Microvascular oxygen tension in the rat mesentery.

Longitudinal Po(2) profiles in the microvasculature of the rat mesentery were studied using a novel phosphorescence quenching microscopy technique that minimizes the accumulated photoconsumption of oxygen by the method. Intravascular oxygen tension (Po(2), in mmHg) and vessel diameter (d, in microm) were measured in mesenteric microvessels (n = 204) of seven anesthetized rats (275 g). The excitation parameters were as follows: 7 x 7-microm spot size; 410 nm laser; and 100 curves at 11 pulses/s, with pulse parameters of 2-micros duration and 80-pJ/microm(2) energy density. The mean Po(2) (+/- SE) was 65.0 +/- 1.4 mmHg (n = 78) for arterioles (d = 18.8 +/- 0.7 microm), 62.1 +/- 2.0 mmHg (n = 38) at the arteriolar end of capillaries (d = 7.8 +/- 0.3 microm), and 52.0 +/- 1.0 mmHg (n = 88) for venules (d = 22.5 +/- 1.0 microm). There was no apparent dependence of Po(2) on d in arterioles and venules. There were also no significant deviations in Po(2) based on d (bin width, 5 microm) from the general mean for both of these types of vessels. Results indicate that the primary site of oxygen delivery to tissue is located between the smallest arterioles and venules (change of 16.3 mmHg, P = 0.001). In conclusion, oxygen losses from mesenteric arterioles and venules are negligible, indicating low metabolic rates for both the vascular wall and the mesenteric tissue. Capillaries appear to be the primary site of oxygen delivery to the tissue in the mesenteric microcirculation. In light of the present results, previously reported data concerning oxygen consumption in the mesenteric microcirculation can be explained as artifacts of accumulated oxygen consumption due to the application of instrumentation having a large excitation area for Po(2) measurements in slow moving and stationary media.

PO2 profiles near arterioles and tissue oxygen consumption in rat mesentery.

A scanning phosphorescence quenching microscopy technique, designed to prevent accumulated O(2) consumption by the method, was applied to Po(2) measurements in mesenteric tissue. In an attempt to further increase the accuracy of the measurements, albumin-bound probe was topically applied to the tissue and an objective-mounted pressurized bag was used to reduce the oxygen transport bypass through the thin layer of fluid over the mesentery. Po(2) was measured at multiple sites perpendicular to the blood/wall interface in the vicinity of 84 mesenteric arterioles (7-39 microm in diameter) at distances of 5, 15, 30, 60, 120, and 180 microm in seven anesthetized Sprague-Dawley rats, thereby creating Po(2) profiles. Interstitial Po(2) above and immediately beside arterioles was found to agree with known intravascular values. No significant difference in Po(2) profiles was found between small and large arterioles, indicating a small longitudinal Po(2) gradient in the precapillary mesenteric microvasculature. In addition, the Po(2) profiles were used to calculate oxygen consumption in the mesenteric tissue (56-65 nl O(2) x cm(-3) x s(-1)). Correction of these values for contamination with ambient oxygen yielded an oxygen consumption rate of 60-68 nl O(2) x cm(-3) x s(-1), the maximal limit for consumption in the mesentery. The results were compared with measurements made by other workers in regard to the employed techniques.

Erythrocyte-associated transients in capillary PO2: an isovolemic hemodilution study in the rat spinotrapezius muscle.

Mathematical simulations of oxygen delivery to tissue from capillaries that take into account the particulate nature of blood flow predict the existence of oxygen tension (Po(2)) gradients between erythrocytes (RBCs). As RBCs and plasma alternately pass an observation point, these gradients are manifested as rapid fluctuations in Po(2), also known as erythrocyte-associated transients (EATs). The impact of hemodilution on EATs and oxygen delivery at the capillary level of the microcirculation has yet to be elucidated. Therefore, in the present study, phosphorescence quenching microscopy was used to measure EATs and Po(2) in capillaries of the rat spinotrapezius muscle at the following systemic hematocrits (Hct(sys)): normal (39%) and after moderate (HES1; 27%) or severe (HES2; 15%) isovolemic hemodilution using a 6% hetastarch solution. A 532-nm laser, generating 10-micros pulses concentrated onto a 0.9-microm spot, was used to obtain plasma Po(2) values 100 times/s at points along surface capillaries of the muscle. Mean capillary Po(2) (Pc(O(2)); means +/- SE) significantly decreased between conditions (normal: 56 +/- 2 mmHg, n = 45; HES1: 47 +/- 2 mmHg, n = 62; HES2: 27 +/- 2 mmHg, n = 52, where n = capillary number). In addition, the magnitude of Po(2) transients (DeltaPo(2)) significantly decreased with hemodilution (normal: 19 +/- 1 mmHg, n = 45; HES1: 11 +/- 1 mmHg, n = 62; HES2: 6 +/- 1 mmHg, n = 52). Results suggest that the decrease in Pc(O(2)) and DeltaPo(2) with hemodilution is primarily dependent on Hct(sys) and subsequent microvascular compensations.

Kv1 currents mediate a gradient of principal neuron excitability across the tonotopic axis in the rat lateral superior olive.

Principal neurons of the lateral superior olive (LSO) detect interaural intensity differences by integration of excitatory projections from ipsilateral bushy cells and inhibitory inputs from the medial nucleus of the trapezoid body. The intrinsic membrane currents active around firing threshold will form an important component of this binaural computation. Whole cell patch recording in an in vitro brain slice preparation was employed to study conductances regulating action potential (AP) firing in principal neurons. Current-clamp recordings from different neurons showed two types of firing pattern on depolarization, one group fired only a single initial AP and had low input resistance while the second group fired multiple APs and had a high input resistance. Under voltage-clamp, single-spiking neurons showed significantly higher levels of a dendrotoxin-sensitive, low threshold potassium current (ILT). Block of ILT by dendrotoxin-I allowed single-spiking cells to fire multiple APs and indicated that this current was mediated by Kv1 channels. Both neuronal types were morphologically similar and possessed similar amounts of the hyperpolarization-activated nonspecific cation conductance (Ih). However, single-spiking cells predominated in the lateral limb of the LSO (receiving low frequency sound inputs) while multiple-firing cells dominated the medial limb. This functional gradient was mirrored by a medio-lateral distribution of Kv1.1 immunolabelling. We conclude that Kv1 channels underlie the gradient of LSO principal neuron firing properties. The properties of single-spiking neurons would render them particularly suited to preserving timing information.

Presynaptic rat Kv1.2 channels suppress synaptic terminal hyperexcitability following action potential invasion.

Voltage-gated K+ channels activating close to resting membrane potentials are widely expressed and differentially located in axons, presynaptic terminals and cell bodies. There is extensive evidence for localisation of Kv1 subunits at many central synaptic terminals but few clues to their presynaptic function. We have used the calyx of Held to investigate the role of presynaptic Kv1 channels in the rat by selectively blocking Kv1.1 and Kv1.2 containing channels with dendrotoxin-K (DTX-K) and tityustoxin-Kalpha (TsTX-Kalpha) respectively. We show that Kv1.2 homomers are responsible for two-thirds of presynaptic low threshold current, whilst Kv1.1/Kv1.2 heteromers contribute the remaining current. These channels are located in the transition zone between the axon and synaptic terminal, contrasting with the high threshold K+ channel subunit Kv3.1 which is located on the synaptic terminal itself. Kv1 homomers were absent from bushy cell somata (from which the calyx axons arise); instead somatic low threshold channels consisted of heteromers containing Kv1.1, Kv1.2 and Kv1.6 subunits. Current-clamp recording from the calyx showed that each presynaptic action potential (AP) was followed by a depolarising after-potential (DAP) lasting around 50 ms. Kv1.1/Kv1.2 heteromers had little influence on terminal excitability, since DTX-K did not alter AP firing. However TsTX-Kalpha increased DAP amplitude, bringing the terminal closer to threshold for generating an additional AP. Paired pre- and postsynaptic recordings confirmed that this aberrant AP evoked an excitatory postsynaptic current (EPSC). We conclude that Kv1.2 channels have a general presynaptic function in suppressing terminal hyperexcitability during the depolarising after-potential.

Two heteromeric Kv1 potassium channels differentially regulate action potential firing.

Low-threshold voltage-gated potassium currents (I(LT)) activating close to resting membrane potentials play an important role in shaping action potential (AP) firing patterns. In the medial nucleus of the trapezoid body (MNTB), I(LT) ensures generation of single APs during each EPSP, so that the timing and pattern of AP firing is preserved on transmission across this relay synapse (calyx of Held). This temporal information is critical for computation of sound location using interaural timing and level differences. I(LT) currents are generated by dendrotoxin-I-sensitive, Shaker-related K+ channels; our immunohistochemistry confirms that MNTB neurons express Kv1.1, Kv1.2, and Kv1.6 subunits. We used subunit-specific toxins to separate I(LT) into two components, each contributing approximately one-half of the total low-threshold current: (1) I(LTS), a tityustoxin-Kalpha-sensitive current (TsTX) (known to block Kv1.2 containing channels), and (2) I(LTR), an TsTX-resistant current. Both components were sensitive to the Kv1.1-specific toxin dendrotoxin-K and were insensitive to tetraethylammonium (1 mm). This pharmacological profile excludes homomeric Kv1.1 or Kv1.2 channels and is consistent with I(LTS) channels being Kv1.1/Kv1.2 heteromers, whereas I(LTR) channels are probably Kv1.1/Kv1.6 heteromers. Although they have similar kinetic properties, I(LTS) is critical for generating the phenotypic single AP response of MNTB neurons. Immunohistochemistry confirms that Kv1.1 and Kv1.2 (I(LTS) channels), but not Kv1.6, are concentrated in the first 20 microm of MNTB axons. Our results show that heteromeric channels containing Kv1.2 subunits govern AP firing and suggest that their localization at the initial segment of MNTB axons can explain their dominance of AP firing behavior.