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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 5, 2003 as doi:10.1096/fj.02-0333fje. |
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,2
Department of Pharmacology and Anatomy, Rudolf Magnus Institute of Neuroscience;
* Department of Neonatology, Wilhelmina Children’s Hospital, University Medical Center, Utrecht, The Netherlands; and
Department Pharmacological Sciences, University of Milan, Milan, Italy
3Correspondence: Department of Pharmacology and Anatomy, Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht, P.O. Box 85060, 3508 AB Utrecht, The Netherlands. E-mail: V.M.Wiegant{at}med.uu.nl
SPECIFIC AIMS
The aim of this study was to investigate long-term effects of neonatal treatment with the synthetic glucocorticoid (GC) hormone dexamethasone (DEX) on hippocampus functions in the rat. The questions we asked were 1) Does neonatal treatment with DEX lead to impaired hippocampus-dependent spatial learning later in life? 2) If so, is this impairment associated with a deficit in hippocampal synaptic plasticity, a putative neuronal substrate of learning? and 3) Does neonatal DEX treatment affect the postsynaptic hippocampal NMDA receptor complex, known to be critically involved in spatial learning and synaptic plasticity?
PRINCIPAL FINDINGS
1. Spatial learning is impaired in adult rats after neonatal DEX treatment
Newborn rats were i.p. injected with DEX on postnatal day 1 (0.5 µg/g body weight), 2 (0.3 µg/g), and 3 (0.1 µg/g) or with equal volumes of saline (SAL). Male animals were used for experimentation at an age of 3-4 months. When tested for place learning with multiple trials in the Morris water maze (3 acquisition trials/day for 5 consecutive days), DEX and SAL rats showed a significant decrease in latency to reach the submerged platform over days (P<0.001). Latencies of DEX rats were significantly longer than of SAL rats (P<0.05), whereas swimming speed did not differ. In a single-trial learning version of the maze (1 acquisition and 1 retention trial/day for 6 days with a different location of the platform each day), only SAL rats showed a significant decrease in latency and distance swum between the acquisition and retention trial (P<0.05), whereas there was no difference between groups during the acquisition trial. In the visible platform version of the maze (3 trials/day for 3 days), there were no differences between DEX and SAL rats, indicating that sensorimotor and motivational functions were not affected.
2. Long-term potentiation is impaired and long-term depression facilitated in hippocampal slices from adult rats after neonatal DEX treatment
Hippocampal slices from adult DEX and SAL rats were used to study long-term potentiation (LTP) and long-term depression (LTD) in the CA1 field by stimulation of afferent fibers at stimulus intensity yielding an fEPSP of half-maximal amplitude. Paired-pulse facilitation was not different between the treatment groups (Fig. 1
A, B). There was a significant reduction of the slope of the fEPSP after a low frequency conditioning train of 900 stimuli (1, 5, or 10 Hz) in slices of DEX compared with SAL rats (Fig. 1C, D
), whereas the average baseline fEPSP slope was not different. Potentiation after high frequency stimulation (50 or 100 Hz) was significantly reduced in DEX vs. SAL rats (Fig. 2C, D
).
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3. Reduced presence of the NR2B subunit in hippocampal postsynaptic densities of adult rats after neonatal DEX treatment
Western blot analysis of purified hippocampal postsynaptic densities (PSD) showed a reduction in immunostaining of the NR2B subunit of the NMDA receptor in DEX rats (-48.3%; P<0.01). Immunostaining of the NR1 and NR2A subunits of the NMDA receptor, the Glu2/3 subunits of the AMPA receptor, and several other proteins known to be highly enriched in PSD (actin, PSD-95, 
CaMKII) was not affected (Fig. 2A
). This reduction in NR2B subunit protein was observed only in PSD and not in the hippocampal homogenate and P2 fraction (Fig. 2B, C
).
4. Increased CaMKII activity in hippocampal postsynaptic densities of adult rats after neonatal DEX treatment
When hippocampal PSD were phosphorylated under conditions known to maximally activate CaMKII and immunoprecipitated with anti-NR2A antiserum, an increase of 32P-phosphate incorporation (P<0.01) was observed in a 170 kDa (+112.4%) and a 50 kDa band (+123.9%) corresponding to NR2A and CaMKII, respectively. Basal p286-CaMKII staining was increased (+88.3%; P<0.01) in DEX vs. SAL rats, but there was no difference in CaMKII staining. Phosphorylation of an exogenous CaMKII substrate (syntide-2) was also increased in DEX rats (+103.2%; P<0.01).
CONCLUSIONS AND SIGNIFICANCE
Our results that treatment with DEX for a brief period in neonatal life leads to a spatial learning deficit at adult age confirm and extend literature data. Our most important novel findings are that this cognitive deficit is associated with 1) attenuated LTP and facilitated LTD in the hippocampal CA1 field in the presence of normal paired-pulse facilitation, indicating reduced synaptic plasticity likely as a consequence of a postsynaptic defect; and 2) selective depletion of the NR2B subunit and increase of CaMKII activity in hippocampal PSD, indicating altered makeup and function of the hippocampal NMDA receptor complex in DEX rats.
There is ample evidence that spatial learning depends on hippocampal synaptic efficacy and that LTP and LTD contribute bidirectionally to synaptic reinforcement and memory storage. Spatial learning and synaptic plasticity in the hippocampal CA1 field are known to require activation of NMDA receptors. NMDA receptors are heteromeric assemblies of one NR1 subunit and various NR2 subunits, and their functional properties depend on subunit composition. An increase in the ratio NR2A/NR2B reduces the chances for LTP, increases those for LTD induction, and impairs learning. The significance of the NR2B subunit in this respect is indicated by findings that overexpression of the subunit in the forebrain of mice leads to facilitation of synaptic plasticity and spatial learning tasks and that selective knockdown of NR2B subunit expression in the hippocampus is sufficient to induce deficits in plasticity and learning in rats. Our present findings that NR2B subunit is reduced in hippocampal PSD of DEX rats whereas NR2A levels are not affected therefore likely explain the impaired hippocampal synaptic plasticity and the spatial learning deficit in these animals. We found the NR2B subunit depleted only in hippocampal PSD of DEX rats, not in the homogenate or crude synaptosomal fraction. This points to altered NMDA receptor assembly and/or trafficking rather than to reduced NR2B subunit expression as the mechanism underlying the reduction in NR2B in PSD.
CaMKII is closely associated with the NMDA receptor and its activity is another essential component of the molecular machinery of hippocampal synaptic plasticity. We found that the activity of CaMKII is increased along with autophosphorylation of the enzyme in hippocampal PSD of DEX rats. Since autophosphorylation of CaMKII is known to result in increased, autonomous activity of the enzyme, this suggests that in PSD of DEX rats, the enzyme is locked in the activated, Ca2+-independent state. Transgenic mice overexpressing a form of CaMKII with autonomous activity display a downward shift of the frequency response curve favoring LTD over LTP and spatial learning disabilities. The elevated activity of CaMKII in PSD of DEX rats may therefore be pertinent to the plasticity and learning deficits in these animals.
Our findings indicate that neonatal treatment with DEX results in structural and functional alterations in the hippocampal NMDA receptor complex that persist into adulthood and, likely as a consequence thereof, in impaired plasticity and cognition. These effects on the developmental programming of hippocampal neurons may be triggered by interaction of DEX with glucocorticoid receptors (GR) that regulate transcriptional activity or may reflect nongenomic actions (Fig. 3
). Sustained GC action has been associated with an increase in intracellular levels of free Ca2+, and disturbed intracellular Ca2+homeostasis with irreversible neuronal damage. Hippocampal neurons are rich in GR. In neonatal life, when endogenous GCs are regulated at extremely low levels, they may be particularly vulnerable to the effects of sustained GR activation since NR2B is the predominant NR2 subunit of the hippocampal NMDA receptor during that period, and NMDA receptors containing NR2B show enhanced Ca2+gating. Although the exact mechanism remains to be elucidated, our results highlight the post-translational machinery that regulates assembly, trafficking and function of the NMDA receptor as a potential target for regulatory and programming effects of GCs.
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Treatment of prematurely born human neonates with GCs is widely practiced to prevent of chronic lung disease, while the brain of these babies is in a phase of active development. Recent evidence has suggested adverse neurological and cognitive effects in infants of up to 5 years of age and has raised great concern. The present results indicate that, in the rat, neonatal treatment with GCs can alter the molecular makeup of the brain and lead to a lasting cognitive deficit. Although extrapolation of results from animal experimentation to humans is hazardous, our present results call for utmost prudence with the use of GCs in human infants until its long-term safety has been established.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0333fje; to cite this article, use FASEB J. (March 5, 2003) 10.1096/fj.02-0333fje 
2 Both authors equally contributed to the manuscript.
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-; n=6) or SAL rats (-
-; n=6). Symbols indicate significant differences vs. baseline (#P<0.05 within the DEX group; +P<0.05 within the SAL group) and differences in response between DEX and SAL treatment groups (*P<0.05; **P<0.01). D) Traces represent averaged fEPSPs (n=6) recorded before (1) and 30 min after (2) application of a 1 Hz or 100 Hz conditioning stimulus. Horizontal scale bar represents 5 ms, vertical bar 1 mV.
