Cellular and connective organization of slice cultures of the rat hippocampus and fascia dentata

This study examined the cellular and connective organization of hippocampal tissue taken from 6-8-day-old rats and cultured by the roller tube technique for 3-6 weeks. In the cultures containing the fascia dentata and the hippocampus proper (CA1, CA3, CA4) the main cell and neuropil layers were organotypically organized when observed in ordinary cell stains. The normal distribution of smaller cell populations of AChE-positive neurons and somatostatin-reactive neurons was demonstrated by histochemical and immunohistochemical methods. Both cell types were mainly confined to str. oriens of CA3 and CA1 and the dentate hilus (CA4). Individual dentate granule cells and hippocampal pyramidal cells were injected with lucifer yellow and HRP, revealing great stability of the dendritic patterns of these cells in the culture condition. The same was found for the axonal branching and termination of HRP-filled mossy fibers arising from an HRP-injected granule cell. The preservation of organotypic afferent patterns in the cultures was also shown by Timm staining of the terminal distribution of the mossy fiber system. Mossy fiber terminals, with characteristic ultrastructural features verified in the electron microscope, were thus found in the hilus (CA4) and along the CA3 pyramidal cell layer onto the CA3-CA1 transition. Depending on the amount of dentate tissue relative to CA3 the terminals could stop before reaching CA1 (small fascia dentata) or take up additional intra and infrapyramidal locations along CA3 (small CA3). In cultures with a gap in the CA3 pyramidal cell layer some mossy fiber terminals were found in contact with the CA3 pyramidal cells beyond the gap. In all cultures there was an aberrant projection of supragranular mossy fibers. This projection is analogous to the one known from lesion and transplant studies to form in the absence of the entorhinal perforant path input to the dentate molecular layer. Also, in accordance with these studies the Timm staining pattern of the outer parts of the dentate molecular layer and the entire molecular layer of the hippocampus was altered corresponding to the spread of afferents normally confined to the inner zone of the dentate and str. radiatum of CA3 and CA1. Possibly as a consequence of the lack of normal targets for projections from CA1, this subfield contained an unusually dense Timm staining suggestive of autoinnervation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Connections of the hippocampal formation in humans: I. The mossy fiber pathway

The hippocampal formation has been one of the most extensively studied cortical regions in rats, yet little is known about the anatomical connections of the hippocampus in primates, especially humans. With the use of an antibody against the calcium-binding protein, calbindin-D28K, in normal autopsy tissue and the neuronal tracers biocytin or biotinylated dextrans in in vitro slice preparations from tissue removed during surgery for intractable epilepsy, we examined the human hippocampal mossy fiber pathway. The injections of biocytin into the dentate granule cell layer labeled neurons in a Golgi-like manner, revealing the presence of basal dendrites on about 30% of the granule cells. The granule cell axons, the mossy fibers, initially formed a diffuse plexus of fibers in the polymorphic layer before organizing into fiber fascicles in the hilar pyramidal region. These fiber fascicles were much more prominent rostrally than caudally. Within the hilus and proximal portions of the extrahilar CA3 field, the mossy fibers ran through the pyramidal cell layer, and while near the transition to field CA2, the fibers turned superficially and crossed the pyramidal layer to run in the stratum lucidum. All of these features, seen following injections of tracer into hippocampal slices from the brains of epileptics, were confirmed by calbindin-staining of mossy fibers in normal brains. Biocytin-labeled mossy fiber axons revealed two characteristic types of enlargements: small varicosities and larger expansions. The expansions were found throughout the neuropil and were highly irregular, diaminobenzidine-dense profiles that had pleiomorphic modes of attachment to the parent axon. Electron microscopic images of these biocytin labeled expansions revealed that they were large synaptic boutons bearing asymmetric synapses. This study indicates that the human mossy fiber pathway shows some minor deviations from the rodent brain but little difference from monkeys. We argue that these changes mirror a phylogenetic growth of the CA3 pyramidal neurons (subfield CA3c) into the hilus rather than an evolutionary change of the mossy fiber pathway. This growth of subfield CA3c and the increase in mossy fibers running through the pyramidal layer (and a presumed accompanying increase in proximal basal dendritic contacts) may reflect a growing role of the projection from the dentate granule cells to subfield CA3c and from there to field CA1 in the primate hippocampus. J. Comp. Neurol. 385:325–351, 1997. © 1997 Wiley-Liss, Inc.     

Origin of microglia in the quail retina: Central-to-peripheral and vitreal-to-scleral migration of microglial precursors during development

Collateral sprouting of dentate granule cell axons, the mossy fibers, occurs in response to denervation, kindling, or excitotoxic damage to the hippocampus. Organotypic slice culture of rodent hippocampal tissue is a model system for the controlled study of collateral sprouting in vitro. Organotypic roller-tube cultures were prepared from hippocampal slices derived from postnatal day 7 mice. The Timm heavy metal stain and densitometry were used to assay the degree of mossy fiber collateral sprouting in the molecular layer of the hippocampal dentate gyrus. Factors influencing mossy fiber collateral sprouting were time in culture, positional origin of the slice culture along the septotemporal axis of the hippocampus, and presence of attached subicular-entorhinal cortical tissues. Collateral sprouting in the molecular layer was first detected after 6 days in culture and increased steadily thereafter. By 2 weeks considerable sprouting was apparent, and at 3 weeks intense sprouting was observed within the molecular layer. An intrinsic septal-to-temporal gradient of collateral sprouting was apparent at 14 days in culture. To determine whether differential damage to the mossy fibers was the basis for the differences in collateral sprouting along the septotemporal axis, we made complete transections of the mossy fiber projection as it exited the dentate hilus at various levels along the septotemporal axis; no differences were found on subsequent collateral sprouting in the dentate molecular layer. Timm-stained hippocampal cultures with an attached entorhinal cortex, a major source of afferent innervation to the dentate granule cells, displayed significantly less collateral sprouting at 10 days in culture compared to that in cultures from adjacent sections without attached subicular-entorhinal tissues present. Thus, time in culture, position along the septotemporal axis, and presence of afferent cortical tissues influence aberrant neurite collateral sprouting in organotypic slice cultures of neonatal mouse hippocampus. © Wiley-Liss, Inc.     

Roles of afadin in the formation of the cellular architecture of the mouse hippocampus and dentate gyrus

The hippocampal formation with tightly packed neurons, mainly at the dentate gyrus, CA3, CA2, and CA1 regions, constitutes a one-way neural circuit, which is associated with learning and memory. We previously showed that the cell adhesion molecules nectins and its binding protein afadin play roles in the formation of the mossy fiber synapses which are formed between the mossy fibers of the dentate gyrus granule cells and the dendrites of the CA3 pyramidal cells. We showed here that in the afadin-deficient hippocampal formation, the dentate gyrus granules cells and the CA3, CA2, and CA1 pyramidal cells were abnormally located; the mossy fiber trajectory was abnormally elongated; the CA3 pyramidal cells were abnormally differentiated; and the densities of the presynaptic boutons on the mossy fibers and the apical dendrites of the CA3 pyramidal cells were decreased. These results indicate that afadin plays roles not only in the formation of the mossy fiber synapses but also in the formation of the cellular architecture of the hippocampus and the dentate gyrus.     

Lesion-induced rerouting of hippocampal mossy fibers in developing but not in adult rats

The organization of the hippocampal mossy fiber system which projects from the granule cells of fascia dentata to the pyramidal cells of the hippocampal regio inferior (CA3) was studied after chronic lesions had been applied to CA3 in neonatal (0–3 day old), 5 and 21 day old, and adult rats. The distribution of terminals was monitored by the histochemical Timm sulphide silver method, and the fibers were demonstrated by the original Nauta silver method. Complete transections of CA3 in neonatal rats induced a layer of aberrant, infrapyramidal mossy fiber terminals in CA3 up to 1,300 μm septal to the transection followed by a corresponding expansion of the suprapyramidal mossy fiber layer for at least 720 μm. At increasing distance above the lesion, the induced changes moved laterally in CA3, affecting more distal parts of the mossy fibers. Corresponding to the layer of aberrant infrapyramidal terminals were aberrant bundles of mossy fibers, with an abnormally ascending septal course. No aberrant mossy fibers or terminals were induced temporal to the complete transections. Partial CA3 lesions in neonates induced changes corresponding to those observed after complete transections septal to the lesions. In addition, a layer of aberrant infrapyramidal mossy fiber terminals was induced in CA3 as far as 900 μm temporal to the partial lesions, accompained by an expansion of the suprapyramidal layer. The infrapyramidal terminals were related to aberrant bundles of fibers which followed an abnormally steep, temporal course, from which they in turn leveled off to join the suprapyramidal bundles. Compared to neonatal lesions, hippocampal transections performed on day 5 induced a slightly less dense layer of aberrant infrapyramidal mossy fiber terminals in CA3, and although a transection on day 21 still induced aberrant infrapyramidal terminals, these were far less abundant than after lesions at day 5. Transections in adult rats induced no such changes at all, and additional denervation of CA3 by simultaneous removal of the commisural projection from the contralateral hippocampus did not have any effect. The lesion-induced, but age-dependent redistribution of mossy fibers to CA3 is discussed in terms of reactive reinnervation following partial deafferentation, and compensatory sprouting and rerouting due to pruning-like effects of the lesions. A rerouting of developing, not fully matured mossy fibers is found to be most likely: Deprived of normal target areas, the fibers have been forced to grow in abnormal directions, ending up hyperinnervating adjacent, accessible levels through the formation of aberrant infrapyramidal terminals and expansion of the suprapyramidal zone.     

Enhanced acetylcholinesterase staining in hippocampal area CA3 after lesion of granule cells by infusion of colchicine

Unilateral infusion of colchicine into the lateral ventricle produced relatively selective destruction of dentate granule cells in the ipsilateral dorsal hippocampal formation of the rat. Timms silver sulfide stain is markedly reduced in the mossy fiber layer on the colchicine treated side but is normal contralaterally. After colchicine treatment, an increase in acetylcholinesterase staining is apparent in the apical dendritic zone of CA3 pyramidal cells. This enhanced staining is localized in the proximal apical dendritic layer of CA3, a region normally occupied by the mossy fiber terminals of dentate granule cells. These results suggest that cholinergic fibers proliferate in CA3 after granule cell lesion and may participate in reinnervation of the denervated area.     

K252a, an inhibitor of Trk, disturbs pathfinding of hippocampal mossy fibers

Hippocampal mossy fibers, which are the axons of dentate granule cells, are continuously generated owing to adult neurogenesis of granule cells. They extend exclusively into the stratum lucidum, a proximal layer of the CA3 pyramidal cells. We visualized the mossy fiber tracts by Timm histochemical staining and DiI labeling in the cultured hippocampal slices from newborn rats. The fibers were abnormally expanded when the slices were cultured in the presence of K252a, an inhibitor of the neurotrophin receptor Trk. Similar defasciculation was observed with an inhibitor of MEK, which is one of the signaling molecules downstream of Trk. This study suggests for the first time that Trk and the MEK pathway are required for mossy fiber pathfinding.     

Morphological organization of rat hippocampal slice cultures.

Using various histological methods, we investigated the cellular and morphological organization of rat hippocampal slice cultures. Many of the typical features of the hippocampus were retained in vitro over a long period of time. The principal cell types of the hippocampus and dentate gyrus, the pyramidal cells and granule cells, were well preserved and matured in vitro. Nonpyramidal cells and gamma-aminobutyric-acid (GABA) cells were also present in slice cultures and exhibited a strikingly similar dendritic appearance at the light microscopic level. Moreover, GABA-immunoreactive cell bodies and presynaptic terminals could be identified at the electron microscopic level; they expressed typical symmetric synaptic contacts with cell bodies and dendrites. The course of the intrinsic hippocampal fiber pathways--the mossy fibers, Schaffer collaterals, and alveus--was generally retained in vitro. Additional aberrant fiber projections could be identified. Finally, three types of nonneuronal cells could be distinguished on the basis of immunocytochemical methods.     

Ultrastructural description of taurine-like immunoreactive cells and processes in the rat hippocampus

A monoclonal antibody against taurine conjugated to KLH was used to identify and describe taurine-like immunoreactive processes in the rat hippocampus. Tissue from perfused rats was processed for immunohistochemical visualization of taurine and embedded for electron microscopy. Representative tissue samples from three regions, the dentate gyrus, CA3, and CA1, were sectioned, examined, and photographed. In the dentate gyrus, both granule cells and pyramidal basket cells were taurine-like immunoreactive. Some axon terminals in the dentate gyrus molecular layer as well as some mossy fiber boutons in the hilus were also taurine-like immunoreactive. In the CA3 region both pyramidal neurons and glial cells were taurine-like immunoreactive A few small-diameter axon terminals in stratum radiatum and some mossy fiber boutons in stratum lucidum were taurine-like immunoreactive. In CA1, pyramidal neurons and some glia were intensely taurine-like immunoreactive. A few immunoreactive axon terminals were seen in stratum radiatum and stratum oriens. In all regions, dendritic staining predominated. Our results support the hypothesis that while taurine may act as a neurotransmitter in a small portion of hippocampal terminals, its main function is probably as a neuromodulator or ionic regulator.     

The comparative immunocytochemical distribution of 28 kDa cholecalcin (CaBP) in the hippocampus of rat, guinea pig and hedgehog

The distribution of 28 kDa cholecalcin (calcium-binding protein, CaBP) in the hippocampal formation of the rat, guinea pig and European hedgehog was examined by immunocytochemistry. The extension of the mossy fibers (the axons of the granule cells of the dentate gyrus) was also studied using the Timm's sulfide-silver method. Cholecalcin was present in all mossy fibers. In the rat, only those pyramidal cells not reached by the labeled mossy fibers displayed cholecalcin immunoreactivity. Immunocytochemical staining of the hedgehog hippocampus showed that contacts between cholecalcin-containing mossy fibers and cholecalcin-containing pyramidal cells are possible. Consequently, the protein is probably not involved in the control of mossy fiber extension. Strikingly, no guinea pig pyramidal cells showed cholecalcin immunoreactivity. The possible involvement of cholecalcin in the differential excitability of pyramidal cells in the CA3 and CA1 areas of the hippocampus could therefore be tested in a comparative study of rat, guinea pig and hedgehog.     

Increased excitatory synaptic input to granule cells from hilar and CA3 regions in a rat model of temporal lobe epilepsy

One potential mechanism of temporal lobe epilepsy is recurrent excitation of dentate granule cells through aberrant sprouting of their axons (mossy fibers), which is found in many patients and animal models. However, correlations between the extent of mossy fiber sprouting and seizure frequency are weak. Additional potential sources of granule cell recurrent excitation that would not have been detected by markers of mossy fiber sprouting in previous studies include surviving mossy cells and proximal CA3 pyramidal cells. To test those possibilities in hippocampal slices from epileptic pilocarpine-treated rats, laser-scanning glutamate uncaging was used to randomly and focally activate neurons in the granule cell layer, hilus, and proximal CA3 pyramidal cell layer while measuring evoked EPSCs in normotopic granule cells. Consistent with mossy fiber sprouting, a higher proportion of glutamate-uncaging spots in the granule cell layer evoked EPSCs in epileptic rats compared with controls. In addition, stimulation spots in the hilus and proximal CA3 pyramidal cell layer were more likely to evoke EPSCs in epileptic rats, despite significant neuron loss in those regions. Furthermore, synaptic strength of recurrent excitatory inputs to granule cells from CA3 pyramidal cells and other granule cells was increased in epileptic rats. These findings reveal substantial levels of excessive, recurrent, excitatory synaptic input to granule cells from neurons in the hilus and proximal CA3 field. The aberrant development of these additional positive-feedback circuits might contribute to epileptogenesis in temporal lobe epilepsy.     

A novel form of low-frequency hippocampal mossy fiber plasticity induced by bimodal mGlu1 receptor signaling

Mossy fiber synapses act as the critical mediators of highly dynamic communication between hippocampal granule cells in the dentate gyrus and CA3 pyramidal neurons. Excitatory synaptic strength at mossy fiber to CA3 pyramidal cell synapses is potentiated rapidly and reversibly by brief trains of low-frequency stimulation of mossy fiber axons. We show that slight modifications to the pattern of stimulation convert this short-term potentiation into prolonged synaptic strengthening lasting tens of minutes in rodent hippocampal slices. This low-frequency potentiation of mossy fiber EPSCs requires postsynaptic mGlu1 receptors for induction but is expressed presynaptically as an increased release probability and therefore impacts both AMPA and NMDA components of the mossy fiber EPSC. A nonconventional signaling pathway initiated by mGlu1 receptors contributes to induction of plasticity, because EPSC potentiation was prevented by a tyrosine kinase inhibitor and only partially reduced by guanosine 5'-O-(2-thiodiphosphate). A slowly reversible state of enhanced synaptic efficacy could serve as a mechanism for altering the integrative properties of this synapse within a relatively broad temporal window.     

Inhibition of presynaptic activity by zinc released from mossy fiber terminals during tetanic stimulation

Zinc exists in high densities in the giant boutons of hippocampal mossy fibers. On the basis of the evidence that zinc decreases extracellular glutamate concentration in the hippocampus, the presynaptic action of zinc released from mossy fibers during high-frequency (tetanic) stimulation was examined using hippocampal slices. The increase in zinc-specific fluorescent signals was observed in both extracellular and intracellular compartments in the mossy fiber terminals during the delivery of tetanic stimuli (100 Hz, 1 sec) to the dentate granule cell layer, suggesting that zinc released from mossy fibers is immediately retaken up by mossy fibers. When mossy fiber terminals were preferentially double-stained with zinc and calcium indicators and tetanic stimuli (100 Hz, 1 sec) were delivered to the dentate granule cell layer, the increase in calcium orange signal during the stimulation was enhanced in mossy fiber terminals by addition of CaEDTA, a membrane-impermeable zinc chelator, and was suppressed by addition of zinc. The decrease in FM4-64 signal (vesicular exocytosis) during tetanic stimulation (10 Hz, 180 sec), which induced mossy fiber long-term potentiation, was also enhanced in mossy fiber terminals by addition of CaEDTA and was suppressed by addition of zinc. The present study demonstrates that zinc released from mossy fibers may be a negative-feedback factor against presynaptic activity during tetanic stimulation.     

Long-term low-protein diet reduces the number of hippocampal mossy fiber synapses

Previously we have reported a loss of the dentate granule cells and hippocampal CA3 pyramidal cells in adult animals after lengthy periods of low-protein diet. In this study we examined the effects of this cell loss upon the synaptic connections between the granule cell axons (the mossy fibers) and CA3 pyramidal cell dendrites. Three groups of five rats each were given a low-casein (8%) diet for 6, 12, and 18 months, respectively, and the results of the ultrastructural morphometric analysis compared with similarly processed control rats kept on a control diet. The numerical density of synapses was decreased in undernourished rats and the fraction of the mossy fiber terminal membrane occupied by synaptic specializations was reduced. It can be inferred that the synaptic connectivity pattern between mossy fiber terminals and CA3 dendrites is altered due to a reduction in the number of contacts. Besides, as the synapses of low-protein-treated animals do not display any increase in the length of their active zones, evidence is not provided for the existence of morphological synaptic plasticity, contrary to what has been recently described in other experimental circumstances.     

Extensive target cell loss during development results in mossy fibers in the regio superior (CA1) of the rat hippocampal formation

The axons of dentate granule cells (mossy fibers) have been reported to appear in the regio superior (CA1) of the rat hippocampal formation following destruction of the pyramidal cells in the regio inferior (CA3). We undertook the present experiments to confirm this finding and to determine the requirements for this dramatic neuronal rearrangement. We found that extensive (greater than 80%) loss of CA3 cells, as well as the presence of surviving CA1 neurons within a narrow period of development (postnatal days 3-5) is necessary, however apparently not sufficient, for the appearance of CA1 mossy fibers. That the absence of normal target cells during a restricted period of mossy fiber development will lead to their association with novel targets suggests that much of the specificity of this developing connection depends on the presence of normal targets during a critical period.     

Fetal Hippocampal Cells Grafted to Kainate-Lesioned CA3 Region of Adult Hippocampus Suppress Aberrant Supragranular Sprouting of Host Mossy Fibers

Selective lesion of the rat hippocampus using an intracerebroventricular administration of kainic acid (KA) represents an animal model for studying both lesion recovery and temporal lobe epilepsy. This KA lesion leads initially to loss of CA3 hippocampal neurons, the postsynaptic target of mossy fibers, and later results in aberrant mossy fiber sprouting into the dentate supragranular layer (DSGL). Because of the close association of this aberrant mossy fiber sprouting with an increase in the seizure susceptibility of the dentate gyrus, delayed therapeutic strategies capable of suppressing the sprouting of mossy fibers into the DSGL are of significant importance. We hypothesize that neural grafting can restore the disrupted hippocampal mossy fiber circuitry in this model through the establishment of appropriate mossy fiber projections onto grafted pyramidal neurons and that these appropriate projections will lead to reduced inappropriate sprouting into the DSGL. Large grafts of Embryonic Day 19 hippocampal cells were transplanted into adult hippocampus at 4 days post-KA lesion. Aberrant mossy fiber sprouting was quantified after 3–4 months survival using three different measures of Timm's staining density. Grafts located near the degenerated CA3 cell layer showed dense ingrowth of host mossy fibers compared to grafts elsewhere in the hippocampus. Aberrant mossy fiber sprouting throughout the dentate gyrus was dramatically and specifically reduced in animals with grafts near the degenerated CA3 cell layer compared to “lesion only” animals and those with ectopic grafts away from the CA3 region. These results reveal the capability of appropriately placed fetal hippocampal grafts to restore disrupted hippocampal mossy fiber circuitry by attracting sufficient host mossy fibers to suppress the development of aberrant circuitry in hippocampus. Thus, providing an appropriate postsynaptic target at early postlesion periods significantly facilitates lesion recovery. The graft-induced long-term suppression of aberrant sprouting shown here may provide a new avenue for amelioration of hyperexcitability that occurs following hippocampal lesions.     

Intracellular recording and labeling of mossy cells and proximal CA3 pyramidal cells in macaque monkeys

Little is known about the morphological characteristics and intracellular electrophysiological properties of neurons in the primate hippocampus and dentate gyrus. We have therefore begun a program of studies using intracellular recording and biocytin labeling in hippocampal slices from macaque monkeys. In the current study, we investigated mossy cells and proximal CA3 pyramidal cells. As in rats, macaque mossy cells display fundamentally different traits than proximal CA3 pyramidal cells. Interestingly, macaque mossy cells and CA3 pyramidal neurons display some morphological differences from those in rats. Macaque monkey mossy cells extend more dendrites into the molecular layer of the dentate gyrus, have more elaborate thorny excrescences on their proximal dendrites, and project more axon collaterals into the CA3 region. In macaques, three types of proximal CA3 pyramidal cells are found: classical pyramidal cells, neurons with their dendrites confined to the CA3 pyramidal cell layer, and a previously undescribed cell type, the "dentate" CA3 pyramidal cell, whose apical dendrites extend into and ramify within the hilus, granule cell layer, and molecular layer of the dentate gyrus. The basic electrophysiological properties of mossy cells and proximal CA3 cells are similar to those reported for the rodent. Mossy cells have a higher frequency of large amplitude spontaneous depolarizing postsynaptic potentials, and proximal CA3 pyramidal cells are more likely to discharge bursts of action potentials. Although mossy cells and CA3 pyramidal cells in macaque monkeys display many morphological and electrophysiological features described in rodents, these findings highlight significant species differences, with more heterogeneity and the potential for richer interconnections in the primate hippocampus.     

Coexistence of GABA and glutamate in mossy fiber terminals of the primate hippocampus: an ultrastructural study.

One of the links in the trisynaptic circuit of the hippocampus is the synapse between the mossy fibre terminals of dentate granule cells and CA3 pyramidal cells of Ammon's horn. This synapse has been physiologically characterized as excitatory, and there is pharmacological and immunohistochemical evidence that mossy fibre terminals utilize glutamate as a neurotransmitter. This study demonstrates the presence of GABA-immunoreactivity in mossy fibre axons and terminals of the monkey at the electron microscopic level. We combined Golgi impregnation to identify CA3 pyramidal neurones, with postembedding immunocytochemistry to characterize the inputs to the identified cells. GABA immunoreactivity was present in mossy fibre terminals that made synaptic contact with complex embedded spines of identified Golgi-impregnated CA3 pyramidal neurones. GABA immunoreactivity could be demonstrated in serial sections of the same mossy fibre terminals by using 3 different antisera raised against GABA. In serial sections, the mossy fibre terminals were shown to be immunoreactive for both glutamate and GABA. In contrast, glutamate immunoreactivity but not GABA immunoreactivity was found in other terminals that did not have the morphological characteristics of mossy fibre terminals. GABA immunoreactivity in mossy fibre terminals was also demonstrated in a human surgical specimen of hippocampus. The coexistence of an "excitatory" amino acid and of an "inhibitory" amino acid in the same "excitatory" nerve terminal raises the possibility of corelease of the two transmitters, suggesting that the control of hippocampal neural activity is more complex than hitherto suspected.     

Selective fura-2 loading of presynaptic terminals and nerve cell processes by local perfusion in mammalian brain slice

We describe a method for filling presynaptic terminals and cell dendrites in adult brain slices with the fluorescent calcium indicator fura-2 by localized perfusion of the acetoxymethyl (AM) ester derivative. The method provides labeling selectivity, similar to that produced by intracellular microinjection of fura-2, with the simplicity of bath application of membrane-permeant AM esters. Application of the method to mossy fiber tracts in hippocampal region CA3 and parallel fiber tracts in cerebellum resulted in distant presynaptic terminals well labelled with fura-2 without concomitant postsynaptic labelling, allowing optical measurements of calcium concentration in individual presynaptic terminals. Application of the method to CA1 pyramidal cells produced intracellular loading of apical dendrites with fura-2. Dendritic calcium changes produced by afferent fiber stimulation were similar to those determined from cells filled with fura-2 by intracellular microinjection. The method appears to be general, and should provide a means to fill projecting axons and dendritic processes in many areas of the brain with fluorescent indicators, allowing optical measurements of ion concentration dynamics to be performed in brain slice that were previously impractical.