Rapid gene transfer into cultured hippocampal neurons and acute

?.

Molecular Brain Research441997171–177

Short communication

Rapid gene transfer into cultured hippocampal neurons and acute

hippocampal slices using adenoviral vectors

Oliver Griesbeck a,),Martin Korte b,Claude Gravel c,Tobias Bonhoeffer b,Hans Thoenen a

a Abteilung Neurochemie,Max-Planck-Institut fur Psychiatrie,Am Klopferspitz18a,82152Martinsried,Germany

¨

b Abteilung Neurophysiologie,Max-Planck-Institut fur Psychiatrie,Am Klopferspitz18a,82152Martinsried,Germany

¨

c Laboratoire de Transfert de Genes,Uni?ersite La?al,2601,de la Canardiere,Beauport,Que.G1J2G3,Canada

`′`

Accepted10September1996

Abstract

Primary cultures of hippocampal neurons were infected with an adenovirus coding for b-galactosidase.Expression could be detected as early as4h after infection and steadily increased to high levels at24h without evidence for a functional impairment of the infected neurons.Similarly,adenovirus-mediated gene transfer into acute hippocampal slices was detectable4h after infection and could be localized to discrete areas of the CA1region by microinjection of the virus stock solution.Infected slices were still suitable for electrophysiological experiments.

Keywords:Adenovirus;Hippocampus;Long-term potentiation;Synaptic plasticity

Adenoviral vectors have been shown to transfer genes with high efficiency into a variety of neuronal cells in vitro w x

and in vivo1,3,6,10,17,22,23,33,34.Most of these early studies focused on the potential use of these vectors for therapeutic applications,with initial time points of recom-binant gene expression rarely examined before24–48h w x

post infection21.However,there is increasing interest in the genetic manipulation of neuronal circuits to study the involvement of specific gene products in processes of synaptic transmission and neuronal plasticity.The require-ments for adenovirus-mediated gene transfer for the analy-sis of subtle physiological phenomena are rather different from its use in gene therapy.The latter aims at long-term replacement of defective inherited genes or,potentially also,the local expression of trophic factors to exert a neuroprotective effect in experimental systems,in which neuronal damage is initiated by diverse mechanical or neurotoxic lesion procedures.In contrast,for physiological studies high levels of recombinant gene expression should

Abbreviations:pfu,plaque forming units;Ad,adenovirus;CMV, cytomegalovirus;EPSP,excitatory postsynaptic potential;LTP,long-term potentiation.

)Corresponding author.Fax:q4985783749;E-mail: griesbec@alf.biochem.mpg.de be achieved within a short period of time,while it has to be excluded that the infection procedure as such interferes with the physiological parameters to be analyzed.

Several groups started to explore the use of viral vectors

w x for gene transfer into slices of nervous tissue2,7,27,33. Adenovirus offers the advantage of being safer to be handled and less cytotoxic than other vectors infecting neurons such as vectors derived from herpes simplex or

w x

vaccinia virus30,31.Moreover,vectors such as vaccinia virus are not suitable to transfer genes efficiently into pure cultures of hippocampal neurons and hippocampal slices of

w x

adult rodents20,28,a prerequisite to approach many questions related to synaptic plasticity.In the present study we examine the ability of a replication-deficient aden-ovirus to introduce a foreign gene rapidly into dissociated cultures of hippocampal neurons and acute hippocampal slices and we assess the suitability of slices that have undergone the infection procedure for electrophysiological studies.

The replication-deficient E1-deleted adenovirus Ad-CMV-lacZ contained the b-galactosidase gene as a re-

?. porter under control of the cytomegalovirus CMV pro-

w x moter and was constructed as previously described26,32.

?99. Two stocks of virus5=10pfu r ml and3=10pfu r ml

w x were independently prepared by standard methods14. Briefly,virus was extracted from cells by homogenization

0169-328X r97r$17.00Copyright q1997Elsevier Science B.V.All rights reserved.

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PII S0169-328X9600246-X

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O.Griesbeck et al.r Molecular Brain Research441997171–177 172

and purified by two subsequent rounds of CsCl density gradient centrifugation.Banded virus was then dialyzed against10mM Tris pH8and10%glycerol.The virus titer was determined by plaque assay on293cells.

In a first series of experiments we examined the charac-teristics of gene transfer into cultured hippocampal neu-rons prepared from embryonic day17Wistar rats.Neurons were infected in a reduced volume of300m l complete w x

medium5with different quantities of AdCMV-lacZ, stained for b-galactosidase24h later and finally counted ?.

4grids per well.Hippocampal neurons were efficiently

?.6 infected by AdCMV-lacZ Fig.1A.By using2.5=10 pfu of virus for105cells,about70%of the neurons

?. expressed b-galactosidase after24h Fig.1A.The ex-pression rate could be even further increased to more than

?. 95%b-galactosidase expressing neurons data not shown by further increasing the quantity of virus added to the ?7.

wells 2.5=10pfu,demonstrating that potentially all the cells in the dish were susceptible to infection by aden-ovirus.The number of b-galactosidase expressing cells also steadily increased until day5after infection when105

5?cells had been infected with10pfu of AdCMV-lacZ Fig.

.

1B,suggesting that all this time infectious particles were persisting in the medium and continuing to infect cells. Indeed,it was possible to use supernatant from infected cultures2days after addition of the virus to infect new

?. cultures,albeit with lower efficiency data not shown.To ensure that cytotoxicity of our viral preparations was not prohibitive for the envisaged application,we examined cell viability after adenoviral infection by labeling living and dead neurons using fluorescein diacetate and propidium

w x iodide according to the procedure of Jones and Senft18. While no adverse effects could be detected24h after

?infection even with the highest virus concentrations5= 6.

10pfu,cell viability was clearly compromised3days after infection,if high amounts of virus were added to the ?.

dishes Fig.1D.In order to assess whether adenoviral infection affects the physiological properties of

hippocam-Fig.1.Gene transfer into cultured hippocampal neurons.A:efficiency of gene transfer into cultured hippocampal neurons.100000neurons were infected using increasing amounts of AdCMV-lacZ and stained24h later.The values are expressed as means"S.E.M.of three independent determinations.B:the number of b-galactosidase expressing cells increases until5days after infection.100000hippocampal neurons had been infected with100000pfu of

?.

AdCMV-lacZ and stained at the indicated day after infection n s3.C:calcium transients in hippocampal neurons infected with AdCMV-lacZ or uninfected controls.The values are expressed as means"S.E.M.The dashed vertical line marks the addition of10m M glutamate.D:cell viability3days after infection of100000hippocampal neurons using increasing amounts of AdCMV-lacZ.Living and dead cells were labeled using fluorescein diacetate and propidium iodide and counted.The number of living cells in infected cultures is expressed as percentage of the number of living cells in uninfected ?.

controls"S.E.M.n s3.

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O.Griesbeck et al.r Molecular Brain Research441997171–177173

pal neurons,we compared Ca2q homeostasis and response to glutamate of control and adenovirus infected neurons

w x

using fura-2fluorescence microscopy16.For this pur-pose,hippocampal neurons were cultured for6days on

?.

UV-light permissive dishes Bachofer,and eventually in-

?74.

fected at day71=10pfu for5=10cells.The next

?. day,neurons were loaded for40min378C,10%CO

2

?.

with2m M fura-2r AM Calbiochem,rinsed and incu-bated in fresh culture medium prior to the measurement.

w2q x

During Ca imaging neurons were kept in medium

i

consisting of142mM NaCl,5.4mM KCl,1.8mM CaCl,

2 0.8mM MgSO,1mM NaH PO,5mM glucose,25mM

424

HEPES and0.1%bovine serum albumin,adjusted to pH 7.4.Cells were visualized with a Zeiss Fluar40x r1.3oil

?

objective using an inverted microscope Axiovert100, .

Zeiss,Jena.Fluorescence was determined at the excitation wavelengths of340and380nm with an ICCD camera ?.

C2400-87,Hamamatsu,and images were processed with

?.

the Argus50r CA software Hamamatsu to calculate the

w2q x respective fluorescence ratios.The basal Ca of in-

i fected neurons were indistinguishable from that of control neurons indicating that the mechanisms responsible for the maintenance of Ca2q homeostasis were intact in infected neurons.Application of10m M glutamate to infected

w2q x

neurons resulted in a rise in Ca.There was no signifi-

i cant difference between the rate and extent of calcium increase in neurons infected with adenovirus and non-in-fected control neurons,demonstrating that neither the basic mechanism for calcium homeostasis nor the responsive-

?ness to glutamate is impaired in infected neurons Fig.

.

1C.

We then examined the onset of transgene expression after adenoviral infection,because especially for experi-ments in acute slices significant expression has to occur within its usual lifetime that hardly exceeds16h.In a first set of experiments we infected dissociated cultures of hippocampal neurons because the quantity of virus added to each well could be precisely controlled.Already4h after infection first cells expressed active b-galactosidase ?.

Fig.2A,demonstrating that the whole infection proce-dure from the initial attachment of the virus particle to an individual cell to the successful expression of a foreign protein occurs within a very short period of time.Between 6and12h after infection a steadily increasing number of

?. neurons expressed b-galactosidase Fig.2B,C.In accor-dance with these histochemical observations transgene mRNA was also detectable by Northern blot analysis as early as4h after infection of1000000cells with6=106 pfu recombinant adenoviral vector per well in1ml of complete medium.There was a further steady increase

in Fig.2.Time course of b-galactosidase appearance in cultured hippocampal neurons after infection with AdCMV-lacZ.Per well,100000neurons in300 m l of medium were infected with3=106pfu of vector and stained using X-gal histochemistry at the indicated time point after infection.Already4h after

?.?.?. infection,single neurons express active b-galactosidase.Arrows mark individual b-galactosidase positive neurons A.From6h B to12h C after infection,the number of b-galactosidase expressing neurons increased further.However,no sign of b-galactosidase expression was detectable12h after

?.

infection,when neurons had been incubated at238C D.Scale bar:100m m.

()O.Griesbeck et al.r Molecular Brain Research 441997171–177

174Fig.3.Gene transfer into acute hippocampal slices.A:first cells express b -galactosidase 4h after bath application of virus.B:localized gene transfer into the CA1region of an acute hippocampal slice.The arrow marks the site where the concentrated virus stock had been microinjected into the slice.After staining for lacZ,the slice was sectioned on a cryostat ?.and sections counterstained using Cresyl violet.Scale bars:125m m A ,?.250m m B .

mRNA levels until 12h after infection,the latest time ?.point studied data not shown .As slices used for electro-physiology are preferably incubated at lower temperatures prior to recording we tested whether a successful infection could also be accomplished under these conditions.We therefore placed cultured hippocampal neurons in an incu-?.bator at room temperature 238C in 10%CO after infec-2tion and stained for b -galactosidase expression at different time points after infection.However,even 12h after infection there was no b -galactosidase expression de-tectable when neurons had been kept under these condi-?.tions Fig.2D .

?.For gene transfer into hippocampal slices Fig.3we ?.prepared acute slices from 129Sv mice P18-P32,a strain commonly used for gene targeting experiments.Hip-pocampi were quickly dissected,cut at 400m m thickness w x using an egg-slicer 19and transferred into oxygenated ?.artificial cerebrospinal fluid ACSF consisting of 124mM NaCl,3mM KCl,1.25mM KH PO ,2mM Mg SO ,262424mM NaHCO ,2.5mM CaCl and 10mM glucose.To 32verify that infection in slices occurs as rapidly as infection of cultures,we placed slices in 600m l ACSF,added

1=107pfu of AdCMV-lacZ and incubated them at 378C in a submerged chamber for 4h.Similar to dissociated ?.cultures Fig.2A ,first cells in slices were found to ?.express b -galactosidase after this time Fig.3A .In gen-eral between 40and 60cells per slice were found to be infected after 4h.The number of b -galactosidase express-ing cells was increasing steadily during longer incubation periods,again reflecting the fact that the virus particles are diluted in the medium and gradually more and more cells become infected.

In order to obtain more spatial control on recombinant gene expression we microinjected virus into the slices.For injections slices were placed in an oxygenated interface w x chamber 12on ACSF.Slices were only handled on tea paper soaked in fresh ACSF and always kept wet.For ?.local gene transfer Fig.3B concentrated virus-stock solu-tion was pressure-injected into the CA1stratum pyrami-dale via a patch pipette that was connected to a pico pump ?.World Precision Instruments .Injections were done under a dissection microscope through a hole in the lid of the interface chamber.Pressure of 4psi was applied for sev-eral periods,each of a duration of 10ms,thereby injecting an estimated 25–50nl into the slice.To control the success ?.of the injections the vital dye Cochenille Red Sigma was added to the virus stock at a concentration of 0.1%.Thereafter slices were incubated for up to 14h at 35–378C before starting electrophysiological recordings.Microinjec-tion of virus was in general more efficient than bath application,probably because the actual concentration of virus in the immediate vicinity of the cells is much higher.?.Use of small diameter pipettes 1m m resulted in reliable delivery of solution into the slice.Accordingly,areas with a high infection rate could be https://www.360docs.net/doc/a117555999.html,e of pipettes of larger diameter or broken tips resulted in a greater spill of liquid and therefore a more scattered infection of cells within a given area.Upon morphological criteria both neurons and glia were infected.The use of neuron-specific ?promoters is,however,possible in these vectors C.Gravel,.unpublished results .As the cytomegalovirus promoter is also active in 293cells and large amounts of b -galacto-sidase are produced during propagation of the vector,it was important to evaluate whether our purified stocks were still contaminated with b -galactosidase which would result in a false positive signal after injection of virus solution into slices.However,neither immediate staining of slices after virus injection nor addition of staining solution to viral stocks resulted in a positive signal,indicating that our stocks were sufficiently pure for our purposes.

We then examined the electrophysiological properties of slices that had undergone the local infection procedure.For stimulation monopolar tungsten electrodes in the CA3Schaffer collateral region were used.Synaptic field poten-tials were elicited with a frequency of 0.1Hz.Extracellular field potentials were recorded with glass electrodes placed ?.in the apical dendritic region stratum radiatum of the CA1pyramidal neurons.The slope of the EPSP was used

()O.Griesbeck et al.r Molecular Brain Research 441997171–177175

as a measure for synaptic strength.For tetanic stimulation ?.3=30pulses 100Hz,50m s duration,5s ISI were applied with the strength of the test stimulus.Data were ?collected with a program written in LabView National .Instruments .To evaluate if synaptic transmission was changed by the infection procedure,we first tested paired ?.pulse facilitation PPF .As shown in Fig.4A there is no significant difference between slices infected with Ad-?CMV-lacZ and non-infected controls P )0.35,t -test,.two-tailed .In addition we investigated if equivalent synaptic responses were evoked with similar stimulus strength and that the area under the field EPSP produced ?.by a single stimulus was equal data not shown .There ?was no statistical difference in these parameters too P

).0.35,t -test,two-tailed .Next we tested if processes of synaptic plasticity were still intact.As a paradigm for synaptic plasticity we used the CA3–CA1hippocampal ?.w x long-term potentiation LTP 4.LTP is based on an increased synaptic efficiency and is a complex phe-nomenon involving various pre-or postsynaptic mecha-nisms.Intactness of LTP induction seemed to be an appro-priate parameter to assess the electrophysiological proper-ties of slices after the infection procedure.Fig.4B shows group data for infected and untreated slices.It demon-

Table 1

Percentage of successful LTPs for infected and control slices from mice P17-P52Treatment Successful induction of LTP Yes No ?.?.Infected slices 61%n s 1439%n s 9?.

?.

Control slices

87%n s 4713%n s 7LTP was judged successful if 60min after the tetanus the slope of the field EPSP was increased by more than 20%in comparison to baseline values.n s number of slices.All experiments were performed blindly.

strates that it was possible to induce LTP in adenovirus infected slices.Single examples are shown in Fig.4C.However,the degree of potentiation in infected slices is slightly reduced in comparison to untreated ones.More-over,the number of incidences,in which LTP could be successfully induced,was reduced in infected slices com-?.pared to untreated slices Table 1.

Our data show that the onset of expression of a trans-gene after adenoviral infection occurs fast enough to allow rapid gene transfer into cultured hippocampal neurons and acute hippocampal slices,provided the infection is per-formed at an incubation temperature of 378C.By micro-injection of virus stock solution expression could be local-

?.Fig.4.Electrophysiology of infected slices.A:paired pulse facilitation PPF as a measure of normal synaptic transmission is not affected in slices infected with AdCMV-lacZ.The percentages denote the ratio of second EPSP slope to first EPSP slope.PPF was tested with 20,30,50and 100ms ISI ?.interstimulus interval .B:comparison of LTP in slices infected with AdCMV-lacZ and untreated slices,group data "S.E.M.The potentiation is slightly reduced in infected slices.C:single examples of LTP in untreated control slices and slices infected with AdCMV-lacZ.Shown is the size of the EPSP ?.slope before and after tetanic stimulation average over 6"S.E.M..

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O.Griesbeck et al.r Molecular Brain Research441997171–177 176

ized to discrete areas of CA1in hippocampal slices.Al-though retrograde transport of adenovirus taken up by

w x

nerve terminals has been described13,24,29,we found no evidence that such a transport did occur within the observation period.Neurons infected by an adenoviral vector were found to be suitable for physiological experi-ments,which is in agreement with other recent reports w x

8,9,34.The observed decrease in the rate of LTP induc-tion and the magnitude of potentiation is most probably due to the necessity for mechanical manipulation of the slices and incubation at higher temperatures,conditions which are known by themselves to compromise slice phys-iology.In accordance with this,slices that were locally injected with PBS and incubated for several hours at378C show a decrease in potentiation similar to slices injected

?.

with AdCMV-lacZ data not shown.Cytopathic effects do not seem to play a role within the short incubation period in vitro as most of the cytotoxicity of E1-deleted adenovi-ral vectors–apart from a toxicity mediated by the pentons –is thought to arise from a low level transcription of viral genes slowly leading to an accumulation of viral gene

w x

products in infected cells11,35,36.

Genetic methods have been introduced recently into the

w x study of nervous system function and plasticity15.Inter-pretation of phenotypes resulting from gene-targeting ex-periments is hampered by the fact that physiological de-fects in mutant animals may reflect the lack of expression of a given gene during embryonic development.More subtle techniques to genetically modify brain functions in

w x transgenic animals are being envisaged25.The use of viral vectors allows a considerable degree of temporal and spatial control of transgene expression which can be fur-ther increased by the use of cell-type specific and in-ducible promoters or,possibly,by targeting viral particles to specific cell types.Adenoviral vectors may be used to complement studies involving transgenic animals,for ex-ample,to rescue physiological functions such as LTP in hippocampal slices of knock-out mice by local and acute transfer of the targeted gene back into the slice.Alterna-tively,these vectors allow the acute reexpression of a given gene in cultures of dissociated hippocampal neurons originating from genetically engineered animals or the expression of mutated forms of a protein in wildtype neurons,followed by an analysis of its physiological con-sequences.In this way,adenovirus will be an interesting tool in the molecular dissection of processes underlying neuronal function and plasticity.

Acknowledgements

We would like to thank Benedikt Berninger and Volker Staiger for help in the imaging experiments and electro-physiology,and Theologos Michaelides for helpful discus-sions.References

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