Long-term behavioural, molecular and morphological effects of neonatal NMDA receptor antagonism

Brief N-methyl-D-aspartate (NMDA) receptor blockade in neonatal rats has been reported to increase neuronal apoptosis. We replicated this finding using MK-801 (0.5 mg/kg) administered twice on postnatal day 7, and then studied the long-term consequences. In adulthood, treated rats showed reduced volume and neuronal number within the hippocampus, and altered hippocampal NMDA receptor (NR1 subunit) expression. Synaptophysin mRNA was decreased in the thalamus (laterodorsal nucleus). Adult MK-801-treated females had prepulse inhibition deficits and increased locomotor activity. The data show that a transient and limited glutamatergic intervention during development can have chronic behavioural, structural and molecular effects. The effects are reminiscent of alterations reported in schizophrenia and, as such, are consistent with hypotheses advocating a role for NMDA receptor hypofunction, and aberrant apoptosis, in the neurodevelopmental pathogenesis of the disorder.     

Prenatal protein deprivation in rats induces changes in prepulse inhibition and NMDA receptor binding

Epidemiological studies suggest that prenatal malnutrition increases the risk of developing schizophrenia. Animal models indicate that prenatal protein deprivation (PPD) affects many aspects of adult brain function. We tested the hypothesis that PPD in rats would alter prepulse inhibition (PPI), which is an operational measure of sensorimotor gating that is deficient in schizophrenia patients. Additionally, we examined dopaminergic and glutaminergic receptor binding in the striatum and hippocampus, which have been suggested to play a role in the etiology of schizophrenia. Rat dams were fed normal (25%) or low (6%) protein diets beginning 5 weeks prior to, and throughout pregnancy. The pups were tested at postnatal days (PND) 35 and 56 for PPI. Striatal and hippocampal NMDA receptor, and striatal dopamine receptor binding were quantified post-mortem in a subset of these rats. Female rats exposed to PPD had reduced levels of PPI at PND 56, but not PND 35, suggesting the emergence of a sensorimotor gating deficit in early adulthood. Striatal NMDA receptor binding was increased in PPD females. A decrease in initial startle response (SR) was also observed in all PPD rats relative to control rats. These results suggest that PPD causes age- and sex-dependent decreases in PPI and increases in NMDA receptor binding. This animal model may be useful for the investigation of neurodevelopmental changes that are associated with schizophrenia in humans.     

大鼠生后发育中听皮层NR1 mRNA的表达

目的：研究大鼠生后发育过程中，听皮层神经元NMDA受体NR1 mRNA的表达。方法：采用特异性DIG标记的寡核苷酸探针，分别在动物出生后第7，14，2l，28，35，42，49，56天和成年，检测听皮层NR1 mRNA阳性神经元的分布。结果：NR1亚单位mRNA阳性神经元从出生后第7天即可检测到，之后，随着天龄增加，阳性神经元分布密度呈明显递增趋势。期间，从生后第7天到第14天阳性神经元密度增加最快，增加了33．80％。到生后第35天NR1 mRNA表达达到高峰，一直保持到成年。对称的两侧听皮层阳性神经元表达模式完全一致。结论：听皮层NR1 mRNA表达与动物年龄相关。研究结果为进一步在皮层水平上探讨生后听觉功能发育可塑性的分子机制提供了重要资料。     

大鼠听皮质NMDA受体亚单位NR2BmRNA年龄-依赖性表达

应用原位杂交技术 ,研究了大鼠生后发育过程中 ,听皮质神经元NMDA受体亚单位NR2BmRNA年龄 依赖性的表达变化。特异性DIG标记寡核苷酸探针检测显示 ,NR2B亚单位mRNA阳性神经元数量从出生后即有高水平表达 ,之后 ,随着天龄增长逐渐递减 ,在出生后 14d出现一过性表达高峰 ,14～ 2 1d时表达水平急剧降低(>5 0 .0 % ) ,2 1d后保持低水平表达至成年。研究结果为进一步在皮质水平上探讨出生后听觉功能发育可塑性的分子机制提供了重要资料     

A developmental influence of the N-methyl-D-aspartate receptor NR3A subunit on prepulse inhibition of startle.

BACKGROUND: The N-methyl-D-aspartate (NMDA) receptor is composed of various conformations of multiple subunits (including NR1, NR2A-D, and NR3A-B). Peak expression of the NR3A subunit occurs approximately 2-3 weeks postnatal, with low levels in adulthood. In the brain, the NR3A subunit is localized primarily in the amygdala, hippocampus, striatum, and cortex. These regions are involved in the modulation of prepulse inhibition of startle (PPI), an operational measure of sensorimotor gating that is modulated by NMDA receptors. NR3A reduces NMDA current in native neurons expressing NR1 and NR2 subunits and forms glycine receptors when expressed with NR1 in the absence of NR2 in both oocyte and mammalian expression systems. METHODS: To examine the role of NR3A in vivo, NR3A knockout (KO), and overexpressing transgenic mice were generated. Adult NR3A overexpressing mice exhibited normal PPI; PPI in NR3A KO mice was tested repeatedly from weaning through adulthood. RESULTS: Male NR3A KO mice exhibited an increase in PPI at 3 and 4 weeks postnatal, whereas female NR3A KO mice did not differ from their WT counterparts at any age tested. CONCLUSIONS: This sex-specific increase in PPI is consistent with the antagonistic role of the NR3A subunit in NMDA receptor function and with the observation that estrogen modulates NMDA receptor function.     

Deletion of the NMDA‐NR1 receptor subunit gene in the mouse nucleus accumbens attenuates apomorphine‐induced dopamine D1 receptor trafficking and acoustic startle behavior

The nucleus accumbens (Acb) contains subpopulations of neurons defined by their receptor content and potential involvement in sensorimotor gating and other behaviors that are dysfunctional in schizophrenia. In Acb neurons, the NMDA NR1 (NR1) subunit is coexpressed not only with the dopamine D1 receptor (D1R), but also with the µ‐opioid receptor (µ‐OR), which mediates certain behaviors that are adversely impacted by schizophrenia. The NMDA‐NR1 subunit has been suggested to play a role in the D1R trafficking and behavioral dysfunctions resulting from systemic administration of apomorphine, a D1R and dopamine D2 receptor agonist that impacts prepulse inhibition to auditory‐evoked startle (AS). Together, this evidence suggests that the NMDA receptor may regulate D1R trafficking in Acb neurons, including those expressing µ‐OR, in animals exposed to auditory startle and apomorphine. We tested this hypothesis by combining spatial‐temporal gene deletion technology, dual labeling immunocytochemistry, and behavioral analysis. Deleting NR1 in Acb neurons prevented the increase in the dendritic density of plasma membrane D1Rs in single D1R and dual (D1R and µ‐OR) labeled dendrites in the Acb in response to apomorphine and AS. Deleting NR1 also attenuated the decrease in AS induced by apomorphine. In the absence of apomorphine and startle, deletion of Acb NR1 diminished social interaction, without affecting novel object recognition, or open field activity. These results suggest that NR1 expression in the Acb is essential for apomorphine‐induced D1R surface trafficking, as well as auditory startle and social behaviors that are impaired in multiple psychiatric disorders. Synapse, 2013. © 2013 Wiley Periodicals, Inc.     

Long-term behavioral and NMDA receptor effects of young-adult corticosterone treatment in BDNF heterozygous mice

Psychiatric illnesses, such as schizophrenia, are most likely caused by an interaction between genetic predisposition and environmental factors, including stress during development. The neurotrophin, brain-derived neurotrophic factor (BDNF) has been implicated in this illness as BDNF levels are decreased in the brain of patients with schizophrenia. The aim of the present study was to assess the combined effect of reduced BDNF levels and postnatal stress, simulated by chronic young-adult treatment with the stress hormone, corticosterone. From 6 weeks of age, female and male BDNF heterozygous mice and their wild-type controls were chronically treated with corticosterone in their drinking water for 3 weeks. At 11 weeks of age, male, but not female BDNF heterozygous mice treated with corticosterone exhibited a profound memory deficit in the Y-maze. There were no differences between the groups in baseline prepulse inhibition (PPI), a measure of sensorimotor gating, or its disruption by treatment with MK-801. However, an increase in startle caused by MK-801 treatment was absent in male, but not female BDNF heterozygous mice, irrespective of corticosterone treatment. Analysis of protein levels of the NMDA receptor subunits NR1, NR2A, NR2B and NR2C, showed a marked increase of NR2B levels in the dorsal hippocampus of male BDNF heterozygous mice treated with corticosterone. In the ventral hippocampus, significantly reduced levels of NR2A, NR2B and NR2C were observed in male BDNF heterozygous mice. The NMDA receptor effects in hippocampal sub-regions could be related to the spatial memory deficits and the loss of the effect of MK-801 on startle in these mice, respectively. No significant changes in NMDA receptor subunit levels were observed in any of the female groups. Similarly, no significant changes in levels of BDNF or its receptor, TrkB, were found other than the expected reduced levels of BDNF in heterozygous mice. In conclusion, the data show differential interactive effects of reduced levels of BDNF expression and corticosterone treatment on spatial memory and startle in male and female mice, accompanied by significant, but region-specific changes in NMDA receptor subunit levels in the dorsal and ventral hippocampus. These results could be important for our understanding of the interaction of neurodevelopmental stress and BDNF deficiency in cognitive and anxiety-related symptoms of psychiatric illnesses, such as schizophrenia.     

Altered N-methyl-D-aspartate receptor function in reelin heterozygous mice: male-female differences and comparison with dopaminergic activity

The aim of this study was to investigate the in vivo relationship between reelin and NMDA receptor function in schizophrenia. We assessed the effect of reelin deficiency in behavioral models of aspects of this illness, NMDA receptor subunit levels, and NMDA receptor, dopamine D₂ receptor, and dopamine transporter density. Male, but not female, reelin heterozygous mice showed significantly enhanced MK-801-induced locomotor hyperactivity compared to wildtype controls (7.4-fold vs. 5.2-fold effect of MK-801 over saline, respectively) but there were no genotype differences in the response to amphetamine. Both male and female reelin heterozygous mice showed enhanced effects of MK-801 on startle, but not prepulse inhibition (PPI) of startle. There were no group differences in the effect of apomorphine on startle or PPI. The levels of NMDA receptor subunits were not altered in the striatum. In the frontal cortex, male and female reelin heterozygous mice showed significant up-regulation of NR1 subunits, but down-regulation of NR2C subunits, which was associated with significantly elevated NR1/NR2A and NR1/NR2C ratios. However, there were no differences in [³H]MK-801 binding density in the nucleus accumbens or caudate nucleus, nor in the density of [³H]YM-09151 or [³H]GBR12935 in these brain regions. The enhanced effects of MK-801 in reelin heterozygous mice in this study could be reflective of the role of reelin deficiency in schizophrenia. This genotype effect was male-specific for locomotor hyperactivity, a model of psychosis, but was seen in male and female mice for startle, which could be an indication of changes in anxiety. Changes in NMDA receptor subunit levels and ratios were also seen in both male and female mice. These results suggest that the role of reelin deficiency in schizophrenia may be particularly mediated by altered NMDA receptor responses, with some of these effects being strictly sex-specific.     

Age-specific effects of early MK-801 treatment on working memory in female rats

The N-methyl-D-aspartate receptor plays a crucial role in developmental plasticity. Evidence shows that neonatal exposure to N-methyl-D-aspartate receptor antagonists impairs cognition in adult rats. This study investigated whether neonatal MK-801 treatment would produce long-term and age-specific effects on working memory and sensorimotor gating in adolescent and adult female rats. After treatment with MK-801 at postnatal days (PND) 5-14, female rats exhibited slightly impaired working memory during adolescence (PND: 35-42). In contrast, working memory was remarkably disrupted in adult (PND: 63-70) female rats. However, prepulse inhibition and startle amplitudes were not significantly affected at both ages. These findings indicate that neonatal MK-801 elicits working memory deficits, especially in the postpuberty female rats.     

Long-lasting effects of elevated neonatal leptin on rat hippocampal function, synaptic proteins and NMDA receptor subunits

The high circulating levels of leptin in neonatal rodents do not seem to be regulating energy balance at this age, but rather may play an important role for brain development. We tested the hypothesis that high neonatal leptin levels modify hippocampal function and production of synaptic proteins with possible long-term consequences on long-term potentiation (LTP) in adulthood. We first showed that in postnatal day (PND) 10 neonates, acute leptin treatment functionally activated leptin receptors (ObR) in the CA1 and DG regions of the hippocampus through the induction of phosphoERK1/2, but not phosphoSTAT3 protein although both phospho-proteins were induced in the arcuate nucleus. We next examined whether chronic leptin administration (3 mg/kg BW, intraperitoneally) during the first 2 weeks of life (postnatal day, PND 2-14) produces a functional signal in the hippocampus that alters the expression of NMDA receptor subunits (NR1, NR2A, NR2B), synaptic proteins and LTP in the short and long-term. In PND 10 as in adults (PND 70) rats, chronic leptin treatment increased NR1 expression in the hippocampus while reducing NR2B protein levels. Elevated hippocampal concentrations of synapsin2A and synaptophysin were detected during leptin treatment on PND 10 suggesting increased neurotransmitter release. In adults, only SNAP-25 expression was increased after neonatal leptin treatment. LTP was reduced dramatically by leptin treatment in preweaning rats although the changes did not persist until adulthood. Elevated exposure to leptin during a critical period of neonatal hippocampal development might serve to enhance NMDA-dependent functions other than LTP and have important effects on synaptogenesis and neurotransmitter release.     

Amphetamine-induced disruption of prepulse inhibition in mice with reduced NMDA receptor function

Genetically altered mice with reductions in the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor have been proposed as a model for the intrinsic NMDA hypofunction hypothesized for schizophrenia. The following study investigated whether NR1-deficient mice have enhanced susceptibility for the effects of amphetamine, similar to the exaggerated responsivity to dopamine agonists observed in many schizophrenia patients. NR1-/- mice and wild-type controls were tested for the effects of amphetamine (2-10 mg/kg) on prepulse inhibition of acoustic startle responses. The results showed that mice with reduced NMDA receptor function demonstrated consistent deficits in prepulse inhibition (PPI), as well as higher startle response amplitudes. In comparison to normal controls, the NR1-/- mice were more sensitive to the disruptive effects of amphetamine on PPI, but not to the drug effects on startle magnitude without a prepulse stimulus. Wild-type mice only showed decreased PPI at the highest dose of amphetamine tested (10 mg/kg) and demonstrated small increases in PPI at lower amphetamine doses (2 and 6 mg/kg). The NR1-/- mice did not show enhanced PPI in response to amphetamine at low doses, with reductions in PPI apparent at doses of 4-10 mg/kg. Overall, these findings suggest that the NR1-/- mouse may provide a model for enhanced sensitivity to dopamine agonist-induced disruption of PPI.     

Differential ontogenic development of three receptors comprising the NMDA receptor/channel complex in the rat hippocampus

The postnatal development of the three receptor binding sites that constitute the N-methyl-D-aspartate (NMDA) receptor channel/complex was examined in six hippocampal regions of rats using quantitative receptor autoradiography. NMDA-sensitive [3H]-glutamate binding, strychnine-insensitive [3H]glycine binding, and [3H]N-(1-[2-thienyl]cyclohexyl)-3,4-piperidine [( 3H]TCP) binding were measured to examine the ontogeny of NMDA recognition sites, glycine modulatory sites, and PCP receptors, respectively. NMDA-sensitive [3H]glutamate binding transiently exceeded adult levels by 50 to 120% in all regions examined, with peak densities generally occurring between postnatal days (PND) 10 and 28. Stratum radiatum CA1 binding increased slowly from 49 to 61% of the adult value between PND 1 and 7, after which, binding rapidly rose to 151% of adult values at PND 14, remained elevated through PND 28, and then decreased to adult levels. The ontogenic profile of NMDA recognition site binding was similar in other hippocampal regions, although the initial age of maximal binding and the period of stabilization varied. The ontogenic profiles of glycine modulatory site binding and PCP receptor binding were very similar to each other. Development was delayed, however, with respect to NMDA recognition site binding. The rapid development of binding observed between PND 7 and 14 with NMDA receptors in stratum radiatum CA1 was contrasted by a much slower increase in glycine and PCP receptor binding. Furthermore, maximal glycine and PCP receptor binding densities were not reached until PND 28 and were lower than NMDA recognition site binding densities. The observed developmental patterns of binding to each of the receptor components of the NMDA receptor channel/complex are consistent with postnatal changes in cytoarchitecture, synaptogenesis, afferent lamination, and functional development of the hippocampus. However, the relative overexpression of NMDA recognition sites with respect to glycine and PCP receptors between PND 7 and 21 suggests that there is differential expression of these binding sites during development.     

Corticosterone alters N-methyl-D-aspartate receptor subunit mRNA expression before puberty

Stress and stress hormones alter the expression of mRNA for the NR1, NR2A and NR2B subunits of the N-methyl-D-aspartate (NMDA) receptor in brain regions associated with the stress response. Early life stress contributes to the risk and pathophysiology of mental illness. Examining how stress hormones modulate NMDA receptor subunit gene expression before and after pubertal onset will further contribute to the understanding of how stress during adolescence relates to adult mental illness. Using in situ hybridization histochemistry, we measured NR1, NR2A and NR2B mRNA expression in the hippocampus and in the hypothalamic paraventricular nucleus (PVN) of rats that had undergone adrenalectomy (ADX) or sham surgery before or after puberty. Some ADX rats received corticosterone pellets that released either normal or stress levels of corticosterone for 14 days prior to sacrifice. There was a significant increase in NR1 subunit mRNA expression throughout the subfields of the hippocampus and in the PVN of ADX prepubertal rats. However, similar changes in hippocampal NR1 expression were not observed in postpubertal ADX rats. Pre- and postpubertal ADX rats implanted with a high-dose corticosterone pellet had decreased expression of PVN NR1 mRNA. Only prepubertal rats had an increase in dentate gyrus NR2A mRNA and CA3 region NR2B mRNA following high-dose replacement. These results provide evidence that glucocorticoids have differential effects on the regional expression of mRNA NMDA receptor subunits and elucidate a window during adolescence in which the NR1, NR2A and NR2B genes are responsive to glucocorticoids.     

A lasting effect of postnatal sevoflurane anesthesia on the composition of NMDA receptor subunits in rat prefrontal cortex

Sevoflurane is widely used in pediatric anesthesia and studies have shown that it is capable of inducing neurodegeneration and subsequent cognitive disorders in the developing brain. However, the evidence that anesthetics are toxic to the human brain is insufficient. N-Methyl-d-aspartate (NMDA) receptors, critical for learning and memory, display expression changes with age and can be modulated by inhalation anesthetics. Generally, NMDA receptor (NR) type 1 is expressed at birth, peaks around the third postnatal week, and then declines slightly to adult levels. NR2Bs slowly decrease and NR2As gradually increase during postnatal development. These developmental switches of NMDA receptor subunits composition mark the transition from immature to adult neural processing and allow for the final maturation of associative learning abilities. In this study, we aimed to evaluate the effect of repeated sevoflurane anesthesia on NMDA receptor subunits composition in the developing rat brain and related behavioral disorders. Six-day-old male Sprague Dawley rats were randomly allocated into either a control group (group con) or a sevoflurane group (group sevo). Group sevo inhaled 2.1% sevoflurane carried by 70% oxygen for 2 h each day from postnatal day (PND) 6 to PND 8. The same procedure, without applying the sevoflurane, was executed in group con. The membrane protein expression of NR1, NR2A and NR2B in the prefrontal cortex (PFC) and hippocampus was assessed at the end of the three days of anesthesia and at PND 21. An open field test was carried out to assess spontaneous locomotion on PNDs 21, 28 and 35. Y maze performance was used to assess attention and working memory on PND 28. Sevoflurane induced upregulation of NR1 and NR2B in the PFC at the end of anesthesia. On PND 21, NR1 and NR2B receptors were significantly increased whereas NR2A receptors were significantly decreased in the PFC in group sevo. Sevoflurane-treated rats showed hyper-locomotion and impairment of working memory in the behavior tests. These results indicate that repeated sevoflurane anesthesia at early stage of life can induce a long lasting effect of interfering with NMDA receptor subunits composition in rat PFC. These changes may contribute to the effects of sevoflurane on neuronal development and subsequent neurobehavioral disorders.     

Deficits in sensorimotor gating and tests of social behavior in a genetic model of reduced NMDA receptor function

Reduced NMDA receptor function is hypothesized to contribute to the pathophysiology of schizophrenia. In order to model chronic and developmental NMDA receptor hypofunction, a mouse line was developed that expresses low levels of the NMDA R1 subunit (NR1) of the NMDA receptor. The present study tested the hypothesis that these NR1 hypomorphic mice would exhibit deficits in sensorimotor and conspecific interactions, analogous to deficits observed in schizophrenic patients. F1 hybrid mice homozygous for the NR1 hypomorphic mutation (NR1 -/-) were generated by crossing heterozygous mice (NR1 +/-) from C57BL/6 and 129 Sv/Ev backgrounds. To assess sensorimotor gating, mice were tested in the paradigm of prepulse inhibition of acoustic startle. The NR1 hypomorphic mice exhibited increased acoustic startle responses and also showed deficits in prepulse inhibition. Startle responses were differentially altered by predator odor exposure in the male NR1 -/- mice, in comparison to control mice. In a test of social affiliation, the wild type mice spent significantly more time investigating a novel mouse in comparison to the NR1 -/- mice. In a resident-intruder test, marked deficits were found in sex-specific aggressive behavior between the wild type and mutant mice. These data support the contention that the NR1 hypomorphic mice exhibit alterations in sensorimotor gating and typical conspecific interactions, reminiscent of behavioral disturbances associated with schizophrenia. The NR1 hypomorphic mice could represent a model system to explore novel treatment and preventative strategies for certain symptoms of schizophrenia.     

Primary and secondary neural networks of auditory prepulse inhibition: a functional magnetic resonance imaging study of sensorimotor gating of the human acoustic startle response

Feedforward inhibition deficits have been consistently demonstrated in a range of neuropsychiatric conditions using prepulse inhibition (PPI) of the acoustic startle eye-blink reflex when assessing sensorimotor gating. While PPI can be recorded in acutely decerebrated rats, behavioural, pharmacological and psychophysiological studies suggest the involvement of a complex neural network extending from brainstem nuclei to higher order cortical areas. The current functional magnetic resonance imaging study investigated the neural network underlying PPI and its association with electromyographically (EMG) recorded PPI of the acoustic startle eye-blink reflex in 16 healthy volunteers. A sparse imaging design was employed to model signal changes in blood oxygenation level-dependent (BOLD) responses to acoustic startle probes that were preceded by a prepulse at 120 ms or 480 ms stimulus onset asynchrony or without prepulse. Sensorimotor gating was EMG confirmed for the 120-ms prepulse condition, while startle responses in the 480-ms prepulse condition did not differ from startle alone. Multiple regression analysis of BOLD contrasts identified activation in pons, thalamus, caudate nuclei, left angular gyrus and bilaterally in anterior cingulate, associated with EMG-recorded sensorimotor gating. Planned contrasts confirmed increased pons activation for startle alone vs 120-ms prepulse condition, while increased anterior superior frontal gyrus activation was confirmed for the reverse contrast. Our findings are consistent with a primary pontine circuitry of sensorimotor gating that interconnects with inferior parietal, superior temporal, frontal and prefrontal cortices via thalamus and striatum. PPI processes in the prefrontal, frontal and superior temporal cortex were functionally distinct from sensorimotor gating.     

Contributions of hippocampal cellular damage and NMDA receptor dysfunction to behavioral markers of schizophrenia.

Pathological changes in the hippocampal formation have been noted in schizophrenic patients and manipulation of neurochemical functions within the limbic system has been shown to yield behavioral changes consistent with schizophrenia. The present study evaluated the impact of kainic acid induced hippocampal cellular damage and manipulation of NMDA receptor function (agonism and antagonism) on common behavioral markers of schizophrenia (habituation and prepulse inhibition of the acoustic startle response in rats). Cellular damage significantly impaired habituation and NMDA antagonism disrupted prepulse inhibition. Damage induced impairment of habituation is consistent with effects on latent inhibition (which is also unaffected by NMDA antagonism) while the antagonist disruption of prepulse inhibition is consistent with effects on associative plasticity. The current findings provide further support for a diverse neurobiological substrate of schizophrenic symptoms suggesting that pharmacologic intervention may need to be multifaceted and could involve competing mechanisms. Cognitive impairments may reflect diminished NMDA receptor function whereas positive symptoms may reflect heightened engagement of anatomically disturbed cellular elements.     

The potential role of phrenic nucleus glutamate receptor subunits in mediating spontaneous crossed phrenic activity in neonatal rat

Cervical spinal cord hemisection rostral to the phrenic nucleus leads to paralysis of the ipsilateral hemidiaphragm in adult rats. Respiratory function can be restored to the paralyzed hemidiaphragm by activating a latent respiratory motor pathway. The latent pathway is called the crossed phrenic pathway. In adult rats, the pathway can be activated by drug-induced upregulation of NMDA receptor NR2A subunit and AMPA receptor GluR1 subunit in the phrenic nucleus following hemisection. In neonatal rats, this pathway is not latent as shown by the spontaneous expression of activity in the ipsilateral hemidiaphragm following hemisection. We hypothesized that the NR2A and GluR1 subunits may be highly expressed naturally on phrenic motoneurons of neonatal rats and may play a potential role in mediating the spontaneous expression of activity in the ipsilateral hemidiaphragm after hemisection. To test this hypothesis, the protein levels of NR2A and GluR1 in different age rats were assessed via Western blot analysis immediately following C2 hemisection and EMG recording of crossed phrenic activity. The protein levels of NR2A and GluR1 were transiently high in postnatal day 2 (P2) rats and then was significantly reduced in P7 and P35 animals. An immunofluorescence study qualitatively supported these findings. The present results indicate that the developmental downregulation of the phrenic nucleus glutamate receptor subunits correlates with the conversion of the crossed phrenic pathway in older postnatal animals from an active state to a latent state.     

Roles of the glutamate receptor epsilon2 and delta2 subunits in the potentiation and prepulse inhibition of the acoustic startle reflex

We examined the regulation of the acoustic startle response in mutant mice of the N-methyl-D-aspartate (NMDA)- and delta-subtypes of the glutamate receptor (GluR) channel, which play important roles in neural plasticity in the forebrain and the cerebellum, respectively. Heterozygous mutant mice with reduced GluRepsilon2 subunits of the NMDA receptor showed strongly enhanced startle responses to acoustic stimuli. On the other hand, heterozygous and homozygous mutation of the other NMDA receptor GluRepsilon subunits exerted no, or only small effects on acoustic startle responses. The threshold of the auditory brainstem response of the GluRepsilon2-mutant mice was comparable to that of the wild-type littermates. The primary circuit of the acoustic startle response is a relatively simple oligosynaptic pathway located in the lower brainstem, whilst the expression of GluRepsilon2 is restricted to the forebrain. We thus suggest that the NMDA receptor GluRepsilon2 subunit plays a role in the regulation of the startle reflex. Ablation of the cerebellar Purkinje cell-specific delta2 subunit of the GluR channel exerted little effect on the acoustic startle response but resulted in the enhancement of prepulse inhibition of the reflex. Because inhibition of the acoustic startle response by a weak prepulse is a measure of sensorimotor gating, the process by which an organism filters sensory information, these observations indicate the involvement of the cerebellum in the modulation of sensorimotor gating.