Isoflurane Disrupts Postsynaptic Density-95 Protein Interactions Causing Neuronal Synapse Loss and Cognitive Impairment in Juvenile Mice via Canonical NO-mediated Protein Kinase-G Signaling.

BACKGROUND: Inhalational anesthetics are known to disrupt PDZ2 domain-mediated protein-protein interactions of the postsynaptic density (PSD)-95 protein. The aim of this study is to investigate the underlying mechanisms in response to early isoflurane exposure on synaptic PSD-95 PDZ2 domain disruption that altered spine densities and cognitive function. The authors hypothesized that activation of protein kinase-G by the components of nitric oxide (NO) signaling pathway constitutes a mechanism that prevents loss of early dendritic spines and synapse in neurons and cognitive impairment in mice in response to disruption of PDZ2 domain of the PSD-95 protein. METHODS: Postnatal day 7 mice were exposed to 1.5% isoflurane for 4 h or injected with 8 mg/kg active PSD-95 wild-type PDZ2 peptide or soluble guanylyl cyclase activator YC-1 along with their respective controls. Primary neurons at 7 days in vitro were exposed to isoflurane or PSD-95 wild-type PDZ2 peptide for 4 h. Coimmunoprecipitation, spine density, synapses, cyclic guanosine monophosphate-dependent protein kinase activity, and novel object recognition memory were assessed. RESULTS: Exposure of isoflurane or PSD-95 wild-type PDZ2 peptide relative to controls causes the following. First, there is a decrease in PSD-95 coimmunoprecipitate relative to N-methyl-d-aspartate receptor subunits NR2A and NR2B precipitate (mean ± SD [in percentage of control]: isoflurane, 54.73 ± 16.52, P = 0.001; and PSD-95 wild-type PDZ2 peptide, 51.32 ± 12.93, P = 0.001). Second, there is a loss in spine density (mean ± SD [spine density per 10 µm]: control, 5.28 ± 0.56 vs. isoflurane, 2.23 ± 0.67, P < 0.0001; and PSD-95 mutant PDZ2 peptide, 4.74 ± 0.94 vs. PSD-95 wild-type PDZ2 peptide, 1.47 ± 0.87, P < 0.001) and a decrease in synaptic puncta (mean ± SD [in percentage of control]: isoflurane, 41.1 ± 14.38, P = 0.001; and PSD-95 wild-type PDZ2 peptide, 50.49 ± 14.31, P < 0.001). NO donor or cyclic guanosine monophosphate analog prevents the spines and synapse loss and decline in the cyclic guanosine monophosphate-dependent protein kinase activity, but this prevention was blocked by soluble guanylyl cyclase or protein kinase-G inhibitors in primary neurons. Third, there were deficits in object recognition at 5 weeks (mean ± SD [recognition index]: male, control, 64.08 ± 10.57 vs. isoflurane, 48.49 ± 13.41, P = 0.001, n = 60; and female, control, 67.13 ± 11.17 vs. isoflurane, 53.76 ± 6.64, P = 0.003, n = 58). Isoflurane-induced impairment in recognition memory was preventable by the introduction of YC-1. CONCLUSIONS: Activation of soluble guanylyl cyclase or protein kinase-G prevents isoflurane or PSD-95 wild-type PDZ2 peptide-induced loss of dendritic spines and synapse. Prevention of recognition memory with YC-1, a NO-independent activator of guanylyl cyclase, supports a role for the soluble guanylyl cyclase mediated protein kinase-G signaling in countering the effects of isoflurane-induced cognitive impairment.

Neonatal Isoflurane Anesthesia or Disruption of Postsynaptic Density-95 Protein Interactions Change Dendritic Spine Densities and Cognitive Function in Juvenile Mice.

BACKGROUND: Experimental evidence shows postnatal exposure to anesthesia negatively affects brain development. The PDZ2 domain, mediating protein-protein interactions of the postsynaptic density-95 protein, serves as a molecular target for several inhaled anesthetics. The authors hypothesized that early postnatal disruption of postsynaptic density-95 PDZ2 domain interactions has persistent effects on dendritic spines and cognitive function. METHODS: One-week-old mice were exposed to 1.5% isoflurane for 4 h or injected with 8 mg/kg active postsynaptic density-95 wild-type PDZ2 peptide along with their respective controls. A subset of these mice also received 4 mg/kg of the nitric oxide donor molsidomine. Hippocampal spine density, long-term potentiation, novel object recognition memory, and fear learning and memory were evaluated in mice. RESULTS: Exposure of 7-day-old mice to isoflurane or postsynaptic density-95 wild-type PDZ2 peptide relative to controls causes: (1) a long-term decrease in mushroom spines at 7 weeks (mean ± SD [spines per micrometer]): control (0.8 ± 0.2) versus isoflurane (0.4 ± 0.2), P < 0.0001, and PDZ2MUT (0.7 ± 0.2) versus PDZ2WT (0.4 ± 0.2), P < 0.001; (2) deficits in object recognition at 6 weeks (mean ± SD [recognition index]): naïve (70 ± 8) versus isoflurane (55 ± 14), P = 0.010, and control (65 ± 13) versus isoflurane (55 ± 14), P = 0.045, and PDZ2MUT (64 ±11) versus PDZ2WT (53 ± 18), P = 0.045; and (3) deficits in fear learning at 7 weeks and memory at 8 weeks (mean ± SD [% freezing duration]): Learning, control (69 ± 12) versus isoflurane (52 ± 13), P < 0.0001, and PDZ2MUT (65 ± 14) versus PDZ2WT (55 ± 14) P = 0.011, and Memory, control (80 ± 17) versus isoflurane (56 ± 23), P < 0.0001 and PDZ2MUT (73 ± 18) versus PDZ2WT (44 ± 19) P < 0.0001. Impairment in long-term potentiation has fully recovered here at 7 weeks (mean ± SD [% baseline]): control (140 ± 3) versus isoflurane (137 ± 8), P = 0.560, and PDZ2MUT (136 ± 17) versus PDZ2WT (128 ± 11), P = 0.512. The isoflurane induced decrease in mushroom spines was preventable by introduction of a nitric oxide donor. CONCLUSIONS: Early disruption of PDZ2 domain-mediated protein-protein interactions mimics isoflurane in decreasing mushroom spine density and causing learning and memory deficits in mice. Prevention of the decrease in mushroom spine density with a nitric oxide donor supports a role for neuronal nitric oxide synthase pathway in mediating this cellular change associated with cognitive impairment.

Nitric Oxide Donor Prevents Neonatal Isoflurane-induced Impairments in Synaptic Plasticity and Memory.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: Some general anesthetics have been shown to have adverse effects on neuronal development that affect neural function and cognitive behavior.Clinically relevant concentrations of inhalational anesthetics inhibit the postsynaptic density (PSD)-95, discs large homolog, and zona occludens-1 (PDZ) domain-mediated protein-protein interaction between PSD-95 or PSD-93 and N-methyl-D-aspartate receptors or neuronal NO synthase. WHAT THIS ARTICLE TELLS US THAT IS NEW: Neonatal PSD-95 PDZ2WT peptide treatment mimics the effects of isoflurane (~1 minimum alveolar concentration) by altering dendritic spine morphology, neural plasticity, and memory without inducing detectable increases in apoptosis or changes in synaptic density.These results indicate that a single dose of isoflurane (~1 minimum alveolar concentration) or PSD-95 PDZ2WT peptide alters dendritic spine architecture and functions important for cognition in the developing brain. This impairment can be prevented by administration of the NO donor molsidomine. BACKGROUND: In humans, multiple early exposures to procedures requiring anesthesia constitute a significant risk factor for development of learning disabilities and disorders of attention. In animal studies, newborns exposed to anesthetics develop long-term deficits in cognition. Previously, our laboratory showed that postsynaptic density (PSD)-95, discs large homolog, and zona occludens-1 (PDZ) domains may serve as a molecular target for inhaled anesthetics. This study investigated a role for PDZ interactions in spine development, plasticity, and memory as a potential mechanism for early anesthetic exposure-produced cognitive impairment. METHODS: Postnatal day 7 mice were exposed to 1.5% isoflurane for 4 h or injected with 8 mg/kg active PSD-95 PDZ2WT peptide. Apoptosis, hippocampal dendritic spine changes, synapse density, long-term potentiation, and cognition functions were evaluated (n = 4 to 18). RESULTS: Exposure of postnatal day 7 mice to isoflurane or PSD-95 PDZ2WT peptide causes a reduction in long thin spines (median, interquartile range [IQR]: wild type control [0.54, 0.52 to 0.86] vs. wild type isoflurane [0.31, 0.16 to 0.38], P = 0.034 and PDZ2MUT [0.86, 0.67 to 1.0] vs. PDZ2WT [0.55, 0.53 to 0.59], P = 0.028), impairment in long-term potentiation (median, IQR: wild type control [123, 119 to 147] and wild type isoflurane [101, 96 to 118], P = 0.049 and PDZ2MUT [125, 119 to 131] and PDZ2WT [104, 97 to 107], P = 0.029), and deficits in acute object recognition (median, IQR: wild type control [79, 72 to 88] vs. wild type isoflurane [63, 55 to 72], P = 0.044 and PDZ2MUT [81, 69 to 84] vs. PDZ2WT [67, 57 to 77], P = 0.039) at postnatal day 21 without inducing detectable differences in apoptosis or changes in synaptic density. Impairments in recognition memory and long-term potentiation were preventable by introduction of a NO donor. CONCLUSIONS: Early disruption of PDZ domain-mediated protein-protein interactions alters spine morphology, synaptic function, and memory. These results support a role for PDZ interactions in early anesthetic exposure-produced cognitive impairment. Prevention of recognition memory and long-term potentiation deficits with a NO donor supports a role for the N-methyl-D-aspartate receptor/PSD-95/neuronal NO synthase pathway in mediating these aspects of isoflurane-induced cognitive impairment.

Molecular Mechanisms of Anesthetic Neurotoxicity: A Review of the Current Literature.

Data from epidemiologic studies and animal models have raised a concern that exposure to anesthetic agents during early postnatal life may cause lasting impairments in cognitive function. It is hypothesized that this is due to disruptions in brain development, but the mechanism underlying this toxic effect remains unknown. Ongoing research, particularly in rodents, has begun to address this question. In this review we examine currently postulated molecular mechanisms of anesthetic toxicity in the developing brain, including effects on cell death pathways, growth factor signaling systems, NMDA and GABA receptors, mitochondria, and epigenetic factors. The level of evidence for each putative mechanism is critically evaluated, and we attempt to draw connections between them where it is possible to do so. Although there are many promising avenues of research, at this time no consensus can be reached as to a definitive mechanism of injury.

Neuroinflammation in primary blast neurotrauma: Time course and prevention by torso shielding.

Mechanisms of primary blast injury caused by overpressure are not fully understood. In particular, the presence and time course of neuroinflammation are unknown and so are the signatures of reactive inflammatory cells, especially the neuroprotective versus injurious roles of microglia. In general, chronic microglial activation in the injured brain suggests a pro-degenerative role for these reactive cells. In this study, we investigated the temporal dynamics of microglial activation in the brain of mice exposed to mild-moderate blast in a shock tube. Because, in our previous work, we had found that torso shielding with rigid Plexiglas attenuates traumatic axonal injury in the brain, we also evaluated neuroinflammatory microglial responses in animals with torso protection at 7 days post blast injury. Because of the prominent involvement of the visual system in blast TBI in rodents, activated microglial cells were counted in the optic tract at various time points post-injury with stereological methods. Cell counts (activated microglial cell densities) from subjects exposed to blast TBI were compared with counts from corresponding sham animals. We found that mild-moderate blast injury causes focal activation of microglia in certain white matter tracts, including the visual pathway. In the optic tract, the density of activated microglial profiles gradually intensified from 3 to 15 days post-injury and then became attenuated at 30 days. Torso protection significantly reduced microglial activation at 7 days. These findings shed light into mechanisms of primary blast neurotrauma and may suggest novel diagnostic and monitoring methods for patients. They leave open the question of whether microglial activation post blast is protective or detrimental, although response is time limited. Finally, our findings confirm the protective role of torso shielding and stress the importance of improved or optimized body gear for warfighters or other individuals at risk for blast exposure.

Manganese-Enhanced Magnetic Resonance Imaging as a Diagnostic and Dispositional Tool after Mild-Moderate Blast Traumatic Brain Injury.

Traumatic brain injury (TBI) caused by explosive munitions, known as blast TBI, is the signature injury in recent military conflicts in Iraq and Afghanistan. Diagnostic evaluation of TBI, including blast TBI, is based on clinical history, symptoms, and neuropsychological testing, all of which can result in misdiagnosis or underdiagnosis of this condition, particularly in the case of TBI of mild-to-moderate severity. Prognosis is currently determined by TBI severity, recurrence, and type of pathology, and also may be influenced by promptness of clinical intervention when more effective treatments become available. An important task is prevention of repetitive TBI, particularly when the patient is still symptomatic. For these reasons, the establishment of quantitative biological markers can serve to improve diagnosis and preventative or therapeutic management. In this study, we used a shock-tube model of blast TBI to determine whether manganese-enhanced magnetic resonance imaging (MEMRI) can serve as a tool to accurately and quantitatively diagnose mild-to-moderate blast TBI. Mice were subjected to a 30 psig blast and administered a single dose of MnCl2 intraperitoneally. Longitudinal T1-magnetic resonance imaging (MRI) performed at 6, 24, 48, and 72 h and at 14 and 28 days revealed a marked signal enhancement in the brain of mice exposed to blast, compared with sham controls, at nearly all time-points. Interestingly, when mice were protected with a polycarbonate body shield during blast exposure, the marked increase in contrast was prevented. We conclude that manganese uptake can serve as a quantitative biomarker for TBI and that MEMRI is a minimally-invasive quantitative approach that can aid in the accurate diagnosis and management of blast TBI. In addition, the prevention of the increased uptake of manganese by body protection strongly suggests that the exposure of an individual to blast risk could benefit from the design of improved body armor.

Using the olfactory system as an in vivo model to study traumatic brain injury and repair.

Loss of olfactory function is an early indicator of traumatic brain injury (TBI). The regenerative capacity and well-defined neural maps of the mammalian olfactory system enable investigations into the degeneration and recovery of neural circuits after injury. Here, we introduce a unique olfactory-based model of TBI that reproduces many hallmarks associated with human brain trauma. We performed a unilateral penetrating impact to the mouse olfactory bulb and observed a significant loss of olfactory sensory neurons (OSNs) in the olfactory epithelium (OE) ipsilateral to the injury, but not contralateral. By comparison, we detected the injury markers p75(NTR), ß-APP, and activated caspase-3 in both the ipsi- and contralateral OE. In the olfactory bulb (OB), we observed a graded cell loss, with ipsilateral showing a greater reduction than contralateral and both significantly less than sham. Similar to OE, injury markers in the OB were primarily detected on the ipsilateral side, but also observed contralaterally. Behavioral experiments measured 4 days after impact also demonstrated loss of olfactory function, yet following a 30-day recovery period, we observed a significant improvement in olfactory function and partial recovery of olfactory circuitry, despite the persistence of TBI markers. Interestingly, by using the M71-IRES-tauLacZ reporter line to track OSN organization, we further determined that inducing neural activity during the recovery period with intense odor conditioning did not enhance the recovery process. Together, these data establish the mouse olfactory system as a new model to study TBI, serving as a platform to understand neural disruption and the potential for circuit restoration.

Mouse urinary biomarkers provide signatures of maturation, diet, stress level, and diurnal rhythm.

Body fluids such as urine potentially contain a wealth of information pertaining to age, sex, social and reproductive status, physiologic state, and genotype of the donor. To explore whether urine could encode information regarding environment, physiology, and development, we compared the volatile compositions of mouse urine using solid-phase microextraction and gas chromatography-mass spectrometry (SPME-GC/MS). Specifically, we identified volatile organic compounds (VOCs) in individual urine samples taken from inbred C57BL/6J-H-2(b) mice under several experimental conditions-maturation state, diet, stress, and diurnal rhythms, designed to mimic natural variations. Approximately 1000 peaks (i.e., variables) were identified per comparison and of these many were identified as potential differential biomarkers. Consistent with previous findings, we found groups of compounds that vary significantly and consistently rather than a single unique compound to provide a robust signature. We identified over 49 new predictive compounds, in addition to identifying several published compounds, for maturation state, diet, stress, and time-of-day. We found a considerable degree of overlap in the chemicals identified as (potential) biomarkers for each comparison. Chemometric methods indicate that the strong group-related patterns in VOCs provide sufficient information to identify several parameters of natural variations in this strain of mice including their maturation state, stress level, and diet.

Variable selection using iterative reformulation of training set models for discrimination of samples: application to gas chromatography/mass spectrometry of mouse urinary metabolites.

The paper discusses variable selection as used in large metabolomic studies, exemplified by mouse urinary gas chromatography of 441 mice in three experiments to detect the influence of age, diet, and stress on their chemosignal. Partial least squares discriminant analysis (PLS-DA) was applied to obtain class models, using a procedure of 20,000 iterations including the bootstrap for model optimization and random splits into test and training sets for validation. Variables are selected using PLS regression coefficients on the training set using an optimized number of components obtained from the bootstrap. The variables are ranked in order of significance, and the overall optimal variables are selected as those that appear as highly significant over 100 different test and training set splits. Cost/benefit analysis of performing the model on a reduced number of variables is also illustrated. This paper provides a strategy for properly validated methods for determining which variables are most significant for discriminating between two groups in large metabolomic data sets avoiding the common pitfall of overfitting if variables are selected on a combined training and test set and also taking into account that different variables may be selected each time the samples are split into training and test sets using iterative procedures.

Emerging views on the distinct but related roles of the main and accessory olfactory systems in responsiveness to chemosensory signals in mice.

In rodents, the nasal cavity contains two separate chemosensory epithelia, the main olfactory epithelium, located in the posterior dorsal aspect of the nasal cavity, and the vomeronasal/accessory olfactory epithelium, located in a capsule in the anterior aspect of the ventral floor of the nasal cavity. Both the main and accessory olfactory systems play a role in detection of biologically relevant odors. The accessory olfactory system has been implicated in response to pheromones, while the main olfactory system is thought to be a general molecular analyzer capable of detecting subtle differences in molecular structure of volatile odorants. However, the role of the two systems in detection of biologically relevant chemical signals appears to be partially overlapping. Thus, while it is clear that the accessory olfactory system is responsive to putative pheromones, the main olfactory system can also respond to some pheromones. Conversely, while the main olfactory system can mediate recognition of differences in genetic makeup by smell, the vomeronasal organ (VNO) also appears to participate in recognition of chemosensory differences between genetically distinct individuals. The most salient feature of our review of the literature is that there are no general rules that allow classification of the accessory olfactory system as a pheromone detector and the main olfactory system as a detector of general odorants. Instead, each behavior must be considered within a specific behavioral context to determine the role of these two chemosensory systems. In each case, one system or the other (or both) participates in a specific behavioral or hormonal response.

Dissecting adrenal and behavioral responses to stress by targeted gene inactivation in mice.

To define the molecular pathways modulating adrenal and behavioral responses to stress, we have generated mice with inactivation of hypothalamic neuropeptides and signaling pathways. Studies in mice deficient in corticotropin-releasing hormone (CRH) have revealed the essential role for CRH in adrenal glucocorticoid production in response to many physiological and psychological stressors. Immune system activation in CRH-deficient mice provides a unique exception to the necessity for CRH in stimulating adrenal glucocorticoid production. By analyzing mice deficient in interleukin-6 (IL-6) and CRH, we find that restoration of glucocorticoid output with inflammation is largely mediated by dysregulated IL-6 production. Current studies focus on identifying cellular and gene targets by which glucocorticoids regulate immune system function. In contrast to impaired adrenocortical responses to stress, CRH-deficient mice exhibit normal behavioral responses to stress. To determine signaling pathways that may contribute to the behavioral responses to stress, we have generated and analyzed mice deficient in adenylyl cyclase type 8 (AC8). AC8 deficient mice have intact adrenocortical responses to stress, but an inability to undergo stress-induced alterations in behavior.

Olfactory fingerprints for major histocompatibility complex-determined body odors II: relationship among odor maps, genetics, odor composition, and behavior.

The olfactory system detects small differences in the composition of natural odorants, made up of hundreds of molecules. Odorous quality is hypothetically represented by a combinatorial code: activation of distinct but overlapping subsets of olfactory receptors resulting in activation of a distinct subset of glomeruli in the main olfactory bulb (MOB). Here we show that modification of a single gene (the K gene of the major histocompatibility locus), which results in a subtle change in the odiferous quality of urine, causes a small but significant change in the composition of urine volatiles and consequently the evoked glomerular activation pattern in the MOB. The magnitude of disparity between urine-evoked glomerular activation patterns is predictive of the extent of (1) the genetic difference among the urine donors, (2) the difference in the chemical composition of urine, and (3) the odor detector's ability to discriminate. These data on natural odors are consistent with the combinatorial code hypothesis and identify subsets of glomeruli that are apt to play a significant role in mediating individual recognition.

Trigeminal collaterals in the nasal epithelium and olfactory bulb: a potential route for direct modulation of olfactory information by trigeminal stimuli.

The nasal epithelium is richly invested with peptidergic (substance P and calcitonin gene-related peptide [CGRP]) trigeminal polymodal nociceptors, which respond to numerous odorants as well as irritants. Peptidergic trigeminal sensory fibers also enter the glomerular layer of the olfactory bulb. To test whether the trigeminal fibers in the olfactory bulb are collaterals of the epithelial trigeminal fibers, we utilized dual retrograde labeling techniques in rats to identify the trigeminal ganglion cells innervating each of these territories. Nuclear Yellow was injected into the dorsal nasal epithelium, and True Blue was injected into the olfactory bulb of the same side. Following a survival period of 3-7 days, the trigeminal ganglion contained double-labeled, small (11.8 x 8.0 microm), ellipsoid ganglion cells within the ethmoid nerve region of the ganglion. Tracer injections into the spinal trigeminal complex established that these branched trigeminal ganglion cells also extended an axon into the brainstem. These results indicate that some trigeminal ganglion cells with sensory endings in the nasal epithelium also have branches reaching directly into both the olfactory bulb and the spinal trigeminal complex. These trigeminal ganglion cells are unique among primary sensory neurons in having two branches entering the central nervous system at widely distant points. Furthermore, the collateral innervation of the epithelium and bulb may provide an avenue whereby nasal irritants could affect processing of coincident olfactory stimuli.