Sensitivities of eupnea and gasping to alterations in temperature of in vivo and perfused rat preparations

Severe hypoxia or ischemia causes an alteration from eupnea to gasping. At body temperatures approximating 37 degrees C in vivo, the frequency of gasping is much less than that of eupnea. However in a perfused juvenile rat preparation, which is maintained at 30-31 degrees C, the frequency of eupnea and gasping is the same. We hypothesized that brainstem mechanisms responsible for the neurogenesis of eupnea and gasping might have different sensitivities to alterations in temperature. In both decerebrate adult rats in vivo and in a perfused juvenile rat preparation, eupnea and gasping had different frequencies at rectal or perfusate temperatures in excess of 34 degrees C, whereas, at lower temperatures, eupnea and gasping had identical frequencies in both preparations. These findings support the conclusion that different brainstem mechanisms underlie the neurogenesis of eupnea and gasping. In addition, these findings have implications for interpretation of results from in vitro mammalian preparations, which are examined at temperatures at which the frequency of eupnea and gasping are indistinguishable.     

Rostral medullary respiratory neuronal activities of decerebrate cats in eupnea, apneusis and gasping.

Eupnea is generated by mechanisms within the pons and medulla. Following removal of pons or exposure to anoxia, gasping is elicited. Eupnea and gasping are markedly different ventilatory patterns. The genesis of gasping is dependent upon rostral medullary neuronal activities. To generate the gasp, these activities should commence before the phrenic burst. In decerebrate, vagotomized, paralyzed and ventilated cats, eupnea was altered to gasping in anoxia. Rostral medullary neuronal activities had inspiratory, expiratory and phase-spanning patterns in eupnea. During gasping, some inspiratory neuronal activities commenced before the phrenic gasp; these same neurons had commenced activities after the onset of the eupneic phrenic burst. Expiratory and phase-spanning neurons did not discharge. Neuronal activities which are consonant with a role in the neurogenesis of gasping had very different discharge patterns in eupnea. Results support the concept that medullary mechanisms for gasping are incorporated in the ponto-medullary circuit responsible for the neurogenesis and expression of eupnea.     

Characterizations of eupnea, apneusis and gasping in a perfused rat preparation

In vivo mammalian preparations can exhibit eupnea, apneusis and gasping. In vitro mammalian preparations exhibit only a single invariant pattern, which appears identical to gasping. We characterized the patterns of ventilatory activity of a perfused heart-brainstem preparation of the juvenile rat. In this preparation, phrenic activity has a 'ramp-like' rise similar to eupnea in vivo. Peak phrenic activity declines and ultimately disappears in hypocapnia. In hypercapnia, both frequency and peak of phrenic bursts increase. In hypoxia, such increases are transient. The phrenic burst is terminated by electrical stimulation of the pontile 'pneumotaxic center' and, as in apneusis, is prolonged by lesions in this region. With severe hypoxia or ischemia, the 'ramp-like' phrenic activity is replaced by the 'decrementing' pattern of gasping. Variables of phrenic activity in gasping produced in hypoxia and ischemia are identical. We conclude that the perfused juvenile rat preparation exhibits patterns of eupnea, apneusis and gasping which are similar to in vivo mammalian preparations.     

Functions of the retrofacial nucleus in chemosensitivity and ventilatory neurogenesis

The hypothesis was evaluated that neurons within the retrofacial nucleus of medulla integrate afferent stimuli from the central chemoreceptors. In decerebrate, vagotomized, paralyzed and ventilated cats, activity of the phrenic nerve was monitored. Peak integrated phrenic activity increased in hypercapnia; the frequency of phrenic bursts typically declined slightly. The retrofacial nucleus was ablated by radio-frequency lesions or neurons within this nucleus were destroyed by microinjections of kainic acid. Results were similar following lesions or injections. Following unilateral ablations, peak phrenic activity was greatly reduced at normocapnia and hypercapnia; the frequency of phrenic bursts typically rose. Both frequency and peak phrenic activity fell further after the contralateral destruction with a cessation of all phasic phrenic discharge being observed in most animals. Injections of kainic acid in regions rostral, caudal or medial to the retrofacial nucleus produced no consistent changes in phrenic activity. We conclude that neuronal activities in the region of the retrofacial nucleus are important both in the integration of stimuli from the central chemoreceptors and in defining the discharge patterns of respiratory neurons.     

Influence of levels of carbon dioxide and oxygen upon gasping in perfused rat preparation.

In vivo, the augmenting pattern of integrated phrenic nerve discharge of eupnea is altered to the decrementing pattern of gasping in severe hypoxia or ischaemia. Identical alterations in phrenic discharge are found in perfused in situ preparations of the juvenile rat. In this preparation, gasping was produced by equilibration of the perfusate with various levels of carbon dioxide and oxygen. The duration of the phrenic burst, the interval between bursts and the burst amplitude were not significantly different following equilibration with 21-6%O(2) at 5% CO(2) or with 0-9% CO(2) at 6% O(2), with the exception that the burst amplitude was significantly greater in hypercapnic-hypoxia (9% CO(2) at 6% O(2)). It is proposed that hypoxia-induced gasping results from the release of an endogenous pacemaker activity of rostral medullary neurons. This release is caused by cellular mechanisms that change the balance between membrane ionic currents. Moreover, these cellular mechanisms may be explicitly induced by alterations in the ionic and metabolic homeostasis.     

Retinoic acid is required early during adult neurogenesis in the dentate gyrus

Retinoic acid (RA) is commonly used in vitro to differentiate stem cell populations including adult neural stem cells into neurons; however, the in vivo function of RA during adult neurogenesis remains largely unexplored. We found that depletion of RA in adult mice leads to significantly decreased neuronal differentiation within the granular cell layer of the dentate gyrus. RA contribution to neurogenesis occurs early, for RA deficiency also results in a decrease in newborn cells expressing an immature neuronal marker. Furthermore, although proliferation is unaffected during RA absence, cell survival is significantly reduced. Finally, a screen for retinoid-induced genes identifies metabolic targets including the lipid transporters, CD-36 and ABCA-1, the lipogenic master regulator SREBP1c as well as components of the Wnt signaling pathway. Our results reveal RA as a crucial contributor to early stages of adult neurogenesis and survival in vivo.     

Active glottal closure during anoxic gasping in lambs.

The present study was aimed at assessing laryngeal dynamics and their consequences during anoxic gasping in ketamine-sedated lambs. We first verified that the glottis was closed between gasps during anoxic gasping in seven chronically instrumented lambs, aged 11-15 days. Recording of glottal constrictor muscle electrical activity, subglottal pressure and lung volume, together with endoscopic observation, confirmed the presence of active glottal closure with maintenance of a high lung volume between gasps. Secondly, we tested whether maintenance of a high lung volume between gasps improved autoresuscitation efficiency. Six sedated lambs aged 8-11 days underwent two anoxic runs, including one with an open tracheostomy to prevent maintenance of a high lung volume. Access back to air was allowed for gasping. No significant difference was found in time to eupnea resumption, hemodynamic parameters or arterial blood gases. We conclude that a high lung volume is actively maintained by glottal closure between anoxic gasps in sedated lambs. Further studies are however needed to define the importance of laryngeal dynamics during gasping.     

Prion protein (PrPc) positively regulates neural precursor proliferation during developmental and adult mammalian neurogenesis

The misfolding of the prion protein (PrP(c)) is a central event in prion diseases, yet the normal function of PrP(c) remains unknown. PrP(c) has putative roles in many cellular processes including signaling, survival, adhesion, and differentiation. Given the abundance of PrP(c) in the developing and mature mammalian CNS, we investigated the role of PrP(c) in neural development and in adult neurogenesis, which occurs constitutively in the dentate gyrus (DG) of the hippocampus and in the olfactory bulb from precursors in the subventricular zone (SVZ)/rostral migratory stream. In vivo, we find that PrP(c) is expressed immediately adjacent to the proliferative region of the SVZ but not in mitotic cells. In vivo and in vitro studies further find that PrP(c) is expressed in multipotent neural precursors and mature neurons but is not detectable in glia. Loss- and gain-of-function experiments demonstrate that PrP(c) levels correlate with differentiation of multipotent neural precursors into mature neurons in vitro and that PrP(c) levels positively influence neuronal differentiation in a dose-dependent manner. PrP(c) also increases cellular proliferation in vivo; in the SVZ, PrP(c) overexpresser (OE) mice have more proliferating cells compared with wild-type (WT) or knockout (KO) mice; in the DG, PrP(c) OE and WT mice have more proliferating cells compared with KO mice. Our results demonstrate that PrP(c) plays an important role in neurogenesis and differentiation. Because the final number of neurons produced in the DG is unchanged by PrP(c) expression, other factors must control the ultimate fate of new neurons.     

Hormonal and synaptic influences of serotonin on adult neurogenesis

New neurons are incorporated into the adult brains of a variety of organisms, from humans and higher vertebrates, to non-vertebrates such as crustaceans. In virtually all of these systems serotonergic pathways appear to provide important regulatory influences over the machinery producing the new neurons. We have developed an in vitro preparation where adult neurogenesis can be maintained under highly controlled conditions, and are using this to test the influence of hormones on the production of neurons in the crustacean (Homarus americanus) brain. Serotonin levels have been manipulated in this in vitro preparation, and the resulting effects on the rate of neurogenesis have been documented. In addition we have compared in vitro influences of serotonin with results acquired from in vivo exposure of whole animals to serotonin. These experiments suggest that there are multiple mechanisms and pathways by which serotonin may regulate neurogenesis in the crustacean brain: (1) serotonin is effective in regulating neurogenesis at levels as low as 10⁻¹⁰ M, suggesting that circulating serotonin may have hormonal influences on neuronal precursor cells residing in a vascular niche or the proliferation zones; (2) contrasting effects of serotonin on neurogenesis (up- vs. down-regulation) at high concentrations (10⁻⁴ M), dependent upon whether eyestalk tissue is present or absent, indicate that serotonin elicits the release of substances from the sinus glands that are capable of suppressing neurogenesis; (3) previously demonstrated (Beltz, B.S., Benton, J.L., Sullivan, J.M., 2001. Transient uptake of serotonin by newborn olfactory projection neurons. Proc. Natl. Acad. Sci. USA 98, 12730-12735) serotonergic fibers from the dorsal giant neuron project directly into the proliferation zone in Cluster 10, suggest synaptic or local influences on neurogenesis in the proliferation zones where the final cell divisions and neuronal differentiation occur. Serotonin therefore regulates neurogenesis by multiple pathways, and the specific mode of influence is concentration-dependent.     

Function in ventilatory control of respiratory neurons at the pontomedullary junction

Previous studies have demonstrated that electrolytic lesions placed in the midline at the pontomedullary junction result in increased respiratory frequency. The increase in frequency is greater during hypercapnia. The present study sought to determine whether the effects of the lesions were mediated, at least in part, by destruction of neurons. Alternatively, the lesions may have interrupted fiber tracts. Both destruction by kainic acid and inhibition by selective cooling of neurons on the midline at the pontomedullary junction in decerebrate, vagotomized, paralyzed and artificially ventilated cats produced results similar to those engendered by the lesions; i.e., an increased respiratory frequency. Microinjections of glutamate produced a slowing of respiration. In additional cats, extracellular single unit recordings were obtained from 48 neurons located in the medial areas (0-1 mm lateral to midline) at the pontomedullary junction. Of these, 41 neurons were tonically active and 38 were judged to be respiratory-modulated by both the F-test and the Friedman test. The activity of these neurons was sensitive to changes in CO2. Therefore, neurons located at the pontomedullary junction may play a primary role in the integration of central chemoreceptor afferent stimuli.     

The deleterious effects of aging and kainic acid may be selective for similar striatal neuronal populations.

The present experiments were performed to determine whether the age-related loss of striatal D2 receptors could be localized to a kainic acid-sensitive neuronal population. This neurotoxin selectively destroys intrinsic neurons. Thus, if kainic acid reduced striatal D2 receptor concentrations such that age differences in this parameter were no longer observed, it would be a good indication that the D2 receptors lost through aging are also sensitive to kainic acid. Mature (6 months) and senescent (24 months) rats were stereotaxically, unilaterally injected with 3 micrograms/0.5 microliter kainic acid into the right striatum. Seven days later striatal D2 receptors were assessed with [3H]-spiperone in one group of mature and senescent rats. A second group of mature and senescent unilaterally lesioned rats was anesthetized and perfused. Brains were dissected and processed for striatal cell counts using cresyl violet staining, tyrosine hydroxylase and met-enkephalin using immunocytochemistry, and acetylcholinesterase using histochemistry. Age-related differences in D2-receptor concentrations were observed in intact, but not lesioned, striata. Kainic acid was less effective in reducing D2-receptor concentrations in senescent animals, suggesting that some proportion of the receptors was already lost prior to lesioning. Kainic acid also reduced total neuronal numbers, as well as Met-Enk and AChE positive staining, to approximately the same extent in mature and senescent rats. No age differences were seen in any of the other parameters following kainic acid administration.     

Transgenic mice neuronally expressing baculoviral p35 are resistant to diverse types of induced apoptosis, including seizure-associated neurodegeneration

Apoptosis is a cell-suicide process that appears to play a central role not only during normal neuronal development but also in several neuropathological disease states. An important component of this process is a proteolytic cascade involving a family of cysteine proteases called caspases. Caspase inhibitors have been demonstrated to be effective in inhibiting neuronal cell death in various apoptotic paradigms. We have created transgenic mice that neuronally express the baculoviral caspase inhibitor p35. Neuronal expression of the p35 protein was found to confer functional caspase inhibitory activity and prevent apoptosis in isolated cerebellar granular cultures induced to undergo apoptosis either via staurosporine treatment or through withdrawal of extracellular potassium. Neuronal expression of p35 was also found to attenuate neurodegeneration associated with the excitotoxic glutamate analogue kainic acid (KA) in vitro and in vivo. Organotypic hippocampal cultures isolated from p35 transgenics demonstrated lowered caspase activity and decreased apoptosis compared with wild type when exposed to KA. In vivo injection of KA also produced decreased caspase activity and cell death in p35 transgenics vs. wild type. These results suggest that the presence of p35 in neurons in vivo is protective against various types of apoptosis, including seizure-related neurodegeneration, and that caspases may be attractive potential targets for preventing neuronal injury associated with diseases such as epilepsy. These mice also provide a valuable tool for exploring the role of caspases in other neuropathological conditions in which apoptosis has been implicated.     

In vivo imaging of juxtaglomerular neuron turnover in the mouse olfactory bulb

As a consequence of adult neurogenesis, the olfactory bulb (OB) receives a continuous influx of newborn neurons well into adulthood. However, their rates of generation and turnover, the factors controlling their survival, and how newborn neurons intercalate into adult circuits are largely unknown. To visualize the dynamics of adult neurogenesis, we produced a line of transgenic mice expressing GFP in approximately 70% of juxtaglomerular neurons (JGNs), a population that undergoes adult neurogenesis. Using in vivo two-photon microscopy, time-lapse analysis of identified JGN cell bodies revealed a neuronal turnover rate of approximately 3% of this population per month. Although new neurons appeared and older ones disappeared, the overall number of JGNs remained constant. This approach provides a dynamic view of the actual appearance and disappearance of newborn neurons in the vertebrate central nervous system, and provides an experimental substrate for functional analysis of adult neurogenesis.     

FGF-2 regulation of neurogenesis in adult hippocampus after brain injury

Fibroblast growth factor-2 (FGF-2) promotes proliferation of neuroprogenitor cells in culture and is up-regulated within brain after injury. Using mice genetically deficient in FGF-2 (FGF-2(-/-) mice), we addressed the importance of endogenously generated FGF-2 on neurogenesis within the hippocampus, a structure involved in spatial, declarative, and contextual memory, after seizures or ischemic injury. BrdUrd incorporation was used to mark dividing neuroprogenitor cells and NeuN expression to monitor their differentiation into neurons. In the wild-type strain, hippocampal FGF-2 increased after either kainic acid injection or middle cerebral artery occlusion, and the numbers of BrdUrd/NeuN-positive cells significantly increased on days 9 and 16 as compared with the controls. In FGF-2(-/-) mice, BrdUrd labeling was attenuated after kainic acid or middle cerebral artery occlusion, as was the number of neural cells colabeled with both BrdUrd and NeuN. After FGF-2(-/-) mice were injected intraventricularly with a herpes simplex virus-1 amplicon vector carrying FGF-2 gene, the number of BrdUrd-labeled cells increased significantly to values equivalent to wild-type littermates after kainate seizures. These results indicate that endogenously synthesized FGF-2 is necessary and sufficient to stimulate proliferation and differentiation of neuroprogenitor cells in the adult hippocampus after brain insult.     

Therapeutic neurogenesis for CNS disorders

Although neurogenesis is observed in the human adult brain, its regulation and role are unknown. Among lots of factors promoting neurogenesis, we focused on fibroblast growth factor-2 (FGF-2), because it is known to be an important factor for neural stem cell culture. In our study, neurogenesis was upregulated in the dentate gyrus (DG) following cerebral ischemia and kainic acid-induced seizure in wild type animals, but it was reduced in FGF-2-/- mice. When FGF-2 was overexpressed using gene transfer technique with herpes virus vector, neurogenesis was upregulated, and, furthermore, degenerative changes of the hippocampus after traumatic brain injury were also reduced. These results suggested that FGF-2 is a critical factor to regulate neurogenesis in the DG after brain injury. Administration of growth factors after brain injury may provide a strategy for repair of the brain following neuronal injury and other CNS disorders.     

Stimulation of endogenous neurogenesis by anti-EFRH immunization in a transgenic mouse model of Alzheimer's disease

Neurogenesis is a subject of intense interest and extensive research, but it stands at the center of a bitter debate over ethical and practical problems. Neurodegenerative diseases, such as Alzheimer's disease (AD), accompanied by a shifting balance between neurogenesis and neurodegeneration, are suitable for stimulation of neurogenesis for the benefit of diseased patients. We have previously shown that Abs against the EFRH sequence of beta-amyloid peptide (AbetaP) prevent aggregation and disaggregate AbetaP both in vitro and in vivo. EFRH, located in the soluble tail of the N-terminal region, acts as a regulatory site controlling both solubilization and disaggregation processes in the AbetaP molecule. Here we show that anti-EFRH immunotherapy of a platelet-derived amyloid precursor protein transgenic mouse model of AD stimulates endogenous neurogenesis, suggested by elevated numbers of BrdU-incorporated cells, most of which are colocalized with a marker of mature neurons, NeuN. These newly born neurons expressed the activity-dependent gene Zif268, indicating their functional integration and participation in response to synaptic input in the brain. These findings suggest that anti-amyloid immunotherapy may promote recovery from AD or other diseases related to AbetaP overproduction and neurotoxicity by restoring neuronal population, as well as cognitive functions in treated patients.     

Segregation of on- and off-center afferents in mink visual cortex.

In the lateral geniculate nucleus of the mink, on-center and off-center neurons occupy separate layers [LeVay, S. & McConnell, S.K. (1982) Nature (London) 300, 350-351]. To study the mode of termination of geniculate afferents in area 17, we recorded from their terminal arborizations in layer IV after the destruction of cortical neurons by injection of kainic acid. At the majority of recording sites, multifiber responses were entirely or predominantly of one type: on-center or off-center. Responses obtained during perpendicular penetrations showed the same preferred sign of contrast throughout the thickness of layer IV. During tangential penetrations through the layer, we encountered sequences of on- and off-center activity separated by stretches of mixed responses. We conclude that on- and off-center afferents terminate in separate, alternating patches that occupy the full thickness of layer IV. These coexist with another set of patches in which the same afferents are segregated by eye of origin.     

Activation of orexin neurons through non-NMDA glutamate receptors evidenced by c-Fos immunohistochemistry

Orexin neuropeptides participate in the regulation of feeding as well as the regulation and maintenance of wakefulness and the cognitive functions. Orexin A and B share a common precursor, prepro-orexin and neurons are localized in the lateral hypothalamus. Physiological studies showed that these neurons are regulated by glutamatergic innervations. We aimed to assess the effects of kainic acid as a potent agonist for non-NMDA glutamate receptors in the activation of orexin neurons. We also analyzed the effect of glutamate antagonist CNQX, injected prior to kainic acid, on this activation. Expression of c-Fos protein was used as a marker for neuronal activation. Dual immunohistochemical labeling was performed for prepro-orexin and c-Fos and the percentages of c-Fos-expressing orexin neurons were obtained for control, kainic acid, and CNQX groups. Kainic acid injection caused statistically significant increase in the number of c-Fos-positive neurons when compared to control group (62.69 and 36.31%, respectively). Activation of orexin neurons was blocked, in part, by CNQX (43.36%). In the light of these results, it is concluded that glutamate takes part in the regulation of orexin neurons and partially exerts its effects through non-NMDA glutamate receptors and that orexin neurons express functional non-NMDA receptors.     

Pyrithiamine-induced thiamine deficiency alters proliferation and neurogenesis in both neurogenic and vulnerable areas of the rat brain

Thiamine deficiency (TD) leads to Wernicke’s encephalopathy (WE), in which focal histological lesions occur in periventricular areas of the brain. Recently, impaired neurogenesis has been reported in the hippocampus during the dietary form of TD, and in pyrithiamine-induced TD (PTD), a well-characterized model of WE. To further characterize the consequences of PTD on neural stem/progenitor cell (NSPC) activity, we have examined the effect of this treatment in the rat on both the subventricular zone (SVZ) of the rostral lateral ventricle and subgranular layer (SGL) of the hippocampus, and in the thalamus and inferior colliculus, two vulnerable brain regions in this disorder. In both the SVZ and SGL, PTD led to a decrease in the numbers of bromodeoxyuridine-stained cells, indicating that proliferation of NSPCs destined for neurogenesis in these areas was reduced. Doublecortin (DCX) immunostaining in the SGL was decreased, indicating a reduction in neuroblast formation, consistent with impaired NSPC activity. DCX labeling was not apparent in focal areas of vulnerability. In the thalamus, proliferation of cells was absent while in the inferior colliculus, numerous actively dividing cells were apparent, indicative of a differential response between these two brain regions. Exposure of cultured neurospheres to PTD resulted in decreased proliferation of NSPCs, consistent with our in vivo findings. Together, these results indicate that PTD considerably affects cell proliferation and neurogenesis activity in both neurogenic areas and parts of the brain known to display structural and functional vulnerability, confirming and extending recent findings on the effects of TD on neurogenesis. Future use of NSPCs in vitro may allow a closer and more detailed examination of the mechanism(s) underlying inhibition of these cells during TD.