Detouring destruction: a role for inhibitory neuronal activity in preventing neuronal loss--implications for Alzheimer's disease.

Recent results from the author's efforts and others have indicated approaches that may eventually lead to new therapeutic strategies for combating neurodegenerative effects associated with dementia and Alzheimer's disease (AD). These strategies include unraveling mechanisms that decrease senile plaque accumulation and delay or slow the neurodegenerative progression associated with AD. Recent work addresses whether normally functioning inhibitory brain circuitry can protect and detour neurodegeneration. The aim of these research efforts is that one or a combination of these approaches will develop into an applied therapy that will enhance brain protection mechanisms and add to quality of life for patients with AD.

Regulation of a Ca2+-sensitive adenylyl cyclase in an excitable cell. Role of voltage-gated versus capacitative Ca2+ entry.

In nonexcitable cells, we had previously established that Ca(2+)-sensitive adenylyl cyclases, whether expressed endogenously or heterologously, were regulated exclusively by capacitative Ca(2+) entry (Fagan, K. A., Mahey, R. and Cooper, D. M. F. (1996) J. Biol. Chem. 271, 12438-12444; Fagan, K. A., Mons, N., and Cooper, D. M. F. (1998) J. Biol. Chem. 273, 9297-9305). Relatively little is known about how these enzymes are regulated by Ca(2+) in excitable cells, where they predominate. Furthermore, no effort has been made to determine whether the prominent voltage-gated Ca(2+) entry, which typifies excitable cells, overwhelms the effect of any capacitative Ca(2+) entry that may occur. In the present study, we placed the Ca(2+)-stimulable, adenylyl cyclase type VIII in an adenovirus vector to optimize its expression in the pituitary-derived GH(4)C(1) cell line. In these cells, a modest degree of capacitative Ca(2+) entry could be discerned in the face of a dramatic voltage-gated Ca(2+) entry. Nevertheless, both modes of Ca(2+) entry were equally efficacious at stimulating adenylyl cyclase. A striking release of Ca(2+) from intracellular stores, triggered either by ionophore or thyrotrophin-releasing hormone, was incapable of stimulating the adenylyl cyclase. It thus appears as though the intimate colocalization of adenylyl cyclase with capacitative Ca(2+) entry channels is an intrinsic property of these molecules, regardless of whether they are expressed in excitable or nonexcitable cells.

Prolonged cytosolic calcium elevations in growth cones contacting muscle.

Encounters by growth cones or neurites of motor neurons with target muscle cells evoke prolonged elevations in the concentrations of neuronal cytosolic free calcium ([Ca(2+)](c)). These calcium elevations are initiated at the point of contact and spread throughout the neuron over a period of tens of minutes. In this study, we addressed how target muscle cells initiate this unique presynaptic response. Primary questions regarding the nature of the muscle signal are whether it is diffusible and whether it must first be induced by a growth cone as part of reciprocal interaction. We addressed whether the signal was strictly target-contact dependent by fixing C2 mouse myotubes with formaldehyde, rinsing extensively and then allowing processes of chick ciliary ganglion neurons to interact with them. We observed frequent sustained elevations in [Ca(2+)](c) in ciliary ganglion processes contacting the fixed myotubes. As a control, ciliary neurons were allowed to interact with fixed myotubes of the S27 variant line. S27 cells were isolated from the parent C2 line on the basis of a defect in glycosaminoglycan biosynthesis and previously shown to be defective in supporting synaptic vesicle localization in contacting neurites. Few elevations in [Ca(2+)](c) were detected in encounters between ciliary processes and fixed S27 cells. In addition, neuron-neuron encounters never elicited prolonged increases in [Ca(2+)](c). These observations demonstrate contact dependence in the neuronal response and rule out reciprocal cellular interactions, diffusible factors or electrical activity in the muscle. The defect in carbohydrate biosynthesis in S27 cells further suggests that cell surface carbohydrates are essential to the signal on the myotube surface that triggers the presynaptic elevation in [Ca(2+)](c). We conclude that growth cone contact with preexisting cell surface structures on target muscle cells induces changes in presynaptic [Ca(2+)](c) that are associated with retrograde signaling, and that proper carbohydrate biosynthesis is required for this signal.

Inhibitory neuronal activity can compensate for adverse effects of beta-amyloid in hippocampal neurons.

One of the most prominent effects of Alzheimer disease is the disruption of finely tuned neuronal circuitry of discrete brain regions associated with learning and memory. Results from the present study support a role for the intrinsic inhibitory component of neuronal circuitry in determining the magnitude of beta-amyloid peptide induced cell death in the highly vulnerable pyramidal neurons of the hippocampus. Previous efforts have mostly focused on direct effects on excitatory neurons. By contrast, less emphasis has been placed on addressing a role for the intrinsic inhibitory component of cell-cell interactions of neuronal networks in response to Abeta. The present study provides evidence demonstrating that blockage of the intrinsic inhibitory component between Abeta exposed neurons leads to destabilization of calcium homeostasis and exacerbated neuronal death compared to Abeta treated cultures. Neuronal electrical activity was first silenced by exposing cultures to tetrodotoxin (TTX; 100 nM) plus Abeta, followed by survival counts. Cell death, unexpectedly, did not significantly differ from Abeta-exposed neurons. The intrinsic inhibition in Abeta-exposed cultures was then pharmacologically removed with picrotoxin (40 microM) or bicuculline (25 microM) resulting in significantly greater death than Abeta-exposed neurons alone. From these observations, it is proposed that intrinsic functional inhibition in hippocampal circuits can reduce adverse effects of Abeta on the excitatory component. By considering not just the excitatory component of electrical activity, but the intrinsic balance between excitation and inhibition, new strategies for the treatment of Alzheimer disease may emerge.

Outgrowth morphology and intracellular calcium of crustacean neurons displaying distinct morphologies in primary culture.

Peptide-secreting neurons from crustacean X-organ regenerating in defined culture possess different ionic current profiles correlated with two distinct morphological types, veiling and branching; voltage-dependent Ca2+ current is prominent in neurons consistently extending large veils, but is small in neurons that repetitively branch. Intracellular free calcium levels ([Ca2+]i) have been implicated in the regulation of neurite outgrowth underlying the establishment of distinct morphologies. Here, basal [Ca2+]i was measured by fura-2 fluorescence ratio imaging from these morphologically distinct neurons and compared. Both morphological types can extend out processes over a [Ca2+]i range (approximately 50 to 300 nM) that is much greater than that reported for neurons of other phyla. Application of high K+ saline led to increases in [Ca2+]i in soma, neurite, and lamellipodium of veiling neurons. Increases were greater for veiling than branching neurons. These observations were consistent with the previous voltage clamp data for calcium currents. Media altered to perturb [Ca2+]i were used to assess the role of [Ca2+]i in veiling or branching outgrowth programs. Outgrowth of veiling cells was arrested by addition of 100 microM Cd2+, a calcium channel blocker. Outgrowth resumed following brief exposures to Cd2+. Branching neurons were unaffected by Cd2+. Cd2+ at lower levels (10 microM) had no effect on outgrowth of either neuronal type, whereas at higher levels (1 mM), outgrowth of both types was arrested. Reduction of extracellular sodium to 0.001 of normal concentration stopped veiling outgrowth, but branching outgrowth continued, although it was less robust. Addition of tetrodotoxin (1 microM) did not alter outgrowth of either neuronal type relative to controls. Thus, peptidergic neurons of differing intrinsic morphologies maintain similar basal [Ca2+]i levels under identical culture conditions, yet show differing sensitivities to manipulations influencing [Ca2+]i with respect to regenerative outgrowth, but not its form.

Ionic currents of morphologically distinct peptidergic neurons in defined culture.

1. The X-organ sinus gland is a major peptidergic neurosecretory system in Crustacea, analogous to the vertebrate hypothalamoneurohypophyseal system. Neuronal somata isolated from the crab (Cardisoma carnifex) X-organ and maintained in primary culture in unconditioned, fully defined medium show immediate regenerative outgrowth. Outgrowth occurring as broad lamellipodia ("veiled") distinguishes neurons consistently showing crustacean hyperglycemic hormone immunoreactivity. Neurons that are immunoreactive against molt-inhibiting hormone and red pigment concentrating hormone antisera give rise to branched neurites ("branched"). 2. The whole-cell variation of the patch-clamp technique was used to study the electrophysiology of these two cell types 24-48 h after plating. Under current clamp, only veiled neurons fired overshooting action potentials either spontaneously or in response to depolarization. 3. Under voltage clamp, net current was predominantly outward. When solutions that suppressed outward current were used, only veiled neurons showed significant inward current. These included a tetrodotoxin (TTX)-sensitive Na current and a slow (time to peak 6-10 ms at 0 mV) Cd-sensitive Ca current (ICa) that was activated at potentials less than -30 mV, was maximal at 0 to +20 mV, and did not reverse at potentials up to +60 mV. 4. In TTX, the form of the Ca current I(V) curve was unchanged by changes of holding potential between -40 and -80 mV, and 75-100% of ICa was available from -40 mV. 5. ICa inactivated slowly and incompletely. Analysis with two-pulse regimes suggested that both inactivation and facilitation mechanisms were present. 6. Outward current was examined in the presence and absence of 0.5 mM Cd2+ (1 microM TTX was always present in the external medium). Cd2+ ions slightly reduced the peak outward current, usually by less than 10% (Vc = -10 to +20 mV; Vh = -80 mV). All additional observations were in the presence of TTX and Cd2+. 7. Both cell types expressed a 4-aminopyridine (4-AP)-sensitive transient current, analogous to IA, and a slower-rising (minimum time to peak 20 ms), sustained current that was partially sensitive to tetraethylammonium, analogous to IK. 8. The mean Vh at which IA was half inactivated was -46 mV, and the mean time constant for removal of inactivation was 46 ms.(ABSTRACT TRUNCATED AT 400 WORDS)

Trends in preterm labor.

Preterm birth has been identified by the National Commission to Prevent Infant Mortality (1988) as the primary cause of the increased infant mortality rate in the United States. An analysis of what is currently known about four areas of preterm labor including (1) definition and causes, (2) identification of patients at risk, (3) management techniques, and (4) use of patient education in labor is presented in this paper. Preterm labor is defined as uterine contractions that occur between 20 and 37 weeks' gestation with progressive cervical dilatation or effacement or both. Directions for future research are discussed.