Proliferation in the auditory receptor epithelium mediated by a cyclic AMP-dependent signaling pathway.

Loss of receptor hair cells in the cochlea accounts for a significant proportion of hearing impairment in the population. Hair cells can be lost as a consequence of viral or bacterial insult, aging, and damage from intense sound or aminoglycoside antibiotics. The generation of replacement hair cells following damage by sound or drugs has been clearly demonstrated in birds; the chick is the best-studied model for auditory hair cell regeneration. New hair cells arise as progeny from an otherwise nondividing supporting cell population induced to proliferate by the damage. Functional recovery of hearing accompanies this cellular recovery process. The signals and pathways responsible for regenerative proliferation are unknown. Here we show that proliferation is induced in the undamaged receptor epithelium by agents that increase cyclic AMP levels, and that following this stimulation hair cells become labeled with proliferation markers. This remarkable proliferative response is blocked by inhibitors of the cAMP-regulated protein kinase A (PKA). In addition we show that the proliferative response induced by in vitro gentamicin damage is also significantly blocked by PKA inhibitors. These observations are the first to identify a signaling pathway that plays a role in regenerative proliferation in the auditory receptor epithelium.

A circadian output in Drosophila mediated by neurofibromatosis-1 and Ras/MAPK.

Output from the circadian clock controls rhythmic behavior through poorly understood mechanisms. In Drosophila, null mutations of the neurofibromatosis-1 (Nf1) gene produce abnormalities of circadian rhythms in locomotor activity. Mutant flies show normal oscillations of the clock genes period (per) and timeless (tim) and of their corresponding proteins, but altered oscillations and levels of a clock-controlled reporter. Mitogen-activated protein kinase (MAPK) activity is increased in Nf1 mutants, and the circadian phenotype is rescued by loss-of-function mutations in the Ras/MAPK pathway. Thus, Nf1 signals through Ras/MAPK in Drosophila. Immunohistochemical staining revealed a circadian oscillation of phospho-MAPK in the vicinity of nerve terminals containing pigment-dispersing factor (PDF), a secreted output from clock cells, suggesting a coupling of PDF to Ras/MAPK signaling.

Response of the timeless protein to light correlates with behavioral entrainment and suggests a nonvisual pathway for circadian photoreception.

The period (per) and timeless (tim) genes are required for circadian behavioral rhythms in Drosophila. The current model for how these rhythms entrain to light is based upon the light induced decrease in timeless protein (TIM) levels. We show here that the TIM response to light correlates with the effect of light on the behavioral rhythm. To identify components of the entrainment pathway, we also assayed the TIM response in flies with mutant visual systems. Flies that lacked eyes displayed a normal response in lateral neurons. The TIM response to a light pulse was attenuated in flies that were mutant for the transient receptor potential (trp) and trp-like (trpl) genes, which are required for calcium conductance in the visual transduction cascade. The reduced TIM response was accompanied by a reduced phase shift in the behavioral rhythm, but neither response was completely eliminated, and the trpl;trp flies entrain to light-dark cycles, suggesting that these genes perturb some aspect of circadian entrainment when mutated but are not essential for it. The TIM response was also unaffected in ninaE flies that lack the rhodopsin protein (rh1). These results support the hypothesis that circadian entrainment does not rely on the visual system and likely involves a dedicated pathway for photoreception.

Thalamic midline cell populations projecting to the nucleus accumbens, amygdala, and hippocampus in the rat.

The organization of the thalamic midline efferents to the amygdaloid complex, hippocampal formation, and nucleus accumbens was investigated in the rat by means of multiple retrograde fluorescent tracing. The present findings indicate that these connections derive from separate cell populations of the thalamic midline, with a low degree of divergent collateralization upon more than one of the targets examined. The neural populations projecting to the amygdala, hippocampus, or accumbens are highly intermingled throughout the thalamic midline, but display some topographical prevalence. Midline thalamo-hippocampal cells are concentrated in the nucleus reuniens; thalamo-accumbens neurons prevail in the ventral portion of the paraventricular nucleus, and in the central medial nucleus. Thalamo-amygdaloid cells display a topographical prevalence in the rostral third of the thalamic midline and are concentrated in the dorsal part of the paraventricular nucleus and in the medial part of the nucleus reuniens. Both dorsally in the paraventricular nucleus and ventrally in the nucleus reuniens, thalamo-amygdaloid cells are located closer to the ependymal lining than the neurons projecting to the hippocampus or nucleus accumbens. Further, thalamo-amygdaloid cells, especially in the paraventricular nucleus, extend their dendritic processes in the vicinity of the ependymal lining, where they arborize profusely. These features indicate a close topographical relationship of neurons projecting to the amygdala with ependymal cells. The fairly discrete origin of midline outputs to the amygdala, hippocampus, and accumbens indicates that the flow of information is conveyed through separate channels from the thalamic midline to limbic and limbic-related targets. Together with the literature on the limbic afferents to the thalamus, these findings emphasize the relationships between the thalamus and the limbic system subserved by parallel input-output routes. However, because of the overlap of the projection cell populations, the thalamic midline may represent a locus of interaction among neurons connected with different parts of the limbic system. The functional implications of these findings are discussed in relation to the "nonspecific" thalamic system, as well as to the circuits involved in memory formation.

Selective labeling by propidium iodide injected into the lateral cerebral ventricle of the rat.

Injections of propidium iodide (PI) into the lateral cerebral ventricle of the rat resulted in a bilateral labeling in the septohippocampal nuclei, substantia nigra (SN), ventral tegmental area (VTA), retrorubral nuclei (rr), dorsal and median raphe nuclei, regions within and dorsal to the medial lemniscus of the caudal midbrain, and Purkinje cells in the cerebellum. No labeled neurons were seen in other areas of the brain. The data suggest that PI appears to exhibit selective labeling, and the mechanism underlying the selective labeling is discussed. Combined with Faglu histofluorescence, it was found that all PI-labeled cells in SN-VTA-rr were catecholamine (CA) neurons. After a transection of the medial forebrain bundle immediately before the PI injection, an accumulation of PI was only seen in the distal segments of severed nigrostriatal CA fibers. This provides a strong evidence that PI labeling of CA cells in SN-VTA-rr is due to axonal uptake and retrograde transport.

Afferent connections of the thalamic paraventricular and parataenial nuclei in the rat--a retrograde tracing study with iontophoretic application of Fluoro-Gold.

The origin of the afferents of the thalamic anterior paraventricular and parataenial nuclei (PVA-PT) in the rat was studied with fluorescent retrograde tracing method. After iontophoretic application of Fluoro-Gold into PVA-PT, many labeled cells were found in the medial frontal cortex, ventral subiculum, claustrum, septum, ventral pallidum, thalamic reticular nuc., zona incerta, hypothalamus, central gray, dorsal raphe nuc., parabrachial nuc., and the laterodorsal tegmental nuc. A few cells were labeled in the medial amygdaloid nuc., amygdalohippocampal area, median raphe nuc., and the locus coeruleus. Occasional neurons were labeled in the ventral lateral geniculate nuc., pretectal area, and in the solitary nuc. and the lateral reticular nuc. in the medulla.

Distribution of catecholamine-containing cell bodies in the human diencephalon.

The distribution and morphological characteristics of catecholamine (CA)-containing cell bodies in the diencephalon of 7-9-month-old fetuses were studied by means of the Faglu histofluorescence method. Although the general distribution of CA cell bodies in human diencephalon observed in the present study is similar to that in other mammals, there are some significant differences. A great number of CA neurons lie within the lateroventral hypothalamic zone. There are more numerous larger CA neurons distributed outside the periventricular gray in the human diencephalon.

Photoconversion of fluorescent retrograde tracers.

Photoconversion of fluorescent dyes, retrogradely transported through axons to their parent cell bodies, into a stable diaminobenzidine (DAB) reaction product was tested in the nigrostriatal and thalamocortical systems of rats. Satisfactory results were obtained with Propidium Iodide (PI), Fluoro-Gold (FG), Fast Blue (FB), Diamidino Yellow (DY), and rhodamine-labeled latex microspheres (RLM); some photoconversion was also observed in Evans Blue (EB)-labeled neurons. The red fluorescent tracers PI, EB and RLM were photoconverted under the excitation wavelength appropriate for eliciting their fluorescent emission. With the yellow or blue fluorescent tracers FG, FB, and DY satisfactory results could instead be obtained using an excitation wavelength which did not elicit visible fluorescent emission. This finding indicates that the latter is not a critical factor for obtaining photoconversion. The features and subcellular localization of photoconverted DAB were different from those of the fluorescent labeling: photoconversion resulted in the appearance of brown granules of DAB reaction products in the cytoplasm, independently from the occurrence of fluorescent labeling in the neuronal cytoplasm or nucleus. Photoconversion may enable new applications of fluorescent retrograde tracing and, in particular, its electron microscopic visualization.

Frequency of adequate contrast opacification of the major intracranial venous structures with CT angiography in the setting of intracerebral hemorrhage: comparison of 16- and 64-section CT angiography techniques.

BACKGROUND AND PURPOSE: DVST is an important cause of ICH because its treatment may require anticoagulation or mechanical thrombectomy. We aimed to determine the frequency of adequate contrast opacification of the major intracranial venous structures in CTAs performed for ICH evaluation, which is an essential factor in excluding DVST as the ICH etiology. MATERIALS AND METHODS: Two readers retrospectively reviewed CTAs performed in 170 consecutive patients with ICH who presented to our emergency department during a 1-year period to determine by consensus whether qualitatively, contrast opacification in each of the major intracranial venous structures was adequate to exclude DVST. "Adequate contrast opacification" was defined as homogeneous opacification of the venous structure examined. "Inadequate contrast opacification" was defined as either inhomogeneous opacification or nonopacification of the venous structure examined. Delayed scans, if obtained, were reviewed by the same readers blinded to the first-pass CTAs to determine the adequacy of contrast opacification in the venous structures according to the same criteria. In patients who did not have an arterial ICH etiology, the same readers determined if thrombosis of an inadequately opacified intracranial venous structure could have potentially explained the ICH by correlating the presumed venous drainage path of the ICH with the presence of inadequate contrast opacification within the venous structure draining the venous territory of the ICH. CTAs were performed in 16- or 64-section CT scanners with bolus-tracking, scanning from C1 to the vertex. Patients with a final diagnosis of DVST were excluded. We used the Pearson χ(2) test to determine the significance of the differences in the frequency of adequate contrast opacification within each of the major intracranial venous structures in scans obtained using either a 16- or 64-section MDCTA technique. RESULTS: Fifty-eight patients were evaluated with a 16-section MDCTA technique (34.1%) and 112 with a 64-section technique (65.9%). Adequate contrast opacification within all major noncavernous intracranial venous structures was significantly less frequent in first-pass CTAs performed with a 64-section technique (33%) than in those performed with a 16-section technique (60%, P value < .0001). Delayed scans were obtained in 50 patients, all of which demonstrated adequate contrast opacification in the major noncavernous intracranial venous structures. In 142 patients with supratentorial or cerebellar ICH without an underlying arterial etiology, we found that thrombosis of an inadequately opacified major intracranial venous structure could have potentially explained the ICH in 38 patients (26.8%), most examined with a 64-section technique (86.8%). CONCLUSIONS: Inadequate contrast opacification of the major intracranial venous structures is common in first-pass CTAs performed for ICH evaluation, particularly if performed with a 64-section technique. Acquiring delayed scans appears necessary to confidently exclude DVST when there is strong clinical or radiologic suspicion.

Case report. Appearance of nephrogenic fibrosing dermopathy on a bone scan.

We report a case of nephrogenic systemic fibrosis involving the thighs bilaterally in a patient who received multiple MRI scans with gadolinium. A recent bone scan demonstrated uptake of radiotracer in a region that correlates with CT and dermatopathological findings.

L-serine degradation in Escherichia coli K-12: cloning and sequencing of the sdaA gene.

A new mutant of Escherichia coli K-12 unable to grow with L-serine, glycine, and L-leucine has been isolated by lambda plac Mu insertion and shown to be deficient in L-serine deaminase activity. The corresponding gene, sdaA, has been cloned from a prototrophic strain, and the clone has been characterized and sequenced. The evidence is consistent with the hypothesis that sdaA is the structural gene for L-serine deaminase. However, other possibilities are also considered. No significant homology with previously reported DNA or protein sequences was detected.

CIITA-dependent and -independent class II MHC expression revealed by a dominant negative mutant.

The MHC class II transactivator gene (CIITA) coordinately controls the expression of the three major human class II genes, HLA-DR, HLA-DQ, and HLA-DP. Indeed, patients with one form of MHC class II immunodeficiency disease, due to defective CIITA genes, lack expression of all three isotypes. Nevertheless, there is substantial evidence that human class II genes are not always coordinately regulated, raising the possibility that CIITA-independent, isotype-specific class II regulatory pathways exist. To address this issue, we have generated a dominant negative mutant of CIITA that lacks the acidic transcription-activating N terminus, but retains the proline/serine/threonine-rich domain. Three newly produced anti-CIITA mAbs revealed that this mutant protein lacked N-terminal epitopes. In this study, we report that this CIITA dominant negative mutant repressed the constitutive expression of all three class II isotypes in human EBV-B cell lines, as well as IFN-gamma-induced class II transcription in HeLa cells. However, in a CIITA-deficient, EBV-transformed B cell line, clone 13, the dominant negative mutant did not alter the endogenous expression of the HLA-DQ gene. Taken together, these data demonstrate the existence of both CIITA-dependent and -independent class II regulatory pathways. Furthermore, our data provide evidence that the latter pathways can be isotype specific.