Molecular tolerance of voltage-gated calcium channels is evident after short exposures to alcohol in vasopressin-releasing nerve terminals.

BACKGROUND: Voltage-gated calcium channels (VGCCs) in rat neurohypophysial terminals exhibit molecular tolerance to alcohol, including desensitization to the drug and increased current density, after 3 weeks of alcohol drinking. Moreover, after this time, terminals from drinking rats exhibit diminished alcohol inhibition of vasopressin (AVP) release. METHODS: We took advantage of organotypic cultures (explants) of the hypothalamo-neurohypophysial system (HNS) to extend our analysis of molecular tolerance to 2 classes of the VGCC. The isolated HNS explant allows much finer temporal resolution of molecular tolerance than do voluntary drinking paradigms. After exposure of the HNS explant to alcohol, terminals are isolated by mechanical treatment and plated in a dish. Patch clamp recording techniques are used to obtain VGCC currents, and immunohistochemistry is used to determine VGCC distribution. A release assay is used to provide functional readout of AVP release. RESULTS: We show that even a brief, 1-hour exposure to a clinically relevant concentration of alcohol is sufficient to evoke similar changes to those observed after several weeks of exposure. Acute ethanol (EtOH) exposure inhibits high K(+) -induced AVP release from naïve terminals. However, terminals pre-exposed to 20 mM EtOH for 1 hour become tolerant to EtOH, and subsequent exposure has significantly less effect on high K(+) -induced AVP release. Electrophysiological recordings indicate that among different types of VGCCs present in the neuronal terminal, the L-type is the most affected by alcohol. The current density of L-type current is significantly increased (approximately 50%), while its responsiveness to alcohol is significantly diminished (approximately 50%), after brief alcohol exposure. Fluorescent imaging results were consistent with the electrophysiology and suggest that the increased current density of VGCCs after brief exposure is attributable to combined synthesis of 1.2 and 1.3 subtypes of the L-type VGCC and redistribution of channel protein into terminal plasma membrane. CONCLUSIONS: These data indicate that a brief alcohol exposure affects subsequent alcohol sensitivity of VGCCs and neuropeptide release from presynaptic terminals.

Olfactory discrimination largely persists in mice with defects in odorant receptor expression and axon guidance.

BACKGROUND: The defining feature of the main olfactory system in mice is that each olfactory sensory neuron expresses only one of more than a thousand different odorant receptor genes. Axons expressing the same odorant receptor converge onto a small number of targets in the olfactory bulb such that each glomerulus is made up of axon terminals expressing just one odorant receptor. It is thought that this precision in axon targeting is required to maintain highly refined odor discrimination. We previously showed that ß3GnT2(-/-) mice have severe developmental and axon guidance defects. The phenotype of these mice is similar to adenylyl cyclase 3 (AC3) knockout mice largely due to the significant down-regulation of AC3 activity in ß3GnT2(-/-) neurons. RESULTS: Microarray analysis reveals that nearly one quarter of all odorant receptor genes are down regulated in ß3GnT2(-/-) mice compared to controls. Analysis of OR expression by quantitative PCR and in situ hybridization demonstrates that the number of neurons expressing some odorant receptors, such as mOR256-17, is increased by nearly 60% whereas for others such as mOR28 the number of neurons is decreased by more than 75% in ß3GnT2(-/-) olfactory epithelia. Analysis of axon trajectories confirms that many axons track to inappropriate targets in ß3GnT2(-/-) mice, and some glomeruli are populated by axons expressing more than one odorant receptor. Results show that mutant mice perform nearly as well as control mice in an odor discrimination task. In addition, in situ hybridization studies indicate that the expression of several activity dependent genes is unaffected in ß3GnT2(-/-) olfactory neurons. CONCLUSIONS: Results presented here show that many odorant receptors are under-expressed in ß3GnT2(-/-) mice and further demonstrate that additional axon subsets grow into inappropriate targets or minimally innervate glomeruli in the olfactory bulb. Odor evoked gene expression is unchanged and ß3GnT2(-/-) mice exhibit a relatively small deficit in their ability to discriminate divergent odors. Results suggest that despite the fact that ß3GnT2(-/-) mice have decreased AC3 activity, decreased expression of many ORs, and display many axon growth and guidance errors, odor-evoked activity in cilia of mutant olfactory neurons remains largely intact.

ß3GnT2 maintains adenylyl cyclase-3 signaling and axon guidance molecule expression in the olfactory epithelium.

In the olfactory epithelium (OE), odorant receptor stimulation generates cAMP signals that function in both odor detection and the regulation of axon guidance molecule expression. The enzyme that synthesizes cAMP, adenylyl cyclase 3 (AC3), is coexpressed in olfactory sensory neurons (OSNs) with poly-N-acetyllactosamine (PLN) oligosaccharides determined by the glycosyltransferase ß3GnT2. The loss of either enzyme results in similar defects in olfactory bulb (OB) innervation and OSN survival, suggesting that glycosylation may be important for AC3 function. We show here that AC3 is extensively modified with N-linked PLN, which is essential for AC3 activity and localization. On Western blots, AC3 from the wild-type OE migrates diffusely as a heavily glycosylated 200 kDa band that interacts with the PLN-binding lectin LEA. AC3 from the ß3GnT2(-/-) OE loses these PLN modifications, migrating instead as a 140 kDa glycoprotein. Furthermore, basal and forskolin-stimulated cAMP production is reduced 80-90% in the ß3GnT2(-/-) OE. Although AC3 traffics normally to null OSN cilia, it is absent from axon projections that aberrantly target the OB. The cAMP-dependent guidance receptor neuropilin-1 is also lost from ß3GnT2(-/-) OSNs and axons, while semaphorin-3A ligand expression is upregulated. In addition, kirrel2, a mosaically expressed adhesion molecule that functions in axon sorting, is absent from ß3GnT2(-/-) OB projections. These results demonstrate that PLN glycans are essential in OSNs for proper AC3 localization and function. We propose that the loss of cAMP-dependent guidance cues is also a critical factor in the severe axon guidance defects observed in ß3GnT2(-/-) mice.

Endogenous ATP potentiates only vasopressin secretion from neurohypophysial terminals.

Exogenous ATP induces inward currents and causes the release of arginine-vasopressin (AVP) from isolated neurohypophysial terminals (NHT); both effects are inhibited by the P2X2 and P2X3 antagonists, suramin and PPADS. Here we examined the role of endogenous ATP in the neurohypophysis. Stimulation of NHT caused the release of both AVP and ATP. ATP induced a potentiation in the stimulated release of AVP, but not of oxytocin (OT), which was blocked by the presence of suramin. In loose-patch clamp recordings, from intact neurohypophyses, suramin or PPADS produces an inhibition of action potential currents in a static bath, that can be mimicked by a hyperpolarization of the resting membrane potential (RMP). Correspondingly, in a static versus perfused bath there is a depolarization of the RMP of NHT, which was reduced by either suramin or PPADS. We measured an accumulation of ATP (3.7 +/- 0.7 microM) released from NHT in a static bath. Applications of either suramin or PPADS to a static bath decreased burst-stimulated capacitance increases in NHT. Finally, only vasopressin release from electrically stimulated intact neurohypophyses was reduced in the presence of Suramin or PPADS. These data suggest that there was sufficient accumulation of ATP released from the neurohypophysis during stimulations to depolarize its nerve terminals. This would occur via the opening of P2X2 and P2X3 receptors, inducing an influx of Ca2+. The subsequent elevation in [Ca2+](i) would further increase the stimulated release of only vasopressin from NHT terminals. Such purinergic feedback mechanisms could be physiologically important at most CNS synapses.

Identification of the neuropeptide content of individual rat neurohypophysial terminals.

The objective of this study was to develop a method that could reliably determine the arginine vasopressin (AVP) and/or oxytocin (OT) content of individual rat neurohypophysial terminals (NHT) >or=5 microm in diameter, the size used for electrophysiological recordings. We used a commercially available, highly sensitive enzyme-linked immunoassay (ELISA) kit with a sensitivity of 0.25 pg to AVP and of 1.0pg to OT. The NHT content of AVP (2.21+/-0.10 pg) was greater than OT (1.77+/-0.08 pg) and increased with terminal size. AVP-positive terminals (10.2+/-0.21 microm) were larger in diameter than OT-positive terminals (9.1+/-0.24 microm). Immunocytochemical techniques indicated that a higher percentage (58%) of smaller terminals contained OT, and that a higher percentage (42%) of larger NHTs were colabeled. Similar percentages of AVP-positive terminals were obtained between immunocytochemical (73%) and ELISA (72%) methods when NHTs were assayed for AVP alone, but there was a higher percentage of OT terminals when using immunocytochemistry (43%) compared to ELISA (26%). The percent of AVP-positive (60%) and OT-positive (18%) terminals decreased when NHT were assayed for both AVP and OT. Therefore, the best method to reliably identify AVP-positive NHTs is to assay only for AVP, since this allows the conclusion that AVP-negative terminals contain only OT.

Endogenous adenosine inhibits CNS terminal Ca(2+) currents and exocytosis.

Bursts of action potentials (APs) are crucial for the release of neurotransmitters from dense core granules. This has been most definitively shown for neuropeptide release in the hypothalamic neurohypophysial system (HNS). Why such bursts are necessary, however, is not well understood. Thus far, biophysical characterization of channels involved in depolarization-secretion coupling cannot completely explain this phenomenon at HNS terminals, so purinergic feedback mechanisms have been proposed. We have previously shown that ATP, acting via P2X receptors, potentiates release from HNS terminals, but that its metabolite adenosine, via A(1) receptors acting on transient Ca(2+) currents, inhibit neuropeptide secretion. We now show that endogenous adenosine levels are sufficient to cause tonic inhibition of transient Ca(2+) currents and of stimulated exocytosis in HNS terminals. Initial non-detectable adenosine levels in the static bath increased to 2.9 microM after 40 min. These terminals exhibit an inhibition (39%) of their transient inward Ca(2+) current in a static bath when compared to a constant perfusion stream. CPT, an A(1) adenosine receptor antagonist, greatly reduced this tonic inhibition. An ecto-ATPase antagonist, ARL-67156, similarly reduced tonic inhibition, but CPT had no further effect, suggesting that endogenous adenosine is due to breakdown of released ATP. Finally, stimulated capacitance changes were greatly enhanced (600%) by adding CPT to the static bath. Thus, endogenous adenosine functions at terminals in a negative-feedback mechanism and, therefore, could help terminate peptide release by bursts of APs initiated in HNS cell bodies. This could be a general mechanism for controlling transmitter release in these and other CNS terminals.

ATP elicits inward currents in isolated vasopressinergic neurohypophysial terminals via P2X2 and P2X3 receptors.

Effects of extracellular adenosine tri-phosphate (ATP) on ionic currents were investigated using the perforated-patch whole-cell recording technique on isolated terminals of the Hypothalamic Neurohypophysial System (HNS). ATP induced a current response in 70% of these isolated terminals. This inwardly-rectifying, inactivating current had an apparent reversal near 0 mV and was dose-dependent on ATP with an EC50=9.6+/-1.0 microM. In addition, current amplitudes measured at maximal ATP concentrations and optimum holding potentials had a current density of 70.8 pA pF(-1) and were greatly inhibited by suramin and PPADS. Different purinergic receptor agonists were tested, with the following efficacy: ATP > or = 2-methylthioATP > ATP-gamma-S > Bz-Bz-ATP > alpha,beta-methylene-ATP > beta,gamma-methylene-ATP. However, UTP and ADP were ineffective. These data suggest the involvement of a P2X purinergic receptor in the ATP-induced responses. Immunocytochemical labeling in vasopressinergic terminals indicates the existence of P2X(2,3,4, and 7), but not P2X6 receptors. Additionally, P2X(2 and 3) were not found in terminals which labeled for oxytocin. In summary, the EC50, decay, inactivation, and pharmacology indicate that a functional mixture of P2X(2 and 3) homomeric receptors mediate the majority of the ATP responses in vasopressinergic HNS terminals. We speculate that the characteristics of these types of receptors reflect the function of co-released ATP in the terminal compartment of these and other CNS neurons.

Alcohol tolerance in large-conductance, calcium-activated potassium channels of CNS terminals is intrinsic and includes two components: decreased ethanol potentiation and decreased channel density.

Tolerance is an important element of drug addiction and provides a model for understanding neuronal plasticity. The hypothalamic-neurohypophysial system (HNS) is an established preparation in which to study the actions of alcohol. Acute application of alcohol to the rat neurohypophysis potentiates large-conductance calcium-sensitive potassium channels (BK), contributing to inhibition of hormone secretion. A cultured HNS explant from adult rat was used to explore the molecular mechanisms of BK tolerance after prolonged alcohol exposure. Ethanol tolerance was intrinsic to the HNS and consisted of: (1) decreased BK potentiation by ethanol, complete within 12 min of exposure, and (2) decreased current density, which was not complete until 24 hr after exposure, indicating that the two components of tolerance represent distinct processes. Single-channel properties were not affected by chronic exposure, suggesting that decreased current density resulted from downregulation of functional channels in the membrane. Indeed, we observed decreased immunolabeling against the BK alpha-subunit on the surface of tolerant terminals. Analysis using confocal microscopy revealed a reduction of BK channel clustering, likely associated with the internalization of the channel.

Evidence for the disproportionate mapping of olfactory airspace onto the main olfactory bulb of the hamster.

Olfactory receptor neurons (ORNs) project to the rodent main olfactory bulb (MOB) from spatially distinct air channels in the olfactory recesses of the nose. The relatively smooth central channels of the dorsal meatus map onto the dorsal MOB, whereas the highly convoluted peripheral channels of the ethmoid turbinates project to the ventral MOB. Medial and lateral components of each projection stream innervate the medial and lateral MOB, respectively. To ascertain whether such topography entails the disproportionate representation seen in other sensory maps, we used disector-based stereological techniques in hamsters to estimate the number of ORNs associated with each channel in the nose and the number of their targets (glomeruli and mitral and tufted cells) in corresponding divisions of the MOB. Each circumferential half of the MOB (dorsal/ventral, medial/lateral) contained about 50% of the 3,100 glomeruli and about 50% of the 160,000 mitral and tufted cells per bulb. We found equivalent numbers of ORNs with dendritic knobs in the medial and lateral channels (4.5 million each). However, the central channels had only 2 million knobbed ORNs, whereas the peripheral channels had 7 million. Thus, there is a disproportionate mapping of the central-peripheral axis of olfactory airspace onto the dorsal-ventral axis of the MOB, encompassing a greater than threefold variation in the average convergence of ORNs onto MOB secondary neurons. We hypothesize that the disproportionate projections help to optimize chemospecific processing by compensating, with differing sensitivity, for significant variation in the distribution and concentration of odorant molecules along the olfactory air channels during sniffing.

NADPH diaphorase activity in olfactory receptor neurons and their axons conforms to a rhinotopically-distinct dorsal zone of the hamster nasal cavity and main olfactory bulb.

NADPH diaphorase histochemical protocols were optimized for the histochemical labeling of olfactory receptor neurons (ORNs) in the nasal cavity and their axon terminals in glomeruli of the main olfactory bulb (MOB) in the Syrian hamster. This labeling was then used to map and quantify the spatial distribution of ORNs and their central projections. Diaphorase-positive ORNs were found to be rhinotopically restricted to dorsal-medially situated segments of sensory mucosa associated with central air channels in the nose, together constituting about 25% of the total receptor sheet. This topography closely resembles the zonal expression patterns of putative odorant receptor genes and cell surface glycoconjugates in the nose. Moreover, the projections of ORNs in the diaphorase-positive dorsal/central zone were found to expand onto the entire dorsal half of the MOB, consistent with spatial patterns discerned in retrograde tract-tracing studies. These boundaries indicate that dorsal/central zone ORNs project to a disproportionately larger region of the MOB than do those in the more ventral/peripheral zones. The demonstration of NADPH diaphorase activity in ORNs is inconsistent with the expression of the best-known NADPH-dependent enzymes, such as nitric oxide synthase (neuronal and endothelial isoforms) and NADPH cytochrome P450 oxidoreductase. Understanding the spatial patterning of histochemical labeling in ORNs should facilitate the biochemical identification of this diaphorase.

Integrated channel plasticity contributes to alcohol tolerance in neurohypophysial terminals.

Short-term ethanol challenge results in the reduction of peptide hormone release from the rat neurohypophysis. However, rats that have been maintained on an ethanol-containing diet for 3 to 4 weeks exhibit tolerance to this effect. Mechanistic underpinnings of this tolerance were probed by examining four ion channel conductances critical for neurohormone release. The voltage-gated L-type calcium channel and the functionally linked calcium-activated BK channel represent a functional dyad. Although these channels show opposite drug responses in the naive terminal (i.e., the L-type Ca2+ channel is inhibited whereas the BK channel is potentiated), the effect of long-term alcohol exposure is to decrease sensitivity to the short-term administration of drug in both instances. In addition to the shift in sensitivity, current density increased for the L-type Ca2+ current and decreased for the BK current, consistent with a compensatory change. Sensitivity to alcohol was also altered for two other channel types studied. Inhibition of the voltage-gated transient Ca2+ current was lessened after long-term treatment. I(A,) which is not sensitive to the drug at clinically relevant concentrations in terminals from the naive rat, acquires sensitivity after long-term exposure, representing a potentially novel type of tolerance. However, neither the transient Ca2+ current nor I(A) shows a change in current density, demonstrating the selectivity of this aspect of tolerance. Overall, these results demonstrate that channel plasticity can explain at least a portion of the behavioral tolerance resulting from changes in sensitivity of peptide hormone release. Furthermore, they suggest that an understanding of tolerance requires the examination of dynamically coupled channel populations.