Long-term maintenance of mature hippocampal slices in vitro

ELSEVIER

Journal of NeurosCIence Methods 98 (2000) 145-154

JOURNAL OF NEUROSCIENCE METHODS www.elsevier.com!1ocate/jneumeth

Long-term maintenance of mature hippocampal slices in vitro Zhongmin Xiang a.l, Sabina Hrabetova a,2, Shaye 1. Moskowitz a, Patrizia Casaccia-Bonnefil a,3, Steven R. Young a, Volker C. Nimmrich a, Henri Tiedge a, Stephen Einheber b, Sergei Karnup a, Riccardo Bianchi a, Peter J. Bergold a.* d

Department of Physiology and Pharmacology. State Unwerslty oj Nell' York-Downstate Medical Center, Box 29, 450 Clarkson Avenue, Brooklyn, Nell' York 11203. USA b Department of Cell Biology, New York University School of Medicine, 550 First Avenue, Nell' York, NY 10016, USA

Received 23 August 1999; received in reVised form 22 February 2000; accepted 23 February 2000

Abstract

Cultures of primary neurons or thin brain slices are typically prepared from immature animals. We introduce a method to prepare hippocampal slice cultures from mature rats aged 20-30 days. Mature slice cultures retain hippocampal cytoarchitecture and synaptic connections up to 3 months in vitro. Spontaneous epileptiform activity is rarely observed suggesting long-term retention of normal neuronal excitability and of excitatory and inhibitory synaptic networks. Picrotoxin. a GABAergic Clchannel antagonist, induced characteristic interictal-like bursts that originated in the CA3 region, but not in the CAl region. These data suggest that mature slice cultures displayed long-term retention of GABAergic inhibitory synapses that effectively suppressed synchronized burst activity via recurrent excitatory synapses of CA3 pyramidal cells. Mature slice cultures lack the reactive synaptogenesis, spontaneous epileptiform activity. and short life span that limit the use of slice cultures isolated from immature rats. Mature slice cultures are anticipated to be a useful addition for the in vitro study of normal and pathological hippocampal function. © 2000 Elsevier Science B.V. All rights reserved. Keprord:,.. Rat; Mossy fiber; Hippocampal slice culture; Neuron; Glia; Electrophysiology

1. Introduction

In vivo studies of brain function have been constrained by the limited access of the brain, the bloodbrain barrier, and other systemic effects. To overcome these limitations, neuroscientists have developed a variety of in vitro approaches. In vitro neurons are either used acutely or cultured for days to weeks. Despite their great utility, acute preparations rarely remain alive

* Correspondmg author Tel.: + 1-718-2703927; fax: + 1-7182702241. E-mail address: [email protected] (P.J. Bergold) 1 Present address' Department of Psychiatry. Mount Sinal School of Medicine, Gustave Levy Place, New York, NY 10029. USA. 2 Present address: Department of PhYSIOlogy; New York University School of Medicine, 550 First Avenue, New York, NY 10016. USA. 3 Present address. Department of Neuroscience, Robert Wood Johnson Medical School, University of Medlcme and Dentistry of New Jersey. 675 Hoes Lane, Piscataway. NJ 08854. USA.

for more than 24 h and are unsuitable for long-term studies. An alternative approach uses cultures of embryonic or perinatal neurons. Embryonic or perinatal neuronal cultures remain viable for many weeks, yet never acquire the anatomical and functional stability of mature neurons. These observations suggest that longterm neuronal stability is only established after in vivo neurogenesis and synaptogenesis is completed. Mature neurons and synapses have been maintained for hours to days in vitro (DIV) in thin brain slices. The hippocampus is a preferred brain region for the preparation of thin slices due to the large number ofaxons and synapses maintained in a 400 11m transverse slice of tissue (Giihwiler et al., 1997). Hippocampal slice cultures are most successfully prepared from animals no older than post-natal day 10 (PlO) (Giihwiler et al., 1997). To date, there has been no reliable method to place hippocampal slices isolated from rats older than 12-15 DIV into long-term, stable culture.

0165-0270/00/$ - see front matter (9 2000 ElseVier SCience B.V. All rights reserved. PH: SOI65-0270(00)00197-7

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Hippocampal slices isolated from rats older than P15 have been typically maintained for no more than 4 days (Kleinberger-Doron and Schramm, 1990; Kantor et al., 1996). We developed the means to place acute hippocampal slices into long-term culture from mature rats aged 20-30 days. The histology and physiology of these cultures have been analyzed for up to 3 months in vitro. These studies have provided strong evidence that mature slice cultures display the longevity and stability that is characteristic of the adult brain. These cultures provide a valuable in vitro preparation to study longterm changes in neuronal function. 2. Methods 2.1. Adult hippocampal slice cultures

Sprague - Dawley rats aged 20- 30 days were anaesthetized with ketamine (100 J.!g/g) and decapitated. The skull was exposed and the brain isolated . Using a razor blade, the hemispheres were separated with a mid-sagittal cut. All remaining manipulations were done using sterile technique. The hemispheres were transferred to a hood and immersed in modified Gey's balanced salt solution (mGBSS) for 20- 30 min that had been prechilled to 4°C and bubbled vigorously with a 95% O 2/5% CO 2 gas mixture. mGBSS is (in mM): CaCI 2 , 1.5; KCl, 4.9; KH 2 P0 4 , 0.2; MgCI 2 , 11.0; MgS0 4 , 0.3; NaCl, 138.0; NaHC0 3 , 2.7; Na 2HP0 4 , 0.8; NaHEPES, 25; glucose 6% (w/v), pH 7.2. An individual hippocampus was isolated and 400 J.!m thick transverse slices prepared using a Mc Illwain tissue chopper (Brinkman Instruments, Westbury, NY). The hippocampus was oriented on the chopper so that the blade cut slices perpendicular to the long axis of the septal hippocampus. The slices were placed in ice-cold mGBSS and observed using a dissection microscope. Only slices with uninterrupted bright transparent neuronal layers were plated onto Millicell CM filters (Millipore, Bedford, MA). One to three slices were plated onto each filter and the filters were placed into a six-well dish. For 2 days, cultures were maintained in 1 ml of elevated potassium slice culture media (25% horse serum (GIBCO-Life Technologies), 50% Basal Essential Media-Eagles, 25% Earle's balanced salt solution (EBSS), 25 mM NaHEPES, 1 mM glutamine, 28 mM glucose, pH 7.2) and were incubated at 32°C in a 5% CO 2 atmosphere. The slice cultures were switched to physiological potassium slice culture media (25% horse serum, 50% Basal Essential Media-Eagles, and EBSS modified so that the potassium concentration was 2.66 mM). After 7 DIV, cultures were maintained in physiological potassium slice culture medium containing 5% horse serum. Cultures were fed weekly if the insert contained one slice, every three days for two slices, and every two

days for three slices. Slice cultures from post-natal day 9-11 were prepared following the method of Stoppini et al. (1991). Slice culture thickness was determined by recutting the slice cultures on a Mc Illwain tissue chopper perpendicular to the membrane insert followed by direct measurement. 2.2. Timm stain

Timm stain was performed with slight modifications of the method of Cavazos et al. (1991). Slice cultures were treated with 0.1 M phosphate-buffered 1% sodium sulfide solution for 5-7 min, fixed in 95% ethanol for 15 min, and left in 70% ethanol overnight. After hydration, cultures were removed from the Millicell-CM insert using a rubber policeman. The slices were put in the dark for 15- 30 min in developing solution that contained 50% Gum Arabic, 5.6% hydroquinone, and citrate buffer (24% sodium citrate and 25% citric acid), in the proportion of 6:3:1. Immediately before use, 0.4 ml 1.7% (w/v) silver nitrate was added to 50 ml of developing solution. The staining was stopped with distilled water, then counter stained with cresyl violet. Mossy fiber sprouting was evaluated in the supragranule region by the criteria of Cavazos et al. (1991). 2.3. DiI staining and biocytin studies

Small crystals of DiI (1 ,I'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-5,5'-disulfonic acid, Molecular Probes, Eugene, OR) were applied onto the slices using a glass pipette under dissection microscope. The cultures were fixed in 0.2 M phosphate-buffered 4% formaldehyde and stored in the dark at room temperature for 3- 7 weeks. DiI epifluorescence was observed using an Axiovert 100 microscope (Carl Zeiss Inc., Thornwood, NY) with a Rhodamine filter set. To fill cells with biocytin, sharp glass electrodes (60- 70 M?) were filled with 1.5 M potassium acetate and 1.5110 biocytin and placed into the pyramidal cell layers of CA3 or CAlor the granule cell layer of the dentate gyrus. 2.4. Extracellular recording

Slice cultures attached to a Millicell-CM insert were transferred to an air-interface slice recording chamber (Fine Science Tools, Foster City, CA) that was modified to fit the membrane insert. Slice cultures were perfused with artificial cerebral spinal fluid (aCSF, in mM: NaCI, 124; KCI , 3; MgCI 2 , 1.6; CaCI 2 1.7; NaH 2P0 4 1.2; NaHC0 3 , 25; D-glucose, 11; pH 7.3) and bubbled with 95% O 2 and 5% CO 2 at 32°C. Mossy fibers or Schaffer collaterals were stimulated with bipolar tungsten electrodes. Glass electrodes filled with aCSF (4-8 Mn) were placed in the pyramidal cell layer

Z . Xiang et al. 'Journal of NeuroSClence Methods 98 (2000) 145- 154

or stratum radiatum of the CA3 or CAl regions to record field potentials. Amplified signals were digitized with a McAdios digitizer (GW Instruments, Somerville, MA) and processed with a Macintosh IIsi computer using Superscope 2.0 software. Baseline recording was started at least 30 min after placing the slices in the recording chamber and another 10 min after positioning the electrodes. To detect spontaneous epileptiform activity, at least 20 min of unstimulated recording was collected. The value of maximal stimulation was determined by assaying the input - output relationship before baseline recording. Maximal stimulation (0.1 ms, 300 ~A) was delivered every 30 s. In some experiments, 3-(2-carboxypiperazin-4yl)propyl-l-phosphonic acid (CPP, 50~M), 6-cyano-7-nitroquinoxaline-2,J-dione (CNQX, 10 ~M), or picrotoxin (25 ~M) was added to the aCSF. The effects of CPP and CNQX on extracellular responses were assessed by measuring the amplitude of the field EPSP. All drugs were purchased from Sigma (St. Louis, MO). 2.5. Intracellular and optical recordings

A culture in the Millicell-CM insert was submerged in a coverslip-bottomed recording chamber and superfused at 3-5 ml/min at 30 - 32°C with (in mM): NaCl, 124; NaHC0 3 , 26; KCI, 2.5; MgCI~, 1.6; CaCI 2 , 2.0 and D-glucose, 10 (pH 7.4) that was gassed with 95 0ft, O 2 and 5% CO 2 , The chamber was mounted on a Nikon Diaphot inverted microscope with optical access to the slice through the glass coverslip and the culture insert membrane. Intracellular recordings were performed in CAl and CA3 pyramidal cells with sharp electrodes containing potassium acetate (0.4 M) and calcium green-l (0.5 mM; C3010, Molecular Probes, Eugene, OR). The electrode resistance was 80-120 MO. Current-clamp recordings were amplified (Axoclamp-2A; Axon Instruments, Foster City, CAl, displayed on an oscilloscope (DSO 400; Gould. Valley View, OH) and on a chart recorder (Gould T A240). A digital stimulator (PG 4000; Neuro Data Instruments, New York, NY) timed intracellular square-wave current pulses. Hyperpolarizing pulses ( - 0.1 to - 1.0 nA, 150-300 ms) were applied to monitor the condition of the cell membrane and the input resistance (Rm; calculated from the amplitudes of the voltage responses divided by the intensity values of the injected current steps). Depolarizing pulses (0.5 - 1.5 nA, 500 ms) were applied to evaluate the distribution and magnitude of the calcium responses from the recorded cell. When recorded with synchronized optical recordings, membrane voltage signals were sampled at 6 kHz and filtered at 1500 Hz for responses to intracellular current pulse injections. Optical recording of picrotoxin-induced bursts (e.g. Fig. 4) required lengthy periods of data acquisition. To keep storage requirements within the limits of the software

147

(see below) the sampling rate was reduced to 800 Hz and the trace was filtered at 250 Hz. Eighteen neurons with stable resting membrane potential and overshooting action potentials were used for this study. Cells were filled with calcium green-1 ejected from the intracellular recording electrodes using 350 ms, - 0.5 nA current pulses at 50% duty cycle for 15-25 min. Epi-illumination was provided by a 150 W xenon short gap bulb (XBO 150 W CR OFR; Osram, Germany) linked to the microscope by a fused silica fiber optic bundle (77578; Oriel, Stratford. CT). Filled cells were viewed through a Nikon XIO Fluor objective (n.a. = 0.5) and standard fluorescein filter set (Nikon B-2E). Simultaneous acquisition of calcium green-l fluorescence and membrane potential was performed as described previously (Lasser-Ross et aI., 1991; Bianchi et aI., 1999). Fluorescence images were acquired from a thermoelectrically cooled CCD camera (CH230; Photometrics, Tucson, Arizona) run in frame-transfer mode. A software-driven timer/pulser (Master-8; AMPI, Israel) synchronized optical and electrical recordings. Fluorescence values were averaged over an area of interest selected within the fluorescence image. Relative intracellular Ca 2 + levels were expressed as the change in fluorescence divided by the resting fluorescence (flF/ F). The applied equation was flF/F (%) = 100 x (F,Fb)/(Fb - Fa), where F, is the average fluorescence of the area of interest in each image, Fb is the baseline fluorescence of the area of interest averaged over at least five images prior to stimulation, and Fa is the autofluorescence measured at an equivalent location in the slice where no stimulus-associated signal was detected. Normalized in this way, dye fluorescence serves as a monotonic measure of intracellular Ca 2 + concentration (Callaway et aI., 1993; Young et aI., 2000). To eliminate bleaching and phototoxic damage during an experimental run, neutral density filters were used in the excitation pathway. Optically recorded cells were considered acceptable when the fluorescence responses (flF/F) to brief trains of action potentials were greater than 10%. The pyramidal cell layer and stratum oriens and stratum radiatum were easily distinguished and Ca 2 + signals were clearly detected from the somatic and dendritic areas of recorded cells. Picrotoxin was added to the perfusate at a final concentration of 25 ~M. Data were analyzed using pClamp (Axon Instruments), Transform (Fortner Software LLC, Sterling, VA), and SigmaPlot (SPSS Inc., Chicago, IL) software. Values are mean ± S.E.M. 2.6. Electron microscopy

Cultures were fixed essentially as described by Buchs et al. (1993). Slices were fixed in a solution containing 1.5% glutaraldehyde and 1% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 for 2 h at 4°C. After

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washing in 0.1 M phosphate buffer, the cultures were removed carefully from the insert membrane and incubated in 2% osmium tetroxide in 0.1 M phosphate buffer for 1 h and embedded in EMbed 812 (Electron Microscopy Sciences, F ort Washington, PA; Milner and Bacon, 1989). Ultrathin sections (50~65 nm) of the embedded slices were collected on copper grids and counterstained with uranyl acetate and Reynold's lead citrate. Sections were analyzed on a Philips CMIO electron microscope. 3. Results 3.1. Long-term retention of hippocampus structure

Transverse slices (400 )lm) were prepared from hippocampi isolated from P20~30 rats and placed into long-term culture. Neuronal damage resulting from loss of cerebral blood flow, trauma, hypoxia, and denervation during dissection was minimized by the use of ketamine anesthesia, rapid isolation followed by prolonged chilling of the brain, and slicing perpendicular to the long axis of the hippocampus. Cultures were initially maintained at 32°C in media containing elevated potassium. Slice cultures degenerated within days if immediately placed into media containing physiological potassium (data not shown). After 7 DIV, cultures were shifted to 35°C in media containing physiological potassium. Spontaneous neuronal loss following slice preparation was analyzed using propidium iodide (PI) and quantitative video microscopy. PI is a cell impermeable dye that specifically enters dying cells and stains DNA. PI epifluorescence was assayed by quantitative video microscopy. At 1~2 DIV, spontaneous neuronal loss was maximal in the CAl and CA3 pyramidal cell layers and the granule cell layer of the dentate gyrus (CAl, 63.1 ± 5.2; CA3, 63.3 ± 6.3; DG 66.1 ± 6.9; values ar~ mean arbitrary fluorescence units ± S.E.M.). By 6 DIV, PI epifluorescence was no longer detectable in the pyramidal cell layers. By 10 DIV, PI epifluorescence was no longer detected in the granule cell layer. Between 10 and 80 DIV, PI epifluorescence was never observed. Necrotic tissue appeared in the slice cultures as a result of isolation and slicing of the hippocampus. Initially, necrotic tissue was present on the cut surfaces of the slice culture and in areas containing extrinsic axonal tracts (fimbria-fornix, angular bundle, and alveus). Despite these areas of degeneration, cell loss as a result of dissection was complete by 10 DIV. Neuronal loss remained undetectable by the PI assay for an additional 70 DIV. Adult slice cultures maintained the laminar structure of the hippocampus for months in vitro. The slice cultures showed modest thinning from 400 to ~ 250 )lm at 30 DIV. Synaptic areas remained opaque and the

pyramidal and granule cell layers remained transparent. Cresyl violet stain of cultures at 30 DIV revealed the pyramidal cell and granule cell layers as dense, thin lamina (Fig. 1). In some cultures, there was some dislocation of cells from the lower limb of the granule cell layer (data not shown). At 66 DIV, there was some spreading of the pyramidal cell layers, yet neurons remained densely packed. These data suggest that neuronal lamina of the hippocampus are well maintained in culture. Biocytin-filled CAl and CA3 pyramidal cells retained elaborate dendritic arborization at 30 DIV (Fig. I C). The axons of two filled CA3 neurons were observed to project in stratum radiatum to the CAl region (data not shown). Granule cells resembled granule cells in vivo (data not shown). Slice culture ultrastructure was also examined in CAl pyramidal cell layer and in stratum radiatum at 21 DIV. In the CAl pyramidal cell layer, neuronal nuclei were densely packed and contained a nucleus consisting largely of euchromatin. In CAl stratum radiatum, numerous synaptic connections were observed. Synapses on dendritic spines were commonly observed (Fig. ID). Astrocytic processes were observed that isolated individual synapses. These data suggest that neuronal morphology and ultrastructure were maintained in slice cultures. 3.2. Long-term maintenance of hippocampal synaptic networks in vitro

The long-term maintenance of neuronal laminae suggested that the laminar arrangement of hippocampal synapses would be preserved as well. We tested this by analyzing the projection of the mossy fiber pathway in mature slice cultures. Mossy fibers project from dentate granule cells to hilar interneurons and to CA3 pyramidal cells and form en passant synapses in stratum lucidum on the proximal dendrites of CA3 pyramidal cells (Shepherd, 1998). After perforant path lesion or severe epileptiform activity, mossy fiber axons form new synapses in the inner molecular layer of the dentate gyrus and in pyramidal cell layer of CA3, a process termed mossy fiber sprouting (Laurberg and Zimmer, 1980; Sutula et aI., 1988). Mossy fiber sprouting is a common feature of immature slice cultures (Robain et aI., 1994; Sakaguchi et aI., 1994; Casaccia-Bonnefil et aI., 1995; Gutierrez and Heinemann, 1999). The projection of the mossy fiber pathway in mature slice cultures at different DIV was studied using Timm stain and the fluorescent tracing dye DiI (Fig. 2). Regardless of the time after plating, mossy fiber termini were observed in the hilus and stratum lucidum. A small number of mossy fiber termini were observed in the granule cell layer of the lower limb of the dentate gyrus. This synaptic reorganization was observed by 10 DIV. The projection of the mossy fiber path was unchanged between 10 and 80 DIV (Table 1). This synaptic reorga-

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Fig. I. Mature slice cultures retain the tissue architecture and ultrastructure of a transverse hippocampal slice. (A) A hippocampal slice cultured for 30 DIV stallled with cresyl vIOlet. Pnnclpal neuronal areas are present as dense narrow lamina. Bar, 250 ~m. (B) A hippocampal slice cultured for 66 DIV and stained with cresyl violet. Bar, 250 ~m. (Cl A CA3 pyramidal cell filled with biocytlll. Bar, 100 ~m. A presumptive axon from another biocytin-filled CA3 pyramidal cell is observed. (D) An electron micrograph of CAl stratum radiatum. Shown are examples of dendrites (D), a presynaptic terminal filled with synaptic vesicles (PT), and a presumptive astrocytic process in close proximity to a presynaptic termlllal and a dendnte (arrow). Bar, 500 nm.

nization differed from mossy fiber sprouting described in vivo or in slice cultures since terminals were observed in the granule cell layer rather than the inner molecular layer and only the lower limb of the dentate gyrus was affected (Laurberg and Zimmer, 1980; Sutula et al., 1988; Robain et aI., 1994; Sakaguchi et aI., 1994; Casaccia-Bonnefil et aI., 1995; Gutierrez and Heinemann, 1999). We then examined the long-term stability of electrophysiological responses in mature slice cultures. Schaffer collateral stimulation evoked a field excitatory post-synaptic potential (fEPSP) in CAl stratum radiatum and a single population spike and fEPSP in the CAl pyramidal cell layer. fEPSP amplitude in the CAl pyramidal cell layer was examined from 1 DIV to 80 DIV (Fig. 3). fEPSP amplitude in CAl decreased steadily from 1 to 4 DIV, increased to ~ 2 mV at 5 DIV and remained unaltered until 80 DIV. These data suggest a transient synaptic depression during the first 4 DIV. After recovery at 5 DIV, synaptic responses re-

Table I Mlllimal mossy fiber sproutlllg Days 10-20 20-30 30-40 40-50 50-80

III

vitro

III

P25 shce cultures"

Mossy fiber llldex

N

0.5 ± 0.2 0.9 ± 0.2 0.6 ± 0.3 0.9 ± 0.2 0.6 ± 0.2

7 II 9 9 7

" Slice cultures were maintained for the indicated days in vitro and the mossy fiber projection visualized using Timm's stain. Mossy fiber sprouting was scored based on the criteria of Cavazos, et al.: 0, no staimng; I, sparse granular staining; 2, more numerous stained granules III a continuous distribution: 3,promlllent granules in a continuous dlstnbutlOn With occasional patches of confluent stallling; 4, prominent, confluent granular stainlllg; 5, confluent dense laminar stallling that extends to the inner molecular layer. Values are mean ± S.E.M. The number of cultures analyzed (N) for each time III vitro is indicated. The mossy fiber projectIOn did not Significantly change from 10 to 80 DIV (ANOVA, F= 1.54, P=0.19).

150

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Juurnal ot Nell fu s('[encc Methud" 98 (2000) 145- 154

A

-

Fig. 2

A2

Ai Control

2mV

81

A3 Picrotoxin

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1 min

SO

tJFlF

~

82

SOOms

83

Co>-

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<"'I

-...;: ........

SO

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<40

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0

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Z. Xiang et al. Journal of Neuroscience Methods 98 (2000) 145-154

A

21 DIV

63 DIV

BODIV

L

B

6·9

DIV

c

Control

Cpp

10·19 20·40 60·80 30 19 14

CNQX

~

L Fig. 3. Mature slice cultures maintam the synaptic responses of a transverse hippocampal slice. (A) Representative recordings of Schaffer collateral-evoked responses from the CAl pyramidal cell layer at 21 DIV and 80 DIV and from CAl stratum radiatum at 63 DIV. Scale bar: vertical, 0.5 mY; horizontal,S ms. (B) Average population spike amplitudes recorded m the CAl pyramidal cell layer in response to maximal stimulation of the Schaffer collaterals from I to 80 DIV. The field potential amplitude from cultures 3 DIV and 4 DIV are slgmficantly reduced from I DIV (ANOVA F=4.6747, P
main stable until 80 DIV. The contributions of NMDA and AMP A receptor activation to the fEPSP were examined using the NMDA receptor antagonist CPP

lSI

(50 11M) or the AMPA receptor antagonist CNQX (10 11M) (Fig. 3C). CPP treatment resulted in a small reduction (4.7 ± 2.7%, n = 3) in the fEPSP while CNQX resulted in a large reduction in the fEPSP (73.9 ± 1.7'Yo, n = 3). These data suggest that most of the Schaffer collateral-evoked fEPSP results from AMPA receptor activation. The appearance of spontaneous epileptiform activity in slice cultures is another indication of changes in synaptic networks or in intrinsic neuronal excitability. Epileptiform activity was examined by recording extracellulariy in the pyramidal cell layer of CAl in mature slice cultures. Spontaneous epileptiform activity was defined as any unstimulated event greater than 0.5 mV in amplitude and longer than 50 ms in duration. In recordings from 278 mature slice cultures between 10 and 28 DIV, only two cultures displayed spontaneous epileptiform activity. Between 30 and 80 DIV such activity was observed in one out of 24 cultures. The absence of spontaneous activity suggests that mature slice cultures retain a normal balance of excitation and inhibition for many months in vitro. Immature slice cultures also rarely show spontaneous epileptiform activity before 21 DIV. However, spontaneous epileptiform activity was observed in ten out of 31 of immature slice cultures between 21 and 28 DIV. After 28 DIV. spontaneous epileptiform activity was observed in 36 out of 39 cultures. These data suggest that epileptiform activity is a rare event in mature slice cultures. In contrast, spontaneous epileptiform activity was frequently observed in immature slice cultures following 28 DIV. A possibility exists that absence of spontaneous epileptiform activity could be due to an inability of mature cultures to retain a synaptic network that supports epileptiform activity. Picrotoxin. a GABA A Clchannel antagonist and convulsant. blocks synaptic inhibition and induces rhythmic synchronized bursting of CAl and CA3 pyramidal cells in acutely prepared hippocampal slices (Miles and Wong, 1987). We tested

Fig. 2. Mature slice cultures main tam the synaptic connections of a transverse hippocampal slice. Panel A, a hippocampal slice cultured for 30 DIV. The mossy fiber path was visualized usmg Tlmm stam and neuronal cell soma was stained with cresyl violet. Minimal mossy fiber sprouting was observed. Bar, 250 Ilm. Panel C, the mossy fiber pathway and three CA3 pyramidal cells stamed With DII in a slice culture maintained 14 DIV. Bar, 50 Ilm. Panel B, a high-power view of the dentate gyrus. No mossy fiber sprouting is observed in the upper limb of the dentate gyrus. In the lower limb, there is both sparse granular staimng (arrowheads) and more numerous granules m a continuous distribution (arrow). Bar. 50 Ilm. Fig. 4. Blockade of GABAA-mediated inhibitIOn mduced epileptiform interictal discharges in mature slice cultures (A) extracellular recordings from the CAl pyramidal cell layer before (AI) and 60 mm after (A2 and A3) addition of picrotoxin (25 IlM). No electrical activity was recorded in the control solution (AI), while plcrotoxm induced spontaneous epileptiform discharges (A2). A discharge is shown in A3 at a faster time scale. (B) mtracellular Ca2+ and voltage recordings from a CA I pyramidal cell in the presence of picrotoxin (25 IlM). BI, image of a CAl pyramidal neuron filled with the Ca 2 + -sensitive dye calcium green-I showing normalized [Ca 2 + 1, increase at the peak of the first burst discharge Illustrated m panel B2. Stratum oriens (or), the pyramidal cell layer (pel), and stratum radiatum (rad) were Identified m higher resolutIOn Images of the same field (not shown). The somatic (s. solid line) and dendritic (d, dashed Ime) areas are boxed where fluorescence changes were measured m panels B2 (somatic) and B3 (somatic and dendntlc). B2, simultaneous intracellular somatic calcium (Ca 2 + ) and voltage (Vm) recordings from the cell m panel BI. Fluorescence was integrated over successive l25-ms time penods and normalized to restmg fluorescence. Low pass filtering reduced action potential amplitudes in the voltage recordmg The resting membrane potential was - 63 mY. Epileptiform burst discharges are associated with transient intracellular Ca 2 + mcreases B3, shown on a faster time scale are the first burst illustrated in B I (Vm) and the associated Ca2 + mcreases (Ca 2 +) in the somatic area (s, solid line) and the prOXimal region of apical dendrites (d, dashed Ime).

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if mature slice cultures retained the ability to support picrotoxin-induced epileptiform activity. Bath application of picrotoxin (25 flM) to mature slice cultures maintained for 21 DIV induced rhythmic bursting in CA3 and CA 1 pyramidal cells (Fig. 4). In six slices, bursts occurred at a frequency of 0.079 Hz and consisted of a primary burst followed by five to eight afterdischarges with half-width of 0.76 ± 0.023 s (n = 8). Picrotoxintreated cultures maintained for 42 DIV displayed similar rhythmic burst activity in CA3 and CAl (data not shown). The size, shape, and duration of the bursts did not significantly change between 21 and 42 DIV. These data suggest that mature slice cultures retained the ability to support picrotoxin-induced bursts. In acute hippocampal slices, picrotoxin-induced bursts originate from the CA3 region and spread to the CAl region (Wong and Traub, 1983). These bursts arise from synchronized activity of CA3 pyramidal neurons that results from recurrent excitatory synapses among CA3 neurons. To test if picrotoxin-induced bursts originated in the CA3 region of mature slice cultures, a cut was made across the Schaffer collateral resulting in isolation of the CA3 and CAl regions. Burst activity remained in the isolated CA3 region but was no longer recorded in CAL These data suggest that CA3 pyramidal cells retained the synaptic connections needed for picrotoxin-induced bursts. Intracellular recordings were performed from CA 1 and CA3 neurons. The average resting membrane potential (Vrest ) and input resistance (Rm) of CAl cells were - 63.4 ± 2.3 m V (n = 8) and 63.8 ± 4.6 MQ, respectively. In CA3 cells, Vres t was - 66.6 ± 2.9 mV and Rln was 74.1 ± 6.8 MQ (n = 10). Changes in intracellular free Ca 2 + were monitored in pyramidal cells filled with the indicator dye, calcium green-I. Picrotoxin-induced bursts were accompanied with increases in somatic and dendritic Ca + 2 concentration (Fig. 4B) These data illustrate the presence of voltage-gated calcium channels that activate during bursting activity.

4. Discussion

4.1. Long-term retention of hippocampal structure in adult hippocampal slice cultures In this study, we characterized hippocampal slices prepared from 20- to 30-day-old rats. Previous to this work, neither dissociated neurons nor slice cultures of this age have been efficiently placed into long-term culture. A striking feature of these slice cultures is their long-term in vitro stability - the laminar structure (Fig. 1) and synaptic networks (Figs. 2 and 3; Table 1) of a hippocampal slice are retained for up to three months. Schaffer collateral-evoked responses remain unchanged from 5 to 80 DIV and are largely mediated by AMPA

receptors (Fig. 3). Recurrent CA3 excitatory synapses are retained (Fig. 4). Spontaneous epileptiform activity was rarely observed up to three months in vitro which is consistent with a long-term balance of excitatory and inhibitory synaptic transmission. The maintenance of a balance of excitatory and inhibitory transmission is further supported by the uniformity of picrotoxin-induced bursts in the hippocampal region of cultures between 14 and 42 DIV (Figs. 3 and 4). However, the levels of excitability in the dentate gyrus were not assessed. In addition, the possibility remains that other properties of mature cultures changed in vitro but did not result in changes in fEPSP amplitude (Fig. 2) nor in the appearance of spontaneous epileptiform activity. Despite the absence of spontaneous epileptiform activity in mature slice cultures, exposure to picrotoxin induced epileptiform activity between 14 and 42 DIV. In acutely prepared hippocampal slices, following blockade of GABA A receptors, recurrent excitatory CA3 synapses are thought to synchronize CA3 bursts and the synchronized bursts can be recorded in CAlor CA3 pyramidal cells (Miles and Wong, 1987). Picrotoxin-induced bursts originate in CA3 and are transmitted via the Schaffer collaterals to CAl (Wong and Traub, 1983). In mature slice cultures, picrotoxin-induced bursts have similar duration, inter-burst intervaL and burst frequency as those recorded in acute slices (Fig. 4). In intact slice cultures, bursts were recorded in both the CA3 and CAl regions. After the Schaffer collaterals were cut, bursts continued in CA3 , but were absent in CAL The presence of picrotoxin-induced bursts in the isolated CA3 region suggests the long-term maintenance of excitatory and inhibitory circuits in the CA3 region. The absence of bursting activity in the isolated CAl region suggests that the CAl region does not develop the synaptic circuitry to sustain picrotoxin-induced bursting. The similarity of the response to picrotoxin in slice cultures maintained 42 DIV and in acutely prepared slices suggests long-term in vitro maintenance of hippocampal circuitry. A minor rearrangement of the mossy fiber pathway was observed in mature slice cultures (Fig. 2). Mossy fiber termini were found in the hilus, stratum lucidum and the granule cell layer in the lower blade of the dentate gyrus. Termini were rarely found in the inner molecular layer (Fig. 2, Table 1). Termini in the granule cell layer were observed as early as 10 DIV and may result from the displacement of granule cells occurred either during slicing or during the first few days in vitro. The termini observed in the granule cell layer of mature slice cultures differ from the mossy fiber sprouting induced by epileptiform activity or by deafferentation (Laurberg and Zimmer, 1980; Sutula et aI., 1988). In these circumstances, mossy fibers sprout to the inner molecular layer of both blades of the dentate gyrus and the sprouting is often associated with altered hippocampal excitability (Sutula et aI., 1988). In mature slice cultures, mossy fiber

Z. Xrang ct al. Journal oj Neuroscience Methuds 98 (2000) 145- 154

terminals in the granule cell layer appear rapidly, but the density of termini does not increase over time nor do termini appear in the inner molecular layer. In addition, only the lower blade of the dentate gyrus was affected and slice culture excitability did not change (Fig. 3; Table 1). The lack of change of the mossy fiber projection between 10 and 80 DIV provides additional evidence for the long-term stability of synaptic networks in hippocampal slice cultures. Mossy fibers in mature slice cultures do not sprout despite the lesion of their fimbria-fornix during the culture preparation. In vivo fimbria-fornix lesion readily induces mossy fiber sprouting (Laurberg and Zimmer, 1980). Mossy fibers may not sprout due to differences between in vitro and in vivo conditions. Alternatively, mature slice cultures may lack the ability to support lesion-induced sprouting.

4.2. Comparison of mature and immature slice cultures Mature slice cultures differ from slice cultures prepared from younger animals. Immediately after plating, mature slice cultures contain functional synapses (Fig. 3). There is a transient depression of evoked synaptic responses between 2 and 5 DIY, after 6 DIV responses remain stable for up to 80 DIV (Fig. 3). The cause of this transient synaptic depression is unknown. After 10 DIV, the projection of the mossy fiber pathway did not change (Table I) and spontaneous epileptiform activity was absent (Fig. 4, AI). These data suggest that, after a 3-day period of adaptation, mature slice cultures are structurally and functionally stable and maintain a balance of neuronal excitation and inhibition comparable to that present in vivo. The long-term stability of mature slice cultures differs from immature slice cultures. Immature slice cultures are isolated from PIO rats during a period of in vivo process outgrowth and synaptogenesis. The mossy fiber pathway develops in vitro (Casaccia-Bonnefil et aI., 1995) and evoked responses increase as a function of the time in culture (Collin et al. 1997). Mossy fiber sprouting occurs in immature slice cultures prepared from the septal and medial hippocampus but not in slices cultured from the temporal end (Casaccia-Bonnefil et aI., 1995). While major excitatory synaptic connections in vivo are faithfully preserved in immature slice cultures, aberrant synaptic connections have been observed by others (Sakaguchi et a1. 1994; Gutierrez and Heinemann, 1999). For the first 21 DIY, the immature slice cultures that we have prepared rarely display spontaneous epileptiform activity. After 28 DIY, most immature slice cultures display spontaneous epileptiform activity. This differs from the lack of spontaneous epileptiform activity in mature slice cultures at 28 DIV. The cause of this difference is not yet known. The increased stability of mature slice cultures may result from the additional brain development that occurs between days 10 and 20 in vivo.

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Another possibility is that differences in the preparation and maintenance of immature and mature slice cultures may also be responsible for the increased stability of mature slice cultures. Immature slice cultures will remain a preferred preparation for the study of important questions of process outgrowth, synapse formation and the neuropathology of the perinatal brain. Mature slice cultures may be advantageous when more adult physiology or pathology is studied.

4.3. Future directions The long-term in vitro retention of the neurons and their synaptic connections is a clear, advantageous feature of mature slice cultures. The uniformity of evoked Schaffer collateral responses is also a clear benefit (Fig. 3). Mature slice cultures would be well suited for studies of epileptogenesis since they lack spontaneous epileptiform activity. Mature slice cultures could potentially be used to model changes in neuronal function that occur during chronic neurodegeneration or as the result of long-term drug exposure. It is not known if brain regions other than hippocampus can be effectively cultured using this method. The maximum age of the rat brain that can be efficiently cultured has not yet been studied systematically. Modifications of this procedure have resulted in efficient culture ofP40 hippocampal slices (Ding Ding and P.J.B., unpublished results). Mature mouse cultures have also been prepared (S.M. and P.J.B .. unpublished results). The longevity and stability of mature hippocampal slices will likely provide a valuable tool for the study of central nervous system function.

Acknowledgements We thank Dr Teresa Milner for her help with the electron microscopy and Dr Todd Sacktor for critically reading an earlier version of this manuscript. This work was supported by NS31 00 10 and the American Heart Association (to P.J.B.).

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