Early stages of sensorimotor map acquisition: neurochemical signature in primary motor cortex and its relation to functional connectivity.

The earliest stages of sensorimotor learning involve learning the correspondence between movements and sensory results-a sensorimotor map. The present exploratory study investigated the neurochemical underpinnings of map acquisition by monitoring 25 participants as they acquired a new association between movements and sounds. Functional magnetic resonance spectroscopy was used to measure neurochemical concentrations in the left primary motor cortex during learning. Resting-state functional magnetic resonance imaging data were also collected before and after training to assess learning-related changes in functional connectivity. There were monotonic increases in γ-aminobutyric acid (GABA) and decreases in glucose during training, which extended into the subsequent rest period and, importantly, in the case of GABA correlated with the amount of learning: participants who showed greater behavioral learning showed greater GABA increase. The GABA change was furthermore correlated with changes in functional connectivity between the primary motor cortex and a cluster of voxels in the right intraparietal sulcus: greater increases in GABA were associated with greater strengthening of connectivity. Transiently, there were increases in lactate and reductions in aspartate, which returned to baseline at the end of training, but only lactate showed a statistical trend to correlate with the amount of learning. In summary, during the earliest stages of sensorimotor learning, GABA levels are linked on a subject-level basis to both behavioral learning and a strengthening of functional connections that persists beyond the training period. The findings are consistent with the idea that GABA-mediated inhibition is linked to maintenance of newly learned information.NEW & NOTEWORTHY Learning the mapping between movements and their sensory effects is a necessary step in the early stages of sensorimotor learning. There is evidence showing which brain areas are involved in early motor learning, but their role remains uncertain. Here, we show that GABA, a neurotransmitter linked to inhibitory processing, rises during and after learning and is involved in ongoing changes in resting-state networks.

Short echo-time Magnetic Resonance Spectroscopy in ALS, simultaneous quantification of glutamate and GABA at 3 T.

Cortical hyperexcitability has been found in early Amyotrophic Lateral Sclerosis (ALS) and is hypothesized to be a key factor in pathogenesis. The current pilot study aimed to investigate cortical inhibitory/excitatory balance in ALS using short-echo Magnetic Resonance Spectroscopy (MRS). Patients suffering from ALS were scanned on a 3 T Trio Siemens MR scanner using Spin Echo Full Intensity Acquired Localized (SPECIAL) Magnetic Resonance Spectroscopy in primary motor cortex and the occipital lobe. Data was compared to a group of healthy subjects. Nine patients completed the scan. MRS data was of an excellent quality allowing for quantification of a range of metabolites of interest in ALS. In motor cortex, patients had Glutamate/GABA and GABA/Cr- ratios comparable to healthy subjects. However, Glutamate/Cr (p = 0.002) and the neuronal marker N-acetyl-aspartate (NAA/Cr) (p = 0.034) were low, possibly due to grey-matter atrophy, whereas Glutathione/Cr (p = 0.04) was elevated. In patients, NAA levels correlated significantly with both hand strength (p = 0.027) and disease severity (p = 0.016). In summary SPECIAL MRS at 3 T allows of reliable quantification of a range of metabolites of interest in ALS, including both excitatory and inhibitory neurotransmitters. The method is a promising new technique as a biomarker for future studies on ALS pathophysiology and monitoring of disease progression.

Structural and functional imaging of the hippocampus in young people at familial risk of depression.

BACKGROUND: Major depression is associated with abnormalities in the function and structure of the hippocampus. However, it is unclear whether these abnormalities might also be present in people 'at risk' of illness. METHOD: We studied 62 young people (mean age 18.8 years) at familial risk of depression (FH+) but who had never been depressed themselves. Participants underwent magnetic resonance imaging to assess hippocampal structure and neural responses to a task designed to activate hippocampal memory networks. Magnetic resonance spectroscopy was used to measure levels of a combination of glutamine and glutamate (Glx) in the right hippocampus. A total of 59 matched controls with no history of mood disorder in a first-degree relative underwent the same investigations. RESULTS: Hippocampal volume did not differ between FH+ participants and controls; however, relative to controls, during the memory task, FH+ participants showed increased activation in brain regions encompassing the insular cortices, putamen and pallidum as well as the dorsal anterior cingulate cortex (ACC). FH+ participants also had increased hippocampal levels of Glx. CONCLUSIONS: Euthymic individuals with a parental history of depression demonstrate increased activation of hippocampal-related neural networks during a memory task, particularly in brain regions involved in processing the salience of stimuli. Changes in the activity of the ACC replicate previous findings in FH+ participants using different psychological tasks; this suggests that task-related abnormalities in the ACC may be a marker of vulnerability to depression. Increased levels of Glx in the hippocampus might also represent a risk biomarker but follow-up studies will be required to test these various possibilities.

Effect of interferon-α on cortical glutamate in patients with hepatitis C: a proton magnetic resonance spectroscopy study.

BACKGROUND: The development of depressive symptomatology is a recognized complication of treatment with the cytokine interferon-α (IFN-α) and has been seen as supporting inflammatory theories of the pathophysiology of major depression. Major depression has been associated with changes in glutamatergic activity and recent formulations of IFN-induced depression have implicated neurotoxic influences that could also lead to changes in glutamate function. The present study used magnetic resonance spectroscopy (MRS) to measure glutamate and its major metabolite glutamine in patients with hepatitis C who received treatment with pegylated IFN-α and ribavirin. METHOD: MRS measurements of glutamate and glutamine were taken from a 25 × 20 × 20 mm voxel including the pregenual anterior cingulate cortex in 12 patients before and after 4-6 weeks of treatment with IFN. RESULTS: IFN treatment led to an increase in cortical levels of glutamine (p = 0.02) and a significant elevation in the ratio of glutamine to glutamate (p < 0.01). Furthermore, changes in glutamine level correlated significantly with ratings of depression and anxiety at the time of the second scan. CONCLUSIONS: We conclude that treatment with IFN-α is associated with MRS-visible changes in glutamatergic metabolism. However, the changes seen differ from those reported in major depression, which suggests that the pathophysiology of IFN-induced depression may be distinct from that of major depression more generally.

Relationship between physiological measures of excitability and levels of glutamate and GABA in the human motor cortex.

Magnetic resonance spectroscopy (MRS) allows measurement of neurotransmitter concentrations within a region of interest in the brain. Inter-individual variation in MRS-measured GABA levels have been related to variation in task performance in a number of regions. However, it is not clear how MRS-assessed measures of GABA relate to cortical excitability or GABAergic synaptic activity. We therefore performed two studies investigating the relationship between neurotransmitter levels as assessed by MRS and transcranial magnetic stimulation (TMS) measures of cortical excitability and GABA synaptic activity in the primary motor cortex. We present uncorrected correlations, where the P value should therefore be considered with caution. We demonstrated a correlation between cortical excitability, as assessed by the slope of the TMS input-output curve and MRS-assessed glutamate levels (r = 0.803, P = 0.015) but no clear relationship between MRS-assessed GABA levels and TMS-assessed synaptic GABA(A) activity (2.5 ms inter-stimulus interval (ISI) short-interval intracortical inhibition (SICI); Experiment 1: r = 0.33, P = 0.31; Experiment 2: r = -0.23, P = 0.46) or GABA(B) activity (long-interval intracortical inhibition (LICI); Experiment 1: r = -0.47, P = 0.51; Experiment 2: r = 0.23, P = 0.47). We demonstrated a significant correlation between MRS-assessed GABA levels and an inhibitory TMS protocol (1 ms ISI SICI) with distinct physiological underpinnings from the 2.5 ms ISI SICI (r = -0.79, P = 0.018). Interpretation of this finding is challenging as the mechanisms of 1 ms ISI SICI are not well understood, but we speculate that our results support the possibility that 1 ms ISI SICI reflects a distinct GABAergic inhibitory process, possibly that of extrasynaptic GABA tone.

Vesicular Ca(2+) participates in the catalysis of exocytosis.

Effects of vesicular monoamine transporter inhibitors on catecholamine release from bovine chromaffin cells have been examined at the level of individual exocytotic events. As expected for a depletion of vesicular stores, release evoked by depolarizing agents was decreased following 15-min incubations with reserpine and tetrabenazine, as evidenced by a decrease in exocytotic frequency and amount released per event. In contrast, two reserpine derivatives, methyl reserpate and reserpic acid, were much less effective. Surprisingly, the incubations also decreased the accompanying rise in intracellular Ca(2+) evoked by depolarizing agents. Subcellular studies revealed that reserpine and tetrabenazine at concentrations near their K(i) values not only could increase cytoplasmic catecholamines but also could displace Ca(2+) from vesicles. Furthermore, transient exposure to tetrabenazine and reserpine, but not methyl reserpate and reserpic acid, induced exocytotic release of catecholamines. Reserpine induced a rise in intracellular Ca(2+), as detected by whole-cell measurements with Fura-2. It could induce exocytosis, albeit at a lower frequency, in Ca(2+)-free solutions, supporting an internal Ca(2+) source. Depletion of endoplasmic reticulum and mitochondrial Ca(2+) pools did not eliminate the reserpine-activated release. These results indicate that vesicular Ca(2+) can play an important role in exocytosis and under some conditions may be involved in initiating this process.

Lead enhances NGF-induced neurite outgrowth in PC12 cells by potentiating ERK/MAPK activation.

Although the neurotoxicity of lead exposure is well documented, the cellular and molecular mechanisms underlying lead neurotoxicity have not been well defined. We have investigated the effect of lead on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells and the role in this process of extracellular signal regulated protein kinase (ERK), a key component of NGF-induced differentiation. We found that exposure of cells to lead acetate (0.1-100 microM) resulted in enhanced NGF-induced neurite outgrowth. Lead exposure also promoted formation of multiple neurites per cell in NGF-treated cells. However, lead alone did not cause neurite outgrowth. Lead also enhanced NGF-induced ERK phosphorylation and activation, but lead alone did not stimulate ERK. The MAP kinase kinase (MEK) inhibitor, PD98059, significantly decreased the effect of lead on NGF-induced neurite outgrowth and ERK activation. These findings indicate that exposure of cells to low, toxic levels of lead amplifies growth factor-induced neurite outgrowth by means of an ERK-dependent signaling pathway.

Effects of nerve growth factor on dihydrotetrabenazine binding to PC12 cells.

Tetrabenazine and dihydrotetrabenzaine (TBZOH) are potent inhibitors of substrate transport by the predominant forms of the vesicular monoamine transporter (VMAT) present in bovine brain synaptic vesicles and bovine adrenal medullary chromaffin vesicles. Radiolabeled TBZOH binds to these preparations with apparent dissociation constants in the low nanomolar range. However, tetrabenazine is a much less potent inhibitor of transport by rVMAT1, a form of the transporter cloned from a rat pheochromocytoma (PC12) cDNA library and expressed in CHO cells. Reported attempts to observe binding of [3H]TBZOH to rVMAT1 have not been successful. We examined binding of [3H]TBZOH to a crude membrane fraction from PC12 cells. Computerized nonlinear least squares curve fitting revealed two classes of binding sites (Kd1 = 1.5 nM, R1 = 0.2 pmol/mg protein, Kd2 = 340 nM, R2 = 15.2 pmol/mg protein). While the identity of the higher affinity sites is not certain, their high affinity for TBZOH suggests that they may be associated with rVMAT2. The lower affinity sites are likely to be associated with rVMAT1 on the basis of their affinity for TBZOH and sensitivity to inhibition of TBZOH binding by transporter substrates and inhibitors. NGF-treated PC12 cells also exhibited two classes of sites (Kd1 = 1.9 nM, R1 = 0.18 pmol/mg protein; Kd2 = 370 nM, R2 = 23.7 pmol/mg protein). While there were no significant differences between control and NGF-treated cells in binding capacity of the higher affinity sites, nor in apparent dissociation constants for either class of sites, there was a highly significant increase in number of lower affinity binding sites in the NGF-treated cells (p = 0.001). These results provide direct evidence that the differential sensitivity of rat brain and adrenal catecholamine stores to depletion by tetrabenazine and its derivatives is due to a much lower affinity of rVMAT1 for these compounds, and that NGF treatment may increase levels of rVMAT1 expression in PC12 cells.

Monoclonal antibodies reactive with a monoamine transporter preparation purified from bovine adrenal chromaffin granule membranes.

There have been no reports of monoclonal antibodies reactive with vesicular monoamine transporters from any source. Western blotting and ELISA data obtained using polyclonal serum from a mouse immunized with a highly purified bovine chromaffin granule monoamine transporter preparation yielded data consistent with the presence of antibodies to the transporter. Hybridomas produced by polyethylene glycol fusion of spleen cells from the mouse with X63/Ag8.653 myeloma cells were screened in an ELISA using partially purified transporter as coating agent. Of the 1142 wells containing colonies, 14 were positive in the initial screen. Hybridomas from wells testing positive were transferred to 24-well plates, grown up, and rescreened. Those still testing positive were subcloned, and the resulting positive wells containing single colonies were grown up and stocked. Of the 6 positive clones that tolerated freeze/thaw (0.5% of the wells tested), 1 was IgG1 kappa, 2 were IgG2a kappa, and 3 were IgG2b kappa isotypes. Ascites fluid was generated in pristane-primed BALB/c mice using hybridomas that had been cloned 2-3 times, and antibodies purified on immobilized Protein A. Immunoreactivity with a mixture of these antibodies, or with only one of them, coincided with dihydrotetrabenazine (TBZOH) binding activity in fractions eluted from all columns employed in the transporter purification. Antibody from at least one of the clones was capable of removing [3H]TBZOH binding activity from a partially purified preparation of transporter. Monoclonal antibodies exhibiting these properties have not been reported previously.

Effect of chronic haloperidol treatment on stimulated synaptic overflow of dopamine in the rat striatum.

In vivo voltammetry was used to assess the change in stimulated striatal dopamine overflow in response to various treatments with the dopamine receptor antagonist haloperidol. Dopamine overflow was induced with stimulating electrodes implanted in the medial forebrain bundle of anesthetized rats while dopamine concentrations were monitored with Nafion-coated, carbon-fiber microelectrodes implanted in the striatum. An acute challenge of haloperidol (0.5 mg kg-1, i.p.) given to naive animals caused stimulated overflow to increase at all stimulation frequencies (10-60 Hz), with the greatest change, 5-fold, occurring at 30 Hz. These results have been compared to those obtained in a different group of rats given daily injections of haloperidol (0.5 mg kg-1, s.c.) for 30 consecutive days. On the 30th day, dopamine striatal tissue levels and uptake kinetics were not altered by this treatment, but 3,4-dihydroxyphenylacetic acid tissue levels were elevated almost 2-fold. A challenge dose of haloperidol (0.5 mg kg-1, i.p.) administered to the animals treated with chronic haloperidol did not elicit a change in stimulated dopamine overflow. In two other groups, rats were withdrawn from 30-day haloperidol treatment for 3 days or 14 days before experimentation. Stimulated dopamine overflow concentrations in both groups were not significantly different from naive animals. When the withdrawn animals were given a haloperidol challenge (0.5 mg kg-1, i.p.), 15- and 12-fold increases in overflow for 3-day and 14-day withdrawal groups, respectively, were observed at a stimulation frequency of 30 Hz. Thus, chronic treatment with haloperidol induces long-lasting effects on the capacity of dopamine receptors to modulate dopamine release.

Cloning and expression of a human ATP-citrate lyase cDNA.

A full-length cDNA clone of 4.3 kb encoding the human ATP-citrate lyase enzyme has been isolated by screening a human cDNA library with the recently isolated rat ATP-citrate lyase cDNA clone [Elshourbagy et al. (1990) J. Biol. Chem. 265, 1430]. Nucleic-acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1105 amino acids in length with a calculated molecular mass of 121,419 Da. Comparison of the human and rat ATP-citrate lyase cDNA sequences reveals 96.3% amino acid identity throughout the entire sequence. Further sequence analysis identified the His765 catalytic phosphorylation site, the ATP-binding site, as well as the CoA binding site. The human ATP-citrate lyase cDNA clone was subcloned into a mammalian expression vector for expression in African green monkey kidney cells (COS) and Chinese hamster ovary cells (CHO) cells. Transfected COS cells expressed detectable levels of an enzymatically active recombinant ATP-citrate lyase enzyme. Stable, amplified expression of ATP-citrate lyase in CHO cells as achieved by using coamplification with dihydrofolate reductase. Resistant cells expressed high levels of enzymatically active ATP-citrate lyase (3 pg/cell/d). Site-specific mutagenesis of His765----Ala diminishes the catalytic activity of the expressed ATP-citrate lyase protein. Since catalysis of ATP-citrate lyase is postulated to involve the formation of phosphohistidine, these results are consistent with the pattern of earlier observations of the significance of the histidine residue in catalysis of the human ATP-citrate lyase.

Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells.

Secretion of catecholamines from single bovine chromaffin cells in culture was elicited by brief pressure ejections from a micropipette containing nicotine, carbamoylcholine, or potassium ions or by mechanical stimulation. Release was monitored electrochemically with a carbon-fiber microelectrode placed adjacent to the cell. Cyclic voltammetry was used to identify secreted species, whereas constant potential amperometry was used for improved temporal resolution (millisecond range) of catecholamine detection. During secretion, brief current spikes were observed, which were shown to be due to detection of catecholamines by electrooxidation. The spikes have the physical characteristics of multimolecular packets of catecholamines released at random times and locations from the surface of the single cell. The half-width of the spikes was found to increase with an increase in cell-electrode spacing. The properties of the catecholamine spikes correlate well with expectations based on secretion from individual storage vesicles. Spikes do not occur in the absence of Ca2+ in the buffer, and the majority of spikes are found to be distributed between 0.2 and 2 picocoulombs, corresponding to 1-10 attomoles of catecholamine detected. The frequency of the spikes increases with the intensity of the stimulus, but the average quantity of catecholamine in each spike is independent of the stimulus. Thus, these measurements represent time-resolved observation of quantal secretion of catecholamines and provide direct evidence for the exocytotic hypothesis.

Purification of a [3H]dihydrotetrabenazine-binding protein from bovine adrenal medulla.

A high affinity binding site for [3H]dihydrotetrabenazine is thought to be present on the monoamine transport protein from chromaffin granules. We describe a procedure for purification of this binding activity from frozen bovine adrenal tissue, and we partially characterize the purified preparation. Binding activity solubilized with sodium cholate and soybean lecithin was fractionated on wheat germ lectin-Sepharose, phenyl-Sepharose, Mono Q, and hydroxylapatite. Denaturing electrophoresis of the purified binding activity, followed by silver staining, revealed a single broad band centered at an apparent molecular weight of 85,000. This preparation bound [3H]dihydrotetrabenazine with an apparent dissociation constant of 2.7 nM and had a site density of 10 nmol/mg. Treatment of the purified protein with neuraminidase reduced the apparent molecular weight by 9000, indicating the presence of terminal sialic acids on the oligosaccharide portion of this molecule.

Secretion of catecholamines from individual adrenal medullary chromaffin cells.

Catecholamine secretion has been measured with electrochemical techniques from isolated, single adrenal medullary chromaffin cells with carbon-fiber microelectrodes. The electrode tip, which is of similar dimensions to the cell, is placed adjacent to the cell to enable the measurement of local secretion. Secretion is caused by exposing the cell to nanoliter volumes of solution containing nicotinic receptor agonists or depolarizing agents. The identification of secreted substances is made with cyclic voltammetry at both bare electrodes and electrodes coated with a perfluorinated cation-exchange polymer. Catecholamine secretion is induced by nicotine (10-500 microM), carbamylcholine (1 mM), and K+ (60 mM). All agents that induce secretion lead to a broad envelope of secreted catecholamines on which sharp concentration spikes are superimposed. The concentration spikes can be monitored with a time resolution of tens of milliseconds when the electrodes are used in the amperometric mode. Release induced by nicotine and K+ is inhibited by Cd2+ (0.5 mM), and hexamethonium selectively blocks the nicotine-induced secretion. The actions of nicotine are found to continue for a longer period of time than those of the other secretagogues tested.

Nicotinic receptor-mediated catecholamine secretion from individual chromaffin cells. Chemical evidence for exocytosis.

Nicotinic receptor-mediated secretion of catecholamines from individual cultured bovine adrenal medullary chromaffin cells was measured and characterized with a voltametric microelectrode placed adjacent to the cells. Nicotine-induced secretion is associated with a large increase in chemical spikes that is temporally resolved into the apparent secretion of discrete packets of attomole quantities of easily oxidized molecules. These data are consistent with direct chemical measurement of single exocytotic events.

Rat ATP citrate-lyase. Molecular cloning and sequence analysis of a full-length cDNA and mRNA abundance as a function of diet, organ, and age.

ATP citrate-lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA in many tissues. We have isolated a full-length cDNA copy of 4.3 kilobase pairs encoding the ATP-citrate lyase mRNA by screening rat liver cDNA library using oligonucleotide probes designed from peptide sequences obtained from the purified rat enzyme. Expression of this cDNA in bacteria, followed by immunoblotting with antibody directed against the ATP citrate-lyase, further demonstrated the identity of this clone. Nucleic acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1100 amino acids in length with a calculated molecular weight of 121,293. RNA blot analysis indicated an mRNA species of about 4.3 kilobase pairs in livers of chow-fed rats. Rats maintained on low fat, high carbohydrate diets exhibited a striking increase (50-fold) in the level of liver ATP citrate-lyase mRNA as compared with the control animals maintained on a normal diet. The tissue distribution of this mRNA in chow-fed animals revealed a relatively high abundance of the message in liver and adrenal, moderate levels were found in lung, brain, and large intestine with only trace amounts of the message in small intestine, stomach, testis, spleen, pancreas, kidney, and heart. During rat development, the ATP citrate-lyase mRNA was relatively high in the liver at parturition, followed by a reduction in its level during suckling. Higher amounts of the mRNA were detected again in adult animals. The isolation and characterization of the mRNA for ATP citrate-lyase will allow further studies on the reaction mechanism and metabolic regulation of this key enzyme in lipogenesis and cholesterogenesis.

Genetic and tissue-specific variation in the expression of a closely linked murine multigene family on chromosome 15 that encodes salivary and lacrimal proteins.

The murine submandibular gland (SMG) produces a novel class of highly acidic salivary proteins encoded by one or more highly abundant mRNA transcripts. In inbred mice, these transcripts are encoded by members of a multigene family comprising approximately 8-12 homologues. Most, and probably all, of these homologues are clustered at a new locus near belted (bt) on chromosome 15, which we designate Spt (salivary protein). Although physically closely linked, Spt genes differ in their patterns of expression both in strains of mice and in their tissues. One gene, Spt-1, is expressed at high levels in the SMG of all inbred strains examined. This gene is also expressed at significant levels in the lacrimal gland. A second gene, Spt-2, appears to be present as a single copy in some strains and as two copies in others. This gene is expressed at high levels only in the SMG of those strains carrying two copies, and Spt-2 mRNA is not detectable in the SMG of strains carrying only one copy. In contrast to Spt-1, the Spt-2 gene is not expressed at detectable levels in the lacrimal gland.

Interaction of dihydrotetrabenazine binding activity with immobilized lectins.

High-affinity dihydrotetrabenazine binding activity was solubilized by treatment of crude bovine striatal synaptosomes with cholate and incubated with lectins immobilized on sepharose. Both concanavalin A and wheat germ lectin sepharose removed dihydrotetrabenazine binding activity from the cholate extract, and this removal was prevented by inclusion of the appropriate hapten sugars. These results imply that the dihydrotetrabenazine binding subunit of the synaptic vesicle catecholamine/serotonin (5-HT) transporter, or a vesicle component tightly associated with it, is glycosylated.

Comparison of uptake of dopamine in rat striatal chopped tissue and synaptosomes.

Uptake of dopamine (DA) by chopped tissue prepared from rat corpus striatum has been examined to determine whether one or two kinetically distinct uptake sites exist. Two methods were used: direct measurement of accumulated [3H]DA and determination of the rate of formation of 3,4-dihydroxyphenylacetic acid (DOPAC) after exposure to DA. The rate of formation of DOPAC in the latter experiments is a direct function of the rate of DA uptake. The rates of [3H]DA uptake and DOPAC formation are both linear with time in the presence of 10 microM substrate. Studies of [3H]DA accumulation into chopped tissue reveal two apparent components with Km values of 160 nM and 3.8 microM, whereas similar experiments with striatal homogenate or synaptosomes yield a single uptake component with a Km equivalent to the lower value found in chopped tissue. Evaluation of DA uptake via the rate of DOPAC formation gave a Km value of 2.3 microM. (High substrate values were used, so a lower value for Km is not apparent in the data.) The high Km-value component was absent in animals with a lesioned striatum induced by prior nigral injections of 6-hydroxydopamine. Several pharmacologic agents (benztropine, amfonelic acid, bupropion, nomifensine and ouabain) were evaluated. All reduced the uptake of DA in chopped tissue, but with reduced potencies compared with the effect of these agents in synaptosomes. The high Km activity in chopped tissue, as well as the apparent reduced potency of uptake inhibitor, appears to arise from the diffusional barrier present inside more intact tissue. This barrier is not present in homogenates or synaptosomes, and, thus, a single uptake process is seen.