Gene expression profile after intense second messenger activation in cortical primary neurones

Authors


Address correspondence and reprint requests to Volker Höllt, Institute for Pharmacology and Toxicology, Otto von Guericke University Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany. E-mail: hoellt@medizin.uni-magdeburg.de

Abstract

Numerous stimuli induce immediate early gene (IEG) expression in neurones, but a comprehensive overview of the late-response genes is lacking. Therefore we aimed to identify changes in the neuronal gene expression profile following intense stimulation. Forskolin and 12-O-tetradecanoylphorbol-13-acetate (TPA), direct activators of intracellular second messengers, were applied to primary cultured cortical neurones. The gene expression profiles were analyzed on Affymetrix DNA chips which cover around 8000 rat genes. Out of these, 95 genes (1.2%) were increased at least three-fold, and 43 genes (0.5%) were at least three-fold decreased. The gene chip results were verified by testing 15 of the altered genes by quantitative real-time PCR. The majority of the up-regulated genes were transcription factors, neurotrophic factors or (putative) neuropeptides. Furthermore, there were marked changes in intracellular signal processing enzymes and in postsynaptic structural proteins (e.g. vesl, arc, narp), which have been implicated in synaptic plasticity. Notably, classical players in neurotransmission or plasticity such as glutamate and GABA receptors or voltage-gated ion channels were not increased. It is likely that the increased production of components of intracellular signalling and of postsynaptic proteins is involved in neuronal plasticity.

Abbreviations used
CRH

corticotropin releasing hormone

DMEM

Dulbecco's modified Eagle's medium

DMSO

dimethylsulphoxide

EST

expressed sequence tag

FCS

fetal calf serum

FGF

fibroblast growth factor

IEG

immediate early gene

IGF

insulin-like growth factor

LTP

long-term potentiation

TPA

12-O-tetradecanoylphorbol-13-acetate

Activation of neurones in the brain by a wide variety of stimuli is accompanied by transient or sometimes permanent changes in gene expression. Alterations in gene expression are most often identified by the induction of immediate early genes (IEGs) such as c-fos, Fos-related antigens, krox-20 (also called egr-2) and krox-24 (also called egr-1, NGFI-A, zif268) in distinct brain areas. These genes were reported to become induced under very different conditions such as drug application (Vaccarino et al. 1993; Simpson and Morris 1994; Erdtmann-Vourliotis et al. 1999a), electrical stimulation including long-term potentiation (LTP) (Ingi et al. 2001; Petrov et al. 1994), learning tests (Cammarota et al. 2000; Guzowski et al. 2001), induction of seizures (Gass et al. 1993; Chiasson et al. 1995), stress (Watanabe et al. 1994) and neuronal damage (Anderson et al. 1994; Belluardo et al. 1995; Whitfield and Pickard 2000). Studies aimed to detect differential gene expression revealed several other inducible genes (for example Fosnaugh et al. 1995), but a comprehensive overview of the neuronal response to stimulation is missing. In order to produce a very general and intense kind of neuronal activation, primary cultured cortical neurones were employed and stimulated with forskolin and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Pilot studies revealed that stimulation of neurotransmitter receptors yields much less gene induction, most likely because of receptor desensitization.

Forskolin increases intracellular cAMP production and thereby mimics receptor stimulation but avoids receptor desensitization. Physiologically, cAMP is increased for example by dopamine D1 and D5 receptors, β-adrenergic receptors, 5-hydroxytryptamine (HT)4,6,7 serotonin receptors, A2a purine receptors and several others. TPA directly activates protein kinase C which is part of the phosphoinositide pathway and which is activated for example by metabotropic glutamate receptors, 5-HT2 serotonin receptors, V1, NK2, OT and CCKA neuropeptide receptors, P2Y purine receptors and also several others (Bloom 1996).

Having at hand a basic collection of genes which respond to a very general kind of neuronal activation will enable future investigations of more specific neuronal processes.

Materials and methods

Preparation of primary cortical neurones

Cortical neurones from 18- to 19-day-old fetal rats were prepared according to Schousboe et al. (1985). In brief, the cortices were dissected from the fetal brain and transferred to Dulbecco's modified Eagle's medium (DMEM) cell culture medium (Bio Whittaker, Verviers, Belgium). The tissue was disassembled to yield single neurones by repeatedly forcing it through a 21 G × 1.5 cannula attached to a syringe. Thereafter, the cells were washed twice in DMEM and plated into a six-well tissue culture dish coated with Matrigel (Beckton Dickinson, Heidelberg, Germany), 1.5 × 105 cells per well. The neurones were kept for 24 h in DMEM supplemented with 10% fetal calf serum (FCS, Biochrom, Berlin, Germany) and then for 5 days in Start V medium without FCS until use. The cells were stimulated with 10 µm forskolin (Sigma-Aldrich, Germany) and 10 µm TPA (Sigma-Aldrich, Germany) in Start V medium for 1 and 4 hours. Control cells received vehicle [2 µL dimethylsulphoxide (DMSO)] only. Incubation was stopped by removing the medium and adding cell lysis buffer from the Qiagen RNeasy kit. Stimulation experiments were performed in triplicate.

RNA extraction and sample preparation

Total RNA was extracted using RNeasy kit (Qiagen, Hilden, Germany) and following the manufacturer's protocols. The integrity of the RNA and the absence of larger amounts of contaminating genomic DNA was checked by gel electrophoresis. The integrity of the primary neurones during the experiment was confirmed by measuring a pronounced (c. 30-fold) c-fos mRNA increase after one hour stimulation using real-time PCR (data not shown).

Gene chip analysis

Total RNA from three wells was pooled. Sample preparation and DNA chip hybridization was performed by a service provider (German Proteome Center, Rostock, Germany). The samples were hybridized to a U34A rat genome array (Affymetrix, Santa Clara, CA, USA) containing probes for around 8000 rat genes, most of them annotated ones.

Real-time RT-PCR

Complementary DNA was created from total RNA using TrueScript MMLV reverse transcriptase (Hybaid, Heidelberg, Germany) and dN6 random primers. 0.2 µg RNA were included in each reaction in a total volume of 20 µL. The reaction was performed at 42°C for 2 h. Thereafter, the mix was diluted five-fold, and 2 µL were added to the PCR reaction mix to yield a total volume of 20 µL. The PCR reagents were from the Light Cycler Fast Start DNA Master SYBR Green I kit from Roche, Mannheim, Germany. The reaction buffer contained 3.1 mm MgCl2. The amplification reaction consisted of 50 cycles of denaturation (95°C, 15 s), annealing (10 s) and elongation (72°C, 20 s). The annealing temperature was determined by the primers used and is indicated in Table 1 along with the nucleotide sequences of the primers. PCR quantification was performed in triplicate.

Table 1.  Oligonucleotide primers used for real-time PCR
Gene 5′-primer 3′-primerAnnealing
temperature
  1. Fifteen genes were selected from the panel of up- or down-regulated genes. Fourteen of the selected ones were up-regulated and one, the transcription factor HES, was down-regulated. The abbreviations of the genes used here are explained in Table 2. The nucleotide sequences of the respective up- and downstream primers are indicated along with the annealing temperature used for PCR amplification.

CREM5′-GCTGCCACAGGTGACATGCCA5′-GTTCTGAACTTCCAGCACTGCGACTC70°C
CRHBP5′-TGGGACACCTGCATGGCCTTC5′-CTCGAGTGGTTCCAGCTGACGATAC70°C
EMP5′-TTCTCTATAATCTCCCTCGTGGTCTTCGT5′-CCAGGTCAGGATGAAACAGTAGCCTTG70°C
FIL5′-CATGGCTCCTGGTAACTACCTCATTGC5′-TTGCTGGCATCGGAGGAGAAGTTG70°C
HES5′-CCCTCGCTGTAGTCCTGGTG5′-GAGATGCTCAGTCCCAAGGAGA64°C
HGF5′-CACGGAGAGTGCAGATACCTGAAGGAG5′-TACCTGAACATGAGAAGTCCCACGATGA72°C
IL65′-ACTGATGTTGTTGACAGCCACTGCCT5′-GTGCATCATCGCTGTTCATACAATCAGA70°C
MHC5′-CTCAAGATCGAGGCCCGTTCTGC5′-CTCCTTCTTCAGCTTCTCCACCTCCA70°C
NARP5′-CCGGCAGAAGACAGAGAACACACTGA5′-CACAGGCAGATGGTGAAGGCATACA72°C
ROK5′-CAACGGAAGAACAGCAGAAATGG5′-CTTGTGGATGGAAGAATATGATCACC67°C
RYU5′-CCGCTGCTGCTGTTGCTCCTC5′-GTTATCTAGTGGCACCAAGGGTGAAATC70°C
SGR5′-AGCCTTCCACACAATATAAGACAGA5′-GATGCCTTGGTAGGGATTGTAGT62°C
SLC5′-CATGGATCTGCAAACCTCGT5′-CTTCACCACAGCAAAGATGACC62°C
STC5′-GTGCATCGCCAATGGGATCACC5′-CCCGATCTTCTCCATCAGGCTGTCT72°C
VGF5′-CACCTCCCGCCGTCGTGACA5′-GCTCGGACCTCTGGGACGCGT72°C

Cocaine application

Approval was sought according to the National Act on the Use of Experimental Animals (Germany) prior to treatment. Male Wistar rats were housed under controlled laboratory conditions. A group of five animals was treated with ascending doses of cocaine for 10 days, from 10 mg/kg to 20 mg/kg per injection. Two intraperitoneal (i.p.) injections per day were applied. On day 11, the animals received another dose of 20 mg/kg cocaine and were killed 1 h later. Brains were removed, and in-situ hybridization was performed as described (Erdtmann-Vourliotis et al. 1999b).

Results

Gene chip analysis yielded several differentially regulated genes which fulfilled the criteria of high reliability for being increased or decreased based on internal controls on the chip. Only genes with an alteration of more than three-fold will be discussed here because the variability of the DNA chips is known to be around factor two (information provided by the manufacturer).

According to the definitions above, out of the 8000 genes spotted on the chip 95 genes (1.2%) appeared to be increased (see Table 2 for overview) and 43 genes (0.5%) were indicated as decreased. Notably, several of the ESTs indicated as being increased were found to correspond to an annotated gene which also was indicated as being increased further underscoring the reliability of the results. These redundant genes are indicated in Table 2. To verify the chip hybridization results by an independent method, 15 genes (11%) out of the 138 differentially regulated genes were selected and examined by quantitative real-time RT-PCR. Figure 1 shows the results of 14 representative real-time PCR runs. Determinations were performed threefold from the samples that have been used for chip hybridization after pooling (see Materials and methods section). Results for 1 h stimulation are also included in the figure (in the bar diagrams). Expression was nearly always higher after 4 h than after 1 h. This is in contrast to the behaviour of the mRNA for the immediate early gene c-fos which peaked after one hour (not shown). Therefore, most genes identified here obviously belong to the delayed early or late response gene class.

Table 2.  Overview of the up-regulated genes
NumberGene Bank
accession
DescriptionFold induction
(chip)
Fold induction
(PCR)
Redundant
  1. CRH, corticotropin releasing hormone; EST, expressed sequence tag; FGF, fibroblast growth factor; IGF, insulin-like growth factor; NGF, nerve growth factor; NF-κB, nuclear factor-κB; TGF, transforming growth factor; nd, not determined. The up-regulated genes were divided into seven functional groups (A–G). Seven genes did not fit any of these groups (H) and no function is known for the genes listed in part I. For the genes that were verified by real-time PCR the induction rate as derived from the PCR results is indicated in the column ‘fold induction (PCR)’.The abbreviations used for these verified genes are typed in bold. Genes that appeared more than once, for example as ESTs, are marked with a ‘+’ sign in the ‘Redundant’ column.

A. Transcription factors
1AA900476EST, corresponds to transcription factor MRG1 3.0nd 
2U75398Krox-24 3.0nd 
3AA875032EST, corresponds to Fos-related antigen-2 (fra-2) 3.1nd 
4M63282Leucin zipper protein 3.4nd 
5X06769c-fos 3.5nd 
6M84176MyoD (involved in myocyte differentiation) 3.8nd 
7L35271AML1, DNA binding protein from leukaemia cells 3.9nd 
8AI172476TGFβ-induced transcription factor homologue 3.9nd 
9D16102Glucocorticoid-receptor translocation promoter 4.0nd 
10L26267NFκB subunit 4.5nd 
11AI045030EST, corresponds to C/EBP 5.1nd 
AI176662EST, corresponds to egr-1, also called krox-24 5.6nd+
12Y00396c-myc oncogene 6.0nd 
13X54686junB 7.0nd 
AF023087NGF-induced factor A, also called egr-1, krox-24 7.1nd+
U75397krox-24 7.1nd+
14U04835CREM (splicing variant) 7.2nd 
15AF104399Melanocyte-specific gene-1 7.7nd 
16S77528C/EBP-related transcription factor 7.9nd 
17U17254NGF-induced factor B 7.9nd 
M18416NGF-induced factor A, also called egr-1, krox-24 9.1nd+
18L14610Transcription factor RZR-β10.4nd 
AA891041EST, corresponds to junB13.8nd+
19U78102krox-2018.9nd 
20S66024CREM (cAMP response element modulator)20.96.8 
B. Neuropeptides and transmitter-related genes
1Y16188Endothelin converting enzyme 3.0nd 
2AF030358Chemokine CX3C 3.2nd 
3AF053312CC chemokine ST38 3.4nd 
AA800602EST, corresponds to chemokine CX3C 3.5nd+
4X56306δ-Preprotachykinin (splice variant of substance P precursor) 3.5nd 
5S67722Cyclooxygenase-2 3.8nd 
6D90219C-type natriuretic peptide 4.4nd 
7AI043796EST, corresponds to monoamine transporter 5.6nd 
8D11445Neutrophil chemoattractant 5.8nd 
9L25633Neuroendocrine-specific protein (RESP18) 7.4nd 
10M26744Interleukin-6 (IL6) 8.44.5 
11U62667Stanniocalcin (STC) 9.311.5 
12M93669Secretogranin (SGR) 9.926.7 
13X53231Preoptic regulatory factor-1 (putative neuropeptide)10.0nd 
14M74223VGF (putative neuropeptide)11.526.2 
15AF003904CRH binding protein (CRHBP)29.3170.9 
C. Growth factors/neurotrophic factors and their signalling pathways
1AA800671Similar to Ras-GAP 2 3.2nd 
2D12498FGF receptor 3.4nd 
3D14014Cyclin D1 3.5nd 
4AI009405IGF binding protein 3.6nd 
5S54008FGF receptor 1β 3.6nd 
6U42627Protein tyrosine phospatase rVH6 3.6nd 
7X55183Schwannoma-derived growth factor 4.4nd 
8AB002561FGF-16 4.5nd 
9D38222Protein tyrosine phoshatase-like protein 4.8nd 
10AF083418Insulin receptor substrate-2 5.0nd 
11AI070721Glial cell line derived growth factor 5.0nd 
12X62875NHMGP1 (nuclear protein in rapidly dividing cells) 5.3nd 
13U04319fit-1, Fos-responsive gene related to IL-1 receptor 6.9nd 
14L05489Heparin-binding EGF-like growth factor (HGF)18.434.0 
D. Structural proteins
1U38481RhoA-binding kinase (ROK, involved in reorganization
of cytoskeleton, Leung et al. 1996)
 3.01.2 
2S61868Heparan sulfate proteoglycan core protein (RYU) 3.12.8 
3X00975Myosin light chain 2 3.4nd 
4U60416Myr6 myosin heavy chain (MHC) 4.01.8 
5U19866Arc, an inducible cytoskeleton-associated protein 5.6nd 
6S82649Neuronal activity-regulated pentraxin (NARP) 7.28.5 
7AA799773EST, corresponds to filamin (FIL) 7.324.4 
8AB003726Vesl, highly homologous to Homer 7.7nd 
E. G-protein coupled receptors and related signalling pathways
1AF030089CaM kin related peptide ania-4 3.0nd 
2AF015728Cyclic nucleotide-gated cation channel α subunit 3.1nd 
3S77867Putative neuropeptide receptor 3.7nd 
4J03627S-100 related protein (putatively calcium binding) 3.9nd 
5X94185Dual specificity phosphatase MKP-3 5.3nd 
6AF013144MAP kinase phosphatase cpg21 6.3nd 
7AF008650Somatostatin receptor-like protein (SLC) 9.056.4 
F. Proteases, their inhibitors and related proteins
1M23697Tissue-type plasminogen activator (t-PA) 3.8nd 
2U61373Proteinase-activated receptor-2 4.3nd 
3X63434Urokinase-type plasminogen activator 6.8nd 
4AI169327EST, corresponds to inhibitor of metalloproteinase-1 7.9nd 
5S43408Endopeptidase-24 8.0nd 
6X71898Urinary plasminogen activator receptor-113.2nd 
G. Metabolism
1U66723Sodium/nucleoside cotransporter 3.0nd 
2D26393Type II hexokinase 3.4nd 
3AJ001290Sodium myo-inositol transporter 3.6nd 
4AA945442EST, corresponds to glucokinase regulatory protein 4.1nd 
S56464Hexokinase II 8.3nd+
H. Others
1X03347Murine osteosarcoma provirus 3.1nd 
2D14437Calponin 3.2nd 
3U07619Tissue factor protein 3.2nd 
4U90121Thrombomodulin 3.7nd 
5J02962IgE binding protein 3.9nd 
6L12025Tumour-associated glycoprotein11.4nd 
7Z54212Epithelial membrane protein 1 (EMP)13.63.8 
I. Unknown
1AI070295Clone from a differential screening experiment 3.8nd 
2AI169756EST 4.3nd 
3H31287EST 5.0nd 
4AA800853EST 6.6nd 
5X96437Clone from a differential screening experiment 7.2nd 
6AI639185EST8.5nd 
7AI639331EST8.5nd 
Figure 1.

Confirmation of gene regulation by real-time PCR. Fifteen genes were selected from the panel of up- or down-regulated genes which was obtained by gene chip hybridization. RhoA-binding kinase (ROK) is not shown since it was up-regulated only weakly according to the PCR results (factor 1.2). The oligonucleotide primers used for amplification are listed in Table 1. The abbreviations of the genes displayed in this figure are given in Table 2. HES is not included in Table 2 because it was indicated as down-regulated on the chip (note that this could be confirmed by PCR). HES codes for the transcription factor Hes-5 (Gene Bank accession D12516). For each gene, the course of the amplification reaction of three control and three 4 h-stimulated dishes is shown (top panel). The traces corresponding to 1 h stimulation were omitted for clarity. The lower panels are displaying the quantification which is derived from the course of amplification. Note that quantification is performed by regarding the PCR cycle in which the amplification starts its exponential phase. Which quantity of PCR product is finally produced is not considered, thus, the more left the sigmoid curves are situated, the more template was present in the reaction. Traces corresponding to the controls are in grey and traces of the stimulated samples are in black.

The increased genes could be divided into the following functional groups (Table 2 shows the members of the groups in detail): group A, transcription factors (20 members); group B, neuropeptides and transmitter-related genes (15 members); group C, growth factors/neurotrophic factors and their signalling pathways (14 members); group D, structural proteins (eight members); group E, G-protein coupled receptors and related signalling pathways (seven members); group F, proteases and their inhibitors (six members); and group G, metabolism (four members).

Notably, only seven genes did not fit any of these groups (part H in Table 2); seven further genes had no known function (part I in Table 2).

The majority of the up-regulated genes were related to transcription factors, neurotrophins or neuropeptides (groups A–C). Up-regulation of these genes implies merely limited conclusions because the function of most of these genes is not defined in sufficient detail. Induction of group F genes may represent a cellular stress response and an increase in metabolic proteins (group G) can easily be explained by the increased metabolic demands of the cells due to intense stimulation.

Groups D and E provide insight into functional and structural responses of the neurone. Up-regulation of group D genes demonstrates that neuronal activation induces the synthesis of building blocks of the cytoskeleton and of the postsynaptic site. By this means synaptic strength is likely to become enhanced. Furthermore, as revealed by group E, intracellular signal transduction becomes altered.Intriguingly, classical players in neurotransmission or neuroplasticity such as GABA-receptors, NMDA, metabotropic or other glutamate receptors, voltage-dependent calcium or potassium channels did not become increased in response to intense stimulation. DNA chip hybridization revealed that the expression of these genes was not altered upon stimulation. Table 3 shows a list of ion channels, glutamate receptors and GABA receptors, along with associated proteins, which were represented on the DNA array used here. Several of these genes may play a role in neurotransmission and synaptic plasticity, but all of these were identified as ‘not changed’ by chip hybridization. Note that being indicated as ‘not changed’ does not rule out that the respective gene is subjected to subtle changes which would be detected by more sensitive methods. However, our study identified several genes that were profoundly regulated, indicating that changes in gene expression take place upon stimulation, but the classical players in neurotransmission or neuronal plasticity are subjected to changes to a much lesser extent.

Table 3.  List of selected genes that were not altered upon forskolin/TPA stimulation
AccessionDescription
  1. Genes from three families, glutamate receptors (part A), GABA receptors (part B) and ion channels (part C) along with associated proteins, were selected and included in the table. Based on their involvement in neurotransmission, changes in the expression level could be expected in response to neuronal stimulation. However, each of these genes was represented on the DNA array but was indicated as not changed.

A. Glutamate signalling
M92076Metabotropic glutamate receptor 3
U08255Glutamate receptor δ1 subunit
M92075Meotropic glutamate receptor 2
X17184Glutamate receptor, AMPA subtype, GluR1
Z11548Glutamate receptor subunit (GluR6), kainate subtype
X96790Meotropic glutamate receptor subtype 7b
M61099Meotropic glutamate receptor (MGLUR1)
U08256Glutamate receptor δ2
M83561Glutamate receptor subunit 5–2 (GluR5-2), kainate subtype
D10891Metabotropic glutamate receptor mGluR5
S61973NMDA receptor glutamate-binding subunit
M38061Glutamate receptor (GluR-B)
M90518Meotropic glutamate receptor (GLUR4)
M36418Glutamate receptor (GluR-A)
D63772Neuronal high affinity glutamate transporter
U15098GluT and GluT-R glutamate transporter
M36421Glutamate receptor (GluR-D)
S56679AMPA-selective glutamate receptor-A
S94371Glutamate receptor subunit 4c (alternatively spliced)
U08257Glutamate receptor KA1 subunit
X54656GluR-K3 gene for the glutamate receptor
D16817Metabotropic glutamate receptor mGluR7
M36420Glutamate receptor (GluR-C)
U11418NMDAR1 glutamate receptor subunit
U53513Glycine-, glutamate-, thienylcyclohexylpiperidine- binding protein
U63288Metabotropic glutamate receptor 8 (mGluR)
B. GABA signalling
AF058795GABA(B) receptor gb2
AB016161GABA(B) receptor 1d
U92284GABA(A) receptor epsilon subunit
X51992GABA(A) receptor α5 subunit
L08495GABA(A) receptor α6 subunit
M81142GABA(A) receptor γ3 subunit
X15466GABA(A) receptor β1 subunit
X15468GABA(A) receptor β3 subunit
D50671GABA receptor rho-3 subunit
L08490GABA(A) receptor α1 subunit
L08491GABA(A) receptor α2 subunit
L08497GABA(A) receptor γ2 subunit
X51991GABA(A) receptor α3 subunit
AF030253Vesicular GABA transporter (VGAT)
S42358GABA transporter = βalanine-sensitive protein
U95368GABA(A) receptor pi subunit
AB016160GABA(B) receptor 1c
X15467GABA(A) receptor β2 subunit
X57514GABA(A) receptor γ1 subunit
M35162GABA(A) receptor δ subunit
X95579GABA rho-1 subunit
M95762GABA transporter GAT-2
L08493GABA(A) receptor α4 subunit
D38494GABA(A) receptor rho-2 subunit
C. Ion channels
Z96106Potassium channel r-ERG
D86039ATP-sensitive inwardly rectifying K + channel, BIR(Kir6.2)
AF032872Potassium channel regulatory protein KChAP
AF049239Voltage-gated sodium channel rPN4
U37147Sodium channel β2 subunit (SCNB2)
Z67744CLC-7 chloride channel protein
AF016191Potassium channel (erg3)
U92655Potassium channel subunit (KvLQT1)
AF089730Potassium channel subunit (Slack)
U55995Calcium activated potassium channel
M27158Potassium channel-Kv1
AF081365ATP-regulated K + channel ROMK1.1 isoform
M59211Potassium channel Kv3.2b
M59980Voltage-gated K + channel protein (RK5)
X17621RCK2 mRNA for potassium channel protein
AF081366ATP-regulated K + channel ROMK2.1 isoform
L04684Dihydropyridine-sensitive l-type calcium channel α1 subunit (CCHL1A3)
M80545l-Type calcium channel β2 subunit
AF087454Potassium channel (KCNQ3)
J04731Potassium channel protein (BK2)
M32867Potassium channel protein (RHK1)
Y00766Brain sodium channel III
AJ007632ELK channel 3
M22254Sodium channel II
U49126Neuroendocrine calcium channel α1 subunit
X16476drk1 gene mRNA for potassium channel protein
AF020712Maxi potassium channel β subunit
AF055477Pore-forming calcium channel α1B subunit variant a
L39018Sodium channel protein 6 (SCP6)
M27159Potassium channel-Kv2
M57682Brain calcium channel α1 subunit
M59313Potassium channel Kv3.2c
M88751Calcium channel β subunit-III
AB013890Inward rectifier potassium channel Kir 1.4
AF051526Class A calcium channel variant riA-I (BCCA1)
AF073891Potassium channel (eag2)
D26111Chloride channels (ClC-K2L and ClC-K2S)
D50497Chloride channel (ClC-5)
L15453Voltage-activated calcium channel α1 subunit (rbe-ii)
U09243GIRK1/KGA inwardly rectifying potassium channel
X62840Potassium channel protein
X92184Voltage-gated sodium channel (SNS)
D13985Chloride channel RCL1
17521Protein kinase C-regulated chloride channel
D42145ATP-sensitive potassium channel uKATP-1
M22253Sodium channel I
M31433Voltage-dependent sodium channel type II
U27558Brain-specific inwardly rectifying K + channel 1
U79661Calcium-activated potassium channel β subunit
AF027984Low voltage-activated, T-type calcium channel α subunit (CACNA1G)
M86621Dihydropyridine-sensitive l-type calcium channel α2 subunit (CCHL2A)
Y09453Calcium channel γ subunit
Y14635Proton-gated cation channels modulatory subunit
Y17607Potassium channel, α subunit (Kv9.3)
AF048828Voltage dependent anion channel (RVDAC1)
AF061957Potassium channel (elk1)
X98564Neuronal potassium channel α subunit
AF005720Chloride channel (ClC-2) gene, alternatively spliced products
AF048828Voltage dependent anion channel (RVDAC1)
AF073892Potassium channel (elk2)
AF087453Potassium channel (KCNQ2)
M77482Potassium channel (Kv2.2; CDRK)
Y17606Potassium channel, α subunit (Kv9.1)
U79568Voltage-dependent sodium channel PN1
AF021137Inward rectifier potassium channel (IRK1)
U78090Potassium channel regulator 1
X70662K+ channel protein, β subunit
D38101l-Type voltage-dependent calcium channel α 1 subunit
U42975Shal-related potassium channel Kv4.3
U69884Calcium-activated potassium channel rSK3 (SK)
M30312Voltage-gated potassium channel protein (RGK5)
AF051527Class A calcium channel variant riA-II (BCCA1)
U31772Calcium channel α1D subunit (ROB3)
U31815Calcium channel α1C subunit (ROB2)
U94403Proton gated cation channel ASIC1
U69882Calcium-activated potassium channel rSK2 (SK)
AF083341Calcium-activated potassium channel (SLON-1)
X77933Sodium channel, γ subunit
U31816Calcium channel α1S subunit (ROB1)
X77932Sodium channel, β subunit
AF059030Voltage-gated Na channel αsubunit NaN
AJ003065Kir2.4, inwardly rectifying potassium channel
M91808Sodium channel β1 subunit

In order to test whether the genes identified as being regulated in vitro may become induced in the brain, too, we investigated rats treated repeatedly with cocaine. Cocaine blocks dopamine, norepinephrine and serotonin re-uptake. Thereby it enhances the concentration of these transmitters in the synaptic cleft and, in turn, indirectly induces the formation of intracellular second messenger molecules (e.g. cAMP). Thus, in neurones expressing catecholamine or serotonin receptors the response to cocaine stimulation and forskolin/TPA application, respectively, should be similar. Insitu hybridization revealed that VGF becomes induced in the caudate-putamen (but notably not in the nucleus accumbens) and, to a lesser extent, in the frontal cortex and in the lateral septum after chronic cocaine stimulation (Fig. 2). More intense stimulation such as seizure induction was necessary to induce several of the other genes in the brain (data not shown). Ongoing studies will reveal which conditions are needed to induce a given gene in the brain.

Figure 2.

Up-regulation of VGF by repeated cocaine administration. Autoradiographs of an in situ hybridization experiment for VGF are shown. (a) A control animal; (b) an animal treated with ascending doses of cocaine, from 10 mg/kg up to 20 mg/kg injected twice daily for 10 consecutive days. The coronal sections are taken at the level of the caudate-putamen. Note the marked induction of VGF mRNA in the dorsal and lateral striatum.

Discussion

Nerve cell stimulation with direct activators of intracellular signalling revealed the up-regulation of distinct gene clusters. It became obvious that signal transduction is markedly subjected to modification upon intense stimulation. Two novel putative G-protein coupled receptors, two types of intracellular protein phosphatases and a putative calcium-binding protein were induced along with several other genes involved in signalling (Table 2E). Furthermore, structural proteins (Table 2D) were up-regulated, most pronounced a gene of the Vesl/Homer family, a filamin, a pentraxin, a cytoskeleton-associated protein (Arc) and an atypical myosin heavy chain (myr6, myosin VI; Zhao et al. 1996) along with a myosin light chain type. Homer proteins are known to represent scaffolds for postsynaptic glutamate receptors (Ango et al. 2000; Ciruela et al. 2000), and this function was also reported for filamin (Lin et al. 2001) which is known as a actin-binding protein (Wallach et al. 1978). Arc is associated with the cytoskeleton in neuronal dendrites (Lyford et al. 1995). The atypical myosin type VI is known to cause deafness when lacking (Avraham et al. 1995) and may be involved in intracellular trafficking which was already shown for the closely related myosin V (Prekeris and Terrian 1997).

It is reasonable to assume that intense and prolonged neuronal stimulation may trigger events of neuronal plasticity. On the other hand, it is known that neuroplasticity depends on gene transcription and protein synthesis (Krug et al. 1984; Stanton and Sarvey 1984; Nguyen and Kandel 1996). Induction of functional clusters of genes suggests that long-term activation of second messengers in neurones leads to a sharply enhanced transcription of building blocks for the postsynaptic site and of transport molecules which are needed to get the newly synthesized proteins to their destination. Accumulation of NARP and Arc in the postsynaptic density was already reported (Kato et al. 2001; Steward and Worley 2001). Thus, the idea emerges that postsynaptic structural proteins, induced in response to stimulation, may play a prominent role in increasing synaptic transmission. This view is clearly supported by our findings. No glutamate receptor types or voltage-gated ion channels, which confer neurotransmission and play a role in plasticity (Voronin et al. 1995; Hölscher et al. 1999; Johnston et al. 2000; Kullmann et al. 2000), were increased. Instead, the scaffolds for them appear to become up-regulated. Receptors and ion channels may be altered at the post-translational level, but this is beyond the scope of the present study.

A paper was published recently which also comprehensively described the response of neurones to activation. Berke et al. (1998) investigated the effects of dopaminergic stimulation in the striatum. The rats were pretreated with 6-hydroxydopamine to deprive the striatum of dopamine, thereby making it hypersensitive to dopamine stimulation. The dominant excitatory receptors for this transmitter in the striatum are of the D1 type which activate adenylate cyclase. Thirty-two up-regulated genes were described, and there is considerable overlap between these findings and the gene regulation observed in our work. Namely, krox-20, zif268 (=krox-24), fra-2, junB, CREM, COX-2, ania-4, homer, NARP and Arc were detected in both studies. Several genes identified here as being up-regulated were found also to be induced under other, very different, conditions. Examples are the transcription factors (see introduction) as well as VGF (Hawley et al. 1992), NARP (Tsui et al. 1996), Vesl/homer (Kato et al. 1997) and Arc (Lyford et al. 1995). Further studies will show which genes are induced rather uniformly under several different conditions and which genes are morespecifically linked to a defined neurophysiological event.

Acknowledgement

This work was supported by the Deutsche Forschungsgemeinschaft (SFB 426).

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