Tonic Activation of NR2D-Containing NMDARs Exacerbates Dopaminergic Neuronal Loss in MPTP-Injected Parkinsonian Mice.

NR2D subunit-containing NMDA receptors (NMDARs) gradually disappear during brain maturation but can be recruited by pathophysiological stimuli in the adult brain. Here, we report that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication recruited NR2D subunit-containing NMDARs that generated an Mg2+-resistant tonic NMDA current (INMDA) in dopaminergic (DA) neurons in the midbrain of mature male mice. MPTP selectively generated an Mg2+-resistant tonic INMDA in DA neurons in the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA). Consistently, MPTP increased NR2D but not NR2B expression in the midbrain regions. Pharmacological or genetic NR2D interventions abolished the generation of Mg2+-resistant tonic INMDA in SNpc DA neurons, and thus attenuated subsequent DA neuronal loss and gait deficits in MPTP-treated mice. These results show that extrasynaptic NR2D recruitment generates Mg2+-resistant tonic INMDA and exacerbates DA neuronal loss, thus contributing to MPTP-induced Parkinsonism. The state-dependent NR2D recruitment could be a novel therapeutic target for mitigating cell type-specific neuronal death in neurodegenerative diseases.SIGNIFICANCE STATEMENT NR2D subunit-containing NMDA receptors (NMDARs) are widely expressed in the brain during late embryonic and early postnatal development, and then downregulated during brain maturation and preserved at low levels in a few regions of the adult brain. Certain stimuli can recruit NR2D subunits to generate tonic persistent NMDAR currents in nondepolarized neurons in the mature brain. Our results show that MPTP intoxication recruits NR2D subunits in midbrain dopaminergic (DA) neurons, which leads to tonic NMDAR current-promoting dopaminergic neuronal death and consequent abnormal gait behavior in the MPTP mouse model of Parkinson's disease (PD). This is the first study to indicate that extrasynaptic NR2D recruitment could be a target for preventing neuronal death in neurodegenerative diseases.

High Salt Intake Recruits Tonic Activation of NR2D Subunit-Containing Extrasynaptic NMDARs in Vasopressin Neurons.

In addition to producing a classical excitatory postsynaptic current via activation of synaptic NMDA receptors (NMDARs), glutamate in the brain also induces a tonic NMDAR current (INMDA) via activation of extrasynaptic NMDARs (eNMDARs). However, since Mg2+ blocks NMDARs in nondepolarized neurons, the potential contribution of eNMDARs to the overall neuronal excitatory/inhibitory (E/I) balance remains unknown. Here, we demonstrate that chronic (7 d) salt loading (SL) recruited NR2D subunit-containing NMDARs to generate an Mg2+-resistant tonic INMDA in nondepolarized [Vh (holding potential) -70 mV] vasopressin (VP; but not oxytocin) supraoptic nucleus (SON) neurons in male rodents. Conversely, in euhydrated (EU) and 3 d SL mice, Mg2+-resistant tonic INMDA was not observed. Pharmacological and genetic intervention of NR2D subunits blocked the Mg2+-resistant tonic INMDA in VP neurons under SL conditions, while an NR2B antagonist unveiled Mg2+-sensitive tonic INMDA but not Mg2+-resistant tonic INMDA In the EU group VP neurons, an Mg2+-resistant tonic INMDA was not generated by increased ambient glutamate or treatment with coagonists (e.g., d-serine and glycine). Chronic SL significantly increased NR2D expression but not NR2B expression in the SON relative to the EU group or after 3 d under SL conditions. Finally, Mg2+-resistant tonic INMDA selectively upregulated neuronal excitability in VP neurons under SL conditions, independent of ionotropic GABAergic input. Our results indicate that the activation of NR2D-containing NMDARs constitutes a novel mechanism that generates an Mg2+-resistant tonic INMDA in nondepolarized VP neurons, thus causing an E/I balance shift in VP neurons to compensate for the hormonal demands imposed by a chronic osmotic challenge.SIGNIFICANCE STATEMENT The hypothalamic supraoptic nucleus (SON) consists of two different types of magnocellular neurosecretory cells (MNCs) that synthesize and release the following two peptide hormones: vasopressin (VP), which is necessary for regulation of fluid homeostasis; and oxytocin (OT), which plays a major role in lactation and parturition. NMDA receptors (NMDARs) play important roles in shaping neuronal firing patterns and hormone release from the SON MNCs in response to various physiological challenges. Our results show that prolonged (7 d) salt loading generated a Mg2+-resistant tonic NMDA current mediated by NR2D subunit-containing receptors, which efficiently activated nondepolarized VP (but not OT) neurons. Our findings support the hypothesis that NR2D subunit-containing NMDARs play an important adaptive role in adult brain in response to a sustained osmotic challenge.

Enhanced function of NR2C/2D-containing NMDA receptor in the nucleus accumbens contributes to peripheral nerve injury-induced neuropathic pain and depression in mice.

N-methyl-d-aspartate receptors (NMDARs) dysfunction in the nucleus accumbens (NAc) participates in regulating many neurological and psychiatric disorders such as drug addiction, chronic pain, and depression. NMDARs are heterotetrameric complexes generally composed of two NR1 and two NR2 subunits (NR2A, NR2B, NR2C and NR2D). Much attention has been focused on the role of NR2A and NR2B-containing NMDARs in a variety of neurological disorders; however, the function of NR2C/2D subunits at NAc in chronic pain remains unknown. In this study, spinal nerve ligation (SNL) induced a persistent sensory abnormity and depressive-like behavior. The whole-cell patch clamp recording on medium spiny neurons (MSNs) in the NAc showed that the amplitude of NMDAR-mediated excitatory postsynaptic currents (EPSCs) was significantly increased when membrane potential held at -40 to 0 mV in mice after 14 days of SNL operation. In addition, selective inhibition of NR2C/2D-containing NMDARs with PPDA caused a larger decrease on peak amplitude of NMDAR-EPSCs in SNL than that in sham-operated mice. Appling of selective potentiator of NR2C/2D, CIQ, markedly enhanced the evoked NMDAR-EPSCs in SNL-operated mice, but no change in sham-operated mice. Finally, intra-NAc injection of PPDA significantly attenuated SNL-induced mechanical allodynia and depressive-like behavior. These results for the first time showed that the functional change of NR2C/2D subunits-containing NMDARs in the NAc might contribute to the sensory and affective components in neuropathic pain.

Development of a Dihydroquinoline-Pyrazoline GluN2C/2D-Selective Negative Allosteric Modulator of the N-Methyl-d-aspartate Receptor.

Subunit-selective inhibition of N-methyl-d-aspartate receptors (NMDARs) is a promising therapeutic strategy for several neurological disorders, including epilepsy, Alzheimer's and Parkinson's disease, depression, and acute brain injury. We previously described the dihydroquinoline-pyrazoline (DQP) analogue 2a (DQP-26) as a potent NMDAR negative allosteric modulator with selectivity for GluN2C/D over GluN2A/B. However, moderate (<100-fold) subunit selectivity, inadequate cell-membrane permeability, and poor brain penetration complicated the use of 2a as an in vivo probe. In an effort to improve selectivity and the pharmacokinetic profile of the series, we performed additional structure-activity relationship studies of the succinate side chain and investigated the use of prodrugs to mask the pendant carboxylic acid. These efforts led to discovery of the analogue (S)-(-)-2i, also referred to as (S)-(-)-DQP-997-74, which exhibits >100- and >300-fold selectivity for GluN2C- and GluN2D-containing NMDARs (IC50 0.069 and 0.035 µM, respectively) compared to GluN2A- and GluN2B-containing receptors (IC50 5.2 and 16 µM, respectively) and has no effects on AMPA, kainate, or GluN1/GluN3 receptors. Compound (S)-(-)-2i is 5-fold more potent than (S)-2a. In addition, compound 2i shows a time-dependent enhancement of inhibitory actions at GluN2C- and GluN2D-containing NMDARs in the presence of the agonist glutamate, which could attenuate hypersynchronous activity driven by high-frequency excitatory synaptic transmission. Consistent with this finding, compound 2i significantly reduced the number of epileptic events in a murine model of tuberous sclerosis complex (TSC)-induced epilepsy that is associated with upregulation of the GluN2C subunit. Thus, 2i represents a robust tool for the GluN2C/D target validation. Esterification of the succinate carboxylate improved brain penetration, suggesting a strategy for therapeutic development of this series for NMDAR-associated neurological conditions.

Glutamatergic synaptic currents of nigral dopaminergic neurons follow a postnatal developmental sequence.

The spontaneous activity pattern of adult dopaminergic (DA) neurons of the substantia nigra pars compacta (SNc) results from interactions between intrinsic membrane conductances and afferent inputs. In adult SNc DA neurons, low-frequency tonic background activity is generated by intrinsic pacemaker mechanisms, whereas burst generation depends on intact synaptic inputs in particular the glutamatergic ones. Tonic DA release in the striatum during pacemaking is required to maintain motor activity, and burst firing evokes phasic DA release, necessary for cue-dependent learning tasks. However, it is still unknown how the firing properties of SNc DA neurons mature during postnatal development before reaching the adult state. We studied the postnatal developmental profile of spontaneous and evoked AMPA and NMDA (N-Methyl-D-aspartic acid) receptor-mediated excitatory postsynaptic currents (EPSCs) in SNc DA neurons in brain slices from immature (postnatal days P4-P10) and young adult (P30-P50) tyrosine hydroxylase (TH)-green fluorescent protein mice. We found that somato-dendritic fields of SNc DA neurons are already mature at P4-P10. In contrast, spontaneous glutamatergic EPSCs show a developmental sequence. Spontaneous NMDA EPSCs in particular are larger and more frequent in immature SNc DA neurons than in young adult ones and have a bursty pattern. They are mediated by GluN2B and GluN2D subunit-containing NMDA receptors. The latter generate long-lasting, DQP 1105-sensitive, spontaneous EPSCs, which are transiently recorded during this early period. Due to high NMDA activity, immature SNc DA neurons generate large and long lasting NMDA receptor-dependent (APV-sensitive) bursts in response to the stimulation of the subthalamic nucleus. We conclude that the transient high NMDA activity allows calcium influx into the dendrites of developing SNc DA neurons.