Macro-connectomics and microstructure predict dynamic plasticity patterns in the non-human primate brain.

The brain displays a remarkable ability to adapt following injury by altering its connections through neural plasticity. Many of the biological mechanisms that underlie plasticity are known, but there is little knowledge as to when, or where in the brain plasticity will occur following injury. This knowledge could guide plasticity-promoting interventions and create a more accurate roadmap of the recovery process following injury. We causally investigated the time-course of plasticity after hippocampal lesions using multi-modal MRI in monkeys. We show that post-injury plasticity is highly dynamic, but also largely predictable on the basis of the functional connectivity of the lesioned region, gradients of cell densities across the cortex and the pre-lesion network structure of the brain. The ability to predict which brain areas will plastically adapt their functional connectivity following injury may allow us to decipher why some brain lesions lead to permanent loss of cognitive function, while others do not.

The Rhesus Monkey Hippocampus Critically Contributes to Scene Memory Retrieval, But Not New Learning.

Humans can recall a large number of memories years after the initial events. Patients with amnesia often have lesions to the hippocampus, but human lesions are imprecise, making it difficult to identify the anatomy underlying memory impairments. Rodent studies enable great precision in hippocampal manipulations, but not investigation of many interleaved memories. Thus it is not known how lesions restricted to the hippocampus affect the retrieval of multiple sequentially encoded memories. Furthermore, disagreement exists as to whether hippocampal inactivations lead to temporally graded or ungraded amnesia, which could be a consequence of differences between rodent and human studies. In the current study, rhesus monkeys of both sexes received either bilateral neurotoxic hippocampal lesions or remained unoperated controls and were tested on recognition and new learning of visual object-in-place scenes. Monkeys with hippocampal lesions were significantly impaired at remembering scenes that were encoded before the lesion. We did not observe any temporal gradient effect of the lesion on memory recognition, with recent and remote memories being equally affected by the lesion. Monkeys with hippocampal lesions showed no deficits in learning new scenes. Thus, the hippocampus, like other cortical regions, may be engaged in the acquisition and storage of new memories, but the role of the damaged hippocampus can be taken over by spared hippocampal tissue or extra-hippocampal regions following a lesion. These findings illustrate the utility of experimental paradigms for studying retrograde and anterograde amnesia that make use of the capacity of nonhuman primates to rapidly acquire many distinct visual memories.SIGNIFICANCE STATEMENT Recalling old memories, creating new memories, and the process by which memories transition from temporary to permanent storage all may rely on the hippocampus. Whether the hippocampus is necessary for encoding and retrieval of multiple related visual memories in primates is not known. Monkeys that learned many visual memory problems before precise lesions of the hippocampus were impaired at recalling those memories after hippocampal damage regardless of when the memories were formed, but could learn new memory problems at a normal rate. This suggests the hippocampus is normally vital for retrieval of complex visual memories regardless of their age, and also points to the importance of investigating mechanisms by which memories may be acquired in the presence of hippocampal damage.

Exposure to sevoflurane anesthesia during development does not impair aspects of attention during adulthood in rats.

Exposure to general anesthetic agents during development has been associated with neurotoxicity and long-term behavioral impairments in rodents and non-human primates. The phenotype of anesthetic-induced cognitive impairment has a robust learning and memory component, however less is known about other psychological domains. Data from retrospective human patient studies suggest that children undergoing multiple procedures requiring general anesthesia are at increased risk of attention deficit hyperactivity disorder. We therefore assessed whether single or repeated exposures of neonatal rats to general anesthesia caused long-term attentional impairments. Female or male Long-Evans pups were exposed to 2.5% sevoflurane for 2h on postnatal day (P) 7, or for 2h each on P7, P10 and P13. Rats were behaviorally tested in late adolescence on the sustained attention task and on the attentional set shifting task. There was no compelling evidence for anesthetic-induced impairment in attentional processing in adult rats exposed to general anesthesia as neonates. These results suggest that, at least at the developmental stage tested here, the phenotype of anesthetic-induced cognitive impairment does not involve disruptions to attentional processing.

Multiple sevoflurane exposures in infant monkeys do not impact the mother-infant bond.

Exposure to general anesthesia during the postnatal period is associated with death of brain cells as well as long-term impairments in cognitive and emotional behavior in animal models. These models are critical for investigating mechanisms of pediatric anesthetic neurotoxicity as well as for testing potential strategies for preventing or mitigating this toxicity. Control conditions for anesthesia exposure involve separation of conscious infants from their mothers for variable periods of time, which could have its own effect on subsequent behavior because of stress to the mother and/or infant as a consequence of separation.We are conducting a long-term study of infant rhesus monkeys exposed three times for 4h each to sevoflurane anesthesia during the first six postnatal weeks, with a comparison condition of control infant monkeys that undergo brief maternal separations on the same schedule, to equate the period of time each infant is conscious and separated from its mother. Because mothers are separated from their infants longer for infants in the anesthesia condition, this could modify maternal behavior toward the infant, which may influence subsequent socioemotional behavior in the infants. In this study, we analyzed maternal behavior immediately after the first post-anesthesia (or control) reunion, as well as during reintroduction of the mother-infant pair to the larger social group 24 hpost-anesthesia or control separation, and found no differences between the conditions with mothers spending most of their time in contact with infants in all conditions analyzed. This indicates that the different durations of maternal separation in this study design do not impact the mother-infant bond, strengthening conclusions that subsequent differences in behavior between monkeys exposed to anesthesia compared to controls are a consequence of anesthesia exposure and not differential maternal behavior in the two conditions.

Multiple Anesthetic Exposure in Infant Monkeys Alters Emotional Reactivity to an Acute Stressor.

BACKGROUND: Retrospective studies in humans have shown a higher prevalence of learning disabilities in children that received multiple exposures to general anesthesia before the age of 4 yr. Animal studies, primarily in rodents, have found that postnatal anesthetic exposure causes neurotoxicity and neurocognitive deficits in adulthood. The authors addressed the question of whether repeated postnatal anesthetic exposure was sufficient to cause long-term behavioral changes in a highly translationally relevant rhesus monkey model, allowing study of these variables against a background of protracted nervous system and behavioral development. METHODS: Rhesus monkeys of both sexes underwent either three 4-h exposures to sevoflurane anesthesia (anesthesia group n = 10) or brief maternal separations (control group n = 10) on postnatal day 6 to 10 that were repeated 14 and 28 days later. Monkeys remained with their mothers in large social groups at all times except for overnight observation after each anesthetic/control procedure. At 6 months of age, each monkey was tested on the human intruder paradigm, a common test for emotional reactivity in nonhuman primates. RESULTS: The frequency of anxiety-related behaviors was significantly higher in monkeys that were exposed to anesthesia as neonates as compared with controls: anesthesia 11.04 ± 1.68, controls 4.79 ± 0.77, mean ± SEM across all stimulus conditions. CONCLUSION: Increased emotional behavior in monkeys after anesthesia exposure in infancy may reflect long-term adverse effects of anesthesia.

Long-term effects of single or multiple neonatal sevoflurane exposures on rat hippocampal ultrastructure.

BACKGROUND: Neonatal exposure to general anesthetics may pose significant neurocognitive risk. Human epidemiological studies demonstrate higher rates of learning disability among children with multiple, but not single, exposures to anesthesia. The authors employ a rat model to provide a histological correlate for these population-based observations. The authors examined long-term differences in hippocampal synaptic density, mitochondrial density, and dendritic spine morphology. METHODS: Twenty male rat pups (n = 5/condition) were exposed to 2.5% sevoflurane under one of four conditions: single 2-h exposure on postnatal day 7 (P7); single 6-h exposure on P7; repeated 2-h exposures on P7, P10, and P13 for a cumulative 6 h of general anesthetics; or control exposure to 30% oxygen on P7, P10, and P13. RESULTS: Repeated exposure to general anesthetics resulted in greater synaptic loss relative to a single 2-h exposure (P < 0.001). The magnitude of synaptic loss induced by three 2-h exposures (1.977 ± 0.040 µm [mean ± SEM]) was more profound than that of a single 6-h exposure (2.280 ± 0.045 µm, P = 0.022). Repeated exposures did not alter the distribution of postsynaptic density length, indicating a uniform pattern of loss across spine types. In contrast, mitochondrial toxicity was best predicted by the cumulative duration of exposure. Relative to control (0.595 ± 0.017), both repeated 2-h exposures (0.479 ± 0.015) and a single 6-h exposure (0.488 ± 0.013) were associated with equivalent reductions in the fraction of presynaptic terminals containing mitochondria (P < 0.001). CONCLUSION: This suggests a "threshold effect" for general anesthetic-induced neurotoxicity, whereby even brief exposures induce long-lasting alterations in neuronal circuitry and sensitize surviving synapses to subsequent loss.

Discovery of 2,5-diarylnicotinamides as selective orexin-2 receptor antagonists (2-SORAs).

The orexin (or hypocretin) system has been identified as a novel target for the treatment of insomnia due to the wealth of biological and genetic data discovered over the past decade. Recently, clinical proof-of-concept was achieved for the treatment of primary insomnia using dual (OX1R/OX2R) orexin receptor antagonists. However, elucidation of the pharmacology associated with selective orexin-2 receptor antagonists (2-SORAs) has been hampered by the lack of orally bioavailable, highly selective small molecule probes. Herein, the discovery and optimization of a novel series of 2,5-diarylnicotinamides as potent and orally bioavailable orexin-2 receptor selective antagonists is described. A compound from this series demonstrated potent sleep promotion when dosed orally to EEG telemetrized rats.

Long-term effects of neonatal single or multiple isoflurane exposures on spatial memory in rats.

General anesthetics are neurotoxic to neonatal rodents and non-human primates. Neonatal exposure to general anesthetics has been associated with long-term cognitive deficits in animal models. Some data from humans are consistent with long-term deleterious effects of anesthetic exposure early in life on cognitive development, with multiple exposures to general anesthetics being particularly damaging. We sought to determine whether repeated exposure of neonatal rats to anesthesia was associated with long-term cognitive impairments and whether the magnitude of impairments was greater than that resulting from a single exposure. Male or female Long-Evans rat pups were exposed to 1.8% isoflurane for 2 h on postnatal day (P) 7, or for 2 h each on P7, P10, and P13. Testing in a spatial working memory task began on P91. Rats that were repeatedly exposed to isoflurane were impaired relative to controls in the spatial working memory task. Male rats that received a single exposure to isoflurane showed an unexpected facilitation in spatial memory performance. These results support the hypothesis that multiple neonatal exposures to general anesthesia are associated with greater long-term cognitive impairment than a single exposure. The findings are congruent with human epidemiological studies reporting long-term cognitive impairments following multiple but not single general anesthetics early in life.

Indazole derivatives as novel bradykinin B1 receptor antagonists.

A new class of indazole-derived bradykinin B(1) antagonists and their structure-activity relationships (SAR) is reported. A number of compounds were found to have low-nanomolar affinity for the human B(1) receptor and possess acceptable P-gp and pharmacokinetics properties.

Discovery of the dual orexin receptor antagonist [(7R)-4-(5-chloro-1,3-benzoxazol-2-yl)-7-methyl-1,4-diazepan-1-yl][5-methyl-2-(2H-1,2,3-triazol-2-yl)phenyl]methanone (MK-4305) for the treatment of insomnia.

Despite increased understanding of the biological basis for sleep control in the brain, few novel mechanisms for the treatment of insomnia have been identified in recent years. One notable exception is inhibition of the excitatory neuropeptides orexins A and B by design of orexin receptor antagonists. Herein, we describe how efforts to understand the origin of poor oral pharmacokinetics in a leading HTS-derived diazepane orexin receptor antagonist led to the identification of compound 10 with a 7-methyl substitution on the diazepane core. Though 10 displayed good potency, improved pharmacokinetics, and excellent in vivo efficacy, it formed reactive metabolites in microsomal incubations. A mechanistic hypothesis coupled with an in vitro assay to assess bioactivation led to replacement of the fluoroquinazoline ring of 10 with a chlorobenzoxazole to provide 3 (MK-4305), a potent dual orexin receptor antagonist that is currently being tested in phase III clinical trials for the treatment of primary insomnia.

Anaesthesia with a combination of ketamine and medetomidine in the rabbit: effect of premedication with buprenorphine.

OBJECTIVE: To assess the effects of premedication with buprenorphine on the characteristics of anaesthesia induced with ketamine/medetomidine. STUDY DESIGN: Prospective crossover laboratory study. ANIMALS: Six female New Zealand White rabbits. METHODS: Rabbits received, on occasions separated by 7 days, either buprenorphine (0.03 mg kg(-1)) or saline subcutaneously (SC) as premedication, followed 1 hour later by SC ketamine (15 mg kg(-1)) and medetomidine (0.25 mg kg(-1)) (K/M). At pre-determined time points reflex responses and cardiopulmonary parameters were recorded and arterial blood samples taken for analysis. Total sleep time was the duration of loss of the righting reflex. Duration of surgical anaesthesia was the time of suppression of the ear pinch and pedal withdrawal reflexes. Wilcoxon signed-ranks tests were used to compare data before (T(0)) and 10 minutes after (T(10)) injection with K/M. RESULTS: All animals lost all three reflex responses within 10 minutes of injection of K/M. The duration of loss of these reflexes significantly increased in animals that received buprenorphine. At induction, animals that had received buprenorphine tended to have a lower respiration rate but there were no significant differences in arterial PCO(2), PO(2) or pH between treatments. Hypoxaemia [median PaO(2) < 6.0 kPa (45 mmHg)] developed in both treatments at T(10) but there was no significant difference between treatments. Mean arterial pressure (MAP) was lower at T(10) in animals that had received buprenorphine. CONCLUSION AND CLINICAL RELEVANCE: Premedication with buprenorphine significantly increased the duration of anaesthesia induced by K/M, with no significant depression of respiration further to the control treatment within the first 10 minutes of anaesthesia. The MAP decreased but this was not reflected in a difference in other physiological parameters. These data show that premedication with buprenorphine, before K/M anaesthesia in the rabbit, has few negative effects and may provide beneficial analgesia.

Bradykinin B1 receptor antagonists: an alpha-hydroxy amide with an improved metabolism profile.

A series of carbo- and heterocyclic alpha-hydroxy amide-derived bradykinin B1 antagonists was prepared and evaluated. A 4,4-difluorocyclohexyl alpha-hydroxy amide was incorporated along with a 2-methyl tetrazole in lieu of an oxadiazole to afford a suitable compound with good pharmacokinetic properties, CNS penetration, and clearance by multiple metabolic pathways.

2-Aminobenzophenones as a novel class of bradykinin B1 receptor antagonists.

Selective bradykinin (BK) B 1 receptor antagonists could be novel therapeutic agents for the treatment of pain and inflammation. Elucidation of the structure activity relationships of the structurally novel HTS lead compound 1 provided potent hBK B 1 receptor antagonists with excellent receptor occupancy in the CNS of hBK B 1 transgenic rats.

A new class of bradykinin B1 receptor antagonists with high oral bioavailability and minimal PXR activity.

The design and synthesis of a novel class of human bradykinin B1 antagonists featuring difluoroethyl ether and isoxazole carboxamide moieties are disclosed. Compound 7g displayed excellent pharmacokinetic properties, efficient ex vivo receptor occupancy, and low potential for P450 induction via PXR activation.

Proline bis-amides as potent dual orexin receptor antagonists.

A series of OX(2)R/OX(1)R dual orexin antagonists was prepared based on a proline bis-amide identified as a screening lead. Through a combination of classical and library synthesis, potency enhancing replacements for both amide portions were discovered. N-methylation of the benzimidazole moiety within the lead structure significantly reduced P-gp susceptibility while increasing potency, giving rise to good brain penetration. A compound from this series has demonstrated in vivo central activity when dosed peripherally in a pharmacodynamic model of orexin activity.

Different behavioral effects of neurotoxic dorsal hippocampal lesions placed under either isoflurane or propofol anesthesia.

Anesthetic protocols for behavioral neuroscience experiments are evolving as new anesthetics are developed and surgical procedures are refined to improve animal welfare. We tested whether neurotoxic dorsal hippocampal lesions produced under two different anesthetic protocols would have different behavioral and/or histo-pathological effects. Rats were anesthetized with either propofol, an intravenous anesthetic, or isoflurane, a gaseous anesthetic, and multiple injections of an excitotoxin (N-methyl-D-aspartate) were stereotaxically placed in the dorsal hippocampus bilaterally. Intraoperative physiological parameters were similar in the two surgical groups, as were the volumes of the lesions, although the profile of postoperative impairment in a spatial learning task differed between the lesion groups depending on the anesthetic regimen used. These results show that the choice of anesthetic protocol is a critical variable in designing behavioral neuroscience experiments using neurosurgical procedures. This factor should be considered carefully in experimental design and in cross-study comparisons of lesion effects on behavior.

Alpha-hydroxy amides as a novel class of bradykinin B1 selective antagonists.

Antagonism of the bradykinin B(1) receptor represents a potential treatment for chronic pain and inflammation. Novel antagonists incorporating alpha-hydroxy amides were designed that display low-nanomolar affinity for the human bradykinin B(1) receptor and good bioavailability in the rat and dog. In addition, these functionally active compounds show high passive permeability and low susceptibility to phosphoglycoprotein mediated efflux, predictive of good CNS exposure.

Bradykinin B1 antagonists: biphenyl SAR studies in the cyclopropanecarboxamide series.

SAR study of the biphenyl region of cyclopropanecarboxamide derived bradykinin B(1) antagonists was examined. Incorporation of a pyridine in place of the proximal phenyl ring and chlorination of the distal phenyl ring proved to be well tolerated and provided compounds with improved pharmacokinetic profiles, CNS penetration, and enhanced receptor occupancy.

Potent bradykinin B1 receptor antagonists: 4-substituted phenyl cyclohexanes.

Selective bradykinin (BK) B(1) receptor antagonists have been shown to be antinociceptive in animal models and could be novel therapeutic agents for the treatment of pain and inflammation. Elucidation of the structure-activity relationships of the biphenyl moiety of the lead compound 1 provided a potent new structural class of BK B(1) receptor antagonists.