Calcium channels coupled to glutamate release identified by omega-Aga-IVA.

Presynaptic calcium channels are crucial elements of neuronal excitation-secretion coupling. In mammalian brain, they have been difficult to characterize because most presynaptic terminals are too small to probe with electrodes, and available pharmacological tools such as dihydropyridines and omega-conotoxin are largely ineffective. Subsecond measurements of synaptosomal glutamate release have now been used to assess presynaptic calcium channel activity in order to study the action of peptide toxins from the venom of the funnel web spider Agelenopsis aperta, which is known to inhibit dihydropyridine and omega-conotoxin-resistant neuronal calcium currents. A presynaptic calcium channel important in glutamate release is shown to be omega-Aga-IVA sensitive and omega-conotoxin resistant.

Calcium as a coagonist of inositol 1,4,5-trisphosphate-induced calcium release.

Inositol 1,4,5-trisphosphate (IP3)-induced calcium release from intracellular stores is a regulator of cytosolic-free calcium levels. The subsecond kinetics and regulation of IP3-induced calcium-45 release from synaptosome-derived microsomal vesicles were resolved by rapid superfusion. Extravesicular calcium acted as a coagonist, potentiating the transient IP3-induced release of calcium-45. Thus, rapid elevation of cytosolic calcium levels may trigger IP3-induced calcium release in vivo. Extravesicular calcium also produced a more slowly developing, reversible inhibition of IP3-induced calcium-45 release. Sequential positive and negative feedback regulation by calcium of IP3-induced calcium release may contribute to transients and oscillations of cytosolic-free calcium in vivo.

Multiple calcium channel types control glutamatergic synaptic transmission in the hippocampus.

N-type calcium channels play a dominant role in controlling synaptic transmission in many peripheral neurons. Transmitter release from mammalian central nerve terminals, however, is relatively resistant to the N channel antagonist omega-conotoxin GVIA. We studied the sensitivity of glutamatergic synaptic transmission in rat hippocampal slices to omega-conotoxin and to omega-Aga-IVA, a P channel antagonist. Both toxins reduced the amplitude of excitatory postsynaptic potentials in CA1 pyramidal neurons, but omega-Aga-IVA was the more rapid and efficacious. These results were corroborated by biochemical studies measuring subsecond, calcium-dependent [3H]glutamate release from hippocampal synaptosomes. Thus, at least two calcium channel types trigger glutamate release from hippocampal neurons, but P-type plays a more prominent role. Eliminating synaptic transmission in the CNS, therefore, may require inhibiting more than a single calcium channel type.

Exocytotic Ca2+ channels in mammalian central neurons.

Intracellular Ca2+ initiates physiological events as diverse as gene transcription, muscle contraction, cell division and exocytosis. Predictably, the metabolic machinery that elicits and responds to changes in intracellular Ca2+ is correspondingly heterogeneous. This review focuses on one element of this complex web that is of particular importance to neurobiologists: identifying which members of the voltage-dependent Ca(2+)-channel superfamily are responsible for the Ca2+ that enters nerve terminals and elicits vesicular release of chemical transmitters.

Galaxy evolution. Black hole feedback in the luminous quasar PDS 456.

The evolution of galaxies is connected to the growth of supermassive black holes in their centers. During the quasar phase, a huge luminosity is released as matter falls onto the black hole, and radiation-driven winds can transfer most of this energy back to the host galaxy. Over five different epochs, we detected the signatures of a nearly spherical stream of highly ionized gas in the broadband x-ray spectra of the luminous quasar PDS 456. This persistent wind is expelled at relativistic speeds from the inner accretion disk, and its wide aperture suggests an effective coupling with the ambient gas. The outflow's kinetic power larger than 10(46) ergs per second is enough to provide the feedback required by models of black hole and host galaxy coevolution.

Basic emotions: can conflicting criteria converge?

The authors discuss some of the key points raised by Ekman (1992), Izard (1992), and Panksepp (1992) in their critiques of Ortony and Turner's (1990) suggestion that there are and probably can be no objective and generally acceptable criteria for what is to count as a basic emotion. A number of studies are discussed that are relevant to the authors' contention that a more promising approach to understanding the huge diversity among emotions is to think in terms of emotions being assemblages of basic components rather than combinations of other basic emotions. The authors stress that their position does not deny that emotions are based on "hardwired" biological systems. On the other hand, the existence of such systems does not mean that some emotions (such as those that appear on lists of basic emotions) have a special status. Finally, the authors note that Ekman, Izard, and Panksepp, in adopting different starting points for their research, arrive at rather different conclusions as to what basic emotions are and which emotions are basic. It is concluded that converging resolutions of these questions are improbable.

What's basic about basic emotions?

A widespread assumption in theories of emotion is that there exists a small set of basic emotions. From a biological perspective, this idea is manifested in the belief that there might be neurophysiological and anatomical substrates corresponding to the basic emotions. From a psychological perspective, basic emotions are often held to be the primitive building blocks of other, nonbasic emotions. The content of such claims is examined, and the results suggest that there is no coherent nontrivial notion of basic emotions as the elementary psychological primitives in terms of which other emotions can be explained. Thus, the view that there exist basic emotions out of which all other emotions are built, and in terms of which they can be explained, is questioned, raising the possibility that this position is an article of faith rather than an empirically or theoretically defensible basis for the conduct of emotion research. This suggests that perhaps the notion of basic emotions will not lead to significant progress in the field. An alternative approach to explaining the phenomena that appear to motivate the postulation of basic emotions is presented.

Pharmacological characterization of presynaptic calcium channels using subsecond biochemical measurements of synaptosomal neurosecretion.

The recent development of peptide antagonists that selectively block subtypes of neuronal calcium channel has provided tools to study the role of presynaptic calcium channels in triggering exocytosis. A variety of methods have consistently demonstrated that multiple channel types participate in exocytosis. We have studied the subsecond kinetics of [3H]glutamate release from rat cortical synaptosomes as an assay for presynaptic calcium channel activity. The system has been characterized over a broad range of conditions in an effort to compare biochemical measurements of transmitter release with electrophysiological measurements of synaptic currents. The efficacies of omega-agatoxin IVA and omega-conotoxins GVIA and MVIIC were increased when Ca2+ influx was decreased by: (1) decreasing the KCl concentration to diminish the extent of depolarization, (2) decreasing the Ca2+ concentration, or (3) partially blocking Ca2+ influx with one of the other antagonists. By using these toxins in combination, we found that at least three types of pharmacologically distinct channel participate in exocytosis. The largest fraction of glutamate release is blocked by omega-agatoxin IVA (IC50 = 12.2 nM) and by omega-conotoxin MVIIC (IC50 = 35 nM), consistent with the pharmacology of a P type channel. The effects of saturating concentrations (1 microM) of omega-agatoxin IVA or omega-conotoxin MVIIC occlude each other, suggesting that these peptides overlap completely. The specific N type antagonist omega-conotoxin GVIA inhibits a significant portion of release (IC50 less than 1 nM) but only under conditions of reduced Ca2+ concentration. These results suggest that the N type channel in nerve terminals is distinct from that found in hippocampal somata, since it appears to be resistant to by omega-conotoxin MVIIC. The combination of omega-conotoxin GVIA (100 nM) and either omega-agatoxin IVA or omega-conotoxin MVIIC (1 microM each) blocked approx 90% of release when the Ca2+ concentration was reduced (0.46 mM or less), but 30-40% of release remained when the concentration of Ca2+ in the stimulus buffer was 1 mM or greater, indicating that a resistant channel type(s) also participates in exocytosis. Specific inhibitors of this resistant phenotype will be useful for further refinement of our understanding of the role of presynaptic calcium channels in mediating neurosecretion.

Calcium influx through presynaptic 5-HT3 receptors facilitates GABA release in the hippocampus: in vitro slice and synaptosome studies.

Serotonin 5-hydroxytryptamine type 3 receptors (5HT3R) are Ca2+-permeant, non-selective cation channels that have been localized to presynaptic terminals and demonstrated to modulate neurotransmitter release. In the present study the effect of 5-HT on GABA release in the hippocampus was characterized using both electrophysiological and biochemical techniques. 5-HT elicited a burst-like, 6- to 10-fold increase in the frequency of GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) measured with whole-cell voltage-clamp recordings of CA1 neurons in hippocampal slices. When tetrodotoxin was used to block action potential propagation, the 5-HT-induced burst of IPSCs was still observed. Stimulation of hippocampal synaptosomes with 5-HT resulted in a significant increase in the amount of [3H]GABA released by hyperosmotic saline. In both preparations, the 5-HT effect was shown to be mediated by 5HT3Rs, as it was mimicked by the selective 5HT3R agonist m-chlorophenyl biguanide and blocked by the selective 5HT3R antagonist 3-tropanylindole-3-carboxylate hydrochloride. The 5HT3R-mediated increase in GABA release was blocked by 100 microM cadmium or by omitting Ca2+ in external solutions, indicating the Ca2+-dependence of the effect. The high voltage-activated Ca2+ channel blockers omega-conotoxin GVIA and omega-conotoxin MVIIC and 10 microM cadmium had no significant effect on the 5-HT3R-mediated enhancement of GABA release, indicating that Ca2+ influx through the 5-HT3R facilitates GABA release. Taken together, these data provide direct evidence that Ca2+ entry via presynaptic 5HT3Rs facilitates the release of GABA from hippocampal interneurons.

Selective attenuation of psychostimulant-induced behavioral responses in mice lacking A(2A) adenosine receptors.

A(2A) adenosine receptors are highly expressed in the striatum where they modulate dopaminergic activity. The role of A(2A) receptors in psychostimulant action is less well understood because of the lack of A(2A)-selective compounds with access to the central nervous system. To investigate the A(2A) adenosinergic regulation of psychostimulant responses, we examined the consequences of genetic deletion of A(2A) receptors on psychostimulant-induced behavioral responses. The extent of dopaminergic innervation and expression of dopamine receptors in the striatum were indistinguishable between A(2A) receptor knockout and wild-type mice. However, locomotor responses to amphetamine and cocaine were attenuated in A(2A) knockout mice. In contrast, D(1)-like receptor agonists SKF81297 and SKF38393 produced identical locomotor stimulation and grooming, respectively, in wild-type and A(2A) knockout mice. Similarly, the D(2)-like agonist quinpirole produced motor-depression and stereotypy that were indistinguishable between A(2A) knockout and wild-type mice. Furthermore, attenuated amphetamine- (but not SKF81297-) induced locomotion was observed in pure 129-Steel as well as hybrid 129-SteelxC57BL/6 mice, confirming A(2A) receptor deficiency (and not genetic background) as the cause of the blunted psychostimulant responses in A(2A) knockout mice. These results demonstrate that A(2A) receptor deficiency selectively attenuates psychostimulant-induced behavioral responses and support an important role for the A(2A) receptor in modulating psychostimulant effects.

Three-dimensional imaging and image analysis of hippocampal neurons: confocal and digitally enhanced wide field microscopy.

The microscopy of biological specimens has traditionally been a two-dimensional imaging method for analyzing what are in reality three-dimensional (3-D) objects. This has been a major limitation of the application of one of science's most widely used tools. Nowhere has this limitation been more acute than in neurobiology, which is dominated by the necessity of understanding both large- and small-scale 3-D anatomy. Fortunately, recent advances in optical instrumentation and computational methods have provided the means for retrieving the third dimension, making full 3-D microscopic imaging possible. Optical designs have concentrated on the confocal imaging mode while computational methods have made 3-D imaging possible with wide field microscopes using deconvolution methods. This work presents a brief review of these methods, especially as applied to neurobiology, and data using both approaches. Specimens several hundred micrometers thick can be sampled allowing essentially intact neurons to be imaged. These neurons or selected components can be contrasted with either fluorescent, absorption, or reflection stains. Image analysis in 3-D is as important as visualization in 3-D. Automated methods of cell counting and analysis by nuclear detection as well as tracing of individual neurons are presented.

Pertussis toxin-sensitive GTP-binding proteins characterized in synaptosomal fractions of embryonic avian cerebral cortex.

Pertussis toxin (PTX)-sensitive GTP-binding proteins (G proteins) are essential intermediaries subserving neuronal signal transduction pathways that regulate excitation-secretion coupling. Despite this established role, relatively little is known regarding the identity, subcellular distribution, and relative abundance of this class of G proteins in synaptic nerve endings. Here, sucrose density gradient centrifugation was combined with 1- and 2-dimensional gel electrophoresis to characterize PTX-sensitive G protein alpha subunits in synaptosomal fractions of embryonic (day 12) chick cerebral cortical homogenates. These findings demonstrate multiple isoforms of M(r) 40-41 kDa Gi alpha and G(o) alpha subunits that can be identified on the basis of PTX-catalyzed ADP-ribosylation and immunoblot analysis.

Obesity in children and adolescents.

The literature regarding etiology and consequences of childhood and juvenile obesity is briefly surveyed with special reference to recent trends in behavioral research. Major emphasis is focused on issues of differential diagnosis, and treatment. The objective is to offer a logical and coherent plan for approaching and managing obesity in childhood and adolescence. The importance of an awareness and understanding of both the patient's and therapist's attitudes toward the problem of obesity is addressed. Furthermore, the necessity for a cooperative approach, utilizing nonmedical professionals, in developing a coordinated treatment plan for obese young people is discussed.

Poisoning: effective clinical intervention.

Poisoning accounts for 40-60% of suicides, is the commonest medical emergency in small children, and an important source of occupational injury. Prevention of unintentional poisoning involves primarily education of parents. In intervention, the patient-not the poison-must be treated. Self-poisoners require supportive but firm handling. Treatment is directed towards prevention of further absorption, removal of absorbed poison, symptomatic or supportive therapy, and administration of systemic antidotes. Careful attention should be paid to the physician's legal responsibilities in cases of poisoning.

Prolonged time course of glutamate release from nerve terminals: relationship between stimulus duration and the secretory event.

The kinetics of synaptosomal [3H]glutamate release were measured on a subsecond time scale to study the relationship between the length of depolarization and the duration of the secretory event. The time course of release evoked by elevated K+ was complex, proceeding for several seconds after a 200-ms depolarization. We developed a protocol for depolarizing excitable membranes on a millisecond time scale to deliver brief depolarizations, termed the synthetic action potential, by using batrachotoxin to activate Na+ channels. Depolarization is achieved by superfusing with solutions containing elevated concentrations of Na+, and the duration of the depolarization is limited by including tetrodotoxin (TTX) in the superfusion solution to block Na+ current and membrane depolarizations were made in batrachotoxin-treated sensory neurons using patch clamp recording methods. Rapid increases in Na+ and TTX concentrations produced transient increases in inward Na+ current that decayed with a time course proportional to TTX concentration. Current clamp measurements indicated that, with 10 microM TTX, depolarizations last approximately 30 ms. Nonetheless, synaptosomal release of [3H]glutamate triggered by the synthetic action potential remained prolonged. Brief neuronal action potentials at some synapses may trigger transmitter release that persists for several seconds.