Chronic Basal Forebrain Activation Improves Spatial Memory, Boosts Neurotrophin Receptor Expression, and Lowers BACE1 and Aβ42 Levels in the Cerebral Cortex in Mice

The etiology of Alzheimer’s dementia has been hypothesized in terms of basal forebrain cholinergic decline, and in terms of reflecting beta-amyloid neuropathology. To study these different biological elements, we activated the basal forebrain in 5xFAD Alzheimer’s model mice and littermates. Mice received 5 months of 1 h per day intermittent stimulation of the basal forebrain, which includes cholinergic projections to the cortical mantle. Then, mice were behaviorally tested followed by tissue analysis. The 5xFAD mice performed worse in water-maze testing than littermates. Stimulated groups learned the water maze better than unstimulated groups. Stimulated groups had 2–3-fold increases in frontal cortex immunoblot measures of the neurotrophin receptors for nerve growth factor and brain-derived neurotrophic factor, and a more than 50% decrease in the expression of amyloid cleavage enzyme BACE1. Stimulation also led to lower Aβ42 in 5xFAD mice. These data support a causal relationship between basal forebrain activation and both neurotrophin activation and reduced Aβ42 generation and accumulation. The observation that basal forebrain activation suppresses Aβ42 accumulation, combined with the known high-affinity antagonism of nicotinic receptors by Aβ42, documents bidirectional antagonism between acetylcholine and Aβ42

Longitudinal two-photon imaging in somatosensory cortex of behaving mice reveals dendritic spine formation enhancement by subchronic administration of low-dose ketamine

Ketamine, a well-known anesthetic, has recently attracted renewed attention as a fast-acting antidepressant. A single dose of ketamine induces rapid synaptogenesis, which may underlie its antidepressant effect. To test whether repeated exposure to ketamine triggers sustained synaptogenesis, we administered a sub-anesthetic dose of ketamine (10 mg/kg i.p.) once-daily for 5 days, and repeatedly imaged dendritic spines of the YFP-expressing pyramidal neurons in somatosensory cortex of awake female mice using in vivo two-photon microscopy. We found that the spine formation rate became significantly higher at 72-132 h after the first ketamine injection (but not at 6-24 h), while the rate of elimination of pre-existing spines remained unchanged. In contrast to the net gain of spines observed in ketamine-treated mice, the vehicle-injected control mice exhibited a net loss typical for young-adult animals undergoing synapse pruning. Ketamine-induced spinogenesis was correlated with increased PSD-95 and phosphorylated actin, consistent with formation of new synapses. Moreover, structural synaptic plasticity caused by ketamine was paralleled by a significant improvement in the nest building behavioral assay. Taken together, our data show that subchronic low-dose ketamine induces a sustained shift towards spine formation.Peer reviewe

Questioning Glutamate Excitotoxicity in Acute Brain Damage: The Importance of Spreading Depolarization

Background Within 2 min of severe ischemia, spreading depolarization (SD) propagates like a wave through compromised gray matter of the higher brain. More SDs arise over hours in adjacent tissue, expanding the neuronal damage. This period represents a therapeutic window to inhibit SD and so reduce impending tissue injury. Yet most neuroscientists assume that the course of early brain injury can be explained by glutamate excitotoxicity, the concept that immediate glutamate release promotes early and downstream brain injury. There are many problems with glutamate release being the unseen culprit, the most practical being that the concept has yielded zero therapeutics over the past 30 years. But the basic science is also flawed, arising from dubious foundational observations beginning in the 1950s Methods Literature pertaining to excitotoxicity and to SD over the past 60 years is critiqued. Results Excitotoxicity theory centers on the immediate and excessive release of glutamate with resulting neuronal hyperexcitation. This instigates poststroke cascades with subsequent secondary neuronal injury. By contrast, SD theory argues that although SD evokes some brief glutamate release, acute neuronal damage and the subsequent cascade of injury to neurons are elicited by the metabolic stress of SD, not by excessive glutamate release. The challenge we present here is to find new clinical targets based on more informed basic science. This is motivated by the continuing failure by neuroscientists and by industry to develop drugs that can reduce brain injury following ischemic stroke, traumatic brain injury, or sudden cardiac arrest. One important step is to recognize that SD plays a central role in promoting early neuronal damage. We argue that uncovering the molecular biology of SD initiation and propagation is essential because ischemic neurons are usually not acutely injured unless SD propagates through them. The role of glutamate excitotoxicity theory and how it has shaped SD research is then addressed, followed by a critique of its fading relevance to the study of brain injury. Conclusions Spreading depolarizations better account for the acute neuronal injury arising from brain ischemia than does the early and excessive release of glutamate

The Critical Role of Spreading Depolarizations in Early Brain Injury: Consensus and Contention

Background: When a patient arrives in the emergency department following a stroke, a traumatic brain injury, or sudden cardiac arrest, there is no therapeutic drug available to help protect their jeopardized neurons. One crucial reason is that we have not identified the molecular mechanisms leading to electrical failure, neuronal swelling, and blood vessel constriction in newly injured gray matter. All three result from a process termed spreading depolarization (SD). Because we only partially understand SD, we lack molecular targets and biomarkers to help neurons survive after losing their blood flow and then undergoing recurrent SD. Methods: In this review, we introduce SD as a single or recurring event, generated in gray matter following lost blood flow, which compromises the Na+/K+ pump. Electrical recovery from each SD event requires so much energy that neurons often die over minutes and hours following initial injury, independent of extracellular glutamate. Results: We discuss how SD has been investigated with various pitfalls in numerous experimental preparations, how overtaxing the Na+/K+ ATPase elicits SD. Elevated K+ or glutamate are unlikely natural activators of SD. We then turn to the properties of SD itself, focusing on its initiation and propagation as well as on computer modeling. Conclusions: Finally, we summarize points of consensus and contention among the authors as well as where SD research may be heading. In an accompanying review, we critique the role of the glutamate excitotoxicity theory, how it has shaped SD research, and its questionable importance to the study of early brain injury as compared with SD theory. © 2022, The Author(s)

The continuum of spreading depolarizations in acute cortical lesion development: Examining Leao's legacy

A modern understanding of how cerebral cortical lesions develop after acute brain injury is based on Aristides Leao's historic discoveries of spreading depression and asphyxial/anoxic depolarization. Treated as separate entities for decades, we now appreciate that these events define a continuum of spreading mass depolarizations, a concept that is central to understanding their pathologic effects. Within minutes of acute severe ischemia, the onset of persistent depolarization triggers the breakdown of ion homeostasis and development of cytotoxic edema. These persistent changes are diagnosed as diffusion restriction in magnetic resonance imaging and define the ischemic core. In delayed lesion growth, transient spreading depolarizations arise spontaneously in the ischemic penumbra and induce further persistent depolarization and excitotoxic damage, progressively expanding the ischemic core. The causal role of these waves in lesion development has been proven by real-time monitoring of electrophysiology, blood flow, and cytotoxic edema. The spreading depolarization continuum further applies to other models of acute cortical lesions, suggesting that it is a universal principle of cortical lesion development. These pathophysiologic concepts establish a working hypothesis for translation to human disease, where complex patterns of depolarizations are observed in acute brain injury and appear to mediate and signal ongoing secondary damage

Dibucaine Mitigates Spreading Depolarization in Human Neocortical Slices and Prevents Acute Dendritic Injury in the Ischemic Rodent Neocortex

Spreading depolarizations that occur in patients with malignant stroke, subarachnoid/intracranial hemorrhage, and traumatic brain injury are known to facilitate neuronal damage in metabolically compromised brain tissue. The dramatic failure of brain ion homeostasis caused by propagating spreading depolarizations results in neuronal and astroglial swelling. In essence, swelling is the initial response and a sign of the acute neuronal injury that follows if energy deprivation is maintained. Choosing spreading depolarizations as a target for therapeutic intervention, we have used human brain slices and in vivo real-time two-photon laser scanning microscopy in the mouse neocortex to study potentially useful therapeutics against spreading depolarization-induced injury.We have shown that anoxic or terminal depolarization, a spreading depolarization wave ignited in the ischemic core where neurons cannot repolarize, can be evoked in human slices from pediatric brains during simulated ischemia induced by oxygen/glucose deprivation or by exposure to ouabain. Changes in light transmittance (LT) tracked terminal depolarization in time and space. Though spreading depolarizations are notoriously difficult to block, terminal depolarization onset was delayed by dibucaine, a local amide anesthetic and sodium channel blocker. Remarkably, the occurrence of ouabain-induced terminal depolarization was delayed at a concentration of 1 µM that preserves synaptic function. Moreover, in vivo two-photon imaging in the penumbra revealed that, though spreading depolarizations did still occur, spreading depolarization-induced dendritic injury was inhibited by dibucaine administered intravenously at 2.5 mg/kg in a mouse stroke model.Dibucaine mitigated the effects of spreading depolarization at a concentration that could be well-tolerated therapeutically. Hence, dibucaine is a promising candidate to protect the brain from ischemic injury with an approach that does not rely on the complete abolishment of spreading depolarizations

Slices have more synapses than perfusion-fixed hippocampus from both young and mature rats

Hippocampal slices have long been used to investigate properties of synaptic transmission and plasticity. Here, for the first time, synapses in slices have been compared quantitatively with synapses occurring in perfusion-fixed hippocampus, which is presumed to represent the natural in vivo state. Relative to perfusion-fixed hippocampus, a remarkable 40–50% increase in spine number occurs in adult hippocampal slices, and a 90 % increase occurs in slices from postnatal day 21 rats. Serial EM shows that all of the dendritic spines have normal synapses with presynaptic and postsynaptic elements; however, not all spine types are affected uniformly. Stubby and mushroom spines increase in the adult slices, and thin, mushroom, and branched spines increase in the immature slices. More axonal boutons with multiple synapses occur in the slices, suggesting that the new synapses form on preexisting axonal boutons. The increase in spine and synapse number is evident within a couple of hours after preparing the slices. Once the initial spine induction has occurred, no further change occurs for up to 13 hr in vitro, the longest time investigated. Thus, the spine increase is occurring during a period when there is little or no synaptic activity during the first hour, and the subsequent stabilization in spine synapse numbers is occurring after synaptic activity returns in the slice. These findings suggest that spines form in response to the loss of synaptic activity when slices are removed from the rest of the brain and during the subsequent 1 hr recovery period. Key words: plasticity; dendritic spines; CA1 pyramidal cell; multiple-synapse boutons; serial electron microscopy Hippocampal slices are widely used to study cellular mechanisms of synaptic transmission and plasticity (Bliss and Collingridge

Chloride Cotransporters as a Molecular Mechanism underlying Spreading Depolarization-Induced Dendritic Beading

Spreading depolarizations (SDs) are waves of sustained neuronal and glial depolarization that propagate massive disruptions of ion gradients through the brain. SD is associated with migraine aura and recently recognized as a novel mechanism of injury in stroke and brain trauma patients. SD leads to neuronal swelling as assessed in real time with two-photon laser scanning microscopy (2PLSM). Pyramidal neurons do not express aquaporins and thus display low inherent water permeability, yet SD rapidly induces focal swelling (beading) along the dendritic shaft by unidentified molecular mechanisms. To address this issue, we induced SD in murine hippocampal slices by focal KCl microinjection and visualized the ensuing beading of dendrites expressing EGFP by 2PLSM. We confirmed that dendritic beading failed to arise during large (100 mOsm) hyposmotic challenges, underscoring that neuronal swelling does not occur as a simple osmotic event. SD-induced dendritic beading was not prevented by pharmacological interference with the cytoskeleton, supporting the notion that dendritic beading may result entirely from excessive water influx. Dendritic beading was strictly dependent on the presence of Cl(−), and, accordingly, combined blockade of Cl(−)-coupled transporters led to a significant reduction in dendritic beading without interfering with SD. Furthermore, our in vivo data showed a strong inhibition of dendritic beading during pharmacological blockage of these cotransporters. We propose that SD-induced dendritic beading takes place as a consequence of the altered driving forces and thus activity for these cotransporters, which by transport of water during their translocation mechanism may generate dendritic beading independently of osmotic forces. SIGNIFICANCE STATEMENT Spreading depolarization occurs during pathological conditions such as stroke, brain injury, and migraine and is characterized as a wave of massive ion translocation between intracellular and extracellular space in association with recurrent transient focal swelling (beading) of dendrites. Numerous ion channels have been demonstrated to be involved in generation and propagation of spreading depolarization, but the molecular machinery responsible for the dendritic beading has remained elusive. Using real-time in vitro and in vivo two-photon laser scanning microscopy, we have identified the transport mechanisms involved in the detrimental focal swelling of dendrites. These findings have clear clinical significance because they may point to a new class of pharmacological targets for prevention of neuronal swelling that consequently will serve as neuroprotective agents

Potent inhibition of anoxic depolarization by the sodium channel blocker dibucaine

Recurring waves of peri-infarct depolarizations (PIDs) propagate across gray matter in the hours and days following stroke, expanding the primary site of injury. Ischemic depolarization (termed anoxic depolarization or AD in live brain slices) is PID-like but immediately arises in the more metabolically compromised ischemic core. This causes dramatic neuronal and astrocyte swelling and dendritic beading with spine loss within minutes, resulting in acute cell death. AD is evoked in rodent neocortical slices by suppressing the Na+/K+-ATPase pump with either oxygen/glucose deprivation (OGD) or exposure to ouabain. The process driving AD and PIDs remains poorly understood. Here we show that dibucaine is a potent drug inhibiting AD because of its high binding affinity to the Na+ channel. Field recording reveals that, when superfused with ouabain (5 min), neocortical slices pretreated with 1 μM dibucaine for 45 min display either no AD or delayed AD onset compared with untreated controls. If ouabain exposure is extended to 10 min, 1 μM dibucaine is still able to delay AD onset by ∼60%. Likewise, it delays OGD-evoked AD onset by ∼54% but does not depress action potentials (APs) or evoked orthodromic field potentials. Increasing dibucaine to 10 μM inhibits AP firing, gradually putting the slice into a stasis that inhibits AD onset but also renders the slice functionally quiescent. Two-photon microscopy reveals that 10 μM dibucaine pretreatment prevents or helps reverse ouabain-induced structural neuronal damage. Although the therapeutic range of dibucaine is quite narrow, dibucaine-like drugs could prove therapeutically useful in inhibiting PIDs and their resultant neuronal damage