Activity-dependent depression of local excitatory connections in the CA1 region of mouse hippocampus

The existence of recurrent excitatory synapses between pyramidal cells in the hippocampal CA1 region has been known for some time yet little is known about activity-dependent forms of plasticity at these synapses. Here we demonstrate that under certain experimental conditions, Schaffer collateral/commissural fiber stimulation can elicit robust polysynaptic excitatory postsynaptic potentials due to recurrent synaptic inputs onto CA1 pyramidal cells. In contrast to CA3 pyramidal cell inputs, recurrent synapses onto CA1 pyramidal cells exhibited robust paired-pulse depression and a sustained, but rapidly reversible, depression in response to low-frequency trains of Schaffer collateral fiber stimulation. Blocking GABA(B) receptors abolished paired-pulse depression but had little effect on low-frequency stimulation (LFS)-induced depression. Instead, LFS-induced depression was significantly attenuated by an inhibitor of A1 type adenosine receptors. Blocking the postsynaptic effects of GABA(B) and A1 receptor activation on CA1 pyramidal cell excitability with an inhibitor of G-protein-activated inwardly rectifying potassium channels had no effect on either paired-pulse depression or LFS-induced depression. Thus activation of presynaptic GABA(B) and adenosine receptors appears to have an important role in activity-dependent depression at recurrent synapses. Together, our results indicate that CA3-CA1 and CA1-CA1 synapses exhibit strikingly different forms of short-term synaptic plasticity and suggest that activity-dependent changes in recurrent synaptic transmission can transform the CA1 region from a sparsely connected recurrent network into a predominantly feedforward circuit.     

Effects of neonatal gamma-ray irradiation on rat hippocampus--I. Postnatal maturation of hippocampal cells.

The axons of dentate granule cells, the mossy fibres, establish synaptic contacts with the thorny excrescences of the apical dendrite of CA3 pyramidal neurons. Dentate granule granule cells develop postnatally in rats, whereas the CA3 pyramidal cells are generated before birth. In the present studies, using unilateral neonatal gamma-ray irradiation to destroy the granule cells in one hemisphere, we have studied the effect of mossy fibre deprivation on the development of their targets. We show that such "degranulation" prevents the normal development of giant thorny excrescences, suggesting that the development of thorny excrescences in CA3 pyramidal neurons is under the control of mossy fibres. In contrast, irradiation of the hippocampus of the neonatal rat does not affect the development of the dendritic arborization of CA3 pyramidal cells and their non-mossy dendritic spines.     

Timing and efficacy of transmitter release at mossy fiber synapses in the hippocampal network

It is widely accepted that the hippocampus plays a major role in learning and memory. The mossy fiber synapse between granule cells in the dentate gyrus and pyramidal neurons in the CA3 region is a key component of the hippocampal trisynaptic circuit. Recent work, partially based on direct presynaptic patch-clamp recordings from hippocampal mossy fiber boutons, sheds light on the mechanisms of synaptic transmission and plasticity at mossy fiber synapses. A high Na⁺ channel density in mossy fiber boutons leads to a large amplitude of the presynaptic action potential. Together with the fast gating of presynaptic Ca²⁺ channels, this generates a large and brief presynaptic Ca²⁺ influx, which can trigger transmitter release with high efficiency and temporal precision. The large number of release sites, the large size of the releasable pool of vesicles, and the huge extent of presynaptic plasticity confer unique strength to this synapse, suggesting a large impact onto the CA3 pyramidal cell network under specific behavioral conditions. The characteristic properties of the hippocampal mossy fiber synapse may be important for pattern separation and information storage in the dentate gyrus-CA3 cell network.     

Characteristics of local excitatory circuits studied with glutamate microapplication in the CA3 area of rat hippocampal slices

1. Local neuronal circuits in CA3 of hippocampal slices were studied by recording excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) intracellularly during glutamate microapplication in CA3. Control experiments validated this approach by providing evidence that glutamate microdrops stimulated neurons but not axons-of-passage or axon terminals in CA3. 2. Glutamate microdrops (10-20 mM, 10-20 microns diam) increased the firing frequency of extracellularly recorded dentate granule cells for 5-10 s when applied to their somata but not when applied to their mossy fiber axons and terminals in the hilus and in CA3. 3. Glutamate microapplications to granule cell somata, but not to mossy fiber axons, also increased the frequency of intracellularly recorded EPSPs in CA3 pyramidal cells for 5-10 s. This provided a second line of evidence that glutamate did not cause firing in mossy fiber axons synapsing in CA3. 4. In slices where the CA3 region was surgically separated from the dentate gyrus and CA2, glutamate microdrops placed in the CA3 stratum pyramidale within 400 microns of intracellularly recorded pyramidal cells increased the frequency of EPSPs and IPSPs. Tetrodotoxin (1 microgram/ml) blocked these increases in PSP frequency, indicating that they did not result from glutamate-induced depolarization and associated transmitter release from presynaptic terminals. Increases in PSP frequency were interpreted to reflect glutamate activations of CA3 neurons with local synaptic connections to recorded cells. 5. Low concentrations of picrotoxin (PTX, 5-10 microM) blocked glutamate-induced increases in IPSP frequency and often revealed increases in EPSP frequency where they were not previously observed. This suggests that recurrent inhibitory circuits normally mask or block transmission through recurrent excitatory pathways in CA3. 6. In five experiments following PTX treatment (7.5-10 microM), large and prolonged (up to 2 min) increases in EPSP frequency were observed in CA3 pyramidal cells to glutamate microapplications in CA3. Rhythmic epileptiform bursts eventually occurred in two of these cases, suggesting that the protracted increases in EPSP frequency represent a form of reverberating excitation during a transition from normal to epileptic states. 7. Sixteen CA3 pyramidal cells were recorded in PTX (5-10 microM) during glutamate microapplications at 200 and 400 microns on each side of the recording site. The most consistent glutamate-induced increases in EPSP frequency occurred to microapplications 200 microns from recording sites on the hilar side.(ABSTRACT TRUNCATED AT 400 WORDS)     

Contributions of mossy fiber and CA1 pyramidal cell sprouting to dentate granule cell hyperexcitability in kainic acid-treated hippocampal slice cultures

Axonal sprouting like that of the mossy fibers is commonly associated with temporal lobe epilepsy, but its significance remains uncertain. To investigate the functional consequences of sprouting of mossy fibers and alternative pathways, kainic acid (KA) was used to induce robust mossy fiber sprouting in hippocampal slice cultures. Physiological comparisons documented many similarities in granule cell responses between KA- and vehicle-treated cultures, including: seizures, epileptiform bursts, and spontaneous excitatory postsynaptic currents (sEPSCs) >600 pA. GABAergic control and contribution of glutamatergic synaptic transmission were similar. Analyses of neurobiotin-filled CA1 pyramidal cells revealed robust axonal sprouting in both vehicle- and KA-treated cultures, which was significantly greater in KA-treated cultures. Hilar stimulation evoked an antidromic population spike followed by variable numbers of postsynaptic potentials (PSPs) and population spikes in both vehicle- and KA-treated cultures. Despite robust mossy fiber sprouting, knife cuts separating CA1 from dentate gyrus virtually abolished EPSPs evoked by hilar stimulation in KA-treated but not vehicle-treated cultures, suggesting a pivotal role of functional afferents from CA1 to dentate gyrus in KA-treated cultures. Together, these findings demonstrate striking hyperexcitability of dentate granule cells in long-term hippocampal slice cultures after treatment with either vehicle or KA. The contribution to hilar-evoked hyperexcitability of granule cells by the unexpected axonal projection from CA1 to dentate in KA-treated cultures reinforces the idea that axonal sprouting may contribute to pathologic hyperexcitability of granule cells.     

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.     

Neuropeptide Y regulates recurrent mossy fiber synaptic transmission less effectively in mice than in rats: Correlation with Y2 receptor plasticity

A unique feature of temporal lobe epilepsy is the formation of recurrent excitatory connections among granule cells of the dentate gyrus as a result of mossy fiber sprouting. This novel circuit contributes to a reduced threshold for granule cell synchronization. In the rat, activity of the recurrent mossy fiber pathway is restrained by the neoexpression and spontaneous release of neuropeptide Y (NPY). NPY inhibits glutamate release tonically through activation of presynaptic Y2 receptors. In the present study, the effects of endogenous and applied NPY were investigated in C57Bl/6 mice that had experienced pilocarpine-induced status epilepticus and subsequently developed a robust recurrent mossy fiber pathway. Whole cell patch clamp recordings made from dentate granule cells in hippocampal slices demonstrated that, as in rats, applied NPY inhibits recurrent mossy fiber synaptic transmission, the Y2 receptor antagonist (S)-N2-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6H)-oxodibenz[b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cyclopentyl]acetyl]-N-[2-[1,2-dihydro-3,5(4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]-argininamide (BIIE0246) blocks its action and BIIE0246 enhances synaptic transmission when applied by itself. Y5 receptor agonists had no significant effect. Thus spontaneous release of NPY tonically inhibits synaptic transmission in mice and its effects are mediated by Y2 receptor activation. However, both NPY and BIIE0246 were much less effective in mice than in rats, despite apparently equivalent expression of NPY in the recurrent mossy fibers. Immunohistochemistry indicated greater expression of Y2 receptors in the mossy fiber pathway of normal mice than of normal rats. Pilocarpine-induced status epilepticus markedly reduced the immunoreactivity of mouse mossy fibers, but increased the immunoreactivity of rat mossy fibers. Mossy fiber growth into the inner portion of the dentate molecular layer was associated with increased Y2 receptor immunoreactivity in rat, but not in mouse. These contrasting receptor changes can explain the quantitatively different effects of endogenously released and applied NPY on recurrent mossy fiber transmission in mice and rats.     

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.     

Blockade of excitation reveals inhibition of dentate spiny hilar neurons recorded in rat hippocampal slices.

1. Extracellular and intracellular recordings in rat hippocampal slices were used to compare the synaptic responses to perforant path stimulation of granule cells of the dentate gyrus, spiny "mossy" cells of the hilus, and area CA3c pyramidal cells of hippocampus. Specifically, we asked whether aspects of the local circuitry could explain the relative vulnerability of spiny hilar neurons to various insults to the hippocampus. 2. Spiny hilar cells demonstrated a surprising lack of inhibition after perforant path activation, despite robust paired-pulse inhibition and inhibitory postsynaptic potentials (IPSPs) in adjacent granule cells and area CA3c pyramidal cells in response to the same stimulus in the same slice. However, when the slice was perfused with excitatory amino acid antagonists [6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), or CNQX with 2-amino-5-phosphonovaleric acid (APV)], IPSPs could be observed in spiny hilar cells in response to perforant path stimulation. 3. The IPSPs evoked in spiny hilar cells in the presence of CNQX were similar in their reversal potentials and bicuculline sensitivity to IPSPs recorded in dentate granule cells or hippocampal pyramidal cells in the absence of CNQX. 4. These results demonstrate that, at least in slices, perforant path stimulation of spiny hilar cells is primarily excitatory and, when excitation is blocked, underlying inhibition can be revealed. This contrasts to the situation for dentate and hippocampal principal cells, which are ordinarily dominated by inhibition, and only when inhibition is compromised can the full extent of excitation be appreciated.(ABSTRACT TRUNCATED AT 250 WORDS)     

The GABAergic phenotype of the "glutamatergic" granule cells of the dentate gyrus

The granule cells of the dentate gyrus (DG), origin of the mossy fibers (MFs), have been considered to be glutamatergic. However, data obtained with different experimental approaches in recent years may be calling for a redefinition of their phenotype. Although they indeed release glutamate for fast neurotransmission, immunohistological and molecular biology evidence has revealed that these glutamatergic cells also express GABAergic markers. The granule cell expression of a GABAergic phenotype is developmentally regulated. Electrophysiological studies reveal that during the first 3 weeks of age, mossy fiber stimulation provokes monosynaptic fast inhibitory transmission mediated by GABA, besides the monosynaptic excitatory glutamatergic transmission, onto their targets in CA3. After this age, mossy fiber GABAergic transmission abruptly disappears and the GABAergic markers are undetected. In the adult, the GABAergic markers are upregulated and GABA-mediated transmission emerges after induction of hyperexcitability. The simultaneous glutamate- and GABA-mediated signals share the same plastic and pharmacological characteristics that correspond to neurotransmission of mossy fiber origin. This intriguing evidence gives rise to two fundamental points of discussion. The first is the plausible fact that glutamate and GABA, two neurotransmitters of opposing actions, are coreleased from the mossy fibers. The second relates to its functional implications that can be immediately inferred, as the dentate gyrus can exert direct GABA-mediated excitatory actions early in life and inhibitory actions in young and adult hippocampus. This evidence poses the need to reevaluate and reinterpret some aspects of the physiology of the mossy fiber pathway under normal and pathological conditions. This work reviews the recent evidence that supports the assumption that glutamate and GABA can be coreleased from a single pathway, the mossy fibers, and makes some considerations about its functional implications.     

Electrophysiological evidence of monosynaptic excitatory transmission between granule cells after seizure-induced mossy fiber sprouting

Mossy fiber sprouting is a form of synaptic reorganization in the dentate gyrus that occurs in human temporal lobe epilepsy and animal models of epilepsy. The axons of dentate gyrus granule cells, called mossy fibers, develop collaterals that grow into an abnormal location, the inner third of the dentate gyrus molecular layer. Electron microscopy has shown that sprouted fibers from synapses on both spines and dendritic shafts in the inner molecular layer, which are likely to represent the dendrites of granule cells and inhibitory neurons. One of the controversies about this phenomenon is whether mossy fiber sprouting contributes to seizures by forming novel recurrent excitatory circuits among granule cells. To date, there is a great deal of indirect evidence that suggests this is the case, but there are also counterarguments. The purpose of this study was to determine whether functional monosynaptic connections exist between granule cells after mossy fiber sprouting. Using simultaneous recordings from granule cells, we obtained direct evidence that granule cells in epileptic rats have monosynaptic excitatory connections with other granule cells. Such connections were not obtained when age-matched, saline control rats were examined. The results suggest that indeed mossy fiber sprouting provides a substrate for monosynaptic recurrent excitation among granule cells in the dentate gyrus. Interestingly, the characteristics of the excitatory connections that were found indicate that the pathway is only weakly excitatory. These characteristics may contribute to the empirical observation that the sprouted dentate gyrus does not normally generate epileptiform discharges.     

Sexual dimorphism in the mossy fiber synapses of the rat hippocampus

Summary The presence of sexual dimorphism in the hippocampal formation has long been recognized. Differences between male and female rats have been detected with respect to the number of dentate granule cells and branching patterns of dentate granule and hippocampal pyramidal cell dendrites. Groups of 6 male and 6 female Sprague-Dawley rats were studied at 180 days of age. Based on light microscopical Timm-staining and Golgi-impregnation and electron microscopy, and applying morphometric techniques, we now report that the total number of synapses between mossy fibers and the apical dendritic excrescences of CA3 pyramidal cells is the same in male and female rats, despite a higher numerical density in the latter. Moreover, the volume of the mossy fiber system was found to be smaller in females. Because the number of dentate granule cells is smaller in females than in males, the increased numerical density of synapses may be thought of as a compensatory mechanism to equalize the number of synaptic contacts between dentate granule and CA3 pyramidal cells in the two sexes. We demonstrate that an increase in the number of mossy fiber boutons in female rats is a determining factor for the sexual differences found.     

Lack of effect of mossy fiber-released zinc on granule cell GABA(A) receptors in the pilocarpine model of epilepsy

The recurrent mossy fiber pathway of the dentate gyrus expands dramatically in the epileptic brain and serves as a mechanism for synchronization of granule cell epileptiform activity. It has been suggested that this pathway also promotes epileptiform activity by inhibiting GABA(A) receptor function through release of zinc. Hippocampal slices from pilocarpine-treated rats were used to evaluate this hypothesis. The rats had developed status epilepticus after pilocarpine administration, followed by robust recurrent mossy fiber growth. The ability of exogenously applied zinc to depress GABA(A) receptor function in dentate granule cells depended on removal of polyvalent anions from the superfusion medium. Under these conditions, 200 microM zinc reduced the amplitude of the current evoked by applying muscimol to the proximal portion of the granule cell dendrite (23%). It also reduced the mean amplitude (31%) and frequency (36%) of miniature inhibitory postsynaptic currents. Nevertheless, repetitive mossy fiber stimulation (10 Hz for 1 s, 100 Hz for 1 s, or 10 Hz for 5 min) at maximal intensity did not affect GABA(A) receptor-mediated currents evoked by photorelease of GABA onto the proximal portion of the dendrite, where recurrent mossy fiber synapses were located. These results could not be explained by stimulation-induced depletion of zinc from the recurrent mossy fiber boutons. Negative results were obtained even during exposure to conditions that promoted transmitter release and synchronized granule cell activity (6 mM [K(+)](o), nominally Mg(2+)-free medium, 33 degrees C). These results suggest that zinc released from the recurrent mossy fiber pathway did not reach a concentration at postsynaptic GABA(A) receptors sufficient to inhibit agonist-evoked activation.     

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.     

Mu-opioid receptors facilitate the propagation of excitatory activity in rat hippocampal area CA1 by disinhibition of all anatomical layers

Hippocampal mu-opioid receptors (MORs) have been implicated in memory formation associated with opiate drug abuse. MORs modulate hippocampal synaptic plasticity acutely, when chronically activated, and during drug withdrawal. At the network level, MORs increase excitability in area CA1 by disinhibiting pyramidal cells. The precise inhibitory interneuron subtypes affected by MOR activation are unknown; however, not all subtypes are inhibited, and specific interneuron subtypes have been shown to preferentially express MORs. Here we investigate, using voltage-sensitive dye imaging in brain slices, the effect of MOR activation on the patterns of inhibition and on the propagation of excitatory activity in rat hippocampal CA1. MOR activation augments excitatory activity evoked by stimulating inputs in stratum oriens [i.e., Schaffer collateral and commissural pathway (SCC) and antidromic], stratum radiatum (i.e., SCC), and stratum lacunosum-moleculare (SLM; i.e., perforant path and thalamus). The augmented excitatory activity is further facilitated as it propagates through the CA1 network. This was observed as a proportionately larger increase in amplitudes of excitatory activity at sites distal from where the activity was evoked. This facilitation was observed for excitatory activity propagating from all three stimulation sites. The augmentation and facilitation were prevented by GABAA receptor antagonists (bicuculline, 30 microM), but not by GABAB receptor antagonists (CGP 55845, 10 microM). Furthermore, MOR activation inhibited IPSPs in all layers of area CA1. These findings suggest that MOR-induced suppression of GABA release onto GABAA receptors augments all inputs to CA1 pyramidal cells and facilitates the propagation of excitatory activity through the network of area CA1.     

Differences in baclofen-sensitivity between CA3 neurons and granule cells of the guinea pig hippocampus in vitro

In the slice preparation of the guinea pig hippocampus, the effects of (+/-) baclofen added to the Krebs-Ringer solution on dentate granule cells and CA3 pyramidal cells were investigated by means of intracellular recording techniques. In a 10-25 microM concentration, baclofen reduces the inhibitory postsynaptic potentials of the granule cells evoked by electrical stimulation of the perforant path and hyperpolarizes the granule cell membrane slightly. The reduction of both, the excitatory and inhibitory postsynaptic potentials of CA3 pyramidal cells evoked by mossy fiber stimulation, however, is accompanied by a strong hyperpolarization and conductance increase. Further, repetitive discharges of granule cells elicited in the presence of the convulsant bicuculline (25 microM) are hardly affected by baclofen (50 microM), whereas those of CA3 neurons are blocked.     

Limbic seizures cause pronounced changes in the expression of neurokinin B in the hippocampus of the rat.

Immunohistological and in situ hybridization techniques were used to study the influence of kainic acid-induced seizures and of pentylenetetrazol kindling on neurokinin B immunoreactivity and neurokinin B mRNA in the rat hippocampus. Pronounced increases in neurokinin B immunoreactivity were observed in the terminal field of mossy fibres 10-60 days after intraperitoneal injection of kainic acid. These slow but persistent increases in immunoreactivity were accompanied by markedly enhanced expression of neurokinin B mRNA in the granule cells and in hilar interneurons adjacent to the granule cell layer. These changes were preceded by transient increases in neurokinin B mRNA and immunoreactivity in CA1 pyramidal cell layer two and 10 days after kainic acid, which, however, subsided later on. Pentylenetetrazol kindling caused similar increases in neurokinin B mRNA expression in granule cells and in CA1 pyramidal cells, but not in hilar interneurons. In CA1, increased neurokinin B message was present two days after termination of the kindling procedure but not after 10 days. Sixty days after kainic acid injection, neurokinin B immunoreactivity extended to the inner-third of the molecular layer of the dentate gyrus. After pentylenetetrazol kindling, a neurokinin B-immunoreactive band was observed in the infrapyramidal region of CA3. Lesions of the dentate granule cells by local injection of colchicine in kainic acid-treated rats abolished the supragranular neurokinin B-positive staining, whereas it was almost unchanged after transection of the ventral hippocampal commissure. These observations suggest that neurokinin B immunoreactivity may be located in ipsilateral mossy fibres undergoing collateral sprouting to the inner molecular layer or to the infrapyramidal region in CA3, respectively. Preprotachykinin A mRNA, which encodes for neurokinin A and substance P, and substance P immunoreactivity were not changed in the hippocampus of epileptic rats compared with untreated animals. The observed changes in neurokinin B immunoreactivity and mRNA indicate that specific functional and morphological changes may be induced in hippocampal neurons by recurrent limbic seizures.     

Optical recording study of granule cell activities in the hippocampal dentate gyrus of kainate-treated rats

In the epileptic hippocampus, newly sprouted mossy fibers are considered to form recurrent excitatory connections to granule cells in the dentate gyrus and thereby increase seizure susceptibility. To study the effects of mossy fiber sprouting on neural activity in individual lamellae of the dentate gyrus, we used high-speed optical recording to record signals from voltage-sensitive dye in hippocampal slices prepared from kainate-treated epileptic rats (KA rats). In 14 of 24 slices from KA rats, hilar stimulation evoked a large depolarization in almost the entire molecular layer in which granule cell apical dendrites are located. The signals were identified as postsynaptic responses because of their dependence on extracellular Ca(2+). The depolarization amplitude was largest in the inner molecular layer (the target area of sprouted mossy fibers) and declined with increasing distance from the granule cell layer. In the inner molecular layer, a good correlation was obtained between depolarization size and the density of mossy fiber terminals detected by Timm staining methods. Blockade of GABAergic inhibition by bicuculline enlarged the depolarization in granule cell dendrites. Our data indicate that mossy fiber sprouting results in a large and prolonged synaptic depolarization in an extensive dendritic area and that the enhanced GABAergic inhibition partly masks the synaptic depolarization. However, despite the large dendritic excitation induced by the sprouted mossy fibers, seizure-like activity of granule cells was never observed, even when GABAergic inhibition was blocked. Therefore, mossy fiber sprouting may not play a critical role in epileptogenesis.     

Endogenous zinc inhibits GABA(A) receptors in a hippocampal pathway

Depending on their subunit composition, GABA(A) receptors can be highly sensitive to Zn(2+). Although a pathological role for Zn(2+)-mediated inhibition of GABA(A) receptors has been postulated, no direct evidence exists that endogenous Zn(2+) can modulate GABAergic signaling in the brain. A possible explanation is that Zn(2+) is mainly localized to a subset of glutamatergic synapses. Hippocampal mossy fibers are unusual in that they are glutamatergic but have also been reported to contain GABA and Zn(2+). Here, we show, using combined Timm's method and post-embedding immunogold, that the same mossy fiber varicosities can contain both GABA and Zn(2+). Chelating Zn(2+) with either calcium-saturated EDTA or N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine had no effect on stratum-radiatum-evoked inhibitory postsynaptic currents (IPSCs), but enhanced IPSCs evoked by stimuli designed to recruit dentate granule cells. We also show that IPSCs recorded in CA3 pyramidal neurons in acute hippocampal slices are depressed by exogenous Zn(2+). This depression was of similar amplitude whether the IPSCs were evoked by stimulation in s. radiatum (to recruit local interneurons) or in the s. granulosum of the dentate gyrus (to recruit mossy fibers). These results show for the first time that GABAergic IPSCs can be modulated by endogenous Zn(2+) and are consistent with GABA release at Zn(2+)-containing mossy fiber synapses.