Early development of an identified serotonergic neuron in Helisoma trivolvis embryos: Serotonin expression,de-expression,and uptake

In early-stage embryos of Helisoma trivolvis, a bilateral pair of identified neurons (ENC1) express serotonin and project primary descending neurites that ramify in the pedal region of the embryo prior to the formation of central ganglia. Pharmacological studies suggest that serotonin released from ENC1 acts in an autoregulatory pathway to regulate its own neurite branching and in a paracrine or synaptic pathway to regulate the activity of pedal ciliary cells. In the present study, several key features of early ENC1 development were characterized as a necessary foundation for further experimental studies on the mechanisms underlying ENC1 development and its physiological role during embryogenesis. ENC1 morphology was determined by confocal microscopy of serotonin-immunostained embryos and by differential-interference contrast (DIC) microscopy of live embryos. The soma was located at an anteriolateral superficial position and contained several distinguishing features, including a large spherical nucleus with prominent central nucleolus, large granules in the apical cytoplasm, a broad apical dendrite ending in a sensory-like structure at the embryonic surface, and a ventral neurite. ENC1 first expressed serotonin immunoreactivity around stage E13, followed immediately by the appearance of an immunoreactive neurite (stage E14). Both the intensity of immunoreactivity and primary neurite length were consistently greater in the right ENC1 at early stages. Serotonin uptake, as indicated by 5,7-dihydroxytryptamine-induced fluorescence, first occurred between stages E18 and E25. At later stages of embryogenesis (after stage E65), serotonin immunoreactivity disappeared, whereas serotonin uptake and normal cell morphology were retained. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 361–376, 1998     

Behavior of a single neuron in a recurrent excitatory loop

A recurrent excitation loop was constructed by enabling each impulse from the slowly adapting stretch receptor organ SAO (crayfish) to trigger through an electronic circuit a brief stretch, or “tug,” of the receptor. When applied independently, each tug influenced the discharge as would an EPSP. Recurrent excitation led to characteristic discharge timings; hence, even an isolated neuron can have intrinsic mechanisms that prevent positive feedback from freezing it in an extreme non-operational state. Such timings depended critically on the “phase”, i.e., on the time elapsed between an SAO impulse and the tug. When the control discharge was stationary (because the SAO length remained invariant), phases of a few ms simply changed the pattern to one of doublets, and affected little the average rate. As the phase increased, bursts appeared, bursts and interburst intervals became more prolonged, and average rates increased. With the largest phases examined (40 ms), the discharge consisted of a slow alternation of high rate bursts, separated by long intervals. When the discharge was modulated (by 0.2/s sinusoidal length variation) with recurrent excitation, the peak-to-peak rate swing, i.e., the sensitivity, and the proportion of the cycle without afferent discharges increased, and the rate vs. length display was distorted even though remaining “loop-plus-extension.” Changes were phase-dependent: for example, loops could have a sharp high peak at one phase and be flat-topped at another. When the interspike interval variability was exaggerated (by a length jitter superimposed upon either invariant or sinusoidally varying lengths), recurrent excitation exerted fewer, weaker and somewhat different effects: e.g., it reduced the overall intensity of the invariant cases and the peak-to-peak swing in the modulated one. The precise mechanisms of these results can only be conjectured at but are likely to involve an electrogenic pump, electromechanical interactions, topographical issues, as well as their interplays. The functional implications involve, for instance, the modulation of the intensity, duration and occurrence of the bursting patterns in oscillating functions (e.g., breathing, chewing, etc.).     

Serotonin modulation of caudal photoreceptor in crayfish

The sixth abdominal ganglion (6th AG) of the crayfish contains two photosensitive neurons. This caudal photoreceptor (CPR) displays spontaneous electrical activity and phasic-tonic responses to light pulses. In this paper, we analyzed the presence of serotonin in the 6th AG and its effects in the modulation of the activity of CPR. In the first part of our study, we identified serotonergic neurons in the 6th AG by immunostaining using an antibody against serotonin. Next, we quantified the serotonin contents in the 6th AG by using liquid chromatography. Finally, we searched for serotonergic modulation of the CPR electrical activity by using conventional extracellular recordings. We found 13 immunopositive neurons located in the ventral side of the 6th AG. The mean diameter of their somata was 23+/-9 microm. In addition, there was immunopositive staining in neuropilar fibers and varicosities. The contents of serotonin and its precursors in the 6th AG varied along the 24-h cycle. Its maximum value was reached by midday. Topic application of serotonin to ganglia kept in darkness increased the CPR spontaneous firing rate and reduced its light responsiveness. Both effects were dose-dependent within ED(50) approximately 1 microM and were blocked by the 5-HT antagonist methysergide. These observations support the role of serotonin as a neurotransmitter or neuromodulator in the CPR of the two species of crayfish Procambarus clarkii and Cherax quadricarinatus.     

The self-tuning neuron: synaptic scaling of excitatory synapses

Homeostatic synaptic scaling is a form of synaptic plasticity that adjusts the strength of all of a neuron's excitatory synapses up or down to stabilize firing. Current evidence suggests that neurons detect changes in their own firing rates through a set of calcium-dependent sensors that then regulate receptor trafficking to increase or decrease the accumulation of glutamate receptors at synaptic sites. Additional mechanisms may allow local or network-wide changes in activity to be sensed through parallel pathways, generating a nested set of homeostatic mechanisms that operate over different temporal and spatial scales.     

Sprouting and functional regeneration of an identified serotonergic neuron following axotomy

An identified serotonergic neuron (C1) in the cerebral ganglion of Helisoma trivolvis sprouts following axotomy and rapidly (seven to eight days) regenerates to recover its regulation of feeding motor output from neurons of the buccal ganglia. The morphologies of normal and regenerated neurons C1 were compared. Intracellular injection of the fluorescent dye, Lucifer Yellow, into neuron C1 was compared with serotonin immunofluorescent staining of the cerebral and buccal ganglia. The two techniques revealed different and complimentary representations of the morphology of neuron C1. Lucifer Yellow provided optimal staining of the soma, major axon branches, and dendritic arborization. Immunocytochemical staining revealed terminal axon branches on distant targets and showed an extensive plexus of fine fibers in the sheaths of ganglia and nerve trunks. In addition to C1, serotonin-like immunoreactivity was localized in approximately 30 other neurons in each of the paired cerebral ganglia. Only cerebral neurons C1 had axons projecting to the buccal ganglia. No neuronal somata in the buccal ganglia displayed serotonin-like immunoreactivity. Observations of regenerating neurons C1 demonstrated: Actively growing neurites, both in situ and in cell culture, displayed serotonin-like immunoreactivity; severed distal axons of C1 retained serotonin-like immunoreactivity for up to 28 days; axotomized neurons C1 regenerated to restore functional control over the feeding motor program.     

Further evidence for a possible excitatory serotonergic synapse on raphe neurons of pons and lower midbrain

Iontophoretic techniques were employed to investigate the response of neurons in the nucleus raphe pontis and median raphe nucleus to 5HT. Neurons were identified by response to stimulation of nucleus paragigantocellularis lateralis (NPL) as driven (D), inhibited (I) or non-responsive (NR) cells. Of 35 D cells tested, 33 were excited by 5HT and 2 inhibited. Of 10 I cells tested, 7 were inhibited by 5HT and 3 excited. Of 56 NR cells tested, 5HT excited 30, inhibited 23 and produced no response for 3. Combining this and previous work, 100 D cells have been tested of which 97 were excited by 5HT. This 97% excitatory response of D cells to 5HT provides a strong suggestion that the NPL-to-raphe projection represents an excitatory serotonergic pathway in the central nervous system.     

Afferent modulation of dopamine neuron firing patterns

In recent studies examining the modulation of dopamine (DA) cell firing patterns, particular emphasis has been placed on excitatory afferents from the prefrontal cortex and the subthalamic nucleus. A number of inconsistencies in recently published reports, however, do not support the contention that tonic activation of NMDA receptors is the sole determinate of DA neuronal firing patterns. The results of work on the basic mechanism of DA firing and the action of apamin suggest that excitatory projections to DA neurons from cholinergic and glutamatergic neurons in the tegmental pedunculopontine nucleus, and/or inhibitory GABAergic projections, are also involved in modulating DA neuron firing behavior.     

Molecular mechanism of modulation of nociceptive neuron membrane excitability by a tripeptide

Using the whole-cell patch-clamp method, the ability of arginine-containing tripeptide Ac-RER-NH₂, dipeptide Ac-RR-NH₂, and free Arg molecule to modulate the membrane excitability of nociceptors was studied. Extracellular Ac-RER-NH₂ upon interaction with the outer membrane of the nociceptive neuron decreases the Z_eff value of the activation gating system of Na_v1.8 channels. Thus, the tripeptide Ac-RER-NH₂ can be considered as a new effective and safe analgesic.     

Antioxidants induce different phenotypes by a distinct modulation of signal transduction

Antioxidants are known to exert a preventive activity against degenerative diseases. Here, we investigated the mechanism of action of three antioxidants: resveratrol, which causes differentiation of HL-60 cells, and hydroxytyrosol and pyrrolidine dithiocarbamate which, in the same model system, activate apoptosis. The expression profile of hydroxytyrosol-treated cells showed the up-regulation of several genes, including c-jun and egr1. Pyrrolidine dithiocarbamate activates both genes, while resveratrol increases uniquely egr1. A selective modulation of signalling pathway explained this finding. All antioxidants up-regulate Erk1/2, while only hydroxytyrosol and pyrrolidine dithiocarbamate activate c-Jun N-terminal kinase (JNK). Since JNK induces apoptosis by Bcl-2 phosphorylation, we investigated this event. Bcl-2 phosphorylation was increased by hydroxytyrosol and pyrrolidine dithiocarbamate and not by resveratrol. Our results indicate that the different phenotypical effects of antioxidants correlate with modulation of selective transduction pathways.     

Mechanism of gain modulation at single neuron and network levels

Gain modulation, in which the sensitivity of a neural response to one input is modified by a second input, is studied at single-neuron and network levels. At the single neuron level, gain modulation can arise if the two inputs are subject to a direct multiplicative interaction. Alternatively, these inputs can be summed in a linear manner by the neuron and gain modulation can arise, instead, from a nonlinear input–output relationship. We derive a mathematical constraint that can distinguish these two mechanisms even though they can look very similar, provided sufficient data of the appropriate type are available. Previously, it has been shown in coordinate transformation studies that artificial neurons with sigmoid transfer functions can acquire a nonlinear additive form of gain modulation through learning-driven adjustment of synaptic weights. We use the constraint derived for single-neuron studies to compare responses in this network with those of another network model based on a biologically inspired transfer function that can support approximately multiplicative interactions.     

Extrasynaptic release of dopamine in a retinal neuron: activity dependence and transmitter modulation

Extrasynaptic release of dopamine is well documented, but its relation to the physiological activity of the neuron is unclear. Here we show that in absence of presynaptic active zones, solitary cell bodies of retinal dopaminergic neurons release by exocytosis packets of approximately 40,000 molecules of dopamine at irregular intervals and low frequency. The release is triggered by the action potentials that the neurons generate in a rhythmic fashion upon removal of all synaptic influences and therefore depends upon the electrical events at the neuronal surface. Furthermore, it is stimulated by kainate and abolished by GABA and quinpirole, an agonist at the D(2) dopamine receptor. Since the somatic receptors for these ligands are extrasynaptic, we suggest that the composition of the extracellular fluid directly modulates extrasynaptic release.     

Long-lasting potentiation of excitatory synaptic signaling to the crayfish lateral giant neuron

The neural circuit that underlies the lateral giant fiber (LG)-mediated reflex escape in crayfish has provided findings relating synaptic change to nonassociative learning such as sensitization and habituation. The LGs receive sensory inputs from the primary sensory afferents and a group of mechanosensory interneurons (MSIs). An increase of excitability by suprathreshold repetitive excitation of this circuit, which is similar to Hebbian long-term potentiation (LTP), has been reported [Miller et al. (1987) J Neurosci 7:1081]. This potentiation was previously thought to result from the enhancement of transmission at cholinergic synapses between primary afferents and MSIs but not the electrical synapses onto LG. In this study, we found that potentiation of synaptic signaling at the electrical synapse onto LG can also be induced when the synapse was activated with subthreshold repetitive pulses or with a few strong suprathreshold shocks. LG LTP was induced in the preparation which had received pulses at limited frequency range. Although whether this LTP is involved in the learning process of escape behavior in crayfish is not clear, the intensity and amount of sensory stimulation used here mimicked those that could easily be produced by a predator trying to catch a crayfish and could be of adaptive significance in life.     

Unusual biochemistry of changes in neuron membrane permeability evoked by cAMP

Influence of different metabolic poisons on cAMP-evoked neuron membrane permeability is investigated. Drugs preventing cAMP binding with R subunits of protein kinase decrease the cAMP-evoked current, but the inhibitor of the C subunit. H8, has no effect. The cAMP-dependent current is increased by uncouplers and decreased by inhibitors of glycolysis and oxidative phosphorylation. The mechanism of cAMP action on neuron permeability is discussed.     

Electrical current and cAMP induce lateral branching in the stolon of hydroids

Pulses of cAMP injected ionophoretically or mechanically into the epidermis of the stolon of the hydroid Hydractinia induce lateral branching at the site of stimulation. Up to 72% of the punctured loci developed a bud 6–24 hr after stimulation. Only pulsatile injection in periods of, e.g., 5 min is effective in inducing lateral buds. Controls provided evidence that in the ionophoretic mode the inducing effect derives not only from the cAMP signal but also, in part, from the positive electric current passed through the micropipet during the retention interval: DC (e.g., 8 nA × 1 hr or 20 nA × 2 hr) entering the tissue also evokes a positive response. Additional pulses of cAMP, but not of AMP, enhance the current effect. The threshold dose for a significant amplification has been determined to be 3.6 × 10^?13M (18 pulses à 2 × 10^?14M).     

Study of neuronal gain in a conductance-based leaky integrate-and-fire neuron model with balanced excitatory and inhibitory synaptic input

Neurons receive a continual stream of excitatory and inhibitory synaptic inputs. A conductance-based neuron model is used to investigate how the balanced component of this input modulates the amplitude of neuronal responses. The output spiking rate is well described by a formula involving three parameters: the mean and variance of the membrane potential and the effective membrane time constant _Q. This expression shows that, for sufficiently small _Q, the level of balanced excitatory-inhibitory input has a nonlinear modulatory effect on the neuronal gain.     

Thermal dependence of serotonergic modulation of neural activity in the hamster

1. The modulatory effect of serotonin on CA1 pyramidal cells in the hamster (Mesocricetus auratus) hippocampus was examined over a range of temperatures. 2. Following repetitive Schaffer collateral/commissural stimulation, changes in the amplitude of population spikes (the synchronous firing of CA1 pyramidal cells) were recorded in the hamster, a hibernator. Amplitudes were measured after 10 microM serotonin was added to and then withdrawn from the perfusing medium with the temperature of the bath fixed at different temperatures. 3. Between 35 degrees C and 15 degrees C a depression in population spike amplitude of at least 10% was seen in 36 of 43 trials, with an average depression of 68%. No significant temperature dependence of the depressive effect was seen. 4. Following the removal of serotonin from the perfusate, the spike amplitude was enhanced over the same range of temperatures, averaging 33% higher than control values. The enhancement was most pronounced at 35 degrees C and 15 degrees C and smallest at 25 degrees C. 5. Thus, over the entire temperature range of 35 degrees C to 15 degrees C, serotonin exerted a dual modulatory effect on the spike amplitude, a depression followed by an enhancement. Serotonin's modulatory effects on pyramidal cell excitation persist over temperatures encountered as the hamster enters hibernation.