Circadian glucocorticoid oscillations preserve a population of adult hippocampal neural stem cells in the aging brain.

A decrease in adult hippocampal neurogenesis has been linked to age-related cognitive impairment. However, the mechanisms involved in this age-related reduction remain elusive. Glucocorticoid hormones (GC) are important regulators of neural stem/precursor cells (NSPC) proliferation. GC are released from the adrenal glands in ultradian secretory pulses that generate characteristic circadian oscillations. Here, we investigated the hypothesis that GC oscillations prevent NSPC activation and preserve a quiescent NSPC pool in the aging hippocampus. We found that hippocampal NSPC populations lacking expression of the glucocorticoid receptor (GR) decayed exponentially with age, while GR-positive populations decayed linearly and predominated in the hippocampus from middle age onwards. Importantly, GC oscillations controlled NSPC activation and GR knockdown reactivated NSPC proliferation in aged mice. When modeled in primary hippocampal NSPC cultures, GC oscillations control cell cycle progression and induce specific genome-wide DNA methylation profiles. GC oscillations induced lasting changes in the methylation state of a group of gene promoters associated with cell cycle regulation and the canonical Wnt signaling pathway. Finally, in a mouse model of accelerated aging, we show that disruption of GC oscillations induces lasting changes in dendritic complexity, spine numbers and morphology of newborn granule neurons. Together, these results indicate that GC oscillations preserve a population of GR-expressing NSPC during aging, preventing their activation possibly by epigenetic programming through methylation of specific gene promoters. Our observations suggest a novel mechanism mediated by GC that controls NSPC proliferation and preserves a dormant NSPC pool, possibly contributing to a neuroplasticity reserve in the aging brain.

Adult-born dentate granule cell excitability depends on the interaction of neuron age, ontogenetic age and experience.

Early during their maturation, adult-born dentate granule cells (aDGCs) are particularly excitable, but eventually develop the electrophysiologically quiet properties of mature cells. However, the stability versus plasticity of this quiet state across time and experience remains unresolved. By birthdating two populations of aDGCs across different animal ages, we found for 10-month-old rats the expected reduction in excitability across cells aged 4-12 weeks, as determined by Egr1 immunoreactivity. Unexpectedly, cells 35 weeks old (after genesis at an animal age of 2 months) were as excitable as 4-week-old cells, in the dorsal hippocampus. This high level of excitability at maturity was specific for cells born in animals 2 months of age, as cells born later in life did not show this effect. Importantly, excitability states were not fixed once maturity was gained, but were enhanced by enriched environment exposure or LTP induction, indicating that any maturational decrease in excitability can be compensated by experience. These data reveal the importance of the animal's age for aDGC excitability, and emphasize their prolonged capability for plasticity during adulthood.

Serotonergic modulation of respiratory motoneurons and interneurons in brainstem slices of perinatal rats.

Respiration-related membrane potential fluctuations were recorded in hypoglossal (XII) motoneurons and pre-Bötzinger complex (pre-BötC) interneurons in medullary slices from perinatal rats. Bath application of serotonin (5-HT) evoked a ketanserine-sensitive depolarization (approximately 11 mV) and tonic spike discharge in XII motoneurons, whereas pre-BötC neurons responded with a <6 mV depolarization and no tonic discharge. The membrane effects were accompanied by an increase in respiratory frequency by up to 260% in 64% of preparations. A frequency decrease leading to block of respiratory activity could also occur (20%) as well as an initial acceleration that turned into a frequency depression (16%). In contrast, iontophoresis of 5-HT into the pre-BötC exclusively increased respiratory frequency by 30-220%, whereas iontophoresis into the XII nucleus did not change respiratory frequency but induced tonic nerve discharge. The effects of local iontophoretic administration of 5-HT on membrane properties of XII and pre-BötC cells were very similar to those upon bath application. Bath application and iontophoresis of the 5-HT2 receptor agonist -methyl-hydroxytryptamine mimicked the effects of 5-HT. Bath application of the 5-HT1A receptor agonist 8-hydroxydipropylaminotetralin hydrobromide did not affect XII nerve bursting or pre-BötC neurons. Iontophoresis of 8-hydroxydipropylaminotetralin hydrobromide had almost no effect on respiratory frequency and induced in the interneurons either a depolarization or hyperpolarization (<5 mV) which was blocked by the 5-HT1A receptor antagonist N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)N-2-pyridinylcyclohexane carboxamide. In conclusion, 5-HT-evoked tonic excitation of respiratory XII motoneurons is mediated by postsynaptic 5-HT2 receptors. The excitatory effects on respiratory rhythm are also primarily attributable to postsynaptic 5-HT2 receptors of pre-BötC neurons. Additional modulatory effects on the interneurons appear to be mediated by postsynaptic 5-HT1A receptors.

Subcellular vesicular aggregations of GABAB R1a and R1b receptors increase with age in neurons of the developing mouse brain.

GABA(B) receptors play a critical neuromodulatory role in the central nervous system. It has been suggested that both the functional role and the cellular distribution of GABA(B) receptors in the neuronal network change during post-natal maturation. In the present study, the cellular and subcellular distribution patterns of the GABA(B) R1a/b receptors have been analysed in different brain regions of the mouse using immunocytochemistry with isoform-specific antisera. GABA(B) R1-immunoreactivity (IR) was present from the first post-natal day (P0) on in most regions of the brain. Neurones exhibited diffuse GABA(B) R1-IR labelling throughout somata and larger proximal dendrites as well as some fine neuronal processes. After P5, distinct punctuated staining was apparent. The number of such GABA(B) IR granules per cell increased with age in a sigmoidal manner from P5 to P60. Electron microscopy revealed GABA(B) IR as clusters of small clear vesicles of 30-50 nm diameter within the cytoplasm and close to the cell membrane at extrasynaptic locations, as well as at pre-synaptic and post-synaptic specialisations. The increase in GABA(B) R1-IR punctuate staining during brain maturation points to increasing functional participation and heterogeneity of GABA(B) receptors as the complexity of the central nervous system expands with growth and development.

Complex anomalies of the human aortic arch system: unique case with both vertebral arteries as additional branches of the aortic arch.

We describe a case of an aortic arch with five primary branches arising in a sequence that has previously never been reported. From right to left, the brachiocephalic trunk and left common carotid, left vertebral, and subclavian arteries originated from the convexity of the aortic arch. The last branch was the right vertebral artery that arose from the dorsal aspect of the aortic arch opposite the ligamentum arteriosum. Presenting a dilatation at its commencement, the right vertebral ran to the right behind the esophagus and entered the foramen transversarium of the seventh cervical vertebra, whereas the left vertebral passed to the foramen transversarium of the fifth cervical vertebra. The left vertebral artery gave off the left thyroid artery; a thyrocervical trunk was absent on the left side. A thyroidea ima arose from the brachiocephalic trunk. The embryology of this complex anomaly as well as its potential clinical significance are discussed.

Pre-Bötzinger complex in the cat.

1. Patterns of respiratory neuronal activity were examined in pentobarbitone anesthetized adult cats in a circumscribed area of the ventrolateral medulla, which has previously been defined as the pre-Bötzinger complex (pre-BOTC) from electrophysiological and morphological criteria in the brain stem-spinal cord preparation of the neonatal rat. The pre-BOTC has been proposed to play a critical role in respiratory rhythm generation in mammals, but electrophysiological properties of the region have not been thoroughly characterized in the adult brain stem in vivo. 2. From intra- and extracellular recordings, we verified the existence of a well-defined zone with a distinct profile of neuronal activity between the rostral Bötzinger complex containing expiratory neurons and the more caudal medullary pool of inspiratory neurons of the ventral respiratory group (VRG) in the para-ambigual region. This zone corresponds to the pre-BOTC. It was characterized by a concentration of the various types of respiratory neurons, particularly those proposed to be involved in respiratory phase transitions, including neurons discharging immediately before the onset of inspiratory phase activity (pre-inspiratory neurons), early-inspiratory, and postinspiratory neurons. The majority of these neurons were presumed interneurons because they were not antidromically activated by spinal cord or cranial nerve stimulation. 3. The locus of the pre-BOTC corresponded histologically to the rostral part of the nucleus ambiguus and ventrolateral reticular formation. It was located caudal to the retrofacial nucleus and rostral to the lateral reticular nucleus, extending 3.0-3.5 mm rostral to the obex, and 3.2-4.0 mm lateral from the midline. This location was homologous to that established in the neonatal rat. 4. Pre-inspiratory neurons (pre-I neurons) were specifically found in the pre-BOTC. Intracellular recordings from these neurons revealed two types of activity patterns. Type 1 of pre-I neurons exhibited a steady membrane depolarization during expiration and a steep membrane depolarization with a high-frequency burst of action-potential discharge during the phase transition from expiration to inspiration. This was followed by a decline of depolarization and spike discharge during the remainder of the inspiratory phase. A second type of pre-I neurons exhibited a secondary graded membrane depolarization and burst discharge during the late-inspiratory period. 5. Synaptic events were examined in other respiratory neurons during the 40-160 ms preceding the onset of phrenic nerve activity when pre-I neurons exhibited peak spike discharge. Early-inspiratory, throughout-respiratory, and postinspiratory neurons were disinhibited during this period, whereas stage-2 expiratory neurons exhibited a decrease in spike activity and repolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

The medullary respiratory network in the rat.

1. In urethane or Nembutal anaesthetized and artificially ventilated Wistar rats, respiratory neurones of the ventrolateral medulla oblongata were analysed in extracellular (n = 74) and intracellular (n = 43) recordings. 2. Some respiratory neurones were identified as bulbospinal by their antidromic response to spinal cord stimulation at the C4 level. The neurones examined were not antidromically excited by vagal nerve stimulation. 3. Based on their discharge pattern in relation to efferent phrenic and vagal nerve activity, six types of respiratory neurones were classified: early-inspiratory, throughout-inspiratory, late-inspiratory, post-inspiratory, expiratory, and phase-spanning expiratory-inspiratory neurones. 4. Analysis of postsynaptic activities and IPSP reversal following chloride injection revealed post-inspiratory and expiratory inhibition in inspiratory neurones a pronounced early-inspiratory and a relatively weak expiratory inhibition in post-inspiratory neurones, and an early-inspiratory and post-inspiratory inhibition in expiratory neurones. 5. In phase-spanning expiratory-inspiratory neurones the post-inspiratory inhibition was strong and effectively blocked action potential discharge. Expiratory-inspiratory neurones were quite similar to the group of inspiratory neurones, but seemed to receive tonic excitatory inputs not shunted by weak expiratory inhibition. This pre-inspiratory discharge was readily blocked by weak negative DC injection. 6. Under conditions of experimental hypoxia, or long lasting lung inflation and non-inflation, post-inspiratory neurones displayed a second burst of discharge at the end of the expiratory phase in addition to their longer lasting post-inspiratory discharge. 7. We conclude that in the rat the central respiratory rhythm is organized in three (inspiratory, post-inspiratory, expiratory) phases, and that synaptic interaction within the medullary respiratory network of the rat occurs similarly to that described for the cat.

Blockade of synaptic inhibition within the pre-Bötzinger complex in the cat suppresses respiratory rhythm generation in vivo.

1. The role of synaptic inhibition in respiratory rhythm generation was analysed by microinjections of GABAA and glycine receptor antagonists into the bilateral pre-Botzinger complex (PBC) of anaesthetized cats. Central respiratory activity was monitored by phrenic nerve recordings. 2. Bilateral injections of bicuculline (50 or 100 microM) irreversibly slowed respiratory frequency and induced apneustic patterns. 3. Bilateral injections of strychnine (50 or 100 microM) greatly reduced phrenic burst amplitudes leading to increased burst frequency or irreversibly blocked rhythmic phrenic discharges. After unilateral tetrodotoxin (TTX) blockade in the PBC, strychnine injection into the contralateral PBC blocked rhythmic phrenic discharges. 4. Bilateral blockade of both GABAergic and glycinergic inhibition abolished rhythmic burst discharges and only tonic phrenic activity remained. Such tonic activity was blocked only by TTX (1 microM). 5. Potentiation of synaptic inhibition by the serotonin 1A receptor agonist 8-hydroxydipropylaminotetralin (8-OH-DPAT; 50 microM) restored rhythmic activity only when given shortly after strychnine and bicuculline applications. It was, however, ineffective after blockade of synaptic inhibition was complete. 6. The study demonstrates the significance of synaptic inhibition in the process of respiratory generation in the adult cat in vivo.

5-HT2 receptor-controlled modulation of medullary respiratory neurones in the cat.

1. The effects of the 5-HT2 receptor agonist alpha-methyl-5-HT were studied on the membrane of expiratory (E2) and post-inspiratory (PI) neurones, by intracellular recordings in the caudal medulla of anaesthetized cats. 2. Ionophoresis of alpha-Me-5-HT depolarized membrane potential and increased action potential frequency in a majority of neurones tested. Depolarization of neurones by alpha-Me-5-HT was accompanied by increased input resistance throughout all phases of the respiratory cycle. These effects were antagonized by ionophoresis of cinanserin, a receptor-blocking agent with high affinity for 5-HT2 receptors. 3. E2 neurones were voltage clamped to measure membrane current changes induced by alpha-Me-5-HT ionophoresis. alpha-Me-5-HT induced a net inward current by reducing inspiratory-phase outward currents and increasing expiratory-phase inward currents. These changes were equivalent with steady membrane depolarization, decreased inspiratory phase membrane hyperpolarization and increased expiratory drive potential recorded from the same neurones in current clamp. 4. The effects of alpha-Me-5-HT are consistent with activation of 5-HT2 receptors on E2 and PI neurones leading to blockade of synaptically activated and persistent conductances to potassium ions.

Selective lesioning of the cat pre-Bötzinger complex in vivo eliminates breathing but not gasping.

1. To examine the functional importance of the pre-Bötzinger complex for breathing we micro-injected, under in vivo conditions, the calcium channel blocker omega-conotoxin GVIA and the sodium channel blocker tetrodotoxin (TTX) into the ventrolateral medulla of adult cats, while monitoring respiratory rhythmic motor output in the phrenic nerve. 2. omega-Conotoxin GVIA caused a highly localized synaptic ablation by blocking presynaptic N-type calcium channels. When injecting 5-60 fmol omega-conotoxin GVIA unilaterally, the amplitude of phrenic nerve activity decreased bilaterally and sometimes disappeared, indicating central apnoea. These effects were reversible and could only be induced in a very localized area of the pre-Botzinger complex. By injecting omega-conotoxin GVIA several times during an experiment and analysing the areas where injections affected respiratory activity, it was possible to map exactly the anatomical extent of the area critical for respiratory rhythm generation. 3. Following the precise localization of the pre-Bötzinger complex with omega-conotoxin GVIA, we injected TTX to induce an irreversible inactivation of this region. TTX injected unilaterally into the pre-Bötzinger complex irreversibly reduced the amplitude of phrenic nerve activity. Bilateral TTX injections eliminated respiratory rhythmic activity, causing a persistent central apnoea. 4. After bilateral lesioning of the pre-Bötzinger complex, it was still possible to induce gasping during hypoxia or asphyxia, indicating that respiration and gasping are generated by two different neuronal networks. 5. We propose that omega-conotoxin GVIA as employed in this study to investigate the functional role of the pre-Bötzinger complex can also be used as a general pharmacological approach to map other neuronal networks. We call this the 'omega-conotoxin GVIA tracing' method.