Galanin inhibits long-term potentiation at Schaffer collateral-CA1 synapses in guinea-pig hippocampal slices

CNSs AND OTHER Master's-prepared nurses are usually expected to lead research utilization (RU) efforts in organizations, but they often feel unprepared to do so. In this article, the need for clinical RU is discussed and ideas for implementation, using an adaptation of the Conduct and Utilization of Research in Nursing model, are described. The RU process is applied to research about once-a-day temperatures in afebrile patients. To assist nurses who are implementing research-based practice, we list studies that were used for the practice change, summarize the research base on the topic, and offer an example of clinical use of practice guidelines.     

Pharmacological analysis of the mode of interaction of McN-A-343 at atrial muscarinic M2 receptors

The consensus sequence for single-base deletions in non-reiterated runs during in vitro DNA-dependent DNA polymerisation is refined using data available in the literature. This leads to the observation that chain termination codons are hotspots for single-base deletions. The evolutionary implications are discussed in two models which differ in whether polymerases evolved while the genetic code emerged or after the genetic code was fixed. A possible answer to the question 'Why are stop codons just what they are?' is suggested.     

All-or-none potentiation at CA3-CA1 synapses

The molecular mechanisms underlying long-term potentiation in the hippocampus have received much attention because of the likely functional importance of synaptic plasticity for information storage and the development of neuronal connectivity. Surprisingly, it remains unclear whether activity modifies the strength of individual synapses in a digital (all-or-none) or analog (graded) manner. Here we characterize step-like all-or-none transitions from baseline synaptic transmission to potentiated states following protocols for inducing potentiation at putative single CA3-CA1 synaptic connections. Individual synapses appear to have all-or-none potentiation indicative of highly cooperative processes but different thresholds for undergoing potentiation. These results raise the possibility that some forms of synaptic memory may be stored in a digital manner in the brain.     

Induction of long-term potentiation at perforant path dentate synapses does not affect place learning or memory

B lymphocytes are crucial participants in pulmonary immune defense. However, excess local antibody production is associated with accelerated lung destruction in several types of lung disease. The purpose of the current study was to study the potential role of alveolar macrophages (AM) in the local regulation of immunoglobulin (Ig) production in the lung in response to a direct B cell mitogen, Staphylococcus aureus Cowan strain (SAC). AM, when added to peripheral blood mononuclear cells, caused a dose-dependent inhibition of IgG and IgM, while not affecting IgA production in response to SAC. The mechanism of the AM-induced inhibition included both membrane-bound and soluble signals. The inhibition was not abrogated by indocin and catalase, or reversed by blocking antibodies to transforming growth factor-beta or interferon-gamma. Mononuclear cells isolated from human lung parenchyma displayed a reduced response to SAC compared with blood cells. However, depletion of macrophages from the parenchymal cells was associated with a restoration of IgG production in response to SAC. The results demonstrate that AM inhibit B cell responses to direct B cell mitogen and suggest that the effect of AM is selective for IgM and IgG.     

Decreased probability of neurotransmitter release underlies striatal long-term depression and postnatal development of corticostriatal synapses

Changes in synaptic efficacy are crucial for the development of appropriate neural circuits and brain information storage. We have investigated mechanisms underlying long-term depression (LTD) at glutamatergic synapses in the striatum, a brain region important in motor performance and cognition, and a target for Huntington and Parkinson diseases. Induction of striatal LTD is dependent on postsynaptic depolarization and calcium influx through L-type channels. Surprisingly, LTD maintenance appears to involve a decrease in the probability of neurotransmitter release from presynaptic terminals as evidenced by increases in paired-pulse facilitation and the coefficient of variation of synaptic responses that are tightly associated with LTD expression. Furthermore, both the apparent probability of neurotransmitter release and the magnitude of LTD decrease concomitantly during postnatal development, consistent with the idea that striatal LTD is involved in a developmental decrease in the probability of neurotransmitter release at corticostriatal synapses. The presynaptic changes that underlie striatal LTD may also be important for motor performance and certain forms of learning and memory.     

Endogenous adenosine mediates the presynaptic inhibition induced by aglycemia at corticostriatal synapses

Energy deprivation, as a result of aglycemia, leads to depression of the central synaptic transmission. Endogenous adenosine has been implicated in this depressant effect. We have studied the possible involvement of endogenous adenosine in the depression of corticostriatal excitatory transmission induced by glucose deprivation by using intracellular recordings in brain slices. After stimulation of corticostriatal fibers, EPSPs were recorded from striatal spiny neurons. Adenosine (3-300 microM) or brief periods (5-10 min) of aglycemia reduced the EPSP amplitude but did not alter the membrane potential and the resistance of the recorded cells. These inhibitory effects were not associated with an alteration of the postsynaptic sensitivity to exogenous glutamate but were coupled with an increased paired-pulse facilitation, suggesting the involvement of presynaptic mechanisms. A delayed postsynaptic membrane depolarization/inward current was detected after 15-20 min of glucose deprivation. The presynaptic inhibitory effects induced by adenosine and aglycemia were both antagonized either by the nonselective adenosine receptor antagonist caffeine (2.5 mM) or by the A1 receptor antagonists 8-cyclopentyl-1,3-dimethylxanthine (CPT, 1 microM) and 1,3-dipropyl-8-cyclopentylxanthine (CPX, 300 nM). Conversely, these antagonists affected neither the delayed membrane depolarization/inward current nor the underlying conductance increase produced by glucose deprivation. The ATP-sensitive potassium channel blockers tolbutamide (1 mM) and glipizide (100 nM) had no effect on the aglycemia-induced decrease of EPSP amplitude. Our data demonstrate that endogenous adenosine acting on A1 receptors mediates the presynaptic inhibition induced by aglycemia at corticostriatal synapses, whereas ATP-dependent potassium channels do not play a significant role in this presynaptic inhibition.     

Brain-derived neurotrophic factor enhances long-term potentiation in rat visual cortex

Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), members of the nerve growth factor (NGF) gene family, have been suggested to play a role in experience-dependent modification of neural networks in the developing nervous system. In this study we addressed the question of whether these neurotrophins are involved in long-term potentiation (LTP) in developing visual cortex. We recorded layer II/III field potentials and whole-cell currents evoked by test stimulation of layer IV at 0.1 Hz in visual cortical slices prepared from young rats (postnatal day 15-25) and observed effects of BDNF, NT-3, and NGF on these responses. Then we analyzed the effects of these neurotrophins on LTP induced by tetanic (Theta-burst type) stimulation of layer IV. We found that BDNF at 200 ng/ml potentiated field potentials and EPSCs in most cases and that this potentiation lasted after cessation of the BDNF application. At the concentration of 20 ng/ml, BDNF did not show such an effect, but it enhanced the magnitude of expressed LTP. On the other hand, NT-3 and NGF had none of these effects. Immunohistochemical staining of slices with antibody against BDNF showed that exogenous BDNF penetrated into the whole slice within approximately 5 min of its application. The actions of BDNF were blocked by preincubation of slices with TrkB-IgG fusion protein, a BDNF scavenger, or coapplication of K252a, an inhibitor for receptor tyrosine kinases. TrkB-IgG or K252a itself completely blocked LTP, suggesting that endogenous BDNF or another TrkB ligand plays a role in LTP in the developing visual cortex.     

Contractile roles of the M2 and M3 muscarinic receptors in the guinea pig colon

Ubiquitin-dependent proteolytic systems underlie many processes, including the cell cycle, cell differentiation and responses to stress. One such system is the N-end rule pathway, which targets proteins bearing destabilizing N-terminal residues. Here we report that Ubr1p, the main recognition component of this pathway, regulates peptide import in the yeast Saccharomyces cerevisiae through degradation of Cup9p, a 35 kDa homeodomain protein. Cup9p was identified using a screen for mutants that bypass the previously observed requirement for Ubr1p in peptide import. We show that Cup9p is a short-lived protein (t1/2 approximately 5 min) whose degradation requires Ubr1p. Cup9p acts as a repressor of PTR2, a gene encoding the transmembrane peptide transporter. In contrast to engineered N-end rule substrates, which are recognized by Ubr1p through their destabilizing N-terminal residues, Cup9p is targeted by Ubr1p through an internal degradation signal. The Ubr1p-Cup9p-Ptr2p circuit is the first example of a physiological process controlled by the N-end rule pathway. An earlier study identified Cup9p as a protein required for an aspect of resistance to copper toxicity in S.cerevisiae. Thus, one physiological substrate of the N-end rule pathway functions as both a repressor of peptide import and a regulator of copper homeostasis.     

Concurrent blockade of beta-adrenergic and muscarinic receptors suppresses synergistically long-term potentiation of population spikes in the rat hippocampal CA1 region

The muscarinic acetylcholine receptor antagonist scopolamine, but not the beta-adrenoceptor antagonist propranolol or atenolol, suppressed tetanus-induced long-term potentiation (LTP) of population spikes in the rat hippocampal CA1 region. When scopolamine was coapplied with propranolol or atenolol, a synergistic effect in preventing LTP generation was observed. On the other hand, the coapplication of scopolamine and atenolol failed to affect tetanus-induced LTP of field EPSP. These findings suggest that cooperative mechanisms via muscarinic and beta-adrenergic receptor activation might contribute to LTP induction in terms of the EPSP-spike potentiation, i.e., an increase in the excitability of hippocampal CA1 pyramidal cells after tetanic stimulation, but are independent of the tetanus-evoked potentiation of a synaptic component.     

In vitro long-term potentiation of electrotonic responses of goldfish mauthner cells is accompanied by ultrastructural changes at afferent mixed synapses

The potentiated afferent mixed synapses of the Mauthner cells of fry and adult goldfish in stumps of the medulla oblongata incubated long-term in vitro were studied by electrophysiological and electron microscopic methods. It was shown that brief high-frequency stimulation of posterior branches of the eighth nerve induced a long-term potentiation of electrotonic transmission at large and small mixed club endings. It was about 135% upon subthreshold stimulation and about 200% upon suprathreshold stimulation. The ultrastructural analysis of ultrathin sections of potentiated mixed synaptic endings revealed an increase in the dimensions of desmosome-like contacts which was proportional to the degree of potentiation, about 135% or 200%, depending on the type of stimulation. The dimensions of gap junctions remained unchanged. The dimensions of active zones at potentiated synapses were reduced two-fold as compared with their unpotentiated counterparts, irrespective of the type of stimulation. Considering that desmosome-like contacts consist predominantly of F-actin, a molecule which possesses electroconductivity, it can be assumed that this cytoskeletal protein is involved in the process of potentiation. The increase in the synapse electrical conductivity can be mediated either directly, by shunting the synaptic junction with polymer actin filaments in the region of desmosome-like contacts, or indirectly, via the interaction of actin with gap junction connections situated nearby.     

Involvement of pre- and postsynaptic mechanisms in posttetanic potentiation at Aplysia synapses

Posttetanic potentiation (PTP) is a common form of short-term synaptic plasticity that is generally thought to be entirely presynaptic. Consistent with that idea, PTP of evoked excitatory postsynaptic potentials at Aplysia sensory-motor neuron synapses in cell culture was reduced by presynaptic injection of a slow calcium chelator and was accompanied by an increase in the frequency but not the amplitude of spontaneous excitatory postsynaptic potentials. However, PTP was also reduced by postsynaptic injection of a rapid calcium chelator or postsynaptic hyperpolarization. Thus, PTP at these synapses is likely to involve a postsynaptic induction mechanism in addition to the known presynaptic mechanisms.     

Regulatory phosphorylation of AMPA-type glutamate receptors by CaM-KII during long-term potentiation

Long-term potentiation (LTP), a cellular model of learning and memory, requires calcium-dependent protein kinases. Induction of LTP increased the phosphorus-32 labeling of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPA-Rs), which mediate rapid excitatory synaptic transmission. This AMPA-R phosphorylation appeared to be catalyzed by Ca2+- and calmodulin-dependent protein kinase II (CaM-KII): (i) it correlated with the activation and autophosphorylation of CaM-KII, (ii) it was blocked by the CaM-KII inhibitor KN-62, and (iii) its phosphorus-32 peptide map was the same as that of GluR1 coexpressed with activated CaM-KII in HEK-293 cells. This covalent modulation of AMPA-Rs in LTP provides a postsynaptic molecular mechanism for synaptic plasticity.     

Calcium-permeable AMPA receptors mediate long-term potentiation in interneurons in the amygdala

Fear conditioning is a paradigm that has been used as a model for emotional learning in animals. The cellular correlate of fear conditioning is thought to be associative N-methyl-D-aspartate (NMDA) receptor-dependent synaptic plasticity within the amygdala. Here we show that glutamatergic synaptic transmission to inhibitory interneurons in the basolateral amygdala is mediated solely by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. In contrast to AMPA receptors at inputs to pyramidal neurons, these receptors have an inwardly rectifying current-voltage relationship, indicative of a high permeability to calcium. Tetanic stimulation of inputs to interneurons caused an immediate and sustained increase in the efficacy of these synapses. This potentiation required a rise in postsynaptic calcium, but was independent of NMDA receptor activation. The potentiation of excitatory inputs to interneurons was reflected as an increase in the amplitude of the GABA(A)-mediated inhibitory synaptic current in pyramidal neurons. These results demonstrate that excitatory synapses onto interneurons within a fear conditioning circuit show NMDA-receptor independent long-term potentiation. This plasticity might underlie the increased synchronization of activity between neurons in the basolateral amygdala after fear conditioning.     

When are class I metabotropic glutamate receptors necessary for long-term potentiation?

The involvement of metabotropic glutamate receptors (mGluRs) in hippocampal long-term potentiation (LTP) is a matter of controversial debate. Using [Ca2+]i measurements by confocal laser scanning microscopy and field recordings of EPSPs (fEPSPs) in the hippocampal CA1-region, we found that the efficacy of the broad-spectrum mGluR-antagonist (S)-alpha-methyl-4-carboxyphenylglycine (MCPG) and of (S)-4-carboxy-phenylglycine (4-CPG), a selective antagonist at class I mGluRs, in LTP is contingent on the tetanization strength and the resulting [Ca2+]i response. As indicated by experiments in which we blocked voltage-dependent calcium channels (VDCCs) and intracellular Ca2+ stores (ICSs), the functional significance of class I mGluRs in LTP is confined to certain types of potentiation, which are induced by weak tetanization protocols and require the release of Ca2+ from ICSs for induction. During strong tetanic stimulation, this Ca2+ source is functionally bypassed by activating VDCCs.     

Molecular rigidity and potency of bispyridinium type allosteric modulators at muscarinic M2-receptors

Several bispyridinium compounds have been shown to be potent allosteric modulators of ligand binding to muscarinic M2-receptors. ,,Uno compounds" are benzyl derivatives of the bispyridinium "TMB4" (trimethylene-bis-[4-hydroxy-iminomethyl-pyridinium]). To gain more insight into structure activity relationships, eleven derivatives with varying structure of the oxime-linked aromatic substituent were tested for their ability to inhibit the equilibrium-binding of [3H]N-methylscopolamine ([3H]NMS) in guinea pig cardiac membranes and to retard [3H]NMS-dissociation allosterically. At a concentration of 3 microM, all compounds reduced [3H]NMS-binding to about 40 % of the control level, indicating a similar potency to inhibit the association of [3H]NMS. Allosteric retardation of [3H]NMS-dissociation required higher concentrations. Comparing the effects of the compounds at 30 and 300 microM, respectively, revealed considerable differences in potency. Therefore, the concentration-dependency of the delay of [3H]NMS-dissociation was determined for selected compounds. The results indicate that introduction of a benzyl-moiety into TMB4 leads to a 20-fold increase in allosteric potency. A further increment by a factor of 10 is obtained with the 2,6-dichlorobenzyl-substitution and with the naphthyl-derivative. The other compounds were less potent. An inverse correlation was found between the rotational freedom of the aromatic substituent and the allosteric potency. In conclusion, the aromatic moiety of non-symmetric bispyridinium-type modulators does not seem to be part of the pharmacophore involved in the inhibitory effect on the association of [3H]NMS. In contrast, a rigid aromatic lateral moiety appears to be essential for the interaction with the recognition site mediating the allosteric delay of [3H]NMS dissociation from muscarinic M2-receptors.     

Synapse specificity of long-term potentiation breaks down at short distances

Long-term potentiation (LTP), the long-lasting increase in synaptic transmission, has been proposed to be a cellular mechanism essential for learning and memory, neuronal development, and circuit reorganization. In the original theoretical and experimental work it was assumed that only synapses that had experienced concurrent pre- and postsynaptic activity are subject to synaptic modification. It has since been shown, however, that LTP is also expressed in synapses on neighbouring neurons that have not undergone the induction procedure. Yet, it is still believed that this spread of LTP is limited to adjacent postsynaptic cells, and does not occur for synapses on neighbouring input fibres. However, for technical reasons, tests for 'input specificity' were always done for synapses relatively far apart. Here we have used a new local superfusion technique, which allowed us to assess the synaptic specificity of LTP with a spatial resolution of approximately 30 microm. Our results indicate that there is no input specificity at a distance of less than 70 microm. Synapses in close proximity to a site of potentiation are also potentiated regardless of their own history of activation, whereas synapses far away show no potentiation.     

Functional role of M2 and M3 muscarinic receptors in the urinary bladder of rats in vitro and in vivo

1. Urinary bladder smooth muscle is enriched with muscarinic receptors, the majority of which are of the M2 subtype whereas the remaining minority belong to the M3 subtype. The objective of the present study was to assess the functional role of M2 and M3 receptors in the urinary bladder of rat in vitro and in vivo by use of key discriminatory antagonists. 2. In the isolated bladder of rat, (+)-cis-dioxolane produced concentration-dependent contractions (pEC50 = 6.3) which were unaffected by tetrodotoxin (0.1 microM). These contractions were antagonized by muscarinic antagonists with the following rank order of affinity (pA2) estimates: atropine (9.1) > 4-diphenyl acetoxy-methyl piperidine methiodide (4-DAMP) (8.9) > darifenacin (8.5) > para fluoro hexahydrosiladifenidol (p-F-HHSiD) (7.4) > pirenzepine (6.8) > methoctramine (5.9). These pA2 estimates correlated most favourably (r = 0.99, P < 0.001) with the binding affinity (pKi) estimates of these compounds at human recombinant muscarinic m3 receptors expressed in Chinese hamster ovary cells, suggesting that the receptor mediating the direct contractile responses to (+)-cis-dioxolane equates with the pharmacologically defined M3 receptor. 3. As M2 receptors in smooth muscle are negatively coupled to adenylyl cyclase, we sought to determine whether a functional role of M2 receptors could be unmasked under conditions of elevated adenylyl cyclase activity (i.e., isoprenaline-induced relaxation of KCl pre-contracted tissues). Muscarinic M3 receptors were preferentially alkylated by exposing tissues to 4-DAMP mustard (40 nM, 1 h) in the presence of methoctramine (0.3 microM) to protect M2 receptors. Under these conditions, (+)-cis-dioxolane produced concentration-dependent reversal (re-contraction) of isoprenaline-induced relaxation (pEC50 = 5.8) but had marginal effects on pinacidil-induced, adenosine 3':5'-cyclic monophosphate (cyclic AMP)-independent, relaxation. The re-contractions were antagonized by methoctramine and darifenacin, yielding pA2 estimates of 6.8 and 7.6, respectively. These values are intermediate between those expected for these compounds at M2 and M3 receptors and were consistent with the involvement of both of these subtypes. 4. In urethane-anaesthetized rats, the cholinergic component (approximately 55%) of volume-induced bladder contractions was inhibited by muscarinic antagonists with the following rank order of potency (ID35%inh, nmol kg-1, i.v.): 4-DAMP (8.1) > atropine (20.7) > methoctramine (119.9) > darifenacin (283.3) > pirenzepine (369.1) > p-F-HHSiD (1053.8). These potency estimates correlated most favourably (r = 0.89, P = 0.04) with the pKi estimates of these compounds at human recombinant muscarinic m2 receptors. This is consistent with a major contribution of M2 receptors in the generation of volume-induced bladder contractions, although the modest potency of darifenacin does not exclude a role of M3 receptors. Pretreatment with propranolol (1 mg kg-1, i.v.) increased the ID35%inh of methoctramine significantly from 95.9 to 404.5 nmol kg-1 but had no significant effects on the inhibitory responses to darifenacin. These data suggest an obligatory role of beta-adrenoceptors in M2 receptor-mediated bladder contractions in vivo. 5. The findings of the present study suggest that both M2 and M3 receptors can cause contraction of the rat bladder in vitro and may also mediate reflex bladder contractions in vivo. It is proposed that muscarinic M3 receptor activation primarily causes direct contraction of the detrusor whereas M2 receptor activation can contract the bladder indirectly by reversing sympathetically (i.e. beta-adrenoceptor)-mediated relaxation. This dual mechanism may allow the parasympathetic nervous system, which is activated during voiding, to cause more efficient and complete emptying of the bladder.     

Pharmacology of long-term potentiation

The exotic or non-indigenous species model for deliberately introduced genetically engineered organisms (GEOs) has often been misunderstood or misrepresented. Yet proper comparisons of of ecologically competent GEOs to the patterns of adaptation of introduced species have been highly useful among scientists in attempting to determine how to apply biological theory to specific GEO risk issues, and in attempting to define the probabilities and scale of ecological risks with GEOs. In truth, the model predicts that most projects may be environmentally safe, but a significant minority may be very risky. The model includes a history of institutional follies that also should remind workers of the danger of oversimplifying biological issues, and warn against repeating the sorts of professional misjudgements that have too often been made in introducing organisms to new settings. We once expected that the non-indigenous species model would be refined by more analysis of species eruptions, ecological genetics, and the biology of select GEOs themselves, as outlined. But there has been political resistance to the effective regulation of GEOs, and a bureaucratic tendency to focus research agendas on narrow data collection. Thus there has been too little promotion by responsible agencies of studies to provide the broad conceptual base for truly science-based regulation. In its presently unrefined state, the non-indigenous species comparison would overestimate the risks of GEOs if it were (mis)applied to genetically disrupted, ecologically crippled GEOs, but in some cases of wild-type organisms with novel engineered traits, it could greatly underestimate the risks. Further analysis is urgently needed.     

Binding of serotonin to receptors at multiple sites is required for structural plasticity accompanying long-term facilitation of Aplysia sensorimotor synapses

Long-term changes in the efficacy of Aplysia sensorimotor synapses accompany nonassociative and associative forms of behavioral plasticity. This synapse expresses long-term facilitation either with repeated applications of 5-hydroxytryptamine (5-HT) or with a single pairing of tetanus in the sensory neuron (SN) and bath application of 5-HT. We examined whether structural changes in the SN accompany all forms of long-term synaptic enhancement and the locations at which 5-HT must bind receptors to evoke long-term functional and/or structural changes. Pairing tetanus with one application of 5-HT evoked both functional and structural changes after 24 hr only when 5-HT application was temporally paired with the tetanus and activated receptors on both the SN cell body and terminal region. Repeated application of 5-HT to the terminal region alone failed to evoke any long-term change. Repeated applications of 5-HT to the SN cell body alone evoked a change in synaptic efficacy at 24 hr but failed to increase SN varicosities. Repeated applications of 5-HT to both the SN cell body and the terminal region evoked increases in both synaptic efficacy and the number of SN varicosities at 24 hr. The results indicate that different external stimuli can evoke equivalent forms of long-term synaptic facilitation with or without structural changes in the SNs. Changes in the number of SN varicosities can accompany different forms of long-term facilitation and require the activation of 5-HT receptors at multiple sites.