Electrical signaling in cochlear efferents is driven by an intrinsic neuronal oscillator.

Efferent neurons are believed to play essential roles in maintaining auditory function. The lateral olivocochlear (LOC) neurons-which project from the brainstem to the inner ear, where they release multiple transmitters including peptides, catecholamines, and acetylcholine-are the most numerous yet least understood elements of efferent control of the cochlea. Using in vitro calcium imaging and patch-clamp recordings, we found that LOC neurons in juvenile and young adult mice exhibited extremely slow waves of activity (∼0.1 Hz). These seconds-long bursts of Na+ spikes were driven by an intrinsic oscillator dependent on L-type Ca2+ channels and were not observed in prehearing mice, suggesting an age-dependent mechanism underlying the intrinsic oscillator. Using optogenetic approaches, we identified both ascending (T-stellate cells of the cochlear nucleus) and descending (auditory cortex) sources of synaptic excitation, as well as the synaptic receptors used for such excitation. Additionally, we identified potent inhibition originating in the glycinergic medial nucleus of trapezoid body (MNTB). Conductance-clamp experiments revealed an unusual mechanism of electrical signaling in LOC neurons, in which synaptic excitation and inhibition served to switch on and off the intrinsically generated spike burst mechanism, allowing for prolonged periods of activity or silence controlled by brief synaptic events. Protracted bursts of action potentials may be essential for effective exocytosis of the diverse transmitters released by LOC fibers in the cochlea.

Renal injury in cardiorenal syndrome type 1 is mediated by albumin.

Cardiorenal syndrome type 1 (CRS-1) acute kidney injury (AKI) is a critical complication of acute cardiovascular disease but is poorly understood. AKI induces acute albuminuria. As chronic albuminuria is associated with worsening kidney disease and albumin has been implicated in tubular epithelial injury, we investigated whether albumin participates in CRS-1, and whether CRS-1 alters renal albumin handling. We report the role of albumin in in vivo and in vitro CRS-1 models. An established translational model, cardiac arrest and cardiopulmonary resuscitation (CA/CPR) induced severe acute albuminuria which correlated with tubular epithelial cell death. In vivo microscopy demonstrated CA/CPR-induced glomerular filtration of exogenous albumin, while administration of exogenous albumin after CA/CPR worsened AKI compared to iso-oncotic control. Increased albumin signal was observed in the proximal tubules of CA/CPR mice compared to sham. Comparison of albumin flux from tubular lumen to epithelial cells revealed saturated albumin transport within minutes of albumin injection after CA/CPR. In vitro, HK2 cells (human kidney tubular epithelial cells), exposed to oxygen-glucose deprivation were injured by albumin in a dose dependent fashion. This interference was unchanged by the tubular endocytic receptor megalin. In conclusion, CRS-1 alters albumin filtration and tubular uptake, leading to increased tubular exposure to albumin, which is injurious to tubular epithelial cells, worsening AKI. Our findings shed light on the pathophysiology of renal albumin and may guide interventions such as albumin resuscitation to improve CRS-1 outcomes. This investigation may have important translational relevance for patients that receive exogenous albumin as part of their CRS-1 treatment regimen.

Role of endothelium-pericyte signaling in capillary blood flow response to neuronal activity.

Local blood flow in the brain is tightly coupled to metabolic demands, a phenomenon termed functional hyperemia. Both capillaries and arterioles contribute to the hyperemic response to neuronal activity via different mechanisms and timescales. The nature and specific signaling involved in the hyperemic response of capillaries versus arterioles, and their temporal relationship are not fully defined. We determined the time-dependent changes in capillary flux and diameter versus arteriolar velocity and flow following whisker stimulation using optical microangiography (OMAG) and two-photon microscopy. We further characterized depth-resolved responses of individual capillaries versus capillary networks. We hypothesized that capillaries respond first to neuronal activation, and that they exhibit a coordinated response mediated via endothelial-derived epoxyeicosatrienoates (EETs) acting on pericytes. To visualize peri-capillary pericytes, we used Tie2-GFP/NG2-DsRed mice, and to determine the role of endothelial-derived EETs, we compared cerebrovascular responses to whisker stimulation between wild-type mice and mice with lower endothelial EETs (Tie2-hsEH). We found that capillaries respond immediately to neuronal activation in an orchestrated network-level manner, a response attenuated in Tie2-hsEH and inhibited by blocking EETs action on pericytes. These results demonstrate that capillaries are first responders during functional hyperemia, and that they exhibit a network-level response mediated via endothelial-derived EETs' action on peri-capillary pericytes.

When the air hits your brain: decreased arterial pulsatility after craniectomy leading to impaired glymphatic flow.

OBJECTIVECranial neurosurgical procedures can cause changes in brain function. There are many potential explanations, but the effect of simply opening the skull has not been addressed, except for research into syndrome of the trephined. The glymphatic circulation, by which CSF and interstitial fluid circulate through periarterial spaces, brain parenchyma, and perivenous spaces, depends on arterial pulsations to provide the driving force for bulk flow; opening the cranial cavity could dampen this force. The authors hypothesized that a craniectomy, without any other pathological insult, is sufficient to alter brain function due to reduced arterial pulsatility and decreased glymphatic flow. Furthermore, they postulated that glymphatic impairment would produce activation of astrocytes and microglia; with the reestablishment of a closed cranial compartment, the glymphatic impairment, astrocytic/microglial activation, and neurobehavioral decline caused by opening the cranial compartment might be reversed.METHODSUsing two-photon in vivo microscopy, the pulsatility index of cortical vessels was quantified through a thinned murine skull and then again after craniectomy. Glymphatic influx was determined with ex vivo fluorescence microscopy of mice 0, 14, 28, and 56 days following craniectomy or cranioplasty; brain sections were immunohistochemically labeled for GFAP and CD68. Motor and cognitive performance was quantified with rotarod and novel object recognition tests at baseline and 14, 21, and 28 days following craniectomy or cranioplasty.RESULTSPenetrating arterial pulsatility decreased significantly and bilaterally following unilateral craniectomy, producing immediate and chronic impairment of glymphatic CSF influx in the ipsilateral and contralateral brain parenchyma. Craniectomy-related glymphatic dysfunction was associated with an astrocytic and microglial inflammatory response, as well as with the development of motor and cognitive deficits. Recovery of glymphatic flow preceded reduced gliosis and return of normal neurological function, and cranioplasty accelerated this recovery.CONCLUSIONSCraniectomy causes glymphatic dysfunction, gliosis, and changes in neurological function in this murine model of syndrome of the trephined.

Cholinergic Interneurons Underlie Spontaneous Dopamine Release in Nucleus Accumbens.

The release of dopamine from terminals in the NAc is regulated by a number of factors, including voltage-gated ion channels, D2-autoreceptors, and nAChRs. Cholinergic interneurons (CINs) drive dopamine release through activation of nAChRs on dopamine terminals. Using cyclic voltammetry in mouse brain slices, nAChR-dependent spontaneous dopamine transients and the mechanisms underlying the origin were examined in the NAc. Spontaneous events were infrequent (0.3 per minute), but the rate and amplitude were increased after blocking Kv channels with 4-aminopyridine. Although the firing frequency of CINs was increased by blocking glutamate reuptake with TBOA and the Sk blocker apamin, only 4-aminopyridine increased the frequency of dopamine transients. In contrast, inhibition of CIN firing with the µ/δ selective opioid [Met5]enkephalin (1 µm) decreased spontaneous dopamine transients. Cocaine increased the rate and amplitude of dopamine transients, suggesting that the activity of the dopamine transporter limits the detection of these events. In the presence of cocaine, the rate of spontaneous dopamine transients was further increased after blocking D2-autoreceptors. Blockade of muscarinic receptors had no effect on evoked dopamine release, suggesting that feedback inhibition of acetylcholine release was not involved. Thus, although spontaneous dopamine transients are reliant on nAChRs, the frequency was not strictly governed by the activity of CINs. The increase in frequency of spontaneous dopamine transients induced by cocaine was not due to an increase in cholinergic tone and is likely a product of an increase in detection resulting from decreased dopamine reuptake.SIGNIFICANCE STATEMENT The actions of dopamine in the NAc are thought to be responsible for endogenous reward and the reinforcing properties of drugs of abuse, such as psychostimulants. The present work examines the mechanisms underlying nAChR-induced spontaneous dopamine release. This study demonstrates that spontaneous dopamine release is (1) dependent of the activation of nicotinic receptors, (2) independent on the spontaneous activity of cholinergic interneurons, and (3) that cocaine increased the detection of dopamine transients by prolonging the presence and increasing the diffusion of dopamine in the extracellular space. The release of acetylcholine is therefore responsible for spontaneous dopamine transients, and cocaine augments dopamine tone without altering activity of cholinergic interneurons.

Association of Perivascular Localization of Aquaporin-4 With Cognition and Alzheimer Disease in Aging Brains.

IMPORTANCE: Cognitive impairment and dementia, including Alzheimer disease (AD), are common within the aging population, yet the factors that render the aging brain vulnerable to these processes are unknown. Perivascular localization of aquaporin-4 (AQP4) facilitates the clearance of interstitial solutes, including amyloid-ß, through the brainwide network of perivascular pathways termed the glymphatic system, which may be compromised in the aging brain. OBJECTIVES: To determine whether alterations in AQP4 expression or loss of perivascular AQP4 localization are features of the aging human brain and to define their association with AD pathology. DESIGN, SETTING, AND PARTICIPANTS: Expression of AQP4 was analyzed in postmortem frontal cortex of cognitively healthy and histopathologically confirmed individuals with AD by Western blot or immunofluorescence for AQP4, amyloid-ß 1-42, and glial fibrillary acidic protein. Postmortem tissue and clinical data were provided by the Oregon Health and Science University Layton Aging and Alzheimer Disease Center and Oregon Brain Bank. Postmortem tissue from 79 individuals was evaluated, including cognitively intact "young" individuals aged younger than 60 years (range, 33-57 years), cognitively intact "aged" individuals aged older than 60 years (range, 61-96 years) with no known neurological disease, and individuals older than 60 years (range, 61-105 years) of age with a clinical history of AD confirmed by histopathological evaluation. Forty-eight patient samples (10 young, 20 aged, and 18 with AD) underwent histological analysis. Sixty patient samples underwent Western blot analysis (15 young, 24 aged, and 21 with AD). MAIN OUTCOMES AND MEASURES: Expression of AQP4 protein, AQP4 immunoreactivity, and perivascular AQP4 localization in the frontal cortex were evaluated. RESULTS: Expression of AQP4 was associated with advancing age among all individuals (R2 = 0.17; P = .003). Perivascular AQP4 localization was significantly associated with AD status independent of age (OR, 11.7 per 10% increase in localization; z = -2.89; P = .004) and was preserved among eldest individuals older than 85 years of age who remained cognitively intact. When controlling for age, loss of perivascular AQP4 localization was associated with increased amyloid-ß burden (R2 = 0.15; P = .003) and increasing Braak stage (R2 = 0.14; P = .006). CONCLUSIONS AND RELEVANCE: In this study, altered AQP4 expression was associated with aging brains. Loss of perivascular AQP4 localization may be a factor that renders the aging brain vulnerable to the misaggregation of proteins, such as amyloid-ß, in neurodegenerative conditions such as AD.

Impairment of glymphatic pathway function promotes tau pathology after traumatic brain injury.

Traumatic brain injury (TBI) is an established risk factor for the early development of dementia, including Alzheimer's disease, and the post-traumatic brain frequently exhibits neurofibrillary tangles comprised of aggregates of the protein tau. We have recently defined a brain-wide network of paravascular channels, termed the "glymphatic" pathway, along which CSF moves into and through the brain parenchyma, facilitating the clearance of interstitial solutes, including amyloid-ß, from the brain. Here we demonstrate in mice that extracellular tau is cleared from the brain along these paravascular pathways. After TBI, glymphatic pathway function was reduced by ∼60%, with this impairment persisting for at least 1 month post injury. Genetic knock-out of the gene encoding the astroglial water channel aquaporin-4, which is importantly involved in paravascular interstitial solute clearance, exacerbated glymphatic pathway dysfunction after TBI and promoted the development of neurofibrillary pathology and neurodegeneration in the post-traumatic brain. These findings suggest that chronic impairment of glymphatic pathway function after TBI may be a key factor that renders the post-traumatic brain vulnerable to tau aggregation and the onset of neurodegeneration.

Impairment of paravascular clearance pathways in the aging brain.

OBJECTIVE: In the brain, protein waste removal is partly performed by paravascular pathways that facilitate convective exchange of water and soluble contents between cerebrospinal fluid (CSF) and interstitial fluid (ISF). Several lines of evidence suggest that bulk flow drainage via the glymphatic system is driven by cerebrovascular pulsation, and is dependent on astroglial water channels that line paravascular CSF pathways. The objective of this study was to evaluate whether the efficiency of CSF-ISF exchange and interstitial solute clearance is impaired in the aging brain. METHODS: CSF-ISF exchange was evaluated by in vivo and ex vivo fluorescence microscopy and interstitial solute clearance was evaluated by radiotracer clearance assays in young (2-3 months), middle-aged (10-12 months), and old (18-20 months) wild-type mice. The relationship between age-related changes in the expression of the astrocytic water channel aquaporin-4 (AQP4) and changes in glymphatic pathway function was evaluated by immunofluorescence. RESULTS: Advancing age was associated with a dramatic decline in the efficiency of exchange between the subarachnoid CSF and the brain parenchyma. Relative to the young, clearance of intraparenchymally injected amyloid-ß was impaired by 40% in the old mice. A 27% reduction in the vessel wall pulsatility of intracortical arterioles and widespread loss of perivascular AQP4 polarization along the penetrating arteries accompanied the decline in CSF-ISF exchange. INTERPRETATION: We propose that impaired glymphatic clearance contributes to cognitive decline among the elderly and may represent a novel therapeutic target for the treatment of neurodegenerative diseases associated with accumulation of misfolded protein aggregates.

Cerebral arterial pulsation drives paravascular CSF-interstitial fluid exchange in the murine brain.

CSF from the subarachnoid space moves rapidly into the brain along paravascular routes surrounding penetrating cerebral arteries, exchanging with brain interstitial fluid (ISF) and facilitating the clearance of interstitial solutes, such as amyloid ß, in a pathway that we have termed the "glymphatic" system. Prior reports have suggested that paravascular bulk flow of CSF or ISF may be driven by arterial pulsation. However, cerebral arterial pulsation could not be directly assessed. In the present study, we use in vivo two-photon microscopy in mice to visualize vascular wall pulsatility in penetrating intracortical arteries. We observed that unilateral ligation of the internal carotid artery significantly reduced arterial pulsatility by ~50%, while systemic administration of the adrenergic agonist dobutamine increased pulsatility of penetrating arteries by ~60%. When paravascular CSF-ISF exchange was evaluated in real time using in vivo two-photon and ex vivo fluorescence imaging, we observed that internal carotid artery ligation slowed the rate of paravascular CSF-ISF exchange, while dobutamine increased the rate of paravascular CSF-ISF exchange. These findings demonstrate that cerebral arterial pulsatility is a key driver of paravascular CSF influx into and through the brain parenchyma, and suggest that changes in arterial pulsatility may contribute to accumulation and deposition of toxic solutes, including amyloid ß, in the aging brain.

α1-Adrenergic receptors mediate coordinated Ca2+ signaling of cortical astrocytes in awake, behaving mice.

Astrocyte Ca2+ signals in awake behaving mice are widespread, coordinated and differ fundamentally from the locally restricted Ca2+ transients observed ex vivo and in anesthetized animals. Here we show that the synchronized release of norepinephrine (NE) from locus coeruleus (LC) projections throughout the cerebral cortex mediate long-ranging Ca2+ signals by activation of astrocytic α1-adrenergic receptors. When LC output was triggered by either physiological sensory (whisker) stimulation or an air-puff startle response, astrocytes responded with fast Ca2+ transients that encompassed the entire imaged field (positioned over either frontal or parietal cortex). The application of adrenergic inhibitors, including α1-adrenergic antagonist prazosin, potently suppressed both evoked, as well as the frequently observed spontaneous astroglial Ca2+ signals. The LC-specific neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), which reduced cortical NE content by >90%, prevented nearly all astrocytic Ca2+ signals in awake mice. The observations indicate that in adult, unanesthetized mice, astrocytes do not respond directly to glutamatergic signaling evoked by sensory stimulation. Instead astrocytes appear to be the primary target for NE, with astrocytic Ca2+ signaling being triggered by the α1-adrenergic receptor. In turn, astrocytes may coordinate the broad effects of neuromodulators on neuronal activity.

General anesthesia selectively disrupts astrocyte calcium signaling in the awake mouse cortex.

Calcium signaling represents the principle pathway by which astrocytes respond to neuronal activity. General anesthetics are routinely used in clinical practice to induce a sleep-like state, allowing otherwise painful procedures to be performed. Anesthetic drugs are thought to mainly target neurons in the brain and act by suppressing synaptic activity. However, the direct effect of general anesthesia on astrocyte signaling in awake animals has not previously been addressed. This is a critical issue, because calcium signaling may represent an essential mechanism through which astrocytes can modulate synaptic activity. In our study, we performed calcium imaging in awake head-restrained mice and found that three commonly used anesthetic combinations (ketamine/xylazine, isoflurane, and urethane) markedly suppressed calcium transients in neocortical astrocytes. Additionally, all three anesthetics masked potentially important features of the astrocyte calcium signals, such as synchronized widespread transients that appeared to be associated with arousal in awake animals. Notably, anesthesia affected calcium transients in both processes and soma and depressed spontaneous signals, as well as calcium responses, evoked by whisker stimulation or agonist application. We show that these calcium transients are inositol 1,4,5-triphosphate type 2 receptor (IP(3)R2)-dependent but resistant to a local blockade of glutamatergic or purinergic signaling. Finally, we found that doses of anesthesia insufficient to affect neuronal responses to whisker stimulation selectively suppressed astrocyte calcium signals. Taken together, these data suggest that general anesthesia may suppress astrocyte calcium signals independently of neuronal activity. We propose that these glial effects may constitute a nonneuronal mechanism for sedative action of anesthetic drugs.

Norepinephrine: a neuromodulator that boosts the function of multiple cell types to optimize CNS performance.

Norepinephrine (NE) is a neuromodulator that in multiple ways regulates the activity of neuronal and non-neuronal cells. NE participates in the rapid modulation of cortical circuits and cellular energy metabolism, and on a slower time scale in neuroplasticity and inflammation. Of the multiple sources of NE in the brain, the locus coeruleus (LC) plays a major role in noradrenergic signaling. Processes from the LC primarily release NE over widespread brain regions via non-junctional varicosities. We here review the actions of NE in astrocytes, microglial cells, and neurons based on the idea that the overarching effect of signaling from the LC is to maximize brain power, which is accomplished via an orchestrated cellular response involving most, if not all cell types in CNS.