Elsevier

Neuroscience

Volume 226, 13 December 2012, Pages 145-155
Neuroscience

The distribution of phosphodiesterase 2A in the rat brain

https://doi.org/10.1016/j.neuroscience.2012.09.011Get rights and content

Abstract

The phosphodiesterases (PDEs) are a superfamily of enzymes that regulate spatio-temporal signaling by the intracellular second messengers cAMP and cGMP. PDE2A is expressed at high levels in the mammalian brain. To advance our understanding of the role of this enzyme in regulation of neuronal signaling, we here describe the distribution of PDE2A in the rat brain. PDE2A mRNA was prominently expressed in glutamatergic pyramidal cells in cortex, and in pyramidal and dentate granule cells in the hippocampus. Protein concentrated in the axons and nerve terminals of these neurons; staining was markedly weaker in the cell bodies and proximal dendrites. In addition, in both hippocampus and cortex, small populations of non-pyramidal cells, presumed to be interneurons, were strongly immunoreactive. PDE2A mRNA was expressed in medium spiny neurons in neostriatum. Little immunoreactivity was observed in cell bodies, whereas dense immunoreactivity was found in the axon tracts of these neurons and their terminal regions in globus pallidus and substantia nigra pars reticulata. Immunostaining was dense in the medial habenula, but weak in other diencephalic regions. In midbrain and hindbrain, immunostaining was restricted to discrete regions of the neuropil or clusters of cell bodies. These results suggest that PDE2A may modulate cortical, hippocampal and striatal networks at several levels. Preferential distribution of PDE2A into axons and terminals of the principal neurons suggests roles in regulation of axonal excitability or transmitter release. The enzyme is also in forebrain interneurons, and in mid- and hindbrain neurons that may modulate forebrain networks and circuits.

Highlights

► PDE2A is highly expressed in principal neurons of cortex and hippocampus. ► PDE2A is also highly expressed by striatal medium spiny neurons. ► In all these neurons, protein concentrated in the axons and terminals. ► PDE2A is evident in non-pyramidal neurons, interneuron terminals, and hindbrain. ► PDE2A is positioned to modulate cortical, hippocampal and striatal networks at several levels.

Introduction

The cyclic nucleotides cAMP and cGMP are ubiquitous intracellular second messengers that regulate a remarkable diversity of biological processes. Utilization of only two molecules for diverse functions is dependent upon the ability to maintain the spatial and temporal specificity of these signals. Key to this specificity is the phosphodiesterase (PDE) superfamily of enzymes, which hydrolyze cAMP and cGMP to terminate their action (Conti and Beavo, 2007, Omori and Kotera, 2007). Eleven families of mammalian PDEs are encoded by 21 genes that, through alternative RNA splicing, give rise to well over 60 isoforms that differ in substrate specificity and allosteric regulation. Each PDE gene has a unique expression pattern at the tissue and cellular level (Bingham et al., 2006). Furthermore, the subcellular distribution of isoforms is restricted and may be subject to local regulation, as has been shown for PDE4 (Lynch et al., 2006). Thus, the precise localization of specific PDEs may be crucial for control of cyclic nucleotide signaling. PDE families in the brain exhibit distinct but overlapping regional and circuit distributions (Menniti et al., 2006, Lakics et al., 2010, Kelly, 2012); the distribution of specific isoforms can provide important clues regarding their roles in brain function. The goal of the present study was to identify the cellular and subcellular distribution of PDE2A, an isoform notable for its exceptionally high expression in the brain.

PDE2A is a dual substrate enzyme, capable of degrading both cGMP and cAMP (Sonnenburg et al., 1991, Rosman et al., 1997). The enzymatic activity of PDE2A may be regulated allosterically by cGMP binding to an N-terminal GAF (cGMP-specific and -stimulated PDEs, Anabaena adenylyl cyclases, and Escherichia coli FhlA) domain to increase hydrolytic activity (Stroop and Beavo, 1991, Juilfs et al., 1999). This provides a potential mechanism by which PDE2A may exert regulatory feedback inhibition of cGMP signaling, as well as cross-talk regulatory inhibition of cAMP signaling by cGMP (MacFarland et al., 1991, Stroop and Beavo, 1991). In a survey of the expression pattern of PDE2A protein in multiple mammalian species (Stephenson et al., 2009), we found that by far the highest levels of protein were in the brain, as suggested by previous data on mRNA expression (Sonnenburg et al., 1991, Rosman et al., 1997, Van Staveren et al., 2003). In our 2009 study, we reported that the expression pattern of PDE2A in the brain is generally consistent across species from rodent to primate. We here present a more detailed analysis of the distribution of this enzyme in the brain, using rat as a representative species.

Section snippets

Animals

Crl:CD(SD)IGS BR rats (strain code 001) were obtained from Charles River Laboratories (Kingston, NY, USA). All rats used in this study were housed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, and were used in accordance with an approved animal care and use protocol, the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals (National Research Council, 1996), and all applicable regulations.

PDE2A mRNA

In situ hybridization demonstrated that message for PDE2A was present at high levels in gray matter throughout the forebrain (Fig. 1; sense control (not shown) revealed no significant signal). PDE2A mRNA was highly expressed in the internal granule layer of the olfactory bulb, and in the anterior olfactory nucleus (Fig. 1A, B). In neocortex, signal was stronger in superficial than deep layers, and was especially intense over the tightly packed layer of neurons in piriform cortex and the

Discussion

The dual substrate phosphodiesterase PDE2A is highly expressed in the brain (Repaske et al., 1993, Yang et al., 1994, Van Staveren et al., 2003, Stephenson et al., 2009, Lakics et al., 2010, Kelly, 2012), suggesting a significant role for the enzyme in regulating neuronal signaling, and fostering interest in developing selective pharmacological inhibitors as potential neurotherapeutics (Menniti et al., 2006, Conti and Beavo, 2007, Omori and Kotera, 2007, Schmidt, 2010). Unique insights into

Acknowledgements

We thank Wilma C. G. van Staveren (Institute of Interdisciplinary Research (IRIBHM), Université Libre de Bruxelles, Faculty of Medicine, Campus Erasme, 808 Route de Lennik, B-1070 Brussels, Belgium) for early input into this study.

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    Present address: University of South Carolina School of Medicine, Department of Pharmacology, Physiology & Neuroscience, VA Building 1, Third Floor, Columbia, SC 29208, USA.

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    Present address: Selventa, One Alewife Center, Cambridge, MA 02140, USA.

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