D4 Dopamine Receptors Modulate NR2B NMDA Receptors and LTP in Stratum Oriens of Hippocampal CA1

Abstract Dopamine plays an important role in synaptic plasticity and learning and is involved in the pathogenesis of various neurological and psychiatric disorders. Here, we reveal staining of dopaminergic fibers in stratum oriens of the mouse hippocampal CA1 region, a finding that is consistent with earlier reports. Furthermore, we examined the effect of dopamine agonists on NMDAR-dependent early long-term potentiation (LTP) (40 min) during γ-aminobutyric acid (GABA)A-mediated blockade. LTP of the AMPA component was strongly reduced in stratum oriens but barely affected in stratum radiatum. This layer-specific effect was caused by D4 receptor activation, which augmented the inactivation of synaptic NMDAR-mediated currents (NMDA EPSCs) during LTP induction through a Ca2+-dependent G-protein–independent mechanism. A similar dopaminergic modulation of both NMDA EPSCs and LTP was also observed in mice constitutively lacking NR2A but was absent in mice lacking NR2B in principal forebrain neurons. Together, these experiments strongly indicate that dopaminergic modulation of early LTP in stratum oriens occurs through NMDARs containing NR2B subunits via D4Rs. Thus, a dopamine hyperfunction in stratum oriens may result in NMDAR hypofunction that could affect both normal and pathological conditions. L-745,870, PD168077, schizophrenia, SKF38393, stratum radiatum Introduction The dopamine system is thought to play an important role in hippocampus-mediated learning and memory (Lisman and Grace 2005; O'Carroll et al. 2006) and seems also highly relevant to the pathogenesis of several psychiatric disorders including attention-deficit/hyperactivity disorder (ADHD) and schizophrenia (Lewis and Lieberman 2000; Winterer and Weinberger 2004). The hippocampal manifestation of schizophrenia may be characterized by dysfunctions of 2 neurotransmitter pathways: a dopamine (DA) hyperfunction caused by an increased firing rate of dopaminergic neurons localized to the ventral tegmental area (VTA) and an NMDA hypofunction of the glutamatergic system (reviewed in Lisman et al. 2008). The NMDA hypofunction appears to comprise the main features of schizophrenia: positive symptoms like hallucinations, negative symptoms like social withdrawal and cognitive defects including deficits in association and working memory (Greene 2001). The characterization of the functional link between the dopaminergic and the glutamatergic systems in the hippocampus is still incomplete. The VTA and the substantia nigra supply the hippocampal formation with dopaminergic fibers. These fibers are mainly localized in the subiculum, hilus, and the stratum lacunosum-moleculare (SLM) (Gasbarri et al. 1997). This dopaminergic innervation could modulate glutamatergic synaptic transmission and synaptic plasticity within the hippocampal CA1 region through both D1-like and D2-like receptor families (Tarazi and Baldessarini 2000). Electrophysiological recordings performed in distinct CA1 layers have demonstrated a dopaminergic modulation of the direct cortical pathway to CA1 in SLM with a possible contribution from the inhibitory network (Otmakhova and Lisman 1999; Romo-Parra et al. 2005; Ito and Schuman 2007). At the Schaffer collateral–commissural synapses in stratum radiatum (RAD), the application of DA agonists or antagonists have indicated that D1-like receptors (D1R, D5R) increase the magnitude of early long-term potentiation (LTP) (Otmakhova and Lisman 1996) and stabilize late LTP both in vitro (Frey et al. 1991; Huang and Kandel 1995) and in vivo (Swanson-Park et al. 1999; Lemon and Mnahan-Vaughan 2006). Furthermore, selective genetic inactivation of the D1R suggested that D1Rs but not D5Rs are critical for both LTP at CA1 synapses and spatial learning (Granado et al. 2008; Ortiz et al. 2010). In contrast, the effects of D2-like receptors (D2R, D3R, D4R) are less explored in CA3-to-CA1 synapses and observations are still inconclusive. A D3R agonist has been shown to enhance LTP (Swant and Wagner 2006), whereas D3R mutant mice exhibited normal learning abilities (Xing et al. 2010). Notably, a D4R agonist depotentiated LTP in response to neuregulin-1 and theta-patterned stimuli (Kwon et al. 2008). Most studies have investigated the dopaminergic modulation of synaptic transmission and synaptic plasticity in CA1 with intact inhibition, which precluded an attribution of dopamine-mediated effects to either glutamatergic or γ-aminobutyric acid (GABA)ergic synapses. Moreover, very few studies have examined the dopaminergic modulation in stratum oriens (OR), in spite of the anatomical evidence for a stronger dopaminergic innervation of OR than RAD (Verney et al. 1985; Gasbarri et al. 1994; Kwon et al. 2008). Here, we compared dopaminergic modulation of NMDAR-dependent early LTP in OR and RAD in acute hippocampal slices using D1R/D5R and D4R agonists. We blocked GABAARs to avoid dopaminergic effects on inhibition and examined the AMPA component of glutamatergic transmission. Even though any NMDAR subtype constellation is capable of inducing NMDAR-dependent LTP in hippocampal RAD synapses (Berberich et al. 2005), dopaminergic modulation may lead to a preferential involvement of certain NMDAR subtypes. NMDARs containing NR2A have been proposed to interact with D1R/D5R (Lee et al. 2002; Varela et al. 2009), whereas NMDARs containing NR2B seem to interact with D2Rs (Liu et al. 2006). Therefore, we explored NMDAR subtype dependency for both NMDA and AMPA components of excitatory postsynaptic currents (EPSCs) in constitutive NR2A knockout (NR2A−/−) and forebrain-specific NR2B knockout mice (NR2BΔFb) in comparison with wild-type mice. We found that during LTP induction DA agonists activating D4Rs reduced the activity of NR2B-containing NMDARs in OR, causing input-specific modulation of LTP. Materials and Methods Experimental procedures were in accordance with the animal welfare guidelines of the Max Planck Society, the “Norwegian Animal Welfare Act” and the “European Union’s Directive 86/609/EEC.” Immunohistochemistry Z/EG mice (Novak et al. 2000) were crossbred with transgenic DATCre mice (Parlato et al. 2006) to generate Z/EGDATcre reporter mice. Four-week-old Z/EGDATcre mice were sacrificed with CO2, perfused through heart with 4% paraformaldehyde/phosphate buffered saline (PFA/PBS) and brains were isolated and postfixed with 4% PFA/PBS at 4 °C overnight. Vibratome sections (50 μm thick) were stained for green fluorescent protein (GFP) with anti-GFP antibody (1:500, Invitrogen), biotinylated secondary anti-Rabbit antibody (Vector Laboratories), amplified by VECTASTAIN ABC kit (Vector Laboratories) and developed with 3,3′-diaminobenzidine (Sigma). Sections were mounted with Eukitt (Fluka). Images were taken on an Axioplan microscope (Zeiss) and processed with Adobe Creative Suite. Extracellular Field Experiments Wild-type (C57BL/6) mice (P27–P30) were killed with isoflurane. Transverse slices (400 μm) from the dorsal half (...truncated)