Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators'' choice.We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein ^1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA ^6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).     

Generation of the mitochondrial permeability transition does not involve inhibition of lysophospholipid acylation. Acyl-coenzyme A:1-acyllysophospholipid acyltransferase activity is not found in rat liver mitochondria

The possible involvement of acyl-coenzyme A:1-acyllysophospholipid acyltransferase activity and phospholipid acylation-deacylation cycles in regulating the mitochondrial permeability transition have been examined by direct methods. 1-Acyllysophospholipid acyltransferase activity found in mitochondrial preparations obtained by differential centrifugation is inhibited by several transition-inducing agents and by glutathione disulfide. However, marker enzyme analysis employing mitochondria prepared by Percoll density gradient centrifugation or fractionated by a shear force-dependent method indicate that this activity is associated with contaminating microsomes and not with mitochondria. The absence of phospholipid acylation-deacylation cycles in isolated mitochondria is demonstrated by the absence of 18O incorporation from H2(18)O into phospholipid acylester carbonyl groups, confirming conclusions arrived at from marker enzyme data by a definitive independent approach. Mitochondria prepared by differential centrifugation and Percoll density gradient centrifugation are shown to be equivalent in requirements for induction of the permeability transition and the apparent rate of this process. It is concluded that 1-acyllysophospholipid acyltransferase activity and phospholipid acylation-deacylation cycles are not factors regulating the transition in isolated mitochondria. However, mitochondrial phospholipase A2 activity remains as a potential regulating factor, whereas the action of transition-inducing agents on microsomal 1-acyllysophospholipid acyltransferase may be important in mechanisms of cell injury.     

Carotid body excision significantly changes ventilatory control in awake rats

We determined the effects of carotid body excision (CBX) on eupneic ventilation and the ventilatory responses to acute hypoxia, hyperoxia, and chronic hypoxia in unanesthetized rats. Arterial PCO2 (PaCO2) and calculated minute alveolar ventilation to minute metabolic CO2 production (VA/VCO2) ratio were used to determine the ventilatory responses. The effects of CBX and sham operation were compared with intact controls (PaCO2 = 40.0 +/- 0.1 Torr, mean +/- 95% confidence limits, and VA/VCO2 = 21.6 +/- 0.1). CBX rats showed 1) chronic hypoventilation with respiratory acidosis, which was maintained for at least 75 days after surgery (PaCO2 = 48.4 +/- 1.1 Torr and VA/VCO2 = 17.9 +/- 0.4), 2) hyperventilation in response to acute hyperoxia vs. hypoventilation in intact rats, 3) an attenuated increase in VA/VCO2 in acute hypoxemia (arterial PO2 approximately equal to 49 Torr), which was 31% of the 8.7 +/- 0.3 increase in VA/VCO2 observed in control rats, 4) no ventilatory acclimatization between 1 and 24 h hypoxia, whereas intact rats had a further 7.5 +/- 1.5 increase in VA/VCO2, 5) a decreased PaCO2 upon acute restoration of normoxia after 24 h hypoxia in contrast to an increased PaCO2 in controls. We conclude that in rats carotid body chemoreceptors are essential to maintain normal eupneic ventilation and to the process of ventilatory acclimatization to chronic hypoxia.     

Suppression of insertions in the complex pdxJ operon of Escherichia coli K-12 by lon and other mutations.

Complementation analyses using minimal recombinant clones showed that all known pdx point mutations, which cause pyridoxine (vitamin B6) or pyridoxal auxotrophy, are located in the pdxA, pdxB, serC, pdxJ, and pdxH genes. Antibiotic enrichments for chromosomal transposon mutants that require pyridoxine (vitamin B6) or pyridoxal led to the isolation of insertions in pdxA, pdxB, and pdxH but not in pdxJ. This observation suggested that pdxJ, like pdxA, pdxB, and serC, might be in a complex operon. To test this hypothesis, we constructed stable insertion mutations in and around pdxJ in plasmids and forced them into the bacterial chromosome. Physiological properties of the resulting insertion mutants were characterized, and the DNA sequence of pdxJ and adjacent regions was determined. These combined approaches led to the following conclusions: (i) pdxJ is the first gene in a two-gene operon that contains a gene, temporarily designated dpj, essential for Escherichia coli growth; (ii) expression of the rnc-era-recO and pdxJ-dpj operons can occur independently, although the pdxJ-dpj promoter may lie within recO; (iii) pdxJ encodes a 26,384-Da polypeptide whose coding region is preceded by a PDX box, and dpj probably encodes a basic, 14,052-Da polypeptide; (iv) mini-Mud insertions in dpj and pdxJ, which are polar on dpj, severely limit E. coli growth; and (v) three classes of suppressors, including mutations in lon and suppressors of lon, that allow faster growth of pdxJ::mini-Mud mutants can be isolated. A model to account for the action of dpj suppressors is presented, and aspects of this genetic analysis are related to the pyridoxal 5'-phosphate biosynthetic pathway.     

Oxygen cost of exercise hyperpnea: implications for performance.

We addressed two questions concerned with the metabolic cost and performance of respiratory muscles in healthy young subjects during exercise: 1) does exercise hyperpnea ever attain a "critical useful level"? and 2) is the work of breathing (WV) at maximum O2 uptake (VO2max) fatiguing to the respiratory muscles? During progressive exercise to maximum, we measured tidal expiratory flow-volume and transpulmonary pressure- (Ptp) volume loops. At rest, subjects mimicked their maximum and moderate exercise Ptp-volume loops, and we measured the O2 cost of the hyperpnea (VO2RM) and the length of time subjects could maintain reproduction of their maximum exercise loop. At maximum exercise, the O2 cost of ventilation (VE) averaged 10 +/- 0.7% of the VO2max. In subjects who used most of their maximum reserve for expiratory flow and for inspiratory muscle pressure development during maximum exercise, the VO2RM required 13-15% of VO2max. The O2 cost of increasing VE from one work rate to the next rose from 8% of the increase in total body VO2 (VO2T) during moderate exercise to 39 +/- 10% in the transition from heavy to maximum exercise; but in only one case of extreme hyperventilation, combined with a plateauing of the VO2T, did the increase in VO2RM equal the increase in VO2T. All subjects were able to voluntarily mimic maximum exercise WV for 3-10 times longer than the duration of the maximum exercise. We conclude that the O2 cost of exercise hyperpnea is a significant fraction of the total VO2max but is not sufficient to cause a critical level of "useful" hyperpnea to be achieved in healthy subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen cost of exercise hyperpnea: measurement.

To quantitate the O2 cost of maximal exercise hyperpnea, we required eight healthy adult subjects to mimic, at rest, the important mechanical components of submaximal and maximal exercise hyperpnea. Expired minute ventilation (VE), transpulmonary and transdiaphragmatic (Pdi) pressures, and end-expiratory lung volume (EELV) were measured during exercise at 70 and 100% of maximal O2 uptake. At rest, subjects were given visual feedback of their exercise transpulmonary pressure-tidal volume loop (WV), breathing frequency, and EELV, which they mimicked repeatedly for 5 min per trial over several trials, while hypocapnia was prevented. The change in total body O2 uptake (VO2) was measured and presumed to represent the O2 cost of the hyperpnea. In 61 mimicking trials with VE of 115-167 l/min and WV of 124-544 J/min, VE, WV, duty cycle of the breath, and expiratory gastric pressure (Pga) integrated with respect to time (integral of Pga.dt/min) were not different from those observed during maximum exercise. integral of Pdi.dt/min was 14% less and EELV was 6% greater during maximum exercise than during mimicking. The O2 cost measurements within a subject were reproducible over 3-12 trials (coefficient of variation +/- 10% range 5-16%). The O2 costs of hyperpnea correlated highly and positively with VE and WV and less, but significantly, with integral of Pdi.dt and integral of Pga.dt. The O2 cost of VE rose out of proportion to the increasing hyperpnea, so that between 70 and 100% of maximal VO2 delta VO2/delta VE increased 40-60% (1.8 +/- 0.2 to 2.9 +/- 0.1 ml O2/l VE) as VE doubled.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the progeny of pre-T cells maintained in vitro by IL-3: expression of the IL-2 receptor and CD3 during thymic development.

We have recently described a bone marrow culture system which is able to maintain, for at least 2 weeks, cells which have the capacity to repopulate the thymus of irradiated recipient mice (pre-T cells). Because this culture system depends upon the addition of an exogenous growth factor (IL-3) which may potentially influence the differentiation of the cultured pre-T cells, it is important to determine whether or not the progeny of cultured marrow cells are able to develop within the thymus in a kinetically normal fashion. Here we report the results of an analysis of the progeny of those cultured progenitor cells at 2, 3, and 4 weeks following intrathymic transfer. The passage of cultured donor-derived cells through critical early (expression of the IL-2 receptor) and late (expression of high levels of CD3) intrathymic events was assessed in these studies and compared with the pattern observed in the progeny of fresh bone marrow cells. The results of these studies showed that the progeny of cultured pre-T cells were able to develop expression of the IL-2 receptor and CD3 surface antigen during their residency within the thymus. In addition, both the timing and levels of expression of these surface markers were virtually identical on the progeny of fresh and cultured pre-T cells. These data suggest that cultured pre-T cells are not dramatically altered by their passage in vitro and are able to give rise to normally developing thymocytes upon in vivo transfer.     

Mechanical impedance as determinant of inspiratory neural drive during exercise in humans

Five healthy males exercised progressively with small 2-min increments in work load. We measured inspiratory drive (occlusion pressure, P0.1), pulmonary resistance (RL), dynamic pulmonary compliance (Cdyn), transdiaphragmatic pressure (Pdi), and diaphragmatic electromyogram (EMGdi). Minute ventilation (VE), mean inspiratory flow rate (VT/TI), and P0.1 all increased exponentially with increased work load, but P0.1 increased at a faster rate than did VT/TI or VE. Thus effective impedance (P0.1/VT/TI) rose throughout exercise. The increasing P0.1 was mostly due to augmented Pdi and coincided with increased EMGdi during this initial portion of inspiration. We found no consistent change in RL or Cdyn throughout exercise. With He breathing (80% He-20% O2), RL was reduced at all work loads; P0.1 fell in comparison with air-breathing values and VE, VT, and VT/TI rose in moderate and heavy work; and P0.1/VT/TI was unchanged with increasing exercise loads. Step reductions in gas density at a constant work load of any intensity showed an immediate reduction in the rate of rise of EMGdi and Pdi followed by increased VT/TI, breathing frequency, and hypocapnia. These changes were maintained during prolonged periods of unloading and were immediately reversible on return to air breathing. These data are consistent with the existence of a reflex effect on the magnitude of inspiratory neural drive during exercise that is sensitive to the load presented by the normal mechanical time constant of the respiratory system. This "load" is a significant determinant of the hyperpneic response and thus of the maintenance of normocapnia during exercise.     

Airway resistance and respiratory muscle function in snorers during NREM sleep

The effect of non-rapid-eye-movement (NREM) sleep on total pulmonary resistance (RL) and respiratory muscle function was determined in four snorers and four nonsnorers. RL at peak flow increased progressively from wakefulness through the stages of NREM sleep in all snorers (3.7 +/- 0.4 vs. 13.0 +/- 4.0 cmH2O X 0.1(-1) X s) and nonsnorers (4.8 +/- 0.4 vs. 7.5 +/- 1.1 cmH2O X 1(-1) X s). Snorers developed inspiratory flow limitation and progressive increase in RL within a breath. The increased RL placed an increased resistive load on the inspiratory muscles, increasing the pressure-time product for the diaphragm between wakefulness and NREM sleep. Tidal volume and minute ventilation decreased in all subjects. The three snorers who showed the greatest increase in within-breath RL demonstrated an increase in the contribution of the lateral rib cage to tidal volume, a contraction of the abdominal muscles during a substantial part of expiration, and an abrupt relaxation of abdominal muscles at the onset of inspiration. We concluded that the magnitude of increase in RL leads to dynamic compression of the upper airway during inspiration, marked distortion of the rib cage, recruitment of the intercostal muscles, and an increased contribution of expiratory muscles to inspiration. This increased RL acts as an internal resistive load that probably contributes to hypoventilation and CO2 retention in NREM sleep.     

The CDK4/6 Inhibitor Abemaciclib Induces a T Cell Inflamed Tumor Microenvironment and Enhances the Efficacy of PD-L1 Checkpoint Blockade

1. Download high-res image (224KB)
2. Download full-size image