Rapid enrichment of presynaptic protein in boutons undergoing classical conditioning is mediated by brain-derived neurotrophic factor.

Presynaptic structural modifications are thought to accompany activity-dependent synaptic plasticity and learning. This may involve the conversion of nonfunctional synapses into active ones or the generation of entirely new synapses. Here, using an in vitro neural analog of classical conditioning, we investigated presynaptic structural changes restricted to auditory nerve synapses that convey the conditioned stimulus (CS) by tract tracing using fluorescent tracers combined with immunostaining for the synaptic vesicle-associated protein synaptophysin. The results show that the size of presynaptic auditory boutons increased and the area and fluorescence intensity of punctate staining for synaptophysin were enhanced after conditioning. This occurred only for auditory nerve boutons apposed to the dendrites but not the somata of abducens motor neurons. Conditioning increased the percentage of boutons that were immunopositive for synaptophysin and enhanced the number of synaptophysin puncta they contained. Pretreatment with antibodies against brain-derived neurotrophic factor (BDNF) inhibited these conditioning-induced structural changes. There was also a net increase in the number of boutons apposed to abducens motor neurons after conditioning or BDNF treatment. These data indicate that the rapid enrichment of presynaptic boutons with proteins required for neurotransmitter recycling and release occurs during classical conditioning and that these processes are mediated by BDNF.

AMPA receptor trafficking and learning.

In the last few years it has become clear that AMPA-type glutamate neurotransmitter receptors are rapidly transported into and out of synapses to strengthen or weaken their function. The remarkable dynamics of AMPA receptor (AMPAR) synaptic localization provides a compelling mechanism for understanding the cellular basis of learning and memory, as well as disease states involving cognitive dysfunction. Here, we summarize the evidence for AMPAR trafficking as a mechanism underlying a variety of learned responses derived from both behavioral and cellular studies. Evidence is also reviewed supporting synaptic dysfunction related to impaired AMPAR trafficking as a mechanism underlying learning and memory deficits in Alzheimer's disease. We conclude that emerging data support the concept of multistage AMPAR trafficking during learning and that a broad approach to include examination of all of the AMPAR subunits will provide a more complete view of the mechanisms underlying multiple forms of learning.

Protein kinase C-dependent and independent signaling pathways regulate synaptic GluR1 and GluR4 AMPAR subunits during in vitro classical conditioning.

Protein kinase C (PKC) signal transduction pathways have been implicated in mechanisms of synaptic plasticity and learning, however, the roles of the different PKC family isoforms remain to be clarified. Previous studies showed that NMDAR-mediated trafficking of GluR4-containing AMPARs supports conditioning and that the mitogen-activated protein kinases (MAPKs) have a central role in the synaptic delivery of GluR4 subunits. Here, an in vitro model of classical conditioning in pond turtles, Pseudemys scripta elegans, was used to assess the role of PKC isoforms in mechanisms underlying this form of learning. We show that the PKC antagonists chelerythrine and bisindolylmaleimide I attenuated conditioned response (CR) acquisition and expression, as did the PKCzeta pseudosubstrate peptide inhibitor ZIP. Analysis of protein expression revealed that PKCzeta is activated in early stages of conditioning followed shortly afterward by increased levels of PKCalpha/beta and activation of ERK MAPK. Data also suggest that PKCzeta is upstream from and activates ERK. Finally, protein localization studies using confocal imaging indicate that inhibitors of ERK, but not PKC, suppress colocalization of GluR1 with synaptophysin while inhibitors of PKC and ERK attenuate colocalization of GluR4 with synaptophysin. Together, these data suggest that acquisition of conditioning proceeds by two stages of AMPAR trafficking. The first is PKC-independent and ERK-dependent synaptic delivery of GluR1 subunits to activate silent synapses. This is followed by PKC-dependent and ERK-dependent synthesis and delivery of GluR4 subunits that supports the acquisition of CRs. Therefore, there is a selective role for PKC and MAPK signaling pathways in multistep AMPAR trafficking that mediates acquisition of classical conditioning.

Coordinate action of pre- and postsynaptic brain-derived neurotrophic factor is required for AMPAR trafficking and acquisition of in vitro classical conditioning.

Brain-derived neurotrophic factor (BDNF) has been implicated in mechanisms of synaptic plasticity such as long-term potentiation (LTP), but its role in associative learning remains largely unknown. In the present study, we investigated the function of BDNF and its receptor tropomyosin-related kinase B (TrkB) in an in vitro model of classical conditioning using pond turtles, Pseudemys scripta elegans. Conditioning resulted in a significant increase in BDNF and phospho (p)-Trk expression. Bath application of antibodies directed against TrkB, but not TrkA or TrkC, abolished acquisition of conditioning, as did a receptor tyrosine kinase inhibitor K252a and an inhibitor of nitric oxide synthase 7-nitroindazole. Significantly, injections of BDNF Ab into the nerve roots of presynaptic axonal projections or postsynaptic motor neurons prevented acquisition of conditioning, suggesting that BDNF is required on both sides of the synapse for modification to occur. The presynaptic proteins synaptophysin and synapsin I were increased upon conditioning or BDNF application. Furthermore, BDNF application alone mimicked conditioning-induced synaptic insertion of GluR1 and GluR4 AMPAR subunits into synapses, which was inhibited by co-application of BDNF and K252a. Data also show that extracellular signal-regulated kinase (ERK) was activated in BDNF-treated preparations. We conclude that coordinate pre- and postsynaptic actions of BDNF are required for acquisition of in vitro classical conditioning.

The desaturation response time of finger pulse oximeters during mild hypothermia.

Pulse oximeters may delay displaying the correct oxygen saturation during the onset of hypoxia. We investigated the desaturation response times of pulse oximeter sensors (forehead, ear and finger) during vasoconstriction due to mild hypothermia and vasodilation caused by glyceryl trinitrate. Ten healthy male volunteers were given three hypoxic challenges of 3 min duration under differing experimental conditions. Mild hypothermia increased the mean response time of finger oximeters from 130 to 215 s. Glyceryl trinitrate partly offset this effect by reducing the response time from 215 to 187 s. In contrast, the response times of the forehead and ear oximeters were unaffected by mild hypothermia, but the difference between head and finger oximeters was highly significant (p < 0.0001). The results suggest that the head oximeters provide a better monitoring site for pulse oximeters during mild hypothermia.

Social stress and corticosterone regionally upregulate limbic N-methyl-D-aspartatereceptor (NR) subunit type NR(2A) and NR(2B) in the lizard Anolis carolinensis.

Social aggression in the lizard Anolis carolinensis produces dominant and subordinate relationships while elevating corticosterone levels and monoaminergic transmitter activity in hippocampus (medial and mediodorsal cortex). Adaptive social behavior for dominant and subordinate male A. carolinensis is learned during aggressive interaction and therefore was hypothesized to involve hippocampus and regulation of N-methyl-d-aspartate (NMDA) receptors. To test the effects of social stress and corticosterone on NMDA receptor subunits (NR), male lizards were either paired or given two injections of corticosterone 1 day apart. Paired males were allowed to form dominant-subordinate relationships and were killed 1 day later. Groups included isolated controls, dominant males, subordinate males and males injected with corticosterone. Brains were processed for glutamate receptor subunit immunohistochemistry and fluorescence was analyzed by image analysis for NR(2A) and NR(2B) in the small and large cell divisions of the medial and mediodorsal cortex. In the small granule cell division there were no significant differences in NR(2A) or NR(2B) immunoreactivity among all groups. In contrast, there was a significant upregulation of NR(2A) and NR(2B) subunits in the large pyramidal cell division in all three experimental groups as compared with controls. The results revealed significantly increased NR(2A) and NR(2B) subunits in behaving animals, whereas animals simply injected with corticosterone showed less of an effect, although they were significantly increased over control. Upregulation of NR(2) subunits occurs during stressful social interactions and is likely to be regulated in part by glucocorticoids. The data also suggest that learning social roles during stressful aggressive interactions may involve NMDA receptor-mediated mechanisms.

Targeting of GLUR4-containing AMPA receptors to synaptic sites during in vitro classical conditioning.

The synaptic delivery of GluR4-containing AMPA receptors during in vitro classical conditioning of a neural correlate of an eyeblink response was examined by fluorescence imaging of punctate staining for glutamate receptor subunits and the presynaptic marker synaptophysin. There was a significant increase in GluR4-containing AMPA receptors to synaptic sites after conditioning as determined by colocalization of GluR4 subunit puncta with synaptophysin. Moreover, the trafficking of these receptor subunits requires NMDA receptor activation as it was blocked by D,L-2-amino-5-phosphonovaleric acid (AP-5). In contrast, colocalization of NR1 subunits with synaptophysin was stable regardless of whether the preparations had undergone conditioning or had been treated by AP-5. The enhanced colocalization of GluR4 and synaptophysin was accompanied by an increase in both the total number and size of puncta for both proteins, suggesting greater synthesis and aggregation during conditioning. Western blot analysis confirmed upregulation of synaptophysin and GluR4 following conditioning. These data support the hypothesis that GluR4-containing AMPA receptors are delivered to synaptic sites during conditioning. Further, they suggest coordinate presynaptic and postsynaptic modifications during in vitro classical conditioning.

Inhibition of NF-kappa B activity by thalidomide through suppression of IkappaB kinase activity.

The sedative and anti-nausea drug thalidomide, which causes birth defects in humans, has been shown to have both anti-inflammatory and anti-oncogenic properties. The anti-inflammatory effect of thalidomide is associated with suppression of cytokine expression and the anti-oncogenic effect with inhibition of angiogenesis. It is presently unclear whether the teratogenic properties of thalidomide are connected in any way to the beneficial, anti-disease characteristics of this drug. The transcription factor NF-kappaB has been shown to be a key regulator of inflammatory genes such as tumor necrosis factor-alpha and interleukin-8. Inhibition of NF-kappaB is associated with reduced inflammation in animal models, such as those for rheumatoid arthritis. We show here that thalidomide can block NF-kappaB activation through a mechanism that involves the inhibition of activity of the IkappaB kinase. Consistent with the observed inhibition of NF-kappaB, thalidomide blocked the cytokine-induced expression of NF-kappaB-regulated genes such as those encoding interleukin-8, TRAF1, and c-IAP2. These data indicate that the therapeutic potential for thalidomide may be based on its ability to block NF-kappaB activation through suppression of IkappaB kinase activity.

In vitro eye-blink classical conditioning is NMDA receptor dependent and involves redistribution of AMPA receptor subunit GluR4.

The classically conditioned vertebrate eye-blink response is a model in which to study neuronal mechanisms of learning and memory. A neural correlate of this response recorded in the abducens nerve can be conditioned entirely in vitro using an isolated brainstem-cerebellum preparation from the turtle by pairing trigeminal and auditory nerve stimulation. Here it is reported that conditioning requires that the paired stimuli occur within a narrow temporal window of <100 msec and that it is blocked by the NMDA receptor antagonist d,l-2-amino-5-phosphonovaleric acid. Moreover, there is a significant positive correlation between the levels of conditioning and greater immunoreactivity with the glutamate receptor 4 (GluR4) AMPA receptor subunit in the abducens motor nuclei, but not with NMDAR1 or GluR1. It is concluded that in vitro classical conditioning of an abducens nerve eye-blink response is generated by NMDA receptor-mediated mechanisms that may act to modify the AMPA receptor by increasing GluR4 subunits in auditory nerve synapses.

Postoperative sleep disturbance: influences of opioids and pain in humans.

STUDY OBJECTIVES: To test the hypothesis that opioids and pain contribute independently to postoperative sleep disturbance, 10 women undergoing surgery requiring a low abdominal incision for treatment of benign gynecologic conditions were randomized to receive either epidural opioid (fentanyl) (n=6) or epidural local anesthetic (bupivacaine) (n=4) for intraoperative and postoperative analgesia. DESIGN: N/A. SETTING: N/A. PATIENTS OR PARTICIPANTS: N/A. INTERVENTIONS: N/A. MEASUREMENTS: Polysomnography was performed in a standard patient room on the preoperative and first three postoperative nights. Pain at rest and with coughing was evaluated using a visual-analogue pain scale each evening and morning. RESULTS: On the first postoperative night, rapid eye movement (REM) sleep was abolished in all patients. On the third postoperative night, the mean +/- SE REM sleep time increased significantly (p=.003) to 9.8% +/- 3.1% in the fentanyl group, and 12.9% +/- 3.8% in the bupivacaine group. Conversely, light non-REM (NREM) sleep (%stage 1 + %stage 2) was higher on the first postoperative night and significantly lower on the third postoperative night (p=0.011). Between group comparison revealed only that the mean % slow-wave sleep (SWS) in the fentanyl group (6.0%, 2.0%, and 14.7%) was different from the bupivacaine group (7.8%, 9.1%, and 10.6%) in the postoperative period after adjusting for the preoperative night % SWS (p=0.021). Pain was well controlled in all patients, but was slightly better controlled in the fentanyl group than in the bupivacaine group on postoperative night 2 (p=0.024). There was no statistically significant association between pain score and any polysomnographically defined stage. CONCLUSION: Postoperative patients suffer a profound sleep disturbance even when opioids are avoided and pain is well controlled.

Melatonin secretion after surgery.

Sleep disturbance is common postoperatively. We examined whether melatonin concentrations were related to this disturbance in seven postoperative patients. Nocturnal concentrations of melatonin were significantly (p=0.005) lower on the first than on the second or third nights after surgery. This finding raises the possibility that melatonin suppression and associated sleep disturbance might be prevented by melatonin replacement.

Immunocytochemical localization of glutamate receptor subunits in the brain stem and cerebellum of the turtle Chrysemys picta.

The regional distribution of ionotropic (AMPA and NMDA) and metabotropic (mGluR1alpha) glutamate receptor subunits was examined in the brain stem and cerebellum of the pond turtle, Chrysemys picta, by using immunocytochemistry and light microscopy. Subunit-specific antibodies that recognize NMDAR1, GluR1, GluR4, and mGluR1alpha were used to identify immunoreactive nuclei in the brain stem and cerebellum. Considerable immunoreactivity in the turtle brain stem and cerebellum was observed with regional differences occurring primarily in the intensity of staining with the antibodies. The red nucleus, lateral reticular nucleus and cerebellum labeled intensely for NMDAR1 and moderately for GluR1. The cerebellum also labeled strongly for mGluR1alpha. All of the cranial nerve nuclei labeled intensely for NMDAR1 and to varying degrees for GluR1, GluR4, and mGluR1alpha. Counterstaining revealed the presence of neuronal somata where there were no immunoreactive neurons in individual nuclei. This finding suggests that there are subpopulations of immunoreactive neurons within a given nucleus that bear different glutamate receptor subunit compositions. The results suggest that the glutamate receptor subunit distribution in the brain stem and cerebellum of turtles is similar to that reported for rats. Additionally, there is considerable colocalization of NMDA and AMPA receptors as revealed by light microscopy. These results have implications for the organization of neural circuits that control motor behavior in turtles, and, generally, for the function of brain stem and cerebellar neural circuits in vertebrates.

Comparison of cortically and subcortically controlled motor systems. II. Distribution of anterogradely labeled terminal boutons on intracellularly filled rubrospinal neurons in rat and turtle.

The present study examined the circuitry of the red nucleus of the Sprague-Dawley rat and the freshwater pond turtle, Chrysemys picta, by using intracellular cell filling combined with anterograde tract tracing. Although both species have a well-developed cerebellorubral system, they differ in that the red nucleus of rats receives direct input from the motor areas of the cerebral cortex, whereas turtles do not. However, a direct descending projection from the hypothalamus to the red nucleus of turtles has been described. The aim of this study was to elucidate the relative functional contributions of the cerebellum and descending inputs to motor signal generation in the red nucleus. The results show that the cellular distribution of cerebellar inputs on rubrospinal neurons is similar between the rat and turtle; these projections are observed on the soma and the proximal and distal dendrites. In contrast, the hypothalamic inputs in turtles occupy mainly the more distally located dendrites, similar to the position of the cortical inputs in rats. These findings suggest that, first, the cerebellar inputs are not spatially segregated from the cortical or hypothalamic inputs in rats or turtles, as far as can be determined by light microscopy. Second, there is specificity of input from the cortex in rats and hypothalamus in turtles onto the distal portions of the dendrites. The similarity in the organizational features of the mammalian and non-mammalian cerebellorubrospinal systems has implications for interpretations of the relative roles of the cerebellum and cerebral cortex in motor control.

Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice.

Platelet activation is characterized by shape change, induction of fibrinogen receptor expression and release of granular contents, leading to aggregation and plug formation. While this response is essential for hemostasis, it is also important in the pathogenesis of a broad spectrum of diseases, including myocardial infarction, stroke and unstable angina. Adenosine 5'-diphosphate (ADP) induces platelet aggregation, but the mechanism for this has not been established, and the relative contribution of ADP in hemostasis and the development of arterial thrombosis is poorly understood. We show here that the purinoceptor P2Y1 is required for platelet shape change in response to ADP and is also a principal receptor mediating ADP-induced platelet aggregation. Activation of P2Y1 resulted in increased intracellular calcium but no alteration in cyclic adenosine monophosphate (cAMP) levels. P2Y1-deficient platelets partially aggregated at higher ADP concentrations, and the lack of P2Y1 did not alter the ability of ADP to inhibit cAMP, indicating that platelets express at least one additional ADP receptor. In vivo, the lack of P2Y1 expression increased bleeding time and protected from collagen- and ADP-induced thromboembolism. These findings support the hypothesis that the ATP receptor P2Y1 is a principal receptor mediating both physiologic and pathological ADP-induced processes in platelets.

Properties of conditioned abducens nerve responses in a highly reduced in vitro brain stem preparation from the turtle.

Previous work suggested that the cerebellum and red nucleus are not necessary for the acquisition, extinction, and reacquistion of the in vitro classically conditioned abducens nerve response in the turtle. These findings are extended in the present study by obtaining conditioned responses (CRs) in preparations that received a partial ablation of the brain stem circuitry. In addition to removing all tissue rostral to and including the midbrain and cerebellum, a transection was made just caudal to the emergence of the IXth nerve. Such ablations result in a 4-mm-thick section of brain stem tissue that functionally eliminates the sustained component of the unconditioned response (UR) while leaving only a phasic component. We refer to this region of brain stem tissue caudal to the IXth nerve as the "caudal premotor blink region." Neural discharge was recorded from the abducens nerve following a single shock unconditioned stimulus (US) applied to the ipsilateral trigeminal nerve. When the US was paired with a conditioned stimulus (CS) applied to the posterior eighth, or auditory, nerve using a delay conditioning paradigm, a positive slope of CR acquisition was recorded in the abducens nerve, and CR extinction was recorded when the stimuli were alternated. Resumption of paired stimuli resulted in reacquisition. Quantitative analysis of the CRs in preparations in which the caudal premotor blink region had been removed and those with cerebellar/red nucleus lesions showed that both types of preparations had abnormally short latency CR onsets compared with preparations in which these regions were intact. Preparations with brain stem transections had significantly earlier CR offsets as more CRs terminated as short bursts when compared with intact or cerebellar lesioned preparations. These data suggest that a highly reduced in vitro brain stem preparation from the turtle can be classically conditioned. Furthermore, the caudal brain stem is not a site of acquisition in this reduced preparation, but it contributes to the sustained activity of both the UR and CR. Finally, the unusually short CR onset latencies following lesions to the cerebellum are not further exacerbated by removal of the caudal brain stem. These studies suggest that convergence of CS and US synaptic inputs onto the abducens nerve reflex circuitry may underlie acquisition in this reduced preparation, but that mechanisms that control learned CR timing arise from the cerebellorubral system.

Morbidity and mortality for alternate-site anesthesia care.

The bibliography contains very few references from anesthesia literature and a preponderance of citations from emergency medicine, radiology and pediatrics journals. The reviewed publications include studies addressing the relation of drug type and dosages to morbidity and mortality in alternate site locations. These studies approach the issue of safety through retrospective analysis of techniques that are associated with acceptably low morbidity. The occurrence of adverse anesthetic events within formalized sedation programs is also reviewed. The low morbidity suggests that the trend of out of the operating room anesthetic and sedative use will continue. The ability of formalized sedation programs to establish and enforce standards of practice as well as to continually collect quality assurance data is a strong endorsement for their continuation.

Context-sensitive half-time and anesthesia: how does theory match reality?

The pharmacokinetic model, which describes context-sensitive decrement times, has received validation through direct measurement. Further validation has come from the ability of targeted infusion schemes to obtain reliable and stable concentrations of a variety of intravenous drugs. A complete understanding of the concept will enable the appropriate selection of drugs and an appreciation of the strengths and limitations of current drug delivery systems.

Comparison of cortically and subcortically controlled motor systems: I. Morphology of intracellularly filled rubrospinal neurons in rat and turtle.

The rat and turtle differ markedly in major structural features of the corticocerebellorubrospinal circuitry. Although both species have a well-developed cerebellorubrospinal system, they differ in that a direct cerebral cortical input to the red nucleus is present only in the rat. The aim of the present study was to compare features of the soma and dendritic morphology of rubrospinal neurons that receive cortical input, as in rats, with those that do not, as in turtles. Intracellular Lucifer Yellow injections of neurons retrogradely labeled with Fast Blue in the rat or activity-dependent sulforhodamine-labeled neurons in the turtle were used to fill rubrospinal neurons in 150-200-microm-thick fixed sections. Images of filled neurons were imported into a computer to analyze quantitatively soma and dendritic morphology. The results show that rubrospinal soma size is slightly larger in the rat than in the turtle. However, analysis of the dendritic morphology, including total dendritic length, length of primary, secondary, and tertiary dendritic branches, and a Scholl analysis of dendritic branch intersections across concentric rings, demonstrated no significant differences between the two species. These findings suggest that the basic dendritic morphology of rubrospinal neurons may have been established early in phylogeny, preceding the evolution of cortical inputs. Alternatively, similar dendritic morphologies may have arisen due to the presence of other synapses in the turtle that occupy the sites of the cortical input in the rat. This comparative approach provides insights into the information processing capabilities of cortically versus subcortically controlled motor systems.

Distribution of hypoglossal motor neurons innervating the prehensile tongue of the African pig-nosed frog, Hemisus marmoratum.

Using retrograde neuronal tracers, a study of the distribution of hypoglossal motor neurons innervating the tongue musculature was performed in the African pig-nosed frog, Hemisus marmoratum. This species is a radically divergent anuran amphibian with a prehensile tongue that can be aimed in three dimensions relative to the head. The results illustrate a unique rostrocaudal distribution of the ventrolateral hypoglossal nucleus and an unusually large number of motor neurons within this cell group. During the evolution of the long, prehensile tongue of Hemisus, the motor neurons innervating the tongue have greatly increased in number and have become more caudally distributed in the brainstem and spinal cord compared to other anurans. These observations have implications for understanding neuronal reconfiguring of motoneurons for novel morphologies requiring new muscle activation patterns.