Angiotensinergic and noradrenergic neurons in the rat and human heart.

Although the physiological and pharmacological evidences suggest a role for angiotensin II (Ang II) with the mammalian heart, the source and precise location of Ang II are unknown. To visualize and quantitate Ang II in atria, ventricular walls and interventricular septum of the rat and human heart and to explore the feasibility of local Ang II production and function, we investigated by different methods the expression of proteins involved in the generation and function of Ang II. We found mRNA of angiotensinogen (Ang-N), of angiotensin converting enzyme, of the angiotensin type receptors AT(1A) and AT(2) (AT(1B) not detected) as well as of cathepsin D in any part of the hearts. No renin mRNA was traceable. Ang-N mRNA was visualized by in situ hybridization in atrial ganglial neurons. Ang II and dopamine-β-hydroxylase (DβH) were either colocalized inside the same neuronal cell or the neurons were specialized for Ang II or DβH. Within these neurons, the vesicular acetylcholine transporter (VAChT) was neither colocalized with Ang II nor DβH, but VAChT-staining was found with synapses en passant encircle these neuronal cells. The fibers containing Ang II exhibited with blood vessels and with cardiomyocytes supposedly angiotensinergic synapses en passant. In rat heart, right atrial median Ang II concentration appeared higher than septal and ventricular Ang II. The distinct colocalization of neuronal Ang II with DβH in the heart may indicate that Ang II participates together with norepinephrine in the regulation of cardiac functions: Produced as a cardiac neurotransmitter Ang II may have inotropic, chronotropic or dromotropic effects in atria and ventricles and contributes to blood pressure regulation.

Changes in volume, surface estimate, three-dimensional shape and total number of neurons of the human primary visual cortex from midgestation until old age.

Macroscopic features such as volume, surface estimate, thickness and caudorostral length of the human primary visual cortex (Brodman's area 17) of 46 human brains between midgestation and 93 years were studied by means of camera lucida drawings from serial frontal sections. Individual values were best fitted by a logistic function from midgestation to adulthood and by a regression line between adulthood and old age. Allometric functions were calculated to study developmental relationships between all the features. The three-dimensional shape of area 17 was also reconstructed from the serial sections in 15 cases and correlated with the sequence of morphological events. The sulcal pattern of area 17 begins to develop around 21 weeks of gestation but remains rather simple until birth, while it becomes more convoluted, particularly in the caudal part, during the postnatal period. Until birth, a large increase in cortical thickness (about 83% of its mean adult value) and caudorostral length (69%) produces a moderate increase in cortical volume (31%) and surface estimate (40%) of area 17. After birth, the cortical volume and surface undergo their maximum growth rate, in spite of a rather small increase in cortical thickness and caudorostral length. This is due to the development of the pattern of gyrification within and around the calcarine fissure. All macroscopic features have reached the mean adult value by the end of the first postnatal year. With aging, the only features to undergo significant regression are the cortical surface estimate and the caudorostral length. The total number of neurons in area 17 shows great interindividual variability at all ages. No decrease in the postnatal period or in aging could be demonstrated.

Nerve growth factor (NGF) stimulation of cholinergic telencephalic neurons in aggregating cell cultures.

The addition of nerve growth factor (2.5S NGF) to serum-free aggregating cell cultures of fetal rat telencephalon greatly stimulated the developmental increase in choline acetyltransferase activity. Two other neuronal enzymes, acetylcholinesterase and glutamic acid decarboxylase, showed only slightly increased activities after NGF treatment whereas the total protein content of the cultures and the activity of 2',3'- cyclic nucleotide phosphodiesterase remained unchanged. The stimulation of choline acetyltransferase was dependent on the NGF media concentrations, showing a 50% maximum effect (120% increase) at approximately 3 ng/ml (10-10 M 2.5S NGF). NGF treatments during different culture periods showed that the cholinergic neurons remained responsive for at least 19 days. The continued treatment was the most effective; however, an initial treatment for only 5 days still caused a significant stimulation of choline acetyltransferase on day 19. The observed stimulation appeared to be specific to NGF. Univalent antibody fragments (Fab) against 2.5S NGF completely abolished the NGF-dependent increase in choline acetyltransferase activity, whereas Fab fragments of control IgG were ineffective. Furthermore, angiotensin II, added in high amounts to the cultures, showed no stimulatory effect. The present results suggest that certain populations of rat brain neurons are responsive to nerve growth factor.

P2Y1 receptor-evoked glutamate exocytosis from astrocytes: control by tumor necrosis factor-alpha and prostaglandins.

ATP, released by both neurons and glia, is an important mediator of brain intercellular communication. We find that selective activation of purinergic P2Y1 receptors (P2Y1R) in cultured astrocytes triggers glutamate release. By total internal fluorescence reflection imaging of fluorescence-labeled glutamatergic vesicles, we document that such release occurs by regulated exocytosis. The stimulus-secretion coupling mechanism involves Ca2+ release from internal stores and is controlled by additional transductive events mediated by tumor necrosis factor-alpha (TNFalpha) and prostaglandins (PG). P2Y1R activation induces release of both TNFalpha and PGE2 and blocking either one significantly reduces glutamate release. Accordingly, astrocytes from TNFalpha-deficient (TNF(-/-)) or TNF type 1 receptor-deficient (TNFR1(-/-)) mice display altered P2Y1R-dependent Ca2+ signaling and deficient glutamate release. In mixed hippocampal cultures, the P2Y1R-evoked process occurs in astrocytes but not in neurons or microglia. P2Y1R stimulation induces Ca2+ -dependent glutamate release also from acute hippocampal slices. The process in situ displays characteristics resembling those in cultured astrocytes and is distinctly different from synaptic glutamate release evoked by high K+ stimulation as follows: (a) it is sensitive to cyclooxygenase inhibitors; (b) it is deficient in preparations from TNF(-/-) and TNFR1(-/-) mice; and (c) it is inhibited by the exocytosis blocker bafilomycin A1 with a different time course. No glutamate release is evoked by P2Y1R-dependent stimulation of hippocampal synaptosomes. Taken together, our data identify the coupling of purinergic P2Y1R to glutamate exocytosis and its peculiar TNFalpha- and PG-dependent control, and we strongly suggest that this cascade operates selectively in astrocytes. The identified pathway may play physiological roles in glial-glial and glial-neuronal communication.

Thrombin-induced ischemic tolerance is prevented by inhibiting c-jun N-terminal kinase.

We have studied ischemic tolerance induced by the serine protease thrombin in two different models of experimental ischemia. In organotypic hippocampal slice cultures, we demonstrate that incubation with low doses of thrombin protects neurons against a subsequent severe oxygen and glucose deprivation. L-JNKI1, a highly specific c-jun N-terminal kinase (JNK) inhibitor, and a second specific JNK inhibitor, SP600125, prevented thrombin preconditioning (TPC). We also show that the exposure to thrombin increases the level of phosphorylated c-jun, the major substrate of JNK. TPC, in vivo, leads to significantly smaller lesion sizes after a 30-min middle cerebral artery occlusion (MCAo), and the preconditioned mice were better off in the three tests used to evaluate functional recovery. In accordance with in vitro results, TPC in vivo was prevented by administration of L-JNKI1, supporting a role for JNK in TPC. These results, from two different TPC models and with two distinct JNK inhibitors, show that JNK is likely to be involved in TPC.

Neurons in the corpus callosum of the cat during postnatal development.

The corpus callosum (CC) is a major telencephalic commissure containing mainly cortico-cortical axons and glial cells. We have identified neurons in the CC of the cat and quantified their number at different postnatal ages. An antibody against microtubule-associated protein 2 was used as a marker of neurons. Immunocytochemical double-labelling with neuron-specific enolase or gamma-aminobutyric acid antibodies in the absence of glial fibrillary acidic protein positivity confirmed the neuronal phenotype of these cells. CC neurons were also stained with anti-calbindin and anti-calretinin antibodies, typical for interneurons, and with an anti-neurofilament antibody, which in neocortex detects pyramidal neurons. Together, these findings suggest that the CC contains a mixed population of neuronal types. The quantification was corrected for double counting of adjacent sections and volume changes during CC development. Our data show that CC neurons are numerous early postnatally, and their number decreases with age. At birth, about 570 neurons are found within the CC boundaries and their number drops to about 200 in the adult. The distribution of the neurons within the CC also changes in development. Initially, many neurons are found throughout the CC, while at later ages they become restricted to the boundaries of the CC, and in the adult to the rostrum of the CC close to the septum pellucidum or to the indusium griseum. Although origin and function of transient CC neurons in development and in adulthood remain unknown, they are likely to be interstitial neurons. Some of them have well-developed and differentiated processes and resemble pyramidal cells or interneurons. An axon-guiding function during the early postnatal period can not be excluded.

Building hippocampal circuits to learn and remember: insights into the development of human memory

The hippocampal formation is essential for the processing of episodic memories for autobiographical events that happen in unique spatiotemporal contexts. Interestingly, before 2 years of age, children are unable to form or store episodic memories for recall later in life, a phenomenon known as infantile amnesia. From 2 to 7 years of age, there are fewer memories than predicted based on a forgetting function alone, a phenomenon known as childhood amnesia. Here, we discuss the postnatal maturation of the primate hippocampal formation with the goal of characterizing the development of the neurobiological substrates thought to subserve the emergence of episodic memory. Distinct regions, layers and cells of the hippocampal formation exhibit different profiles of structural and molecular development during early postnatal life. The protracted period of neuronal addition and maturation in the dentate gyrus is accompanied by the late maturation of specific layers in different hippocampal regions that are located downstream from the dentate gyrus, particularly CA3. In contrast, distinct layers in several hippocampal regions, particularly CA1, which receive direct projections from the entorhinal cortex, exhibit an early maturation. In addition, hippocampal regions that are more highly interconnected with subcortical structures, including the subiculum, presubiculum, parasubiculum and CA2, mature even earlier. These findings, together with our studies of the development of human spatial memory, support the hypothesis that the differential maturation of distinct hippocampal circuits might underlie the differential emergence of specific "hippocampus-dependent" memory processes, culminating in the emergence of episodic memory concomitant with the maturation of all hippocampal circuits.

Coagulation Factor Xa Activates Thrombin and Signals Ischemic Neuronal Death Via Par-1 and JNK in Ischemic Neural Tissue

Background: The coagulation factor thrombin mediates ischemic neuronal deathand, at a low concentration, induces tolerance to ischemia.We investigated its modeof activation in ischemic neural tissue using an in vitro approach to distinguish therole of circulating coagulation factors from endogenous cerebral mechanisms. Wealso studied the signalling pathway downstream of thrombin in ischemia and afterthrombin preconditioning.Methods: Rat organotypic hippocampal slice cultures to 30 minute oxygen (5%)and glucose (1 mmol/L) deprivation (OGD).Results: Selective factor Xa (FXa) inhibition by fondaparinux during and afterOGD significantly reduced neuronal death in the CA1 after 48 hours. Thrombinactivity was increased in the medium 24 hours after OGD and this increasewas prevented by fondaparinux suggesting that FXa catalyzes the conversion ofprothrombin to thrombin in neural tissue after ischemia in vitro. Treatment withSCH79797, a selective antagonist of the thrombin receptor protease activatedreceptor-1 (PAR-1), significantly decreased neuronal cell death indicating thatthrombin signals ischemic damage via PAR-1. The JNK pathway plays an importantrole in cerebral ischemia and we observed activation of the JNK substrate,c-Jun in our model. Both the FXa inhibitor, fondaparinux and the PAR-1 antagonistSCH79797, decreased the level of phospho-c-Jun Ser73. After thrombin preconditioningc-Jun was activated by phosphorylation in the nuclei of neurons of the CA1.Treatment with a synthetic thrombin receptor agonist resulted in the same c-Junactivation profile and protection against subsequent OGD indicating that thrombinalso signals via PAR-1 and c-Jun in cell protection.Conclusion: These results indicate that FXa activates thrombin in cerebral ischemia,leading via PAR-1 to the activation of the JNK pathway resulting in neuronal death.Thrombin induced tolerance also involves PAR-1 and JNK, revealing commonfeatures in cell death and survival signalling.

Deficit in memory consolidation (abnormal forgetting rate) in childhood temporal lobe epilepsy. Pre and postoperative long-term observation.

Deficits in memory consolidation have been reported in adult patients with epilepsy but, not to our knowledge, in children. We report the long-term follow-up (9 y. o. to 18 y. o.) of a boy who suffered from temporal lobe epilepsy and underwent a left temporal lobectomy with amygdalo-hippocampal resection at the age of 10. He showed an abnormal forgetting rate when trying to encode new information and a significant deficit for retrieving remote episodic memories (when compared with his twin brother), both consistent with a consolidation disorder. His memory condition slightly improved after cessation of the epilepsy, nevertheless did not normalize. No standard memory assessment could pinpoint his memory problem, hence an adapted methodology was needed. We discuss the nature of the memory deficit, its possible causes and its clinical implications.

Induction of brain aquaporin 9 (AQP9) in catecholaminergic neurons in diabetic rats.

Aquaporin 9 facilitates the diffusion of water but also glycerol and monocarboxylates, known as brain energy substrates. AQP9 was recently observed in catecholaminergic neurons that are implicated in energy homeostasis and also possibly in neuroendocrine effects of diabetes. Recently it has been observed that the level of AQP9 expression in hepatocytes is sensitive to the blood concentration of insulin. Furthermore, insulin injection in the brain is known to be related to the energy homeostasis. Based on these observations, we investigated if the concentration of insulin affects the level of brain AQP9 expression and if so, in which cell types. This study has been carried out, in a model of the diabetic rat generated by streptozotocin injection and on brainstem slices. In diabetic rats showing a decrease in systemic insulin concentration, AQP9 is only increased in brain areas containing catecholaminergic neurons. In contrast, no significant change is detected in the cerebral cortex and the cerebellum. Using immunocytochemistry, we are able to show that the increase in AQP9 expression is specifically present in catecholaminergic neurons. In brainstem slice cultures, 2 microM insulin induces a significant decrease in AQP9 protein levels 6 h after application, suggesting that brain AQP9 is also regulated by the insulin. These results show that the level of expression of brain AQP9 is affected by variations of the concentration of insulin in a diabetic model and in vitro.

Macrophage migration inhibitory factor is critically involved in basal and fluoxetine-stimulated adult hippocampal cell proliferation and in anxiety, depression, and memory-related behaviors.

Intensive research is devoted to unravel the neurobiological mechanisms mediating adult hippocampal neurogenesis, its regulation by antidepressants, and its behavioral consequences. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that is expressed in the CNS, where its function is unknown. Here, we show, for the first time, the relevance of MIF expression for adult hippocampal neurogenesis. We identify MIF expression in neurogenic cells (in stem cells, cells undergoing proliferation, and in newly proliferated cells undergoing maturation) in the subgranular zone of the rodent dentate gyrus. A causal function for MIF in cell proliferation was shown using genetic (MIF gene deletion) and pharmacological (treatment with the MIF antagonist Iso-1) approaches. Behaviorally, genetic deletion of MIF resulted in increased anxiety- and depression-like behaviors, as well as of impaired hippocampus-dependent memory. Together, our studies provide evidence supporting a pivotal function for MIF in both basal and antidepressant-stimulated adult hippocampal cell proliferation. Moreover, loss of MIF results in a behavioral phenotype that, to a large extent, corresponds with alterations predicted to arise from reduced hippocampal neurogenesis. These findings underscore MIF as a potentially relevant molecular target for the development of treatments linked to deficits in neurogenesis, as well as to problems related to anxiety, depression, and cognition.

Carbonic anhydrase activity in primary sensory neurons. II. Influence of environmental factors on the phenotypic expression of the enzyme in dissociated cultures of chicken dorsal root ganglion cells.

Neuronal subpopulations of dorsal root ganglion (DRG) cells in the chicken exhibit carbonic anhydrase (CA) activity. To determine whether CA activity is expressed by DRG cells maintained in in vitro cultures, dissociated DRG cells from 10-day-old chick embryos were cultured on a collagen substrate. The influence exerted by environmental factors on the enzyme expression was tested under various conditions of culture. Neuron-enriched cell cultures and mixed DRG-cell cultures (including numerous non-neuronal cells) were performed either in a defined medium or in a horse serum-supplemented medium. In all the tested conditions, subpopulations of cultured sensory neurons expressed CA activity in their cell bodies, while their neurites were rarely stained; in each case, the percentage of CA-positive neurons declined with the age of the cultures. The number and the persistence of neurons possessing CA activity as well as the intensity of the reaction were enhanced by addition of horse serum. In contrast, the expression of the neuronal CA activity was not affected by the presence of non-neuronal cells or by the rise of CO2 concentration. Thus, the appearance and disappearance of neuronal subpopulations expressing CA activity may be decisively influenced by factors contained in the horse serum. The loss of CA-positive neurons with time could result from a cell selection or from genetic repression. Analysis of the time curves does not support a preferential cell death of CA-positive neurons but suggests that the eventual conversion of CA-positive neurons into CA-negative neurons results from a loss of the enzyme activity. These results indicate that the phenotypic expression of cultured sensory neurons is dependent on defined environmental factors.

Hippocampal atrophy predicts conversion to dementia after STN-DBS in Parkinson's disease.

BACKGROUND: Hippocampal atrophy (HA) is a known predictor of dementia in Alzheimer's disease. HA has been found in advanced Parkinson's disease (PD), but no predicting value has been demonstrated yet. The identification of such a predictor in candidates for subthalamic deep brain stimulation (STN-DBS) would be of value. Our objective was to compare preoperative hippocampal volumes (HV) between PD patients who subsequently converted to dementia (PDD) after STN-DBS and those who did not (PDnD). METHODS: From a cohort of 70 consecutive STN-DBS treated PD patients, 14 converted to dementia over 25.6+/-20.2 months (PDD). They were compared to 14 matched controls (PDnD) who did not convert to dementia after 43.9+/-11.7 months. On the preoperative 3D MPRAGE MRI images, HV and total brain volumes (TBV) were measured by a blinded investigator using manual and automatic segmentation respectively. RESULTS: PDD had smaller preoperative HV than PDnD (1.95+/-0.29 ml; 2.28+/-0.33 ml; p<0.01). This difference reinforced after normalization for TBV (3.28+/-0.48, 3.93+/-0.60; p<0.01). Every 0.1 ml decrease of HV increased the likelihood to develop dementia by 24.6%. A large overlap was found between PD and PDnD HVs, precluding the identification of a cut-off score. CONCLUSIONS: As in Alzheimer's disease, HA may be a predictor of the conversion to dementia in PD. This preoperative predictor suggests that the development of dementia after STN-DBS is related to the disease progression, rather then the procedure. Further studies are needed to define a cut-off score for HA, in order to affine its predictive value for an individual patient.

Activity-dependent phosphorylation of SNAP-25 in hippocampal organotypic cultures.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is thought to play a key role in vesicle exocytosis and in the control of transmitter release. However, the precise mechanisms of action as well as the regulation of SNAP-25 remain unclear. Here we show by immunoprecipitation that activation of protein kinase C (PKC) by phorbol esters results in an increase in SNAP-25 phosphorylation. In addition, immunochemical analysis of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels shows that SNAP-25 focuses as three or four distinct spots in the expected range of molecular weight and isoelectric point. Changing the phosphorylation level of the protein by incubating the slices in the presence of either a PKC agonist (phorbol 12,13-dibutyrate) or antagonist (chelerythrine) modified the distribution of SNAP-25 among these spots. Phorbol 12,13-dibutyrate increased the intensity of the spots with higher molecular weight and lower isoelectric point, whereas chelerythrine produced the opposite effect. This effect was specific for regulators of PKC, as agonists of other kinases did not produce similar changes. Induction of long-term potentiation, a property involved in learning mechanisms, and production of seizures with a GABA(A) receptor antagonist also increased the intensity of the spots with higher molecular weight and lower isoelectric point. This effect was prevented by the PKC inhibitor chelerythrine. We conclude that SNAP-25 can be phosphorylated in situ by PKC in an activity-dependent manner.