High-speed Amperometry Research Articles

Hypoxic Effects of Heroin and Fentanyl and Their Basic Physiological Mechanisms.

Respiratory depression that diminishes oxygen delivery to the brain is the most dangerous effect of opioid drugs. While plethysmography is a valuable tool to examine drug-induced changes in respiration, the primary cause of brain abnormalities induced by opioids is the global decrease in brain oxygen levels. The primary goal of this review is to provide an overview and discussion on fluctuations in brain oxygen levels induced by opioids, with a focus on heroin and fentanyl. To evaluate fluctuations in brain oxygen levels we used oxygen sensors coupled with high-speed amperometry in awake, freely moving rats. First, we provide an overview of brain oxygen responses induced by natural physiological stimuli and discuss the mechanisms regulating oxygen entry into brain tissue. Then, we present data on brain oxygen responses induced by heroin and fentanyl and review their underlying mechanisms. These data allowed us to compare the effects of these drugs on brain oxygen regarding their latency, potency, time-dependency, and potential lethality at high doses as well as their relationships with peripheral oxygen responses. We also discuss data on the effects of naloxone on brain oxygen responses induced by heroin and fentanyl in the paradigms of both the pre-treatment and treatment, when naloxone is administered at different times after the primary opioid drug. Although most data discussed were obtained in rats, they may have clinical relevance for understanding the mechanisms underlying the physiological effects of opioids and developing rational treatment strategies to decrease acute lethality and long-term health complications of opioid misuse.

Brain oxygen responses induced by opioids: focus on heroin, fentanyl, and their adulterants.

Opioids are important tools for pain management, but abuse can result in serious health complications. Of these complications, respiratory depression that leads to brain hypoxia is the most dangerous, resulting in coma and death. Although all opioids at large doses induce brain hypoxia, danger is magnified with synthetic opioids such as fentanyl and structurally similar analogs. These drugs are highly potent, act rapidly, and are often not effectively treated by naloxone, the standard of care for opioid-induced respiratory depression. The goal of this review paper is to present and discuss brain oxygen responses induced by opioids, focusing on heroin and fentanyl. In contrast to studying drug-induced changes in respiratory activity, we used chronically implanted oxygen sensors coupled with high-speed amperometry to directly evaluate physiological and drug-induced fluctuations in brain oxygen levels in awake, freely moving rats. First, we provide an overview of brain oxygen responses to physiological stimuli and discuss the mechanisms regulating oxygen entry into brain tissue. Next, we present data on brain oxygen responses induced by heroin and fentanyl and review underlying mechanisms. These data allowed us to compare the effects of these drugs on brain oxygen in terms of their potency, time-dependent response pattern, and potentially lethal effect at high doses. Then, we present the interactive effects of opioids during polysubstance use (alcohol, ketamine, xylazine) on brain oxygenation. Finally, we consider factors that affect the therapeutic potential of naloxone, focusing on dosage, timing of drug delivery, and contamination of opioids by other neuroactive drugs. The latter issue is considered chiefly with respect to xylazine, which strongly potentiates the hypoxic effects of heroin and fentanyl. Although this work was done in rats, the data are human relevant and will aid in addressing the alarming rise in lethality associated with opioid misuse.

Combined treatment with naloxone and the alpha2 adrenoceptor antagonist atipamezole reversed brain hypoxia induced by a fentanyl-xylazine mixture in a rat model.

Xylazine, a veterinary tranquillizer known by drug users as "Tranq", is being increasingly detected in people who overdose on opioid drugs, indicating enhanced health risk of fentanyl-xylazine mixtures. We recently found that xylazine potentiates fentanyl- and heroin-induced brain hypoxia and eliminates the rebound-like post-hypoxic oxygen increases. Here, we used oxygen sensors coupled with high-speed amperometry in rats of both sexes to explore the treatment potential of naloxone plus atipamezole, a selective α2-adrenoceptor antagonist, in reversing brain (nucleus accumbens) and periphery (subcutaneous space) hypoxia induced by a fentanyl-xylazine mixture. Pretreatment with naloxone (0.2 mg/kg, IV) fully blocked brain and peripheral hypoxia induced by fentanyl (20 μg/kg, IV), but only partially decreased hypoxia induced by a fentanyl-xylazine mixture. Pretreatment with atipamezole (0.25 mg/kg, IV) fully blocked the hypoxic effects of xylazine (1.0 mg/kg, IV), but not fentanyl. Pretreatment with atipamezole + naloxone was more potent than naloxone alone in blocking the hypoxic effects of the fentanyl-xylazine mixture. Both naloxone and naloxone + atipamezole, delivered at the peak of brain hypoxia (3 min post fentanyl-xylazine exposure), reversed the rapid initial brain hypoxia, but only naloxone + atipamezole decreased the prolonged weaker hypoxia. There were no sex differences in the effects of the different drugs and their combinations on brain and peripheral oxygen responses. Results indicate that combined treatment with naloxone and atipamezole is more effective than naloxone alone in reversing the hypoxic effects of fentanyl-xylazine mixtures. Naloxone + atipamezole treatment should be considered in preventing overdoses induced by fentanyl-xylazine mixtures in humans.

Basic metabolic and vascular effects of ketamine and its interaction with fentanyl

Ketamine is a short-acting general anesthetic with hallucinogenic, analgesic, and amnestic properties. In addition to its anesthetic use, ketamine is commonly abused in rave settings. While safe when used by medical professionals, uncontrolled recreational use of ketamine is dangerous, especially when mixed with other sedative drugs, including alcohol, benzodiazepines, and opioid drugs. Since synergistic antinociceptive interactions between opioids and ketamine were demonstrated in both preclinical and clinical studies, such an interaction could exist for the hypoxic effects of opioid drugs. Here, we focused on the basic physiological effects of ketamine as a recreational drug and its possible interactions with fentanyl—a highly potent opioid that induces strong respiratory depression and robust brain hypoxia. By using multi-site thermorecording in freely-moving rats, we showed that intravenous ketamine at a range of human relevant doses (3, 9, 27 mg/kg) dose-dependently increases locomotor activity and brain temperature, as assessed in the nucleus accumbens (NAc). By determining temperature differentials between the brain, temporal muscle, and skin, we showed that the brain hyperthermic effect of ketamine results from increased intracerebral heat production, an index of metabolic neural activation, and decreased heat loss due to peripheral vasoconstriction. By using oxygen sensors coupled with high-speed amperometry we showed that ketamine at the same doses increases NAc oxygen levels. Finally, co-administration of ketamine with intravenous fentanyl results in modest enhancement of fentanyl-induced brain hypoxia also enhancing the post-hypoxic oxygen increase. Therefore, in contrast to fentanyl, ketamine increases brain oxygenation but potentiates brain hypoxia induced by fentanyl.

Rapid fluctuations in brain oxygenation during glucose-drinking behavior in trained rats.

Proper inflow of oxygen into brain tissue is essential for maintaining normal neural functions. Although oxygen levels in the brain's extracellular space depend upon a balance between its delivery from arterial blood and its metabolic consumption, the use of high-speed electrochemical detection revealed rapid increases in brain oxygen levels elicited by various salient sensory stimuli. These stimuli also increase intrabrain heat production, an index of metabolic neural activation, but these changes are slower and more prolonged than changes in oxygen levels. Therefore, under physiological conditions, the oxygen inflow into brain tissue exceeds its loss due to consumption, thus preventing any metabolic deficit. Here, we used oxygen sensors coupled with amperometry to examine the pattern of real-time oxygen fluctuations in the nucleus accumbens during glucose-drinking behavior in trained rats. Following the exposure to a glucose-containing cup, oxygen levels rapidly increased, peaked when the rat initiated drinking, and relatively decreased during consumption. Similar oxygen changes but more episodic drinking occurred when Stevia, a calorie-free sweet substance, was substituted for glucose. When water was substituted for glucose, rats tested the water but refused to consume all of it. Although the basic pattern of oxygen changes during this water test was similar to that with glucose drinking, the increases were larger. Finally, oxygen increases were significantly larger when rats were exposed to concealed glucose and made multiple unsuccessful attempts to obtain and consume it. Based on these data, we discuss the mechanisms underlying behavior-related brain oxygen fluctuations and their functional significance.NEW & NOTEWORTHY Oxygen sensors coupled with high-speed amperometry were used to examine brain oxygen fluctuations during glucose-drinking behavior in trained rats. Oxygen levels rapidly increased following presentation of a glucose-contained cup, peaking at the initiation of glucose drinking, and relatively decreasing during drinking. Oxygen increases were larger when rats were exposed to concealed glucose and made multiple attempts to obtain it. We discuss the mechanisms underlying behavior-related brain oxygen fluctuations and their functional significance.

Cocaine added to heroin fails to affect heroin-induced brain hypoxia

Heroin and cocaine are both highly addictive drugs that cause unique physiological and behavioral effects. These drugs are often co-administered and cocaine has been found in ~20% of cases of opioid overdose death. Respiratory depression followed by brain hypoxia is the most dangerous effect of high-dose opioids that could result in coma and even death. Conversely, cocaine at optimal self-administering doses increases brain oxygen levels. Considering these differences, it is unclear what pattern of oxygen changes will occur when these drugs are co-administered. Here, we used high-speed amperometry with oxygen sensors to examine changes in oxygen concentrations in the nucleus accumbens (NAc) induced by intravenous (iv) cocaine, heroin, and their mixtures in freely-moving rats. Cocaine delivered at a range of doses, both below (0.25 mg/kg) and within the optimal range of self-administration (0.5 and 1.0 mg/kg) modestly increased NAc oxygen levels. In contrast, heroin increased oxygen levels at a low reinforcing dose (0.05 mg/kg), but induced a biphasic down-up change at higher reinforcing doses (0.1 and 0.2 mg/kg), and caused a strong monophasic oxygen decrease during overdose (0.6 mg/kg). When combined at moderate doses, cocaine (0.25, 0.5 mg/kg) slightly increased and prolonged oxygen increases induced by heroin alone (0.5 and 0.1 mg/kg), but oxygen decreases were identical when cocaine (1 mg/kg) was combined with heroin at large doses (0.2 and 0.6 mg/kg). Therefore, health dangers of speedball may result from de-compensation of vital functions due to diminished intra-brain oxygen inflow induced by high-dose heroin coupled with enhanced oxygen use induced by cocaine.

Counting the Number of Glutamate Molecules in Single Synaptic Vesicles

Analytical tools for direct quantitative measurements of glutamate, the principal excitatory neurotransmitter in brain have been lacking. Here, we introduce a new method for direct quantification of the number of glutamate molecules stored inside single synaptic vesicles. An ultra-fast enzyme-based glutamate sensor is placed into a solution of isolated synaptic vesicles, which stochastically rupture at the sensor surface in a potential dependent manner when applying a constant negative potential. High-speed amperometry is used to record sub-millisecond current spikes, which represent glutamate release from single vesicles that burst open at the sensor surface and is amperometrically quantified. Our measurements show that a single synaptic vesicle encapsulates about the same amount of glutamate molecules and sometimes the partial amount as we monitor being released at single exocytotic events and at in the nucleus accumbens of mouse brain tissue. Hence, this new methodology introduces the means to quantify ultra-small amounts of glutamate and to study synaptic vesicle physiology, pathogenesis and drug treatments for neuronal disorders where glutamate is involved.

Interactions of benzodiazepines with heroin: Respiratory depression, temperature effects, and behavior

Benzodiazepines are important therapeutic drugs, but they are often abused and co-abused with opioids. Clinical evidence suggests that benzodiazepines can inhibit respiration, and when combined with the respiratory-depressive effects of opioids, may increase likelihood of death. In this study we used oxygen sensors coupled with high-speed amperometry and multi-site thermorecording to examine how intravenous (iv) midazolam, a potent benzodiazepine, modulates the brain hypoxic and temperature effects of iv heroin in freely-moving rats. Oxygen levels and brain temperature were assessed with high temporal resolution in the nucleus accumbens (NAc), an important structure in the motivational-reinforcement circuit. When administered alone, midazolam (2 mg/kg) modestly decreased NAc temperature but had no evident effects on oxygen levels in this structure. In contrast, heroin (0.4 mg/kg) induced a strong decrease in NAc oxygen that was followed by a weaker, rebound-like oxygen increase. Midazolam pretreatment did not affect heroin-induced brain hypoxia but potentiated the initial hypothermia induced by heroin. However, co-administration of these drugs potentiated the heroin-induced oxygen decrease and enhanced heroin-induced brain hypothermia. Co-administration of heroin and midazolam also resulted in enhanced locomotor inhibition and loss of motor control. This effect caused some rats to collapse, resulting in nose and mouth occlusion, which caused a secondary hypoxic phase. These results could have important implications for human drug users, as the combined use of benzodiazepines with potent opioids not only results in sustained brain hypoxia but creates conditions of loss of motor control which could result in asphyxia and death.This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

Central and Peripheral Mechanisms Underlying Physiological and Drug-Induced Fluctuations in Brain Oxygen in Freely-Moving Rats.

The goal of this work is to consider physiological fluctuations in brain oxygen levels and its changes induced by opioid drugs. This review article presents, as a comprehensive story, the most important findings obtained in our laboratory by using high-speed amperometry with oxygen sensors in awake, freely moving rats; most of these findings were separately published elsewhere. First, we show that oxygen levels in the nucleus accumbens (NAc) phasically increase following exposure to natural arousing stimuli. Since accumbal neurons are excited by arousing stimuli and NAc oxygen levels increase following glutamate (GLU) microinjections in the NAc, local neural activation with subsequent cerebral vasodilation appears to mediate the rapid oxygen increases induced by arousing stimuli. While it is established that intra-cerebral entry of oxygen depends on brain metabolism, physiological increases in NAc oxygen occurred more rapidly than increases in metabolic activity as assessed by intra-brain heat production. Therefore, due to neural activation and the subsequent rise in local cerebral blood flow (CBF), the brain receives more oxygen in advance of its metabolic requirement, thus preventing potential metabolic deficits. In contrast to arousing stimuli, three opioid drugs tested (heroin, fentanyl and oxycodone) decrease oxygen levels. As confirmed by our recordings in the subcutaneous space, a densely vascularized location with no metabolic activity of its own, these decreases result from respiratory depression with subsequent fall in blood oxygen levels. While respiratory depression was evident for all tested drugs, heroin was ~6-fold more potent than oxycodone, and fentanyl was 10-20-fold more potent than heroin. Changes in brain oxygen induced by respiratory depression appear to be independent of local vascular and blood flow responses, which are triggered, via neuro-vascular coupling, by the neuronal effects of opioid drugs.

Opposing mechanisms underlying differential changes in brain oxygen and temperature induced by intravenous morphine

Morphine remains widely used in clinical settings due to its potent analgesic properties. However, one of the gravest risks of all opioids is their ability to induce respiratory depression and subsequent brain hypoxia that can lead to coma and death. Due to these life-threatening effects, our goal was to examine the effects of intravenous morphine at a wide range of doses (0.1-6.4 mg/kg) on changes in brain oxygen levels in freely moving rats. We used oxygen sensors coupled with high-speed amperometry and conducted measurements in the nucleus accumbens (NAc) and subcutaneous (SC) space, the latter serving as a proxy for blood oxygen levels that depend on respiratory activity. We also examined the effects of morphine on NAc, muscle, and skin temperature. Morphine induced dose-dependent decreases in SC oxygen levels, suggesting respiratory depression, but differential effects on NAc oxygen: increases at low and moderate doses (0.1-1.6 mg/kg) and decreases at the highest dose tested (6.4 mg/kg). Morphine also increased brain temperature at low and moderate doses but induced a biphasic, down-up change at high doses. The oxygen increases appear to result from a neurovascular coupling mechanism via local vasodilation and enhanced oxygen entry into brain tissue to compensate for blood oxygen drops caused by modest respiratory depression. At high morphine doses, this adaptive mechanism is unable to compensate for the enhanced respiratory depression, resulting in brain hypoxia. Hence, morphine appears to be safe when used as an analgesic at clinically relevant doses but poses great risks at high doses, likely to be abused by drug users. NEW & NOTEWORTHY With the use of oxygen sensors coupled with amperometry, we show that morphine induces differential effects on brain oxygen levels, slightly increasing them at low doses and strongly decreasing them at high doses. In contrast, morphine dose dependently decreases oxygen levels in the SC space. Therefore, morphine engages opposing mechanisms affecting brain oxygen levels, enhancing them through neurovascular coupling at low, clinically relevant doses and decreasing them due to dramatic respiratory depression at high doses, likely to be abused.

Changes in brain oxygen and glucose induced by oxycodone: Relationships with brain temperature and peripheral vascular tone

Oxycodone is a semi-synthetic opioid drug that is used to alleviate acute and chronic pain. However, oxycodone is often abused and, when taken at high doses, can induce powerful CNS depression that manifests in respiratory abnormalities, hypotension, coma, and death. Here, we employed several techniques to examine the effects of intravenous oxycodone at a wide range of doses on various metabolism-related parameters in awake, freely-moving rats. High-speed amperometry was used to assess how oxycodone affects oxygen and glucose levels in the nucleus accumbens (NAc). These measurements were supplemented by recordings of locomotor activity and temperature in the NAc, temporal muscle, and skin. At low doses, which are known to maintain self-administration behavior (0.15–0.3 mg/kg), oxycodone transiently decreased locomotor activity, induced modest brain and body hyperthermia, and monotonically increased NAc oxygen and glucose levels. While locomotor inhibition became stronger with higher oxycodone doses (0.6–1.2 mg/kg), NAc oxygen and glucose transiently decreased and subsequently increased. High-dose oxycodone induced similar biphasic down-up changes in brain and body temperature, with the initial decreases followed by increases. While cerebral vasodilation induced by neural activation appears to be the underlying mechanism for the correlative increases in brain oxygen and glucose levels, respiratory depression and the subsequent drop in blood oxygen likely mediate the brain hypoxia induced by large-dose oxycodone injections. The initial inhibitory effects induced by large-dose oxycodone injections could be attributed to rapid and profound CNS depression—the most dangerous health complication linked to opioid overdose in humans.

Inflow of oxygen and glucose in brain tissue induced by intravenous norepinephrine: relationships with central metabolic and peripheral vascular responses.

As an essential part of sympathetic activation that prepares the organism for "fight or flight," peripheral norepinephrine (NE) plays an important role in regulating cardiac activity and the tone of blood vessels, increasing blood flow to the heart and the brain and decreasing blood flow to the organs not as necessary for immediate survival. To assess whether this effect is applicable to the brain, we used high-speed amperometry to measure the changes in nucleus accumbens (NAc) levels of oxygen and glucose induced by intravenous injections of NE in awake freely moving rats. We found that NE at low doses (2-18 μg/kg) induces correlative increases in NAc oxygen and glucose, suggesting local vasodilation and enhanced entry of these substances in brain tissue from the arterial blood. By using temperature recordings from the NAc, temporal muscle, and skin, we show that this central effect is associated with strong skin vasoconstriction and phasic increases in intrabrain heat production, indicative of metabolic neural activation. A tight direct correlation between NE-induced changes in metabolic activity and NAc levels of oxygen and glucose levels suggests that local cerebral vasodilation is triggered via a neurovascular coupling mechanism. Our data suggest that NE, by changing vascular tone and cardiac activity, triggers a visceral sensory signal that rapidly reaches the central nervous system via sensory nerves and induces neural activation. This neural activation leads to a chain of neurovascular events that promote entry of oxygen and glucose in brain tissue, thus preventing any possible metabolic deficit during functional activation. NEW & NOTEWORTHY Using high-speed amperometry and thermorecording in freely moving rats, we demonstrate that intravenous norepinephrine at physiological doses induces rapid correlative increases in nucleus accumbens oxygen and glucose levels coupled with increased intrabrain heat production. Although norepinephrine cannot cross the blood-brain barrier, by changing cardiac activity and vascular tone, it creates a sensory signal that reaches the central nervous system via sensory nerves, induces neural activation, and triggers a chain of neurovascular events that promotes intrabrain entry of oxygen and glucose.

Heroin Contaminated with Fentanyl Dramatically Enhances Brain Hypoxia and Induces Brain Hypothermia.

While opioid abuse is an established medical and public health issue, the increased availability of highly potent synthetic opioids, such as fentanyl, has given rise to acute health complications, including a comatose state and death during drug overdose. Since respiratory depression that leads to acute hypoxia is the most dangerous complication of opioid drug use, we examined the effects of intravenous heroin and heroin contaminated with 10% fentanyl on oxygen levels in the nucleus accumbens (NAc) monitored using high-speed amperometry in freely moving rats. Additionally, we examined the effects of heroin, fentanyl, and their mixture on locomotion and temperatures in the NAc, temporal muscle, and skin. Both fentanyl and heroin at human-relevant doses (400 and 40 μg/kg, respectively) induced rapid, strong and transient decreases in NAc oxygen, indicative of brain hypoxia. When the heroin-fentanyl mixture was injected, the NAc hypoxic response was greatly potentiated in its duration, suggesting sustained hypoxia. In contrast to modest, monophasic brain temperature increases caused by heroin alone, the heroin-fentanyl mixture induced a biphasic temperature response, with a prominent postinjection decrease resulting from peripheral vasodilation. This hypothermic effect, albeit much smaller and more transient, was typical of fentanyl injected alone. Our findings indicate that accidental use of fentanyl instead of heroin, or even a relatively minor contamination of "street heroin" with fentanyl, poses great danger for acute health complications, including a comatose state and death.

Fentanyl-Induced Brain Hypoxia Triggers Brain Hyperglycemia and Biphasic Changes in Brain Temperature.

Fentanyl is a potent synthetic opioid used extensively in humans for general anesthesia and analgesia. Fentanyl has emerged as a recreational drug, often in combination with heroin, and can result in lethality during overdose. Fentanyl is well characterized as an anesthetic, but the basic physiological effects of fentanyl in the brain when taken as a drug of abuse are largely unknown. We used high-speed amperometry in freely moving rats to examine the effects of intravenous fentanyl at doses within the range of possible human intake (3-40 μg/kg) on oxygen and glucose levels in nucleus accumbens (NAc). Fentanyl induced a rapid, dose-dependent decrease in NAc oxygen followed by a more delayed and prolonged increase in NAc glucose. Fentanyl induced similar oxygen decreases in the basolateral amygdala, indicating that brain hypoxia could be a generalized phenomenon. We used oxygen recordings in the subcutaneous space to confirm that fentanyl-induced brain hypoxia results from decreases in blood oxygen levels caused by drug-induced respiratory depression. Temperature recordings in the NAc, muscle, and skin showed that fentanyl induces biphasic changes in brain temperature, with an initial decrease that results primarily from peripheral vasodilation, and a subsequent increase driven by metabolic brain activation. The initial vasodilation appears caused by respiratory depression-induced hypoxia and a subsequent rise in CO2 that drives fentanyl-induced increases in NAc glucose. Together, these data suggest that fentanyl-induced respiratory depression triggers brain hypoxia and subsequent hyperglycemia, both of which precede slower changes in brain temperature and metabolic brain activity.

Intravenous Heroin Induces Rapid Brain Hypoxia and Hyperglycemia that Precede Brain Metabolic Response.

Heroin use and overdose have increased in recent years as people transition from abusing prescription opiates to using the cheaper street drug. Despite a long history of research, many physiological effects of heroin and their underlying mechanisms remain unknown. Here, we used high-speed amperometry to examine the effects of intravenous heroin on oxygen and glucose levels in the nucleus accumbens (NAc) in freely-moving rats. Heroin within the dose range of human drug use and rat self-administration (100-200 μg/kg) induced a rapid, strong, but transient drop in NAc oxygen that was followed by a slower and more prolonged rise in glucose. Using oxygen recordings in the subcutaneous space, a densely-vascularized site with no metabolic activity, we confirmed that heroin-induced brain hypoxia results from decreased blood oxygen, presumably due to drug-induced respiratory depression. Respiratory depression and the associated rise in CO2 levels appear to drive tonic increases in NAc glucose via local vasodilation. Heroin-induced changes in oxygen and glucose were rapid and preceded the slow and prolonged increase in brain temperature and were independent of enhanced intra-brain heat production, an index of metabolic activation. A very high heroin dose (3.2 mg/kg), corresponding to doses used by experienced drug users in overdose conditions, caused strong and prolonged brain hypoxia and hyperglycemia coupled with robust initial hypothermia that preceded an extended hyperthermic response. Our data suggest heroin-induced respiratory depression as a trigger for brain hypoxia, which leads to hyperglycemia, both of which appear independent of subsequent changes in brain temperature and metabolic neural activity.

Rapid Physiological Fluctuations in Nucleus Accumbens Oxygen Levels Induced by Arousing Stimuli: Relationships with Changes in Brain Glucose and Metabolic Neural Activation.

Proper entry of oxygen from arterial blood into the brain is essential for maintaining brain metabolism under normal conditions and during functional neural activation. However, little is known about physiological fluctuations in brain oxygen and their underlying mechanisms. To address this issue, we employed high-speed amperometry with platinum oxygen sensors in freely moving male rats. Recordings were conducted in the nucleus accumbens (NAc), a critical structure for sensorimotor integration. Rats were exposed to arousing stimuli of different nature (brief auditory tone, a 1-min novel object presentation, a 3-min social interaction with a conspecific, and a 3-min tail-pinch). We found that all arousing stimuli increased NAc oxygen levels. Increases were rapid (4–10-s onset latencies), modest in magnitude (1–3 μM or 5%–15% over baseline) and duration (5–20 min), and generally correlated with the arousing potential of each stimulus. Two strategies were used to determine the mechanisms underlying the observed increases in NAc oxygen levels. First, we showed that NAc oxygen levels phasically increase following intra-NAc microinjections of glutamate (GLU) that excite accumbal neurons. Therefore, local neural activation with subsequent local vasodilation is involved in mediating physiological increases in NAc oxygen induced by arousing stimuli. Second, by employing oxygen monitoring in the subcutaneous space, a highly-vascularized area with no metabolic activity, we determined that physiological increases in NAc oxygen also depend on the rise in blood oxygen levels caused by respiratory activation. Due to the co-existence of different mechanisms governing oxygen entry into brain tissue, NAc oxygen responses differ from fluctuations in NAc glucose, which, within a normal behavioral continuum, are regulated exclusively by neuro-vascular coupling due to glucose’s highly stable levels in the blood. Finally, we discuss the relationships between physiological fluctuations in NAc oxygen, glucose and metabolic brain activation assessed by intra-brain heat production.

Brain Hyperglycemia Induced by Heroin: Association with Metabolic Neural Activation.

Glucose enters the brain extracellular space from arterial blood, and its proper delivery is essential for metabolic activity of brain cells. By using enzyme-based biosensors coupled with high-speed amperometry in freely moving rats, we previously showed that glucose levels in the nucleus accumbens (NAc) display high variability, increasing rapidly following exposure to various arousing stimuli. In this study, the same technology was used to assess NAc glucose fluctuations induced by intravenous heroin. Heroin passively injected at a low dose optimal for maintaining self-administration behavior (100 μg/kg) induces a rapid but moderate glucose rise (∼150-200 μM or ∼15-25% over resting baseline). When the heroin dose was doubled and tripled, the increase became progressively larger in magnitude and longer in duration. Heroin-induced glucose increases also occurred in other brain structures (medial thalamus, lateral striatum, hippocampus), suggesting that brain hyperglycemia is a whole-brain phenomenon but changes were notably distinct in each structure. While local vasodilation appears to be the possible mechanism underlying the rapid rise in extracellular glucose levels, the driving factor for this vasodilation (central vs peripheral) remains to be clarified. The heroin-induced NAc glucose increases positively correlated with increases in intracerebral heat production determined in separate experiments using multisite temperature recordings (NAc, temporal muscle and skin). However, glucose levels rise very rapidly, preceding much slower increases in brain heat production, a measure of metabolic activation associated with glucose consumption.

Development of Microelectrode Arrays Using Electroless Plating for CMOS-Based Direct Counting of Bacterial and HeLa Cells.

The development of two new types of high-density, electroless plated microelectrode arrays for CMOS-based high-sensitivity direct bacteria and HeLa cell counting are presented. For emerging high-sensitivity direct pathogen counting, two technical challenges must be addressed. One is the formation of a bacteria-sized microelectrode, and the other is the development of a high-sensitivity and high-speed amperometry circuit. The requirement for microelectrode formation is that the gold microelectrodes are required to be as small as the target cell. By improving a self-aligned electroless plating technique, the dimensions of the microelectrodes on a CMOS sensor chip in this work were successfully reduced to 1.2 μm × 2.05 μm. This is 1/20th of the smallest size reported in the literature. Since a bacteria-sized microelectrode has a severe limitation on the current flow, the amperometry circuit has to have a high sensitivity and high speed with low noise. In this work, a current buffer was inserted to mitigate the potential fluctuation. Three test chips were fabricated using a 0.6- μm CMOS process: two with 1.2 μm × 2.05 μm (1024 × 1024 and 4 × 4) sensor arrays and one with 6- μm square (16 × 16) sensor arrays; and the microelectrodes were formed on them using electroless plating. The uniformity among the 1024 × 1024 electrodes arranged with a pitch of 3.6 μm × 4.45 μm was optically verified. For improving sensitivity, the trenches on each microelectrode were developed and verified optically and electrochemically for the first time. Higher sensitivity can be achieved by introducing a trench structure than by using a conventional microelectrode formed by contact photolithography. Cyclic voltammetry (CV) measurements obtained using the 1.2 μm × 2.05 μm 4 × 4 and 6- μm square 16 × 16 sensor array with electroless-plated microelectrodes successfully demonstrated direct counting of the bacteria-sized microbeads and HeLa cells.

Behavior-associated and post-consumption glucose entry into the nucleus accumbens extracellular space during glucose free-drinking in trained rats.

Glucose is the primary energetic substrate for the metabolic activity of brain cells and its proper delivery from the arterial blood is essential for neural activity and normal brain functions. Glucose is also a unique natural reinforcer, supporting glucose-drinking behavior without food or water deprivation. While it is known that glucose enters brain tissue via gradient-dependent facilitated diffusion, it remains unclear how glucose levels are changed during natural behavior and whether the direct central action of ingested glucose can be involved in regulating glucose-drinking behavior. Here, we used glucose biosensors with high-speed amperometry to examine the pattern of phasic and tonic changes in extracellular glucose in the nucleus accumbens (NAc) during unrestricted glucose-drinking in well-trained rats. We found that the drinking behavior is highly cyclic and is associated with relatively large and prolonged increases in extracellular glucose levels. These increases had two distinct components: a highly phasic but relatively small behavior-related rise and a larger tonic elevation that results from the arrival of consumed glucose into the brain’s extracellular space. The large post-ingestion increases in NAc glucose began minutes after the cessation of drinking and were consistently associated with periods of non-drinking, suggesting that the central action of ingested glucose could inhibit drinking behavior by inducing a pause in activity between repeated drinking bouts. Finally, the difference in NAc glucose responses found between active, behavior-mediated and passive glucose delivery via an intra-gastric catheter confirms that motivated behavior is also associated with metabolic glucose use by brain cells.

Central and peripheral contributions to dynamic changes in nucleus accumbens glucose induced by intravenous cocaine.

The pattern of neural, physiological and behavioral effects induced by cocaine is consistent with metabolic neural activation, yet direct attempts to evaluate central metabolic effects of this drug have produced controversial results. Here, we used enzyme-based glucose sensors coupled with high-speed amperometry in freely moving rats to examine how intravenous cocaine at a behaviorally active dose affects extracellular glucose levels in the nucleus accumbens (NAc), a critical structure within the motivation-reinforcement circuit. In drug-naive rats, cocaine induced a bimodal increase in glucose, with the first, ultra-fast phasic rise appearing during the injection (latency 6–8 s; ~50 μM or ~5% of baseline) followed by a larger, more prolonged tonic elevation (~100 μM or 10% of baseline, peak ~15 min). While the rapid, phasic component of the glucose response remained stable following subsequent cocaine injections, the tonic component progressively decreased. Cocaine-methiodide, cocaine's peripherally acting analog, induced an equally rapid and strong initial glucose rise, indicating cocaine's action on peripheral neural substrates as its cause. However, this analog did not induce increases in either locomotion or tonic glucose, suggesting direct central mediation of these cocaine effects. Under systemic pharmacological blockade of dopamine transmission, both phasic and tonic components of the cocaine-induced glucose response were only slightly reduced, suggesting a significant role of non-dopamine mechanisms in cocaine-induced accumbal glucose influx. Hence, intravenous cocaine induces rapid, strong inflow of glucose into NAc extracellular space by involving both peripheral and central, non-dopamine drug actions, thus preventing a possible deficit resulting from enhanced glucose use by brain cells.