comparison of the distribution of parvalbumin-immunoreactive and other synapses onto the somata of...

Comparison of the Distribution ofParvalbumin-Immunoreactive and OtherSynapses Onto the Somata of CallosalProjection Neurons in Mouse Visual

and Somatosensory Cortex

DAVID CZEIGER AND EDWARD L. WHITE*

Department of Morphology, Zlotowski Center for Neuroscience, 3-D Brain Imaging Group,Ben-Gurion University of the Negev, Beer Sheva, Israel

ABSTRACTThe distribution of synapsesmade by parvalbumin-immunoreactive (pv-ir) and nonimmu-

noreactive terminals was determined for the cell bodies of callosal projection neurons in thesomatosensory and visual areas of mouse cerebral cortex. Callosal neurons were labeled bythe retrograde transport of horseradish peroxidase applied to the contralateral hemisphere.The surface areas of somata belonging to callosal cells in somatosensory cortex ranged from230 to 243 µm2 in size and received roughly one-third of their synapses from pv-ir terminals.Visual cortex, in contrast, contained two populations of callosal cell bodies: relatively largeones ranging in size from 255 to 279 µm2 that received 3–9% of their synapses from pv-irterminals and smaller cell bodies that both in size (232–237 µm2) and in the proportion ofsynapses received from pv-ir terminals resemble the callosal cells examined in somatosensorycortex. That different functional areas of the cortex have populations of callosal cells similar insize, and displaying similar patterns of somatic synapses, supports the notion that a commonplan of synaptic connectivity characterizes different functional areas. Results in visual cortexindicate that functional areas contain, in addition, area-specific patterns of synapses. J. Comp.Neurol. 379:198–210, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: cerebral cortex; callosum; horseradish peroxidase; silver/gold intensification;

immunolabeling

In the somatosensory system, the corpus callosum isinvolved mainly with continuity of sensation across themidline (i.e., midline fusion; Innocenti, 1986; Manzoni etal., 1989) and presumably with bimanual function (Guille-mot et al., 1992). In the visual system, the corpus callosumfunctions in binocular vision, in depth perception, and inthe integration of the two visual hemifields (see review byInnocenti, 1986). Despite observations that some nonpyra-midal cells project into the corpus callosum in rat, cat, andmonkey (Innocenti and Fiore, 1976; Schwartz and Gold-man-Rakic, 1984; Peters et al., 1990), the corpus callosumin mouse is reported to be composed entirely of axonsbelonging to pyramidal neurons (see, e.g., discussion inCzeiger and White, 1993).It is generally accepted that a principal factor influenc-

ing the function of a neuron is the number and types ofsynapses impinging on it. It has been shown that differenttypes of projection cells receive different, characteristicpatterns of synapses onto their dendrites (see, e.g., White,

1989a). Farinas and DeFelipe (1991) have shown thatdifferent patterns of synapses characterize callosal vs.corticothalamic projection cells. Despite the fact that eachprimary sensory and motor area in all mammalian speciespossesses a strong callosal component, it is not knownwhether a particular synaptic pattern characterizes callo-sal neurons in different cortical areas. One main purposeof the present study is to compare synaptic patterns oncallosal neurons in two different cortical areas. Part of ourapproach includesdifferentiatingbetweenterminalspresynap-tic to callosal neurons by their content of parvalbumin.

Contract grant sponsor: Israel Science Foundation; Contract grantnumber: 618/93.*Correspondence to: Edward L. White, Department of Morphology,

Faculty of Health Sciences, Ben-Gurion University, P.O.B. 653, Beer Sheva,Israel. E-mail: [email protected] 5 April 1996; Revised 6 September 1996; Accepted 19 Septem-

ber 1996

THE JOURNAL OF COMPARATIVE NEUROLOGY 379:198–210 (1997)

r 1997 WILEY-LISS, INC.

Cell bodies of callosal neurons, like those of otherpyramidal cells in the cerebral cortex, receive only sym-metrical-type synapses from terminals that most likelycontain g-aminobutyric acid (GABA); see reviews by Feld-man, 1984; Keller, 1989). Some of the terminals that formsynapses onto pyramidal somata are immunoreactive forparvalbumin (pv-ir), a calcium binding protein. GABAer-gic terminals typically contain either parvalbumin oranother calcium binding protein such as calbindin. Onrare occasions, the terminals contain both of these calciumbinding proteins or neither of them (Celio, 1986, 1990;Hendry et al., 1989; Van Brederode et al., 1990; Hendryand Jones, 1991; Hogan and Berman, 1994; Wilson, 1994).Pv-ir cells have been implicated in the development ofneurological diseases such as epilepsy, ischemia, and Alz-heimer’s disease (Ferrer et al., 1992, 1993, 1994; Inagumaet al., 1992; Ribak, 1992; Adamas and Munoz, 1993;DeFelipe et al., 1993; Leifer and Kowall, 1993) and havebeen shown to have more intense inhibitory effects on cellbodies postsynaptic to them than do GABAergic cellscontaining calbindin (Kawaguchi and Kubota, 1993;Kawaguchi, 1995).

MATERIALS AND METHODS

Young adult, male CD/1 mice were anesthetized withsodium pentobarbitol (80 mg/kg), and horseradish peroxi-dase (HRP) was applied to the cut ends of callosal axons asdescribed in detail previously (White and Czeiger, 1991;Czeiger and White, 1993). Three days later, the mice wereanesthetized deeply with chloral hydrate and perfusedwith a fixative containing 4% paraformaldehyde, 0.3%glutaraldehyde, and 15% of a saturated picric acid solutionin 0.1 M phosphate buffer. Within 2 hours, the brains wereremoved from the skull. The advantage of this fixative isthat it preserves the antigenicity of the tissue for parvalbu-min, as well as the activity of the HRP, while preservingthe fine structure of the tissue for subsequent electronmicroscopic examination (Somogyi and Takagi, 1982).

Retrograde HRP transport and silverintensification

Immediately after removal from the skull, the visualand somatosensory areas contralateral to the injection sitewere sliced coronally with a vibratome at 50 µm thickness.The sections were collected in a 0.9% solution of NaCl andprocessed for HRP by using diaminobenzidine (DAB) asthe chromagen (see, e.g., Warr et al., 1982). Gold-substituted silver intensification was used to intensify theperoxidase reaction product (GSSP; Van den Pol, 1988) asfollows: Sections containing callosal cell bodies labeled bythe retrograde transport of HRP were washed in 0.1 Mphosphate buffer, left overnight in 2% thioglycolic aciddiluted in distilled water at 4°C to reduce the argyrophiliaof the tissue (Gallyas et al., 1982; Leranth and Pickel,1989), and washed in an isotonic solution of 2% sodiumacetate, pH 8.7. Then, the sections were transferred byusing a glass rod to freshly made developer, for 3–10minutes, that had been prepared by mixing equal volumesof an aqueous solution of 5% anhydrous sodium carbonatewith a solution containing 0.2% ammonium nitrate, 0.2%silver nitrate, 1% tungstosilicic acid, and 0.4% formalinfrom a saturated (37%) stock solution (Liposits et al.,1984). During the process, the sections were examinedwith a light microscope, and the development was stopped

when black precipitate appeared around the DAB, bytransferring the sections to 1% acetic acid for 10 minutes.The sections were transferred to 2% sodium acetate for 10minutes, 0.05% gold chloride for 15 minutes, 2% sodiumacetate for 5minutes, 3% sodium thiosulfate for 5minutes,and finally 2% sodium acetate for 10 minutes followed bysoaking in 0.1 M phosphate buffer.

Immunohistochemistry

Sections containing well-labeled callosal cell bodies werereacted for parvalbumin antigen. The sections were incu-bated for 2 hours in 10% sucrose dissolved in phosphatebuffer and then transferred to bottles containing 30%sucrose until they sank. The sections were then frozen/thawed by transfering the bottles to liquid nitrogen untilthe sucrose froze, then removing the bottles and allowingthem to reach room temperature (Somogyi and Takagi,1982; Zaborszky and Heimer, 1989). For the immunologi-cal reaction we used as primary antibody IgG1 monoclonalantibody against parvalbumin (Sigma, St. Louis, MO) and,for rest of the reaction, avidin-biotinylated HRPmacromo-lecular complex (Vectastain Elite ABC Kit; Hsu et al.,1981).

Electron microscopy preparationand analysis

The sections were then osmicated in aqueous 1% os-mium for 30 minutes, dehydrated, and embedded in epon-araldite for electron microscopy (EM). During dehydra-tion, the sections were placed for 2 hours in 1% uranylacetate dissolved in 70% methanol. Each section wasexamined with the light microscope to select labeledcallosal cells that occurred just under the surface of thesection in order to select tissue for EM examination thatcontained immunolabeled terminals throughout its fullthickness. This was verified by subsequent EM examina-tion as described below. Selected callosal cell bodies wereserially thin sectioned and mounted onto formvar-coated,copper-slotted grids for examination with the electronmicroscope.Callosal cell bodies labeled with HRP and silver/gold

deposits were followed in serial thin sections in order tocompare the proportion of synapses made onto them bypv-ir-labeled terminals vs. nonlabeled terminals. Examina-tion was limited to only those callosal cell bodies that wereentirely, or nearly so, included within the series of sections.This was indicated by the fact that in every case the seriesthrough the cell body began and ended with sections inwhich the cell membrane was tangentially sectioned, enface, such that synapses could not be identified. Specialcare was taken to exclude the possibility that low penetra-tion of the immunoreagents affected the results. Theexistence of pv-ir terminals was confirmed in every thinsection in regions adjacent to the serially sectioned callosalcell body. The surface areas of the cell bodies were calcu-lated from measurements of the largest profiles (Petersand Harriman, 1992) by using a formula for prolatespheroids, the shape most closely approximating the callo-sal cell bodies. The surfacemembrane was considered to becontinuous and to pass unbroken across the origins ofprocesses emitted from the cell body. Each cell bodyexamined contained a proportion of cell membrane thatwas sectioned tangentially, complicating the identificationof synapses. For this reason, values for the number ofsynapses per surface area of cell body were obtained by

PARVALBUMIN SYNAPSES ONTO CALLOSAL NEURONS 199

dividing the surface area of membrane sectioned in thenormal plane by the numbers of synapses observed inthese regions. In several instances, the complement ofparvalbumin-positive axons and terminals surroundingcallosal cell bodies was reconstructed by using a computer-ized 3-D reconstruction system to determine the three-dimensional relationsip of pv-ir axonal ramifications in theimmediate vicinity of the postsynaptic cell bodies.

Controls

Performing the silver/gold reaction on sections fromnonoperated brains showed no labeling, indicating thatonly the injected HRP deposits were labeled with thesilver/gold reaction. In addition, no immunological label-ing was observed when the primary anti-pv antibody wasomitted, indicating that the immunological labeling ob-served was of pv-ir elements. Experiments were carriedout in accordance with NIH guidelines for animal care asprovided in publication no. 86-23, revised 1987.

RESULTS

Light microscopy

Parvalbumin immunoreactivity. In both visual andsomatosensory areas of cortex, pv-ir cell bodies and pro-cesses emitted from them were stained a homogeneousdark brown color (Fig. 1A). Pv-ir cell labeling was uniformover the sections, and there were no partially labeled cellsthat might have made it difficult to recognize pv-ir cellbodies. Pv-ir cells were morphologically heterogeneous,ranging in size from 9 to 16 µm in diameter, and displayeda wide range of shapes, the most common being sphericaland multipolar. Pv-ir cell bodies were distributed in allcortical layers; they were particularly numerous in thedeeper part of layer II/III in somatosensory cortex and inlayers IV and VI but were uncommon in layer I in bothareas.Callosal cell labeling with HRP. Neurons labeled by

the retrograde transport of HRP were particularly numer-ous in the border regions of areas 1 and 40 with area 3 andin the border regions of area 17 with areas 18a and 18b.Light brown HRP reaction product and dark (GSSP)granules filled the cell bodies and at least the proximalportions of the apical and basal dendrites of callosal cells(Fig. 1A). Cells thus labeled were identified as pyramidalneurons on the basis of the shapes of their cell bodies anddendritic trees. In both visual and somatosensory areas,callosal cells were found primarily in layers II/III and V,with some cells also occurring in layer IV. GSSP-labeledcallosal fibers were observed in both hemispheres and inthe corpus callosum.In all layers, but mainly in layers II/III, V, and VI, dark

brown granules were observed both in the neuropil andsituated around unlabeled cell bodies (Fig. 1B). Thesegranules could be the axon terminals of callosal cellslabeled with HRP and gold substitution or, alternatively,terminals belonging to pv-ir cells. Although the identity ofthe granules is uncertain at the light microscopic level,subsequent electron microscopic examination suggestedthat the neuropil contained both types of terminal but thatthose abutting cell bodies were only pv-ir terminals. Alsoobserved with the electron microscope were ‘‘nests’’ of pv-irterminals that contacted both unlabeled pyramidal neu-rons and callosal cells such as that shown in Figure 1C.

Electron microscopy

Visual cortex. In thin sections, the cytoplasm of pv-ircell bodies was heavily labeled with a dark reactionproduct (Fig. 2A). The heavy labeling obscured the cell’sorganelles, and it was difficult to identify the type ofsynapses made with the cell as symmetrical or asymmetri-cal (cf. Colonnier, 1968). None of the terminals presynapticto pv-ir cell bodies was labeled with any of the labelingprocedures used in this study. The nuclei of pv-ir cells filledmost of the cell volume. Pv-ir dendrites were occasionallyobserved; these dendrites, uniformly labeled with darkreaction product, lacked spines and were postsynaptic atboth symmetrical and asymmetrical synapses. Pv-ir termi-nalsmade only symmetrical synapses with dendritic shaftsand cell bodies; compare a symmetrical synapse of anunlabeled terminal (Fig. 2B) with that of pv-ir labeledterminals (Figs. 2C, 3).Callosal cell bodies labeled by retrogradely transported

HRP and gold substitution were identified with the lightmicroscope and then examined in serial thin sections todetermine the distribution of the different types of syn-apses onto them. As seen with the electron microscope,labeling of callosal cell bodies and their terminals (Figs. 4and 5) differed from pv-ir labeling of cell bodies andterminals in that the pv-ir label was darker and moreuniform than the GSSP label (cf. Figs. 2A,C, 3 and Figs. 4,5). In all instances, examination of serial thin sectionsconfirmed that pv-ir labeled axon terminals consistentlyformed synapses of the symmetrical type; no dense band ofpostsynaptic material was adherent to the cytoplasmicsurface of the postsynaptic membrane (cf. Fig. 5). Termi-nals of both GSSP-labeled local axon collaterals of callosalneurons and degenerating axon terminals presumed tooriginate from the contralateral hemisphere made onlyasymmetrical synapses, primarily onto spines (see, e.g.,Fig. 5; cf. Czeiger and White, 1993). No callosal axonterminals were observed to form synapses onto callosal orother types of cell bodies. Callosal cell bodies were followedin serial thin sections, and all terminals that synapsedonto them were counted and identified as pv-ir-labeled or-unlabeled, symmetrical or asymmetrical.In the visual areas, seven callosal cells were examined in

serial thin sections, and two subpopulations of callosalneurons were identified: Three cell bodies had 60–69symmetrical synapses each and received no asymmetricalsynapses; 3.3–8.7% of the terminals presynaptic to thesecell bodies were pv-ir. These cell bodies had relatively largesurface areas ranging from 255 to 279 µm2. Four other cellbodies had 57–95 symmetrical synapses each, of which26–35% were pv-ir. These cells had relatively smallersurface areas ranging from 232 to 237 µm2. For bothpopulations of callosal cells, synapses of pv-ir terminalswere not observed to be preferentially located with respectto any particular region of the cell body. Values for thenumber of synapses per somatic surface area were calcu-lated for each cell (see Table 1).Somatosensory cortex. The appearances and distribu-

tion of pv-ir and callosal elements in the somatosensoryareas were identical to those described above for the visualcortex. Four somata of callosal cells in somatosensorycortex were examined in serial thin sections: These cellbodies, whose surface areas ranged from 230 to 243 µm2,formed from 48 to 92 symmetrical synapses each, of whichone-third to one-half were pv-ir (Table 1). Thus, the size

200 D. CZEIGER AND E.L. WHITE

Fig. 1. A,B: Light micrographs obtained by video microscopythat show a parvalbumin immunoreactive (PV) neuron and, atright, a callosal projection cell (C) labeled by the retrograde trans-port of horseradish peroxidase intensified with silver/gold. B: Lightmicrograph that shows three unlabeled neurons (NL) contacted byaxon terminals (arrows) presumed to be parvalbumin-immunoreac-tive (pv-ir). C: Three-dimensional reconstruction depicting thecentral relationship of a callosal cell body in somatosensory cortexto a surrounding ‘‘nest’’ of pv-ir axons that in thin sections areobserved to form synapses onto the cell body. Scale bars 5 10 µm inA, B.


Fig. 2. Electron micrographs that show a pv-ir cell A; a symmetri-cal synapse onto an unlabeled cell body made by an unlabeled axonterminal (t) B; and a symmetrical synapse between a pv-ir axonterminal (p) and a callosal cell body (cl) in somatosensory cortex (C). In

all instances, synapse type was confirmed by examination of serialthin sections. Arrow in C indicates silver-intensified/gold-substitutedhorseradish peroxidase (HRP) reaction product in the cell cytoplasm.Scale bars 5 1.5 µm inA, 0.2 µm in B, C.


and synaptic patterns of callosal cell bodies in somatosen-sory cortex resembled those of one population identified invisual cortex.Statistical analyses using Mann-Whitney tests showed

no significant difference among the sizes of neurons withineach of the three populations examined (large and small invisual cortex and small in somatosensory cortex), norbetween the small populations of the somatosensory andvisual cortices. The analysis showed that the small celltypes in both somatosensory and visual cortices differedsignificantly in size (P , 0.05) from the large cells in visualcortex. In addition, the small cell bodies in both corticalareas received significantly greater proportions of theirsynapses from pv-ir terminals (P , 0.05).In both somatosensory and visual cortices, unlabeled

(noncallosal) pyramidal cells were observed that werepostsynaptic at variable proportions to pv-ir terminals:Some of these neurons received many synapses from pv-irterminals on each thin-sectioned profile (Fig. 6); otherunlabeled pyramids received no synapses on any of theprofiles within the series examined.Three-dimensional reconstructions showed that pv-ir

axonal ramifications surrounding small callosal cell bodiesin both the visual and the somatosensory areas formed‘‘nests’’ or ‘‘baskets’’ around the postsynaptic cell bodies(Fig. 1C), an organization reminiscent of that reported for

the axon terminals of basket cells in higher species (Marin-Padilla and Stibitz, 1974; and see discussions in Fairen etal., 1984;White, 1989b).Within the limits of the reconstruc-tions, it was observed that the pv-ir terminals originatedfrom three to five axonal branches. A multiple axonalorigin for the axonal baskets around pyramidal cells inhigher species has been noted previously (Marin-Padillaand Stibitz, 1974).

DISCUSSION

Pyramidal cells in somatosensory cortical areas 1 and40, and in visual areas 17/18a and 17/18b, were labeled bythe retrograde transport of HRP that was injected intosevered callosal fibers in the contralateral hemisphere.HRP reaction product was intensified with silver/gold,prior to reacting the tissue with parvalbumin antibody.The number and spatial distribution of synapses with thelabeled callosal cells made by pv-ir and nonreactive termi-nals were assessed in serial thin sections. Callosal cells invisual cortex were of two types, larger cells having rela-tively few synapses from pv-ir terminals and smaller cellsthat received roughly one-third of their synapses frompv-ir terminals. In both size and synaptic patterns, thecallosal cells in somatosensory cortex resemble the smallpopulation in visual cortex.

Fig. 3. Electron micrograph that shows a pv-ir axon terminal (p) forming a symmetrical synapse ontoa callosal cell body (cl) from visual cortex. Arrow indicates silver-intensified/gold-substituted HRPreaction product in the cell cytoplasm. Scale bar 5 0.2 µm.


Fig. 4. Electron micrograph of a thin section through the cell body of a callosal neuron labeled by theretrograde transport of HRP. Gold particles associated with the HRP reaction product are particularlyprominent in the dendrite directed towards the upper left. Scale bar 5 1.0 µm.

Labeling methods

A disadvantage of HRP retrograde labeling is that theenzyme is transported both retrogradely and antero-gradely so that the target site contains not only HRP-labeled neuronal cell bodies and their local (intrinsic)axonal ramifications but also extrinsic terminals belong-ing to axons originating at the injection site. In the presentstudy, axons originating at the injection site were lesioned,and their degenerating terminals were easily distin-guished from terminals of local origin. Another potentialsource for the misidentification of labeled elements is thecombination of HRP intraaxonal transport with ap-proaches to immunolabeling that employ HRP to visualizethe antigen. The possibility of differentiating neuronslabeled by transported HRP from those labeled by HRPused as part of an immunological procedure has been

reported previously (e.g., Zaborszky and Leranth, 1985;Farinas and DeFelipe, 1991).To avoid confusion between pv-ir terminals and termi-

nals labeled by the retrograde transport of HRP, a proce-dure was employed in the present study whereby goldparticles were used to label selectively all elements contain-ing retrogradely transported HRP (GSSP; Van den Pol,1988; Leranth and Pickel, 1989). Labeling with gold waseffected before immunoreacting the tissue for parvalbu-min. Thus, callosal cell bodies and local axon terminalswere labeled with HRP and with gold particles, whereaspv-ir cell bodies and terminals were labeled only withHRP.This approach has been applied previously to differentiateneuronal processes labeled, in a single specimen, by reac-tion with two different antibodies (Gallyas et al., 1982;Liposits et al., 1984, 1985; Gorcs et al., 1986; Leranth andPickel, 1989).Finally, the approach used in which retrograde HRP

labeling was combined with immunolabeling suffers fromthe limitation that careful selection procedures (see Mate-rials and Methods) must be followed to ascertain that thetissue examined contains both labels. A balance must bestruck between the limited penetration of immunore-agents within thick slices of tissue and adequate HRPlabeling. We overcame this difficulty by 1) selecting forelectron microscopic examination only relatively superfi-cial regions of the sections showing good immunostainingand 2) employing silver-gold enhancement of the retro-gradely transported HRP.

Distribution of callosal and pv-ir neurons

The presence of large numbers of callosal projectionneurons in somatosensory areas 1 and 40, and in visualareas 17/18a and 17/18b (see, e.g., Yorke and Caviness,1975), is consistent with previous findings in the mouseand in other species (Yorke and Caviness, 1975; Innocenti,1986; Frost and Moy, 1989; Malach, 1989). Axon terminalsof callosal cells made only asymmetrical synapses, hasbeen reported for the somatosensory and motor cortices ofthe mouse (Porter and White, 1986; White and Czeiger,1991), cat (Jones and Powell, 1970a), and monkey (Sloperand Powell, 1979); the visual cortex of the mouse (Czeigerand White, 1993), rat (Lund and Lund, 1970), and cat(Voigt et al., 1988); the auditory cortex of the rat (Cipolloniand Peters, 1983); and the motor cortex of the monkey(Sloper and Powell, 1979). In the present study, all callosalcells were identified as pyramidal neurons; no nonpyrami-dal neurons were observed to be labeled by the retrogradetransport of HRP. In contrast, nonpyramidal callosal neu-rons have been identified in the visual cortex both of therat, where they may form symmetrical synapses (Hughesand Peters, 1990; Martınez-Garcıa et al., 1994), and of thecat (Innocenti and Fiore, 1976; Hornung and Garey, 1980,1981; Innocenti, 1986; Buhl and Singer, 1989; Peters et al.,1990) and also in the rat somatosensory cortex (Gonchar etal., 1995).Pv-ir cells in the present study were observed primarily

in layer IV of somatosensory cortex and in layer II/III ofvisual cortex, results in accordance with previous findingsin the mouse (Del Rio et al., 1994). In other species pv-irterminals in visual cortex are most prominent in layer IV(rat; Celio, 1990; Ren et al., 1992; cat; Hogan and Berman,1994; Alcantara and Ferrer, 1994; monkey; Hendry et al.,1989; Blumcke et al., 1990; Van Brederode et al., 1990).

Fig. 5. Electron micrograph that shows an asymmetrical synapsebetween a callosal (cl) axon terminal of intrinsic origin and anunlabeled dendritic spine (S). Scale bar 5 0.2 µm.

TABLE 1. Surface Area and Synaptic Connectivities of Callosal ProjectionCell Bodies

CellSurface

area (µm2)Synapses/10 µm2*

No. ofsynapses

Percent-age pv-ir

Visual areas 17/18a and 17/18b1 255 2.35 60 6.62 279 2.36 60 3.33 270 2.76 69 8.7

4 232 2.76 60 355 237 4.11 93 266 235 4.32 95 287 232 2.61 57 26

Somatosensory areas 1 and 401 242 2.73 64 482 243 3.96 88 383 238 2.23 48 274 230 4.31 92 34

*Values calculated by dividing the surface area of normal sectioned cell membrane bythe number of synapses (see Materials and Methods).


Fig. 6. Electron micrograph of a thin section through an unlabeled neuron that is abutted bynumerous pv-ir axon terminals (arrows); cf. Figure 1B,C. Scale bar 5 1.5 µm.


Size of callosal cell bodies

Serial thin sections through four callosal cells fromsomatosensory cortex and seven callosal cells from visualcortex were examined to determine the surface areas of thecallosal cell bodies and the distribution of synapses ontothem. Results showed that callosal cell bodies in somatosen-sory cortex and one group of callosal cells in visual cortexwere significantly smaller than a second group of callosalcell bodies in visual cortex. These findings are interpretedto mean that callosal cells in the visual area fall into twosubpopulations; additional support for this conclusion isprovided by differences in the synaptic patterns on the cellbodies, as discussed below.Callosal neurons examined in this work are consider-

ably smaller than pyramidal cells described in previouspapers. For example, callosal cell bodies in the visualcortex of the rat average 558 µm2 in size (Peters andHarriman, 1992), whereas those in visual cortex of the catrange from 1,010 to 1,417 µm2 (Farinas and DeFelipe,1991). Differences in the sizes of pyramidal cells in differ-ent species are not surprising when taking into consider-ation that the cortices of smaller brains tend to be com-posed of smaller neurons (White, 1989b).

Synapses onto callosal cell bodies

All the synapses on the surface of the callosal cell bodieswere symmetrical, which is in agreement with previousresults from a variety of species (Colonnier, 1968; Jonesand Powell, 1970b; Peters, Kaiserman-Abramof, 1970;LeVay, 1973; Davis and Sterling, 1979; Sloper et al., 1979;Winfield et al., 1981; DeGroot, 1984; Peters and Kara,1985; Peters and Harriman, 1990; Farinas and DeFelipe,1991; Liu et al., 1991; DeFelipe and Farinas, 1992; Tiggeset al., 1992; White et al., 1994). The concentration ofsomatic synapses is considered to be an important factor indetermining the physiological responses of the postsynap-tic neuron (see discussions in Farinas and DeFelipe, 1991;White et al., 1994).An accurate assessment of the distribu-tion of synapses on cell bodies can be obtained by recon-structing the cell body from unbroken series of thinsections and plotting the locations of synapses onto thesurface of the soma (see, e.g., Davis and Sterling, 1979;White and Rock, 1980; White et al., 1993). In the presentstudy, examination of entire callosal cell bodies in serialthin sections showed them to form roughly two to foursynapses per 10 µm2 of surface area. Clearly, this value isan underestimate because of the inability to observesynapses on tangentially sectioned regions of cell mem-brane. For example, White and Rock (1980) estimated that16% of the somatic synapses onto the reconstruction of aspiny stellate cell in mouse somatosensory cortex were notobserved because they occurred in tangentially sectionedregions of the cell membrane.The value of 4 synapses/10 µm2 of surface area observed

for some callosal neurons is among the highest reported forpyramidal cells in the cortex. For example, in layer III ofcat visual cortex, callosal cell bodies form 1.5–3.1 syn-apses/10 µm2 of surface area, whereas corticothalamiccells form 0.7–1.4 synapses/10 µm2 (Farinas and DeFelipe,1991). In layer IV of the cat, pyramidal cells whoseprojection type was not identified form 1.1 synapses/10µm2 (Davis and Sterling, 1979). In the visual cortex of therat, layer III pyramidal cell bodies form 1.1 synapses/10µm2 (Peters and Harriman, 1992), and Betz cells in the cat

and monkey form, respectively, 1.3 and 0.4 synapses/10µm2 (Kaiserman-Abramof and Peters, 1972; Tigges et al.,1992). Finally, layer V pyramidal cells in the somatosen-sory cortex of rat form 1.7–3.1 synapses/10 µm2 of surfacearea (White et al., 1994). Differences in the concentrationof synapses onto pyramidal neurons are presumably re-lated to species and areal differences.Synapses onto the cell bodies of cortical pyramids have

been shown to be GABAergic (Ribak, 1978; Peters andProskauer, 1980; Freund et al., 1983; Hendry et al., 1983;Houser et al., 1984; DeFelipe et al., 1986; Somogyi andSoltesz, 1986; Farinas and DeFelipe, 1991; Berman et al.,1992; Peters and Harriman, 1992). GABAergic terminalsusually contain, in addition, a calcium binding protein:typically parvalbumin or calbindin. Rarely, they containboth proteins or neither of them (Hendry et al., 1989; Celio,1990; Akil and Lewis, 1992; Ren et al., 1992). Somepyramidal cell bodies receive numerous pv-ir terminals,but others do not (see, e.g., Van Brederode et al., 1990;Williams et al., 1990; Akil and Lewis, 1992; Ren et al.,1992; Ribak, 1992).Correlations have made between the distribution of

synapses made by pv-ir terminals and other characteris-tics of pyramidal neurons. For instance, pyramidal cells inmonkey motor cortex that contain nonphosphorylatedneurofilament protein (NFP) tend also to have on their cellbodies many synapses from pv-ir terminals (Akil andLewis, 1992). The same is true for pyramidal cells inhuman temporal cortex that contain SmI32 (Del Rıo andDeFelipe, 1994). Pyramidal neurons in specific layers ofthe hippocampus of the monkey have been shown toreceive characteristic proportions of synapses made bypv-ir terminals (Ribak et al., 1993). Results of the presentstudy indicate that neurons in cerebral cortex also can bedifferentiated on the basis of the distribution onto them ofsynapses made by pv-ir terminals.Pv-ir terminals have been reported to contact callosal

cell bodies in the prefrontal cortex of the monkey (Williamset al., 1992). In the present study, we observed thatrelatively large callosal cell bodies in visual cortex form alower proportion of their synapses with pv-ir terminals (bya factor of approximately 7) than do smaller callosal cellbodies in the visual and somatosensory areas. Thus, thevisual cortex of the mouse contains two subpopulations ofcallosal neurons that can be differentiated on the basis ofthe size of their cell bodies and on the specific pattern ofsynapses that each forms with pv-ir terminals.Quantitative data regarding the proportion of GABAer-

gic terminals in the neuropil that are pv-ir are notavailable, but information on the proportion of GABAergicneurons that are pv-ir does exist. Considering values forthe proportion of GABAergic/pv-ir neurons vs. GABAergic/pv-ir-negative neurons might provide insight into theproportion of GABAergic/pv-ir terminals in the neuropil.In the somatosensory cortex of the rat, 50% of the GABAer-gic cells in layer III, the layer containing the callosal cellsexamined in the present study, are pv-ir (Ren et al., 1992).In the visual cortex of the monkey, the proportion ofGABAergic cells in layer III that are pv-ir is also about50% (Van Brederode et al., 1990). Except for one instance(see Table 1), callosal cell bodies examined in the presentstudy received one-third or far fewer of their synapsesfrom pv-ir terminals. This suggests that callosal cells inmouse typically receive a considerably lower proportion ofsynapses from GABAergic/pv-ir terminals than would be


expected from the proportion of GABAergic/pv-ir neuronsin the neuropil. Such a result could be associated with arelative paucity of GABAergic/pv-ir terminals from therebeing fewer or shorter parent axons, or fewer terminalsper axon of this type, compared to GABAergic/pv-ir-negative axons.Alternatively, itmight also be thatGABAer-gic/pv-ir terminals synapse selectively onto elements otherthan the cell bodies of callosal projection cells. Support forthe notion that pv-ir terminals tend to synapse at greaterfrequency with some postsynaptic elements than withothers is provided by the observation that some noncallo-sal, pyramidal cell bodies receive many more synapsesfrom pv-ir terminals than do the callosal cells examined inthis study.

Generalizations of synaptic organization

Based on data derived mainly from studies of mousesomatosensory cortex, but also on considerable data fromother species and other areas of cortex (White, 1989c), a setof organizational rules has been proposed for the cerebralcortex. The search for connectional rules that apply gener-ally is important because it makes it possible to developbroad functional interpretations of data from differentspecies and disciplines. The rules as thus far constitutedrelate mainly to the issue of specificity of synaptic connec-tions. For instance, evidence from a variety of corticalareas has been judged to support the contention thataxonal pathways of both extrinsic and intrinsic originprovide, and their postsynaptic elements receive, specific,quantitatively different patterns of synapses. The issue ofwhether similar targets in different functional areas re-ceive similar synaptic patterns has so far received littleattention. In part, this is due to the difficulty of arriving ata clear definition for neuronal types that would allowmeaningful comparisons to be made across disparatefunctional areas. The present study represents an initialeffort to explore the issue of synaptic patterns onto ‘‘simi-lar’’ neurons. The emphasis of this study is on the synapticconnectivity of callosal projection cells; these neuronsdisplay similar morphologies and can be considered to playsimilar functional roles in the sense that they are involvedsignificantly in processes requiring interhemispheric com-munication.Results showing that groups of callosal cells in visual

and somatosensory cortices display similar synaptic pat-terns suggest that a common plan of synaptic connectivitymight exist for callosal neurons. In contrast, the discoveryof a group of callosal neurons in visual cortex having adifferent pattern of synaptic connectivity with regard topv-ir terminals serves as an important caveat regardingthe drawing of generalizations about synaptic connectivityin different functional areas. Consistent with this notionare conclusions from a recent study in which differences inparvalbumin staining in different auditory areas of themonkey were interpreted to reflect differential innervationpatterns from the medial geniculate (Jones et al., 1995).For callosal functions, the implication is that the coordina-tion of interhemispheric communication and processingmay be mediated in different functional areas by specificsynaptic arrangements.

ACKNOWLEDGEMENTS

The authors acknowledge the expert technical assis-tance of Ms. Liza Weinfeld and the care and dedication to

our work of Ms. Rachel Ben Ishai. We thank Dr. William F.Silverman for his advice with immunological procedures.We are grateful to Martin and Rena Blackman, and to Dr.Avishai Braverman, for their generous support of ourefforts. Funds were provided by Israel Science Foundationgrant 618/93 to E.L.W.

LITERATURE CITED

Adamas, L.A., and D.G. Munoz (1993) Differential incorporation of pro-cesses derived from different classes of neurons into senile plaques inAlzheimer’s disease. Acta Neuropathol. (Berlin) 86:365–370.

Akil, M., and D.A. Lewis (1992) Differential distribution of parvalbumin-immunoreactive pericellular clusters of terminal boutons in developingand adult monkey neocortex. Exp. Neurol. 115:239–249.

Alcantara, S., and I. Ferrer (1994) Postnatal development of parvalbuminimmunoreactivity in the cerebral cortex of the cat. J. Comp. Neurol.348:133–149.

Berman, N.J., R.J. Douglas, and K.A.C. Martin (1992) GABA-mediatedinhibition in the neural networks of visual cortex. Progr. Brain Res.90:443–476.

Blumcke, I., P.R. Hof, J.H. Morrison, and M.R. Celio (1990) Distribution ofparvalbumin immunoreactivity in the visual cortex of old world mon-keys and humans. J. Comp. Neurol. 301:417–432.

Buhl, E.H., and W. Singer (1989) The callosal projection in cat visual cortexas revealed by a combination of retrograde tracing and intracellularinjection. Exp. Brain Res. 75:470–476.

Celio, M.R. (1986) Parvalbumin in the most gamma-aminobutyric acid-containing neurons of the rat cerebral cortex. Science 231:995–997.

Celio, M.R. (1990) Calbindin D-28 and parvalbumin in the rat nervoussystem. Neuroscience 35:375–475.

Cipolloni, P.B., andA. Peters (1983) The termination of callosal fibres in theauditory cortex of the rat. A combined Golgi-electron microscope anddegeneration study. J. Neurocytol. 12:713–726.

Colonnier, M. (1968) Synaptic patterns on different cell types in thedifferent laminae of the cat visual cortex. An electron microscope study.Brain Res. 9:268–287.

Czeiger, D., and E.L. White (1993) Synapses of extrinsic and intrinsic originmade by callosal projection neurons in mouse visual cortex. J. Comp.Neurol. 330:502–513.

Davis, T.L., and P. Sterling (1979) Microcircuitry of cat visual cortex:Classification of neurons in layer IV of area 17, and identification of thepatterns of lateral geniculate input. J. Comp. Neurol. 188:599–628.

DeFelipe, J., and I. Farinas (1992) The pyramidal neuron of the cerebralcortex: Morphological and chemical characteristics of the synapticinputs. Progr. Neurobiol. 39:563–607.

DeFelipe, J., S.H.C. Hendry, and E.G. Jones (1986) A correlative electronmicroscopic study of basket cells and large GABAergic neurons in themonkey sensory-motor cortex. Neuroscience 17:991–1009.

DeFelipe, J., R. Garcıa Sola, P. Marco, M.R. Del Rıo, P. Pulido, and S.Ramon y Cajal (1993) Selective changes in the microorganization of thehuman epileptogenic neocortex revealed by parvalbumin immunoreac-tivity. Cerebral Cortex 3:39–48.

DeGroot, D.M.G. (1984) Improvements of the serial section method inrelation to the estimation of the numerical density of complex-shapedsynapses. In A. Reith and T.M. Mayhew (eds): Stereology in ElectronMicroscopy. NewYork: Hemisphere/McGraw-Hill.

Del Rıo, M.R., and J. DeFelipe (1994) A study of SMI 32-stained pyramidalcells, parvalbumin-immunoreactive chandelier cells, and presumptivethalamocortical axons in the human temporal neocortex. J. Comp.Neurol. 342:389–408.

Del Rıo, J.A., L. De Lecea, I. Ferrer, and E. Soriano (1994) The developmentof parvalbumin-immunoreactivity in the neocortex of the mouse. Dev.Brain Res. 81:247–259.

Fairen, A., J. DeFelipe, and J. Regidor (1984) Nonpyramidal neurons:General account. In A. Peters and E.G. Jones (eds): Cerebral Cortex.Vol. 1. Cellular Components of the Cerebral Cortex. New York: PlenumPress, pp. 201–253.

Farinas, I., and J. DeFelipe (1991) Patterns of synaptic input on corticocor-tical and corticothalamic cells in the cat visual cortex. I. The cell body. J.Comp. Neurol. 304:53–69.

Feldman, M.L. (1984) Morphology of the neocortical pyramidal neuron. InA. Peters, and E.G. Jones (eds): Cerebral Cortex. Vol. 1. Cellular


Components of the Cerebral Cortex. New York: Plenum Press, pp.123–200.

Ferrer, I.B., M. Pineda, M. Tallada, B. Oliver, A. Russi, R. Oller, R. Noboa,M.J. Zuyar, and S. Alcantara (1992) Abnormal local-circuit neurons inthe epilepsia partialis continua associated with focal cortical dysplasia.Acta Neuropathol. (Berlin) 83:647–652.

Ferrer, I.B., M.J. Zuyar, R. Rivera, M. Soria, A. Vidal, and R. Cassas (1993)Parvalbumin-immunoreactive dystrophic neurites and aberrant sproutsin the cerebral cortex of patients with Alzheimer’s disease. Neurosci.Lett. 158:163–166.

Ferrer, I.B., Oliver, A. Russi, R. Casas, and R. Rivera (1994) Parvalbuminand calbindin-D28k immunocytochemistry in human neocortical epilep-tic foci. J. Neurol. Sci. 123:18–25.

Freund, T.F., K.A.C. Martin, A.D. Smith, and P. Somogyi (1983) Glutamatedecarboxylase-immunoreactive terminals of Golgi-impregnated axoax-onic cells and of presumed basket cells in synaptic contact withpyramidal neurons of the cat’s visual cortex. J. Comp. Neurol. 221:263–278.

Frost, D.O., and Y.P. Moy (1989) Effects of dark rearing on the developmentof visual callosal connections. Exp. Brain Res. 78:203–213.

Gallyas, F., T. Gorcs, and I. Merchenthalar (1982) High-grade intensifica-tion of the end-product of the diaminobenzidine reaction for peroxidasehistochemistry. J. Histochem. Cytochem. 30:183–184.

Gorcs, T.J., C. Leranth, and N.J. MacLusky (1986) The use of gold-substituted silver-intensified diaminobenzidine (DAB) and non-intensi-fied DAB for simultaneous electron microscopic immunoperoxidaelabeling of tyrosine hydroxylase and glutamic acid decarboxylaseimmunoreaxtivity in the rat medial preoptic area. J. Histochem.Cytochem. 34:1439–1447.

Gonchar, Y.A., P.B. Johnson, and R.J. Weinberg (1995) GABA-immunoposi-tive neurons in rat neocortex with contralateral projections to S-I. BrainRes. 697:27–34.

Guillemot, J.-P., L. Richer, M. Ptito, M. Guilbert, and F. Lepore (1992)Somatosensory receptive field properties of corpus callosum fibres inthe raccoon. J. Comp. Neurol. 321:124–132.

Hendry, S.H.C., and E.G. Jones (1991) GABA neuronal subpopulations incat primary auditory cortex: Co-localization with calcium bindingproteins. Brain Res. 543:45–55.

Hendry, S.H.C., C.R. Houser, E.G. Jones, and J.E. Vaughn (1983) Synapticorganization of immunocytochemically identified GABA neurons inmonkey sensory-motor cortex. J. Neurocytol. 12:639–660.

Hendry, S.H.C., E.G. Jones, P.C. Emson, D.E.M. Lawson, C.W. Weizmann,and P. Streit (1989) Two classes of cortical GABA neurons defined bydifferential calcium binding protein immunoreactivities. Exp. BrainRes. 76:467–472.

Hogan, D., and N.E.J. Berman (1994) The development of parvalbumin andcalbindin-D28k immunoreactive interneurons in kitten visual corticalareas. Dev. Brain Res. 77:1–21.

Hornung, J.P., and L.J. Garey (1980)Adirect pathway from the thalamus tovisual callosal neurons in cat. Exp. Brain Res. 38:121–123.

Hornung, J.P., and L.J. Garey (1981) Ultrastructure of visual callosalneurons in cat identified by retrograde axonal transport of horseradishperoxidase. J. Neurocytol. 10:297–314.

Houser, C.R., J.E. Vaughn, S.H.C. Hendry, E.G. Jones, andA. Peters (1984)GABAneurons in the cerebral cortex. In E.G. Jones andA. Peters (eds):Cerebral Cortex. Vol. 2: Functional Properties of Cortical Cells. NewYork: Plenum Press, pp. 63–89.

Hsu, S.M., L. Raine, and H. Fanger (1981) Use of avidin biotin peroxidasecomplex (ABC) in immunoperoxidase techniques. J. Histochem. Cyto-chem. 29:577–580.

Hughes, C.M., and A. Peters (1990) Morphological evidence for callosallyprojecting nonpyramidal neurons in rat visual cortex. Anat. Embryol.182:591–603.

Hughes, C.M., andA. Peters (1992) Symmetric synapses formed by callosalafferents in rat visual cortex. Brain Res. 583:271–278.

Inaguma, Y., H. Shinohara, T. Inagaki, and K. Kato (1992) Immunoreactiveparvalbumin concentrations in parahippocampal gyrus decrease inpatients withAlzheimer’s disease. J. Neurol. Sci. 110:57–61.

Innocenti, G.M. (1986) General organization of callosal connections in thecerebral cortex. In: Cerebral Cortex. Vol. 5. Sensory-Motor Areas andAspects of Cortical Connectivity. NewYork: Plenum Press, pp. 291–353.

Innocenti, G.M., and L. Fiore (1976) Morphological correlates of visual fieldtransformation in the corpus callosum. Neurosci. Lett. 2:245–252.

Jones, E.G., and T.P.S. Powell (1970a) An electron microscopic study of thelaminar pattern and mode of terminaton of afferent fibre pathways in

the somatic sensory cortex of the cat. Phil. Trans. R. Soc. London [Biol.]257:45–62.

Jones, E.G., and T.P.S. Powell (1970b) Electron microscopy of the somaticsensory cortex of the cat. I. Cell types and synaptic organization. Phil.Trans. R. Soc. London [Biol.] 257:1–11.

Jones, E.G., M.E. Dell’anna, M. Molinri, E. Rausell, and T. Hashikawa(1995) Subdivisions of macaque monkey auditory cortex revealed bycalcium-binding protein immunoreactivity. J. Comp. Neurol. 362:153–170.

Kaiserman-Abramof, I.R., and A. Peters (1972) Some aspects of themorphology of Betz cells in the cerebral cortex of the cat. Brain Res.43:527–546.

Kawaguchi, Y. (1995) Physiological subgroups of nonpyramidal cells withspecific morphological characteristics in layer II/III of rat frontal cortex.J. Neurophysiol. 15:2638–2655.

Kawaguchi, Y., and Y. Kubota (1993) Correlation of physiological subgroup-ings of nonpyramidal cells with parvalbumin- and calbindin D28K-immunoreactive neurons in layer V of rat frontal cortex. J. Neuro-physiol. 70:387–396.

Keller, A. (1989) GABAergic inhibition in the cerebral cortex. In: CorticalCircuits. Synaptic Organization of the Cerebral Cortex; Structure,Function, and Theory. Boston: Birkhauser, pp. 150–158.

Leifer, D., and N.W. Kowall (1993) Immunohistochemical patterns ofselective cellular vulnerability in human cerebral ischemia. J. Neurol.Sci. 119:217–228.

Leranth, C., and V.M. Pickel (1989) Electron microscopic preembeddingdouble-immunostaining methods. In L. Heiner and L. Zaborszky (eds):Neuroanatomical Tract-Tracing Methods 2. New York: Plenum Press,pp. 129–172.

LeVay, S. (1973) Synaptic patterns in the visual cortex of the cat andmonkey. Electron microscopy of Golgi preparations. J. Comp. Neurol.150:53–86.

Liposits, Z., Gy. Setalo, and B. Flerko (1984) Application of the silver-goldintensified 3,38-diaminobenzidine chromogen to the light and electronmicroscopic detection of the luteinizing hormone-releasing hormonesystem of the rat brain. Neuroscience 13:513–525.

Liposits, Z., T. Gorcs, and K. Trombitas (1985) Ultrastructural analysis ofcentral serotoninergic neurons immunolabeled by silver-gold-intensi-fied diaminobenzidine chromogen. Completion of immunocytochemis-try with X-ray microanalysis. J. Histochem. Cytochem. 33:604–610.

Liu, X.-B., Z.-H. Zheng, M.-C. Xi, and C.-P. Wu (1991) Distribution ofsynapses on fast and slow pyramidal tract neurons in the cat. Anelectron microscopic study. Brain Res. 545:239–247.

Lund, J.S., and R.D. Lund (1970) The termination of callosal fibers in theparavisual cortex of the rat. Brain Res. 17:25–45.

Malach, R. (1989) Patterns of connections in rat visual cortex. J. Neurosci.9:3741–3752.

Manzoni, T., P. Barbaresi, F. Conti, and M. Fabri (1989) The callosalconnections of the primary somatosensory cortex and the neural basesof midline fusion. Exp. Brain Res. 76:251–266.

Marin-Padilla, M., and G.R. Stibitz (1974) Three-dimensional reconstruc-tion of the basket cell of the human motor cortex. Brain Res. 70:511–514.

Martınez-Garcıa, F., T. Gonzales-Hernandez, and L.Martınez-Millan (1994)Pyramidal and nonpyramidal callosal cells in the striate cortex of theadult rat. J. Comp. Neurol. 350:439–451.

Peters, A., and K.M. Harriman (1990) Different kinds of axon terminalsforming symmetric synapses with the cell bodies and initial axonsegments of layer II/III pyramidal cells. I. Morphometric analysis. J.Neurocytol. 19:154–174.

Peters, A., and K.M. Harriman (1992) Different kinds of axon terminalsforming symmetric synapses with the cell bodies and initial axonsegments of layer II/III pyramidal cells. III. Origins and frequency ofoccurrence of the terminals. J. Neurocytol. 21:679–692.

Peters, A., and I.R. Kaiserman-Abramof (1970) The small pyramidalneuron of the rat cerebral cortex. The perikaryon, dendrites and spines.Am. J. Anat. 127:321–356.

Peters, A., and D.A. Kara (1985) The neuronal composition of area 17 of ratvisual cortex. I. The pyramidal cells. J. Comp. Neurol. 234:218–241.

Peters, A., and C.C. Proskauer (1980) Synaptic relations between amultipolar stellate cell and a pyramidal neuron in the rat visual cortex.A combined Golgi-electron microscope study. J. Neurocytol. 9:163–183.

Peters, A., B.R. Payne, K. and Josephson (1990) Transcallosal non-pyramidal cell projections from visual cortex in the cat. J. Comp.Neurol. 302:124–142.


Porter, L., and E.L. White (1986) Synaptic connections of callosal projectionneurons in the vibrissal region of mouse primary motor cortex: Anelectron microscopic/horseradish peroxidase study. J. Comp. Neurol.248:573–587.

Ren, J.Q., Y. Aika, C.W. Heizmann, and T. Kosaka (1992) Quantitativeanalysis of neurons and glial cells in the rat somatosensory cortex, withspecial reference to GABAergic neurons and parvalbumin-containingneurons. Exp. Brain Res. 92:1–14.

Ribak, C.E. (1978) Aspinous and sparsely-spinous stellate neurons in thevisual cortex of rats contain glutamic acid decarboxylase. J. Neurocytol.7:461–478.

Ribak, C.E. (1992) Local circuitry of GABAergic basket cells in the dentategyrus. Epilepsy Res. Suppl. 7:29–47.

Ribak, C.E., L. Seress, and C. Leranth (1993) Electronmicroscopic immuno-cytochemical study of the distribution of parvalbumin-containing neu-rons and axon terminals in the primate dentate gyrus and Ammon’shorn. J. Comp. Neurol. 327:298–321.

Schwartz, M.L., and P.S. Goldman-Rakic (1984) Callosal and interhemi-spheric connectivity of the prefrontal association cortex in the rhesusmonkey: Relation between intraparietal and principle sulcus cortex. J.Comp. Neurol. 226:403–420.

Sloper, J.J., and T.P.S. Powell (1979) An experimental electron microscopicstudy of afferent connections to the primate motor and somatic sensorycortices. Phil. Trans. R. Soc. London [Biol.] 285:199–225.

Sloper, J.J., R.W. Hiorns, and T.P.S. Powell (1979) A qualitative andquantitative electron microscopic study of the neurons in the primatemotor and somatic sensory cortices. Phil. Trans. R. Soc. London [Biol.]285:141–171.

Somogyi, P., and I. Soltesz (1986) Immunogold demonstration of GABA insynaptic terminals of intracellularly recorded, horseradish peroxidase-filled basket cells and clutch cells in the cat’s visual cortex. Neurosci-ence 19:1051–1065.

Somogyi, P., and H. Takagi (1982) A note on the use of picric acid-paraformaldehyde-glutaraldehyde fixative for correlated light and elec-tron microscopic immunocytochemistry. Neuroscience 7:1779–1783.

Tigges, J., J.G. Herndon, and A. Peters (1992) Axon terminals on Betz cellsomata of area 4 in rhesus monkey throughout adulthood. Anat. Rec.232:305–315.

VanBrederode, J.F.M., K.S.Mulligan, andA.E.Hendrickson (1990) Calcium-binding proteins as markers for subpopulations of GABAergic neuronsin monkey striate cortex. J. Comp. Neurol. 298:1–22.

Van den Pol, A.N. (1988) Silver intensification of colloidal gold or horserad-ish peroxidase for dual ultrastructural immunocytochemistry. In F.W.van Leeuwen, R.M. Buijs, C.W. Pool, and O. Pach (eds): MolecularNeuroanatomy. NewYork: Elsevier, pp. 315–336.

Voigt, T., S. LeVay, andM.A. Stamnes (1988) Morphological and immunocy-tochemical observations on the visual callosal projections in the cat. J.Comp. Neurol. 272:450–460.

Warr, W.B., J.S. de Olmos, and L. Heimer (1982) Horseradish peroxidase:the basic procedure. In: Neuroanatomical Tract-Tracing Methods. NewYork: Plenum Press, pp. 207–262.

White, E.L. (1989a) Synaptic connections between identified elements. In:Cortical Circuits. Synaptic Organization of the Cerebral Cortex; Struc-ture, Function and Theory. Boston: Birkhauser, pp. 46–82.

White, E.L. (1989b) Cell types. In: Cortical Circuits. Synaptic Organizationof the Cerebral Cortex; Structure, Function and Theory. Boston:Birkhauser, pp. 19–45.

White, E.L. (1989c) Synaptic connections between identified elements. In:Cortical Circuits. Synaptic Organization of the Cerebral Cortex; Struc-ture, Function and Theory. Boston: Birkhauser, p. 82.

White, E.L., and D. Czeiger (1991) Synapses made by axons of callosalprojection neurons in mouse somatosensory cortex: Emphasis on intrin-sic connections. J. Comp. Neurol. 303:233–244.

White, E.L., and M.P. Rock (1980) Three dimensional aspects and synapticrelationships of a Golgi impregnated spiny stellate cell reconstructedfrom serial thin sections. J. Neurocytol. 9:615–636.

White, E.L., Y. Amitai, and M.J. Gutnick (1993) A novel approach tocorrelative studies of neuronal structure and function. Israel J. Med.Sci. 29:549–555.

White, E.L., Y. Amitai, and M.J. Gutnick (1994) A comparison of synapsesonto the somata of intrinsically bursting and regular spiking neurons inlayer V of rat SmI cortex. J. Comp. Neurol. 342:1–14.

Williams, S.M., P.S. Goldman-Rakic, and C. Leranth (1990) Characteriza-tion of parvalbumin neurons and axons in the primate prefrontalcortex. Soc. Neurosci. Abstr. 16:241.

Williams, S.M., P.S. Goldman-Rakic, and C. Leranth (1992) The synaptol-ogy of parvalbumin-immunoreactive neurons in the primate prefrontalcortex. J. Comp. Neurol. 320:353–369.

Wilson, C.J. (1994) Understanding the neostriatal microcircuitry: High-voltage electron microscopy. Microsc. Res. Tech. 29:368–380.

Winfield, D.A., R.N.L. Brooke, J.J. Sloper, and T.P.S. Powell (1981) Acombined Golgi-electron microscopic study of the synapses made by theproximal axon and recurrent collaterals of a pyramidal cell in thesomatic sensory cortex of the monkey. Neuroscience 6:1217–1230.

Yorke, C.H., Jr., and V.S. Caviness, Jr. (1975) Interhemispheric neocorticalconnections of the corpus callosum in the normal mouse: A study basedon anterograde and retrogrademethods. J. Comp. Neurol. 164:233–246.

Zaborszky, L., and C. Leranth (1985) Simultaneous ultrastructural demon-stration of retrogradely transported horseradish peroxidase and cholineacetyltransferase immunoreactivity. Histochemistry 82:529–537.

Zaborszky, L., and L. Heimer (1989) Combinations of Tracer Techniques,Especially HRP and PHA-L, with Transmitter Identification for Corre-lated Light and Electron Microscopic Studies. In: NeuroanatomicalTract-Tracing Methods 2. NewYork: Plenum Press, pp. 49–96.


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