77:1075-1086, 1997. J NeurophysiolDarrell A. Henze, J. Patrick Card, German Barrionuevo and Yezekiel Ben-Ari

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Large Amplitude Miniature Excitatory Postsynaptic Currents inHippocampal CA3 Pyramidal Neurons Are of Mossy Fiber Origin

DARRELL A. HENZE,1 J. PATRICK CARD,1 GERMAN BARRIONUEVO,1 AND YEZEKIEL BEN-ARI2

1Department of Neuroscience and Center for the Neural Basis of Cognition, University of Pittsburgh, Pittsburgh,Pennsylvania 15260; and 2Institut National de la Sante et de la Recherche Medicale, Unite 29, 75014, Paris, France

Henze, Darrell A., J. Patrick Card, German Barrionuevo, and the case for CA3 pyramidal neurons (Amaral and WitterYezekiel Ben-Ari. Large amplitude miniature excitatory postsyn- 1989; Blackstad 1956; Blackstad et al. 1970; Hjorth-Si-aptic currents in hippocampal CA3 pyramidal neurons are of mossy monsen 1973; Hjorth-Simonsen and Jeune 1972).fiber origin. J. Neurophysiol. 77: 1075–1086, 1997. Neonatal (P0) CA3 pyramidal cells receive a major glutamatergic synap-g-irradiation was used to lesion selectively the mossy fiber (MF) tic input from the granule cells of the dentate gyrus via thesynaptic input to CA3 pyramidal cells. This lesion caused a ú85%

mossy fibers (MFs) (Neuman et al. 1988). The MFs makereduction in the MF input as determined by quantitative assessmentsynaptic contacts on to the proximal dendrites of the CA3of the number of dynorphin immunoreactive MF boutons. Thepyramidal cells and, therefore, are believed to play a criticalg-irradiation lesion caused a reduction in the mean number of

miniature excitatory postsynaptic currents (mEPSCs) recorded role in modulating activity in these cells. Numerous studiesfrom CA3 pyramidal cells (2,292 vs. 1,429/3-min period; n Å already have demonstrated that the MF to CA3 pyramidal10). The lesion also caused a reduction in the mean mEPSC peak cell synapse has unique features that distinguish it from otheramplitude from 19.1 { 0.45 to 14.6 { 0.49 pA (mean { SE; peak cortical synapses. For example, the MF synaptic terminalconductance 238.8 { 5.6 to 182.0 { 6.1 pS). Similarly, there was has a large presynaptic bouton (3–10 mm diam) with smalla reduction in the mean 10–85% rise time from 1.72 { 0.02 ms

finger-like extensions (Amaral 1979; Blackstad and Kjaer-to 1.42 { 0.04 ms. The effects of the g-irradiation on both mEPSCheim 1961; Hamlyn 1962) that completely envelopes a large,amplitude and 10–85% rise time were significant at P õ 0.002complex, postsynaptic ‘‘thorn.’’ Ultrastructural studies haveand P õ 0.005 (2-tailed Kolmogorov-Smirnov test) . Based on therevealed that a single presynaptic MF bouton has °35 sepa-selectivity of the g-irradiation, MF and non-MF mEPSC amplitude

and 10–85% rise-time distributions were calculated. Both the am- rate active sites (Chicurel and Harris 1992). The MF presyn-plitude and 10–85% rise-time distributions showed extensive over- aptic terminal also has a distinctive complement of neuro-lap between the MF and non-MF mediated mEPSCs. The MF transmitters with the opioid dynorphin being colocalizedmEPSC distributions had a mean peak amplitude of 24.6 pA (307.5 with glutamate (Gall 1988). Finally, the presynaptic termi-pS) and a mean 10–85% rise time of 2.16 ms. The non-MF mEPSC

nal of MFs is unique in that it can express at least twodistributions had a mean peak amplitude of 12.2 pA (152.5 pS)distinctive forms of plasticity: a prolonged posttetanic poten-and 10–85% rise time of 1.26 ms. The modes of the amplitudetiation (Langdon et al. 1995) and a N-methyl-D-aspartatedistributions were the same at 5 pA (62 pS). The MF and non-(NMDA) receptor independent form of long-term potentia-MF mEPSC amplitude and 10–85% rise-time distributions were

significantly different at P ! 0.001 (1-tailed, large sample Kolmo- tion (Castillo et al. 1994; Johnston et al. 1992; Katsuki etgorov-Smirnov test) . The data demonstrate that the removal of the al. 1991; Langdon et al. 1995; Urban and Barrionuevo 1996;MF synaptic input to CA3 pyramidal cells leads to the absence of Xiang et al. 1994; Zalutsky and Nicoll 1990).the large amplitude mEPSCs that are present in control recordings. Coincident with the presence of MF synapses, mEPSCs

recorded from CA3 pyramidal cells are often an order ofmagnitude larger in amplitude than those observed fromI N T R O D U C T I O Nother cells that are similar anatomically and physiologically(e.g., CA1 pyramidal cells) (compare Jonas et al. 1993 withOne useful tool in investigations of the basic mechanismsMalgaroli and Tsien 1992 or Manabe et al. 1992). To deter-of neurotransmission is the study of the spontaneous minia-mine whether the MF synapses are the source of the largeture excitatory postsynaptic currents (mEPSCs). mEPSCsmEPSCs recorded from CA3 pyramidal cells, in the presentare signals produced by the sodium action potential indepen-study, we have taken advantage of a hippocampal slice prep-dent spontaneous release of neurotransmitter from the pre-aration in which the granule cells and their axons, the MFs,synaptic terminal. The properties of mEPSCs, in particularare lesioned selectively (Dessi et al. 1991; Repressa et al.frequency of occurrence and relative amplitude, offer a1991). This selective lesion of the MFs was accomplishedmeans to study the otherwise inaccessible presynaptic termi-by focal g-irradiation of neonatal rats; this has been shown tonals within the CNS (Malgaroli and Tsien 1992; Manabe etremove the majority (ú85%) of MFs from the hippocampalal. 1992). However, the major drawback in the use offormation. The selectivity of the g-irradiation induced lesionmEPSCs to study the presynaptic element is the difficultyis supported by a number of studies: CA3 dendritic arborsin identifying the synaptic origin of an individual synapticand spine density are not affected (Gaiarsa et al. 1992; Re-current. This is particularly problematic when studying com-pressa et al. 1991), the number of NMDA binding sites inplex neurons of the CNS that receive anatomically and physi-

ologically heterogeneous populations of synapses such as is area CA3 is not affected (Dessi et al. 1991), and bicuculline

10750022-3077/97 $5.00 Copyright q 1997 The American Physiological Society

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induces ‘‘normal’’ epileptic bursting (Gaiarsa et al. 1994).Using this approach, we have determined that the presenceof the large mEPSCs is correlated with the presence of aMF synapse onto CA3 pyramidal cells.

M E T H O D S

Irradiation

Newborn Wistar rats (postnatal day P0) were irradiated at 6 GyFIG. 1. Areas examined to quantify dynorphin labeling of mossy fibers(600 rad) by a cobalt bomb (Centre d’etudes nucleaires, Fontenay-

(MFs). A schematic diagram of the transverse hippocampal slice illustratingau-Roses) using parameters previously shown to induce pro-3 regions used to quantify the effect of g-irradiation on dynorphin labelednounced degeneration of granule cells in the dentate gyrus (DessiMF boutons.et al. 1991; Repressa et al. 1991). The g-rays were collimated to

irradiate only one side of the brain. The nonirradiated contralateralside was used for control recordings. Tissue analysis

Cresyl violet stained preparations were used to determine thequalitative extent of cell loss in the g-irradiated hippocampus, andLesion characterizationdynorphin immunoreactivity was used to evaluate the degree ofMF loss in the afflicted stratum lucidum. In each case, the dentateTISSUE PROCESSING. The extent of g-irradiation-induced cellgyrus ipsilateral and contralateral to the irradiated side of the brainand fiber loss in the hippocampus was characterized in six animalswere compared within each animal. As noted above, dynorphinthat were irradiated unilaterally as neonates (see above) and pre-immunoreactivity in the stratum lucidum was used as the measurepared for morphological analysis. These animals were anesthetizedof MF bouton loss in the irradiated hippocampus. Subjective deter-and killed by transcardiac infusion of buffered aldehyde fixativeminations of the extent of the lesion were made by comparing the28 days after g-irradiation exposure. The perfusion procedure in-density of staining on the control and lesion sides. Quantitativecluded an initial infusion of phosphate buffered saline (PBS; 0.1determinations of the magnitude of MF bouton loss on the lesionedM, pH 7.4) into the ascending aorta followed by 200–300 ml ofside also were conducted using the following procedure. Threeparaformaldehyde-lysine-periodate fixative (McLean and Nakanecomparable 100 mm2 segments of stratum lucidum were identified1974). The brain then was removed, postfixed for 2 h, washed inin the control and irradiated sides of the brain, midway throughPBS, and cryoprotected in 20% phosphate buffered sucrose forthe rostro-caudal extent of the hippocampus. The relative positionÇ72 h, all at 47C. The forebrain was sectioned (35 mm/section)of these three regions are illustrated in the schematic diagramin the coronal plane through the rostrocaudal extent of the hippo-shown in Fig. 1. Varicose boutons in each of these regions dis-campus using a freezing microtome. Sections were collected seri-playing dynorphin immunoreactivity were mapped using a drawingally in six wells of phosphate buffer such that each well containedtube and a 140 objective on a Nikon Optiphot microscope. Thea one-in-six series of sections through the hippocampus. One wellnumber of boutons were counted in each area and quantitativeof tissue was mounted immediately on gelatin-coated slides, stainedcomparisons of counts of lesion and control sides were made towith cresyl violet, dehydrated, cleared, and coverslipped using stan-determine the degree of MF bouton loss in the irradiated hippocam-dard procedures. This tissue was used to determine the relativepus. No effort was made to quantitate the degree of cell loss withinamount of cell loss in the dentate gyrus of the irradiated hippocam-the g-irradiated dentate gyrus.pus. Remaining tissue was transferred to an ethylene glycol-based

cryopreservant (Watson et al. 1986) and stored at 0207C to pre-serve antigenicity for subsequent immunohistochemical pro- In vitro slice preparationcessing.

The extent of MF degeneration in the stratum lucidum of the g- Slices were prepared from the g-irradiated rats 27 to 40 daysold. Briefly, animals were anesthetized with equithesin and theirirradiated hippocampus was evaluated by immunohistochemical

localization of dynorphin immunoreactivity. Previous studies have chest cavities were opened rapidly. The rats then were trans-cardially perfused with a cold oxygenated sucrose solution withdemonstrated that MF boutons in the rat hippocampus contain high

levels of dynorphin and that this peptide is a reliable marker of the following composition (in mM): 229 sucrose, 1.9 KCl, 1.2Na2PO4, 25 NaHCO3, 10 glucose, 10 N-2-hydroxyethylpiperazine-the MF projection pathway (Gall 1988). A series of sections from

each of the six brains was washed thoroughly at room temperature N*-2-ethanesulfonic acid (HEPES), and 6.0 MgCl2 . Before thetranscardial perfusion, the sucrose solution was bubbled with 95%in multiple changes of PBS to remove the cryopreservant, and the

tissue then was processed for immunohistochemical localization O2-5% CO2 to adjust the pH to 7.4 at 47C. After 1 min of trans-cardial perfusion, the rats were decapitated and the hippocampiof dynorphin using a rabbit polyclonal antiserum generated against

dynorphin 1-8 (Peninsula Laboratories) . In the first stage of this dissected from the brain. Each hippocampus then was sliced into400-mm-thick slices perpendicular to its long axis using a vibra-procedure, the tissue was incubated in primary antiserum diluted

to a final concentration of 1:2,000 with PBS, normal donkey serum tome. This sucrose perfusion technique originally was describedas a method to improve the viability of facial motoneurons in in(1%), and Triton X-100 (0.3%). The incubation was conducted

at 47C for 24–48 h. Antigen localization was visualized with the vitro slices taken from rat brain stem (Aghajanian and Rasmussen1989). We found that the sucrose perfusion also improves theavidin-biotin modification of the immunoperoxidase procedure

(Hsu et al. 1981) using an affinity-purified donkey anti-rabbit sec- viability neurons of area CA3 in hippocampal slices.After sectioning, slices were maintained at room temperatureondary antibody (Jackson ImmunoResearch) and Vectastain Elite

reagents (Vector Laboratories) . Further details regarding applica- (19–237C) in an artificial cerebrospinal fluid (ACSF) consistingof (in mM) 126 NaCl, 3.5 KCl, 1.2 Na2PO4, 11 glucose, 25tion of this procedure in our laboratory have been published (Card

and Enquist 1994). Processed tissue was mounted on gelatin coated NaHCO3, 3.0 MgCl2 , and 3.0 CaCl2 . The ACSF was bubbled with95% O2-5% CO2 (pH to 7.4) . Individual slices were transferred asslides, dehydrated, cleared, and coverslipped.

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MOSSY FIBER MEPSCS 1077

sis of all mEPSCs included ‘‘smoothing’’ the raw data and itssubsequent derivatives using boxcar averaging with an 11-pointwindow. The detection algorithm was developed and run usingIgor Pro (Wavemetrics) on an accelerated Macintosh IIci. Thisalgorithm is similar to a published automated detection algorithmfor detecting miniature inhibitory postsynaptic potentials (Ankri etal. 1994). All values given in the text are means { SE, unlessotherwise indicated. The accuracy of the mEPSC detection algo-rithm was evaluated by comparing the automated results with re-sults collected manually. We considered three types of error: 1)the number of false positives. The false positives were determinedfor a single 60-s period where the algorithm found 1,341 events.Of the detected 1,341 events, only 7 of those events (0.52%) couldbe rejected as events not having an mEPSC-like appearance. 2)

FIG. 2. Method for detecting miniature excitatory postsynaptic currents The number of overlapping doublets that were not resolved. The(mEPSCs). Schematic illustration of method used to automate detection overlapping doublets for the same 60-s period was 57. The lackand quantification of spontaneous mEPSCs. Raw data are smoothed using

of detection of these events can be explained by the fact that theseboxcar averaging. First and 3rd derivatives then are found, each also beingdoublets, although inflected on the rising phase, did not reverseboxcar averaged. Peaks in 1st derivative exceeding 5 pA/ms indicate andirection and therefore did not have a 0 crossing in the first deriva-event has occurred (1) . After detection of an mEPSC, its amplitude and risetive. 3) The number of false negatives. There were numerous smalltime is determined as follows: peak of mEPSC is determined by scanning the

1st derivative forward for a 0 crossing (2) . Foot of mEPSC is determined events that had an mEPSC-like appearance that were not detectedby scanning backward in the 3rd derivative for a 0 crossing (3) . Amplitude by the algorithm. These undetected events were always very smallchange between points 2 and 3 represents mEPSC amplitude; 10–85% rise (õ5 pA) and, by definition, had rising slopesõ5 pA/ms. Althoughtimes also are calculated using points 2 and 3 as 0 and 100%. many of these small mEPSCs are recognizable by eye, it is impossi-

ble to establish a set of criteria to identify all of them becausethey recede into the noisy baseline and therefore this error is notneeded (between 2 and 12 h after slice preparation) to a submergedquantifiable.chamber where they were constantly superfused with oxygenated

ACSF at 337C.

R E S U L T SElectrophysiological recording

Anatomic effects of g-irradiation lesionSpontaneous mEPSCs were recorded using the ‘‘blind’’ whole

Clear changes in the cellular organization of the hippo-cell recording technique (Blanton et al. 1989). Recordings for bothcontrol and g-irradiated groups were from CA3 pyramidal cells campus were apparent in cresyl violet-stained coronal sec-(verified in some cases by filling cells with biocytin) in the same tions passing through the same portion of the rostrocaudalarea used to quantify the amount of dynorphin immunoreactivity axis that was selected for electrophysiological analysis in(see Fig. 1) . The mEPSCs were recorded using a voltage-clamp other animals. As previously reported (Dessi et al. 1991;amplifier, filtered at 3 kHz, digitized at 10 kHz, and then stored on Gaiarsa et al. 1992, 1994; Repressa et al. 1991), the mostcomputer disk in 3-min epochs. The recordings also were collected obvious alterations were a profound cell loss in the dentatesimultaneously to DAT tapes, which provided a continuous record

gyrus and a decreased packing density of pyramidal cells inof the experiment. The analysis presented is based on one 3-minthe CA1 subfield of each g-irradiated hippocampus. Theseepoch per cell selected for having a relatively stable baseline andchanges were quite consistent between animals and werestable series resistance. Whole cell pipettes were filled with a solu-distinguished easily when comparing the g-irradiated andtion consisting of (in mM) 50 CsF, 80 CsCl, 10 HEPES, and 1.0

bis-(o-aminophenoxy)-N,N,N *,N *-tetraacetic acidrK4 with a pH control hippocampus within the same animal (Fig. 3, A andof 7.0 and final osmolarity of 270 mOsm. Pipettes had a resistance B) . Below, we focus on the g-irradiation induced cell lossof 3–6 MV. Membrane potentials were voltage clamped at 080 in the dentate gyrus and the resultant alterations of the MFmV. Data were rejected if the access resistance was ú20 MV or projection to stratum lucidum.a significant change (ú15%) in whole cell series resistance was g-Irradiated tissue exhibited extensive cell loss in theobserved. All recordings were carried out in the presence of 1 mM granule cell layer of the dentate gyrus and a correspondingtetrodotoxin and 10 mM bicuculline methiodide (Sigma).

decrease in the thickness of the molecular cell layer, changesthat were not present in the contralateral, nonirradiated tissue

Automated mEPSC detection algorithm from the same animals. The cell loss was apparent in boththe supra- and infrapyramidal blades of the granule cell layerThe detection and measurement of the mEPSCs was accom-

plished using an automated routine to permit the rapid analysis of and occurred throughout the rostrocaudal axis of hippocam-long recording epochs. The algorithm first calculated the first and pus. The characteristic pattern and magnitude of cell loss inthird derivatives of the raw data trace with respect to time. Then the dentate gyrus of all experimental animals is apparent inmEPSCs were detected by scanning for peaks in the first derivative comparing Fig. 3, C and D . These photomicrographs ofwhich corresponds to the rising slopes of the mEPSCs. For all cresyl violet-stained preparations from a single animal illus-recordings, an mEPSC was detected if its rising slope ú5 pA/ms. trate the extensive cell loss in the irradiated hippocampusAfter the detection of a mEPSC, the mEPSC peak was determined

(Fig. 3D) compared with the nonirradiated control hippo-from the first 0 crossing after the peak in the first derivative. Thecampus (Fig. 3C) . In contrast, the immediately adjacentfoot of the mEPSC was determined from the 0 crossing in the thirdpolymorphic neurons of the hilus and the pyramidal cells ofderivative preceding the peak in the first derivative (Fig. 2) . Thearea CA3 exhibited no apparent g-irradiation-inducedamplitude and 10–85% rise time were determined using the

mEPSC foot and peak detected by the search algorithm. The analy- changes in either cell morphology, number, or distribution,

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FIG. 3. Effect of g-irradiation on dentate gyrus and stratum lucidum of hippocampus. Morphology of control (A and C)and irradiated (B and D) hippocampus are illustrated in cresyl violet stained coronal sections. The same, which receivedunilateral neonatal irradiation, was the source for all of photomicrographs. Comparison of A and B reveals profound g-irradiation-induced cell loss in dentate gyrus and a splaying of the CA1 pyramidal cell layer (small open block arrows inB) . The extent of granule cell loss and the accompanying reduction in thickness of the molecular layer (demarcated by barswith arrowheads) resulting from irradiation is more readily apparent in comparing C and D . Scale bars: 440 mm in B for Aand B ; 160 mm in D for C and D .

a finding consistent with previous reports (Dessi et al. 1991; Electrophysiological effects of g-irradiation lesionGaiarsa et al. 1992, 1994; Repressa et al. 1991).

The present analysis compares recordings from cells ing-Irradiation-induced changes in the number of dynor-10 slices from control and 10 slices from g-irradiated ani-phin immunoreactive MF boutons in the s. lucidum weremals. All recordings were collected under voltage-clampdramatic and consistent across animals. In the nonirradi-conditions with the holding potential set to 080 mV. Appli-ated control hippocampus, a broadband of dynorphin im-cation of 10 mM 6-cyano-7-nitroquinoxaline-2,3-dione com-munoreactive varicose fibers filled the stratum lucidumpletely abolished all observed mEPSCs, indicating that theyadjacent to the CA3 pyramidal neurons (Fig. 4, A andare mediated by glutamatergic a-amino-3-hydroxy-5-C ) . Scattered individual immunoreactive fibers also weremethyl-4-isoxazolepropionic acid (AMPA) receptor activa-observed within the stratum pyramidale. This was intion (data not shown). Figure 5 illustrates the effect of g-marked contrast to the decrease in immunoreactive vari-irradiation on the overall frequency of mEPSCs recordedcosities in comparable regions of the contralateral, g-irra-from two selected CA3 cells. When the results from the 10diated hippocampus (Fig. 4, B and D ) . The immunoreac-recordings from each condition are considered together, thetive boutons in the g-irradiated hippocampus were re-mean frequency of mEPSCs decreased from Ç12.5 to 7.8duced substantially throughout the full extent of CA3.Hz. This decrease was not statistically significant for P õComparative counts of the number of immunoreactive var-0.05 (1-tailed, Student’s t-test, P Å 0.082) primarily due toicosities within each of three 100-mm2 areas ( see Fig. 1 ) ofthe large variation in mEPSC frequency between recordingsthe stratum lucidum in control and irradiated hippocampus(standard deviations were 7.5 and 7.0 Hz for control andconfirmed that there was a substantial decrease of theseg-irradiated tissue, respectively) . The number of mEPSCsprofiles on the irradiated side. These counts revealed thatdetected at all amplitudes and rise times were reduced bythere was an Ç84% decrease in the number of immunore-the g-irradiation treatment, however, larger amplitude (Fig.active varicosities irrespective of whether the data were6A) and slower rising (Fig. 6B) mEPSCs showed a greateranalyzed in individual 100 mm2 bins or grouped together

(Table 1 ) . decrease. The mean amplitude decreased from 19.1 { 0.45

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MOSSY FIBER MEPSCS 1079

FIG. 4. Effect of g-irradiation on dynorphin labeled MF boutons. Dynorphin immunoreactivity in MFs of control (A andC) and irradiated (B and D) tissue is illustrated. The extent of dynorphin immunoreactive profiles in coronal sections throughcontrol and irradiated hippocampus are shown in A and B , respectively. C and D are higher magnification views of dynorphin-immunoreactive fibers in stratum lucidum of control and irradiated tissue. A thick band of dynorphin immunoreactivityadjacent to proximal portions of apical dendrites of CA3 pyramidal cells defines stratum lucidum in control tissue (A andC) . In contrast, irradiation-induced degeneration of dentate granule cells causes a pronounced reduction in both density andthickness of stratum lucidum, as defined by dynorphin immunoreactive fibers (B and D) . The densest concentration ofdynorphin immunoreactive fibers in control and irradiated tissue is defined by regions between arrowheads on right-handborder of C and D . Scattered immunoreactive boutons outside of this zone in irradiated animal are indicated by open blockarrows in D . Scale bars: 440 mm in B for A and B ; 14 mm in D for C and D .

pA in control to 14.6 { 0.49 pA (P õ 0.05, 1-tailed Stu- respectively) . The median peak amplitude also was de-creased in irradiated records, going from 13.9 to 11.2 pAdent’s t-test) in g-irradiated tissue (corresponding to mean

peak conductances of 238.8 { 5.6 pS and 182 { 6.1 pS, (173.8 to 140 pS). The distributions of mEPSC amplitudes

TABLE 1.

Control Irradiated % Control

Rat 1 2 3 Total 1 2 3 Total 1 2 3 Total

4329 287 298 271 856 17 31 31 79 0.059 0.104 0.114 0.0924330 268 289 282 839 49 51 50 150 0.183 0.176 0.177 0.1794331 411 352 275 1,038 114 108 85 307 0.277 0.307 0.309 0.2964332 424 307 335 1,066 51 63 59 173 0.120 0.205 0.176 0.1624333 379 502 454 1,335 62 30 33 125 0.164 0.060 0.073 0.0944334 368 399 472 1,239 67 77 57 201 0.182 0.193 0.121 0.162

Average 0.164 0.174 0.162 0.164

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D. A. HENZE, J. P. CARD, G. BARRIONUEVO, AND Y. BEN-ARI1080

FIG. 5. Effect of g-irradiation on mEPSCs recorded from hippocampal CA3 cells. Examples of raw data collected fromcontrol and g-irradiated slices. A, top : 3 min of a voltage-clamp (VH Å 080 mV) whole cell recording from a control CA3pyramidal cell (1 mM tetrdotoxin and 10 mM bicuculline present) . A, bottom : similar recording from an g-irradiated CA3pyramidal cell. B : an example of raw data from a control recording at a higher gain. Synaptic currents were abolished byapplication of 10 mM 6-cyano-7-nitroquinoxaline-2,3-dione (data not shown).

are not normally distributed as reflected by the high skew arising from MF synapses removed by the lesion (see DIS-

CUSSION) . Figure 8 illustrates the general appearance of thevalues of 4.3 and 5.5 for control and g-irradiated tissue,respectively. The mean distribution of mEPSC amplitudes large and/or slow mEPSCs that are reduced greatly or ab-

sent from g-irradiated tissue. The examples shown in Fig.from g-irradiated animals was significantly different fromthe mean distribution from control animals (P õ 0.002, 2- 8 are also representative of the few large mEPSCs that

were observed in g-irradiated tissue.tailed Kolmogorov-Smirnov test, 1 pA bins) . It can be seenfrom both the standard and cumulative probability histo-grams that the relative differences between the amplitudes D I S C U S S I O Nof mEPSCs from control and g-irradiated tissue are greatest

Previous work has shown that destruction of the granulefor larger amplitudes. With respect to the 10–85% rise-timecells by postnatal g-irradiation selectively reduces the MFdistributions, the mean 10–85% rise time decreased frominput to area CA3 (Dessi et al. 1991; Gaiarsa et al. 1992,1.72 { 0.02 ms in control to 1.42 { 0.04 ms (P õ 0.05,1994; Repressa et al. 1991). Using this preparation, we have1-tailed Student’s t-test ) in g-irradiated tissue. g-Irradia-demonstrated that the large amplitude and/or slow risingtion also decreased the median 10–85% rise time from 1.44mEPSCs recorded from CA3 pyramidal cells are correlatedto 1.16 ms. The distributions of mEPSC 10–85% rise timewith the presence of normal, intact MFs.also are not normally distributed as reflected by the high

skew values of 2.3 and 2.5 for control and g-irradiatedtissue, respectively. The mean distribution of mEPSC 10– Specificity of the g-irradiation-induced lesion85% rise times from g-irradiated animals was significantlydifferent from the mean distribution from control animals In the brain of the postnatal rat, there are only a few

populations of cells that have not finished their final division(P õ 0.005, 2-tailed Kolmogorov-Smirnov test, 0.1-msbins) . It can be seen from both the standard and cumulative and therefore are sensitive to g-irradiation. These cells in-

clude the granule cells of the olfactory bulb, the granuleprobability histograms that the relative differences between10–85% rise times of mEPSCs from control and g-irradi- cells of the cerebellum, and the granule cells of the dentate

gyrus (Bayer and Altman 1975b). Focal application of theated tissue differ the most for longer rise times. Figure 7illustrates the amplitude and rise-time distributions for radiation permits the specific lesioning of the granule cells

of the dentate gyrus (Bayer and Altman 1975a). Other stud-those mEPSCs that were absent from recordings from g-irradiated tissue. These distributions were obtained by sim- ies have shown that within the g-irradiated zone, only the

dentate gyrus granule cells and their MF input to area CA3ply subtracting the respective irradiated histogram fromthe respective control histogram. To the extent that the g- pyramidal cells are affected, whereas all other ionotropic

synaptic inputs, both excitatory and inhibitory, remain un-irradiation-induced lesion is selective for MFs, these histo-grams nominally represent the distributions for mEPSCs changed (Dessi et al. 1991; Gaiarsa et al. 1994). The best

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FIG. 6. Effect of g-irradiation on observed mEPSC amplitude and rise-time distributions. Average histogram of mEPSCamplitudes (A) and 10–85% rise times (B) recorded from control and g-irradiated tissue (n Å 10/group; {SE). Note g-irradiation affects all amplitudes and rise times. Both histograms are truncated for presentation purposes as illustrated bybroken axes. For amplitude, single events measuring °400 pA were recorded (an average of 16.9 mEPSCs with amplitudesú100 pA in control and 5.4 mEPSCs in g-irradiated recordings, single bins after axis break). For rise time, single eventsmeasuring °14 ms were recorded (an average of 7.6 mEPSCs with 10–85% rise times ú7 ms in control and 0.7 mEPSCsin g-irradiated recordings, single bins after axis break). Amplitude and 10–85% rise-time histograms are significantlydifferent at P õ 0.002 and P õ 0.005, respectively (2-tailed Kolmogorov-Smirnov test) . Insets : control vs. g-irradiatedcumulative probability histograms. mEPSCs from g-irradiated slices display narrower cumulative distributions of amplitudeand rise time.

evidence that sprouting of associational/commissural syn- sion that large and/or slow mEPSCs arise from the MFsynapses. Sprouting of associational synapses would onlyapses is not significant is the observation that there is no

change in NMDA receptor density (as measured by immuno- serve to counter the observed decrease in the frequency ofmEPSCs and minimize the apparent effect of the g-irradia-histochemistry) in the stratum oriens (Dessi et al. 1991).

Other reports also suggest that non-MF synapses do not tion lesion. With respect to the MFs, the 84% reduction inthe number of dynorphin positive boutons in the stratumsprout as reflected by the loss of all spinelike structures on

the CA3 pyramidal cell in the stratum lucidum (Gaiarsa et lucidum is exactly what is predicted given the reduction ingranule cells, and can be taken as a strong indication thatal. 1992; Repressa et al. 1991). It is important to point out

that a small amount of sprouting of glutamatergic inputs there is no MF sprouting. Although it is possible that the g-irradiation treatment may affect the ability of residual MFsfrom the associational collaterals does not affect our conclu-

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FIG. 7. Amplitude and rise-time distributions for mEPSCs removed by g-irradiation. Amplitude (A) and 10–85% rise-time (C) distributions of the mEPSCs removed by g-irradiation-induced lesions were obtained by subtracting the distributionsfor g-irradiated tissue from the distributions for control tissue. Respective amplitude and 10–85% rise-time cumulativeprobability distributions are shown in B and D . These distributions nominally represent distribution of MF mEPSC amplitudeand rise time.

to express dynorphin, this is unlikely because the residual et al. (1994) have shown that both excitatory AMPA- andNMDA-receptor-mediated neurotransmission at non-MF syn-MF boutons in g-irradiated tissue appear to express dynor-

phin at control levels. These data are in agreement with apses are unchanged with respect to control. In addition, theyhave shown that both g-aminobutyric acid A (GABAA)- andprevious reports showing a drastic reduction in Timm stain-

ing in the stratum lucidum after g-irradiation (Repressa et GABAB-mediated neurotransmission are unaffected. Second,the irradiation treatment induces the lesion by disrupting theal. 1991). Timm staining reveals Zn2/ , which has been

shown to exist at elevated levels in the mossy fiber terminals. mitotic process of granule cells (Hicks and D’Amato 1966).Because most of the non-MF synapses are formed after the g-With respect to changes in nonglutamatergic neurotrans-

mission, we have observed an increase in cholinergic in- irradiation treatment, there can be no direct effect of the treat-ment on the synapses. Finally, previous work also has shownnervation of the g-irradiated hippocampus (data not shown),

as has been observed for colchicine-induced DG lesions (Re- that the only change in the dendritic morphology of CA3 pyra-midal cells after the g-irradiation lesion is the loss of the largepressa and Ben-Ari 1989). We determined that the increased

cholinergic innervation did not play a role in causing the thorny excrescences that normally receive the MF input (Gai-arsa et al. 1992; Repressa et al. 1991). It is not likely that theobserved changes in the mEPSC amplitude and rise-time

histograms because adding the cholinergic antagonist sco- loss of the thorny excrescences could, in and of itself, accountfor the observations in this study because the loss of the extrapolamine had no effect on mEPSCs recorded from g-irradi-

ated tissue (data not shown). membrane and channels within that membrane would onlyserve to increase input resistance and therefore increase theA possible nonspecific effect of the g-irradiation treatment

is that it could decrease the amplitude of non-MF mEPSCs. observed amplitude and decrease the observed rise time ofdistal synaptic signals.This is highly unlikely for at least three reasons. First, Gaiarsa

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sioned MFs (see figure legend for details of calculations) .The relative probability of an mEPSC of a given amplitudeor rise time arising from either the MF or non-MF synapsesis illustrated in Fig. 9, B and D .

Examination of these calculated histograms indicate that,on average, MF mEPSCs are larger and slower than non-MF mEPSCs although there is extensive overlap of the distri-butions for these two classes of synapse. The mean MFmEPSC peak amplitude is twice that of non-MF mEPSCs(24.6 vs. 12.2 pA corresponding to 307.5 vs. 152.5 pS).The median MF mEPSC amplitude is also twice that of thenon-MF mEPSC (17.5 vs. 9.0 pA corresponding to 218 vs.112 pS). The two amplitude distributions are significantlydifferent (P ! 0.001) using a one-tailed, large sample Kol-mogorov-Smirnov test (1-pA bin size) . The distributionsalso indicate that, on average, the MF mEPSCs are slowerrising than non-MF mEPSCs. The mean MF mEPSC 10–85% rise time is twice that of the non-MF mEPSCs (2.16vs. 1.26 ms). The median MF mEPSC 10–85% rise timeis also twice that of the non-MF mEPSC (1.8 vs. 0.97 ms).The two amplitude distributions are significantly different(P ! 0.001) using a one-tailed, large sample Kolmogorov-Smirnov test (0.1-ms bin size) .

The mean MF mEPSC amplitude reported above is largerthan that (16.1 pA) previously reported by Jonas et al.(1993). This difference is best explained by the fact that inthat report, mEPSCs where determined to be of MF originusing a 20–80% rise-time criteria ofõ0.8 ms. This approachto identify MF mEPSCs would exclude many MF mEPSCsthat have longer rise times, which can be very large in ampli-tude (see Fig. 8) .

As expected, the mEPSC amplitude histogram for non-MF synapses is very similar to published values for mEPSCsrecorded from CA1 pyramidal cells (Malgaroli and Tsien1992; Manabe et al. 1992). This was expected for two rea-FIG. 8. Examples of monophasic mEPSCs of large amplitude. Three

examples of large mEPSCs arising from putative MF synapses. A : 2 events sons. First, collaterals of the same CA3 pyramidal cell axonsdetected, larger measured at 229 pA and 10–85% rise time of 2.2 ms. B : provide input to both CA1 and CA3, and second, the mor-2 events detected, larger measured at 277 pA and 10–85% rise time of 5.5 phological structure of CA1 and CA3 pyramidal cells, whilems. C : 1 event detected, measured at 207 pA and 10–85% rise time of 5.5

not identical, is similar with respect to the overall electronicms. D : 1 event detected, measured at 100 pA and 10–85% rise time of 1.5ms. All 4 examples are from a recording with a 12-MV series resistance. locations of synapses, particularly for proximal synapses.

We found that the maximal non-MF mEPSC amplitude wasÇ30 pA, similar to values reported for recordings from areaCalculated MF and non-MF mEPSC amplitude and 10–CA1 pyramidal cells (Chavez-Noriega and Stevens 1994;85% rise-time distributionsMalgaroli and Tsien 1992; Manabe et al. 1992). Thus it isimpossible that the approximate 10-fold difference in maxi-We have taken advantage of the specificity of the g-irradi-

ation induced lesion to estimate the separate distributions of mal mEPSC amplitude observed between MF and non-MFmEPSCs is strictly due to the dendritic locations of the syn-mEPSC amplitudes and 10–85% rise times for the MF and

non-MF synapses. The MF mEPSCs are represented by the apses. Instead, large mEPSCs recorded from CA3 pyramidalcells are due to some special property of neurotransmissionmEPSCs that were subject to removal by the irradiation

treatment (see subtracted histograms of Fig. 7) . The non-MF at the MF synapse (see below).mEPSCs are represented by the mEPSCs that are observedfollowing the irradiation treatment (see g-irradiated histo- Properties of mossy fiber mEPSCsgrams in Fig. 6) . Unfortunately, these two histograms donot account for the 15% of the MF synapses that are not The apparent quantitative mismatch between an 85% re-

duction in MF synaptic input and the lack of a statisticallyremoved by the g-irradiation treatment. As a result, the sub-tracted histograms of Fig. 7 are 15% smaller than would be significant change in overall mEPSC frequency can be ex-

plained by the anatomy and biophysics of CA3 pyramidalexpected and the g-irradiated histograms of Fig. 6 containthe 15% of the mEPSCs missing from the histograms of Fig. cells and their inputs. Each CA3 pyramidal cell receives

Ç46 MF boutons (Amaral et al. 1990) and each bouton has7. Figure 9, A and C, illustrates what the mossy and non-MF mEPSC amplitude and 10–85% rise-time histograms Ç15 release sites (Chicurel and Harris 1992); thus each

CA3 pyramidal cell receives Ç700 MF release sites. Thiswould look like if they are corrected for the residual nonle-

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FIG. 9. Amplitude and 10–85% rise time distributions for MF- and non-MF-mediated mEPSCs. Distributions of mEPSCamplitude and 10–85% rise time were calculated by subtracting raw distributions from all 10 g-irradiated recordings fromraw distributions from all 10 control recordings. Difference distributions then were scaled by multiplying by 1.176 to correctfor MF mEPSCs that were not removed by g-irradiation. Individual non-MF distributions were calculated by subtracting15% of scaled MF distributions from raw g-irradiated distributions. This was to correct for those MFs that were not removedby g-irradiation. MF and non-MF mEPSC amplitude (A) and rise-time (C) distributions are significantly different (P !0.001, 1-tailed large sample Kolmogorov-Smirnov test using x 2 sampling distribution with 2 df) . Relative contribution ofboth MF and non-MF synapses to mEPSCs of a given amplitude or rise time are shown in B and D . MF mEPSCs (B , ●)are Ç80% of the mEPSCs recorded in 30-pA bin whereas non-MF mEPSCs comprise 20%. For mEPSC amplitudes ú45pA, ¢95% of the mEPSCs are of MF origin. MF mEPSCs (D , ●) are Ç80% of mEPSCs recorded in 3-ms bin whereasnon-MF mEPSCs comprise 20%. For mEPSC 10–85% rise times ú4.5 ms, ¢95% of the mEPSCs are of MF origin.

is in contrast to the estimated 12,000 release sites from the rise to the slowest rising mEPSCs. Because of its close elec-trotonic location, it has long been held that evoked MF syn-commissural /associational collaterals and the perforant path

(Amaral et al. 1990). This works out to be that õ4% of the aptic EPSCs should be of large amplitude and fast rise time,whereas evoked non-MF EPSCs should be smaller andtotal synaptic input to a typical CA3 pyramidal cell is of

MF origin. In light of the anatomically small contribution slower (Brown and Johnston 1983; Johnston and Brown1983; Rall et al. 1967). It is important to point out that theof the MFs to the total number of synapses on a CA3 cell,

one might also question why any effect of the g-irradiation mEPSCs we attribute to the MFs are not necessarily largeand slow because both large and fast, as well as, large andwas seen at all. Whereas synapse numbers favor the detection

of non-MF mEPSCs, the electrotonic filtering properties of slow mEPSCs are reduced by g-irradiation treatment. Thisobservation is supported by the lack of a correlation betweenthe dendrites favor the detection of MF mEPSCs by reducing

the contribution of the distal non-MF synaptic inputs to be- mEPSC amplitude and rise time when plotted against oneanother (data not shown). The lack of a significant shape-low detectable levels. The interaction between the number

of synapses for MFs and non-MFs and the differential elec- index correlation suggests that the contribution of electro-tonic filtering to variations in mEPSC size and shape istrotonic filtering leads to the observed relative contribution

by each of these two types of synapses to the overall level relatively minor. Due to the problems inherent in wholecell recording from large neurons of the CNS (e.g., accessof mEPSC activity.

An unexpected outcome of this study is that MFs give resistance and dendritic cable filtering), the reported abso-

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AMARAL, D. G., ISHIZUKA, N., AND CLAIBORNE, B. Neurons, numbers, andlute values for mEPSC rise times should be considered withthe hippocampal network. Prog. Brain Res. 83: 1–11, 1990.care. However, due to the comparative nature of our study,

AMARAL, D. G. AND WITTER, M. P. The three-dimensional organization ofthe validity of the conclusion that g-irradiation removes a the hippocampal formation: a review of anatomical data. Neurosciencepopulation of slower rising mEPSCs is unaffected by these 31: 571–591, 1989.

ANKRI, N., LEGENDRE, P., FABER, D. S., AND KORN, H. Automatic detectiontechnical issues.of spontaneous synaptic responses in central neurons. J. Neurosci. Meth-Another explanation for the presence of long MF riseods 52: 87–100, 1994.times is that our automated detection method cannot resolve

BAYER, S. A. AND ALTMAN, J. Radiation-induced interference with postnatalindividual mEPSCs that have overlapping rising phases, hippocampal cytogenesis in rats and its long-term effects on the acquisi-which it then would count as a single large slow event. This, tion of neurons and glia. J. Comp. Neurol. 163: 1–20, 1975a.

BAYER, S. A. AND ALTMAN, J. The effects of X-irradiation on the postna-however, cannot account for the data because we found thattally-forming granule cell populations in the olfactory bulb, hippocampus,the detection algorithm misses relatively few mEPSCs (57and cerebellum of the rat. Exp. Neurol. 48: 167–174, 1975b.missed, 1,341 detected per 60-s period) that we would have

BLACKSTAD, T. W. Commissural connections of the hippocampal region inincluded when inspected by eye with no bias toward missing the rat, with special reference to their mode of termination. J. Comp.overlapping events. Although the algorithm does fail to re- Neurol. 105: 417–537, 1956.

BLACKSTAD, T. W., BRINK, K., HEM, J., AND JEUNE, B. Distribution ofsolve some overlapping mEPSCs, when the data traces arehippocampal mossy fibers in the rat. An experimental study with silverinspected visually, there do exist many monophasic mEPSCsimpregnation methods. J. Comp. Neurol. 138: 433–450, 1970.with amplitudesú250 pA and/or rise timesú5 ms as shown

BLACKSTAD, T. W. AND KJAERHEIM, A. Special axo-dendritic synapses inin Fig. 8. the hippocampal cortex: electron and light microscopic studies on the

A possible presynaptic mechanism by which the MF syn- layer of mossy fibers. J. Comp. Neurol. 117: 133–146, 1961.BLANTON, M. G., LOTURCO, J. J., AND KRIEGSTEIN, A. R. Whole cell re-apse could give rise to the observed large and/or slow

cording from neurons in slices of reptilian and mammalian cerebral cor-mEPSCs (‘‘maxi’’-mEPSCs) could involve multiquantal re-tex. J. Neurosci. Methods 30: 203–210, 1989.lease from the multiple active zones of the presynaptic bou- BROWN, T. H. AND JOHNSTON, D. Voltage-clamp analysis of mossy fiber

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CARD, J. P. AND ENQUIST, L. W. The use of pseudorabies virus for definitionof synaptically linked population of neurons. In: Methods in MolecularGenetics , edited by K. W. Adolph. Orlando, FL: Academic, 1994, p.Summary363–382.

CASTILLO, P. E., WEISSKOPF, M. G., AND NICOLL, R. A. The role of Ca2/The major goal of this study was to test the hypothesischannels in hippocampal mossy fiber synaptic transmission and long-that the large mEPSCs observed in recordings from CA3term potentiation. Neuron 12: 261–269, 1994.

pyramidal cell arise from synapses made by the MFs of the CHAVEZ-NORIEGA, L. AND STEVENS, C. F. Increased transmitter release atdentate gyrus granule cells. We have shown that ¢95% of excitatory synapses produced by direct activation of adenylate cyclase

in rat hippocampal slices. J. Neurosci. 14: 310–317, 1994.mEPSCs with amplitudes ú45 pA are from the g-irradia-CHICUREL, M. E. AND HARRIS, K. M. Three-dimensional analysis of thetion-sensitive MF synapses. The data also suggest that 95%

structure and composition of CA3 branched dendritic spines and theirof the events with 10–85% rise times ú4.5 ms are from thesynaptic relationships with mossy fiber boutons in the rat hippocampus.MF synapses. These data provide evidence for yet another J. Comp. Neurol. 325: 169–182, 1992.

unique feature of neurotransmission at the MF synapse, spe- DESSI, F., REPRESSA, A., AND BEN-ARI, Y. Effects of neonatal gamma-raycifically that the MFs give rise to a population of unusually irradiation on rat hippocampus. II. Development of excitatory amino acid

binding sites. Neuroscience 42: 151–157, 1991.large and/or slow mEPSCs. Further work elucidating theGAIARSA, J. L., BEAUDOIN, M., AND BEN-ARI, Y. Effect of neonatal degran-mechanisms that give rise to these maxi-mEPSCs will deter-

ulation on the morphological development of rat CA3 pyramidal neurons:mine the relevance of these events. inductive role of mossy fibers on the formation of thorny excrescences.J. Comp. Neurol. 321: 612–625, 1992.

GAIARSA, J.-L., ZAGREAN, L., AND BEN-ARI, Y. Neonatal irradiation pre-We gratefully acknowledge the contribution of J. Cabarlo to the anatomicvents the formation of hippocampal mossy fibers and the epileptic actionportion of this investigation.of kainate on rat CA3 pyramidal neurons. J. Neurophysiol. 71: 204–215,This work was supported by National Institutes of Health Grants NS-1994.24288 and predoctoral fellowship MH-10474, Institut National de la Sante

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