Article Figures & Data
Figures
- Figure 1.
Overexpression of PDE4A5 in hippocampal excitatory neurons increases PDE4A5 protein levels and PDE4 activity. A, Mice were bilaterally injected with AAV9-CaMKII-eGFP or AAV9-CaMKII-PDE4A5-VSV into the hippocampus. B, Viral PDE4A5 protein expression was restricted to the hippocampus. C–E, Viral expression of PDE4A5 was observed in all three major hippocampal subregions. F–H, Transgene expression was not observed in hippocampal astrocytes (PDE4A5-VSV, red; GFAP, green; bottom). I, A representative immunoblot for PDE4A5 protein levels. Viral overexpression of PDE4A5 leads to significantly higher PDE4A5 protein levels in hippocampal lysates. J, Overexpression of PDE4A5 significantly increases PDE4 activity without affecting non-PDE4 activity. All error bars denote SEM. ***p = 0.001, ****p = 0.0001.
- Figure 2.
Overexpression of PDE4A5 in hippocampal excitatory neurons reduces cAMP levels in the hippocampus. A, Viral expression of PDE4A5 reduces cAMP levels in the hippocampus, but not in the prefrontal cortex or cerebellum. B, C, Representative cAMP immunoreactivity images in mice expressing eGFP or overexpressing PDE4A5. Note the reduction in cAMP immunoreactivity in the major hippocampal subregions, but not in the amygdala. All error bars denote SEM. *p < 0.05, **p = 0.01.
- Figure 3.
Overexpression of PDE4A5 in hippocampal excitatory neurons reduces forskolin-induced potentiation. A, Input–output curves relating the amplitude of the presynaptic fiber volley to the initial slope of the corresponding fEPSP at various stimulus intensities was not altered due to viral overexpression of PDE4A5 in hippocampal neurons. B, PDE4A5 overexpression does not change paired-pulse facilitation, a short-term form of synaptic plasticity, in hippocampal slices. C, Elevated expression of PDE4A5 in hippocampal neurons attenuates LTP induced by bath application with the adenylate cyclase activator forskolin. The mean fEPSP slope over the last 20 min of the recording was significantly reduced in PDE4A5 mice. In all sample sweeps, black traces indicate baseline, and red traces were acquired at 1 h after tetanus injection. All error bars denote SEM.
- Figure 4.
Localization of PDE4A5 by the unique N-terminal domain plays a central role in the memory deficits associated with the overexpression of PDE4A5. A, Overexpression of PDE4A5 in hippocampal neurons impairs the formation of long-term memories for context–fear associations without affecting freezing levels during training. B, In contrast, elevated PDE4A5 protein levels in hippocampal neurons do not change the formation of short-term contextual fear memories. C, PDE4A5 overexpression in hippocampal neurons does not alter the consolidation of amygdala-dependent tone-cued fear memories and also did not affect freezing levels during training in either fear-conditioning paradigm. D, Overexpression of a truncated form of PDE4A5, PDE4A5Δ4, that lacks the isoform-unique N-terminal domain or the short isoform PDE4A1 in hippocampal neurons does not alter the formation of long-term contextual fear memories. E, Increasing expression of PDE4A5 in hippocampal excitatory neurons impairs memory consolidation for object–location. F, In contrast, increasing PDE4A5 protein levels in the hippocampus leaves the consolidation of long-term memories for object identity undisturbed. G, Overexpression of the N-terminal-lacking PDE4A5Δ4 or the short isoform PDE4A1 in hippocampal neurons does not modulate memory consolidation for object location. H, I, Elevated expression of PDE4A5 in hippocampal excitatory neurons does not alter exploratory behavior in an open field task or anxiety levels in the zero maze task. The dotted line indicates no preference. All error bars denote SEM. **p = 0.01 ***p = 0.005.
- Figure 5.
Full-length but not truncated PDE4A5 attenuates FK-mediated cAMP responses assessed by a FRET indicator. A, FRET cAMP responses were measured in cultured hippocampal neurons expressing a control construct, PDE4A5, or truncated N-terminal domain-lacking PDE4A5Δ4 after consecutive bath application with the adenylate cyclase activator FK and phosphodiesterase inhibitor IBMX. Forskolin treatment significantly elevated cAMP levels in control neurons and neurons expressing PDE4A5Δ4, whereas the cAMP response was attenuated in neurons overexpressing full-length PDE4A5. B, Average FRET sensor responses for the different treatment conditions. Baseline FRET responses were not affected by the overexpression of either construct. The expression of PDE4A5, but not PDE4A5Δ4, attenuated the forskolin-mediated FRET response. Consecutive bath applications with the PDE inhibitor IBMX normalized the FRET responses. CT = 20 cells from three preparations; PDE4A5 = 11 cells from three preparations; PDE4A5Δ4 = 14 cells from three preparations. All error bars denote SEM. ***p ≤ 0.001.
- Figure 6.
Loss of the isoform-unique N-terminal domain alters the compartmentalization of PDE4A5 in hippocampal neurons. Full-length PDE4A5 is compartmentalized to both a discrete perinuclear area and dendritic compartments (left panels). In contrast, truncated PDE4A5 lacking the N-terminal domain that is unique for the isoform was located predominantly in the perinuclear region (middle panels). PDE4A5 expression is absent from the nucleus (right panels).
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