Calcium dynamics in the extracellular space of mammalian neural tissue.
(17/4009)
In the brain, hundreds of intracellular processes are known to depend on calcium influx; hence any substantial fluctuation in external calcium ([Ca2+]o) is likely to engender important functional effects. Employing the known scales and parameters of mammalian neural tissue, we introduce and justify a computational approach to the hypothesis that large changes in local [Ca2+]o will be part of normal neural activity. Using this model, we show that the geometry of the extracellular space in combination with the rapid movement of calcium through ionic channels can cause large external calcium fluctuations, up to 100% depletion in many cases. The exact magnitude of a calcium fluctuation will depend on 1) the size of the consumption zone, 2) the local diffusion coefficient of calcium, and 3) the geometrical arrangement of the consuming elements. Once we have shown that using biologically relevant parameters leads to calcium changes, we focus on the signaling capacity of such concentration fluctuations. Given the sensitivity of neurotransmitter release to [Ca2+]o, the exact position and timing of neural activity will delimit the terminals that are able to release neurotransmitter. Our results indicate that mammalian neural tissue is engineered to generate significant changes in external calcium concentrations during normal activity. This design suggests that such changes play a role in neural information processing. (+info)
Presynaptic strontium dynamics and synaptic transmission.
(18/4009)
Strontium can replace calcium in triggering neurotransmitter release, although peak release is reduced and the duration of release is prolonged. Strontium has therefore become useful in probing release, but its mechanism of action is not well understood. Here we study the action of strontium at the granule cell to Purkinje cell synapse in mouse cerebellar slices. Presynaptic residual strontium levels were monitored with fluorescent indicators, which all responded to strontium (fura-2, calcium orange, fura-2FF, magnesium green, and mag-fura-5). When calcium was replaced by equimolar concentrations of strontium in the external bath, strontium and calcium both entered presynaptic terminals. Contaminating calcium was eliminated by including EGTA in the extracellular bath, or by loading parallel fibers with EGTA, enabling the actions of strontium to be studied in isolation. After a single stimulus, strontium reached higher peak free levels than did calcium (approximately 1.7 times greater), and decayed more slowly (half-decay time 189 ms for strontium and 32 ms for calcium). These differences in calcium and strontium dynamics are likely a consequence of greater strontium permeability through calcium channels, lower affinity of the endogenous buffer for strontium, and less efficient extrusion of strontium. Measurements of presynaptic divalent levels help to explain properties of release evoked by strontium. Parallel fiber synaptic currents triggered by strontium are smaller in amplitude and longer in duration than those triggered by calcium. In both calcium and strontium, release consists of two components, one more steeply dependent on divalent levels than the other. Strontium drives both components less effectively than does calcium, suggesting that the affinities of the sensors involved in both phases of release are lower for strontium than for calcium. Thus, the larger and slower strontium transients account for the prominent slow component of release triggered by strontium. (+info)
Hypothalamic hypocretin (orexin): robust innervation of the spinal cord.
(19/4009)
Hypocretin (orexin) is synthesized by neurons in the lateral hypothalamus and has been reported to increase food intake and regulate the neuroendocrine system. In the present paper, long descending axonal projections that contain hypocretin were found that innervate all levels of the spinal cord from cervical to sacral segments, as studied in mouse, rat, and human spinal cord and not previously described. High densities of axonal innervation are found in regions of the spinal cord related to modulation of sensation and pain, notably in the marginal zone (lamina 1). Innervation of the intermediolateral column and lamina 10 as well as strong innervation of the caudal region of the sacral cord suggest that hypocretin may participate in the regulation of both the sympathetic and parasympathetic parts of the autonomic nervous system. Double-labeling experiments in mice combining retrograde transport of diamidino yellow after spinal cord injections and immunocytochemistry support the concept that hypocretin-immunoreactive fibers in the cord originate from the neurons in the lateral hypothalamus. Digital-imaging physiological studies with fura-2 detected a rise in intracellular calcium in response to hypocretin in cultured rat spinal cord neurons, indicating that spinal cord neurons express hypocretin-responsive receptors. A greater number of cervical cord neurons responded to hypocretin than another hypothalamo-spinal neuropeptide, oxytocin. These data suggest that in addition to possible roles in feeding and endocrine regulation, the descending hypocretin fiber system may play a role in modulation of sensory input, particularly in regions of the cord related to pain perception and autonomic tone. (+info)
Vesicular monoamine transporter-2 and aromatic L-amino acid decarboxylase enhance dopamine delivery after L-3, 4-dihydroxyphenylalanine administration in Parkinsonian rats.
(20/4009)
Medical therapy in Parkinson's disease (PD) is limited by the short-duration response and development of dyskinesia that result from chronic L-3,4-dihydroxyphenylalanine (L-DOPA) therapy. These problems occur partly because the loss of dopamine storage sites leads to erratic dopamine delivery. Vesicular monoamine transporter-2 (VMAT-2) plays a critical role in dopamine storage by packaging dopamine into synaptic vesicles and regulating sustained release of dopamine. To restore the capacity to produce and store dopamine in parkinsonian rats, primary skin fibroblast cells (PF) were genetically modified with aromatic L-amino acid decarboxylase (AADC) and VMAT-2 genes. After incubation with L-DOPA in culture, the doubly transduced fibroblast cells (PFVMAA) produced and stored dopamine at a much higher level than the cells with either gene alone. PFVMAA cells in culture released dopamine gradually in a constitutive manner. Genetically modified fibroblast cells were grafted in parkinsonian rat striata, and L-DOPA was systemically administered. Higher dopamine levels were sustained for a longer duration in rats grafted with PFVMAA cells than in those grafted with either control cells or cells with AADC alone. These findings underscore the importance of dopamine storage capacity in determining the efficacy of L-DOPA therapy and illustrate a novel method of gene therapy combined with precursor administration to overcome the major obstacles of PD treatment. (+info)
Clozapine preferentially increases dopamine release in the rhesus monkey prefrontal cortex compared with the caudate nucleus.
(21/4009)
Despite substantial differences between species in the organization and elaboration of the cortical dopamine innervation, little is known about the pharmacological response of cortical or striatal sites to antipsychotic medications in nonhuman primates. To examine this issue, rhesus monkeys were chronically implanted with guide cannulae directed at the principal sulcus, medial prefrontal cortex, premotor cortex, and caudate nucleus. Alterations in dopamine release in these discrete brain regions were measured in response to administration of clozapine or haloperidol. Clozapine produced significant and long-lasting increases in dopamine release in the principal sulcus, and to a lesser extent, in the caudate nucleus. Haloperidol did not produce a consistent effect on dopamine release in the principal sulcus, although it increased dopamine release in the caudate. Clozapine's preferential augmentation of dopamine release in the dorsolateral prefrontal cortex supports the idea that clozapine exerts its therapeutic effects in part by increasing cortical dopamine neurotransmission. (+info)
The influence of the colon on postprandial glucagon-like peptide 1 (7-36) amide concentration in man.
(22/4009)
Glucagon-like peptide (7-36) amide (GLP-1) is an incretin hormone of the enteroinsular axis released rapidly after meals despite the fact that GLP-1 secreting cells (L-cells) occur predominantly in the distal gut. The importance of these colonic L-cells for postprandial GLP-1 was determined in healthy control subjects and in ileostomy patients with minimal small bowel resection (<5 cm). Subjects were fed a high complex carbohydrate test meal (15.3 g starch) followed by two carbohydrate-free, high fat test meals (25 g and 48.7 g fat respectively). Circulating levels of glucose, insulin, glucagon, glucose insulinotrophic peptide (GIP) and GLP-1 were measured over a 9-h postprandial period. For both subject groups the complex carbohydrate test meal failed to elicit a rise in either GIP or GLP-1. However, both hormones were elevated after the fat load although the GLP-1 concentration was significantly reduced in the ileostomist group when compared with controls (P=0.02). Associated with this reduction in circulating GLP-1 was an elevation in glucagon concentration (P=0.012) and a secondary rise in the plasma glucose concentration (P=0.006). These results suggest that the loss of colonic endocrine tissue is an important determinant in the postprandial GLP-1 concentration. Ileostomists should not be assumed to have normal enteroinsular function as the colon appears to have an important role in postprandial metabolism. (+info)
Glutamate regulates IP3-type and CICR stores in the avian cochlear nucleus.
(23/4009)
Neurons of the avian cochlear nucleus, nucleus magnocellularis (NM), are activated by glutamate released from auditory nerve terminals. If this stimulation is removed, the intracellular calcium ion concentration ([Ca2+]i) of NM neurons rises and rapid atrophic changes ensue. We have been investigating mechanisms that regulate [Ca2+]i in these neurons based on the hypothesis that loss of Ca2+ homeostasis causes the cascade of cellular changes that results in neuronal atrophy and death. In the present study, video-enhanced fluorometry was used to monitor changes in [Ca2+]i stimulated by agents that mobilize Ca2+ from intracellular stores and to study the modulation of these responses by glutamate. Homobromoibotenic acid (HBI) was used to stimulate inositol trisphosphate (IP3)-sensitive stores, and caffeine was used to mobilize Ca2+ from Ca2+-induced Ca2+ release (CICR) stores. We provide data indicating that Ca2+ responses attributable to IP3- and CICR-sensitive stores are inhibited by glutamate, acting via a metabotropic glutamate receptor (mGluR). We also show that activation of C-kinase by a phorbol ester will reduce HBI-stimulated calcium responses. Although the protein kinase A accumulator, Sp-cAMPs, did not have an effect on HBI-induced responses. CICR-stimulated responses were not consistently attenuated by either the phorbol ester or the Sp-cAMPs. We have previously shown that glutamate attenuates voltage-dependent changes in [Ca2+]i. Coupled with the present findings, this suggests that in these neurons mGluRs serve to limit fluctuations in intracellular Ca2+ rather than increase [Ca2+]i. This system may play a role in protecting highly active neurons from calcium toxicity resulting in apoptosis. (+info)
Contribution of single-channel properties to the time course and amplitude variance of quantal glycine currents recorded in rat motoneurons.
(24/4009)
The amplitude of spontaneous, glycinergic miniature inhibitory postsynaptic currents (mIPSCs) recorded in hypoglossal motoneurons (HMs) in an in vitro brain stem slice preparation increased over the first 3 postnatal weeks, from 42 +/- 6 pA in neonate (P0-3) to 77 +/- 11 pA in juvenile (P11-18) HMs. Additionally, mIPSC amplitude distributions were highly variable: CV 0.68 +/- 0.05 (means +/- SE) for neonates and 0.83 +/- 0.06 for juveniles. We wished to ascertain the contribution of glycine receptor (GlyR)-channel properties to this change in quantal amplitude and to the amplitude variability and time course of mIPSCs. To determine whether a postnatal increase in GlyR-channel conductance accounted for the postnatal change in quantal amplitude, the conductance of synaptic GlyR channels was determined by nonstationary variance analysis of mIPSCs. It was 48 +/- 8 pS in neonate and 46 +/- 10 pS in juvenile HMs, suggesting that developmental changes in mIPSC amplitude do not result from a postnatal alteration of GlyR-channel conductance. Next we determined the open probability (Popen) of GlyR channels in outside-out patches excised from HMs to estimate the contribution of stochastic channel behavior to quantal amplitude variability. Brief (1 ms) pulses of glycine (1 mM) elicited patch currents that closely resembled mIPSCs. The GlyR channels' Popen, calculated by nonstationary variance analysis of these currents, was approximately 0.70 (0.66 +/- 0.09 in neonates and 0.72 +/- 0.05 in juveniles). The decay rate of patch currents elicited by brief application of saturating concentrations of glycine (10 mM) increased postnatally, mimicking previously documented changes in mIPSC time course. Paired pulses of glycine (10 mM) were used to determine if rapid GlyR-channel desensitization contributed to either patch current time course or quantal amplitude variability. Because we did not observe any fast desensitization of patch currents, we believe that fast desensitization of GlyRs underlies neither phenomenon. From our analysis of glycinergic patch currents and mIPSCs, we draw three conclusions. First, channel deactivation is the primary determinant of glycinergic mIPSC time course, and postnatal changes in channel deactivation rate account for observed developmental changes in mIPSC decay rate. Second, because GlyR-channel Popen is high, differences in receptor number between synapses rather than stochastic channel behavior are likely to underlie the majority of quantal variability seen at glycinergic synapses throughout postnatal development. We estimate the number of GlyRs available at a synapse to be on average 27 in neonate neurons and 39 in juvenile neurons. Third, this change in the calculated number of GlyRs at each synapse may account for the postnatal increase in mIPSC amplitude. (+info)