Layer-specific interhemispheric functional connectivity in the somatosensory cortex of rats: resting state electrophysiology and fMRI studies.

The spontaneous cerebral hemodynamic fluctuations observed during the resting state have been frequently visualized using functional magnetic resonance imaging (rsfMRI). However, the neuronal populations and neuroelectric characteristics underlying the functional connectivity of cerebrohemodynamic activities are poorly understood. We investigated the characteristics of bi-hemispheric functional connectivity via electrophysiology and rsfMRI in the primary sensory cortex of rats anesthetized by α-chloralose. Unlike the evoked responses, the spontaneous electrophysiological activity was concentrated in the infragranular layers and could be classified into subtypes with distinctive current sources and sinks. Both neuroelectric and rsfMRI signals were interhemispherically correlated in a layer-specific manner, suggesting that there are independent neural inputs to infragranular and granular/supragranular layers. The majority of spontaneous electrophysiological activities were bilaterally paired with delays of up to ~50 ms between each pair. The variable interhemispheric delay implies the involvement of indirect, multi-neural pathways. Our findings demonstrated the diverse activity patterns of layer-specific electrophysiological substrates and suggest the recruitment of multiple, non-specific brain regions in construction of interhemispheric functional connectivity.

Stability of the acetic acid-induced bladder irritation model in alpha chloralose-anesthetized female cats.

Time- and vehicle-related variability of bladder and urethral rhabdosphincter (URS) activity as well as cardiorespiratory and blood chemistry values were examined in the acetic acid-induced bladder irritation model in α-chloralose-anesthetized female cats. Additionally, bladder and urethra were evaluated histologically using Mason trichrome and toluidine blue staining. Urodynamic, cardiovascular and respiratory parameters were collected during intravesical saline infusion followed by acetic acid (0.5%) to irritate the bladder. One hour after starting acetic acid infusion, a protocol consisting of a cystometrogram, continuous infusion-induced rhythmic voiding contractions, and a 5 min "quiet period" (bladder emptied without infusion) was precisely repeated every 30 minutes. Administration of vehicle (saline i.v.) occurred 15 minutes after starting each of the first 7 cystometrograms and duloxetine (1mg/kg i.v.) after the 8(th). Acetic acid infusion into the bladder increased URS-EMG activity, bladder contraction frequency, and decreased contraction amplitude and capacity, compared to saline. Bladder activity and URS activity stabilized within 1 and 2 hours, respectively. Duloxetine administration significantly decreased bladder contraction frequency and increased URS-EMG activity to levels similar to previous reports. Cardiorespiratory parameters and blood gas levels remained consistent throughout the experiment. The epithelium of the bladder and urethra were greatly damaged and edema and infiltration of neutrophils in the lamina propria of urethra were observed. These data provide an ample evaluation of the health of the animals, stability of voiding function and appropriateness of the model for testing drugs designed to evaluate lower urinary tract as well as cardiovascular and respiratory systems function.

Vasomotion and neurovascular coupling in the visual thalamus in vivo.

Spontaneous contraction and relaxation of arteries (and in some instances venules) has been termed vasomotion and has been observed in an extensive variety of tissues and species. However, its functions and underlying mechanisms are still under discussion. We demonstrate that in vivo spectrophotometry, measured simultaneously with extracellular recordings at the same locations in the visual thalamus of the cat, reveals vasomotion, measured as an oscillation (0.14 hz) in the recorded oxyhemoglobin (OxyHb) signal, which appears spontaneously in the microcirculation and can last for periods of hours. During some non-oscillatory periods, maintained sensory stimulation evokes vasomotion lasting ~30s, resembling an adaptive vascular phenomenon. This oscillation in the oxyhaemoblobin signal is sensitive to pharmacological manipulation: it is inducible by chloralose anaesthesia and it can be temporarily blocked by systemic administration of adrenaline or acetylcholine (ACh). During these oscillatory periods, neurovascular coupling (i.e. the relationship between local neural activity and the rate of blood supply to that location) appears significantly altered. This raises important questions with regard to the interpretation of results from studies currently dependent upon a linear relationship between neural activity and blood flow, such as neuroimaging.

Comparison of alpha-chloralose, medetomidine and isoflurane anesthesia for functional connectivity mapping in the rat.

Functional connectivity measures based upon low-frequency blood-oxygenation-level-dependent functional magnetic resonance imaging (BOLD fMRI) signal fluctuations have become a widely used tool for investigating spontaneous brain activity in humans. Still unknown, however, is the precise relationship between neural activity, the hemodynamic response and fluctuations in the MRI signal. Recent work from several groups had shown that correlated low-frequency fluctuations in the BOLD signal can be detected in the anesthetized rat - a first step toward elucidating this relationship. Building on this preliminary work, through this study, we demonstrate that functional connectivity observed in the rat depends strongly on the type of anesthesia used. Power spectra of spontaneous fluctuations and the cross-correlation-based connectivity maps from rats anesthetized with alpha-chloralose, medetomidine or isoflurane are presented using a high-temporal-resolution imaging sequence that ensures minimal contamination from physiological noise. The results show less localized correlation in rats anesthetized with isoflurane as compared with rats anesthetized with alpha-chloralose or medetomidine. These experiments highlight the utility of using different types of anesthesia to explore the fundamental physiological relationships of the BOLD signal and suggest that the mechanisms contributing to functional connectivity involve a complicated relationship between changes in neural activity, neurovascular coupling and vascular reactivity.

Intracortical microcirculatory change induced by anesthesia in rat somatosensory cortex.

The present study aimed to characterize microcirculatory responses to anesthesia in brain tissue. With multi-photon excitation fluorescence microscopy, intra-cortical capillary dimension and red blood cell (RBC) flow were successfully visualized up to a depth of approximately 0.6 mm from the cortical surface in rats anesthetized with either isoflurane or alpha-chloralose. We observed that the diameter of the major cerebral artery was approximately 100 microm under isoflurane, but approximately 75 microm under alpha-chloralose. The capillary diameter was observed to be larger under alpha-chloralose than isoflurane: 5.1 +/- 1.2 microm vs. 4.8 +/- 1.1 microm, respectively. A significant difference in the mean RBC speed measured in single capillaries was observed: 0.4 +/- 0.4 mm/s under alpha-chloralose vs. 1.5 +/- 0.4 mm/s under isoflurane. In agreement with these observations, arterio-venous transit-time and laser-Doppler flowmetry consistently showed a significant reduction of the RBC and plasma blood speed under alpha-chloralose relative to isoflurane. These findings may indicate that local blood flow regulatory mechanisms exist at the capillary level for the balance of oxygen supply and demand induced by anesthesia in the brain tissue.

Isoflurane anesthesia is a valuable alternative for alpha-chloralose anesthesia in the forepaw stimulation model in rats.

Isoflurane (ISO) can be a valuable alternative for alpha-chloralose (ACL) anesthesia in functional MRI (fMRI) studies. Therefore, we compared the efficacy of the blood oxygen level dependent (BOLD) effect in fMRI studies during ISO and ACL anesthesia sequentially in the same animals. After non-invasive instrumentation for ventilation and monitoring, series of T2* weighted MR images were acquired during forepaw stimulation, first under ISO, then followed by ACL anesthesia. The results demonstrated that ISO and ACL were both suitable to perform this fMRI experiment. The center of activation was at the same stereotactic position for both anesthetics and matched the primary somatosensory cortex (S1). Under the applied conditions, the BOLD response during ISO anesthesia declined in magnitude during the first stimulation period, as compared to ACL. From this study, we conclude that since ISO has several positive properties in comparison to ACL, including fast pharmacokinetics and suitability for repeated measurements, it is a valuable alternative for anesthesia in fMRI studies of rats.

Is urethane-chloralose anaesthesia appropriate for pharmacokinetic-pharmacodynamic assessment? Studies with carvedilol.

INTRODUCTION: The aim of the work was to establish the impact of urethane-chloralose anaesthesia on pharmacokinetic-pharmacodynamic (PK-PD) properties of carvedilol in control rats and L-NAME hypertensive animals. METHODS: Male Wistar Rats were randomly divided into: control (n=12) with tap water to drink and L-NAME rats (n=12) with L-NAME solution (40 mg/kg/day) to drink for 2 weeks. Effects of carvedilol (1 mg kg(-1), i.v.) on blood pressure and heart rate were recorded during 3 h in conscious and urethane (500 mg kg(-1), i.p.) - chloralose (50 mg kg(-1), i.p.) anaesthetized rats. Carvedilol plasma pharmacokinetics was studied by means of traditional blood sampling. PK-PD modeling of carvedilol was made by means of an effect compartment model. RESULTS: Neither urethane-chloralose nor L-NAME modified estimation of pharmacokinetic parameters of carvedilol. Although urethane-chloralose did not modify potency of carvedilol comparing with awake animals in control and hypertensive group, maximal negative chronotropic response was significantly greater in anaesthetized L-NAME rats in comparison to awake animals. Conversely, anaesthesia did not modify maximal chronotropic response to carvedilol in control rats. Whilst no differences were found in the estimated potency of carvedilol hypotensive response comparing control and L-NAME rats in both awake and anaesthetized conditions, maximal hypotensive effect of carvedilol was significantly greater in anaesthetized control and L-NAME animals in comparison to conscious rats. L-NAME rats showed a greater maximal hypotensive response comparing to control group. DISCUSSION: Urethane-chloralose anaesthesia is an acceptable experimental condition for the evaluation of PK-PD properties of carvedilol, considering that it does not affect the potency of carvedilol for its chronotropic and hypotensive effect. Conclusions obtained from urethane-chloralose anaesthetized animals, regarding the impact of l-NAME treatment on PK-PD properties of carvedilol, did not differ from those obtained from conscious animals. Anaesthesia did not modify pharmacokinetic behaviour of carvedilol in both normotensive and L-NAME hypertensive rats.

State-dependent amygdala stimulation-induced cardiovascular effects in rats.

Stimulation of the amygdala is known to produce pressor, depressor, or has no effects. The present study was performed to test whether amygdala cardiovascular effects are influenced by consciousness states and by different types of anesthetics. Adult rats were set up for stimulation amygdala and measurement of blood pressure in a chronic preparation. After recovery, same sites of the amygdala were stimulated electrically for several trials with the rat under conscious or anesthetic states induced by pentobarbital, urethane, ketamine, alpha-chloralose and urethane plus alpha-chloralose, respectively. The interval between any two stimulation trials was at least 2 days. The stimulation was an 80-Hz, 0.5-ms, 100-micro A square wave pulse train lasting for 15 s. Cardiovascular responsive sites were found in the central, medial, and basolateral nuclei of the amygdala. In stimulating these responsive sites, significantly different cardiovascular effects were induced under a conscious state and an anesthetized state of the animal, yet no significant differences were found among the various anesthetic agents. We conclude, that the cardiovascular influence of the amygdala is state-dependent in the rat.

Effect of galanin on substance P- and vasoactive intestinal polypeptide-induced nociceptive trigemino-hypoglossal reflex in rats.

Substance P (SP), vasoactive intestinal polypeptide (VIP) and galanin (GAL), present in primary sensory neurons, are involved in transmission of nociceptive signaling from the peripheral to central nervous system. In this study we investigated the effect of GAL on SP-induced or VIP-induced evoked tongue jerks (ETJ) in response to noxious tooth pulp stimulation during perfusion of the cerebral ventricles with SP or VIP solutions. The experiments were carried out on rats under chloralose anesthesia. It was shown that both, SP and VIP, perfused through the cerebral ventricles enhanced the ETJ amplitude as compared with control, but the effect produced by SP was stronger. The intracerebroventricular perfusion of GAL 5 minutes before SP caused a dose-dependent inhibition of SP-induced ETJ, whereas GAL perfused through the cerebral ventricles 5 minutes before VIP did not reduce the excitatory effect of VIP on ETJ. These results indicate that the antinociceptive effect of GAL perfused through the cerebral ventricles, tested on the trigemino-hypoglossal reflex in rats, is specifically mediated by the SP-ergic system.

Successful treatment of capture myopathy in three wild greater sandhill cranes (Grus canadensis tabida).

Two adult and 1 juvenile free-flying greater sandhill cranes (Grus canadensis tabida) were diagnosed with capture myopathy after alpha-chloralose baiting and physical capture during a banding and feeding ecologic study. Blood samples were collected for serum biochemical analysis at the time of capture for the 2 adults, and at 24 hours postcapture, at various intervals during treatment, and at the time of release for all 3 birds. Concentrations of creatine kinase, aspartate transaminase, and lactate dehydrogenase were high within 1 hour of capture and peaked approximately 3 days after capture. By days 10-17 after capture, creatine kinase and lactate dehydrogenase concentrations both decreased to within the reference range measured for cranes at capture, but aspartate transaminase concentrations remained 2-5 times higher than the measured reference range. Treatment consisted of corticosteroids, selenium/vitamin E, parenteral fluids, and gavage feedings. Physical therapy consisted of assisting the cranes to stand and walk 2-8 times a day, massaging leg muscles, and moving limbs manually through the range of motion. The adults were released when they were able to stand up independently and were pacing in the pen. The juvenile was released 12 hours after it was able to stand independently but was returned to the pen when it fell and could not rise. It was treated supportively for an additional 3 days and then successfully released. Both adult cranes were observed on their territories with their original mates after release and returned to their territories for the subsequent 8 years, raising chicks most years. After release, the juvenile was observed in a flock of cranes near its natal territory for the next 2 days.

Exploring nociceptive response by BOLD fMRI in alpha-chloralose anesthetized rats.

The technique of blood oxygenation level dependent (BOLD) functional magnetic resonance imaging (fMRI) was used to provide a spatial-temporal mapping of nociceptive activation in brain. We contrived to obtain an illustration of the pain related regions by injecting formalin at the hindpaw using a 4.7 T MR system in alpha-chloralose anesthetized rats. In order to obtain the pain response, we avoided any invasive surgery on animals to purify the signal of nociception. The dynamic data were analyzed by mapping correlation coefficient and the time activity curves were calculated by atlas-based region of interest selection. The BOLD signals showed obvious difference in anterior cingulated cortex, somatosensory cortex, medial thalamus, and striatum after stimulation. The results not only show the global somatotopic organization of noxious stimulation on hindpaw in rats, but also provided invaluable information for neuroscience research.

Structural analysis of heart rhythm spectrogram in narcotized cats.

Eight types of peaks were revealed in the cardiac rhythm spectrum during acute experiments on vagotomized cats. Some peaks had no physiological nature and resulted from specificity of ECG processing by Fourier analysis, while others reflected myogenic reaction of the sinoatrial node (length-dependence of automaticity) to changes in venous return caused by respiratory-induced and other variations of the blood flow in the cardiovascular system.

In vivo alpha-adrenergic responses and troponin I phosphorylation: anesthesia interactions.

The mechanisms by which alpha-adrenergic stimulation of the heart in vivo can cause contractile dysfunction are not well understood. We hypothesized that alpha-adrenergic-mediated contractile dysfunction is mediated through protein kinase C phosphorylation of troponin I, which in in vitro experiments has been shown to reduce actomyosin Mg-ATPase activity. We studied pressure-volume loops in transgenic mice expressing mutant troponin I lacking protein kinase C phosphorylation sites and hypothesized altered responses to phenylephrine. As anesthesia agents can produce markedly different effects on contractility, we studied two agents: avertin and alpha-chloralose-urethane. With alpha-chloralose-urethane, at baseline, there were no contractile abnormalities in the troponin I mutants. Phenylephrine produced a 50% reduction in end-systolic elastance in wild-type controls, although a 9% increase in troponin I mutants (P <0.05). Avertin was associated with reduced contractility compared with alpha-chloralose-urethane. Avertin anesthesia, at baseline, produced a reduction in end-systolic elastance by 31% in the troponin I mutants compared with wild-type (P <0.05), and this resulted in further marked systolic and diastolic dysfunction with phenylephrine in the troponin I mutants. Dobutamine produced no significant difference in the contractile phenotype of the transgenic mice with either anesthetic regimen. In conclusion, these data (alpha-chloralose-urethane) demonstrate that alpha-adrenergic-mediated force reduction is mediated through troponin I protein kinase C phosphorylation. beta-Adrenergic responses are not mediated through this pathway. Altering the myofilament force-calcium relationship may result in in vivo increased sensitivity to negative inotropy. Thus choice of a negative inotropic anesthetic agent (avertin) with phenylephrine can lead to profound contractile dysfunction.

Comparison of the effects of dexmedetomidine and esmolol on myocardial oxygen consumption in dogs.

BACKGROUND AND OBJECTIVE: The beta-adrenergic blocker esmolol and the alpha 2-adrenergic agonist dexmedetomidine have the potential to decrease perioperative myocardial ischaemia. The pathophysiological mechanisms involved in these anti-ischaemic properties have not been thoroughly studied. We compared the effects of esmolol and dexmedetomidine on two indices of overall myocardial oxygen demand and on directly measured myocardial oxygen consumption of the left anterior coronary artery territory. METHODS: Eleven mongrel dogs were instrumented to measure aortic and left ventricular pressure, aortic and left anterior coronary artery flow and myocardial wall thickening. Variables related to myocardial oxygen metabolism were also determined. Measurements were performed during four sequential experimental conditions in each dog (Control 1: esmolol; Control 2: dexmedetomidine). RESULTS: Esmolol and dexmedetomidine decreased haemodynamic indices of myocardial oxygen demand to a similar extent: esmolol decreased the rate-pressure product by 16+/-3% and the pressure-work index (PWI) by 16+/-3%, dexmedetomidine decreased the rate-pressure product by 26+/-3% and the PWI by 16+/-7%. However, these similar decreases resulted from different haemodynamic effects of the two study drugs. Dexmedetomidine had a more pronounced bradycardic effect than esmolol (P = 0.01) and increased systolic aortic pressure (SAP) by 15+/-4% while esmolol decreased SAP by 8+/-2% (P < 0.01). dP/dt(max) and regional myocardial area decrease were lower after esmolol than after dexmedetomidine. Neither drug had an effect on myocardial oxygen consumption. CONCLUSIONS: Esmolol and dexmedetomidine decreased two haemodynamic indices of overall myocardial oxygen demand to a similar extent but neither drug decreased directly measured myocardial oxygen consumption in the territory of the left anterior descending artery.

Modularity of motor output evoked by intraspinal microstimulation in cats.

We studied the forces produced at the cat's hindpaw by microstimulation of the ipsi- and contralateral lumbar spinal cord in spinal intact alpha-chloralose anesthetized (n = 3) or decerebrate (n = 3) animals. Isometric force and EMG responses were measured at 9-12 limb configurations, with the paw attached to a force transducer and with the hip and femur fixed. The active forces elicited at different limb configurations were summarized as force fields representing the sagittal plane component of the forces produced at the paw throughout the workspace. The forces varied in amplitude over time but the orientations were stable, and the pattern of an active force field was invariant through time. The active force fields divided into four distinct types, and a few of the fields showed convergence to an equilibrium point. The fields were generally produced by coactivation of the hindlimb muscles. In addition, some of the fields were consistent with known spinal reflexes and the stimulation sites producing them were in laminae where the interneurons associated with those reflexes are known to be located. Muscle activation produced by intraspinal stimulation, as assessed by intramuscular EMG activity, was modified with limb configuration, suggesting that the responses were not fixed, but were modified by position-dependent sensory feedback. The force responses may represent basic outputs of the spinal circuitry and may be related to similar spinal primitives found in the frog and rat.

Hemodynamic and electrocardiographic effects of graded doses of amiodarone in healthy dogs anesthetized with morphine/alpha chloralose.

This research was designed to study acute hemodynamic and electrocardiographic effects of amiodarone and to determine an IV dose of amiodarone that minimally affects left ventricular function and does not increase the tendency for ventricular arrhythmia.

Alpha-chloralose immobilization of rock doves in Ohio.

To improve capture efficacy of rock doves (Columba livia) in nuisance situations, we reevaluated the effectiveness of three dosages (60, 120 and 180 mg/kg) of alpha-chloralose (AC). Responses to immobilization using 180 mg/kg AC also were compared in rock doves deprived of food for 16 hr and not food deprived. Mean (+/- SE) time to first effects (33 +/- 2 min) and mean time to capture (94 +/- 5 min) were significantly less for rock doves receiving 180 mg/kg than for rock doves receiving lower dosages (> or = 53 +/- 3 min and > or = 153 +/- 17 min, respectively). Ten, 10, and eight rock doves immobilized with 60, 120, and 180 mg/kg AC recovered within 24 hr, respectively; all rock doves recovered within 29 hr. Although food-deprived rock doves showed effects of AC immobilization earlier than did rock doves with food, time to capture was similar between these two groups. For capturing rock doves, we recommend treating corn with 3 mg AC/kernel and using 180 mg/kg as the effective dose. This modified formulation and dosage should improve capture success of rock doves substantially and improve the ability to resolve nuisance rock dove problems.

A simple method, using 2-hydroxypropyl-beta-cyclodextrin, of administering alpha-chloralose at room temperature.

Effective long term stable anaesthesia is a goal of many drug regimens employed in neuroscience in which procedures carried out are not practical in awake animals. A particular problem is the study of nociceptive mechanisms where good anaesthesia is essential. Similarly studies of cardiovascular or cerebrovascular mechanisms require that normal physiological reflexes be preserved as much as is practical. For non-recovery anaesthesia alpha-chloralose is a good choice since it provides good anaesthesia without excess depression of physiological reflexes. However, alpha-chloralose is sparingly soluble so that its use is not straightforward. We describe the characterisation of a simple procedure to solubilise alpha-chloralose in a solution of 2-hydroxypropyl-beta-cyclodextrin. The resulting solution is stable at room temperature and gives a high concentration of alpha-chloralose making it easier to administer regularly during longer time course experiments.

Comparison of three formulations of alpha-chloralose for immobilization of Canada geese.

The effectiveness of an alpha-chloralose (AC)-corn oil suspension, an AC-margarine mixture, and AC tablets were compared for immobilizing Canada geese (Branta canadensis). Responses to AC immobilization also were compared in male and female Canada geese. There was no difference in mean time to first effects or mean time to capture between male and female geese dosed with 30 mg/kg AC in orally-administered bread baits. Recovery times (< or = 24 hr) also were similar between sexes. Mean (+/-SE) time to first effects for geese immobilized with AC tablets in bread baits (19 +/- 3 min) was significantly less than mean time to first effects for geese immobilized with AC in margarine (28 +/- 6 min) or AC in corn oil (32 +/- 7 min) applied to bread baits (n = 12 geese/treatment). Respective mean times to capture geese immobilized with AC tablets, AC-margarine, and AC-corn oil were not significantly different at 62 +/- 25, 89 +/- 48, and 88 +/- 30 min, respectively. Ten, 11 and, seven geese immobilized with AC tablets, AC-margarine, and AC-corn oil recovered within 24 hr, respectively; all geese recovered within 28 hr. Male and female Canada geese respond similarly to AC immobilization, at least during molt, and AC mixed with margarine or AC in tablet form is at least as effective as the presently used AC-corn oil suspension. AC tablets or AC-margarine also would be more practical for some field situations. Additional development of AC tablets will be required prior to field use for capturing nuisance waterfowl.