Pedro Mestres

Posttraumatic GABAA-Mediated [Ca2+]i Increase Is Essential for the Induction of Brain-Derived Neurotrophic Factor-Dependent Survival of Mature Central Neurons

A shift of GABAA-mediated responses from hyperpolarizing to depolarizing after neuronal injury leads to GABAA-mediated increase in [Ca2+]i. In addition, central neurons become dependent on BDNF for survival. Whether these two mechanisms are causally interrelated is an open question. Here, we show in lesioned CA3 hippocampal neurons in vitro and in axotomized corticospinal neurons in vivo that posttraumatic downregulation of the neuron-specific K–Cl cotransporter KCC2 leads to intracellular chloride accumulation by the Na–K–2Cl cotransporter NKCC1, resulting in GABA-induced [Ca2+]i transients. This mechanism is required by a population of neurons to survive in a BDNF-dependent manner after injury, because blocking GABAA-depolarization with the NKCC1 inhibitor bumetanide prevents the loss of neurons on BDNF withdrawal. The resurgence of KCC2 expression during recovery coincides with loss of BDNF dependency for survival. This is likely mediated through BDNF itself, because injured neurons reverse their response to this neurotrophin by switching the BDNF-induced downregulation of KCC2 to upregulation.

Single chloride channels in colon mucosa and isolated colonic enterocytes of the rat

SummaryChloride channels from rat colonic enterocytes were studied using the patch-clamp technique. After removal of mucus, inside-out patches were excised from the apical membrane of intact epithelium located at the luminal surface. They contained spontaneously switching Cl− channels with a conductance of 35–40 pS. The channels were blocked reversibly by anthracene-9-carboxylic acid (1mm).In excised patches from single enterocytes, isolated by calcium removal, the Cl− channels were studied in more detail. TheI–V relation was linear between ±80 mV. The selectivity was I−>Br−>Cl−=NO3−>F−=HCO3−.Thirty pS Cl− channels were also found on the basolateral membrane of crypts isolated by brief calcium removal. TheI–V curve of these Cl− channels was also linear.The results provide direct evidence for the existence of Cl− channels in the apical membrane of surface cells in colonic mucosa. The properties of these channels are similar to those previously observed when incorporating membrane vesicles into planar lipid bilayers. Both results support the validity of the theoretical models describing intestinal secretion.

GDNF is a trophic factor for adult rat corticospinal neurons and promotes their long-term survival after axotomy in vivo.

Glial cell line‐derived neurotrophic factor (GDNF) is a trophic factor for several neuronal populations involved in motor control. The present study evaluates the trophic actions of GDNF on corticospinal neurons, an important central nervous system motor projection into the spinal cord. Death of spinal motoneurons and corticospinal neurons is observed in the neurodegenerative disease amyotrophic lateral sclerosis. Axotomy of adult rat corticospinal neurons at internal capsule levels induces half of them to die, and the surviving population displays severe atrophy. To examine the trophic effects of GDNF on corticospinal neurons, Fast Blue‐labelled corticospinal neurons were stereotaxically axotomized at internal capsule levels and GDNF was infused intracortically to lesioned corticospinal neurons at total doses of 2, 4, 10, 20, 40, 100 and 300 μg for 7 days. GDNF prevented axotomy‐induced death of corticospinal neurons at doses between 2 and 40 μg and abolished or attenuated their atrophy at all doses examined. In addition, treatment with 8 μg GDNF for the first 2 weeks after axotomy resulted in the long‐term survival of corticospinal neurons for 42 days. With regard to the development of treatment strategies for upper motoneuron degeneration in amyotrophic lateral sclerosis, application of GDNF via the cerebrospinal fluid may be more relevant than intracortical delivery as its diffusion within the brain parenchyma is limited. lntraventricular as well as intracisternal infusion of GDNF (300 μg over 7 days) completely prevented corticospinal neuron death. These results show that GDNF promotes the long‐term survival of corticospinal neurons and has a positive effect on their size in vivo Furthermore, the survival‐promoting effect of GDNF on corticospinal neurons after delivery via cerebrospinal fluid has important clinical implications for potential treatment of the upper motoneuron degeneration seen in amyotrophic lateral sclerosis.

BDNF, but not NT-3, promotes long-term survival of axotomized adult rat corticospinal neurons in vivo

Axotomy-induced death of corticospinal neurons (CSN) is prevented by intracotrical infusions of BDNF or NT-3 within the first week after axotomy. The present study examined whether this represents merely a delay of CSN death or whether BDNF and NT-3 can promote long-term survival of these neurons in vivo. The neurotrophins were infused for an initial period of 14 days to lesioned CSN which was followed by 28 days without treatment. BDNF was able to promote CSN survival for at least 42 days while NT-3 had no significant effect. These results suggest that initial BDNF treatment induces an endogamous mechanism that promotes survival of axotomized CSN without further exogenous neurotrophic factor supply. These findings may be important for the design of therapeutic strategies for motoneuron disease.

The GDNF-induced neurite outgrowth and neuronal survival in dissociated myenteric plexus cultures of the rat small intestine decreases postnatally

Abstract Glial cell-line-derived neurotrophic factor (GDNF), a member of the transforming growth-factor- (TGF-) β-family, is an essential factor for the development of the enteric nervous system (ENS) during embryogenesis. In the present study, the effects of GDNF on postnatal ENS development were investigated using cultures of myenteric plexus from the small intestine of newborn albino rats of different developmental phases (P1, P7, P14). Myenteric plexus was dissociated and cultivated as mixed cultures of enteric neurons and glial cells. After seeding, the cultures were kept for 24 h or 7 days in serum-free medium containing various doses (1, 10, 100 ng/ml) of GDNF. The effect of the neurotrophic factor was evaluated using parameters such as cell size, neuronal survival, or neurite elongation. While neither glial-cell nor neuronal size was influenced by GDNF, there was an observable effect upon neuronal survival and neurite elongation. The cultures treated with GDNF displayed increased neurite outgrowth. The promoting effect was dose- and age-dependent, decreasing clearly during the early postnatal period. Already after 24 h, neuronal survival was increased in P1 and P7, but not in P14 cultures. In long-term cultures, a marked tendency to form cell aggregates and dense fiber networks was observed when treated with GDNF. These observations suggest that GDNF plays an important role not only in pre-, but also in postnatal development of the enteric nervous system.

The endogenous survival promotion of axotomized rat corticospinal neurons by brain-derived neurotrophic factor is mediated via paracrine, rather than autocrine mechanisms

The previously reported rescue of corticospinal neurons (CSN) from axotomy-induced death by intracortical glial cell line-derived neurotrophic factor (GDNF)- and neurotrophin-3 (NT-3)-infusions depends on endogenous cortical brain-derived neurotrophic factor (BDNF). The present study examines whether BDNF, GDNF, or NT-3 can stimulate an autocrine or paracrine BDNF-support of lesioned CSN. BDNF-infusions increase BDNF mRNA-expression throughout cortical layers 2-5 and NT-3-treatment results in upregulation of BDNF-transcripts in the upper cortical layers. In contrast, GDNF-treatment had no effect. While virtually all CSN express the BDNF-receptor trkB, less than half of them express BDNF, and these expression patterns are unchanged after axotomy and the different neurotrophic factor treatments. The findings suggest that axotomized CSN are supported via a paracrine BDNF-mechanism which can be stimulated by BDNF- and NT-3-, but not by GDNF.

An axotomy model for the induction of death of rat and mouse corticospinal neurons in vivo.

To study trophic dependencies of rat and mouse corticospinal neurons (CSN), we established a lesion model for the induction of death of analogous populations of CSN in these rodent species. Before lesion, CSN were retrogradely labeled with Fast Blue (FB). A stereotaxic cut lesion through the entire internal capsule (ICL) was used to axotomize CSN. The extent of axotomy was determined by application of a control tracer. In both species, FB-labeled CSN were localized in three major areas: (1) the sensory motor cortex; (2) the supplementary motor and medial prefrontal cortex; and (3) the somatosensory cortex. ICL does not lead to complete axotomy of CSN of the rat and mouse somatosensory cortex. In rats, ICL results in complete axotomy of CSN of the sensory motor cortex and incomplete axotomy of the caudal portion of the supplementary motor and medial prefrontal cortex. In mice, the area of axotomized CSN extends significantly further frontally. In both species, axotomy-induced death of CSN is observed in the center of the sensory motor cortex. This lesion model is useful for investigations on the response of CSN of the sensory motor cortex to lesion and therapeutic drugs.

Infusion of Gdnf into the cerebral spinal fluid through two different routes: effects on body weight and corticospinal neuron survival

SURVIVAL of axotomized adult rat corticospinal neurons (CSN) is supported by glial cell line-derived neurotrophic factor (GDNF). We have evaluated the trophic effects of intrathecally applied GDNF on CSN survival and rat body weight. Body weight reduction is the major side effect of intracerebral neurotrophic factor treatment. GDNF was tested at total doses of 30, 100 and 300 mg over 7 days after axotomy via different application routes: intracerebroventricularly (i.c.v.) and cisternally (cis). Animals injected i.c.v. displayed severe body weight reduction at all doses tested but CSN rescueonly at the highest dose. In contrast, cis-infusion of GDNF promoted CSN survival at all doses and only the highest dose reduced the body weight. These results show that intracisternal, but not i.c.v., GDNF infusion at low doses canpromote CSN survival without negatively affecting rat body weight. This finding may have implications for the clinic use of GDNF.

Direct and indirect actions of HgCl2 and methyl mercury chloride on permeability and chloride secretion across the rat colonic mucosa.

The actions of two mercury compounds, the inorganic HgCl2 and the organic methyl mercury chloride (MeHg), and of CdCl2 on ion transport across the rat colon were studied with the Ussing chamber and the everted sac method. The mercury compounds (5-50 microM), but not CdCl2, administered to the luminal side, induced a large, concentration-dependent increase of tissue conductance (Gt). The transepithelial movement of the extracellular marker, mannitol, was enhanced in the presence of the mercury compounds, indicating that they cause an increase in the permeability of the epithelium. Morphological studies revealed that at least for HgCl2 this increase of the permeability was associated with a loosening of the tight junctions, severe alterations of the enterocytes, and a loss of the continuity of the epithelium. After washing out HgCl2, cells neighboring the altered enterocytes developed lateral processes and in this way restored the continuity of the epithelial layer. In parallel, the mercury compounds induced an increase of short-circuit current (Isc), which is indicative of an induction of Cl- secretion. The increase of Isc, but not that of Gt, was suppressed by indomethacin and, in the case of MeHg, also by tetrodotoxin. These results suggest that MeHg and HgCl2 induce Cl- secretion by an indirect effect on the epithelium, which is mediated by prostaglandins.

A New Method to Assess Drug Sensitivity on Breast Tumor Acute Slices Preparation

Abstract:  A method for assessing tumor drug sensitivity is described that is based on preparation of tissue slices and use of silicon chips equipped with electrochemical sensors (multisensor array). The tumor slices (200–300 μM thick) are prepared after surgery and incubated in a medium for recovery after slicing. The advantage, compared to other preparations, is that the original three‐dimensional structure is retained. Multisensor arrays measure: (a) pericellular acidification (anaerobic metabolism) and (b) oxygen consumption (respiration). The innovative aspect is that such measurements can be made online, as opposed to using a large battery of endpoint tests on cell vitality and proliferation. Electron microscopy of slices serves to determine cell density and structure and induction of apoptosis/necrosis. Slices of more than 200 breast tumors were used. Metabolic activity was inhibited by sodium fluoride, which reduces glycolysis, and potassium cyanide, which inhibits respiration. These changes are thus reflected in the curves of acidification and oxygen consumption. In other experiments the cytostatic Taxol, an anticytoskeletal agent, was used showing dose and time‐dependent effects on acidification and oxygen consumption. In conclusion, the method presented here, is able to provide information on drug sensitivity of a tumor, which aids in designing individualized therapy and is used for drug screening.