A cDNA library was constructed from the brains of 18 day old rats, and was screened with a synthetic DNA probe to yield clones representing myelin basic protein (MBP). One 1.9 kb clone was sequenced and found to encode the 14 kd MBP. Using this clone as a hybridization probe, cosmid clones from a library of wild type mouse DNA were selected and characterized. One clone was shown to carry five exons which encode 14 kd MBP, distributed over a 32 kb region. A sixth exon was detected with a synthetic DNA probe, and was found to encode the 41 amino acids which distinguish 18.5 kd from 14 kd MBP. The 5' end ot the gene was mapped with S1 nuclease protection and primer extension experiments to a position 47 bp 5' of the initator codon for MBP synthesis. It was shown that the gene cloned is probably the only MBP gene in the mouse genome.
\r\n\r\nCloned DNAs were used to analyze the MBP gene and its expression in the myelin deficient mutant mouse shiverer. It was shown that a deletion has removed five out of six MBP exons, leaving only the 5'-most exon and 13 kb of the first intervening sequence. The deletion completely prevents expression of normal 2.1 kb MBP mRNAs, but a 16-fold lower number of transcripts are observed which initiate correctly at the 5' end of the first exon, are not correctly spliced, and are rarely polyadenylated. If translated, they would direct synthesis of a 61 amino acid peptide containing the first 56 amino acids of MBP. The MBP gene was mapped to mouse chromosome 18 by hybridization of MBP probes with DNA from Chinese hamster-mouse hybrid cell lines, showing it to be linked to the shiverer mutation. It is proposed that the partial deletion of the MBP gene is the primary lesion of the shiverer mutation.
", "doi": "10.7907/4d7s-zs52", "publication_date": "1985", "thesis_type": "phd", "thesis_year": "1985" }, { "id": "thesis:10891", "collection": "thesis", "collection_id": "10891", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05152018-142224321", "primary_object_url": { "basename": "Jennings_KR_1982.pdf", "content": "final", "filesize": 71550175, "license": "other", "mime_type": "application/pdf", "url": "/10891/1/Jennings_KR_1982.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Studies of Excitability in a Model Peptidergic System: The Roles of Cyclic AMP, Protein Phosphorylation and Serotonin During Afterdischarge in the Bag Cell Neurons of Aplysia californica", "author": [ { "family_name": "Jennings", "given_name": "Kent Richard", "clpid": "Jennings-Kent-Richard" } ], "thesis_advisor": [ { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" } ], "thesis_committee": [ { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" }, { "family_name": "Brockes", "given_name": "Jeremy P.", "orcid": "0000-0002-3395-5159", "clpid": "Brockes-Jeremy-P" }, { "family_name": "Hudspeth", "given_name": "A. James", "clpid": "Hudspeth-A-J" }, { "family_name": "Konopka", "given_name": "Ronald J.", "clpid": "Konopka-Ronald-J" }, { "family_name": "Lazarides", "given_name": "Elias", "clpid": "Lazarides-E" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The polypeptide hormone-secreting bag cell neurons from the abdominal ganglion of Aplysia can be induced to fire repetitively when triggered by a brief electrical stimulus to the afferent pathway. This thesis investigates the mechanism of this afterdischarge by employing biochemical, pharmacological and electrophysiological approaches.
\r\n\r\nThe description of bag cell afterdischarge, its modulation by the transmitters serotonin and dopamine and evidence for the role of cyclic AMP in the genesis of afterdischarge is presented in Chapter 1. Bag cell afterdischarge is shown to be inhibited by the application of serotonin and lengthened by the application of dopamine or the methylxanthine phosphodiesterase inhibitors. Cyclic AMP undergoes a 2-3 fold increase in the bag cell clusters during an electrically-stimulated afterdischarge but not in matched controls where equivalent electrical stimulation did not elicit afterdischarge. As further evidence for a role for cyclic AMP in the genesis of afterdischarge, afterdischarges were obtained in unstimulated preparations by the extracellular application of the cyclic AMP analogues, 8-benzylthio-cyclic AMP and 8-methylthio-cyclic AMP.
\r\n\r\nChapter 2 describes protein phosphorylation in bag cell tissues under a number of different conditions. The presence of an endogenous, cyclic AMP-dependent protein kinase activity is demonstrated in crude membranes prepared from bag cells and the substrate specificity for this activity is shown to be similar to that of protein kinase catalytic subunit prepared from bovine heart. Increases in phosphorylation of a 33,000 dalton and 21,000 dalton phosphoprotein are shown to occur during electrically-stimulated afterdischarge in bag cells. The 21,000 dalton substrate is shown to be apparently specific to bag cell tissues and an amino acid composition and partial amino acid sequence of this protein is presented.
\r\n\r\nChapter 3 presents evidence that serotonin, within the physiological range reported by other workers for Aplysia (0.1-1.0 \u03bcM) brings about a rapid inhibition of an ongoing afterdischarge. This inhibition is antagonized by the stereospecific blocker of serotonin action, D-butaclamol but not its inactive isomer, L-butaclamol. Serotonergic inhibition is shown to be associated with decreased bag cell action potential duration and height and an increased threshold to spike generation. Evidence is presented that the second, calcium-dependent phase of bag cell afterdischarge is most sensitive to the action of the transmitter and that the potassium channel blocker, tetraethylammonium can overcome serotonin's inhibitory effect. This raises the possibility that serotonin may cause inhibition of bag cell afterdischarge by increasing potassium conductance. The possible functional role of serotonin inhibition of egg-laying is discussed.
", "doi": "10.7907/8k0z-ft54", "publication_date": "1982", "thesis_type": "phd", "thesis_year": "1982" }, { "id": "thesis:11843", "collection": "thesis", "collection_id": "11843", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10222019-143344602", "type": "thesis", "title": "Behavioral Neurogenetic Studies of a Circadian Clock in Drosophila melanogaster", "author": [ { "family_name": "Orr", "given_name": "Dominic Ping-Yim", "clpid": "Orr-Dominic-Ping-Yim" } ], "thesis_advisor": [ { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" }, { "family_name": "Konopka", "given_name": "Ronald J.", "clpid": "Konopka-Ronald-J" } ], "thesis_committee": [ { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Konopka", "given_name": "Ronald J.", "clpid": "Konopka-Ronald-J" }, { "family_name": "Konishi", "given_name": "Masakazu", "clpid": "Konishi-M" }, { "family_name": "Hopfield", "given_name": "John J.", "clpid": "Hopfield-J-J" }, { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The circadian clock controlling the locomotor activity of the adult fruitfly, Drosophila melanogaster, is studied in one wild-type and five clock mutant strains. Locomotive activity of individual flies are monitored using arrays of infra-red beams and detectors. It is found that the temperature compensation mechanism is intact in the mutants And and ClkK06, is slightly defective in the mutant pers and is grossly defective in the mutants perl1 and perl2. In the pers and perl1 mutants, this defect is enhanced when both eyes and major parts of both optic lobes are eliminated by a genetic mutation (sine oculus). The inter-individual variation of periods in a strain is found to increase much more than linearly with the average period of the same strain. The interaction between the And and the per loci and that between the And and ClkK06 loci are found to be either very weak or non-existent (effects of mutations additive), whereas the interactions among the various alleles in the per locus are found to be strong (effects of mutations non-additive).
\r\n\r\nTen 'Phase Resetting Curves' (PRC) obtained with saturating light pulses for six strains of flies at various temperatures are presented. All the ten cases exhibit basically 'type-1' resetting behavior (average slope = 1). Comparisons of the PRC's for pers, perl1 and wild-type at 17\u00b0C suggest that the mutations pers and perl1 change the period of the circadian clock by differentially shortening and lengthening, respectively, the duration of the 'subjective day' phase of the oscillation. Comparisons between the PRC's for pers at 17\u00b0C, 22\u00b0C, and 25\u00b0C and comparison between the wild-type PRC's at 17\u00b0C and 22\u00b0C do not reveal major changes in the temporal structure of these two circadian clocks over the stated temperature ranges.
\r\n\r\nThe responses of one wild-type and five mutant circadian clocks to sustained dim light of the range 5 x 10-4 lux to 50 lux at 22\u00b0C are studied. In each strain, a critical 'window' of light intensity is found within which a variety of unstable clock features, including arrhythmia, are observed. The light intensity at which this critical window occurs in each of the mutant is 5 to 10 times lower than that in the wild-type. Responses from a ERG-defective mutant (norpA) are found to be qualitatively, but not quantitatively, similar to that of the wild-type. Responses from an eyeless and ocelli-less mutant (sine oculus) indicate that both period changes and arrhythmicity can be elicited by light in the absence of the compound eyes and ocelli. However, the sharp dependence of the occurences of these phenomena on light intensity is lost in this mutant.
\r\n\r\nArguments are presented to suggest that none of the four mutations -- And, ClkK06, pers, and perl1 -- cause changes of period by mimicking the effects of tonic light on the Drosophila circadian system.
\r\n\r\nThe phase resetting curves (PRC) and the dim light responses described above are found to be incompatible with a particular model of the Velocity Response Curve (VRC) theory to inter-relate the phasic to tonic effects of light, in which the tonic effect of light is assumed to be the result of a summation of the effects of a contiguous series of single Light pulses, taking into account adaptation.
", "doi": "10.7907/wp4e-5054", "publication_date": "1982", "thesis_type": "phd", "thesis_year": "1982" }, { "id": "thesis:3806", "collection": "thesis", "collection_id": "3806", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09272005-141028", "type": "thesis", "title": "Biochemical and Immunohistochemical Studies of the Egg-Laying Hormone of Aplysia californica: Purification, Primary Structure, Neurosecretion and Morphological Distribution", "author": [ { "family_name": "Chiu", "given_name": "Arlene Yuen-Chin", "clpid": "Chiu-Arlene-Yuen-Chin" } ], "thesis_advisor": [ { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" } ], "thesis_committee": [ { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" }, { "family_name": "Hudspeth", "given_name": "A. James", "clpid": "Hudspeth-A-J" }, { "family_name": "Lazarides", "given_name": "Elias", "clpid": "Lazarides-E" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" }, { "family_name": "Brockes", "given_name": "Jeremy P.", "orcid": "0000-0002-3395-5159", "clpid": "Brockes-Jeremy-P" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Egg-laying behavior in Aplysia californica can be triggered by the introduction of a neuropeptide, the Egg-Laying Hormone (ELH) into the circulation. ELH is synthesized by the neurosecretory bag cells of the abdominal ganglion and released when these neurons are induced to fire repetitively. In this thesis, biochemical and immunohistoehemical techniques have been employed to study the primary structure, release and distribution of ELH in the nervous system of Aplysia.
\r\n\r\nThe purification of ELH to homogeneity from extracts of bag cell clusters, and the analysis of its primary structure are discussed in Chapter 1. A 100-fold enrichment of bioactive material was obtained by cation exchange chromatography (Sephadex SP C25) followed by gel filtration (BioRad P-6). This purified material was determined to be homogeneous by four lines of analysis: (i) SDS polyacrylamide gel electrophoresis, (ii) isoelectric focussing, (iii) microsequence analysis, and (iv) comparison of the amino acid compositions from acid hydrolysis and from microsequence data. ELH is a 36 amino acid, basic peptide with a calculated molecular weight of 4385 and an apparent isoelectric point of 9.0-9.2. Its amino acid sequence was determined as:
\r\n\r\nH-lle-Ser-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-Leu-Thr-Glu-Gln-lleArg-Glu-Arg-Gln-Arg-Tyr-Leu-Ala-Asp-Leu-Arg-Gln-Arg-Leu-Leu-Glu-Lys-OH
\r\n\r\nChapter 2 demonstrates the release of ELH, identified by molecular weight, pI and bioactivity, when bag cell clusters afterdischarge in vitro. During such synchronous and prolonged electrical activity, bag cell clusters, which have been pulsed in 35S-Met, secrete at least four labeled presumed peptides of different molecular weights. One of these comigrates with 3H-Leu labeled, purified ELH on gel filtration chromatography and causes egg laying when injected into a test animal. This material also comigrates with 3H-ELH on isoelectrifocussing gels. A second released peptide has a molecular weight of approximately 5-6 K and a pI of 4.8; its function, and the functions of the other released molecules, are unknown.
\r\n\r\nIn order to study the distribution of ELH in the nervous system of Aplysia, antibodies were generated against the purified neuropeptide, coupled to a carrier molecule, thyroglobulin (Tg). Immune sera, enriched for anti-ELH antibodies by passage through an affinity column to remove antibodies which bound to Tg, was used for localizing ELH-like immunoreactivity in frozen sections of Aplysia ganglia. These results were discussed in Chapters 3 and 4.
\r\n\r\nWhen sections of the abdominal ganglion were stained by the PAP method for ELH, all neurons within the bag cell clusters were found to be immunoreactive. Except for occasionally displaced bag cells, all neurons within the ganglion remained unreactive, reflecting the specificity of the antiserum. Immunopositive processes from bag cells proliferate in the vascularized connective tissue capsule which serves as a neurohemal organ facilitating release of neurohormones. Some processes form a spiralling cuff around the nerve trunks of the pleuro-visceral connective and the vulvar nerves; others invade the ganglion in association with connective tissue septa which form partitions between groups of neurons. Immunoreactive fibers with varicosities are also found within the neuropile and the commissure between the two hemiganglia. This light microscopic visualization of the bag cell neuroendocrine system provides morphological support for the model of local hormone action of ELH upon other neurons in the abdominal ganglion. The immunoreactivity of all neurons within the clusters provides the strongest evidence to date of the homogeneity of the bag cell population.
\r\n\r\nAntibodies generated against ELH from A. californica selectively stained the bag cell systems of three other species of Aplysia - A. braziliana, A. vaccaria and A. dactylomela - which also share cross bioactivity. It is likely that receptor binding sites and antigenic determinants are conserved in their ELHs.
\r\n\r\nThe fourth chapter describes the organization of cells and fiber tracts with ELH-like immunoreactivity, endogenous to the head ganglia. Each pleural ganglion has 1-5 immunopositive somata which are strikingly similar to bag cells in cell and nuclear sizes, process morphology and location. These similarities, coupled with the close developmental association of the pleural and abdominal ganglia, suggest a common heritage for both populations of ELH+ cells.
\r\n\r\nThe ELH immunoreactive system in the cerebral ganglion consists of two laterally located clusters of small cells on the dorsal surface of the ganglion and extensive fiber tracts throughout the neuropile. The nature of immunoreactive molecules and the function of these systems within the cerebral and pleural ganglia are unknown. However, perfusion of ELH is known to induce long-term changes in the electrical activity of head ganglia neurons in vitro, and some of these changes may be linked to the suppression of feeding and locomotion during egg laying. The presence of these immunopositive systems in the pleural and cerebral ganglia raises the possibility that ELH target neurons in head ganglia may respond to local sources of ELH or ELH-like molecules.
", "doi": "10.7907/50RT-EZ43", "publication_date": "1981", "thesis_type": "phd", "thesis_year": "1981" }, { "id": "thesis:18370", "collection": "thesis", "collection_id": "18370", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02172026-214230970", "primary_object_url": { "basename": "Stuart_DK_1978.pdf", "content": "final", "filesize": 36944860, "license": "other", "mime_type": "application/pdf", "url": "/18370/1/Stuart_DK_1978.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "The Neurosecretion of the Polypeptide Egg-Laying Hormone (ELH) from the Bag Cells, Neuronal Sites of Action of ELH, and Circadian Release of Polypeptides from the Eye of Aplysia californica", "author": [ { "family_name": "Stuart", "given_name": "Duncan Knight", "clpid": "Stuart-Duncan-Knight" } ], "thesis_advisor": [ { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" } ], "thesis_committee": [ { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" }, { "family_name": "Hudspeth", "given_name": "A. James", "clpid": "Hudspeth-A-J" }, { "family_name": "Konishi", "given_name": "Masakazu", "clpid": "Konishi-M" }, { "family_name": "Owen", "given_name": "Ray David", "clpid": "Owen-R-D" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The thesis is about the neurosecretion of Aplysia peptides and demonstrates\r\nthat one of them acts directly upon the nervous system. Such neuronal\r\neffects of neurosecretory substances may prove to be a general phenomenon; this is\r\ndiscussed in the introduction.
\r\n\r\nIn chapter 1, radiolabeled peptides released from electrically active bag\r\ncell neurons in isolated bag cell clusters were compared with the polypeptide egg-laying\r\nhormone (ELH), 6,000 daltons, pl 9.0-9.3, as purified from homogenates of\r\nbag cell clusters. A labeled peptide which is selectively released from electrically\r\nactive bag cell clusters comigrates with ELH from cluster homogenates on P-6 gel\r\nfiltration columns and subsequent isoelectric focusing gels. When bag cells are\r\nactivated, a released factor(s) induces egg-laying and comigrates with ELH from\r\ncluster homogenates on P-6 columns. At least three other presumed peptides of\r\nunknown function are also released. These experiments demonstrate that ELH\r\n(6,000 m.w., pl 9.0-9.3) as purified from bag cell cluster homogenates is the major,\r\nactive form secreted from bag cells.
\r\n\r\nIn chapter 2, the effects of ELH on neuronal activity of the attached head\r\nganglia (buccal, cerebral, pleural, and pedal), on the isolated buccal ganglia, as well\r\nas on feeding in intact Aplysia were studied. Starved animals (n = 7) injected at\r\n20\u00b0C with a crude- extract containing ELH stopped eating algae at 17 \u00b1 4 min and\r\ntheir eggs first appeared at 29 \u00b1 4 min after injection. These data clearly indicate\r\nthat a suppression of feeding activity occurs before the appearance of eggs. ELH\r\napplied to the paired buccal ganglia in vitro activates a pair of neurons into a tonic\r\npacing mode (~1 spike/sec). The time for the full appearance of this activity in\r\nvitro correlates well with the time for suppression of feeding in vivo. These\r\nneurons each have an ipsilateral axon in buccal nerve 3. ELH increases the rate of\r\nfiring of a second pair of buccal neurons, each with an ipsilateral axon in the\r\ncerebrobuccal connective. ELH when applied to the attached head ganglia causes\r\nlarge bursts of neuronal activity in pedal nerves to the foot, and increased activity\r\nin the nerve to the penis. These in vitro effects were produced by ELH partially\r\npurified from bag cell cluster homogenates using ammonium sulfate precipitation,\r\nan anion exchange column, and a gel filtration column or by ELH released from\r\nactivated bag cells in isolated abdominal ganglia and then frationated by gel\r\nfiltration. The ELH effects upon the in vitro nervous system support the working ~\r\nhypothesis that ELH in vivo acts directly on the nervous system to suppress feeding\r\nactivity. ELH may also activate neural circuits in the pedal and probably cerebral\r\nganglia that produce characteristic movements of the head during egg-laying; the\r\nrelevant neurons remain to be identified.
\r\n\r\nIn chapter 3, a circadian rhythm (CR) of release of presumed peptides\r\nfrom the isolated eye of Aplysia is demonstrated. This isolated eye is known to\r\nhave a CR of compound action potentials (CAPs) as recorded from its optic nerve.\r\nSubstances labeled with radioactive amino acids and released into the perfusate\r\nwere separated on gel filtration columns and SDS polyacrylamide gels. In the CR\r\nexperiments, the perfusate of a single, labeled, dark-maintained eye was collected\r\nevery 3 h for two days while simultaneously recording the CR of CAPs. Each 3-h\r\nperfusate was applied to a P-2 gel filtration column. Excluded substances\r\n(m.w. ~ 2000) and material fractionated in the region of m.w. ~ 1000 showed a CR\r\nwhich was in phase with the CR of CAPs. Much of these labeled substances can be\r\nprecipitated with trichloroacetic acid. Their release is stimulated by a high\r\npotassium solution and inhibited by a low calcium solution that also inhibits CAP\r\nactivity. This and other previously published evidence suggests that the CAPs and\r\nthe peptide release are directly produced by electrically coupled neurosecretory\r\ncells which may also contain the CR oscillator. One or more of these peptides may\r\nbe a neurohormone and/or transmitter used for synchronizing, entraining and/or\r\ndriving the rest of the animal's CRs.
", "doi": "10.7907/c981-k394", "publication_date": "1978", "thesis_type": "phd", "thesis_year": "1978" }, { "id": "thesis:17849", "collection": "thesis", "collection_id": "17849", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01302026-191212444", "primary_object_url": { "basename": "Rothman_BS_1976.pdf", "content": "final", "filesize": 91033690, "license": "other", "mime_type": "application/pdf", "url": "/17849/1/Rothman_BS_1976.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Electrophysiological and Biochemical Studies on the Effects of RNA and Protein Synthesis Inhibitors on the Circadian Rhythm of the Isolated Aplysia Eye", "author": [ { "family_name": "Rothman", "given_name": "Barry Samuel", "clpid": "Rothman-Barry-Samuel" } ], "thesis_advisor": [ { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" } ], "thesis_committee": [ { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" }, { "family_name": "Benzer", "given_name": "Seymour", "clpid": "Benzer-S" }, { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" }, { "family_name": "Wiersma", "given_name": "Cornelis A. G.", "clpid": "Wiersma-C-A-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The isolated eye of Aplysia californica produces a circadian rhythm\r\nof optic nerve activity. In filtered sea water, at 15\u00b0C, in constant\r\ndarkness, the free-running period of the circadian rhythm is 23.4 hrs.\r\nOptic nerve activity is recorded by means of suction electrode, and is\r\nin the form of spontaneous compound action potentials (CAPs) which vary\r\nin frequency from 0 to 200 per hr.
\r\n\r\nExperiments were designed to test the necessity of macromolecular\r\nsynthesis for the production of the circadian rhythm. Eyes were given\r\na pulse of an inhibitor of RNA or protein synthesis and the effects on\r\nthe circadian rhythm, biochemistry or electrophysiology assayed.
\r\n\r\nWhen eyes were given a 3 hr pulse of actinomycin D (AMD)(4 ug/ml\r\nthe circadian rhythm was inhibited without blocking spontaneous\r\nactivity altogether. Eyes receiving a 3 hr pulse of aflatoxin B1(AFTX)\r\n(16 ug/ml) revealed similar effects in half the cases studied, while in\r\nthe other half a reduced amplitude phase delayed circadian rhythm was\r\nfound. Eyes given a 12 hr pulse of puromycin (PURO) (20-134 ug/ml) or\r\ncycloheximide (CHX) (500-2000 ug/ml) beginning in mid-subjective night\r\nhad their circadian rhythms phase delayed by 12-16 hrs and 6-12 hrs,\r\nrespectively, after the drug pulse was washed out. A phase-response\r\ncurve determined for the effects of a 6 hr PURO (125 ug/ml) pulse showed\r\nthat maximum phase delays were caused by pulses given in late subjective\r\nnight, and maximum phase advances caused by pulses given in early subjective\r\nday.
\r\n\r\nIn biochemical studies, incorporation of 3H-uridine and 14C-leucine\r\nwere measured 1-9, 9-17, 49-57 and 73-81 hrs after the removal of a 3 hr\r\npulse of AFTX (16 ug/ml) or AMD (4 ug/ml). Uridine incorporation was\r\ninhibited by 50-75% from 1 to 17 hrs after an AFTX or AMD pulse, while\r\nleucine incorporation was inhibited by 40-70% from 1 to 81 hrs after an\r\nAFTX pulse, and by about 20% from 49 to 57 hrs after an AMD pulse. At\r\nall other times measured, uridine and leucine incorporation were not\r\nsignificantly different from controls. In other biochemical studies the\u00b7\r\neffects of ?URO and CHX on leucine incorporation were tested by means of\r\na double-label 303-polyacrylamide gel system. When eyes were labeled\r\nduring the last 5 hrs of a 12 hr pulse of PUR.O (20 ug/ml) or CHX (500\r\nug/ml), incorporation was inhibited by about 50%. The distribution of\r\nlabel in the gels of PURO-treated eyes showed increasing inhibition of\r\nincorporation with increasing molecular weight above 75,000 daltons,\r\nwhile in the gels of CHX-treated eyes, incorporation was almost equally\r\ninhibited at all molecular weights. A 12 hr PURO (125 ug/ml) inhibited\r\nleucine incorporation by about 85%, while the distribution of label in\r\nthe gels showed increasing inhibition of incorporation with increasing\r\nmolecular weight above 12,000 daltons. At 12-20 hrs and 20-28 hrs after\r\nthe end of the PURO pulse, incorporation was normal except for a small\r\npeak at 20,000 daltons.
\r\n\r\nThe electrophysiological properties of eyes were tested by recording\r\nspontaneous CAP activity and responses to light pulses at various\r\ntimes before, during and after the administration of a drug pulse.\r\nEight electrophysiological parameters were measured and compared quantitatively\r\nbetween experimental and control eyes. They were the latency,\r\namplitude and frequency of both the phasic and tonic light responses;\r\nand the amplitude and frequency of spontaneous CAP activity. AFTX (3\r\nhrs, 16 ug/ml) induced multiphasic tonic light responses during the\r\ndrug pulse; and when applied during the peak of an activity cycle,\r\nincreased the frequency of spontaneous CAP activity by 35% for the\r\nremainder of the cycle. AMD (3 hrs, 4 ug/ml) caused a 13% increase in\r\nspontaneous CAP amplitude and a 10% decrease in tonic light response\r\nlatency subsequent to its removal. PURO (12 hrs, 20 ug/ml) increased\r\nthe amplitude of the tonic light response by 23% when measured more\r\nthan. 7 hrs after the end of the pulse. CHX (12 hrs, 500 ug/ml) caused\r\na 32% increase in the tonic light response frequency measured 0-7 hrs\r\nafter the end of the pulse, and a 33% decrease in the duration of spontaneous\r\nCAP bursts during the pulse.
\r\n\r\nThe results of these experiments indicate that doses of four inhibitors\r\nof macromolecular synthesis capable of modifying the circadian\r\nrhythm of the eye reduce the incorporation of uridine and/or leucine\r\nand cause only small changes in the electrophysiology of the eye.\r\nThese data suggest that the production of the circadian rhythm of the\r\nAplysia eye is dependent on macromolecular synthesis.
", "doi": "10.7907/ay8x-wp67", "publication_date": "1976", "thesis_type": "phd", "thesis_year": "1976" }, { "id": "thesis:10584", "collection": "thesis", "collection_id": "10584", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12052017-094450464", "type": "thesis", "title": "Part I. Studies on the Organization of the Eye of Aplysia californica. Part II. Studies on the Interrelationship between Two Neuronal Circadian Oscillators in Aplysia californica", "author": [ { "family_name": "Audesirk", "given_name": "Gerald Joseph", "clpid": "Audesirk-Gerald-Joseph" } ], "thesis_advisor": [ { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" } ], "thesis_committee": [ { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" }, { "family_name": "Wiersma", "given_name": "Cornelis A. G.", "clpid": "Wiersma-C-A-G" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Pettigrew", "given_name": "John D.", "clpid": "Pettigrew-J-D" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Part I
\r\n\r\n\r\nThe isolated eye of Aplysia californica produces a bursting\r\npattern of spontaneous compound action potentials (CAPs) when recordings \r\nare made from the optic nerve in darkness. The CAP frequency varies\r\nwith a circadian rhythm. The light response, also composed of CAPs,\r\nmay be separated into an initial phasic response and a late tonic \r\nresponse similar in form to the dark discharge. Solutions containing\r\nLa+++ or high Mg++ with low Ca++, which are expected to block chemical\r\nsynapses, stop the dark discharge and tonic light response but not the \r\nphasic light response. The suppression of dark discharge by high Mg++\r\nwith low Ca++ is usually temporary, lasting about 0.5 to 4 hours.\r\nSynchrony of the CAPs is not affected by either La+++ or high Mg++, low \r\nCa++. These results indicate that the dark discharge is driven through \r\nchemical synapses, but the light response is not. Replacement of \r\nchloride in the bathing medium by propionate, which uncouples electrical \r\njunctions in the crayfish septate axon, abolishes all CAPs for varying \r\nperiods of time, usually several hours. Propionate leaves the ERG\r\nintact and the optic nerve electrically excitable. A model for inter-neuronal \r\nconnections in the Aplysia eye is constructed from these data. \r\nIt is postulated that the light response is initiated in the photoreceptors, \r\nwith the receptor depolarization passing through electrical \r\nsynapses to higher order cells. Spikes are produced in these cells and \r\npass down their axons in the optic nerve. Spontaneous dark activity\r\nalso represents spiking in these higher order cells, but is initiated\r\nthrough chemical synapses by pacemaker cell(s). Synchrony of the CAPs \r\nis facilitated by electrical synapses between higher order cells. In\r\nlow Ca++ media, these higher order cells may become hyperexcitable to\r\nthe point of autoactivity.
\r\n\r\n\r\n\r\nPart II
\r\n\r\n\r\nThe circadian rhythm of spike output of the single neuron R15 \r\nin the isolated PVG of Aplysia californica can be entrained in vivo \r\nby light. The timing of the rhythm depends not only on the lighting\r\nschedule to which the animal was exposed prior to dissection, but also \r\non the time of dissection relative to that light schedule. Entrainment \r\nof the rhythm by light proceeds very slowly, if at all, in Aplysia \r\nwith their eyes removed. An indirect inhibitory neural pathway is\r\nshown to exist between the eyes and R15, but cutting nervous \r\nconnections containing this and any other neural paths from the eyes \r\nto R15 does not prevent entrainment by light in a majority of animals. \r\nIn vitro experiments show that the eyes can influence the activity \r\nof R15 even when the eyes and the PVG are not neurally connected.\t\r\nThe eyes therefore must release a water soluble factor which can affect\r\nR15, either directly or through some other neurons in the PVG. If the \r\neyes and PVGs from different animals are incubated together for\r\nseveral days and then separated, the subsequent spiking behavior of R15 \r\nis similar to that observed after in vivo entrainment to a light \r\nschedule equivalent in phase to the circadian rhythm of the eyes in \r\nvitro. It is a strong possibility that the factor released by the\r\neyes can entrain the circadian rhythm of R15.
\r\n\r\n \r\n\r\n", "doi": "10.7907/BT9J-NK43", "publication_date": "1975", "thesis_type": "phd", "thesis_year": "1975" }, { "id": "thesis:14432", "collection": "thesis", "collection_id": "14432", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11202021-011429389", "primary_object_url": { "basename": "RAM_JL_1974.pdf", "content": "final", "filesize": 74580335, "license": "other", "mime_type": "application/pdf", "url": "/14432/1/RAM_JL_1974.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Effects of High K\u207a Media on Leucine Incorporation into Aplysia Nervous Tissue", "author": [ { "family_name": "Ram", "given_name": "Jeffrey Lewis", "clpid": "Ram-Jeffrey-Lewis" } ], "thesis_advisor": [ { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" } ], "thesis_committee": [ { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" }, { "family_name": "Olds", "given_name": "James", "clpid": "Olds-J" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Russell", "given_name": "Richard L.", "clpid": "Russell-R-L" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "To study possible coupling between membrane polarization and protein synthesis, elevated external K+ levels were used to depolarize the cell membranes in isolated Aplysia californica parieto-visceral ganglia (PVG). The effect of this treatment on the incorporation of labeled leucine into proteins in the ganglion was analyzed on sodium dodecyl sulfate polyacrylamide gels. PVGs were preincubated 3 hours and then incubated 4 hours in either control medium (14C-leucine, 10 mM [ K+]) or experimental medium (3H-leucine, 10 + x mM [K+] with equimolar [Na+] reduction). These were homogenized together, separated into aqueous soluble and aqueous insoluble fractions, and run on gels.
\r\n\r\nIn the aqueous soluble fraction of the PVG High [K+] (90-110 mM [K+]} caused relative increases in incorporation in distinct peaks at 50K (K = 1000 daltons) and 40K. The larger peak, at 50K, was studied further.
\r\n\r\nThe relative increase at 50K occurred when 14c-leucine (instead of the usual 3H-leucine) was incorporated in High [K+]. The relative increase at 50K did not occur (1) when [K+] was raised to only 50 mM; (2) when [Na+] was reduced by 80 mM, and tris+ (HCl to neutralize) was substituted instead of K+; (3) in pleura-visceral connective (PVC) nerve; and (4) in the aqueous insoluble fraction of the PVG.
\r\n\r\nThe effect of High [K+] on incorporation into the giant cell (R2) of the PVG was examined by first labeling the PVG in control medium, rinsing it, and then labeling it in experimental medium. High [K+] in the experimental medium caused a significant relative increase at 50K in whole PVGs, half PVGs, and R2s dissected from the PVG following incubation. The results in R2 were marred by great variability in the control patterns.
\r\n\r\nAutoradiography of identified cells (R2 and R15) dissected from PVGs labeled with 3H-leucine in normal [K+] showed that contaminating cells {mostly glia), which always adhere to such dissected cells, generally account for less than 20% of the total incorporated formalin-fixed label. This contamination is large enough so that a glial origin of the High [K+] effect on incorporation at 50K cannot be positively excluded. However, the presence of this effect in dissected R2s and its absence in PVC nerves, which contain axons, glia, and connective tissue, but no nerve cell bodies, lend support to the notion that the effect is neuronal in origin.
\r\n\r\nHigh [K+] caused a reduction of approximately SO% in total incorporation into both aqueous soluble and aqueous insoluble proteins of the PVG. Similar decreases of 35% were seen in dissected R2s. [Na+] reduction (by 80 mM, tris+ substitution) had no significant effect on total incorporation (measured only in the aqueous soluble fraction of the PVG). High [K+] caused a reduction of approximately 85% in total incorporation into PVC nerve. Autoradiography of the nerve showed that this reduction occurred in both the connective tissue sheath and the axonal-glial region. High [K+] caused no significant change in non-volatile TCA soluble label in either the ganglion or the nerve.
\r\n\r\nOther effects of High [K+] on the PVG: (1) a small (not large enough to have caused the relative increase at 50K) decrease in the relative amount of label in the aqueous soluble, TCA insoluble fraction compared to the aqueous insoluble fraction, and (2) a relative decrease in incorporation in higher molecular weight peptides compared to lower molecular weight peptides in both aqueous soluble and aqueous insoluble fractions.
\r\n\r\nThese results suggest, but do not prove, that High [K+] caused an increase in the synthesis of a neuronal peptide of approximately 50,000 daltons molecular weight. The possibility that this peptide may be a tubulin subunit is briefly discussed.
", "doi": "10.7907/qtf0-tz26", "publication_date": "1974", "thesis_type": "phd", "thesis_year": "1974" }, { "id": "thesis:9123", "collection": "thesis", "collection_id": "9123", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08282015-154034894", "primary_object_url": { "basename": "Toevs_ls_1970.pdf", "content": "final", "filesize": 28811814, "license": "other", "mime_type": "application/pdf", "url": "/9123/1/Toevs_ls_1970.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Identification and Characterization of the Egg-Laying Hormone from the Neurosecretory Bag Cells of Aplysia", "author": [ { "family_name": "Toevs", "given_name": "Lois Schloemer", "clpid": "Toevs-Lois-Schloemer" } ], "thesis_advisor": [ { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" } ], "thesis_committee": [ { "family_name": "Unknown", "given_name": "Unknown" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "This investigation has resulted in the chemical identification\r\nand isolation of the egg-laying hormone from Aplysia californica,\r\nAplysia vaccaria, and Aplysia dactylomela. The hormone, which was\r\noriginally identified as the Bag Cell-Specific protein (BCS protein)\r\non polyacrylamide gels, is a polypeptide of molecular weight \u2248 6000,\r\nwhich is localized in the neurosecretory bag cells of the parietovisceral\r\nganglion and the surrounding connective tissue sheath which\r\ncontains the bag cell axons. All three species produce a hormone of\r\nsimilar molecular weight, but varying electrophoretic mobility as determined\r\non polyacrylamide gels. As tested, the hormone is completely\r\ncross-reactive among the three species.
\r\n\r\nAlthough the bag cells of sexually immature animals contain the\r\nactive hormone, sexual maturation of the animal results in a 10-fold\r\nincrease in the BCS protein content of these neurons.
\r\n\r\nA seasonal variation in the BCS protein content was also observed,\r\nwith 150 times more hormone contained in the bag cells of\r\nAplysia californica in August than in January. This correlates well\r\nwith the variation in the animals' ability to lay eggs throughout the\r\nyear (Strumwasser et al., 1969). There are some indications that the\r\nreceptivity of the animal to the available hormone also fluctuates\r\nduring the year, being lower in winter than in swmner. The seasonal\r\nrhythm of the other species, Aplysia vaccaria and Aplysia dactylomela,\r\nhas not been investigated.
\r\n\r\nA polyacrylamide gel electrophoresis analysis of water-soluble\r\nproteins in Aplysia californica revealed several other nerve-specific\r\nproteins. One of these is also located in the bag cell somas and stains\r\nturquoise with Amido Schwarz. The function of this protein has not been\r\ninvestigated.
", "doi": "10.7907/70RQ-P642", "publication_date": "1970", "thesis_type": "phd", "thesis_year": "1970" } ]