submitted to unilateral nanoinjection of BMI (10pmol/100nl) into RDMH (n=7) or LDMH (n=7). In group 1, BMI was injected into RDMH and in group 2, BMI was injected into LDMH. In both groups, injection of BMI into the DMH produced increases in HR (Δ RDMH: 141 ± 15 bpm and LDMH: 125 ± 15 bpm) accompanied by changes in MAP (Δ RDMH: 18 ± 2 mmhg and LDMH: 16 ± 2 mmHg). No significant difference between groups was found. However, when we compared the time course of the tachycardia evoked by BMI, we observed that the duration of effect evoked by RDMH activation lasted almost twice as long as evoked by the LDMH (RDMH: 38 ± 3 min and LDMH: 20 ± 3 min; P=0.0033). These results corroborate our previous data suggesting that the RDMH exert a prominent control on chronotropism. Support: CAPES, FAPEMIG, CNPq.

Intracerebroventricular injection of liposome‐entrapped GABA attenuates the renal sympathetic nerve activity response evoked by central administration of bicuculline in anesthetized rats

Liposomes are lipid vesicles that allow a sustained release of entrapped substances. GABA is the most prevalent inhibitory neurotransmitter of the central nervous system. In this study we tested the efficacy of liposome‐entrapped GABA (GL) in attenuating the cardiovascular response evoked by intracerebroventricular (icv) administration of GABAA antagonist bicuculline methiodide (BMI). Liposomes were prepared by the freeze‐thawing method followed by extrusion (200‐nm final vesicle diameter). Under tribromoethanol anesthesia (250 mg/Kg i.p.), guide cannula was implanted into the lateral ventricle of Wistar rats. In two groups of animals (n=6–8), GL or empty liposomes (EL, 0.9% NaCl) were injected icv 48 hours before the GABAA antagonist (BMI, 0.5 nmol/2μL). The icv injection of BMI produced similar increases in HR in both groups (EL: 49 ± 15 and GL: 30 ± 10 bpm). Minor changes in blood pressure were observed. However, the evoked increase in renal sympathetic nerve activity showed in EL group was greatly attenuated in GL group (43 ± 8 vs. 12 ± 9 %, P=0.034). Current data extends our previous findings suggesting that liposome‐entrapped GABA is a promising tool for long‐term intervention in central nervous system. Support: CNPq/FAPEMIG.

The Prefrontal Cortex and Oculomotor Delayed Response: A Reconsideration of the “Mnemonic Scotoma”

The concept of the "mnemonic scotoma," a spatially circumscribed region of working memory impairment produced by unilateral lesions of the PFC, is central to the view that PFC is critical for the short-term retention of information. Presented here, however, are previously unpublished data that offer an alternative, nonmnemonic interpretation of this pattern of deficit. In their study, Wajima and Sawaguchi [Wajima, K., & Sawaguchi, T. The role of GABAergic inhibiton in suppressing perseverative responses in the monkey prefrontal cortex. Neuroscience Research, 50(Suppl. 1), P3-P317, 2004] applied the GABA(A) antagonist bicuculline methiodide unilaterally to the PFC of two monkeys while they performed an oculomotor delayed-response task. Consistent with previous studies, errors for the initial memory-guided saccade were markedly higher when the cued location fell into the region of the visual field affected by the infusion. These erroneous saccades tended to select an alternative target location (out of a possible 16) that had not been cued on that trial. By extending the analysis window, however, it was observed that the second, "corrective" saccade often acquired the location that had been cued on that trial. Further analysis of the erroneous initial saccades indicated that they tended to be directed to a location that had been relevant on the previous trial. Thus, the deficit was not one of "forgetting" the cued location. Rather, it was one of selecting between currently and previously relevant locations. These findings suggest a need for a reconsideration of the concept of the mnemonic scotoma, which in turn invites a reconsideration of functional interpretations of sustained neuronal activity in PFC.

Role of medullary GABA signal transduction on parasympathetic reflex vasodilatation in the lower lip

In the orofacial area, noxious stimulation of the orofacial structure in the trigeminal region evokes parasympathetic reflex vasodilatation, which occurs via the trigeminal spinal nucleus (Vsp) and the inferior/superior salivatory nucleus (ISN/SSN). However, the neurotransmitter involved in the inhibitory synaptic inputs within these nuclei has never been described. This parasympathetic reflex vasodilatation is suppressed by GABAergic action of volatile anesthetics, such as isoflurane, sevoflurane, and halothane, suggesting that medullary GABAergic mechanism exerts its inhibitory effect on the parasympathetic reflex via an activation of GABA receptors. The aim of the present study was to determine the role of GABAA and GABAB receptors in the Vsp and the ISN in regulating the lingual nerve (LN)-evoked parasympathetic reflex vasodilatation in the lower lip. Under urethane anesthesia (1g/kg), change in lower lip blood flow elicited by electrical stimulation of the LN was recorded in cervically vago-sympathectomized rats. Microinjection of GABA (10μM; 0.3μl/site) into the Vsp or the ISN significantly and reversibly attenuated the LN-evoked parasympathetic reflex vasodilatation. Microinjection of the GABAA receptor-selective agonist muscimol (100μM; 0.3μl/site) or the GABAB receptor-selective agonist baclofen (100μM; 0.3μl/site) into the Vsp or the ISN significantly and irreversibly reduced this reflex vasodilatation, and these effects were attenuated by pretreatment with microinjection of each receptor-selective antagonists [GABAA receptor selective antagonist bicuculline methiodide (1mM; 0.3μl/site) or GABAB receptor selective antagonist CGP-35348 (1mM; 0.3μl/site)] into the Vsp or the ISN. Microinjection of these antagonists alone into the Vsp or the ISN had no significant effect on this reflex vasodilatation. In addition, microinjection (0.3μl/site) of the mixture of muscimol (100μM) and baclofen (100μM) into the Vsp or the ISN also significantly reduced this reflex vasodilatation. These results suggest that medullary GABA signal transduction inhibits the parasympathetic reflex vasodilatation in the rat lower lip via GABAA and GABAB receptors in the Vsp and the ISN.