The hippocampus, prefrontal cortex, and perirhinal cortex are critical to incidental order memory.

Considerable research in rodents and humans indicates the hippocampus and prefrontal cortex are essential for remembering temporal relationships among stimuli, and accumulating evidence suggests the perirhinal cortex may also be involved. However, experimental parameters differ substantially across studies, which limits our ability to fully understand the fundamental contributions of these structures. In fact, previous studies vary in the type of temporal memory they emphasize (e.g., order, sequence, or separation in time), the stimuli and responses they use (e.g., trial-unique or repeated sequences, and incidental or rewarded behavior), and the degree to which they control for potential confounding factors (e.g., primary and recency effects, or order memory deficits secondary to item memory impairments). To help integrate these findings, we developed a new paradigm testing incidental memory for trial-unique series of events, and concurrently assessed order and item memory in animals with damage to the hippocampus, prefrontal cortex, or perirhinal cortex. We found that this new approach led to robust order and item memory, and that hippocampal, prefrontal and perirhinal damage selectively impaired order memory. These findings suggest the hippocampus, prefrontal cortex and perirhinal cortex are part of a broad network of structures essential for incidentally learning the order of events in episodic memory.

Retrieval-induced NMDA receptor-dependent Arc expression in two models of cocaine-cue memory.

The association of environmental cues with drugs of abuse results in persistent drug-cue memories. These memories contribute significantly to relapse among addicts. While conditioned place preference (CPP) is a well-established paradigm frequently used to examine the modulation of drug-cue memories, very few studies have used the non-preference-based model conditioned activity (CA) for this purpose. Here, we used both experimental approaches to investigate the neural substrates of cocaine-cue memories. First, we directly compared, in a consistent setting, the involvement of cortical and subcortical brain regions in cocaine-cue memory retrieval by quantifying activity-regulated cytoskeletal-associated (Arc) protein expression in both the CPP and CA models. Second, because NMDA receptor activation is required for Arc expression, we investigated the NMDA receptor dependency of memory persistence using the CA model. In both the CPP and CA models, drug-paired animals showed significant increases in Arc immunoreactivity in regions of the frontal cortex and amygdala compared to unpaired controls. Additionally, administration of a NMDA receptor antagonist (MK-801 or memantine) immediately after cocaine-CA memory reactivation impaired the subsequent conditioned locomotion associated with the cocaine-paired environment. The enhanced Arc expression evident in a subset of corticolimbic regions after retrieval of a cocaine-context memory, observed in both the CPP and CA paradigms, likely signifies that these regions: (i) are activated during retrieval of these memories irrespective of preference-based decisions, and (ii) undergo neuroplasticity in order to update information about cues previously associated with cocaine. This study also establishes the involvement of NMDA receptors in maintaining memories established using the CA model, a characteristic previously demonstrated using CPP. Overall, these results demonstrate the utility of the CA model for studies of cocaine-context memory and suggest the involvement of an NMDA receptor-dependent Arc induction pathway in drug-cue memory interference.

Running wheel exercise before a binge regimen of methamphetamine does not protect against striatal dopaminergic damage.

Repeated administration of methamphetamine (mAMPH) to rodents in a single-day "binge" dosing regimen produces long-lasting damage to forebrain dopaminergic nerve terminals as measured by decreases in tissue dopamine (DA) content and levels of the plasmalemmal DA transporter (DAT). However, the midbrain cell bodies from which the DA terminals arise survive, and previous reports show that striatal DA markers return to control levels by 12 months post-mAMPH, suggesting long-term repair or regrowth of damaged DA terminals. We previously showed that when rats engaged in voluntary aerobic exercise for 3 weeks before and 3 weeks after a binge regimen of mAMPH, exercise significantly ameliorated mAMPH-induced decreases in striatal DAT. However, these data left unresolved the question of whether exercise protected against the initial neurotoxicity from the mAMPH binge or accelerated the repair of the damaged DA terminals. The present experiments were designed to test whether exercise protects against the mAMPH-induced injury. Adult male Sprague-Dawley rats were allowed to run in wheels for 3 weeks before an acute binge regimen of mAMPH or saline, then placed into nonwheel cages for an additional week before autoradiographic determination of striatal DAT binding. The autoradiographic findings showed that prior exercise provided no protection against mAMPH-induced damage to striatal DA terminals. These results, together with analyses from our previous experiments, suggest that voluntary exercise may accelerate the repair of mAMPH-damaged DA terminals and that voluntary exercise may be useful as therapeutic adjunct in the treatment mAMPH addicts.

Methamphetamine influences on brain and behavior: unsafe at any speed?

Methamphetamine damages monoamine-containing nerve terminals in the brains of both animals and human drug abusers, and the cellular mechanisms underlying this injury have been extensively studied. More recently, the growing evidence for methamphetamine influences on memory and executive function of human users has prompted studies of cognitive impairments in methamphetamine-exposed animals. After summarizing current knowledge about the cellular mechanisms of methamphetamine-induced brain injury, this review emphasizes research into the brain changes that underlie the cognitive deficits that accompany repeated methamphetamine exposure. Novel approaches to mitigating or reversing methamphetamine-induced brain and behavioral changes are described, and it is argued that the slow spontaneous reversibility of the injury produced by this drug may offer opportunities for novel treatment development.

Running wheel exercise ameliorates methamphetamine-induced damage to dopamine and serotonin terminals.

Repeated administration of methamphetamine (mAMPH) to rodents in a single-day "binge" produces long-lasting damage to dopaminergic and serotonergic terminals. Because previous research has demonstrated that physical activity can ameliorate nigrostriatal injury, this study investigated whether voluntary exercise in rats can alter the monoaminergic damage resulting from a neurotoxic mAMPH binge. Adult male rats were allowed constant access to running wheels or kept in nonwheel cages for three weeks, then given a binge dosing regimen of mAMPH or saline. The rats were returned to their original environments for three additional weeks post-mAMPH. [(125) I]RTI-55 binding and autoradiography was used to quantify dopamine transporters (DAT), and radioimmunocytochemistry was used to quantify striatal tyrosine hydroxylase (TH). Binge mAMPH treatment significantly reduced striatal DAT and TH in a regionally specific pattern; with greatest effects in ventral caudate-putamen (CP) and relative sparing of the nucleus accumbens septi (NAc). The effects of mAMPH on striatal DAT and TH were ameliorated in the running, compared to the sedentary, animals. Also, mAMPH was found to reduce [(125) I]RTI-55 binding to serotonin transporters (SERT) in frontoparietal cortex, and this too was significantly attenuated by exercise. Additional correlational analyses showed that the post-mAMPH running of individual animals predicted the amelioration of striatal DAT and TH as well as frontoparietal SERT. Overall, voluntary exercise significantly diminished mAMPH-induced forebrain monoaminergic damage. The significant correlations between post-mAMPH exercise and markers of monoaminergic terminal integrity provide novel evidence that voluntary exercise may exert beneficial effects on behavior in recovering mAMPH addicts.

A neurotoxic regimen of methamphetamine impairs novelty recognition as measured by a social odor-based task.

Repeated administration of methamphetamine (mAMPH) to rodents in a single-day "binge" regimen damages forebrain monoaminergic nerve terminals and produces subsequent cognitive deficits. Here we investigate performance on a social odor-based task, demonstrating enduring mAMPH-induced deficits in recognition memory. Three weeks after a neurotoxic mAMPH regimen, singly-housed male Long-Evans rats had four wooden beads placed in their home cage: three beads containing odors from their home cage (HC beads) and one bead from a cage of a rat not present in the colony room (N1 bead). Exploration times for each bead were recorded during three 1-min habituation trials separated by 1-min intertrial intervals. Twenty-four hours later, a 1-min memory test was conducted, in which animals were presented with two HC beads, one N1 bead, and one bead from another novel animal (N2). Saline- and mAMPH-treated rats showed similar, progressive decreases in exploration time for the N1 bead during the habituation trials, indicating equivalent short-term olfactory habituation to the novel odor. By contrast, during the subsequent memory test, saline-treated rats showed a strong preference for the N2 bead over the N1 bead while mAMPH-treated rats showed no preference. The use of the rats' primary sensory modality (olfaction) coupled with the social significance (from conspecifics) of the odors produces strong, long-lasting memories. Our results show that prior treatment with a neurotoxic regimen of mAMPH impairs long-term memory for the previously experienced odors. As compared with previously employed object recognition tasks, this test may be advantageous for investigating mAMPH-induced memory impairments in rodents.

Neurotoxic methamphetamine regimens produce long-lasting changes in striatal G-proteins.

Animals repeatedly dosed with methamphetamine during a single day suffer damage to brain dopamine and serotonin terminals and show behavioral deficits. These methamphetamine regimens also produce long-term reductions in dopamine agonist-stimulated immediate-early gene responses both in striatum and several cortical areas, but the mechanism(s) underlying these long-lasting effects of methamphetamine remain uncertain. Six weeks after a neurotoxic regimen of methamphetamine (4 × 4 mg/kg) or saline, α subunit levels of striatal G-proteins that couple dopamine receptors to second messenger systems were measured. Because the damage to striatal monoamine terminals produced by methamphetamine is regionally heterogeneous, we used radioimmunocytochemistry, which combines quantification with regional resolution. We found significant increases in G(iα) and G(olfα) expression in the ventral striatum (but not in the dorsolateral striatum or nucleus accumbens) of methamphetamine-pretreated rats, a regional pattern similar to that reported for methamphetamine effects on dopamine terminal markers. By contrast, G(qα) expression was unaffected in all striatal subregions. The central roles of G(i) and G(olf) in modulating the activity of a series of interlinked intracellular signaling pathways suggest that methamphetamine-induced changes in G(i) and G(olf) can have lasting effects on striatal neuronal function.

A sensitizing D-amphetamine dose regimen induces long-lasting spinophilin and VGLUT1 protein upregulation in the rat diencephalon.

Numerous studies in this lab and others have reported psychostimulant-induced alterations in both synaptic protein expression and synaptic density in striatum and prefrontal cortex. Recently we have shown that chronic D-amphetamine (D-AMPH) administration in rats increased synaptic protein expression in striatum and limbic brain regions including hippocampus, amygdala, septum, and paraventricular nucleus of the thalamus (PVT). Potential synaptic changes in thalamic nuclei are interesting since the thalamus serves as a gateway to cerebral cortex and a nodal point for basal ganglia influences. Therefore we sought to examine drug-induced differences in synaptic protein expression throughout the diencephalon. Rats received an escalating (1-8 mg/kg) dosing regimen of D-AMPH for five weeks and were euthanized 28 days later. Radioimmunocytochemistry (RICC) revealed significant upregulation of both spinophilin and the vesicular glutamate transporter, VGLUT1, in PVT, mediodorsal (MD), and ventromedial (VM) thalamic nuclei as well as in lateral hypothalamus (LH) and habenula. Strong positive correlations were observed between VGLUT1 and spinophilin expression in PVT, medial habenula, MD, VM and LH of D-AMPH-treated rats. No significant D-AMPH effect was seen in sensorimotor cortices for either protein. Additionally, no significant differences in the general vesicular protein synaptophysin were observed for any brain region. These findings add to evidence suggesting that long-lasting stimulant-induced synaptic alterations are widespread but not ubiquitous. Moreover, they suggest that D-AMPH-induced synaptic changes may occur preferentially in excitatory synapses.

Reversal-specific learning impairments after a binge regimen of methamphetamine in rats: possible involvement of striatal dopamine.

A growing body of evidence indicates that protracted use of methamphetamine (mAMPH) causes long-term impairments in cognitive function in humans. Aside from the widely reported problems with attention, mAMPH users exhibit learning and memory deficits, particularly on tasks requiring response control. Although binge mAMPH administration to animals results in cognitive deficits, few studies have attempted to test behavioral flexibility in animals after mAMPH exposure. The aim of this study was to evaluate whether mAMPH would produce impairments in two tasks assessing flexible responding in rats: a touchscreen-based discrimination-reversal learning task and an attentional set shift task (ASST) based on a hallmark test of executive function in humans, the Wisconsin Card Sort. We treated male Long-Evans rats with a regimen of four injections of 2 mg/kg mAMPH (or vehicle) within a single day, a dosing regimen shown earlier to produce object recognition impairments. We then tested them on (1) reversal learning after pretreatment discrimination learning or (2) the ASST. Early reversal learning accuracy was impaired in mAMPH-treated rats. MAMPH pretreatment also selectively impaired reversal performance during ASST testing, leaving set-shifting performance intact. Postmortem analysis of [(125)I]RTI-55 binding revealed small (10-20%) but significant reductions in striatal dopamine transporters produced by this mAMPH regimen. Together, these results lend new information to the growing field documenting impaired cognition after mAMPH exposure, and constitute a rat model of the widely reported decision-making deficits resulting from mAMPH abuse seen in humans.

Long-term changes in dopamine-stimulated gene expression after single-day methamphetamine exposure.

Methamphetamine (mAMPH) is a highly addictive psychostimulant drug that injures monoaminergic neurons and results in behavioral impairments in humans and animals. Although evidence exists for changes in cortical volume, metabolism, and blood oxygenation levels in human mAMPH abusers, animal models have instead emphasized this drug's long-lasting influence on ascending monoaminergic (dopamine, serotonin) projections. The aim of this study was to investigate cortical and subcortical function in rats long after administration of a single-day mAMPH regimen known to damage monoaminergic systems, at a time point when behavioral impairments are still evident. Rats were given either saline or a neurotoxic (4 x 4 mg/kg, sc) mAMPH regimen. Five weeks later, they were given pharmacological treatments that stimulate cortical gene expression: either the dopaminergic agonist apomorphine (3 mg/kg, sc) or the muscarinic acetylcholine agonist pilocarpine (25 mg/kg, ip). Cortical and subcortical immediate early gene (IEG) responses were measured by immunocytochemical analysis of Fos or JunB, protein products of the IEGs, c-fos and junB. Compared with saline-pretreated controls, mAMPH-pretreated animals had about 50-70% fewer Fos- and JunB-immunoreactive cells in anterior cingulate, infralimbic, orbital, somatosensory, and rhinal cortices as well as caudate-putamen and nucleus accumbens, 90 min after apomorphine challenge. By contrast, mAMPH-pretreated rats had no reductions in the numbers of Fos or JunB-positive cells following pilocarpine challenge. This study demonstrates the profound and enduring effects of mAMPH administration on dopamine-stimulated cortical function in animals.

Methamphetamine influences on recognition memory: comparison of escalating and single-day dosing regimens.

Methamphetamine (mAMPH) is an addictive drug that produces memory and recall impairments in humans. Animals subjected to a binge mAMPH dosing regimen that damages brain dopamine and serotonin terminals show impairments in an object recognition (OR) task. Earlier research demonstrated that preceding a single-day mAMPH binge regimen with several days of increasing mAMPH doses greatly attenuates its neurotoxicity in rats. The escalating dose (ED) paradigm appears to mimic the human pattern of escalating drug intake. The current aim was to test whether an ED plus binge mAMPH regimen produces OR impairments. In addition to its translational value, this experiment helps address whether monoaminergic neurotoxicity accounts for OR impairments seen after mAMPH administration. To further address this issue, a separate experiment investigated both OR impairments and monoamine transporter integrity in groups of rats treated with a range of mAMPH doses during a single day. An ED mAMPH regimen attenuated the acute hyperthermic response to the subsequent mAMPH binge and prevented the OR impairments and reductions in [125 I]RTI-55 binding to monoamine transporters in striatum, hippocampus (HC), and perirhinal cortex (pRh) that otherwise occur 1 week after the mAMPH binge. Single-day mAMPH regimens (4 x 1mg/kg to 4 x 4 mg/kg, s.c.) dose-dependently produced acute hyperthermia and, 1 week post-mAMPH, produced dose-dependent impairments in OR and reductions in monoamine transporter binding. The OR impairments of single-day mAMPH-treated rats correlated with monoaminergic transporter loss in ventral caudate-putamen, HC, and pRh. In aggregate, these findings suggest a correspondence between mAMPH-induced monoaminergic injury and the resulting OR deficits.

Methamphetamine-induced neural and cognitive changes in rodents.

AIMS: Although psychostimulant drug abuse carries with it several potential health risks, the chronic abuse of amphetamines carries the danger of permanent brain injury. The purpose of these experiments is to develop animal models to understand the long-lasting influences of methamphetamine exposure on cerebral cortex and cognitive function. METHODS: The approach taken is to administer a regimen of methamphetamine known to be neurotoxic to dopamine and serotonin nerve terminals in the rat, and to investigate the influences of that dosing regimen on (i) cortical neuron integrity and function using anatomical stains and (ii) novel object recognition memory. RESULTS: In rodents, repeated administration of methamphetamine during a single day produces long-lasting damage to striatal dopamine and forebrain serotonin terminals as well as degeneration of somatosensory cortical neurons. The degeneration of somatosensory cortical neurons may represent only the most visible form of long-term deleterious effects on cerebral cortex, as exposure of rats to methamphetamine can reduce the immediate early gene responses of neurons in widespread cortical areas, even long after exposure to the drug. Together with the death and long-lasting functional impairments of cortical neurons, rats exposed to methamphetamine have impaired cognitive function. When tested for object recognition memory, methamphetamine-treated rats show deficiencies lasting for at least 3 weeks after drug exposure. CONCLUSIONS: Using a rodent model, these findings provide an avenue to study the cortical influences of methamphetamine and their cognitive sequelae.

A sensitizing regimen of methamphetamine causes impairments in a novelty preference task of object recognition.

A neurotoxic regimen of methamphetamine impairs object recognition (OR) in rats. The present study investigated whether neurotoxicity is a necessary component of methamphetamine's effect on OR. Animals were exposed to a sensitizing regimen of methamphetamine, and were tested for OR one week, and locomotor behavior two weeks, later. Quantitative autoradiography was used to measure [(125)I]RTI-55 binding to forebrain dopaminergic and serotonergic transporters. Methamphetamine treatment produced significant OR impairments (and increased locomotion), without reducing dopamine or serotonin transporter binding. This study supports the conclusion that factors other than monoamine terminal injury contribute to the methamphetamine-induced cognitive impairments.

Impaired object recognition memory following methamphetamine, but not p-chloroamphetamine- or d-amphetamine-induced neurotoxicity.

Repeated moderate doses of methamphetamine (mAMPH) damage forebrain monoaminergic terminals and nonmonoaminergic cells in somatosensory cortex, and impair performance in a novelty preference task of object recognition (OR). This study aimed to determine whether the memory deficit seen after a neurotoxic mAMPH regimen results from damage to dopamine (DA) and/or serotonin (5-HT) terminals. Animals were given a neurotoxic regimen of mAMPH, p-chloroamphetamine (PCA, preferentially damages 5-HT terminals), d-amphetamine (d-AMPH, preferentially damages DA terminals), or saline. After 1 week, animals were trained and tested for OR memory. Rats treated with mAMPH showed no recognition memory during the short-term memory (STM) test, whereas both PCA- and d-AMPH-treated rats showed OR STM scores comparable to controls. After behavioral testing, the specificity of monoaminergic lesions was determined by postmortem [125I]RTI-55 binding to dopamine (DAT) and serotonin (SERT) transporter proteins. Tissue from a separate group of animals killed 3 days after drug treatment was processed for Fluoro-Jade (F-J) fluorescence histochemistry to detect damaged cortical neurons. mAMPH-treated rats showed reductions in striatal DAT and hippocampal (HC) and perirhinal (pRh) SERT, as well as degeneration of neurons in primary somatosensory cortex. In PCA-treated rats, HC and pRh SERT were substantially depleted, but striatal DAT and cortical neuron survival were unaffected. By contrast, d-AMPH-treated animals showed marked depletions in striatal DAT and cortical neurodegeneration, but HC and pRh SERT were unaffected. This pattern of results indicates that no single feature of mAMPH-induced neurotoxicity is sufficient to produce the OR impairments seen after mAMPH treatment.

Neurotoxic regimens of methamphetamine induce persistent expression of phospho-c-Jun in somatosensory cortex and substantia nigra.

Repeated systemic administration of moderate doses of methamphetamine (mAMPH) can result in neuronal damage. In addition to the prominent damage of forebrain dopamine and serotonin terminals, mAMPH also injures certain non-monoaminergic neuronal somata in the cerebral cortex. In previous studies, we have localized the damaged neurons to the "whisker barrels" in primary somatosensory cortex, reported the time course of their appearance, and found that sensory inputs from the mystacial vibrissae appear to play a crucial role in the mechanism of their injury by mAMPH. One common feature of these studies is that they used a single marker for neuronal injury, the fluorochrome dye Fluoro-Jade, which stains neurons injured by disparate mechanisms. Here we compare mAMPH-induced damage to somatosensory cortical neurons as assessed by Fluoro-Jade and immunohistochemical staining for phospho-c-Jun. A neurotoxic regimen of mAMPH induced phospho-c-Jun-positive neurons in both cortical whisker barrels and the substantia nigra. Neurons in the barrel cortex can be sufficiently damaged by mAMPH that they become Fluoro-Jade-positive within 2 hr after the final mAMPH injection. By contrast, phospho-c-Jun immunoreactivity does not appear until 12-24 hr after mAMPH. As reported in an earlier study, unilateral removal of vibrissae prior to mAMPH treatment affords partial protection from injury in the hemisphere contralateral to the vibrissotomy. The vibrissotomized animals show similar decreases in Fluoro-Jade staining and phospho-c-Jun immunoreactivity in the protected hemisphere. Since phospho-c-Jun indicates activation of Jun N-terminal kinase pathways, which have been implicated in apoptosis, we conclude that phospho-c-Jun provides a useful new marker for mAMPH-induced damage to cortical neurons.

Neurotoxic methamphetamine regimen severely impairs recognition memory in rats.

Methamphetamine (mAMPH), when administered repeatedly to rodents or primates, is neurotoxic to some cortical neurons and to forebrain dopaminergic and serotonergic axon terminals. The aim of the present study was to investigate the effects of a neurotoxic regimen of mAMPH on two hippocampus-dependent memory tasks: object recognition, a nonspatial memory task, and the Morris water maze, a spatial memory task. Male rats were treated with mAMPH (4 x 4.0 mg/kg, s.c.) or saline and trained in the object recognition task 1 week and 3 weeks later. During training, animals explored two identical copies of the same object. In retention test sessions one of the objects was replaced by a novel object. mAMPH-treated rats showed no recognition memory during the short-term memory (STM) test, given 90 min after the training session, and showed marked impairments in the long-term memory (LTM) test, given 24 h after training. Even 3 weeks after drug injections, the mAMPH-treated animals were unable to discriminate between the novel and familiar objects during both STM and LTM tests. Despite the severe deficits observed in the recognition memory, no effects of prior mAMPH treatment were seen in the water maze task. Damage to monoamine terminals was confirmed by significant 30-40% losses of [(125)I]RTI-55 binding to striatal dopamine transporter and hippocampal serotonin transporter sites at both 1 and 3 weeks after mAMPH treatments. Thus, administration of mAMPH restricted to a single day can produce a profound, persistent, and selective deficit in a nonspatial hippocampus-dependent memory.

Effects of vibrissae removal on methamphetamine-induced damage to rat somatosensory cortical neurons.

Repeated methamphetamine (mAMPH) damages forebrain monoamine terminals and causes degeneration of nonmonoaminergic cell bodies in rat primary somatosensory cortex (S1). These degenerating cortical neurons can be labeled with the fluorochrome dye Fluoro-Jade (FJ) and are found almost exclusively in layers II/III and IV of the vibrissae representation in S1. Within S1, layer IV is organized into discrete, anatomically identifiable units termed barrels, each of which receives information from a single whisker. We previously reported that mAMPH-damaged neurons in S1 were located within the whisker barrels, suggesting that the prolonged mAMPH-induced whisking contributes to S1 neuronal injury. Here, we investigate effects of vibrissae removal on mAMPH-induced damage to S1 neurons. Rats were anesthetized and vibrissae were trimmed from either the left, right, or neither side of the snout. The next day they were given four injections of either saline (1 ml/kg, s.c.) or mAMPH (4 mg/kg, s.c.) at 2-h intervals. Three days later, cortical sections were processed for FJ histochemistry. The hemivibrissotomy produces a hemispheric asymmetry in FJ-positive neurons in barrel cortex, with fewer damaged neurons contralateral than ipsilateral to whisker removal. Taken together with the demonstration that acute injection of this dose of mAMPH induces the immediate early gene zif/268 and Fos protein in barrel cortex, these data suggest that the prolonged behavioral activity involving the vibrissae contributes to the mAMPH-induced damage to S1 neurons. Thus, some of the injurious effects of drugs may depend on afferent activity occurring as a result of the abnormal behaviors evoked by their administration.

UPTAKE OF AMINO ACIDS BY PAREURYTHOE CALIFORNICA: SUBSTRATE INTERACTION MODIFIES NET INFLUX FROM THE ENVIRONMENT.

Pareurythoe californica is capable of simultaneous net uptake of 18 amino acids, each present at an initial concentration of 200 nM. Rates of uptake are comparable for all amino acids tested. Kinetics of uptake are well described by the Michaelis-Menten equation. Neither bacteria nor other epifauna play a significant role in the observed uptake. The net entry of the amino acids tested was inhibited in the presence of equimolar concentrations of representatives of all major classes of amino acids (i.e., polar and nonpolar neutral, dicarboxylic, polybasic) at concentrations comparable to those found in the environment. Thus, a mixture mimicking the composition and concentration of substrates normally present in the environment was used to obtain a more realistic estimate of entry rates under natural conditions. When this was done, the contribution of exogenous amino acids to the nitrogen needs (based on ammonia excretion) and the requirement for reduced carbon (based on oxygen consumption) of Pareurythoe ranged from 10% to 50%, depending on levels of substrate available in the environment.