Differences in frontal cortical activation by a working memory task after substitution of risperidone for typical antipsychotic drugs in patients with schizophrenia

Antipsychotic drug treatment of schizophrenia may be complicated by side effects of widespread dopaminergic antagonism, including exacerbation of negative and cognitive symptoms due to frontal cortical hypodopaminergia. Atypical antipsychotics have been shown to enhance frontal dopaminergic activity in animal models. We predicted that substitution of risperidone for typical antipsychotic drugs in the treatment of schizophrenia would be associated with enhanced functional activation of frontal cortex. We measured cerebral blood oxygenation changes during periodic performance of a verbal working memory task, using functional MRI, on two occasions (baseline and 6 weeks later) in two cohorts of schizophrenic patients. One cohort (n = 10) was treated with typical antipsychotic drugs throughout the study. Risperidone was substituted for typical antipsychotics after baseline assessment in the second cohort (n = 10). A matched group of healthy volunteers (n = 10) was also studied on a single occasion. A network comprising bilateral dorsolateral prefrontal and lateral premotor cortex, the supplementary motor area, and posterior parietal cortex was activated by working memory task performance in both the patients and comparison subjects. A two-way analysis of covariance was used to estimate the effect of substituting risperidone for typical antipsychotics on power of functional response in the patient group. Substitution of risperidone increased functional activation in right prefrontal cortex, supplementary motor area, and posterior parietal cortex at both voxel and regional levels of analysis. This study provides direct evidence for significantly enhanced frontal function in schizophrenic patients after substitution of risperidone for typical antipsychotic drugs, and it indicates the potential value of functional MRI as a tool for longitudinal assessment of psychopharmacological effects on cerebral physiology.

Suppression of protein synthesis in brain during hibernation involves inhibition of protein initiation and elongation

Protein synthesis (PS) has been considered essential to sustain mammalian life, yet was found to be virtually arrested for weeks in brain and other organs of the hibernating ground squirrel, Spermophilus tridecemlineatus. PS, in vivo, was below the limit of autoradiographic detection in brain sections and, in brain extracts, was determined to be 0.04% of the average rate from active squirrels. Further, it was reduced 3-fold in cell-free extracts from hibernating brain at 37°C, eliminating hypothermia as the only cause for protein synthesis inhibition (active, 0.47 ± 0.08 pmol/mg protein per min; hibernator, 0.16 ± 0.05 pmol/mg protein per min, P < 0.001). PS suppression involved blocks of initiation and elongation, and its onset coincided with the early transition phase into hibernation. An increased monosome peak with moderate ribosomal disaggregation in polysome profiles and the greatly increased phosphorylation of eIF2α are both consistent with an initiation block in hibernators. The elongation block was demonstrated by a 3-fold increase in ribosomal mean transit times in cell-free extracts from hibernators (active, 2.4 ± 0.7 min; hibernator, 7.1 ± 1.4 min, P < 0.001). No abnormalities of ribosomal function or mRNA levels were detected. These findings implicate suppression of PS as a component of the regulated shutdown of cellular function that permits hibernating ground squirrels to tolerate “trickle” blood flow and reduced substrate and oxygen availability. Further study of the factors that control these phenomena may lead to identification of the molecular mechanisms that regulate this state.

Impaired metabolic modulation of α-adrenergic vasoconstriction in dystrophin-deficient skeletal muscle

The neuronal isoform of nitric oxide synthase (nNOS) is highly expressed in mammalian skeletal muscle, but its functional role has not been defined. NO has been implicated in the local metabolic regulation of blood flow in contracting skeletal muscle in part by antagonizing sympathetic vasoconstriction. We therefore hypothesized that nNOS in skeletal muscle is the source of the NO mediating the inhibition of sympathetic vasoconstriction in contracting muscle. In the mdx mouse, a model of Duchenne muscular dystrophy in which dystrophin deficiency results in greatly reduced expression of nNOS in skeletal muscle, we found that the normal ability of skeletal muscle contraction to attenuate α-adrenergic vasoconstriction is defective. Similar results were obtained in mutant mice that lack the gene encoding nNOS. Together these data suggest a specific role for nNOS in the local metabolic inhibition of α-adrenergic vasoconstriction in active skeletal muscle.

Noninvasive measurement of gene expression in skeletal muscle

We have developed a noninvasive detection method for expression of viral-mediated gene transfer. A recombinant adenovirus was constructed by using the gene for arginine kinase (AK), which is the invertebrate correlate to the vertebrate ATP-buffering enzyme, creatine kinase. Gene expression was noninvasively monitored using 31P-magnetic resonance spectroscopy (31P-MRS). The product of the AK enzyme, phosphoarginine (PArg), served as an MRS-visible reporter of AK expression. The recombinant adenovirus coding for arginine kinase (rAdCMVAK) was injected into the right hindlimbs of neonatal mice. Two weeks after injection of rAdCMVAK, a unique 31P-MRS resonance was observed. It was observable in all rAdCMVAK injected hindlimbs and was not present in the contralateral control or the vehicle injected limb. PArg and phosphocreatine (PCr) concentrations were calculated to be 11.6 ± 0.90 and 13.6 ± 1.1 mM respectively in rAdCMVAK injected limbs. AK activity was demonstrated in vivo by monitoring the decreases in PArg and ATP resonances during prolonged ischemia. After 1 h of ischemia intracellular pH was 6.73 ± 0.06, PCr/ATP was decreased by 77 ± 8%, whereas PArg/ATP was decreased by 50 ± 15% of basal levels. PArg and PCr returned to basal levels within 5 min of the restoration of blood flow. AK activity persisted for at least 8 mo after injection, indicating that adenoviral-mediated gene transfer can produce stable expression for long periods of time. Therefore, the cDNA encoding AK provides a useful reporter gene that allows noninvasive and repeated monitoring of gene expression after viral mediated gene transfer to muscle.

Portosystemic shunting and persistent fetal vascular structures in aryl hydrocarbon receptor-deficient mice

A physiological examination of mice harboring a null allele at the aryl hydrocarbon (Ah) locus revealed that the encoded aryl hydrocarbon receptor plays a role in the resolution of fetal vascular structures during development. Although the aryl hydrocarbon receptor is more commonly studied for its role in regulating xenobiotic metabolism and dioxin toxicity, a developmental role of this protein is supported by the observation that Ah null mice display smaller livers, reduced fecundity, and decreased body weights. Upon investigating the liver phenotype, we found that the decrease in liver size is directly related to a reduction in hepatocyte size. We also found that smaller hepatocyte size is the result of massive portosystemic shunting in null animals. Colloidal carbon uptake and microsphere perfusion studies indicated that 56% of portal blood flow bypasses the liver sinusoids. Latex corrosion casts and angiography demonstrated that shunting is consistent with the existence of a patent ductus venosus in adult animals. Importantly, fetal vascular structures were also observed at other sites. Intravital microscopy demonstrated an immature sinusoidal architecture in the liver and persistent hyaloid arteries in the eyes of adult Ah null mice, whereas corrosion casting experiments described aberrations in kidney vascular patterns.

In vivo magnetic resonance vascular imaging using laser-polarized 3He microbubbles

Laser-polarized gases (3He and 129Xe) are currently being used in magnetic resonance imaging as strong signal sources that can be safely introduced into the lung. Recently, researchers have been investigating other tissues using 129Xe. These studies use xenon dissolved in a carrier such as lipid vesicles or blood. Since helium is much less soluble than xenon in these materials, 3He has been used exclusively for imaging air spaces. However, considering that the signal of 3He is more than 10 times greater than that of 129Xe for presently attainable polarization levels, this work has focused on generating a method to introduce 3He into the vascular system. We addressed the low solubility issue by producing suspensions of 3He microbubbles. Here, we provide the first vascular images obtained with laser-polarized 3He. The potential increase in signal and absence of background should allow this technique to produce high-resolution angiographic images. In addition, quantitative measurements of blood flow velocity and tissue perfusion will be feasible.

Integrin activation controls metastasis in human breast cancer

Metastasis is the primary cause of death in human breast cancer. Metastasis to bone, lungs, liver, and brain involves dissemination of breast cancer cells via the bloodstream and requires adhesion within the vasculature. Blood cell adhesion within the vasculature depends on integrins, a family of transmembrane adhesion receptors, and is regulated by integrin activation. Here we show that integrin αvβ3 supports breast cancer cell attachment under blood flow conditions in an activation-dependent manner. Integrin αvβ3 was found in two distinct functional states in human breast cancer cells. The activated, but not the nonactivated, state supported tumor cell arrest during blood flow through interaction with platelets. Importantly, activated αvβ3 was expressed by freshly isolated metastatic human breast cancer cells and variants of the MDA-MB 435 human breast cancer cell line, derived from mammary fat pad tumors or distant metastases in severe combined immunodeficient mice. Expression of constitutively activated mutant αvβ3D723R, but not αvβ3WT, in MDA-MB 435 cells strongly promoted metastasis in the mouse model. Thus breast cancer cells can exhibit a platelet-interactive and metastatic phenotype that is controlled by the activation of integrin αvβ3. Consequently, alterations within tumors that lead to the aberrant control of integrin activation are expected to adversely affect the course of human breast cancer.

Neuroimaging evidence implicating cerebellum in the experience of hypercapnia and hunger for air

Recent neuroimaging and neurological data implicate cerebellum in nonmotor sensory, cognitive, vegetative, and affective functions. The present study assessed cerebellar responses when the urge to breathe is stimulated by inhaled CO2. Ventilation changes follow arterial blood partial pressure CO2 changes sensed by the medullary ventral respiratory group (VRG) and hypothalamus, entraining changes in midbrain, pons, thalamus, limbic, paralimbic, and insular regions. Nearly all these areas are known to connect anatomically with the cerebellum. Using positron emission tomography, we measured regional brain blood flow during acute CO2-induced breathlessness in humans. Separable physiological and subjective effects (air hunger) were assessed by comparisons with various respiratory control conditions. The conjoint physiological effects of hypercapnia and the consequent air hunger produced strong bilateral, near-midline activations of the cerebellum in anterior quadrangular, central, and lingula lobules, and in many areas of posterior quadrangular, tonsil, biventer, declive, and inferior semilunar lobules. The primal emotion of air hunger, dissociated from hypercapnia, activated midline regions of the central lobule. The distributed activity across the cerebellum is similar to that for thirst, hunger, and their satiation. Four possible interpretations of cerebellar function(s) here are that: it subserves implicit intentions to access air; it provides predictive internal models about the consequences of CO2 inhalation; it modulates emotional responses; and that while some cerebellar regions monitor sensory acquisition in the VRG (CO2 concentration), others influence VRG to adjust respiratory rate to optimize partial pressure CO2, and others still monitor and optimize the acquisition of other sensory data in service of air hunger aroused vigilance.

Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability

Nitric oxide (NO) plays a critical role in vascular endothelial growth factor (VEGF)-induced angiogenesis and vascular hyperpermeability. However, the relative contribution of different NO synthase (NOS) isoforms to these processes is not known. Here, we evaluated the relative contributions of endothelial and inducible NOS (eNOS and iNOS, respectively) to angiogenesis and permeability of VEGF-induced angiogenic vessels. The contribution of eNOS was assessed by using an eNOS-deficient mouse, and iNOS contribution was assessed by using a selective inhibitor [l-N6-(1-iminoethyl) lysine, l-NIL] and an iNOS-deficient mouse. Angiogenesis was induced by VEGF in type I collagen gels placed in the mouse cranial window. Angiogenesis, vessel diameter, blood flow rate, and vascular permeability were proportional to NO levels measured with microelectrodes: Wild-type (WT) ≥ WT with l-NIL or iNOS−/− > eNOS−/− ≥ eNOS−/− with l-NIL. The role of NOS in VEGF-induced acute vascular permeability increase in quiescent vessels also was determined by using eNOS- and iNOS-deficient mice. VEGF superfusion significantly increased permeability in both WT and iNOS−/− mice but not in eNOS−/− mice. These findings suggest that eNOS plays a predominant role in VEGF-induced angiogenesis and vascular permeability. Thus, selective modulation of eNOS activity is a promising strategy for altering angiogenesis and vascular permeability in vivo.

Behind the scenes of functional brain imaging: A historical and physiological perspective

At the forefront of cognitive neuroscience research in normal humans are the new techniques of functional brain imaging: positron emission tomography and magnetic resonance imaging. The signal used by positron emission tomography is based on the fact that changes in the cellular activity of the brain of normal, awake humans and laboratory animals are accompanied almost invariably by changes in local blood flow. This robust, empirical relationship has fascinated scientists for well over a hundred years. Because the changes in blood flow are accompanied by lesser changes in oxygen consumption, local changes in brain oxygen content occur at the sites of activation and provide the basis for the signal used by magnetic resonance imaging. The biological basis for these signals is now an area of intense research stimulated by the interest in these tools for cognitive neuroscience research.

Event-related brain potentials in the study of visual selective attention

Event-related brain potentials (ERPs) provide high-resolution measures of the time course of neuronal activity patterns associated with perceptual and cognitive processes. New techniques for ERP source analysis and comparisons with data from blood-flow neuroimaging studies enable improved localization of cortical activity during visual selective attention. ERP modulations during spatial attention point toward a mechanism of gain control over information flow in extrastriate visual cortical pathways, starting about 80 ms after stimulus onset. Paying attention to nonspatial features such as color, motion, or shape is manifested by qualitatively different ERP patterns in multiple cortical areas that begin with latencies of 100–150 ms. The processing of nonspatial features seems to be contingent upon the prior selection of location, consistent with early selection theories of attention and with the hypothesis that spatial attention is “special.”

In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function.

To examine the hypothesis that surface P-selectin-positive (degranulated) platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo. Washed platelets prepared from nonhuman primates (baboons) were labeled with PKH2 (a lipophilic fluorescent dye), thrombin-activated, washed, and reinfused into the same baboons. Three-color whole blood flow cytometry was used to simultaneously (i) identify platelets with a mAb directed against glycoprotein (GP)IIb-IIIa (integrin alpha 11b beta 3), (ii) distinguish infused platelets by their PKH2 fluorescence, and (iii) analyze platelet function with mAbs. Two hours after infusion of autologous thrombin-activated platelets (P-selectin-positive, PKH2-labeled), 95 +/- 1% (mean +/- SEM, n = 5) of the circulating PKH2-labeled platelets had become P-selectin-negative. Compared with platelets not activated with thrombin preinfusion, the recovery of these circulating PKH2-labeled, P-selectin-negative platelets was similar 24 h after infusion and only slightly less 48 h after infusion. The loss of platelet surface P-selectin was fully accounted for by a 67.1 +/- 16.7 ng/ml increase in the plasma concentration of soluble P-selectin. The circulating PKH2-labeled, P-selectin-negative platelets were still able to function in vivo, as determined by their (i) participation in platelet aggregates emerging from a bleeding time wound, (ii) binding to Dacron in an arteriovenous shunt, (iii) binding of mAb PAC1 (directed against the fibrinogen binding site on GPIIb-IIIa), and (iv) generation of procoagulant platelet-derived microparticles. In summary, (i) circulating degranulated platelets rapidly lose surface P-selectin to the plasma pool, but continue to circulate and function; and (ii) we have developed novel three-color whole blood flow cytometric methods for tracking of platelets and measurement of platelet function in vivo.

Conscious recollection and the human hippocampal formation: evidence from positron emission tomography.

We used positron emission tomography (PET) to examine the role of the hippocampal formation in implicit and explicit memory. Human volunteers studied a list of familiar words, and then they either provided the first word that came to mind in response to three-letter cues (implicit memory) or tried to recall studied words in response to the same cues (explicit memory). There was no evidence of hippocampal activation in association with implicit memory. However, priming effects on the implicit memory test were associated with decreased activity in extrastriate visual cortex. On the explicit memory test, subjects recalled many target words in one condition and recalled few words in a second condition, despite trying to remember them. Comparisons between the two conditions showed that blood-flow increases in the hippocampal formation are specifically associated with the conscious recollection of studied words, whereas blood-flow increases in frontal regions are associated with efforts to retrieve target words. Our results help to clarify some puzzles concerning the role of the hippocampal formation in human memory.