Microfabricated sampling probes for in vivo monitoring of neurotransmitters.

Microfabricated fluidic systems have emerged as a powerful approach for chemical analysis. Relatively unexplored is the use of microfabrication to create sampling probes. We have developed a sampling probe microfabricated in Si by bulk micromachining and lithography. The probe is 70 µm wide by 85 µm thick by 11 mm long and incorporates two buried channels that are 20 µm in diameter. The tip of the probe has two 20 µm holes where fluid is ejected or collected for sampling. Utility of the probe was demonstrated by sampling from the brain of live rats. For sampling, artificial cerebral spinal fluid was infused in through one channel at 50 nL/min while sample was withdrawn at the same flow rate from the other channel. Analysis of resulting fractions collected every 20 min from the striatum of rats by liquid chromatography with mass spectrometry demonstrated reliable detection of 17 neurotransmitters and metabolites. The small probe dimensions suggest it is less perturbing to tissue and can be used to sample smaller brain nuclei than larger sampling devices, such as microdialysis probes. This sampling probe may have other applications such as sampling from cells in culture. The use of microfabrication may also enable incorporation of electrodes for electrochemical or electrophysiological recording and other channels that enable more complex sample preparation on the device.

Effects of ß-hydroxybutyrate and different calcium and potassium concentrations on the membrane potential and motility of abomasal smooth muscle cells in cattle.

The left displacement of the abomasum (LDA) is a common disease in periparturient dairy cows. Plasma ß-hydroxybutyrate (BHBA) levels above the reference range are regarded as risk factors for the occurrence of LDA. Additionally, hypokalemia and hypocalcemia have been observed in LDA cows. The aim of the present study was to characterize the membrane potential and the slow waves in abomasal smooth muscle cells by a microelectrode technique and to determine possible effects of BHBA and of various calcium and potassium concentrations on the membrane potential. Subsequently, the results obtained by the microelectrode technique were combined with in vitro motility experiments of abomasal smooth muscles. Strips of the abomasal circular smooth muscles were prepared and incubated in different buffer solutions. For the microelectrode technique, healthy bulls and cows that underwent surgery for LDA were sampled. These measurements showed a frequency of the slow waves between 3.5 and 10.9 per minute (for amplitudes ≥ 3mV) and between 0.6 and 4.5 per minute (for amplitudes ≥ 5mV). The frequency of contractions (1.8 to 3.1 per minute) were in the same order as the frequency of the slow waves with amplitudes ≥ 5 mV. Blocking potassium conductance with barium chloride induced a depolarization of the basal membrane potential (from -43±2.9 to -37±4.1mV; mean ± standard error of the mean) without affecting the frequency or the height of the slow waves. The reduction in the potassium concentration from 5.4 to 2 mmol/L resulted in a nominal decrease in the activity of contractions (from 22.2 to 18.6 mN/min). The subsequent addition of 1 mmol of KCl/L induced a nominal increase in contraction activity (from 18.6 to 25.7 mN/min). An effect of BHBA (5 mmol/L) could not be demonstrated, neither on the electric nor on the motility parameters. A simulated hypocalcemia (1.2 mmol/L total, 0.9 mmol/L ionized Ca) did not change slow waves and motility. In conclusion, changes in membrane potential ≥ 5 mV correlated with contractions of abomasal muscles. Hypokalemic conditions may reduce abomasal contraction activity via an effect on the membrane potential. An exclusive increase in BHBA or a slight hypocalcemia (0.9 mmol/L ionized Ca) had no effects on the motility of healthy abomasal smooth muscle cells. A prolonged or severe exposition of muscles to a combination of low Ca and high BHBA concentrations might nevertheless be able to affect abomasal motility.

Retinal oxygenation and oxygen metabolism in Abyssinian cats with a hereditary retinal degeneration.

PURPOSE: To investigate the effects of a hereditary retinal degeneration on retinal oxygenation and determine whether it is responsible for the severe attenuation of retinal circulation in hereditary photoreceptor degenerations. METHODS: Seven adult Abyssinian cats affected by hereditary retinal degeneration were studied. Oxygen microelectrodes were used to collect spatial profiles of retinal oxygenation in anesthetized animals. A one-dimensional model of oxygen diffusion was fitted to the data to quantify photoreceptor oxygen utilization (Qo(2)). RESULTS: Photoreceptor Qo(2) progressively decreased until it reached zero in the end stage of the disease. Average inner retinal oxygen tension remained within normal limits at all disease stages, despite the observed progressive retinal vessel attenuation. Light affected photoreceptors normally, decreasing Qo(2) by approximately 50% at all stages of the disease. CONCLUSIONS: Loss of photoreceptor metabolism allows choroidal oxygen to reach the inner retina, attenuating the retinal circulation in this animal model of retinitis pigmentosa (RP) and probably also in human RP. As the degeneration progresses, there is a strong relationship between changes in the a-wave of the ERG and changes in rod oxidative metabolism, indicating that these two functional measures change together.

Types of serotonergic receptors involved in the control of reticulo-ruminal myoelectric activity in sheep.

The effects of peripheral (intravenous, i.v.) and central (intracerebroventricular, ICV) administration of agonists of 5-HT1A, 5-HT2, 5-HT3 and 5-HT4 receptors were investigated in conscious sheep chronically fitted with intraparietal electrodes on the reticulum and the dorsal, ventral and caudo-ventral rumen. The 5-HT1A agonist 8-hydroxydipropylaminotetralin increased reticular and decreased ruminal spike burst frequency when given i.v. (80 micrograms/kg) and ICV (8 micrograms/kg). The 5-HT2 and 5-HT3 agonists, alpha-methylserotonin and 2-methylserotonin, induced a moderate inhibition of rumino-reticular contractions when given i.v. at 100 and 150 micrograms/kg, respectively, while marked inhibition was observed after ICV administration at doses of 10 and 5 micrograms/kg, respectively. The 5-HT4 agonist 5-methoxytryptamine strongly stimulated rumino-reticular motility by the ICV (10 micrograms/kg) route, whereas it induced a moderate inhibition when administered i.v. (200 micrograms/kg). The selective antagonist of 5-HT1A, 5-HT2, 5-HT3 and 5-HT4 receptors, spiroxatrine, ritanserin, granisetron and DAU 6285, respectively, blocked the responses of the respective agonists given by the same route. Moreover, the antagonists given ICV blocked the effects of the agonists given i.v. except for DAU 6285 ICV, which did not antagonize the inhibition induced by 5-methoxytryptamine i.v. It is concluded that the four types of serotonergic receptors investigated control rumino-reticular motility at the central level. However, according to the receptor type and the forestomach area (reticulum or rumen) this control may be stimulatory or inhibitory, demonstrating a pleiotropic role of serotonin in the control of rumino-reticular motility in sheep.

Use of a conductance catheter to assess the effect of endotoxemia on left ventricular end-systolic pressure-volume relationships in anesthetized swine.

Effects of endotoxemia on left ventricular contractility and systemic hemodynamics were determined in pentobarbital-anesthetized swine. A multielectrode conductance (volume) catheter and a high-fidelity pressure transducer catheter were passed retrograde into the left ventricle to continuously measure pressure and volume. End-systolic pressure-volume relationships were determined during transient (8 to 10 s) caudal vena caval balloon occlusion. Lactated Ringer's solution was administered at a rate sufficient to maintain left ventricular end-diastolic pressure > or = 6 mm of Hg. Following baseline measurements, Escherichia coli endotoxin (O55-B5) was infused IV at 2.5 micrograms/kg of body weight/h for 3 hours. Left ventricular end-systolic elastance (Ees), the slope of the end-systolic pressure-volume relationship; end-systolic elastance normalized for left ventricular end-diastolic volume (Ees norm); the rate of increase of left ventricular pressure (dP/dt max); and preload recruitable stroke work (PRSW, stroke work-to-end-diastolic volume relationship) did not change in endotoxemic swine, compared with baseline measurements or with values from control (physiologic saline solution-treated) swine. Left ventricular pressures and volumes had marked pig-to-pig variability in the control and endotoxin-treated groups. Determination of Ees, Ees norm, and PRSW was further confounded by development of frequent premature ventricular contractions during caudal vena caval balloon occlusion. Endotoxin significantly (P < 0.05) decreased left ventricular end-diastolic pressure, compared with that in control swine, and significantly (P < 0.01) decreased left ventricular end-diastolic volume, compared with baseline. Endotoxin decreased cardiac index and arterial blood pressure, whereas heart rate, central venous pressure, and mean pulmonary arterial pressure increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the teleost Edinger-Westphal nucleus by retrograde horseradish peroxidase labeling and by electrophysiological criteria.

A homolog of the Edinger-Westphal nucleus of other vertebrates is described in two species of serranid basses of the genus Paralabrax, a group possessing a wide range of ocular accommodation but lacking a pupillary reflex to light. The nucleus was found by retrograde labeling from the ciliary ganglion and lies dorsolateral to the ipsilateral oculomotor nucleus. The nucleus consists of 60 to 100 neurons with an average soma diameter of about 20 microns in animals weighing 70 to 150 g. Electrophysiological experiments support the identification. Microstimulation of the nucleus evokes contraction of the ipsilateral lens retractor muscle and slight constriction of the caudal ipsilateral iris. Multi- and single-unit recordings in the nucleus reveal spontaneous firing (about 30 spikes/s in single units), the rate of which decreases during visually-evoked lens retractor relaxations (accommodation to near stimuli). Recordings of muscle fiber activity in the lens retractor show essentially the same behavior, which suggests that the ciliary ganglion and neuromuscular junctions simply relay impulses with little if any synaptic integration. The existence of a discrete Edinger-Westphal nucleus devoted largely to accommodation makes Paralabrax a good model system for the further tracing of central accommodation control pathways.

Mechanoreceptor activity in the gills of the carp. I. Gill filament and gill raker mechanoreceptors.

Physiological properties of gill filament and gill raker mechanoreceptors in the gills of spontaneously breathing carp, Cyprinus carpio L., were analysed. Stroking stimuli applied to gill filaments elicited a phasic mechanoreceptive response, which was recorded from neurons in the epibranchial ganglia. Sustained deflection resulted in a short on-off response. The same neurons were also activated by slight movements of lamellae on a gill filament. The receptive field extended over all the lamellae of one filament at most, but generally covered a small part of it, including both dorsal and ventral lamellae. Deflection of gill rakers also elicited a brief response in epibranchial ganglion neurons. The threshold of both filament-related and gill raker mechanoreceptors was relatively high. They did not respond during normal respiration. It was therefore argued that these receptors do not function in normal respiratory control, but rather serve against mechanical damage from excessive pressure or particles in the water.

Effects of secretagogues on membrane potential and input resistance of pancreatic acinar cells of sheep.

The effects of acetylcholine, short-chain fatty acids and peptide hormones on membrane potential and input resistance of the pancreatic acinar cells of sheep were investigated using intracellular glass microelectrodes. The resting membrane potential and input resistance were -28.0 +/- 4.3 mV (mean +/- SD, inside negative, n = 23) and 2.4 +/- 1.7 M omega, respectively. The application of acetylcholine, short-chain fatty acids (acetate, butyrate and caprylate) and peptide hormones (caerulein and bombesin) always caused depolarisation accompanied by a marked reduction in input resistance. The equilibrium potentials of acetylcholine and caprylate were -13.5 and -12.8 mV, respectively.

Functional roles and circuitry in an inhibitory pathway to feeding command neurones in Pleurobranchaea.

The paracerebral neurones (PCNs) of the brain of Pleurobranchaea californica serve a command role in the initiation of feeding behaviour (Gillette, Kovac & Davis, 1978). The PCNs are synaptically excited by food stimuli applied to the oral veil of hungry, naive animals. In food avoidance-conditioned animals, the PCNs are inhibited by a barrage of inhibitory postsynaptic potentials concomitant with the suppression of feeding (Davis & Gillette, 1978). In this paper, an interneuronal pathway is described which causes inhibition of the PCNs and potentially mediates the effects of learning. The inhibitory pathway consists of three serially connected interneurones. One population, designated the Interneurone 1s (Int-1s), monosynaptically inhibits the PCNs. A second population, the Interneurone 2s (Int-2s), excites the Int-1 population. They also excite other neurones of the brain including the metacerebral giant neurones. A third population, the Interneurone 3s (Int-3s), monosynaptically excites the Interneurone 2 population. Dual intracellular recordings and current injection show that ipsilateral members of the Int-2 population are electrically coupled via a nonrectifying connection. Contralateral members of the Int-2 population are excitatorily coupled via a polysynaptic pathway. The Int-1 population is phasically active during the rhythmic motor activity that underlies feeding. In the isolated nervous system Int-1 activity is phase-locked with rhythmic PCN activity; Int-1 activity occurs maximally at the end of a PCN burst, during the retraction phase of the cycle. Int-2 activity also occurs during the retraction phase. During actual feeding in the whole animal preparation, the Int-2s are also phasically active; maximal excitation occurs during buccal mass retraction and maximal inhibition during protraction and the bite. Stimulated activity in a single Int-2 can entirely suppress the rhythmic motor activity of the feeding network evoked by electrical stimulation of the stomatogastric nerve. The suppressant effects of Int-2 activity must be mediated widely within the feeding network because the rhythmic motor output so driven is not dependent on PCN spiking. Application of an appetitive chemosensory stimulus to whole and semi-intact animal preparations initiated feeding and elicited excitation of the Int-1 and Int-2 populations. Noxious chemosensory stimuli, such as a dilute soap solution or ethanol, elicited oral veil withdrawal and inhibition of the Int-2s by multiple inhibitory postsynaptic potentials.(ABSTRACT TRUNCATED AT 400 WORDS)