H.E. Scharfman (Ed.)

Progress in Brain Research, Vol. 163

ISSN 0079-6123

Copyright r 2007 Elsevier B.V. All rights reserved

CHAPTER 6

Mossy fiber synaptic transmission: communicationfrom the dentate gyrus to area CA3

$

David B. Jaffe1,� and Rafael Gutierrez2

1Department of Biology, University of Texas at San Antonio, One UTSA Circle, San Antonio, TX 78249, USA2Department of Physiology, Biophysics and Neurosciences, Center for Research and Advanced Studies, Mexico City,

Apartado Postal 14-740, Mexico D.F. 07000, Mexico

Abstract: Communication between the dentate gyrus (DG) and area CA3 of the hippocampus proper istransmitted via axons of granule cells — the mossy fiber (MF) pathway. In this review we discuss andcompare the properties of transmitter release from the MFs onto pyramidal neurons and interneurons. Anexamination of the anatomical connectivity from DG to CA3 reveals a surprising interplay between ex-citation and inhibition for this circuit. In this respect it is particularly relevant that the major targets of theMFs are interneurons and that the consequence of MF input into CA3 may be inhibitory or excitatory,conditionally dependent on the frequency of input and modulatory regulation. This is further complicatedby the properties of transmitter release from the MFs where a large number of co-localized transmitters,including GABAergic inhibitory transmitter release, and the effects of presynaptic modulation finely tunetransmitter release. A picture emerges that extends beyond the hypothesis that the MFs are simply ‘‘det-onators’’ of CA3 pyramidal neurons; the properties of synaptic information flow from the DG have moresubtle and complex influences on the CA3 network.

Keywords: mossy fibers; synaptic transmission; CA3; co-transmitters; plasticity

Neural output from the dentate gyrus (DG) istransmitted via axons of granule cells called themossy fiber (MF) pathway. The MFs not onlyproject into area CA3 of the hippocampus, but alsosynapse proximally onto DG basket cells (provid-ing local recurrent inhibition in the dentate) andpyramidal-like neurons in the hilus (Johnston andAmaral, 2004). They are so named because of thelarge (up to 5mm diameter) varicosities along theaxon. Ramon y Cajal (who named the pathway

$Both authors contributed equally to this work.

�Corresponding author. Tel.: +1 210 458 5843;

Fax: +1 210 458 5658; E-mail: david.jaffe@utsa.edu

DOI: 10.1016/S0079-6123(07)63006-4 109

originally) and Lorente de No both suggested thatthe large varicosities made synaptic contacts ontoCA3 pyramidal neurons (Ramon y Cajal, 1911;Lorente de No, 1934), later to be confirmed withelectron microscopy (Blackstad and Kjaerheim,1961), thereby making this pathway an integralcomponent within the so-called, and overly sim-plistic, ‘‘tri-synaptic’’ circuit.

In this review we will discuss and compare theproperties of transmitter release at the most well-characterized MF synapses, those in area CA3onto pyramidal neurons and interneurons. Ourdiscussion will focus on the potential roles ofco-localized transmitters and modulators as wellas how the properties of synaptic transmission

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from the DG influence the primary target ofDG output — the CA3 region of the hippo-campus.

MF anatomy: does form follow function?

The MFs form three types of synaptic contactsonto its target neurons. First, and most notably,are the large expansions that synapse onto CA3pyramidal neurons. The large boutons appear atapproximately 150 mm intervals (Blackstad et al.,1970) and a single granule cell contacts approxi-mately 15 pyramidal neurons (each terminalsynapses onto a single pyramidal neuron). OneCA3 pyramidal neuron may receive up to a totalof approximately 50 MF inputs only (Claiborneet al., 1986; Amaral et al., 1990). Second, from thelarge expansions there may extend 2–3 filopodiathat make synaptic contacts onto interneurons(Amaral, 1979; Acsady et al., 1998). Theseso-called filopodia are motile and regulated byglutamatergic neuromodulation (Tashiro et al.,2003). Third, small terminals, resembling boutonsof other hippocampal neurons, also contact CA3interneurons. As a result, it appears that by sheernumbers the major target of the MFs in CA3 is

Fig. 1. Interneurons are the primary target of DG granule cells. Diag

by granule cells. Within the dentate/hilar region collaterals of the mos

cells. In area CA3, the MFs contact pyramidal neurons via large ex

extensions from the large boutons or smaller en passant expansions a

onto inhibitory interneurons, rather than pyram-idal neurons (Fig. 1).

The large terminals of the MFs are uniquestructures in a variety of ways. In addition to thefilopodial-like synaptic contacts onto interneu-rons, mentioned above, the large boutons arefilled with a high density of vesicles (Blackstad andKjaerheim, 1961), express more than one activezone, and encase a complex branched dendriticspine emanating from CA3 pyramidal neurons —generally referred to as a thorny excrescence(Amaral and Dent, 1981; Chicurel and Harris,1992). Putative synaptic sites on these boutonswere originally identified as both asymmetric andsymmetric, suggesting differences in their respec-tive properties of transmission (Hamlyn, 1961).

Another important anatomical aspect of the MFpathway is that they are generally restricted toa narrow band running within stratum lucidum

where the large terminals make contacts onto themost proximal portions of pyramidal neuronapical dendrites (Brown and Johnston, 1983). Ad-ditionally, there is a less extensive infrapyramidalMF projection onto the proximal basal dendritesof pyramidal neurons in CA3b and CA3c.

The first recordings of MF synaptic transmis-sion were extracellular field potential recordings

ram representing the multiple types of excitatory contacts made

sy fibers (MFs) innervate both mossy cells and inhibitory basket

pansions, but also excite interneurons through either filopodial

long MF axons. (See Color Plate 6.1 in color plate section.)

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made in vivo. Stimulation of the DG triggered acurrent sink restricted to s. lucidum (Gloor et al.,1963), confirming the anatomical evidence thatMF input was restricted to a discrete layer,and supporting the view that the synapse wasexcitatory.

Two conflicting ideas emerge out of the ana-tomical connectivity from the DG to area CA3,discussed above. On the one hand, the proximallocation of the MF large boutons, and the fact thattheir large terminals are copiously supplied withvesicles, suggests that a single action potentialfrom a granule cell might have a strong excitatoryinfluence on CA3 pyramidal neurons. That is, asingle spike invading a single terminal might becapable of triggering an action potential in a CA3pyramidal neuron. This is the so-called ‘‘detonatorsynapse’’ hypothesis. The circuitry of area CA3resembles that of an auto-associative network(Marr, 1971) reflected in the large number ofrecurrent excitatory connections between CA3pyramidal neurons (Johnston and Amaral, 2004).The concept of the MFs as detonators led to thehypothesis that these sparse inputs might serve as a‘‘teacher’’ signal underlying forms of associativelearning (McNaughton and Morris, 1987; Rolls,1989). The presence of a large supply of vesiclessuggests a large readily releasable pool (Hallermannet al., 2003), and that the degree of transmitterrelease could be maintained over time by the largereserve pool of vesicles. The proximal locationof these synapses relative to the spike generatingzone also would limit any loss of depolarizationdue to cable filtering (Johnston and Brown, 1983).That individual MF synapses would have largeunitary synaptic strength would also be a require-ment if the pathway as a whole were to be effectiveat driving the postsynaptic cell, because — asdiscussed above — each CA3 pyramidal neuronreceives only about 50 mossy inputs (Amaral et al.,1990).

On the other hand, the MFs also make directsynaptic contact onto inhibitory interneurons inarea CA3 (Fig. 1). Moreover, the ratio of synapticcontacts onto interneurons is approximately four-fold higher than onto pyramidal neurons. Assum-ing that (i) a single granule cell contacts 15pyramidal neurons, (ii) each large expansion

contacts directly or via its filopodia up to threeinterneurons, and (iii) 15 interneurons are inner-vated by the smaller boutons one-granule cell willcontact approximately 60 interneurons comparedwith only 15 pyramidal neurons (a 4:1 ratio).Therefore, it is conceivable that under certain con-ditions the firing of granule cells would have a netinhibitory effect on pyramidal neurons in CA3(Bragin et al., 1995a, b; Penttonen et al., 1997;Acsady et al., 1998).

Excitatory–inhibitory conductance sequence

Yamamoto (1972) was the first to utilize brain slicemethods and intracellular recording to study MFsynaptic transmission onto CA3 pyramidal neu-rons. Stimulation of the granule cell layer triggereda biphasic response composed of a small com-pound excitatory postsynaptic potential (EPSP)followed by a larger, overlapping compound in-hibitory postsynaptic potential (IPSP), presumablymediated either by feed-forward/feed-back inhibi-tion or the direct stimulation of inhibitory inter-neurons (Yamamoto, 1972). The IPSP itself wasbiphasic, comprised of an initial GABAA receptorand later GABAB receptor-mediated phase (Ogataand Ueno, 1976; Knowles et al., 1984).

One of Yamamoto’s most important earlyobservations was that paired stimulation of thefibers at short intervals (20–100ms) markedlypotentiated the second EPSP. While low-frequencystimulation (LFS) rarely triggered spikes,frequency facilitation of the compound EPSPwas generally capable of reaching spike threshold,in spite of the overlapping IPSPs. Frequencyfacilitation occurred over a very wide range offrequencies of stimulation (0.05–1Hz), and couldpotentiate responses as much as threefold (Salinet al., 1996).

Johnston and Brown (1983) applied voltage-clamp methods to the study of MF synaptictransmission, recognizing that the advantageousproximal location of MFs relative to the somapermitted reasonable voltage-control and, in turn,space clamp issues were minimized (Johnston andBrown, 1983; Spruston and Johnston, 1992; Sprus-ton et al., 1993). Measuring synaptic currents

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under voltage-clamp also allowed better resolutionof the mixed excitatory–inhibitory conductancesequence, even though it was recognized that therewas still considerable overlap between the excita-tion and inhibition (Brown and Johnston, 1983;Barrionuevo et al., 1986). Two important obser-vations were made in these early studies. First, forcompound postsynaptic currents, the GABAA-mediated inhibitory conductance was almostfivefold larger than the excitatory conductance.Second, the onset of the inhibitory conductanceappeared to be very close to that of the excitatoryresponse. A disynaptic GABAergic response, froma feed-forward or feed-back circuit, should retardthe onset of the inhibitory response relative to theexcitatory conductance. It is therefore possiblethat the paradigms used to stimulate the MFpathway might directly trigger the release ofGABA, most likely from inhibitory neurons, ontoCA3 pyramidal neurons.

Properties of transmitter release from MF synapses

Granule cells discharge action potentials downthe MFs at basal rates less than 0.5Hz (Jung andMcNaughton, 1993), though firing rates may reachup to 50Hz during certain types of behaviors(Skaggs et al., 1996; Wiebe and Staubli, 1999;Henze et al., 2002b) and conduction velocity isapproximately 7m/s, consistent with the MFsbeing an unmyelinated pathway (Langdon et al.,1993). Upon reaching synaptic terminals, presy-naptic action potentials trigger synaptic transmis-sion by eliciting Ca2+ influx through multipletypes of voltage-gated Ca2+ channels. Blockadeof N-, P-, and R-type Ca2+ channels decreases fasttransmitter release, while dihydropyridine-sensi-tive L-type Ca2+ channels do not (Kamiya et al.,1988; Castillo et al., 1994; Nicoll et al., 1994;Yamamoto et al., 1994; Wu et al., 1998; Miyazakiet al., 2005). That said, L-type channels are presentin MF presynaptic terminals and allow Ca2+ entrywhen MFs are stimulated (Tokunaga et al., 2004).Although L-channels do not appear to participatein fast transmitter release, they may play a role intransmission during repetitive stimulation (Reuter,1995).

To directly study presynaptic action potentialsand their interaction with voltage-gated ion chan-nels, patch-clamp methods have recently beenapplied to the large boutons of the MFs (Bischof-berger et al., 2006). One of the first notableobservations was that the action potential, whileof short duration during LFS, widens duringhigh-frequency stimulation (HFS) due to the rapidinactivation of a voltage-gated K+ conductance(Geiger and Jonas, 2000). Frequency-dependentspike broadening, and the associated increase inCa2+ influx, provides for, in part, a mechanismunderlying frequency-facilitation (Salin et al.,1996). The density of Na+ channels in the boutonsappears relatively large, and may be importantfor the efficient propagation of action potentials(Engel and Jonas, 2005). The load resulting fromthe greater surface area of the large boutons couldpotentially extinguish or slow down spike propa-gation in these unmyelinated fibers. Computersimulations suggested that the high density andproperties of these Na+ channels ensures thereliability of synaptic transmission.

Ca2+ in the presynaptic terminal is also affectedby other mechanisms. Intracellular Ca2+ storesregulate presynaptic Ca2+ concentration (Lianget al., 2002). Ca2+-induced Ca2+ release maywork in concert with spike-mediated Ca2+ influxto raise intraterminal Ca2+ concentration. Inter-estingly, although Ca2+-induced Ca2+ release ac-counts for a large proportion of the presynapticsignal (Scott and Rusakov, 2006), depletion ofCa2+ stores has no effect on fast trans-mitter release. Release of Ca2+ may, however,play a role in frequency facilitation (Lauri et al.,2003).

In a recent study, it was reported that EPSPs indentate granule cells, surprisingly, propagateddown hundreds of microns along the MFs andwere capable of modulating synaptic transmission(Alle and Geiger, 2006). Furthermore, spike-evoked EPSCs in CA3 pyramidal neurons wereenhanced when paired with a presynaptic wave-form that mimicked an EPSP in the bouton. Themechanism underling this type of modulation maybe related to a similar phenomenon observedat the Calyx of Held (Awatramani et al., 2005),but other studies suggest it may also be due to

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voltage and not solely due to resting Ca2+ levels(Ruiz et al., 2003).

Quantal nature of transmission at the MF-CA3

pyramidal neuron synapse

The strong frequency-dependent facilitation ofMF synaptic transmission onto CA3 pyramidalneurons implies that the initial probability oftransmitter release is small. If the probability ofrelease were high, either a ceiling effect would limitan increase in release (assuming that there is alarge reserve pool of primed vesicles) or, alterna-tively, one could observe synaptic depression dueto the refractory period produced by a lack ofprimed vesicles. Indeed, quantal analysis of uni-tary synaptic responses onto CA3 pyramidal neu-rons is consistent with the hypothesis that theinitial release probability is in fact low (vonKitzing et al., 1994; Lawrence et al., 2004). Thisis in spite of the fact that the number of releasesites at a single MF synapse is very high (Chicureland Harris, 1992), and that release at thesesynapses is most likely multiquantal (Henzeet al., 1997). Indeed, in the study by Jonas et al.(1993) quantal content (the number of quantareleased per action potential) ranged from 2–16,with a mean of 8, values similar to that firstreported by Yamamoto (Yamamoto et al., 1991).Others have found that the probability of releasewas much lower, o0.3 (Lawrence et al., 2004).There is also evidence, however, that large synap-tic currents arise from the release of single quanta(Henze et al., 2002a). This is in contrast to com-missural/associational (C/A) synapses, where theprobability of release is higher and, concomitantly,frequency facilitation is weaker (Salin et al., 1996).

Mean quantal size at MF-CA3 pyramidalneuron synapses is approximately 150 pS, corre-sponding to about 17 AMPA receptors. Thesenumbers are consistent with unitary responses(unitary conductance ¼ quantal size� quantalcontent), which generally have magnitudes ofapproximately 1 nS. Corresponding unitary EPSPamplitudes can be up to 10mV (Jonas et al., 1993),in contrast to C/A unitary responses that aretypically o1mV (Debanne et al., 1998; Pavlidis

and Madison, 1999). Similarly large unitary EPSPshave been demonstrated for MF-mossy cellsynapses (Scharfman et al., 1990).

Evidence for multiple neurotransmitters/modulators

in the granule cells

Glutamate is believed to be the primary excitatoryneurotransmitter released from the MFs (Crawfordand Connor, 1973; Terrian et al., 1988), and MFEPSPs are blocked by glutamate receptor antago-nists (Sawada et al., 1983). The neuronal gluta-mate transporter (EAAC1) is the most abundantuptake mechanism and is selectively enriched inhippocampal principal neurons, including DGgranule cells (Rothstein et al., 1994). The presy-naptic and postsynaptic actions of glutamate re-leased from the MFs are discussed below.

Granule cells also contain and release severalneuromodulators of different chemical composi-tion (Fig. 2). The most prominent are those ofpeptidic nature, which are contained and releasedfrom large dense-core vesicles. Dynorphin andenkephalin, as well as their receptors, are presentin the MFs in rodents and humans, but there isvariation between mammalian species (Gall et al.,1981; McGinty et al., 1983; Chavkin et al., 1985;McLean et al., 1987; Terrian et al., 1988; Houseret al., 1990; Chavkin, 2000). The actions of opioidpeptides in the hippocampus are inhibitory, medi-ated by inhibition of Ca++ currents, in the caseof dynorphin, or activation of K+ currents, inthe case of enkephalins, and are a consequenceof activation of G-protein-coupled receptors(Zieglgansberger et al., 1979). In the rat MF sys-tem, activation of m receptors facilitates MF LTPin an indirect fashion. Activation of m receptorsdepresses GABA release from interneurons, whichin turn leads to a failure of GABA to inhibitglutamate release from MFs (Jin and Chavkin,1999). On the other hand, the m opioid receptoragonist DAMGO inhibits low-frequency stimu-lated MF responses in Sprague-Dawley rats, as itdoes in the guinea pig (Salin et al., 1995). Directactions of dynorphin on MF transmission havebeen described in the guinea pig, but not in the rat.Indeed the MFs possess k receptors, which, upon

Fig. 2. Summary of the pre- and post-synaptic constituents of the different MF terminals and of their plasticity. (A) Schematic

representation of the giant MF boutons, which contact CA3 pyramidal cells. These terminals are characterized by low probability of

basal release and multiple release sites. (B) Schematic representation of filopodial extensions and en passant contacts, which synapse on

to interneurons. These have a high probability of basal release and single release sites. Both types of MF terminals contain several

neuromodulators and the neurotransmitters glutamate and GABA. Note that both the presynaptic and postsynaptic sites contain

several ionotropic GluRs and metabotropic GluRs, which confer to the MF a high degree of plasticity and the capacity for synaptic

integration. (C) Relative expression of the different releasable contents and of the receptors and transporters during development, in

the adult (D), and after epileptic activity (E). Arrows and font size indicate relative differences between the three states. (See Color

Plate 6.2 in color plate section.)

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activation, depress neurotransmitter release. Thus,for example, in the guinea pig high frequencystimulation of the MF causes a transient heterosy-naptic inhibition of neighboring MF or perforantpath synapses in the dentate, which is mediated bythe synaptic release of dynorphin that activatespresynaptic k receptors (Weisskopf et al., 1993).

However, in the rat, neither exogenous nor en-dogenous dynorphin affect MF neurotransmis-sion, which is consistent with the finding thatk-receptor binding in this projection is dense in theguinea pig and sparse in the Sprague-Dawley rat(see Salin et al., 1995). Dynorphin significantlyinhibits MF responses in the hamster, mouse, and

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in Long-Evans rats (Salin et al., 1995). Interest-ingly, hippocampal opioid peptides are upregulat-ed after seizure activity (McGinty et al., 1983;Gall, 1988; Gall et al., 1990), whereas a decrease ink receptors has been observed in epileptic humancells (Jeub et al., 1999). Thus, opioid peptides arelocalized in the MF, presumably for effective con-trol of neurotransmitter release (Weisskopf et al.,1993; Castillo et al., 1996; Drake et al., 1996;Simmons and Chavkin, 1996).

Other peptides present in the MF are neuropep-tide Y (NPY), neurokinin B and cholecystokinin(Gall, 1984; Gall et al., 1990; Holm et al., 1993;Tonder et al., 1994; Chandy et al., 1995; Schwarzerand Sperk, 1995; Makiura et al., 1999). Like opioidpeptides, these peptides have been shown to exertinhibitory actions. Neuropeptide Y is synthesized,stored in, and released from the MFs, and inhibitsMF transmission by a presynaptic mechanism(McQuiston and Colmers, 1996; McCarthy et al.,1998) and their receptors are normally present in theMFs themselves (Widdowson, 1993; Jacqueset al., 1997). Seizures increase the release of NPYwhich in turn, tonically inhibits MF synaptic trans-mission (Tu et al., 2005). Besides, seizures upregu-late NPY Y2 receptors, which appear to mediate theeffects on MF (Vezzani and Sperk, 2004),although Y5 receptors could play a role (Marshet al., 1999; Ho et al., 2000). This upregulation re-flects a neuroprotective function of NPY that hasbeen shown in animal models of epilepsy and in theepileptic human (Schwarzer et al., 1998; Patryloet al., 1999; Furtinger et al., 2001; Vezzani andSperk, 2004). By contrast, substance P, which iscontained in and released from the MFs activatesits receptors in the same MF, increases glutamaterelease (Borhegyi and Leranth, 1997; Liu et al.,1999).

The MFs also contain high levels of Zn++,which can be released together with glutamatein an activity-dependent manner (Stengaard-Pedersen et al., 1981; Wenzel et al., 1997; Molnarand Nadler, 2001; Qian and Noebels, 2005).Exogenously applied Zn++ blocks glutamateand GABA receptors (Westbrook and Mayer,1987; Draguhn et al., 1990; Smart et al., 1994).Indeed, Zn++ occupies a high-affinity binding siteon NMDA receptors (Vogt et al., 2000) and can

block GABAA receptors composed by a and b butnot g subunits (Draguhn et al., 1990). It has beenshown that synaptically released Zn++ from theMF pathway strongly modulates NMDA (Vogtet al., 2000) and GABAA receptors in CA3 (Ruizet al., 2004). Because MF release, besides gluta-mate, GABA and Zn++ (Gutierrez, 2005), thissynapse is most suitable to study the effects of thesynaptically released Zn++ on GABA responseselicited by the same terminals. Indeed, it wasfound that Zn++ tonically depresses the inhibitoryactions of GABA, as it reaches a high concentra-tion in the vicinity of the GABAA receptor onCA3 pyramidal cells. Consequently, chelation ofZn++ relieves this inhibition (Ruiz et al., 2004).This is particularly important for developmentaland pathological processes, in particular, epilepsy.Indeed, a higher input of Zn++ from sproutedMFs after epileptic activity contributes to inhibitreverberating excitatory activity in the DG(Nadler, 2003; Tu et al., 2005).

Other interesting signaling molecules present inthe MF are brain derived neurotrophic factor(BDNF) and nerve growth factor (NGF) (Gall andIsackson, 1989; Gall and Lauterborn, 1992;Lowenstein and Arsenault, 1996; Conner et al.,1997; Smith et al., 1997). Granule cells containrelatively high concentrations of BDNF and NGF,which can be released in vivo by the MFs anddendrites to affect other MFs or other cells, re-spectively (Blochl and Thoenen, 1995; Lowensteinand Arsenault, 1996; Conner et al., 1997). Den-dritic targeting of BDNF mRNA and its transla-tion to protein can account for the release of thisneurotrophin to neighboring granule cell dendrites(Tongiorgi et al., 2004). This suggests that the lig-and–receptor interaction occurs by means of anautocrine/paracrine mechanism. On the otherhand, anterograde transport of BDNF (Conneret al., 1997) would effectively provide a mechanismto release BDNF from MF terminals, where itcan increase neurotransmitter release (Altar andDiStefano, 1998; Elmer et al., 1998). As with mostsignaling molecules in the granule cells, BDNFexpression is increased after limbic seizures (Isack-son et al., 1991). In the MF, a consequence of thiswould be the activation of TrkB receptors locatedin the same fibers (He et al., 2002; Scharfman,

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2005). TrkB activation is thought to promoteCa++ influx into terminals (Berninger et al., 1993)and this is the mechanism that leads to enhancedneurotransmitter release. Eventually, this producesan enhancement of excitability that can lead to thegeneration of epileptiform activity (Thoenen,1995; Huang and Reichardt, 2001; Scharfmanet al., 2002).

Importantly, it has been shown that BDNF canexert a rapid depolarization in hippocampal neu-rons by promoting Na+ influx through TTX-insensitive channels (Kafitz et al., 1999; Rose et al.,2003). Another important issue is that BDNF maybe packaged preferentially in the large MFboutons, although it does reside in the small ter-minals also, so it can potentially modulate bothexcitation and feed-forward inhibition in CA3(Danzer and McNamara, 2004).

Besides a possible involvement of hippocampalBDNF in excitability and synaptic plasticity(Thoenen, 1995, 2000), neurotrophins have adifferentiating effect on interneurons in the hip-pocampus (Marty et al., 1996a, b) and cortex(Marty et al., 1997; Patz et al., 2003), by regulatingthe expression of GABAergic markers. Neuronalactivity is the main activator of GAD expressionby neurotrophins, differentially modulating tran-scription and translation in a context-dependentmanner (Patz et al., 2003). Interestingly, this is themechanism shown to underlie the maturation ofthe GABAergic phenotype in hippocampal inter-neurons (Marty et al., 1996a, b) and, as recentlyshown, the expression of all GABAergic markersin granule cells (Gomez-Lira et al., 2005).

Granule cells also contain high levels of adeno-sine (Braas et al., 1986) and its A1 receptor(Rivkees et al., 1995; Swanson et al., 1995). Ithas been suggested that endogenous tonic activa-tion of A1 receptors underlies the low basal prob-ability of neurotransmitter release from the MFs(Moore et al., 2003; also see Kukley et al., 2005).Their activation produces a Gi/o protein-depend-ent sustained inhibition of neurotransmitterrelease (Moore et al., 2003). Other set of recep-tors present in the MF terminals, and whichactivation controls neurotransmitter release, arethe ATP receptors P2X (Armstrong et al., 2002)and a adrenergic receptors (Scanziani et al., 1993).

Finally, the inhibitory amino acid GABA isprobably the most important and controversialaddition to the list of modulators and transmittersthat are co-localized in the granule cells. Sandlerand Smith (1991) provided the first data that sug-gested the presence GABA in the ‘‘glutamatergic’’MF in monkey and human hippocampi (Sandlerand Smith, 1991). They found GABA immunore-activity in MF terminals that made asymmetricsynaptic contacts with spines arising from largedendrites of CA3 pyramidal cells. They alsoshowed the colocalization of GABA and gluta-mate within the same terminals with electronmicroscopy. From this anatomical evidence, andthe idea that GAD was not present in the MF,they concluded that GABA had to be eitherincorporated from the extracellular space or orig-inated from an alternative route of synthesis suchas g-hydroxybutyrate. Second, they suggested thatat least a component of the inhibitory synapticpotentials evoked in pyramidal cells by DG stim-ulation had to be of MF origin. In this way,GABA released by the MF could modulatethe normal MF glutamatergic responses. On theother hand, studies carried out on MF synapto-somes provided the first neurochemical evidenceshowing that MF terminals contained and re-leased GABA (Terrian et al., 1988; Taupin et al.,1994a, b). These authors, in agreement withSandler and Smith (1991), suggested that theamino acid could be synthesized in the granulecells from a route different from the GAD-dependent pathway.

Evidence clarifying the presence and origin ofGABA in the MF was provided by Sloviter et al.(1996). These authors conclusively demonstratedthat immunoreactivity for the amino acid and itssynthesizing enzyme, GAD, was normally presentin the MFs of rats, monkeys, and humans (Sloviteret al., 1996). Therefore, if the granule cells had thenecessary enzyme for the synthesis of GABA andGABA itself, the granule cells indeed synthesizedGABA that could probably be used as a neuro-transmitter. Complementing and extending theinitial findings of Sandler and Smith (1991) andSloviter et al. (1996), Bergersen et al. (2003)recently confirmed with immunogold the coexist-ence of glutamate and GABA in MF synapses,

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which also contained the respective receptors inapposition to the presynaptic terminal (Bergersenet al., 2003). This study demonstrated that bothamino acids coexist in all MF terminals examinedand that they have a close spatial relation tosynaptic vesicles. Indeed, both GABA and gluta-mate were shown to be located at a distance thatsuggests their presence inside vesicles and in therelease zones. The estimated concentration ofGABA, however, was much lower than thatof glutamate within the MF terminals and evenlower than that of inhibitory types of terminals(Bergersen et al., 2003).

Interestingly, it was demonstrated that provok-ing seizures by stimulation of the perforant path-way for 24 upregulated the content of bothisoforms of GAD and GABA in the MFs, whereasno changes were observed in area CA1 (Sloviteret al., 1996). Other authors (Schwarzer and Sperk,1995; Lehmann et al., 1996) showed that GAD67

and its mRNA (but not GAD65) were transientlyupregulated after seizures provoked by kainic acid(KA) or by the kindling method in the rat. On theother hand, the upregulation of GAD67 and itsmRNA in granule cells was further confirmed withother seizure- and epilepsy-induction methods thatuse chemical convulsants or electrical stimulation(Ding et al., 1998; Makiura et al., 1999; Szaboet al., 2000; Ramirez and Gutierrez, 2001). It wasalso established that GAD67 could be upregulatedin an activity-dependent manner, in the absence ofepileptiform activity (Ramirez and Gutierrez,2001; Gutierrez, 2002). Recently, a direct link be-tween the presence of seizures and the concomitantupregulation of GAD and endogenous GABA wasfound in MF terminals of epileptic rats (Gomez-Lira et al., 2002). An analysis of the expression ofGAD67 at different ages has revealed that GAD67

(but not GAD65 or GAD65 RNA) is also devel-opmentally regulated in the MFs (Maqueda et al.,2003). Indeed, it was shown that GAD67 isexpressed in the MFs early in life and then down-regulated by days 23–24, after completion ofdevelopment (Gutierrez, 2003; Maqueda et al.,2003). Likewise, GABA-immunoreactive cells withcharacteristics of granule cells are found in thestratum granulare of the DG of developing ratsbut not of adults.

Contrary to the data showing the upregulationof both isoforms of GAD by seizures (Sloviteret al., 1996), several reports have shown thatGAD67 (and its mRNA) and not GAD65 is reg-ulated in granule cells by increased activity(Schwarzer and Sperk, 1995; Szabo et al., 2000;Ramirez and Gutierrez, 2001; Maqueda et al.,2003), Ca++ entry and BDNF activation (Gomez-Lira et al., 2005). It has been proposed that thecellular distribution of both enzymes differed andthis could be reflected in distinctive functionswithin neurons (Erlander and Tobin, 1991), i.e.,GAD65 may be in synaptic terminals, whileGAD67 is present in terminals, somata and dend-rites (Kaufman et al., 1991). This suggests that oneisoform synthesizes a metabolic pool of GABA(in the soma) and the other the releasable pool(in the terminals). Despite the possible differencesin GAD65- and GAD67-originated GABA, there isa strong correspondence of the expression ofGAD67 to that of GABA present within the gran-ule cells and their MFs (Gomez-Lira et al., 2002).All these studies suggest that GABA synthesis inthe MFs is a means to counteract the enhancedexcitability caused by epileptic activity.

Presynaptic modulation

As mentioned above, a number of neuromodula-tors control transmitter release from the MFs.Glutamate and GABA are also important presy-naptic modulators of MF synaptic transmissiontransmission. In particular, one of the uniqueproperties of MF synaptic transmission, in con-trast to transmitter release from recurrent synapsesonto CA3 pyramidal neurons, is its sensitivity tometabotropic glutamate receptor (mGluR) ago-nists; mGluR agonists depress MF synaptic trans-mission (Manzoni et al., 1995; Yoshino et al.,1996). Because of this response, sensitivity tomGluR agonists is a widely used assay to deter-mine if a synaptic response is of MF origin. Thereare species-specific differences with respect to pre-synaptic mGluRs. MF glutamatergic neurotrans-mission in the rat is sensitive to the group IImGluR agonist, DCG-IV, but not to the group IIImGluR agonist, L-AP4 (Lanthorn et al., 1984;

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Kamiya et al., 1996; Maccaferri et al., 1998). Theopposite is true for the guinea pig where L-AP4strongly depresses MF transmission (Lanthornet al., 1984; Manzoni et al., 1995; Tong et al., 1996;Min et al., 1998). Interestingly, the granule cellsand MF of the rat DG express groups II/IIImGluR mRNA (Ohishi et al., 1993; Ohishi et al.,1995) and mGluR2,4,7 (Bradley et al., 1996;Shigemoto et al., 1997; Lie et al., 2000). Electronmicroscopy has revealed immunolabeling for thegroup III mGluR predominantly in presynapticactive zones of asymmetrical and symmetricalsynapses, whereas mGluR-II immunolabelingwas found in preterminal rather than terminalportions of axons (Shigemoto et al., 1996, 1997).

Although it has been well established that acti-vation of group II mGluR almost completely de-presses MF glutamatergic transmission in the rat,several reports show that MF GABAergic trans-mission is strongly inhibited through activation ofgroup III mGluR (with L-AP4) both in the guineapig and the rat (Gutierrez, 2000; Walker et al.,2001; Gutierrez, 2002, 2003; Romo-Parra et al.,2003; Kasyanov et al., 2004; Safiulina et al., 2006).This pharmacological profile is not only consistentwith neurotransmission of MF origin but it alsodemonstrates that the modulation of MFGABAergic transmission differs from MF glut-amatergic transmission. This physiological evi-dence gives a functional significance to theanatomical findings showing that both types ofreceptors are present in rat granule cells with adistinct localization and function (Ohishi et al.,1995; Shigemoto et al., 1996, 1997). These data,together with the finding that activation of mGluRproduces a downregulation of the exocytoticmachinery (Kamiya and Ozawa, 1999), suggestthat group III mGluR are associated with mech-anisms of GABA release, and also that thereis presynaptic segregation of mGluR receptorsaccording to the class of neurotransmitter to bereleased.

Kainic acid has long been used as an epilepto-genic agent and was recognized that it binds to theMF with high affinity (Monaghan and Cotman,1982; Represa et al., 1987). It is now clear that theseveral KA receptors that are present in the MF(Wisden and Seeburg, 1993; Darstein et al., 2003)

modulate plasticity at this synapse in a complexmanner (Schmitz et al., 2000).

Presynaptic GABAB receptors effectively inhibitglutamate release from the MFs (Thompson andGahwiler, 1992), and it has recently shown thationotropic GABAA receptors also inhibit gluta-mate (Ruiz et al., 2003) and GABA release(Trevino and Gutierrez, 2005) and control theexcitability of this pathway (Kullmann et al., 2005;Trevino and Gutierrez, 2005). Other subcorticalmodulators also control the release of transmitterfrom the MFs. Muscarinic acetylcholine receptorsinhibit transmitter release (Williams and Johnston,1988, 1990) while nicotinic receptor activationraises presynaptic Ca2+ levels and thereby mayenhance transmitter release (Gray et al., 1996; alsosee Vogt and Regehr, 2001).

Co-localization of plasma membrane transporters

of glutamate and GABA

Glutamate transport is the major mechanism con-trolling extracellular glutamate levels, preventingexcitotoxicity, and averting neural damage associ-ated with hyperexcitability. As mentioned above,the neuronal glutamate transporter (EAAC1) isexpressed in granule cells and, surprisingly, in anumber of GABAergic neurons (Rothstein et al.,1994; He et al., 2002; Sepkuty et al., 2002). There-fore, it has been suggested that besides controllingextracellular glutamate levels, its function is linkedto GABA metabolism, because capture of gluta-mate is essential for GABA synthesis (Sepkutyet al., 2002). It is not surprising that seizures andepilepsy have direct consequences on EAAC1 ex-pression and function, as has been demonstrated(Gorter et al., 2002; Zhang et al., 2004). On theother hand, it has been proposed that the presenceof the membrane transporter of GABA, GAT-1, isrestricted to neurons that synthesize and releaseGABA, and glial cells (Iversen and Kelly, 1975;Radian et al., 1990; Ribak et al., 1996). SomeGABAergic cells, immunocytochemically charac-terized by the presence of GAD67 or GABA, donot contain or contain traces of GAT-1, but notvice versa (Rattray and Priestley, 1993). However,it has been found that GAT-1 is also localized in

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the glutamatergic granule cells (Frahm et al., 2000;Sperk et al., 2003), and it controls GABA uptaketo the MF terminals (Gomez-Lira et al., 2002) butnot GABA release at this synapse (Vivar andGutierrez, 2005; Safiulina et al., 2006).

Co-localization of the vesicular transporters for

glutamate (VGlut-1) and GABA (VGAT)

Because glutamate is a general metabolic substrateand serves as the precursor of inhibitory transmit-ter GABA, glutamate immunoreactivity is notspecific to glutamatergic neurons. Therefore, thedetection of glutamate vesicular transporter(s) hasbeen used to establish the glutamatergic phenotypeof neurons. As expected for glutamatergic neu-rons, the MF terminals of the granule cells containthe glutamate vesicular transporter VGlut-1(Bellocchio et al., 1998; Kaneko et al., 2002). Inaccordance with immunohistological data showingthat the granule cells express GAD and GABA, itwas found that they express VGAT mRNA in anactivity-dependent manner (Lamas et al., 2001;Gutierrez, 2003; Gomez-Lira et al., 2005). How-ever, these cells do not contain the transporterprotein (Chaudhry et al., 1998; Sperk et al., 2003;also see Safiulina et al., 2006). Thus, the lack ofdetection of VGAT indicates that its expression istoo low to be revealed with immunohistochemis-try, or the existence of a yet unidentified trans-porter. Recent experiments show that glutamateand GABA are in close relation to vesicles in MFsterminals (Bergersen et al., 2003) strongly suggest-ing that VGAT or another related transporterprotein must be present in these terminals(Chaudhry et al., 1998; Gomez-Lira et al., 2005).

Postsynaptic responses of CA3 pyramidal neurons

to MF glutamatergic input

As mentioned above, glutamate is believed to be theprimary excitatory neurotransmitter released fromthe MFs (Crawford and Connor, 1973; Terrianet al., 1988). The primary ionotropic glutamate re-ceptors mediating the fast synaptic response at theMF synapse are a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type receptors

(Lanthorn et al., 1984; Neuman et al., 1988; Itoand Sugiyama, 1991; Jonas et al., 1993). Voltage-clamp analysis of unitary and compound excitatorypostsynaptic currents (EPSC) onto CA3 pyramidalneurons finds the equilibrium potential close to0mV, consistent with ionotropic glutamate recep-tors (Brown and Johnston, 1983). Based on the fastrise times and decay kinetics of the current, the re-sponses are consistent with the properties of AMPAreceptors and therefore reflects an electrotonicallyclose synapse (Williams and Johnston, 1991; Jonaset al., 1993).

Early studies using radio-labeled NMDA foundthat the MF terminal field contains a low densityof NMDA receptors relative to other regions ofthe hippocampal formation (Monaghan andCotman, 1985). Although lower than at othersynaptic sites, glutamate release from the MFsactivates a small, but measurable, NMDA com-ponent that, as expected, exhibits voltage-depend-ence and slower kinetics, which are characteristicsof NMDA receptor-mediated responses (Jonaset al., 1993; Weisskopf and Nicoll, 1995).

In contrast to NMDA receptors, the MF ter-minal field contains a sizable density of high-affinity sites for KA binding (Foster et al., 1981;Monaghan and Cotman, 1982) and KA channelsare expressed in CA3 pyramidal neurons (Egebjerget al., 1991; Werner et al., 1991). Focal applicationof kainate into the MF terminal field induces astrong depolarization in CA3 pyramidal neurons(Sawada et al., 1988). Postsynaptic activation ofkainate receptors requires repetitive stimulationdue to their slow kinetics (Castillo et al., 1997;Bortolotto et al., 2003). This, combined with thestrong frequency-dependent facilitation of release,suggests that KA receptors contribute to theactivity-dependent enhancement of transmissionfrom granule cells to CA3 pyramidal neuronsdiscussed above.

Because of the arguments above that MFs re-lease GABA and it acts as a classical neurotrans-mitter, one would expect that GABA receptorswould be located in apposition to MF terminals.In support of this prediction, there is evidence thatGABAA receptors cluster with glutamatergicreceptors in pyramidal cells in apposition toboth glutamatergic and GABAergic terminals

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(Rao et al., 2000). It was later found, with im-munogold detection, that AMPA and GABAA

receptors also colocalize at MF synapses in thehilar region (Bergersen et al., 2003).

Finally, glutamate released from MFs triggersthe release of Ca++ from intracellular stores ofCA3 pyramidal cells via mGluR receptor activa-tion (Miller et al., 1996; Jaffe and Brown, 1997).Tetanic stimulation of the MFs triggers waves ofspike-independent increases in Ca++ concentra-tion in the proximal apical dendrites of CA3pyramidal neurons mediated by the activation oftype I mGluR receptors (Kapur et al., 2001).Single presynaptic spikes appear to be insuffi-cient to elicit Ca++ release (Reid et al., 2001).Frequency-dependent mGluR release of Ca++ fromintracellular stores may be important for long-lasting changes in MF synaptic efficacy, discussedbelow (Yeckel et al., 1999; Wang et al., 2004).

Are MF synapses onto CA3 pyramidal neurons

detonators?

It is not surprising that LFS of the MFs fails toroutinely trigger spikes, given many of the pointsmade above. For example, the major target of theMFs in area CA3 involves the activation of feed-forward inhibitory circuits. Glutamate is co-released with GABA, and a wide-array of otherneuromodulators, which potentially inhibit MFtransmission. CA3 pyramidal cells also have athreshold that is 10–15mV from resting potential(Podlogar and Dietrich, 2006). There is a largeamplitude unitary EPSP (Jonas et al., 1993), so theMFs may not relay synapses with a high safetyfactor. An elegant demonstration of the ‘‘condi-tional’’ nature of MF excitation of CA3 pyramidalneurons was shown by Henze et al. (2002b). Withlow frequency stimulation (o40Hz), the likeli-hood that a CA3 pyramidal neuron would dis-charge following a granule cell was low. Only athigher frequencies did CA3 pyramidal neuronsfollow granule cell firing. Interestingly, this maynot be the case for all MF targets. Scharfman andcolleagues showed faithful following of granulecell firing when the targets of MFs were hilarmossy cells or hilar interneurons (Scharfman et al.,

1990), a difference that may contribute to hilar cellvulnerability to excitotoxicity.

The MF-to-CA3 synapse can therefore be con-sidered as ‘‘conditional detonators’’ in two do-mains. The first, as described above, is thetemporal domain where presynaptic facilitation,the slow membrane time constant of CA3 pyram-idal neurons (Spruston and Johnston, 1992), andpossibly postsynaptic amplification by KA recep-tors combine to boost EPSP amplitude and fire thecell. The second domain reflects the spatial sum-mation of inputs concomitantly with the MFs.For example, if a MF EPSP occurred coincidentlywith a perforant path EPSP, the summation ofthese two would then be suprathreshold (Urbanand Barrionuevo, 1998). Although all synapsescould be considered as conditional detonatorsgiven this definition, the mean strength of unitaryMF EPSPs endows them with a greater ability tomove the membrane potential closer to threshold.

The granule cells simultaneously release glutamate

and GABA: electrophysiological evidence

Indirect but compelling evidence has accumulatedover the last years of the co-release of glutamateand GABA from the MFs. The first electrophys-iological evidence of GABAergic transmissionfrom the MFs to CA3 agreed with theimmunohistochemical observations showing thatseizures transiently upregulated the expression ofGAD65 and GAD67 (Schwarzer and Sperk, 1995;Sloviter et al., 1996). Indeed, it was shown thatstimulation of the MFs produced monosynapticGABA-mediated transmission in pyramidal cellsof CA3 in kindled epileptic but not in controlhealthy rats (Gutierrez, 2000; Gutierrez andHeinemann, 2001).

As mentioned above, feed-forward inhibitionresults from activation of local inhibitory inter-neurons by MF glutamatergic, excitatory synapses(Crawford and Connor, 1973; Acsady et al.),which in turn inhibit pyramidal cells (Yamamoto,1972; Brown and Johnston, 1983; Buzsaki, 1984).Thus, activation of the DG leads to mono-synaptic excitation and disynaptic inhibition onCA3 pyramidal neurons that, with intracellular

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recordings, are observed as an EPSP/IPSPsequence. Both these conductances are typicallyblocked in the presence of blockers of glut-amatergic transmission. In kindled epileptic ani-mals, however, a bicuculline-sensitive IPSP is stillelicited in the presence of the glutamate receptorblockers. This IPSP had the same latency as thecontrol EPSP and could be inhibited by activationof metabotropic glutamate receptors (mGluR).Expression of the monosynaptic IPSP was tran-sient because it could be observed 24–48 h after thelast kindled seizure but was not present if theexperiment was carried out a month after the lastseizure. The kindled epileptic state is not necessaryfor this monosynaptic IPSP, because a single sei-zure (Gutierrez, 2000) or repeated LTP-like stim-ulation could elicit the response (Gutierrez, 2002).Moreover, the monosynaptic IPSP produced bythe activation of the DG persisted when perfusinga medium with a low [Ca++] or the GABAB ago-nist, baclofen. These manipulations depressed theDG-evoked IPSP amplitude without altering itsonset latency or slope. This unequivocally estab-lished that the IPSP was a monosynaptic responsebecause, under these conditions, the ability torecruit local interneurons quickly enough totrigger such a short-latency IPSP is unlikely. Thepossibility of recruiting inhibitory interneuronsby activating electrical synapses was also dis-carded (Gutierrez, 2000). Independently, Walkeret al. (2001) demonstrated that monosynapticGABAergic responses could be normally evokedin CA3 pyramidal cells by MF activation in slicesof young guinea pigs (Walker et al., 2001). Theyshowed that these MF GABAergic responses hadthe same pharmacological and plastic properties asthose reported for glutamatergic MF transmission,i.e., there was strong frequency-dependentpotentiation, and they were sensitive to anantagonist of the metabotropic glutamate recep-tor that is expressed preferentially by MFs, L-AP4.They were able to show that minimal stimulationof the MF evoked glutamate only, GABA only,and compound glutamate-GABA currents in py-ramidal cells. This indicated that both responses,glutamatergic and GABAergic, had a commonorigin. In addition, both neurotransmitters couldbe released synchronously or asynchronously,

discarding the possibility that they are packagedin single vesicles. Moreover, the evidence of MFGABA transmission onto both pyramidal cells(Gutierrez, 2000; Gutierrez and Heinemann, 2001;Walker et al., 2001), and interneurons of CA3(Romo-Parra et al., 2003; Safiulina et al., 2006)and onto the mossy cells in the hilar region(Bergersen et al., 2003) excludes the possiblesegregation of neurotransmitters according to thetarget cell.

It has been shown that the putative release ofGABA from the MF also occurs during develop-ment, when the granule cells express all the mark-ers of the GABAergic phenotype, and provokeGABA receptor-mediated responses in pyramidalcells and interneurons of CA3. This occurs duringthe first weeks of age, after which the GABAergicphenotype shuts off in a clear-cut manner. Sup-porting this notion, recent work by Gutierrez et al.(2003), Kasyanov et al. (2004) and Safiulina et al.(2006) have shown that GABA is the main neuro-transmitter released from MF terminals during thefirst postnatal days (Kasyanov et al., 2004;Safiulina et al., 2006). Thus, MFs contain twodifferent sets of low- and high-threshold fibers thatrelease GABA and GABA plus glutamate, respec-tively. The first would disappear with maturation,whereas the second would persist longer or wouldreappear in pathological conditions, such as in ep-ilepsy (Gutierrez, 2003; Safiulina et al., 2006). It ispossible that signaling by both, synaptic and non-synaptic release of GABA, may play a crucial rolein tuning hippocampal network during postnataldevelopment (Gutierrez, 2003, 2005; Safiulinaet al., 2006). While the molecular mechanismsthat shut off the GABAergic phenotype at matu-rity have not been disclosed, upregulation ofthe GABAergic phenotype in the adult dependson protein synthesis, Ca++ entry, and acti-vation of TrkB receptors by BDNF (Gutierrez,2002; Romo-Parra et al., 2003; Gomez-Lira et al.,2005).

Long-term plasticity at the MF CA3 synapse

Like other excitatory synapses in the hippo-campal formation, the MF synapse expresses

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LTP in response to a brief episode of HFS(Yamamoto et al., 1980). As described above, theMF terminal field contains a lower density ofNMDA receptors compared with other areas of thehippocampus. This observation motivated Harrisand Cotman (1986) to test whether LTP at the MFsynapse was dependent on NMDA receptors. Theyfound that in the presence of NMDA receptorantagonists, HFS of the MFs was still capable oftriggering LTP (Harris and Cotman, 1986).

Although there is agreement that Ca++ is nec-essary for the induction process, the mechanismunderlying the induction of NMDA-independentLTP has been a source of controversy (Nicoll andSchmitz, 2005). Briefly, many experiments point toan induction mechanism that is solely presynaptic.Here the working hypothesis is that HFS triggersan increase in presynaptic Ca++ that, via calmod-ulin, activates adenylyl cyclase (Zalutsky andNicoll, 1992; Weisskopf et al., 1994; Villacreset al., 1998). An alternative presynaptic mecha-nism is that presynaptic KA receptor activationleads to Ca++ influx that triggers release of Ca++

from intracellular stores (Contractor et al., 2001;Lauri et al., 2001; Bortolotto et al., 2003; Lauriet al., 2003), although this remains controversial(Breustedt and Schmitz, 2004).

Alternatively, there is evidence for a postsynapticcomponent to the induction mechanism (Williamsand Johnston, 1989; Jaffe and Johnston, 1990).Here, an interaction between Ca++ entry throughvoltage-gated Ca++ channels and the activationof mGluRs takes place (Kapur et al., 1998; Yeckelet al., 1999; Kapur et al., 2001; Wang et al., 2004). Itmay be that depending on stimulus conditions, in-duction may have either a presynaptic or post-synaptic locus (Urban and Barrionuevo, 1996).

In contrast to the induction process, there is lit-tle controversy regarding the locus for the expres-sion of MF LTP. Numerous studies indicate thatsynaptic potentiation is mediated by a presynapticchange in transmitter release (Zalutsky and Nicoll,1990; Yamamoto et al., 1992; Zalutsky and Nicoll,1992; Xiang et al., 1994; Reid et al., 2004). Ifinduction of MF LTP has a postsynaptic locus,then expression must require a retrograde com-munication. One possible mechanism involvespostsynaptic ephrinB receptors and presynaptic

B-ephrins reverse signaling (Contractor et al.,2002; Armstrong et al., 2006).

‘‘What goes up must go down’’ is an apt phraseto describe MF synaptic plasticity. LFS triggerslong-term depression (LTD) of MF synaptic in-puts onto CA3 pyramidal neurons (Kobayashiet al., 1996; Yokoi et al., 1996) where presynapticactivation of mGluR2 receptors depresses cAMPlevels, countering the expression of LTP.

CA3-interneuron synapses

As discussed above, the majority of MF synapticcontacts are onto GABAergic interneurons(Acsady et al., 1998) of which there are a widevariety of subtypes, typically characterized basedon a combination of parameters including cellbody location, axonal projection, morphology, co-localized peptides, and calcium binding proteincontent (Parra et al., 1998). Of particular interestare a class of bipolar interneurons whose dendritesprimarily reside along and within s. lucidum

(Spruston et al., 1997; Vida and Frotscher, 2000),in contrast to other interneurons where the den-dritic tree projects orthogonally against the MFsthat receive most of their innervation from otherpathways. The studies discussed below were madeprimarily from this specific subtype of interneuron.

Like the pyramidal cell synapse, MF transmis-sion onto interneurons is reduced by the activationof presynaptic group II mGluR receptors(Maccaferri et al., 1998; Toth et al., 2000); Pelkeyet al., 2005), although other types of mGluRs mayplay a role in synaptic plasticity at this synapse(discussed below). In contrast to the pyramidalneuron synapse, at MF-interneuron synapsesAMPA receptor expression is heterogeneous.Glutamate released from the MFs may activatereceptors with or without GluR2 subunits, therebyforming receptors with either Ca++ permeable orimpermeable subunits (Toth and McBain, 1998;Toth et al., 2000).

Also different from the pyramidal neuron MFsynapse is the frequency response of the interneu-ron, which is highly variable. The response canrange from a weak frequency-dependent facilita-tion (much less pronounced than at the pyramidal

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neuron synapse) to synaptic depression (Tothet al., 2000).

Quantal transmission at interneuron synapses isalso different than at the pyramidal neuronsynapse. As expected from anatomy, thesesynapses express a low number of release sites(Acsady et al., 1998). The probability of release atthese sites, however, appears higher than at thepyramidal neuron synapse (Lawrence et al., 2004).This difference in probability of release is alsoconsistent with differences frequency facilitationobserved between the two synapses (i.e., a low in-itial probability of release predicts large facilita-tion). Interestingly, quantal size at interneuronsynapses is within the same ranges, if not higher,than for pyramidal neuron synapse. As a result,and combined with a more depolarized restingpotenial, unitary responses of interneuronsynapses are as capable of triggering action po-tentials as MF inputs onto pyramidal neurons(Lawrence et al., 2004), and this also appears to bethe case for MF transmission to hilar interneurons(Scharfman et al., 1990).

Long-lasting plasticity at MF-interneuronsynapses also differ from their pyramidal neuroncounterparts. Most notably, HFS does not elicitLTP at the MF-interneuron synapse (Maccaferriet al., 1998). Rather, HFS triggers LTDat synapses expressing Ca++-permeable AMPAreceptors, while NMDA receptors trigger theinduction of LTD at synapses containing Ca++-impermeant AMPA receptors (Lei and McBain,2002). Activation of mGluR7 receptors by L-AP4produces a chemical version of LTD at thissynapse that is distinct from pyramidal neuroncontacts (Pelkey et al., 2005) and differentiallymodulates voltage-gated Ca++ channels in thepresynaptic filopodia (Pelkey et al., 2006). If HFStriggers LTD at the MF-interneuron synapse,feed-forward inhibition should be depressed ontoCA3 pyramidal neurons following HFS. However,fast synaptic inhibition onto CA3 pyramidal neu-rons is not affected by LTP of the MFs (Griffithet al., 1986) suggesting that a decrease in feed-for-ward inhibition through interneurons of s. lucidumis compensated by enhanced recurrent inhibitionor other forms of synaptic plasticity within theCA3 network.

Communication from DG to CA3: exciting

yet inhibiting

A picture is emerging that relates the contributionof MF transmission to excitation and inhibitionin area CA3, both in terms of the repertoire ofneurotransmitters/modulators released from thefibers but also with respect to circuitry that isrequired to take into account inhibitory neurons.At low frequencies (o0.5Hz), frequency facilitationof the MF-CA3 pyramidal neuron synapse is weakand the probability of eliciting a spike is low (Henzeet al., 2002b). Furthermore, in this frequency do-main granule cells are likely to be firing inhibitoryinterneurons preferentially relative to pyramidalneurons CA3 (Bragin et al., 1995a, b; Penttonenet al., 1997). As such, concomitant extrinsic inputfrom entorhinal cortex (via the perforant path) orvia recurrent excitation via the C/A pathway shouldfail to excite sets of CA3 pyramidal neurons. But asgranule cell firing rates increase, such as when ananimal traverses a place field, frequency facilitationcan then depolarize CA3 pyramidal neuronsto spike threshold. Interestingly, after seizures,MFs tonically inhibit b/g field oscillations andspontaneous subthreshold membrane oscillationsof CA3 interneurons in the CA3 area throughGABA-medated signaling. Coincident stimulationof the MFs at y and b/g frequencies produces afrequency-dependent excitation of interneurons andthe inhibition of pyramidal cells. Indeed, theseeffects are maximal at the b frequency, suggestinga resonance phenomenon (Trevino et al., 2007).Because of the properties of synaptic plasticitywithin the auto-associative circuit of CA3, patternseparation, pattern completion, and the encodingof new information will then be dependent on theprecise timing of granule cell and pyramidal neuronfiring (Kohonen, 1977; Chattarji et al., 1989;Zalutsky and Nicoll, 1990; August and Levy,1999; Nakazawa et al., 2002; Rolls and Kesner,2006).

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Plate 5.8. (A) Timm stained sections of hippocampus from adult rat, subjected to hippocampal x-irradiation as newborn, stop

odentate granule cell formation and lead to few MF terminals. (B) Timm stained section from the contralateral hemisphere of the rat

shown in A, depicting a well-integrated dentate transplant (tpl), derived from a small block of fascia dentata, grafted just after the x-

irradiation. From the graft, an apparently normal, laminar-specific MF projection (mf) developed, connecting dentate granule cells of

the graft (g) with the host CA3. The molecular layer of the graft (m) received a comparably laminar-specific projection of host

entorhinal perforant path projections, normalizing the Timm stained laminar appearance of the layer. (C) Slightly displaced dentate

transplant (tpl), grafted to x-irradiated newborn hippocampus just after irradiation as part of same experiment (Sunde et al., 1984).

Encroaching on the recipient CA3, MFs from the granule cells of this graft has entered adjacent parts of CA3 and project ‘‘down-

stream’’, but appear to stop at the border with CA1. Surprisingly, no MFs from the graft projected in the ‘‘upstream’’ direction, i.e.,

toward the host fascia dentata. Scale bar: 500mm (For B/W version, see page 100 in the volume.)

Plate 6.1. Interneurons are the primary target of DG granule cells. Diagram representing the multiple types of excitatory contacts

made by granule cells. Within the dentate/hilar region collaterals of the mossy fibers (MFs) innervate both mossy cells and inhibitory

basket cells. In area CA3, the MFs contact pyramidal neurons via large expansions, but also excite interneurons through either

filopodial extensions from the large boutons or smaller en passant expansions along MF axons. (For B/W version, see page 110 in the

volume.)

Plate 6.2. Summary of the pre- and post-synaptic constituents of the different MF terminals and of their plasticity. (A) Schematic

representation of the giant MF boutons, which contact CA3 pyramidal cells. These terminals are characterized by low probability of

basal release and multiple release sites. (B) Schematic representation of filopodial extensions and en passant contacts, which synapse on

to interneurons. These have a high probability of basal release and single release sites. Both types of MF terminals contain several

neuromodulators and the neurotransmitters glutamate and GABA. Note that both the presynaptic and postsynaptic sites contain

several ionotropic GluRs and metabotropic GluRs, which confer to the MF a high degree of plasticity and the capacity for synaptic

integration. (C) Relative expression of the different releasable contents and of the receptors and transporters during development, in

the adult (D), and after epileptic activity (E). Arrows and font size indicate relative differences between the three states. (For B/W

version, see page 114 in the volume.)