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Anti-glucocorticoid gene therapy reverses the impairing effects of elevated corticosterone on spatial memory, hippocampal neuronal excitability, and synaptic plasticity - PubMed

  • ️Fri Jan 01 2010

Anti-glucocorticoid gene therapy reverses the impairing effects of elevated corticosterone on spatial memory, hippocampal neuronal excitability, and synaptic plasticity

Theodore C Dumas et al. J Neurosci. 2010.

Abstract

Moderate release of the major stress hormones, glucocorticoids (GCs), improves hippocampal function and memory. In contrast, excessive or prolonged elevations produce impairments. Enzymatic degradation and reformation of GCs help to maintain optimal levels within target tissues, including the brain. We hypothesized that expressing a GC-degrading enzyme in hippocampal neurons would attenuate the negative impact of an excessive elevation in GC levels on synaptic physiology and spatial memory. We tested this by expressing 11-beta-hydroxysteroid dehydrogenase (type II) in dentate gyrus granule cells during a 3 d GC treatment followed by examination of synaptic responses in hippocampal slices or spatial performance in the Morris water maze. In adrenalectomized rats with basal GC replacement, additional GC treatments for 3 d reduced synaptic strength and promoted the expression of long-term depression at medial perforant path synapses, increased granule cell and CA1 pyramidal cell excitability, and impaired spatial reference memory (without influencing learning). Expression of 11-beta-hydroxysteroid dehydrogenase (type II), mostly in mature dentate gyrus granule cells, reversed the effects of high GC levels on granule cell and pyramidal cell excitability, perforant path synaptic plasticity, and spatial memory. These data demonstrate the ability of neuroprotective gene expression limited to a specific cell population to both locally and trans-synaptically offset neurophysiological disruptions produced by prolonged increases in circulating stress hormones. This report supplies the first physiological explanation for previously demonstrated cognitive sparing by anti-stress gene therapy approaches and lends additional insight into the hippocampal processes that are important for memory.

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Figures

Figure 1.
Figure 1.

Plasmid map for 11βHSDII and eGFP expression. A, Diagram of the 11βHSDII plasmid showing the herpes oriS origin of replication flanked by herpes α4 and α22 promoters. The eGFP gene is driven by the α22 promoter and ends with a simian virus poly(A) termination sequence (SV40). 11βHSDII was ligated to a cytomegalovirus poly(A) termination sequence (data not shown) and inserted at the HindIII (2484) site just downstream of the α4 promoter. Also shown are the ampicillin resistance (ampR)-positive selection gene and packaging “a” sequence that is necessary for inclusion of the plasmid into viral capsids. B, eGFP expression from an animal in the MWM experiment that received the 11βHSDII vector. Photos show eGFP-positive granule cells in a 33 μm hippocampal section. Digital photo was taken at 10× magnification. Scale bar, 200 μm. C, Photos show eGFP-positive granule cells in 40 μm sections (11βHSDII, left; eGFP, right) from hippocampal slices used for electrophysiology. Digital photos were taken at 20× magnification. Scale bar, 100 μm.

Figure 2.
Figure 2.

Effects of CORT and 11βHSDII expression on PP–DG baseline synaptic strength and granule cell excitability. A, I/O curve shows mean EPSP slope values plotted against mean FP amplitude values for all groups (low CORT/11βHSDII, n = 19; low CORT/eGFP, n = 19; high CORT/11βHSDII, n = 16; high CORT/eGFP, n = 18). Inset shows averaged waveforms of responses recorded in the middle molecular layer at all stimulation intensities (5 sweeps per level). Calibration: 1 mV, 10 ms. B, Bar graph shows mean EPSP to FP ratios plotted by stimulation intensity for each condition. C, ISI curve shows mean percentage PPF values plotted against ISI values. D, Bar graph shows PS/EPSP ratio across stimulation intensities for each group. *p < 0.025 by ANOVA. Inset shows averaged waveforms (10 sweeps each) of responses recorded in the granule cell layer at all stimulation intensities. Brackets and asterisk denote significant post hoc two-way repeated-measures ANOVA.

Figure 3.
Figure 3.

Effects of CORT and 11βHSDII expression on LTD. A, Timelines for mean normalized responses before and after LTD induction in low-CORT animals. Inset shows overlaid averaged waveforms representing responses before and after LTD induction (baseline, 20 sweeps; after induction, 10 sweeps). Calibration: 1 mV, 10 ms. B, Timelines for mean normalized responses before and after LTD induction in high-CORT animals. Inset shows overlaid averaged waveforms representing responses before and after LTD induction. *p < 0.0001, significant difference from baseline at 20–25 min after termination of induction.

Figure 4.
Figure 4.

Learning curves and probe trial dwell times in the MWM. A, Learning curves show mean escape latencies across training block for each group. B, Histograms show mean dwell time for each quadrant (g, goal; a, adjacent; o, opposite) during the immediate probe trial. *p < 0.05, **p < 0.005, significant difference from chance (15 s) in post hoc one-group t tests. C, Histograms show mean dwell time for each quadrant during the 24 h probe trial.

Figure 5.
Figure 5.

Fluorescent images showing eGFP in infected dentate gyrus granule cells and immunohistochemistry results for BrdU and DCX. All images are of the granule cell layer in the superior blade with dendrites pointing upward (40× magnification). A1, Infected neurons (green) do not colocalize with BrdU (red). A2, There is no red fluorescence in sections not treated with anti-BrdU primary antibody. B1, In most cases, infected neurons (green) do not colocalize with DCX (red). Instances of colocalization between eGFP and DCX could be found in low-CORT (B1a) and high-CORT (B1b) animals. B2, There is no red fluorescence in sections not treated with anti-DCX primary antibody. αMs, Anti-mouse; αGt, anti-goat.

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