Antidepressants that inhibit both serotonin and norepinephrine reuptake impair long-term potentiation in hippocampus

Abstract

Rationale: Monoamine reuptake inhibitors can stimulate expression of brain-derived neurotrophic factor (BDNF) and alter long-term potentiation (LTP), a widely used model for the synaptic mechanisms that underlie memory formation. BDNF expression is upregulated during LTP, and BDNF in turn positively modulates LTP. Previously, we found that treatment with venlafaxine, a serotonin and norepinephrine reuptake inhibitor (SNRI), but not citalopram, a selective serotonin reuptake inhibitor (SSRI), reduced LTP in hippocampal area CA1 without changing hippocampal BDNF protein expression.

Objectives: We tested the hypothesis that combined serotonin and norepinephrine reuptake inhibition is necessary for LTP impairment, and we reexamined the potential role of BDNF by testing for region-specific changes in areas CA1, CA3, and dentate gyrus. We also tested whether early events in the LTP signaling pathway were altered to impair LTP.

Methods: Animals were treated for 21 days with venlafaxine, imipramine, fluoxetine, or maprotiline. In vitro hippocampal slices were used for electrophysiological measurements. Protein expression was measured by enzyme-linked immunosorbent assay (ELISA) and Western blotting.

Results: LTP was impaired only following treatment with combined serotonin and norepinephrine reuptake inhibitors (venlafaxine, imipramine) but not with selective serotonin (fluoxetine) or norepinephrine (maprotiline) reuptake inhibitors. BDNF protein expression was not altered by venlafaxine or imipramine treatment, nor were postsynaptic depolarization during LTP inducing stimulation or synaptic membrane NMDA receptor subunit expression affected.

Conclusions: LTP is impaired by chronic treatment with antidepressant that inhibit both serotonin and norepinephrine reuptake; this impairment results from changes that are downstream of postsynaptic depolarization and calcium influx.

Figure 1. LTP was significantly reduced in slices from venlafaxine (Ven) and imipramine (Imi) treated animals, but not in slices from maprotiline (Map) and fluoxetine (Fluo) treated animals

Graphs shows normalized extracellular field EPSP slopes evoked every 15 s, with HFS was delivered at time 0. A. At 55-60 min post-HFS, normalized EPSP slopes for slices in the Ven group were significantly reduced (p < 0.03) compared to the Con group. Insets at top show EPSPs from representative slices from the Con and Ven groups; for each example, EPSPs were averaged over the final 5 min of baseline recording period, and are shown superimposed with averaged EPSPs from the final 5 min of the post-HFS recording period. B. At 55-60 min post-HFS, normalized EPSP slopes for slices in the Imi group were significantly reduced (p < 0.02) compared to the Con group. Insets (top) show superimposed, averaged EPSPs (final 5 min of baseline and final 5 min of post-HFS recording) from representative slices from the Con and Imi groups. C. Normalized EPSP slopes at 55-60 min post-HFS were not significantly different between the Map and Con groups (p > 0.65). Insets show superimposed, averaged EPSPs (baseline, final 5 min) from representative slices from the Con and Map groups. D. Normalized EPSP slopes at 55-60 min post-HFS were not significantly different between the Fluo and Con groups (p > 0.40). Insets show superimposed, averaged EPSPs (baseline, final 5 min) from representative slices from the Con and Map groups.

Figure 2. BDNF and TrkB protein expression in micro-dissected areas of hippocampus were not affected by treatment with venlafaxine (Ven) or imipramine (Imi)

A1. BDNF protein levels in CA1, CA3, and dentate gyrus (DG) did not differ between Ven and control (Con) groups (all p-values > 0.70). A2. No difference in BDNF protein levels in area CA1 between Imi and Con groups (p > 0.70). B1. Representative TrkB western blot from one replication. Blot with proteins from micro-dissected CA1, CA3 and DG regions from one Ven and one Con animal were probed with TrkB antibody (top), then stripped and reprobed with -actin antibody (bottom) for loading control. Positions of molecular weight markers are shown at right, expected positions of full-length TrkB (145 kD) and -actin proteins are shown at left. The lower molecular weight (approximately 95 kD) protein recognized by the TrkB antibody is most likely the truncated TrkB receptor lacking the kinase domain (Klein et al 1990; Middlemas et al 1991). B2. For each protein sample, TrkB expression was normalized to the β-actin loading control, then all samples on the same blot were normalized relative to control, and results were averaged across blots. There were no significant differences (p-values > 0.35; n = 7 for both Con and Ven groups).

Figure 3. LTP impairment in slices from venlafaxine (Ven) and imipramine (Imi) treated animals could be overcome

A. Normalized EPSP slopes at 55-60 min following theta burst stimulation were not significantly different between the Ven and control (Con) groups (p > 0.55). Insets show superimposed, averaged EPSPs (baseline, final 5 min) from representative slices from the Con and Ven groups. B. Normalized EPSP slopes at 55-60 min following HFS in the presence of bicuculline (Bic; application period indicated by labeled horizontal bar) were not significantly different between the Imi and Con groups (p > 0.45). Insets show superimposed, averaged EPSPs (baseline, final 5 min) from representative slices from the Con and Imi groups.

Figure 4. Antidepressant effects on synaptic transmission

A. Imipramine (Imi), but not other antidepressants, increased the input-output function. A1. Example input-output curve from control (Con) slice. Field EPSPs were evoked by stimuli of varying intensity (1-10 V), and EPSP slopes were plotted against stimulus intensity to generate input-output curve. Area under the curve (AUC) was calculated then averaged across all slices from the same treatment condition. A2. AUC for slices from antidepressant treated and control animals. AUC was significantly greater for Imi group compared to Con (p < 0.005), but not for venlafaxine (Ven), maprotiline (Map), or fluoxetine (Fluo) groups relative to their controls (all p-values > 0.75). B, C. No effect of imipramine treatment on postsynaptic membrane potential during HFS. B1,2. Whole cell current clamp recordings of membrane potentials in representative CA1 pyramidal cells from imipamine-treated and control animals. Recordings were made first in normal ACSF (B1) and then following application of 100 μM picrotoxin (Ptx; B2); HFS was 100 Hz for 1 s. For each cell, membrane potentials during HFS were compared over 2 time periods: early during HFS (mean membrane potential measured immediately before EPSPs 2-4), and at the end of HFS (mean membrane potential measured immediately before EPSPs 91-100). C1. Membrane potentials during HFS in normal ACSF. C2. Membrane potentials during HFS after blocking GABA_A receptors with Ptx. There were no significant differences in C1 or C2 between the Imi and Con groups (all p-values > 0.12).

Figure 5. No effect of imipramine (Imi) treatment on NMDA receptor subunit proteins in synaptic membranes

A. Representative blot showing synaptic membrane proteins from two Imi and two control (Con) animals; proteins were probed for NMDA receptor subunits (NR1, NR2A, NR2B), PSD-95, and β-actin (loading control). B. For each protein, expression was normalized to β-actin, and then all values on the same blot were normalized relative to the mean control value. Results were averaged across blots. There were no significant differences in NMDA receptor subunit expression (all p-values > 0.15) or PSD-95 (p > 0.60).