Chemical composition and biological effects of kratom (Mitragyna speciosa): In vitro studies with implications for efficacy and drug interactions.

The safety and efficacy of kratom (Mitragyna speciosa) for treatment of pain is highly controversial. Kratom produces more than 40 structurally related alkaloids, but most studies have focused on just two of these, mitragynine and 7-hydroxymitragynine. Here, we profiled 53 commercial kratom products using untargeted LC-MS metabolomics, revealing two distinct chemotypes that contain different levels of the alkaloid speciofoline. Both chemotypes were confirmed with DNA barcoding to be M. speciosa. To evaluate the biological relevance of variable speciofoline levels in kratom, we compared the opioid receptor binding activity of speciofoline, mitragynine, and 7-hydroxymitragynine. Mitragynine and 7-hydroxymitragynine function as partial agonists of the human µ-opioid receptor, while speciofoline does not exhibit measurable binding affinity at the µ-, δ- or ƙ-opioid receptors. Importantly, mitragynine and 7-hydroxymitragynine demonstrate functional selectivity for G-protein signaling, with no measurable recruitment of ß-arrestin. Overall, the study demonstrates the unique binding and functional profiles of the kratom alkaloids, suggesting potential utility for managing pain, but further studies are needed to follow up on these in vitro findings. All three kratom alkaloids tested inhibited select cytochrome P450 enzymes, suggesting a potential risk for adverse interactions when kratom is co-consumed with drugs metabolized by these enzymes.

Identifying rare variants for genetic risk through a combined pedigree and phenotype approach: application to suicide and asthma.

Suicidal behavior is a complex disorder, with evidence for genetic risk independent of other genetic risk factors including psychiatric disorders. Since 1996, over 3000 DNA samples from Utah suicide decedents have been collected and banked for research use through the Utah Medical Examiner. In addition, over 12,000 Utah suicides were identified through examination of death certificates back to 1904. By linking this data with the Utah Population Database, we have identified multiple extended pedigrees with increased risk for suicide completion. A number of medical conditions co-occur with suicide, including asthma, and this study was undertaken to identify genetic risk common to asthma and suicide. This study tests the hypothesis that a particular comorbid condition may identify a more homogeneous genetic subgroup, facilitating the identification of specific genetic risk factors in that group. From pedigrees at increased risk for suicide, we identified three pedigrees also at significantly increased familial risk for asthma. Five suicide decedents from each of these pedigrees, plus an additional three decedents not from these pedigrees with diagnosed asthma, and 10 decedents with close relatives with asthma were genotyped. Results were compared with 183 publicly available unaffected control exomes from 1000 Genomes and CEPH (Centre d'etude du polymorphisme humain) samples genotyped on the same platform. A further 432 suicide decedents were also genotyped as non-asthma suicide controls. Genotyping was done using the Infinium HumanExome BeadChip. For analysis, we used the pedigree extension of Variant Annotation, Analysis and Search Tool (pVAAST) to calculate the disease burden of each gene. The Phenotype Driven Variant Ontological Re-ranking tool (Phevor) then re-ranked our pVAAST results in context of the phenotype. Using asthma as a seed phenotype, Phevor traversed biomedical ontologies and identified genes with similar biological properties to those known to result in asthma. Our top associated genes included those related to neurodevelopment or neural signaling (brain-derived neurotrophic factor (BDNF), neutral sphingomyelinase 2 (SMPD2), homeobox b2 (HOXB2), neural cell adhesion molecule (NCAM2), heterogeneous nuclear ribonucleoprotein A0 (HNRNPA0)), inflammation (free fatty acid receptor 2 (FFAR2)) and inflammation with additional evidence of neuronal involvement (oxidized low density lipoprotein receptor 1 (OLR1), toll-like receptor 3 (TLR3)). Of particular interest, BDNF has been previously implicated in both psychiatric disorders and asthma. Our results demonstrate the utility of combining pedigree and co-occurring phenotypes to identify rare variants associated with suicide risk in conjunction with specific co-occurring conditions.

Developmental outcome in preterm infants <29 weeks gestation with ≤ Stage 3 retinopathy of prematurity (ROP): relationship to severity of ROP.

We have determined the influence of the severity of retinopathy of prematurity (ROP) on development at 3 years of age in infants <29 weeks gestation from a population-based cohort. Primary analysis of surviving infants born <29 weeks gestational age (GA) from 1998 to 2001 in New South Wales and the Australian Capital Territory were grouped according to stage of ROP. Infants with periventricular leukomalacia, Grade III or IV intraventricular haemorrhage, hydrocephalus, major congenital abnormalities, Stage 4 or 5 ROP, cerebral palsy or a severe hearing impairment were excluded. Infants with Stage 3 ROP were matched for GA, birthweight and gender to those with no ROP, Stage 1 and Stage 2 ROP. The four groups were then compared for their 3-year-old developmental outcome, using the Griffiths Mental Development Scale. Development was also compared for those infants with Stage 3 ROP who were either treated or not treated with laser therapy. A secondary multivariate regression analysis on developmental outcome was performed with all infants included in the analysis. In neurologically comparable groups and in the multivariate analysis, there was no association between ROP and developmental outcome. There was also no difference in the Griffiths assessment at 3 years between those who were or were not treated for severe ROP. Neither severity of ROP nor treatment for severe ROP were related to developmental outcome at 3 years of age in a large population-based cohort of infants born <29 weeks gestation.

Citicoline enhances frontal lobe bioenergetics as measured by phosphorus magnetic resonance spectroscopy.

Citicoline supplementation has been used to ameliorate memory disturbances in older people and those with Alzheimer's disease. This study used MRS to characterize the effects of citicoline on high-energy phosphate metabolites and constituents of membrane synthesis in the frontal lobe. Phosphorus ((31)P) metabolite data were acquired using a three-dimensional chemical-shift imaging protocol at 4 T from 16 healthy men and women (mean +/- SD age 47.3 +/- 5.4 years) who orally self-administered 500 mg or 2000 mg Cognizin Citicoline (Kyowa Hakko Kogyo Co., Ltd, Ibaraki, Japan) for 6 weeks. Individual (31)P metabolites were quantified in the frontal lobe (anterior cingulate cortex) and a comparison region (parieto-occipital cortex). Significant increases in phosphocreatine (+7%), beta-nucleoside triphosphates (largely ATP in brain, +14%) and the ratio of phosphocreatine to inorganic phosphate (+32%), as well as significant changes in membrane phospholipids, were observed in the anterior cingulate cortex after 6 weeks of citicoline treatment. These treatment-related alterations in phosphorus metabolites were not only regionally specific, but tended to be of greater magnitude in subjects who received the lower dose. These data show that citicoline improves frontal lobe bioenergetics and alters phospholipid membrane turnover. Citicoline supplementation may therefore help to mitigate cognitive declines associated with aging by increasing energy reserves and utilization, as well as increasing the amount of essential phospholipid membrane components needed to synthesize and maintain cell membranes.

Severe retinopathy of prematurity in infants <30 weeks' gestation in New South Wales and the Australian Capital Territory from 1992 to 2002.

BACKGROUND: Retinopathy of prematurity (ROP) significantly increased in New South Wales (NSW) from 1986 to 1994, but more recent data suggest that there has now been a decrease. OBJECTIVE: To study the incidence and treatment of severe ROP (stage >or=3) in NSW and the Australian Capital Territory (ACT) from 1992 to 2002. METHODS: Data collected prospectively from the Neonatal Intensive Care Units' (NICUS) Data Collection over an 11-year period in infants <30 weeks' gestation were divided into four epochs and analysed retrospectively. The incidence and treatment of severe ROP were compared for gestational ages or=3: from 17 (41.5%) to 41 (53.9%), p=0.052; treated: from 8 (19.5%) to 25 (32.9%), p<0.05 (first and fourth epoch)). In infants 25-26 weeks' gestation the incidence of severe ROP decreased significantly whereas there was a non-significant increase in those treated (stage >or=3: from 55 (26.2%) to 46 (19.3%), p<0.05; treated: from 19 (9.0%) to 32 (13.4%)). In infants 27-29 weeks' gestation, there was no significant change in the incidence of severe ROP or those treated (stage >or=3: from 30 (4.1%) to 17 (2.4%); treated: from 14 (1.9%) to 8 (1.1%)). CONCLUSION: In infants

Eye-hand co-ordination skills in very preterm infants <29 weeks gestation at 3 years: Effects of preterm birth and retinopathy of prematurity.

INTRODUCTION: Preterm infants are known to have low gross motor and fine motor skills. We questioned whether poor eye-hand coordination skills are associated with moderate to severe stages of Retinopathy of Prematurity (ROP). AIMS: The aim of this study was to examine development, with specific reference to eye-hand coordination skills, among preterm infants <29 weeks gestation with different stages of ROP at 3 years of age. METHODS AND MATERIALS: Fifteen preterm infants (<29 weeks gestation) who developed Stage 3 ROP were matched for gestation, birthweight and gender with infants who developed Stage 2 and Stage 1/no ROP. Developmental (Griffiths Mental Development Scales and Peabody Developmental Motor Scales) and ophthalmic assessments in the 3 matched groups of 15 were performed at 3 years of age. RESULTS: 1) Whilst the eye-hand coordination scores and Peabody fine motor scores were lower in the Stage 3 ROP group, they were not significantly lower than the other ROP groups. 2) Locomotor, Peabody gross motor skills and hearing and speech were significantly lower in the infants with Stage 3 ROP. The other developmental domains were not significantly different to the severe ROP group. 3) All 3 groups (of preterm infants) had lower eye-hand coordination and Peabody fine motor scores compared to test norms. 4) There were 8 of 15 infants with Stage 3 ROP who developed moderate visual problems by 3 years of age. CONCLUSION: In preterm infants, low eye-hand coordination/fine motor scores are likely to be due to their extreme prematurity.

Basal ganglia volumetric studies in affective disorder: what did we learn in the last 15 years?

Until today, morphometric neuroimaging studies on affective disorders concentrate on the limbic system, especially the hippocampus, amygdala, and anterior cingulate. In most of the studies and reviews available today, the basal ganglia are of secondary interest. It seems that the basal ganglia are interest of neurologist, whereas the limbic system is reserved for psychiatric neuroimaging studies. We follow a different approach in this review, studying all available papers on MRI research of the basal ganglia in unipolar depression and bipolar disorder. We found a possibly larger neostriatum in bipolar and possibly smaller one in unipolar patients. None of the unipolar studies found any larger basal ganglion, and only one out of 12 bipolar studies found smaller basal ganglia. Both findings seemed to depend on age (tendency toward smaller volumes in unipolar and bipolar with older age), sex (men tending to pathology in both disorders) and bipolar patients show a possible influence of medication, which is not assessed so far in unipolar depression. We conclude that several methodological shortcomings in volumetric MRI research on the basal ganglia in affective disorders make it necessary to imply more research in this area. We suggest (a) better MRI methods (we do not have a single volumetric 3 Tesla study in this patient group); (b) studies of medication-naïve patients (thus ruling out the medication effect); (c) Studies that directly compare unipolar depressed and bipolar patients are needed to determine whether these apparent differences in morphometric abnormalities, as observed through the mediating comparison with healthy subjects, are real.

Neural correlates of different types of deception: an fMRI investigation.

Deception is a complex cognitive activity, and different types of lies could arise from different neural systems. We investigated this possibility by first classifying lies according to two dimensions, whether they fit into a coherent story and whether they were previously memorized. fMRI revealed that well-rehearsed lies that fit into a coherent story elicit more activation in right anterior frontal cortices than spontaneous lies that do not fit into a story, whereas the opposite pattern occurs in the anterior cingulate and in posterior visual cortex. Furthermore, both types of lies elicited more activation than telling the truth in anterior prefrontal cortices (bilaterally), the parahippocampal gyrus (bilaterally), the right precuneus, and the left cerebellum. At least in part, distinct neural networks support different types of deception.

Chronic citicoline increases phosphodiesters in the brains of healthy older subjects: an in vivo phosphorus magnetic resonance spectroscopy study.

RATIONALE: Phosphatidylcholine (PtdCho) in brain cell membranes decreases with age. Evidence from both animal and in vitro studies indicates that CDP-choline (citicoline) administration may increase phosphatidylcholine (PtdCho) synthesis and might reverse PtdCho loss. OBJECTIVES: We investigated whether oral citicoline can increase PtdCho synthesis in the brains of older subjects by measuring levels of phosphorus-containing metabolites using proton-decoupled phosphorus magnetic resonance spectroscopy ((31)P-MRS) before and after citicoline treatment. METHODS: All subjects took 500 mg citicoline once orally each day for 6 weeks, then took either citicoline or placebo once orally per day for a second 6-week period. Subjects underwent a (31)P-MRS scan at baseline and following 6 and 12 weeks of treatment. RESULTS: Treatment with citicoline for 6 weeks was associated with a 7.3% increase from baseline levels in brain phosphodiesters ( P=0.008), including an 11.6% increase in glycerophosphoethanolamine ( P=0.002) and a 5.1% increase in glycerophosphocholine ( P=0.137). Subjects who continued to take citicoline for the second 6-week period did not show significant additional increases in the levels of these metabolites. No changes were seen in other phosphorus-containing metabolites. There was a correlation between improvement on the California Verbal Learning Test and increase in phosphodiesters. CONCLUSIONS: The increases in phosphodiesters seen in this study indicate that phospholipid synthesis and turnover were stimulated by 6 weeks of oral citicoline. These results in humans support previous in vitro and animal studies and suggest that the administration of oral citicoline may be of use in reversing age-related changes in the brain.

Sex differences in amygdala activation during the perception of facial affect.

The cognitive and affective systems of the cerebral cortex are often more lateralized in males than females, but it is unclear whether these differences extend to subcortical systems. We used fMRI to examine sex differences in lateralized amygdala activity during happy and fearful face perception. Amygdala activation differed for men and women depending on the valence of the expression. Overall, males were more lateralized than females, but the direction differed between valence conditions. Happy faces produced greater right than left amygdala activation for males but not females. Both sexes showed greater left amygdala activation for fearful faces. These findings suggest that the lateralization of affective function may extend beyond the cortex to subcortical regions such as the amygdala.

Morphometry of individual cerebellar lobules in schizophrenia.

OBJECTIVE: Previous imaging studies have described focal cortical changes in schizophrenia, with predominant findings of abnormalities in the temporal and frontal regions. The current study hypothesized that cerebellar regions involved in feedback and feed-forward loops with cortical regions affected in schizophrenia would also demonstrate structural changes. METHOD: Using magnetic resonance imaging, the authors measured the volume of individual cerebellar lobules in 19 patients with schizophrenia and 19 healthy comparison subjects. RESULTS: The inferior vermis was significantly smaller in the schizophrenic group than in the comparison group. Patients with schizophrenia also demonstrated a significantly smaller cerebellar asymmetry than the comparison subjects. CONCLUSIONS: The authors hypothesize that these morphometric changes may be developmental in origin and possibly related to cortical abnormalities.

Sex-specific developmental changes in amygdala responses to affective faces.

It is hypothesized that adolescent development involves a redistribution of cerebral functions from lower subcortical structures to higher regions of the prefrontal cortex to provide greater self-control over emotional behavior. We further hypothesized that this redistribution is likely to be moderated by sex-specific hormonal changes. To examine developmental sex differences in affective processing, 19 children and adolescents underwent fMRI while viewing photographs of faces expressing fear. Males and females differed in the pattern of their amygdala vs prefrontal activation during adolescent maturation. With age, females showed a progressive increase in prefrontal relative to amygdala activation in the left hemisphere, whereas males failed to show a significant age related difference. There appear to be sex differences in the functional maturation of affect-related prefrontal-amygdala circuits during adolescence.

Umbilical cord blood lactate in normal infants: comparison between two methods of measurement.

OBJECTIVES: Firstly, to determine the accuracy of the Radiometer ABL 625 lactate electrode (Radiometer Medical Pty Ltd, Nunawading, Victoria, Australia) by comparing the lactate values obtained by this method to those obtained with the Hitachi 917 lactate analyser (Boehringer Mannheim Corporation, Charlottetown, Prince Edward Island, Canada). Secondly, to determine the effect of delay in measurement on blood lactate levels. METHODOLOGY: Umbilical venous (UCV) blood samples were obtained from healthy term infants delivered vaginally. Lactate levels were measured with the Radiometer ABL 625 lactate electrode in the Neonatal Intensive Care Unit, Westmead Hospital and with the Hitachi 917 lactate analyser in 49 paired samples. In addition 26 UCV blood samples were placed in ice slurry and a further 26 samples at room temperature and blood lactate was measured at 5-min intervals for 30 min to determine the change of lactate levels with time. RESULTS: The lactate levels obtained from the Radiometer ABL 625 lactate electrode were consistently lower than the levels obtained from the Hitachi 917 lactate analyser (mean difference - 0.24), but the correlation was high (r = 0.97). The blood lactate levels increased at the rate of 0.012 mmol/L per min if the blood was left at room temperature. The lactate levels remained stable for 20 min if the blood was placed in ice slurry. CONCLUSION: The Radiometer ABL 625 lactate electrode was easy to use and there was high correlation with the values obtained by the standard laboratory method. The blood specimen must be place in an ice slurry if a delay in analysis is anticipated.

Inspired gas humidity during mechanical ventilation: effects of humidification chamber, airway temperature probe position and environmental conditions.

OBJECTIVES: To determine the inspired gas humidity during mechanical ventilation with: (i) four different humidification chambers; (ii) two airway temperature probe (ATP) positions; (iii) five different humidicrib temperatures; and (iv) insulating the inspiratory limb with bubble wrap. METHODOLOGY: An observational study in the Neonatal Laboratory and Neonatal Intensive Care Unit, Westmead Hospital. The humidity of the inspired gas was measured at the proximal end of the endotracheal tube (ETT) during mechanical ventilation. Inspired humidity measurements were made with four different humidification chambers (Fisher & Paykel (F&P Healthcare Pty Ltd, Auckland, New Zealand) auto refill MR290. F&P manual refill MR310, Suruga (Suruga Inc. Humidifiers, Vincent Medical, Dongguan, China) manual refill MI-20 and MI-10F) with the humidity control (relative humidity setting) set at - 2. Measurements were made with the ATP positioned either; (A) at the distal end of the inspiratory tube inside the humidicrib or (B) outside the humidicrib 50 cm proximal to the ETT. The inspired gas temperatures were set at 36.5 degrees C and at 39.0 degrees C, respectively. For each of the different humidification chambers and ATP positions, inspired humidity measurements were made with the humidicrib temperature set at 30.8, 32.9, 35.2, 36.2, or 37.2 degrees C. Two further sets of measurements were made, one with the inspiratory limb insulated with bubble wrap and the second set without bubble wrap. RESULTS: There were significant differences in inspired humidity with the four humidification chambers at both ATP positions at all humidicrib temperatures. Both Suruga humidification chambers produced significantly higher inspired gas humidities under most conditions. Positioning the ATP outside the humidicrib produced significantly higher inspired gas humidities than with the ATP inside the humidicrib. Insulating the inspiratory tubing with bubble wrap also significantly improved the inspired gas humidity. CONCLUSIONS: Significant differences in inspired gas humidity were found with the humidification chambers tested. The position of the ATP and the set temperature had a significant impact on the absolute humidity of the inspired gas. In general, higher inspired gas humidities were obtained with the ATP outside the humidicrib. However, condensation of water close to the ETT appeared at low humidicrib temperatures (< 36.2 degrees C) with the ATP outside the humidicrib and extreme care should be taken that particulate water does not enter the lungs under these conditions.

Inspired gas temperature during mechanical ventilation: effects of environmental temperature and airway temperature probe position.

OBJECTIVES: To determine the inspired gas temperature during mechanical ventilation with: (i) five different humidicrib temperatures; (ii) two airway temperature probe (ATP) positions; and (iii) four ATP adaptors. METHODOLOGY: An observational study in the Neonatal Intensive Care Laboratory, Westmead Hospital. The inspired gas temperature was measured at the proximal end of the endotracheal tube (ETT) during conventional mechanical ventilation using a Tele-thermometer. Inspired gas temperature measurements were made with: (i) the humidicrib temperature set at 30.8. 32.9, 35.2, 36.2. or 37.2 degrees C; (ii) the ATP either (A) positioned inside the humidicrib at the distal end of the inspiratory tubing or (B) positioned outside the humidicrib 50 cm proximal to the ETT, with the inspired gas temperatures set at 36.5 and 39.0 degrees C, respectively; and (iii) the measurements repeated with four different ATP adaptors at each humidicrib temperature and each ATP position. RESULTS: With the ATP inside the humidicrib, there were no significant differences between set and actual inspired gas temperature. However, with the ATP outside the humidicrib, there were significant decreases in inspired gas temperature at each humidicrib temperature. For instance, with the ATP outside the humidicrib and set at 39.0 degrees C, the inspired gas temperature decreased to 34.7+/-0.2 degrees C at a humidicrib temperature of 30.8 degrees C and to 37.7+/-0.2 degrees C at a humidicrib temperature of 37.2 degrees C. The type of ATP adaptor also had a significant effect on inspired gas temperature. CONCLUSIONS: With the ATP placed outside the humidicrib and with variations of humidicrib temperature, infants are likely to have inspired gas temperatures that are significantly different to the desired temperature. Certain ATP adaptors cause these variations in inspired gas temperature to be more pronounced. Extreme care must be used to avoid suboptimal inspired gas temperatures with these environmental variations and the ATP positioned outside the humidicrib.

Increased orbitofrontal cortex levels of choline in depressed adolescents as detected by in vivo proton magnetic resonance spectroscopy.

BACKGROUND: The frontal lobe has been implicated in the pathology of depression in adults. Through the use of magnetic resonance spectroscopy, altered brain choline levels have also been linked to the pathophysiology of affective disorders. METHODS: To identify possible alterations in orbitofrontal cortex levels of cytosolic choline in adolescents with and without depression, 22 depressed and 43 control adolescents were recruited. Of those recruited, usable proton magnetic resonance spectra were acquired from a voxel in the left anterior medial frontal lobe of 17 depressed (mean age 15.8+/-1.6) and 28 healthy adolescents (mean age 14.5+/-1.7). RESULTS: Orbitofrontal cytosolic choline/creatine (Cho/Cr) ratios (p =.032) and cytosolic choline/N-acetyl aspartate (Cho/NAA) ratios (p =.043) were significantly higher in the depressed subjects than in the control subjects. There were no significant differences between depressed and control subjects in gray or white matter content within the voxel. CONCLUSIONS: These findings suggest that brain cytosolic choline may be increased in depressed adolescents in comparison with control subjects and independent of a corresponding structural change. These results are consistent with similar, previously reported findings in adults and suggest that depression in adolescents is associated with alterations in orbitofrontal metabolism.

Neuroimaging in bipolar disorder: what have we learned?

New technologies are offering increasingly powerful means to obtain structural, chemical, and functional images of the brain during life, often without the use of ionizing radiation. Bipolar disorder, with its clear physiologic features, would appear to be a prime candidate for the application of current brain imaging; however, only a modest number of studies have been reported to date, and most studies have small sample sizes and heterogeneous subject groups. Nonetheless, there are a few consistent findings among these studies, including the following: 1) Structural imaging studies suggest an increased number of white matter hyperintensities in patients with bipolar disorder. These may be lesions unique to bipolar disorder and its treatment, or related to cardiovascular risk factors, which are more common in bipolar patients. Decreased cerebellar size and anomalies of cerebellar blood volume have also been reported. Increased sulcal prominence and enlargement of the lateral and third ventricles are less consistently observed findings. 2) Spectroscopic imaging suggests abnormalities of metabolism of choline-containing compounds in symptomatically ill bipolar patients and, possibly, treatment-induced changes in choline- and myoinositol-containing compounds. Each of these groups of metabolites serves as a component of membrane phospholipids and cellular second-messenger cycles. 3) Metabolic and blood flow studies provide evidence for decreased activity of the prefrontal cortex (PFC) in bipolar patients during depression. It is not clear if these changes are restricted to particular subregions of the PFC, nor if they are reversed with mania. No single pathophysiologic mechanism yet explains these findings, although all might be due to regional alterations in cellular activity and metabolism or changes in cell membrane composition and turnover. The development of imaging technologies has far outpaced their use in bipolar disorder. The promise of future studies is great, with more powerful magnetic resonance scanners, additional ligands for positron emission tomography and single photon emission computed tomography imaging, and improved image generation and processing already available.

Functional activation of the left amygdala and hippocampus during associative encoding.

The human hippocampus is critical to episodic encoding, but the role of the amygdala in memory is less clear. Animal research suggests a role for the amygdala in associative memory, but this has not been examined systematically in humans. Using fMRI, we compared amygdala and hippocampus activation for seven healthy subjects during two visual encoding tasks: serially presented single faces and faces presented as pairs. Single faces activated bilateral hippocampi, but not the amygdala. Paired faces activated bilateral amygdala, but only the left hippocampus. Subtraction of the two conditions revealed greater activation within the left amygdala and hippocampus during paired face encoding, suggesting that associative encoding activates a left-lateralized limbic network including the hippocampus and amygdala.