Caffeine effects on buccal muscles of Aplysia

1. Caffeine (35-70 mM) elicited contractions of Aplysia buccal muscle El. In a Ca2+-free medium, in which ACh-elicited contractions rapidly fail, caffeine elicited contractions of approximately the same size as in normal medium. 2. 5-HT (10(-8) M and 10(-7) M) did not enhance caffeine-elicited contractions. 3. Lower concentrations (1-10 mM) of caffeine inhibited ACh-elicited contractions. Caffeine (7 mM) reduced the contraction by 80%. 4. Caffeine (7 mM) reduced ACh-elicited depolarization by 60%. 5. Caffeine (7 mM) increased 45Ca2+ influx into Aplysia buccal muscle I5. The stimulation of influx of 45Ca2+ by 10(-3) M ACh was non-additive with the stimulation caused by caffeine, and 7 mM caffeine reduced the influx caused by 10(-3) M ACh.     

Morphological differences between excitatory and inhibitory nerve terminals in cockroach coxal muscles

Two morphologically distinct types of neuromuscular junction on the coxal leg muscles of the cockroach, Periplaneta americana, which have been physiologically described as innervated by fast, slow and inhibitory nerve fibers, have been found. In one type of neuromuscular junction the axon terminal contains many round clear synaptic vesicles and contacts several sarcoplasmic extensions from the muscle fiber. The muscle processes adhere to the axon terminal for a short distance (short contact or SC type). The axon terminal of the other type of neuromuscular junction directly contacts the muscle fiber and no extensions of the muscle fiber are formed. The contact region is comparatively long (long contact or LC type). The nerve terminal contains many polymorphic synaptic vesicles. From a correlation of the present morphological findings and the previous physiological results, it may be suggested that the SC type of nerve terminal represents both fast and slow nerve terminals and the inhibitory terminal is of the LC type.     

Adenylate cyclase-mediated modulation of interaction between excitatory and inhibitory synaptic influences on smooth muscles

We found that nonadrenergic inhibitory synaptic potentials (ISP) induced by intramural stimulation in atropine-treated smooth muscles of the guinea-pig large intestine demonstrated no changes upon the influence of an activator of adenylate cyclase, forskolin. This indicates that cAMP-dependent pathways are not involved in the generation of ISP. However, in these muscles with no atropine pretreatment ISP were suppressed by forskolin; intramural stimulation evoked in these smooth muscle cells M-cholinergic excitatory synaptic potentials (ESP) instead of ISP. An increase in the intracellular cAMP concentration due to application of its membrane-penetrating form, dibutyryl-cAMP, did not mimic the above-described effect of forskolin. Hence, it can be supposed that the effect of forskolin on inhibitory synaptic transmission in the atropine-untreated smooth muscles is not related to changes in the intracellular cAMP level; this effect is determined by other mechanisms. The above differences between the effects of forskolin on ISP in the atropine-treated and atropine-untreated smooth muscle strips indicate that the interaction of intracellular signal pathways (probably, through protein G_q/11), which is observed with activation of adenylate cyclase, occurs under conditions of simultaneous activation of M cholinoreceptors and purinoreceptors. The pattern of adenylate cyclase-mediated modulation of inhibitory effects of purinergic neurons on smooth muscles does not allow us to rule out the possibility of involvement of interstitial cells of Cajal as a relay link providing this synaptic effect. Transmission of excitation from cholinergic nerve terminals to smooth muscles is realized without the participation of the interstitial cells of Cajal.Neirofiziologiya/Neurophysiology, Vol. 36, Nos. 5/6, pp. 438–445, September–December, 2004.This revised version was published online in April 2005 with a corrected cover date and copyright year.     

Serotonin induced protein phosphorylation in the Aplysia eye

Serotonin (5-HT) increases the phosphorylation of two low molecular weight phosphoproteins of 23,000 and 15,000 daltons molecular weight and decreases the phosphorylation of a 20,000 dalton phosphoprotein in the isolated Aplysia eye. The cAMP analog 8-benzylthio cAMP increases and decreases the phosphorylation of the 23,000 and 20,000 dalton 5-HT sensitive phosphoproteins, respectively. The effect of 5-HT on protein phosphorylation is not affected by the phase of the circadian rhythm of spontaneous compound action potentials generated in the eye.     

Modulation of swimming in Tritonia: excitatory and inhibitory effects of serotonin

1. In the mollusc Tritonia escape swimming is produced by a network of central pattern generator (CPG) neurons. The purpose of this study was to determine which neurotransmitters might be involved in the swim system.
2. Injection of serotonin (5HT) into whole animals elicited swimming followed by a long-lasting inhibition of swimming. In isolated brain preparations, bath-applied 5HT elicited a swim pattern at short latency and also caused a long-lasting inhibition of the swim pattern. The activation of swimming by 5HT was associated with a tonic depolarization of cerebral cell 2 (C2) and the dorsal swim interneurons (DSI) which form part of the swim CPG network.
3. In isolated brain preparations, bath applied glycine, histamine, proctolin, and FMFRamide had no effect on the swim motor pattern elicited by electrical stimulation of a peripheral nerve. Aspartate, carbacol, dopamine, glutamate, octopamine, pilocarpine, and small cardioactive peptide-B (SCP_B) inhibited the activation of swimming by nerve stimulation.
4. The 5HT antagonists cyproheptidine, tryptamine, and 7-methyltryptamine had no effect on swimming, but methysergide and fenfluramine inhibited swimming to both normal sensory stimuli and exogenously applied 5HT.
5. Staining with a polyclonal antibody indicated that one class of CPG neurons, the dorsal swim interneurons (DSI), was immunoreactive for 5HT.
6. Taken together, the data suggest that pattern generator interneurons, particularly the DSIs, use 5HT as a neurotransmitter.

     

TNF‐α has both stimulatory and inhibitory effects on mouse monocyte‐derived osteoclastogenesis

Phenotypically different osteoclasts may be generated from different subsets of precursors. To what extent the formation of these osteoclasts is influenced or mediated by the inflammatory cytokine TNF‐α, is unknown and was investigated in this study. The osteoclast precursors early blasts (CD31^hiLy‐6C^?), myeloid blasts (CD31⁺Ly‐6C⁺), and monocytes (CD31^?Ly‐6C^hi) were sorted from mouse bone marrow using flow cytometry and cultured with M‐CSF and RANKL, with or without TNF‐α. Surprisingly, TNF‐α prevented the differentiation of TRAcP⁺ osteoclasts generated from monocytes on plastic; an effect not seen with early blasts and myeloid blasts. This inhibitory effect could not be prevented by other cytokines such as IL‐1β or IL‐6. When monocytes were pre‐cultured with M‐CSF and RANKL followed by exposure to TNF‐α, a stimulatory effect was found. TNF‐α also stimulated monocytes’ osteoclastogenesis when the cells were seeded on bone. Gene expression analysis showed that when TNF‐α was added to monocytes cultured on plastic, RANK, NFATc1, and TRAcP were significantly down‐regulated while TNF‐αR1 and TNF‐αR2 were up‐regulated. FACS analysis showed a decreased uptake of fluorescently labeled RANKL in monocyte cultures in the presence of TNF‐α, indicating an altered ratio of bound‐RANK/unbound‐RANK. Our findings suggest a diverse role of TNF‐α on monocytes’ osteoclastogenesis: it affects the RANK‐signaling pathway therefore inhibits osteoclastogenesis when added at the onset of monocyte culturing. This can be prevented when monocytes were pre‐cultured with M‐CSF and RANKL, which ensures the binding of RANKL to RANK. This could be a mechanism to prevent unfavorable monocyte‐derived osteoclast formation away from the bone.

     

Serotonin immunoreactivity of neurons in the gastropod Aplysia californica

Serotonergic neurons and axons were mapped in the central ganglia of Aplysia californica using antiserotonin antibody on intact ganglia and on serial sections. Immunoreactive axons and processes were present in all ganglia and nerves, and distinct somata were detected in all ganglia except the buccal and pleural ganglia. The cells stained included known serotonergic neurons: the giant cerebral neurons and the RB cells of the abdominal ganglion. The area of the abdominal ganglion where interneurons are located which produce facilitation during the gill withdrawal reflex was carefully examined for antiserotonin immunoreactive neurons. None were found, but two bilaterally symmetric pairs of immunoreactive axons were identified which descend from the contralateral cerebral or pedal ganglion to abdominal ganglion. Because of the continuous proximity of this pair of axons, they could be recognized and traced into the abdominal ganglion neuropil in each preparation. If serotonin is a facilitating transmitter in the abdominal ganglion, these and other antiserotonin immunoreactive axons in the pleuroabdominal connectives may be implicated in this facilitation.     

Serotonin and forskolin enhance both magnitude of contraction and relaxation rate of Aplysia dorsal extrinsic muscle independently of acetylcholine receptor

Hexamethonium bromide (Hex. Br.) blocks acetylcholine (ACh) elicited contractions but not electrically elicited contractions of isolated preparations of Aplysia californica dorsal extrinsic muscle. Serotonin (5-hydroxytryptamine, 5-HT) enhances both magnitude and relaxation rate of ACh and electrically elicited contractions. In the presence of Hex. Br., 5-HT still exhibits its modulatory effects on electrically elicited contractions. Forskolin enhances both magnitude and relaxation rate of ACh and electrically elicited contractions. Forskolin (10(-5) M) increases the cyclic AMP content of the accessory radula closer and dorsal extrinsic muscles.     

Serotonin depresses feeding behaviour in ants

Feeding behaviour is a complex functional system that relies on external signals and the physiological state of the animal. This is also the case in ants as they vary their feeding behaviour according to food characteristics, environmental conditions and - as they are social insects - to the colony's requirements. The biogenic amine serotonin (5-HT) was shown to be involved in the control and modulation of many actions and processes related to feeding in both vertebrates and invertebrates. In this study, we investigated whether 5-HT affects nectar feeding in ants by analysing its effect on the sucking-pump activity. Furthermore, we studied 5-HT association with tissues and neuronal ganglia involved in feeding regulation. Our results show that 5-HT promotes a dose-dependent depression of sucrose feeding in Camponotus mus ants. Orally administered 5-HT diminished the intake rate by mainly decreasing the volume of solution taken per pump contraction, without modifying the sucrose acceptance threshold. Immunohistochemical studies all along the alimentary canal revealed 5-HT-like immunoreactive processes on the foregut (oesophagus, crop and proventriculus), while the midgut and hindgut lacked 5-HT innervation. Although the frontal and suboesophageal ganglia contained 5-HT immunoreactive cell bodies, serotonergic innervation in the sucking-pump muscles was absent. The results are discussed in the frame of a role of 5-HT in feeding control in ants.     

Secondary hair cells and afferent neurones of the squid statocyst receive both inhibitory and excitatory efferent inputs

Summary Intracellular recordings were obtained from the hair cells and afferent neurones of the angular acceleration receptor system of the statocyst of the squid,Alloteuthis subulata. Electrical stimulation of the efferent fibres in the crista nerve (minor) evoked responses in all of the secondary hair cells recorded from (n=211). 48% of the secondary air cells responded with a small depolarization, 15% with a hyperpolarization, and 37% with a depolarization followed by a hyperpolarization. The depolarizations and hyperpolarizations had mean stimulus to response delays of 6.7 ms and 24 ms, and reversal potentials of about –1 mV and –64 mV, respectively. Both types of potential increased in amplitude, up to a point, when the stimulus shock was increased and facilitation and/or summation effects could be obtained by applying multiple shocks. These data, together with the fact that both responses could be blocked by bath application of cobalt or cadmium, indicate that the secondary hair cells receive both inhibitory and excitatory efferent inputs and that these are probably mediated via chemical synapses. No efferent responses were seen in the primary hair cells but both depolarizing and hyperpolarizing efferent responses were obtained from the afferent neurones.     

Photoaffinity Labeling of Adenylate Cyclase-Linked Serotonin Receptors in Aplysia Neurons

Serotonin stimulated adenylate cyclase in Aplysia neurons with a Kact of 0.7 microM. Under the same conditions, 1-[2-(4-aminophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine stimulated adenylate cyclase with a Kact of 20 microM. The azido derivative of this compound, 1-[2-(4-azidophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine, or of serotonin, (4-amino, 3-nitrophenylazido-serotonin), also stimulated the cyclase in the dark, but with lower efficiency (Kact greater than 10(-4) M). Irradiation of the membranes in the presence of 100 microM 1-[2-(4-azidophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine abolished 75% of the cyclase activity stimulated by 5 microM serotonin. Under the same conditions, 100 microM 4-amino, 3-nitrophenylazido-serotonin did not inhibit serotonin-stimulated adenylate cyclase activity. When [3H]1-[2-(4-azidophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine (20 microM) was irradiated with membranes for 5 min at 4 degrees C, a dozen peptides were labeled, as revealed by a fluorogram of sodium dodecyl sulfate-polyacrylamide gels. Among them, the labeling of five polypeptides (molecular weights of 45,000, 55,000, 63,000, 80,000, and 94,000) was protected by the presence of 0.2 mM serotonin during photolysis. These peptides may be related to serotonin receptors.     

Dynamical responses to external stimuli for both cases of excitatory and inhibitory synchronization in a complex neuronal network

For studying how dynamical responses to external stimuli depend on the synaptic-coupling type, we consider two types of excitatory and inhibitory synchronization (i.e., synchronization via synaptic excitation and inhibition) in complex small-world networks of excitatory regular spiking (RS) pyramidal neurons and inhibitory fast spiking (FS) interneurons. For both cases of excitatory and inhibitory synchronization, effects of synaptic couplings on dynamical responses to external time-periodic stimuli S(t) (applied to a fraction of neurons) are investigated by varying the driving amplitude A of S(t). Stimulated neurons are phase-locked to external stimuli for both cases of excitatory and inhibitory couplings. On the other hand, the stimulation effect on non-stimulated neurons depends on the type of synaptic coupling. The external stimulus S(t) makes a constructive effect on excitatory non-stimulated RS neurons (i.e., it causes external phase lockings in the non-stimulated sub-population), while S(t) makes a destructive effect on inhibitory non-stimulated FS interneurons (i.e., it breaks up original inhibitory synchronization in the non-stimulated sub-population). As results of these different effects of S(t), the type and degree of dynamical response (e.g., synchronization enhancement or suppression), characterized by the dynamical response factor \(D_f\) (given by the ratio of synchronization degree in the presence and absence of stimulus), are found to vary in a distinctly different way, depending on the synaptic-coupling type. Furthermore, we also measure the matching degree between the dynamics of the two sub-populations of stimulated and non-stimulated neurons in terms of a “cross-correlation” measure \(M_c\). With increasing A, based on \(M_c\), we discuss the cross-correlations between the two sub-populations, affecting the dynamical responses to S(t).     

Optimal learning with excitatory and inhibitory synapses

Characterizing the relation between weight structure and input/output statistics is fundamental for understanding the computational capabilities of neural circuits. In this work, I study the problem of storing associations between analog signals in the presence of correlations, using methods from statistical mechanics. I characterize the typical learning performance in terms of the power spectrum of random input and output processes. I show that optimal synaptic weight configurations reach a capacity of 0.5 for any fraction of excitatory to inhibitory weights and have a peculiar synaptic distribution with a finite fraction of silent synapses. I further provide a link between typical learning performance and principal components analysis in single cases. These results may shed light on the synaptic profile of brain circuits, such as cerebellar structures, that are thought to engage in processing time-dependent signals and performing on-line prediction.     

Differences in the cytoskeleton in inhibitory and excitatory synapses

The cytoskeleton of afferent chemical synapses, with various ultrastructure of contact zones, was examined in the Mauthner cells of the goldfish. The synapses with combined active zones and desmosome-like specialized contacts possessed a well developed cytoskeleton consisting of filaments and microtubules oriented towards the synaptic apposition. Regular arrays of synaptic vesicles oriented in the same direction were observed beyond and near the active zones. The cytoskeleton of the synapses lacking desmosome-like formations was diffusely organized throughout the boutons. The distribution of vesicles in the vicinity of active zones was also not ordered. The role of cytoskeleton in organization of the two morphologically distinct synapses is discussed. A special function of cytoskeleton as an intermediary between synaptoplasm and membrane is regarded as a necessary basis for plasticity of excitatory rather than inhibitory synapses.     

Serotonin and cAMP induce excitatory modulation of a serotonergic neuron

Serotonin (5-HT) is an excitatory neurotransmitter and neuromodulator. In the Aplysia nervous system it increases excitability and induces spike broadening in sensory neurons. It is released at the synaptic terminals of the metacerebral cells (MCCs) and modulates the feeding neural circuit and buccal muscles during the aroused feeding state. We report that MCC itself is depolarized by 5-HT and becomes excitable. 5-HT induces tonic spike activity and even spike-burst activity. Conceivably, this sensitivity to its own transmitter could provide positive feedback excitation of MCC. Voltage clamp analysis of isolated cultured MCCs shows that 5-HT reduces a calcium-dependent outward current at the resting potential (-60 mV), and enhances steady state inward currents between -55 and -30 mV and between -75 and -100 mV. 8-Br-cAMP has similar effects, suggesting that cAMP mediates the 5-HT effects, in part. A transient calcium current is enhanced at voltages more positive than -40 mV. Barium and cesium selectively block the 5-HT-induced inward current between -75 and -100 mV. Substitution of N-methyl-D-glucamine for sodium and adding cobalt block this current, also indicating that it is a hyperpolarization-activated cation current. The 5-HT-induced inward current between -55 and -30 mV is also blocked by sodium substitution and added cobalt, suggesting that 5-HT increases a depolarization-activated cation current. The outward current that remains when sodium and calcium currents are blocked is reduced by 5-HT. Thus, 5-HT enhances two different cation currents and reduces potassium currents.     

Familiarity breeds plasticity: distinct effects of experience on putative excitatory and inhibitory neurons in inferior temporal cortex

Primates have a remarkable capacity to recognize a vast array of visual objects, an ability that depends on experience. In this issue of Neuron, Woloszyn and Sheinberg (2012) report that putative excitatory and inhibitory neurons in inferior temporal cortex exhibit distinct influences long-term visual experience.     

Protein kinase C has both stimulatory and suppressive effects on macrophage superoxide production.

Unlike resident peritoneal macrophages (RPM) or tumor necrosis factor alpha (TNF alpha)-primed bone marrow-derived macrophages (BMM), unprimed BMM do not generate superoxide in response to the protein kinase C (PKC) activator, phorbol myristate acetate (PMA). However, these cells do contain significant levels of PKC activity. In contrast to PMA, zymosan induces the generation of superoxide in unprimed BMM, as well as in TNF alpha-primed BMM and RPM. Staurosporine, a potent PKC inhibitor, failed to affect the zymosan-induced production of superoxide by unprimed and TNF alpha-primed BMM and RPM, in spite of substantial inhibition of PMA-induced superoxide production by the primed BMM and RPM. However, when PKC was depleted from unprimed BMM by prolonged (24 h) treatment with phorbol dibutyrate (PdBt) (10(-7) M) the ability of zymosan to induce the production of superoxide was greatly diminished. Such a result could be interpreted as suggesting a role for PKC in the zymosan-induced response, a conclusion which contrasts with the inhibitor data. However, PKC depletion, in this case, is achieved via the PdBt-induced activation of PKC. It is thus possible that it is the initial activation of PKC, rather than its depletion, that suppresses superoxide production. Consistent with this interpretation, the co-stimulation of unprimed BMM with both zymosan and PMA resulted in a reduced superoxide release compared to zymosan alone. The activation of PKC therefore appears to have a suppressive effect on the generation of superoxide by unprimed cells. We thus conclude that PKC is not required for zymosan-induced superoxide production by either primed or unprimed macrophages and suggest that PKC may be involved in regulatory mechanisms restricting superoxide production by macrophages. However, since PMA alone can initiate the release of superoxide from primed BMM and RPM, it would appear that PKC can mediate both stimulatory and suppressive signals for macrophage superoxide production.