Astroglial Control of the Antidepressant-Like Effects of Prefrontal Cortex Deep Brain Stimulation.

Although deep brain stimulation (DBS) shows promising efficacy as a therapy for intractable depression, the neurobiological bases underlying its therapeutic action remain largely unknown. The present study was aimed at characterizing the effects of infralimbic prefrontal cortex (IL-PFC) DBS on several pre-clinical markers of the antidepressant-like response and at investigating putative non-neuronal mechanism underlying DBS action. We found that DBS induced an antidepressant-like response that was prevented by IL-PFC neuronal lesion and by adenosine A1 receptor antagonists including caffeine. Moreover, high frequency DBS induced a rapid increase of hippocampal mitosis and reversed the effects of stress on hippocampal synaptic metaplasticity. In addition, DBS increased spontaneous IL-PFC low-frequency oscillations and both raphe 5-HT firing activity and synaptogenesis. Unambiguously, a local glial lesion counteracted all these neurobiological effects of DBS. Further in vivo electrophysiological results revealed that this astrocytic modulation of DBS involved adenosine A1 receptors and K(+) buffering system. Finally, a glial lesion within the site of stimulation failed to counteract the beneficial effects of low frequency (30 Hz) DBS. It is proposed that an unaltered neuronal-glial system constitutes a major prerequisite to optimize antidepressant DBS efficacy. It is also suggested that decreasing frequency could heighten antidepressant response of partial responders.

[Escitalopram: a selective inhibitor and allosteric modulator of the serotonin transporter]. / L'escitalopram: un inhibiteur sélectif et un modulateur allostérique du transporteur de la sérotonine.

Citalopram (Séropram) is an antidepressant of the selective serotonin (5-HT) reuptake inhibitor (SSRI) class, composed of equal amounts of S-enantiomer, escitalopram, and R-enantiomer, R-citalopram. Both clinical and preclinical studies have reported that escitalopram is a potent SSRI that possesses a faster onset of antidepressant activity in comparison with citalopram. Conversely, R-citalopram, although devoid of 5-HT reuptake inhibition property, was reported to counteract the effect of the S-enantiomer in several in vitro and in vivo experiments. For instance, microdialysis studies have shown that escitalopram increased the extracellular 5-HT levels in the frontal cortex and the ventral hippocampus, and this effect was prevented by concomitant injection of R-citalopram. The in vivo relevance of the antagonistic effect of R-citalopram on escitalopram efficacy was confirmed in dorsal raphe nucleus, a brain region known to be a target for SSRIs. In the later region, escitalopram was four times more potent than citalopram in suppressing the firing activity of 5-HT neurons and this effect of escitalopram was significantly prevented by R-citalopram. The antagonizing effect of R-citalopram on escitalopram efficacy was also observed in behavioural tests predictive of anxiolytic or antidepressant properties. In adult rats, R-citalopram reduced the anxiolytic-like effect of escitalopram obtained in the footshock-induced ultrasonic vocalization model, the conditioned fear model or the Vogel conflict and elevated plus maze tests. In validated chronic models with high predictive value for antidepressant activity, when escitalopram was administered for five weeks, either alone or with twice as much R-citalopram, the effect of the treatment regimens on reversal of hedonic deficit was significantly different. Importantly, chronic treatment with escitalopram reversed the decrease in cytogenesis in the rat dentate gyrus, induced by chronic mild stress. However, in naïve rats, while chronic treatment with R-citalopram did not modify the basal proliferation rate in the dentate gyrus, it blocked the increase induced by escitalopram when coadministered. This suggests that neuronal adaptive changes, which are essential for antidepressant response, are rapidly induced by escitalopram but prevented by R-citalopram coadministration. The attenuating effect of R-citalopram was suggested to underlie the delayed recovery of 5-HT neuronal activity following long-term treatment with citalopram versus escitalopram. This is confirmed since a treatment with R-citalopram antagonized the recovery of firing observed in escitalopram-treated rats. The exact mechanism by which R-citalopram exerts its action is not yet fully defined; however, an allosteric interaction between the enantiomers and the 5-HT transporter (SERT) has been proposed. In this context, in vitro studies have revealed the existence of at least two binding sites on SERT: (1) a primary high-affinity binding site or orthosteric site that mediates the inhibition of 5-HT reuptake and (2) an allosteric low-affinity binding site that modulates the binding of ligands at the primary site. In presence of escitalopram alone, both the primary and the allosteric sites are occupied. Thus, escitalopram exerts a stabilizing effect on this association to SERT, resulting in an effective inhibition of 5-HT reuptake activity. On the other hand, in the presence of the two enantiomers, R-citalopram binds to the allosteric site and decreases the escitalopram action on SERT. Such an innovative mechanism of action can constitute a basis for development of new allosteric antidepressants that demonstrate higher efficacy and earlier onset of therapeutic effect.

Restraint stress and adrenalectomy do not affect the level of rat cerebral enolase.

Gamma gamma (gamma gamma) enolase amount and enolase isozyme activities were measured in brain structures involved in stress response (hypothalamic paraventricular and supraoptic nuclei, locus coeruleus, hippocampus, A1 and A2 regions, hypophysis). Analyses were made in rats after immobilization or adrenalectomy, two experimental conditions that have been reported to increase brain glucose consumption and protein synthesis. In control rats enolase content was highest in the A1 region and hippocampus and lowest in hypophysis. A positive correlation was found between the enzyme level and its activity in the brain regions studied. However, in stressed or adrenalectomized rats, the level of glycolytic enzyme enolase was not significantly modified.

Further characterization of the tyrosine hydroxylase induction elicited by reserpine in the rat locus coeruleus and adrenals.

This study shows that reserpine causes a long lasting increase in the amount of protein and activity of tyrosine hydroxylase (TH) in central noradrenergic neurons of the locus coeruleus (LC) and in peripheral cells of the adrenal gland. In these structures, the drug effect appears to be reversible (maximum at day 2-4 after injection) and is entirely dose-dependent. Variations in TH activity were measured both in vitro in optimal enzymatic conditions and in vivo. Reserpine did not modify the ratio between activities measured in vivo and in vitro. But it decreased the specific activity of the enzyme as measured in homogenates of LC. The reversibility of the reserpine effect on all parameters studied appeared faster in adrenal than in central LC neurons. Reserpine did not change the activity and amount of TH in the region of the substantia nigra where dopaminergic neurons are located, except at a high dose (10 mg/kg): a significant elevation (+36%) of the TH protein amount was then observed 2 days after treatment. Moreover, reserpine did not influence the amount of neuron specific enolase in the LC region at a time when maximal effect was observed for TH, thus showing the biochemical specificity of this regulation. Concomitant injection of clonidine at two doses 30 min before (100?g/kg) and 3 h after (50?g/kg) reserpine administration did not affect the induction by reserpine alone. Thus, stimulation of LC noradrenergic cell firing resulting from reserpine administration is probably of negligible importance in the genesis of TH induction by this drug.

Serum neuron-specific enolase in senile dementia of the Alzheimer type.

The level of neuron-specific enolase (NSE), a glycolytic enzyme localized in neurons, was measured in the serum of patients with senile dementia of the Alzheimer type (SDAT). No difference was observed between NSE levels in SDAT and in healthy elderly controls of the same age range. No correlation was found between NSE levels and severity of the cognitive deficits. There was a marginally significant negative correlation between age and NSE, younger patients having higher NSE levels. The present results suggest that serum NSE is not a useful biological marker in the senile form of the dementia of the Alzheimer type.

An enzymoimmunoassay for rat gamma enolase using two monoclonal antibodies.

Monoclonal antibodies specific for rat ? enolase were prepared by fusion between ?? enolase immunized mouse spleen cells and mouse myeloma cells. Isolated hybrid cell lines were screened against both ?? and ?? enolase with an enzymoimmunoassay. Two types of antibodies were identified: one type, corresponding to 21% of the generated clones, recognized both ? and ? subunits, the other type, representing 3% of the clones, was specific for ? enolase. The specificity of antibodies was ascertained by immunofixation of ?? enolase activity. Two monoclonal antibodies (2H12 and 8A9) were used for quantitative determination of rat ? enolase in a "sandwich type" enzymoimmunoassay. The ? enolase has been shown to be a specific neuronal and neuroendocrine product. The development of a ? enolase assay will facilitate the study of the regulation of ? enolase expression.

[Merkel cell carcinoma of the skin. Anatomoclinical, ultrastructural and immunohistochemical study of 14 cases]. / Carcinome à cellules de Merkel de la peau. Etude anatomo-clinique, ultrastructurale et immunohistochimique de 14 cas.

The clinical and pathological features of 14 cases of Merkel cell carcinoma are reported. They commonly arise in the skin of elderly patients, particularly on the face and pelvis. They have a loco-regional aggressivity (nodal metastases in 4 cases) but some patients die with disseminated metastases (2 cases). These tumors are composed of round cells with scanty cytoplasm, arranged in solid or trabecular sheets. Mitotic figures are usually numerous. The ultrastructural study reveal secretory granules and paranuclear collection of intermediate filaments. Immunohistochemical phenotype is highly characteristic: cytoplasmic diffuse positivity with an anti-neuron-specific enolase polyclonal antibody; polar and/or diffuse positivity with anti-cytokeratin, anti-epithelial membrane antigen and anti-S100 protein monoclonal antibodies; polar positivity with an anti-neurofilament monoclonal antibody. The negativity with an anti-common leucocyte antigen monoclonal antibody is helpful to differentiate Merkel cell carcinoma from cutaneous malignant lymphoma.

Serum neuron-specific enolase. A marker for disease extent and response to therapy for small-cell lung cancer.

Serum neuron-specific enolase (S-NSE) levels in 43 newly diagnosed untreated patients with small-cell lung cancer (SCLC) were compared with levels in 35 adult controls, 14 patients with non-small cell lung cancer (N-SCLC), and nine patients with noncancerous lung disease (N-CLD). The S-NSE level was raised (greater than or equal to 16 ng/ml) in 28 of 43 patients with SCLC, six of 16 patients with limited stage SCLC, and 22 of 27 of those with extensive stage SCLC. Extensive stage patients with SCLC had a significantly higher mean S-NSE level (50 ng/ml) than did limited stage patients with SCLC (16 ng/ml). Mean S-NSE levels in patients with N-SCLC and in patients with N-CLD were respectively 11 and 7 ng/ml. Serial measurements performed on 19 patients between the three-day-courses of chemotherapy showed an excellent correlation between S-NSE and clinical evolution. In addition, S-NSE was measured during the first three-day course of chemotherapy in 13 other patients; among them, seven had S-NSE levels greater than or equal to 100 ng/ml (mean: 490 ng/ml); these seven patients were responders; the remaining six had S-NSE levels less than 100 ng/ml (mean 28 ng/ml): among them, only two were responders. Such S-NSE measurements during the first cytostatic course seem to reflect the importance of tumor burden and may be valuable as early indicators of the response rate to chemotherapy.

Experimental brain ischemia: neuron-specific enolase level in cerebrospinal fluid as an index of neuronal damage.

Levels of neuron-specific enolase (NSE) were measured in rat CSF following occlusion of the four major arteries to the brain for 10, 20, or 30 min. In the CSF of rats submitted to 30 min of total ischemia, an up to nine-fold increase of NSE level occurred within the first few hours and then slowly diminished. Significant levels were seen for as long as 8 days. Histological observations 3 days after ischemia showed neuronal loss as well as neuronal damage in several forebrain regions such as hippocampus, striatum, and thalamus. Ischemia was followed by transient decreases in exploration behavior and neurological states that were no longer visible 24 h later. After 10 or 20 min ischemia, NSE levels were increased to a lesser degree and fewer damaged neurons were observed. The positive correlation between duration of ischemia and amount of NSE release in CSF indicates that the measurement of NSE in the CSF is a sensitive and reliable index of neuronal lesions.

Neuron-specific enolase in cerebrospinal fluid: a possible indicator of neuronal damage in kainic acid lesions.

The presence of the neuron specific enolase (NSE) has been tested by sandwich enzyme immunoassay in cerebrospinal fluid (CSF) of rats after intrastriatal injection of various amounts of kainic acid (KA). Incremental release of NSE was observed in CSF for increasing concentrations of injected KA. Still, a significant decrease of NSE striatal content was detected only with the two maximal amounts of KA infused. These data indicate that measurements of NSE in CSF might be a more sensitive index of neuronal damage than the actual assay within the tissue, and also present the advantage of being a non-invasive method of investigation.

Release of neuron specific enolase (NSE) in cerebrospinal fluid following experimental lesions of the rat brain.

Neuron-specific enolase (NSE) levels were measured by sandwich enzymo-immunoassay as well as by enzymatic assay in rat cerebrospinal fluid (CSF), following mechanical lesions of the brain tissue. Significant increases of NSE were observed in CSF, with a peak 2 h following lesions located near the lateral ventricle. Values returned to normal around 48 h later. In another experimental group, lesions were realized further away from the lateral ventricle; the elevation of NSE in CSF reached the maximal value 11 h later. In addition, measurements which were performed following lesions at the same location but of various sizes, indicated that the quantity of NSE released is proportional to the extent of brain damage. The possible factors which govern the time course and amount of NSE release in CSF are discussed. These results suggest that NSE could be a useful and easily detected marker of neuronal damage.

Neuron-specific enolase as a marker of neuronal lesions during various comas in man.

The detection of neuron-specific enolase in biological fluids has been investigated as an indirect marker of neuronal damage in man. This protein was measured by a sandwich enzymoimmunoassay in serums and cerebrospinal fluids from patients with consciousness disorders of various aetiologies. Neuron-specific enolase level was significantly increased in sera from patients with comas resulting from anoxemia, head injury, septic state, cirrhosis and fulminant hepatitis. On the other hand, patients with meningitis (affection not normally accompanied with neuronal lesion) exhibited no change of this marker level. The statistical analysis of our results suggests that, in neurological disorders, the neuron-specific enolase levels in cerebrospinal fluid could have some prognostic value. The correlation between its level in cerebrospinal fluid and in serum was also demonstrated. Neuron-specific enolase increase in biological fluids thus represents a useful and promising marker to biochemically characterize various strokes possibly resulting in neuronal damage.

Developmental studies with the 14-3-2 antigen and the neuron specific enolase (NSE) associated activities-I.

We have compared the rodent developmental pattern of the 14-3-2 antigen estimated by a microcomplement fixation technique with that of the cerebral enolases. Chromatographic separation of enolase isozymes on microcolumns demonstrates that the embryonic neuron specific enolase is firstly and mostly represented by the ?? isozyme. The most important increase in 14-3-2 antigen and ?? enolase occurs between post-natal days 7th and 15th. By post-natal day 30, adult levels have been reached. An interesting observation is-during embryonic development-the decrease in the specific activity of the cerebral enolase isozyme ??. This could be explained by the replacement-in neuroblasts-of ?? enolase by neuron specific enolase. A comparison between 14-3-2 antigen and neuron specific enolase (??) purified by completely different methods is presented. The 14-3-2 antigen exhibits an enolase specific activity comparable to that of purified enzyme and has the same electrophoretic mobility. Antibodies raised against either antigen have an identical specificity. Pre and post-natal developmental pattern in rodent brains are similar for both proteins. Thus neuron specific 14-3-2 antigen is identical to neuron specific enolase. Thus we have precisely described the ontogenic transition between the three cerebral enolase isozymes at the tissue level. This study is completed by the analysis of these transitions at the neuronal cell level, using homogenous cell lines (Part II of this paper).

Transition between isozymic forms of enolase during in vitro differentiation of neuroblastoma cells-II.

An analysis of enolase expression during differentiation of neuroblastoma clones in homogeneous culture is presented. The enolases expressed in these neuroblast-like cells are identical to those of mouse brain with respect to the examined properties. Our biochemical investigation has premitted us to demonstrate formally that neuroblastoma cells undergo a transition from the embryonic ?? form to the neuronal ?? form and contain both enolases as well as the ?? hybrid form during maturation. These results suggest that the same phenomenon must exist in vivo for neuroblasts. In neuroblastoma cells, an increase in both ?? and ?? neuron specific enolases is related to cell maturation and expression of the ?? form precedes that of the ?? form during differentiation. Modulation of neuronal enolase activities is similar in the various conditions of differentiation studied and appears not to be necessarily related with morphological differentiation, although concomitant with an arrest of cell division. The evolution of specific neuronal enolases in neuroblastoma cells parallels that observed in vivo, in brain from embryonic day 15 to post-natal day 7. Moreover, at least one treatment (dimethylsulfoxide) causes an important decrease in the high specific ?? activity of these cells as occurs in vivo. This enolase can therefore also be considered as a biochemical marker for neuroblastoma maturation. As observed with other markers and other cell types, neuroblastoma cells in culture express an immature biochemical differentiation of the enolase isozymes.

[Micromethod of measuring the 3 brain isoenzymes of enolase]. / Microméthode de mesure des trois isoenzymes cérébrales de l'énolase.

The brain of the vertebrates contains three isozymic forms of the glycolytic enzyme enolase, two of which are neuron specific. These proteins could therefore be utilized as biochemical markers for various studies of neurons. We have devised a separation procedure which allows the measurement of the three enolase forms from minute amounts of tissue (50 to 100 microgram total protein). We show that this method can be applied to studies of brain microstructures, in vivo and in vitro (clonal cell lines) neural differentiation, and in measurements in biological fluids.