Pituitary adenylate cyclase activating polypeptide reduces expression of Kv1.4 and Kv4.2 subunits underlying A-type K(+) current in adult mouse olfactory neuroepithelia.

A-type K(+) currents (I(A)) in olfactory receptor neurons have been characterized electrophysiologically but the molecular identities of the underlying channel subunits have not been determined. Using RT-PCR, immunoblot and immunohistochemistry, we found that the two candidate channel families underlying I(A), shaker and shal, are expressed in olfactory epithelia of Swiss Webster mice. Specifically, Kv1.4, the only I(A) candidate from the shaker family, and Kv4.2 and Kv4.3 from the shal family were expressed, but Kv4.1 mRNA was not amplified from the olfactory epithelia. Immunoblot and immunohistochemical studies confirmed the existence of Kv1.4 and Kv4.2/3 subunits. Furthermore, quantitative RT-PCR showed that pituitary adenylate cyclase activating polypeptide (PACAP) reduced the expression of Kv1.4 and Kv4.2 but did not reduce the already low expression of Kv4.3. The PACAP-induced reduction of Kv4.1 and Kv4.2 expression was completely blocked by inhibiting the phospholipase C (PLC) pathway but was still significantly downregulated by PACAP when the cyclic AMP pathway was inhibited. In addition, downstream of the PLC pathway, calcium mediated the reduction of both Kv1.4 and Kv4.2 expression and I(A) current density. Phosphokinase C (PKC) activation did not affect Kv1.4 and Kv4.2 mRNA expression, even though PKC reduced I(A) current density. Together with our previous studies, our data suggest that A-type K(+) currents in olfactory receptor neurons are composed of multiple K(+) channel subunits, among which Kv1.4 and Kv4.2 are subject to transcriptional modulation by PACAP. We also found that PACAP predominately uses a PLC-calcium pathway to modulate Kv4.1 and Kv4.2 expression. Modulation of A-type K(+) current expression may contribute to the previously observed neuroprotective effects of PACAP on olfactory receptor neurons.

Pituitary adenylate cyclase activating polypeptide reduces A-type K+ currents and caspase activity in cultured adult mouse olfactory neurons.

Pituitary adenylate cyclase activating polypeptide has been shown to reduce apoptosis in neonatal cerebellar and olfactory receptor neurons, however the underlying mechanisms have not been elucidated. In addition, the neuroprotective effects of pituitary adenylate cyclase activating polypeptide have not been examined in adult tissues. To study the effects of pituitary adenylate cyclase activating polypeptide on neurons in apoptosis, we measured caspase activation in adult olfactory receptor neurons in vitro. Interestingly, we found that the protective effects of pituitary adenylate cyclase activating polypeptide were related to the absence of a 4-aminopyridine (IC50=144 microM) sensitive rapidly inactivating potassium current often referred to as A-type current. In the presence of 40 nM pituitary adenylate cyclase activating polypeptide 38, both A-type current and activated caspases were significantly reduced. A-type current reduction by pituitary adenylate cyclase activating polypeptide was blocked by inhibiting the phospholipase C pathway, but not the adenylyl cyclase pathway. Our observation that 5 mM 4-aminopyridine mimicked the caspase inhibiting effects of pituitary adenylate cyclase activating polypeptide indicates that A-type current is involved in apoptosis. This work contributes to our growing understanding that potassium currents are involved with the activation of caspases to affect the balance between cell life and death.

Modulation by PKA of the hyperpolarization-activated current (Ih) in cultured rat olfactory receptor neurons.

The hyperpolarization-activated Ih channel is modulated by neurotransmitters acting through the cAMP messenger system. In rat olfactory receptor neurons (ORNs), dopamine, by inhibition of adenylyl cyclase, shifts the voltage of half-maximal activation (V1/2) of Ih to more negative potentials and decreases Ih maximal relative conductance. Whether these effects result from a phosphorylation-dependent mechanism is unclear. Therefore, we used whole-cell patch-clamp recording techniques to study cAMP-dependent phosphorylation via PKA on Ih in rat ORNs. General protein kinase inhibition (50 nM K252a) produced a hyperpolarizing shift in Ih V1/2 and decreased Ih maximal conductance. Specific inhibition of PKA with H-89 (500 nM) also shifted the V1/2 of Ih to more negative potentials, and, in some cells, decreased Ih maximal conductance. PKA-mediated phosphorylation (cBIMPS, 50 mM) shifted Ih V1/2 more positive, modulated the kinetics of Ih channel activation and increased Ih peak current amplitude. Internal perfusion of the catalytic subunit of PKA (84 nM) also shifted Ih V1/2 positive and this shift was blocked by co-perfusion with PKI (50 nM). These results show that in rat ORNs, the voltage dependence of Ih activation can be modulated by PKA-dependent phosphorylation. We also show that PKA and other protein kinases may be involved in the regulation of Ih maximal conductance. Our findings suggest that changes in the phosphorylation state of ORNs may affect resting properties as well as modulate odor sensitivity.

Identification and characterization of neuronal precursors and their progeny from human fetal tissue.

We have examined primary human neuronal precursors (HNPs) from 18-22-week-old fetuses. We showed that E-NCAM/MAP2/beta-III tubulin-immunoreactive neuronal precursors divide in vitro and could be induced to differentiate into mature neurons in 2 weeks. HNPs did not express nestin and differentiated slowly compared to rodent neuronal restricted precursors (NRPs, 5 days). Immunocytochemical and physiological analyses showed that HNPs could generate a heterogeneous population of neurons that expressed neurofilament-associated protein and various neurotransmitters, neurotransmitter synthesizing enzymes, voltage-gated ion channels, and ligand-gated neurotransmitter receptors and could fire action potentials. Undifferentiated and differentiated HNPs did not coexpress glial markers. Only a subset of cells that expressed GFP under the control of the Talpha1 tubulin promoter was E-NCAM/beta-III tubulin-immunoreactive, indicating nonexclusive overlap between these two HNP cell populations. Overall, HNPs resemble NRPs isolated from rodent tissue and appear to be a neuronal precursor population.

Immunohistochemical evidence for the Na+/Ca2+ exchanger in squid olfactory neurons.

The olfactory organs from the squid Lolliguncula brevis are composed of a pseudostratified epithelium containing five morphological subtypes of chemosensory neurons and ciliated support cells. Physiological recordings have been made from two of the subtypes and only the type 4 neuron has been studied in detail. Odour-stimulated increases in intracellular calcium and rapid activation of an electrogenic Na+/Ca2+ exchanger current in type 4 neurons suggest that the exchanger proteins are localized very close to the transduction machinery. Electrophysiological studies have shown that olfactory signal transduction takes place in the apical ciliary regions of olfactory neurons. Using polyclonal antiserum against squid Na+/Ca2+ proteins, we observed specific staining in the ciliary region of cells that resemble type 2, 3, 4 and 5 neurons. Staining was also observed in axon bundles, and in muscle tissue. Collectively, these data support the model that Na+/Ca2+ exchanger proteins are localized to transduction machinery in cilia of type 4 neurons and suggest that the other olfactory subtypes also use Ca2+ during chemosensory responses.

Immunocytochemical and physiological characterization of a population of cultured human neural precursors.

Human neural precursor cells (HNPC) have recently become commercially available. In an effort to determine the usefulness of these cells for in vitro studies, we have grown cultured HNPCs (cHNPCs) according to the supplier specifications. Here we report our characterization of cHNPCs under nondifferentiating and differentiating growth conditions and make a comparison to primary HNPCs (pHNPCs) obtained at the same developmental time point from a different commercial supplier. We found that under nondifferentiating conditions, cHNPCs expressed nestin, divided rapidly, expressed few markers of differentiated cells, and displayed both 4-aminopyridine (4-AP)-sensitive and delayed-rectifier type K(+) currents. No inward currents were observed. On changing to differentiating culture conditions, a majority of the cells expressed neuronal markers, did not divide, expressed inward and outward time- and voltage-dependent currents, and responded to the application of the neurotransmitters acetylcholine and glutamate. The outward current densities were indistinguishable from those in undifferentiated cells. The inward currents included TTX-sensitive and -resistant Na(+) currents, sustained Ca(2+) currents, and an inwardly rectifying K(+) current. Comparison of the properties of differentiated cells from cHNPCs with neurons obtained from primary fetal cultures (pHNPCs) revealed two major differences: the differentiated cHNPCs did not express embryonic neural cell adhesion molecule (E-NCAM) immunoreactivity but did co-express GFAP immunoreactivity. The co-expression of neuronal and glial markers was likely due to the growth of cells in serum containing medium as the pHNPCs that were never exposed to serum did express E-NCAM and did not co-express glial fibrillary acidic protein (GFAP). The relevance of these results is discussed and compared with results from other neuronal progenitor populations and cultured human neuronal cells.

Electrogenic Na(+)/Ca(2+) exchange. A novel amplification step in squid olfactory transduction.

Olfactory receptor neurons (ORNs) from the squid, Lolliguncula brevis, respond to the odors l-glutamate or dopamine with increases in internal Ca(2+) concentrations ([Ca(2+)](i)). To directly asses the effects of increasing [Ca(2+)](i) in perforated-patched squid ORNs, we applied 10 mM caffeine to release Ca(2+) from internal stores. We observed an inward current response to caffeine. Monovalent cation replacement of Na(+) from the external bath solution completely and selectively inhibited the caffeine-induced response, and ruled out the possibility of a Ca(2+)-dependent nonselective cation current. The strict dependence on internal Ca(2+) and external Na(+) indicated that the inward current was due to an electrogenic Na(+)/Ca(2+) exchanger. Block of the caffeine-induced current by an inhibitor of Na(+)/Ca(2+) exchange (50-100 microM 2',4'-dichlorobenzamil) and reversibility of the exchanger current, further confirmed its presence. We tested whether Na(+)/Ca(2+) exchange contributed to odor responses by applying the aquatic odor l-glutamate in the presence and absence of 2', 4'-dichlorobenzamil. We found that electrogenic Na(+)/Ca(2+) exchange was responsible for approximately 26% of the total current associated with glutamate-induced odor responses. Although Na(+)/Ca(2+) exchangers are known to be present in ORNs from numerous species, this is the first work to demonstrate amplifying contributions of the exchanger current to odor transduction.

Mixture interactions of glutamate and betaine in single squid olfactory neurons.

We used nystatin-patch techniques to characterize the responses of squid olfactory receptor neurons to the attractive odorant, L-glutamate, and to study mixture interactions between glutamate and the aversive odorant, betaine. We report that glutamate activates a cation-selective conductance that is permeable to Ca2+, K+, and Na+ and which would depolarize squid olfactory receptor neurons under physiological conditions. The responses to glutamate were concentration dependent. The EC50 of individual cells ranged from 0.3 mmol x l(-l) to 85.0 mmol x l(-l). We found that individual cells were capable of responding to both glutamate and betaine, and that the relative magnitudes of these responses varied from cell to cell. Finally, we report that current responses to binary mixtures of glutamate and betaine are suppressed relative to the sum of the responses to the individual odors in single squid olfactory receptor neurons.

Calcium signalling in squid olfactory receptor neurons.

Isolated squid olfactory receptor neurons respond to dopamine and betaine with hyperpolarizing conductances. We used Ca(2+) imaging techniques to determine if changes in intracellular Ca(2+) were involved in transducing the hyperpolarizing odor responses. We found that dopamine activated release of Ca(2+) from intracellular stores while betaine did not change internal Ca(2+) concentrations. Application of 10 mM caffeine also released Ca(2+) from intracellular stores, suggesting the presence of ryanodine-like receptors. Depletion of intracellular stores with 100 microM thapsigargin revealed the presence of a Ca(2+) store depletion-activated Ca(2+) influx. The influx of Ca(2+) through the store-operated channel was reversibly blocked by 10 mM Cd(2+). Taken together, these data suggest a novel odor transduction system in squid olfactory receptor neurons involving Ca(2+) release from intracellular stores.

Lineage-restricted neural precursors can be isolated from both the mouse neural tube and cultured ES cells.

We have previously identified multipotent neuroepithelial (NEP) stem cells and lineage-restricted, self-renewing precursor cells termed NRPs (neuron-restricted precursors) and GRPs (glial-restricted precursors) present in the developing rat spinal cord (A. Kalyani, K. Hobson, and M. S. Rao, 1997, Dev. Biol. 186, 202-223; M. S. Rao and M. Mayer-Proschel, 1997, Dev. Biol. 188, 48-63; M. Mayer-Proschel, A. J. Kalyani, T. Mujtaba, and M. S. Rao, 1997, Neuron 19, 773-785). We now show that cells identical to rat NEPs, NRPs, and GRPs are present in mouse neural tubes and that immunoselection against cell surface markers E-NCAM and A2B5 can be used to isolate NRPs and GRPs, respectively. Restricted precursors similar to NRPs and GRPs can also be isolated from mouse embryonic stem cells (ES cells). ES cell-derived NRPs are E-NCAM immunoreactive, undergo self-renewal in defined medium, and differentiate into multiple neuronal phenotypes in mass culture. ES cells also generate A2B5-immunoreactive cells that are similar to E9 NEP-cell-derived GRPs and can differentiate into oligodendrocytes and astrocytes. Thus, lineage restricted precursors can be generated in vitro from cultured ES cells and these restricted precursors resemble those derived from mouse neural tubes. These results demonstrate the utility of using ES cells as a source of late embryonic precursor cells.

Common components of patch-clamp internal recording solutions can significantly affect protein kinase A activity.

Common components of whole-cell internal recording solutions were tested both in vitro and in patch-clamp experiments for their effects on the activity of cAMP-dependent protein kinase. Potassium fluoride (KF), 440 mM trimethylamine chloride and exclusion of bovine serum albumin (BSA) decreased the activity of the enzyme, while ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) and the potassium salts of aspartate, gluconate, methylsulfate and monobasic phosphate increased its activity. Addition of KF to the internal solution produced a hyperpolarizing shift in the V1/2 of Ih channel activation, consistent with the KF-induced reduction of protein kinase A activity. Therefore, consideration of the composition of internal solutions is warranted when studying channel physiology by patch-clamp techniques.

A method for maintaining odor-responsive adult rat olfactory receptor neurons in short-term culture.

We report a culture system requiring the addition of freshly made ascorbic acid to the medium, that supports the short-term survival of adult rat olfactory receptor neurons. The cultured neurons exhibit typical voltage-gated currents and are responsive to application of odorants.

Characterization of a Na+-dependent betaine transporter with Cl- channel properties in squid motor neurons.

Most marine invertebrates, including squids, use transporters to accumulate organic osmolytes such as betaine, to prevent water loss when exposed to elevated salinity. Although a limited number of flux studies have shown the Na+ dependence of betaine transport, nothing is known about the electrogenic properties of osmolyte transporters. We used whole cell and perforated-patch voltage-clamp techniques to characterize the electrical properties of the betaine transporter in giant fiber lobe motor neurons of the squid Lolliguncula brevis. Betaine activated a large, Cl--selective current that was reversibly blocked by 100 microM niflumic acid (97 +/- 2% block after 40 s, SD; n = 7) and partially inhibited by 500 microM SITS (29 +/- 11%; n = 5). The Cl- current was Na+ dependent and was virtually eliminated by isotonic replacement of Na+ with Li+, NMDG+, or Tris+. Concentration-response data revealed an EC50 in a physiologically relevant range for these animals of 5.1 +/- 0.9 mM (n = 11). In vertebrates, the betaine transporter is structurally related to the GABA transporter, and although GABA did not directly activate the betaine-induced current, it reversibly reduced betaine responses by 34 +/- 14% (n = 8). Short-term changes in osmolality alone did not activate the Cl- current, but when combined with betaine, Cl- currents increased in hypertonic solutions and decreased in hypotonic solutions. Activation of the betaine transporter and Cl- current in hypertonic conditions may affect both volume regulation and excitability in L. brevis motor neurons. This study is the first report of a novel betaine transporter in neurons that can act as a Cl- channel.

Transient and persistent tetrodotoxin-sensitive sodium currents in squid olfactory receptor neurons.

Squid olfactory receptor neurons are primary bipolar sensory neurons capable of transducing water-born odorant signals into electrical impulses that are transmitted to the brain. In this study, we have identified and characterized the macroscopic properties of voltage-gated Na+ channels in olfactory receptor neurons from the squid Lolliguncula brevis. Using whole-cell voltage-clamp techniques, we found that the voltage-gated Na+ channels were tetrodotoxin sensitive and had current densities ranging from 5 to 169 pA pF-1. Analyses of the voltage dependence and kinetics revealed interesting differences from voltage-gated Na+ channels in olfactory receptor neurons from other species; the voltage of half-inactivation was shifted to the right and the voltage of half-activation was shifted to the left such that a "window-current" occurred, where 10-18% of the Na+ channels activated and did not inactivate at potentials near action potential threshold. Our findings suggest that in squid olfactory neurons, a subset of voltage-gated Na+ channels may play a role in generating a pacemaker-type current for setting the tonic levels of electrical activity required for transmission of hyperpolarizing odor responses to the brain.

Dopamine modulates inwardly rectifying hyperpolarization-activated current (Ih) in cultured rat olfactory receptor neurons.

The presence of dopamine receptors in olfactory receptor neurons (ORNs) suggests that odor sensitivity may be modulated by neurotransmitters at the level of primary sensory neurons. Using standard patch-clamp techniques on rat ORNs, we found that 1 microM dopamine, 500 microM SQ 22536 (SQ, an adenylyl cyclase inhibitor), 20 and 50 microM quinpirole (a selective dopamine D2 receptor agonist), and 1 mM adenosine 3', 5'-cyclic monophosphate (cAMP) modulate the hyperpolarization-activated current Ih. On hyperpolarizing from a holding potential of -58 mV, a small Cs+-sensitive inwardly rectifying current (Ih) was observed. Increases in extracellular K+ increased Ih amplitude without shifting its voltage dependence of activation, whereas increases in temperature produced an increase in Ih amplitude and a hyperpolarizing shift in the activation curve. Application of 1 microM dopamine reversibly shifted Ih activation to more negative potentials and decreased Ih current amplitudes. These effects were blocked by concomitant application of dopamine with sulpiride, a selective dopamine D2 receptor antagonist. The effects of dopamine were mimicked by quinpirole. Quinpirole (20 microM) decreased Ih current amplitude, but was without effect on Ih voltage dependence of activation. However, 50 microM quinpirole produced both a reduction of Ih peak currents and a hyperpolarizing shift in the activation curve for Ih. External application of the adenylyl cyclase inhibitor SQ 22536 produced a reversible decrease in peak currents but had no effect on Ih voltage dependence of activation, whereas internal application of cAMP shifted Ih activation to more depolarized potentials. Because Ih modulates cell excitability and spike frequency adaptation, our findings support a role for dopamine in modulating the sensitivity and output of rat ORNs to odorants.

Catecholamine concentrations in rat nasal mucus are modulated by trigeminal stimulation of the nasal cavity.

Olfactory mucus provides the perireceptor environment in which the initial steps of olfactory signal transduction occur [5]. Extrinsic autonomic and trigeminal innervation controls mucus secretion and may release neurotransmitters into nasal mucus [13]. We quantitated catecholamines in rat nasal mucus and found that catecholamine levels first increased and then declined with trigeminal stimulation. These data indicate that catecholamine levels are regulated in nasal mucus and could modulate the odor sensitivity of olfactory sensory neurons.

Spinal cord neuronal precursors generate multiple neuronal phenotypes in culture.

Neuronal restricted precursors (NRPs) () can generate multiple neurotransmitter phenotypes during maturation in culture. Undifferentiated E-NCAM+ (embryonic neural cell adhesion molecule) immunoreactive NRPs are mitotically active and electrically immature, and they express only a subset of neuronal markers. Fully mature cells are postmitotic, process-bearing cells that are neurofilament-M and synaptophysin immunoreactive, and they synthesize and respond to different subsets of neurotransmitter molecules. Mature neurons that synthesize and respond to glycine, glutamate, GABA, dopamine, and acetylcholine can be identified by immunocytochemistry, RT-PCR, and calcium imaging in mass cultures. Individual NRPs also generate heterogeneous progeny as assessed by neurotransmitter response and synthesis, demonstrating the multipotent nature of the precursor cells. Differentiation can be modulated by sonic hedgehog (Shh) and bone morphogenetic protein (BMP)-2/4 molecules. Shh acts as a mitogen and inhibits differentiation (including cholinergic differentiation). BMP-2 and BMP-4, in contrast, inhibit cell division and promote differentiation (including cholinergic differentiation). Thus, a single neuronal precursor cell can differentiate into multiple classes of neurons, and this differentiation can be modulated by environmental signals.

Betaine activates a hyperpolarizing chloride conductance in squid olfactory receptor neurons.

Isolated olfactory receptor neurons from the squid Lolliguncula brevis respond to betaine, a repellent odorant, with hyperpolarizing receptor potentials. Using perforated-patch techniques, we determined that the hyperpolarizing conductance was selective for Cl- and could be reversibly blocked by the Cl- channel blockers 4-acetamido-4'-isothio-cyanatistilbene-2,2'disulfonic acid and niflumic acid. Gramicidin-patch recordings revealed that [Cl-]i in squid olfactory receptor neurons is normally very low compared to vertebrate olfactory receptor neurons, and that activating a Cl- conductance would hyperpolarize the cell in vivo. The lack of dependence on internal or external K+ or Na+ ruled out the possibility that the Cl- conductance was generated by a cation-dependent cotransporter or pump. Common G-protein-dependent signalling pathways, including phospholipase C, arachidonic acid, and cyclic nucleotides, do not appear to be involved. Ca2+ imaging experiments showed that betaine did not affect [Ca2+]i, suggesting that the Cl- current is not Ca2+ dependent. Our findings represent the first report of an odorant-activated, hyperpolarizing chloride conductance in olfactory receptor neurons.

Characterization of voltage- and Ca(2+)-activated K+ channels in squid olfactory receptor neurons.

We performed whole-cell voltage-clamp experiments on isolated olfactory neurons from the squid Lolliguncula brevis. Total outward currents were composed of three identifiable K+ currents: a delayed rectifier K+ current that showed slow inactivation and was sensitive to 5 mmol l-1 tetraethylammonium; a rapidly inactivating, 4-aminopyridine (4-AP)-sensitive, A-type K+ current and a Ca(2+)-sensitive K+ current that was blocked by 200 nmol l-1 charybdotoxin and 10 mmol l-1 Cd2+ but was insensitive to apamin. The proportion of each current type varied from cell to cell, suggesting that responses to a given odorant would depend of the complement of channels present. The kinetics of the K+ currents were affected by temperature, with Q10 values ranging from 2 to 6. The identification and characterization of these K+ currents will greatly aid our understanding of action potential generation in these cells and will facilitate modelling of how odor responses are transduced and coded in squid olfactory receptor neurons.

Quantification of L-Dopa and Dopamine in Squid Ink: Implications for Chemoreception.

Squid ink is an alarm substance that both confuses predators and alerts conspecifics to the presence of danger. Although the ejection of ink is a powerful visual stimulus, studies also indicate a chemical component to the signal. Squid ink is composed mainly of melanin pigments, but the nonpigmented portion of the ink contains the enzymes and precursors of melanin synthesis. Our previous behavioral studies showed that squid olfactory organs detect L-dopa, a key chemical in melanogenesis. Squid olfactory neurons also respond to dopamine, a biogenic amine not previously described in squid ink. We performed HPLC on ink taken from the ink sacs of adult Loligo opalescens. The ink was conjugated with orthophthaldialdehyde (OPA) and injected into the HPLC, and amine-containing compounds were detected fluorometrically. Standard curves constructed for L-dopa and dopamine allowed quantitation from individual ink sacs. We found that L-dopa was present in undiluted ink at a mean concentration of 1.15 mM and was significantly greater than the mean dopamine concentration of 0.19 mM. These values are greater than those at which both compounds are effective in behavioral and electrophysiological experiments. In addition we found that an unidentified antioxidant in the ink may prevent rapid oxidation of L-dopa and dopamine following dilution in seawater.