Potentiation of excitatory postsynaptic potentials during and after repetitive stimulation in thin hippocampal sections

Summary Excitatory postsynaptic potentials (EPSPs) elicited by mossy fiber stimulation were recorded intracellularly from neurons in the CA₃ region in thin hippocampal sections in vitro and potentiation of the EPSPs was examined during and after repetitive stimulation. Inhibitory postsynaptic potentials (IPSPs) and seizure discharges were blocked by bicuculline and high concentrations of Mg²⁺. When two shocks were applied at short intervals, the second EPSP was markedly potentiated. This potentiation declined exponentially with a time-constant of about 180 ms and was unaffected by changes in ambient temperature. The amount of potentiation during a pulse train was explained by summation of potentiation by individual pulses. Post tetanic potentiation lasted longer in media containing Ca²⁺ at higher concentrations and Mg²⁺ at lower concentrations. At high Ca²⁺ concentrations, tetanic stimulation induced long-term potentiation which was occasionally preceded by a long-lasting suppression. Tetanus to a bundle of mossy fibers potentiated EPSPs elicited by stimulation of a separate bundle of mossy fibers (heterosynaptic potentiation) but did not augment EPSPs elicited by fimbrial stimulation.     

Multiple forms of LTP in hippocampal CA3 neurons use a common postsynaptic mechanism.

We investigated long-term potentiation (LTP) at mossy fiber synapses on CA3 pyramidal neurons in the hippocampus. Using Ca2+ imaging techniques, we show here that when postsynaptic Ca2+ was sufficiently buffered so that [Ca2+]i did not rise during synaptic stimulation, the induction of mossy fiber LTP was prevented. In addition, induction of mossy fiber LTP was suppressed by postsynaptic injection of a peptide inhibitor of cAMP-dependent protein kinase. Finally, when ionotropic glutamate receptors were blocked, LTP depended on the postsynaptic release of Ca2+ from internal stores triggered by activation of metabotropic glutamate receptors. These results support the conclusion that mossy fiber LTP and LTP at other hippocampal synapses share a common induction mechanism involving an initial rise in postsynaptic [Ca2+].     

Recurrent mossy fiber pathway in rat dentate gyrus: synaptic currents evoked in presence and absence of seizure-induced growth

A common feature of temporal lobe epilepsy and of animal models of epilepsy is the growth of hippocampal mossy fibers into the dentate molecular layer, where at least some of them innervate granule cells. Because the mossy fibers are axons of granule cells, the recurrent mossy fiber pathway provides monosynaptic excitatory feedback to these neurons that could facilitate seizure discharge. We used the pilocarpine model of temporal lobe epilepsy to study the synaptic responses evoked by activating this pathway. Whole cell patch-clamp recording demonstrated that antidromic stimulation of the mossy fibers evoked an excitatory postsynaptic current (EPSC) in approximately 74% of granule cells from rats that had survived >10 wk after pilocarpine-induced status epilepticus. Recurrent mossy fiber growth was demonstrated with the Timm stain in all instances. In contrast, antidromic stimulation of the mossy fibers evoked an EPSC in only 5% of granule cells studied 4-6 days after status epilepticus, before recurrent mossy fiber growth became detectable. Notably, antidromic mossy fiber stimulation also evoked an EPSC in many granule cells from control rats. Clusters of mossy fiber-like Timm staining normally were present in the inner third of the dentate molecular layer at the level of the hippocampal formation from which slices were prepared, and several considerations suggested that the recorded EPSCs depended mainly on activation of recurrent mossy fibers rather than associational fibers. In both status epilepticus and control groups, the antidromically evoked EPSC was glutamatergic and involved the activation of both AMPA/kainate and N-methyl-D-aspartate (NMDA) receptors. EPSCs recorded in granule cells from rats with recurrent mossy fiber growth differed in three respects from those recorded in control granule cells: they were much more frequently evoked, a number of them were unusually large, and the NMDA component of the response was generally much more prominent. In contrast to the antidromically evoked EPSC, the EPSC evoked by stimulation of the perforant path appeared to be unaffected by a prior episode of status epilepticus. These results support the hypothesis that recurrent mossy fiber growth and synapse formation increases the excitatory drive to dentate granule cells and thus facilitates repetitive synchronous discharge. Activation of NMDA receptors in the recurrent pathway may contribute to seizure propagation under depolarizing conditions. Mossy fiber-granule cell synapses also are present in normal rats, where they may contribute to repetitive granule cell discharge in regions of the dentate gyrus where their numbers are significant.     

Noradrenergic enhancement of long-term potentiation at mossy fiber synapses in the hippocampus

1. We tested several hypotheses related to the modulation of long-term potentiation (LTP) by norepinephrine (NE) at the mossy fiber synapses in the rat hippocampal slice preparation using extracellular and intracellular recording techniques. 2. NE exerted frequency-dependent effects on mossy fiber synaptic transmission. It had little effect on extracellular population excitatory postsynaptic potentials (pEPSPs) sampled during low-frequency stimulation, whereas it had marked effects on the duration, magnitude, and probability of induction of LTP at these synapses. 3. The beta-adrenoceptor agonist isoproterenol mimicked all of the effects of NE, whereas the beta-adrenoceptor antagonists propranolol and timolol reversibly blocked the induction of LTP, suggesting the effects of NE are mediated by a beta-adrenoceptor and that beta-adrenoceptor activation may be an important constituent for the expression of LTP at these synapses. 4. Frequency-dependent effects of NE and isoproterenol on mossy fiber pEPSPs were also observed in the presence of the gamma-aminobutyric acid (GABA) antagonist, picrotoxin, suggesting that NE can enhance LTP by a mechanism that does not depend on intact inhibition. However, propranolol did not block LTP in these disinhibited slices and did not affect LTP magnitude. 5. The adenylate cyclase activator forskolin augmented pEPSPs sampled during low-frequency stimulation in disinhibited slices and significantly enhanced LTP. Forskolin, however, did not produce LTP in the absence of tetanic stimulation. This supports the hypothesis that NE and isoproterenol augment features of LTP by stimulating adenosine 3',5'-cyclic monophosphate (cAMP) production and that cAMP plays a modulatory role in the induction of LTP. 6. The postsynaptic injection of the cAMP analogue 8-bromoadenosine 3',5'-cyclic monophosphate (8-bromo-cAMP) significantly increased the probability of induction of LTP measured intracellularly under voltage-clamp conditions with intact inhibition. An analysis of the inhibitory synaptic slope conductance during these experiments indicated that changes in this measure could neither account for the increase in mossy fiber synaptic slope conductance in those cells that displayed it nor account for the group differences in this variable. 7. The amplitude and duration of the postsynaptic depolarization during tetanic stimulation in the cells that displayed LTP in the 8-bromo-cAMP-injected group were significantly greater than in the cells that did not display LTP in the adenosine 5'-monophosphate-injected group.(ABSTRACT TRUNCATED AT 400 WORDS)     

NMDA receptor-dependent metaplasticity at hippocampal mossy fiber synapses

Hippocampal mossy fiber synapses have been reported to lack NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) of AMPA excitatory postsynaptic currents (EPSCs), unlike conventional glutamatergic synapses. An explanation for this difference may reside in the relatively low number of NMDARs at these synapses. Because mossy fiber synapses display LTP selective for NMDARs, we examined whether this would affect the plasticity rules at mossy fiber-CA3 synapses in mouse hippocampal slices. We found that LTP of NMDARs serves as a metaplastic switch making mossy fiber synapses competent for generating NMDAR-dependent LTP of AMPA EPSCs.     

Metabotropic glutamate receptor antagonist AIDA blocks induction of mossy fiber-CA3 LTP in vivo

Metabotropic glutamate receptors (mGluR) are implicated in long-term memory storage. mGluR-I and mGluR-II antagonists impede various forms of learning and long-term potentiation (LTP) in animals. Despite the evidence linking mGluR to learning mechanisms, their role in mossy fiber-CA3 long-term potentiation (LTP) is not yet clear. To explain the involvement of mGluR-I in memory mechanisms, we examined the function of the mGluR-I antagonist 1-aminoindan-1, 5-dicarboxylic acid (AIDA) on the induction of mossy fiber-CA3 LTP in vivo in male Sprague Dawley and Fischer 344 (F344) rats. Acute extracellular mossy fiber (MF) responses were evoked by stimulation of the MF bundle and recorded in the stratum lucidum of CA3. The excitatory postsynaptic potential (EPSP) magnitude was measured by using the initial slope of the field EPSP slope measured 2-3 ms after response onset. After collection of baseline MF-CA3 responses at 0.05 Hz, animals received either ((+/-))-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (N-methyl-D-aspartate-R antagonist, 10 mg/kg ip), naloxone (opioid-R antagonist, 10 mg/kg ip), or AIDA (mGluR antagonist, 1 mg/kg ip or 37.5 nmol ic). LTP was induced by two 100-Hz trains at the intensity sufficient to evoke 50% of the maximal response. Responses were collected for an additional 1 h. AIDA blocked induction of LTP in the mossy fiber pathway (P < 0.05) in both strains of rats after systemic and in Sprague Dawley rats after intrahippocampal injection.     

Short- and long-term plasticity of the perforant path synapse in hippocampal area CA3

The direct perforant path (PP) projection to CA3 is a major source of cortical input to the hippocampal region, yet relatively little is known about the basic properties of physiology and plasticity in this pathway. We tested whether PP long-term potentiation (LTP) in CA3 possesses the Hebbian property of associativity; i.e., whether the firing of fibers of different orders can induce PP LTP. We stimulated PP with weak trains of high-frequency stimulation (HFS), which by itself was below the threshold for LTP induction. The identical HFS was effective in inducing LTP when the mossy fiber pathway (MF) was activated simultaneously, thus demonstrating associative plasticity between the two pathways. We also demonstrated associative LTP between PP and recurrent collateral fibers (RC). PP LTP was blocked by the N-methyl-D-aspartate receptor (NMDAR) antagonist 2-amino-5-phosphonovaleric acid in both the associative and homosynaptic induction conditions. Neither MF nor RC fiber HFS alone resulted in permanent changes in PP field excitatory postsynaptic potential (fEPSP) amplitude. However, HFS delivered to either MF or RC alone led to transient heterosynaptic depression of the PP fEPSP. Our results support the conceptual framework that regards CA3 as an autoassociative memory network in which efficient retrieval of previously stored activity patterns is mediated by associative plasticity of the PP synapse.     

Mechanisms underlying LTP of inhibitory synaptic transmission in the deep cerebellar nuclei

Whole-cell recordings were used to investigate long-term potentiation of inhibitory synaptic currents (IPSCs) in neurons of deep cerebellar nuclei (DCN) in slices. IPSCs were evoked by electrical stimulation of the white matter surrounding the DCN in the presence of non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (20 microM). High-frequency stimulation induced a long-term potentation (LTP) of the IPSC amplitude without changing its reversal potential, rise time, and decay-time constant. This LTP did not require the activation of postsynaptic gamma-aminobutyric acid-A (GABA(A)) receptors but depended on the activation of NMDA receptors. LTP of IPSCs in DCN neurons could also be induced by voltage-depolarizing pulses in postsynaptic neurons and appeared to depend on an increase in intracellular calcium as the LTP was blocked when the cells were loaded with a calcium chelator, 1,2-bis-(2-amino-phenoxy)-N,N,N', N'-tetraacetic acid (BAPTA, 10 mM). LTP of IPSCs was accompanied by an increase in the frequency of spontaneous IPSCs and miniature IPSCs (recorded in the presence of tetrodotoxin 1 microM), but there was no significant change in their amplitude. In addition, during the LTP, the amplitude of response to exogenously applied GABA(A) receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride was increased. Intracellular application of tetanus toxin, a powerful blocker of exocytosis, in DCN neuron prevented the induction of LTP of IPSCs. Our results suggest that the induction of LTP of IPSCs in the DCN neurons likely involves a postsynaptic locus. Plasticity of inhibitory synaptic transmission in DCN neurons may play a crucial role in cerebellar control of motor coordination and learning.     

Recurrent excitatory connectivity in the dentate gyrus of kindled and kainic acid-treated rats

Repeated seizures induce mossy fiber axon sprouting, which reorganizes synaptic connectivity in the dentate gyrus. To examine the possibility that sprouted mossy fiber axons may form recurrent excitatory circuits, connectivity between granule cells in the dentate gyrus was examined in transverse hippocampal slices from normal rats and epileptic rats that experienced seizures induced by kindling and kainic acid. The experiments were designed to functionally assess seizure-induced development of recurrent circuitry by exploiting information available about the time course of seizure-induced synaptic reorganization in the kindling model and detailed anatomic characterization of sprouted fibers in the kainic acid model. When recurrent inhibitory circuits were blocked by the GABA(A) receptor antagonist bicuculline, focal application of glutamate microdrops at locations in the granule cell layer remote from the recorded granule cell evoked trains of excitatory postsynaptic potentials (EPSPs) and population burst discharges in epileptic rats, which were never observed in slices from normal rats. The EPSPs and burst discharges were blocked by bath application of 1 microM tetrodotoxin and were therefore dependent on network-driven synaptic events. Excitatory connections were detected between blades of the dentate gyrus in hippocampal slices from rats that experienced kainic acid-induced status epilepticus. Trains of EPSPs and burst discharges were also evoked in granule cells from kindled rats obtained after > or = 1 wk of kindled seizures, but were not evoked in slices examined 24 h after a single afterdischarge, before the development of sprouting. Excitatory connectivity between blades of the dentate gyrus was also assessed in slices deafferented by transection of the perforant path, and bathed in artificial cerebrospinal fluid (ACSF) containing bicuculline to block GABA(A) receptor-dependent recurrent inhibitory circuits and 10 mM [Ca(2+)](o) to suppress polysynaptic activity. Low-intensity electrical stimulation of the infrapyramidal blade under these conditions failed to evoke a response in suprapyramidal granule cells from normal rats (n = 15), but in slices from epileptic rats evoked an EPSP at a short latency (2.59 +/- 0.36 ms) in 5 of 18 suprapyramidal granule cells. The results are consistent with formation of monosynaptic excitatory connections between blades of the dentate gyrus. Recurrent excitatory circuits developed in the dentate gyrus of epileptic rats in a time course that corresponded to the development of mossy fiber sprouting and demonstrated patterns of functional connectivity corresponding to anatomic features of the sprouted mossy fiber pathway.     

Control of the subthalamic innervation of the rat globus pallidus by D2/3 and D4 dopamine receptors

The effects of activating dopaminergic D(2/3) and D(4) receptors during activation of the subthalamic projection to the globus pallidus (GP) were explored in rat brain slices using the whole cell patch-clamp technique. Byocitin labeling and both orthodromic and antidromic activation demonstrated the integrity of some subthalamopallidal connections in in vitro parasagittal brain slices. Excitatory postsynaptic currents (EPSCs) that could be blocked by CNQX and AP5 were evoked onto pallidal neurons by local field stimulation of the subthalamopallidal pathway in the presence of bicuculline. Bath application of dopamine and quinpirole, a dopaminergic D(2)-class receptor agonist, reduced evoked EPSCs by about 35%. This effect was only partially blocked by sulpiride, a D(2/3) receptor antagonist. The sulpiride-sensitive reduction of the subthalamopallidal EPSC was associated with an increase in the paired-pulse ratio (PPR) and a reduction in the frequency but not the mean amplitude of spontaneous EPSCs (sEPSCs), indicative of a presynaptic site of action, which was confirmed by variance-mean analysis. The sulpiride-resistant EPSC reduction was mimicked by PD 168,077 and blocked by L-745,870, selective D(4) receptor agonist and antagonist, respectively, suggesting the involvement of D(4) receptors. The reduction of EPSCs produced by PD 168,077 was not accompanied by changes in PPR or the frequency of sEPSCs; however, it was accompanied by a reduction in mean sEPSC amplitude, indicative of a postsynaptic site of action. These results show that dopamine modulates subthalamopallidal excitation by presynaptic D(2/3) and postsynaptic D(4) receptors. The importance of this modulation is discussed.     

NO enhances presynaptic currents during cerebellar mossy fiber-granule cell LTP

Nitric oxide (NO) is a candidate retrograde messenger in long-term potentiation (LTP). The NO metabolic pathway is expressed in the cerebellar granule cell layer but its physiological role remained unknown. In this paper we have investigated the role of NO in cerebellar mossy fiber-granule cell LTP, which has postsynaptic N-methyl-d-aspartate (NMDA) receptor-dependent induction. Pre- and postsynaptic current changes were simultaneously measured by using extracellular focal recordings, and NO release was monitored with an electrochemical probe in P21 rat cerebellar slices. High-frequency mossy fiber stimulation induced LTP and caused a significant NO release (6.2 +/- 2.8 nM; n = 5) in the granular layer that was dependent on NMDA receptor as well as on nitric oxide synthase (NOS) activation. Preventing NO production by perfusing the NOS inhibitor 100 microM NG-nitro-l-arginine (L-NNA), blocking extracellular NO diffusion by 10 microM MbO2, or inhibiting the NO target guanylyl cyclase (sGC) with 10 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-dione (ODQ) prevented LTP. Moreover, the NO donor 10 microM 2-(N,N-diethylamino)-diazenolate-2-oxide.Na (DEA-NO) induced LTP, which was mutually occlusive with LTP generated by high-frequency stimulation, prevented by ODQ, and insensitive to NMDA channel blockade (50 microM APV + 25 microM 7-Cl-kyn) or interruption of mossy fiber stimulation. Thus NO is critical for LTP induction at the cerebellar mossy fiber-granule cell relay. Interestingly, LTP manipulations were accompanied by consensual changes in the presynaptic current, suggesting that NO acts as a retrograde signal-enhancing presynaptic terminal excitability.     

Seizures induce simultaneous GABAergic and glutamatergic transmission in the dentate gyrus-CA3 system

Monosynaptic and polysynaptic responses of CA3 pyramidal cells (PC) to stimulation of the dentate gyrus (DG) are normally blocked by glutamate receptor antagonists (GluRAs). However, after kindled seizures, GluRAs block the monosynaptic excitatory postsynaptic potential (EPSP) and isolate a monosynaptic inhibitory postsynaptic potential (IPSP), suggesting that mossy fibers release GABA. However, kindling epilepsy induces neuronal sprouting, which can underlie this fast inhibitory response. To explore this possibility, the synaptic responses of PC to DG stimulation were analyzed in kindled epileptic rats, with and without seizures, and in nonepileptic rats, immediately after a single pentylenetetrazol (PTZ)-induced seizure, in which sprouting is unlikely to have occurred. Excitatory and inhibitory synaptic responses of PC to DG stimulation were blocked by GluRAs in control cells and in cells from kindled nonseizing rats, confirming that inhibitory potentials are disynaptically mediated. However, a fast IPSP could be evoked in kindled epileptic rats and in nonepileptic rats after a single PTZ-induced seizure. The same response was induced after rekindling the epileptic nonseizing rats. This IPSP has an onset latency that parallels that of the control EPSP and is not altered under low Ca(2+) medium or halothane perfusion. In addition, it was reversibly depressed by L(+)-2-amino-4-phosphonobutyric acid (L-AP4), which is known to inhibit transmitter release from mossy fibers. These results demonstrate that seizures, and not the synaptic rearrangement due to an underlying epileptic state, induce the emergence of fast inhibition in the DG-CA3 system, and suggest that the mossy fibers underlie this plastic change.     

Subthreshold contribution of N-methyl-d-aspartate receptors to long-term potentiation induced by low-frequency pairing in rat hippocampal CA1 pyramidal cells

Long-term potentiation (LTP) is a use-dependent and persistent enhancement of synaptic strength. In the CA1 region of the hippocampus, LTP has Hebbian characteristics and requires precisely timed interaction between presynaptic firing and postsynaptic depolarization. Although depolarization is an absolute requirement for plasticity, it is still not clear whether the postsynaptic response during LTP induction should be subthreshold or suprathreshold for the generation of somatic action potential. Here, we use the whole-cell patch-clamp technique and different pairing protocols to examine systematically the postsynaptic induction requirements for LTP. We induce LTP by changes only in membrane potential while keeping the afferent stimulation constant and at minimal levels. This approach permits differentiation of two types of LTP: LTP induced with suprathreshold synaptic responses (LTP(AP)) and LTP induced with subthreshold excitatory postsynaptic current (EPSCs; LTP(EPSC)). We found that LTP(AP) (>40%) required pairing of depolarization (V(m)>or=-40 mV, for 40-60 s) with four to six (0.1 Hz) single synaptically initiated action potentials. LTP(EPSC) was of smaller magnitude (<30%) and required pairing of depolarization to -50 mV (60 s) with six subthreshold EPSCs. The N-methyl-d-aspartate receptor (NMDAR) antagonists aminophosphonovaleric acid and 7-chlorokynurenic acid consistently blocked LTP(EPSC) but were ineffective in preventing LTP(AP). Robust, NMDAR-independent LTP is obtained by stronger postsynaptic depolarization that converts the EPSCs to suprathreshold somatic action potentials. Purely NMDAR-dependent LTP is obtained by pairing mild somatic depolarization with subthreshold afferent pulses to the postsynaptic cell. Our results indicate that the degree of postsynaptic depolarization in the presence of single afferent pulses determines the type and magnitude of LTP.     

Excitatory synaptic currents in lumbosacral parasympathetic preganglionic neurons elicited from the lateral funiculus

Excitatory postsynaptic currents (EPSCs) in parasympathetic preganglionic neurons (PGNs) were examined using the whole cell patch-clamp recording technique in L6 and S1 spinal cord slices from neonatal rats (6-16 days old). PGNs were identified by labeling with retrograde axonal transport of a fluorescent dye (Fast Blue) injected into the intraperitoneal space 3-7 days before the experiment. Synaptic responses were evoked in PGNs by field stimulation of the lateral funiculus (LF) in the presence of bicuculline methiodide (10 microM) and strychnine (1 microM). In approximately 40% of the cells (total, 100), single-shock electrical stimulation of the LF elicited short, relatively constant latency [3.0 +/- 0.1 (SE) ms] fast EPSCs consistent with a monosynaptic pathway. The remainder of the cells did not respond to stimulation. At low intensities of stimulation, the EPSCs often occurred in an all-or-none manner, indicating that they were mediated by a single axonal input. Most cells (n = 33) exhibited only fast EPSCs (type 1), but some cells (n = 8) had fast EPSCs with longer, more variable latency polysynaptic EPSCs superimposed on a slow inward current (type 2). Type 1 fast synaptic EPSCs were pharmacologically dissected into two components: a transient component that was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 microM), a non-NMDA glutamatergic antagonist, and a slow decaying component that was blocked by 2-amino-5-phosphonovalerate (APV, 50 microM), a NMDA antagonist. Type 2 polysynaptic currents were reduced by 5 microM CNQX and completely blocked by combined application of 5 microM CNQX and 50 microM APV. The fast monosynaptic component of type 1 EPSCs had a linear current-voltage relationship and reversed at a membrane potential of 5.0 +/- 5.9 mV (n = 5), whereas the slow component exhibited a negative slope conductance at holding potentials greater than -20 mV. The type 1, fast synaptic EPSCs had a time to peak of 1.4 +/- 0.1 ms and exhibited a biexponential decay (time constants, 5.7 +/- 0.6 and 38.8 +/- 4.0 ms). In the majority of PGNs (n = 11 of 15 cells), EPSCs evoked by electrical stimulation of LF exhibited paired-pulse inhibition (range; 25-33% depression) at interstimulus intervals ranging from 50 to 120 ms. These results indicate that PGNs receive monosynaptic and polysynaptic glutamatergic excitatory inputs from axons in the lateral funiculus.     

Long-term potentiation of guinea pig mossy fiber responses is not blocked by N-methyl D-aspartate antagonists

Receptors preferentially activated by the excitatory amino acid N-methyl-D-aspartate (NMDA) do not mediate synaptic transmission in the hippocampus but are involved in initiating long-term potentiation (LTP) in hippocampal region CA1. We have examined the role of NMDA receptors in LTP of the commissural/associational and mossy fiber pathways to region CA3 pyramidal neurons. In the commissural/associational pathway, NMDA receptor blockers did not reduce synaptic responses but reversibly blocked the induction of LTP. In contrast, NMDA receptor blockers had no effect on mossy fiber LTP. These results suggest that induction of commissural/associational LTP differs from mossy fiber LTP, although the mechanisms underlying expression of LTP along these pathways could be similar. Kynurenate and L-2-amino-4-phosphonobutyrate, which potently reduce mossy fiber responses, also did not block induction of mossy fiber LTP.     

保留后根的新型脊髓旁矢状面切片技术及其在脊髓突触传递研究中的应用

目的:对保留脊神经后根的脊髓切片技术进行改良,增加所保留的脊神经后根纤维投射到脊髓后角浅层的完整性,以提高实验效率。方法:选用4～5周的SD大鼠,应用振动切片机对其脊髓腰骶膨大分别进行横断面或矢状面切片,在全细胞模式记录下,给予后根刺激观察两者中诱发出兴奋性突触后电流(EPSCs)的成功率,并对其进行比较;调整后根刺激参数分别刺激Aβ、Aδ和C纤维以诱发EPSCs,并对不同纤维诱发的EPSCs进行鉴别。结果:在保留后根的横断面和矢状面切片上诱发出EPSCs的成功率分别是38.43±9.97%和86.36±5.32%,具有显著的统计学差异(P<0.0001);与非保留后根的脊髓切片上诱发出的EPSCs相比,应用保留后根的脊髓横断面切片和矢状面切片所诱发的EPSCs均可通过刺激强度和潜伏期的差异,对不同纤维诱发的EPSCs进行有效的区分。结论:保留后根的脊髓矢状面切片刺激后根反应率显著高于横断面切片,且可对不同纤维诱发的EPSCs进行有效区分。因此,保留后根的脊髓矢状面切片比横断面切片更完整的保留了后根到脊髓后角浅层的投射,可提高实验效率,是研究脊髓中枢突触传递及其可塑性的可靠离体模型。     

Mossy fiber-granule cell synapses in the normal and epileptic rat dentate gyrus studied with minimal laser photostimulation.

Dentate granule cells become synaptically interconnected in the hippocampus of persons with temporal lobe epilepsy, forming a recurrent mossy fiber pathway. This pathway may contribute to the development and propagation of seizures. The physiology of mossy fiber-granule cell synapses is difficult to characterize unambiguously, because electrical stimulation may activate other pathways and because there is a low probability of granule cell interconnection. These problems were addressed by the use of scanning laser photostimulation in slices of the caudal hippocampal formation. Glutamate was released from a caged precursor with highly focused ultraviolet light to evoke action potentials in a small population of granule cells. Excitatory synaptic currents were recorded in the presence of bicuculline. Minimal laser photostimulation evoked an apparently unitary excitatory postsynaptic current (EPSC) in 61% of granule cells from rats that had experienced pilocarpine-induced status epilepticus followed by recurrent mossy fiber growth. An EPSC was also evoked in 13-16% of granule cells from the control groups. EPSCs from status epilepticus and control groups had similar peak amplitudes ( approximately 30 pA), 20-80% rise times (approximately 1.2 ms), decay time constants ( approximately 10 ms), and half-widths (approximately 8 ms). The mean failure rate was high (approximately 70%) in both groups, and in both groups activation of N-methyl-D-aspartate receptors contributed a small component to the EPSC. The strong similarity between responses from the status epilepticus and control groups suggests that they resulted from activation of a similar synaptic population. No EPSC was recorded when the laser beam was focused in the dentate hilus, suggesting that indirect activation of hilar mossy cells contributed little, if at all, to these results. Recurrent mossy fiber growth increases the density of mossy fiber-granule cell synapses in the caudal dentate gyrus by perhaps sixfold, but the new synapses appear to operate very similarly to preexisting mossy fiber-granule cell synapses.     

Low-threshold Ca2+ channels mediate induction of long-term potentiation in kitten visual cortex.

1. The induction mechanism of long-term potentiation (LTP) in developing visual cortex was studied by recording intracellular responses from layer III-IV cells in slice preparations of kitten visual cortex at 30-40 days after birth. 2. Strong stimulation of white matter produced a late depolarizing response after an orthodromic action potential. This depolarizing response was abolished by membrane depolarization or hyperpolarization caused by current injection through the recording electrode. In addition, this response was reduced by bath application of a low concentration (100 microM) of Ni2+ without any changes in the rising slope of the excitatory postsynaptic potential (EPSP) or orthodromic action potential. This suggests that this response is mediated by low-threshold Ca2+ channels (LTCs). 3. The involvement of LTCs in the induction of LTP was tested. White matter was stimulated at 2 Hz for 15 min as a conditioning stimulus to induce LTP, and the resultant changes in EPSPs were tested by low-frequency (0.1 Hz) stimulation of white matter. Conditioning stimulation produced a large N-methyl-D-aspartate (NMDA) receptor-mediated depolarizing response in these cells, which obscured the presence of the late depoliarzation. Therefore the test was conducted in a solution containing an NMDA antagonist 2-amino-5-phosphonovalerate (APV). 4. Weak conditioning stimulation, which evoked no LTC responses, never induced LTP; whereas strong conditioning stimulation, which evoked LTC responses, always induced LTP. Strong conditioning stimulation failed to induce LTP when LTC responses were prevented either by membrane depolarization or hyperpolarization or by a bath application of 100 microM Ni2+. 5. In a solution without APV, the application of Ni2+ also prevented the induction of LTP. 6. When cells were impaled by an electrode containing a Ca2+ chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), LTP was never induced, even though LTC responses were evoked by conditioning stimulation. These results indicate that Ca2+ influx into postsynaptic cells through LTCs induces the LTP. 7. The responses mediated by LTCs, which were evoked by the injection of current pulses into the cells, were maximum at the critical period of visual cortical plasticity, suggesting that LTCs in postsynaptic cells regulate the plastic changes in developing visual cortex.     

Mu opiates inhibit long-term potentiation induction in the spinal cord slice

Long-term potentiation (LTP) involves a prolonged increase in neuronal excitability following repeated afferent input. This phenomenon has been extensively studied in the hippocampus as a model of learning and memory. Similar long-term increases in neuronal responses have been reported in the dorsal horn of the spinal cord following intense primary afferent stimulation. In these studies, we utilized the spinal cord slice preparation to examine effects of the potently antinociceptive mu opioids in modulating primary afferent/dorsal horn neurotransmission as well as LTP of such transmission. Transverse slices were made from the lumbar spinal cord of 10- to 17-day-old rats, placed in a recording chamber, and perfused with artificial cerebrospinal fluid also containing bicuculline (10 microM) and strychnine (1 microM). Primary afferent activation was achieved in the spinal slice by electrical stimulation of the dorsal root (DR) or the tract of Lissauer (LT) which is known to contain a high percentage of small diameter fibers likely to transmit nociception. Consistent with this anatomy, response latencies of LT-evoked field potentials in the dorsal horn were considerably slower than the response latencies of DR-evoked potentials. Only LT-evoked field potentials were found to be reliably inhibited by the mu opioid receptor agonist [D-Ala(2), N-Me-Phe(4), Gly(5)] enkephalin-ol (DAMGO, 1 microM), although evoked potentials from both DR and LT were blocked by the AMPA/kainate glutamate receptor antagonist 6-cyano-7-nitroquinoxalene-2,3-dione. Moreover repeated stimulation of LT produced LTP of LT- but not DR-evoked potentials. In contrast, repeated stimulation of DR showed no reliable LTP. LTP of LT-evoked potentials depended on N-methyl-D-aspartate (NMDA) receptor activity, in that it was attenuated by the NMDA antagonist APV. Moreover, such LTP was inhibited by DAMGO interfering with LTP induction mechanisms. Finally, in whole cell voltage-clamp studies of Lamina I neurons, DAMGO inhibited excitatory postsynaptic current (EPSC) response amplitudes from LT stimulation-evoked excitatory amino acid release but not from glutamate puffed onto the cell and increased paired-pulse facilitation of EPSCs evoked by LT stimulation. These studies suggest that mu opioids exert their inhibitory effects presynaptically, likely through the inhibition of glutamate release from primary afferent terminals, and thereby inhibit the induction of LTP in the spinal dorsal horn.     

Enhancement of postsynaptic responsiveness during long-term potentiation of mossy fiber synapses in guinea pig hippocampus.

The primary site responsible for the long-lasting enhancement of synaptic transmission during long-term potentiation (LTP) was examined by quantal analysis of excitatory postsynaptic potentials in thin sections of the guinea pig hippocampus. With induction of LTP in mossy fiber synapses, estimated values of quantal amplitude (q) and Pascal parameters p and r were increased significantly. No increases in quantal content (m) were detected. The magnitude of increases in q was almost equal to that of LTP. These results indicate that LTP in mossy fiber synapses results from increases in responsiveness of postsynaptic neurons.