Outcome-Locked Cholinergic Signaling Suppresses Prefrontal Encoding of Stimulus Associations.

Acetylcholine (ACh) is thought to control arousal, attention, and learning by slowly modulating cortical excitability and plasticity. Recent studies, however, discovered that cholinergic neurons emit precisely timed signals about the aversive outcome at millisecond precision. To investigate the functional relevance of such phasic cholinergic signaling, we manipulated and monitored cholinergic terminals in the mPFC while male mice associated a neutral conditioned stimulus (CS) with mildly aversive eyelid shock (US) over a short temporal gap. Optogenetic inhibition of cholinergic terminals during the US promoted the formation of the CS-US association. On the contrary, optogenetic excitation of cholinergic terminals during the US blocked the association formation. The bidirectional behavioral effects paralleled the corresponding change in the expression of an activity-regulated gene, c-Fos in the mPFC. In contrast, optogenetic inhibition of cholinergic terminals during the CS impaired associative learning, whereas their excitation had marginal effects. In parallel, photometric recording from cholinergic terminals in the mPFC revealed strong innate phasic responses to the US. With subsequent CS-US pairings, cholinergic terminals weakened the responses to the US while developing strong responses to the CS. The across-session changes in the CS- and US-evoked terminal responses were correlated with associative memory strength. These findings suggest that phasic cholinergic signaling in the mPFC exerts opposite effects on aversive associative learning depending on whether it is emitted by the outcome or the cue.SIGNIFICANCE STATEMENT Drugs compensating for the decline of acetylcholine (ACh) are used for cognitive impairment, such as Alzheimer's disease. However, their beneficial effects are limited, demanding new strategies based on better understandings of how ACh modulates cognition. Here, we report that by manipulating ACh signals in the mPFC, we can control the strength of aversive associative learning in mice. Specifically, the suppression of ACh signals during an aversive outcome facilitated its association with a preceding cue. In contrast, the suppression of ACh signals during the cue impaired learning. Considering that this paradigm depends on the brain regions affected in Alzheimer's disease, our findings indicate that precisely timed control of ACh signals is essential to refine ACh-based strategies for cognitive enhancement.

Influence of cortical synaptic input on striatal neuronal dendritic arborization and sensitivity to excitotoxicity in corticostriatal coculture.

Corticostriatal cocultures are utilized to recapitulate the cortex-striatum connection in vitro as a convenient model to investigate the development, function, and regulation of synapses formed between cortical and striatal neurons. However, optimization of this dissociated neuronal system to more closely reproduce in vivo circuits has not yet been explored. We studied the effect of varying the plating ratio of cortical to striatal neurons on striatal spiny projection neuron (SPN) characteristics in primary neuronal cocultures. Despite the large difference in cortical-striatal neuron ratio (1:1 vs. 1:3) at day of plating, by 18 days in vitro the difference became modest (∼25% lower cortical-striatal neuron ratio in 1:3 cocultures) and the neuronal density was lower in the 1:3 cocultures, indicating enhanced loss of striatal SPNs. Comparing SPNs in cocultures plated at a 1:1 vs. 1:3 ratio, we found that resting membrane potential, input resistance, current injection-induced action potential firing rates, and input-output curves were similar in the two conditions. However, SPNs in the cocultures plated at the lower cortical ratio exhibited reduced membrane capacitance along with significantly shorter total dendritic length, decreased dendritic complexity, and fewer excitatory synapses, consistent with their trend toward reduced miniature excitatory postsynaptic current frequency. Strikingly, the proportion of NMDA receptors found extrasynaptically in recordings from SPNs was significantly higher in the less cortical coculture. Consistently, SPNs in cocultures with reduced cortical input showed decreased basal pro-survival signaling through cAMP response element binding protein and enhanced sensitivity to NMDA-induced apoptosis. Altogether, our study indicates that abundance of cortical input regulates SPN dendritic arborization and survival/death signaling.

Impaired Replenishment of Cortico-Striatal Synaptic Glutamate in Huntington's Disease Mouse Model.

BACKGROUND: Huntington's disease (HD) is an inherited neurodegenerative disorder caused by expansion of CAG repeats in the Huntingtin gene (HTT). Studies suggest cortical to striatal (C-S) projections, which regulate movement and provide cell survival signals to SPNs, are altered in the pre-manifest and early symptomatic stages of HD. But whether and how presynaptic cortical terminals are affected in HD is not well explored. OBJECTIVE: Test size and replenishment of readily releasable pool (RRP), and assess glutamate refill of C-S synapses in HD models. METHODS: Immunocytochemistry was applied in C-S co-cultures generated from FVB/N (WT: wildtype) mice and YAC128, an HD mouse model expressing human HTT with  128 CAG repeats on the FVB/N background; Whole-cell patch clamp recordings from striatal neurons were performed both in cultures, with or without osmotic stimuli, and in acute brain slices from 6-month-old early symptomatic YAC128 mice and WT following prolonged trains of electrical stimuli in corpus callosum. RESULTS: We found no change in the average size or vesicle replenishment rate of RRP in C-S synapses of YAC128, compared with WT, cultures at day in vitro 21, a time when immunocytochemistry showed comparable neuronal survival between the two genotypes. However, YAC128 C-S synapses showed a slowed rate of recovery of glutamate release in co-cultures as well as in acute brain slices. CONCLUSION: Mutant HTT expression impairs glutamate refill but not RRP size or replenishment in C-S synapses. This work provides a foundation for examining the contribution of deficits in presynaptic cortical terminals on HD progression.

NAMPT inhibitor protects ischemic neuronal injury in rat brain via anti-neuroinflammation.

Nicotinamide phosphoribosyltransferase (NAMPT) is an important neuroprotective factor in cerebral ischemia, and it has been reported that NAMPT inhibitors can aggravate neuronal injury in the acute phase. However, because it is a cytokine, NAMPT participates in many inflammatory diseases in the peripheral system, and its inhibitors have therapeutic effects. Following cerebral ischemia, the peripheral and resident inflammatory and immune cells produce many pro-inflammatory mediators in the ischemic area, which induce neuroinflammation and impair the brain. However, the effects of NAMPT inhibitors in the neuroinflammation after ischemic brain injury remain unknown. Here, we found that FK866, a potent NAMPT inhibitor, decreased the level of TNF-α, NAMPT and IL-6 in the ischemic brain tissue one day after middle-cerebral-artery occlusion and reperfusion (MCAO/R), improved neurological dysfunction, decreased infarct volume and neuronal loss, and inhibited microgliosis and astrogliosis 14days after MCAO/R. The expression of NAMPT protein was induced in Iba1-positive microglia/macrophages in the ischemia core 14days after MCAO/R. In vitro studies show that oxygen-glucose deprivation and recovery (OGD/R) activate microglia. Activated microglia increased the activity of NF-κB, increased the mRNA synthesis of TNF-α, NAMPT and IL-6, and increased the secretion of TNF-α, NAMPT and IL-6. On the other hand, NAMPT can act synergistically with other cytokines and activate microglia. FK866 strongly inhibited these changes and alleviated OGD/R-induced activation of microglia. As such, NAMPT is a crucial determinant of cellular inflammation after cerebral ischemia. NAMPT inhibitors are novel compounds to protect neuronal injury from ischemia via anti-inflammatory effects.