The critical period: neurochemical and synaptic mechanisms shared by the visual cortex and the brain stem respiratory system.

The landmark studies of Wiesel and Hubel in the 1960's initiated a surge of investigations into the critical period of visual cortical development, when abnormal visual experience can alter cortical structures and functions. Most studies focused on the visual cortex, with relatively little attention to subcortical structures. The goal of the present review is to elucidate neurochemical and synaptic mechanisms common to the critical periods of the visual cortex and the brain stem respiratory system in the normal rat. In both regions, the critical period is a time of (i) heightened inhibition; (ii) reduced expression of brain-derived neurotrophic factor (BDNF); and (iii) synaptic imbalance, with heightened inhibition and suppressed excitation. The last two mechanisms are contrary to the conventional premise. Synaptic imbalance renders developing neurons more vulnerable to external stressors. However, the critical period is necessary to enable each system to strengthen its circuitry, adapt to its environment, and transition from immaturity to maturity, when a state of relative synaptic balance is attained. Failure to achieve such a balance leads to neurological disorders.

Chronic intermittent hypoxia alters the dendritic mitochondrial structure and activity in the pre-Bötzinger complex of rats.

Mitochondrial bioenergetics is dynamically coupled with neuronal activities, which are altered by hypoxia-induced respiratory neuroplasticity. Here we report structural features of postsynaptic mitochondria in the pre-Bötzinger complex (pre-BötC) of rats treated with chronic intermittent hypoxia (CIH) simulating a severe condition of obstructive sleep apnea. The subcellular changes in dendritic mitochondria and histochemistry of cytochrome c oxidase (CO) activity were examined in pre-BötC neurons localized by immunoreactivity of neurokinin 1 receptors. Assays of mitochondrial electron transport chain (ETC) complex I, IV, V activities, and membrane potential were performed in the ventrolateral medulla containing the pre-BötC region. We found significant decreases in the mean length and area of dendritic mitochondria in the pre-BötC of CIH rats, when compared to the normoxic control and hypoxic group with daily acute intermittent hypoxia (dAIH) that evokes robust synaptic plasticity. Notably, these morphological alterations were mainly observed in the mitochondria in close proximity to the synapses. In addition, the proportion of mitochondria presented with enlarged compartments and filamentous cytoskeletal elements in the CIH group was less than the control and dAIH groups. Intriguingly, these distinct characteristics of structural adaptability were observed in the mitochondria within spatially restricted dendritic spines. Furthermore, the proportion of moderately to darkly CO-reactive mitochondria was reduced in the CIH group, indicating reduced mitochondrial activity. Consistently, mitochondrial ETC enzyme activities and membrane potential were lowered in the CIH group. These findings suggest that hypoxia-induced respiratory plasticity was characterized by spatially confined mitochondrial alterations within postsynaptic spines in the pre-BötC neurons. In contrast to the robust plasticity evoked by dAIH preconditioning, a severe CIH challenge may weaken the local mitochondrial bioenergetics that the fuel postsynaptic activities of the respiratory motor drive.

Acute and chronic changes in the control of breathing in a rat model of bronchopulmonary dysplasia.

Infants born very prematurely (<28 wk gestation) have immature lungs and often require supplemental oxygen. However, long-term hyperoxia exposure can arrest lung development, leading to bronchopulmonary dysplasia (BPD), which increases acute and long-term respiratory morbidity and mortality. The neural mechanisms controlling breathing are highly plastic during development. Whether the ventilatory control system adapts to pulmonary disease associated with hyperoxia exposure in infancy remains unclear. Here, we assessed potential age-dependent adaptations in the control of breathing in an established rat model of BPD associated with hyperoxia. Hyperoxia exposure ( FIO2 ; 0.9 from 0 to 10 days of life) led to a BPD-like lung phenotype, including sustained reductions in alveolar surface area and counts, and modest increases in airway resistance. Hyperoxia exposure also led to chronic increases in room air and acute hypoxic minute ventilation (V̇e) and age-dependent changes in breath-to-breath variability. Hyperoxia-exposed rats had normal oxygen saturation ( SpO2 ) in room air but greater reductions in SpO2 during acute hypoxia (12% O2) that were likely due to lung injury. Moreover, acute ventilatory sensitivity was reduced at P12 to P14. Perinatal hyperoxia led to greater glial fibrillary acidic protein expression and an increase in neuron counts within six of eight or one of eight key brainstem regions, respectively, controlling breathing, suggesting astrocytic expansion. In conclusion, perinatal hyperoxia in rats induced a BPD-like phenotype and age-dependent adaptations in V̇e that may be mediated through changes to the neural architecture of the ventilatory control system. Our results suggest chronically altered ventilatory control in BPD.

Uncovering a critical period of synaptic imbalance during postnatal development of the rat visual cortex: role of brain-derived neurotrophic factor.

KEY POINTS: With daily electrophysiological recordings and neurochemical analysis, we uncovered a transient period of synaptic imbalance between enhanced inhibition and suppressed excitation in rat visual cortical neurons from the end of the fourth toward the end of the fifth postnatal weeks. The expression of brain-derived neurotrophic factor (BDNF), which normally enhances excitation and suppresses inhibition, was down-regulated during that time, suggesting that this may contribute to the inhibition/excitation imbalance. An agonist of the BDNF receptor tropomyosin-related kinase B (TrkB) partially reversed the imbalance, whereas a TrkB antagonist accentuated the imbalance during the transient period. Monocular lid suture during the transient period is more detrimental to the function and neurochemical properties of visual cortical neurons than before or after this period. We regard the period of synaptic imbalance as the peak critical period of vulnerability, and its existence is necessary for neurons to transition from immaturity to a more mature state of functioning. ABSTRACT: The mammalian visual cortex is immature at birth and undergoes postnatal structural and functional adjustments. The exact timing of the vulnerable period in rodents remains unclear. The critical period is characterized by inhibitory GABAergic maturation reportedly dependent on brain-derived neurotrophic factor (BDNF). However, most of the studies were performed on experimental/transgenic animals, questioning the relationship in normal animals. The present study aimed to conduct in-depth analyses of the synaptic and neurochemical development of visual cortical neurons in normal and monocularly-deprived rats and to determine specific changes, if any, during the critical period. We found that (i) against a gradual increase in excitation and inhibition with age, a transient period of synaptic and neurochemical imbalance existed with suppressed excitation and enhanced inhibition at postnatal days 28 to 33/34; (ii) during this window, the expression of BDNF and tropomyosin-related kinase B (TrkB) receptors decreased, along with glutamatergic GluN1 and GluA1 receptors and the metabolic marker cytochrome oxidase, whereas that of GABAA Rα1 receptors continued to rise; (iii) monocular deprivation reduced both excitatory and inhibitory synaptic activity and neurochemicals mainly during this period; and (iv) in vivo TrkB agonist partially reversed the synaptic imbalance in normal and monocularly-deprived neurons during this time, whereas a TrkB antagonist accentuated the imbalance. Thus, our findings highlight a transitory period of synaptic imbalance with a negative relationship between BDNF and inhibitory GABA. This brief critical period may be necessary in transitioning from an immature to a more mature state of visual cortical functioning.

Transcriptional Regulation of Brain-derived Neurotrophic Factor Coding Exon IX: ROLE OF NUCLEAR RESPIRATORY FACTOR 2.

Brain-derived neurotrophic factor (BDNF) is an active neurotrophin abundantly expressed throughout the nervous system. It plays an important role in synaptic transmission, plasticity, neuronal proliferation, differentiation, survival, and death. The Bdnf gene in rodents has eight non-coding exons and only a single coding exon (IX). Despite its recognized regulation by neuronal activity, relatively little is known about its transcriptional regulation, and even less about the transcription factor candidates that may play such a role. The goal of the present study was to probe for such a candidate that may regulate exon IX in the rat Bdnf gene. Our in silico analysis revealed tandem binding sites for nuclear respiratory factor 2 (NRF-2) on the promoter of exon IX. NRF-2 is of special significance because it co-regulates the expressions of mediators of energy metabolism (cytochrome c oxidase) and mediators of neuronal activity (glutamatergic receptors). To test our hypothesis that NRF-2 also regulates the Bdnf gene, we performed electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), promoter cloning, and site-directed mutagenesis, real-time quantitative PCR (RT-qPCR), and Western blotting analysis. Results indicate that NRF-2 functionally regulates exon IX of the rat Bdnf gene. The binding sites of NRF-2 are conserved between rats and mice. Overexpressing NRF-2 up-regulated the expression of Bdnf exon IX, whereas knocking down NRF-2 down-regulated such expression. These findings are consistent with our hypothesis that NRF-2, in addition to regulating the coupling between neuronal activity and energy metabolism, also regulates the expression of BDNF, which is intimately associated with energy-demanding neuronal activity.

Specificity protein 4 (Sp4) transcriptionally regulates inhibitory GABAergic receptors in neurons.

Previous studies in our laboratory have shown that the neuron-specific specificity protein 4 (Sp4) transcriptionally regulates many excitatory neurotransmitter receptor subunit genes, such as those for GluN1, GluN2A, and GluN2B of N-methyl-d-aspartate (NMDA) receptors and Gria2 of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. It also regulates Atp1a1 and Atp1b1 subunit genes of Na(+)/K(+)-ATPase, a major energy-consuming enzyme, as well as all 13 subunits of cytochrome c oxidase (COX), an important energy-generating enzyme. Thus, there is a tight coupling between energy consumption, energy production, and excitatory neuronal activity at the transcriptional level in neurons. The question is whether inhibitory neurotransmitter receptors are also regulated by Sp4. In the present study, we tested our hypothesis that Sp4 regulates receptor subunit genes of a major inhibitory neurotransmitter, GABA, specifically GABAA receptors. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, real-time quantitative PCR, chromatin immunoprecipitation, promoter mutational analysis, over-expression and shRNA of Sp4, functional assays, and western blots, we found that Sp4 functionally regulates the transcription of Gabra1 (GABAA α1) and Gabra2 (GABAA α2), but not Gabra3 (GABAA α3) subunit genes. The binding sites of Sp4 are conserved among rats, humans, and mice. Thus, our results substantiate our hypothesis that Sp4 plays a key role in regulating the transcription of GABAA receptor subunit genes. They also indicate that Sp4 is in a position to transcriptionally regulate the balance between excitatory and inhibitory neurochemical expressions in neurons.

Nuclear respiratory factor 2 regulates the transcription of AMPA receptor subunit GluA2 (Gria2).

Neuronal activity is highly dependent on energy metabolism. Nuclear respiratory factor 2 (NRF-2) tightly couples neuronal activity and energy metabolism by transcriptionally co-regulating all 13 subunits of an important energy-generating enzyme, cytochrome c oxidase (COX), as well as critical subunits of excitatory NMDA receptors. AMPA receptors are another major class of excitatory glutamatergic receptors that mediate most of the fast excitatory synaptic transmission in the brain. They are heterotetrameric proteins composed of various combinations of GluA1-4 subunits, with GluA2 being the most common one. We have previously shown that GluA2 (Gria2) is transcriptionally regulated by nuclear respiratory factor 1 (NRF-1) and specificity protein 4 (Sp4), which also regulate all subunits of COX. However, it was not known if NRF-2 also couples neuronal activity and energy metabolism by regulating subunits of the AMPA receptors. By means of multiple approaches, including electrophoretic mobility shift and supershift assays, chromatin immunoprecipitation, promoter mutations, real-time quantitative PCR, and western blot analysis, NRF-2 was found to functionally regulate the expression of Gria2, but not of Gria1, Gria3, or Gria4 genes in neurons. By regulating the GluA2 subunit of the AMPA receptor, NRF-2 couples energy metabolism and neuronal activity at the transcriptional level through a concurrent and parallel mechanism with NRF-1 and Sp4.

Specificity protein 4 (Sp4) regulates the transcription of AMPA receptor subunit GluA2 (Gria2).

The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are important glutamatergic receptors mediating fast excitatory synaptic transmission in the brain. The regulation of the four subunits of AMPA receptors, GluA1-4, is poorly understood. Excitatory synaptic transmission is highly energy-demanding, and this energy is derived mainly from the oxidative pathway. Recently, we found that specificity factor regulates all subunits of cytochrome c oxidase (COX), a critical energy-generating enzyme. COX is also regulated by nuclear respiratory factor 1 (NRF-1), which transcriptionally controls the Gria2 (GluA2) gene of AMPA receptors. The goal of the present study was to test our hypothesis that Sp-factors (Sp1, Sp3, and/or Sp4) also regulate AMPA subunit genes. If so, we wish to determine if Sp-factors and NRF-1 function via a complementary, concurrent and parallel, or a combination of complementary and concurrent/parallel mechanism. By means of multiple approaches, including electrophoretic mobility shift and supershift assays, chromatin immunoprecipitation, promoter mutations, real-time quantitative PCR, and western blot analysis, we found that Sp4, but not Sp1 or Sp3, regulates the Gria2, but not Gria1, 3, or 4, subunit gene of the AMPA receptor in a concurrent and parallel manner with NRF-1. Thus, Sp4 and NRF-1 both mediate the tight coupling between neuronal activity and energy metabolism at the transcriptional level.

Nuclear respiratory factor 2 regulates the expression of the same NMDA receptor subunit genes as NRF-1: both factors act by a concurrent and parallel mechanism to couple energy metabolism and synaptic transmission.

Neuronal activity and energy metabolism are tightly coupled processes. Previously, we found that nuclear respiratory factor 1 (NRF-1) transcriptionally co-regulates energy metabolism and neuronal activity by regulating all 13 subunits of the critical energy generating enzyme, cytochrome c oxidase (COX), as well as N-methyl-d-aspartate (NMDA) receptor subunits 1 and 2B, GluN1 (Grin1) and GluN2B (Grin2b). We also found that another transcription factor, nuclear respiratory factor 2 (NRF-2 or GA-binding protein) regulates all subunits of COX as well. The goal of the present study was to test our hypothesis that NRF-2 also regulates specific subunits of NMDA receptors, and that it functions with NRF-1 via one of three mechanisms: complementary, concurrent and parallel, or a combination of complementary and concurrent/parallel. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation of mouse neuroblastoma cells and rat visual cortical tissue, promoter mutations, real-time quantitative PCR, and western blot analysis, NRF-2 was found to functionally regulate Grin1 and Grin2b genes, but not any other NMDA subunit genes. Grin1 and Grin2b transcripts were up-regulated by depolarizing KCl, but silencing of NRF-2 prevented this up-regulation. On the other hand, over-expression of NRF-2 rescued the down-regulation of these subunits by the impulse blocker TTX. NRF-2 binding sites on Grin1 and Grin2b are conserved among species. Our data indicate that NRF-2 and NRF-1 operate in a concurrent and parallel manner in mediating the tight coupling between energy metabolism and neuronal activity at the molecular level.

Specificity protein 4 functionally regulates the transcription of NMDA receptor subunits GluN1, GluN2A, and GluN2B.

N-Methyl-d-aspartate (NMDA) receptors are major glutamatergic receptors involved in most excitatory neurotransmission in the brain. The transcriptional regulation of NMDA receptors is not fully understood. Previously, we found that the GluN1 and GluN2B subunits of the NMDA receptor are regulated by nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2). NRF-1 and NRF-2 also regulate all 13 subunits of cytochrome c oxidase (COX), a critical energy-generating enzyme, thereby coupling neuronal activity and energy metabolism at the transcriptional level. Specificity protein (Sp) is a family of transcription factors that bind to GC-rich regions, with Sp1, Sp3, and Sp4 all binding to the same cis- motifs. Sp1 and Sp3 are ubiquitously expressed, whereas Sp4 expression is restricted to neurons and testicular cells. Recently, we found that the Sp1 factor regulates all subunits of COX. The goal of the present study was to test our hypothesis that the Sp factors also regulate specific subunits of NMDA receptors, and that they function with NRF-1 and NRF-2 via one of three mechanisms: complementary, concurrent and parallel, or a combination of complementary and concurrent/parallel. By means of multiple approaches we found that Sp4 functionally regulated GluN1, GluN2A, and GluN2B, but not GluN2C. On the other hand, Sp1 and Sp3 did not regulate these subunits as previously thought. Our data suggest that Sp4 operates in a complementary and concurrent/parallel manner with NRF-1 and NRF-2 to mediate the tight coupling between energy metabolism and neuronal activity at the molecular level.

Regulation of Na(+)/K(+)-ATPase by neuron-specific transcription factor Sp4: implication in the tight coupling of energy production, neuronal activity and energy consumption in neurons.

A major source of energy demand in neurons is the Na(+)/K(+)-ATPase pump that restores the ionic gradient across the plasma membrane subsequent to depolarizing neuronal activity. The energy comes primarily from mitochondrial oxidative metabolism, of which cytochrome c oxidase (COX) is a key enzyme. Recently, we found that all 13 subunits of COX are regulated by specificity (Sp) factors, and that the neuron-specific Sp4, but not Sp1 or Sp3, regulates the expression of key glutamatergic receptor subunits as well. The present study sought to test our hypothesis that Sp4 also regulates Na(+)/K(+)-ATPase subunit genes in neurons. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, chromatin immunoprecipitation, promoter mutational analysis, over-expression, and RNA interference studies, we found that Sp4, with minor contributions from Sp1 and Sp3, functionally regulate the Atp1a1, Atp1a3, and Atp1b1 subunit genes of Na(+)/K(+)-ATPase in neurons. Transcripts of all three genes were up-regulated by depolarizing KCl stimulation and down-regulated by the impulse blocker tetrodotoxin (TTX), indicating that their expression was activity-dependent. Silencing of Sp4 blocked the up-regulation of these genes induced by KCl, whereas over-expression of Sp4 rescued them from TTX-induced suppression. The effect of silencing or over-expressing Sp4 on primary neurons was much greater than those of Sp1 or Sp3. The binding sites of Sp factors on these genes are conserved among mice, rats and humans. Thus, Sp4 plays an important role in the transcriptional coupling of energy generation and energy consumption in neurons.

Reduced levels of brain-derived neurotrophic factor contribute to synaptic imbalance during the critical period of respiratory development in rats.

Previously, our electrophysiological studies revealed a transient imbalance between suppressed excitation and enhanced inhibition in hypoglossal motoneurons of rats on postnatal days (P) 12-13, a critical period when abrupt neurochemical, metabolic, ventilatory and physiological changes occur in the respiratory system. The mechanism underlying the imbalance is poorly understood. We hypothesised that the imbalance was contributed by a reduced expression of brain-derived neurotrophic factor (BDNF), which normally enhances excitation and suppresses inhibition. We also hypothesised that exogenous BDNF would partially reverse this synaptic imbalance. Immunohistochemistry/single-neuron optical densitometry, real-time quantitative PCR (RT-qPCR) and whole-cell patch-clamp recordings were done on hypoglossal motoneurons in brainstem slices of rats during the first three postnatal weeks. Our results indicated that: (1) the levels of BDNF and its high-affinity tyrosine receptor kinase B (TrkB) receptor mRNAs and proteins were relatively high during the first 1-1.5 postnatal weeks, but dropped precipitously at P12-13 before rising again afterwards; (2) exogenous BDNF significantly increased the normally lowered frequency of spontaneous excitatory postsynaptic currents but decreased the normally heightened amplitude and frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) during the critical period; (3) exogenous BDNF also decreased the normally heightened frequency of miniature IPSCs at P12-13; and (4) the effect of exogenous BDNF was partially blocked by K252a, a TrkB receptor antagonist. Thus, our results are consistent with our hypothesis that BDNF and TrkB play an important role in the synaptic imbalance during the critical period. This may have significant implications for the mechanism underlying sudden infant death syndrome.

Regulation of Na(+)/K(+)-ATPase by nuclear respiratory factor 1: implication in the tight coupling of neuronal activity, energy generation, and energy consumption.

BACKGROUND: NRF-1 regulates mediators of neuronal activity and energy generation. RESULTS: NRF-1 transcriptionally regulates Na(+)/K(+)-ATPase subunits α1 and ß1. CONCLUSION: NRF-1 functionally regulates mediators of energy consumption in neurons. SIGNIFICANCE: NRF-1 mediates the tight coupling of neuronal activity, energy generation, and energy consumption at the molecular level. Energy generation and energy consumption are tightly coupled to neuronal activity at the cellular level. Na(+)/K(+)-ATPase, a major energy-consuming enzyme, is well expressed in neurons rich in cytochrome c oxidase, an important enzyme of the energy-generating machinery, and glutamatergic receptors that are mediators of neuronal activity. The present study sought to test our hypothesis that the coupling extends to the molecular level, whereby Na(+)/K(+)-ATPase subunits are regulated by the same transcription factor, nuclear respiratory factor 1 (NRF-1), found recently by our laboratory to regulate all cytochrome c oxidase subunit genes and some NMDA and AMPA receptor subunit genes. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation, promoter mutational analysis, and real-time quantitative PCR, NRF-1 was found to functionally bind to the promoters of Atp1a1 and Atp1b1 genes but not of the Atp1a3 gene in neurons. The transcripts of Atp1a1 and Atp1b1 subunit genes were up-regulated by KCl and down-regulated by tetrodotoxin. Atp1b1 is positively regulated by NRF-1, and silencing of NRF-1 with small interference RNA blocked the up-regulation of Atp1b1 induced by KCl, whereas overexpression of NRF-1 rescued these transcripts from being suppressed by tetrodotoxin. On the other hand, Atp1a1 is negatively regulated by NRF-1. The binding sites of NRF-1 on Atp1a1 and Atp1b1 are conserved among mice, rats, and humans. Thus, NRF-1 regulates key Na(+)/K(+)-ATPase subunits and plays an important role in mediating the tight coupling between energy consumption, energy generation, and neuronal activity at the molecular level.

Neuron-specific specificity protein 4 bigenomically regulates the transcription of all mitochondria- and nucleus-encoded cytochrome c oxidase subunit genes in neurons.

Neurons are highly dependent on oxidative metabolism for their energy supply, and cytochrome c oxidase (COX) is a key energy-generating enzyme in the mitochondria. A unique feature of COX is that it is one of only four proteins in mammalian cells that are bigenomically regulated. Of its thirteen subunits, three are encoded in the mitochondrial genome and ten are nuclear-encoded on nine different chromosomes. The mechanism of regulating this multisubunit, bigenomic enzyme poses a distinct challenge. In recent years, we found that nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) mediate such bigenomic coordination. The latest candidate is the specificity factor (Sp) family of proteins. In N2a cells, we found that Sp1 regulates all 13 COX subunits. However, we discovered recently that in primary neurons, it is Sp4 and not Sp1 that regulates some of the key glutamatergic receptor subunit genes. The question naturally arises as to the role of Sp4 in regulating COX in primary neurons. The present study utilized multiple approaches, including chromatin immunoprecipitation, promoter mutational analysis, knockdown and over-expression of Sp4, as well as functional assays to document that Sp4 indeed functionally regulate all 13 subunits of COX as well as mitochondrial transcription factors A and B. The present study discovered that among the specificity family of transcription factors, it is the less known neuron-specific Sp4 that regulates the expression of all 13 subunits of mitochondrial cytochrome c oxidase (COX) enzyme in primary neurons. Sp4 also regulates the three mitochondrial transcription factors (TFAM, TFB1M, and TFB2M) and a COX assembly protein SURF-1 in primary neurons.

Expression of phospho-Ca(2+) /calmodulin-dependent protein kinase II in the pre-Bötzinger complex of rats.

The pre-Bötzinger complex (pre-BötC) in the ventrolateral medulla oblongata is a presumed kernel of respiratory rhythmogenesis. Ca(2+) -activated non-selective cationic current is an essential cellular mechanism for shaping inspiratory drive potentials. Ca(2+) /calmodulin-dependent protein kinase II (CaMKII), an ideal 'interpreter' of diverse Ca(2+) signals, is highly expressed in neurons in mediating various physiological processes. Yet, less is known about CaMKII activity in the pre-BötC. Using neurokinin-1 receptor as a marker of the pre-BötC, we examined phospho (P)-CaMKII subcellular distribution, and found that P-CaMKII was extensively expressed in the region. P-CaMKII-ir neurons were usually oval, fusiform, or pyramidal in shape. P-CaMKII immunoreactivity was distributed within somas and dendrites, and specifically in association with the post-synaptic density. In dendrites, most synapses (93.1%) examined with P-CaMKII expression were of asymmetric type, occasionally with symmetric type (6.9%), whereas in somas, 38.1% were of symmetric type. P-CaMKII asymmetric synaptic identification implicates that CaMKII may sense and monitor Ca(2+) activity, and phosphorylate post-synaptic proteins to modulate excitatory synaptic transmission, which may contribute to respiratory modulation and plasticity. In somas, CaMKII acts on both symmetric and asymmetric synapses, mediating excitatory and inhibitory synaptic transmission. P-CaMKII was also localized to the perisynaptic and extrasynaptic regions in the pre-BötC.

Alterations in synapses and mitochondria induced by acute or chronic intermittent hypoxia in the pre-Bötzinger complex of rats: an ultrastructural triple-labeling study with immunocytochemistry and histochemistry.

Introduction: The pre-Bötzinger complex (pre-BötC), a kernel of inspiratory rhythmogenesis, is a heterogeneous network with excitatory glutamatergic and inhibitory GABAergic and glycinergic neurons. Inspiratory rhythm generation relies on synchronous activation of glutamatergic neuron, whilst inhibitory neurons play a critical role in shaping the breathing pattern, endowing the rhythm with flexibility in adapting to environmental, metabolic, and behavioral needs. Here we report ultrastructural alterations in excitatory, asymmetric synapses (AS) and inhibitory, symmetric synapses (SS), especially perforated synapses with discontinuous postsynaptic densities (PSDs) in the pre-BötC in rats exposed to daily acute intermittent hypoxia (dAIH) or chronic (C) IH. Methods: We utilized for the first time a combination of somatostatin (SST) and neurokinin 1 receptor (NK1R) double immunocytochemistry with cytochrome oxidase histochemistry, to reveal synaptic characteristics and mitochondrial dynamic in the pre-BötC. Results: We found perforated synapses with synaptic vesicles accumulated in distinct pools in apposition to each discrete PSD segments. dAIH induced significant increases in the PSD size of macular AS, and the proportion of perforated synapses. AS were predominant in the dAIH group, whereas SS were in a high proportion in the CIH group. dAIH significantly increased SST and NK1R expressions, whereas CIH led to a decrease. Desmosome-like contacts (DLC) were characterized for the first time in the pre-BötC. They were distributed alongside of synapses, especially SS. Mitochondria appeared in more proximity to DLC than synapses, suggestive of a higher energy demand of the DLC. Findings of single spines with dual AS and SS innervation provide morphological evidence of excitation-inhibition interplay within a single spine in the pre-BötC. In particular, we characterized spine-shaft microdomains of concentrated synapses coupled with mitochondrial positioning that could serve as a structural basis for synchrony of spine-shaft communication. Mitochondria were found within spines and ultrastructural features of mitochondrial fusion and fission were depicted for the first time in the pre-BötC. Conclusion: We provide ultrastructural evidence of excitation-inhibition synapses in shafts and spines, and DLC in association with synapses that coincide with mitochondrial dynamic in their contribution to respiratory plasticity in the pre-BötC.

The kinesin superfamily protein KIF17 is regulated by the same transcription factor (NRF-1) as its cargo NR2B in neurons.

The kinesin superfamily of motor proteins is known to be ATP-dependent transporters of various types of cargoes. In neurons, KIF17 is found to transport vesicles containing the N-methyl-D-aspartate receptor NR2B subunit from the cell body specifically to the dendrites. These subunits are intimately associated with glutamatergic neurotransmission as well as with learning and memory. Glutamatergic synapses are highly energy-dependent, and recently we found that the same transcription factor, nuclear respiratory factor 1 (NRF-1), co-regulates energy metabolism (via its regulation of cytochrome c oxidase and other mitochondrial enzymes) and neurochemicals of glutamatergic transmission (NR1, NR2B, GluR2, and nNOS). The present study tested our hypothesis that NRF-1 also transcriptionally regulates KIF17. By means of in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation assays, promoter mutations, and real-time quantitative PCR, we found that NRF-1 (but not NRF-2) functionally regulates Kif17, but not Kif1a, gene. NRF-1 binding sites on Kif17 gene are highly conserved among mice, rats, and humans. Silencing of NRF-1 with small interference RNA blocked the up-regulation of Kif17 mRNA and proteins (and of Grin1 and Grin2b) induced by KCl-mediated depolarization, whereas over-expressing NRF-1 rescued these transcripts and proteins from being suppressed by TTX. Thus, NRF-1 co-regulates oxidative enzymes that generate energy and neurochemicals that consume energy related to glutamatergic neurotransmission, such as KIF17, NR1, and NR2B, thereby ensuring that energy production matches energy utilization at the molecular and cellular levels.

Synaptic relationship between somatostatin- and neurokinin-1 receptor-immunoreactive neurons in the pre-Bötzinger complex of rats.

The pre-Bötzinger complex (pre-BötC) in the ventrolateral medulla oblongata is critical for the generation of respiratory rhythm in mammals. Somatostatin (SST) and neurokinin 1 receptor (NK1R) immunoreactivity have been used as markers of the pre-BötC. SST immunoreactivity almost completely overlaps with small fusiform NK1R-immunoreactive (ir) neurons, the presumed rhythmogenic neurons, but not with large multipolar NK1R-ir neurons. Understanding the neurochemical characteristics, especially the synaptic relationship of SST/NK1R-ir neurons within the pre-BötC network is essential in providing cellular and structural bases for understanding their physiological significance. This work has not been documented so far. We found that SST immunoreactivity was highly expressed in terminals, somas, and primary dendrites in the pre-BötC. Besides the small fusiform neurons, a small population of medium-sized NK1R-ir neurons also colocalized with SST. Large NK1R-ir neurons were not SST-ir, but received somatostatinergic inputs. SST-ir terminals were glutamatergic or GABAergic, and synapsed with NK1R-ir neurons. Most of synapses between them were of the symmetric type, indicating their inhibitory nature. Asymmetric synapses were evident between SST-ir terminals and NK1R-ir dendrites, strongly suggesting an excitatory innervation from the presumed rhythmogenic neurons as these neurons are glutamatergic. We speculate that SST-mediated excitatory and inhibitory synaptic transmission onto NK1R-ir rhythmogenic and follower neurons synchronizes their activity to contribute to respiratory rhythmogenesis and control.

Bigenomic regulation of cytochrome c oxidase in neurons and the tight coupling between neuronal activity and energy metabolism.

Cytochrome c oxidase is the terminal enzyme of the mitochondrial electron transport chain, without which oxidative metabolism cannot be carried to completion. It is one of only four unique, bigenomic proteins in mammalian cells. The holoenzyme is made up of three mitochondrial-encoded and ten nuclear-encoded subunits in a 1:1 stoichiometry. The ten nuclear subunit genes are located in nine different chromosomes. The coordinated regulation of such a multisubunit, multichromosomal, bigenomic enzyme poses a challenge. It is especially so for neurons, whose mitochondria are widely distributed in extensive dendritic and axonal processes, resulting in the separation of the mitochondrial from the nuclear genome by great distances. Neuronal activity dictates COX activity that reflects protein amount, which, in turn, is regulated at the transcriptional level. All 13 COX transcripts are up- and downregulated by neuronal activity. The ten nuclear COX transcripts and those for Tfam and Tfbms important for mitochondrial COX transcripts are transcribed in the same transcription factory. Bigenomic regulation of all 13 transcripts is mediated by nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2). NRF-1, in addition, also regulates critical neurochemicals of glutamatergic synaptic transmission, thereby ensuring the tight coupling of energy metabolism and neuronal activity at the molecular level in neurons.

Excitatory-inhibitory imbalance in hypoglossal neurons during the critical period of postnatal development in the rat.

Hypoglossal motoneurons (HMs) innervate tongue muscles and are critical in maintaining patency of the upper airway during respiration. Abnormalities in HMs have been implicated in sudden infant death syndrome (SIDS) and obstructive sleep apnoea. Previously, we found a critical period in respiratory network development in rats around postnatal day (P) 12-13, when abrupt neurochemical, metabolic and physiological changes occurred. To test our hypothesis that an imbalance between inhibitory and excitatory synaptic transmission exists during the critical period, whole-cell patch-clamp recordings of HMs were done in brainstem slices of rats daily from P0 to P16. The results indicated that: (1) the amplitude and charge transfer of miniature excitatory postsynaptic currents (mEPSCs) were significantly reduced at P12-13; (2) the amplitude, mean frequency and charge transfer of miniature inhibitory postsynaptic currents (mIPSCs) were significantly increased at P12-13; (3) the kinetics (rise time and decay time) of both mEPSCs and mIPSCs accelerated with age; (4) the amplitude and frequency of spontaneous EPSCs were significantly reduced at P12-13, whereas those of spontaneous IPSCs were significantly increased at P12-13; and (5) both glycine and GABA contributed to mIPSCs. However, GABAergic currents fluctuated within a narrow range during the first three postnatal weeks, whereas glycinergic ones exhibited age-dependent changes comparable to those of total mIPSCs, indicating a reversal in dominance from GABA to glycine with development. Thus, our results provide strong electrophysiological evidence for an excitatory-inhibitory imbalance in HMs during the critical period of postnatal development in rats that may have significant implications for SIDS.