Acceptability of Neuroscientific Interventions in Education.

Researchers are increasingly applying neuroscience technologies that probe or manipulate the brain to improve educational outcomes. However, their use remains fraught with ethical controversies. Here, we investigate the acceptability of neuroscience applications to educational practice in two groups of young adults: those studying bioscience who will be driving future basic neuroscience research and technology transfer, and those studying education who will be choosing among neuroscience-derived applications for their students. Respondents rated the acceptability of six scenarios describing neuroscience applications to education spanning multiple methodologies, from neuroimaging to neuroactive drugs to brain stimulation. They did so from two perspectives (student, teacher) and for three recipient populations (low-achieving, high-achieving students, students with learning disabilities). Overall, the biosciences students were more favorable to all neuroscience applications than the education students. Scenarios that measured brain activity (i.e., EEG or fMRI) to assess or predict intellectual abilities were deemed more acceptable than manipulations of mental activity by drug use or stimulation techniques, which may violate body integrity. Enhancement up to the norm for low-achieving students and especially students with learning disabilities was more favorably viewed than enhancement beyond the norm for high-achieving students. Finally, respondents rated neuroscientific applications to be less acceptable when adopting the perspective of a teacher than that of a student. Future studies should go beyond the acceptability ratings collected here to delineate the role that concepts of access, equity, authenticity, agency and personal choice play in guiding respondents' reasoning.

Teaching neuroscience to science teachers: facilitating the translation of inquiry-based teaching instruction to the classroom.

In science education, inquiry-based approaches to teaching and learning provide a framework for students to building critical-thinking and problem-solving skills. Teacher professional development has been an ongoing focus for promoting such educational reforms. However, despite a strong consensus regarding best practices for professional development, relatively little systematic research has documented classroom changes consequent to these experiences. This paper reports on the impact of sustained, multiyear professional development in a program that combined neuroscience content and knowledge of the neurobiology of learning with inquiry-based pedagogy on teachers' inquiry-based practices. Classroom observations demonstrated the value of multiyear professional development in solidifying adoption of inquiry-based practices and cultivating progressive yearly growth in the cognitive environment of impacted classrooms.

Age-related changes in regional brain mitochondria from Fischer 344 rats.

Brain mitochondrial function has been posited to decline with aging. In order to test this hypothesis, cortical and striatal mitochondria were isolated from Fischer 344 rats at 2, 5, 11, 24 and 33 months of age. Mitochondrial membrane potential remained stable through 24 months, declining slightly in mitochondria from both brain regions at 33 months. The ability of calcium to induce mitochondrial swelling and depolarization, characteristics of the permeability transition, was remarkably stable through 24 months of age and increased at advanced ages only for cortical, but not striatal, mitochondria. Striatal mitochondria were more sensitive to calcium than were cortical mitochondria throughout the first 2 years of life. A two-fold increased resistance to calcium was observed in striatal mitochondria between 5 and 11 months. Although these measurements do demonstrate changes in mitochondrial function with aging, the changes in polarization are relatively small and the increased cortical susceptibility to the permeability transition only occurred at very advanced ages. Thus mitochondrial decline with advanced age depends upon brain region.

Age-dependent changes in the calcium sensitivity of striatal mitochondria in mouse models of Huntington's Disease.

Striatal and cortical mitochondria from knock-in and transgenic mutant huntingtin mice were examined for their sensitivity to calcium induction of the permeability transition, a cause of mitochondrial depolarization and ATP loss. The permeability transition has been suggested to contribute to cell death in Huntington's Disease. Mitochondria were examined from slowly progressing knock-in mouse models with different length polyglutarnine expansions (Q20, Q50, Q92, Q111) and from the rapidly progressing transgenic R6/2 mice overexpressing exon I of human huntingtin with more than 110 polyglutamines. As previously observed in rats, striatal mitochondria from background strain CD1 and C57BL/6 control mice were more sensitive to calcium than cortical mitochondria. Between 5 and 12 months in knock-in Q92 mice and between 8 and 12 weeks in knock-in Q111 mice, striatal mitochondria developed resistance, becoming equally sensitive to calcium as cortical mitochondria, while those from Q50 mice were unchanged. Cortical mitochondrial calcium sensitivity did not change. In R6/2 mice striatal and cortical mitochondria were equally resistant to Ca2+ while striatal mitochondria from littermate controls were more susceptible. No increases in calcium sensitivity were observed in the mitochondria from Huntington's Disease (HD) mice compared to controls. Neither motor abnormalities, nor expression of cyclophilin D corresponded to the changes in mitochondrial sensitivity. Polyglutamine expansions in huntingtin produced an early increased resistance to calcium in striatal mitochondria suggesting mitochondria undergo compensatory changes in calcium sensitivity in response to the many cellular changes wrought by polyglutamine expansion.

Cyclooxygenase-2 inhibitor ns-398 protects neuronal cultures from lipopolysaccharide-induced neurotoxicity.

BACKGROUND AND PURPOSE: The prostanoid-synthesizing enzyme cyclooxygenase (COX)-2 is markedly upregulated after cerebral ischemia and may participate in the mechanisms by which postischemic inflammation contributes to the late stages of ischemic brain injury. In the present study, we sought to provide additional evidence for a role of COX-2 in the mechanisms of neurotoxicity associated with inflammation. METHODS: Nine-day-old neuronal-glial cultures, prepared from the cerebral cortex of newborn C57BL/6J mice, were exposed to lipopolysaccharide (LPS), a potent proinflammatory agent. The contribution of COX-2 was investigated by using the COX-2 inhibitor NS-398. RESULTS: LPS produced a dose-dependent (0.001 to 10 microg/mL) and selective neuronal death that was well developed 72 hours after treatment. The effect was associated with a marked increase in the concentration of the COX reaction product prostaglandin E(2) (PGE(2)) and of the cytokine tumor necrosis factor-alpha (TNF-alpha). NS-398 (10 micromol/L) blocked the PGE(2) increase, attenuated the TNF-alpha increase, and prevented the neuronal death produced by LPS. TNF-alpha-blocking antibodies attenuated LPS-induced neuronal death, but the protection was less pronounced than that afforded by NS-398. LPS failed to elevate PGE(2) or to produce cell death in neuron-enriched cultures, suggesting that glial cells are required for these effects. CONCLUSIONS: COX-2, in part through TNF-alpha-related mechanisms, contributes to LPS-induced neuronal death. The data support the hypothesis that COX-2, in addition to its role in glutamate excitotoxicity, participates in the cytotoxicity associated with inflammation.

Neurons are protected from excitotoxic death by p53 antisense oligonucleotides delivered in anionic liposomes.

The potential of anionic liposomes for oligonucleotide delivery was explored because the requirement for a net-positive charge on transfection-competent cationic liposome-DNA complexes is ambiguous. Liposomes composed of phosphatidylglycerol and phosphatidylcholine were monodisperse and encapsulated oligonucleotides with 40-60% efficiency. Ionic strength, bilayer charge density, and oligonucleotide chemistry influenced encapsulation. To demonstrate the biological efficacy of this vector, antisense oligonucleotides to p53 delivered in anionic liposomes were tested in an in vitro model of excitotoxicity. Exposure of hippocampal neurons to glutamate increased p53 protein expression 4-fold and decreased neuronal survival to approximately 35%. Treatment with 1 microm p53 antisense oligonucleotides in anionic liposomes prevented glutamate-induced up-regulation of p53 and increased neuronal survival to approximately 75%. Encapsulated phosphorothioate p53 antisense oligonucleotides were neuroprotective at 5-10-fold lower concentrations than when unencapsulated. Replacing the anionic lipid with phosphatidylserine significantly decreased neuroprotection. p53 antisense oligonucleotides complexed with cationic liposomes were ineffective. Neuroprotection by p53 antisense oligonucleotides in anionic liposomes was comparable with that by glutamate receptor antagonists and a chemical inhibitor of p53. Anionic liposomes were also capable of delivering plasmids and inducing transgene expression in neurons. Anionic liposome-mediated internalization of Cy3-labeled oligonucleotides by neurons and several other cell lines demonstrated the universal applicability of this vector.

On the mechanisms of neuroprotection by creatine and phosphocreatine.

Creatine and phosphocreatine were evaluated for their ability to prevent death of cultured striatal and hippocampal neurons exposed to either glutamate or 3-nitropropionic acid (3NP) and to inhibit the mitochondrial permeability transition in CNS mitochondria. Phosphocreatine (PCr), and to a lesser extent creatine (Cr), but not (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK801), dose-dependently ameliorated 3NP toxicity when applied simultaneously with the 3NP in Mg2+-free media. Pre-treatment of PCr for 2 or 5 days and Cr for 5 days protected against glutamate excitotoxicity equivalent to that achieved by MK801 post-treatment. The combination of PCr or Cr pre-treatment and MK801 post-treatment did not provide additional protection, indicating that both prevented the toxicity attributable to secondary glutamate release. To determine if Cr or PCr directly inhibited the permeability transition, mitochondrial swelling and depolarization were assayed in isolated, purified brain mitochondria. PCr reduced the amount of swelling induced by calcium by 20%. Cr decreased mitochondrial swelling when inhibitors of creatine kinase octamer-dimer transition were present. However, in brain mitochondria prepared from rats fed a diet supplemented with 2% creatine for 2 weeks, the extent of calcium-induced mitochondrial swelling was not altered. Thus, the neuroprotective properties of PCr and Cr may reflect enhancement of cytoplasmic high-energy phosphates but not permeability transition inhibition.

Limitations of cyclosporin A inhibition of the permeability transition in CNS mitochondria.

Activation of the mitochondrial permeability transition may contribute to excitotoxic neuronal death (Ankarcrona et al., 1996; Dubinsky and Levi, 1998). However, cyclosporin A (CsA), a potent inhibitor of the permeability transition in liver mitochondria, only protects against neuronal injury by limited doses of glutamate and selected ischemic paradigms. The lack of consistent CsA inhibition of the mitochondrial permeability transition was analyzed with the use of isolated brain mitochondria. Changes in the permeability of the inner mitochondrial membrane were evaluated by monitoring mitochondrial membrane potential (Deltapsi), using the distribution of tetraphenylphosphonium, and by monitoring mitochondrial swelling, using light absorbance measurements. Metabolic impairments, large Ca(2+) loads, omission of external Mg(2+), or low doses of palmitic acid or the protonophore FCCP exacerbated Ca(2+)-induced sustained depolarizations and swelling and eliminated CsA inhibition. BSA restored CsA inhibition in mitochondria challenged with 50 microm Ca(2+), but not with 100 microm Ca(2+). CsA failed to prevent Ca(2+)-induced depolarization or to repolarize mitochondria when mitochondria were depolarized excessively. Similarly, CsA failed to prevent mitochondrial swelling or PEG-induced shrinkage after swelling when the Ca(2+) challenge produced a strong, sustained depolarization. Thus in brain mitochondria CsA may be effective only as an inhibitor of the permeability transition and the Ca(2+)-activated low permeability state under conditions of partial depolarization. In contrast, ADP plus oligomycin inhibited both permeabilities under all of the conditions that were tested. In situ, the neuroprotective action of CsA may be limited to glutamate challenges sufficiently toxic to induce the permeability transition but not so severe that mitochondrial depolarization exceeds threshold.

Dual responses of CNS mitochondria to elevated calcium.

Isolated brain mitochondria were examined for their responses to calcium challenges under varying conditions. Mitochondrial membrane potential was monitored by following the distribution of tetraphenylphosphonium ions in the mitochondrial suspension, mitochondrial swelling by observing absorbance changes, calcium accumulation by an external calcium electrode, and oxygen consumption with an oxygen electrode. Both the extent and rate of calcium-induced mitochondrial swelling and depolarization varied greatly depending on the energy source provided to the mitochondria. When energized with succinate plus glutamate, after a calcium challenge, CNS mitochondria depolarized transiently, accumulated substantial calcium, and increased in volume, characteristic of a mitochondrial permeability transition. When energized with 3 mM succinate, CNS mitochondria maintained a sustained calcium-induced depolarization without appreciable swelling and were slow to accumulate calcium. Maximal oxygen consumption was also restricted under these conditions, preventing the electron transport chain from compensating for this increased proton permeability. In 3 mM succinate, cyclosporin A and ADP plus oligomycin restored potential and calcium uptake. This low conductance permeability was not effected by bongkrekic acid or carboxyatractylate, suggesting that the adenine nucleotide translocator was not directly involved. Fura-2FF measurements of [Ca(2+)](i) suggest that in cultured hippocampal neurons glutamate-induced increases reached tens of micromolar levels, approaching those used with mitochondria. We propose that in the restricted substrate environment, Ca(2+) activated a low-conductance permeability pathway responsible for the sustained mitochondrial depolarization.

The mitochondrial permeability transition: the brain's point of view.

The mitochondrial permeability transition (mPT) has been implicated in both central nervous system ischaemia/reperfusion injury and excitotoxic neuronal death. To characterize the mPT of brain mitochondria, fluorescent mitochondrial dyes were applied to cultured neurons and astrocytes and isolated brain mitochondria were prepared. In astrocytes, mPT induction was observed as calcium-induced mitochondrial swelling following permeabilization by digitonin or introduction of a calcium ionophore. In hippocampal neurons, mPT induction was observed upon introduction of calcium and ionophore or application of toxic doses of glutamate. In isolated brain mitochondria, calcium dose-dependently produced calcium accumulation and mitochondrial swelling that was prevented by pretreatment with ADP or cyclosporin A. Additionally, when mitochondrial substrates were limited, calcium dose-dependently produced mitochondrial depolarization without swelling or calcium accumulation that was reversed by ADP, cyclosporin A or Ruthenium Red. The degree of mitochondrial depolarization was modulated by free fatty acids, magnesium, calcium concentration and protonophore Repolarization of mitochondria and closure of this low-conductance manifestation of the mPT pore by cyclosporin A was modulated by the degree of depolarization.

Calcium-induced activation of the mitochondrial permeability transition in hippocampal neurons.

The mitochondrial permeability transition (mPT) has been implicated in both excitotoxic and apoptotic neuronal cell death, despite the fact that it has not been previously identified in neurons. To study the mPT in hippocampal neurons, cultures were loaded with the mitochondrial dye JC-1 and observed with confocal and conventional microscopy. After pretreatment with 4Br-A23187 and subsequent calcium addition, the initially rodlike mitochondria increased in diameter until mitochondria became rounded in appearance. Morphological changes reversed when calcium was removed by EGTA. When neurons were loaded with both fura-2-AM and rhodamine 123, calcium loading produced an increase in cytosolic calcium, mitochondrial depolarization, and similar alterations in mitochondrial morphology. Smaller calcium challenges produced calcium cycling, delaying morphological changes until after secondary depolarization and calcium release to the cytosol. In neurons exposed to glutamate, confocal observation of JC-1 fluorescence revealed comparable changes in mitochondrial morphology that were prevented when barium was substituted for calcium, or following pretreatment with the mPT inhibitor, cyclosporin A. These experiments establish conditions in which the mPT could be observed in situ in neurons in response to calcium loading. In addition, the timing of changes suggested that induction of the permeability transition in situ represents a sequence of multiple events that may reflect the multiple open conformations of the mPT pore.

Cerebral ischemia enhances polyamine oxidation: identification of enzymatically formed 3-aminopropanal as an endogenous mediator of neuronal and glial cell death.

To elucidate endogenous mechanisms underlying cerebral damage during ischemia, brain polyamine oxidase activity was measured in rats subjected to permanent occlusion of the middle cerebral artery. Brain polyamine oxidase activity was increased significantly within 2 h after the onset of ischemia in brain homogenates (15.8 +/- 0.9 nmol/h/mg protein) as compared with homogenates prepared from the normally perfused contralateral side (7.4 +/- 0.5 nmol/h/mg protein) (P <0.05). The major catabolic products of polyamine oxidase are putrescine and 3-aminopropanal. Although 3-aminopropanal is a potent cytotoxin, essential information was previously lacking on whether 3-aminopropanal is produced during cerebral ischemia. We now report that 3-aminopropanal accumulates in the ischemic brain within 2 h after permanent forebrain ischemia in rats. Cytotoxic levels of 3-aminopropanal are achieved before the onset of significant cerebral cell damage, and increase in a time-dependent manner with spreading neuronal and glial cell death. Glial cell cultures exposed to 3-aminopropanal undergo apoptosis (LD50 = 160 microM), whereas neurons are killed by necrotic mechanisms (LD50 = 90 microM). The tetrapeptide caspase 1 inhibitor (Ac-YVAD-CMK) prevents 3-aminopropanal-mediated apoptosis in glial cells. Finally, treatment of rats with two structurally distinct inhibitors of polyamine oxidase (aminoguanidine and chloroquine) attenuates brain polyamine oxidase activity, prevents the production of 3-aminopropanal, and significantly protects against the development of ischemic brain damage in vivo. Considered together, these results indicate that polyamine oxidase-derived 3-aminopropanal is a mediator of the brain damaging sequelae of cerebral ischemia, which can be therapeutically modulated.

Mitochondrial permeability transition in the central nervous system: induction by calcium cycling-dependent and -independent pathways.

Isolated rat CNS mitochondria and cultured cortical astrocytes were examined for behavior indicative of a mitochondrial permeability transition (mPT). Exposure of isolated CNS mitochondria to elevated calcium or phosphate or both produced loss of absorbance indicative of mitochondrial swelling. The absorbance decreases were prevented by ADP and Mg2+ and reduced by cyclosporin A, dithiothreitol, and N-ethylmaleimide. Ruthenium red prevented calcium cycling-induced, but only attenuated phosphate-induced losses of absorbance. In cultured astrocytes permeabilized with digitonin or treated with the calcium ionophore, 4-bromo-A23187, elevations of external calcium altered mitochondrial morphology visualized with the dye, JC-1, from rod-like to rounded, swollen structures. Similar changes were observed in digitonin-permeabilized astrocytes exposed to phosphate. The incidence of calcium-induced changes in astrocyte mitochondria was prevented by Mg2+ and pretreatment with dithiothreitol and N-ethylmaleimide, and was reduced by cyclosporin A, ADP, and butacaine alone or in combinations. Ruthenium red and the Na+/Ca2+ exchange inhibitor CGP 37157 blocked calcium cycling and prevented mitochondrial shape changes in digitonin-treated, but not ionophore-treated astrocytes. Thus, the demonstrated induction conditions and pharmacological profile indicated the existence of an mPT in brain mitochondria. The mPT occurred consequent to activation of calcium cycling-dependent and -independent pathways. Induction of an mPT could contribute to neuronal injury following ischemia and reperfusion.

Effect of a prolonged glutamate challenge on plasmalemmal calcium permeability in mammalian central neurones. Mn2+ as a tool to study calcium influx pathways.

The rate of Mn(2+)-induced fluorescence quenching (RFQ) was used as a relative measure of plasma membrane Ca2+ permeability (PCa) in fura-2-loaded cultured hippocampal neurons and cerebellar granule cells during and after protracted (15-30 min) glutamate (GLU) treatment. Some limitations of this method were evaluated using a kinetic model of a competitive binding of Mn2+ and Ca2+ to fura-2 in the cell. In parallel experiment a contribution of Ca2+ influx to the cytoplasmic Ca2+ ([Ca2+]i) was repeatedly examined during and following a prolonged GLU challenge by short-duration "low-Ca2+ trials" (50 microM EGTA) and by measurements of 45Ca2+ uptake. Experiments failed to reveal a putative persistent increase in PCa that earlier was thought to underlie Ca2+ overload of the neuron caused by its toxic GLU treatment. By contrast, a sustained increase of [Ca2+]i was found to be associated with a progressive decrease in PCa and Ca2+ influx both in the period of GLU application and after its termination. These findings give new evidence in favour of the hypothesis that the GLU-induced Ca2+ overload of the neuron mainly from an impairment of its Ca2+ extrusion systems.

An obligate role for oxygen in the early stages of glutamate-induced, delayed neuronal death.

In vitro models of hypoxic/hypoglycemic injury reveal common mechanisms with glutamate excitotoxicity, but glutamate-induced toxicity in the absence of oxygen has never been directly addressed. Therefore, we assessed neuronal survival and intracellular calcium concentrations ([Ca2+]i) in neonatal hippocampal cultures in response to glutamate in the presence and absence of oxygen. Twenty-four hours of hypoxia alone killed 40% of the initial population, attributable to glutamate receptor-stimulated osmotic lysis. A 5 min glutamate exposure in ambient air killed 80% of the initial population by 24 hr later. When cultures were deprived of oxygen during and for 2-24 hr after excitotoxin exposure, glutamate did not cause additional neuronal death beyond that induced by oxygen depletion alone. Toxicities caused by activation of NMDA, AMPA, or kainate receptors were each ameliorated by oxygen depletion. In the absence of oxygen, glutamate evoked normal increases in [Ca2+]i, indicating that glutamate receptors functioned normally. The glutamate-induced increases in [Ca2+]i were not toxic in the absence of oxygen. In a similar manner, oxygen-depletion prevented neuronal killing by the calcium ionophore, ionomycin. Neuronal death produced by hydrogen peroxide or iron sulfate was not ameliorated by oxygen removal. These oxidants maximally produced only a slow increase in [Ca2+]i as the plasma membrane permeability increased nonspecifically. Therefore, oxygen-based reactions were an essential component of calcium-mediated, delayed neuronal death.

On the probabilistic nature of excitotoxic neuronal death in hippocampal neurons.

In an attempt to distinguish hypothesized rapid and slow components, we have systematically studied the time course of hippocampal neuronal death in an in vitro model of excitotoxicity. In all paradigms involving glutamate, NMDA or AMPA as toxins, the population of trypan-blue excluding (live) neurons progressively declined over 48 hr. The percent survival over time could be fit mathematically using single exponential decay curves, implying that the death of any individual neuron was a stochastic event. One or two hours after glutamate exposure, prevention of further glutamate-receptor interactions by addition of MK-801 or MK-801 plus CNQX resulted in the survival of 60-80% of the original population at 24 hr. Thus delayed, continuous blockade of secondary glutamate receptor stimulation was protective, apparently interrupting the cyclic nature of the toxicity cascade. Twelve hours of MK-801 immediately following glutamate removal protected the majority of cells during the period of active receptor blockade. As soon as MK-801 was removed, the progressive decay in population size resumed, indicating that short term receptor blockade was insufficient to prevent expression of the initial injury. A kinetic model is proposed to place these experimental results into a framework for discussion and formulation of future experimentation.

Excitotoxicity as a stochastic process.

1. Neuronal death following excitotoxic insult appears to be a stochastic process involving transition through an intermediate biochemical state. 2. Hydrogen ion accumulation in the hours after toxic glutamate exposure may indicate that this transition has occurred.

The ability of diphenylpiperazines to prevent neuronal death in dorsal root ganglion neurons in vitro after nerve growth factor deprivation and in vivo after axotomy.

The mechanism of neuroprotection by the calcium channel antagonist flunarizine against neuronal death is unknown. We investigated the ability of other calcium channel antagonists (cinnarizine, nimodipine, nicardipine, diltiazem, and verapamil), calmodulin antagonists, and calpain inhibitors to prevent neuronal death in rat dorsal root ganglion neurons in vitro after nerve growth factor (NGF) deprivation and the ability of cinnarizine and diltiazem to protect in vivo after axotomy. In vitro, only neurons treated with cinnarizine or flunarizine were protected from death after withdrawal. In vivo, cinnarizine, but not diltiazem, protected dorsal root ganglion neurons in rats after unilateral sciatic nerve crush. Intracellular calcium concentration ([Ca2+]i) was evaluated with fura 2 after NGF deprivation in vitro. Neurons "committed to die" 24 h after NGF deprivation displayed a decline in [Ca2+]i before visible morphological deterioration consistent with cell death. The influx of extracellular calcium was not necessary to produce neuronal death. Neurons deprived of NGF gradually lost the ability to respond to elevated external potassium with an increase in [Ca2+]i during the first 24 h after trophic factor deprivation. After 24 h, neurons deprived of NGF could not be rescued by readministration of NGF. Neurons protected from cell death with diphenylpiperazines maintained their response to high external potassium, suggesting continued membrane integrity. We speculate that diphenylpiperazines may protect sensory neurons via an unknown mechanism that stabilizes cell membranes.

Colocalization of excitatory and inhibitory neurotransmitter markers in striatal projection neurons in the rat.

The principle neuronal output of the neostriatum comes from medium spiny neurons that project from the caudate/putamen to the globus pallidus and substantia nigra. Although current evidence generally indicates that gamma-aminobutyric acid (GABA) is the principal neurotransmitter in this pathway, this cannot account for the excitatory synaptic activity present among cultures of striatal neurons or the short latency excitatory postsynaptic potentials which often proceed or obscure inhibitory activity evoked by striatal stimulation. In this study, retrograde transport of [3H]D-aspartate has been used to demonstrate striato-pallidal and striato-nigral neurons that possess a high-affinity uptake system for glutamate and aspartate and are therefore putatively glutamatergic. Injections of [3H]D-aspartate into the globus pallidus or substantia nigra, pars reticularis of the rat retrogradely labeled medium-sized neurons throughout the rostral-caudal extent of the neostriatum. To characterize this population further, adjacent sections were immunoreacted with antibodies to either GABA, glutamic acid decarboxylase (GAD), calbindin, or parvalbumin prior to autoradiographic processing. Under these conditions, autoradiographically labeled neurons displayed positive immunoreactivity for GABA, GAD, or calbindin. Autoradiographic label did not colocalize with parvalbumin immunoreactivity. The colocalization of anatomical markers of GABAergic and glutamatergic neurotransmission raises the possibility that both neurotransmitters are functionally expressed within single striatal projection neurons.