Activation of endogenous neural stem cells in the adult human brain following subarachnoid hemorrhage.

In the adult human brain, the presence of neural stem cells has been documented in the subgranular layer of the dentate gyrus of the hippocampus and in the subventricular zone of the lateral ventricles. Neurogenesis has also been reported in rodent models of ischemic stroke, traumatic brain injury, epileptic seizures, and intracerebral or subarachnoid hemorrhage. However, only sparse information is available about the occurrence of neurogenesis in the human brain under similar pathological conditions. In the present report, we describe neural progenitor cell proliferation in the brain of patients suffering from subarachnoid hemorrhage (SAH) resulting from ruptured aneurysm. Ten cerebral samples from both SAH and control patients obtained, respectively, during aneurysm clipping and deep brain tumor removal were analyzed by reverse transcription followed by polymerase chain reaction (RT-PCR) and/or immunohistochemistry (IHC). In tissue specimens from SAH patients, RT-PCR and IHC revealed the expression of a variety of markers consistent with CNS progenitor cells, including nestin, vimentin, SOX-2, and Musashi1 and -2. In the same specimens, double immunohistochemistry followed by confocal analysis revealed that Musashi2 consistently colocalized with the proliferation marker Ki67. By contrast, no such gene or protein expression profiles were detected in any of the control specimens. Thus, activation of neural progenitor cell proliferation may occur in adult human brain following subarachnoid hemorrhage, possibly contributing to the promotion of spontaneous recovery, in this pathological condition.

Extensive training in a maze task reduces neurogenesis in the adult rat dentate gyrus probably as a result of stress.

It has recently been shown that hippocampal neurogenesis can be modulated either directly or indirectly by ascending cholinergic inputs from the basal forebrain. In the present work, we sought to address whether extended training in a spatial navigation task would affect hippocampal neurogenesis in the presence of a severe and selective cholinergic depletion. Young female rats received stereotaxic injections of the immunotoxin 192 IgG-saporin into the basal forebrain nuclei and/or the cerebellar cortex. Starting from 4 to 5 weeks post-lesion, and for the subsequent 2 weeks, the animals were trained on paradigms of reference and working memory in the water maze and received single daily i.p. injections of bromodeoxyuridine (BrdU) at the end of each testing session. In line with previous observations, a dramatic 80% decrease in neuron proliferation was seen in the dentate gyrus of lesioned animals, as compared to vehicle-injected or intact controls. Interestingly, however, rats subjected to maze training over 2 weeks, irrespective of their learning success, exhibited significantly fewer newborn neurons than matched controls with no maze exposure. Thus, at least for the type of task used here, which has previously been shown to impose a certain degree of stress, extended training and learning does not appear to affect proliferation in the dentate gyrus.

Selective cholinergic immunolesioning affects synaptic plasticity in developing visual cortex.

Cholinergic neurotransmission is known to affect activity-dependent plasticity in various areas, including the visual cortex. However, relatively little is known about the exact role of subcortical cholinergic inputs in the regulation of plastic events in this region during early postnatal development. In the present study, synaptic transmission and plasticity in the developing visual cortex were studied following selective immunotoxic removal of the basal forebrain cholinergic afferents in 4-day-old rat pups. The lesion produced dramatic cholinergic neuronal and terminal fibre loss associated with decreased mRNA levels for the M1 and M2 muscarinic receptors, as well as clear-cut impairments of long-term potentiation (LTP) in visual cortex slices. Indeed, after theta burst stimulation of layer IV a long-term depression (LTD) instead of an LTP was induced in immunolesioned slices. This functional change appears to be due to the lack of cholinergic input as exogenous application of acetylcholine prevented the shift from LTP to LTD. In addition, lesioned rats showed an increased sensitivity to acetylcholine (ACh). While application of 20 microm ACh produced a depression of the field potential in immunolesioned rat slices, in order to observe the same effect in control slices we had to increase ACh concentration to up to 200 microm. Taken together, our results indicate that deprivation of cholinergic input affects synaptic transmission and plasticity in developing visual cortex, suggesting that the cholinergic system could play an active role in the refinement of the cortical circuitry during maturation.

The neuronal nicotinic acetylcholine receptor in some hereditary epilepsies.

Recent advances in human genetics and in the neurobiology of neurotransmitter receptors and channels have led to the discovery of specific genes associated with hereditary epileptic phenotypes. All the genes identified to date code for ligand- and voltage-gated ion channels. Some clinically rare idiopathic epilepsies are associated with mutations in genes coding for different neuronal nicotinic acetylcholine receptor (AChR) subunits. Distinct alpha subunits are found in the brain and in the peripheral nervous system, and structural, non-alpha subunits like beta2 and beta4 confer different properties to neuronal receptors. Thus, the final properties of the oligomeric AChR depend on the different combinations of alpha and beta subunits. Most mutations found so far occur in the alpha4 chain, the most abundant subunit in the central nervous system. Specifically, the identification of mutations in the alpha4 subunit of neuronal AChR in human benign familial neonatal convulsions (BFNC) and autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) raise the possibility that the observed gene defects are linked (causatively) with these two diseases or, alternatively, that AChR alpha4 mutants increase the probability of epileptic discharges. We discuss testable hypotheses for unraveling the pathophysiology of these two disorders associated with AChR mutations.

Expression of a neuronal nicotinic acetylcholine receptor in insect and mammalian host cell systems.

Different mammalian and insect somatic host cell systems were tested in their ability to express, fold, and assemble alpha7-type neuronal acetylcholine receptor (AChR) both at the transcriptional and translational level. For this purpose we employed clonal cell lines derived from the neural crest, such as PC12 cells from a rat adrenal pheochromocytoma, and GH3 cells isolated from a rat pituitary tumor, as well as non-neuronal cells such as NIH-3T3 fibroblasts from embryonic NIH Swiss mouse and Sf9 cells from ovary tissue of the Spodoptera frugiperda butterfly. Total RNA, isolated from either transfected or non-transfected PC12, GH3 or 3T3 cells, or recombinant AcNPV-infected and mock-infected Sf9 cells was analyzed by Northern blot. PC12 cells, which endogenously express alpha7 AChR, and all its heterologous alpha7-transfectant clones, exhibited variable but generally high amounts of a single transcript. GH3 and NIH-3T3 transfectant clones and recombinant AcNPV-infected Sf9 cells expressed variable levels of alpha7-mRNA, with a single transcript that co-migrated with the 28S rat rRNA. Only the neural crest-derived cell lines appeared to functionally express the alpha7 AChR, as measured by their [125I]alpha-bungarotoxin binding ability. The results suggest that heterologous expression of alpha7 is regulated not at the transcriptional, but at the postranslational level and that not all host cell systems appear to express the cellular factors needed for the correct postranslational modifications leading to mature and functional alpha7 AChR. Furthermore, the results suggest that tightly controlled expression mechanisms have evolved in parallel with this ancient cholinergic sequence.

Cells defective in sphingolipids biosynthesis express low amounts of muscle nicotinic acetylcholine receptor.

The properties of the nicotinic acetylcholine receptor (AChR) are modulated by its lipid microenvironment. Studies of such modulation are hampered by the cell's homeostatic mechanisms that impede sustained modification of membrane lipid composition. We have devised a novel strategy to circumvent this problem and study the effect of changes in plasma membrane lipid composition on the functional properties of AChR. This approach is based on the stable transfection of AChR subunit cDNAs into cells defective in a specific lipid metabolic pathway. In the present work we illustrate this new strategy with the successful transfection of a temperature-sensitive Chinese hamster ovary (CHO) cell line, SPB-1, with the genes corresponding to the four adult mouse AChR subunits. The new clone, SPB-1/SPH, carries a mutation of the gene coding for serine palmitoyl transferase, the enzyme that catalyses the first step in sphingomyelin (Sph) biosynthesis. This defect causes a decrease of Sph de novo synthesis at non-permissive temperatures. The IC50 for inhibition of alpha-BTX binding with the agonist carbamoylcholine exhibited values of 3.6 and 2.7 microm in the wild-type and Sph-deficient cell lines, respectively. The corresponding IC50 values for the competitive antagonist D-tubocurarine (D-TC) were 2.8 and 3.4 microm, respectively. No differences in single-channel properties were observed between wild-type and mutant cell lines grown at the non-permissive, lipid defect-expressing temperature using the patch-clamp technique. Both cells exhibited two open times with mean values of 0.35 +/- 0.05 and 1.78 +/- 0.2 ms at 12 degrees C. Taken together, these results suggest that the AChR is expressed as the complete heteroligomer. However, only 10-20% of the total AChR synthesized reached the surface membrane in the mutant cell line and exhibited a higher metabolic turnover, with a half-life about 50% shorter than the wild-type cells. When control CHO-K1/A5 cells were treated with fumonisin B1, an inhibitor of sphingosine (sphinganine) N-acetyltransferase (ceramide synthase), a 45.5% decrease in cell surface AChR expression was observed. The results suggest that sphingomyelin deficiency conditions AChR targeting to the plasma membrane.

Nicotinic acetylcholine receptor channels are influenced by the physical state of their membrane environment.

We investigated the effect of the physical state of the cell membrane on the activity of the nicotinic acetylcholine receptor (AChR) in various clonal cell lines transfected with the cDNAs of embryonic or adult AChR by measuring single-channel properties and some membrane physicochemical properties as a function of temperature. Unitary conductance and channel closing rate, alpha, had Q(10) values of 1.2 and 2.2, respectively. Using Eyring's transition state theory, it was calculated that both embryonic and adult-type AChR had relatively low thermal sensitivity of ionic conductance and activation energy (E(a) of 3.0-5.0 kcal-mol(-1) at 20 degrees C), indicating that once the AChR channel opens, ion movement is dominated by diffusional processes. Channel closure exhibited higher energy requirements, with E(a) values of about 13 kcal-mol(-1). This process appears to be more endothermic (higher delta H(a) values) than ion permeation, and it is plausible that the energy acquired by the system can be used in the maintenance of its degree of order, as revealed by the delta S(a) 0 calculated for channel closure. The influence of the membrane environment on AChR function is reinforced by the observation that the conductance of the same, embryonic-type AChR protein, expressed in qualitatively different cellular lipid environments, appeared to have different energetic requirements. A correlation between the electrophysiological and thermodynamic parameters of the AChR and physicochemical properties of the membrane bilayer in which the protein is embedded could be established using measurements of the so-called generalized polarization (GP) of the lipophilic probe laurdan. Both embryonic and adult AChR exhibited a higher GP and a higher sensitivity to temperature-dependent changes in GP when heterologously expressed in stable form in Chinese hamster ovary (CHO)-derived cells than did the native embryonic AChR in BC3H-1 cells, indicating that these two properties are determined by the host membrane and are not inherent properties of the AChR type. In addition, the differences in the macroscopic physical states of the lipids and membrane-associated solvent (water) dipolar relaxation between BC3H-1 and CHO-derived cells indicated by the spectroscopic properties of laurdan suggest that both lipid and associated water may influence the microscopic activity of individual AChR molecules embedded in the lipid bilayer. Finally, the different dependence of AChR channel conductance and mean open time as a function of GP observed between the different AChR subtypes in clonal cell lines suggests the importance of specific lipid-protein interactions in addition to bulk membrane properties.