Developmental changes in plasticity, synaptic, glia and connectivity protein levels in rat dorsal hippocampus

Abstract

Thus far the identification and functional characterization of the molecular mechanisms underlying synaptic plasticity, learning, and memory have not been particularly dissociated from the contribution of developmental changes. Brain plasticity mechanisms have been largely identified and studied using in vitro systems mainly derived from early developmental ages, yet they are considered to be general plasticity mechanisms underlying functions -such as long-term memory- that occurs in the adult brain. Although it is possible that part of the plasticity mechanisms recruited during development is then re-recruited in plasticity responses in adulthood, systematic investigations about whether and how activity-dependent molecular responses differ over development are sparse. Notably, hippocampal-dependent memories are expressed relatively late in development, and the hippocampus undergoes and extended developmental post-natal structural and functional maturation, suggesting that the molecular mechanisms underlying hippocampal neuroplasticity may actually significantly change over development. Here we quantified the relative basal expression levels of sets of plasticity, synaptic, glia and connectivity proteins in rat dorsal hippocampus, a region that is critical for the formation of long-term explicit memories, at two developmental ages, postnatal day 17 (PN17) and PN24, which correspond to a period of relative functional immaturity and maturity, respectively, and compared them to adult age. We found that the levels of numerous proteins and/or their phosphorylation, known to be critical for synaptic plasticity underlying memory formation, including immediate early genes (IEGs), kinases, transcription factors and AMPA receptor subunits, peak at PN17 when the hippocampus is not yet able to express long-term memory. It remains to be established if these changes result from developmental basal activity or infantile learning. Conversely, among all markers investigated, the phosphorylation of calcium calmodulin kinase II α (CamKII α and of extracellular signal-regulated kinases 2 (ERK-2), and the levels of GluA1 and GluA2 significantly increase from PN17 to PN24 and then remain similar in adulthood, thus representing correlates paralleling long-term memory expression ability.

Keywords: Connectivity; Development; Hippocampus; Memory; Myelin; Plasticity; Protein; Synapse.

Figure 1. PN17 rats show IA memory only shortly after training, PN24 and PN80 form strong and long-lasting memories

Experimental schedule is shown above the panel. Rats were trained at PN17, PN24 or PN80 (n=8/group) and tested either immediately after training, 1 day (d) or 7 d after training. Acquisition (Acq.) and memory retention are expressed as mean latency ± s.e.m (in seconds, s). Two-way ANOVA followed by Bonferroni post hoc tests, ***p < 0.001.

Figure 2. The levels of immediate early genes c-Fos, Zif268 and Arc are higher in early development and decrease in adulthood

Examples and densitometric western blot analyses of dHC total extracts from naïve rats euthanized at PN17 (white), PN24 (gray) or PN80 (adult; black) (n=4–8). Data are expressed as mean percentage ± s.e.m. of adult naïve rats. One-way ANOVA followed by Newman–Keuls post hoc tests, *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 3. Opposite developmental correlation between CREB vs. CaMKIIα and ERK1/2 phosphorylation

Examples and densitometric western blot analyses of dHC total extracts from untrained rats euthanized at PN17 (white), PN24 (gray) or PN80 (adult; black) (n=4–8). Data are expressed as mean percentage ± s.e.m. of adult naïve rats. One-way ANOVA followed by Newman–Keuls post hoc tests, *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4. Significant increase of GluA1 and GluA2 AMPA receptor subunit concentrations and significant decrease of their phosphorylation over development

Examples and densitometric western blot analyses of dHC total extracts from naïve rats euthanized at PN17 (white), PN24 (gray) or PN80 (adult; black) (n=4–8). Data are expressed as mean percentage ± s.e.m. of adult naïve rats. One-way ANOVA followed by Newman–Keuls post hoc tests, *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 5. GAD65 and GAD67 increase over development

Examples and densitometric western blot analyses of dHC total extracts from untrained rats euthanized at PN17 (white), PN24 (gray) or PN80 (adult; black) (n=4–8). Data are expressed as mean percentage ± s.e.m. of adult naïve rats. One-way ANOVA followed by Newman–Keuls post hoc tests, *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 6. Structural synapse maturation proteins increase with development

Examples and densitometric western blot analyses of dHC total extracts from naïve rats euthanized at PN17 (white), PN24 (gray) or PN80 (adult; black) (n=4–8). Data are expressed as mean percentage ± s.e.m. of adult naïve rats. One-way ANOVA followed by Newman–Keuls post hoc tests, *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 7. Neurite connectivity marker levels decrease over development

Examples and densitometric western blot analyses of dHC total extracts from naïve rats euthanized at PN17 (white), PN24 (gray) or PN80 (adult; black) (n=4–8). Data are expressed as mean percentage ± s.e.m. of adult naïve rats. One-way ANOVA followed by Newman–Keuls post hoc tests, *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 8. Changes in myelination markers over development

Examples and densitometric western blot analyses of dHC total extracts from naïve rats euthanized at PN17 (white), PN24 (gray) or PN80 (adult; black) (n=4–8). Data are expressed as mean percentage ± s.e.m. of adult naïve rats. One-way ANOVA followed by Newman–Keuls post hoc tests, *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 9. Synopsis of plasticity, synaptic, glia and connectivity protein levels in rat dorsal hippocampus over development

Graphic representations of the identified temporal molecular changes occurring in the rat dorsal hippocampus from PN17 to PN24 and to PN80 (adult). The ontogenic progression is characterized by a decrease in markers of neural activation and an increase in proteins supporting structural and functional maturations, in particular those involved in growth of dendrites, axons and synapses. The distinct molecular background of PN17 compared to PN24 and adult rat dorsal hippocampus may reflect the period of infantile amnesia.