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The enzyme glutamate carboxypeptidase II (GCP II) has been cloned from rat brain and human prostate. This enzyme, which catabolizes the neuropeptide N-acetylaspartylglutamate, has also been known as N-acetylated alpha-linked acidic dipeptidase (NAALADase), and is identical to the prostate-specific membrane antigen and to the jejunal folylpoly-gamma-glutamate carboxypeptidase. The goals of the present study were to elucidate the cell specificity and regional pattern of GCP II expression in the rat nervous system by using Northern blots and enzymatic assays of brain and subfractionated primary neuronal and glial cultures together with in situ hybridization histochemistry (ISHH) in sections of adult rat tissue. GCP II activity was assayed in astrocyte cultures (4.4 pmol/mg protein per minute), neuronal-glial cocultures (2.5 pmol/mg protein per minute) and neuron-enriched cultures (0.38 pmol/mg protein per minute), with the activity in each preparation correlating to its astrocytic content (r = 0.99). No activity was detected in cultured oligodendrocytes or microglia. Northern blots probed with a GCP II cDNA detected mRNAs exclusively in activity-positive cell preparations. ISHH results show that GCP II is expressed by virtually all astrocytes, by Bergmann glial cells in cerebellum, by Müller cells in retina and by the satellite cells in dorsal root ganglia. Astrocytes in select groups of nuclei (e.g., habenula, supraoptic nucleus, pontine nucleus) contained pronounced levels of GCP II message. The data of the present study suggest that GCP II is expressed in the adult rat nervous system exclusively in astrocytic glial cells.
Copyright 1999 Wiley-Liss, Inc.
Embryonic (E19-E20) and early postnatal (P2) spinal cords with intact saphenous and sciatic nerves were isolated and placed in aerated artificial cerebral spinal fluid (CSF). Intracellular recordings were made from cells in the L2-L6 dorsal root ganglia using microelectrodes filled with 3 M potassium acetate or 5% neurobiotin (NB) in 1 M potassium acetate. Several physiological properties of adequately impaled cells were measured, including peripheral conduction velocity, action potential (AP) amplitude and duration, duration of afterhyperpolarization (AHP), input impedance, rheobase, presence of inward rectifying current, and maximum somal firing frequency. The extent to which these properties are correlated also was determined. One cell per ganglion was injected with NB. Stained somata and their central projections in the spinal cord were visualized in serial 50 microm sections. Cell size was determined and the central morphology of the central projections examined. Although some fibers were in the process of growing into the spinal cord, others had established projections over several millimeters in the dorsal columns. Although most of these fibers supported projections in the gray matter, 22% only maintained fibers in the dorsal columns. Fibers with projections in the dorsal horn exhibited three types of morphology: projections confined to the superficial dorsal horn (laminae I, II); terminals confined to laminae III-V; and projections spanning laminae II-V. In addition, some embryonic fibers maintained projections to the dorsal horn that extended over five lumbar segments. Somal APs could be divided into two groups: broad spikes with inflections on their falling phase and narrow spikes without inflections. On average, cells with broad spikes (BS) had the following characteristics: slower peripheral conduction velocity, larger amplitude, higher rheobase and input impedance, longer AHP duration, and lower maximum firing frequency. There were significant correlations between conduction velocity and several of the physiological properties. Conduction velocity was negatively correlated with AP duration, rheobase, and input impedance and positively correlated with maximum firing frequency. Comparisons between spike shape and central morphology revealed that cells lacking collaterals in the gray matter and those with projections in the superficial dorsal horn always had broad somal spikes with inflections. Those with projections confined to laminae III-V always had narrow somal spikes (NS).
Neuronal cell firing is crucial to nerve-nerve communication. The ability to produce consecutive action potentials is related to the activation of inward currents after each upstroke. If fast Na current is indeed responsible for the overshoot, it is still unclear which current drives membrane voltage to the Na threshold. In this study we present evidence that in adult rat sensory neurones a dihydropyridine-sensitive Ca channel exists in addition to the well characterized L-type, or high-threshold Ca channel. During stimulated action potential trains, L-type Ca channels open during the excitation wave, whereas activity of the other dihydropyridine-sensitive Ca channel was observed primarily between action potentials. This second Ca pathway shows remarkably long openings at negative potentials after a series of positive prepulses. The nerve action potential and the repetitive firing work as a physiological Ca channel facilitation mechanism. Therefore, we suggest that this novel Ca conductance provides inward current, between two consecutive action potentials, able to modulate the frequency of neuronal bursts.
1. Voltage-gated Ca2+ currents (ICa) and Ca(2+)-activated Cl- currents (ICl(Ca)) were recorded from cultured rat dorsal root ganglion (DRG) neurones using the whole-cell configuration of the patch clamp technique. Intracellular photorelease of Ca2+ by flash photolysis of DM-nitrophen elicited transient inward currents only in those cells which possessed Ca(2+)-activated Cl- tail currents following ICa. The reversal potential of the flash responses was hyperpolarized when extracellular Cl- was replaced by SCN-. The flash responses and the Ca(2+)-activated Cl- tail currents were inhibited by the Cl- channel blockers niflumic acid (10-100 microM) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (10 microM). 2. After activation by ICa, the Ca(2+)-activated Cl- current could be reactivated during its decay by photorelease of caged Ca2+. Experiments carried out on neurones held at 0 mV demonstrated that ICl(Ca) could be chronically activated due to residual Ca2+ influx. These data directly demonstrated that the decay of ICl(Ca) is not due to inactivation but rather to deactivation as a result of removal of the Ca2+ load from the cell cytoplasm. 3. Photorelease of caged inositol 1,4,5-trisphosphate (IP3) failed to activate any Ca(2+)-dependent current responses in cultured DRG neurones, although application of caffeine elicited transient inward currents, and responses to photoreleased IP3 could be obtained from freshly dissociated smooth muscle cells. 4. Photorelease of Ca2+ provides a useful method for investigating the properties of ICl(Ca) independently from other physiological parameters. In addition, we have directly demonstrated that ICl(Ca) in DRG neurones does not inactivate, and so may continue to modulate membrane excitability as long as the intracellular Ca2+ concentration ([Ca2+]i) close to the cell membrane is elevated.(ABSTRACT TRUNCATED AT 250 WORDS)
In brain and nerves the phosphorylation of glucose, rather than its transport, is generally considered the major rate-limiting step in metabolism. Since little is known regarding the kinetic coupling between these processes in neuronal tissues, we investigated the transport and phosphorylation of [2-3H]glucose in two neuronal cell models: a stable neuroblastoma cell line (NCB20), and a primary culture of isolated rat dorsal root ganglia cells. When transport and phosphorylation were measured in series, phosphorylation was the limiting step, because intracellular glucose concentrations were the same as those outside of cells, and because the apparent Km for glucose utilization was lower than expected for the transport step. However, the apparent Km was still severalfold higher than the Km of hexokinase I. When [2-3H]glucose efflux and phosphorylation were measured from the same intracellular glucose pool in a parallel assay, rates of glucose efflux were three- to-fivefold greater than rates of phosphorylation. With the parallel assay, we observed that activation of glucose utilization by the sodium channel blocker veratridine caused a selective increase in glucose phosphorylation and was without effect on glucose transport. In contrast to results with glucose, both cell types accumulated 2-deoxy-D-[14C]glucose to concentrations severalfold greater than extracellular concentrations. We conclude from these studies that glucose utilization in neuronal cells is phosphorylation-limited, and that the coupling between transport and phosphorylation depends on the type of hexose used.
Neurotrophins activate the Trk tyrosine kinase receptors, which subsequently initiate signaling pathways that have yet to be fully resolved, resulting in neuronal survival and differentiation. The ability of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) to activate GTP binding to p21ras was investigated using cultured embryonic chick neurons. In both sympathetic and sensory neurons, the addition of NGF markedly increased the formation of Ras-GTP. The magnitude of the effect was found to depend upon the developmental stage, peaking at embryonic day 11 in sympathetic neurons and at embryonic day 9 in sensory neurons, times when large numbers of neurons depend on NGF for survival. Surprisingly, following the addition of BDNF, no formation of Ras-GTP could be observed in neurons cultured with BDNF. When sensory neurons were cultured with NGF alone, both NGF and BDNF stimulated GTP binding to Ras. In rat cerebellar granule cells, while the acute exposure of these cells to BDNF resulted in the formation Ras-GTP, no response was observed following previous exposure of the cells to BDNF, as was observed with sensory neurons. However, this desensitization was not observed in a transformed cell line expressing TrkB. In neurons, the mechanism underlying the loss of the BDNF response appeared to involve a dramatic loss of binding to cell-surface receptors, as determined by cross-linking with radiolabeled BDNF. Receptor degradation could not account for the desensitization since cell lysates from neurons pretreated with BDNF revealed that the levels of TrkB were comparable to those in untreated cells. These results indicate that in neurons, the pathways activated by NGF and BDNF are differentially regulated and that prolonged exposure to BDNF results in the inability of TrkB to bind its ligand.
These studies were designed to determine the pattern of initial afferent fiber ingrowth into the prenatal spinal gray matter and the establishment of the topographic organization of the presynaptic neuropil in the dorsal horn. A total of 113 lumbar dorsal root ganglia were labeled with carbocyanine fluorescent dye DiI or DiA in 67 rat embryos and neonatal pups aged embryonic day 13 to postnatal day 0 (E13-P0). The initial fiber penetration of the lumbar spinal gray began at E15 and was restricted to the segments of entry. Subsequent growth of fibers into gray matter of adjacent segments began approximately one day later, and this delay was continued, about one day for each successive segment. A second wave of ingrowth of putative small-diameter afferents into the substantia gelatinosa began at E19 and also displayed the same rostrocaudal delay. Fiber ingrowth was specific and occupied the somatotopic area appropriate for the adult, from the earliest stages (E18) in which dorsal horn laminae could be adequately defined. The somatotopic organization of the presynaptic neuropil in laminae III and IV did not change significantly throughout embryonic development as the amount of overlap between adjacent and non-adjacent ganglion projections remained constant throughout embryonic development. In addition, it was found that fibers innervating the proximal and distal hindlimb entered the spinal gray simultaneously at E15 before the innervation of the distal toes was established. The results of these studies indicate that the somatotopic organization of the presynaptic neuropil is established very early in development and requires little refinement to match that seen in the adult. The simultaneous penetration of the fibers originating from the proximal and distal areas of the limb before innervation is complete suggests that this ingrowth may be independent of the establishment of specific peripheral connections.
Development of the peripheral innervation patterns of the L1-S1 lumbosacral ganglia and motor segments in embryonic day 12-17 (E12-17) rat embryos was examined using carbocyanine dyes. Individual dorsal root ganglia (DRGs) and/or isolated ventral horn (VH) segments, or individual peripheral nerves, were isolated in rat embryos fixed at different stages and filled with one of three carbocyanine dyes; DiI, DiA, and DiO. Individual experimental preparations included labeling of 1) single DRGs; 2) multiple DRGs with alternating dyes, DiO, DiI, and DiA; 3) single isolated VH segments; 4) multiple VH segments with alternating dyes; 5) single VH segments and the corresponding segmental DRGs with different dyes; and 6) two or more individual peripheral nerves labeled with different dyes. Results from these preparations have shown that the first fibers exited the lumbar ventral horn and DRGs at E12. At E13 major nerve trunks (e.g., femoral and sciatic) were visible as they exited the plexus region. By E14 afferent fibers were present in the epidermis of the proximal hindlimb, and the major nerve trunks extended into the leg. Fibers originating from L3 to L5 (DRG and VH) reached the paw by E14.5-E15, and the epidermis of the most distal toes was innervated by E16-E16.5. While afferent fibers and motor axons of the same segmental origin mixed extensively in the spinal nerve, fibers of different segmental origin combined in the plexus and major nerve trunks with little or no interfascicular mixing. Dermatomes observed at E14 were in general spotty and non-contiguous. However, by E16 the dermatomes resembled mature forms with substantial overlap only between adjacent ones. Thus the adult pattern of spatial relationships between cutaneous afferent fibers in the periphery is established early in development.
HPLC analysis of rat spinal cord revealed a uniform distribution of N-acetyl-aspartate (NAA) across both longitudinal and dorsoventral axes. In contrast, ventral cord N-acetyl-aspartylglutamate (NAAG) levels were significantly higher than those measured in dorsal halves of cervical, thoracic, and lumbar segments. Immunocytochemical studies using an affinity-purified antiserum raised against NAAG-bovine serum albumin revealed an intense staining of motoneurons within rat spinal cord. Along with the considerable NAAG content in ventral roots, these results suggest that NAAG may be concentrated in motoneurons and play a role in motor pathways. NAAG was also present in other peripheral neural tissues, including dorsal roots, dorsal root ganglia, superior cervical ganglia, and sciatic nerve. It is interesting that NAA levels in peripheral nervous tissues were lower than those in CNS structures and that NAA levels in ventral roots and sciatic nerve were lower than NAAG levels. These findings further document a lack of correlation between NAAG and NAA levels in both central and peripheral nervous tissues. Taken together, these data demonstrate the presence of NAAG in nonglutamatergic neuronal systems and suggest a more complex role of NAAG in neuronal physiology than previously postulated.
We examined, in vitro, the effects of changing the free-running period (tau) of one oscillator on the phase relationship between the circadian rhythms of impulse activity in the optic nerves that are driven by the bilaterally paired ocular pacemakers in Bulla gouldiana. One eye of the coupled pair was treated either with lithium artificial seawater (to lengthen tau) or with low-chloride artificial seawater (to shorten tau). The results suggested that the coupling is relatively weak, since the majority (9 to 16) of eyes were unable to maintain a stable phase relationship when tau differences between the eyes were only about 1 hr. When stable phase differences were achieved, the tau of the coupled system was intermediate between the tau's of the individual oscillators, and the eye with the shorter intrinsic tau would invariably phase-lead the pair. Interestingly, in a few instances, pairs of eyes that had desynchronized by 9.5-10.5 hr resynchronized within a single cycle via a massive phase advance in the rhythm from the phase-lagging eye. The result suggests the existence of a novel phase-shifting mechanism that is part of the mutual coupling pathway. We found evidence that connection of the eye with the cerebral ganglion increases the tau of the ocular pacemaker, suggesting that efferent signals from the central nervous system influence tau. These signals may also modulate the phase-shifting response.