High-frequency Afferent Stimulation Research Articles

Chronic stress dampens excitatory synaptic gain in the paraventricular nucleus of the hypothalamus.

Glutamatergic synaptic inputs to corticotrophin-releasing hormone (CRH) secreting neurons in the paraventricular nucleus of the hypothalamus (PVN) are required for stress-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis. These synapses also undergo stress-induced plasticity, thereby influencing HPA axis stress adaptation. By using patch clamp electrophysiology, we show that, in adult non-stressed mice, action potentials at these glutamatergic afferents elicit multiquantal transmission to the postsynaptic PVN-CRH neurons (i.e. synaptic multiplicity). Mechanistically, synaptic multiplicity results from multivesicular release at common synaptic sites, which is facilitated upon elevation of release probability, effectively increasing the upper limit of the dynamic range of synaptic transmission. Following chronic variable stress, functional PVN glutamate synapse number increases, although its synaptic multiplicity paradoxically decreases. These two contrasting synaptic changes can, respectively, increase the baseline excitatory drive while also limiting the capacity for potentiation, and may preferentially increase the baseline excitatory drive onto PVN-CRH neurons. The activation of the hypothalamic-pituitary-adrenal (HPA) axis relies on excitation of neuroendocrine neurons in the paraventricular nucleus of the hypothalamus (PVN) that secrete corticotrophin-releasing hormone (CRH). Afferent glutamate synapses onto these PVN-CRH neurons convey critical excitatory inputs during stress, and also undergo stress-induced plasticity, highlighting their roles in both stress activation and adaptation of the HPA axis. In the present study, using whole-cell patch clamp recordings from PVN-CRH neurons in brain slices from adult mice, we found that the amplitude of action potential-dependent spontaneous EPSCs (sEPSCs) was larger than that of action potential independent miniature EPSCs (mEPSCs), suggesting that action potentials at individual axons recruited multiquantal transmission onto the same postsynaptic neurons (i.e. synaptic multiplicity). The large, putative multiquantal sEPSCs had fast rise times similar to mEPSCs, and were abolished by replacing extracellular Ca2+ with Sr2+ , indicating Ca2+ -dependent synchronous release of multiple vesicles. Application of a low affinity, fast dissociating competitive AMPA receptor antagonist γ-d-glutamylglycine revealed that synaptic multiplicity resulted from multivesicular release targeting a common population of postsynaptic receptors. High-frequency afferent stimulation facilitated synaptic multiplicity, effectively increasing the upper limit of the dynamic range of synaptic transmission. Finally, we found that chronic variable stress (CVS), a stress model known to cause basal HPA axis hyperactivity, increased sEPSCs frequency but paradoxically decreased synaptic multiplicity. These results suggest that the CVS-induced synaptic changes may elevate the baseline excitatory drive at the same time as limiting the capacity for potentiation, and may contribute to the basal HPA axis hyperactivity.

The repetition timing of high frequency afferent stimulation drives the bidirectional plasticity at central synapses in the rat medial vestibular nuclei

In this study we show that high frequency stimulation (HFS, 100Hz) of afferent fibers to the medial vestibular nucleus (MVN) can induce opposite long-term modifications of synaptic responses in the type B neurons depending upon the stimulation pattern. Long burst stimulation (LBS: 2s) and short burst stimulation (SBS: 0.55s) were applied with different burst number (BN) and inter-burst intervals (IBI). It results that both LBS and SBS can induce either N-methyl-d aspartate receptors (NMDARs)-mediated long-term potentiation (LTP) or long-term depression (LTD), depending on temporal organization of repetitive bursts. In particular, the IBI plays a relevant role in guiding the shift from LTP to LTD since by using both LBS and SBS LTP is induced by shorter IBI than LTD. By contrast, the sign of long-term effect does not depend on the mean impulse frequency evaluated within the entire stimulation period. Therefore, the patterns of repetitive vestibular activation with different ratios between periods of increased activity and periods of basal activity may lead to LTP or LTD probably causing different levels of postsynaptic Ca2+. On the whole, this study demonstrates that glutamatergic vestibular synapse in the MVN can undergo NMDAR-dependent bidirectional plasticity and puts forward a new aspect for understanding the adaptive and compensatory plasticity of the oculomotor responses.

Decreased afferent excitability contributes to synaptic depression during high-frequency stimulation in hippocampal area CA1

Long-term potentiation (LTP) is often induced experimentally by continuous high-frequency afferent stimulation (HFS), typically at 100 Hz for 1 s. Induction of LTP requires postsynaptic depolarization and voltage-dependent calcium influx. Induction is more effective if the same number of stimuli are given as a series of short bursts rather than as continuous HFS, in part because excitatory postsynaptic potentials (EPSPs) become strongly depressed during HFS, reducing postsynaptic depolarization. In this study, we examined mechanisms of EPSP depression during HFS in area CA1 of rat hippocampal brain slices. We tested for presynaptic terminal vesicle depletion by examining minimal stimulation-evoked excitatory postsynaptic currents (EPSCs) during 100-Hz HFS. While transmission failures increased, consistent with vesicle depletion, EPSC latencies also increased during HFS, suggesting a decrease in afferent excitability. Extracellular recordings of Schaffer collateral fiber volleys confirmed a decrease in afferent excitability, with decreased fiber volley amplitudes and increased latencies during HFS. To determine the mechanism responsible for fiber volley changes, we recorded antidromic action potentials in single CA3 pyramidal neurons evoked by stimulating Schaffer collateral axons. During HFS, individual action potentials decreased in amplitude and increased in latency, and these changes were accompanied by a large increase in the probability of action potential failure. Time derivative and phase-plane analyses indicated decreases in both axon initial segment and somato-dendritic components of CA3 neuron action potentials. Our results indicate that decreased presynaptic axon excitability contributes to depression of excitatory synaptic transmission during HFS at synapses between Schaffer collaterals and CA1 pyramidal neurons.

Heterosynaptic plasticity induced by intracellular tetanization in layer 2/3 pyramidal neurons in rat auditory cortex

Associative Hebbian-type synaptic plasticity underlies the mechanisms of learning and memory; however, Hebbian learning rules lead to runaway dynamics of synaptic weights and lack mechanisms for synaptic competition.Heterosynaptic plasticity may solve these problems by complementing plasticity at synapses that were active during the induction, with opposite-sign changes at non-activated synapses. In visual cortex, a potential candidate mechanism for normalization is plasticity induced by a purely postsynaptic protocol, intracellular tetanization. Here we asked if intracellular tetanization can induce long-term plasticity in auditory cortex. We recorded excitatory postsynaptic potentials (EPSPs) of regular (n =76) and all-or-none (n =24) type in layer 2/3 pyramidal cells in slices from rat auditory cortex. After intracellular tetanization, 32 of 76 regular inputs (42%) showed long-term depression, 21 inputs (28%) showed potentiation and 23 inputs (30%) did not change. The direction of plasticity correlated with the initial release probability: inputs with initially low release probability tended to be potentiated, while inputs with high release probability tended to be depressed. Thus, intracellular tetanization had a normalizing effect on synaptic efficacy. Induction of plasticity by intracellular tetanization required a rise of intracellular [Ca(2+)], because it was impaired by chelating intracellular calcium with EGTA. The long-term changes induced by intracellular tetanization involved both pre and postsynaptic mechanisms. EPSP amplitude changes were correlated with changes of release indices: paired-pulse ratio and the inverse of the coefficient of variation (CV(-2)). Furthermore at some all-or-none synapses, changes of averaged response amplitude were correlated with a change of the failure rate, without a change of the synaptic potency, measured as averaged amplitude of successful responses. Presynaptic components of plastic changes were abolished in experiments with blockade of NO-synthesis and spread, indicating involvement of NO signalling. These results demonstrate that the ability of purely postsynaptic challenges to induce plasticity is a general property of pyramidal neurons of both auditory and visual cortices.

Plasticity of synaptic glun receptors is required for the Src‐dependent induction of long‐term potentiation at CA3‐CA1 synapses

The induction of long-term potentiation (LTP) of CA3-CA1 synapses requires activation of postsynaptic N-methyl-D-aspartate receptors (GluNRs). At resting potential, the contribution of GluNRs is limited by their voltage-dependent block by extracellular Mg(2+). High-frequency afferent stimulation is required to cause sufficient summation of excitatory synaptic potentials (EPSPs) to relieve this block and to permit an influx of Ca(2+). It has been assumed that this relief of Mg(2+) block is sufficient for induction. We postulated that the induction of LTP also requires a Src-dependent plasticity of GluNRs. Using whole-cell recordings, LTP (GluARs) of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors-EPSCS was induced by pairing postsynaptic depolarization with presynaptic stimulation. This LTP was both GluNR and Src-dependent, being sensitive to AP-5, a GluNR selective antagonist, or to SU6656, a Src-selective inhibitor. When CNQX was used to block all GluARs, we observed a long-lasting potentiation of GluNR-mediated EPSCs. This plasticity was prevented by transiently blocking GluNRs during the induction protocol or by chelating intracellular Ca(2+). GluNRs plasticity was also prevented by bath applications of SU6656 or intracellular applications of the Src-selective inhibitory peptide, Src(40-58). It was also blocked by preventing activation of protein kinase C, a kinase that is upstream of Src-kinase-dependent regulation of GluNRs. Both GluN2A and GluN2B receptors were found to contribute to the plasticity of GluNRs. The contribution of GluNRs and, in particular, their plasticity to the maintenance of LTP was explored using AP5 and SU6656, respectively. When applied >20 min after induction neither drug influenced the magnitude of LTP. However, when applied immediately after induction, treatment with either drug caused the initial magnitude of LTP to progressively decrease to a sustained phase of reduced amplitude. Collectively, our findings suggest that GluNR plasticity, although not strictly required for induction, is necessary for the maintenance of a nondecrementing component of LTP.

Cyclic estrogenic fluctuation influences synaptic transmission of the medial vestibular nuclei in female rats

Conclusion: The estrous cycle in female rats influences the basal synaptic responsiveness and plasticity of the medial vestibular nucleus (MVN) neurons through different levels of circulating 17β-estradiol (cE2). Objective: The aim of this study was to verify, in the female rat, whether cyclic fluctuations of cE2 influence long-term synaptic effects induced by high frequency afferent stimulation (HFS) in the MVN, since we found that HFS in the male rat induces fast long-term potentiation (fLTP), which depends on the neural synthesis of E2 (nE2) from testosterone (T). Methods: We analyzed the field potential (FP) evoked in the MVN by vestibular afferent stimulation, under basal conditions, and after HFS, in brainstem slices of female rats during high levels (proestrus, PE) and low levels (diestrus, DE) of cE2. Selective blocking agents of converting T enzymes were used. Results: Unlike in the male rat, HFS induced three effects: fLTP through T conversion into E2, and slow LTP (sLTP) and long-term depression (LTD), through T conversion into DHT. The occurrence of these effects depended on the estrous cycle phase: the frequency of fLTP was higher in DE, and those of sLTP and LTD were higher in PE. Conversely, the basal FP was also higher in PE than in DE.

High-Frequency Afferent Stimulation Induces Long-Term Potentiation of Field Potentials in the Ventral Tegmental Area

Excitatory synapses on dopamine neurons in the VTA can undergo both long-term potentiation and depression. Additionally, drug-induced plasticity has been found at VTA synapses, and is proposed to play a role in reward-related learning and addiction by modifying dopamine cell firing. LTP at these synapses is difficult to generate experimentally in that it requires an undisturbed intracellular milieu and is often small in magnitude. Here, we demonstrate the induction of LTP as a property of evoked field potentials within the VTA. Excitatory field potentials were recorded extracellularly from VTA neurons in acute horizontal midbrain slices. Using extracellular and intracellular recording techniques, we found that evoked field potentials originate within the VTA itself and are largely composed of AMPA receptor-mediated EPSPs and action potentials triggered by activation of glutamatergic synapses on both dopamine and GABA neurons. High-frequency afferent stimulation (HFS) induced LTP of the field potential. The induction of this LTP was blocked by application of the NMDAR antagonist, d-APV, prior to HFS. As reported previously, glutamatergic synapses on GABA neurons did not express LTP while those on dopamine neurons did. We conclude that the potentiation of glutamatergic synapses on dopamine neurons is a major contributor to NMDA receptor-dependent LTP of the field potential. Field potential recordings may provide a convenient approach to explore the basic electrophysiological properties of VTA neurons and the development of addiction-related processes in this brain region.

A delayed response enhancement during hippocampal presynaptic plasticity in mice

High frequency afferent stimulation of chemical synapses often induces short-term increases in synaptic efficacy, due to increased release probability and/or increased supply of readily releasable synaptic vesicles. This may be followed by synaptic depression, often caused by vesicle depletion. We here describe an additional, novel type of delayed and transient response enhancement phase which occurred during prolonged stimulation at 5-20 Hz frequency of excitatory glutamatergic synapses in slices from the adult mouse CA1 hippocampal region. This second enhancement phase, which was most clearly defined at physiological temperatures and essentially absent at 24 degrees C, was dependent on the presence of F-actin filaments and synapsins I and/or II, and could not be ascribed to changes in presynaptic action potentials, inhibitory neurotransmission or glutamate receptor desensitization. Time course studies showed that the delayed response phase interrupted the synaptic decay 3-4 s after stimulus train initiation and continued, when examined at 5-10 Hz frequencies, for approximately 75 stimuli before decay. The novel response enhancement, probably deriving from a restricted pool of synaptic vesicles, may allow maintenance of synaptic efficacy during prolonged periods of excitatory synaptic activity.

Shifting the Balance

The striatum controls motor activity through the balance of the activity of a direct and indirect pathway. Kreitzer and Malenka used mice engineered to express green fluorescent protein in neurons of the direct pathway or indirect pathway to show that the cells of the indirect pathway exhibited (i) higher probability of neurotransmitter release, (ii) increased ratios of NMDA- to AMPA-type glutamate receptors, and (iii) increased rate of synaptic activity compared with cells of the direct pathway. High-frequency afferent stimulation only triggered long-term depression (LTD) in the indirect pathway, and application of an antagonist of the endocannabinoid receptor CB1 or the D2 dopamine receptor blocked this response. However, this difference was not caused by a lack of CB1 receptors in the direct pathway, because application of CB1 agonist increased both direct and indirect pathway excitatory postsynaptic potentials. Instead, endocannabinoid release or synthesis in response to synaptic activity appeared to be limited to the indirect pathway. Mouse models of Parkinson's disease showed a lack of an indirect pathway LTD response that was restored if the slices were exposed to a D2 agonist or an inhibitor of endocannabinoid degradation. In the Parkinson's model mice, treatment with both a D2 agonist and the endocannabinoid degradation inhibitor reduced catalepsy and improved motor activity compared to treatment with the D2 agonist alone. A. C. Kreitzer, R. C. Malenka, Endocannabinoid-mediated rescue of striatal LTD and motor deficits in Parkinson's disease models. Nature 445 , 643-647 (2007). [PubMed]

The role of NMDAR subtypes and charge transfer during hippocampal LTP induction

Activation of NMDA receptors (NMDARs) is a requirement for persistent synaptic alterations, such as long-term potentiation of synaptic transmission (LTP). NMDARs are composed of NR1 and NR2 subunits, and NR2 subunit-dependent gating properties of NMDAR subtypes cause dramatic differences in the timing of charge transfer. These postsynaptic temporal profiles are further influenced by the frequency of synaptic activation. Here, we investigated in the CA1 region of hippocampal slices from P28 mice, whether particular NMDAR subtypes are recruited based on NR2 subunit-specific gating following different induction protocols. For high frequency afferent stimulation (HFS), we found that genetic impairment of NR2A or pharmacological block of NR2A- or NR2B-type NMDARs can reduce field LTP. In contrast, when pairing low frequency synaptic stimulation with postsynaptic depolarization (LFS pairing) in single CA1 neurons, pharmacological antagonism of either subtype modestly reduced the charge transfer during LFS pairing without reducing the LTP magnitude. These results indicate that HFS-triggered LTP is induced by more than one NMDAR subtype, whereas a single subtype is sufficient during LFS pairing. Analysis of charge transfer during LFS pairing in 13 different conditions revealed a threshold for LTP induction, which was independent of the NR2 antagonist tested. Thus, at least for LFS pairing, the amount of charge transfer, and thus Ca 2+ influx, during LTP induction is a factor more critical than the participation of a particular NMDAR subtype.

Postnatal development of intrinsic GABAergic rhythms in mouse hippocampus

The local circuitry of the mammalian limbic cortices, including the hippocampus, is capable of generating spontaneous rhythmic activities of 0.5–4Hz when isolated in vitro. These rhythmic activities are mediated by synchronous inhibitory postsynaptic potentials in pyramidal neurons as the result of repeated discharges of inhibitory interneurons. As such, they are thought to represent an intrinsic inhibitory rhythm. It is unknown at present whether such a rhythm occurs in the immature rodent hippocampus and, if so, the postnatal time window in which it develops. We explored these issues using our recently developed whole mouse hippocampal isolate preparation in vitro. We found that spontaneous rhythmic field potentials started to emerge in mouse hippocampal isolates around postnatal day 10, stabilized after postnatal day 15 and persisted into adulthood. In postnatal days 11–14 mouse hippocampi, the properties of these rhythmic potentials were in keeping with a CA3-driven, IPSP-based intrinsic network activity. The lack of spontaneous field rhythm in neonatal (postnatal days 2–7) hippocampi cannot be attributed to the excitatory activities mediated by gamma-aminobutyric acid type A (GABA-A) receptors, as chloride-dependent hyperpolarizing inhibitory postsynaptic potentials were detectable in neonatal pyramidal neurons at voltages near resting potentials and pharmacological antagonisms of GABA-A receptors produced robust epileptiform discharges in neonatal hippocampi. High frequency afferent stimulation or applications of 4-aminopyridine at low micromolar concentrations failed to induce persistent field rhythm in neonatal hippocampi, suggesting that an overall weak glutamatergic drive is not the sole causing factor. We suggest that the inhibitory postsynaptic potential-based spontaneous rhythmic field potentials develop in a discrete time window during the second postnatal week in the mouse hippocampus due to a fine-tuning in the structure and function of CA3 recurrent circuitry and associated GABAergic inhibitory interneurons.

Regional and postnatal heterogeneity of activity-dependent long-term changes in synaptic efficacy in the dorsal striatum.

High-frequency activation of excitatory striatal synapses produces lasting changes in synaptic efficacy that may contribute to motor and cognitive functions. While some of the mechanisms responsible for the induction of long-term potentiation (LTP) and long-term depression (LTD) of excitatory synaptic responses at striatal synapses have been characterized, much less is known about the factors that govern the direction of synaptic plasticity in this brain region. Here we report heterogeneous activity-dependent changes in the direction of synaptic strength in subregions of the developing rat striatum. Neurons in the dorsolateral region of the anterior striatum tended to express LTD after high-frequency afferent stimulation (HFS) in slices from animals aged P15-P34. However, HFS in dorsolateral striatum from P12-P14 elicited an N-methyl-D-aspartate (NMDA) receptor-dependent form of LTP. Synapses in the dorsomedial anterior striatum exhibited a propensity to express an NMDA-receptor dependent form of LTP across the entire developmental time period examined. The NMDA receptor antagonist (+/-)-2-amino-5-phosphopentanoic acid (APV) inhibited evoked excitatory postsynaptic potentials recorded in striatum obtained from P12-P15 rats but had little effect in striatum from older animals. The expression of multiple forms of synaptic plasticity in the striatum suggests mechanisms by which this brain region plays pivotal roles in the acquisition or encoding of some forms of motor sequencing and stereotypical behaviors.

Dendritically Released Peptides Act as Retrograde Modulators of Afferent Excitation in the Supraoptic Nucleus In Vitro

Oxytocin (OXT) and vasopressin (VP) are known to be released from dendrites of magnocellular neurons. Here, we show that these peptides reduced evoked EPSCs by a presynaptic mechanism, an effect blocked by peptide antagonists and mimicked by inhibition of endogenous peptidases. Dendritic release of peptides, elicited with depolarization achieved by high frequency stimulation of afferents or with current injection into an individual neuron, induced short-term synaptic depression similar to that seen following exogenous peptide application and was prevented by peptide antagonists. Thus, dendritically released peptides depress evoked EPSCs in magnocellular neurons by activating presynaptic OXT and/or VP receptors. Such a retrograde modulatory action on afferent excitation may serve as a feedback mechanism to permit peptidergic neurosecretory neurons to autoregulate their own activity.

Cholecystokinin and response modifications in hippocampal field CA1

This study indicates that cholecystokinin (CCK-8S) and high-frequency afferent stimulation (HFS) modify neuronal responses to stimulation of the perforant pathway in hippocampal field CA1. HFS, previously neutral with regard to modifications, induced depression of both field evoked action potentials (EAPs) and field excitatory postsynaptic potentials (EPSPs) in the presence of 500 nM CCK-8S. However, in the presence of 1 µM CCK-8S, HFS induced a potentiation of both field evoked responses. With intermediate concentrations of CCK-8S, HFS was indifferent to potentiation or depression: 750 nM CCK-8S was not associated with modifications, whereas only potentiation of EAPs occurred with 900 nM CCK-8S. The mechanism underlying HFS/CCK-8S-induced LTP or LTD may involve reduction of γ-aminobutyric acid (GABA) release. LTP and LTD may underlie certain forms of memory, and this study implicates hippocampal CCK-8S/GABAergic pathways in their induction.

The role of nitric oxide in hippocampal long-term potentiation

Long-term potentiation is a long-lasting, use-dependent increase in the strength of synaptic connections. We investigated the role of nitric oxide (NO) in determining the duration of potentiation induced by high frequency stimulation of afferents in the CAI region of the rat hippocampus. The calcium/calmodulin-dependent production of NO can be initiated by activation of excitatory amino acid receptors and results in increased levels of cGMP in target cells. Here we report that only a relatively short-term potentiation can be induced in the presence of nitr- l-arginine methyl ester ( l-NAME), an NO synthase inhibitor. The effects of l-NAME on the duration of potentiation are partially reversed by coadministration of l-arginine, a precursor of neuronal NO, and by dibutyryl cGMP. Hemoglobin, which binds extracellular NO, also shortens the duration of stimulus-induced potentiation. The results suggest a role for NO in the maintenance of activity-dependent synaptic enhancements, possibly via the generation of cGMP.

The contribution of increases in extracellular potassium to primary afferent depolarization in the bullfrog spinal cord

To assess the extent to which depolarization by accumulated K + contributes to the generation of primary afferent depolarization (PAD), the isolated bullfrog spinal cord was superfused with K +-rich Ringer solutions and the resultant dorsal root depolarizations were recorded extracellularly. Action potential blockade (with tetrodotoxin) did not reduce the K +-induced depolarization of primary afferents, indicating that the depolarization was generated locally in the region around the afferents. In this respect superfusion with K +-rich solutions adequately models the localized K + accumulation which occurs physiologically during afferent activity. K +-induced depolarizations were decreased in the presence of 20 mM Mg 2+; this effect was due to a decrease in the membrane response to K + and not to blockade of K +-induced transmitter release onto primary afferents. The depolarization caused by a K + concentration comparable to a maximum estimate of the K + accumulating around afferent terminals following a single afferent volley was found to account for no more than about one-third of the DRP height. However, higher K + levels, comparable to those resulting from high frequency afferent stimulation, caused large depolarizations of primary afferents, sometimes greater than the DRP amplitude. Therefore, K +-induced depolarization may contribute more significantly to PAD evoked by high frequency afferent activity.