Selective cardiorespiratory and catecholaminergic areas express the hypoxia-inducible factor-1alpha (HIF-1alpha) under in vivo hypoxia in rat brainstem.

Under severe oxygen deprivation, all cells are able to express the transcription factor HIF-1, which activates a wide range of genes. Under tolerable hypoxia, chemosensory inputs are integrated in brainstem areas, which control cardiorespiratory responses. However, the molecular mechanisms of this functional acclimatization are unknown. We investigated when and where the inducible HIF-1alpha subunit is expressed in the rat brainstem in vivo, under physiological hypoxia. The regional localization of HIF-1alpha mRNA and protein was determined by in situ hybridization and immunocytochemistry in adult male rats exposed to moderate hypoxia (10% O2) for 1-6 h. HIF-1alpha protein was found in cell types identified by immunocytochemistry as catecholaminergic neurons. Hypoxia induced HIF-1alpha mRNA and protein in only some parts of the brainstem located dorsomedially and ventrolaterally, which are those involved in the cardiorespiratory control. No labelling was detected under normoxia. The protein was detected in glia and neurons after 1 and 6 h of hypoxia, respectively. A subset of A2C2 and A1C1 catecholaminergic neurons colocalized tyrosine hydroxylase and HIF-1alpha proteins under hypoxia, but no HIF-1alpha was detected in more rostral catecholaminergic areas. In contrast to cardiorespiratory areas, HIF-1alpha protein was already present under normoxia in glial cells of brainstem tracts but was not overexpressed under hypoxia, although HIF-1alpha mRNA was up-regulated. In conclusion, there appear to be two regulatory mechanisms for HIF-1alpha expression in the brainstem: hypoxic induction of HIF-1alpha protein in cardiorespiratory-related areas and constitutive protein expression unaffected by hypoxia in brainstem tracts.

O2-sensing after carotid chemodenervation: hypoxic ventilatory responsiveness and upregulation of tyrosine hydroxylase mRNA in brainstem catecholaminergic cells.

Ventilatory responses to acute and long-term hypoxia are classically triggered by carotid chemoreceptors. The chemosensory inputs are carried within the carotid sinus nerve to the nucleus tractus solitarius and the brainstem respiratory centres. To investigate whether hypoxia acts directly on brainstem neurons or secondarily via carotid body inputs, we tested the ventilatory responses to acute and long-term hypoxia in rats with bilaterally transected carotid sinus nerves and in sham-operated rats. Because brainstem catecholaminergic neurons are part of the chemoreflex pathway, the ventilatory response to hypoxia was studied in association with the expression of tyrosine hydroxylase (TH). TH mRNA levels were assessed in the brainstem by in situ hybridization and hypoxic ventilatory responses were measured in vivo by plethysmography. After long-term hypoxia, TH mRNA levels in the nucleus tractus solitarius and ventrolateral medulla increased similarly in chemodenervated and sham-operated rats. Ventilatory acclimatization to hypoxia developed in chemodenervated rats, but to a lesser extent than in sham-operated rats. Ventilatory response to acute hypoxia, which was initially low in chemodenervated rats, was fully restored within 21 days in long-term hypoxic rats, as well as in normoxic animals which do not overexpress TH. Therefore, activation of brainstem catecholaminergic neurons and ventilatory adjustments to hypoxia occurred independently of carotid chemosensory inputs. O2-sensing mechanisms unmasked by carotid chemodenervation triggered two ventilatory adjustments: (i) a partial acclimatization to long-term hypoxia associated with TH upregulation; (ii) a complete restoration of acute hypoxic responsivity independent of TH upregulation.

Reorganization of pontine rhythmogenic neuronal networks in Krox-20 knockout mice.

We have shown previously that the inactivation of the zinc finger gene Krox-20 affects hindbrain segmentation, resulting in the elimination of rhombomeres 3 and 5. We demonstrate here that Krox-20 homozygous mutant mice exhibit abnormally slow respiratory and jaw opening rhythms, indicating that a modification of hindbrain segmentation influences the function of neuronal networks after birth. Central neuronal networks that control respiratory frequency are made predominantly depressant by the elimination of a previously undescribed rhythm-promoting system. Recordings of rhythmic activity from the isolated hindbrain following progressive tissue transections indicate that the reorganization takes place in the caudal pontine reticular formation. The newborn (PO) Krox-20-/- mice, in which apneas are ten times longer than in wild-type animals, may be a valuable model for the study of life-threatening apneas during early infancy.

Plasticity of tyrosine hydroxylase gene expression in the rat nucleus tractus solitarius after ventilatory acclimatization to hypoxia.

The aim of this study was to define the influence of long-term hypoxia on gene expression of tyrosine hydroxylase (TH) in the rat nucleus tractus solitarius (NTS). Animals were exposed to normobaric hypoxia (10% O2 in nitrogen) for 2 weeks. At this time, the hypoxia-induced hyperventilation reached a plateau, indicating ventilatory acclimatization. In horizontal brainstem sections, hypoxia-induced changes in TH protein and TH mRNA were assessed by immunocytochemistry and in-situ hybridization, respectively. Long-term hypoxia increased TH mRNA levels seen as both an increase in the number of grains per cell and an extension of the labeled area. The highest degree of labeling was found selectively located in caudal NTS. Hypoxia also enhanced TH immunoreactivity in the caudal NTS but this labeling extended more rostrally than that of TH mRNA. The data suggest that there is an hypoxia-induced plasticity of gene expression at the gene level in the NTS, which is associated with ventilatory acclimatization. The hypoxia model described in this study may serve as a framework for future regulatory studies.

Autoradiographic localisation of NMDA binding sites in brainstem cardiorespiratory areas of adult and newborn cats.

Quantitative autoradiography was used to determine the distribution of [3H]1-[1-(2-thienyl)cyclohexyl] piperidine ([3H]TCP) binding sites in the brainstem in order to localize the possible targets activated by excitatory amino acids in adult and newborn cats during autonomic rhythmic functions. Medium to high densities of binding sites were found in the nucleus tractus solitarius complex and the laterodorsal part of the pontine tegmentum. In the kitten, there was an apparent higher density in cranial motor nuclei proximal to these structures. It is concluded that there is an overlapping between the high density of NMDA receptors and the localization of cardiorespiratory neurons in cat as well as in kitten.

Mossy fiber sprouting in epileptic rats is associated with a transient increased expression of alpha-tubulin.

Kainate-induced seizures lead to marked increases of alpha-tubulin mRNA and protein immunoreactivity in the rat dentate gyrus. The increase in alpha-tubulin mRNA was restricted to the granule cell bodies. alpha-Tubulin immunoreactivity was enhanced in granule cell dendrites and axons (the mossy fibers), in the molecular layer. These changes peaked 6-12 days after kainate treatment and preceded the collateral sprouting of mossy fibers which occur 12 to 30 days after seizures. The present results suggest that microtubule formation contributes to the synaptic rearrangements which take place in the hippocampus after seizures.