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Sequencing mitochondrial DNA hypervariable regions I and II (HVI and HVII) is useful in forensic missing person and unidentified remains cases. Improvements in ease and sensitivity of testing will yield results from more samples in a timely fashion. Routinely, amplification of HVI and HVII is followed by Sanger sequencing using the BigDye(®) Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) using 4 μL of ready reaction mix (RRM). Each sequencing reaction is then purified through column filtration before capillary electrophoresis. Using lower amounts of RRM (2 μL or 1 μL) and purification using BigDye(®) XTerminator(™) (Applied Biosystems) instead of columns showed no loss of sequence length and increased the quality and the sensitivity of testing, allowing HVI and HVII typing from mitochondrial genome equivalent to 125 fg of nuclear DNA, or 100 pg of HVI/HVII amplicons. Using this methodology, testing can be completed in 1 day, and the cost of testing is reduced.

Familial Mediterranean fever (FMF) is an inherited disease whose manifestations are acute but reversible attacks of sterile inflammation affecting synovial and serosal spaces. The FMF gene (MEFV) was recently cloned, and it codes for a protein (pyrin/marenostrin) homologous to known nuclear factors. We previously reported the deficient activity of a C5a/interleukin (IL)–8 inhibitor, a physiologic regulator of inflammatory processes, in FMF serosal and synovial fluids. We now describe the concomitant expression ofMEFV and C5a/IL-8–inhibitor activity in primary cultures of human fibroblasts. Fibroblasts grown from synovial and peritoneal tissues displayed C5a/IL-8–inhibitor activity that could be further induced with phorbol myristate acetate (PMA) and IL-1β. Very low levels of chemotactic inhibitor were evident in skin fibroblast cultures or in peritoneal and skin fibroblasts obtained from FMF patients. MEFV was expressed in peritoneal and skin fibroblasts at a lower level than in neutrophils and could be further induced by PMA and IL-1β. In the FMF cultures, the MEFV transcript carried the M694V mutation, consistent with the genetic defect found in patients with this disease. MEFV was also expressed in other cell lines that do not produce C5a/IL-8 inhibitor. These findings suggest that human primary fibroblast cultures express MEFV and produce C5a/IL-8–inhibitor activity. The interrelationship between pyrin, the MEFV product, and the C5a/IL-8 inhibitor requires further investigation.

In order to identify genes specific for the sensory neurons of Aplysia, a miniaturized differential screening method based on the polymerase chain reaction and applicable to small amounts of tissue was used. One messenger RNA was isolated that is expressed in every mechanoreceptor sensory cluster of the Aplysia central nervous system. This messenger RNA encodes a peptide that seems to function as an inhibitory cotransmitter. The peptide selectively inhibits certain postsynaptic cells but not others and thereby allows the sensory neurons to achieve target-specific synaptic actions.

The purpose of this research was to characterize morphologically and electrophysiologically tissue slices obtained from the hippocampus of homing and non-homing pigeons. When hippocampal slices from the brain of homing and non-homing pigeons are observed under the dissecting microscope, diffuse fiber paths can be seen. These fiber pathways appeared to be identical with the medial fiber tract (VM) previously described histologically in the hippocampus of homing pigeon. Visualization of these tracts in living slices allowed placement of stimulating and recording electrodes in corresponding locations in these slices in both homing and non-homing pigeons. Extracellular potentials recorded from VM regions of the brains of both homing and non-homing pigeons were sensitive to CNQX indicating that glutamate may be a neurotransmitter in this area of pigeon hippocampus. These potentials could undergo long-term potentiation (LTP) following high frequency stimulation. This LTP was blocked by NMDA receptor antagonist APV in the hippocampus of homing pigeon, but was APV-resistant in the hippocampus of non-homing pigeon. Extracellular potentials from the hippocampus of homing pigeons were increased in amplitude when slices were perfused with Mg2+-free Ringer, while potentials recorded from hippocampal slices from non-homing pigeons were unaffected by Mg2+-free solutions. Intracellular recordings from the hippocampal slices of homing pigeons revealed that about half the cells demonstrated excitatory synaptic potentials evoked by extracellular stimulation. The EPSP was sometimes large enough to trigger an action potential. Neurons filled with the fluorescent dye, Lucifer Yellow, in the hippocampus of homing pigeons showed multipolar structure. The response of these cells to extracellular stimulation provides the activity responsible for the extracellular potentials which can undergo LTP.

Nervous tissue of the marine mollusc, Aplysia californica, generates arachidonic acid metabolites in response to neurotransmitters such as histamine or FMRFamide. In addition, identified neurons of Aplysia respond to the pharmacologic application of some of these products, particularly those of the 12-lipoxygenase pathway. We investigated the chirality of the initial Aplysia 12-lipoxygenase product, 12-HPETE, in preparation for more detailed metabolic studies and for the analysis of the physiological activity of the endogenous lipid. Neural homogenates and intact ganglia exclusively generate 12(S)-HPETE as do the better characterized mammalian lipoxygenases. The direct application of 12(S)-HPETE to cultured sensory neurons induced a hyperpolarization which averaged 2.6 mV. We did not find any difference between the response to the naturally-occurring 12(S)-HPETE and its diastereomer, 12(R)-HPETE which is not generated in Aplysia. Both isomers were significantly more effective than 15(S)-HPETE. In contrast, 12(S)-HPETE, but not 12(R)-HPETE, was a potent modulator of the action of the molluscan neuropeptide, FMRFamide. Prior application of 12(S)-HPETE to cultured sensory neurons increased the subsequent response to a submaximal dose of FMRFamide by 60%. On the other hand, 12(R)-HPETE reduced the subsequent response to the peptide by 30%. The lack of stereospecificity in the direct effect of the lipids differs markedly from their stereospecific effects as modulators of FMRFamide action. This suggests that there may be an important neurophysiologic role for these lipid modulators which is distinct from their direct effects, and also indicates that there are multiple sites and mechanisms by which lipid hydroperoxides act on neurons in Aplysia.

In mollusks as in other animals, peptides can act as hormones, growth factors, and neurotransmitters. The presence of insulin in vertebrate brain as well as its actions on nerve cells led us to examine the electrophysiological effects of the mammalian hormone on Aplysia neurons. Application of insulin extracellularly causes hyperpolarization of L14 and L10, identified neurons of the abdominal ganglion. This hyperpolarization is associated with a decreased membrane conductance that reverses at -35 mV. We also injected inositol phosphate glycan (IPG) into the identified neurons. This complex sugar, which was purified from rat liver and which is a putative second messenger for insulin in nonneural vertebrate cells (Saltiel and Cuatrecasas, 1986; Saltiel, Osterman, and Darnell, 1988), causes hyperpolarization with decreased membrane conductance in L14 and L10 similar to the effects of insulin. Furthermore, exposure of isolated ganglia to insulin results in the generation of IPG with a compensating decrease in its glycosyl-phosphatidylinositol precursor. We suggest that, in addition to its other roles, insulin may function as a neuropeptide transmitter using IPG as a second messenger.

1. We have identified putative histaminergic neurons in the central nervous system of Aplysia californica by light-microscopic autoradiography after uptake of [3H]histamine and by immunohistochemistry with the use of an antibody specific for histamine. 2. In the cerebral ganglion cells previously shown to contain histamine (C2 and 2 large neighboring cells in the E cluster and a group of smaller cells in the L cluster) were identified both by uptake of [3H]histamine and by histamine immunoreactivity. The identification of C2 was confirmed by experiments in which individual C2s were characterized electrophysiologically and injected with Lucifer yellow before processing for immunohistochemistry. The giant serotonergic neuron did not take up [3H]histamine and was not immunoreactive. 3. In the abdominal ganglion two clusters of cells--one in the left hemiganglion and the other in the right--took up [3H]histamine and were histamine immunoreactive. These clusters are located in the regions occupied by the 30 identified respiratory interneurons, R25 and L25. Individual cells in the R25 and L25 clusters were identified electrophysiologically, marked by injection of Lucifer yellow, and processed for immunocytochemistry. Eleven of the 30 L25 cells examined (from 7 ganglia) and 2 of the 25 R25 cells (from 6 ganglia) that had been marked with Lucifer yellow were also histamine immunoreactive. 4. Also in the abdominal ganglion, identified cells in the L32 cluster were not histamine immunoreactive and did not take up [3H]histamine. These interneurons, which mediate presynaptic inhibition, had previously been considered histaminergic. Neurons in the ganglion known to use transmitters other than histamine (L10, R2, RB cells, and bag cells) were not histamine immunoreactive.(ABSTRACT TRUNCATED AT 250 WORDS)

We have examined the relationships between the modulation of transmitter release and of specific ionic currents by membrane potential in the cholinergic interneuron L10 of the abdominal ganglion of Aplysia californica . The presynaptic cell body was voltage-clamped under various pharmacological conditions and transmitter release from the terminals was assayed simultaneously by recording the synaptic potentials in the postsynaptic cell. When cell L10 was voltage-clamped from a holding potential of -60 mV in the presence of tetrodotoxin, graded transmitter release was evoked by depolarizing command pulses in the membrane voltage range (-35 mV to + 10 mV) in which the Ca 2+ current was also increasing. Depolarizing the holding potential of L10 results in increased transmitter output. Two ionic mechanisms contribute to this form of plasticity. First, depolarization inactivates some K + channels so that depolarizing command pulses recruit a smaller K + current. In unclamped cells the decreased K + conductance causes spike-broadening and increased influx of Ca 2+ during each spike. Second, small depolarizations around resting potential (-55 mV to -35 mV) activate a steady-state Ca 2+ current that also contributes to the modulation of transmitter release, because, even with most presynaptic K + currents blocked pharmacologically, depolarizing the holding potential still increases transmitter release. In contrast to the steady-state Ca 2+ current, the transient inward Ca 2+ current evoked by depolarizing clamp steps is relatively unchanged from various holding potentials.

A subcellular fraction (P3) from Aplysia is enriched in synaptosomes (Chin et al., 1988) and is capable of accumulating 5-HT and choline. At an external 3H-5-HT concentration of 1.8 microM, the P3 fraction took up 0.12 nmol/mg protein in 30 min. Uptake was dependent on external Na+. Electron microscopic autoradiography showed that much of the accumulated 3H-5-HT is localized to synaptosomes. At 0.5 microM 3H- choline, P3 took up 0.11 nmol/mg protein in 30 min and converted 40% to 3H-ACh. This synaptosomal fraction was also capable of releasing transmitter. After 3H-5-HT or 3H-choline was taken up, P3 released about 5% of the total radioactive transmitter in a Ca2+-dependent manner during a 30 sec exposure to a depolarizing concentration of K+ (100 mM). Identified, prelabeled synaptosomes were prepared by injecting 3H-choline into the large cholinergic neuron L10. The abdominal ganglia containing the injected cells were then fractionated, yielding synaptosomes containing radioactivity derived from L10. After this synaptosomal fraction was exposed to high K+, 2% of the radioactivity was released in a Ca2+-dependent manner. This release was completely blocked by 0.1 mM histamine, a modulatory transmitter that has previously been shown to cause presynaptic inhibition in L10.

By voltage clamping presynaptic cell L10 and using pharmacologic separation techniques, we have analyzed the specific ionic currents in the presynaptic cell that correlate with presynaptic inhibition while assaying transmitter release with intracellular recordings from postsynaptic cells. We have found that presynaptic inhibition can be elicited in conditions in which the Na+ and the various K+ channels are pharmacologically blocked and depolarizing current pulses produce only an inward Ca2+ current. Both inward currents and tail currents at and above the K+ reversal potential were always less inward during presynaptic inhibition. The changes in conductance associated with presynaptic inhibition were voltage sensitive and paralleled the voltage sensitivity of the Ca2+ channel. We therefore conclude that presynaptic inhibition is caused by a direct transmitter-mediated decreased of presynaptic Ca2+-channel conductance.

1. A behavioral and electrophysiological analysis of defensive ink release in Aplysia californica was performed to examine the response of this behavior and its underlying neural circuit to various-duration noxious stimuli. 2. Three separate behavioral protocols were employed using electrical shocks to the head as noxious stimuli to elicit ink release. Ink release was found to be selectively responsive to longer duration stimuli, and to increase in a steeply graded fashion as duration is increased. 3. Intracellular stimulation of ink motor neurons revealed that ink release is a linear function of motor neuron spike train duration, indicating that the selective sensitivity of the behavior to long-duration stimuli is not due to a nonlinearity in the glandular secretory process. 4. In contrast, electrophysiological examination of ink motor neuron activity in response to sustained head shock revealed an accelerating spike train. During the later part of the spike train, compound excitatory synaptic potentials show a positive shift in reversal potential. 5. Our results suggest a central locus for the mechanisms that determine sensitivity of inking behavior to stimulus duration. 6. In contrast to ink release, defensive gill withdrawal was found to be extremely sensitive to short-duration stimuli.

12-Hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE), a lipoxygenase product, simulates the synaptic responses produced by the modulatory transmitter, histamine, and the neuroactive peptide, Phe-Met-Arg-Phe-amide (FMRFamide), in identified neurons of the marine mollusk, Aplysia californica (Piomelli, D., Shapiro, E., Feinmark, S. J., and Schwartz, J. H. (1987) J. Neurosci. 7, 3675-3886; Shapiro, E., Piomelli, D., Feinmark, S., Vogel, S., Chin, G., and Schwartz, J. H. (1988) Cold Spring Harbor Symp. Quant. Biol. 53, in press). The 12-lipoxygenase pathway has not yet been fully characterized, but 12-HPETE is known to be metabolized further. We therefore began to search for other metabolites in order to investigate whether the actions of 12-HPETE might require its conversion to other active products. Here we report the identification of 12-keto-5,8,10,14-eicosatetraenoic acid (12-KETE), a metabolite of 12-HPETE formed by Aplysia nervous tissue. This product was identified in incubations of the tissue with arachidonic acid using high performance liquid chromatography, UV spectrometry, and gas chromatography/mass spectrometry. [3H]12-KETE was formed from endogenous lipid stores in nervous tissue, labeled by incubation with [3H]arachidonic acid, when stimulated by application of histamine. In L14 and L10 cells, identified neurons in the abdominal ganglion, applications of 12-KETE elicit changes in membrane potential similar to those evoked by histamine. 12(S)-Hydroxy-5,8,10,14-eicosatetraenoic acid, another metabolite of 12-HPETE, is inactive. These results support the hypothesis that 12-HPETE and its metabolite, 12-KETE, participate in transduction of histamine responses in Aplysia neurons.

Release of arachidonic acid from membrane phospholipids is receptor- mediated and might generate second messengers in neurons. We tested this idea using the simple nervous system of the marine mollusk, Aplysia californica. Aplysia neural components metabolize arachidonic acid through lipoxygenase and cyclo-oxygenase pathways. We identified 2 major lipoxygenase products, 12- and 5-hydroxyeicosatetraenoic acids (12-HETE and 5-HETE), and 2 cyclo-oxygenase products, PGE2 and PGF2 alpha. These metabolites of arachidonic acid are formed in synaptosomes, as well as in identified nerve cell bodies, indicating that both lipoxygenase and cyclo-oxygenase pathways are active within neurons. Application of the modulatory neurotransmitter histamine to cerebral ganglia that had been labeled with 3H-arachidonic acid induced the formation of 3H-12-HETE. This response was inhibited by the histamine antagonist cimetidine. Furthermore, release of radioactive 5- HETE and 12-HETE was observed after intracellular stimulation of the histaminergic cell C2 in cerebral ganglia labeled with 3H-arachidonic acid. Cimetidine also inhibited this response. Application of serotonin or stimulation of the giant serotonergic cell (GCN) in the cerebral ganglion did not cause detectable amounts of the labeled eicosanoids to be released. We found that intracellular stimulation of putative histaminergic neurons in the L32 cluster of the abdominal ganglion, which produces presynaptic inhibition in L10 neurons, also elicited the release of 3H-12-HETE and 3H-PGE2. Thus, for the first time we provide evidence that synaptic stimulation promotes turnover of arachidonic acid in neurons. We suggest that metabolites of arachidonic acid are likely to participate in some postsynaptic responses to histamine and may be second messengers for presynaptic inhibition.

The lipoxygenase product 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE), stimulates the synaptic response produced by the modulatory transmitter histamine and the neuroactive peptide Phe-Met-Arg-Phe-amide (FMRFamide) in identified neurons of the marine mollusk Aplysia californica. The 12-lipoxygenase pathway has not yet been fully characterized, but 12-HPETE is known to be metabolized further. Therefore, we began to search for other metabolites in order to investigate whether the actions of 12-HPETE might require its conversion to other active products. We have identified 12-keto-5,8,10,14-eicosatetraenoic acid (12-KETE) as a metabolite of 12-HPETE formed by Aplysia nervous tissue. 12-KETE was identified in incubations of the tissue with arachidonic acid using HPLC, UV spectrometry, and gas-chromatography/mass spectrometry. [3H]12-KETE is formed from endogenous lipid stores in nervous tissue, labeled with [3H]arachidonic acid upon stimulation by application of histamine. In L14 and L10 cells, identified neurons in the abdominal ganglion, applications of 12-KETE elicit changes in membrane potential similar to those evoked by histamine. Another metabolite of 12-HPETE, 12(s)-hydroxy-5,8,10,14-eicosatetraenoic acid [12(S)-HETE], is inactive. These results support the hypothesis that 12-HPETE and its metabolite, 12-KETE, participate in transduction of histamine responses in Aplysia neurons.

1. The release of ink from the ink gland of Aplysia californica in response to noxious stimuli is mediated by three electrically coupled motor neurons, L14A, L14B, L14C, whose cell bodies are located in the abdominal ganglion. The initial synaptic input to the ink motor neurons is relatively ineffective in firing the cells. As a result, a pause of 1--3 s often occurs before the cells attain their maximum firing frequency and cause the release of ink. Using current and voltage-clamp techniques we have analyzed the mechanisms underlying the firing pattern of these cells. 2. The presence of a fast transient K+ current appears to play an important role in mediating the firing pattern of the ink motor neurons. Their high resting potential (-75 mV) ensures that the steady-state level of inactivation of the conductance channels for the fast K+ current will normally be low. Thus a train of EPSPs or a depolarizing current pulse can activate this current maximally, thereby reducing the initial effectiveness of the excitatory input. 3. In addition to the fast transient K+ current, four other currents were identified: 1) a fast transient tetrodotoxin-sensitive inward current, presumed to be carried by Na+; 2) a slower tetrodotoxin-insensitive inward current, presumed to be carried by Ca2+; 3) a slow transient outward tetraethylammonium- (TEA) sensitive current; and 4) a very slow TEA-insensitive outward current. 4. A decreased conductance EPSP, which turns on over a several-second period, contributes to a late acceleration of spike discharge in the L14 cells. 5. The results suggest that a unique combination of biophysical properties of the L14 cells and the features of the synaptic input cause them to act as a low-pass filter in the reflex pathway for inking.Their high resting potential, which ensures minimal inactivation of the fast transient K+ current channel, makes these cells preferentially responsive to strong and long-lasting stimuli. The delayed recruitment of a decreased conductance EPSP augments the tendency of the L14 cells to fire in an accelerating burst pattern.

During the last decade a variety of pharmacological and physiological studies have indicated that receptors to chemical transmitter substances are located not only on the cell body and dendrites, but also on presynaptic terminals of neurons (Fig. 1). The finding of receptors on presynaptic terminals is interesting and important for three reasons.

In Aplysia neural tissue, the release and metabolism of arachidonic acid are stimulated by histamine or by activation of the identified L32 nerve cell circuit of the abdominal ganglion. Previously we found that histamine and intracellular stimulation of L32 cells, which are putatively histaminergic neurons, cause the production of 12-hydroxy-5,8,10,14-icosatetraenoic acid (12-HETE), a product of the 12-lipoxygenase pathway formed through 12-hydroperoxy-5,8,10,14-icosatetraenoic acid (12-HPETE). 12-HPETE, but not 12(S)-HETE, mimics the dual-action response of L14 ink motor neurons to histamine and stimulation of L32. 12-HPETE can also be further metabolized to 8-hydroxy-11,12-epoxy-5,9,14-icosatrienoic acid (8-HEpETE) which was identified by HPLC, enzymatic hydrolysis, and GC/MS. Production of 8-HEpETE is specific, as its positional isomer 10-hydroxy-11,12-epoxy-5,8,14-icosatrienoic acid is not formed after physiologic stimulation. 8-HEpETE can elicit the late component (hyperpolarization) of the dual-action response in L14 cells, suggesting that it may be a second messenger in Aplysia.

We prepared and characterized subcellular membrane fractions from the CNS of Aplysia californica that are enriched in isolated nerve terminals (synaptosomes). Ganglia were homogenized in 1.1 M sucrose and fractionated on a 2-step sucrose gradient, yielding 50 micrograms protein/animal in the synaptosomal fraction (P3), which was enriched 3- fold in plasma membrane as compared with the initial homogenate. Quantitative morphometry of electron micrographs revealed that P3 contained 25% intact synaptosomes, a 5-fold enrichment over the homogenate. Although fractionation on a 5-step sucrose gradient reduced the yield of protein in the synaptosomal fraction to 40 micrograms/animal, this fraction (the 0.35 M/0.75 M interface) was more enriched in plasma membrane than P3 and was less contaminated by lysosomes and free mitochondria. By electron microscopy, the 0.35 M/0.75 M interface contained up to 50% synaptosomes. Synaptosomal fractions contained cAMP-, Ca2+/calmodulin-, and Ca2+/phospholipid- dependent protein kinase activities and were enriched in a Mr 40,000 pertussis toxin substrate, Gi/o. In the accompanying paper, we show that these synaptosomes retain the ability to release transmitters.