Cyclic AMP stimulates neurite outgrowth of lamprey reticulospinal neurons without substantially altering their biophysical properties.

Reticulospinal (RS) neurons are critical for initiation of locomotor behavior, and following spinal cord injury (SCI) in the lamprey, the axons of these neurons regenerate and restore locomotor behavior within a few weeks. For lamprey RS neurons in culture, experimental induction of calcium influx, either in the growth cone or cell body, is inhibitory for neurite outgrowth. Following SCI, these neurons partially downregulate calcium channel expression, which would be expected to reduce calcium influx and possibly provide supportive conditions for axonal regeneration. In the present study, it was tested whether activation of second messenger signaling pathways stimulates neurite outgrowth of lamprey RS neurons without altering their electrical properties (e.g. spike broadening) so as to possibly increase calcium influx and compromise axonal growth. First, activation of cAMP pathways with forskolin or dbcAMP stimulated neurite outgrowth of RS neurons in culture in a PKA-dependent manner, while activation of cGMP signaling pathways with dbcGMP inhibited outgrowth. Second, neurophysiological recordings from uninjured RS neurons in isolated lamprey brain-spinal cord preparations indicated that dbcAMP or dbcGMP did not significantly affect any of the measured electrical properties. In contrast, for uninjured RS neurons, forskolin increased action potential duration, which might have increased calcium influx, but did not significantly affect most other electrical properties. Importantly, for injured RS neurons during the period of axonal regeneration, forskolin did not significantly alter their electrical properties. Taken together, these results suggest that activation of cAMP signaling by dbcAMP stimulates neurite outgrowth, but does not alter the electrical properties of lamprey RS neurons in such a way that would be expected to induce calcium influx. In conclusion, our results suggest that activation of cAMP pathways alone, without compensation for possible deleterious effects on electrical properties, is an effective approach for stimulating axonal regeneration of RS neuron following SCI.

Interproximal cervical lesions caused by incorrect flossing technique.

UNLABELLED: This case report describes an interproximal cervical lesion caused by the incorrect use of dental floss. A 58-year-old man who was asymptomatic, presented with unusual notch-like cervical lesions. After clinical and radiographical examinations, it was concluded that the aetiology of these lesions was an incorrect flossing technique. The treatment plan included extraction of maxillary 3rd molars and re-education of the patient in oral hygiene technique. CONCLUSION: These lesions are irreversible and often go undiagnosed; therefore, it is important for the clinician to be familiar with the clinical presentation and aetiology.

Homeostatic control of conjunctival mucosal goblet cells by NKT-derived IL-13.

Although the effects of the interleukin 13 (IL-13) on goblet cell (GC) hyperplasia have been studied in the gut and respiratory tracts, its effect on regulating conjunctival GC has not been explored. The purpose of this study was to determine the major IL-13-producing cell type and the role of IL-13 in GC homeostasis in normal murine conjunctiva. Using isolating techniques, we identified natural killer (NK)/natural killer T (NKT) cells as the main producers of IL-13. We also observed that IL-13 knockout (KO) and signal transducer and activator of transcription 6 knockout (STAT6KO) mice had a lower number of periodic acid Schiff (PAS)+GCs. We observed that desiccating stress (DS) decreases NK population, GCs, and IL-13, whereas it increases interferon-γ (IFN-γ) mRNA in conjunctiva. Cyclosporine A treatment during DS maintained the number of NK/NKT cells in the conjunctiva, increased IL-13 mRNA in NK+ cells, and decreased IFN-γ and IL-17A mRNA transcripts in NK+ and NK- populations. C57BL/6 mice chronically depleted of NK/NKT cells, as well as NKT cell-deficient RAG1KO and CD1dKO mice, had fewer filled GCs than their wild-type counterparts. NK depletion in CD1dKO mice had no further effect on the number of PAS+ cells. Taken together, these findings indicate that NKT cells are major sources of IL-13 in the conjunctival mucosa that regulates GC homeostasis.

Localization, pharmacology, and organization of brain locomotor areas in larval lamprey.

In larval lamprey, spinal locomotor activity can be initiated by pharmacological microstimulation from the following higher order brain locomotor areas [Paggett et al. (2004) Neuroscience 125:25-33; Jackson et al. (2007) J Neurophysiol 97:3229-3241]: rostrolateral rhombencephalon (RLR); ventromedial diencephalon (VMD); or dorsolateral mesencephalon (DLM). In the present study, pharmacological microstimulation with excitatory amino acids (EAAs) or their agonists in the brains of in vitro brain/spinal cord preparations was used to determine the sizes, pharmacology, and organization of these locomotor areas. First, the RLR, DLM and VMD locomotor areas were confined to relatively small areas of the brain, and stimulation as little as 50 µm outside these areas was ineffective or elicited tonic or uncoordinated motor activity. Second, pharmacological stimulation with NMDA, kainate, or AMPA in the VMD or DLM reliably initiated well-coordinated spinal locomotor activity. In the RLR, stimulation with all three ionotropic EAA receptor agonists could initiate spinal locomotor activity, but NMDA or AMPA was more reliable than kainate. Third, with synaptic transmission blocked only in the brain, stimulation in the RLR, VMD, or DLM no longer initiated spinal locomotor activity, suggesting that these locomotor areas do not directly activate spinal locomotor networks. Fourth, following a complete transection at the mesencephalon-rhombencephalon border, stimulation in the RLR no longer initiated spinal motor activity. Thus, the RLR locomotor area does not appear able to initiate spinal locomotor activity by neural circuits confined entirely within the rhombencephalon but requires more rostral neural centers, such as those in the VMD and DLM, as previously proposed [Paggett et al. (2004) Neuroscience 125:25-33].

Methodological considerations for the calculation of cumulative compression exposure of the lumbar spine: a sensitivity analysis on joint model and time standardization approaches.

Cumulative lumbar spine loading has attracted much attention as a factor associated with the development of low back pain. While evidence supports cumulative loading to be a plausible mechanism in explaining several workplace injuries, research establishing a threshold limit value (TLV) for cumulative spine loading has been challenging. The lack of a TLV or even a trend towards harmful cumulative load values may suggest that methodological considerations are greatly influencing the results. This paper examines the impact of different joint models (single muscle equivalent, an electromyography-based third order polynomial, a modified version of the polynomial and a hybrid approach) to determine cumulative spine compression, as well as the importance of time standardization in the calculation of a daily cumulative loading dose. Findings demonstrated that the polynomial predicted cumulative compression values were 43-53% higher than those with all other models tested and the single muscle equivalent predicted loads 18% higher than loads predicted using a modified polynomial. Profound differences between modelling approaches suggest that caution should be taken when selecting a muscle model to determine cumulative spine compressive loading. Time standardized cumulative compression values were found to be 28.3% greater than non-standardized estimates, illustrating the importance of selecting a standard time frame in the calculation of cumulative spine compression.

Organization of higher-order brain areas that initiate locomotor activity in larval lamprey.

In the lamprey, spinal locomotor activity can be initiated by pharmacological microstimulation in several brain areas: rostrolateral rhombencephalon (RLR); dorsolateral mesencephalon (DLM); ventromedial diencephalon (VMD); and reticular nuclei. During DLM- or VMD-initiated locomotor activity in in vitro brain/spinal cord preparations, application of a solution that focally depressed neuronal activity in reticular nuclei often attenuated or abolished the locomotor rhythm. Electrical microstimulation in the DLM or VMD elicited synaptic responses in reticulospinal (RS) neurons, and close temporal stimulation in both areas evoked responses that summated and could elicit action potentials when neither input alone was sufficient. During RLR-initiated locomotor activity, focal application of a solution that depressed neuronal activity in the DLM or VMD abolished or attenuated the rhythm. These new results suggest that neurons in the RLR project rostrally to locomotor areas in the DLM and VMD. These latter areas then appear to project caudally to RS neurons, which probably integrate the synaptic inputs from both areas and activate the spinal locomotor networks. These pathways are likely to be important components of the brain neural networks for the initiation of locomotion and have parallels to locomotor command systems in higher vertebrates.

High-speed solution switching using piezo-based micropositioning stages.

Motion-induced vibration is a critical limitation in high-speed micropositioning stages used to achieve solution switching. Controlled rapid solution switching is used to study the fast activation and deactivation kinetics of ligand-gated ion-channel populations isolated in excised membrane patches--such studies are needed to understand fundamental mechanisms that mediate synaptic excitation and inhibition in the central nervous system. However, as the solution-switching speed is increased, vibration induced in the piezo-based positioning stages can result in undesired, repeated, ligand application to the excised patch. The article describes a method to use knowledge of the piezo-stage's vibrational dynamics to compensate for and reduce these unwanted vibrations. The method was experimentally verified using an open-electrode technique, and fast solution switching (100 micros range) was achieved.

Mutation of the ATP-binding pocket of SSA1 indicates that a functional interaction between Ssa1p and Ydj1p is required for post-translational translocation into the yeast endoplasmic reticulum.

The translocation of proteins across the yeast ER membrane requires ATP hydrolysis and the action of DnaK (hsp70) and DnaJ homologues. In Saccharomyces cerevisiae the cytosolic hsp70s that promote post-translational translocation are the products of the Ssa gene family. Ssa1p maintains secretory precursors in a translocation-competent state and interacts with Ydj1p, a DnaJ homologue. Although it has been proposed that Ydj1p stimulates the ATPase activity of Ssa1p to release preproteins and engineer translocation, support for this model is incomplete. To this end, mutations in the ATP-binding pocket of SSA1 were constructed and examined both in vivo and in vitro. Expression of the mutant Ssa1p's slows wild-type cell growth, is insufficient to support life in the absence of functional Ssa1p, and results in a dominant effect on post-translational translocation. The ATPase activity of the purified mutant proteins was not enhanced by Ydj1p and the mutant proteins could not bind an unfolded polypeptide substrate. Our data suggest that a productive interaction between Ssa1p and Ydj1p is required to promote protein translocation.

Attachment theory and violence toward women by male intimate partners.

PURPOSE: Explore whether understanding of violence toward women by their male intimate partners is enhanced by attachment theory. ORGANIZING FRAMEWORK: This review was focused on the evidence that men prone to intimate partner violence were insecurely attached as infants, and as a consequence, their internal working models of attachment relationships include the use of aggression to gain power and control. These internal working models are brought to their adult relationships with intimate partners. The intergenerational transmission of violence occurs when children who witness episodes of violence in their homes, or experience violence directly as victims, become aggressive in adult relationships. Parallels between concepts of infant attachment and adult romantic attachment, such as maintaining proximity to attachment figures, were examined. METHODS: The literature on attachment theory and intimate partner violence was reviewed. References were identified from electronic databases and from a manual search of the literature. CONCLUSIONS: Evidence about intimate partner violence is consistent with attachment theory. This framework is a base for extension of knowledge of intimate partner violence. Changes in practice, research, and policy to support an attachment perspective are discussed.

Coordination of locomotor activity in the lamprey: role of descending drive to oscillators along the spinal cord.

In the lamprey and most fish, locomotion is characterized by caudally propagating body undulations that result from a rostrocaudal phase lag for ipsilateral burst activity. One of the mechanisms that might contribute to rostrocaudal phase lags is a gradient of oscillator burst frequencies along the spinal cord that presumably are produced, in part, by descending drive from the brain. The purpose of the present study was to test whether a gradient of oscillator frequencies does exist along the lamprey spinal cord. First, during brain-initiated locomotor activity in in vitro brain/spinal cord preparations, the cycle times (=1/frequency) of locomotor activity generated by the functionally isolated rostral spinal cord (activity blocked in middle and caudal cord) were significantly shorter than control cycle times when the entire spinal cord was generating locomotor activity. Second, the cycle times of locomotor activity generated by the functionally isolated caudal cord (activity blocked in rostral and middle cord) were significantly longer than control cycle times for activity generated by the entire spinal cord. Thus, no one region of the spinal cord appears to dictate the overall cycle times of locomotor activity generated by the entire spinal cord, although overall cycle times tended to be closest to those of the isolated rostral spinal cord. Finally, although short- and long-distance coupling as well as oscillator frequency gradients probably contribute to rostrocaudal phase lags of spinal locomotor activity, the asymmetrical nature of short-distance coupling, in which the descending component dominates, appears to be the main factor.

Axonal regeneration of descending brain neurons in larval lamprey demonstrated by retrograde double labeling.

In larval lamprey, the large, identified descending brain neurons (Müller and Mauthner cells) are capable of axonal regeneration. However, smaller, unidentified descending brain neurons, such as many of the reticulospinal (RS) neurons, probably initiate locomotion, and it is not known whether the majority of these neurons regenerate their axons after spinal cord transection. In the present study, this issue was addressed by using double labeling of descending brain neurons. In double-label control animals, in which Fluoro-Gold (FG) was applied to the spinal cord at 40% body length (BL; measured from anterior to posterior from tip of head) and Texas red dextran amine (TRDA) was applied later to the spinal cord at 20% BL, an average of 98% of descending brain neurons were double labeled. In double-label experimental animals, FG was applied to the spinal cord at 40% BL; two weeks later the spinal cord was transected at 10% BL; and, eight weeks or 16 weeks after spinal cord transection, TRDA was applied to the spinal cord at 20% BL. At eight weeks and 16 weeks after spinal cord transection, an average of 49% and 68%, respectively, of descending brain neurons, including many unidentified RS neurons, were double labeled. These results in larval lamprey are the first to demonstrate that the majority of descending brain neurons, including small, unidentified RS neurons, regenerate their axons after spinal cord transection. Therefore, in spinal cord-transected lamprey, axonal regeneration of descending brain neurons probably contributes significantly to the recovery of locomotor function.

Coordination of spinal locomotor activity in the lamprey: long-distance coupling of spinal oscillators.

The extent and strength of long-distance coupling between locomotor networks in the rostral and caudal spinal cord of larval lamprey were examined with in vitro brain/spinal cord preparations, in which spinal locomotor activity was initiated by chemical microstimulation in the brain, as well as with computer modeling. When locomotor activity and short-distance coupling were blocked in the middle spinal cord for at least 40 segments, burst activity in the rostral and caudal spinal cord was still coupled 1:1, indicating that long-distance coupling is extensive. However, in the absence of short-distance coupling, intersegmental phase lags were not constant but decreased significantly with increasing cycle times, suggesting that long-distance coupling maintains a relatively constant delay rather than a constant phase lag between rostral and caudal bursts. In addition, under these conditions, intersegmental phase lags, measured between rostral and caudal burst activity, were significantly less than normal, and the decrease was greater for longer distances between rostral and caudal locomotor networks. The above result could be mimicked by a computer model consisting of pairs of oscillators in the rostral, middle, and caudal spinal cord that were connected by short- and long-distance coupling. With short-distance coupling blocked in the middle spinal cord, strychnine was applied to either the rostral or caudal spinal cord to convert the pattern locally from right-left alternation to synchronous burst activity. Synchronous burst activity in the rostral spinal cord resulted in a reduction in right-left phase values for burst activity in the caudal cord. These results also could be mimicked by the computer model. Strychnine-induced synchronous burst activity in the caudal spinal cord did not appear to alter the right-left phase values of rostral burst activity. Taken together, the experimental and modeling results suggest that the descending and ascending components of long-distance coupling, although producing qualitatively different effects, are comparatively weak. In particular, the descending component of long-distance coupling appears to become progressively weaker with increasing distance between two given regions of spinal cord. Therefore, short-distance coupling probably contributes substantially to normal rostrocaudal phase lags for locomotor activity along the spinal cord. However, short-distance coupling may be more extensive than "nearest neighbor coupling."

Receptor system response kinetics reveal functional subtypes of native murine and recombinant human GABAA receptors.

1. Regional distinctions in GABA type A (GABAA) miniature IPSC responses are thought to be determined by postsynaptic receptor composition. The kinetics of receptor activation and deactivation were studied using rapid exchange (100 micros) of GABA at excised patches containing recombinant (alpha1beta1gamma2 or alpha2beta1gamma2) and native (cortical) GABAA receptors. 2. Receptors activated by brief (< 1 ms) pulses of GABA demonstrated a characteristic current response, hereby referred to as the 'receptor system response'. System response properties included agonist concentration-dependent peak amplitudes and concentration-independent maximal rates of activation and deactivation. Receptor subtypes were characterized functionally and phenotyped using the system response characteristics. 3. System responses obtained for alpha1beta1gamma2 receptors exhibited a single phenotype while alpha2beta1gamma2 receptors exhibited either a predominant slow deactivation (type I) or a relatively infrequent faster (type II) phenotype. Receptor system responses of alpha2beta1gamma2 receptors reached peak currents twice as fast as those of alpha1beta1gamma2 receptors (0.5 versus 1.0 ms) but decayed 2 or 6 times more slowly (taulong of approximately 190 and 62 ms for type I and II alpha2beta1gamma2, and approximately 34 ms for alpha1beta1gamma2 receptors). 4. Receptor system responses from cultured fetal mouse cortical neurons could be statistically separated and classified into five major types with little intragroup variability, primarily based on variations in the current deactivation phases. 5. Receptors subjected to pharmacological modulation exhibited alterations in system response properties consistent with known mechanisms of action, such that distinctions between binding and gating modulations were possible. 6. Brief agonist exposure places limits on receptor activation and deactivation response kinetics. Consequently, receptor system responses may be used to characterize and functionally phenotype an excised patch receptor population. Furthermore, since synaptic exposure to transmitter is postulated to be similarly brief, IPSC kinetics may reflect a functional fingerprint of synaptic receptors.

Specific molecular chaperone interactions and an ATP-dependent conformational change are required during posttranslational protein translocation into the yeast ER.

The posttranslational translocation of proteins across the endoplasmic reticulum (ER) membrane in yeast requires ATP hydrolysis and the action of hsc70s (DnaK homologues) and DnaJ homologues in both the cytosol and ER lumen. Although the cytosolic hsc70 (Ssa1p) and the ER lumenal hsc70 (BiP) are homologous, they cannot substitute for one another, possibly because they interact with specific DnaJ homologues on each side of the ER membrane. To investigate this possibility, we purified Ssa1p, BiP, Ydj1p (a cytosolic DnaJ homologue), and a GST-63Jp fusion protein containing the lumenal DnaJ region of Sec63p. We observed that BiP, but not Ssa1p, is able to associate with GST-63Jp and that Ydj1p stimulates the ATPase activity of Ssa1p up to 10-fold but increases the ATPase activity of BiP by <2-fold. In addition, Ydj1p and ATP trigger the release of an unfolded polypeptide from Ssa1p but not from BiP. To understand further how BiP drives protein translocation, we purified four dominant lethal mutants of BiP. We discovered that each mutant is defective for ATP hydrolysis, fails to undergo an ATP-dependent conformational change, and cannot interact with GST-63Jp. Measurements of protein translocation into reconstituted proteoliposomes indicate that the mutants inhibit translocation even in the presence of wild-type BiP. We conclude that a conformation- and ATP-dependent interaction of BiP with the J domain of Sec63p is essential for protein translocation and that the specificity of hsc70 action is dictated by their DnaJ partners.

Mitochondrial Hsp70 cannot replace BiP in driving protein translocation into the yeast endoplasmic reticulum.

To determine whether mitochondrial hsp70 (mHsp70) could substitute for the endoplasmic retuculum (ER) Hsp70 (BiP) during protein translocation, we assembled ER-derived reconstituted proteoliposomes supplemented with either protein. We found that only BiP restored translocation in kar2 mutant vesicles and stimulated translocation approximately 3-fold in wild type proteoliposomes. mHsp70 associated poorly with both a BiP binding (DnaJ) domain of Sec63p and an ER precursor, and its ATPase activity was poorly enhanced upon incubation with the DnaJ domain. In contrast, BiP bound to the Sec63p-DnaJ domain in an ATP-dependent manner and its ATPase activity was stimulated significantly by this polypeptide. We conclude that mHsp70 is unable to support protein translocation into the ER because it fails to associate productively with Sec63p and a precursor.

Anatomical regeneration and behavioral recovery following crush injury of the trigeminal root in lamprey.

In normal larval lamprey, bilateral application of horseradish peroxidase (HRP) to the dorsal part of the anterior oral hood labeled subpopulations of trigeminal components on both sides of the brain; peripherally projecting motoneurons, medullary dorsal cells (sensory), and spinal dorsal cells (sensory), as well as centrally projecting afferents in the trigeminal descending tracts. Following unilateral crush injury of the right trigeminal root, HRP labeling of sensory and motor trigeminal components on the right side gradually increased with increasing recovery time, between 2 weeks and 12 weeks postcrush (PC). Axons of trigeminal motoneurons appeared to exhibit robust regeneration, whereas restoration of projections in the descending trigeminal tract ipsilateral to the injury was incomplete. Control experiments indicated that motor and sensory axons from the intact side of the oral hood did not sprout across the midline to the denervated side. Several results suggested that regenerated trigeminal sensory fibers made synapses with brain neurons that have direct or indirect inputs to reticulospinal (RS) neurons. Following a unilateral crush injury of the right trigeminal root, escape behavior in response to stimulation of the right side of the oral hood gradually returned to normal. Muscle recordings at various recovery times confirmed that anatomical regeneration of trigeminal sensory axons was functional. In addition, at 8 or 12 weeks PC, brief stimulation of the oral hood ipsilateral or contralateral to the crush injury elicited synaptic responses in RS neurons on either side of the brain, similar to that in normal animals. In the lamprey, compensatory mechanisms probably allow recovery of behavioral function despite incomplete regeneration of trigeminal sensory axons within the central nervous system.

Adaptive variations of undulatory behaviors in larval lamprey: comparison of swimming and burrowing.

In larval lamprey, movements and muscle activity during swimming and burrowing behaviors were compared. Burrowing consisted of two components: an initial component in which the head was driven into the burrowing medium; and a final component in which the animal pulled the rest of its body into the burrowing medium. The initial component of burrowing was characterized by large undulatory movements and rhythmic muscle burst activity that were similar in form to those during fast swimming, but more intense. During the initial component of burrrowing, burst durations, burst amplitudes, and burst proportions of motor activity were larger than those during swimming, while cycle time was slightly shorter than during swimming. Intersegmental phase lags and right-left phase values were similar for swimming and initial burrowing. The final component of burrowing was characterized by sharp, long-duration flexures on one side of the body, sometimes followed by similar flexures on the other side. Each flexure was produced by long-duration, large-amplitude muscle burst activity on the same side of the body or several shorter sequential bursts with slightly smaller amplitudes. During the final component of burrowing, burst durations and burst amplitudes of motor activity were much larger than those during swimming or during the initial component of burrowing. It is suggested that the motor patterns for swimming and the initial component of burrowing are produced by a common spinal locomotor network. The final component of burrowing may use some of the same neurons in the spinal locomotor networks, but the networks are probably configured differently than the situation during swimming.