Synaptogenesis in the Prefrontal Cortex of Rhesus Monkeys

Since the turn of the century, the prefrontal association areasof the cerebral cortex have been thought to be among the lastregions of the cortical mantle to develop. We have examinedthe course of synaptogenesis in the macaque prefrontal cortexby quantitative electron microscopic analysis in 25 rhesus monkeysranging in age from embryonic day 47 (E47) to 20 years of age.A series of overlapping electron micrographs spanning the wholecortical thickness in each animal provided data on the number,the proportion, and the density of synapses per unit area (N_A)and per unit volume (N_V) of neuropil. The tempo and kinetics of synapse formation in prefrontal cortexclosely resemble those described for sensory and motor areas,particularly during the stages of synapse acquisition and overproduction(Rakic et al., 1986). In young embryos, we describe a precorticalphase (E47-E78), when synapses are found only above and below,but not within, the cortical plate. Following that, there isan early cortical phase, from E78 to E104, during which synapsesaccumulate within the cortical plate, initially exclusivelyon dendritic shafts. The next rapid phase of synaptogenesisbegins at 2 months before birth and ends approximately at 2months after birth, culminating with a mean density of 750 millionsynapses per cubic micrometer. This accumulation is largelyaccounted for by a selective increase in axospine synapses inthe supragranular layers. The period of explosive synaptic densityis followed by a protracted plateau stage that lasts from 2months to 3 years of age when synaptic density remains relativelyconstant. The final period of decline, from 3 years throughover 20 years of age, is marked by a slight but statisticallysignificant decline in synaptic density. Concurrent recruitment of synapses with that of sensory andmotor areas supports the concept that the initial establishmentof cortical circuitry is governed by general mechanisms commonto all areas, independent of their specific functional domain.The finding that synaptic density is relatively stable fromearly adolescence through puberty (the plateau period) is indicativeof the importance, in primates, of a consistent and high synapticdensity during the formative years when learning experiencesare most intense.     

Beneficial effect of a novel pentadecapeptide BPC 157 on gastric lesions induced by restraint stress,ethanol, indomethacin,and capsaicin neurotoxicity

Very recently, the integrity of capsaicin somatosensory neurons and their protection were suggested to be related to the activity in nociception of a newly discovered 15-amino acid peptide, BPC 157, shown to have strong beneficial effect on intestinal and liver lesions. Therefore, from this viewpoint, we have studied the gastroprotective effect of the pentadecapeptide BPC 157, on gastric lesions produced in rats by 96% ethanol, restraint stress, and indomethacin. The possible involvement of sensory neurons in the salutary actions of BPC 157 (10µg/kg, 10 ng/kg intraperitoneally) was studied with capsaicin, which has differential effects on sensory neurons: a high dose in adult (125 mg/kg subcutaneously, 3 months old) or administration (50 mg/kg subcutaneously) to neonatal animals (age of the 7 days) destroys sensory fibers, whereas a low dose (500µg/kg intraperitoneally) activates neurotransmitter release and protective effects on the mucosa. In the absence of capsaicin, BPC 157 protected gastric mucosa against ethanol, restraint, and indomethacin application. In the presence of neurotoxic doses of capsaicin, the negative influence of capsaicin on restraint, ethanol, or indomethacin lesions consistently affected salutary activity of BPC 157. However, BPC 157 protection was still evident in the capsaicin-treated rats (either treated as adults or as newborns) in all of these assays. Interestingly, after neonatal capsaicin treatment, a complete abolition of BPC gastroprotection was noted if BPC 157 was applied as a single nanogram-regimen, but the mucosal protection was fully reversed when the same dose was used daily. In line with the excitatory dose of capsaicin the beneficial effectiveness of BPC 157 appears to be increased as well. Taken together, these data provide evidence for complex synergistic interaction between the beneficial effectiveness of BPC 157 and peptidergic sensory afferent neuron activity.     

A critical role of neural-specific JNK3 for ischemic apoptosis

c-Jun N-terminal kinase (JNK) signaling is an important contributor to stress-induced apoptosis, but it is unclear whether JNK and its isoforms (JNK1, JNK2, and JNK3) have distinct roles in cerebral ischemia. Here we show that JNK1 is the major isoform responsible for the high level of basal JNK activity in the brain. In contrast, targeted deletion of Jnk3 not only reduces the stress-induced JNK activity, but also protects mice from brain injury after cerebral ischemia-hypoxia. The downstream mechanism of JNK3-mediated apoptosis may include the induction of Bim and Fas and the mitochondrial release of cytochrome c. These results suggest that JNK3 is a potential target for neuroprotection therapies in stroke.     

Distinct abnormalities of the primate prefrontal cortex caused by ionizing radiation in early or midgestation

Prenatal exposure of the brain to environmental insult causes different neurological symptoms and behavioral outcomes depending on the time of exposure. To examine the cellular bases for these differences, we exposed rhesus macaque fetuses to x‐rays during early gestation (embryonic day [E]30–E42), i.e., before the onset of corticogenesis, or in midgestation (E70–E81), when superficial cortical layers are generated. Animals were delivered at term (～E165), and the size and cellular composition of prefrontal association cortex (area 46) examined in adults using magnetic resonance imaging (MRI) and stereologic analysis. Both early and midgestational radiation exposure diminished the surface area and volume of area 46. However, early exposure spared cortical thickness and did not alter laminar composition, and due to higher cell density, neuron number was within the normal range. In contrast, exposure to x‐rays at midgestation reduced cortical thickness, mainly due to elimination of neurons destined for the superficial layers. A cell‐sparse gap, observed within layer III, was not filled by the later‐generated neurons destined for layer II, indicating that there is no subsequent replacement of the lost neurons. The distinct areal and laminar pathology consequent to temporally segregated irradiation is consistent with basic postulates of the radial unit hypothesis of cortical development. In addition, we show that an environmental disturbance inflicted in early gestation can induce subtle cytoarchitectonic alterations without loss of neurons, such as those observed in schizophrenia, whereas midgestational exposure causes selective elimination of neurons and cortical thinning as observed in some forms of mental retardation and fetal alcohol syndrome. J. Comp. Neurol. 521:1040–1053, 2013. © 2012 Wiley Periodicals, Inc.     

Antibodies to cannabinoid type 1 receptor co‐react with stomatin‐like protein 2 in mouse brain mitochondria

Anti‐cannabinoid type 1 receptor (CB₁) polyclonal antibodies are widely used to detect the presence of CB₁ in a variety of brain cells and their organelles, including neuronal mitochondria. Surprisingly, we found that anti‐CB₁ sera, in parallel with CB₁, also recognize the mitochondrial protein stomatin‐like protein 2. In addition, we show that the previously reported effect of synthetic cannabinoid WIN 55,212‐2 on mitochondrial complex III respiration is not detectable in purified mitochondrial preparations. Thus, our study indicates that a direct relationship between endocannabinoid signaling and mitochondrial functions in the cerebral cortex seems unlikely, and that caution should be taken interpreting findings obtained using anti‐CB₁ antibodies.     

Primary cilia regulate hippocampal neurogenesis by mediating sonic hedgehog signaling

Primary cilia are present on mammalian neurons and glia, but their function is largely unknown. We generated conditional homozygous mutant mice for a gene we termed Stumpy. Mutants lack cilia and have conspicuous abnormalities in postnatally developing brain regions, including a hypoplasic hippocampus characterized by a primary deficiency in neural stem cells known as astrocyte-like neural precursors (ALNPs). Previous studies suggested that primary cilia mediate sonic hedgehog (Shh) signaling. Here, we find that loss of ALNP cilia leads to abrogated Shh activity, increased cell cycle exit, and morphological abnormalities in ALNPs. Processing of Gli3, a mediator of Shh signaling, is also altered in the absence of cilia. Further, key mediators of the Shh pathway localize to ALNP cilia. Thus, selective targeting of Shh machinery to primary cilia confers to ALNPs the ability to differentially respond to Shh mitogenic signals compared to neighboring cells. Our data suggest these organelles are cellular “antennae” critically required to modulate ALNP behavior.     

Antibacterial,antioxidant and anti-proliferative properties and zinc content of five south Portugal herbs

Context: Crataegus monogyna L. (Rosaceae) (CM), Equisetum telmateia L. (Equisataceae) (ET), Geranium purpureum Vil. (Geraniaceae) (GP), Mentha suaveolens Ehrh. (Lamiaceae) (MS), and Lavandula stoechas L. spp. luisieri (Lamiaceae) (LS) are all medicinal.

Objective: To evaluate the antioxidant, antiproliferative and antimicrobial activities of plant extracts and quantify individual phenolics and zinc.

Material and methods: Aerial part extracts were prepared with water (W), ethanol (E) and an 80% mixture (80EW). Antioxidant activity was measured with TAA, FRAP and RP methods. Phenolics were quantified with a HPLC. Zinc was quantified using voltammetry. Antibacterial activity (after 48?h) was tested using Enterococcus faecalis, Bacillus cereus, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Listeria monocytogenes. Antiproliferative activity (after 24?h) was tested using HEP G2 cells and fibroblasts.

Results: Solvents influenced results; the best were E and 80EW. GP had the highest antioxidant activity (TAA and FRAP of 536.90?mg AAE/g dw and 783.48?mg TE/g dw, respectively). CM had the highest zinc concentration (37.21?mg/kg) and phenolic variety, with neochlorogenic acid as the most abundant (92.91?mg/100?g dw). LS was rich in rosmarinic acid (301.71?mg/100?g dw). GP and LS inhibited the most microorganisms: B. cereus, E. coli and S. aureus. GP also inhibited E. faecalis. CM had the lowest MIC: 5830?μg/mL. The antibacterial activity is explained by the phenolics present. LS and CM showed the most significant anti-proliferative activity, which is explained by their zinc content.

Conclusion: The most promising plants for further studies are CM, LS and GP.     

Mitotic spindle rotation and mode of cell division in the developing telencephalon

The mode of neural stem cell division in the forebrain proliferative zones profoundly influences neocortical growth by regulating the number and diversity of neurons and glia. Long-term time-lapse multiphoton microscopy of embryonic mouse cortex reveals new details of the complex three-dimensional rotation and oscillation of the mitotic spindle before stem cell division. Importantly, the duration and amplitude of spindle movement predicts and specifies the eventual mode of mitotic division. These technological advances have provided dramatic data and insights into the kinetics of neural stem cell division by elucidating the involvement of spindle rotation in selection of the cleavage plane and the mode of neural stem cell division that together determine the size of the mammalian neocortex.     

Improving the delivery of continuous renal replacement therapy using regional citrate anticoagulation

AIMS: Regional citrate anticoagulation during acute renal replacement therapy (RRT) effectively prevents extracorporeal thrombosis and avoids bleeding risk. There have been a number of citrate anticoagulation protocols published; but a simple and predictable scheme with standardized components and procedures, as well as clearly defined citrate pharmacokinetics, is needed for continuous RRT (CRRT) that is now used frequently in the critical care setting. The present study sets forth methodology with standardized blood flow and dialysate composition, and with citrate and calcium infusions that are quantitatively linked to extracorporeal blood flow rate--a predictable and easily replicated CRRT paradigm. MATERIALS AND METHODS: CRRT using continuous venovenous hemofiltration with dialysis (CVVHD) was standardized using 150-200 ml/min blood flow, calcium-free dialysate with only moderate sodium (135 mEq/l) and bicarbonate (28 mEq/l) concentrations, and ultrafiltration limited to that needed for overall fluid balance in the intensive care unit. Citrate infusion (ACD-A solution) into the extracorporeal blood and calcium repletion in blood returned to the patient were proportional to blood flow. Anticoagulation was accomplished by keeping extracorporeal ionized calcium below 0.4 mM/l. Filter performance, citrate removal and changes in calcium, sodium and alkali were evaluated longitudinally. RESULTS: CVVHD using this protocol delivered urea clearance exceeding 2 l/h (48 l/d) when filter function was sustained. Filter longevity was markedly improved using citrate when compared with standard heparin anticoagulation, and nursing time spent on initiating and troubleshooting CRRT was approximately halved using this protocol. Sieving coefficients for urea, creatinine and citrate were approximately 0.9 and were sustained through nearly 3 days of filter use. Citrate clearance and removal were quantitatively linked to dialysate and ultrafiltration flow, resulting in 35-50% direct removal of the citrate-calcium chelate and reduced systemic citrate load. Serum tonicity and acid-base status were not problematic. The only notable side effect was modest calcium accumulation that necessitated reduction in calcium repletion rate. CONCLUSIONS: CVVHD is well suited to regional citrate anticoagulation. The present protocol is straightforward and predictable, with minor metabolic consequences that can be anticipated and adjusted. These results commend regional citrate anticoagulation to wider application.