High Frequency Tetanic Stimulation Research Articles

Role of N-methyl-d-aspartate receptor subunits GluN2A and GluN2B in auditory thalamocortical long-term potentiation in adult mice

Cortical neurons undergo continuous remodelling throughout development and into adulthood, associated with long-term changes in the synaptic transmission of thalamocortical pathways, i.e., long-term potentiation (LTP); such plasticity is input-specific, reflected in the frequency-specificity of the auditory system. It is well established that thalamocortical LTP is dependent on the activation of N-methyl-d-aspartate (NMDA) receptors. In this study, the roles of NMDA receptor subunits GluN2A and GluN2B in LTP induction were examined in thalamocortical pathways of the auditory system using subunit-selective pharmacological inhibition and in vivo tetanic stimulation of the auditory thalamus, while recording neural response in the primary auditory cortex. Long-term enhancement of thalamocortical field excitatory postsynaptic potentials (i.e., thalamocortical LTP) were induced by high frequency tetanic stimulation of the ventral division of the medial geniculate body. Such enhancement in thalamocortical fEPSPs was decreased when a GluN2A blocker (NVP-M077) was applied to the recording site in the primary auditory cortex and was increased when a GluN2B blocker (Ro25-6981) was applied. Our data suggest that the induction of thalamocortical LTP is dependent on the differential expression of the GluN2A and GluN2B subunits of NMDA receptors in thalamocortical circuits.

Initiation of spreading depression by synaptic and network hyperactivity: Insights into trigger mechanisms of migraine aura.

Background Cortical spreading depression (SD) is thought to underlie migraine aura but mechanisms of triggering SD in the structurally normal, well-nourished cortex of migraine patients remain unknown. Synaptic and network dysfunctions appear to underlie episodic neurological disorders, including migraine. The narrative review summarizes old and recent experimental evidence for triggering SD by synaptic/network mechanisms and discusses the relevance of the data to migraine pathogenesis. Our hypothesis is that under some conditions synaptic/network hyperactivity may reliably ignite SD, and this mechanism may underlie triggering migraine aura in patients. Findings High-frequency tetanic stimulation of the cortex reliably triggers SD in synaptically connected regions; SD is a reliable cortical response to acute hyperexcitability (epileptic seizures), though chronic epilepsy prevents triggering SD; in the hyperexcitable cortex, SD may be triggered by sensory stimulation; compromised glutamatergic transmission plays the critical role in triggering SD. Conclusion SD may be triggered by dynamic network instability produced by dysfunction of calcium-dependent glutamate release. Synaptic drive from subcortical sensory processing structures (brainstem and/or thalamocortical networks) is able to evoke depolarization of hyperexcitable cortical neurons sufficient to initiate the regenerative SD process. Studying SD initiation by synaptic/network hyperexcitability may provide insights into basic mechanisms underlying SD generation in migraine brain.

Age-dependent uncoupling of mitochondria from Ca2+ release units in skeletal muscle

Calcium release units (CRUs) and mitochondria control myoplasmic [Ca2+] levels and ATP production in muscle, respectively. We recently reported that these two organelles are structurally connected by tethers, which promote proximity and proper Ca2+ signaling.Here we show that disposition, ultrastructure, and density of CRUs and mitochondria and their reciprocal association are compromised in muscle from aged mice. Specifically, the density of CRUs and mitochondria is decreased in muscle fibers from aged (>24 months) vs. adult (3-12 months), with an increased percentage of mitochondria being damaged and misplaced from their normal triadic position. A significant reduction in tether (13.8 ± 0.4 vs. 5.5 ± 0.3 tethers/100 µm2) and CRU-mitochondrial pair density (37.4 ± 0.8 vs. 27.0 ± 0.7 pairs/100 µm2) was also observed in aged mice. In addition, myoplasmic Ca2+ transient (1.68 ± 0.08 vs 1.37 ± 0.03) and mitochondrial Ca2+ uptake (9.6 ± 0.050 vs 6.58 ± 0.54) during repetitive high frequency tetanic stimulation were significantly decreased. Finally oxidative stress, assessed from levels of 3-nitrotyrosine (3-NT), Cu/Zn superoxide-dismutase (SOD1) and Mn superoxide dismutase (SOD2) expression, were significantly increased in aged mice. The reduced association between CRUs and mitochondria with aging may contribute to impaired cross-talk between the two organelles, possibly resulting in reduced efficiency in activity-dependent ATP production and, thus, to age-dependent decline of skeletal muscle performance.

Role of Mitofusin-2 in mitochondrial localization and calcium uptake in skeletal muscle

As muscle contraction requires ATP and Ca2+, skeletal muscle function is highly dependent on communication between two major intracellular organelles: mitochondria and sarcoplasmic reticulum (SR). In adult skeletal muscle, mitochondria located within the I-band of the sarcomere are connected to the SR by small ∼10nm electron dense tethers that bridge the outer mitochondrial membrane to the region of SR that is ∼130nm from the site of Ca2+ release. However, the molecular composition of tethers and their precise impact on mitochondrial Ca2+ uptake in skeletal muscle is unclear. Mitofusin-2 (Mfn2) is a transmembrane GTPase present in both mitochondria and ER/SR membranes that forms trans-dimers and participates in mitochondrial fusion. Here we evaluated the role Mfn2 plays in mitochondrial morphology, localization, and functional SR-mitochondrial Ca2+ crosstalk in adult skeletal muscle. Compared to a non-targeting (CTRL) siRNA, in vivo electroporation of 400nM Mfn2 siRNA (Mfn2 KD) into mouse footpads resulted in a marked acute reduction (67±3%) in Mfn2 protein levels in flexor digitorum brevis (FDB) muscles that occurred without a change in other key Ca2+ regulatory proteins. Electron microscopy analyses revealed that Mfn2 knockdown resulted in a change in mitochondria morphology and mis-localization of some mitochondria from the I-band to the A-band region of the sarcomere. To assess the role of Mfn2 in SR-mitochondrial crosstalk, we measured mitochondrial Ca2+ uptake and myoplasmic Ca2+ transients with rhod-2 and mag-fluo-4, respectively, during repetitive high frequency tetanic stimulation (5×100Hz tetani, 500ms/tetani, 0.2 duty cycle) in CTRL and Mfn2 KD fibers. Mitochondrial Ca2+ uptake during repetitive tetanic stimulation was significantly reduced (40%) in Mfn2 KD FDB fibers, which was accompanied by a parallel elevation in the global electrically evoked myoplasmic Ca2+ transient. Mfn2 KD also resulted in a reduction of the mitochondrial membrane potential, which contributed to the observed decrease in activity-dependent mitochondrial Ca2+ uptake. Consistent with this idea, a similar decrease in mitochondrial Ca2+ uptake was also observed in wild type fibers following a comparable reduction in mitochondrial membrane potential induced by acute exposure to a low concentration (50nM) of carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). In addition, both global and mitochondrial Ca2+ transients during repetitive tetanic stimulation were similarly reduced by both slow (EGTA) and fast (BAPTA) Ca2+ chelating agents. Together, these results indicate that Mfn2 promotes proper mitochondrial morphology, localization, and membrane potential required for optimal activity-dependent mitochondrial Ca2+ uptake and buffering of the global myoplasmic Ca2+ transient in adult skeletal muscle.

(R)‐alpha‐methylhistamine Suppresses Inhibitory Neurotransmission in Hippocampal CA1 Pyramidal Neurons Counteracting Propofol‐Induced Amnesia in Rats

Propofol is a short-acting, intravenous general anesthetic that is widely used in clinical practice for short procedures; however, it causes depressed cognitive function for several hours thereafter. (R)-alpha-methylhistamine (RAMH), a selective histamine H3 receptor agonist, can enhance memory retention and attenuates memory impairment in rats. In this study, we investigated whether RAMH could rescue propofol-induced memory deficits and the underlying mechanisms partaking in this process. In the modified Morris water maze (MWM) test, rats were randomized into the following groups: control, propofol (25mg/kg, i.p., 30min before training), RAMH (10mg/kg, i.p., 60min before training), and propofol plus RAMH. All randomized rats were subjected to 2days of training, and a probe test was conducted on day 3. Field excitatory postsynaptic potentials were recorded from CA1 neurons in rat hippocampal slices, and long-term potentiation (LTP) was induced by either theta-burst stimulation (TBS) or high-frequency tetanic stimulation (HFS). Spontaneous and miniature inhibitory (sIPSCs, mIPSCs) or excitatory (sEPSCs, mEPSCs) postsynaptic currents were recorded from CA1 pyramidal neurons by whole-cell patch clamp. In the MWM task, propofol injection significantly impaired spatial memory retention. Pretreatment with RAMH reversed propofol-induced memory retention. In hippocampal CA1 slices, propofol perfusion markedly inhibited TBS- but not HFS-induced LTP. Co-perfusion of RAMH reversed the inhibitory effect of propofol on TBS-induced LTP reduction. Furthermore, in hippocampal CA1 pyramidal neurons, RAMH significantly suppressed the frequency but not the amplitude of sIPSCs and mIPSCs and had little effects on both the frequency and amplitude of sEPSCs and mEPSCs. Our results suggest that RAMH, by inhibiting presynaptic GABAergic neurotransmission, suppresses inhibitory neurotransmission in hippocampal CA1 pyramidal neurons, which in turn reverses inhibition of CA1 LTP and the spatial memory deficits induced by propofol in rats.

Bacterial melanin increases electrical activity of neurons in Substantia Nigra pars compacta.

Bacterial melanin (BM) has been used in different series of experiments as a neuroprotector. It facilitates recovery and regeneration processes after CNS lesions. The action of BM after Substantia Nigra destruction is of major interest. Electrophysiological study tries to reveal the effects of this substance on the electrical activity of Substantia Nigra pars compacta (SNc) neurons. The substance significantly increases the firing rate of SN cdopaminergic neurons. BM increases the rate of excitatory responses after high frequency tetanic stimulation of ipsilateral caudate–putamen. Overall increase in firing rate of SN neurons can contribute to recovery processes after neuronal degeneration in SN.

Membrane excitability and excitation–contraction uncoupling in muscle fatigue

High-frequency tetanic stimulation is associated with an increase in extracellular and T-tubular K(+) and changes of Na(+) and Cl(-) concentrations, membrane depolarization as well as inactivation of voltage-gated Na(+) channels. These alterations are expected to lead to fiber inexcitability, which is largely prevented by mechanisms intrinsic or extrinsic to muscle fibers. They act by adapting electrical membrane properties or by accelerating the reconstitution of ionic homeostasis. The high Cl(-) conductance of muscle fibers supports the K(+) conductance in fast and complete repolarization and creates a mechanism for the fast reuptake of K(+), thereby reducing the T-tubular K(+) accumulation. Excitability is increased by a Ca(2+) and proteinkinase C dependent inhibition of the Cl(-) conductance which is efficient especially in the T-tubular system. Several mediators activate the Na(+)/K(+)-ATPase and thus enhance the restoration of ionic homeostasis. Examples are purines (ATP, ADP), calcitonin-gene related peptide and adrenaline. It is also necessary to adapt the strength of the sarcoplasmic Ca(2+) concentration to the requirements of tetanic contractions. An overwhelming Ca(2+) signal leads to enzymatically driven excitation-contraction uncoupling. This process is most likely driven by the Ca(2+) dependent protease μ-calpain and might lead to the long-lasting fatigue observed after excessive physical activity.

Matrix metalloproteinase-dependent shedding of intercellular adhesion molecule-5 occurs with long-term potentiation

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that can be released or activated in a neuronal activity dependent manner. Although pathologically elevated levels of MMPs may be synaptotoxic, physiologically appropriate levels of MMPs may instead enhance synaptic transmission. MMP inhibitors can block long term potentiation (LTP), and at least one family member can affect an increase in the volume of dendritic spines. While the mechanism by which MMPs affect these changes is not completely understood, one possibility is that the cleavage of specific synaptic cell adhesion molecules plays a role. In the present study, we have examined the ability of neuronal activity to stimulate rapid MMP dependent shedding of the intercellular adhesion molecule-5 (ICAM-5), a synaptic adhesion molecule that is thought to inhibit the maturation and enlargement of dendritic spines. Since such cleavage would likely occur within minutes if it were relevant to a process such as LTP, we focused on post stimulus time points of 30 min or less. We show that NMDA can stimulate rapid shedding of ICAM-5 from cortical neurons in dissociated cell cultures and that such shedding is diminished by pretreatment of cultures with inhibitors that target MMP-3 and -9, proteases thought to influence synaptic plasticity. Additional studies suggest that MMP mediated cleavage of ICAM-5 occurs at amino acid 780, so that the major portion of the ectodomain is released. Since reductions in ICAM-5 have been linked to changes in dendritic spine morphology that are associated with LTP, we also examined the possibility that MMP dependent ICAM-5 shedding occurs following high frequency tetanic stimulation of murine hippocampal slices. Results show that the shedding of ICAM-5 occurs in association with LTP, and that both LTP and the associated ICAM-5 shedding are reduced when slices are pretreated with an MMP inhibitor. Together, these findings suggest that neuronal activity is linked to the shedding of a molecule that may inhibit dendritic spine enlargement and that MMPs can affect this change. While further studies will be necessary to determine the extent to which cleavage of ICAM-5 in particular contributes to MMP dependent LTP, our data support an emerging body of literature suggesting that MMPs are critical mediators of synaptic plasticity.

The Role of Store-Operated Calcium Entry in Store Repletion During Repetitive High Frequency Tetanic Stimulation of Single Skeletal Muscle Fibers

Store-operated Ca2+ Entry (SOCE) involves a trans-sarcolemmal Ca2+ influx mechanism triggered by Ca2+ store depletion. Recently, we demonstrated that SOCE activation in skeletal myotubes involves a functional coupling between STIM1 Ca2+ sensor proteins in the sarcoplasmic reticulum (SR) and Ca2+-permeable Orai1 channels in the sarcolemma. However, the physiological role of SOCE in muscle remains unknown. Here, we monitored myoplasmic Ca2+ transients in mag-fluo-4 loaded mouse flexor digitorum brevis fibres during repetitive high frequency tetanic stimulation (60 consecutive 500ms, 50Hz stimulation trains every 2.5s). In normal Ringer's solution, tetanic Ca2+ transient amplitude decays in three phases: an initial rapid phase (trains 1-10), a second phase of maintained amplitude (trains 10-40), and a final phase of decay (trains 40-60). The maintained phase corresponds to a slightly elevated tail transient integral during each interpulse interval, consistent with activation of Ca2+ influx between tetani. Addition of 0.5mM CdCl2 plus 0.2mM LaCl3 did not alter the initial or final phases of Ca2+ transient decay, but significantly (p<0.01) compromised both the maintained Ca2+ transient (4±3% reduction from trains 10 to 40 in normal Ringer versus 30±3% reduction with Cd/La) and the increase in tail transient integral (which decreased 21±7% with Cd/La) observed during the second phase. Similar results were obtained following addition of either BTP-2 or SKF96365, two known SOCE inhibitors, consistent with SOCE mediating store repletion during the secondary phase of maintained release. Together, these results suggest that repetitive high frequency tetanic stimulation activate a SOCE flux used to replenish SR Ca2+ stores required to maintain subsequent Ca2+ release. Current experiments are testing the validity of this assertion using molecular interventions (transient STIM1 knockdown and dnOrai1 expression) to more selectively inhibit SOCE.

Recording long-term potentiation of synaptic transmission by three-dimensional multi-electrode arrays

BackgroundMulti-electrode arrays (MEAs) have become popular tools for recording spontaneous and evoked electrical activity of excitable tissues. The majority of previous studies of synaptic transmission in brain slices employed MEAs with planar electrodes that had limited ability to detect signals coming from deeper, healthier layers of the slice. To overcome this limitation, we used three-dimensional (3D) MEAs with tip-shaped electrodes to probe plasticity of field excitatory synaptic potentials (fEPSPs) in the CA1 area of hippocampal slices of 129S5/SvEvBrd and C57BL/6J-TyrC-Brd mice.ResultsUsing 3D MEAs, we were able to record larger fEPSPs compared to signals measured by planar MEAs. Several stimulation protocols were used to induce long-term potentiation (LTP) of synaptic responses in the CA1 area recorded following excitation of Schäffer collateral/commissural fibres. Either two trains of high frequency tetanic stimulation or three trains of theta-burst stimulation caused a persistent, pathway specific enhancement of fEPSPs that remained significantly elevated for at least 60 min. A third LTP induction protocol that comprised 150 pulses delivered at 5 Hz, evoked moderate LTP if excitation strength was increased to 1.5× of the baseline stimulus. In all cases, we observed a clear spatial plasticity gradient with maximum LTP levels detected in proximal apical dendrites of pyramidal neurones. No significant differences in the manifestation of LTP were observed between 129S5/SvEvBrd and C57BL/6J-TyrC-Brd mice with the three protocols used. All forms of plasticity were sensitive to inhibition of N-methyl-D-aspartate (NMDA) receptors.ConclusionPrincipal features of LTP (magnitude, pathway specificity, NMDA receptor dependence) recorded in the hippocampal slices using MEAs were very similar to those seen in conventional glass electrode experiments. Advantages of using MEAs are the ability to record from different regions of the slice and the ease of conducting several experiments on a multiplexed platform which could be useful for efficient screening of novel transgenic mice.

Dopaminergic D 1 receptor agonist SKF 38393 induces GAP-43 expression and long-term potentiation in hippocampus in vivo

We evaluated whether activating dopamine D 1 receptors (D 1R) with an agonist will mimic the effects of long-term potentiation (LTP)-inducing electrical stimulation and trigger the expression of the presynaptic growth-associated protein 43 (GAP-43), a putative synaptic plasticity factor. Thus, we conducted GAP-43 protein analyses together with assessments of LTP across CA3/CA1 synapses in guinea pigs administered with SKF38393 (the D 1R agonist) and/or SCH23390 (the D 1R antagonist). Our results showed that guinea pigs treated with SKF38393 coupled with low-frequency stimulation gradually exhibited an LTP-like potentiation in correlation with increased GAP-43 protein expression. However, when SKF38393 treatment was preceded by administration of SCH23390, this antagonized the occurrence of both synaptic potentiation and GAP-43 up-regulation. By comparison, persistent LTP was readily expressed after brief high frequency tetanic stimulation in control guinea pigs, whereas animals injected with SCH23390 and tetanized only developed early-LTP but not late-LTP. Western blot analyses showed GAP-43 up-regulation in the tetanized control guinea pigs but not those injected with SCH23390. We conclude that direct D 1R activations with an agonist can mimic LTP-inducing electrical stimulation to produce GAP-43 up-regulation and synaptic plasticity.

Increased ability to induce long-term potentiation of spinal dorsal horn neurones in monoarthritic rats

Long-term potentiation (LTP) of transmission of impulses in unmyelinated (C-fibre) primary afferents by prior tetanic conditioning stimulation has been demonstrated in the dorsal horn of the spinal cord. Since this potentiation has been proposed to be relevant to the increased responsiveness of spinal neurones associated with peripheral inflammation (central sensitisation), the present experiments compared the induction of LTP in normal rats and rats with monoarthritis. Monoarthritis was induced by injection of complete Freund's adjuvant (CFA) into the left ankle joint of 12 rats. All animals showed behavioural signs of thermal hyperalgesia and were used for electrophysiological experiments after 4–8 days. In each animal, extracellular recordings were obtained from a single, wide dynamic range (WDR) dorsal horn neurone. High frequency tetanic conditioning stimulation of the sciatic nerve gave varying effects on the C-fibre-evoked responses of neurones in the normal rats, with potentiation in two, no change in five and a depression in five. By contrast, conditioning stimulation in rats with inflammation produced a long-lasting potentiation of C-fibre-evoked responses in 11 out of 12 neurones, with no effect in one. The ease with which LTP was induced in animals with inflammation supports the proposal that the underlying mechanisms of LTP are similar to those of the central sensitisation associated with peripheral inflammation.

Potentiation of local field potentials in the anterior cingulate cortex evoked by the stimulation of the medial thalamic nuclei in rats

The limbic thalamus and cingulate cortex are essential components in mediating the affective component of pain responses. In the present study, we examined the excitatory properties of medial thalamus (MT)-evoked field potentials in the anterior cingulated cortex (ACC). We also examined the effects of paired pulses and brief tetanic stimuli of the MT. The aim of this study was to determine whether nociceptive inputs to medial thalamic afferents cause plastic changes in the ACC. In α-chloralose (50 mg/kg, i.v.) anaesthetized rats, tungsten microelectrodes were used to stimulate the MT and to record field potentials in the ACC. The locations of MT were identified by searching and examining their responses to peripheral noxious stimuli. Early negative (about 4.7 ms latency) and late positive (about 11.7 ms) potentials could be evoked in the ACC by MT stimuli. The evoked field potentials were potentiated by prepulse stimulation. Maximal paired pulse facilitation (509±51%) was produced in 80–150 ms interpulse intervals. Evoked field potentials were also potentiated (28.8±6.3% and 29.6±5.9%, respectively) by low (10 Hz/10 s) and high (100 Hz/2s/2×) frequency tetanic stimulation of the MT, with a duration maintained for about 90 s and 120 min, respectively. The potentiation of MT-evoked ACC potentials provides a neural basis for synaptic plasticity, which may be essential for the establishment of pain-initiated conditioning behavior and affective responses to noxious stimuli.

Time-dependent inhibition of hippocampal LTP in vitro following contextual fear conditioning in the rat.

The effects of contextual fear-learning on hippocampal synaptic excitability were investigated by means of high frequency tetanic stimulation (HFS) in rat brain slices (hippocampal CA1 region), prepared at different intervals (immediately, 24 h or 7 days) after a one-trial contextual fear conditioning paradigm session. In the latter, rats that had previously received aversive electrical footshocks in the experimental apparatus exhibited freezing (the conditioned response) when placed again in the same apparatus (retrieval test). It was shown that contextual fear-learning affects the hippocampal synaptic response. In fact, the HFS produced a decrease in the amplitude of short-term (STP) and long-term potentiation (LTP) when compared to control "naïve" subject values. This decrease in STP amplitude could be observed only in slices prepared immediately after the training session. A decrease in the amplitude of long-term but not short-term potentiation was also observed at 24 h. At 7 days, no decreases in amplitude were observed. These modifications may be thought of as specifically associated with the learning process as they were not recorded in brain preparations from "shock-only" rats (i.e. those that received the same number of aversive stimuli of equal intensity as the conditioned group but with the shocks compressed temporally so that the shocked subjects could not associate nociceptive stimulation and surroundings - no conditioned freezing during retention testing). In "exploration" preparations (brain slices from rats having only freely explored the experimental apparatus without receiving any adverse stimulation) a decrease in LTP amplitude was recorded only immediately after the training session, and STP was never modified. The synaptic response modifications do not appear to be due to presynaptic events, as they are not associated with paired-pulse facilitation curve (PPF) modifications. The present results show that contextual fear conditioning and exploration of a novel environment (i) reduce the ability to induce synaptic plasticity; (ii) differentially influence STP and LTP and that (iii) the persistence of synaptic modifications depends on an animal's prior experience.

Activity- and Ca(2+)-dependent modulation of surface expression of brain-derived neurotrophic factor receptors in hippocampal neurons.

Brain-derived neurotrophic factor (BDNF) has been shown to regulate neuronal survival and synaptic plasticity in the central nervous system (CNS) in an activity-dependent manner, but the underlying mechanisms remain unclear. Here we report that the number of BDNF receptor TrkB on the surface of hippocampal neurons can be enhanced by high frequency neuronal activity and synaptic transmission, and this effect is mediated by Ca(2+) influx. Using membrane protein biotinylation as well as receptor binding assays, we show that field electric stimulation increased the number of TrkB on the surface of cultured hippocampal neurons. Immunofluorescence staining suggests that the electric stimulation facilitated the movement of TrkB from intracellular pool to the cell surface, particularly on neuronal processes. The number of surface TrkB was regulated only by high frequency tetanic stimulation, but not by low frequency stimulation. The activity dependent modulation appears to require Ca(2+) influx, since treatment of the neurons with blockers of voltage-gated Ca(2+) channels or NMDA receptors, or removal of extracellular Ca(2+), severely attenuated the effect of electric stimulation. Moreover, inhibition of Ca(2+)/calmodulin-dependent kinase II (CaMKII) significantly reduced the effectiveness of the tetanic stimulation. These findings may help us to understand the role of neuronal activity in neurotrophin function and the mechanism for receptor tyrosine kinase signaling.

Signaling Mechanisms Mediating BDNF Modulation of Synaptic Plasticity in the Hippocampus

Although recent studies indicate that brain-derived neurotrophic factor (BDNF) plays an important role in hippocampal synaptic plasticity, the underlying signaling mechanisms remain largely unknown. Here, we have characterized the signaling events that mediate the BDNF modulation of high-frequency synaptic transmission. Mitogen-associated protein kinase (MAPK), phosphotidylinositol-3 kinase (PI3K), and phospholipase C-gamma (PLC-gamma) are the three signaling pathways known to mediate neurotrophin signaling in other systems. In neonatal hippocampal slices, application of BDNF rapidly activated MAPK and PI3K but not PLC-gamma. BDNF greatly attenuated synaptic fatigue at CA1 synapses induced by a train of high-frequency, tetanic stimulation (HFS). Inhibition of the MAPK and PI3K, but not PLC-gamma, prevented the BDNF modulation of high-frequency synaptic transmission. Neurotrophin-3 (NT-3), a close relative of BDNF, did not activate MAPK or PI3K and had no effect on synaptic fatigue in the neonatal hippocampus. Neither forskolin, which activated MAPK but not PI3 kinase, nor ciliary neurotrophic factor (CNTF), which activated PI3K but not MAPK, affected HFS-induced synaptic fatigue. Treatment of the slices with forskolin together with CNTF still had no effect on synaptic fatigue. Thus, although the activation of MAPK and PI3K is required, the two together are not sufficient to mediate the BDNF effect. Inhibition of new protein synthesis by anisomycin or cycloheximide did not prevent the BDNF effect. These data suggest that BDNF modulation of high-frequency transmission is independent of protein synthesis but requires MAPK and PI3K and yet another signaling pathway to act together in the hippocampus.

Potentiation of phosphoinositide-derived signals during LTP in intact rat brain.

In order to examine the relationship between long-term potentiation (LTP) and phosphoinositide (PI) turnover, we evaluated these throughout anesthetized rat brain using carbon-11-labeled diacylglycerol (11C-DAG). High-frequency tetanic stimulation (400 pulses at 400 Hz) to the perforant pathway induced LTP in rat dentate gyrus. In autoradiograms of rat brains, LTP was associated with the occurrence of multiple highly radioactive spots in many regions distant from the stimulated site. Following i.v. administration of an NMDA receptor antagonist prior to stimulation, however, no high-density spots were found. These findings directly demonstrate that potentiation of phosphoinositide-derived signaling was induced during LTP, and the finding of multiple location suggests the occurrence of polysynaptic neurotransmission through neural networks pertaining to learning and memory.

Activity-dependent regulation of dopamine content in the olfactory bulbs of naris-occluded rats

Several lines of evidence strongly suggest that reduced olfactory nerve activity results in decreased bulb dopamine content. In the present study, high performance liquid chromatography with electrochemical detection was used to assess catecholamine levels in bulbs from postnatal day 60 rats that had undergone either unilateral naris cautery or a sham surgery on day 30. Thirty days of odor deprivation dramatically reduced dopamine and dihydroxyphenylacetic acid levels in functionally-deprived bulbs (ipsilateral to occluded nares) as compared to contralateral controls, while norepinephrine and dihydroxyphenylglycol levels were unchanged. The loss of dopamine was more severe in medial as compared to lateral aspects of experimental bulbs, while the loss of dihydroxyphenylacetic acid was similar on the two sides. To test directly the hypothesis that afferent activity regulates dopamine and dihydroxyphenylacetic acid content, 1 h of high frequency tetanic nerve stimulation was provided to the rostral-medial olfactory nerve layer in deprived olfactory bulbs, and catecholamine levels were assessed from 6 to 192 h later. Partial and temporary recovery of dopamine was observed in medial aspects of the bulb when rats were examined 96 h later, while consistent recovery of dihydroxyphenylacetic acid content was not apparent. These data corroborate evidence that olfactory nerve activity is a potent regulator of bulb dopamine and indicate that continued afferent input is necessary to maintain dopamine levels.

Modification of parallel activity elicited by propagating bursts in developing networks of rat cortical neurones.

Networks of cultured cortical neurones exhibit regular, synchronized, propagating bursts which are synaptically mediated, and which are hypothesized to play a part in activity-dependent formation of connections during development in vivo. The relationship between the strength of synaptic connections and the characteristics of synchronized propagating bursting, however, is unclear. Modification of synchronized activity in cortical cultures in response to electrical stimulation was examined using multisite electrode array recording. By measuring the response of the network to weak, localized, test stimulation (TS), we observed a potentiation of activity following a relatively stronger inducing stimulation (IS). This potentiation was evident as an increased probability of eliciting bursts by TS, an increased frequency of spontaneous bursts and number of spikes per burst, and increased speed of burst propagation, and it lasted for at least 20 min. Changing the parameters of IS revealed that high frequency tetanic stimulation is not necessary to induce potentiation, while it is essential for IS to produce a regeneratively propagating burst. The results provide a direct demonstration of modification of both the spatial and temporal characteristics of synchronized network activity, and suggest an important physiological role for propagating synchronized bursting, as a mechanism for inducing plastic modifications in the developing cortex.

The synergism between metabotropic glutamate receptor activation and arachidonic acid on glutamate release is occluded by induction of long-term potentiation in the dentate gyrus.

In synaptosomes prepared from dentate gyrus, activation of the metabotropic glutamate receptor by the specific agonist, trans-1-amino-cyclopentyl-1,3-dicarboxylate, increases release of glutamate in the presence of a low concentration of arachidonic acid. A similar interaction between trans-1-amino-cyclopentyl-1,3-dicarboxylate and arachidonic acid is observed on inositol phospholipid turnover and on protein kinase C activity. We report here that when long-term potentiation is induced in the dentate gyrus by high frequency tetanic stimulation to the perforant path, the synergism between arachidonic acid and trans-1-amino-cyclopentyl-1,3-dicarboxylate is occluded. The occlusion of the synergistic action between arachidonic acid and trans-1-amino-cyclopentyl-1,3-dicarboxylate on glutamate release extended to occlusion of the effect in inositol phospholipid turnover and protein kinase C activation in synaptosomes prepared from dentate gyrus in which long-term potentiation was induced in vivo. One interpretation of the results presented here is that tetanic stimulation is followed by stimulation of metabotropic glutamate receptors at a time when arachidonic acid concentration in the synaptic region is elevated, and that this interaction triggers the presynaptic changes required for expression of long-term potentiation.