Enhanced Ca(2+) influx with mossy fiber stimulation in hippocampal CA3 neurons of spontaneously epileptic rats

This study was performed to determine whether the intracellular Ca²⁺ concentration ([Ca²⁺]_i) is increased in hippocampal CA3 neurons of spontaneously epileptic rats (SER) which show both absence-like and convulsive seizures using hippocampal slices loaded with Calcium Green-1 when a weak single stimulation is given to the mossy fiber. [Ca²⁺]_i in the CA3 area was significantly increased after a single stimulus to mossy fibers in SER, while no changes were detected in normal Wistar rats. These findings suggest the existence of an abnormality in the Ca²⁺ channel in the SER CA3 region and that this is probably responsible for epileptic seizures.     

Heterosynaptic enhancement of the excitability of hippocampal mossy fibers by long-range spill-over of glutamate

Several classes of ionotropic receptors have been reported to depolarize the axonal membrane of hippocampal mossy fibers. Both kainate receptors and GABA(A) receptors are localized on axons and/or presynaptic terminals, and these receptors have been known to be activated by synaptically released glutamate and GABA which spill out from the synaptic clefts. However the relative contribution of these two receptors in modulating the excitability of mossy fiber axon was not reported so far. In this study, we revealed that glutamate spilled out from commissural/associational synapses evoked the facilitation of antidromic population spikes of mossy fibers. Increase in amplitude and decrease in latency of population spikes suggest that the number of recruited mossy fibers increases by depolarization of axonal membrane. Application of non-NMDA receptor antagonist CNQX (10 μM) almost abolished this effect. TBOA (30 μM), an inhibitor of glutamate transporter, prolonged the duration of heterosynaptic facilitation. These results suggest that glutamate released from distant commissural/associational synapses spills out from synaptic cleft and activates the kainate receptors on the mossy fibers of CA3 region, and plays a major role in modulating presynaptic excitability than GABA.     

Selective release of endogenous zinc from the hippocampal mossy fibers in situ

The release of endogenous zinc was studied in the hippocampus of the anesthetized rat. Push-pull cannulae were bilaterally introduced in the hippocampus and zinc concentrations determined by atomic absorption spectrophotometry. We first studied the regional distribution of K+-evoked release of zinc. A 2 min (30 mM) K+ pulse produced a release of endogenous zinc, when the push-pull cannulae were located in the vicinity of the mossy fibers (CA3 or hilus) but not in other regions (including CA1, fimbria, molecular layer of the fascia dentata, thalamus etc...). In CA3 the maximal release was of 2000 ng/ml (200 times the levels of zinc present in the cerebrospinal fluid (CSF)). Destruction of the mossy fibers by a local unilateral injection of 1.5 micrograms of colchicine dissolved in 0.6 microliter of a saline solution, eliminated this release without affecting the release in the control (contralateral) side. Electrical stimulation of the perforant path at 1 Hz did not evoke a release of zinc. In contrast at 10 Hz this stimulation produced a burst of population spike and a significant release of zinc in the mossy fibers (and not in other regions of the hippocampus). These experiments provide direct evidence that zinc is selectively released from the mossy fibers.     

Homeostatic maintenance in excitability of tree shrew hippocampal CA3 pyramidal neurons after chronic stress

The experience of chronic stress induces a reversible regression of hippocampal CA3 apical neuron dendrites. Although such postsynaptic membrane reduction will obviously diminish the possibility of synaptic input, the consequences for the functional membrane properties of these cells are not well understood. We tested the hypothesis that chronic stress affects the input-output characteristics and excitability of CA3 pyramidal cells. Somatic whole-cell current-clamp recording with parallel intracellular biocytin labeling was performed on CA3 neurons from in vitro hippocampal slices from male tree shrews, which were collected after 28 days of psychosocial stress exposure and compared to recordings obtained from control animals. Post hoc morphometric analysis of biocytin-labeled CA3 cells revealed branch regression, by fewer dendritic crossings and length, limited to a distance of approximately 280-340 microm from the soma only. The results from whole-cell recording indicate that chronic stress surprisingly reduced the apparent membrane time constant and input resistance 20-25%, accompanied by increased amplitude of the hyperpolarization-induced voltage "sag." All active membrane properties, including depolarization-induced action potential kinetics, complex spiking patterns, and afterhyperpolarization voltages, were indistinguishable from control recordings. Although linear association analysis confirmed that differences in geometry, such as apical length or branch number, were correlated to functional variability in properties of the AP current and voltage threshold, these changes were too marginal to be reflected in the group differences. However, the individual adrenal hormone status was associated significantly with the selective changes in subthreshold excitability. Taken together, the data provide evidence that despite long-term stress induces morphological changes, upregulates cortisol release and shifts the intrinsic membrane properties, the efficacy of somatic excitability of CA3 pyramidal neurons is largely preserved.     

Spontaneous epileptiform bursts and long-term potentiation in rat CA3 hippocampal slices induced by chaotic stimulation of mossy fibers

The relation between long-term potentiation (LTP) and spontaneous rhythm in CA3 was investigated using rat hippocampal slices. Field potential response of CA3 to mossy fiber stimulation consisted of a mono-synaptic positive potential and subsequent poly-synaptic negative potentials. LTP of both field potentials was induced by chaotic mossy fiber stimulation. Although CA3 did not show any spontaneous rhythm before LTP induction in a normal perfusing medium, CA3 spontaneously caused epileptiform bursts after LTP induction by chaotic mossy fiber stimulation. The amplitude of those epileptiform bursts and the inter-burst interval were not uniform. After LTP induction, the cross-correlation function of spontaneous field potentials simultaneously recorded at two sites approximately 300 μm apart in CA3 showed a large central peak. This indicates that neuronal activity at two sites is synchronized. These results suggest that epileptiform bursts in CA3 are caused by synchronization of spontaneous CA3 pyramidal cell activity due to LTP induced by chaotic burst stimulation.     

Timm's sulfide silver reaction for zinc during experimental anterograde degeneration of hippocampal mossy fibers

The boutons of the hippocampal mossy fibers stain particularly well with Timm's sulfide silver reaction for zinc and other metals. In the present study anterograde degeneration was produced in the mossy fibers in the rat by placing lesions in the fascia dentata, after which the Timm reaction was checked at different postoperative intervals. No unequivocal changes were seen as early as five hours after operation. Barely discernible blanching of the affected parts of the mossy fiber layer was observed after seven hours, a significant reduction of stainability was present after ten hours, and a nearly complete loss after 24 hours. The latter situation persisted for the rest of the period studied, viz., up to eight days after operation. Topographical coincidence of reduced Timm staining with areas of anterograde degeneration was verified by impregnating alternating sections of the sulfide treated tissue according to the reduced silver method of Fink and Heimer. These sections moreover confirmed that the affected boutons retain their structural identity for at least several days after the disappearance of the stainability with Timm's method. These findings are compatible with the concept that the zinc may be associated with some rapidly metabolized substance directly or indirectly involved in synaptic transmission.     

Circadian rhythm of redox state regulates membrane excitability in hippocampal CA1 neurons

Behaviors, such as sleeping, foraging, and learning, are controlled by different regions of the rat brain, yet they occur rhythmically over the course of day and night. They are aligned adaptively with the day‐night cycle by an endogenous circadian clock in the suprachiasmatic nucleus (SCN), but local mechanisms of rhythmic control are not established. The SCN expresses a ~24‐hr oscillation in reduction‐oxidation that modulates its own neuronal excitability. Could circadian redox oscillations control neuronal excitability elsewhere in the brain? We focused on the CA1 region of the rat hippocampus, which is known for integrating information as memories and where clock gene expression undergoes a circadian oscillation that is in anti‐phase to the SCN. Evaluating long‐term imaging of endogenous redox couples and biochemical determination of glutathiolation levels, we observed oscillations with a ~24 hr period that is 180° out‐of‐phase to the SCN. Excitability of CA1 pyramidal neurons, primary hippocampal projection neurons, also exhibits a rhythm in resting membrane potential that is circadian time‐dependent and opposite from that of the SCN. The reducing reagent glutathione rapidly and reversibly depolarized the resting membrane potential of CA1 neurons; the magnitude is time‐of‐day‐dependent and, again, opposite from the SCN. These findings extend circadian redox regulation of neuronal excitability from the SCN to the hippocampus. Insights into this system contribute to understanding hippocampal circadian processes, such as learning and memory, seizure susceptibility, and memory loss with aging.     

Decreased numerical density of CA3 hippocampal mossy fiber synapses in schizophrenia

The CA3 region of the hippocampus is unique in its connectivity, its role in cognitive maintenance, and its great vulnerability in schizophrenia. The down regulation of the expression and binding activity of glutamate receptors was revealed in the CA3 hippocampal region and may be attributed to cognitive disturbances in schizophrenia. Our previous study demonstrated that only schizophrenics with predominantly positive (but not predominantly negative) symptoms had smaller-sized branched spines (thorny excrescences) of CA3 pyramidal neurons and fewer synaptic contacts formed by dentate mossy fiber terminals (MFT-synapses). In the present study, we used an unbiased stereological physical dissector method to verify whether the numerical density of MFT-synapses is altered in schizophrenia. A morphometric study was performed in 10 normal controls and eight age-matched cases with chronic schizophrenia, including five cases with predominantly positive and three with predominantly negative symptoms. Schizophrenic cases had a significantly reduced numerical density of MFT-synapses (-25%, P < 0.01) compared with the control group. The decrease was similar in schizophrenic subgroups with predominantly positive and predominantly negative symptoms. No effects of postmortem delay, age, duration of disease, and neuroleptic exposure were found. Taken together with our previous results, the data suggest that the decrease of numerical density of MFT-synapses may be the result of different mechanisms in schizophrenics with predominantly positive and predominantly negative symptoms.     

Associative mossy fibre LTP induced by pairing presynaptic stimulation with postsynaptic hyperpolarization of CA3 neurons in rat hippocampal slice

Whole cell recordings of excitatory postsynaptic potentials/currents (EPSPs/EPSCs) evoked by minimal stimulation of commissural-associative (CF) and mossy fibre (MF) inputs were performed in CA3 pyramidal neurons. Paired responses (at 50 ms intervals) were recorded before, during and after hyperpolarization of the postsynaptic membrane (20-30 mV for 15-35 min). Membrane hyperpolarization produced a supralinear increase of EPSPs/EPSCs amplitude in MF-inputs. Synaptic responses remained potentiated for the rest of the recording period (up to 40 min) after resetting the membrane potential to control level (221 +/- 60%, n = 15 and 219 +/- 61%, n = 11 for MF-EPSP and MF-EPSC, respectively). We shall refer to this effect as hyperpolarization-induced LTP (HI-LTP). In the absence of afferent stimulation, membrane hyperpolarization was unable to produce HI-LTP. In contrast to MF-EPSPs, the mean amplitude of CF-EPSPs did not increase significantly after hyperpolarization relative to controls (138 +/- 29%, n = 22). HI-LTP was associated with modifications of classical indices of presynaptic release: paired-pulse facilitation, failures rate, coefficient of variation of EPSP amplitudes and quantal content. The induction of HI-LTP was NMDA independent but was dependent on metabotropic glutamate receptors (mGluRs) activation and calcium release from inositol 1,4,5-triphosphate (IP3)-sensitive intracellular stores: it was prevented by mGluR antagonist, intracellular heparin and BAPTA. We conclude that while the induction of HI-LTP was postsynaptic, its expression was presynaptic.     

Activity dynamics and behavioral correlates of CA3 and CA1 hippocampal pyramidal neurons

The CA3 and CA1 pyramidal neurons are the major principal cell types of the hippocampus proper. The strongly recurrent collateral system of CA3 cells and the largely parallel‐organized CA1 neurons suggest that these regions perform distinct computations. However, a comprehensive comparison between CA1 and CA3 pyramidal cells in terms of firing properties, network dynamics, and behavioral correlations is sparse in the intact animal. We performed large‐scale recordings in the dorsal hippocampus of rats to quantify the similarities and differences between CA1 (n > 3,600) and CA3 (n > 2,200) pyramidal cells during sleep and exploration in multiple environments. CA1 and CA3 neurons differed significantly in firing rates, spike burst propensity, spike entrainment by the theta rhythm, and other aspects of spiking dynamics in a brain state‐dependent manner. A smaller proportion of CA3 than CA1 cells displayed prominent place fields, but place fields of CA3 neurons were more compact, more stable, and carried more spatial information per spike than those of CA1 pyramidal cells. Several other features of the two cell types were specific to the testing environment. CA3 neurons showed less pronounced phase precession and a weaker position versus spike‐phase relationship than CA1 cells. Our findings suggest that these distinct activity dynamics of CA1 and CA3 pyramidal cells support their distinct computational roles. © 2012 Wiley Periodicals, Inc.     

Mixed electrical-chemical transmission between hippocampal mossy fibers and pyramidal cells

Morphological and electrophysiological studies have shown that granule cell axons, the mossy fibers (MFs), establish gap junctions and therefore electrical communication among them. That granule cells express gap junctional proteins in their axons suggests the possibility that their terminals also express them. If this were to be the case, mixed electrical-chemical communication could be supported, as MF terminals normally use glutamate for fast communication with their target cells. Here we present electrophysiological studies in the rat and modeling studies consistent with this hypothesis. We show that MF activation produced fast spikelets followed by excitatory postsynaptic potentials in pyramidal cells (PCs), which, unlike the spikelets, underwent frequency potentiation and were strongly depressed by activation of metabotropic glutamate receptors, as expected from transmission of MF origin. The spikelets, which persisted during blockade of chemical transmission, were potentiated by dopamine and suppressed by the gap junction blocker carbenoxolone. The various waveforms evoked by MF stimulation were replicated in a multi-compartment model of a PC by brief current-pulse injections into the proximal apical dendritic compartment, where MFs are known to contact PCs. Mixed electrical and glutamatergic communication between granule cells and some PCs in CA3 may ensure the activation of sets of PCs, bypassing the strong action of concurrent feed-forward inhibition that granule cells activate. Importantly, MF-to-PC electrical coupling may allow bidirectional, possibly graded, communication that can be faster than chemical synapses and subject to different forms of modulation.     

Maturation of granule cell dendrites after mossy fiber arrival in hippocampal field CA3

Most granule neurons in the rat dentate gyrus are born over the course of the first 2 postnatal weeks. The resulting heterogeneity has made it difficult to define the relationship between dendritic and axonal maturation and to delineate a time course for the morphological development of the oldest granule neurons. By depositing crystals of the fluorescent label Dil in hippocampal field CA3, we retrogradely labeled granule neurons in fixed tissue slices from rats aged 2-9 days. The results showed that all labeled granule cells, regardless of the age of the animal, exhibited apical dendrites. On day 2, every labeled neuron had rudimentary apical dendrites, and a few dendrites on each cell displayed immature features such as growth cones, varicosities, and filopodia. Some cells displayed basal dendrites. By day 4, the most mature granule neurons had longer and more numerous apical branches, as well as various immature features. Most had basal dendrites. On days 5 and 6, the immature features and the basal dendrites had begun to regress on the oldest cells, and varying numbers of spines were present. On day 7, the first few adult-like neurons were seen: immature features and basal dendrites had disappeared, all dendrites reached the top of the molecular layer, and the entire dendritic tree was covered with spines. These data show that dendritic outgrowth occurs before, or concurrent with, axon arrival in the CA3 target region, and that adult-like granule neurons are present by the end of the first week.     

Transient effect of mossy fiber stimulation on spatial firing of CA3 neurons

Strong hippocampal mossy fiber synapses are thought to function as detonators, imposing “teaching” signals onto CA3 neurons during new memory formation. For an empirical test of this long‐standing view, we examined effects of optogenetically stimulating mossy fibers on spatial firing of CA3 neurons in freely‐moving mice. We found that spatially restricted mossy fiber stimulation drives novel place‐specific firing in some CA3 pyramidal neurons. Such neurons comprise only a minority, however, and many more CA3 neurons showed inhibited spatial firing during mossy fiber stimulation. Also, changes in spatial firing induced by mossy fiber stimulation, both activated and inhibited, reverted immediately upon stimulation termination, leaving CA3 place fields unaltered. Our results do not support the traditional view that mossy fibers impose teaching signals onto CA3 network, and show robustness of established CA3 spatial representations.     

Loss of zinc staining from hippocampal mossy fibers during kainic acid induced seizures: a histofluorescence study

A quinoline fluorescence method for staining zinc in axonal boutons was used to study the effects of kainic acid (KA) induced seizures upon zinc in the boutons of hippocampal mossy fibers. Compared to untreated rats, rats given KA (10-12 mg/kg) and undergoing sustained seizures showed a marked loss of zinc fluorescence in the mossy fiber regions. The reduced fluorescence was detectable within 3 h of KA administration, was most pronounced at 12-24 h, and was still noticeable up to 48 h after KA. The findings suggest that zinc is released rapidly from mossy fiber boutons during seizures.     

Plasticity and metaplasticity of adult rat hippocampal mossy fibers induced by neurotrophin‐3

Changes in synaptic efficacy and morphology are considered as the downstream mechanisms of consolidation of memories and other adaptive behaviors. In the last decade, neurotrophin‐3 (NT‐3) has emerged as one potent mediator of synaptic plasticity. In the adult brain, expression of NT‐3 is largely confined to the hippocampal dentate gyrus (DG). Our previous studies show that application of high‐frequency stimulation (HFS) sufficient to elicit long‐term potentiation (LTP) at the DG‐CA3 pathway as well as acute intrahippocampal microinfusion of brain‐derived neurotrophin factor produce mossy fiber (MF) structural reorganization. Here, we show that intrahippocampal microinfusion of NT‐3 induces a long‐lasting potentiation of synaptic efficacy in the DG‐CA3 projection accompanied by an MF structural reorganization of adult rats in vivo. It is considered that the capacity of synapses to express plastic changes is itself subject to variation depending on previous experience; taking into consideration the effects of NT‐3 on MF synaptic plasticity, we thus used intrahippocampal microinfusion of NT‐3 to analyse its effects on functional and structural plasticity induced by subsequent MF‐HFS sufficient to induce LTP in adult rats, in vivo. Our results show that NT‐3 modifies the ability of the MF pathway to present subsequent LTP by HFS, and modifies the structural reorganization pattern. The modifications in synaptic efficacy and morphology elicited by NT‐3 at the MF‐CA3 pathway were blocked by the presence of a Trk receptor inhibitor (K252a). These findings support the idea that NT‐3 actions modify subsequent synaptic plasticity, a homeostatic mechanism thought to be essential for maintaining synapses in the adult mammalian brain.     

Synergistic actions of metabotropic acetylcholine and glutamate receptors on the excitability of hippocampal CA1 pyramidal neurons

A variety of neurotransmitters are responsible for regulating neural activity during different behavioral states. Unique responses to combinations of neurotransmitters provide a powerful mechanism by which neural networks could be differentially activated during a broad range of behaviors. Here, we show, using whole-cell recordings in rat hippocampal slices, that group I metabotropic glutamate receptors (mGluRs) and muscarinic acetylcholine receptors (mAChRs) synergistically increase the excitability of hippocampal CA1 pyramidal neurons by converting the post-burst afterhyperpolarization to an afterdepolarization via a rapidly reversible upregulation of Ca(v)2.3 R-type calcium channels. Coactivation of mAChRs and mGluRs also induced a long-lasting enhancement of the responses mediated by each receptor type. These results suggest that cooperative signaling via mAChRs and group I mGluRs could provide a mechanism by which cognitive processes may be modulated by conjoint activation of two separate neurotransmitter systems.     

Distal infrapyramidal and longitudinal mossy fibers at a midtemporal hippocampal level

Timm's and horseradish peroxidase histochemical methods were used to demonstrate a small, distal infrapyramidal mossy fiber projection at midtemporal hippocampal levels in the rat. The Timm's sulfide silver method revealed sparse granules of dark, mossy fiber-like staining at an infrapyramidal location in an area roughly equivalent to hippocampal subfield CA3a (close to the CA1 border). The use of anterograde transport of horseradish peroxidase enabled the visualization of short mossy fiber axons that left the suprapyramidal bundle, penetrated the pyramidal cell layer, and terminated in a distal infrapyramidal position. The HRP-labeled axons possessed periodic swellings that are characteristic of mossy fiber morphology. The HRP studies also provided evidence for longitudinally oriented mossy fibers at midtemporal hippocampal levels. The distal tip of the suprapyramidal band of mossy fibers turned temporally and could be detected up to 1140 micrometers ventral to the main suprapyramidal band.