Complete protection from impending stroke following permanent middle cerebral artery occlusion in awake, behaving rats.

Using a rodent model of ischemic stroke [permanent middle cerebral artery occlusion (pMCAO)], our laboratory has previously demonstrated that sensory-evoked cortical activation via mechanical single whisker stimulation treatment delivered under an anesthetized condition within 2 h of ischemic onset confers complete protection from impending infarct. There is a limited time window for this protection; rats that received the identical treatment at 3 h following ischemic onset lost neuronal function and sustained a substantial infarct. Rats in these studies, however, were anesthetized with sodium pentobarbital or isoflurane, whereas most human stroke patients are typically awake. To optimize our animal model, the present study examined, using functional imaging, histological, and behavioral analysis, whether self-induced sensorimotor stimulation is also protective in unrestrained, behaving rats that actively explore an enriched environment. Rats were revived from anesthesia either immediately or at 3 h after pMCAO, at which point they were allowed to freely explore an enriched environment. Rats that explored immediately after ischemic onset maintained normal cortical function and did not sustain infarct, even when their whiskers were clipped. Rats that were revived at 3 h post-pMCAO exhibited eliminated cortical function and sustained cortical infarct. Further, the data suggested that the level of individual active exploration could influence the outcome. Thus, early activation of the ischemic cortical area via unrestrained exploration resulted in protection from ischemic infarct, whereas late activation resulted in infarct, irrespective of the level of arousal or whisker-specific stimulation.

Permanent cerebral vessel occlusion via double ligature and transection.

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia). Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex, is the most common site of human stroke, and ischemia within its territory can result in extensive dysfunction or death. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA. Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Early stimulation treatment provides complete sensory-induced protection from ischemic stroke under isoflurane anesthesia.

Using a rodent model of ischemia [permanent middle cerebral artery occlusion (pMCAO)], previous studies demonstrated that whisker stimulation treatment completely protects the cortex from impending stroke when initiated within 2 h following pMCAO. When initiated 3 h post-pMCAO, the identical treatment exacerbates stroke damage. Rats in these studies, however, were anesthetised with sodium pentobarbital, whereas human stroke patients are typically awake. To overcome this drawback, our laboratory has begun to use the anesthetic isoflurane, which allows rats to rapidly recover from pMCAO within minutes, to test stimulation treatment in awake rats and to determine whether isoflurane has an effect upon the pMCAO stroke model. We found no difference in infarct volume between pMCAO in untreated controls under either sodium pentobarbital or isoflurane, and the primary finding was that rats that received treatment immediately post-pMCAO maintain cortical function and no stroke damage, whereas rats that received treatment 3 h post-pMCAO exhibited eliminated cortical activity and extensive stroke damage. The only difference between anesthetics was the broad extent of evoked cortical activity observed during both functional imaging and electrophysiological recording, suggesting that the extent of evoked activity evident under isoflurane anesthesia is supported by underlying neuronal activity. Given the high degree of similarity with previous data, we conclude that the pMCAO stroke model is upheld with the use of isoflurane. This study demonstrated that the isoflurane-anesthetised rat pMCAO model can be used for cerebrovascular studies, and allows for highly detailed investigation of potential novel treatments for ischemic stroke using awake, behaving animals.

A rat's whiskers point the way toward a novel stimulus-dependent, protective stroke therapy.

Stroke is the fourth leading cause of death in the United States and the leading cause of long-term disability. Ischemic stroke, due to an interruption in blood supply, is particularly prevalent; 87% of all strokes are ischemic. Unfortunately, current options for acute treatment are extremely limited and there is a great need for new treatment strategies. This review will discuss evidence that mild sensory stimulation can completely protect the jeopardized brain from an impending stroke in a rodent model. When delivered within the first 2 hours following ischemic onset, this stimulation results in complete protection, including a full reestablishment of cortical function, sensorimotor capabilities, and blood flow. Identical stimulation, however, initiated 3 hours following ischemic onset, results in an increase in damage compared with untreated animals. The protective effect is not specific to a single sensory modality, anesthesia, or age, and increasing evoked cortical activity by increasing stimulation accelerates recovery. Taken together, these findings demonstrate that cortical activity is a critical factor for protection and suggest a new, exciting potential avenue for the development of acute stroke treatment strategies that may produce a noninvasive, drug-free, equipment-free, and side effect-free means of protecting from ischemic stroke.

Sensory Stimulation-Based Complete Protection from Ischemic Stroke Remains Stable at 4 Months Post-Occlusion of MCA.

Previous research from our lab has shown that when using a rodent model of ischemic stroke (permanent middle cerebral artery occlusion), mild sensory stimulation, when delivered within two hours of ischemic onset, completely protects the cortex from impending ischemic stroke damage when assessed 24 hours post-occlusion. However, the long-term stability of this protection remains unclear. Using intrinsic signal optical imaging for assessment of cortical function, laser speckle imaging for assessment of blood flow, a battery of behavioral tests and cresyl violet for histological assessment, the present study examined whether this protection was long-lasting. When assessed 4 months post-occlusion (this length of time being equivalent to 10-15 years in humans), rats receiving sensory stimulation treatment immediately after ischemic onset exhibit normal neuronal and vascular function, and they are behaviorally and histologically equivalent to healthy controls (surgical shams). Thus, the complete neuroprotection due to cortical activation via sensory stimulation remains stable with time. These findings add support to the translational potential of this sensory stimulation-based treatment.

Mild sensory stimulation protects the aged rodent from cortical ischemic stroke after permanent middle cerebral artery occlusion.

BACKGROUND: Accumulated research has shown that the older adult brain is significantly more vulnerable to stroke than the young adult brain. Although recent evidence in young adult rats demonstrates that single-whisker stimulation can result in complete protection from ischemic damage after permanent middle cerebral artery occlusion (pMCAO), it remains unclear whether the same treatment would be effective in older animals. METHODS AND RESULTS: Aged rats (21 to 24 months of age) underwent pMCAO and subsequently were divided into "treated" and "untreated" groups. Treated aged rats received intermittent single-whisker stimulation during a 120-minute period immediately after pMCAO, whereas untreated aged rats did not. These animals were assessed using a battery of behavioral tests 1 week before and 1 week after pMCAO, after which their brains were stained for infarct. An additional treated aged group and a treated young adult group also were imaged with functional imaging. Results demonstrated that the recovery of treated aged animals was indistinguishable from that of the treated young adult animals. Treated aged rats had fully intact sensorimotor behavior and no infarct, whereas untreated aged rats were impaired and sustained cortical infarct. CONCLUSIONS: Taken together, our results confirm that single-whisker stimulation is protective in an aged rodent pMCAO model, despite age-associated stroke vulnerability. These findings further suggest potential for translation to the more clinically relevant older adult human population. (J Am Heart Assoc. 2012;1:e001255 doi: 10.1161/JAHA.112.001255.).

Mild sensory stimulation reestablishes cortical function during the acute phase of ischemia.

When delivered within 1 and in most cases 2 h of permanent middle cerebral artery occlusion (pMCAO), mild sensory stimulation (intermittent single whisker stimulation) was shown to be completely neuroprotective 24 h after pMCAO in a rodent model of ischemic stroke, according to assessment with multiple techniques (Lay et al., 2010). The acute effect of stimulation treatment on the ischemic cortex, however, has yet to be reported. Here we characterize cortical function and perfusion during the 120 min whisker stimulation period in four experimental groups with treatment initiated 0, 1, 2 (protected groups), or 3 h (unprotected group) post-pMCAO using multiple techniques. According to functional imaging, a gradual return of evoked whisker functional representation to baseline levels was initiated with treatment onset and completed within the treatment period. Evoked neuronal activity and reperfusion to the ischemic area also showed a gradual recovery in protected animals. Surprisingly, a similar recovery profile was observed in response to treatment in all protected animals, regardless of treatment onset time. Nonstimulated pMCAO control group data demonstrate that reperfusion is not spontaneous. This makes the complete protection observed in the majority of animals stimulated at 2 h post-pMCAO even more surprising, as these animals recovered despite having been in a severely ischemic state for two full hours. In summary, when delivered within a 2 h window post-pMCAO, whisker stimulation treatment initiated reperfusion and a gradual recovery of cortical function that was completed or nearly completed within the treatment period.

Amount but not pattern of protective sensory stimulation alters recovery after permanent middle cerebral artery occlusion.

BACKGROUND AND PURPOSE: Using a rodent model of ischemia (permanent middle cerebral artery occlusion), our laboratory previously demonstrated that 4.27 minutes of patterned single-whisker stimulation delivered over 120 minutes can fully protect from impending damage when initiated within 2 hours of permanent middle cerebral artery occlusion ("early"). When initiated 3 hours postpermanent middle cerebral artery occlusion ("late"), stimulation resulted in irreversible damage. Here we investigate the effect of altering pattern, distribution, or amount of stimulation in this model. METHODS: We assessed the cortex using functional imaging and histological analysis with altered stimulation treatment protocols. In 2 groups of animals we administered the same number of whisker deflections but in a random rather than patterned fashion distributed either over 120 minutes or condensed into 10 minutes postpermanent middle cerebral artery occlusion. We also tested increased (full-whisker array versus single-whisker) stimulation. RESULTS: Early random whisker stimulation (condensed or dispersed) resulted in protection equivalent to early patterned stimulation. Early full-whisker array patterned stimulation also resulted in complete protection but promoted faster recovery. Late full-whisker array patterned stimulation, however, resulted in loss of evoked function and infarct volumes larger than those sustained by single-whisker counterparts. CONCLUSIONS: When induced early on after ischemic insult, stimulus-evoked cortical activity, irrespective of the parameters of peripheral stimulation that induced it, seems to be the important variable for neuroprotection.

Mild sensory stimulation completely protects the adult rodent cortex from ischemic stroke.

Despite progress in reducing ischemic stroke damage, complete protection remains elusive. Here we demonstrate that, after permanent occlusion of a major cortical artery (middle cerebral artery; MCA), single whisker stimulation can induce complete protection of the adult rat cortex, but only if administered within a critical time window. Animals that receive early treatment are histologically and behaviorally equivalent to healthy controls and have normal neuronal function. Protection of the cortex clearly requires reperfusion to the ischemic area despite permanent occlusion. Using blood flow imaging and other techniques we found evidence of reversed blood flow into MCA branches from an alternate arterial source via collateral vessels (inter-arterial connections), a potential mechanism for reperfusion. These findings suggest that the cortex is capable of extensive blood flow reorganization and more importantly that mild sensory stimulation can provide complete protection from impending stroke given early intervention. Such non-invasive, non-pharmacological intervention has clear translational potential.

Intrinsic signal optical imaging of brain function using short stimulus delivery intervals.

Intrinsic signal optical imaging (ISOI) can be used to map cortical function and organization. Because its detected signal lasts 10+s consisting of three phases, trials are typically collected using a long (tens of seconds) stimulus delivery interval (SDI) at the expense of efficiency, even when interested in mapping only the first signal phase (e.g., ISOI initial dip). It is unclear how the activity profile can change when stimuli are delivered at shorter intervals, and whether a short SDI can be implemented to improve efficiency. The goals of the present study are twofold: characterize the ISOI activity profile when multiple stimuli are delivered at 4s intervals, and determine whether successful mapping can be attained from trials collected using an SDI of 4s (offering >10x increase in efficiency). Our results indicate that four stimuli delivered 4s apart evoke an activity profile different from the triphasic signal, consisting of signal dips in a series at the same frequency as the stimuli despite a strong rise in signal prior to the 2nd to 4th stimuli. Visualization of such signal dips is dependent on using a baseline immediately prior to every stimulus. Use of the 4-s SDI is confirmed to successfully map activity with a similar location in peak activity and increased areal extent and peak magnitude compared to using a long SDI. Additional experiments were performed to begin addressing issues such as SDI temporal jittering, response magnitude as a function of SDI duration, and application for successful mapping of cortical function topography.

Imaging cortical absorption, scattering, and hemodynamic response during ischemic stroke using spatially modulated near-infrared illumination.

We describe a technique that uses spatially modulated near-infrared (NIR) illumination to detect and map changes in both optical properties (absorption and reduced scattering parameters) and tissue composition (oxy- and deoxyhemoglobin, total hemoglobin, and oxygen saturation) during acute ischemic injury in the rat barrel cortex. Cerebral ischemia is induced using an open vascular occlusion technique of the middle cerebral artery (MCA). Diffuse reflected NIR light (680 to 980 nm) from the left parietal somatosensory cortex is detected by a CCD camera before and after MCA occlusion. Monte Carlo simulations are used to analyze the spatial frequency dependence of the reflected light to predict spatiotemporal changes in the distribution of tissue absorption and scattering properties in the brain. Experimental results from seven rats show a 17+/-4.7% increase in tissue concentration of deoxyhemoglobin and a 45+/-3.1, 23+/-5.4, and 21+/-2.2% decrease in oxyhemoglobin, total hemoglobin concentration and cerebral tissue oxygen saturation levels, respectively, 45 min following induction of cerebral ischemia. An ischemic index (I(isch)=ctHHbctO(2)Hb) reveals an average of more then twofold contrast after MCAo. The wavelength-dependence of the reduced scattering (i.e., scatter power) decreased by 35+/-10.3% after MCA occlusion. Compared to conventional CCD-based intrinsic signal optical imaging (ISOI), the use of structured illumination and model-based analysis allows for generation of separate maps of light absorption and scattering properties as well as tissue hemoglobin concentration. This potentially provides a powerful approach for quantitative monitoring and imaging of neurophysiology and metabolism with high spatiotemporal resolution.

Robust and stable drinking behavior following long-term oral alcohol intake in rhesus macaques.

Face validity in animal models of alcohol abuse and dependence is often at odds with robust demonstrations of ethanol-seeking. This study determined the relative influence of ethanol and a flavorant in maintaining ethanol intake in a nonhuman primate model of "cocktail" drinking. Four-year-old male monkeys were maintained on a 6% ethanol/6% Tang solution made available in daily (M-F) 1-h sessions. Experiments determined the effect of (1) a second daily access session, (2) concurrent presentation of the Tang vehicle, (3) sequential presentation of the vehicle in the first daily session and the ethanol solution in the second session, (4) altering the Tang concentration, (5) altering the ethanol concentration, and (6) removal of the flavorant. Mean daily intake (2.7+/-0.2 g/kg/day) was stable over 7 months. Simultaneous availability of a large, but not a low-moderate, volume of the vehicle reduced ethanol intake by about 50%. Decreasing the concentration of Tang in the first daily session reduced ethanol intake, whereas intake of the standard solution was increased in the second session. Ethanol consumption was decreased by only 27% when the flavorant was removed. In summary, alterations that reduced intake in the first daily session resulted in compensatory increases in ethanol intake in the second session, suggesting that animals sought a specific level of ethanol intake per day. It is concluded that models with excellent face validity (flavored beverages) can produce reliable ethanol intake in patterns that are highly consistent with ethanol-seeking behavior.

Impact of ambient temperature on hyperthermia induced by (+/-)3,4-methylenedioxymethamphetamine in rhesus macaques.

The ambient temperature (T(A)) under which rodents are exposed to (+/-)3,4-methylenedioxymethamphetamine (MDMA) affects the direction and magnitude of the body temperature response, and the degree of hypo/hyperthermia generated in subjects can modify the severity of lasting brain changes in 'neurotoxicity' models. The thermoregulatory effects of MDMA have not been well described in non-human primates and it is unknown if T(A) has the potential to affect acute hyperthermia and therefore other lasting consequences of MDMA. The objective of this study was to determine if the temperature alteration produced by MDMA in nonhuman primates depends on T(A) as it does in rats and mice. Body temperature and spontaneous home cage activity were monitored continuously in six male rhesus monkeys via radiotelemetry. The subjects were challenged intramuscularly with 0.56-2.4 mg/kg (+/-)MDMA under each of three T(A) conditions (18, 24, and 30 degrees C) in a randomized order. The temperature was significantly elevated following injection with all doses of MDMA under each ambient temperature. The magnitude of mean temperature change was approximately 1 degrees C in most conditions suggesting a closely controlled thermoregulatory response in monkeys across a range of doses and ambient temperatures. Activity levels were generally suppressed by MDMA; however, a 50% increase over vehicle was observed after 0.56 MDMA under the 30 degrees C condition. It is concluded that MDMA produces a similar degree of hyperthermia in rhesus monkeys across a range of T(A) conditions that result in hypothermia or exaggerated hyperthermia in rodents. Monkey temperature responses to MDMA appear to be more similar to humans than to rodents and therefore the monkey may offer an improved model of effects related to MDMA-induced hyperthermia.

Differential contributions of dopaminergic D1- and D2-like receptors to cognitive function in rhesus monkeys.

RATIONALE: Dopaminergic neurotransmission is critically involved in many aspects of complex behavior and cognition beyond reward/reinforcement and motor function. Mental and behavioral disorders associated with major disruptions of dopamine neurotransmission, including schizophrenia, attention deficit/hyperactivity disorder, Parkinson's disease, Huntington's disease, and substance abuse produce constellations of neuropsychological deficits in learning, memory, and attention in addition to other defining symptoms. OBJECTIVE: To delineate the role dopaminergic D1- and D2-like receptor subtypes play in complex brain functions. MATERIALS AND METHODS: Monkeys (N = 6) were trained on cognitive tests adapted from a human neuropsychological assessment battery (CAmbridge Neuropsychological Test Automated Battery). The battery included tests of spatial working memory (self-ordered spatial search task), visuo-spatial associative memory and learning (visuo-spatial paired associates learning task, vsPAL) and motivation (progressive ratio task, PR). Tests of motor function (bimanual motor skill task, BMS; rotating turntable task, RTT) were also included. The effects of the dopamine D2-like antagonist raclopride (10-56 microg/kg, i.m.) and the D1-like antagonist SCH23390 (SCH, 3.2-56 microg/kg, i.m.) on cognitive performance were then determined. RESULTS: Deficits on PR, RTT, and BMS performance were observed after both raclopride and SCH23390. Spatial working memory accuracy was reduced to a greater extent by raclopride than by SCH, which was unexpected, given prior reports on the involvement of D1 signaling for spatial working memory in monkeys. Deficits were observed on vsPAL performance after raclopride, but not after SCH23390. CONCLUSIONS: The intriguing results suggest a greater contribution of D2- over D1-like receptors to both spatial working memory and object-location associative memory.

Hyperthermia induced by 3,4-methylenedioxymethamphetamine in unrestrained rhesus monkeys.

BACKGROUND: Exposure to (+/-)3,4-methylenedioxymethamphetamine ((+/-)MDMA) results in lasting reductions of many markers for serotonin terminals in a range of species. In rodents, the severity of insult depends in large part on the generation of hyperthermia in the subject. (+/-)MDMA can produce either hyperthermia or hypothermia in rodents depending on the ambient temperature and these effects may be limited to the S(+) enantiomer. Limited prior evidence suggests (+/-)MDMA does not produce hyperthermia in chair-restrained monkeys [Bowyer, J.F., Young, J.F., Slikker, W., Itzak, Y., Mayorga, A.J., Newport, G.D., Ali, S.F., Frederick, D.L., Paule, M.G., 2003. Plasma levels of parent compound and metabolites after doses of either d-fenfluramine or d-3,4-methylenedioxymethamphetamine (MDMA) that produce long-term serotonergic alterations. Neurotoxicology 24, 379-390]. This study was therefore conducted to determine if racemic MDMA and its enantiomers induce hyperthermia and increase spontaneous locomotor activity in unrestrained rhesus monkeys. METHODS: Body temperature and spontaneous home cage activity were monitored continuously in four monkeys via radiotelemetric devices. The subjects were challenged with 1.7 mg/kg, i.m., (+/-)MDMA, S(+)MDMA and R(-)MDMA in pseudorandomized order. RESULTS: Maximum and average temperature in the 4h interval post-dosing was elevated 0.7-0.9 degrees C by (+/-)MDMA and each enantiomer. Reductions in locomotor activity following dosing did not reliably differ from vehicle effects. CONCLUSIONS: MDMA produces an acute hyperthermia in unrestrained rhesus monkeys, much as it does with rats, mice, pigs, rabbits and humans. Hyperthermia occurs despite no increase in locomotor activity thus the effect does not depend on motor activation. Each enantiomer appears to be equivalently active thus primates may differ from rodents in thermoregulatory sensitivity to the R(-) enantiomer. Significant differences in outcome between this and a prior study in monkeys indicate a need for additional study of the thermoregulatory impact of MDMA in nonhuman primates.

Development of a chipscale integrated microelectrode/microelectronic device for brain implantable neuroengineering applications.

An ultralow power analog CMOS chip and a silicon based microelectrode array have been fully integrated to a microminiaturized "neuroport" for brain implantable neuroengineering applications. The CMOS integrated circuit (IC) includes preamplifier and multiplexing circuitry, and a hybrid flip-chip bonding technique was developed to fabricate a functional, encapsulated microminiaturized neuroprobe device. Our neuroport has been evaluated using various methods, including pseudospike detection and local excitation measurement, and showed suitable characteristics for recording neural activities. As a proof-of-concept demonstration, we have measured local field potentials from thalamocortical brain slices of rats, suggesting that the new neuroport can form a prime platform for the development of a microminiaturized neural interface to the brain in a single implantable unit. An alternative power delivery scheme using photovoltaic power converter, and an encapsulation strategy for chronic implantation are also discussed.

A microelectrode/microelectronic hybrid device for brain implantable neuroprosthesis applications.

We have designed, fabricated, and characterized a microminiaturized "neuroport" for brain implantable neuroprosthesis applications, using an analog CMOS integrated circuit and a silicon based microelectrode array. An ultra-low power, low-noise CMOS preamplifier array with integral multiplexing was designed to accommodate stringent thermal and electrophysiological requirements for implantation in the brain, and a hybrid integration approach was developed to fabricate a functional microminiaturized neuroprobe device. Measurements showed that our fully scalable 16-channel CMOS amplifier chip had an average gain of 44 dB, bandwidth from 10 Hz to 7.3 kHz, and an equivalent input noise of approximately 9 microVrms with an average power consumption per preamplifier of 52 microW, which is consistent with simulation results. As a proof-of-concept demonstration, we have measured local field potentials from thalamocortical brain slices of rats, showing oscillatory behavior with an amplitude about 0.5 mV and a period ranging 80-120 ms. The results suggest that the hybrid integrated neuroport can form a prime platform for the development of a next level microminiaturized neural interface to the brain in a single implantable unit.

Controlled and behaviorally relevant levels of oral ethanol intake in rhesus macaques using a flavorant-fade procedure.

BACKGROUND: Flavorant-fading procedures can initiate and maintain oral ethanol intake in rodents. The present study developed a similar procedure to achieve controlled and behaviorally relevant levels of ethanol intake in monkeys. METHODS: Male rhesus macaques (N = 13) were initially given the opportunity to consume 0.5 g/kg of a 1% (w/v) ethanol plus 4% (w/v) Tang solution in 1-hr limited-access sessions without the requirement of an operant response. Once consumption was stable at a particular concentration (%) and/or amount (g/kg), animals were given access to higher concentrations and/or amounts of ethanol. Animals were tested on a bimanual motor skill (BMS) task 20 and 90 min after consumption to assess behavioral impairment. Blood alcohol levels (BALs) were assessed after a session in which animals had the opportunity to consume up to 3.0 g/kg of 6% (w/v) ethanol. RESULTS: The gradual fading up of higher concentrations and amounts of ethanol resulted in controlled and robust levels (>2.0 g/kg) of ethanol intake in half of the subjects. Increasing the concentration of the sweetener from 4 to 6% (w/v) was effective in initiating consumption in three animals. Two monkeys required the additional step of presenting the increased-sweetener solutions after a meal (postprandial consumption) to initiate significant ethanol intake. Animals were significantly impaired on the BMS task after consumption of 2.0, 2.5, and 3.0 g/kg of ethanol. Individual consumption ranging from 0.8 to 3.0 g/kg of ethanol produced BALs of 18 to 269 mg/dl. CONCLUSIONS: The flavorant-fading procedure was effective in producing behaviorally relevant levels of ethanol consumption in rhesus macaques. This model facilitated a randomized-dose procedure to determine the behavioral effects of 0.5 to 3.0 g/kg of ethanol. This procedure therefore is of significant utility in determining behavioral or physiologic effects of specific doses of consumed ethanol in monkeys.