Postnatal development of Na(+)-K(+)-2Cl(-) co-transporter 1 and K(+)-Cl(-) co-transporter 2 immunoreactivity in multiple brain stem respiratory nuclei of the rat.

Previously, we reported that in rats, GABA(A) and glycine receptor immunoreactivity increased markedly in multiple brain stem respiratory nuclei around postnatal days (P) 12-13, a critical period when abrupt neurochemical, metabolic, ventilatory, and electrophysiological changes occur in the respiratory network and when the system is under greater inhibition than excitation. Since Na(+)-K(+)-2Cl(-) co-transporter 1 (NKCC1) and K(+)-Cl(-) co-transporter 2 (KCC2) play pivotal roles in determining the responses of GABA(A) and glycine receptors, we hypothesized that NKCC1 and KCC2 undergo significant changes during the critical period. An in-depth immunohistochemical and single neuron optical densitometric study of neurons in seven respiratory-related nuclei (the pre-Bötzinger complex [PBC], nucleus ambiguus [Amb], hypoglossal nucleus [XII], ventrolateral subnucleus of solitary tract nucleus [NTS(VL)], retrotrapezoid nucleus/parafacial respiratory group [retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG)], dorsal motor nucleus of the vagus nerve [dorsal motor nucleus of the vagus nerve (DMNX)], and inferior olivary nucleus [IO]) and a non-respiratory cuneate nucleus (CN, an internal control) was undertaken in P0-P21 rats. Our data revealed that (1) NKCC1 immunoreactivity exhibited a developmental decrease from P0 to P21 in all eight nuclei examined, being relatively high during the first 1½ postnatal weeks and decreased thereafter. The decrease was abrupt and statistically significant at P12 in the PBC, Amb, and XII; (2) KCC2 immunoreactivity in these eight nuclei showed a developmental increase from P0 to P21; and (3) the significant reduction in NKCC1 and the greater dominance of KCC2 around P12 in multiple respiratory nuclei of the brain stem may form the basis of an enhanced inhibition in the respiratory network during the critical period before the system stabilizes to a more mature state.

Postnatal development of N-methyl-D-aspartate receptor subunits 2A, 2B, 2C, 2D, and 3B immunoreactivity in brain stem respiratory nuclei of the rat.

Previously, we reported that a critical period in respiratory network development exists in rats around postnatal days (P; P12-P13), when abrupt neurochemical, metabolic, and physiological changes occur. Specifically, the expressions of glutamate and N-methyl-d-aspartate (NMDA) receptor (NR) subunit 1 in the pre-Bötzinger complex (PBC), nucleus ambiguus (Amb), hypoglossal nucleus (XII), and ventrolateral subnucleus of solitary tract nucleus (NTS(VL)) were significantly reduced at P12. To test our hypothesis that other NR subunits also undergo postnatal changes, we undertook an in-depth immunohistochemical study of NR2A, 2B, 2C, 2D, and 3B in these four respiratory nuclei in P2-P21 rats, using the non-respiratory cuneate nucleus (CN) as a control. Our results revealed that: (1) NR2A expression increased gradually from P2 to P11, but fell significantly at P12 in all four respiratory nuclei (but not in the CN), followed by a quick rise and a relative plateau until P21; (2) NR2B expression remained relatively constant from P2 to P21 in all five nuclei examined; (3) NR2C expression had an initial rise from P2 to P3, but remained relatively constant thereafter until P21, except for a significant fall at P12 in the PBC; (4) NR2D expression fell significantly from P2 to P3, then plateaued until P12, and declined again until P21; and (5) in contrast to NR2D, NR3B expression rose gradually from P2 to P21. These patterns reflect a dynamic remodeling of NMDA receptor subunit composition during postnatal development, with a distinct reduction of NR2A expression during the critical period (P12), just as NR1 did in various respiratory nuclei. There was also a potential switch between the neonatal NR2D and the more mature NR3B subunit, possibly around the critical period. Thus, during the critical period, NMDA receptors are undergoing greater adjustments that may contribute to attenuated excitatory synaptic transmission in the respiratory network.

Expressions of 5-HT/5-HT(2A) receptors and phospho-protein kinase C theta in the pre-Bötzinger complex in normal and chronic intermittent hypoxic rats.

The pre-Bötzinger complex (pre-BötC), a functionally defined subregion in the ventrolateral medulla oblongata, is a presumed kernel of normal respiratory rhythmogenesis. However, less is known about the pre-BötC's contribution to respiratory neuroplasticity. The most frequently studied model for respiratory neuroplasticity is episodic hypoxia-induced phrenic long-term facilitation, which is 5-HT(2A) receptors (5-HT(2A)R)-dependent. We hypothesized that preconditioning with chronic intermittent hypoxic (CIH) would activate the 5-HT/5-HT(2A)R system and the downstream protein kinase C (PKC) pathway in the pre-BötC. Animals were exposed to alternating 5 min of hypobaric hypoxia and 5 min of normoxia for 10 h/day for 7 days. Hypobaric hypoxia was achieved by continuous air evacuation to reach a pressure of 210-220 mm Hg, corresponding to an altitude of 9000-10000 m. In contrast to the CIH model, a group of animals were pretreated with chronic sustaining hypoxia (CSH), a protocol of continuous hypobaric hypoxia at 360 mm Hg, corresponding to an altitude of about 6000 m, for 10 h/day for 7 days. Immunoreactivity of 5-HT and 5-HT(2A)R was examined in the pre-BötC, identified by the presence of neurokinin-1 receptor (NK1R). We found that 15.5% of 5-HT-immunoreactive (ir) terminals were in contact with NK1R-ir neurons. Asymmetric synapses could be identified between them. 38.7% of NK1R-ir dendrites were also immunoreactive for 5-HT(2A)R, which was distributed along the inner surface of the plasma membrane in control animals. CIH challenge increased the expressions of 5-HT and 5-HT(2A)R in the pre-BötC, an increase in the expressed 5-HT(2A)R that was not detected in this region in CSH animals. Specifically, 5-HT(2A)R was distributed not only along the inner surface, but also along the outer surface, or directly on the plasma membrane, a pattern not detectable in control animals. 5-HT(2A)R was also detectable in the invaginations of the plasma membrane, where receptor endocytosis or exocytosis might occur, indicating CIH-induced higher trafficking of 5-HT(2A)R. Concurrently, there was an up-regulation of phospho-PKC theta (P-PKCtheta) in the pre-BötC, suggesting a 5-HT/5-HT(2A)R-activated PKC mechanism that may contribute to hypoxia-induced respiratory neuroplasticity in the pre-BötC. The close association of P-PKCtheta with the postsynaptic density implicates a postsynaptic mechanism mediating respiratory neuroplasticity in the pre-BötC.

Postnatal changes in the expressions of serotonin 1A, 1B, and 2A receptors in ten brain stem nuclei of the rat: implication for a sensitive period.

A critical period in respiratory network development occurs in the rat around postnatal days (P) 12-13, when abrupt neurochemical, metabolic, and physiological changes were evident. As serotonin and its receptors are involved in respiratory modulation, and serotonergic abnormality is implicated in sudden infant death syndrome, we hypothesized that 5-HT receptors are significantly downregulated during the critical period. This was documented recently for 5-HT(2A)R in several respiratory nuclei. The present study represents a comprehensive analysis of postnatal development of 5-HT(1A)R and 5-HT(1B)R in 10 brain stem nuclei and 5-HT(2A)R in six nuclei not previously examined. Optical densitometric analysis of immunohistochemically-reacted neurons from P2 to P21 indicated four developmental patterns of expression: (1) Pattern I: a high level of expression at P2-P11, an abrupt and significant reduction at P12, followed by a plateau until P21 (5-HT(1A)R and 5-HT(1B)R in raphé magnus [RM], raphé obscurus [ROb], raphé pallidus [RP], pre-Bötzinger complex [PBC], nucleus ambiguus [Amb], and hypoglossal nucleus [XII; 5-HT(1A)R only]). (2) Pattern II: a high level at P2-P9, a gradual decline from P9 to P12, followed by a plateau until P21 (5-HT(1A)R and 5-HT(1B)R in the retrotrapezoid nucleus (RTN)/parafacial respiratory group (pFRG)). (3) Pattern III: a high level at P2-P11, followed by a gradual decline until P21 (5-HT(1A)R in the ventrolateral subnucleus of solitary tract nucleus [NTS(VL)] and the non-respiratory cuneate nucleus [CN]). (4) Pattern IV: a relatively constant level maintained from P2 to P21 (5-HT(1A)R in the commissural subnucleus of solitary tract nucleus (NTS(COM)); 5-HT(1B)R in XII, NTS(VL), NTS(COM), and CN; and 5-HT(2A)R in RM, ROb, RP, RTN/pFRG, NTS(VL), and NTS(COM)). Thus, a significant reduction in the expression of 5-HT(1A)R, 5-HT(1B)R, and 5-HT(2A)R in multiple respiratory-related nuclei at P12 is consistent with reduced serotonergic transmission during the critical period, thereby rendering the animals less able to respond adequately to ventilatory distress.

Near-infrared light via light-emitting diode treatment is therapeutic against rotenone- and 1-methyl-4-phenylpyridinium ion-induced neurotoxicity.

Parkinson's disease is a common progressive neurodegenerative disorder characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta. Mitochondrial dysfunction has been strongly implicated in the pathogenesis of Parkinson's disease. Thus, therapeutic approaches that improve mitochondrial function may prove to be beneficial. Previously, we have documented that near-infrared light via light-emitting diode (LED) treatment was therapeutic to neurons functionally inactivated by tetrodotoxin, potassium cyanide (KCN), or methanol intoxication, and LED pretreatment rescued neurons from KCN-induced apoptotic cell death. The current study tested our hypothesis that LED treatment can protect neurons from both rotenone- and MPP(+)-induced neurotoxicity. Primary cultures of postnatal rat striatal and cortical neurons served as models, and the optimal frequency of LED treatment per day was also determined. Results indicated that LED treatments twice a day significantly increased cellular adenosine triphosphate content, decreased the number of neurons undergoing cell death, and significantly reduced the expressions of reactive oxygen species and reactive nitrogen species in rotenone- or MPP(+)-exposed neurons as compared with untreated ones. These results strongly suggest that LED treatment may be therapeutic to neurons damaged by neurotoxins linked to Parkinson's disease by energizing the cells and increasing their viability.

Activity-dependent transcriptional regulation of nuclear respiratory factor-1 in cultured rat visual cortical neurons.

Nuclear respiratory factor 1 is a transcription factor involved in the regulation of mitochondrial biogenesis by activating the transcription of subunit genes of cytochrome oxidase and other respiratory enzymes. Very little is known of its role in neurons. To determine if neuronal activity regulates nuclear respiratory factor 1 expression, cultured primary neurons from postnatal rat visual cortex were subjected to 20 mM KCl depolarizing treatment for 1, 3, 5, and 7 h, or exposed to 7 h of KCl followed by withdrawal for 1, 3, 5, and 7 h. Nuclear respiratory factor 1 expression was analyzed by immunoblots, immunocytochemistry, quantitative electron microscopy, real-time quantitative PCR, and in situ hybridization. Nuclear respiratory factor 1 protein was expressed at relatively low basal levels in both the nucleus, where it was associated primarily with euchromatin, and in the cytoplasm, where it was localized to free ribosomes and occasionally to the Golgi apparatus and the outer nuclear membrane. Depolarizing treatment progressively up-regulated both nuclear respiratory factor 1 protein and mRNA in a time-dependent manner, increasing above controls after 1 h and remaining high at 3, 5, and 7 h. Both nuclear and cytoplasmic mRNA levels increased with stimulation, and there was an apparent cytoplasmic-to-nuclear translocation of protein. Following the withdrawal of KCl, both nuclear respiratory factor 1 message and protein were significantly reduced after 1 h. The message returned to basal levels by 5 h and the protein by 7 h. These results strongly indicate that the expression and compartmental redistribution of nuclear respiratory factor 1 protein and mRNA in visual cortical neurons are dynamic processes tightly controlled by neuronal activity.

Bigenomic functional regulation of all 13 cytochrome c oxidase subunit transcripts in rat neurons in vitro and in vivo.

Cytochrome c oxidase is a multisubunit, bigenomically encoded inner mitochondrial membrane protein. Its enzymatic activity and amount in the brain vary with metabolic demands, but the precise regulation of all 13 subunits to form a functional holoenzyme in a 1:1 stoichiometry is not well understood. To determine if all 13 subunit transcripts were coordinately regulated by functional alteration in neurons, cultured primary neurons were depolarized by potassium chloride for 5-24 h, or tetrodotoxin inactivated for 2-6 days. In vivo studies were done on rats monocularly enucleated for 4 days to 2 weeks. Expressions of cytochrome c oxidase subunit mRNAs were measured by real-time quantitative polymerase chain reaction. Results showed that in vitro, all 13 transcripts were significantly up-regulated after 5 h of depolarizing stimulation. With tetrodotoxin blockade, however, the three mitochondrial-encoded transcripts were down-regulated earlier than the 10 nuclear ones (2 days versus 4 days). In vivo, all three mitochondrial-encoded subunit mRNAs were also down-regulated earlier than the nuclear ones in deprived visual cortex (4 days versus 1 week after monocular enucleation). Cytochrome c oxidase activity and protein levels were significantly decreased in parallel after 4 days of deprivation in vitro and 1 week in vivo. Our results are consistent with a coordinated mechanism of up-regulation of all 13 transcripts in response to functional stimulation, but an earlier and more severe down-regulation of the mitochondrial transcripts than the nuclear ones in response to functional deprivation. Thus, the mitochondrial subunits may play a more important role in regulating cytochrome c oxidase protein amount and activity in neurons. Our results also point to the need of all 13 subunits to form a functional holoenzyme.

Photobiomodulation partially rescues visual cortical neurons from cyanide-induced apoptosis.

Near-infrared light via light-emitting diode treatment has documented therapeutic effects on neurons functionally inactivated by tetrodotoxin or methanol intoxication. Light-emitting diode pretreatment also reduced potassium cyanide-induced cell death, but the mode of death via the apoptotic or necrotic pathway was unclear. The current study tested our hypothesis that light-emitting diode rescues neurons from apoptotic cell death. Primary neuronal cultures from postnatal rat visual cortex were pretreated with light-emitting diode for 10 min at a total energy density of 30 J/cm2 before exposing to potassium cyanide for 28 h. With 100 or 300 microM potassium cyanide, neurons died mainly via the apoptotic pathway, as confirmed by electron microscopy, Hoechst 33258, single-stranded DNA, Bax, and active caspase-3. In the presence of caspase inhibitor I, the percentage of apoptotic cells in 300microM potassium cyanide was significantly decreased. Light-emitting diode pretreatment reduced apoptosis from 36% to 17.9% (100 microM potassium cyanide) and from 58.9% to 39.6% (300 microM potassium cyanide), representing a 50.3% and 32.8% reduction, respectively. Light-emitting diode pretreatment significantly decreased the expression of caspase-3 elicited by potassium cyanide. It also reversed the potassium cyanide-induced increased expression of Bax and decreased expression of Bcl-2 to control levels. Moreover, light-emitting diode decreased the intensity of 5-(and -6) chloromethy-2', 7-dichlorodihydrofluorescein diacetate acetyl ester, a marker of reactive oxygen species, in neurons exposed to 300 microM potassium cyanide. These results indicate that light-emitting diode pretreatment partially protects neurons against cyanide-induced caspase-mediated apoptosis, most likely by decreasing reactive oxygen species production, down-regulating pro-apoptotic proteins and activating anti-apoptotic proteins, as well as increasing energy metabolism in neurons as reported previously.

Therapeutic photobiomodulation for methanol-induced retinal toxicity.

Methanol intoxication produces toxic injury to the retina and optic nerve, resulting in blindness. The toxic metabolite in methanol intoxication is formic acid, a mitochondrial toxin known to inhibit the essential mitochondrial enzyme, cytochrome oxidase. Photobiomodulation by red to near-IR radiation has been demonstrated to enhance mitochondrial activity and promote cell survival in vitro by stimulation of cytochrome oxidase activity. The present studies were undertaken to test the hypothesis that exposure to monochromatic red radiation from light-emitting diode (LED) arrays would protect the retina against the toxic actions of methanol-derived formic acid in a rodent model of methanol toxicity. Using the electroretinogram as a sensitive indicator of retinal function, we demonstrated that three brief (2 min, 24 s) 670-nm LED treatments (4 J/cm(2)), delivered at 5, 25, and 50 h of methanol intoxication, attenuated the retinotoxic effects of methanol-derived formate. Our studies document a significant recovery of rod- and cone-mediated function in LED-treated, methanol-intoxicated rats. We further show that LED treatment protected the retina from the histopathologic changes induced by methanol-derived formate. These findings provide a link between the actions of monochromatic red to near-IR light on mitochondrial oxidative metabolism in vitro and retinoprotection in vivo. They also suggest that photobiomodulation may enhance recovery from retinal injury and other ocular diseases in which mitochondrial dysfunction is postulated to play a role.

Increase in cytochrome oxidase activity in regenerating nerve fibers of hemitransected spinal cord in the rat.

We explored the possibility of cytochrome oxidase (CO) involvement in spinal cord regeneration in adult rats. The spinal cord was hemitransected at T9. After one month's survival, the animals were deeply anesthetized and perfused. The spinal cord segments including the lesion site were removed and sectioned horizontally for CO histochemistry. Under light microscope, a substantial number of CO-reactive nerve fibers and boutons were identified in the lateral funiculus adjacent to the lesion site. Under electron microscope, moderately to highly CO-reactive mitochondria could be seen within nerve fibers and boutons. Synaptic contacts were identified among them. The increase in CO activity in nerve fibers and boutons may indicate their high-energy demand for synaptic and spontaneous activity following spinal cord hemisection.

Light-emitting diode treatment reverses the effect of TTX on cytochrome oxidase in neurons.

Light close to and in the near-infrared range has documented benefits for promoting wound healing in human and animals. However, mechanisms of its action on cells are poorly understood. We hypothesized that light treatment with a light-emitting diode array at 670 nm (LED) is therapeutic in stimulating cellular events involving increases in cytochrome oxidase activity. LED was administered to cultured primary neurons whose voltage-dependent sodium channels were blocked by tetrodotoxin. The down-regulation of cytochrome oxidase activity by TTX was reverted to control levels by LED. LED alone also up-regulated enzyme activity. Thus, the results are consistent with our hypothesis that LED has a stimulating effect on cytochrome oxidase in neurons, even when they have been functionally silenced by TTX.

Distribution and colocalization of neurotransmitters and receptors in the pre-Bötzinger complex of rats.

The pre-Bötzinger complex (PBC), thought to be the center of respiratory rhythm generation, is a cell column ventrolateral to the nucleus ambiguus. The present study analyzed its cellular and neurochemical composition in adult rats. PBC neurons were mainly oval, fusiform, or multipolar in shape and small to medium in size. Neurokinin-1 receptor, a marker of the PBC, was present in the plasma membrane of mostly medium and small neurons and their associated processes and boutons. Among neurons immunoreactive for different neurotransmitter or receptor candidates, various numbers were colocalized with neurokinin-1 receptor. The highest ratio was with nitric oxide synthase (52.72%), and the lowest was with glycine receptors (31.93%). Glutamic acid decarboxylase- and glycine transporter 2-immunoreactive boutons, as well as GABA(A) receptor-immunoreactive plasma membrane processes and boutons, were also identified in the PBC. PBC neurons exhibited different levels of cytochrome oxidase activity, indicating their various energy demands. Our results suggest that synaptic interactions within the PBC of adult rats involve a variety of neurotransmitter and receptor types and that nitric oxide may play an important role in addition to glutamate, GABA, glycine, and neurokinin.

Developmental study of cytochrome oxidase activity in the brain stem respiratory nuclei of postnatal rats.

We utilized cytochrome oxidase (CO) as a marker of neuronal functional activity to examine metabolic changes in brain stem respiratory nuclei of rats from newborn to 21 day of age. The pre-Bötzinger complex (PBC), upper airway motoneurons of nucleus ambiguus (NA(UAM)), ventrolateral nucleus of solitary tract (NTS(VL)), and medial and lateral parabrachial nuclei (PB(M) and PB(L), respectively) were examined at postnatal days (P) 0, 1, 2, 3, 4, 5, 7, 14, and 21. CO histochemistry was performed, and the intensity of CO reaction product was quantitatively analyzed by optical densitometry. In addition, CO histochemistry was combined with neurokinin-1 receptor (NK1R) immunogold-silver staining to doubly label neurons of PBC in P14 animals. The results showed that levels of CO activity generally increased with age in all of the nuclei examined. However, a significant decrease was found in NA(UAM) at P3 (P < 0.01), and a distinct plateau of CO activity was noted at P3 in PBC and at P3 and P4 in NTS(VL), PB(M), and PB(L). Of the neurons examined in PBC, 83% were doubly labeled with CO and NK1R. Of these, CO activity was high in 33.9%, moderate in 27.3%, and light in 38.8% of neurons, suggesting different energy demands in these metabolic groups that may be related to their physiological or synaptic properties. The transient decrease or plateau in CO activity at P3 and P4 implies a period of synaptic adjustment or reorganization during development, when there may be decreased excitatory synaptic drive or increased inhibitory synaptic drive, or both, in these brain stem respiratory nuclei. The adjustment, in turn, may render the system less responsive to respiratory insults. This may bear some relevance to our understanding of pathological events during postnatal development, such as occurs in sudden infant death syndrome.

Synthesis and degradation of cytochrome oxidase subunit mRNAs in neurons: differential bigenomic regulation by neuronal activity.

Cytochrome oxidase (CO) plays a key role in oxidative capacity of neurons and serves as a sensitive indicator of neuronal activity. The mechanism(s) involved in the regulation of this bigenomic-encoded mitochondrial enzyme is still not clearly understood. Previous studies have shown changes in the level of its subunit mRNAs encoded by the nuclear or mitochondrial genome in response to changing neuronal activity. Our goals in the present study were to determine whether such changes were due to RNA synthesis rate or stability or both. The level of CO activity of neurons in primary cultures assayed histochemically was increased after depolarizing KCl treatment. The steady-state levels of CO subunit II (CO II; mitochondrial-encoded) and IV (CO IV; nuclear-encoded) mRNAs were up-regulated in response to 5 hr of 20 mM KCl treatment. By using gene-specific probes, the relative rates of synthesis of CO II and IV mRNA were elevated significantly after KCl treatment (P < 0.05). The degradation of CO II and IV mRNAs was monitored by (3)H-uridine pulse-chase labeling, which revealed half-lives of 84 min for CO II mRNA and 50 min for CO IV mRNA. Under KCl treatment, the half-life of CO IV was increased to 102 min, but there was no statistically significant change in the half-life of CO II mRNA. These results indicate that mitochondrial subunit CO II mRNA is regulated mainly at the transcriptional level, whereas the nuclear subunit CO IV mRNA is regulated at both the synthetic and the degradative levels. Both subunits, however, are tightly governed by neuronal activity.

Depolarizing stimulation upregulates GA-binding protein in neurons: a transcription factor involved in the bigenomic expression of cytochrome oxidase subunits.

Neurons are unique in having dendrites that extend far away from their cell bodies. Mitochondria located in the dendrites can be separated from the nucleus for long distances. The mechanism of bigenomic coordination is of particular importance to cytochrome oxidase (CO), which has subunits that are encoded in both the nuclear and mitochondrial DNA. GA-binding protein (GABP) is a transcription factor that is required for the promoter activity of mitochondrial transcription factor A as well as several nuclear-encoded CO subunits. Thus, GABP may play a key role in coordinating the transcription of both mitochondrial and nuclear-encoded subunits of CO. The goal of the present study was to determine if GABP was expressed in neurons and whether and how it responded to increased neuronal activity. Using primary neuronal cultures, the beta-subunit of GABP was localized immunocytochemically to both the cytoplasm and the nucleus, whereas the alpha-subunit was expressed mainly in the nucleus. In KCl-treated cultures, immunoreactivity for both alpha- and beta-subunits was significantly increased in the nucleus compared with untreated sister cultures. The induction of GABP preceded that of CO gene expression from the two genomes, which, in turn, preceded that of CO activity. Thus, our data suggest that neuronal activity regulates subunit concentrations of GABP in the nucleus, and GABP may be a critical sensor of changes in neuronal activity. Our data are also consistent with the postulated role of GABP as a coordinator of both mitochondrial and nuclear transcription for subunits of CO in neurons.

Human COX6A1 gene: promoter analysis, cDNA isolation and expression in the monkey brain.

The human COX6A1 gene encodes the ubiquitous isoform of cytochrome c oxidase (COX) subunit VIa (VIa-L), and is located in a CpG island on chromosome 12q24.2. We compared the COX6A1 gene with the published cDNA and several ESTs and concluded that subunit COX VIa-L is synthesized as a preprotein, as are other COX subunits. The same transcription start sites were identified by primer extension analysis of human brain and lymphoblastoid RNA. Analysis of the COX6A1 promoter revealed several conserved sequence elements found in other COX genes, namely binding sites for nuclear respiratory factor 1 (NRF-1), nuclear respiratory factor 2/GA binding protein (NRF-2/GABP), and ying-yang protein 1 (YY1). These conserved elements were shown to bind nuclear proteins from HeLa nuclear extracts. COX6A1 cDNA was isolated from a human brain cDNA library, and the sequence was identical to that of human liver. The expression of this gene was demonstrated by in-situ hybridization in monkey brain sections with our human brain cDNA. Monocular impulse blockade in adult monkeys induced a downregulation of COX6A1 expression in deprived visual neurons, suggesting that this subunit gene is regulated by neuronal activity.

Effects of hindlimb unloading on neuromuscular development of neonatal rats.

We hypothesized that hindlimb suspension unloading of 8-day-old neonatal rats would disrupt the normal development of muscle fiber types and the motor innervation of the antigravity (weightbearing) soleus muscles but not extensor digitorum longus (EDL) muscles. Five rats were suspended 4.5 h and returned 1.5 h to the dam for nursing on a 24 h cycle for 9 days. To control for isolation from the dam, the remaining five littermates were removed on the same schedule but not suspended. Another litter of 10 rats housed in the same room provided a vivarium control. Fibers were typed by myofibrillar ATPase histochemistry and immunostaining for embryonic, slow, fast IIA and fast IIB isomyosins. The percentage of multiple innervation and the complexity of singly-innervated motor terminal endings were assessed in silver/cholinesterase stained sections. Unique to the soleus, unloading accelerated production of fast IIA myosin, delayed expression of slow myosin and retarded increases in standardized muscle weight and fiber size. Loss of multiple innervation was not delayed. However, fewer than normal motor nerve endings achieved complexity. Suspended rats continued unloaded hindlimb movements. These findings suggest that motor neurons resolve multiple innervation through nerve impulse activity, whereas the postsynaptic element (muscle fiber) controls endplate size, which regulates motor terminal arborization. Unexpectedly, in the EDL of unloaded rats, transition from embryonic to fast myosin expression was retarded. Suspension-related foot drop, which stretches and chronically loads EDL, may have prevented fast fiber differentiation. These results demonstrate that neuromuscular development of both weightbearing and non-weightbearing muscles in rats is dependent upon and modulated by hindlimb loading.

Human nuclear respiratory factor 2 alpha subunit cDNA: isolation, subcloning, sequencing, and in situ hybridization of transcripts in normal and monocularly deprived macaque visual system.

Nuclear respiratory factor 2 (NRF-2) has been shown to contribute to the transcriptional regulation of a number of subunits of respiratory chain enzymes, including cytochrome c oxidase (CO). Our recent study demonstrated a parallel distribution of the alpha subunit proteins of NRF-2 (NRF-2 alpha) with CO in the monkey striate cortex, and that it can be regulated by neuronal activity. To determine whether this regulation is at the transcriptional level, the present study examined the expression of NRF-2 alpha mRNA in normal and monocularly deprived adult monkeys. A partial NRF-2 alpha cDNA was isolated from a human brain cDNA library. Sequence analysis revealed that it shared 99% identity with the published sequence from human HeLa cells. Riboprobes of NRF-2 alpha was generated and labeled with digoxigenin-11-UTP for in situ hybridization. The expression pattern of NRF-2 alpha mRNA in the normal striate cortex paralleled that of CO activity. It was highly expressed in layers IVC and VI, which contained high levels of CO, and more densely expressed in puffs of layers II and III than in interpuffs. In monkeys monocularly treated with tetrodotoxin for 1 day to 2 weeks, both NRF-2 alpha expression and CO activity were reduced in deprived ocular dominance columns of the visual cortex and in deprived layers of the lateral geniculate nucleus. These data indicate that, in the normal and visually deprived adult monkeys, NRF-2 alpha is regulated by neuronal activity at the transcriptional level.

Nuclear respiratory factor-2 subunit protein: correlation with cytochrome oxydase and regulation by functional activity in the monkey primary visual cortex.

Previous studies have shown that a transcription factor of the Ets family, nuclear respiratory factor 2 (NRF-2), can activate in vitro the gene expression of cytochrome oxidase (CO), a mitochondrial enzyme of oxidative metabolism. The goals of our present study were to determine whether the distribution of NRF-2 alpha subunit proteins correlated with that of CO activity in the macaque monkey visual cortex and whether the level could be perturbed by visual deprivation. We generated polyclonal antibodies specifically against human NRF-2 alpha subunit. In normal monkeys, patterns of NRF-2 alpha distribution resembled closely that of CO activity: 1) NRF-2 alpha immunoreactivity was localized in both nuclei and cytoplasm of neurons, but the levels differed among various laminae; 2) layers IVA, IVC, and VI, which had high CO activity, were labeled more densely by NRF-2 alpha than layers I, IVB, and V, which contained lower levels of both NRF-2 alpha and CO activity; and 3) CO-rich puffs in layers II and III contained a higher level of NRF-2 alpha than CO-poor interpuffs. From 1 day to 7 days after monocular impulse blockade with tetrodotoxin, there was a progressive reduction of NRF-2 alpha in deprived ocular dominance columns, in parallel with decreases in CO activity. These results suggest that local levels of NRF-2 in the monkey visual cortex closely reflect neuronal physiological and metabolic levels revealed by CO activity and that the expression of NRF-2 alpha, like that of CO, is regulated tightly by neural functional activity.

Expression and regulation of NMDA receptor subunit R1 and neuronal nitric oxide synthase in cortical neuronal cultures: correlation with cytochrome oxidase.

Our previous studies showed a differential distribution of the glutamatergic terminals in cytochrome oxidase-rich and -poor regions of the visual cortex. The NMDA type of glutamate receptors have been proposed to be involved in the activation of nitric oxide synthase to produce nitric oxide, the neurotransmitter. In the present study, we hypothesized that the expressions of glutamate receptor, NMDA receptors (NMDAR1) and neuronal nitric oxide synthase (nNOS) were colocalized and were also correlated with that of cytochrome oxidase (CO) in a subset of neurons. We used primary cultures of postnatal rat visual cortical neurons as a model system, so that we could examine both the somatic and dendritic expressions of these neurochemicals in individual neurons. We found a difference in the sequence of developmental expressions of NMDAR1, nNOS, CO, and Na(+)/K(+) ATPase. Triple labeling showed that all nNOS-positive neurons were immunoreactive for NMDAR1, and a subpopulation of them had high CO activity. The expression of NMDAR1 was positively correlated with CO activity. This is consistent with our previous finding that CO activity is strongly governed by excitatory glutamatergic synapses. After 40 hours of depolarizing potassium chloride treatment, CO activity was increased, and NMDAR1and nNOS levels were up-regulated in parallel. One week of tetrodotoxin significantly decreased the expression of NMDAR1, nNOS, and CO activity. Our results demonstrate that NMDA receptors and nNOS do co-exist in a subset of neurons that have high CO activity and their expressions are under the control of neuronal activity.