Electrical Stimulation Of Slices Research Articles

Spinal Dopaminergic Mechanisms Regulating the Micturition Reflex in Male Rats with Complete Spinal Cord Injury.

Traumatic spinal cord injury (SCI) often causes micturition dysfunction. We recently discovered a low level of spinally-derived dopamine (DA) that regulates recovered bladder and sphincter reflexes in SCI female rats. Considering substantial sexual dimorphic features in the lower urinary tract, it is unknown if the DA-ergic mechanisms act in the male. Histological analysis showed a similar distribution of tyrosine hydroxylase (TH)+ neurons in the lower cord of male rats and the number increased following thoracic SCI. Subsequently, focal electrical stimulation in slices obtained from L6/S1 spinal segments of SCI rats elicited detectable DA release with fast scan cyclic voltammetry. Using bladder cystometrogram and external urethral sphincter (EUS) electromyography in SCI male rats, intravenous (i.v.) administration of SCH 23390, a D1-like receptor (DR1) antagonist, induced significantly increased tonic EUS activity and a trend of increased residual volume, whereas activation of these receptors with SKF 38393 did not influence the reflex. Meanwhile, blocking spinal D2-like receptors (DR2) with remoxipride had no effect but stimulating these receptors with quinpirole elicited EUS bursting to increase voiding volume. Further, intrathecal delivery of SCH 23390 and quinpirole resulted in similar responses to those with i.v. delivery, respectively, which indicates the central action regardless of delivery route. In addition, metabolic cage assays showed that quinpirole increased the voiding frequency and total voiding volume in spontaneous micturition. Collectively, spinal DA-ergic machinery regulates recovered micturition reflex following SCI in male rats; spinal DR1 tonically suppress tonic EUS activity to enable voiding and activation of DR2 facilitates voiding.

Chronic Social Isolation Stress during Peri-Adolescence Alters Presynaptic Dopamine Terminal Dynamics via Augmentation in Accumbal Dopamine Availability.

Chronic peri-adolescent stress in humans increases risk to develop a substance use disorder during adulthood. Rats reared in social isolation during peri-adolescence (aSI; 1 rat/cage) period show greater ethanol and cocaine intake compared to group housed (aGH; 4 rats/cage) rats. In addition, aSI rats have a heightened dopamine response in the nucleus accumbens (NAc) to rewarding and aversive stimuli. Furthermore, single pulse electrical stimulation in slices containing NAc core elicits greater dopamine release in aSI rats. Here, we further investigated dopamine release kinetics and machinery following aSI. Dopamine release, across a wide range of stimulation intensities and frequencies, was significantly greater in aSI rats. Interestingly, subthreshold intensity stimulations also resulted in measurable dopamine release in accumbal slices from aSI but not aGH rats. Extracellular [Ca2+] manipulations revealed augmented calcium sensitivity of dopamine release in aSI rats. The readily releasable pools of dopamine, examined by bath application of Ro-04-1284/000, a vesicular monoamine transporter 2 (VMAT2) inhibitor, were depleted faster in aGH rats. Western blot analysis of release machinery proteins (VMAT2, Synaptogyrin-3, Syntaxin-1, and Munc13-3) showed no difference between the two groups. Tyrosine hydroxylase (TH) protein expression levels, however, were elevated in aSI rats. The greater dopamine release could potentially be explained by higher levels of TH, the rate-limiting step for dopamine synthesis. This augmented responsivity of the dopamine system and heightened dopamine availability post-aSI may lead to an increased risk of addiction vulnerability.

Burst-Dependent Bidirectional Plasticity in the Cerebellum Is Driven by Presynaptic NMDA Receptors.

Numerous studies have shown that cerebellar function is related to the plasticity at the synapses between parallel fibers and Purkinje cells. How specific input patterns determine plasticity outcomes, as well as the biophysics underlying plasticity of these synapses, remain unclear. Here, we characterize the patterns of activity that lead to postsynaptically expressed LTP using both in vivo and in vitro experiments. Similar to the requirements of LTD, we find that high-frequency bursts are necessary to trigger LTP and that this burst-dependent plasticity depends on presynaptic NMDA receptors and nitric oxide (NO) signaling. We provide direct evidence for calcium entry through presynaptic NMDA receptors in a subpopulation of parallel fiber varicosities. Finally, we develop and experimentally verify a mechanistic plasticity model based on NO and calcium signaling. The model reproduces plasticity outcomes from data and predicts the effect of arbitrary patterns of synaptic inputs on Purkinje cells, thereby providing a unified description of plasticity.

α2δ-1 signaling in nucleus accumbens is necessary for cocaine-induced relapse.

Relapse to cocaine seeking is associated with potentiated excitatory synapses in nucleus accumbens. α2δ-1 is an auxiliary subunit of voltage-gated calcium channels that affects calcium-channel trafficking and kinetics, initiates extracellular signaling cascades, and promotes excitatory synaptogenesis. Previous data demonstrate that repeated exposure to alcohol, nicotine, methamphetamine, and morphine upregulates α2δ-1 in reward-related brain regions, but it was unclear whether this alteration generalized to cocaine. Here, we show that α2δ-1 protein was increased in nucleus accumbens after cocaine self-administration and extinction compared with saline controls. Furthermore, the endogenous ligand thrombospondin-1, responsible for the synaptogenic properties of the α2δ-1 receptor, was likewise elevated. Using whole-cell patch-clamp recordings of EPSCs in nucleus accumbens, we demonstrated that gabapentin, a specific α2δ-1 antagonist, preferentially reduced the amplitude and increased the paired-pulse ratio of EPSCs evoked by electrical stimulation in slices from cocaine-experienced rats compared with controls. In vivo, gabapentin microinjected in the nucleus accumbens core attenuated cocaine-primed but not cue-induced reinstatement. Importantly, gabapentin's effects on drug seeking were not due to a general depression of spontaneous or cocaine-induced locomotor activity. Moreover, gabapentin had no effect on reinstatement of sucrose seeking. These data indicate that α2δ-1 contributes specifically to cocaine-reinstated drug seeking, and identifies this protein as a target for the development of cocaine relapse medications. These results also inform ongoing discussion in the literature regarding efficacy of gabapentin as a candidate addiction therapy.

Specific mechanism of use-dependent channel block of calcium-permeable AMPA receptors provides activity-dependent inhibition of glutamatergic neurotransmission

This study examined the blocking action of the selective channel blocker of calcium-permeable (CP) AMPA receptors, N1-(1-phenylcyclohexyl)pentane-1,5-diaminium bromide (IEM-1925), on excitatory postsynaptic currents in rat neostriatal and cortical neurons and in fly neuromuscular junctions. In both preparations, the blocking of CP-AMPA receptor currents increased along with the stimulation frequency. The continuous presence of kainate, which activates AMPA receptors, in the external solution also caused an enhanced blocking effect. Likewise, decrease of the synaptic release by lowering calcium concentration resulted in significant reduction of the blocking action. The activity dependence of the block is explained using the guarded receptor model. The drug molecule can only bind if the channel is open. After the channel has closed, the drug molecule remains trapped inside. However, the trapped molecule slowly egresses from closed channels to the cytoplasm. The total block effect is determined by the equilibrium between accumulation of the drug in the open channels and relief from the closed channels. Therefore, the conditions that favour the open state result in enhanced inhibition. This significant finding reveals a new way to modulate CP-AMPAR-mediated transmission using a physiologically relevant approach. Moreover, it allows the involvement of CP-AMPARs in the physiological and pathological processes – such as high-frequency synaptic activity or increase of the steady-state glutamate concentration – to be examined.

Activation of ATP-sensitive K+ (K(ATP)) channels by H2O2 underlies glutamate-dependent inhibition of striatal dopamine release.

In many cells, ATP-sensitive K+ channels (KATP channels) couple metabolic state to excitability. In pancreatic beta cells, for example, this coupling regulates insulin release. Although KATP channels are abundantly expressed in the brain, their physiological role and the factors that regulate them are poorly understood. One potential regulator is H2O2. We reported previously that dopamine (DA) release in the striatum is modulated by endogenous H2O2, generated downstream from glutamatergic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor activation. Here we investigated whether H2O2-sensitive KATP channels contribute to DA-release modulation by glutamate and gamma-aminobutyric acid (GABA). This question is important because DA-glutamate interactions underlie brain functions, including motor control and cognition. Synaptic DA release was evoked by using local electrical stimulation in slices of guinea pig striatum and monitored in real time with carbon-fiber microelectrodes and fast-scan cyclic voltammetry. The KATP-channel antagonist glibenclamide abolished the H2O2-dependent increase in DA release usually seen with AMPA-receptor blockade by GYKI-52466 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride] and the decrease in DA release seen with GABA-type-A-receptor blockade by picrotoxin. In contrast, 5-hydroxydecanoate, a mitochondrial KATP-channel blocker, was ineffective, as were sulpiride, a D2-receptor antagonist, and tertiapin, a G protein-coupled K+-channel inhibitor. Diazoxide, a sulfonylurea receptor 1 (SUR1)selective KATP-channel opener, prevented DA modulation by H2O2, glutamate, and GABA, whereas cromakalim, a SUR2-selective opener, did not. Thus, endogenous H2O2 activates SUR1-containing KATP channels in the plasma membrane to inhibit DA release. These data not only demonstrate that KATP channels can modulate CNS transmitter release in response to fast-synaptic transmission but also introduce H2O2 as a KATP-channel regulator.

Functional neocortical microcircuitry demonstrated with intrinsic signal optical imaging in vitro

Intrinsic signal optical imaging was used to record the changes in light transmittance evoked by electrical stimulation in slices prepared from sensorimotor cortex of young adult rats. The spatial characteristics of the optical signal evoked by stimulation of layer II/III, IV, V, or VI were clearly different. Layer IV and V stimulation elicited a radially-oriented region of increased light transmittance which was “hourglass” shaped: its tangential extent was greatest in layers II/III and layer V, and least in layer IV. Layer VI stimulation also elicited a radially-oriented signal but the tangential extent of this signal was the same across layers II–VI—that is, it was column-shaped. Upper layer stimulation produced a signal whose tangential extent was much greater in the upper layers than its radial extent to the deeper layers. The spatial form of the stimulus-evoked intrinsic signal was not dependent on the cytoarchitectonic area in which it was elicited. The tangential and radial distribution of the signal evoked by stimulation of different layers appears to reflect the connectivity of cortex, particularly the horizontal connectivity present in layers II/III, V, and VI, and the interlaminar connections that exist between layers II/III and V and from layers VI to IV. The spatial characteristics of the intrinsic signal were independent of the strength of stimulation used. The idea that inhibitory mechanisms restrict the tangential extent of the signal was evaluated in experiments in which the intrinsic signal was recorded before and after the addition of 10 μM bicuculline methiodide. In all slices studied in this way ( n=12), bicuculline methiodide drastically increased the tangential extent of the signal. In 4/12 slices, the tangential spread of the signal was asymmetric with respect to the stimulus site. Asymmetric spread of the signal occurred for both layer V and layer VI stimulation and, in 2/4 of those cases, could be attributed to a cytoarchitectonic border whose presence appeared to restrict the spread of the signal across the border. Although increasing stimulation strength did not change the spatial characteristics of the radially-oriented signal evoked by layer V or VI stimulation, at maximal stimulus intensity the signal evoked from these layers was often accompanied by a band of decreased light transmittance in the most superficial layers (layers I and II). It is concluded that in vitro intrinsic optical signal imaging allows one to image a response attributable to activation of local subsets of cortical connections. In addition, the opposite effects of high-intensity deep layer stimulation on the superficial layers vs layers III–VI of the same column raise the possibility that the most superficial layers may respond differently to repetitive input drive than the rest of the cortical column.

Non-synaptic release of ATP by electrical stimulation in slices of rat hippocampus, cerebellum and habenula.

ATP is thought to be a fast neurotransmitter in the medial habenula region of the brain, and may be coreleased with other transmitters, for example with glutamate in the hippocampus. We monitored ATP release in rat brain slices using the bioluminescent indicator system luciferin-luciferase. Electrical stimulation of the hippocampus, cerebellum or habenula led to ATP release, but this release was calcium-independent and was not blocked by tetrodotoxin, or by other agents found to block ATP release from red blood cells. Although calcium-dependent ATP release may occur in response to electrical stimulation, it appears to be overwhelmed by calcium-independent release, which may result from electroporation of cells close to the stimulating electrode. Consistent with this, uptake into cells of the fluorescent dye Lucifer yellow was promoted by electrical stimulation. Our data undermine a previous suggestion, based on use of the luciferin-luciferase technique, that ATP is synaptically released with glutamate in the hippocampus.

Chapter 14 Nmda-receptor-dependent synaptic plasticity in the visual cortex

Publisher Summary N-methyl-D-aspartate (NMDA) receptors also play an important role in synaptic long-term potentiation (LTP) and long term depression (LTD) that can be evoked using electrical stimulation in slices of visual cortex. The theoretical analysis of receptive field plasticity suggests a connection between these forms of NMDA receptor-dependent synaptic plasticity. The evidence obtained for NMDA receptor involvement in aspects of visual cortical plasticity is briefly reviewed along with the progress that has been made using theory-driven experimentation in brain slices to elucidate the mechanisms of synaptic modification that may underlie receptive field plasticity in visual cortex. Pharmacological manipulations in vivo show that NMDA receptor activation is required for normal receptive field plasticity in visual cortex, including the loss of responsiveness to inputs deprived of patterned stimulation and the acquisition of responsiveness to new stimulation patterns. The data from slice experiments suggest a precise role for NMDA receptors in receptive field plasticity. These data indicate that the NMDA receptor can serve as an analog detector of coincident pre- and postsynaptic activity, triggering synaptic depression when input activity coincides with low postsynaptic responses and triggering synaptic potentiation when input activity coincides with large postsynaptic responses. In conclusion, substantial progress has been made in refining our understanding of how NMDA receptors contribute to synaptic plasticity in the visual cortex.

Intrastriatal dopaminergic grafts restore inhibitory control over striatal cholinergic neurons.

The aim of the study was to examine the influence of intrastriatal dopaminergic grafts on the functioning of striatal cholinergic neurons using an in vitro superfusion method. Rats bearing unilateral 6-hydroxydopamine lesion of the nigrostriatal dopaminergic system received a cell suspension obtained from ED 14 rat embryonic mesencephali which was injected into the denervated striatum. Lesioned animals displayed an ipsilateral rotation in response to amphetamine (5 mg/kg i.p.). This rotational response disappeared following grafting and there was even a significant contralateral rotation in response to the drug. Apomorphine (0.1 mg/kg s.c.) induced a contralateral rotation following the lesion. This latter response was attenuated in the grafted group. Three months after grafting 350 microns thick slices were prepared from striata from the control and experimental sides of lesioned and graft-bearing animals. The slices were preincubated either with 3H-dopamine (10(-7) M) or 3H-choline (10(-7) M) and then superfused with an oxygenated Krebs-Ringer solution. Stimulation with electrical pulses following preincubation with 3H-dopamine elicited a marked increase of tritium outflow from control slices. Stimulation-evoked overflow was of similar magnitude from slices from striata containing the graft, while it was much reduced in slices from lesioned striata. Amphetamine markedly potentiated the effect of electrical stimulation in slices obtained from control and graft-containing striata. Nomifensine (a dopamine uptake blocker) led to a significant decrease of the overflow of 3H-acetylcholine evoked by electrical stimulation from control striatal slices. This inhibition was antagonized by domperidone, a D2 dopamine receptor blocker, a finding which indicates that the action of nomifensine was indeed due to a potentiation of the action of endogenous dopamine released by the electrical stimulation. A similar, although somewhat attenuated, action of nomifensine and domperidone was observed for striatal slices containing the graft. Amphetamine inhibited the stimulation evoked overflow of 3H-acetylcholine in a dose-dependent manner from striatal slices obtained both from the intact and experimental sides of graft-bearing animals, while it had no action on slices from denervated striata. Finally, the dose-response curve for the inhibition of 3H-acetylcholine release by apomorphine was significantly shifted to the left for slices from the lesioned striata as compared with control slices. This leftward shift was totally abolished in the slices from the graft-containing striatum.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition by yohimbine of the calcium-dependent evoked release of [ 3H]GABA in rat and mouse brain slices in vitro

3alcium-dependent release of [ 3H]GABA was elicited by electrical stimulation in slices of rat and mouse cerebral cortex or by potassium stimulation in the mouse brain-stem. The stimulation-evoked release of [ 3H]GABA was inhibited by yohimbine in a concentration-dependent manner. High concentrations of other α-adrenoceptor antagonists such as phentolamine, RS 21361, idazoxan, rauwolscine and corynanthine also inhibited [ 3H]GABA release. This effect was not observed with pseudoyohimbine or prazosin. [ 3H]GABA release was not affected by exposure to the α-adrenoceptor agonists clonidine, M7, noradrenaline or methoxamine. In addition, clonidine did not antagonize the yohimbine-induced inhibition of [ 3H]GABA release. The inhibitory effect of yohimbine did not result from an interaction with endogenously released noradrenaline since the inhibition was still observed in reserpine-pretreated animals. It is concluded that yohimbine and other α-adrenoceptor antagonists inhibit the stimulation-evoked release of [ 3H]GABA through a mechanism which appears to be independent of the blockade of α 2-adrenoceptors and which does not involve an interaction with endogenous noradrenaline. The present results indicate that yohimbine exerts non-specific actions on the release of [ 3H]GABA and that similar effects can be observed with other α-adrenoceptor blocking agents in high concentrations. Consequently, when studying the effects of yohimbine and other α 2-adrenoceptor antagonists on noradrenergic neurotransmission, the possibility of non-specific effects should be taken into consideration, particularly in the high concentrations range.

Factors influencing maintenance and release of acetylcholine in rat cortical brain slices.

Abstract Rat cortical brain slices were incubated under a variety of conditions and the ACh concentration of the brain slices and incubating fluid was measured. A high potassium concentration in the incubating fluid released ACh from slices and newly-synthesized ACh remained primarily unbound. Under low-potassium conditions in media containing eserine, there was some evidence for ACh re-uptake. Electrical stimulation of slices resulted in an enhanced release of ACh from brain slices without a fall in brain slice ACh concentration. In a medium containing scopolamine, electrical stimulation of slices resulted in greater ACh release compared to stimulated controls and a fall in brain slice ACh levels. Scopolamine increased ACh release only with electrical stimulation.

Protein Synthesis in Brain Slices: EFFECTS OF ELECTRICAL STIMULATION AND ACIDIC AMINO ACIDS

Abstract Protein synthesis in brain slices was followed by the incorporation of 14C-amino acid, mostly 14C-valine; it remained steady for at least 4 hours at 37°. When applied for 15 min or longer, electrical stimulation of slices, by use of alternating line current reduced to 0.5 to 3 volts, inhibited protein synthesis increasingly, the inhibition reaching a plateau at 1.5 volts. For comparison, the paired slice technique was used. When stimulation was in the presence of uniformly labeled 14C-glucose, only slight inhibition of 14C incorporation into proteins was found. However, electrical stimulation also increases the conversion of glucose to amino acids. Therefore, the pool of 14C-labeled amino acids increases with stimulation. If this is taken into account, the degree of inhibition of protein synthesis due to electrical stimulation becomes equal whether the amino acid is internally produced from glucose or is added to the suspending fluid. Acidic amino acids that have excitatory effect on nerves in vivo were found to inhibit protein synthesis in brain slices in the following order of decreasing percentage inhibition: dl-homocysteate (47%), l-glutamate (34%), d-aspartate (35%), and dl-α-aminoadipate (17%). Exposure was for 60 min at 37°. The order is approximately parallel to their excitatory potency on nerves. In both electrical and chemical stimulation, the inhibition of protein synthesis was poorly reversible or irreversible. Analogous effects of electrical stimulation or acidic amino acids were not found with kidney slices. Amino acid permeation in brain slices was unaffected by electrical or chemical stimulation.