Morphology of CA3 neurons in hippocampal slices with nonepileptic and epileptic activity: A light and electron microscopic study

0361-9230/93 $6.00 + .OO Copyright 0 1993 Pergamon Press Ltd.

Brain Research Bulletin, Vol. 32, pp. 329-338, 1993 Printed in the USA. All rights reserved.

Morphology of CA3 Neurons in Hippocampal Slices With Nonepileptic and Epileptic Activity: A Light and Electron Microscopic Study C. SCHORMAIR,*’

D. BINGMANN,?

W. WITTKOWSKI*

AND E.-J. SPECKMANN*

*Institut fur Anatomie, Westfdlische Wilhelms-Universittit Miinster, Vesaliusweg 2-4, 4400 Miinster, Germany flnstitut fur Physiologie, Universitiit Essen, Hufelandstr. 55, 4300 Essen, Germany glnstitut fur Physiologie, Westfa’lische Wilhelms-Universitat Miinster, Robert-Koch-Str. 27a, 4400 Miinster, Germany $Institut fur Experimentelle Epilepsieforschung, Westfa’lische Wilhelms-Universitiit Miinster, Htiflerstr. 68, 4400 Miinster, Germany Received 23 May 199 1; Accepted 29 June 1992 SCHORMAIR, C., D. BINGMANN, W. WITTKOWSKl AND E.-J. SPECKMANN. Morphology ofCA3 neurons in hippocampal and epileptic activity: A light and electron microscopic study. BRAIN RES BULL 32(4) 329-338, 1993.In guinea pig hippocampal slices, relations between morphology and spontaneous bioelectric activity of neurons were studied in control saline and with exposure to the epileptogenic drug pentylenetetrazole (PTZ) for 2-3 h. Light and electron microscopic structures of the CA3 region were analyzed after recording the membrane potential. Neurons in slices kept in control saline exhibited spontaneous aperiodic bioelectric activities partly mixed with rhythmically occurring burst discharges. In slices exposed to PTZ, these periodic burst discharges and/or paroxysmal depolarization shifts (PDS) predominated. Light microscopic comparison focussing on tissue preservation showed no significant differences between control and PTZ-treated slices. Ultrastructural morphology revealed, on the one hand, no differences regarding spine and synaptic densities, and on the other hand, significantly more irregular electron translucent vacuoles within dendrites of PTZ-treated slices being either randomly distributed or clustered. The vacuoles are interpreted as early changes during epileptic activity. slices with nonepileptic

Hippocampus Epilepsy Horseradish peroxidase

Pentylenetetrazol

Morphology

Bioelectric activity

Intracellular staining

METHOD

IN patients suffering from chronic epilepsies, morphological changes of brain structures have often been observed. The observations made in humans were confirmed by findings obtained in animal experiments (26,32). However, the causal linkage between the morphological alterations and the occurrence of epi-

of guinea pigs were cut into 0.2-0.5 mm thick slices perpendicular to their longitudinal axis. The slices were preincubated in saline containing (in mmol/l) NaCl 124, KC1 5, NaHCOj 26, KH2P04 0.5, MgS04 1.25, glucose 11, CaC& 0.15 (33). The saline was equilibrated with 5% CO* in 02, and thermostabilized at 28°C. For intracellular recording and injection of horseradish peroxidase (HRP), slices were positioned on the bottom of a glass chamber. The slices were continuously superfused with saline having the same composition as the preincubation solution except for the concentration of CaCl, (1.75 mmol/l). The temperature was kept at 32°C. Microelectrodes were filled with a solution of 4% HRP (Sigma Type VI), 2 mol/l KCl, and 0.05 mol/l Tris buffer. HRP was injected either iontophoretically by current pulses (2 mA, 500 ms, l/s) or pressure (2 bar, 10 to 60 min) in about half of the slices. Only neurons located deeper than about 100 pm below slice surface were impaled in order Hippocampi

leptic activity remains obscure. The present investigations aimed to contribute to a further clarification of the interdependence of morphology of nervous tissue and of bioelectrical activity. Therefore, in hippocampal slice preparations a correlation was made on the one hand of bioelectrical activity that a) occurred spontaneously (burst and nonburst type) and that b) was induced by epileptogenic drug, and, on the other hand, of morphological appearance of a) the general structure of the entire tissue and of b) special structure of single neurons intracellulary stained. To this purpose, histology and ultrastructure of the CA3 region were analyzed after recording the bioelectrical activity by intracellular microelectrodes.

’ To whom requests for reprints should be addressed.

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-1’ABLt I CKlTtRIA __-__ Evaluated structure Grade 1

2

3

4

5

Pyramidal Cell Penkarya

Optimal preservation of cell structure. Slight alterations of nucleus (e.g. irregular nuclear shape, beginning pyknosis) or cytoplasma (e.g. small vacuoles. clodding of ER. irregular cell limits). Severe tissue damage: shrinkage or swelling of entire cells (“coagulative or edematous cell change” (23)). severe alterations of nuclear and cytoplasmic shape. Cell death with maximal shrinkage or swelling of cells and their isolation from the surrounding neuropil; nuclear pyknosis or swelling of karyoplasm with clumping of nuclear chromatin. Tissue entirely destroyed (cell structures not identifiable).

bOR LXH I MI(‘ROS(‘OPIC

Neurop~l Surrounding Pyramidal Cell Perikarya

____

Main Dendrites of Pyramidal Cells

Neuropll 01‘Stratum Radratum

Optimal preservation of tissue structure. Narrow clefts between neuropil and neurons, slight swelling of some neuronal or glial processes.

Optimal preservation of dendrites. Slight pathological changes with little swelling or shrinkage of dendrites.

Optimal preservation of tissue structure. Swelling of some neuronal and glial processes and astrocytes: beginning degradation of myelin sheaths.

Predominant swelling of perineuronal astrocytic processes. but without general disconnection of neurons and neuropil.

Moderate swelling of dendrites, partly irregular delineation by winding, bulging or strangling.

Predominant swelling of astrocytes. glial and neuronal processes, wide perivascular spaces, without loss of tissue coherence.

Damage of neuropil mainly by swelling of processes. Tissue disrupted.

Massive swelling or condensation and fragmentation of processes.

Spongeous

Tissue entirely destroyed

Tissue entirely destroyed

Tissue entirely destroyed.

regions damaged by slice cutting. After injection, slices were incubated for an additional period of at least I h. The slices were divided into two groups. The control group (35 slices, 22 hippocampi) was superfused with control saline for 2 to 3 h. The PTZ group (20 slices, I I hippocampi) was repeatedly superfused with saline containing 5 mmol/l pentylenetetrazole (PTZ) (7). For morphological evaluation, slices were processed in two steps: in a first step, the slices were fixed with 1.5% glutaraldehyde/ 1% paraformaldehyde in 0. I mol/l phosphate buffer. The slices were embedded in 2% agar-agar and cut into 60 Frn thick sections parallelly to the initial sectional plane. Only regions 50200 pm below slices surface were investigated. For HRP staining the tissue was incubated in 0.05% diaminobenzidine (DAB, Sigma) dissolved in 0.1 mol/l phosphate buffer (pH 7.3), cobalt chloride and nickel ammonium sulfate, and H202 (I). Apart from that, other slices were fixed with 4% glutaraldehyde without DAB staining. In a second step, 60 pm sections were postfixed in 1% osmium tetroxide, dehydrated, and embedded in Epon between two glass slides having been wrapped with aluminium foil. After embedding, material was sectioned again using an ultrotome. Semithin sections were stained with toluidine blue; ultrathin sections were double stained with uranyl acetate and lead citrate. Evaluations were performed after the first and second step of tissue processing. After the first step, all HRP filled cells were to avoid

ASSESSMENl

neuropil

investigated by light microscopy, photographed, and drawn using an optical tube. After the second step, semithin sections and ultrathin sections were studied in the following way: semithin sections were assessed by two independent observers. The preservation state of the tissue was evaluated applying a detailed list of morphological criteria describing progressive grades of tissue damage (Table I). A subjective mean grade was calculated from these results. The samples were statistically compared by the Cltest (15). From both groups to be investigated by electron microscopy, slices were chosen randomly without knowing the results in light microscopic tissue assessment. In ultrathin sections, both density and area of spines as well as the synapse density in the stratum radiatum were evaluated in a clearly defined region about 100 to 150 pm beyond the mossy fiber bundles crossing the apical dendrites. Ultrathin sections to be analyzed were stochastically selected. Electronmicrographs (magnification 13,000X) were taken. Fifty-two areas (each 100 kmL) were assayed by applying a point-counting method to the electronmicrographs ( 10,16,17). Synapses were counted when a postsynaptic density and nearby synaptic vesicles in a presynaptic profile were both clearly visible; spines were counted when they were either attached to a parent dendrite or contained a spine apparatus, and lacked any mitochondria or microtubules [according to (IO)]. The dendritic vacuoles were counted and related to dendritic area. For morphometric analysis only, neurons not stained with HRP were taken.

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lOUrns 7

2

mV --LO

3

--50

Mp,

--60

smin

IOUms

FIG. 1, Spontaneous bioelectric activity of CA3 neurons (hippocampai slices, guinea pig)recorded under control conditions. The tracings of the membrane potentials, recorded by an oscilloscope (MPI) and by an inkwriter (MPZ)are related to each other by numbers. (A) Spontaneous aperiodic fluctuations of MP superimposed by single and grouped action potentials (MP I t-3). (B) Initial aperiodic activity (MPI 1) and subsea$tentdevelopment

of periodically occurring burst discharges(MPI 2-T).

RESULTS

Morphological preservation of CA3 neurons was investigated in hippocampal slices with different nonepileptic and drug-induced epileptic bioelectric activities. In a first series of experiments spontaneous bioelectric activity was recorded under control conditions (hippocampal slices of 55 guinea pigs). CA3 neurons selected for evaluation had a membrane potential of at least -50 mV 10 to 60 min after impalement. The majority of neurons showed spontaneous aperiodic activity (Fig. IA). In slices of 34 guinea pigs, this discharge

pattern was the only spontaneous activity observed. In slices of 2 I animals, periodically occurring burst discharges were found at various depths below the surface of the slice beside the aforementioned activity. These bursts consisted of bell-shaped depolarizations superimposed by trains of action potentials. Bursts often started after the stabilization of the intracellular recording (Fig. 1B) and continued until the end of the experiment. During the recordings, bursts of a single cell were rather constant in shape, amplitude and frequency, while bursts of different cells even in one slice showed a wide variability. The spontaneous bioelectrical events of single CA3 neurons often resembled epileptic discharges. The frequency of bursts typically ranged be-

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50 mV

Ne2

6 50 mV

.._

I

50 mV

PTZ FIG. 2. Parallel recordings of the membrane potentials of two CA3 neurons some 100 Nrn remote from each other (Nel, Ne2). The asynchroneous, aperiodic activity of both neurons observed under control conditions (Ne1,2 l-3) was replaced by rhythmically appearing, synchroneous burst discharges when the slice was exposed to pentylenetetrazole (Ne1,2 4-6; PTZ 7.5 mmol/l).

tween 8 and 20/rnin. Bursts were not synchronized even in neighboring cells (Fig. 2). In a second series of experiments, slices of 13 guinea pigs were exposed to the epileptogenic drug pentylenetetrazole (PTZ). After repeated applications of PTZ, in 11 of 13 trials aperiodic bioelectrical activity as well as asynchroneous bursts of CA3 neurons were replaced by rhythmically appearing burst discharges and paroxysmal depolarization shifts (PDS; Fig. 2). These PDS consisted of a steep depolarization accompanied by two to three full-blown action potentials, a 100-200 ms lasting plateaulike diminution of the membrane potential that was superimposed by abortive spikes, and a steep repolarization that most often was followed by a long-lasting afterhyperpolarization. Epileptic bursts and PDS occurred synchroneously at least in adjacent nerve cells (Fig. 2) and persisted as long as the slice was exposed to PTZ-concentrations of 2-10 mmol/l in the bath saline (7).

Light Microscopical

Findings

Stratum pyramidale and stratum radiatum of both control group (i.e., with nonbursting discharge pattern and with spontaneous burst activity; 34 slices) and PTZ group (i.e., with PTZinduced paroxysmal depolarization shifts; 20 slices) showed different stages of tissue preservation. Well-preserved tissue (Fig. 3) looked very compact. The nerve cell pericarya were smoothly delineated from the surrounding neuropil without perineuronal clefts. Cytoplasmic organelles, especially Nissl bodies, were found in typical distribution. Their nuclei lay centrally with clearly visible nucleoli and showed a fine granular dispersion of chromatin. Large apical dendrites emerged from the soma. Astrocytes were easily recognized by the lack of a discernible pericaryon and their small rim of nuclear chromatin. In moderately damaged tissue CAZsomata appeared either swollen or shrunken, their cytoplasm looked cloddy, vacuolated,

FIG. 3. Light microscopic structure of hippocampal CA3 region and corresponding bioelectric activity. Left, middle, and right column: slices with nonbursting discharge pattern (N), with spontaneous burst activity (B), and with PTZ-induced paroxysmal depolarization shifts (PTZ), respectively. Typical bioelectric activity recorded from pyramidal cells is shown above the micrographs. In each group well-preserved tissue (upper row) is set against severely damaged tissue (lower row). Semithin sections of Epon-embedded slices stained with toluidin-blue. X3 10.

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and irregularly bounded. The nuclear envelope was often enfolded and its chromatin clumped. Apical dendrites showed alterations similar to their somata. Besides perineuronal clefts of varying extension large perivascular clefts were found and the neuropil showed vacuolar degeneration. Astrocytes often exhibited swollen perikarya and processes. In severely damaged tissue (Fig. 3) most neurons were destroyed. They were either entirely shrunken or maximally swollen with only cell detritus left in large round spaces. Both forms of cell changes coexisted within one slice, the swollen cells usually outnumbering the shrunken ones by far. The neuropil appeared incoherent and was densely interspersed with vacuoles of different size. Evaluating the histological preservation of each slice by applying the morphological criteria listed in Table I the grades given to slices in the control group ranged from I .7 to 4.0 with a mean of 2.6 (SD = 0.7) and in the PTZ-group from 1.6 to 4.4 with a mean of 2.9 (SD = 0.8). The two samples statistically did not differ significantly according to the Cl-test (p = 0.05). The characteristics of changes were nearly the same in each slice; the range of changes differed in different slices but was fairly homogeneously within each slice in the defined depth below the cutting surface. The somata of CA3 pyramidal cells of the control (n = 6) and PTZ group (n = 14) identified electrophysiologically and filled with HRP showed characteristic shapes (Fig. 4). Their dendritic tree spread out basally and apically as it is typical for pyramidal cells of this region. Both basal and apical dendrites were densely and uniformly studded with spines. No obvious changes of ramification pattern or selective loss of spines in certain segments of the dendritic tree were noticed after 2-3 h of epileptic activity. Neither in the control group nor in the PTZ group any nodulations or fragmentations were observed along the dendrites, Occasionally. axonal varicosities could be seen.

Ultrastructural investigation of slices of control (n = 17) and PTZ group (n = 14) confirmed light microscopical findings and revealed a great variability of preservation of neuronal somata in a single slice as well as in different slices. Even adjacent pyramidal cells often showed quite different stages of preservation. Concerning tissue damage, shrinkage of cells and condensation of cell organelles were found associated with widened extracellular space. But, also, swelling of cells and their organelles occurred frequently. No specific cell changes could be found or be attributed to certain electrophysiological results obtained in the slice. In the stratum radiatum of both groups of slices, intercellular spaces often appeared widened due to shrinkage of both neuronal processes and glial fibres. The structures involved in synaptic contacts looked unaffected. Many dendrites showed a quite normal structure; others were moderately shrunken or swollen. A conspicuous feature were irregular electron lucent cisterns or vacuoles exceeding 150 nm in diameter (Fig. 5). They were especially observed in PTZ-treated slices. The majority was grouped to clusters and sometimes intermingled with dense bodies. Occasionally they almost filled a whole dendrite but they seemed not to bulge its surface. Such focal clusters apparently interrupted the continuity of microtubules. Whereas a connection of these clustered vacuoles with cell organelles usually was not visible, other more randomly distributed vacuoles seemed to be derived from cisterns of the ER or to be connected with microtubules. Lysosomes, and especially multivesicular bodies, were found to occur in the vicinity of vacuoles. The elongated

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mitochondria looked unaltered. The number of these vacuoles differed significantly in both groups of slices: an average of 30.0 vacuoles per 100 pm2 ofdendritic area (SD = 3 I .7) was counted in control slices in contrast to an average of 82.0 vacuoles per 100 pm’ (SD = 25.2) in PTZ slices. The number of tubules per unit area was not significantly different. As a control, in both groups there were slices not treated with DAB. A comparison of the subgroups shows that marked differences between DABtreated control and PTZ group are apparent. There were, however, also minor differences between DAB-treated and not treated control slices, the latter showing the smallest number of vacuoles. The number of spines evaluated by point counting per 100 pm’ was on an average of 26.3 (SD = 7.0) hit by 30.9 (SD = 6.7) dots in the control group and of 28.2 (SD = 12.2) hit by 32.6 (SD = 9.9) dots in the PTZ group, respectively. The relative area of each spine calculated from hits divided by the number of spines was I 18 in the control group and I. I6 in the PTZ group. The number of synaptic profiles per unit area was on an average of 22.2 (SD = 4.3) in the control group and of 2 I .9 (SD = 6. I) in the PTZ group. This difference is also not signihcant. Pericarya, as well as dendrites of HRP-labelled neurons ol control (n = 6) and PTZ group (tt = IO). were clearly separated from the neuropil leaving more or less wide clefts which were bridged by few axo-somatic synapses. A large nucleus with one or two nucleoli lay centrally in the cytoplasm. Its nuclear envelope was often deeply enfolded. Nuclear chromatin often was clumped. Cytoplasm was densely packed with cell organelles embedded in a dark fine granular matrix. Mitochondria stood out by their less electron dense matrix and their cristae. In few cells of both control and PTZ group they were moderately or severely, but uniformly, swollen. Golgi complex and rough ER were rather conspicuous in HRP-labelled neurons by their electron-lucent and often widened cisterns or saccules stacked in parallel arrays. In contrast to unstained pyramidal cells. IHRPlabelled neurons of both groups contained more vacuoles throughout the cytoplasm. They often formed irregular clusters around the Golgr apparatus. IXS(‘IJSSION

A wide range of pathological alterations has been observed in brain of epileptic patients or has been reported from animal experiments applying convulsant drugs or electrical stimulation of nervous tissue. These pathological lesions involve whole neurons as well as parts of neurons like somata, dendrites, spines, or synapses. Neuronal loss, particularly in certain parts of the human hippocampus, has been described by Mouritzen Dam (20), Scheibel et al. (28), Scheibel (29), Brown (9), Babb and Brown (3), and Sagar and Oxburg (27). Frequently the appearance of dendrites is altered in a characteristic way: administration of kainic acid caused damage of CA3 pyramidal neurons with acute swellings of dendritic segments and spines as earliest change and swelling of astrocytes (22,26). Using electrical stimulation of perforant path Sloviter (31) and Olney et al. (23) provoked similar lesions by sustained stimulation for 2 h and intermittent stimulations for 24 h. Fifkova and Harrefeld (11) showed that long-term activation was followed by spine swellings. Following highfrequency stimulation, Lee et al. (14) noticed a decrease in variability of dendritic spines and an increase in number of axodendritic shaft synapses but no significant changes of axospineous synapses. Nitsch (2 1) applied methoxypyridoxine and found changes in synaptic vesicles and membrane recycling in mossy fibre boutons and a deteriorated appearance of postsynaptic CA3 and CA4 pyramidal cells. Thus, dendritic spines and

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PTZ

FIG. 4. Bioelectric activity (upper row) and light microscopic structure (middle row; photographs and drawings) as well as ultrastructure (lower row) of hippocampal CA3 pyramidal cells intracellularly stained with horseradish peroxidase. Electronmicrographs show parts of the periphery of perikarya. Left and right column: slices with spontaneous nonburst activity (N) and with PTZ-induced paroxysmal depolarization shifts (PTZ), respectively. Horizontal and vertical bars in the drawing indicate interruptions of dendrites. Magnifications: electron micrographs X 12,800, light micrographs X260.

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FIG. 5. Ultrastructure of the stratum radiatum of hippocampal CA3 region with various aspects of dendritic vacuoles, (a) Control slice with spontaneous burst activity (B); (b-e) slices with PTZ-induced paroxysmal depolarization shifts (PTZ). (b) HRP filled dendrite with spines, note the vacuoles (t) within dendrites of PTZ-treated slices: (c) vacuoles derived from cisterns of ER (t) or in connection with microtubules (ft). MB multivesicular body (note irregular course of some microtubules); (d) vacuoles of different size with local rarefication of microtubules and focal agglomeration of vesicles (t). DS dendritic spine: (e) groups of vacuoles and waste products within an edematous part of a dendrite. mitochondria seem not to be altered.

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dendritic synapses seem to react in a specific way after electrical stimulation or application of convulsant substances. In the present experiments, in addition to morphometric evaluation applied by several authors (3,5,10,14,20), electrophysiological and morphological investigations were done in one and the same neurons. These combined studies, as well as quantitative evaluation, revealed that light microscopic pathological alterations of nervous tissue found in various degree in our slice preparations after exposure to PTZ cannot be attributed to effects of this drug. The same alterations were also found in control slices and, hence, were interpreted as unspecific changes. These unspecific changes resemble alterations described in various pathological processes including ischemia (4,8,13,18,19,25,30), and seem to represent a uniform reaction pattern to cytotoxic conditions during slice treatment. A causal linkage with epileptic activity is unlikely. PTZ-induced epileptic activity, however, gave rise to quantitative alterations of dendritic ultrastructure: an increased number of irregular electron translucent vacuoles occurred in dendrites of PTZ-treated slices compared with control slices. They partly seemed to originate from or to be connected with different cytoplasmic organelles such as ER or microtubules. Vacuolar changes of mitochondria that are observed in hypoxic brain and probably reflect pronounced osmotic imbalance (24)

however, were missing. Vacuoles were frequently seen in clusters interrupting the tubular system of dendrites and thereby possibly destroying intradendritic transport mechanisms. Large confluent vacuoles-visible by light microscopy-were also found in experiments on snail neurons exposed to PTZ (2). In these studies, vacuoles finally led to disruption of processes. A rise in metabolism caused by epileptic activity might principally lead to 02-deficiency that could contribute to formation of vacuoles. A marked contribution of such metabolic mechanisms, however, is unlikely because vacuoles appeared in zones both with good and poor 02-supply in our earlier experiments (6). An influence of HRP staining can be excluded because labelled neurons had not been taken into account. Moreover, the question remains whether the DAB reaction itself results in poor tissue preservation, e.g., provoking dendritic vacuolization. Frotscher and Misgeld ( 12) describe unaltered preservation in DAB-treated nervous tissue. The differences in subgroups seen in the present investigation can be assumed to be due to different fixation procedures rather than to DAB reaction. As a whole, the bioelectric activity itself seems to induce ultrastructural changes described that may lead to cellular alterations, e.g., by destroying intraaxonal and intradendritic transport mechanisms needed to preserve cellular function.

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