Distribution and developmental change in [3H]MK-801 binding within zebra finch song nuclei

In many songbirds, vocal learning depends upon appropriate auditory experience during a sensitive period that coincides with the formation and reorganization of song-related neural pathways. Because some effects of early sensory experience on neural organization and early learning have been linked to activation of N-methyl-d-aspartate (NMDA) receptors, we measured binding to this receptor within the neural system controlling song behavior in zebra finches. Quantitative autoradiography was used to measure binding of the noncompetitive antagonist [³H]MK-801 (dizocilpine) in the brains of both adult and juvenile male zebra finches, focusing on four telencephalic regions implicated in song learning and production. Overall, the pattern of MK-801 binding in zebra finches was similar to the pattern found in rats (Monaghan and Cotman, 1985, J. Neurosci. 5:2909–2919; Sakurai, Cha, Penney, and Young, 1991, Neuroscience 40:533–543). That is, binding was highest in the telencephalon, intermediate in thalamic regions, and virtually absent from the brain stem and cerebellum. The telencephalic song areas exhibited intermediate levels of binding, and binding in the juveniles was not significantly different from adult levels in most song nuclei. However, in the lateral magnocellular nucleus of the anterior neostriatum (IMAN), binding at 30 days of age was significantly higher than binding in adults. Given the established role of NMDA receptors in other developing neural systems, both their presence in song control nuclei and their developmental regulation within a region implicated in song learning suggest that NMDA receptors play a role in mediating effects of auditory experience on the development of song behavior.     

Initial sex differences in neuron growth and survival within an avian song nucleus develop in the absence of afferent input

Only male zebra finches (Poephila guttata) sing, and nuclei implicated in song behavior exhibit marked sex differences in neuron number. In the robust nucleus of the anterior neostriatum (RA), these sex differences develop because more neurons die in young females than in males. However, it is not known whether the sexually dimorphic survival of RA neurons is a primary event in sexual differentiation or a secondary response to sex differences in the number of cells interacting trophically with RA neurons. In particular, since sexual differentiation of the RA parallels the development of dimorphisms in the numbers of neurons providing afferent input from the lateral magnocellular nucleus of the anterior neostriatum (lMAN) and the high vocal center (HVC), it has been hypothesized that sex differences in the size of these afferent populations trigger differential RA neuron survival and growth. To test this hypothesis, we lesioned either the lMAN or both the lMAN and HVC unilaterally in 12-day-old male and female zebra finches. Subsequently, RA cell death and RA neuron number and size were measured. Unilateral lMAN lesions increased cell death and decreased neuron number and size within the ipsilateral RA of both sexes. However, even in the lMAN-lesioned hemisphere, these effects were less pronounced in males than in females, so that by day 25 the volume, number, and size of neurons were sexually dimorphic in both the contralateral and ipsilateral RA. Similarly, the absence of both lMAN and HVC afferents did not prevent the emergence of sex differences in the number and size of RA neurons by 25 day posthatching. We conclude that these sex differences within the RA are not a secondary response to dimorphisms in the numbers of lMAN or HVC neurons providing afferent input. © 1995 John Wiley & Sons, Inc.     

A sensorimotor area in the songbird brain is required for production of vocalizations in the song learning period of development

Sensory feedback is essential for acquiring and maintaining complex motor behaviors, including birdsong. In zebra finches, auditory feedback reaches the song control circuits primarily through the nucleus interfacialis nidopalii (Nif), which provides excitatory input to HVC (proper name)—a premotor region essential for the production of learned vocalizations. Despite being one of the major inputs to the song control pathway, the role of Nif in generating vocalizations is not well understood. To address this, we transiently inactivated Nif in late juvenile zebra finches. Upon Nif inactivation (in both hemispheres or on one side only), birds went from singing stereotyped zebra finch song to uttering highly variable and unstructured vocalizations resembling sub‐song, an early juvenile song form driven by a basal ganglia circuit. Simultaneously inactivating Nif and LMAN (lateral magnocellular nucleus of the anterior nidopallium), the output nucleus of a basal ganglia circuit, inhibited song production altogether. These results suggest that Nif is required for generating the premotor drive for song. Permanent Nif lesions, in contrast, have only transient effects on vocal production, with song recovering within a day. The sensorimotor nucleus Nif thus produces a premotor drive to the motor pathway that is acutely required for generating learned vocalizations, but once permanently removed, the song system can compensate for its absence. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1213–1225, 2016     

Adenosine receptor-mediated modulation of dopamine release in the nucleus accumbens depends on glutamate neurotransmission and N-methyl-D-aspartate receptor stimulation

Adenosine, by acting on adenosine A(1) and A(2A) receptors, exerts opposite modulatory roles on striatal extracellular levels of glutamate and dopamine, with activation of A(1) inhibiting and activation of A(2A) receptors stimulating glutamate and dopamine release. Adenosine-mediated modulation of striatal dopaminergic neurotransmission could be secondary to changes in glutamate neurotransmission, in view of evidence for a preferential colocalization of A(1) and A(2A) receptors in glutamatergic nerve terminals. By using in vivo microdialysis techniques, local perfusion of NMDA (3, 10 microm), the selective A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 3, 10 microm), the selective A(1) receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 300, 1000 microm), or the non-selective A(1)-A(2A) receptor antagonist in vitro caffeine (300, 1000 microm) elicited significant increases in extracellular levels of dopamine in the shell of the nucleus accumbens (NAc). Significant glutamate release was also observed with local perfusion of CGS 21680, CPT and caffeine, but not NMDA. Co-perfusion with the competitive NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (APV; 100 microm) counteracted dopamine release induced by NMDA, CGS 21680, CPT and caffeine. Co-perfusion with the selective A(2A) receptor antagonist MSX-3 (1 microm) counteracted dopamine and glutamate release induced by CGS 21680, CPT and caffeine and did not modify dopamine release induced by NMDA. These results indicate that modulation of dopamine release in the shell of the NAc by A(1) and A(2A) receptors is mostly secondary to their opposite modulatory role on glutamatergic neurotransmission and depends on stimulation of NMDA receptors. Furthermore, these results underscore the role of A(1) vs. A(2A) receptor antagonism in the central effects of caffeine.     

Horizontal transmission of the father's song in the zebra finch (Taeniopygia guttata)

As is the case for human speech, birdsong is transmitted across generations by imitative learning. Although transfer of song patterns from adults to juveniles typically occurs via vertical or oblique transmission, there is also evidence of horizontal transmission between juveniles of the same generation. Here, we show that a young male zebra finch (Taeniopygia guttata) that has been exposed to its father during the sensitive period for song learning can lead a brother, that has never heard the paternal song, to imitate some sounds of the father. Moreover, song similarity between the two brothers was higher than the similarity measured between the paternal song and the song of the brother that had a week-long exposure to the father. We speculate that the phenomenon of within-generation song learning among juveniles may be more widespread than previously thought and that when a juvenile evaluates potential models for imitative learning, a sibling may be as salient as an adult.     

Role of a telencephalic nucleus in the delayed song learning of socially isolated zebra finches

Male zebra finches normally learn their song from adult models during a restricted period of juvenile development. If song models are not available then, juveniles develop an isolate song which can be modified in adulthood. In this report we investigate the features of juvenile experience that underly the timing of song learning. Juvenile males raised in soundproof chambers or in visual isolation from conspecifics developed stable isolate song. However, whereas visual isolate song notes were similar to those of colony-reared males, soundproof chamber isolates included many phonologically abnormal notes in their songs. Despite having stable isolate songs, both groups copied new notes from tutors presented to them in adulthood (2.7 notes per bird for soundproof chamber isolates, 4.4 notes per bird for visual isolates). Old notes were often modified or eliminated. We infer that social interactions with live tutors are normally important for closing the sensitive period for song learning. Lesions of a forebrain nucleus (IMAN) had previously been shown to disrupt juvenile song learning, but not maintenance of adult song for up to 5 weeks after surgery. In this study, colony-reared adult males given bilateral lesions of IMAN retained all their song notes for up to 4–7.5 months after lesioning. However, similar lesions blocked all song note acquisition in adulthood by both visual and soundproof chamber isolates. Other work has shown that intact hearing is necessary for the maintenance of adult zebra finch song. We infer that auditory pathways used for song maintenance and acquisition differ: IMAN is necessary for auditorily guided song acquisition—whether by juveniles or adults—but not for adult auditorily guided song maintenance. © 1993 John Wiley & Sons, Inc.     

Mitochondrial haplotype does not affect sperm velocity in the zebra finch Taeniopygia guttata

Mitochondrial DNA (mtDNA) variation has been suggested as a possible cause of variation in male fertility because sperm activity is tightly coupled to mitochondrial oxidative phosphorylation and ATP production, both of which are sensitive to mtDNA mutations. Since male‐specific phenotypes such as sperm have no fitness consequences for mitochondria due to maternal mitochondrial (and mtDNA) inheritance, mtDNA mutations that are deleterious in males but which have negligible or no fitness effect in females can persist in populations. How often such mutations arise and persist is virtually unknown. To test whether there were associations between mtDNA variation and sperm performance, we haplotyped 250 zebra finches Taeniopygia guttata from a large pedigreed‐population and measured sperm velocity using computer‐assisted sperm analysis. Using quantitative genetic ‘animal’ models, we found no effect of mtDNA haplotype on sperm velocity. Therefore, there is no evidence that in this system mitochondrial mutations have asymmetric fitness effects on males and females, leading to genetic variation in male fertility that is blind to natural selection.     

Mode of binding of [3H]dibenzocycloalkenimine (MK-801) to the N-methyl-D-aspartate (NMDA) receptor and its therapeutic implication

Binding of the labeled anticonvulsant drug [³H]dibenzocycloalkenimine (³H]MK-801) to the N-methyl-D-aspartate (NMDA) receptor and its dissociation from the receptor at 25°C are slow processes, both of which follow first order kinetics (t_1/270 and 180 min, respectively). Both reactions are markedly accelerated by glutamate and glycine (t_1/22-8 and 4 min, respectively), which allow bimolecular association kinetics of the labeled drug with the receptors whereas equilibrium binding of [³H]MK-801 (K_d 2–4 nM) is hardly affected by glutamate and glycine. The data suggest that MK-801 acts as a steric blocker of the NMDA receptor channel. The competitive antagonist D-(−)-2-amino-5-phosphovaleric acid (AP-5) freezes the receptor in a state which precludes either binding of [³H]MK-801 to the receptor channel or its dissociation from it. These findings have therapeutic implications.     

Expression of splice variants of the NR1 subunit of the N-methyl-D-aspartate receptor in the normal and injured rat spinal cord

Quantitative western blot analysis in laminectomy control spinal cords of adult rats was used to provide the first report of the normal expression patterns of the N1, C1, C2 and C2' cassettes in the cervical, thoracic and lumbar regions of the spinal cord as a percent of total NR1 subunit protein. In all regions studied, the C1 and C2 cassettes were usually contained in less than 10% of total NR1 protein. In contrast, approximately 90% of total NR1 protein contained the C2' cassette. A significant proportion of total NR1 protein (approximately 30%) also contained the N1 cassette. These data are consistent with expression of NR1(000) (NR1-4a) and NR1(100) (NR1-4b) as the dominant splice forms in the spinal cord. Splice variant expression was also studied following incomplete, contusive spinal cord injury (SCI) to the thoracic level 8 (T8) region. This injury did not change expression of the C1 or C2 cassette in any region of the spinal cord acutely at 24 h or chronically at 1 month. There was an increase in expression of the N1 cassette in the lumbar regions 1 month after injury (p < 0.05). These data indicate that SCI induces distal changes in NR1 splice variant expression, which may play a role in the adaptive response of neurons in the chronically injured spinal cord.     

Model structures of the N-methyl-D-aspartate receptor subunit NR1 explain the molecular recognition of agonist and antagonist ligands

Molecular models of the ligand-binding domain of N-methyl-d-aspartate subunit R1 (NR1) were made using the published crystal structures of rat glutamate receptor B (GluRB), the bacterial glutamate receptor (GluR0), and the glutamine-binding protein (QBP) of Escherichia coli. Separate models of NR1 were built to represent the ligand-binding conformation for agonist (glycine, d- and l-isomers of serine and alanine, and the partial agonist ligand d-cycloserine) and antagonist (5,7-dichloro-4-oxo-1,4-dihydroquinoline-2-carboxylic acid (DCKA) and E-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1-H-indole-2-carboxylic acid (MDL 105,519)) ligands. Side-chain conformations of residues within the NR1 ligand-binding site were selected that optimized the hydrophobic packing and hydrogen bonding among residues, while taking into account published data comparing receptor mutants with wild-type NR1. Ligands docked to the model structures provide a rational explanation for the observed differences in binding affinity and receptor activation among agonist and antagonist ligands. NR1 prefers smaller ligands (glycine, serine, and alanine) in comparison with GluRB and GluR0 that bind l-glutamate: the bulky side chain of W731 in NR1 dramatically reduces the size of the ligand-binding site, functioning to selectively restrict recognition to glycine and the d-isomers of serine and alanine. Nevertheless, many of the interactions seen for ligands bound to GluRB, GluR0, and periplasmic-binding proteins are present for the ligands docked to the model structures of NR1.     

Effect of isolation and conspecific presence in a novel environment on corticosterone concentrations in a social avian species, the zebra finch (Taeniopygia guttata)

Zebra finches are a highly social and monogamous avian species. In the present study, we sought to determine the effect of social isolation (separation from the flock) in a novel environment with and without a conspecific present on the adrenocortical activity of paired and unpaired individuals of this species. With regard to paired birds, we hypothesized that the presence of the mate during isolation from the group would act as a social buffer against the stressful effects of isolation. We observed that 10 but not 30 minutes of social isolation resulted in elevated concentrations of corticosterone in unpaired and paired male zebra finches in comparison to baseline concentrations of corticosterone. Furthermore, the presence of a mate during isolation in a novel environment did not have a buffering effect against increases in corticosterone concentrations. Additionally, to compare concentrations of corticosterone in response to isolation (in a novel environment) to a previously well-established stressor, we subjected groups of birds to restraint. We observed that 10 or 30 minutes of restraint led to significantly higher concentrations of corticosterone as compared to baseline. Finally, to rule out the possibility that merely handling a bird would result in significantly elevated concentrations of corticosterone as compared to baseline samples, we measured corticosterone concentrations 10 or 30 minutes after handling involving capture and release only. Our results suggest that handling alone might have contributed to the elevation of corticosterone in birds exposed to 10 minutes but not 30 minutes of restraint. Handling by itself did not account, however, for the elevated corticosterone in birds socially isolated for 10 minutes.     

An investigation into the role of N-glycosylation in the functional expression of a recombinant heteromeric NMDA receptor

The effect of N-glycosylation on the assembly of N-methyl-D-aspartate (NMDA) heteromeric cloned receptors was studied. Thus human embryonic kidney (HEK) 293 cells were cotransfected with N-methyl-D-aspartate R1 (NR1) and N-methyl-d-aspartate R2A (NR2A) clones and the cells grown post-transfection in the presence of tunicamycin (TM). TM treatment resulted in a decrease of the NR1 subunit with M_r 117 000 with a concomitant increase in a M_r 97 000 immunoreactive species previously identified as the non-N-glycosylated NR1 subunit. In parallel, TM caused a dose-dependent inhibition of [³H]MK801 binding to the expressed receptor which was a result of an approximate four-told reduction in the Dissociation Constant (K_D) but with no change in the number of binding sites (B_MAX)-NMDA receptor cell surface expression was unchanged following TM treatment but it did result in a decrease in the percentage cell death post-transfection compared to control samples. The removal of TM from the cell culture media resulted in a return to the control K_D value for [³H]MK801 binding and partial reglycosylation of newly synthesized NR1 subunit. These results demonstrate that N-glycosylation is requisite for the efficient expression of functional NR1/NR2A receptors. Furthermore, they suggest that N-glycosylation may be important for the correct formation of the channel domain of the NR1/NR2A receptor.     

T-cell receptor tetramer binding or the lack there of does not necessitate antigen reactivity in T-cell receptor transduced T cells

Genetic transfer of T-cell receptor (TCR) chains provides a means of transferring tumor antigen specificity onto an alternate T-cell population. To determine which tumor reactive TCRs are best suitable for such adoptive transfer, careful evaluation of the resulting TCR modified populations need to be performed. We have previously cloned, and expressed TCRs from melanoma, EBV, HCV, and HPV reactive T-cell clones and found that several routine indicators of T-cell function do not always predict the relative strength of a TCR. Using a combination of tetramer binding assays and antigen recognition assays, we identified TCRs that fall into three classes. One class of TCR did not bind tetramers yet resulted in cells with high avidity for antigen. A second TCR class bound tetramer but did not secrete cytokines in response to antigen. Finally, the third class of TCRs bound tetramer and reacted to antigen as would be expected. We conclude that tetramer binding is not always a good indicator of the function of a cloned TCR or the avidity of a TCR gene modified T cell. Furthermore, our data indicate that the use of tetramer binding alone to identify antigen reactive TCRs may result in the exclusion of TCRs that may be highly reactive or cross reactive to the relevant tumor antigen.     

Expression of a soluble glycine binding domain of the NMDA receptor in Escherichia coli

Glycine is an essential co-agonist of the excitatory N-methyl-D-aspartate (NMDA) receptor. The glycine binding site of this subtype of ionotropic glutamate receptors is formed by the S1 and S2 regions of the NR1 subunit. Here, different S1S2 fusion proteins were expressed and purified from Escherichia coli cultures, and refolding protocols were established allowing the production of 30 mg of soluble S1S2 fusion protein from 1 liter bacterial culture. After affinity purification and renaturation, two of the fusion proteins (S1S2 and S1S2-V1) bound the competitive glycine site antagonist [3H]MDL105,519 with K(d) values of 9.35 and 3.9 nM, respectively. In contrast, with three other constructs (S1S2M, S1S2-V2, and -V3) saturable ligand binding could not be obtained. These results redefine the S1S2 domains required for high-affinity glycine binding. Furthermore, our high-affinity binding proteins may be used for the large-scale production of the glycine binding core region for future structural studies.     

Deletion of the serotonin 2c receptor from transgenic mice overexpressing leptin does not affect their lipodystrophy but exacerbates their diet-induced obesity

The binding of leptin to hypothalamic neurons elicits inhibition of orexigenic NPY/AgRP neurons and stimulation of anorexigenic POMC/CART neurons. Projections of serotonergic neurons onto POMC neurons suggest that leptin and serotonin converge onto POMC neurons to regulate body weight. We probed the interaction of these pathways by generating transgenic mice overexpressing leptin (LepTg) without 5HT2c receptors. On a chow diet, the lean phenotype of LepTg mice was unaffected by the absence of 5HT2c receptors, whereas on a high fat diet, LepTg/5HT2c receptors knockout mice showed an exacerbation of diet-induced obesity. POMC mRNA levels were low in LepTg, 5HT2c receptors knockout and LepTg/5HT2c receptors knockout mice, demonstrating that perturbations of the 5HT2c receptor and leptin pathways, either alone or in combination, negatively impact on POMC expression. Thus, on a chow diet, leptin action is independent of 5HT2c receptors whereas on a high fat diet 5HT2c receptors are required for the attenuation of obesity.     

Modulation of the levels of NMDA receptor subunit mRNA and the bindings of [3H]MK-801 in rat brain by chronic infusion of subtoxic dose of MK-801

The effects of continuous infusion of NMDA receptor antagonist MK-801 on the modulation of NMDA receptor subunits NR1, NR2A, NR2B, and NR2C were investigated by using in situ hybridization study. Differential assembly of NMDA receptor subunits determines their functional characteristics. Continuous intracerebroventricular (i.c.v.) infusion with MK-801 (1 pmol/10 l/h) for 7 days resulted in significant modulations in the NR1, NR2A, and NR2B mRNA levels without producing stereotypic motor syndromes. The levels of NR1 mRNA were significantly increased (9-20%) in the cerebral cortex, striatum, septum, and CA1 of hippocampus in MK-801-infused rats. The levels of NR2A mRNA were significantly decreased (11-16%) in the CA3 and dentate gyrus of hippocampus in MK-801-infused rats. In contrast to NR2A, NR2B subunit mRNA levels were increased (10-14%) in the cerebral cortex, caudate putamen, and thalamus. However, no changes of NR2C subunits in cerebellar granule layer were observed. Using quantitative ligand autoradiography, the binding of NMDA receptor ligand [³H]MK-801 was increased (12-25%) significantly in almost all brain regions except in the thalamus and cerebellum after 7 days infusion with MK-801. These results suggest that region-specific changes of NMDA receptor subunit mRNA and [³H]MK-801 binding are involved in the MK-801-infused adult rats.     

Effect of marginal vitamin A deficiency during pregnancy on retinoic acid receptors and N-methyl-D-aspartate receptor expression in the offspring of rats

This study examined whether pregnancy-related marginal vitamin A deficiency (MVAD) influences postnatal development of retinoic acid receptors (RARs) and N-methyl-d-aspartate (NMDA) receptor subunit 1 (NR1) in hippocampus of rat pups. Sixteen female rats were randomized equally into control and MVAD groups. Dams and pups were fed with either a normal control diet or one deficient in vitamin A. Eight female pups in each group were killed at 1 day, 2 weeks, 4 weeks and 8 weeks after birth, respectively. Serum retinol levels were monitored. The messenger RNA (mRNA) and protein expressions and subcellular localization of RARα, RARβ and NR1 in postnatal hippocampus were detected. At 1 day, 2 weeks and 8 weeks after birth, serum retinol levels in the MVAD group were significantly lower than those in the control group. Results of Morris water maze test at 7 weeks of age showed that spatial learning and memory in the MVAD group were affected. Vitamin A deficiency resulted in decreased mRNA levels of RARα, RARβ and NR1 (P<.05). The protein level of RARα and NR1 in the MVAD group was lower than that of the control group (P<.05). There was no significant difference in RARβ between the groups (P>.05). A mass of RARα and NR1 colocalized in hippocampal cell cytoplasm on postnatal day 1. Our data suggested that vitamin A deficiency in pregnancy may affect the postnatal expression of RARα and NR1, affecting learning and memory function in the hippocampus and synaptic plasticity of the calcium signaling pathway.