The first week following insemination is the period of major pregnancy failure in pasture-grazed dairy cows.

A 60% pregnancy success for inseminations is targeted to optimize production efficiency for dairy cows within a seasonal, pasture-grazed system. Routine measures of pregnancy success are widely available but are limited, in practice, to a gestation stage beyond the first 28 d. Although some historical data exist on embryonic mortality before this stage, productivity of dairy systems and genetics of the cows have advanced significantly in recent decades. Accordingly, the aim was to construct an updated estimate of pregnancy success at key developmental stages during the first 70 d after insemination. Blood samples were collected for progesterone concentrations on d 0 and 7. A temporal series of 4 groups spanning fertilization through d 70 were conducted on 4 seasonal, pasture-grazed dairy farms (n = 1,467 cows) during the first 21 d of the seasonal breeding period. Morphological examination was undertaken on embryos collected on d 7 (group E7) and 15 (group E15), and pregnancy was diagnosed via ultrasonography on approximately d 28 and 35 (group E35) as well as d 70 (group E70). Fertilization, embryo, and fetal evaluation for viability established a pregnancy success pattern. Additionally, cow and on-farm risk factor variables associated with pregnancy success were evaluated. We estimated pregnancy success rates of 70.9%, 59.1%, 63.8%, 62.3%, and 56.7% at d 7, 15, 28, 35, and 70, respectively. Fertilization failure (15.8%) and embryonic arrest before the morula stage (10.3%) were the major developmental events contributing to first-week pregnancy failures. Embryo elongation failure of 7% contributed to pregnancy failure during the second week. The risk factors for pregnancy success that were related to the cows included interval between calving and insemination, and d-7 plasma progesterone concentrations, whereas insemination sire was associated with pregnancy outcome. Most pregnancy failure occurs during the first week among seasonal-calving pasture-grazed dairy cows.

Combining genetic and physiological data to identify predictors of lifetime reproductive success and the effect of selection on these predictors on underlying fertility traits.

Fertility of the dairy cow relies on complex interactions between genetics, physiology, and management. Mathematical modeling can combine a range of information sources to facilitate informed predictions of cow fertility in scenarios that are difficult to evaluate empirically. We have developed a stochastic model that incorporates genetic and physiological data from more than 70 published reports on a wide range of fertility-related traits in dairy cattle. The model simulates pedigree, random mating, genetically correlated traits (in the form of breeding values for traits such as hours in estrus, estrous cycle length, age at puberty, milk yield, and so on), and interacting environmental variables. This model was used to generate a large simulated data set (200,000 cows replicated 100 times) of herd records within a seasonal dairy production system (based on an average New Zealand system). Using these simulated data, we investigated the genetic component of lifetime reproductive success (LRS), which, in reality, would be impractical to assess empirically. We defined LRS as the total number of times, during her lifetime, a cow calved within the first 42 d of the calving season. Sire estimated breeding values for LRS and other traits were calculated using simulated daughter records. Daughter pregnancy rate in the first lactation (PD_1) was the strongest single predictor of a sire's genetic merit for LRS (R2 = 0.81). A simple predictive model containing PD_1, calving date for the second season and calving rate in the first season provided a good estimate of sire LRS (R2 = 0.97). Daughters from sires with extremely high (n = 99,995 daughters, sire LRS = +0.70) or low (n = 99,635 daughters, sire LRS = -0.73) LRS estimated breeding values were compared over a single generation. Of the 14 underlying component traits of fertility, 12 were divergent between the 2 lines. This suggests that genetic variation in female fertility has a complex and multifactorial genetic basis. When simulated phenotypes were compared, daughters of the high LRS sires (HiFERT) reached puberty 44.5 d younger and calved ∼14 d younger at each parity than daughters from low LRS sires (LoFERT). Despite having a much lower genetic potential for milk production (-400 L/lactation) than LoFERT cows, HiFERT cows produced 33% more milk over their lifetime due to additional lactations before culling. In summary, this simulation model suggests that LRS contributes substantially to cow productivity, and novel selection criteria would facilitate a more accurate prediction at a younger age.

Embryo loss in cattle between Days 7 and 16 of pregnancy.

Embryo loss between embryonic Days 7 and 16 (Day 0=day of IVF) in nonlactating cattle, Bos taurus, was analyzed using transfer of 2449 (in groups of 3 to 30) in vitro-produced (IVP) blastocysts. In 152 transfers, pregnancy losses attributable solely to recipient failings amounted to between 6% (beef heifers) and 16% (parous dairy cows), of which 3% were caused by uterine infections. Neither season, year, nor the age of the embryos on retrieval affected pregnancy rates. The latter observation indicated that the reason that a recipient failed to retain embryos was already present at the time of transfer. Notably, the proportion of embryos recovered decreased (P=0.03) as more embryos were transferred, particularly at later stages (Day 14, P<0.01). The average length of embryos decreased by approximately 5% for every additional embryo transferred (P<0.0001). These effects may be linked to embryonic migration. Embryo mortality inherent to the embryo during the second week of pregnancy was 24%. Additionally, 9% of Day 14 embryos were of inferior quality, as they did not contain an epiblast. Combining embryo and recipient causes but excluding infection effects, embryonic loss of IVP embryos during the second week of pregnancy amounted to 26% (heifers) or 34% (parous dairy cows). The length of embryos doubled every day between Days 9 and 16, with a 4.4-fold range in sizes representing two thirds of the variation in length. Embryos retrieved from heifers were twice the size of those incubated in parous cows (P<0.0001), indicating faster embryonic development/trophoblast proliferation in heifers. Whereas season did not affect embryo recoveries, length was lower (50%) in winter (winter-autumn, P<0.05; winter-spring, P<0.001). Lastly, transuterine migration in cattle, when transferring multiple embryos, commenced at Day 14 (4%) and had occurred in all recipients by Day 16 (38% of embryos found contralaterally).

The temporal relationship between oocyte maturation and early fertilisation events in relation to the pre-ovulatory LH peak and preimplantation embryo development in red deer (Cervus elaphus).

The temporal relationships among oocyte maturation, gamete transport and fertilisation following the pre-ovulatory luteinsing hormone surge in red deer were established; and secondly, early preimplantation development to the blastocyst stage in relation to the onset of oestrus was determined for red deer. In the first series of observations, oestrus was synchronised in April (N=22), for the fixed time recovery of gametes from 0 to 36 h after the estimated pre-ovulatory LH peak. Matings were observed and the time of the LH peak was determined from the retrospective analysis of blood plasma collected at 3h intervals. Gametes were recovered surgically and the meiotic status of follicular and ovulated oocytes assessed. Spermatozoa were recovered from the oviduct and their motility analysed by videomicroscopy. Nineteen of 22 hinds exhibited a pre-ovulatory LH surge and were observed to mate. Oocyte metaphase I occurred between 11 and 18 h, and metaphase II was completed within the follicle between 20 and 25 h following the pre-ovulatory LH peak. Fertilised ova were recovered from 30 to 36 h in both the ampulla and isthmic portions of the oviduct. Motile spermatozoa were first recovered from the isthmus and the ampulla at 13 and 21 h, respectively, after the LH peak. Hyperactive spermatozoa were observed in both the isthmus and the ampulla flushings but only from the eight hinds that had ovulated. In the second series of observations, 16 mature hinds were synchronised and allocated to groups for embryo collection on days 3, 5 and 7 after oestrus. Eight embryos were recovered; an 8-cell at 90 h, 3 morulae at 137, 138 and 186 h, and 4 blastocysts at 180, 182 and 190 h post-mating. Blastocysts were only recovered from the uterine horns and the mean+/-S.E.M. number of nuclei per blastocyst was 93.5+/-10.0 with a range of 66-114 cells. The results of this study will improve the application of assisted reproductive technologies to red deer as they indicate that oocyte maturation, fertilisation and early embryonic development of the red deer is similar to other domestic ruminants with the exception that the red deer embryo enters the uterus at the blastocyst stage.

Nicotinic signal transduction machinery.

Nicotinic synapses employ acetylcholine to activate ligand-gated ion channels that are cation-selective in vertebrates. Although the resulting nicotinic cholinergic transmission is famously excitatory at the neuromuscular junction, it plays many additional roles in the CNS. Most prevalent is that of modulation, usually involving calcium and signal transduction. Because of this, it is becoming increasingly important not only to understand the mechanisms that guide nicotinic receptors to appropriate locations but also to identify the postsynaptic machinery making possible the requisite signal transduction. Clearly, the kinds of components tethered in the vicinity of the receptor will assume a major role in determining the consequences of receptor activation. One of the most abundant and interesting nicotinic receptors in this respect is the species comprised of the alpha7 gene product (Broide and Leslie, 1999). These alpha7 homopentameric nicotinic acetylcholine receptors (alpha7 nAChRs) have a high relative permeability to calcium, rivaling that of NMDA receptors. But unlike NMDA receptors, alpha7 nAChRs promote calcium influx without requiring a coincident event such as membrane depolarization. As a result, the receptors are well equipped to regulate calcium-dependent events in neurons, particularly when depolarization might be occluded.

New developments reproductive technologies in deer.

In vitro embryo production is the platform for advanced reproductive technologies, such as cloning. The in vitro embryo production system developed for farmed red deer (Cervus elaphus) evolved along similar lines to that pioneered by other domestic species researchers. However, applying existing in vitro embryo production methods from these other species resulted in limited success and has necessitated developing a species-specific methodology for red deer based on the their physiology. Analysis of oviduct fluid led to the development of a semi-defined fertilization and culture media system, Deer Synthetic Oviduct Fluid (DSOF), which resulted in successful culture of red deer embryos to the blastocyst stage. Transvaginal ultrasound-guided ovarian examination and ovum pickup has enabled the study of seasonality constraint and propagation from selected female genetics, respectively. During the 4-month breeding season (April-July), 15% of cleaved oocytes developed to blastocysts, whereas no blastocysts developed from oocytes collected after July. The process of developing an in vitro embryo production system for farmed red deer may serve as a beneficial model for the propagation of endangered cervine species.

Ionic calcium levels in oviduct explant-conditioned media from an Australian marsupial, the brushtail possum (Trichosurus vulpecula) and its relevance to in vitro fertilization.

Gametes from the brushtail possum (Trichosurus vulpecula), an Australian marsupial, require exposure to oviductal cells and/or their secretions before sperm binding and penetration of the zona pellucida can occur. Sperm-egg fusion, the next critical step in fertilization has not previously been reported in vitro. Here we describe the refinement of an oviduct epithelial cell (OEC) explant culture system using two different media to obtain in vitro sperm-egg fusion in the brushtail possum for the first time. Conditioned media from OEC explant cultures were supplemented with either 1% fetal calf serum (FCS) or 1 mg/ml polyvinyl alcohol and used for co-culture of epididymal sperm and superovulated eggs. Under these conditions zona penetration rates varied from 0 to 46% and sperm-egg fusion from 0 to 20%. Analysis of explant conditioned media indicated that qualitative and quantitative differences between batches could account, at least partially, for the large variability in zona penetration rates. Conditioned media that contained approximately 1 mM of ionic calcium were most effective for achieving sperm capacitation, zona binding, and penetration and sperm-egg fusion. The reorientation of the sperm head to T-shape, an indicator of capacitation in the brushtail possum, was closely linked with the concentration of calcium present in vitro.

Development of in vitro embryo production systems for red deer (Cervus elaphus). Part 1. Effect of epithelial oviductal monolayers and heparin on in vitro sperm motility and penetration of in vitro matured oocytes.

In vitro fertilisation (IVF) protocols for red deer have yielded low fertilisation rates, with no embryo development beyond the eight-cell stage when heparin was used as the in vitro capacitation agent. As this low fertilisation rate may result from reduced motility, the present study investigated the use of red deer oviduct epithelial cell monolayers (COEM) and conditioned medium (Cm) from the monolayers to maintain red deer sperm motility in vitro. A second experiment compared the fertilisability of red deer sperm pre-incubated for 4-12h on COEM or for 4h in TALP medium supplemented with 20 microg of heparin.COEM was superior in maintaining red deer sperm motility compared with either Sp-TALP alone or Cm (P<0.05). COEM sustained sperm motility at levels comparable to the initial motility over the 24h period. The motility of sperm incubated in Sp-TALP and Cm was similar and had declined to less than 10% by 4h and no motile sperm were observed by 8h. Overall, the penetration rates of in vitro red deer oocytes were low (5-28%) regardless of sperm treatment. Sperm pre-incubated on COEM penetrated more oocytes than sperm incubated with heparin (P<0.001). Penetration rates were similar for 4-12h pre-incubation of sperm on COEM (P>0.50). Penetration rates were greater across all treatments when both sperm and oocytes were co-incubated for 24h compared to 12h (P<0.001). There were no differences in penetration rates among the four donor stags used in the study. It was concluded that COEM sustains red deer sperm motility in vitro during the 24h observation period. Pre-incubating sperm on COEM does increase sperm penetration rates compared with heparin alone, but at a rate too low and variable to be used on a routine basis. Overall, the penetration rates were comparable to those previously reported for red deer even though differences in heparin concentration, fertilisation systems and stags were used.

Development of in vitro embryo production systems for red deer (Cervus elaphus). Part 2. The timing of in vitro nuclear oocyte maturation.

The time course of in vitro red deer nuclear oocyte maturation was determined. Ovaries were obtained at slaughter and oocytes were aspirated from follicles greater than 2mm in diameter. Oocytes with compact cumulus cells were matured in 50 microl microdrops (10 per drop) under mineral oil containing TCM 199 supplemented with 0.33 mM pyruvate, 10 microg LH and FSH, 1 microg oestradiol and 10% foetal bovine serum. Oocytes were matured at 39 degrees C and 5% CO(2) in air. At 3h intervals (0-27 h) oocytes were removed from incubation, cumulus expansion scored and removed, and fixed oocytes in ethanol:acetic acid (3:1) for 48 h. Oocytes were stained with lacmoid (1%) and nuclear maturation assessed. Oocytes were arrested in the germinal vesicle (GV) stage at aspiration and up to 6h of incubation. The nuclear membrane began to disperse after 6h and by 10.6+/-0.6h of incubation 75% of the oocytes exhibited germinal vesicle breakdown (GVBD). The mean time for 50% of the oocytes to reach metaphase one (MI) and metaphase two (MII) was 11.7+/-0.4 and 24.8+/-0.9h, respectively. Cumulus oophorus were tightly compacted at aspiration and did not begin expansion until 12h of culture. Full expansion was complete by 18 h of culture. Corona radiata cells did not begin expansion until 15 h and were fully expanded by 24h. Results indicate that in vitro red deer oocyte maturation follows a similar time course of nuclear maturation as reported for bovine and ovine oocytes.

Development of in vitro embryo production systems for red deer (Cervus elaphus). Part 3. In vitro fertilisation using sheep serum as a capacitating agent and the subsequent birth of calves.

The following experiments investigated the use of sheep serum (SS) as a capacitating agent for red deer (Cervus elaphus) sperm during in vitro fertilisation. Red deer oocytes were collected at slaughter and matured in vitro for 24h in TCM-199 supplemented with 10% foetal calf serum, 10 microg ml(-1) FSH and LH, and 1microg ml(-1) of oestradiol. Fertilisation medium was IVF-SOF modified to contain 5mM Ca(2+) and no glucose. Experiment 1 investigated the addition of heparin, BSA (8 mg ml(-1)) or 20% SS. All oocytes were penetrated when IVF-SOF was supplemented with SS compared to 10 and 0% penetration when either heparin or BSA was present (P<0.01). However, 43.8% of these oocytes were polyspermic when the medium contained SS. In Experiment 2, the effect of sperm concentration on penetration rates during in vitro fertilisation was investigated. Total sperm penetration and monospermic penetration rates increased with increased sperm concentrations in a log linear manner (P<0.001) and both approached an asymptote at 0.4 x 10(6) sperm ml(-1) with 93.6 and 77% for total and monospermic penetration, respectively. Polyspermic fertilisation also increased with increasing sperm concentrations (P<0.05) but was variable (range 3.5+/-4.2 to 42.3+/-10.6%), especially at the lower sperm concentrations. Experiment 3 investigated the viability of these oocytes after transfer into red deer recipients. Fifteen 2- and 4-cell embryos were transferred into the oviducts of synchronized recipients 28 h post in vitro insemination. An additional fourteen embryos (8-10 cell) were transferred into synchronised recipients after 48 h of in vitro culture in either SOFaaBSA (n=10) or on red deer epithelial oviduct monolayers (n=4). Five (33% 5/15) of the recipients that received 2- and 4-cell embryos were pregnant at Day 45 (verified by ultrasonography) and four recipients subsequently calved. One recipient receiving an embryo cultured in SOFaaBSA was pregnant at Day 45 and subsequently calved. The birth of five normal calves indicate that full developmental competence of red deer oocytes matured and fertilised in vitro can be achieved by the techniques described.

Voltage-gated channels block nicotinic regulation of CREB phosphorylation and gene expression in neurons.

Synaptic activation of the transcription factor CREB and downstream gene expression usually depend on calcium influx aided by voltage-gated calcium channels. We find that nicotinic signaling, in contrast, activates CREB and gene expression in ciliary ganglion neurons both in culture and in situ only if voltage-gated channels are silent. The nicotinic response requires calcium influx and release from internal stores and acts through CaMK and MAPK pathways to sustain activated CREB. Voltage-gated channels mobilize CaMK to activate CREB initially, but they also enable calcineurin and PP1 to terminate the activation before transcription is affected. L-type voltage-gated channels dominate the outcome and block the effects of nicotinic signaling on transcription. This demonstrates a novel aspect of activity-dependent gene regulation.

Nicotinic acetylcholine receptors containing alpha 7 subunits on rat cortical neurons do not undergo long-lasting inactivation even when up-regulated by chronic nicotine exposure.

Chronic exposure to (-)nicotine has been widely reported to up-regulate nicotinic acetylcholine receptors on neurons and induce long-term inactivation as a possible cause. Nicotinic receptors containing alpha 7 subunits are among the most abundant in brain and influence diverse cellular events. Whole-cell patch clamp recording from embryonic rat cortical neurons in culture was used to identify responses from alpha 7-containing receptors. Immunochemical staining for glutamic acid decarboxylase (GAD) indicated that both GABAergic and non-GABAergic neurons expressed the receptors. Exposure to micromolar concentrations of nicotine for 1-4 days caused up-regulation of the receptors as measured by [alpha-(125)I]-bungarotoxin binding. Carbachol produced the same up-regulation, and cell counts demonstrated that neuronal survival was unchanged. The up-regulation was accompanied by an increased whole-cell response; no evidence was found for long-lasting inactivation. Autonomic alpha 7-containing receptors also avoided long-lasting inactivation, even though the receptors were down-regulated by nicotine. Blocking protein synthesis or protein glycosylation prevented receptor up-regulation on cortical neurons, suggesting that new synthesis was required. No evidence was found for a pre-existing intracellular pool that supplied receptors to the surface. The results indicate that alpha 7-containing receptors differ from other receptor subtypes in their regulation by nicotine and demonstrate further that long-lasting inactivation is not an obligatory requirement for up-regulation in this case.

beta -Amyloid peptide blocks the response of alpha 7-containing nicotinic receptors on hippocampal neurons.

Alzheimer's disease produces a devastating decline in mental function, with profound effects on learning and memory. Early consequences of the disease include the specific loss of cholinergic neurons in brain, diminished cholinergic signaling, and the accumulation of beta-amyloid peptide in neuritic plaques. Of the nicotinic acetylcholine receptors at risk, the most critical may be those containing the alpha7 gene product (alpha7-nAChRs), because they are widespread, have a high relative permeability to calcium, and regulate numerous cellular events in the nervous system. With the use of whole-cell patch-clamp recording we show here that nanomolar concentrations of beta-amyloid peptides specifically and reversibly block alpha7-nAChRs on rat hippocampal neurons in culture. The block is noncompetitive, voltage-independent, and use-independent and is mediated through the N-terminal extracellular domain of the receptor. It does not appear to require either calcium influx or G protein activation. beta-Amyloid blockade is likely to be a common feature of alpha7-nAChRs because it applies to the receptors at both somato-dendritic and presynaptic locations on rat hippocampal neurons and extends to homologous receptors on chick ciliary ganglion neurons as well. Because alpha7-nAChRs in the central nervous system are thought to have numerous functions and recently have been implicated in learning and memory, impaired receptor function in this case may contribute to cognitive deficits associated with Alzheimer's disease.

Synaptically driven calcium transients via nicotinic receptors on somatic spines.

Dendritic spines commonly receive glutamatergic innervation at postsynaptic densities and compartmentalize calcium influx arising from synaptic signaling. Recently, it was shown that a class of nicotinic acetylcholine receptors containing alpha7 subunits is concentrated on somatic spines emanating from chick ciliary ganglion neurons. The receptors have a high relative calcium permeability and contribute importantly to synaptic currents, although they appear to be excluded from postsynaptic densities. Here we show that low-frequency synaptic stimulation of the alpha7-containing receptors induces calcium transients confined to the spines. High-frequency stimulation induces a transient calcium elevation in the spines and a more sustained cell-wide elevation. The high-frequency transient elevation again depends on alpha7-containing receptors, whereas the sustained elevation can be triggered by other nicotinic receptors and depends on calcium release from internal stores and probably influx through voltage-gated L-type calcium channels as well. Retrograde axonal stimulation of the neurons at high frequency mimics synaptic stimulation in producing sustained cell-wide calcium increases that depend on L-type channels and release from internal stores, but it does not produce calcium transients in the spines. Thus frequent action potentials are sufficient to generate the cell-wide increases, but alpha7-containing receptors are needed for spine-specific effects. Patch-clamp recording indicates that alpha7-containing receptors preferentially desensitize at high-frequency stimulation, accounting for the inability of the stimulation to sustain high calcium levels in the spines. The spatial and temporal differences in the patterns of calcium elevation could enable the neurons to monitor their own firing histories for regulatory purposes.

Cluster formation of alpha7-containing nicotinic receptors at interneuronal interfaces in cell culture.

Nicotinic receptors containing the alpha7 gene product are among the most abundant in the nervous system. Because of their widespread distribution and high relative permeability to calcium, the receptors regulate a diverse array of cellular events. On chick ciliary neurons the receptors are concentrated on somatic spines folded into discrete mats on the cell body and are overlaid by a large presynaptic calyx. The receptors co-localize with filamentous actin and the actin-associated protein drebrin which are concentrated in the spines. We show here that embryonic ciliary ganglion neurons grown in dissociated cell culture express and concentrate the receptors in large clusters or plaques that form at interneuronal interfaces between small clumps of neurons. The receptors resist detergent extraction even after disruption of the actin cytoskeleton, suggesting the importance of additional molecular mechanisms determining receptor location. The cell adhesion molecules N-CAM and N-cadherin are concentrated at the receptor plaques and may influence plaque stability. Although ciliary neurons do not normally contact each other in vivo, they do so in culture and may mimic interactions normally occurring between calyx and soma in vivo. As a result the cultures may prove useful for identifying components shaping development of postsynaptic specializations on neurons.

The neurofilament infrastructure of a developing presynaptic calyx.

Calyx-type synapses appear to be specifically designed to support fast, reliable, high-frequency excitatory transmission. In the chick ciliary ganglion, calyx terminals from preganglionic neurons in the midbrain form early in development on ciliary neurons. We find that labeling the calyx membranes with a lipophilic dye delivered by diffusion down the preganglionic nerve reveals a large membrane structure engulfing the postsynaptic cell by the end of embryogenesis. In contrast, labeling the calyces with a water-soluble dye by diffusion through the preganglionic nerve suggests large discontinuities in the calyx. A similar pattern of discontinuities is seen when presynaptic neurofilaments are labeled with antibodies selective for highly phosphorylated neurofilaments. The neurofilament infrastructure of the calyx first appears as a single thick bundle, which subsequently bifurcates during development and eventually generates a fine meshwork of filaments subdivided by several large neurofilament bundles encircling the postsynaptic cell body. The large bundles probably produce protruding ridges in the otherwise thin calyx cup, accounting for the disparity in staining patterns observed with membrane and cytosolic dyes. The postsynaptic membrane also undergoes restructuring during development with the appearance of large folded mats of somatic spines heavily invested with nicotinic receptors. The large presynaptic neurofilament bundles do not overlap the postsynaptic receptor clusters but do codistribute with large tracks of presynaptic microtubules. The neurofilament bundles may act as girders to provide structural support while at the same time defining conduits for microtubule-dependent transport of materials and rapid propagation of electrical signals throughout the extended calyx.

Storage of semen and artificial insemination in deer.

Methods of collection and freezing of semen of some deer species and aspects of controlled reproduction associated with the use of frozen-thawed semen by artificial insemination (AI) are discussed.

Two distinct classes of functional 7-containing nicotinic receptor on rat superior cervical ganglion neurons.

Nicotinic acetylcholine receptors (nAChRs) that bind alpha-bungarotoxin (alpha Bgt) were studied on isolated rat superior cervical ganglion (SCG) neurons using whole-cell patch clamp recording techniques. Rapid application of ACh onto the soma of voltage clamped neurons evoked a slowly desensitizing current that was reversibly blocked by alpha Bgt (50 nM). The toxin-sensitive current constituted on average about half of the peak whole-cell response evoked by ACh. Nanomolar concentrations of methyllycaconitine blocked the alpha Bgt-sensitive component of the ACh-evoked current as did intracellular dialysis with an anti-alpha 7 monoclonal antibody. The results indicate that the slowly reversible toxin-sensitive response elicited by ACh arises from activation of an unusual class of alpha 7-containing receptor (alpha 7-nAChR) similar to that reported previously for rat intracardiac ganglion neurons. A second class of functional alpha 7-nAChR was identified on some SCG neurons by using rapid application of choline to elicit responses. In these cases a biphasic response was obtained, which included a rapidly desensitizing component that was blocked by alpha Bgt in a pseudo-irreversible manner. The pharmacology and kinetics of the responses resembled those previously attributed to alpha 7-nAChRs in a number of other neuronal cell types. Experiments measuring the dissociation rate of 125I-labelled alpha Bgt from SCG neurons revealed two classes of toxin-binding site. The times for toxin dissociation were consistent with those required to reverse blockade of the two kinds of alpha Bgt-sensitive response. These results indicate that rat SCG neurons express two types of functional alpha 7-nAChR, differing in pharmacology, desensitization and reversibility of alpha Bgt blockade.

Cytoskeletal links of neuronal acetylcholine receptors containing alpha 7 subunits.

Nicotinic acetylcholine receptors serve a variety of signaling functions in the nervous system depending on cellular location, but little is known about mechanisms responsible for tethering them at specific sites. Among the most interesting are receptors containing the alpha7 gene product, because of their abundance and high relative permeability to calcium. On chick ciliary ganglion neurons alpha7-containing receptors are highly concentrated on somatic spines folded into discrete patches on the cell. We show that the spines contain filamentous actin and drebrin. After cell dissociation, the actin slowly redistributes, the spines retract, and the alpha7-containing receptors disperse and are subsequently lost from the surface. Latrunculin A, a drug that depolymerizes filamentous actin, accelerates receptor dispersal, whereas jasplikinolide, a drug that stabilizes the actin cytoskeleton, preserves large receptor clusters and prevents receptor loss from the surface. The receptors are resistant to extraction by nonionic detergent even after latrunculin A treatment. Other, less abundant, nicotinic receptors on the neurons are readily solubilized by the detergent even though these receptors are located in part on the spines. The results demonstrate that the actin cytoskeleton is important for retaining receptor-rich spines and indicate that additional cytoskeletal elements or molecular interactions specific for alpha7-containing receptors influence their fate in the membrane. The cytoskeletal elements involved are not dependent on the architecture of the postsynaptic density because alpha7-containing receptors are excluded from such sites on ciliary ganglion neurons.

Targeting alpha7-containing nicotinic receptors on neurons to distal locations.

Nicotinic receptors containing the alpha7 gene product are widely expressed in the nervous system and have a high relative permeability to Ca(2+). This permits them to influence a variety of Ca(2+)-dependent events in neurons. On chick ciliary ganglion neurons, the receptors are concentrated on somatic spines and contribute directly to postsynaptic signaling. Receptors containing the alpha7 gene product can also be found in the chick sciatic nerve being transported to distal locations. Both motoneurons and dorsal root ganglion neurons are candidate sources of the receptors since both extend processes into the nerve and synthesize alpha7 protein. Immunoprecipitation assays with subunit-specific monoclonal antibodies and pharmacological comparisons fail to detect differences between sciatic nerve and ciliary ganglion alpha7-containing receptors. Cell-specific machinery and receptor posttranslational modifications may determine which sites the receptors populate.