Aidan K. Curran

Potassium Permanganate Can Mark the Site of Microdialysis in Brain Sections

Abstract Microdialysis is a technique used to study the extracellular environment or to deliver minute quantities of drugs into the central nervous system and other tissues in physiological experiments. It may have an expanding role in clinical studies. It can be used to study the microenvironment or to study the systemic effects of extremely localized pharmacological interventions, without interpretation of the results being confounded by systemic distribution of circulating drug. Proper interpretation of experimental results, however, depends on precise histological localization of the site of microdialysis or microinjection. This paper describes a simple method using potassium permanganate in 1% filtered aqueous solution to mark the site of experimental microdialysis or microinjection in brain tissue. Potassium permanganate reacts with brain tissue to produce insoluble, tar-like organic manganese dioxide reaction products that make a more visible mark than staining with either 5% neutral red or 5% fast green. Subsequent tissue processing and histological staining with cresyl violet or hematoxylin, eosin, and Luxol fast blue did not obscure the mark. (The J Histotechnol 23:151, 2000)

Lesion or muscimol in the rostral ventral medulla reduces ventilatory output and the CO2 response in decerebrate piglets

Developmental abnormalities have been described in the arcuate nucleus of sudden infant death syndrome (SIDS) victims. The arcuate nucleus has putative homologues in chemosensitive areas of the ventral medulla in animals. We refer to some of these areas collectively as the rostral ventral medulla (RVM). In the RVM of decerebrate piglets 2-15 days of age, we studied the effects of electrolytic lesions (n=7) or microdialysis of muscimol (n=15), a GABAA receptor agonist, on ventilatory output and the response to hypercapnia. Lesions caused a 66.7+/-17.3% reduction in eupneic phrenic minute activity (MA) and abolished the response to hypercapnia. Muscimol dialysis caused a 32.4+/-10.4% reduction in MA with a significant downward displacement of the response to hypercapnia with no significant effect on the slope. We conclude that the piglet RVM contains neurons of vital importance in the maintenance of normal breathing and the response to systemic CO(2). We hypothesize that dysfunction of homologous regions in the human infant could lead to impaired ability to respond to hypercapnia and could potentially be involved in the pathogenesis of SIDS.

Investigating the complexity of respiratory patterns during the laryngeal chemoreflex

BackgroundThe laryngeal chemoreflex exists in infants as a primary sensory mechanism for defending the airway from the aspiration of liquids. Previous studies have hypothesized that prolonged apnea associated with this reflex may be life threatening and might be a cause of sudden infant death syndrome.MethodsIn this study we quantified the output of the respiratory neural network, the diaphragm EMG signal, during the laryngeal chemoreflex and eupnea in early postnatal (3–10 days) piglets. We tested the hypothesis that diaphragm EMG activity corresponding to reflex-related events involved in clearance (restorative) mechanisms such as cough and swallow exhibit lower complexity, suggesting that a synchronized homogeneous group of neurons in the central respiratory network are active during these events. Nonlinear dynamic analysis was performed using the approximate entropy to asses the complexity of respiratory patterns.ResultsDiaphragm EMG, genioglossal activity EMG, as well as other physiological signals (tracheal pressure, blood pressure and respiratory volume) were recorded from 5 unanesthetized chronically instrumented intact piglets. Approximate entropy values of the EMG during cough and swallow were found significantly (p < 0.05 and p < 0.01 respectively) lower than those of eupneic EMG.ConclusionReduced complexity values of the respiratory neural network output corresponding to coughs and swallows suggest synchronous neural activity of a homogeneous group of neurons. The higher complexity values exhibited by eupneic respiratory activity are the result of a more random behaviour, which is the outcome of the integrated action of several groups of neurons involved in the respiratory neural network.

Complexity measures of the central respiratory networks during wakefulness and sleep

Since sleep is known to influence respiratory activity we studied whether the sleep state would affect the complexity value of the respiratory network output. Specifically, we tested the hypothesis that the complexity values of the diaphragm EMG (EMGdia) activity would be lower during REM compared to NREM. Furthermore, since REM is primarily generated by a homogeneous population of neurons in the medulla, the possibility that REM-related respiratory output would be less complex than that of the awake state was also considered. Additionally, in order to examine the influence of neuron vulnerabilities within the rostral ventral medulla (RVM) on the complexity of the respiratory network output, we inhibited respiratory neurons in the RVM by microdialysis of GABA(A) receptor agonist muscimol. Diaphragm EMG, nuchal EMG, EEG, EOG as well as other physiological signals (tracheal pressure, blood pressure and respiratory volume) were recorded from five unanesthetized chronically instrumented intact piglets (3-10 days old). Complexity of the diaphragm EMG (EMGdia) signal during wakefulness, NREM and REM was evaluated using the approximate entropy method (ApEn). ApEn values of the EMGdia during NREM and REM sleep were found significantly (p < 0.05 and p < 0.001, respectively) lower than those of awake EMGdia after muscimol inhibition. In the absence of muscimol, only the differences between REM and wakefulness ApEn values were found to be significantly different.

Complexity Measures of the Central Respiratory Networks during Wakefulness and Sleep in Piglets

In this study, we examine EMGdi complexity before and after general (muscimol) inhibition of respiratory neurons within the rostral ventral medulla (RVM). We inserted a microdialysis guide tube into the RVM region, allowing us to dialyze muscimol on a daily basis during experiments. Animals were studied using the technique of barometric plethysmography, allowing us to measure ventilation without restraint. The EMGdi signals were recorded from 5 unanesthetized, chronically instrumented and intact piglets (3-10 days old) during eupnea before and after general inhibition of respiratory neurons in the RVM and analyzed using the approximate entropy and fractal analysis methods. Ten consecutive breaths were taken after 6 continuous minutes of unequivocal wakefulness, 3 minutes of NREM and 1 minute of REM sleep. Once the control responses to room air were measured, we dialyzed muscimol. GABAA agonist (10mM) into the RVM and repeated in room air. Note that muscimol is an nonspecific inhibitor and inhibits the neurons in the RVM. The EMGdi signals were recorded from 4 unanesthetized, chronically instrumented piglets (3-10 days old) during eupnea and analyzed using the expectation-maximization (EM) fractal method as in the first set of experiments, The complexity values in NREM were reduced more by the inhibition than were the awake stage. However, the changes in the complexity values due to the inhibition of the RVM were much more prominent during REM. The differences in the complexity measures of EMGdi before and after muscimol dialysis were statistically significant (p<0.01) during REM and NREM, but not during wakefulness. Our findings show that inhibition of the RVM reduced the complexity of the respiratory patterns significantly (p<0.05) during NREM and REM sleep stages. These data indicate that the RVM plays an important role in both the control of sleep and the sudden infant death syndrome (SIDs).