4.2. GluN2D Receptor Subunit Function
Glutamate displays 5–6 times greater potency at GluN2D-containing NMDARs than GluN2A- or GluN2B-containing NMDARs
[25][77][39,132]. Similarly, GluN1 agonists such as glycine are most potent when the GluN2 subunit in the NMDAR is GluN2D
[78][133]. GluN2D-contaning NMDARs also have a weak Mg2+ block that is 10-fold lower than that of GluN2A- or GluN2B-containing receptors and are also reported to have a slightly lower Ca2+ permeability
[79][80][134,135]. The resistance to Mg2+ block suggests that neurons expressing the GluN2D subunit may be more responsive to synaptic glutamate release. The non-competitive NMDAR antagonist ketamine is more potent at, and shows approximately five-fold selectivity for GluN2D-containing receptors, compared with GluN2A or GluN2B subunits
[81][136]. This, combined with the fact that GluN2D-containing receptors are predominantly expressed on interneurons, may suggest that the GluN2D-containing receptors might be involved in the cortical disinhibition induced by certain NMDAR antagonists such as ketamine
[69][82][121,137].
GluN2D-containing receptors play a role in both presynaptic and postsynaptic neurotransmission. Studies have reported GluN2D-containing NMDARs on interneurons in the hippocampus and neocortex where they play a key role in postsynaptic signalling
[70][83][122,141]. GluN2D-containing receptors are thought to enable interneurons to synchronize and coordinate the firing of large groups of cortical pyramidal neurons. A recent study showed that tonic activation of these GluN2D-containing NMDARs on developing cortical interneurons is required for proper intrinsic excitability, dendritic arborization, GABAergic synaptogenesis and inhibitory tone onto excitatory pyramidal cells
[83][141]. GluN2D-containing NMDARs have also been reported on the dendrites of neurons in the subthalamic nucleus, on dopaminergic projection neurons in the substantia nigra pars compacta and in the dorsal horn of the spinal cord, where they contribute to the modulation of the indirect pathway, mediate dopamine release to the striatum and play a role in pain perception, respectively
[84][85][142,143]. GluN2D-containing NMDARs expressed at presynaptic sites are thought to play a modulatory role in the hippocampus and cerebellum. In the hippocampus, GluN2D, along with GluN2B and postsynaptic metabotropic glutamate receptors, have been shown to be critical for the induction of spike-time-dependent LTD
[86][144].
Interestingly, it has been reported that the human
GRIN2D gene contains four estrogen-responsive elements which are highly preserved in the rat, suggesting that the GluN2D subunit might be under neuroendocrine control
[87][147]. In line with this, a study using ovariectomised rats found an upregulation of GluN2D mRNA in the hypothalamus following 17β-estradiol treatment
[87][147]. Given the well-established links between fluctuating levels of 17β-estradiol and schizophrenia onset and symptom severity
[88][148], it is intriguing to consider that one of the actions by which 17β-estradiol may exert its effects is via regulation of GluN2D and other NMDAR subunits
[89][149].
5. Alterations to GluN2D in Schizophrenia
There is evidence to suggest that the GluN2D subunit is altered in schizophrenia. A study of approximately 200 Japanese people with schizophrenia found single nucleotide polymorphisms (SNPs) in the gene for the GluN2D receptor that might contribute to schizophrenia susceptibility
[90][150]. They report that specific combinations of four SNPs within the
GRIN2D gene were significantly associated with schizophrenia. These specific combinations were found in three pairs of SNPs: INT10SNP–EX13SNP2, EX13SNP2–EX13SNP3 and EX6SNP–EX13SNP2
[90][150]. A recent mutation-screening study also identified an ultra-rare, loss-of-function splice-site mutation (c.1412G>A) in the exonic region of the
GRIN2D gene, which may lead to the creation of a truncated, nonfunctional GluN2D receptor, thereby contributing to schizophrenia risk
[91][151]. Th
eis study additionally found four missense mutations in schizophrenia patients in the
GRIN2D gene, and although the actual functional impact of these amino acid substitutions was not examined, in silico analysis classified each of the four variants as disease-causing based on their predicted effect on protein function
[91][151]. An in situ hybridization study on post-mortem human tissue reported a 53% increase in the proportion of GluN2D mRNA expression in the PFC of people with schizophrenia
[92][128]. This increase in the proportion of GluN2D expression was not seen in anti-psychotic-treated control brains, proving that the change was specific to schizophrenia and could not be attributed to treatment with anti-psychotics alone
[92][128].
6. Consequences of Loss of GluN2D Function
6.1. Genetic Models
GluN2D-knockout (KO) mice are viable, reproduce and grow normally, and have no overt changes in neuronal histology
[93][159]. Moreover, mRNA levels of the other NMDAR subunits are unaffected in these mice
[93][159]. These mice, however, exhibit unique behavioural phenotypes, including diminished spontaneous motor movements in open-field tests
[93][94][159,160]. GluN2D-KO mice also display deficits in spatial learning and memory, as well as impaired contextual fear memory, but show no deficits in the novel object recognition task
[82][95][96][137,161,162]. Most studies report no abnormalities in motor function as measured by the rotarod test, nor any differences in anxiety when compared with WT mice during the light–dark compartment test and elevated maze test
[93][97][159,163]. However, Miyamoto et al. report reduced sensitivity to stress induced by the elevated-plus maze, the light–dark compartment test and forced swim tests in these KO mice
[94][160]. The discrepancies in these findings might be due to the differences in the way the tests were performed, which could be sensitive to subtly different anxiety- or fear-related behaviours.
GluN2D-KO mice were reported to have similar basal neural oscillatory power between frequency ranges 30 and 200 Hz when compared with WT mice
[98][168]. However, whilst the administration of the NMDAR antagonists MK-801, ketamine and memantine increased oscillatory power in WT mice, they had very little effect on GluN2D-KO mice, especially at the high gamma frequency range (65–140 Hz)
[98][168].
6.2. Pharmacological Manipulations
Although currently, to the best of our knowledge, there are no GluN2D-selective drugs available, competitive antagonists with 3–10-fold higher selectivity for GluN2C/GluN2D compared with GluN2A/GluN2B-containing NMDARs have been developed. One such compound, (2R*,3S*)-1-(phenanthrenyl-2-carbonyl)piperazine-2,3-dicarboxylic acid (PPDA), resulted in more potent inhibition of LTD than LTP in rat hippocampal slices, suggesting a role for the GluN2D (and GluN2C) NMDAR subunits in hippocampal LTD
[99][169].
7. How Might Alterations to the GluN2D Subunit Contribute to Schizophrenia?
7.1. GluN2D Subunit and Parvalbumin-Positive GABAergic Interneurons
As previously discussed, inhibitory interneurons have been identified as the key locus or point of convergence of the glutamatergic, GABAergic and dopaminergic hypotheses of schizophrenia and are also implicated in the cognitive deficits seen in schizophrenia. Interestingly, in situ hybridization, electrophysiology and immunohistochemistry studies have revealed that GluN2D-containing NMDARs are specifically enriched in the PV-expressing subclass of interneurons in the hippocampus and PFC, two regions that underlie learning and memory function (refer to
Section 4.1)
[68][69][70][83][120,121,122,141]. This makes the GluN2D subunit particularly intriguing in the context of schizophrenia as several studies have shown that the hypofunction of NMDARs at fast-spiking PV-containing interneurons is sufficient to produce schizophrenia-like symptoms, including cognitive dysfunction
[59][100][101][102][84,173,174,175].
Despite there being more than 20 different classes of GABAergic interneurons
[103][176], it is the interneurons containing the calcium-binding protein, PV, that have been proposed to be especially important in schizophrenia
[104][105][177,178]. Not only are PV-containing interneurons crucial for regulating cortical inhibition via the pyramidal neurons they innervate, but also for the generation of synchronous gamma-frequency oscillations
[105][106][107][108][109][110][178,179,180,181,182,183]. Gamma oscillations are synchronous electrophysiological brain rhythms in the gamma frequency range (30–80 Hz) that are crucial for information processing and appropriate cortical function and underpin a wide range of cognitive processes, including those disrupted in schizophrenia like working memory
[111][112][113][184,185,186]. Abnormal gamma-frequency synchrony is a major pathological characteristic of schizophrenia and underlies cognitive deficits
[113][186]. For example, a recent study found lower-amplitude gamma oscillations in people with schizophrenia while they were performing a working memory task
[114][187].
Cortical pyramidal neurons innervated by PV cells stimulate dopaminergic neurons in the midbrain which project to the associative striatum. The associative striatum includes the rostral and dorsal part of the caudate nuclei and is implicated in the pathophysiology of schizophrenia
[115][116][117][118][198,199,200,201]. The associative striatum is rich in dopamine receptors and dopamine afferents and receptors and is thus thought to be the primary site of action of antipsychotics. As such, any disruption to the GluN2D-containing NMDARs on PV cells could also indirectly affect midbrain dopaminergic neurons and lead to enhanced dopamine release in the striatum which has been linked to the positive symptoms of schizophrenia
[119][202]. Following treatment with ketamine, Yamamoto et al. found an increase in locomotor activity and nitric oxide (NO) synthesis in the dendrites of medium spiny neurons in the dorsal striatum and PFC in WT but not GluN2D-KO mice
[120][166]. Postsynaptic neuronal NO synthesis is functionally coupled to the stimulation of NMDARs. The failure of ketamine to induce an increase in striatal NO synthesis in GluN2D-KO mice provides support for the role of GluN2D-containing receptors in the corticostriatal neuronal circuit.
7.2. GluN2D Subunit and Dopaminergic Neurons
Multiple studies have found that the GluN2D subunit forms functional NMDAR channels in the substantia nigra pars compacta dopaminergic neurons
[76][121][122][123][131,203,204,205]. The substantia nigra plays an essential role in modulating motor movement and reward functions. Interestingly, GluN2D-KO mice have a hypolocomotor phenotype and the hyperlocomotor effects of PCP and ketamine are reduced in GluN2D-KO mice
[82][97][120][137,163,166]. The amount of dopamine release in the forebrain following PCP treatment is also reduced in GluN2D-KO mice
[97][163]. This suggests that GluN2D-containing NMDARs might play either a direct or indirect role in modulating dopaminergic function and, consequently, locomotor activity. In people with schizophrenia, there are reports of long-term deficits in basic motor function and control
[124][125][126][206,207,208]. Thus, it is possible that dysfunction of the GluN2D subunit could affect burst-firing in these dopaminergic neurons, disrupting their function in the nigrostriatal circuitry, which is hypothesised to underlie motor symptoms in schizophrenia
[127][209].
8. Conclusions
Precipitating factors, including any combination of genetic predisposition and environmental factors like maternal infection or obstetric complications, can lead to NMDAR hypofunction disproportionately at fast-spiking PV-containing interneurons during development. This results in pathological phenotypes including impaired oscillatory activity and neuronal synchrony, cortical disinhibition and dopaminergic dysfunction, ultimately giving rise to the various symptoms of schizophrenia. Disruption of the GluN2D subunit and alteration to GluN2D neurotransmission could be a molecular pathway contributing to the symptomatology of schizophrenia. This is of importance as it may provide new insights into the aetiology of this disorder and might even lead to the development of novel drugs for the treatment of specific schizophrenia symptoms, including cognitive dysfunction.