The kreisler mutation leads to the loss of intrinsically hypoxia-activated spots in the region of the retrotrapezoid nucleus/parafacial respiratory group.

Acute hypoxia elicits a biphasic respiratory response characterized in the newborn by a transient hyperventilation followed by a severe decrease in respiratory drive known as hypoxic respiratory depression. Medullary O(2) chemosensitivity is known to contribute to respiratory depression induced by hypoxia, although precise involvement of cell populations remains to be determined. Having a thorough knowledge of these populations is of relevance because perturbations in the respiratory response to hypoxia may participate in respiratory diseases in newborns. We aimed to analyze the hypoxic response of ponto-medullary cell populations of kreisler mutant mice. These mice have defects in a gene expressed in two rhombomeres encompassing a part of the medulla oblongata implicated in hypoxic respiratory depression. Central responses to hypoxia were analyzed in newborn mice by measuring respiratory rhythm in ex vivo caudal pons-medullary-spinal cord preparations and c-fos expression in wild-type and kreisler mutants. The homozygous kreisler mutation, which eliminates most of rhombomere 5 and mis-specifies rhombomere 6, abolished (1) an early decrease in respiratory frequency within 10 min of hypoxia and (2) an intrinsic hypoxic activation, which is characterized by an increase in c-fos expression in the region of the ventral medullary surface encompassing the retrotrapezoid nucleus/parafacial respiratory group expressing Phox2b. This increase in c-fos expression persisted in wild-type Phox2b-negative and Phox2b-positive cells after blockade of synaptic transmission and rhythmogenesis by a low [Ca(2+)](0). Another central response was retained in homozygous kreisler mutant mice; it was distinguished by (1) a delayed (10-30 min) depression of respiratory frequency and (2) a downregulation of c-fos expression in the ventrolateral reticular nucleus of the medulla, the nucleus of the solitary tract, and the area of the A5 region. Thus, two types of ponto-medullary cell groups, with distinct anatomical locations, participate in central hypoxic respiratory depression in newborns.

Subcellular compartmentalization of a potassium channel (Kv1.4): preferential distribution in dendrites and dendritic spines of neurons in the dorsal cochlear nucleus.

Voltage-dependent ion channels have specific patterns of distribution along the neuronal plasma membrane of dendrites, cell bodies and axons, which need to be unravelled in order to understand their contribution to neuronal excitability and firing patterns. We have investigated the subcellular compartmentalization of Kv1.4, a transient, fast-inactivating potassium channel, in fusiform cells and related interneurons of the rat dorsal cochlear nucleus. A polyclonal antibody which binds to a region near the N-terminus domain of a Kv1.4 channel was raised in rabbits. Using a high-resolution combination of immunocytochemical methods, Kv1.4 was localized mainly in the apical dendritic trunks and cell bodies of fusiform cells, as well as in dendrites and cell bodies of interneurons of the dorsal cochlear nucleus, likely cartwheel cells. Quantitative immunogold immunocytochemistry revealed a pronounced distal to proximal gradient in the dendrosomatic distribution of Kv1. 4. In plasma membrane localizations, Kv1.4 was preferentially present in dendritic spines, either in the spine neck or in perisynaptic locations, always away from the postsynaptic density. These findings indicate that Kv1.4 is largely distributed in dendritic compartments of fusiform and cartwheel cells of the dorsal cochlear nucleus. Its preferential localization in dendritic spines, where granule cell axons make powerful excitatory synapses, suggests a role for this voltage-dependent ion channel in the regulation of dendritic excitability and excitatory inputs.

Differential expression of alpha-bungarotoxin-sensitive neuronal nicotinic receptors in adrenergic chromaffin cells: a role for transcription factor Egr-1.

Adrenomedullary chromaffin cells express at least two subtypes of acetylcholine nicotinic receptors, which differ in their sensitivity to the snake toxin alpha-bungarotoxin. One subtype is involved in the activation step of the catecholamine secretion process and is not blocked by the toxin. The other is alpha-bungarotoxin-sensitive, and its functional role has not yet been defined. The alpha7 subunit is a component of this subtype. Autoradiography of bovine adrenal gland slices with alpha-bungarotoxin indicates that these receptors are restricted to medullary areas adjacent to the adrenal cortex and colocalize with the enzyme phenylethanolamine N-methyl transferase (PNMT), which confers the adrenergic phenotype to chromaffin cells. Transcripts corresponding to the alpha7 subunit also are localized exclusively to adrenergic cells. To identify possible transcriptional regulatory elements of the alpha7 subunit gene involved in the restricted expression of nicotinic receptors, we isolated and characterized its 5' flanking region, revealing putative binding sites for the immediate early gene transcription factor Egr-1, which is known to activate PNMT expression. In reporter gene transfection experiments, Egr-1 increased alpha7 promoter activity by up to sevenfold. Activation was abolished when the most promoter-proximal of the Egr-1 sites was mutated, whereas modification of a close upstream site produced a partial decrease of the Egr-1 response. Because Egr-1 was found to be expressed exclusively in adrenergic cells, we suggest that this transcription factor may be part of a common mechanism involved in the induction of the adrenergic phenotype and the differential expression of alpha-bungarotoxin-sensitive nicotinic receptors in the adrenal gland.

Neuronal nicotinic acetylcholine receptors on bovine chromaffin cells: cloning, expression, and genomic organization of receptor subunits.

Neuronal nicotinic acetylcholine receptors from bovine adrenomedullary chromaffin cells play a primary role in triggering catecholamine secretion. In the present study, their constituent subunits were characterized. In addition to the alpha 3 subunit, which we have previously cloned, the presence of alpha 5 and beta 4 but not of beta 2 subunits was detected by reverse transcription-PCR analysis of mRNA from adrenal medulla. In situ hybridization indicated that alpha 3, alpha 5, and beta 4 subunits are coexpressed in all chromaffin cells. The primary structure of alpha 5 and beta 4 subunits was determined and functional receptors were obtained upon coinjection of subunit cRNAs into Xenopus oocytes. In contrast to other beta 4-containing nicotinic receptors, the ones formed by the bovine beta 4 subunit are insensitive to the agonist cytisine. Finally, we characterized the intergenic region of alpha 3 and alpha 5 subunits, which together with the beta 4 subunit, form a gene cluster in rats and chickens. RNase assays and the existence of overlapping cDNAs indicate that, in the bovine genome, the alpha 3 and alpha 5 genes overlap at their 3' ends. This fact is probably due to inefficient transcription termination, as a result of weak polyadenylation signals.

Expression of alpha 7 neuronal nicotinic receptors during postnatal development of the rate cerebellum.

Several lines of evidence suggest that alpha-bungarotoxin-sensitive neuronal nicotinic acetylcholine receptors may play a developmental role by modulating plasticity in neuronal circuits. The alpha 7 subunit, a main component of these receptors, is expressed in most regions of the brain, including the cerebellum, where it is present almost exclusively in Purkinje cells and deep cerebellar nuclei. Purkinje cells constitute the only efferent pathway of the cerebellum and their development involves complex interactions, which have been extensively studied. They therefore provide a potentially useful model for analysis of development plasticity which could be influenced by alpha 7 neuronal nicotinic receptors. In the present study a previously characterized monoclonal antibody (mAb 307) has been used to determine the temporal pattern of expression of the alpha 7 subunit in the developing rat cerebellum. No detectable alpha 7 immunoreactivity is found between P0 and P2. Between P3 and P5, however, the Purkinje cell layer shows moderate immunolabeling. alpha 7 expression in this layer increases rapidly between P8 and P15. This increase in alpha 7 staining, which overlaps in time with important developmental and synaptogenic events, is not uniform throughout the cerebellar cortex. Thus, between P3 and P5 all Purkinje cells are weakly labeled, while at later stages (P8-P15) immunolabeling becomes more intense, but at the same time, disappears from Purkinje cells in rostral lobules. In addition, a very well defined pattern for discontinuous or columnar labeling is detected in regions of the Purkinje cell layer where alpha 7 subunits were being expressed. Finally, at P20, alpha 7 subunit labeling is found again in all Purkinje cells, although with lower intensity. These results suggest that alpha 7 receptor expression is developmentally regulated, with a time course that parallels the final differentiation of Purkinje cells. In addition, the heterogeneous spatial distribution of alpha 7-containing nicotinic receptors indicates that, during cerebellar maturation, these cells may receive different signals that modulate receptor gene expression in a very specific way.

Immunocytochemical localization of the alpha 7 subunit of the nicotinic acetylcholine receptor in the rat central nervous system.

Previous molecular cloning studies have revealed that alpha-bungarotoxin binding proteins present in the brain are members of the neuronal nicotinic acetylcholine receptor gene family. The alpha 7 subunit is structurally related to the agonist binding subunits present in the central and peripheral nervous systems and, when expressed in Xenopus oocytes, forms functional channels blockable by alpha-bungarotoxin. In the present study, three different monoclonal antibodies raised against the alpha 7 subunit were used to map its distribution throughout the central nervous system of the rat. Immunohistochemical localization revealed that the alpha 7 subunit is expressed in most regions of the brain, being, overall, well correlated with previous "in situ" localization of alpha 7 transcripts and alpha-bungarotoxin autoradiographic binding studies. Particularly strong immunoreactivity was observed in several sensory and motor nuclei of the brainstem as well as the red nucleus. At the cellular level, alpha 7 immunostaining was usually found both in somata and dendrites, whereas axonal and terminal labeling was not observed. The widespread distribution of the alpha 7 subunit polypeptide is consistent with immunoprecipitation data demonstrating that it is a component of the predominant subtype of brain alpha-bungarotoxin-sensitive nicotinic receptors.