Stimulation-induced Responses Research Articles

Contextual fear memory impairment in Angelman syndrome model mice is associated with altered transcriptional responses

Angelman syndrome (AS) is a rare neurogenetic disorder caused by UBE3A deficiency and characterized by severe developmental delay, cognitive impairment, and motor dysfunction. In the present study, we performed RNA-seq on hippocampal samples from both wildtype (WT) and AS male mice, with or without contextual fear memory recall. There were 281 recall-associated differentially expressed genes (DEGs) in WT mice and 268 DEGs in AS mice, with 129 shared by the two genotypes. Gene ontology analysis showed that extracellular matrix and stimulation-induced response genes were prominently enriched in recall-associated DEGs in WT mice, while nuclear acid metabolism and tissue development genes were highly enriched in those from AS mice. Further analyses showed that the 129 shared DEGs belonged to nuclear acid metabolism and tissue development genes. Unique recall DEGs in WT mice were enriched in biological processes critical for synaptic plasticity and learning and memory, including the extracellular matrix network clustered around fibronectin 1 and collagens. In contrast, AS-specific DEGs were not enriched in any known pathways. These results suggest that memory recall in AS mice, while altering the transcriptome, fails to recruit memory-associated transcriptional programs, which could be responsible for the memory impairment in AS mice.

Disrupting nociceptive information processing flow through transcranial focused ultrasound neuromodulation of thalamic nuclei

BackgroundMRI-guided transcranial focused ultrasound (MRgFUS) as a next-generation neuromodulation tool can precisely target and stimulate deep brain regions with high spatial selectivity. Combined with MR-ARFI (acoustic radiation force imaging) and using fMRI BOLD signal as functional readouts, our previous studies have shown that low-intensity FUS can excite or suppress neural activity in the somatosensory cortex. ObjectiveTo investigate whether low-intensity FUS can suppress nociceptive heat stimulation-induced responses in thalamic nuclei during hand stimulation, and to determine how this suppression influences the information processing flow within nociception networks. FindingsBOLD fMRI activations evoked by 47.5 °C heat stimulation of hand were detected in 24 cortical regions, which belong to sensory, affective, and cognitive nociceptive networks. Concurrent delivery of low-intensity FUS pulses (650 kHz, 550 kPa) to the predefined heat nociceptive stimulus-responsive thalamic centromedial_parafascicular (CM_para), mediodorsal (MD), ventral_lateral (VL_ and ventral_lateral_posteroventral (VLpv) nuclei suppressed their heat responses. Off-target cortical areas exhibited reduced, enhanced, or no significant fMRI signal changes, depending on the specific areas. Differentiable thalamocortical information flow during the processing of nociceptive heat input was observed, as indicated by the time to reach 10% or 30% of the heat-evoked BOLD signal peak. Suppression of thalamic heat responses significantly altered nociceptive processing flow and direction between the thalamus and cortical areas. Modulation of contralateral versus ipsilateral areas by unilateral thalamic activity differed. Signals detected in high-order cortical areas, such as dorsal frontal (DFC) and ventrolateral prefrontal (vlPFC) cortices, exhibited faster response latencies than sensory areas. ConclusionsThe concurrent delivery of FUS suppressed nociceptive heat response in thalamic nuclei and disrupted the nociceptive network. This study offers new insights into the causal functional connections within the thalamocortical networks and demonstrates the modulatory effects of low-intensity FUS on nociceptive information processing.

Parabrachial nucleus circuit governs neuropathic pain-like behavior

The lateral parabrachial nucleus (LPBN) is known to relay noxious information to the amygdala for processing affective responses. However, it is unclear whether the LPBN actively processes neuropathic pain characterized by persistent hyperalgesia with aversive emotional responses. Here we report that neuropathic pain-like hypersensitivity induced by common peroneal nerve (CPN) ligation increases nociceptive stimulation-induced responses in glutamatergic LPBN neurons. Optogenetic activation of GABAergic LPBN neurons does not affect basal nociception, but alleviates neuropathic pain-like behavior. Optogenetic activation of glutamatergic or inhibition of GABAergic LPBN neurons induces neuropathic pain-like behavior in naïve mice. Inhibition of glutamatergic LPBN neurons alleviates both basal nociception and neuropathic pain-like hypersensitivity. Repetitive pharmacogenetic activation of glutamatergic or GABAergic LPBN neurons respectively mimics or prevents the development of CPN ligation-induced neuropathic pain-like hypersensitivity. These findings indicate that a delicate balance between excitatory and inhibitory LPBN neuronal activity governs the development and maintenance of neuropathic pain.

P3752Ablation of the superior left ganglionic plexus is not necessary for effective denervation of the sinoatrial and atrioventricular nodes

Abstract Background Cardioneuroablation by targeting of atrial ganglionic plexi (GP) has been proposed as a new therapeutic option in selected patients with reflex syncope. Contribution of individual GPs to cardiac autonomic regulations is not fully established. Purpose Because consistent vagal responses have been observed during left superior pulmonary vein isolation in patients undergoing ablation for atrial fibrillation, we investigated whether standalone ablation of the superior left GP modifies the vagal input into the sinoatrial (SAN) and atrioventricular node (AVN). Methods Study hypothesis was investigated in otherwise healthy patients undergoing cardioneuroablation for symptomatic bradyarrhythmias. All had preprocedural atropine test suggesting functional disorder. Anatomically-navigated (CARTO-3) radiofrequency (RF) ablation (25–30 W/30 s/20 ml/min) at empirical GP sites was performed in general anaesthesia. Extracardiac high-frequency vagal nerve stimulation (25–60 V/30–50 Hz/0.05–0.1 ms) via right jugular vein was performed at baseline, after initial superior left GP ablation, and after the ablation of remaining septal and inferior GPs. High-frequency vagal nerve stimulation was always done in both sinus rhythm and atrial pacing. The elimination of all stimulation-induced vagal responses was the endpoint of the procedure. Results A study included 8 patients (34±8 years; 5 males). Six of them had recurrent syncope with cardioinhibitory response at the SAN (n=4), AVN (n=1) or both nodes (n=1); and 2 patients had symptomatic sinus bradycardia. At baseline, high-frequency vagal nerve stimulation induced long episodes of sinus arrest and advanced AV block in all patients. Cluster ablation at the superior left GP (RF time: 192±28 s) did not change the sinus rate (59±14 vs 60±15, NS), PQ interval (174±37 vs 173±37, NS), and did not induce any tangible change in SAN/AVN response to high-frequency vagal nerve stimulation. Subsequently, anterior right GP was targeted from the aspect of right atrium (n=7), from the anterior antrum of right pulmonary veins (n=6), and inferior GPs were targeted from the aspect of left atrium (n=7). This lesion set finally resulted in complete non-responsiveness of SAN and AVN to high-frequency vagal nerve stimulation in all patients. Ablation procedure overall (duration: 172±15 min; RF time: 988±306 s; radiation dose: 70±34 μGy·m2) led to sinus rate acceleration by a median of 29 (IQR: 18–38) bpm, increase of Wenckebach point by 21 (IQR: 9–28) bpm, and shortening of AVN effective refractory period by 40 (IQR: 15–73) ms. Conclusions Cardioneuroablation guided by extracardiac high-frequency vagal nerve stimulation can achieve complete denervation of SAN and AVN by ablation of postero(para)septal and inferior GPs only. Ablation of the superior left GP appears unnecessary and can be eliminated from the lesion set design.

Focal stimulation of the sheep motor cortex with a chronically implanted minimally invasive electrode array mounted on an endovascular stent.

Direct electrical stimulation of the brain can alleviate symptoms associated with Parkinson's disease, depression, epilepsy and other neurological disorders. However, access to the brain requires invasive procedures, such as the removal of a portion of the skull or the drilling of a burr hole. Also, electrode implantation into tissue can cause inflammatory tissue responses and brain trauma, and lead to device failure. Here, we report the development and application of a chronically implanted platinum electrode array mounted on a nitinol endovascular stent for the localized stimulation of cortical tissue from within a blood vessel. Following percutaneous angiographic implantation of the device in sheep, we observed stimulation-induced responses of the facial muscles and limbs of the animals, similar to those evoked by electrodes implanted via invasive surgery. Proximity of the electrode to the motor cortex, yet not its orientation, was integral to achieving reliable responses from discrete neuronal populations. The minimally invasive endovascular surgical approach offered by the stent-mounted electrode array might enable safe and efficacious stimulation of focal regions in the brain.

PO-147 Effect of Exercise-induced Fatigue on the Electrical Activity of the External Globus Pallidus Neurons in rats

Objective The motor cortex (MC) stimulation-induced unitary responses of globus pallidus external segment (GPe) neurons in control and exercise induced-fatigue rats were recorded in vivo to examine the role of cortical-striatum-external globus pallidal pathway in the mechanism of central fatigue.
 Methods 32 Clean healthy male Wistar rats (260~300g), were randomly divided into 4 groups: control group (Control), 1-day fatigue group (1FG), 3-day fatigue group (3FG) and 7-day fatigue group (7FG). Rats were subjected to a 5-day adaptive treadmill training. Modified Bedford treadmill exercise with progressively increasing load was used to creat the exercise fatigue model. （3 levels:8.2 m/min, 15 min; 15m/min, 15 min; 20 m/min, lasting till exhaustion） The spontaneous unit activity and responses to MC stimulation of GPe neurons were recorded by the electrophysiological technique of extracellular recording of glass microelectrodes.
 Results The results showed that the firing frequency of high-frequency firing with pause (HFP) and low frequency firing with burst (LFB) in the GPe of 1FG was comparable with that of control group (P>0.05). However in 3FG and 7FG , the percentage of HFP neuron was significantly decreased (P<0.05), while the proportion of LFB was significantly increased (P<0.05), and the average firing rate of LFB was higher and inter spike intervals (ISI) was significantly lower than that of the control group. With 200μA electrical stimulation, the explosive discharge of GPe neurons was attenuated after fatigue in rats. The response of GPe neurons to variable frequency stimulation in exhausted model groups was stronger than that of the control group.MC-stimulation typically induced a triphasic response composed of early excitation, inhibition, and late excitation in GPe neurons. The population of neurons showing a short inhibition slightly increased in 3FG and 7FG.
 Conclusions 1. The results confirmed that GPe is an important nucleus of basal ganglia involved in the regulation of exercise-induced fatigue by the change of spontaneous activity.
 
 Electrical stimulation on the cortex can alter response patterns of GPe neurons in exercise-induced fatigue rats, the results confirmed that the Ctx-Str-GPe neural pathway is involved in the regulation of exercise fatigue, and the indirect pathway is over-activated.

C.06 Somatotopic organization of the human spinothalamic tract: CT-guided mapping in awake patients undergoing cordotomy

Background: After correlating in vivo macrostimulation-induced pain or temperature sensation during percutaneous cervical cordotomy with simultaneous CT imaging of the electrode tip location, we present a modern description of the somatotopy of the human cervical spinothalamic tract Methods: Twenty patients with medically refractory, unilateral, nociceptive pain due to malignancy received contralateral cervical percutaneous cordotomy. In a post-hoc analysis of the data, each individual’s cervical spinal cord was measured from the CT image using PACS software. The location of the electrode tip during each stimulation-induced response was then superimposed on a diagram of their cord Results: The lower limb responses were found more superficial and posterior to those of the upper limb. Interestingly, the region for upper limb responses surrounded that for lower limb primarily anteriorly and medially (deep) but also posteriorly Conclusions: This work simultaneously combined awake physiologic localization of fibers within the human -spinothalamic tract (STT) with neuroimaging documenting their precise anatomical localization within the spinal cord. The resultant map of the STT demonstrates, for the first time, that fibers from the lower limb are located superficially and posteriorly within the anterolateral spinal cord with the fibers from the upper limb surrounding them primarily deep and anteriorly but also posteriorly

Heart Rate Changes in Response to Mechanical Pressure Stimulation of Skeletal Muscles Are Mediated by Cardiac Sympathetic Nerve Activity.

Stimulation of mechanoreceptors in skeletal muscles such as contraction and stretch elicits reflexive autonomic nervous system changes which impact cardiovascular control. There are pressure-sensitive mechanoreceptors in skeletal muscles. Mechanical pressure stimulation of skeletal muscles can induce reflex changes in heart rate (HR) and blood pressure, although the neural mechanisms underlying this effect are unclear. We examined the contribution of cardiac autonomic nerves to HR responses induced by mechanical pressure stimulation (30 s, ~10 N/cm2) of calf muscles in isoflurane-anesthetized rats. Animals were artificially ventilated and kept warm using a heating pad and lamp, and respiration and core body temperature were maintained within physiological ranges. Mechanical stimulation was applied using a stimulation probe 6 mm in diameter with a flat surface. Cardiac sympathetic and vagus nerves were blocked to test the contribution of the autonomic nerves. For sympathetic nerve block, bilateral stellate ganglia, and cervical sympathetic nerves were surgically sectioned, and for vagus nerve block, the nerve was bilaterally severed. In addition, mass discharges of cardiac sympathetic efferent nerve were electrophysiologically recorded. Mechanical stimulation increased or decreased HR in autonomic nerve-intact rats (range: −56 to +10 bpm), and the responses were negatively correlated with pre-stimulus HR (r = −0.65, p = 0.001). Stimulation-induced HR responses were markedly attenuated by blocking the cardiac sympathetic nerve (range: −9 to +3 bpm, p < 0.0001) but not the vagus nerve (range: −75 to +30 bpm, p = 0.17). In the experiments with cardiac sympathetic efferent nerve activity recordings, mechanical stimulation increased, or decreased the frequency of sympathetic nerve activity in parallel with HR (r = 0.77, p = 0.0004). Furthermore, the changes in sympathetic nerve activity were negatively correlated with its tonic level (r = −0.62, p = 0.0066). These results suggest that cardiac sympathetic nerve activity regulates HR responses to muscle mechanical pressure stimulation and the direction of HR responses depends on the tonic level of the nerve activity, i.e., bradycardia occurs when the tonic activity is high and tachycardia occurs when the activity is low.

The effects of hydrogen sulfide on electrical field stimulation-induced neurogenic contractile responses in isolated rabbit lower esophageal sphincter: Contribution of nitrergic and non-adrenergic non-cholinergic transmission.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that, similar to nitric oxide (NO), plays an important role as an inhibitor neurotransmitter in the digestive tract. This study aimed to investigate the effect of H2S and to identify neurogenic contraction responses dependent on the electrical field stimulation (EFS) in the isolated lower esophageal sphincters of rabbits. An isolated lower esophageal sphincter was placed in an organ bath system and mechanical responses were recorded using a force transducer. The nerve-evoked contractile responses were obtained by EFS. The contractile responses were obtained as biphasic "on" and "off" phases seen at the beginning and end of EFS, respectively. Sodium hydrogen sulfide (NaHS) reduced the EFS-mediated "off" phase and the EFS-mediated non-adrenergic non-cholinergic (NANC) "off" phase. NaHS reduced the EFS-mediated "on" phase as well. l-Cysteine ​​reduced the EFS-mediated "off" phase and the EFS-mediated NANC "off" phase. l-Propargylglycine (PAG) did not affect the EFS-mediated "off" phase or the EFS-mediated NANC "off" phase. NaHS, l-cysteine, and PAG reduced the EFS-mediated, NO-independent "off" phase. The effect of NaHS in all of the experiments returned in time. Also, NaHS caused significant relaxation of 80-mM KCl-Krebs solution induced-contractions, while l-cysteine ​​and PAG did not cause a significant relaxation. These findings suggest that H2S has an inhibitory effect on the lower esophageal sphincter muscle. While the effect of H2S on EFS-mediated responses disappeared in time, the effect of H2S sustained the KCl-Krebs solution-induced contractions. This shows that H2S may have an effect on neurotransmission at the nerve terminal.

EP 24. Contralateral smiles induced by deep brain stimulation of the nucleus accumbens region in a patient with major depressive disorder

Introduction Deep brain stimulation (DBS) is a well-established procedure for the treatment of therapy-refractory movement disorders. Limited evidence suggests that DBS of the nucleus accumbens region (NACC) may be effective for the treatment of psychiatric disorders, such as obsessive compulsive disorder and major depressive disorder (MDD). Hitherto, little is known concerning behavioural responses upon acute high-frequency stimulation of the NACC region in patients suffering from MDD. Objective To further characterize behavioural responses and clinical effects elicited upon acute unilateral DBS of the NACC area. Intra- and postoperative observations (including video-sequences) as well as a two-month outcome are presented. Material and Methods We performed bilateral NACC-DBS in a female patient (age: 47) with MDD refractory to psycho-, pharmaco- and electroconvulsive (>50 sessions) therapy on a compassionate-use basis. The stereotactic procedure was performed under local anaesthesia. The NACC was targeted (x) 7 mm lateral to the midline, (y) 3 mm anterior to the anterior commissure, (z) 4 mm below the anterior commissure-posterior commissure plane. A commercially available DBS lead with 4 contacts (model 3391, Medtronic Co., MN, USA; contact length: 3 mm; interelectrode distance: 4 mm) was inserted bilaterally for permanent stimulation. Results Upon test-stimulation in the operating room, acute unilateral NACC-DBS on both hemispheres stereotypically induced a visible facial reaction with a contralateral smile (bipolar stimulation of the ventralmost contacts (0− cathodal vs. 1+ anodal); pulse width: 90 μs; frequency: 130 Hz). This smile could repeatedly be elicited either when stimulation amplitude was slowly ramped up to 7 mA, or abruptly switched on to 4–5 mA. The smile was invariably associated with a significantly improved mood (as assessed by an analog scale). This effect was documented by different examiners and clearly reproducible in the subsequent clinical course. A dramatic improvement of the depression scores, patient’s mood and social behaviour was documented at a first follow-up (6 weeks postoperatively). Conclusion Albeit preliminary, the patient’s beneficial response tentatively suggests that NACC-DBS may be a viable option for selected patients with MDD. In line with previous reports on stimulation-induced smile responses in patients with obsessive compulsive disorder, our intraoperative observations support and expand the notion of the NACC area constituting or containing an important interface between limbic and motor networks, respectively, that may be involved in the pathophysiology of MDD. It is suggested that the results of long-term NACC-DBS in patients with MDD should be evaluated by larger patient samples and randomized, controlled trials.

Brain (Hyper)Excitability Revealed by Optimal Electrical Stimulation of GABAergic Interneurons.

Neurological disorders are often characterized by an excessive and prolonged imbalance between neural excitatory and inhibitory processes. An ubiquitous finding among these disorders is the disrupted function of inhibitory GABAergic interneurons. The objective is to propose a novel stimulation procedure able to evaluate the efficacy of inhibition imposed by GABAergic interneurons onto pyramidal cells from evoked responses observed in local field potentials (LFPs). Using a computational modeling approach combined with in vivo and in vitro electrophysiological recordings, we analyzed the impact of electrical extracellular, local, bipolar stimulation (ELBS) on brain tissue. We implemented the ELBS effects in a neuronal population model in which we can tune the excitation-inhibition ratio and we investigated stimulation-related parameters. Computer simulations led to sharp predictions regarding: i) the shape of evoked responses as observed in local field potentials, ii) the type of cells (pyramidal neurons and interneurons) contributing to these field responses and iii) the optimal tuning of stimulation parameters (intensity and frequency) to evoke meaningful responses. These predictions were tested in vivo (mouse). Neurobiological mechanisms were assessed in vitro (hippocampal slices). Appropriately-tuned ELBS allows for preferential activation of GABAergic interneurons. A quantitative neural network excitability index (NNEI) is proposed. It is computed from stimulation-induced responses as reflected in local field potentials. NNEI was used in four patients with focal epilepsy. Results show that it can readily reveal hyperexcitable brain regions. Well-tuned ELBS and NNEI can be used to locally probe brain regions and quantify the (hyper)excitability of the underlying brain tissue.

Intraoperative smile in a multiple sclerosis patient with medication-refractory tremor

Deep brain stimulation has been utilized to improve disease symptoms in patients with Parkinson’s disease, dystonia, essential tremor, and other neuropsychiatric syndromes such as depression and obsessive compulsive disorder. Deep brain stimulation has also been observed to improve tremor for select patients with multiple sclerosis. During intraoperative stimulation in these multiple sclerosis patients, researchers have observed a wide spectrum of motor and sensory phenomena, but no stimulation-induced emotional responses have been reported. We detailed intraoperative smiling associated with stimulation of the ventralis oralis anterior/ventralis oralis posterior border region of the left thalamus. A single patient with medication-resistant multiple sclerosis tremor experienced smiling, laughing, and subjective euphoria during intraoperative stimulation of the left thalamus. Specifically, during intraoperative stimulation of the left thalamic ventralis oralis anterior border, the patient developed a contralateral smile which progressed to a bilateral smile and was accompanied by a feeling of subjective happiness. The smile habituated in approximately 60 seconds and it was reproducible on a repeat stimulation. The patient could subjectively feel the facial movement, and, at higher voltages, the movement was described as a pulling sensation. Stimulation of the anterior ventralis oralis anterior border of the left thalamus in an multiple sclerosis patient produced a unilateral smile that rapidly developed into a bilateral smile accompanied by euphoria. There were presumed capsular side effects at higher voltages. The exact mechanism by which stimulation of the thalamus produced a smile and mood elevation is unknown, but we speculate that the smile could be induced by stimulation of corticobulbar fibers arising from the caudal cingulate motor area connecting the contralateral facial nerve nucleus.

Blood natural killer cell cytotoxicity enhancement correlates with an increased activity in brain motor structures following chronic stimulation of the bed nucleus of the stria terminalis in rats

The present study indicates that a chronic 14 day electrical stimulation of the bed nucleus of the stria terminalis (BST) increased blood but not spleen natural killer cell (NK) cytotoxicity and a large granular lymphocyte (LGL) number. These immune changes positively correlated with the increased activity in brain cortical and subcortical motor structures that influence the BST stimulation-induced behavioral response. No significant changes in blood and spleen leukocyte population numbers and plasma corticosterone concentration after the stimulation were found. The obtained results suggest that this immunoenhancing effect on blood NK cell function and number is dependent on the behavioral outcome of the BST stimulation rather than endocrine response.

Lutein Inhibits the Function of the Transient Receptor Potential A1 Ion Channel in Different In Vitro and In Vivo Models

Transient receptor potential (TRP) ion channels, such as TRP vanilloid 1 and ankyrin repeat domain 1 (TRPV1 and TRPA1), are expressed on primary sensory neurons. Lutein, a natural tetraterpene carotenoid, can be incorporated into membranes and might modulate TRP channels. Therefore, the effects of the water-soluble randomly methylated-β-cyclodextrin (RAMEB) complex of lutein were investigated on TRPV1 and TRPA1 activation. RAMEB-lutein (100 μM) significantly diminished Ca(2+) influx to cultured rat trigeminal neurons induced by TRPA1 activation with mustard oil, but not by TRPV1 stimulation with capsaicin, as determined with microfluorimetry. Calcitonin gene-related peptide release from afferents of isolated tracheae evoked by mustard oil, but not by capsaicin, was inhibited by RAMEB-lutein. Mustard oil-induced neurogenic mouse ear swelling was also significantly decreased by 100 μg/ml s.c. RAMEB-lutein pretreatment, while capsaicin-evoked edema was not altered. Myeloperoxidase activity indicating non-neurogenic granulocyte accumulation in the ear was not influenced by RAMEB-lutein in either case. It is concluded that lutein inhibits TRPA1, but not TRPV1 stimulation-induced responses on cell bodies and peripheral terminals of sensory neurons in vitro and in vivo. Based on these distinct actions and the carotenoid structure, the ability of lutein to modulate lipid rafts in the membrane around TRP channels can be suggested.

High b -value diffusion-weighted fMRI in a rat forepaw electrostimulation model at 7 T

Spin-echo diffusion-weighted functional MRI (DW-fMRI) was performed on a rat forepaw electrostimulation model at 7 T. This small animal model used electric (rather than visual) stimulation and allowed DW-fMRI experiments to be performed over a broader range of acquisition parameters than previous work on humans and cats. Resting state experiments with injections of ultra-small superparamagnetic iron oxide (USPIO) were also used to investigate the effects of gradient coupling on the signal change. The experiments were performed over five b-values (0, 200, 800, 1400 and 2000s/mm(2)) and three echo-times (30, 60 and 90 ms). Alterations to the stimulation-induced response with respect to TE and b-value were evaluated in two intervals: the positive stimulus-correlated response (5-20s after stimulus onset) and the post-stimulus undershoot (27-40s). There was no strong dependence of the signal change on b-value for any of the intervals or TEs. Similarly, changes to the apparent transverse relaxation rate showed no clear dependence on b-value. In contrast to previous DW-fMRI studies, the simplest explanation for the observed data is a single-compartment signal model with the functional signal changes probably corresponding to extravascular SE-BOLD. Experiments with USPIO suggested that at 7 T and within the range of parameters used, the influence of gradient coupling may be sufficient to explain minor DW-fMRI signal changes.

GABAA receptor-mediated modulation of neuronal activity propagation upon tetanic stimulation in rat hippocampal slices

Tetanic stimulation (100 Hz), which can induce long-term potentiation in synaptic connections in the hippocampal CA1 region, causes γ-aminobutyric acid (GABA)(A) receptor-mediated long-lasting depolarization of postsynaptic neurons. However, it is not clear how this stimulation modulates neuronal activity propagation. We studied tetanic burst-induced neuronal responses in the hippocampal CA1 region by using optical-recording methods employing a voltage-sensitive dye and focused on GABA(A) receptor-mediated modulation. We observed that burst stimulation induced long-lasting depolarization and progressive decrease in individual excitatory postsynaptic potentials (EPSPs). Both these effects were suppressed by picrotoxin, a GABA(A) receptor antagonist. Under whole-cell voltage-clamp conditions, we observed a long-lasting inhibitory current (IPSC) and a prominent progressive decrease in the amplitude of the excitatory postsynaptic current (EPSC). Further, picrotoxin inhibited the IPSC and the progressive decrease in EPSC. The optically recorded long-lasting depolarization and progressive decrease of EPSPs were strongly dependent on the distance between the recording electrode and the stimulation site. Optical recordings performed across a wide swatch of CA1 revealed that the decrease in activity propagation was followed by facilitation of propagation after recovery and that this facilitation also depended on GABA(A) receptors. Intense activation of GABA(A) receptors is a key factor shaping the spatiotemporal patterns of high-frequency stimulation-induced responses in the CA1 region.

Inhibition of field stimulation-induced contractions of rabbit vas deferens by muscarinic receptor agonists: selectivity of McN-A-343 for M1 receptors.

Inhibition of the field stimulation-induced twitch responses of the rabbit vas deferens by the muscarinic receptor agonist, McN-A-343, has been attributed to presynaptic muscarinic receptors of the M1 subtype located on noradrenergic nerve terminals. Stimulation of these receptors causes inhibition of transmitter release and inhibition of the contractile response. However, the selectivity of McN-A-343 for M1 receptors has been questioned and this throws doubt on whether the prejunctional receptors of the rabbit vas deferens are of the M1 subtype. In this study we have undertaken a comprehensive re-evaluation of the inhibition of prostatic and epididymal portions of the rabbit isolated field-stimulated vas deferens by several agonists, including McN-A-343, and quantified the antagonism by M1-selective antagonists, pirenzepine and telenzepine. Prostatic and epididymal portions of vasa deferentia from New Zealand White rabbits were immersed in a low Ca2+ Krebs solution at 32+/-0.5 degrees C gassed with 5% CO2 in oxygen. Yohimbine (1.0mM) was present throughout to block prejunctional alpha2-adrenoceptors. Field stimulation was applied by repeated application of single pulses (30 V, 0.05 Hz, 0.5 ms) and isometric contractions recorded. Carbachol and oxotremorine initially potentiated the epididymal contractions but at higher concentrations there was inhibition. In the prostatic portion, oxotremorine only inhibited. McN-A-343 produced inhibitory responses only in both epididymal and prostatic portions. Pirenzepine shifted the concentration-response curves forthe inhibitory responses to oxotremorine to the right. However, the potentiation of the twitches also became more apparent with the lower concentrations of oxotremorine. Schild plots for the antagonism by pirenzepine yielded pA2 values of 7.96+/-0.004 and 7.7+/-0.02 for the epididymal and prostatic portions, respectively. The concentration-response curves for the inhibition of twitches by McN-A-343 were displaced to the right in a parallel manner by pirenzepine in both prostatic and epididymal portions with no potentiation of the twitches. The Schild plot for this antagonism generated pA2 values of 7.68+/-0.01 and 8.07+/-0.01, respectively. Telenzepine caused parallel shifts of the McN-A-343 concentration-response curves to the right in prostatic portions, the pA2 value being 8.70+/-0.13. Telenzepine (10(-7) M) abolished the inhibitory effect of carbachol to reveal only concentration-dependent potentiation of the contractions. The Schild plot for antagonism of this contractile effect yielded a pA2 value (7.07+/-0.09) that was significantly less by almost two orders of magnitude (1.70) than the value for the antagonism by telenzepine of the McN-A-343-induced inhibitory response. The pA2 values of pirenzepine and telenzepine against the inhibitory responses of the rabbit vas deferens are consistent with the involvement of M1 receptors. This leads to the conclusion that McN-A-343 causes inhibition through this receptor type. The doubts concerning the selectivity of McN-A-343 for M1 receptors are therefore unfounded. The fact that McN-A-343 does not display a selective binding profile suggests that its selectivity does not arise from affinity differences but probably resides in its intrinsic efficacy.

Hypertension alters the function of nitrergic and sensory innervation in mesenteric arteries from female rats

To investigate whether hypertension could modify the function of adrenergic, nitrergic, and sensory innervations involved in the electrical field stimulation-induced response in mesenteric arteries from female rats. Vascular reactivity experiments were performed in endothelium-denuded mesenteric arteries from normotensive, Wistar-Kyoto and spontaneously hypertensive female rats; protein expression was measured by western blot; nitric oxide release was measured by fluorometry; calcitonin gene-related peptide and noradrenaline release were determined by enzyme immunoassay. The electrical field stimulation-induced contractions were significantly lower in segments from spontaneously hypertensive rats than those of Wistar-Kyoto rats. Hypertension did not modify either the response or release of noradrenaline. Preincubation with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester increased the electrical field stimulation-induced contractions only in segments from Wistar-Kyoto rats. The relaxation induced by the nitric oxide donor sodium nitroprusside was similar in segments from both strains. The electrical field stimulation-induced nitric oxide release was decreased in segments from spontaneously hypertensive rats. The calcitonin gene-related peptide receptor antagonist CGRP(8-37) did not alter the electrical field stimulation-induced contractions in segments from Wistar-Kyoto rats but increased them in segments from spontaneously hypertensive rats. The calcitonin gene-related peptide-induced relaxation was increased in segments from spontaneously hypertensive rats. The expression of the 15-kDa active form of RAMP1 was increased in segments from spontaneously hypertensive rats. In contrast to male rats, electrical field stimulation-induced contractions are decreased in hypertensive female rats. Nitrergic innervation plays a role in the development and/or maintenance of hypertension, whereas sensory innervation is a counteracting mechanism through the increased calcitonin gene-related peptide response.

Dynamic map of protein interactions in the Escherichia coli chemotaxis pathway

Protein–protein interactions play key roles in virtually all cellular processes, often forming complex regulatory networks. A powerful tool to study interactions in vivo is fluorescence resonance energy transfer (FRET), which is based on the distance-dependent energy transfer from an excited donor to an acceptor fluorophore. Here, we used FRET to systematically map all protein interactions in the chemotaxis signaling pathway in Escherichia coli, one of the most studied models of signal transduction, and to determine stimulation-induced changes in the pathway. Our FRET analysis identified 19 positive FRET pairs out of the 28 possible protein combinations, with 9 pairs being responsive to chemotactic stimulation. Six stimulation-dependent and five stimulation-independent interactions were direct, whereas other interactions were apparently mediated by scaffolding proteins. Characterization of stimulation-induced responses revealed an additional regulation through activity dependence of interactions involving the adaptation enzyme CheB, and showed complex rearrangement of chemosensory receptors. Our study illustrates how FRET can be efficiently employed to study dynamic protein networks in vivo.

Targeted disruption of the A 2A adenosine receptor reduces in-vitro prostate contractility in mature mice

Prostatic A 2A adenosine receptors mediate varied effects. This study aimed to test whether genetic disruption of this receptor affects prostate contractility. Prostates taken from mice which were homozygous (A 2AR−/−) and heterozygous (A 2AR+/−) for the disrupted A 2A adenosine receptor gene and wild-type littermates (A 2AR +/+) were mounted in organ baths. Contractile responses to nerve stimulation and noradrenaline were measured in the presence of various pharmacological tools. Electrical field stimulation (0.5 ms pulse duration, 60 V, 0.1–20 Hz) yielded frequency-dependent contractions while exogenous administration of noradrenaline (10 nM–1 mM) or tyramine (1 μM–1 mM) produced concentration-dependent responses. Contractile responses to electrical field stimulation from A 2AR−/− and A 2AR+/− prostates were reduced when compared to A 2A+/+ prostates ( P = 0.013, n = 33–36). Prazosin (0.3 μM) inhibited electrical field stimulation-induced responses in prostates from A 2AR+/+ and A 2AR+/− mice ( P ≤ 0.016, n = 5–7) but not A 2AR−/− mice ( P = 0.400, n = 6). Tetrodotoxin abolished electrical field stimulation-induced responses in all prostates ( P < 0.001, n = 5–7). NF 449 and ZM 241385 were without effect ( P ≤ 0.421, n = 4–6). There were no genotype differences in noradrenaline or tyramine concentration–response curves ( P ≥ 0.180, n = 10–13). Prazosin (0.3 μM) and cocaine (10 μM) attenuated the responses induced by noradrenaline ( P < 0.001, n = 6–7) and tyramine ( P < 0.001, n = 5–6), respectively, in all genotypes. Disruption of the A 2A adenosine receptor leads to reduced nerve mediated contractile responses of the prostate in mature mice.