Cation-chloride cotransporters and GABA-ergic innervation in the human epileptic hippocampus.

Intracellular chloride concentration, [Cl(-)](i), determines the polarity of GABA(A)-induced neuronal Cl(-) currents. In neurons, [Cl(-)](i) is set by the activity of Na(+), K(+), 2Cl(-) cotransporters (NKCC) such as NKCC1, which physiologically accumulate Cl(-) in the cell, and Cl(-) extruding K(+), Cl(-) cotransporters like KCC2. Alterations in the balance of NKCC1 and KCC2 activity may determine the switch from hyperpolarizing to depolarizing effects of GABA, reported in the subiculum of epileptic patients with hippocampal sclerosis. We studied the expression of NKCC (putative NKCC1) and KCC2 in human normal temporal neocortex by Western blot analysis and in normal and epileptic regions of the subiculum and the hippocampus proper using immunocytochemistry. Western blot analysis revealed NKCC and KCC2 proteins in adult human neocortical membranes similar to those in rat neocortex. NKCC and KCC2 immunolabeling of pyramidal and nonpyramidal cells was found in normal and epileptic hippocampal formation. In the transition between the subiculum with sclerotic regions of CA1, known to exhibit epileptogenic activity, double immunolabeling of NKCC and KCC2 revealed that approximately 20% of the NKCC-immunoreactive neurons do not express KCC2. In these same areas some neurons were distinctly hyperinnervated by parvalbumin (PV) positive hypertrophic basket formations that innervated mostly neurons expressing NKCC (74%) and to a lesser extent NKCC-immunonegative neurons (26%). Hypertrophic basket formations also innervated KCC2-positive (76%) and -negative (24%) neurons. The data suggest that changes in the relative expression of NKCC1 and KCC2 in neurons having aberrant GABA-ergic hyperinnervation may contribute to epileptiform activity in the subicular regions adjacent to sclerotic areas of the hippocampus.

Water permeability of Na+-K+-2Cl- cotransporters in mammalian epithelial cells.

Water transport properties of the Na+-K+-2Cl- cotransporter (NKCC) were studied in cultures of pigmented epithelial cells (PE) from the ciliary body of the eye. Here, the membrane that faces upwards contains NKCCs and can be subjected to rapid changes in bathing solution composition and osmolarity. The anatomy of the cultured cell layer was investigated by light and electron microscopy. The transport rate of the cotransporter was determined from the bumetanide-sensitive component of 86Rb+ uptake, and volume changes were derived from quenching of the fluorescent dye calcein. The water permeability (Lp) of the membrane was halved by the specific inhibitor bumetanide. The bumetanide-sensitive component of the water transport exhibited apparent saturation at osmotic gradients higher than 200 mosmol l-1. Cell shrinkages produced by NaCl or KCl were smaller than those elicited by equi-osmolar applications of mannitol, indicating reflection coefficients for these salts close to zero. The activation energy of the bumetanide-sensitive component of the Lp was 21 kcal mol-1, which is four times higher than that of an aqueous pore. The data suggest that osmotic transport via the cotransporter involves conformational changes of the cotransporter and interaction with Na+, K+ and Cl-. Similar measurements were performed on immortalized cell cultures from the thick ascending limb of the loop of Henle (TALH). Given similar overall transport rates of bumetanide-sensitive 86Rb+, the NKCCs of this tissue did not contribute any bumetanide-sensitive Lp. This suggests that the cotransporters of the two tissues are either different isoforms or the same cotransporter but in two different transport modes.

Postnatal maturation of Na+, K+, 2Cl- cotransporter expression and inhibitory synaptogenesis in the rat hippocampus: an immunocytochemical analysis.

GABA, a major inhibitory neurotransmitter, depolarizes hippocampal pyramidal neurons during the first postnatal week. These depolarizations result from an efflux of Cl- through GABAA-gated anion channels. The outward Cl- gradient that provides the driving force for Cl- efflux might be generated and maintained by the Na+, K+, 2Cl- cotransporter (NKCC) that keeps intracellular Cl- concentration above electrochemical equilibrium. The developmental pattern of expression of the cotransporter in the hippocampus is not known. We studied the postnatal distribution pattern of NKCC in the hippocampus using a monoclonal antibody (T4) against a conserved epitope in the C-terminus of the cotransporter molecule. We also examined the temporal relationships between the developmental pattern of NKCC expression and the formation of perisomatic GABAergic synapses. This study was aimed at determining, with antivesicular inhibitory amino acid transporter (VIAAT) antibodies, whether perisomatic GABAergic synapses are formed preferentially at the time when GABA is depolarizing. During the first postnatal week, NKCC immunolabelling was restricted to cell bodies in the pyramidal cell layer and in the strata oriens and radiatum. In contrast, at postnatal day 21 (P21) and in adult animals little or no labelling occurred in cell bodies; instead, a prominent dendritic labelling appeared in both pyramidal and nonpyramidal neurons. The ultrastructural immunogold study in P21 rat hippocampi corroborated the light-microscopy results. In addition, this study revealed that a portion of the silver-intensified colloidal gold particles were located on neuronal plasmalemma, as expected for a functional cotransporter. The formation of inhibitory synapses on perikarya of the pyramidal cell layer was a late process. The density of VIAAT-immunoreactive puncta in the stratum pyramidale at P21 reached four times the P7 value in CA3, and six times the P7 value in CA1. Electron microscopy revealed that the number of synapses per neuronal perikaryal profile in the stratum pyramidale of the CA3 area at P21 was three times higher than at P7, even if a concomitant 20% increase in the area of these neuronal perikaryal profiles occurred. It is concluded that, in hippocampal pyramidal cells, there is a developmental shift in the NKCC localization from a predominantly somatic to a predominantly dendritic location. The presence of NKCC during the first postnatal week is consistent with the hypothesis that this transporter might be involved in the depolarizing effects of GABA. The depolarizing effects of GABA may not be required for the establishment of the majority of GABAergic synapses in the stratum pyramidale, because their number increases after the first postnatal week, when GABA action becomes hyperpolarizing.

Biología celular y molecular del envejecimiento neuronal: estado actual y perspectivas / Cellular and molecular biology of neural aging: actual situation and perspectives

En el presente trabajo se analiza el problema del envejecimiento con énfasis en sus causas y en el curso que toma en el sistema nervioso. Se revisa la evidencia experimental que muestra que tanto la esperanza de vida como el límite biológico de la duración de la misma, pueden alargarse por medio de la manipulación genética. Se discute el significado biológico del envejecimiento y de la muerte desde la perspectiva de la selección natural. Se establecen las diferencias objetivas entre envejecimiento normal y enfermedades neurodegenerativas. Este último constituye uno de los puntos más importantes dado que es común, incluso en ambientes médicos, confundir al envejecimiento normal con las enfermedades neurodegenerativas, particularmente con la enfermedad de Alzheimer. Se analizan el colapso de dos viejos dogmas de la neurobiología: 1) la ausencia de neurogénesis en el cerebro humano adulto y 2) que el deterioro característico de las funciones mentales durante el envejecimiento normal resulta de la pérdida en el número de neuronas. Por mucho tiempo se mantuvo que la pérdida en el número de neuronas en el cerebro humano adulto era irreversible. Se sostuvo que la incapacidad neurogénica aunada a la pérdida progresiva de neuronas constituían el devenir de muchas enfermedades neurológicas y en el proceso de envejecimiento cerebral. Estos puntos de vista deben ser revaluados a la luz de descubrimientos recientes que muestran la formación de nuevas neuronas en el cerebro humano adulto a partir de células progenitoras. Estos hallazgos se relacionan con otros que prueban que la escasa muerte neuronal que ocurre durante el envejecimiento normal, no explica el deterioro en las funciones mentales que le es característico. Este declinar se debe a cambios sutiles, morfológicos y funcionales, en ciertos circuitos clave...