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.

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.

Formation of acetylcholine receptor clusters in chick myotubes: migration or new insertion?

Experiments were performed to study the feasibility of two mechanisms of acetylcholine receptor (ACHR) accumulation in chick myotubes: diffusion and trapping of previously dispersed surface receptors and localized insertion of new receptors at accumulation sites. Fluorescence photobleaching recovery (FPR) measurements indicated that the majority of diffusely distributed ACHRs in chick myotube membranes were mobile whereas nearly all receptors within high density clusters were effectively immobile. Unlike previous reports, two rates of ACHR movement characterized the mobile population. Moreover, we found that the estimated diffusion coefficient depended critically on the objective (spot size) used to assay recovery from bleaching. Implications of this finding for mechanisms of receptor immobilization are discussed. Extracts of chick brain, known to increase the number of surface receptors, did not alter receptor mobility. Extracts of Torpedo electric organ that increase the number of receptor aggregates, decreased the mobile fraction of ACHRs. Simulations of the diffusion and trapping mechanism indicated that captured receptors should congregate around the periphery of a receptor patch during the first hour after they were inserted into the membrane. However, newly inserted ACHRs were found to be located centrally within receptor patches under neurites, and this was not consistent with an exclusive diffusion-trapping mechanism. We also studied the mobility of ACHRs near points of contact made by cholinergic growth cones. The rate of receptor movement was increased in the vicinity of growth cones, but the magnitude of this effect was small.

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.

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.

Ionic currents in two strains of rat anterior pituitary tumor cells.

The ionic conductance mechanisms underlying action potential behavior in GH3 and GH4/C1 rat pituitary tumor cell lines were identified and characterized using a patch electrode voltage-clamp technique. Voltage-dependent sodium, calcium, and potassium currents and calcium-activated potassium currents were present in the GH3 cells. GH4/C1 cells possess much less sodium current, less voltage-dependent potassium current, and comparable amounts of calcium current. Voltage-dependent inward sodium current activated and inactivated rapidly and was blocked by tetrodotoxin. A slower-activating voltage-dependent inward calcium current was blocked by cobalt, manganese, nickel, zinc, or cadmium. Barium was substituted for calcium as the inward current carrier. Calcium tail currents decay with two exponential components. The rate constant for the slower component is voltage dependent, while the faster rate constant is independent of voltage. An analysis of tail current envelopes under conditions of controlled ionic gradients suggests that much of the apparent decline of calcium currents arises from an opposing outward current of low cationic selectivity. Voltage-dependent outward potassium current activated rapidly and inactivated slowly. A second outward current, the calcium-activated potassium current, activated slowly and did not appear to reach steady state with 185-ms voltage pulses. This slowly activating outward current is sensitive to external cobalt and cadmium and to the internal concentration of calcium. Tetraethylammonium and 4-aminopyridine block the majority of these outward currents. Our studies reveal a variety of macroscopic ionic currents that could play a role in the initiation and short-term maintenance of hormone secretion, but suggest that sodium channels probably do not make a major contribution.

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.

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.

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.

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.

Effects of calcium chelators on intracellular calcium and excitotoxicity.

In an attempt to probe the relationship between excitotoxicity and increases in intracellular calcium ([Ca2+]i), BAPTA-AM and its analogs were applied to cultured hippocampal neurons. Chelation of [Ca2+]i depressed and prolonged transient responses to glutamate and did not effect elevation of [Ca2+]i by prolonged exposure. This explains the inability of the chelators to prevent glutamate-induced toxicity.

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-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.

Intracellular calcium levels during the period of delayed excitotoxicity.

Intracellular calcium concentrations ([Ca2+]i) among cultured hippocampal neurons were monitored during and in the hours following an excitotoxic glutamate application to determine the time course of changes involved in delayed excitotoxicity. After a 5 min toxic insult, [Ca2+]i increased immediately and remained elevated for an hour. Subsequently, [Ca2+]i declined to normal resting levels and remained so up to 13 hr following insult. Only a few neurons displayed greatly elevated [Ca2+]i at these extended times. Survival experiments in sister cultures indicated that 85% of the neurons died after 24 hr. Therefore, intracellular calcium returned to baseline levels prior to neuronal death. Additionally, during this period when basal calcium levels had recovered, the majority of neurons responded to a second excitatory amino acid application with a second increase in [Ca2+]i.

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.

Development of inhibitory synapses among striatal neurons in vitro.

The development of excitatory and inhibitory synaptic connections has been studied in postnatal neurons from the caudate and putamen maintained in tissue culture. Excitatory postsynaptic potentials which were sensitive to the glutamate antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) appeared between 4 and 8 d in vitro. This is the first indication that glutamatergic excitatory neurons may be intrinsic to the striatum. Spontaneous inhibitory postsynaptic currents appeared at approximately the same time, several days after process outgrowth. Antibodies to glutamic acid decarboxylase (GAD), the synthetic enzyme for GABA, labeled neurons which produce bicuculline-sensitive, inhibitory postsynaptic currents. GAD immunoreactivity and immunoreactivity to synapsin I, a synaptic vesicle-associated protein, became localized to discrete sites along neurites 4-8 d after plating. It is concluded that the punctate GAD immunoreactivity identified possible sites of presynaptic transmitter release.

Changes in intracellular pH associated with glutamate excitotoxicity.

Excitotoxic neuronal injury is known to be associated with increases in cytosolic calcium ion concentrations. However, it is not known if perturbations in other intracellular ions are also associated with glutamate (GLU)-induced neuronal death. Accordingly, intracellular hydrogen ion concentrations were measured in cultured hippocampal neurons with the fluorescent dye BCECF during and after toxic exposures. Five minute GLU applications produced an initial cytosolic acidification. During the hour after GLU removal, intracellular pH (pHi) recovered steadily, resulting in a rebound cytosolic alkalinization. Lowering extracellular calcium depressed the initial GLU-induced acidification, suggesting that the rapid acidification may result partly as a consequence of calcium entry. An acidification-induced rebound alkalinization appeared to be activated by GLU exposure. Inhibitors of intracellular pH regulation, harmaline, 4,4'-disothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and replacement of external Na+ with N-methyl-glucamine+ (NMG+), retarded the rate of recovery from GLU-induced acidification. The rapid acidification and rebound alkalinization could be mimicked by challenging neurons with elevated external K+ or replacement of external Na+ with NMG+. Two or more hours following toxic GLU exposure, hydrogen ion concentration did not stabilize at initial levels but progressively increased. High K+ or Na+ removal did not produce this long-term acidification and were not toxic. The cumulative increase in intracellular hydrogen ion may reflect the declining health of injured neurons and could contribute directly to neuronal death. Therefore, cytosolic acidification may act synergistically with increases in calcium concentration in mediating excitotoxicity.