 |
 |
S.M.A.R.T.
EFFECTS OF AN ESCALATING
DOSE REGIMEN OF METHAMPHETAMINE ON GENE EXPRESSION IN THE PRE-FRONTAL CORTEX USING
DNA CHIPS
L. de Lecea1,2, V. Fabre3, R.A. Gallegos2, M.
Sánchez-Alavez2, S.J. Henriksen2, and J.R. Criado2.
1Dept. Molecular Biology; 2Dept. Neuropharmacology, Scripps
Research Institute, La Jolla, CA.; 3Dept. Molecular Biology, Scripps Research
Institute, La Jolla, CA.
INTRODUCTION
[home]
methods | down regulated genes
| up regulated genes
| figure 1
| figure 2 |
summary
Repeated administration of drugs of abuse activates neuroadaptive processes that
play an important regulatory role in the addiction process. These neuroadaptations
evoke a series of addiction-related neural events resulting in the development of
sensitization, tolerance, withdrawal and craving. However, the cellular and molecular
neuroadaptations leading to these events are still unknown. The present exploratory
study utilized oligonucleotide-based DNA chips to characterize specific changes in
gene expression as a consequence of repeated methamphetamine (METH) administration
and withdrawal. These studies focussed on the prefrontal cortex (PFC).
Although much research has been devoted to the characterization of METH neurotoxicity
[1], determining the molecular basis of the neuroadaptive
mechanisms regulating METH addiction in animal models requires the study of METH
treatment regimens that might provide valuable insight into the neuropsychopharmacological
alterations that occur during the course of high-dose stimulant abuse in humans (see
[2]). Studies have shown that a common usage pattern of abusers
of AMPH, including METH, is to escalate the dose administered over time to very high
levels [3]. This usage pattern results in the development
of tolerance to the sympathomimetic effects of psychostimulants, allowing the abusers
to tolerate higher doses [4] (2,81,236) and to maintain the
high levels of euphoria produced by these drugs [5]. Interestingly,
psychostimulant-induced psychosis has been associated with this pattern of psychostimulant
abuse [6]. Consistent with this, studies have shown that
pre-exposure to an escalating dose regimen (EDR) of METH protects against the neurotoxic
effects of METH [7]. Moreover, pretreatment with gradually
escalating doses has been shown to alter the neurochemical effects of high doses
of psychostimulants [8]. This pattern of drug exposure appears
to be more physiologically and behaviorally relevant. In this study we propose to
characterize in the PFC patterns of gene expression and to identify genes as a consequence
of a physiologically relevant METH EDR.
METHODS
introduction
| down regulated genes | up regulated genes | figure 1 | figure 2 | summary
Poly A-selected cytoplasmic RNA was extracted from pre-frontal cortex of Sprague-Dawley
rats as described [9]. In our hands, cytoplasmic RNA extraction
yields RNA of much better quality (as measured by average length) and is less contaminated
with genomic DNA than total cellular extractions. One µg of mRNA was converted
into cDNA in the presence of biotindUTP with RNaser H-reverse transcriptase (Gibco).
Labeled cDNAs were depleted from repetitive sequences by preincubating with mouse
repetitive DNA. DNA chips were hybridized in the presence of 5xSSC, 0.1%SDS, 5M Urea,
100 µl/ml of Cot 1 mouse genomic DNA, 10 µg/ml of polyA at 42oC for 18
hours. After hybridization, chips were washed in 2xSSC, 0.1%SDS at 55oC and incubated
with streptavidin coupled to the fluorescent dye [10]. After
severall washes in 2xSSC, DNA chips were scanned with an Affymetrix Gene scanner.
For microarray spotting, we used a ScanArray 5000 (GSI Lumonics) confocal scanner
equipped with three HeNe lasers (543 nm, 590 nm and 632 nm) and a 488 nm Argon laser,
10 emission filters (20 nm band pass) with 5, 10, 20, 30 and 50 µm pixel resolution.
Images were analyzed with QuantArray and data were treated with GeneSpring (Scripps
DNA chip Core Facility).
In the present study, we used oligonucleotide based Affymetrix chips to identify
sets of mRNAs whose expression is affected at different stages post METH EDR. We
have hybridized fluorescently labeled cDNA, prepared from pre-frontal cortex cytoplasmic
mRNA of male Sprague-Dawley rats, to a chip containing 1300 rat cDNAs of neurobiological
interest (Rat neurobiology chip; see http://www.affymetrix.com/products/u34_neuro.html
for a complete list). While the chip used in this initial study is admittedly biased
and represents only a fraction of the expressed genes in the rat pre-frontal cortex
and contains very few ESTs or unknown cDNAs, it is an excellent substrate to assay
molecular changes in the experimental conditions tested in the present study. After
hybridization, DNA chips were washed and scanned to an Affymetrix GeneScanner, to
obtain values of fluorescent intensity. Chips were hybridized with samples from:
1) controls (saline injections and protocol similar to group #3); 2) 2 hr post; 3)
2 wks post and 4) 2 hr after an acute METH challenge (2.21 mg/kg), 2 wks post METH
EDR. Fluorescent signals were obtained in 87% of the cDNAs on the chip, which included
several positive (GAPDH, cyclophilin,rat genomic DNA) and negative (bacterial and
yeast genes) hybridization controls. Each of the 1300 cDNAs are represented in the
chip by a set of 20 oligonucleotides (25~mers) that span the coding region (ten of
which are mismatch controls). An average of the hybridization intensity was then
calculated for the positive cDNAs. Hybridization intensities for the experimental
conditions were then compared with the control group.
DOWN-REGULATED GENES 2
HOURS AFTER METHAMPHETAMINE EDR
introduction
| methods
| up regulated genes
| figure 1
| figure 2 |
summary
|
| Treatment |
Control
|
2 hours
|
Gene |
GenBank number |
|
|
Functional category |
|
|
|
Cytoskeletal structural protein |
| U44979 |
1
|
0.03
|
kinesin-related protein 2 |
|
|
|
Release/transport |
| U39549 |
1
|
0.39
|
p29; synaptic vesicle protein |
|
|
|
Transcription factor |
| AF050660 |
1
|
0.08
|
activity and neurotransmitter-induced early gene 8 |
|
|
|
Transmitter metabolism |
| S71597 |
1
|
0.12
|
inducible NO synthase |
|
|
|
Intracellular signaling |
| M55417 |
1
|
0.39
|
protein kinase C-gamma |
| D30734 |
1
|
0.08
|
Ras GTPase-activating protein |
|
|
|
Cytokines, growth factors, peptides, intercellular signaling |
| U03491 |
1
|
0.19
|
transforming growth factor beta-3 |
| X16703 |
1
|
0.48
|
insulin-like growth factor II |
|
|
|
Tansmitter receptors |
| L22558 |
1
|
0.02
|
serotonin 5-HT7 receptor |
| D49395 |
1
|
0.12
|
serotonin 5-HT3 receptor |
| S94371 |
1
|
0.11
|
glutamate receptor subunit 4c |
|
|
|
Ion channels |
| M27158 |
1
|
0.39
|
potassium channel-Kv1 |
|
|
|
EST |
| AA925846 |
1
|
0.03
|
EST |
|
GENES UPREGULATED 2 HOURS,
14 DAYS AND 14 DAYS + INJECTION AFTER METHAMPHETAMINE EDR
introduction
| methods
| down regulated genes | figure 1 | figure 2 | summary
|
| Treatment |
C1
|
2 hours
|
14 days
|
14 days
+
injection
|
Gene |
| GenBank number |
|
|
|
|
Functional category |
|
|
|
|
|
Apoptosis |
| L14680 |
1
|
9.22
|
19.39
|
9.59
|
bcl-2 |
| AF065431 |
1
|
8.68
|
12.38
|
5.08
|
bcl-2 related ovarian death gene product BOD |
|
|
|
|
|
Cytoskeletal structural protein |
| S74265 |
1
|
9.91
|
12.65
|
4.66
|
high molecular weight microtubule-associated protein |
|
|
|
|
|
Protease |
| U89514 |
1
|
1.79
|
2.22
|
5.21
|
calcium dependent cysteine proteinase calpain large subunit |
|
|
|
|
|
Release/Transport |
| L20820 |
1
|
6.80
|
3.68
|
7.82
|
syntaxin 3 |
|
|
|
|
|
Intracellular signaling |
| L13406 |
1
|
6.07
|
4.02
|
3.88
|
calcium/calmodulin-dependent protein kinase II delta subunit |
| X74227 |
1
|
13.36
|
2.34
|
7.38
|
IP3 3-kinase |
| AJ000557 |
1
|
5.09
|
2.78
|
3.68
|
Janus protein tyrosine kinase 2 |
| AJ006855 |
1
|
3.85
|
3.22
|
7.36
|
inositol-5-phosphatase DeltaSAC-synaptojanin1 |
| AF075382 |
1
|
2.83
|
24.90
|
9.16
|
suppressor of cytokine signaling-2 |
|
|
|
|
|
Transmitter metabolism |
| M93257 |
1
|
8.49
|
2.14
|
4.87
|
cathechol-O-methyltransferase |
| M10244 |
1
|
2.72
|
6.81
|
6.39
|
tyrosine hydroxylase |
|
|
|
|
|
Cytokines, growth factors, peptides, intercellular signaling |
| E03082 |
1
|
6.33
|
7.74
|
2.08
|
nerve growth factor |
| M25646 |
1
|
3.51
|
2.36
|
18.75
|
vasopressin/neurophysin precursor |
|
|
|
|
|
Transmitter receptors |
| S67316 |
1
|
5.29
|
3.16
|
7.10
|
alpha 2-adrenergic receptor |
| AB017656 |
1
|
6.44
|
3.72
|
2.74
|
muscarinic receptor m3 |
|
|
|
|
|
Receptors |
| D16443 |
1
|
19.66
|
7.68
|
7.92
|
prostaglandin E2 receptor EP3 subtype isoform |
| M91599 |
1
|
2.38
|
4.77
|
5.51
|
fibroblast growth factor receptor subtype 4 |
| AF091566 |
1
|
117.59
|
115.69
|
40.54
|
isolate HTF-SP1 olfactory receptor |
|
|
|
|
|
Ion channels |
| X16476 |
1
|
2.13
|
5.35
|
2.37
|
potassium channel protein |
| AB010963 |
1
|
3.90
|
9.63
|
20.12
|
calcium activated potassium channel beta subunit spliced variant |
| X83581 |
1
|
11.43
|
9.52
|
5.13
|
inward rectifier potassuim channel 9 |
| X70662 |
1
|
8.61
|
7.15
|
10.53
|
beta subunit potassium channel |
| AF073891 |
1
|
3.72
|
5.12
|
5.50
|
potassium channel (eag2) |
| M27159 |
1
|
1.89
|
5.76
|
9.16
|
potassium channel-Kv2 |
| U72410 |
1
|
1.36
|
2.64
|
8.69
|
C-terminal truncation variant Kir3.1 alternative splice variant deltaB |
|
|
|
|
|
EST |
| AI178835 |
1
|
8.16
|
2.17
|
6.40
|
EST |
| AI012265 |
1
|
6.90
|
4.43
|
5.23
|
EST |
|
FIGURE 1
introduction
| methods
| down regulated genes | up regulated genes | figure 2 | summary
 |
Figure 1. Distribution of mRNAs whose expression is modified
as a consequences of METH treatment schedules.
Top graph shows the distribution of mRNAs whose expression is modified in the
4 experimental groups, compared to control levels. For each group, a Bell curve is
plotted, whose median peak represents the control value. Dots above control values
are plotted in red, whereas downregulated genes are plotted in blue. Bottom graph
is a scattered plot showing comparison of the expression of the 1300 cDNAs in the
DNA chip at 2 hours post METH EDR compared to control levels. Each cross represents
a single cDNA. The green lines show mean ± std dev. More than 80% of the transcripts
are present in this window. Transcripts outside this interval were further analyzed
in replicas. This scattered graph is plotted for each of the experimental groups,
relative to control levels, and the candidate cDNAs showing a robust induction or
inhibition are further analyzed.
(for a much larger view, click on figure)
|
FIGURE 2
introduction
| down regulated genes | up regulated genes | methods | figure 1 | summary
Expression profiles of a cluster of genes down-regulated 2 hr
post METH EDR
(for a much larger view, click on figure)
SUMMARY
introduction
| methods
| down regulated genes | up regulated genes | figure 1 | figure 2
In this study we have generated clusters of self-organizing maps (SOMs) of gene
expression based on the data generated from our four experimental groups. These maps
are profiles that ask the question: Which are the most prominent profiles of gene
expression across all experimental conditions? The following hypotheses emerged from
this initial analysis of functional categories: A) Repeated METH induces the expression
of several molecules involved in inflammation and apoptosis resulting in an immunological
response; B) Repeated METH induces the expression of several K+ channel subunits
resulting in PFC hyperexcitability; and, C) Upregulation of muscarinic and serotonin
receptors were observed immediately after termination of the METH EDR and, in some
cases, during withdrawal. These data suggest a direct involvement of these neurotransmitters
in the consequences of repeated METH in the PFC. These hypotheses and additional
hypotheses involving cDNAs that appear to be implicated during different times of
withdrawal after METH EDR will be further studied by in situ hybridization and other
high resolution methods. (Supported in part by DA-12444)
CITATIONS
introduction
| methods
| down regulated genes | up regulated genes | figure 1 | figure 2 | summary
1. J. Neurochem 70: 190, 1998; J. Neurosci 14: 2260, 1994;
Biol. Psychiatry 2: 189, 1970; Ann. N.Y. Acad Sci, 648: 371, 1992; Brain Res 486:
73, 1989.
2. JPET, 282: 561, 1997; J. Neurosci 17: 2551, 1997.
3. Amphetamine and its analogues. San Diego, Academic Press. 387, 1994; Current
concepts on amphetamine abuse. U.S. Government Printing Office, 143, 1972; Science,
251: 1580, 1991; JAMA, 201: 305, 1967.
4. Fed Proc 34: 1845, 1975; Pharmacol. Biochem. Behav 2: 503-508, 1974.
5. Brain reward system and abuse. New York, Raven Press. 109, 1987.
6. Amphetamine and its analogues. San Diego, Academic Press, 387, 1994; NIDA
Research Monograph Series. U.S. Government Printing Office, 224, 1996; Amphetamine
and related stimulants: Chemical, biological, clinical and social aspects. Boca Raton,
FL, CRC Press, 161, 1980.
7. Brain Res. 343: 305, 1985; J. Neurochemistry 44: 852, 1985.
8. J. Neurochemistry 44: 852, 1985.
9. Proc Natl Acad Sci U S A 93(16): 8733, 1996.
10. Nat. Genet., 21: 20-24, 1999.
11. JPET, 282: 561, 1997.
TOP | HOME
|
|
