Shelter type and birth number influence the birth and death sites of lambs and ewe movement around lambing time.

A significant number of lambs born each yr in Australia die within 72 h of birth. Periods of high wind, combined with rain and low temperatures, can lead to marked increases in the level of mortality. Under these weather conditions mortality levels may be reduced with the provision of shelter, provided it is utilized by lambs. This study used GPS collars to determine the use of shelter by ewes and lambs, to compare the movement of ewes with twin lambs across 2 types of shelter (hedgerows and shrubs), while also comparing ewes with single and twin lambs in a single shelter type (hedgerows). Additionally, the birth sites of 364 lambs and death sites of 252 lambs were recorded across the 3 shelter type and litter size combinations (Twins in shrubs, Twins in hedgerows, Singles in hedgerows) plus an unsheltered group (Singles in unsheltered). A higher (P < 0.001) than randomly expected percentage of ewes lambed in the areas closest to both shelter types; in the shrub shelter 42% of ewes lambed within 2.5 m of shrub rows compared to an expected 11% based on the proportion of the paddock this area constituted. Despite the higher than expected percentage of ewes lambing close to the shelter rows, ewes in both twin lamb shelter types avoided the areas close to the shelter before and after lambing (Hedgerows-2.5 m; Shrubs- 6.25 m) and single bearing ewes showed no preference for or against these areas. With a high proportion of twin bearing ewes lambing close to the shelter, a design that reduces the potential for ewe and offspring separation while providing good shelter will offer the greatest potential reduction in newborn twin lamb mortality arising from exposure.

Ewe and lamb contact at lambing is influenced by both shelter type and birth number.

A significant number of lambs born each year in Australia die within 72 h of birth. Periods of high wind, combined with rain and low temperatures can lead to marked increases in the mortality level. Under these climatic conditions mortality levels may be reduced with the provision of shelter. This study used contact loggers to compare interactions between ewes with twin lambs across two shelter types (Hessian and shrubs), while also comparing ewes with single and twin lambs in a single shelter type (Hessian). The contact loggers record the time of the initial contact (within approximately 4 to 5 m) between collared animals and the duration of each contact. Contact levels between ewes immediately after lambing were only 10% of the initial levels (1 h/day). For single-born lambs, lambs averaged 11 h contact per day with their mother, while for twinborn lambs, each lamb averaged 9.25 h/day with its mother and 14.7 h/day with its sibling. The level of contact between ewes and each of their offspring in the Hessian was 24% lower (P < 0.05) for ewes with twin lambs than with singles. For ewes with twin lambs the level of contact was 17% lower (P < 0.05) in the Hessian shelter compared with shrub shelter. We conclude that shelter type and birth number can affect the level of contact between ewes and their offspring.

Asymmetric voltage dependence of embryonic cardiac gap junction channels.

The voltage dependence of junctional conductance (Gj) and the unitary channel behavior of junctions in most pairs of 3-day, 7-day, and 18-day embryonic chick heart cells are symmetrical, i.e., they are independent of the direction of polarization of junctional potential (Vj). With either cell depolarized relative to its neighbor, unitary channel events have a maximal unit conductance (yj) near 240 pS and five substates at nearly equal 40-pS increments down to near 40 pS (6, 9). Using the dual patch-clamp technique, we demonstrate here that, in a fraction of such cell pairs, Vj-dependent channel kinetics are asymmetric. Depolarization of one cell causes a larger and faster voltage-dependent decline in Gj than the same depolarization of the other cell. In a typical asymmetric preparation, depolarization of the strongly Vj-dependent side caused an immediate series of 47 +/- 16 pS closing steps in single-channel current (ij), followed by virtual cessation of channel activity. After depolarization of the less Vj-sensitive side, channel activity (56 +/- 13 pS) continues for many seconds. The large-conductance states (160-240 pS) observed in the electrically symmetric junctions were absent from the asymmetric preparations. In these cell pairs, connexin (Cx) 42, Cx43, and Cx45 could be immunolocalized at the junctional surfaces. We postulate that the asymmetry of voltage dependence in some cell pairs results from a preponderance of heterochannels formed from these different connexins. The frequency of asymmetric pairs obtained from 3-day, 7-day, and 18-day embryonic hearts was 50% (4/8), 24% (6/25), and 12.5% (1/8), suggesting that the fraction of heterochannels in the junctions decreases with cardiac development.

Specific motifs in the external loops of connexin proteins can determine gap junction formation between chick heart myocytes.

1. Gap junction formation was compared in the absence and presence of small peptides containing extracellular loop sequences of gap junction (connexin) proteins by measuring the time taken for pairs of spontaneously beating embryonic chick heart myoballs to synchronize beat rates. Test peptides were derived from connexin 32. Non-homologous peptides were used as controls. Control pairs took 42 +/- 0.5 min (mean +/- S.E.M.; n = 1088) to synchronize. 2. Connexins 32 and 43, but not 26, were detected in gap junction plaques. The density and distribution of connexin immunolabelling varied between myoballs. 3. Peptides containing conserved motifs from extracellular loops 1 and 2 delayed gap junction formation. The steep portion of the dose-response relation lay between 30 and 300 microM peptide. 4. In loop 1, the conserved motifs QPG and SHVR were identified as being involved in junction formation. In loop 2, the conserved SRPTEK motif was important. The ability of peptides containing the SRPTEK motif to interfere with the formation of gap junctions was enhanced by amino acids from the putative membrane-spanning region. 5. Peptides from loop 1 and loop 2 were equivalently effective; there was no synergism between them. 6. The inclusion of conserved cysteines in test peptides did not make them more effective in the competition assay.

Temperature dependence of embryonic cardiac gap junction conductance and channel kinetics.

We have investigated the effects of temperature on the conductance and voltage-dependent kinetics of cardiac gap junction channels between pairs of seven-day embryonic chick ventricle myocytes over the range of 14-26 degrees C. Records of junctional conductance (Gj) and steady-state unit junctional channel activity were made using the whole-cell double patch-clamp technique while the bath temperature was steadily changed at a rate of about 4 degrees C/min. The decrease in Gj upon cooling was biphasic with a distinct break at 21 degrees C. In 12 cell pairs, Q10 was 2.2 from 26 to 21 degrees C, while between 21 and 14 degrees C it was 6.5. The mean Gj at 22 degrees C (Gj22) was 3.0 +/- 2.1 nS, ranging in different preparations from 0.24 to 6.4 nS. At room temperature, embryonic cardiac gap junctions contain channels with conductance states near 240, 200, 160, 120, 80 and 40 pS. In the present study, we demonstrate that cooling decreases the frequency of channel openings at all conductance levels, and at temperatures below 20 degrees C shifts the prevalence of openings from higher to lower conductance states: all 240 pS openings disappear below 20 degrees C; 200 pS openings are suppressed at 17 degrees C; below 16 degrees C 160 and 120 pS events disappear and only 80 and 40 pS states are seen. Temperature also affected the voltage-dependent kinetics of the channels. Application of a 6 sec, 80 mV voltage step across the junction (Vj80) caused a biexponential decay in junctional conductance. Decay was faster at lower temperatures, whereas the rate of recovery of Gj after returning to Vj0 was slowed. Cooling reduced the fast decay time constant, increased both recovery time constants, and decreased the magnitude of Gj decay, thus leaving a 10-16% larger residual conductance (Gss/Ginit, +/- 80 mV Vj) at 18 than at 22 degrees C. From these results we propose that embryonic chick cardiac gap junctions contain at least two classes of channels with different conductances and temperature sensitivities.

Inter-beat intervals of cardiac-cell aggregates during exposure to 2.45 GHz CW, pulsed, and square-wave-modulated microwaves.

Inter-beat intervals of aggregated cardiac cells from chicken embryos were studied during 190 s exposures to 2.45 GHz microwaves in an open-ended coaxial device. Averaged specific-absorption rates (SARs) and modulation conditions were 1.2-86.9 W/kg continuous-wave (CW), 1.2-12.2 W/kg pulse modulation (PW, duty cycle approximately 11%), and 12.0-43.5 W/kg square-wave modulation (duty cycle = 50%). The inter-beat interval decreased during microwave exposures at 42.0 W/kg and higher when CW or square-wave modulation was used, which is consistent with established effects of elevated temperatures. However, increases in the inter-beat interval during CW exposures at 1.2-12.2 W/kg, and decreases in the inter-beat interval after PW exposures at 8.4-12.2 W/kg, are not consistent with simple thermal effects. Analysis of variance indicated that SAR, modulation, and the modulation-SAR interaction were all significant factors in altering the inter-beat interval. The latter two factors indicated that the cardiac cells were affected by athermal as well as thermal effects of microwave exposure.

Multiple-channel conductance states and voltage regulation of embryonic chick cardiac gap junctions.

We used the double whole-cell voltage-clamp technique on ventricle cell pairs isolated from 7-day chick heart to measure the conductance of their gap junctions (Gj) and junctional channels (gamma j) with a steady-state voltage difference (Vj) applied across the junction. Currents were recorded from single gap junction channels (ij) as symmetrical rectangular signals of equal size and opposite sign in the two cells, and gamma j was measured from ij/Vj. We observed channel openings at six reproducible conductance levels with means of 42.6, 80.7, 119.6, 157.7, 200.4 and 240.3 pS. More than half of all openings were to the 80- and 160-pS conductance levels. The probability that a high conductance event (e.g., 160 or 240 pS) results from the random simultaneous opening of several 40-pS channels is small, based on their frequency of occurrence and on the prevalence of shifts between small and large conductance states with no intervening 40-pS steps. Our results are consistent with three models of embryonic cardiac gap junction channel configuration: a homogeneous population of 40-pS channels that can open cooperatively in groups of up to six; a single population of large channels with a maximal conductance near 240 pS and five smaller substates; or several different channel types, each with its own conductance. Gj was determined from the junctional current (Ij) elicited by rectangular pulses of applied transjunctional voltage as Ij/Vj. It was highest near 0 Vj and was progressively reduced by application of Vj between 20 and 80 mV or -20 and -80 mV. In response to increases in Vj, Gj decayed in a voltage- and time-dependent fashion. After a 6-sec holding period at 0 Vj, the initial conductance (G(init) measured immediately after the onset of an 80-mV step in Vj was nearly the same as that measured by a 10-mV prepulse. However, during 6-sec pulses of Vj greater than +/- 20 mV, Gj declined over several seconds from G(init)to a steady-state value (Gss). At potentials greater than +/- 20 mV the current decay could be fit with biexponential curves with the slow decay time constant (tau 2) 5-20 times longer than tau 1. For the response to a step to 80 mV Vj, for example, tau 1 = 127 msec and tau 2 = 2.6 sec. The rate of current decay in response to smaller positive or negative steps in Vj was slower, the magnitude of the decline was smaller, and the ratio tau 2/tau 1 decreased.(ABSTRACT TRUNCATED AT 400 WORDS)

Developmental changes in the calcium currents in embryonic chick ventricular myocytes.

Using the patch-clamp technique, we recorded whole-cell calcium current from isolated cardiac myocytes dissociated from the apical ventricles of 7-day and 14-day chick embryos. In 70% of 14-day cells after 24 hr in culture, two component currents could be separated from total ICa activated from a holding potential (Vh) of -80 mV. L-type current (IL) was activated by depolarizing steps from Vh -30 or -40 mV. The difference current (IT) was obtained by subtracting IL from ICa. IT could also be distinguished pharmacologically from IL in these cells. IT was selectively blocked by 40-160 microM Ni2+, whereas IL was suppressed by 1 microM D600 or 2 microM nifedipine. The Ni2(+)-resistant and D600-resistant currents had activation thresholds and peak voltages that were near those of IT and IL defined by voltage threshold, and resembled those in adult mammalian heart. In 7-day cells, IT and IL could be distinguished by voltage threshold in 45% (S cells), while an additional 45% of 7-day cells were nonseparable (NS) by activation voltage threshold. Nonetheless, in most NS cells, ICa was partly blocked by Ni2+ and by D600 given separately, and the effects were additive when these agents were given together. Differences among the cells in the ability to separate IT and IL by voltage threshold resulted largely from differences in the position of the steady-state inactivation and activation curves along the voltage axis. In all cells at both ages in which the steady-state inactivation relation was determined with a double-pulse protocol, the half-inactivation potential (V1/2) of the Ni2(+)-resistant current IL averaged -18 mV. In contrast, V1/2 of the Ni2(+)-sensitive IT was -60 mV in 14-day cells, -52 mV in 7-day S cells, and -43 mV in 7-day NS cells. The half-activation potential was near -2 mV for IL at both ages, but that of IT was -38 mV in 14-day and -29 mV in 7-day cells. Maximal current density was highly variable from cell to cell, but showed no systematic differences between 7-day and 14-day cells. These results indicate that the main developmental change that occurs in the components of ICa is a negative shift with embryonic age in the activation and inactivation relationships of IT along the voltage axis.

Analysis of the T-type calcium channel in embryonic chick ventricular myocytes.

T-type calcium channels (IT channels) were studied in cell-attached patch electrode recordings from the ventricular cell membrane of 14-day embryonic chick heart. All experiments were performed in the absence of Ca2+ with Na+ (120 mM) as the charge carrier. IT channels were distinguished from L-type calcium channels (IL) by their more negative activation and inactivation potential ranges; their smaller unitary slope conductance (26 pS), and their insensitivity to isoproterenol or D600. Inactivation kinetics were voltage dependent. The time constant of inactivation was 37 msec when the membrane potential was depolarized 40 mV from rest (R + 40 mV), and 20 msec at R + 60 mV. The frequency histogram of channel open times (tau o) was fit by a single-exponential curve while that of closed times (tau c) was bi-exponential. tau o was the same at R + 40 mV and R + 60 mV whereas tau c was shortened at R + 60 mV. The open-state probability (P o) increased with depolarization: 0.35 at R + 40 mV, 0.8 at R + 60 mV and 0.88 at R + 80 mV. This increase in Po at depolarized potentials could be accounted for by the decrease in tau c.

Low-threshold current is major calcium current in chick ventricle cells.

Single myocytes were dissociated from the apical ventricles of 14-day-old chick embryos and were maintained in culture for 24 h. Isolated rounded cells, 13-18 microns in diameter with input capacitance averaging 5.2 pF, were selected for whole cell patch-clamp analysis of calcium current (ICa). Two current components, T-type (IT) and L-type (IL), were identified by differences in their voltage thresholds and pharmacological sensitivity, IL was activated by depolarizing steps from -40 mV and was selectively blocked by 1 microM D 600 (methoxyverapamil). IT was obtained by subtracting IL from total ICa and was blocked by 120 microM Ni2+. Identified by threshold potential, sensitivity to Ni2+, or resistance to D 600, IT had a greater current density than has been reported in any adult cardiac tissue. It was also larger than IL and was therefore the major contributor to ICa in this preparation.

Development of cardiac beat rate in early chick embryos is regulated by regional cues.

The mesoderm of each of the paired lateral heart-forming regions (HFRs) in the stage 5-7 chick embryo includes prospective conus (pre-C), ventricle (pre-V), and sinoatrial (pre-SA) cells, arranged in a rostrocaudal sequence (C-V-SA). With microsurgery we divided each HFR into three rostrocaudally arranged segments. After 24 hr of further incubation, each segment differentiated into a spontaneously beating vesicle of heart tissue to form a multiheart embryo. The cardiac vesicles in these embryos expressed left-right and rostrocaudal beat rate gradients: the left caudal pre-SA mesoderm produced tissue with the fastest beat rate of the six while the rostral vesicle formed from right pre-C was the slowest. In another operation, we prevented the HFRs from fusing in the midline by cutting through the anterior intestinal portal at stage 8, to produce cardia bifida (CB) embryos with an independently beating half-heart on each side. In these cases, the left half-heart of 87.2% of CB embryos beat faster than the right, confirming the left-right difference in intrinsic beat rate. To assess whether the future beat rate of each region is already determined in the st 5-7 HFR, we exchanged rectangular fragments of left pre-SA mesoderm and attached endoderm with right pre-C fragments to yield a left HFR with the sequence C-V-C and a right HFR with the sequence SA-V-SA. A CB operation was subsequently performed on these exchange embryos to prevent fusion of the lateral HFRs. Preconus mesoderm, transplanted to the pre-SA region, differentiated into tissue with a rapid beat rate, while pre-SA mesoderm relocated to the preconus region formed heart tissue with a slow spontaneous rate typical of the conus. In 73% of the exchange CB embryos, the left half-heart beat faster than the right, despite the origins of its mesoderm. The exchanged mesoderm developed a rate that was appropriate for its new location rather than the site of origin of the mesodermal fragment. In a third set of operations, we implanted a fragment of st 15 differentiated conus tissue into a site lateral to the left caudal HFR in st 5, 6, and 7 embryos, and subsequently performed CB operations on them. The implant caused the adjacent half-heart to develop with a slower beat rate than in unoperated or sham-operated controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Development of the fast sodium current in early embryonic chick heart cells.

Single ventricle cells were dissociated from the hearts of two-, three-, four- or seven-day-old chick embryos, and were maintained in vitro for an additional 6 to 28 hr. Rounded 13 to 18 micron cells with input capacitance of 5 to 10 pF were selected for analysis of fast sodium current (INa). Voltage command protocols designed to investigate the magnitude, voltage dependence, and kinetics of INa were applied with patch electrodes in the whole-cell clamp configuration. INa was present in over half of the 2d, and all 3d, 4d and 7d cells selected. The current showed no systematic differences in activation kinetics, voltage dependence, or tetrodotoxin (TTX) sensitivity with age or culture conditions. Between the 2d and 7d stages, the rate of current inactivation doubled and channel density increased about eightfold. At all stages tested, INa was blocked by TTX at a half-effective concentration of 0.5 to 1.0 nM. We conclude that the lack of Na dependence of the action potential upstroke on the second day of development results from the relatively depolarized level of the diastolic potential, and failure to activate the small available excitatory Na current. The change from Ca to Na dependence of the upstroke during the third to the seventh day of incubation results partly from the negative shift of the diastolic potential during this period, and in part from the increase in available Na conductance.

Cardiac gap junction channel activity in embryonic chick ventricle cells.

We have recorded single-gap junction-channel currents from pairs of 7-day chick embryo ventricle cells, using the double whole cell patch-clamp technique. Junctional conductance (Gj) was variable from one preparation to the next, ranging from 0.15 to 35.0 nS. Single-channel conductance (gamma j) of the main junctional channel was 166 +/- 51 pS and was independent of Gj; a second conductance level of 60-80 pS was also seen in favorable records. The transition time from the closed to the open state was 285 +/- 153 microseconds, with some slow transitions lasting 1-5 ms. Channels opened and closed stochastically; Gj could be defined by the product of the number of active channels in the junction (N), the mean open-state probability (Po) of the channels, and gamma j. Channel activity was unaffected by cell membrane potential or by transjunctional potential. Po and Gj were reversibly reduced to low levels by 1-octanol or by elevated [Cai], whereas gamma j was unchanged by these agents. The 60-80 pS conductance mechanism was octanol- and Ca-resistant, but it is not clear whether this represents a subconductance level of the main channel or a separate class of smaller channels.

Measurement of single channel currents from cardiac gap junctions.

Cardiac gap junctions consist of arrays of integral membrane proteins joined across the intercellular cleft at points of cell-to-cell contact. These junctional proteins are thought to form pores through which ions can diffuse from cytosol to cytosol. By monitoring whole-cell currents in pairs of embryonic heart cells with two independent patch-clamp circuits, the properties of single gap junction channels have been investigated. These channels had a conductance of about 165 picosiemens and underwent spontaneous openings and closings that were independent of voltage. Channel activity and macroscopic junctional conductance were both decreased by the uncoupling agent 1-octanol.

Electrotonic interactions between aggregates of chick embryo cardiac pacemaker cells.

Synchronization of spontaneously active heart cell aggregates occurs shortly after they are brought into contact. The synchronous rate is determined by pacemaker phase resetting and passive subthreshold electrotonic interactions. To further study the effects of passive electrical interactions, we have used 150-microns diameter aggregates prepared from cells of 4d (4-day ventricle + 1 day in vitro), 7d, and 14d embryonic chick ventricle as models of primary, latent, and nonpacemaker tissues, respectively. Coupling of 4d and 7d aggregates (4d/7d pairs) leads to intermediate synchronous rates. We show here that elevating external K+ from 1.3 to 2.8 mM, which has no effect on 4d/4d pairs but selectively reduces the beat rate of 7d/7d pairs by 42%, slows the synchronous beat rate of 4d/7d pairs by 23%. Increases in electrical coupling in newly joined 4d/14d pairs cause the 4d rate to slow to a minimum value (16 +/- 13 beats/min, n = 16) just prior to the onset of synchronous activity. The rate slowly recovers to a final value of 40 +/- 12 beat/min. We conclude that the spontaneous beat rate of a primary pacemaker is modulated by both active and passive interactions with latent or nonpacemaker tissues.

Some limitations of the cell-attached patch clamp technique: a two-electrode analysis.

With two independent patch electrodes sealed to small clusters of electrically coupled chick embryo cardiac cells, we have measured four parameters: true seal and patch resistance, channel conductance, and membrane potential. One electrode was in the cell-attached mode, and recorded current flowing in parallel through the membrane patch and seal. The second electrode, sealed on a different cell in the cluster, was in the whole cell recording configuration, and served to record or control the membrane potential of the cluster. We fit the four measured parameters to a simple electrical model to reveal errors not usually recognized in the patch-clamp technique. Among these are the following: (1) The apparent seal resistance, determined by changing the potential in a patch electrode, may be a poor estimate of true seal resistance, since it includes the parallel combination of seal- and patch-resistance. (2) Patch resistance may be influenced by the electrode filling solution, and is often much lower than is usually assumed. (3) With a small cell preparation that has an input resistance in the gigaohm range, measurements of single-channel conductance using a cell-attached patch electrode may be inaccurate because cell membrane potential does not remain constant as electrode potential is varied.

Channel currents during spontaneous action potentials in embryonic chick heart cells. The action potential patch clamp.

Single-channel currents were recorded with the cell-attached patch-clamp technique from small clusters (2-20 cells) of spontaneously beating 7-d embryo ventricle cells. Because the preparation was rhythmically active, the trans-patch potential varied with the action potential (AP). The total current through the patch membrane was the patch action current (AC). ACs and APs could be recorded simultaneously, with two electrodes, or sequentially with one electrode. Channel activity, which varied depending on the number and type of channels in the patch, was present during normal cell firing. This method can reveal the kinetics and magnitudes of the specific currents that contributed to the AP, under conditions that reflect not only the time and voltage dependence of the channels, but also environmental factors that may influence channel behavior during the AP.