Serotonin shifts the phase of the circadian rhythm from the Aplysia eye.

A putative neurotransmitter, serotonin, may be used to transmit temporal information in the eye of Aplysia, because it can shift the phase of the circadian rhythm of spontaneous optic nerve impulses from the eye and the eye contains a significant quantity of serotonin. Serotonin acts either directly on the cell, or cells, containing the circadian pacemaker or on cells electronically coupled to the pacemaker cells.

Glutamate-induced losses of oligodendrocytes and neurons and activation of caspase-3 in the rat spinal cord.

The toxicity of released glutamate contributes substantially to secondary cell death following spinal cord injury (SCI). In this work, the extent and time courses of glutamate-induced losses of neurons and oligodendrocytes are established. Glutamate was administered into the spinal cords of anesthetized rats at approximately the concentration and duration of its release following SCI. Cells in normal tissue, in tissue exposed to artificial cerebrospinal fluid and in tissue exposed to glutamate were counted on a confocal system in control animals and from 6 h to 28 days after treatment to assess cell losses. Oligodendrocytes were identified by staining with antibody CC-1 and neurons by immunostaining for Neuronal Nuclei (NeuN) or Neurofilament H. The density of oligodendrocytes declined precipitously in the first 6 h after exposure to glutamate, and then relatively little from 24 h to 28 days post-exposure. Similarly, neuron densities first declined rapidly, but at a decreasing rate, from 0 h to 72 h post-glutamate exposure and did not change significantly from 72 h to 28 days thereafter. The nuclei of many cells strongly and specifically stained for activated caspase-3, an indicator of apoptosis, in response to exposure to glutamate. Caspase-3 was localized to the nucleus and may participate in apoptotic cell death. However, persistence of caspase-3 staining for at least a week after exposure to glutamate during little to no loss of oligodendrocytes and neurons demonstrates that elevation of caspase-3 does not necessarily lead to rapid cell death. Beyond about 48 h after exposure to glutamate, locomotor function began to recover while cell numbers stabilized or declined slowly, demonstrating that functional recovery in the experiments presented involves processes other than replacement of oligodendrocytes and/or neurons.

Upregulation of the phosphorylated form of CREB in spinothalamic tract cells following spinal cord injury: relation to central neuropathic pain.

Spinal cord injury (SCI) often leads to the generation of chronic intractable neuropathic pain. The mechanisms that lead to chronic central neuropathic pain (CNP) following SCI are not well understood, resulting in ineffective treatments for pain relief. Studies have demonstrated persistent hyperexcitability of dorsal horn neurons which may provide a substrate for CNP. We propose a number of similarities between CNP mechanisms and mechanisms that occur in long-term potentiation, in which hippocampal neurons are hyperexcitable. One biochemical similarity may be activation of the transcription factor, cyclic AMP response element-binding protein (CREB), via phosphorylation (pCREB). The current study was designed to examine whether tactile allodynia that develops in segments rostral to SCI (at-level pain) correlates with an increase in CREB phosphorylation in specific neurons known to be involved in allodynia, the spinothalamic tract (STT) cells. This study determined that, in animals experiencing at-level allodynia 35 days after SCI, pCREB was upregulated in the spinal cord segment rostral to the injury. In addition, pCREB was found to be upregulated specifically in STT cells in the rostral segment 35 days after SCI. These findings suggest one mechanism of maintained central neuropathic pain following SCI involves persistent upregulation of pCREB expression within STT cells.

Paraquat--a superoxide generator--kills neurons in the rat spinal cord.

Microdialysis was used to administer paraquat into the spinal cord of the anesthetized rat to determine the effects of the in vivo generation of the superoxide anion (O2.-) on neurons. Exposure to paraquat caused blockage of axonal conduction, destruction of the cell bodies of neurons, and a general release of amino acids. Thus, paraquat is quite harmful to neuronal tissue. Similarities between paraquat-induced damage and that previously observed from the hydroxyl radical are consistent with paraquat, causing damage by generation of reactive oxygen species and the widespread belief that generation of O2.- initiates the formation of destructive reactive oxygen species in a wide variety of traumas and other disorders.

Anatomy and ultrastructure of the axons and terminals of neurons R3-R14 in Aplysia.

Using light and electron microscopy and autoradiography, we have traced the axons of neurons R3-R14 in the parietovisceral ganglion (PVG) of Aplysia to terminal fields associated with vascular tissue. The axons are identified by their large size (15-30 micrometer diameter), extensive glial infolding, characteristic dense core vesicles (DCV; approximately 180 nm diameter), and specific, rapid uptake of 3H-glycine. Each neuron in this homogeneous group sends an axon via the branchial nerve to the pericardial region surrounding the junction of the efferent gill vein and the heart. R14 also sends axons to major arteries near the PVG. The R3-R14 axons branch extensively; we estimate that there are at least several hundred per cell. Branches along axons in the branchial nerve exit the nerve, subdivide, and end blindly in the sheath which is bathed by hemolymph. Similar blind endings from R3R14 occur in the sheath of the PVG (Coggeshall, '67). Axonal branches in the pericardial region and the special R14 axons in the arterial walls form both varicose endings near and terminals in contact with vasvular smooth muscle. All R3-R14 endings are free of glia, packed with DCV, show occasional omega-shaped profiles and rapidly take up 3H-glycine. R3-R14 manufacture specific low molecular weight peptides (Gainer and Wollberg, '74), and both the cell bodies (Iliffe et al., '77) and the germinals contain unusually high concentrations of glycine. The presence of peptides as putative neurohormones and sheath endings (neurohormonal release areas) are consistent with R3-R14 being neurosecretory (Coggeshall et al., '66). While glycine could not be a circulating hormone due to its high circulating levels (Iliffe et al., '77), glycine could act as a local chemical messenger between R3-R14 and smooth muscle. The terminal morphology of R3-R14 is consistent with these neurons having both synaptic-type and neurosecretory-type functions.

The distribution of serotonin in the CNS of an elasmobranch fish: immunocytochemical and biochemical studies in the Atlantic stingray, Dasyatis sabina.

The distribution of serotonin (5HT) in the brain of the Atlantic stingray was studied with peroxidase-antiperoxidase immunocytochemistry and high-pressure liquid chromatography. The regional concentrations of 5HT determined for this stingray fell within the range of values previously reported for fishes. A consistent trend in vertebrates for the hypothalamus and midbrain to have the highest concentrations and the cerebellum the lowest was confirmed in stingrays. Neuronal cell bodies and processes exhibiting 5HT-like immunoreactivity were distributed in variable densities throughout the neuraxis. Ten groups of 5HT cells were described: (I) spinal cord, (II-IV) rhombencephalon, (V, VI) mesencephalon, (VII, VIII) prosencephalon, (IX) pituitary, and (X) retina. There were three noteworthy features of the 5HT system in the Atlantic stingray: (1) 5HT cells were demonstrated in virtually every location in which 5HT-containing cells have been described or alluded to in the previous literature. The demonstration of immunopositive cells in the spinal cord, the retina, and the pars distalis of the pituitary suggests that 5HT may be an intrinsic neurotransmitter (or hormone) in these regions. (2) The distribution of 5HT cells in the brainstem shared many similarities with that in other vertebrates. However, there were many 5HT cells outside of the raphe nuclei, in the lateral tegmentum. It appears that the hypothesis that "lateralization" of the 5HT system is an advanced evolutionary trend cannot be supported. (3) 5HT fibers and terminals were more widely distributed in the Atlantic stingray brain than has been reported for other nonmammalian vertebrates on the basis of histofluorescence. It appears that this feature of the 5HT system arose early in phylogeny, and that the use of immunohistochemistry might reveal a more general occurrence of widespread 5HT fibers and terminals.

Neurotoxicity of glutamate at the concentration released upon spinal cord injury.

Damage caused by administering glutamate into the spinal cord was characterized histologically. Glutamate destroyed neurons for several hundred micrometers around the administering microdialysis fiber. At 24 h after treatment, significant (P = 0.036) loss of neurons was observed (75%) relative to control (47%) near the fiber when glutamate was administered for 1 h at a concentration outside the fiber approximating the maximum glutamate released upon spinal cord injury. Significant loss of neurons (P = 0.006, 0.022) was also caused by administering a combination of glutamate at about its average concentration released upon injury over the 1 h period of administration in combination with the maximum aspartate concentration released upon injury. This work provides a direct demonstration that the concentrations of excitatory amino acids released upon spinal cord injury are neurotoxic. The destruction of neurons by exposure to excitatory amino acids when there is also substantial loss of neurons simply from the presence of the microdialysis fiber may reflect sensitization of neurons to excitotoxicity by stress.

Considerations in the determination by microdialysis of resting extracellular amino acid concentrations and release upon spinal cord injury.

The following issues are further addressed: (1) Is there considerable leakage of amino acids from the circulation into the space around microdialysis probes, or are amino acid concentrations naturally much higher in the interstitial space than is generally thought? (2) Do observed high interstitial concentrations or depletion of substances in the intracellular space by microdialysis affect release measurements upon spinal cord injury? Amino acid concentrations around microdialysis fibres in the spinal cord of rats were found to approach those in the circulation and to be much higher than interstitial concentrations previously estimated in the CNS. However, much lower concentrations of amino acids were derived in the hippocampus by analogous experiments. Considerable Evans Blue/albumin leaked from the circulation into the interstitial space in the spinal cord immediately after fibre insertion. However, this movement diminished considerably by 4 h later, demonstrating substantial resealing of the blood-brain barrier, at least to large molecules. There is either substantial damage-induced movement of amino acids from the circulation into the dialysis zone after insertion of a microdialysis probe, or there is much less impediment to movement of amino acids across the blood-brain barrier in the spinal cord than in the brain. At low flow rates through the fibre, adding concentrations of amino acids to the inside of the fibre equal to the concentrations around the fibre to prevent their depletion by removal through the microdialysis fibre did not affect increases in concentrations of amino acids in microdialysates following injury. Thus the high concentrations of amino acids present around microdialysis fibres following their insertion do not seem to disturb measurements of amino acid release upon spinal cord injury.

Ab initio characterization of ring-opening H-transfer in ionized cyclopentanone: Similarity to ion-neutral complex-mediated alkane eliminations.

Ab initio procedures were used to characterize the transition states for α-cleavage and subsequent H-transfer starting with ionized cyclopentanone (1). The objective was to determine whether the parts of the ion disconnected by the bond cleavage remain in close association through the H-transfer. The transition state for C-C bond cleavage is close in energy to the resulting distonic ion, which is a stable species, and far in geometry from any transition state involving H-transfer. We find no evidence for any concertedness or "quasiconcertedness" in this reaction. Ring-opening H-transfer in 1 and ion-neutral complex-mediated alkane eliminations are compared.

The effect of ion size on rate of dissociation: RRKM calculations on model large polypeptide ions.

The larger an ion is, the less likely it is to decompose on mass spectrornetry time scales at given critical and internal energies. This is an obstacle to obtaining structural information on large molecules by mass spectrometry. We performed RRKM calculations on model ions with masses from 0.27 kDa to 102.4 kDa to explore what such calculations predict regarding this limitation. According to the calculations, it is impractical to add enough energy to fragment very large ions unless the decomposition has a low critical energy. It is suggested that ion-molecule reactions that are either very low in their critical energies or exothermic may be a feasible approach to fragmenting ionized macromolecules.

Preference for an ion-neutral complex-mediated pathway over a five-membered-ring H shift in the isomerization of CH3O (+)HCH 2CH 2 (·) to CH 3CH 2CH 2OH (+·) by Ab initio theory.

Ab initio theory is used to explore whether the path from CH3OH(+)CH2CH 2 (·) (1) to CH3CH2CH2OH(+·) (5) goes by way of a conventional 1,4-H shift to form ·CH2OH(+)CH2CH3 (2), or via the ion-neutral complex-mediated H transfer [CH3OHCH2=CH2](+·) (3) → [CH3CH 2 (·) CH2OH(+)] (4). Five levels of theory all place the highest energy point in the complex-mediated reaction 3 → 4 slightly below that for the 1,4-H shift 1 → 2, but both routes appear energetically feasible near the threshold for the dissociation of 1 to CH3CH2 + CH2=OH(+). Thus, 1 may take both paths to 5. It is concluded that when both a conventional and a complex-mediated pathway seem plausible in a given system, the latter should be considered to be as likely as the former. Ab initio descriptions of other species involved in the isomerization of 1 to 2 also are presented.

Elimination of methane from protonated acetaldehyde: The Ab initio transition state.

The transition state (TS) for loss of CH4 from protonated acetaldehyde has been located at the second-order Moller-Plesset (MP2)/6-31G(d, p) level of theory. The activation energy is predicted to be 263.9 kJ/mol starting from the more stable form (methyl and hydrogen E) and 261.6 kJ/mol starting from the less stable form (methyl and hydrogen Z) that is required for reaction. The products (methane and the formyl ion) are predicted to lie 136.6 kJ/mol below the TS for their formation. MP2 methods underestimate the heats of formation of both the TS and the reaction products by about 40 kJ/mol when compared with experiment. Restricted Hartree-Fock (RHF) calculations give much more accurate relative energies. The MP2 TS leads directly to fragmentation and is described as a protonation of the methyl group by the acidic proton on oxygen. Under RHF theory the reaction is stepwise. An RHF TS similar to the MP2 TS leads to a nonclassical intermediate (which is stable at this level of theory) that has one of the C-H bonds protonated. This mechanism (protonation of an alkyl group) appears to be a general one for high energy 1,2 eliminations from organic cations.

Formation of the 3-Pentanone ion from ionized propyl propanoate through Ion-Neutral complexes.

Formation of the 3-pentanone ion (3) from ionized propyl propanoate through the complex [C2H5CO(+) (•)OC3H7] is proposed based on data obtained by photoionization. The threshold and energy dependence for forming 3 relative to those for related processes support this proposal. The threshold for forming 3 coincides with that predicted for forming [CH3CH2CO(+) (•)CH2CH3], suggesting that that complex is also an intermediate in the pathway to 3. 3-Pentanone ion formation is important much further above threshold than is alkane elimination through [RCO(+) (•)R] complexes. This adds to evidence that reactions between the partners in ion-dipole complexes take place over a wider energy range than do such reactions in complexes containing nonpolar neutral partners.

Isomeric ion-neutral complexes generated from ionized 2-Methylpropanol and n-Butanol: the effect of the polarity of the neutral partner on complex-mediated reactions.

Photoionization was used to characterize the energy dependence of C3H 7 (+) , C3H 6 (+) , CH3OH 2 (+) and CH2=OH(+) formation from (CH3)2)CHCH2OH(+•) (1) and CH3CH2CH2CH2OH(+•) (2). Decomposition patterns of labeled ions demonstrate that close to threshold these products are primarily formed through [CH 3 (+) CHCH3 (•)CH2OH] (bd3) from 1 and through [CH3CH2CH2 (•)CH2=OH(+)] (9) from 2. The onset energies for forming the above products from 1 are spread over 85 kJ mol(-1), and are all near thermochemical threshold. The corresponding onsets from 2 are in a 19 kJ mol(-1) range, and all except that of CH2=OH(+) are well above their thermochemical thresholds. Each decomposition of 3 occurs over a broad energy range (> 214 kJ mol(-1)), This demonstrates that ion-permanent dipole complexes can be significant intermediates over a much wider energy range than ion-induced dipole complexes can be. H-exchange between partners in the complexes appears to be much faster than exchange by conventional interconversions of the alcohol molecular ions with their distonic isomers. The onsets for water elimination from 1 and 2 are below the onsets for the complex-mediated processes, demonstrating that the latter are not necessarily the lowest energy decompositions of a given ion when the neutral partner in the complex is polar.

A photoionization study of the ion-neutral complexes CH3CH (+)CH 3 (·) CH 2CH 3] and CH 3CH 2CH (+)CH 3 (·) CH 3 in the gas phase: Formation, H-transfer and C-C bond formation between partners, and channeling of energy into dissociation.

Photoionization mass spectrometry was used to investigate the dynamics of ion-neutral complex-mediated dissociations of the n-pentane ion (1). Reinterpretation of previous data demonstrates that a fraction of ions 1 isomerizes to the 2-methylbutane ion (2) through the complex CH3CH(+)CH 3 (·) CH2CH3 (3), but not through CH3CH(+)CH2CH 3 (·) CH3 (4). The appearance energy for C3Hin 7 (+) formation from 1 is 66 kJ mol(-1) below that expected for the formation of n-C3H 7 (+) and just above that expected for formation of i-C3H 7 (+) . This demonstrates that the H shift that isomerizes C3H 7 (+) is synchronized with bond cleavage at the threshold for dissociation to that product. It is suggested that ions that contain n-alkyl chains generally dissociate directly to more stable rearranged carbenium ions. Ethane elimination from 3 is estimated to be about seven times more frequent than is C-C bond formation between the partners in that complex to form 2, which demonstrates a substantial preference in 3 for H abstraction over C-C bond formation. In 1 → CH3CH(+)CH2CH3 + CH3 by direct cleavage of the C1-C2 bond, the fragments part rapidly enough to prevent any reaction between them. However, 1 → 2 → 4 → C4H 8 (+) + CH4 occurs in this same energy range. Thus some of the potential energy made available by the isomerization of n-C4H9 in 1 is specifically channeled into the coordinate for dissociation. In contrast, analogous formation of 3 by 1 → 3 is predominantly followed by reaction between the electrostatically bound partners.

Size effects in ion-neutral complex-mediated alkane eliminations from ionized aliphatic ethers.

The effects of the size of the ionic and neutral partners on ion-neutral complex-mediated alkane eliminations from ionized aliphatic ethers were determined by obtaining metastable decomposition spectra and photoionization ionization efficiency curves. Increasing the size of the ionic partner decreases the competitiveness of alkane elimination with alkyl loss. This is attributed to decreasing attraction between the partners with increasing distance between the neutral partner and the center of charge in the associated ion. Increasing the size of the neutral partner lowers the threshold for alkane elimination relative to that for simple dissociation when the first threshold is above ΔHf(products). This is attributed to increasing attraction between the partners with increasing polarizability of the radical in the complex. Adding a CH2 to the radical in a complex seems to increase the attraction between the partners by about 24 kJ mol(-1).

An experimental model combining microdialysis with electrophysiology, histology, and neurochemistry for exploring mechanisms of secondary damage in spinal cord injury: effects of potassium.

An experimental model for characterizing effects of agents suspected to cause damage secondary to spinal cord injury is presented. Microdialysis was used to administer candidate damaging agents and to sample the release of other substances in response to administered agents. Damage was assessed by monitoring the amplitudes of evoked potentials during candidate administration and by postmortem histologic examination. This approach enabled the correlation of electrophysiologic, histologic, and neurochemical parameters in the same experiment. Potassium was chosen as a candidate damaging agent, because (1) in high extracellular concentrations it destroys neurons and (2) its extracellular concentration rises substantially following impact injury to the spinal cord. A technique using parallel administering and collecting microdialysis fibers was developed to estimate the in vivo concentration of K+ outside the administering fiber. Administration of 100 mM KCl, 50 mM K2HPO4, or 100 mM potassium glucuronate in artificial cerebrospinal fluid blocked electrical conduction and destroyed cell bodies. Release of the neurotransmitter amino acids aspartate, glutamate, glycine, and gamma-aminobutyric acid (GABA) and the possible neurotransmitter taurine were dramatically increased by administration of 100 mM KCl. Levels of non-neurotransmitter amino acids increased to lesser degrees. Impairment of conduction, destruction of cell bodies, and release of excitotoxins were all consistent with elevated K+ serving as a secondary damaging agent in spinal cord injury.

Norepinephrine and serotonin release upon impact injury to rat spinal cord.

Microdialysis sampling was used to characterize the release of norepinephrine and serotonin upon impact injury to the rat spinal cord. Increases in extracellular norepinephrine concentrations in response to injury were small and of short duration. In contrast, serotonin concentrations quickly rose 35-90 times following injury and took 30-45 min to return to control levels. Bleeding caused by injury was probably the major source of the increased serotonin levels. Our results allow a role for serotonin in secondary damage upon injury to the spinal cord but suggest that norepinephrine is not a very significant contributor to such damage.

Microdialysis studies of the role of chemical agents in secondary damage upon spinal cord injury.

Assorted microdialysis studies of the roles endogenous chemical agents may play in secondary damage upon spinal cord injury (SCI) are described. Issues addressed include the concentrations reached upon injury, mechanisms of release upon injury, and effects of drugs on injury-elicited increases in glutamate concentrations. An important question in identifying an agent of secondary damage upon central nervous system (CNS) trauma is not simply whether the substance is released upon injury, but whether it reaches harmful levels. To resolve this requires establishing the concentration attained and then determining whether administering that level damages neurons. To make microdialysis measurements of amino acids in the CNS more quantitative, we characterized the effects of insertion of a microdialysis fiber on leakage of glutamate from the circulation and explored the effects of depletion by microdialysis on release caused by SCI. Very high glutamate concentrations were found around the fiber for several hours after fiber insertion and 2 days later, and there was substantial leakage of alpha-aminoisobutyric acid from the circulation into the dialysis zone for several hours after fiber insertion. Glutamate concentrations reached upon SCI under nondepleting conditions were similar to those estimated earlier under depleting conditions. Mg2+ release was detectable when microdialysis probes were perfused with Mg2+-free fluid, but not when the concentrations in the perfusing fluid approximated those in the interstitial space. It is concluded (1) that insertion of microdialysis probes into CNS tissue can cause long-lasting leakage of amino acids from the circulation into the space around the fiber, (2) that this leakage can obscure concentration changes that otherwise occur, and (3) that depletion of substances in the fluid around the fiber may cause increases in concentration to be observed that do not normally happen. We also describe demonstrations that administration of methylprednisolone and dihydrokainic acid diminish increases in glutamate concentrations caused by SCI, showing that microdialysis can be used to explore effects of drugs on actions of damaging substances.

Rodent model of chronic central pain after spinal cord contusion injury and effects of gabapentin.

Spinal cord injury (SCI) often results in abnormal pain syndromes in patients. We present a recently developed SCI mammalian model of chronic central pain in which the spinal cord is contused at T8 using the NYU impactor device (10-g rod, 2.0-mm diameter, 12.5-mm drop height), an injury which is characterized behaviorally as moderate. Recovery of locomotor function was assessed with an open field test and scored using the open field test scale (BBB scale). Somatosensory tests of paw withdrawal responses accompanied by supraspinal responses to both mechanical punctate (von Frey hairs) and nonpunctate (4 mm diameter blunt probe) as well as thermal (radiant heat) peripheral stimuli were performed. Comparisons at the level of the individual animal between precontusion and postcontusion responses indicated significant increases in reactions to low threshold punctate mechanical stimuli, non-punctate stimuli and thermal stimuli (p < 0.05). To demonstrate the validity of this model as a central pain model, gabapentin, an agent used clinically for central pain, was given i.p. at 10 or 30 mg/kg. Gabapentin treatment significantly and reversibly changed the responses, consistent with the attenuation of the abnormal sensory behavior, and the attenuated responses lasted for the duration of the drug effect (up to 6 h). These results support the use of the spinal contusion model in the study of chronic central pain after SCI.