Calcium entry through cyclic nucleotide-gated channels in individual cilia of olfactory receptor cells: spatiotemporal dynamics.

Transient elevations of intracellular Ca2+ play an important role in regulating the sensitivity of olfactory transduction, but such elevations have not been demonstrated in the olfactory cilia, which are the site of primary odor transduction. To begin to understand Ca2+ signaling in olfactory cilia, we used high-resolution imaging techniques to study the Ca2+ transients that occur in salamander olfactory receptor neurons (ORNs) as a result of cyclic nucleotide-gated (CNG) channel activation. To visualize ciliary Ca2+ signals, we loaded ORNs with the Ca2+ indicator dye Fluo-3 AM and measured fluorescence with a laser scanning confocal microscope. Application of the phosphodiesterase inhibitor IBMX increased fluorescence in the cilia and other neuronal compartments; the ciliary signal occurred first and was more transient. This signal could be abolished by lowering external Ca2+ or by applying LY83583, a potent blocker of CNG channels, indicating that Ca2+ entry through CNG channels was the primary source of fluorescence increases. Direct activation of CNG channels with low levels of 8-Br-cGMP (1 microM) led to tonic Ca2+ signals that were restricted locally to the cilia and the dendritic knob. Elevated external K+, which depolarizes cell membranes, increased fluorescence signals in the cell body and dendrite but failed to increase ciliary Ca2+ fluorescence. The results demonstrate the existence and spatiotemporal properties of Ca2+ transients in individual olfactory cilia and implicate CNG channels as a major pathway for Ca2+ entry into ORN cilia during odor transduction.

Androgen alters the dendritic arbors of SNB motoneurons by acting upon their target muscles.

In adult male rats, motoneurons of the spinal nucleus of the bulbocavernosus (SNB) have been shown to retract and reextend their dendritic branches in response to systemic androgen deprivation and readministration. Furthermore, other studies have suggested that the dendritic complexity of neurons can be regulated by their targets. To assess whether androgens might act upon the target muscles to mediate changes in SNB dendrites, adult male rats were castrated and implanted with a small capsule filled with testosterone (T) next to the bulbocavernosus and levator ani muscle complex (BC/LA) on one side, while the muscles on the contralateral side were implanted with another capsule containing hydroxyflutamide (hFl), an anti-androgen. We have previously shown that after 30 d of this focused, lateralized androgen treatment the BC/LA complex is significantly larger on the T-treated side. We now report that the total dendritic lengths of SNB motoneurons innervating muscles given androgen blockade are reduced by 44% compared to SNB motoneurons innervating muscles given androgen stimulation. Dendrite lengths within three regions of the spinal cord were altered in a nonuniform manner: large changes occurred in the dorsal and contralateral dendritic fields while there was no difference in the ipsilateral dendritic field. These results suggest that BC/LA muscles, in response to androgen stimulation, produce a trophic substance which regulates the dendritic organization of SNB motoneurons in adulthood.

Blocking Ca2+ mobilization with thapsigargin reduces neurite initiation in cultured adult rat DRG neurons.

Adult rat DRG neurons rapidly extend extensive neuritic arbors after a 1-2-day delay in culture and generate large depolarization-induced calcium signals during this time period that are derived primarily from intracellular calcium release. To assess whether intracellular calcium mobilization is required for neurite initiation, calcium stores were depleted by brief exposure to the irreversible endoplasmic reticulum calcium ATPase inhibitor thapsigargin; cultures were then maintained for 3 days, immunostained for neurofilament and scored for percentage of neurons with neurites at least twice as long as the cell body. Brief thapsigargin treatment (20 min) during the first 24 h in culture resulted in a substantial decrease in neurite initiation frequency without affecting neuronal or nonneuronal cell survival, suggesting that intracellular calcium mobilization is necessary for triggering neurite initiation in these neurons.

Differential role of two Ca(2+)-permeable non-NMDA glutamate channels in rat retinal ganglion cells: kainate-induced cytoplasmic and nuclear Ca2+ signals.

1. The permeability of non-N-methyl-D-aspartate (non-NMDA) glutamate channels to divalent cations and specifically the entry of Ca2+ and subsequent elevations in cytoplasmic and nuclear Ca2+ signals were investigated in cultured neonatal rat retinal ganglion cells using the whole cell patch-clamp technique and Ca2+ imaging with confocal microscopy. In addition, divalent-permeable non-NMDA receptor channels were studied in retinal slices using a Co2+ staining technique. 2. Using Ca2+ (2.5 mM) as the only permeable cation in the external solution, stimulation with 100 microM kainate produced nondesensitizing, nonselective cation currents with either low or high Ca2+ permeability. Both currents were reversibly blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Neurons with the low divalent-permeable currents (type 1) had reversal potentials of -41.5 +/- 4.4 mV (mean +/- SD), and neurons with the high divalent-permeable currents (type 2) had reversal potentials of -22.6 +/- 5.5 mV. The permeability ratio PCa/PCs was 3.3 for the type 1 currents and 8.5 for the type 2 currents, indicating a 2.5-fold greater permeability to Ca2+ for the type 2 non-NMDA glutamate channels. 3. Both types of non-NMDA glutamate channels showed relatively little selectivity between Ca2+ and Co2+. The type 1 neurons had a slightly higher permeability to Co2+ than to Ca2+, whereas the type 2 neurons were equally permeable to both divalent cations. The type 2 neurons had a much higher permeability for both divalent cations compared with the type 1 neurons. 4. Staining for Co2+ uptake through kainate-stimulated non-NMDA glutamate channels in retinal slices provided additional evidence for the presence of the two ganglion cell populations. Activation of the neurons by kainate in conditions isolating the non-NMDA glutamate channel caused differential uptake of Co2+. In contrast, depolarization in the presence of the non-NMDA antagonist CNQX failed to cause Co2+ influx. 5. Imaging experiments using confocal microscopy showed that kainate stimulation induced an increase in intracellular Ca2+ in both types of retinal ganglion cells, but only the type 2 neurons showed a substantial increase in cytoplasmic and nuclear Ca2+ signals. Kainate-induced Ca2+ signals in the type 2 neurons were almost nine times greater than those of the type 1 neurons. 6. When intracellular Ca2+ stores were depleted by brief treatment with thapsigargin, kainate-induced Ca2+ signals in the type 1 neurons were unchanged. However, in the type 2 neurons kainate no longer induced large Ca2+ signals in the cytoplasm and nucleus, despite normal influx of Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular calcium mobilization and neurite outgrowth in mammalian neurons.

Cultured adult rat dorsal root ganglion (DRG) neurons were used to study depolarization-induced Ca2+ mobilization and the effects of intracellular Ca2+ depletion on neurite outgrowth. Cytoplasmic and nuclear Ca2+ signals were visualized in dissociated DRG neurons using confocal scanning laser microscopy and the Ca2+ indicator dye fluo-3. The depolarization-induced Ca2+ signals were highest in neurons during the first few days in culture, prior to neurite extension; during this time nuclear signals exceeded those of the cytoplasm severalfold. After several days in culture, neurons began to arborize, depolarization-induced Ca2+ signals became attenuated, and nuclear signals no longer exceeded those of the cytoplasm. Elevated Ca2+ signals were dependent upon both Ca2+ influx and intact intracellular Ca2+ stores, indicating that the signals are generated by calcium-induced calcium release (CICR). Thapsigargin, an endoplasmic reticulum Ca2+ ATPase inhibitor, depleted intracellular Ca2+ stores and blocked the induction of the large nuclear Ca2+ signals. Treating DRG neurons briefly with thapsigargin (200 nM for 20 min) shortly after plating reduced subsequent neuritogenesis, implying that intact Ca2+ stores are necessary for initiating neurite outgrowth. Immunostaining of DRG neurons with antibodies to Ca2+/calmodulin-dependent kinase II (CaM kinase II) demonstrated that this enzyme is present in the nucleus at early times in culture. These observations are consistent with the idea that CICR triggered by Ca2+ entry subsequent to depolarization may elicit neurite outgrowth by activating nuclear enzymes appropriate for such outgrowth.

Nuclear and cytoplasmic Ca2+ signals in developing rat dorsal root ganglion neurons studied in excised tissue.

Confocal microscopy and the Ca(2+)-sensitive fluorescent dye fluo-3 were used to study subcellular Ca2+ signals in embryonic, neonatal, and adult dorsal root ganglion (DRG) neurons in excised dorsal root ganglia. Optical images obtained from isolated whole embryonic and neonatal ganglia revealed a marked variability in the resting Ca2+ signals of different neurons as compared to signals in adult neurons which were uniformly faint. Many of the embryonic and neonatal neurons displayed nuclear Ca2+ signals at rest which were larger than those in the cytoplasm. Embryonic DRG neurons showed a significant increase in nuclear and cytoplasmic fluorescence in response to depolarization with elevated extracellular potassium or electrical stimulation. A single brief electrical stimulus was sufficient to elicit nuclear Ca2+ signals in a subset of the embryonic neurons. The depolarization-induced Ca2+ signals were blocked by removal of extracellular Ca2+, but not by treatment with 2,5-di (tert-butyl)-1,4 benzohydroquinone (DTBHQ), a compound which depletes intracellular Ca2+ stores. The intensity of the depolarization-induced Ca2+ signals declined significantly between the late embryonic (E18-E20) and early postnatal time periods (P0-P1). The nuclear and cytoplasmic Ca2+ signals of the embryonic DRG neurons in the excised tissue preparation occur at a time of intense target innervation, suggesting a role for Ca2+ signals in the development and maturation of rat DRG neurons.

Transient presence of GABA in astrocytes of the developing optic nerve.

Immunostaining and high-pressure liquid chromatography (HPLC) were used to study the developmental time course of astrocytic gamma-aminobutyric acid (GABA) expression in rat optic nerve. GABA immunostaining was carried out on cultured astrocytes, and on whole optic nerve. Confocal scanning laser microscopy was used to obtain optical sections in excised whole tissue in order to localize the cellular origins of GABA within the relatively intact optic nerve. GABA immunoreactivity was localized in astrocytes identified by GFAP staining; GABA staining was most intense in early neonatal optic nerve and attenuated over 3 weeks of postnatal development. The staining was pronounced in the astrocyte cell bodies and processes but not in the nucleus. There was a paucity of GABA immunoreactivity by postnatal day 20, both in culture and in whole optic nerve. A biochemical assay for optic nerve GABA using HPLC indicated a relatively high concentration of GABA in the neonate, which rapidly attenuated over the first 3 postnatal weeks. Immunoreactivity for the GABA synthesis enzyme glutamic acid decarboxylase (GAD) was pronounced in neonates but also attenuated with development. These results indicate that GABA and the GABA synthesis enzyme GAD are localized in astrocytes of optic nerve, and that their expression is transient during postnatal development.

Kainate elicits elevated nuclear calcium signals in retinal neurons via calcium-induced calcium release.

Intracellular Ca2+ was imaged in cultured neonatal rat retinal neurons using the Ca(2+)-sensitive dye fluo-3 and confocal scanning laser microscopy. Depolarization via elevation of bath K+ concentration resulted in large cytoplasmic and nuclear Ca2+ signals; responses in the nucleus exceeded those of the cytoplasm. Glutamate or kainate application elicited the same intracellular pattern of elevated Ca2+ signals. Kainate stimulation was blocked by the non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and greatly reduced by removing Ca2+ from the bath and adding ethylene glycol-bis (beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid (EGTA). Kainate was equally effective in eliciting Ca2+ signals when bath Na+ was replaced with equimolar concentrations of choline, or in the presence of the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (APV). Caffeine treatment significantly reduced the kainate-induced intracellular Ca2+ response. These results suggest that Ca2+ can enter through the kainate receptor of retinal neurons and amplify the Ca2+ signals in the cytoplasm and nucleus by releasing Ca2+ from intracellular stores.

Androgen locally regulates rat bulbocavernosus and levator ani size.

Adult male rats were gonadectomized, and small Silastic capsules filled with hormone were sutured to each bulbocavernosus and levator ani muscle complex (BC/LA). In the first experiment, one capsule contained testosterone (T), while the capsule on the contralateral muscles contained the antiandrogen hydroxyflutamide (hFl). The intent of this treatment was to provide a focus of androgenic stimulation to the muscles on one side. After 30 days, animals were sacrificed, and the BC/LA muscle pairs were removed, weighed, and compared. BC/LAs receiving T treatment were heavier than those receiving hFl treatment (p less than 0.0001), with an average weight difference of 12%. Muscle fibers from T-treated BCs were significantly larger in diameter than those from contralateral, hFl-treated BCs. These results indicate that androgen exerts its anabolic effect by acting locally upon a cell population within or near the BC/LA. When hFl and blank capsules were implanted in castrated males, the hFl-treated muscles were significantly heavier (by 9%), demonstrating an anabolic effect of hFl in the absence of androgen, and refuting the idea that hFl may have caused local toxic effects in the first experiment. Gonadectomized animals given T versus blank capsules had T-treated muscles that were 8% heavier than the blank-treated side. Muscle weights were also compared in animals receiving bilateral denervation of the BC/LA at the time of T and hFl capsule implantation and gonadectomy; local testosterone treatment failed to affect BC/LA weights in these denervated muscles.

Consensual pupillary light reflex in the pigmented rat.

We measured the pupillary light reflex (PLR) in 5 pigmented, Long Evans rats (under urethan sedation) in three conditions: direct stimulation, consensual stimulation, and a control condition designed to measure the effects of stray light. The average constriction (maximal amplitude) produced by a ganzfeld stimulus delivering 1.6 log quanta absorbed per rod per sec for a duration of 3 sec was measured to be 0.78 +/- 0.07 mm for the direct PLR, 0.67 +/- 0.06 mm for the consensual PLR, and 0.07 +/- 0.029 mm for the control condition. We corrected the consensual measurement for each rat by subtracting the value of the control (stray-light induced) constriction. A comparison of the corrected consensual constriction to the direct constriction showed that, on average, the consensual constriction attained an amplitude of 78% of the direct constriction. Our findings contradict claims that the consensual pupillary light reflex is absent in rodents. Although our results are in agreement with findings showing bilateral projections of the retina to the pretectum (which subserves the pupillary light reflex) in the rat, the consensual-to-direct ratio we report is higher than might be expected from anatomical estimates of the overall proportion of uncrossed to crossed optic fibers in the rat.

Ontogeny of functional innervation of bulbocavernosus muscles in male and female rats.

Electrical stimulation of the sixth lumbar ventral root reliably elicited contractions of the bulbocavernosus muscle (BC) in untreated female rats on the day before birth. This functional contact indicates that testosterone probably does not act to save the spinal nucleus of the bulbocavernosus (SNB) and its BC target muscles through the establishment of an active neuromuscular synapse, since this is already present in animals with an SNB system which is fated to die.