| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 19, 2004 as doi:10.1096/fj.03-0612fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Department of Molecular and Cellular Neurobiology, Graduate School Neurosciences Amsterdam, Research Institute Neurosciences,
Department of Mathematics, Faculty of Sciences, Vrije Universiteit, Amsterdam, The Netherlands; and Departments of
Medical Oncology, and
Medical Pharmacology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands
3Correspondence: Department of Molecular and Cellular Neurobiology, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands. E-mail: sspijker{at}bio.vu.nl
SPECIFIC AIMS
The aim was to gain insight in the temporal profile of gene expression in the nucleus accumbens (NAc), related to development and endurance of morphine-induced neuroplasticity. We used a morphine administration paradigm, which yields a morphologically, biochemically, and behaviorally defined neuro-adapted state. This allowed the study of expression profiles of major gene families with roles in intra- and intercellular signaling and transcriptional regulation that are deemed important for development of drug-induced neuroplasticity. Temporal expression profiles of 159 genes were measured in the nucleus accumbens during a 2 wk exposure to morphine (days 1, 2, 4, 8, and 14) and during a 3 wk morphine-free abstinence period (days 1, 2, 4, 8, 12, and 18) (Fig. 1
). Mapping the temporal expression profile of these genes and their dynamics in the exposure and abstinence phase is a first step to gain insight in their possible role in development of drug-induced neuro-adaptation.
|
PRINCIPAL FINDINGS
1. Real-time quantitative PCR has the sensitivity and accuracy for neuronal gene expression profiling
We used real-time quantitative PCR to monitor gene expression in order to resolve small changes in expression for genes in various abundance classes. We established its overall sensitivity and resolution by assessing several critical parameters, such as quality of RNA isolation and cDNA synthesis, reproducibility of measurements and biological variation. We show with a measured error mathematical model that the methodological error allows detection of 15–20% difference in gene expression levels. When biological error (biological variation, dissection) is included in this paradigm (Fig. 1)
, detection of >35% difference in gene expression levels is feasible.
2. Morphine induces phase-specific temporal gene expression profiles
Daily injections of morphine (Fig. 1)
typically induced strong highly dynamic changes in gene expression, thereby characterizing the morphine exposure phase with many different profiles of fluctuating gene expression. These dynamic expression profiles may indicate a homeostatic response to morphine stimulation in order to reach a semi-stable state. In contrast to the exposure phase, expression profiles in the abstinence phase are highly dynamic but coherent (Figs. 2
, 3
). Abrupt withdrawal of morphine treatment during the first days of the abstinence phase gave rise to a completely new type of response. Typically, compared with the end of the exposure phase, the first two days of drug-abstinence were marked by few down-regulated genes, and some immediate early gene expression (Fig. 2A, D
). After these first two drug-free days most genes showed a down-regulation of expression during the remainder of this period with a major down-regulation at 1 wk of abstinence (Fig. 2)
. A few genes showed a marked up-regulation after 2–3 wk of abstinence. Therefore, 1) exposure to morphine and subsequent abstinence from the drug are typified by phase-specific gene expression; and 2) natural withdrawal from morphine is a potent new stimulus in terms of enduring gene expression in the abstinence phase.
|
|
3. Phase-specific expression profiles of molecules involved in neurotransmission and neuronal plasticity
Early response genes at morphine exposure are several transcription factors (e.g., krox-20), and immediate early genes (e.g., Arc, c-fos) (Fig. 2D
). During this phase dynamic expression is most prominently and coherently observed for G-protein coupled receptors, e.g., dopamine receptors, and several neuropeptides, as previously reported (Fig. 2B
and Fig. 3
). At the introduction of morphine abstinence early response genes are transcription factors, (e.g., fra-2) and immediate early genes (e.g., Arc, c-fos) (Fig. 2D
). Coherent peak activation of transcription factors, together with a resistance to repression at 1 wk abstinence, suggest that the intermittent morphine regimen followed by drug-abstinence involves a new gene expression program. Part of this program could be neuronal plasticity as reflected by dynamic regulation of trophic factors (receptors) and related molecules during abstinence (Fig. 2E
, Fig. 3
). This complies with the idea that the abstinence period is crucial to development of drug-induced behaviors such as sensitization, drug-seeking, and craving, and to observed concomitant changes in neurotransmitter release and neuronal wiring.
Characteristic of the abstinence phase is the down-regulation at 1 wk abstinence and thereafter found for glutamatergic and GABAergic receptor subunits (Fig. 2C
). This suggests long-term changes in receptor stoichiometries arguing for altered synaptic signaling, probably occurring at 95% of GABAergic medium spiny interneurons of the NAc. A shift in relative expression of NMDA receptor subunits that, as previously suggested may change receptor stoichiometry was found. Long-term down-regulation of all AMPA receptor subunits (Fig. 2C
) may be related to altered dendritic morphology observed long after morphine exposure. Tight coregulation in expression of the channel subunits and their postsynaptic interaction partners, such as the postsynaptic density proteins PSD-95 and Homer-1a, indicates that these receptor-protein complexes may be coordinately regulated to yield adapted post-synaptic structures important for long-term morphine-induced neuronal plasticity (Figs. 2
, 3)
.
During abstinence, the groups mostly resistant to repression were transcription factors and several growth-related molecules implicated in growth cone motility and synaptic plasticity. Only a few genes showed up-regulation after 2–3 wk of abstinence, which were mainly transcription factors. In particular molecules such as MEN1, junD, krox-24, Arc, and NAC-1, may play a role in neuronal plasticity (Fig. 2D
). Induction of the transcriptional regulator menin (MEN1 gene), known to repress neuronal outgrowth and to allow generation of central synapses, may play a role at the end of the abstinence phase. The plasticity gene Arc is a marker for recent synaptic activity, and is involved in long-term memory consolidation and impaired maintenance of LTP. A hypothesis worth testing would be that synaptic plasticity may precipitate in stably changed synaptic connections by (long-term) up-regulation of synapse-stabilizing molecules such as Arc and menin. Several trophic factors (receptors) and growth-related molecules show tight coregulation during both exposure and abstinence (Fig. 2E
) (e.g., several ligands and receptor-ligand pairs).
In summary, various functional groups of genes specifically become actively regulated during abstinence, in particular indicating genes involved in aspects of neuronal plasticity (Fig. 3)
.
CONCLUSIONS AND SIGNIFICANCE
We have demonstrated that profound gene expression changes occur not only during exposure but also long after cessation of drug-administration (Fig. 2)
. The phase-specific expression profiles highlight distinct activities of functional groups of genes (Figs. 2
, 3)
. Gene expression changes not only involve elements of synaptic transmission (receptors and interaction partners), but also include proteins implicated in neuronal plasticity (e.g., in outgrowth and synapse formation). The wave of gene expression induced by natural withdrawal and subsequent abstinence might therefore be part of a novel gene program that is essential to previously reported drug-induced morphological reorganization of neuronal networks. In general, persistent changes in gene expression during abstinence may form the molecular basis of drug-induced long-term plasticity. Our findings underscore the notion that discontinuation of drug taking is an important trigger in development of long-term behavioral consequences of repeated drug exposure and is related to behavioral phenomena such as sensitization and addiction.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0612fje; 
2 These authors contributed equally to this work.
This article has been cited by other articles:
|
|
 |
|
  J. Postmus, A. B. Canelas, J. Bouwman, B. M. Bakker, W. van Gulik, M. J. T. de Mattos, S. Brul, and G. J. Smits Quantitative Analysis of the High Temperature-induced Glycolytic Flux Increase in Saccharomyces cerevisiae Reveals Dominant Metabolic Regulation J. Biol. Chem., August 29, 2008; 283(35): 23524 - 23532. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Hawle, D. Horst, J. P. Bebelman, X. X. Yang, M. Siderius, and S. M. van der Vies Cdc37p Is Required for Stress-Induced High-Osmolarity Glycerol and Protein Kinase C Mitogen-Activated Protein Kinase Pathway Functionality by Interaction with Hog1p and Slt2p (Mpk1p) Eukaryot. Cell, March 1, 2007; 6(3): 521 - 532. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
