Persistent decrease in multiple components of the perineuronal net following status epilepticus - PubMed (original) (raw)

Persistent decrease in multiple components of the perineuronal net following status epilepticus

Paulette A McRae et al. Eur J Neurosci. 2012 Dec.

Abstract

In the rodent model of temporal lobe epilepsy, there is extensive synaptic reorganization within the hippocampus following a single prolonged seizure event, after which animals eventually develop epilepsy. The perineuronal net (PN), a component of the neural extracellular matrix (ECM), primarily surrounds inhibitory interneurons and, under normal conditions, restricts synaptic reorganization. The objective of the current study was to explore the effects of status epilepticus (SE) on PNs in the adult hippocampus. The aggrecan component of the PN was studied, acutely (48 h post-SE), sub-acutely (1 week post-SE) and during the chronic period (2 months post-SE). Aggrecan expressing PNs decreased by 1 week, likely contributing to a permissive environment for neuronal reorganization, and remained attenuated at 2 months. The SE-exposed hippocampus showed many PNs with poor structural integrity, a condition rarely seen in controls. Additionally, the decrease in the aggrecan component of the PN was preceded by a decrease in hyaluronan and proteoglycan link protein 1 (HAPLN1) and hyaluronan synthase 3 (HAS3), which are components of the PN known to stabilize the connection between aggrecan and hyaluronan, a major constituent of the ECM. These results were replicated in vitro with the addition of excess KCl to hippocampal cultures. Enhanced neuronal activity caused a decrease in aggrecan, HAPLN1 and HAS3 around hippocampal cells in vivo and in vitro, leaving inhibitory interneurons susceptible to increased synaptic reorganization. These studies are the foundation for future experiments to explore how loss of the PN following SE contributes to the development of epilepsy.

© 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

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Figures

Figure 1

Figure 1. Status epilepticus decreases expression of aggrecan positive PNs in the rat dorsal hippocampus

PN expression was observed in the dentate gyrus/CA4 (A–D), CA3 (E–H), CA1 (I–L), and subiculum (M–P) under control conditions (A, E, I, M) or following pilocarpine induced SE. Aggrecan expression did not differ from control 48 hours post-SE (B, F, J, N). One week post-SE there was a decrease in the expression of Cat-315 in all regions of the hippocampus, (C, G, K, O), which remained attenuated two months following SE (D, H, L, P). Scale bar, 200 μ.m.

Figure 2

Figure 2. Status epilepticus alters the quantity of Aggrecan expressing PNs surrounding parvalbumin interneurons in the rat dorsal hippocampus

In the control CA1 region of the hippocampus aggrecan PNs (green) encapsulated parvalbumin cells (red) (A–C). Two months post-SE there were parvalbumin cells lacking the PN (D–F). Stereological counts of aggrecan positive PNs with normal morphology (G–I) and were performed at 48 hours (G), one week (H), and two months (I) post-SE. There was no change in aggrecan expressing PNs 48 hours after SE (G). There was a significant decrease in aggrecan PNs one week following SE in all regions of the hippocampus Bonferroni’s post-hoc test p<0.001 (H). There was a significant decrease in aggrecan PNs two months post-SE in all regions of the hippocampus, Bonferroni’s post-hoc test CA3 and the subiculum p<0.05, CA1 p<0.01, DG and CA4 p<0.001 (I). Error bars represent the standard error of the mean. Scale bar, 50 μ.m.

Figure 3

Figure 3. Status epilepticus alters the quality of aggrecan expressing PNs surrounding parvalbumin interneurons in the rat dorsal hippocampus

The PN normally ensheathes the soma and proximal processes (A), however after SE the structural integrity of the PN was compromised (B). Stereological counts of aggrecan positive PNs with poor integrity (degraded) (C–E) were performed at 48 hours (C), one week (D), and two months (E) post-SE. There were few degraded PNs 48 hours after SE (C). There was a significant increase in degraded PNs in all regions excluding the dentate gyrus at one week, Bonferroni’s post-hoc test CA4 p<0.05, CA3, CA1 and subiculum p<0.001 (D). There was a significant increase in degraded PNs in all regions excluding the subiculum two months post-SE, Bonferroni’s post-hoc test DG and CA4 p<0.001, CA3 p<0.05, CA1 p<0.01 (E). Error bars represent the standard error of the mean. Scale bar, 40 μ.m.

Figure 4

Figure 4. HAPLN1 expression decreases in the rat dorsal hippocampus following status epilepticus

In the control CA1 region HAPLN1 (A) and aggrecan (B) colocalize (C, HAPLN1 is red, Cat-315 is green). HAPLN1 expression decreased in the CA1 region 48 hours (D), one week (E) and two months (F) following SE. HAPLN1 expression significantly decreased 48 hours post-SE, Bonferroni’s post-hoc test CA4 and CA1 p<0.0001 (G), and remains attenuated one week post-SE, Bonferroni’s post-hoc test CA1 p<0.001 (H), and two months post-SE, Bonferroni’s post-hoc test CA4, CA3, CA1 p<0.001 (I). Error bars represent the standard error of the mean. Scale bar, 100 μ.m.

Figure 5

Figure 5. HAS3 expression is decreased in the rat dorsal hippocampus following status epilepticus

In the DG/CA4 region aggrecan (green) colocalizes with HAS3 (red) (A,B,F). Aggrecan colocalized with HAS3 in CA3 (K,L,P). There was a decrease in HAS3 expression 48 hours post-SE in DG/CA4region (C,H) and CA3(M,R). There was a more pronounced decrease in HAS3 1 week post-SE in the DG/CA4 and CA3 regions (D,I; N,S). HAS3 expression remained suppressed at 2 months in the DG/CA4 and CA3 regions (E,J; O,T). High magnification images (G–J and Q–T). Scale bar, 100 μ.m (A–F, K–P). Scale bar 50 μ.m (G–J, Q–T).

Figure 6

Figure 6. Increased neuronal activity in-vitro decreases aggrecan, HAPLN1 and HAS3 expression

Saline (A1-A4, D1-D4) or 20 mM KCl was added to hippocampal neurons at 30 DIV for 6 hours (B1-B4, E1-E4) or 2 days (C1-C4, F1-F4). In the control condition aggrecan expression was found in the cell body and along the processes of hippocampal neurons (A1, A4, D1, D4), 6 hours after the addition of the addition of KCl aggrecan was still expressed (B1, B4, E1, E4). HAPLN1 expression was significantly decreased throughout the cell (B2, B4, G) and HAS3 expression was limited to the soma and no longer expressed in the processes (E2, E4, H). However, following 2 days of KCl treatment, with cultures fixed at one week, aggrecan expression decreased (C1, C4, F1, F4) while HAPLN1 (C2, C4, G) and HAS3 (F2, F4, H) levels remain depressed. DAPI expression (A3, B3, C3, D3, E,3, F3). The Bonferroni post-test shows a significant decrease in HAPLN1 after KCl treatment for 6 hours (p<0.05) and two days (p<0.05) and HAS3 after 6 hours (p<0.0001) and two days (p<0.0001). Scale bar, 50 μ.m.

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