Growth hormone modulates hippocampal excitatory synaptic transmission and plasticity in old rats

Abstract

Alterations in the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPA-R) and N-methyl-D-aspartate receptor (NMDA-R) have been documented in aged animals and may contribute to changes in hippocampal-dependent memory. Growth hormone (GH) regulates AMPA-R and NMDA-R-dependent excitatory transmission and decreases with age. Chronic GH treatment mitigates age-related cognitive decline. An in vitro CA1 hippocampal slice preparation was used to compare hippocampal excitatory transmission and plasticity in old animals treated for 6-8 months with either saline or GH. Our findings indicate that GH treatment restores NMDA-R-dependent basal synaptic transmission in old rats to young adult levels and enhances both AMPA-R-dependent basal synaptic transmission and long-term potentiation. These alterations in synaptic function occurred in the absence of changes in presynaptic function, as measured by paired-pulse ratios, the total protein levels of AMPA-R and NMDA-R subunits or in plasma or hippocampal levels of insulin-like growth factor-I. These data suggest a direct role for GH in altering age-related changes in excitatory transmission and provide a possible cellular mechanism through which GH changes the course of cognitive decline.

Figure 1. Electrophysiological recordings on hippocampal slices

Anatomical section of hippocampus depicting the major subregions of the hippocampus and the positioning of electrodes for electrophysiological recordings. A bipolar stimulating electrode was positioned in area CA3 for the stimulation of the Schaffer collateral pathway and responses were recorded utilizing a recording electrode positioned in the CA1 region.

Figure 2. Curve fitting analysis of CMPD, AMPA and NMDA responses

Compound input/output (I/O) curves (A), AMPA-R-mediated I/O curves (B), and NMDA-R-mediated I/O curves (C) were generated from hippocampal CA1 field recordings. The stimulus intensity required to elicit the half maximal response in each experimental group was compared as demonstrated by the bar graph. (A) Compound I/O curves were unaffected by age or GH treatment, p = 0.997. (B) AMPA-R-mediated sfEPSPs did not differ between young and old saline-treated rats, but slices from old GH-treated rats showed significant enhancement (p = 0.01; * = Old GH significantly different from Young and Old Saline). (C) NMDA-R-mediated fEPSPs declined with age p < 0.001, and these responses were enhanced by chronic GH treatment to a greater responsiveness than that observed in old p = 0.011 and young saline rats p = 0.008 (* = Old Saline significantly different from Young Saline and Old GH).

Figure 3. Presynaptic facilitation

Bar graph illustrating the paired-pulse ratio (Amplitude Pulse 2/Amplitude Pulse 1, inter-pulse interval: 50 msec) of fEPSPs recorded from young saline rats and in old rats treated with either saline or GH. No group differences in this measure were observed, p > 0.05).

Figure 4. Long-Term Potentiation

The time course of LTP induction in young saline rats and in old rats treated with saline or GH is represented as percent amplitude of enhancement compared to baseline (A). Analysis of the percent potentiation in the last 5 min of LTP (30-35 min) compared to the baseline (1-5 min) (B) for each treatment group revealed that LTP in old GH treated rats was significantly greater than in old saline rats p = 0.016 (*); young saline rats did not differ significantly from either old group.

Figure 5. Representative Western Blots for Synaptic Proteins in the CA1 Region of Hippocampus

Western blot analysis was performed to assess CA1 protein levels of the NMDA subunits NR2A, NR1, and NR2B, and the AMPA subunits GluR1 and GluR2. Actin was assessed on each blot to insure equal protein loading. Experimental groups were the following: Lane 1: young saline-treated, Lane2: old saline-treated, and Lane 3: old GH-treated Fischer 344 × Brown Norway rats.

Figure 6. Assessment of glutamate receptor subunit protein level changes

Densitometric Western blot analysis of subunit proteins in the CA1 region evaluated relative levels of the NR1, NR2A, and NR2B subunits of the NMDA receptor, and the GluR1 and GluR2 subunits of the AMPA-R . The analysis revealed that only NR2B declined significantly with age (p < 0.001), an effect that was not mitigated by GH treatment (* = Young Saline significantly different from Old Saline and Old GH). Chronic GH treatment twice daily for 6-8 months did not produce a significant change in the protein levels of any of the NMDA or AMPA subunits compared to that present in old saline treated animals.

Figure 7. Examination of IGF-I plasma and tissue levels

IGF-I levels were determined in the plasma (A) and hippocampus (B) of young saline rats and old rats treated with either saline or GH. (A) The decrease in plasma IGF-I between young and old saline rats fell short of significance (p = 0.098). Plasma IGF-I levels in old rats treated with GH do not differ significantly from old saline rats, but were significantly lower than in young saline rats p = 0.02 (* = Young Saline significantly different from Old GH). (B) IGF-I levels in hippocampus did not differ with age or GH injection.