Ambient but not local lactate underlies neuronal tolerance to prolonged glucose deprivation

Neurons require a nearly constant supply of ATP. Glucose is the predominant source of brain ATP, but the direct effects of prolonged glucose deprivation on neuronal viability and function remain unclear. In sparse rat hippocampal microcultures, neurons were surprisingly resilient to 16 h glucose removal in the absence of secondary excitotoxicity. Neuronal survival and synaptic transmission were unaffected by prolonged removal of exogenous glucose. Inhibition of lactate transport decreased microculture neuronal survival during concurrent glucose deprivation, suggesting that endogenously released lactate is important for tolerance to glucose deprivation. Tandem depolarization and glucose deprivation also reduced neuronal survival, and trace glucose concentrations afforded neuroprotection. Mass cultures, in contrast to microcultures, were insensitive to depolarizing glucose deprivation, a difference attributable to increased extracellular lactate levels. Removal of local astrocyte support did not reduce survival in response to glucose deprivation or alter evoked excitatory transmission, suggesting that on-demand, local lactate shuttling is not necessary for neuronal tolerance to prolonged glucose removal. Taken together, these data suggest that endogenously produced lactate available globally in the extracellular milieu sustains neurons in the absence of glucose. A better understanding of resilience mechanisms in reduced preparations could lead to therapeutic strategies aimed to bolster these mechanisms in vulnerable neuronal populations

Loss of local astrocyte support disrupts action potential propagation and glutamate release synchrony from unmyelinated hippocampal axon terminals in vitro

Neuron–astrocyte interactions are critical for proper CNS development and function. Astrocytes secrete factors that are pivotal for synaptic development and function, neuronal metabolism, and neuronal survival. Our understanding of this relationship, however, remains incomplete due to technical hurdles that have prevented the removal of astrocytes from neuronal circuits without changing other important conditions. Here we overcame this obstacle by growing solitary rat hippocampal neurons on microcultures that were comprised of either an astrocyte bed (+astrocyte) or a collagen bed (−astrocyte) within the same culture dish. −Astrocyte autaptic evoked EPSCs, but not IPSCs, displayed an altered temporal profile, which included increased synaptic delay, increased time to peak, and severe glutamate release asynchrony, distinct from previously described quantal asynchrony. Although we observed minimal alteration of the somatically recorded action potential waveform, action potential propagation was altered. We observed a longer latency between somatic initiation and arrival at distal locations, which likely explains asynchronous EPSC peaks, and we observed broadening of the axonal spike, which likely underlies changes to evoked EPSC onset. No apparent changes in axon structure were observed, suggesting altered axonal excitability. In conclusion, we propose that local astrocyte support has an unappreciated role in maintaining glutamate release synchrony by disturbing axonal signal propagation. SIGNIFICANCE STATEMENT Certain glial cell types (oligodendrocytes, Schwann cells) facilitate the propagation of neuronal electrical signals, but a role for astrocytes has not been identified despite many other functions of astrocytes in supporting and modulating neuronal signaling. Under identical global conditions, we cultured neurons with or without local astrocyte support. Without local astrocytes, glutamate transmission was desynchronized by an alteration of the waveform and arrival time of axonal action potentials to synaptic terminals. GABA transmission was not disrupted. The disruption did not involve detectable morphological changes to axons of glutamate neurons. Our work identifies a developmental role for astrocytes in the temporal precision of excitatory signals

Differential presynaptic ATP supply for basal and high-demand transmission

The relative contributions of glycolysis and oxidative phosphorylation to neuronal presynaptic energy demands are unclear. In rat hippocampal neurons, ATP production by either glycolysis or oxidative phosphorylation alone sustained basal evoked synaptic transmission for up to 20 min. However, combined inhibition of both ATP sources abolished evoked transmission. Neither action potential propagation failure nor depressed Ca(2+) influx explained loss of evoked synaptic transmission. Rather, inhibition of ATP synthesis caused massive spontaneous vesicle exocytosis, followed by arrested endocytosis, accounting for the disappearance of evoked postsynaptic currents. In contrast to its weak effects on basal transmission, inhibition of oxidative phosphorylation alone depressed recovery from vesicle depletion. Local astrocytic lactate shuttling was not required. Instead, either ambient monocarboxylates or neuronal glycolysis was sufficient to supply requisite substrate. In summary, basal transmission can be sustained by glycolysis, but strong presynaptic demands are met preferentially by oxidative phosphorylation, which can be maintained by bulk but not local monocarboxylates or by neuronal glycolysis. SIGNIFICANCE STATEMENT Neuronal energy levels are critical for proper CNS function, but the relative roles for the two main sources of ATP production, glycolysis and oxidative phosphorylation, in fueling presynaptic function in unclear. Either glycolysis or oxidative phosphorylation can fuel low-frequency synaptic function and inhibiting both underlies loss of synaptic transmission via massive vesicle release and subsequent failure to endocytose lost vesicles. Oxidative phosphorylation, fueled by either glycolysis or endogenously released monocarboxylates, can fuel more metabolically demanding tasks such as vesicle recovery after depletion. Our work demonstrates the flexible nature of fueling presynaptic function to maintain synaptic function

Generation of Human Striatal Neurons by MicroRNA-Dependent Direct Conversion of Fibroblasts

SummaryThe promise of using reprogrammed human neurons for disease modeling and regenerative medicine relies on the ability to induce patient-derived neurons with high efficiency and subtype specificity. We have previously shown that ectopic expression of brain-enriched microRNAs (miRNAs), miR-9/9∗ and miR-124 (miR-9/9∗-124), promoted direct conversion of human fibroblasts into neurons. Here we show that coexpression of miR-9/9∗-124 with transcription factors enriched in the developing striatum, BCL11B (also known as CTIP2), DLX1, DLX2, and MYT1L, can guide the conversion of human postnatal and adult fibroblasts into an enriched population of neurons analogous to striatal medium spiny neurons (MSNs). When transplanted in the mouse brain, the reprogrammed human cells persisted in situ for over 6 months, exhibited membrane properties equivalent to native MSNs, and extended projections to the anatomical targets of MSNs. These findings highlight the potential of exploiting the synergism between miR-9/9∗-124 and transcription factors to generate specific neuronal subtypes

Monocarboxylate shuttling underlies neuronal resilience during prolonged glucose deprivation.

<p>(A) Neuronal survival was measured following prolonged incubation in indicated treatment conditions. The ‘+’ and ‘-’ indicate presence and absence, respectively. Data points represent individual cultures, color coded to indicate siblings (one-way ANOVA with Dunnett’s multiple comparisons test). (B) Basal evoked EPSCs are not affected by the absence (black bar/symbols) or presence (red bar/symbols) of monocarboxylate transport inhibitor, 4-CIN (100 μM for 2 h preceding recording), following prolonged glucose deprivation (p > 0.05 Student’s t-test). (C) Cells from B were subjected to K⁺-induced vesicle depletion, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195520#pone.0195520.g002" target="_blank">Fig 2D</a>. EPSCs subjected to glucose deprivation without (black trace) and with 4-CIN (red traces; p<0.05, Two-way repeated measures ANOVA). Summary data are represented as mean ± SEM. *p<0.05. n.s., non-significant.</p

Ongoing, global lactate release supports neuronal resilience to glucose deprivation.

<p>(A) Schematic of microcultures and high-density mass cultures. Compared with mass cultures (right), microcultures (left) typically contain fewer neurons (black dots) and fewer astrocytes overall (blue circles), but with similar local density of astrocytes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195520#pone.0195520.ref038" target="_blank">38</a>]. (B) Survival of microcultures and mass cultures, incubated in either glucose-free saline (0 glucose, black dots) or in glucose-free depolarizing saline (+KCl, gray dots). Lines connect sibling cultures. Two-way ANOVA with repeated measures for depolarization state showed a significant interaction between culture condition and depolarization state (p < 0.001). Results of post-hoc, Bonferroni-corrected multiple comparisons are indicated above symbols. (C) Summary of lactate concentrations measured from microcultures and mass cultures following prolonged incubation in glucose-free saline (black bars) or glucose-free depolarizing saline (gray bars; Two-way ANOVA with no significant difference between glucose deprivation ± KCl and p < 0.001 between microculture and mass culture). (D) 4-CIN induces neuronal loss in mass cultures at both 100 μM (teal dots) and 300 μM (burgundy dots). Lines connect sibling cultures. One-way repeated measures ANOVA with Dunnett’s multiple comparisons test with glucose deprivation as control. (E) Lactate concentration measured in mass cultures in the absence of neurons does not differ from that measured in the presence of neurons. Black bars indicate prolonged glucose deprivation and gray bars indicate prolonged depolarizing glucose deprivation (+KCl; p > 0.05, Two-way ANOVA). (F) Summary of neuronal survival for microcultures incubated in glucose-free saline with or without 0.35 mM lactate added to mimic mass culture lactate. Two-way ANOVA with repeated measures for depolarization state revealed a significant interaction between lactate and depolarization state (p < 0.05). Results of post-hoc Bonferroni corrected multiple comparisons are indicated above symbols. (G) Summary of lactate concentrations measured from microcultures with or without added lactate, confirming persistence of lactate addition (Two-way ANOVA with no significant difference between glucose deprivation ± KCl and p < 0.0001 between microculture ± lactate). Data are represented as mean ± SEM. *p<0.05. **p<0.01. ***p<0.001. ****p<0.0001. n.s., non-significant.</p

Synaptic transmission is intact following prolonged glucose deprivation.

<p>(A,B) Representative baseline action potential evoked EPSCs (black traces) and subsequent EPSC recovery following vesicle depletion (gray traces) following control incubation in conditioned medium (A) and following prolonged glucose deprivation (B). (C) Summary of baseline charge transfer following control incubation (black bar/symbols) or glucose deprivation (red bar/symbols; p > 0.05, Student’s t-test). (D) Summary of recovery EPSCs following vesicle depletion protocol (90 mM KCl applied for 30 sec) for conditioned medium (Control; black trace) compared to glucose deprivation (red trace; p > 0.05; Two-way repeated measures ANOVA). Data are represented as mean ± SEM. n.s, non-significant.</p

Loss of local astrocyte support does not affect basal or high-demand transmission following prolonged glucose deprivation.

<p>(A,B) Representative baseline evoked EPSCs (black traces) and subsequent recovery following K⁺-evoked vesicle depletion (gray traces) of -astrocyte neurons incubated in conditioned medium (A, Control) and following prolonged glucose deprivation (B, 0 Glucose). (C) Summary of baseline charge transfer following prolonged incubation in either conditioned medium (Control; black bar/symbols) or glucose deprivation (red bar/symbols; p > 0.05, Student’s t-test). (D) Summary of recovery of evoked EPSCs following K⁺-induced vesicle depletion for conditioned medium controls (black trace) compared to glucose deprivation (red trace; p > 0.05; Two-way repeated measures ANOVA). Data are represented as mean ± SEM. n.s., non-significant.</p