Brain Surviving CA1 pyramidal cells receive intact ... · L. Wittner,1 L. Er}ooss, 2 S. Czirja´k,3...

doi:10.1093/brain/awh339 Brain (2005), 128, 138–152

Surviving CA1 pyramidal cells receive intactperisomatic inhibitory input in the humanepileptic hippocampus

L. Wittner,1 L. Er}ooss,2 S. Czirjak,3 P. Halasz,4 T. F. Freund1 and Zs. Magloczky1

Correspondence to: Dr. T. F. Freund, Institute of

Experimental Medicine, Hungarian Academy of Sciences,

POB 67, H-1450 Budapest, Hungary

E-mail: [email protected]

1Institute of Experimental Medicine, Hungarian Academy

of Science, 2Neurosurgical Department, MAV Hospital

Budapest, 3National Institute of Neurosurgery, H-1577 and4National Institute of Psychiatry and Neurology, Epilepsy

Center, Budapest, Hungary

SummaryTemporal lobe epilepsy (TLE) is known to be linked to an

impaired balance of excitation and inhibition. Whether

inhibition is decreased or preserved in the human epileptic

hippocampus, beside the excess excitation, is still a debated

question. In the present study, quantitative light and elec-tron microscopy has been performed to analyse the

distribution, morphology and input–output connections

of parvalbumin (PV)-immunopositive interneurons,

together with the entire perisomatic input of pyramidal

cells, in the human control and epileptic CA1 region.

Based on the degree of cell loss, the patients with therapy-

resistant TLE formed four pathological groups. In the

non-sclerotic CA1 region of TLE patients, where largenumbers of pyramidal cells are preserved, the number

of PV-immunopositive cell bodies decreased, whereas

axon terminal staining, and the distribution of their post-

synaptic targets was not altered. The synaptic coverage of

CA1 pyramidal cell axon initial segments (AISs) remained

unchanged in the epileptic tissue. The somatic inhibitory

input is also preserved; it has been decreased only in the

cases with patchy pyramidal cell loss in the CA1 region

(control, 0.637; epileptic with mild cell loss, 0.642; epilep-

tic with patchy cell loss, 0.424 mm synaptic length/100 mmsoma perimeter). The strongly sclerotic epileptic CA1

region, where pyramidal cells can hardly be seen, contains

a very small number of PV-immunopositive elements. Our

results suggest that perisomatic inhibitory input is pre-

served in the epileptic CA1 region as long as pyramidal

cells are present. Basket and axo-axonic cells survive in

epilepsy if their original targets are present, although

many of them lose their PV content or PV immunoreact-ivity. An efficient perisomatic inhibition is likely to take

part in the generation of abnormal synchrony in the non-

sclerotic epileptic CA1 region, and thus participate in the

maintenance of epileptic seizures driven, for example, by

hyperactive afferent input.

Keywords: perisomatic inhibition; GABA; basket cell; chandelier cell; selective vulnerability

Abbreviations: AIS = axon initial segment; CA1–3 = regions of the cornu Ammonis; PV = parvalbumin;

TLE = temporal lobe epilepsy

Received January 30, 2004. Revised August 19, 2004. Accepted October 4, 2004. Advance Access publication

November 17, 2004

IntroductionEpilepsy is thought to be related to a changed balance between

excitation and inhibition (Avoli, 1983; Mody et al., 1992). In

epilepsy, the CA1 region was shown to receive an enhanced

excitatory input from the CA3 region in the rat hippocampus

(Buzsaki et al., 1989; Rafiq et al., 1993; Bragin et al., 1997),

from the dentate gyrus and/or CA2 region in the human hip-

pocampus (Wittner et al., 2002) and from the sprouting CA1

pyramidal cell axons in rats (Esclapez et al., 1999; Lehmann

et al., 2001) and humans (Lehmann et al., 2000). It is still not

clear whether inhibition is decreased or preserved in the

human epileptic CA1 region. There is evidence for interneur-

onal cell death (de Lanerolle et al., 1988, 1989; Magloczky

et al., 2000), as well as for the preservation of glutamic

acid dehydrogenase (GAD)-positive cells (Babb et al., 1989).

Brain Vol. 128 No. 1 # Guarantors of Brain 2004; all rights reserved

Since CA1 pyramidal cells provide the cortical output con-

nections of the hippocampus (Ramon y Cajal, 1909–1911;

Lorente de No, 1934; Maclean, 1992) and are known to be

the most vulnerable in epilepsy (Sommer, 1880; Corsellis,

1955; Falconer, 1968), alterations in the inhibitory network

in the CA1 region may be crucial in the pathophysiology of

the epileptic temporal lobe.

The calcium-binding protein parvalbumin (PV) is known to

be present exclusively in perisomatic inhibitory interneurons

in the hippocampus of rodents (Kosaka et al., 1987;

Katsumaru et al., 1988b; Holm et al., 1990; Nitsch et al.,

1990), monkeys (Seress et al., 1991; Ribak et al., 1993)

and humans (Braak et al., 1991; Seress et al., 1993). Two

perisomatic inhibitory cell types, basket and chandelier or axo-

axonic cells, were demonstrated to be PV-immunopositive

in all examined species, including humans (Seress et al.,

1993; for a review see Freund and Buzsaki, 1996) and to

control the output of principal cells (Miles et al., 1996).

The survival of PV-immunopositive interneurons in epilepsy,

as well as their role in the pathophysiological mechanisms of

seizures and cell death are still controversial questions. Some

authors have described the preservation (Sloviter et al., 1991;

Arellano et al., 2003) and others the vulnerability (Zhu et al.,

1997; Wittner et al., 2001) of these cells in the human

epileptic hippocampus, and a decrease in the number of

PV-immunopositive interneurons has been found in the epi-

leptic neocortex (DeFelipe et al., 1993; Marco et al., 1997;

DeFelipe, 1999). PV immunoreactivity is very sensitive to

epileptic and ischaemic conditions and might disappear

from neurons that are otherwise preserved (Johansen et al.,

1990; Tortosa and Ferrer, 1993; Bazzett et al., 1994; Scotti

et al., 1997a, b). Since PV immunostaining probably reveals

only part of the originally PV-immunopositive cells, which in

turn represent only one of the at least two basket cell types (the

other being the cholecystokinin-containing cells; for review

see Freund, 2003), perisomatic inhibition cannot be fully

characterized by the analysis of PV-immunopositive inter-

neurons. Controversial results have been found in different

rat models of epilepsy and human studies concerning the

perisomatic inhibitory input of hippocampal principal cells.

In the dentate gyrus, loss of axo-axonic and axo-somatic

inhibitory inputs of granule cells was found in the kindling

(Sayin et al., 2003) and pilocarpine models (Kobayashi and

Buckmaster, 2003), but the preservation of perisomatic inhi-

bitory input (Wittner et al., 2001) and an increased rather than

decreased recurrent inhibition of granule cells (Isokawa-

Akesson et al., 1989) has been shown in the dentate gyrus

of epileptic patients. Perisomatic GABAergic input of CA1

pyramidal cells remained unchanged in the kainate model

(Morin et al., 1999), whereas in the pilocarpine model den-

dritic but not somatic inhibition is decreased (Cossart et al.,

2001), together with a slight decrease also in axo-axonic input

(Dinocourt et al., 2003). These contradictory results also show

that an extensive comparison and correlation with data

obtained from human epileptic tissue is needed to validate

predictions from animal models.

No detailed electron microscopic analyses have been done

to date investigating the perisomatic inhibitory input of CA1

pyramidal cells in the human hippocampus. Here we analysed

the distribution, morphology and input–output characteristics

of PV-immunopositive interneurons, as well as the inhibitory

synaptic input of CA1 pyramidal cell somata and axon initial

segments (AISs) of human control subjects and epileptic

patients.

Material and methodsPV-containing elements were examined in the CA1 region of

32 temporal lobe samples surgically removed from epileptic patients,

and in seven control hippocampi. Patients with intractable TLE were

operated on in the National Institute of Neurosurgery and in the

MAV Hospital in Budapest; standard anterior temporal lobectomies

were performed (Spencer and Spencer, 1985). The anterior third of

the temporal lobe was removed together with the temporomedial

structures. Control brain samples were obtained from a 37-year-

old woman who died by accident, from 47- and 48-year-old

women and 47-, 51- and 56-year-old men who had a cardiac arrest,

and from a 53-year-old man who died by suffocation. None of the

control subjects had a record of any neurological disorder. Brains

were removed 2–4 h after death; the dissection was performed in

the Institute of Pathology and Forensic Medicine, Semmelweis

University, Budapest. In all cases, the patient’s consent was

obtained according to the Declaration of Helsinki, and the ethical

regulations of the Hungarian Ministry of Health were followed.

After surgical removal, the epileptic tissue was immediately dis-

sected into 2 mm thick blocks, and immersed in a fixative containing

4% paraformaldehyde, 0.1% glutaraldehyde and 0.2% picric acid

in 0.1 M phosphate buffer (PB, pH 7.4), as described earlier

(Magloczky et al., 1997). The fixative was changed every hour to

a fresh solution during constant agitation for 6 h, then the blocks were

post-fixed in the same fixative overnight. Four of the control hippo-

campi were subjected to the same procedure. Three of the control

brains (C2, C10 and C11) were removed from the skull 2 h after death

and both internal carotid and vertebral arteries were cannulated; the

brain was perfused with physiological saline (2 l in 30 min) followed

by a fixative solution containing 4% paraformaldehyde and 0.2%

picric acid in 0.1 M PB (5 l in 3.5 h). The hippocampus was removed

after perfusion and cut into 2 mm thick blocks, which were post-fixed

in the same fixative solution overnight.

ImmunocytochemistryVibratome sections (60 mm thick) were cut from the blocks and,

following washing in PB, they were immersed in 30% saccharose

for 1–2 days, then frozen three times over liquid nitrogen. Sections

were processed for immunostaining against PV, as follows. Sections

were transferred to Tris-buffered saline (TBS, pH 7.4), then endo-

genous peroxidase was blocked by 1% H2O2 in TBS for 10 min. TBS

was used for all the washes (3 3 10 min between each sample) and

dilution of the antisera. Non-specific immunostaining was blocked

by 5% milk powder and 2% bovine serum albumin. A monoclonal

mouse antibody against PV (1 : 5000; Sigma, St Louis, MO) was

used for 2 days at 4�C. The specificity of the antibody has been

thoroughly tested by the manufacturer. For visualization of immuno-

positive elements, biotinylated anti-mouse immunoglobulin G (IgG)

(1 : 300, Vector) was applied as secondary antiserum followed by

Parvalbumin-positive cells in human CA1 region 139

avidin-biotinylated horseradish peroxidase complex (ABC; 1 : 300,

Vector). The immunoperoxidase reaction was developed by 3,30-diaminobenzidine tetrahydrochloride (DAB; Sigma) dissolved in

Tris buffer (TB, pH 7.6) as a chromogen. Sections were then osmi-

cated (1% OsO4 in PB, 40 min) and dehydrated in ethanol (1% uranyl

acetate was added at the 70% ethanol stage for 40 min) and mounted

in Durcupan (ACM, Fluka). The control hippocampi were processed

in the same way.

Controls of the method included the incubation of sections in the

same way, but the primary antibody was replaced by normal mouse

serum. No specific immunostaining could be detected under these

conditions. Non-specific staining visualized large numbers of lipo-

fuscin granules in the cells, which were therefore considered as

background.

After light microscopic examination, areas of interest were

re-embedded and sectioned for electron microscopy. Ultrathin serial

sections were collected on Formvar-coated single slot grids, stained

with lead citrate, and examined with a Hitachi 7100 electron

microscope.

Quantitative analysisCell counting was performed in three control (C6, C10 and C11) and

in six epileptic (P16, P22, P31, P40, P75 and P79) brains. Each

PV-immunopositive cell together with the outlines of the CA1 region

were drawn by camera lucida from two to five sections per patient.

The area of the CA1 region was measured using the analySIS pro-

gram, and the number of PV-immunopositive cells per unit area

(mm2) was determined in the same area. The numbers of cells

derived from different sections of the same subject were summed

and divided by the sum of the areas of the same sections. The

averages of the different pathological groups (control, mild, patchy

and sclerotic) were determined by calculating the average of data

from all patients in each group.

The target elements of PV-immunopositive axon terminals were

examined in four control brains (C2, C6, C10 and C11) and in four

epileptic patients (P15, P31, P40 and P54). Based on light micro-

scopic examination, sections parallel to the plane of the CA1 pyram-

idal cell apical dendrites were chosen for the electron microscopic

analysis. The entire width of CA1 stratum pyramidale including the

zone of AISs located at the border of stratum oriens was re-embedded

from PV-immunostained sections; the size of the blocks was similar.

Serial sections were made from the blocks, and PV-containing

terminals were analysed in every tenth section in order, following

the rules of systematic random sampling, to avoid sampling of

the same axon terminals. Photographs were taken from every PV-

immunopositive synaptic terminal in each section, and the distribu-

tion of the postsynaptic target elements of the PV-immunopositive

terminals was determined. In all electron microscopic analyses, the

means of the different pathological groups (control versus epileptic,

mild and patchy groups) were determined by calculating the average

of the data of every patient belonging to that group.

The synaptic inputs of CA1 pyramidal cell AISs and somata were

analysed in three control hippocampi (C2, C6 and C10) and in five

epileptic patients (AIS, P15, P16, P40, P54 and P79; cell body, P15,

P31, P40, P54 and P79). The sampling of the AISs of pyramidal cells

was made as described above; each AIS observed in the stratum

pyramidale and the bordering zone of the stratum oriens was digit-

ized from every tenth section using a MegaView II camera. About

40–80 AISs were analysed in every subject. The border zone of the

stratum oriens was included in the examined area to analyse the

whole length of the pyramidal cell AISs. The AISs were obliquely

cut, and the average perimeter was similar in all control and epileptic

subjects. This varied between 7.38 and 13.64 mm, and no statistical

differences were found between the groups of AISs of different

subjects [Kruskal–Wallis ANOVA (analysis of variance), P >

0.05]. For the analysis of synaptic input of CA1 pyramidal cell

bodies, serial sections were made from two blocks from each subject

containing the entire width of the stratum pyramidale. About

20 pyramidal cells were digitized from each block (�40 cells per

subject). The perimeters of the pyramidal cell AISs and somata and

the length of the synapses they receive were measured by the ana-

lySIS program. The synaptic coverage (mm synaptic length/100 mm

of AIS or soma perimeter) of every AIS and soma has been recorded.

Statistical analysis (Kruskal–Wallis ANOVA and Mann–Whitney

U test) were performed using the Statistica 6.0 program. The number

of synapses (terminals forming simple and perforated synapses)

per 100 mm perimeter was calculated in every patient by dividing

the total number of synapses by the total perimeter recorded.

Asymmetric somatic synapses were only occasionally found, and

were not included in the calculation.

Dependence of PV immunostaining on fixationand post-mortem delayIn a preliminary experiment, we examined 12 control brains with

different post-mortem delays and age in both genders (Wittner et al.,

2002). Briefly, the progression of age influences the quality and

quantity of immunostaining, i.e. most of the antisera showed weaker

staining and a lower number of cells in aged subjects. Therefore, the

subjects older than 60 years have been excluded from the detailed

analysis. For electron microscopic studies, subjects were chosen with

a post-mortem delay of <4 h, because longer post-mortem delays

(6–24 h) resulted in poor ultrastructural preservation, and a decrease

in immunoreactivity. The electron microscopic analysis of these

tissues revealed acceptable ultrastructural preservation even in the

immersion-fixed control (C6), although it was inferior to the perfused

tissue. The preservation of the 2–3 h post-mortem perfused controls

(C2, C10 and C11) was comparable to the immediately fixed epi-

leptic samples and to the perfused animal tissues. The PV staining in

the control subjects included in the study was similar to each other

and to previously described human or monkey PV immunostaining

(Braak et al., 1991; Seress et al., 1991, 1993; Sloviter et al., 1991;

Ribak et al., 1993). Therefore, we concluded that the differences

found between control and epileptic tissues in the present study are

likely to be caused by epilepsy.

Pathological classification of the tissue samplesAll patients examined in the present study had therapy-resistant

epilepsy of temporal lobe origin. The seizure focus was identified

by multimodal studies including video-EEG monitoring, single

photon emission computed tomography (SPECT) and/or PET.

Only patients without gross temporal lobe damage or tumour

were included in the study. The patients had different degrees of

hippocampal atrophy and/or sclerosis. Similarly to recent results

(de Lanerolle et al., 2003), we established four groups of epileptic

patients based on the principal cell loss and interneuronal changes

examined at the light microscopic level as follows (Table 1). (i) Type 1

(mild): similar to control, no considerable cell loss in the CA1 region,

pyramidal cells are present, layers are visible, their borders are

clearly identified. There is a slight loss in certain interneuron

types, mostly in the hilus and the stratum oriens of the CA1 region.

(ii) Type 2 (patchy): pyramidal cell loss in patches in the CA1

140 L. Wittner et al.

pyramidal cell layer, but these segments of the CA1 region are not

atrophic. Interneuron loss is more pronounced. (iii) Type 3 (sclerotic):

the CA1 region is shrunken, atrophic, >90% cell loss, occasion-

ally scattered pyramidal cells remained in the CA1 region, separation

of the layers is impossible. Only the stratum lacunosum-moleculare

is present in the CA1 region as a distinct layer; the other strata could

not be separated from each other due to the lack of pyramidal cells

and shrinkage of the tissue. This remaining part should contain the

layers determined in the control as strata oriens, pyramidale and

radiatum. Mossy fibre sprouting and considerable changes in the

distribution and morphology of interneurons can be observed in

the samples of this group. (iv) Type 4 (gliotic): the whole hippo-

campus is shrunken, atrophic, not only the CA1 region, the loss of all

cell types can be observed, including the resistant neurons (granule

cells, calbindin-positive interneurons). The number of samples

belonging to this group was small and therefore they were not

included in this study.

ResultsThe distribution and morphological features of PV-

immunopositive cells were examined at light and electron

microscopic levels in the CA1 region derived from human

controls and TLE patients. The number and distribution of

cells proved that the antigenicity was retained for PV in the

post-mortem controls included in this study (see Material and

methods: considerations of the quality of post-mortem tissue).

Samples taken from four control brains (three of them were

perfused, see Material and methods) as well as from epileptic

patients with mild and strong sclerosis were analysed further

in the electron microscope to obtain data about the distribution

of PV-positive axon terminals, their postsynaptic targets, the

synaptic input of PV-positive interneuron cell bodies and

dendrites, as well as the perisomatic synaptic input of CA1

pyramidal cells.

Light microscopy of PV-positive interneuronsIn describing different regions of the hippocampal formation,

we used the nomenclature of Lorente de No (1934) and

Rosene and Van Hoesen (1987) with the proposed modifica-

tions of Seress concerning the CA3c region (Seress, 1988)

(see also Fig. 1).

In the human control CA1 region, PV-immunopositive inter-

neurons were located in the strata oriens and pyramidale

(Fig. 2A), as was reported in previous publications (Braak

Table 1 Patient data and general characterization of cell loss of the hippocampus

Path. group Patientnumber

Age(years)

Gender Age at onset ofepilepsy (year)

Duration ofepilepsy (years)

CA1sclerosis

Thickness of the granulecell layer

Type 1: mild cell loss 40 20 M 6 14 1 Normal54 22 F 15 7 1 Local increase58 23 F 12 11 1 Normal67 23 F 17 6 1 Normal79 47 M 8 39 1 Normal

Type 2: patchy cell loss 15 25 M 8 17 2 Normal16 23 M 16 7 2 Normal30 40 F 1 39 1 Increased31 23 F 2 21 2 Normal41 16 M 2 14 2 Normal42 43 F 13 30 2 Normal59 32 M 7 25 2 Normal68 44 M 20 24 2 Normal73 27 F 10 17 2 Normal

Type 3: sclerotic 9 39 M 23 16 3* Increased11 36 M 30 6 3* Increased18 32 M 20 12 3 Normal19 56 M 24 32 3 Local increase21 30 M 21 9 3* Increased22 27 F 22 5 3 Increased29 29 F 5 24 3* Increased32 41 F 19 22 3* Local increase35 25 M 12 13 3* Local increase36 45 F 31 14 3 Increased37 41 M 2 39 3* Local cell loss53 26 M 17 9 3* Increased60 30 M 6 24 3 Local increase62 45 F 25 20 3 Increased75 24 M 0.3 24 3* Increased

Type 4: gliotic 24 30 F 21 9 3* Cell loss39 17 F 5 12 3* Cell loss70 14 F 6 8 3* Cell loss

Semi-quantitative scale: 0 = no damage; 1 = 0–20%; 2 = 20–50%; 3 = >50%; 3* = 100% of the pyramidal cells of the region are missing.Path. group = pathological group; M = male; F = female.


et al., 1991; Sloviter et al., 1991; Seress et al., 1993). Large

multipolar cells can be seen in the stratum pyramidale;

their long dendrites run radially through the region and termi-

nate in the stratum lacunosum-moleculare. Horizontal PV-

immunopositive interneurons are situated in the stratum oriens;

their dendrites arborize mostly in this layer (Fig. 2A). As it was

described in the rat hippocampus (Fukuda and Kosaka, 2000),

we often found long dendritic segments originating from dif-

ferent cells, attached to each other. Such dendritic juxtaposi-

tions were seen most frequently among radially running

dendrites in the strata radiatum and lacunosum-moleculare

(Fig. 2A, arrowheads) and the horizontal dendrites of the stra-

tum oriens. Dense PV-immunopositive terminal staining was

observed in the stratum pyramidale (Fig. 2A, insert). The PV-

immunopositive interneurons in the human control CA1 region

are known to belong to the populations of basket and axo-

axonic cells (Braak et al., 1991; Seress et al., 1993; Sloviter

et al., 1991), and terminate within the stratum pyramidale.

In the non-sclerotic epileptic CA1 region with mild cell loss

(type 1), the distribution and number of PV-immunopositive

interneurons did not change considerably; only a slight

decrease was observed in the number of the horizontal stratum

oriens cells (Fig. 2B, Table 2). Interestingly, the dendrites of

PV-immunopositive interneurons were shorter than in the

control tissue. Dense axonal staining of a density similar

to, if not more than, that of controls was observed in the

stratum pyramidale (Fig 2B, insert); basket-like formations

(small arrow) and vertically oriented bouton rows character-

istic of chandelier cells can also be distinguished (double

arrows). In the CA1 region with patchy cell loss (type 2),

a considerable decrease was found in the number of PV-

immunopositive cells (Fig. 2C, Table 2), with the preservation

of a dense axonal staining (Fig. 2C, insert). The termination

pattern of PV-immunopositive cells did not change; the axo-

nal cloud was confined to the stratum pyramidale. It was

distributed homogeneously in the pyramidal cell layer, and

also covered the patches with no pyramidal cells visible at

the light microscopic level (Fig. 2C, insert). In the sclerotic

epileptic tissue (type 3), the number of PV-immunopositive

elements was dramatically decreased (Fig. 2D, Table 2).

PV-immunopositive cells and axons can hardly be found;

only a few PV-immunopositive dendrites were present in

Fig. 1 Low power light micrographs of PV-immunostained sections from human control (B) and strongly sclerotic epileptic (D)hippocampus. In the strongly sclerotic epileptic CA1 region, PV-positive cells can hardly be found, whereas in the prosubiculum thenumber and distribution of PV-positive elements seem to be unchanged. Parts of the prosubiculum marked with asterisks on the lowpower micrographs are shown in (A) (control) and (C) (epileptic) at higher power. DG = dentate gyrus; CA1 = part of the cornu Ammonis;ProS = prosubiculum; S = subiculum. Scales: A and C, 20 mm; B and D, 0.5 mm.


Fig. 2 Light micrographs showing PV-immunostained elements in the human control (A) and epileptic (B, C and D) CA1 region. (A) In thecontrol CA1 region, large PV-positive multipolar cells are located in the stratum pyramidale and horizontal cells in the stratum oriens(arrows). Long radial dendrites are running in the stratum radiatum and terminate in the stratum lacunosum-moleculare. Long dendritesegments originating from different cells were often attached to each other (arrowheads). A dense terminal staining can be observed in thestratum pyramidale (insert). (B) In the epileptic CA1 region with mild cell loss, a slight loss of PV-positive cells was observed, mostly inthe stratum oriens. PV-positive dendrites seem to be shorter. The axon terminal cloud (insert) is similar to the control in density; basket-(small arrows) and chandelier-like (double arrows) formations can be observed. (C) In the epileptic tissue with patchy cell loss, the decreasein number of PV-immunostained cells is more pronounced. A dense terminal staining (insert), similar to the control, can be found even inthose regions where no PV-positive cells are present. (D) In the strongly sclerotic epileptic tissue, the number of PV-immunoreactivecells is dramatically decreased; usually a few dendrites (arrowheads) are present in the region. Axon terminals can hardly be found (insert).s.o. = stratum oriens; s.p. = stratum pyramidale; s.r. = stratum radiatum. Scales, 50 mm; inserts, 15 mm.


the sclerotic CA1 region (Fig. 2D, insert). The border between

the sclerotic CA1 region and the prosubiculum was clearly

visible; this latter was not sclerotic, a large number of

pyramidal cells were present and the PV staining was similar

to the control (Fig. 1). Since very small numbers of PV-

immunopositive elements were found in the sclerotic CA1

region, samples from only the non-sclerotic epileptic hippo-

campi (with mild or patchy cell loss) were chosen for further

quantitative analyses performed at the electron microscopic

level. Cell counting confirmed the loss of PV-immunopositive

cells in the epileptic CA1 region (Table 2). Three controls and

two patients from each pathological group were chosen for the

analysis. The area of the CA1 region was measured and the

number of PV-immunopositive cells per unit area was deter-

mined (see Material and methods). We found a decrease of

PV-immunopositive cells that correlated well with the degree

of hippocampal damage in general (Table 2), i.e. the number

of PV-immunopositive neurons was similar to control in those

hippocampi where no considerable pyramidal cell loss was

found in the CA1 region (type 1), a severe decrease in the

number of PV-immunopositive somata accompanied by the

preservation of axonal staining in cases with patchy cell loss

(type 2), and severe cell loss with no axonal staining for PV in

the sclerotic hippocampi (type 3).

Electron microscopy of PV-positive elementsCell bodies and dendritesFive PV-immunopositive cells from the CA1 region of human

controls C2, C6 and C11 were analysed at the electron micro-

scopic level (Fig. 3A). Every examined cell body was located

in the stratum pyramidale. PV-immunopositive interneuron

cell bodies received a large number of asymmetrical (excitat-

ory) synapses, and axon terminals forming symmetrical

(inhibitory) synapses were rarely seen (Fig. 3A1–3). Ten to

12 synaptic contacts per section were observed to terminate

on the cell body of PV-immunopositive interneurons, usually

one or two of them giving symmetrical synapses. PV-stained

dendrites were covered by a large number of asymmetrical

synapses. Zonula adhaerentia were frequently observed

between PV-immunopositive dendrites (Fig. 4A).

Four PV-immunopositive interneurons derived from epi-

leptic tissue (P15, P40, P54 and P79) were analysed in the

electron microscope (Fig. 3B). Only PV-immunopositive

cells from the non-sclerotic CA1 region were analysed,

since PV-immunopositive cell bodies can hardly be found

in the sclerotic tissue. In the non-sclerotic epileptic CA1

region, the synaptic input of PV-immunopositive cell

bodies and dendrites was similar to the control, a large

number of terminals forming asymmetrical synapses and a

few others giving symmetrical synapses terminated on the

PV-immunopositive elements (Fig. 3B1–2). A small number

of PV-immunopositive dendrites were also analysed in the

sclerotic CA1 region (P21, P22, type 3, sclerotic tissue).

These dendrites were also covered by asymmetrical synapses,

and by glial elements (Fig. 4B). In the epileptic CA1

region, zonula adhaerentia were frequently found between

PV-immunopositive dendrites, but PV-immunopositive and

-negative dendrites also formed zonula adhaerentia with

each other (Fig. 4B).

Axon terminals and postsynaptic targets

The postsynaptic target distribution of PV-immunopositive

boutons was determined in the CA1 region of four control

(C2, A6, C10 and C11, n = 52–55) and four epileptic samples

(P40 and P54, mild; and P15 and P31, patchy; Fig. 5, n =

52–107). In the CA1 region of patients with strong sclerosis,

PV-immunopositive terminals were too sparse to study; in

most areas they were absent. In the sample from P15, we dis-

tinguished patches containing (P15PC +) and lacking

(P15PC–) pyramidal cells. Blocks were re-embedded from

both regions, and the target distribution of PV-immunoposi-

tive terminals determined separately. Since the results

deriving from the two separate blocks were similar, the

data were pooled. PV-immunopositive terminals gave exclu-

sively symmetrical synapses mostly to pyramidal cells in both

control and epileptic tissue: they terminated on AISs (control,

14.5%; epileptic, 17.3%), pyramidal cell bodies (control,

14.8%; epileptic, 11.1%; Fig. 4D), proximal dendrites (con-

trol, 18.2%; epileptic, 23.1%), distal dendrites of pyramidal

cells (control, 11.6%; epileptic, 6.4%) and spines (control,

17.6%; epileptic, 10.9%). They rarely established synaptic

contacts on interneuron dendrites (control, 2.3%; epileptic,

1.9%), and even less frequently on PV-immunopositive

interneuron dendrites. We also distinguished a category of

unidentified dendrites with small diameter (control, 21.0%;

epileptic, 29.4%). There was a larger variance within the

groups of control and epileptic samples than between the

control and the epileptic groups. Some of the targets were

degenerating profiles in the epileptic samples from P15, P31

and P54 (P15, 54.2%; in P15PC+ , 11.7%; in P15PC–, 92.2%;

P31, 81.5%; P54, 60.7%; Fig. 4C).

Table 2 Density of PV-positive cells in the human controland epileptic CA1 region

Patientnumber

Cell density(no. of cells/mm2)

Control C10 3.85C11 6.81C6 4.97Average 5.21

Epileptic (mild) P40 3.06P79 4.97Average 4.02

Epileptic (patchy) P16 1.01P31 0.89Average 0.95

Epileptic (sclerotic) P75 1.10P22 1.15Average 1.12


Perisomatic inhibition of pyramidal cellsCell bodies. Somatic inhibitory input of CA1 pyramidal cells

was analysed in three control (C2, C6 and C10) and in five

epileptic (P40, P54 and P79, mild; and P15 and P31, patchy)

samples (Table 3, Fig. 6A). From each subject, we analysed on

average 40 pyramidal cells from different areas (two sepa-

rate blocks, �20 cells each). Synaptic coverage (mm synaptic

length/100 mm soma perimeter) and the number of synapses/

100 mm soma perimeter were determined (see Materials and

methods). Every symmetric synapse, irrespective of its

PV content, was recorded and measured. Asymmetric

synapses were only occasionally found and were not

included in the analysis. In the control tissue, 16–29% of

the terminals were PV immunopositive, giving symmetric

Fig. 3 Electron micrographs show PV-immunostained cell bodies from the stratum pyramidale of the CA1 region of control (A) andepileptic (B) subjects. A large number of axon terminals give asymmetric (presumed excitatory) synapses (arrows in A1, A2 and B1) anda few give symmetric (presumed inhibitory) synaptic contacts (arrowheads in A3 and B2) to the cell body of PV-positive interneurons.There was no considerable difference in the number and type of axon terminals arriving at the somata of PV-positive cells originating fromcontrol or epileptic tissue. Scales: A and B, 2 mm; A1–3, and B1 and B2, 0.5 mm.


synapses to the pyramidal cell bodies. This ratio was higher

in certain epileptic cases (45–48%, in P79, mild; P15 and

P31, patchy), and did not change in others (27%, in P40;

P54, mild).

In both control and epileptic tissue, some small differences

were found in the synaptic coverage and the number of

terminals giving synapses to pyramidal cell somata in the

different blocks derived from the same subject. After the

Fig. 4 PV-stained dendrites received a large number of asymmetric (excitatory) synapses (arrows) in both the control and epilepticCA1 region. Zonula adhaerentia (arrowheads) were often observed between PV-immunoreactive dendrites in the human control (A) andepileptic tissue. Zonula adhaerentia were found between PV-positive and -negative dendrites as well in the epileptic CA1 region (B). In thestrongly sclerotic epileptic CA1 region, only a small number of PV-positive dendrites was found (B), and usually they were covered by glialprocesses (g). PV-immunoreactive axon terminals give symmetric synaptic contacts mostly to pyramidal cells in both the control andnon-sclerotic epileptic tissue (with mild or patchy cell loss in the CA1 region). This figure shows two PV-stained boutons terminating ondegenerating (C) and healthy pyramidal cell somata (D) deriving from an epileptic patient with patchy cell loss in the CA1 region.Scales: 1 mm.


data were pooled by subjects, very similar results were found

within the different groups (control, mild and patchy epileptic,

Table 3, Fig. 6A). In the epileptic group with mild cell loss

the synaptic input of pyramidal cells was very similar to the

control (synaptic coverage: 0.637 6 0.043 (mean 6 SEM)

versus 0.642 6 0.048 in control and epileptic with mild cell

loss, respectively).

In the epileptic tissue with patchy cell loss (P15 and

P31), we took samples from light microscopically identified

patches that were rich or poor in (contained hardly any)

pyramidal cells. At the electron microscopic level, we found

that in ‘poor’ patches, pyramidal cells were still present, but

the majority of them showed signs of degeneration (100% in

P15; 81% in P31). Interestingly, all examined parameters

(synaptic coverage, number of synapses/100 mm soma

Postsynaptic target distribution of PV-

positive axon terminals in the CA1 region

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Spine

Dendrite

Cell body

AIS

Control Epileptic

(mild)

Epileptic

(patchy)

Fig. 5 Postsynaptic target distribution of PV-positive axonterminals in the CA1 region of human control and epileptichippocampus. PV-immunostained terminals give symmetricsynapses mostly to pyramidal cells: somata, AIS, proximal anddistal dendrites and spines. The target distribution did not changesystematically in the epileptic tissue; a high variability was foundbetween the different subjects of the same groups (control, andepileptic with mild or patchy cell loss).

Table 3 Synaptic input of CA1 pyramidal cell somata

Synaptic coverage of pyramidal cellsomata (mm synapse/100 mm somaperimeter, mean 6 SEM)

No. of synapses(terminals)/100 mmsoma perimeter

Ratio of PV-positiveterminals (%)

Average synaptic activezone length (mm)

Control C2 (n = 40) 0.676 6 0.086 3.36 29.07 0.168C10 (n = 40) 0.633 6 0.074 3.44 15.85 0.167C6 (n = 40) 0.604 6 0.070 3.59 24.42 0.160Average 0.637 3.462 23.11 0.165

Epileptic P40 (n = 40) 0.730 6 0.097 3.95 26.67 0.168P54 (n = 39) 0.583 6 0.079 3.13 26.92 0.172P79 (n = 40) 0.613 6 0.075 3.63 46.07 0.159Type 1 average 0.642 3.572 33.22 0.166

P15 (n = 39) 0.444 6 0.076* 2.06 45.10 0.198P31 (n = 40) 0.404 6 0.075* 2.18 48.00 0.170

Type 2 average 0.424* 2.117 46.55 0.184Average 0.554 2.990 38.55 0.173

n = number of analysed pyramidal cell bodies, *P < 0.05.

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

C2 C6 C10 P40 P54 P79 P15 P31

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

5

C2 C6 C10 P40 P54 P79 P15 P16

Synaptic coverage ofCA1 pyramidal cell somata

(A) Synaptic coverage =µm synaptic length/100 µm

soma or AIS perimeter

(B)

Subject

Control

Epileptic (mild)

Epileptic (patchy)

Subject

**

p<0,05*

Synaptic coverage ofCA1 pyramidal cell AIS

Fig. 6 Perisomatic synaptic input of CA1 pyramidal cells. (A) Thesynaptic coverage (100 mm synaptic length/100 mm soma or AISperimeter) of CA1 pyramidal cell somata did not change in theepileptic CA1 region with mild cell loss, but it decreased notably inthe epileptic tissue derived from patients with patchy cell loss in theCA1 region. (B) The synaptic coverage of CA1 pyramidal cell AISsdid not change in the epileptic tissue compared with the control.


perimeter, ratio of PV-immunopositive terminals forming

synapses on the pyramidal cell somata and synaptic active

zone length) were similar in the blocks derived from

patches containing degenerating and healthy pyramidal

cells. Therefore, the results were pooled. Thus, in the epi-

leptic tissue with patchy cell loss, the synaptic input was

significantly decreased (synaptic coverage 0.424 6 0.045,

P < 0.05) compared with the control, or with the mildly

epileptic samples (Table 3, Fig. 6A). In the samples contain-

ing a considerable number of degenerating pyramidal cells

(P15, P31 and P54), we determined the synaptic input of

degenerating and non-degenerating pyramidal cells. The

synaptic coverage of the degenerating cells was slightly

increased in all cases, but this difference was not higher

than the variance between different areas or subjects.

Axon initial segments. Synaptic input of CA1 pyramidal

cell AISs was analysed in three control (C2, C6 and C10)

and five epileptic hippocampi (P40, P54 and P79, mild; P15

and P16, patchy; for sampling see Material and methods;

Table 4, Fig. 6A). Synaptic coverage (mm synaptic length/

100 mm AIS perimeter) and number of synapses/100 mm AIS

perimeter were determined. Every axon terminal giving

symmetric synapses to AISs was included in the study, irre-

spective of their PV content. About half of the terminals

(45–46%) forming synaptic contact on pyramidal cell AISs

were PV immunopositive in the control tissue, and this did

not change considerably in the epileptic tissue (36–50%).

The synaptic coverage of AISs did not change significantly

in the epileptic samples (Kruskal–Wallis ANOVA and

Mann–Whitney U tests, P > 0.05; Table 4, Fig. 6B). We

separated the degenerating AISs in samples P15 and P54,

and analysed their synaptic input. Degenerating AISs were

identified on the basis of the following ultrastructural criteria:

they showed signs of degeneration (homogeneous and electro-

ndense cytoplasm, degenerating organelles) and at the same

time the characteristic undercoating of the plasma membrane

and microtubule fascicles was preserved. The synaptic

coverage of degenerating AISs from both samples was

decreased [synaptic coverage in P15, 3.60 6 0.65 (mean 6

SEM) versus 2.20 6 0.53 of non-degenerating and degenerat-

ing AIS, respectively; synaptic coverage in P54, 3.98 6 0.94

versus 3.69 6 0.49 of non-degenerating and degenerating

AIS, respectively]. However, the sample is too small (16

degenerating AISs from P15, and eight non-degenerating

AISs from P54) to evaluate the significance of this result.

We found an increase of active zone length of synapses

terminating on pyramidal cell somata and AISs (Tables 3

and 4), as it was reported earlier in an animal model of

epilepsy (Nusser et al., 1998) and human epileptic tissue

(Wittner et al., 2002).

DiscussionThe number of PV-immunopositive cells isdecreased in the epileptic CA1 regionAnimal models and human studies have provided evidence

for both the vulnerability (Best et al., 1993; Magloczky and

Freund, 1995; Buckmaster and Dudek, 1997; Zhu et al.,

1997) and the preservation (Sloviter et al., 1991) of

PV-immunopositive interneurons in epilepsy. In the

present study, we observed a decrease in the number of

PV-immunopositive interneurons in the human epileptic

CA1 region, and this reduction was correlated with the

degree of sclerotic cell loss. In the non-sclerotic epileptic

CA1 region with mild cell loss (type 1), a slight decrease

was observed in the number of PV-immunopositive inter-

neurons (Fig. 2B), which was more pronounced in the CA1

region with patchy pyramidal cell loss (type 2). This phe-

nomenon was observed in conjunction with the preservation

of the dense terminal staining in the stratum pyramidale

(Fig. 2C, Table 2). The distribution of postsynaptic target

elements of PV-positive axon terminals remained unchanged

Table 4 Synaptic input of CA1 pyramidal cell AISs

Total length ofAISs (mm)

Average of AIS synapticcoverage (mm synapse/100 mm AIS perimeter,mean 6 SEM)

No. of synapses(terminals)/100 mmAIS perimeter

Ratio of PV-positiveterminals (%)

Average synapticactive zone length (mm)

Control C10 (n = 76) 678.93 2.57 6 0.32 12.23 45.88 0.172C2 (n = 60) 450.47 2.28 6 0.33 10.21 45.65 0.163C6 (n = 40) 466.52 2.01 6 0.34 10.07 44.68 0.151Average 2.28 10.84 45.41 0.162

Epileptic P40 (n = 76) 921.61 3.03 6 0.45 13.67 35.71 0.181P54 (n = 41) 407.58 3.75 6 0.66 13.49 41.82 0.208P79 (n = 40) 440.09 2.75 6 0.37 16.81 50.00 0.156Type 1 average 3.17 14.66 42.51 0.181

P16 (n = 39) 532.08 1.53 6 0.37 7.14 40.98 0.187P15 (n = 63) 577.24 3.24 6 0.51 10.57 36.84 0.264

Type 2 average 2.39 8.85 38.91 0.225Average 2.86 12.34 41.07 0.199

n = number of analysed AISs.


(Fig. 5) and the perisomatic inhibitory input of CA1

pyramidal cells was preserved in the non-sclerotic epileptic

CA1 region (Tables 3 and 4, Fig. 6). A reduction of PV

immunoreactivity without basket cell loss was found in ani-

mal models of ischaemia and epilepsy (Johansen et al., 1990;

Tortosa and Ferrer, 1993; Bazzett et al., 1994; Magloczky

and Freund, 1995; Scotti et al., 1997a), as well as in the

human epileptic dentate gyrus (Wittner et al., 2001). We

found that the loss of PV immunostaining from cell bodies

and dendrites was accompanied by the preservation of their

terminals as well as the inhibitory input of pyramidal cells,

suggesting that here again we are dealing with the decrease

of immunoreactivity rather than the death of the cells. In

animal models of epilepsy, slower synthesis of PV or a

change in conformation due to calcium overloading was

suggested as a possible cause for the reduced PV immunos-

taining (Johansen et al., 1990; Scotti et al., 1997a). This

might also operate in the human hippocampus.

In contrast to the non-sclerotic epileptic hippocampus, in

the sclerotic CA1 region PV-immunopositive interneurons are

lost, the number of PV-immunopositive elements has drama-

tically decreased and usually small numbers of dendrites are

visible in the region (Fig. 2D). The nomenclature of the

human hippocampal formation is still controversial, including

the determination of the border between the CA1 region and

subiculum and the existence of the prosubiculum (Rosene and

Van Hoesen, 1987; Amaral and Insausti, 1990; Duvernoy,

1998). This might explain the contradictory observations con-

cerning the preservation (Arellano et al., 2003) or disappear-

ance (present work) of PV-immunopositive elements in the

sclerotic CA1 region.

PV-immunopositive interneurons receive the highest num-

ber of synapses among the different interneuron types in the

rat hippocampus, and they receive the smallest proportion of

GABAergic input (Gulyas et al., 1999). No difference was

observed in the synaptic input of PV-immunopositive cell

bodies in the epileptic CA1 region compared with the control

(Fig. 3), but the sprouting of mossy fibres in the dentate gyrus

(Sutula et al., 1989; Houser et al., 1990; Represa et al., 1990),

as well as the sprouting of a calbindin-positive excitatory

pathway in the CA1 region (Wittner et al., 2002) is likely

to increase the density of excitatory input to PV-immuno-

positive dendrites further (Blasco-Ibanez et al., 2000;

Magloczky et al., 2000). Direct evidence for enhanced exci-

tation in the CA1 region was shown only in animal models

(Buzsaki et al., 1989; Rafiq et al., 1993; Bragin et al., 1997).

The overexcitation of these interneurons might play a role in

the decrease of PV below the level of immunocytochemical

detection (Bazzett et al., 1994), and/or in the excitotoxic death

of these cells, as was shown earlier for CA1 pyramidal cells

(Sano and Kirino, 1990; Meldrum, 1991, 1993). PV-immu-

nopositive cell bodies located in the CA1 region receive a

much larger excitatory input than those in the dentate gyrus

(10–12 versus 4–5 synapses/cell body perimeter in one sec-

tion, in the CA1 region or the dentate gyrus, respectively;

Wittner et al., 2001). It is possible that these two mechanisms

together, i.e. loss of original targets and overexcitation of the

cells, play a role in the disappearance of PV-immunopositive

interneurons in the sclerotic CA1 region.

Perisomatic inhibition is preserved ifpyramidal cells are presentBasket cells and axo-axonic (or chandelier) cells are respon-

sible for the perisomatic input of principal cells in the hippo-

campus of all examined species, including rat and monkey

(Somogyi et al., 1983; Lewis and Lund, 1990; Seress and

Frotscher, 1991; Freund and Buzsaki, 1996; Halasy et al.,

1996; Martinez et al., 1996) and humans as well (Seress

et al., 1993; Braak et al., 1991). The majority of these

cells are PV immunopositive, but cholecystokinin-containing

basket cells (Lotstra and Vanderhaeghen, 1987) and calbindin-

positive chandelier cells (Wittner et al., 2002) have also been

described in the human hippocampus.

Since the PV content of perisomatic inhibitory cells is

sensitive to epileptic and ischaemic conditions (see above),

some surviving PV-immunopositive cells may not be visible

with PV immunostaining. Therefore, we examined the

perisomatic inhibitory synapses on CA1 pyramidal cells

irrespective of their PV immunoreactivity.

Analysis of synaptic coverage of pyramidal cell somata and

AISs showed that perisomatic inhibitory input is preserved in

the epileptic CA1 region, except in the samples belonging to

the pathological group with patchy cell loss, where a slight,

but significant decrease was observed (Table 3, Fig. 6).

Furthermore, the distribution of postsynaptic targets of

PV-positive terminals in the epileptic tissue was similar to

that of the control (Fig. 5). These results suggest that periso-

matic inhibitory cells may survive in epilepsy, if their targets

are present in the region. The lack of profound changes in

perisomatic inhibitory input in the CA1 region suggests that

other factors are likely to account for the selective vulner-

ability of pyramidal cells.

In the rodent hippocampus, electrical coupling plays an

important role in the synchronization of PV-immunopositive

interneurons (Deans et al., 2001; Meyer et al., 2002). Gap

junction-coupled ensembles of PV-immunopositive cells

(Katsumaruetal., 1988a; Fukuda and Kosaka, 2000) synchron-

ize the firing pattern of pyramidal cells and generate gamma

oscillations in the neocortex and hippocampus (Penttonen

et al., 1998; Galarreta and Hestrin, 1999; Gibson et al.,

1999; Tamas et al., 2000). Zonula adhaerentia have been

observed between PV-immunopositive dendrites in the

human control (Seress et al., 1993) and epileptic hippocampus

(Fig. 4A and B), as well as in the vicinity of gap junctions

in rats (Kosaka and Hama, 1985), and they are thought to

ensure the mechanical connection of dendrites. Our results

suggest that similar coupling mechanisms occur between

PV-immunopositive interneurons in human tissue, which

in turn probably lead to a more effective synchronization

of pyramidal cells that may participate in the maintenance

of epileptic seizures.


ConclusionsThe balance of excitation and inhibition in the human CA1

region is considerably altered during epileptic changes. An

excess excitation is provided by the sprouted CA1 pyramidal

cell axons (Lehmann et al., 2000), as well as by the sprouting

of afferents from the dentate gyrus and/or the CA2 region

(Wittner et al., 2002), and/or from extrahippocampal path-

ways (Lehmann et al., 2000). Certain populations of dendritic

inhibitory cells survive in epilepsy, and participate in the

intense synaptic reorganization (Wittner et al., 2002), while

others are lost (de Lanerolle et al., 1988, 1989; Magloczky

et al., 2000). Perisomatic inhibitory input is preserved in the

non-sclerotic epileptic CA1 region, where pyramidal cells

survive. The preserved perisomatic inhibition together with

an excess excitation and an impaired dendritic inhibition

might lead to an abnormal synchrony in output regions of

the hippocampus, which still contain large enough numbers

of projection neurons. This might have an important role in

the generation and spread of epileptic seizures. In this group

of patients, the preservation and possible increase in the effi-

cacy of the perisomatic inhibitory network may be causally

related to hypersynchrony during seizures. This may partly

explain the difficulties in therapies that rely on the modulation

of GABAergic transmission, that does not distinguish

between perisomatic (controlling synchrony) and dendritic

(controlling input plasticity) inhibition.

AcknowledgementsWe wish to thank Drs M. Palkovits, P. Sotonyi and

Zs. Borostyank}ooi (Semmelweis University, Budapest) for

providing control human tissue, and Ms K. Lengyel,

E. Simon and Mr Gy. Goda for excellent technical assistance.

This study was supported by grants from the NIH (MH

54671), the Howard Hughes Medical Institute (55000307)

and OTKA (T032251) Hungary.

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