Serotonergic neurons differentially modulate the efficacy of two motor neurons innervating the same muscle fibers in Aplysia

Feeding behavior in Aplysia shows substantial plasticity. An important site for the generation of this plasticity is the modulation of synaptic transmission between motor neurons and the buccal muscles that generate feeding movements. We have been studying this modulation in the anterior portion of intrinsic buccal muscle 3 (I3a), which is innervated by two excitatory motor neurons and identified serotonergic modulatory neurons, the metacerebral cells (MCCs). We have shown previously that serotonin (5-HT) applied selectively to the muscle potently modulates excitatory junction potentials (EJPs) and contractions. All the effects of 5-HT were persistent, lasting many hours after wash out. We examined whether the release of endogenous 5-HT from the MCC could produce effects similar to the application of 5-HT. Stimulation of the MCCs did produce similar short-term effects to the application of 5-HT. MCC stimulation facilitates EJPs, potentiates contractions, and decreases the latency between the onset of a motor neuron burst and the onset of the evoked contraction. The effects of MCC stimulation reached a maximum at quite low firing frequencies, which were in the range of those previously recorded during feeding behavior. The maximal effects were similar to those produced by superfusion with approximately 0.1 microM 5-HT. Although the effects of MCC stimulation on EJPs were persistent, they were less persistent than the effects of 0.1 microM 5-HT. Mechanisms that may account for differences in the persistence between released and superfused 5-HT are discussed. Thus activity in the MCCs has dramatic short-term effects on the behavioral output of motor neurons, increasing the amplitude and relaxation rate of contractions evoked by both B3 and B38 and shifting the temporal relationship between B38 bursts and evoked contractions.     

Identification of two phosphoproteins affected by serotonin in Aplysia sensory neurons

Although control mechanisms of cochlear blood flow (CBF) have been intensively studied since laser Doppler flowmetry was introduced for CBF measurement in animals and humans, the role of adenosine 5'-triphosphate (ATP) in CBF regulation is not known. Since ATP is a potent vasoactive agent in other organs, the aim of this study is to examine ATP-induced changes in CBF and to test whether the nitric oxide pathway is involved in ATP-induced CBF changes. The anterior inferior cerebellar artery (AICA) of anesthetized pigmented guinea pigs was exposed, and ATP was perfused into the AICA. For CBF measurement, the bulla was opened and the 0.7 mm laser probe of a Perimed PF2B flowmeter was positioned on the basal turn of the cochlea. AICA perfusion of an ATP solution caused dose-dependent transient CBF increases. The maximum CBF increase induced was 220% of the baseline. In some animals, CBF showed a dual effect; a transient decrease followed by a longer-lasting increase. The perfusions of sodium nitroprusside (SNP) also resulted in dose-dependent CBF changes. The intravenous application of N(omega)-nitro-L-arginine methyl ester (L-NAME) significantly attenuated ATP-induced CBF increases, and enhanced ATP-induced decreases, but did not affect SNP-induced CBF changes. The ATP-induced CBF responses indicate that ATP plays a role in CBF regulation. The biphasic characteristic of the ATP-induced CBF change suggests the involvement of both P2x- and P2y-subtype purinoceptors. That L-NAME caused attenuation of the ATP-induced CBF increase implies that the ATP-induced CBF increase is mediated by the release of endothelium-derived relaxing factor, nitric oxide, following activation of endothelial P2y-purinoceptors in the cochlear vascular bed and/or cochlear supplying vessels.     

Task-dependent modulation of inhibitory actions within the primary motor cortex

In 11 healthy subjects motor-evoked potentials (MEPs) and silent periods (SPs) were measured in the right first dorsal interosseus (FDI) and abductor pollicis brevis muscles (APB): (1) when transcranial magnetic cortex stimulation (TMS) was applied at tonic isometric contraction of 20% of maximum force, (2) when TMS was applied during tactile exploration of a small object in the hand, (3) when TMS was applied during visually guided goal-directed isometric ramp and hold finger flexion movements, and (4) when at tonic isometric contraction peripheral electrical stimulation (PES) of the median nerve was delivered at various intervals between PES and TMS. Of the natural motor tasks, duration of SPs of small hand muscles was longest during tactile exploration (APB 205+/-42 ms; FDI 213+/-47 ms). SP duration at tonic isometric contraction amounted to 172+/-35 ms in APB and 178+/-31 ms in FDI, respectively. SP duration in FDI was shortest when elicited during visually guided isometric finger movements (159+/-15 ms). At tonic isometric contraction, SP was shortened when PES was applied at latencies -30 to +70 ms in conjunction with TMS. The latter effect was most pronounced when PES was applied 20 ms before TMS. PES-induced effects increased with increasing stimulation strength up to a saturation level which appeared at the transition to painful stimulation strengths. Both isolated stimulation of muscle afferents and of low-threshold cutaneous afferents shortened SP duration. However, PES of the contralateral median nerve had no effect on SPs. Amplitudes of MEPs did not change significantly in any condition. Inhibitory control of motor output circuitries seems to be distinctly modulated by peripheral somatosensory and visual afferent information. We conclude that somatosensory information has privileged access to inhibitory interneuronal circuits within the primary motor cortex.     

Mutation in the phosphorylation sites of MAP kinase blocks learning-related internalization of apCAM in Aplysia sensory neurons

The synaptic growth that accompanies 5-HT-induced long-term facilitation of the sensory to motor neuron connection in Aplysia is associated with the internalization of apCAM at the surface membrane of the sensory neuron. We have now used epitope tags to examine the fate of each of the two apCAM isoforms (membrane bound and GPI-linked) and find that only the transmembrane form is internalized. This internalization can be blocked by overexpression of transmembrane constructs with a single point mutation in the two MAPK consensus sites, as well as by injection of a specific MAPK antagonist into sensory neurons. These data suggest MAPK phosphorylation at the membrane is important for the internalization of apCAMs and, thus, may represent an early regulatory step in the growth of new synaptic connections that accompanies long-term facilitation.     

Direct glutamate-mediated presynaptic inhibition of sensory afferents by the postsynaptic motor neurons

An in vitro preparation of the crayfish central nervous system was used to study a negative feedback control exerted by the glutamatergic motor neurons (MNs) on to their presynaptic cholinergic sensory afferents. This negative control consists in small amplitude, slowly developing depolarizations of the primary afferents (sdPADs) strictly timed with MN bursts. They were not blocked by picrotoxin, but were sensitive to glutamate non-N-methyl-D-aspartate (NMDA) antagonists. Intracellular recordings were performed within thin branches of sensory terminals while electrical antidromic stimulation were applied to the motor nerves, or while glutamate (the MN neurotransmitter) was pressure-applied close to the recording site. Electrical motor nerve stimulations and glutamate pressure application had similar effects on to sensory terminals issued from the coxo-basipodite chordotonal organ (CBTs): like sdPADs, both stimulation-induced depolarizations were picrotoxin-resistant and were dramatically reduced by non-NMDA antagonist bath application. These results indicate that sdPADs are likely directly produced by MNs during locomotor activity. A functional scheme is proposed.     

Distribution of inhibitory synapses on the somata of pyramidal neurons in cat motor cortex

The mechanisms by which cortical neurons perform spatial and temporal integration of synaptic inputs are dependent, in large part, on the numbers, types, and distributions of their synapses. To further our understanding of these integrative mechanisms, we examined the distribution of synapses on identified classes of cortical neurons. Pyramidal cells in the cat motor cortex projecting either to the ipsilateral somatosensory cortex or to the spinal cord were labeled by the retrograde transport of horseradish peroxidase. Entire soma of selected corticocortical and corticospinal cells were examined using serial-section electron microscopy. The profiles of these somata and the synapses formed with each of these profiles were reconstructed from each thin section with a computer-aided morphometry system. All somatic synapses were of the symmetrical, presumably inhibitory type. For both cell types, these synapses were not homogeneously distributed over the somatic membrane, but were clustered at several discrete zones. The number and density of synapses on the somata of different corticocortical and corticospinal neurons were not significantly different. However, the density of these synapses was inversely correlated with the size of their postsynaptic somata. We discuss the significance of these findings to the integrative properties of cortical neurons.     

Characterization of buccal motor programs elicited by a cholinergic agonist applied to the cerebral ganglion of Aplysia californica

Applying the non-hydrolyzable cholinergic agonist carbachol (CCh) to the cerebral ganglion of Aplysia elicits sustained, regular bursts of activity in the buccal ganglia resembling those seen during biting. The threshold for bursting is approximately 10(-4) M. Bursting begins after a 2 to 5 min delay. The burst frequency increases over the first 5 bursts, reaching a plateau value of approximately 3 per minute. Bursting is maintained for over 10 min. Some of the effects of CCh may be attributed to its ability to depolarize and fire CBI-2, a command-like neuron in the cerebral ganglion that initiates biting. CBI-2 is also depolarized by ACh, and by stimulating peripheral sensory nerves. Excitation of CBI-2 caused by carbachol is partially blocked by the muscarinic antagonist atropine. We examined whether CCh-induced bursting is modified in ganglia taken from Aplysia that previously experienced treatments inhibiting feeding, such as satiation, head shock contingent or non-contingent with food, and training animals with an inedible food. No treatment consistently and repeatedly affected the latency, the peak burst period, the length of time that bursting was maintained, or the threshold CCh concentration for eliciting bursting. However, there was a decrease in the rate of the build-up of the buccal ganglion program in previously satiated animals.     

Activation of protein kinase A contributes to the expression but not the induction of long-term hyperexcitability caused by axotomy of Aplysia sensory neurons

Nociceptive sensory neurons (SNs) in Aplysia provide useful models to study both memory and adaptive responses to nerve injury. Induction of long-term memory in many species, including Aplysia, is thought to depend on activation of cAMP-dependent protein kinase (PKA). Because Aplysia SNs display similar alterations in models of memory and after nerve injury, a plausible hypothesis is that axotomy triggers memory-like modifications by activating PKA in damaged axons. The present study disproves this hypothesis. SN axotomy was produced by (1) dissociation of somata from the ganglion [which is shown to induce long-term hyperexcitability (LTH)], (2) transection of neurites of dissociated SNs growing in vitro, or (3) peripheral nerve crush. Application of the competitive PKA inhibitor Rp-8-CPT-cAMPS at the time of axotomy failed to alter the induction of LTH by each form of axotomy, although the inhibitor antagonized hyperexcitability produced by 5-HT application. Strong activation of PKA in the nerve by coapplication of a membrane-permeant analog of cAMP and a phosphodiesterase inhibitor was not sufficient to induce LTH of either the SN somata or axons. Furthermore, nerve crush failed to activate axonal PKA or stimulate its retrograde transport. Therefore, PKA activation plays little if any role in the induction of LTH by axotomy. However, the expression of LTH was reduced by intracellular injection of the highly specific PKA inhibitor PKI several days after nerve crush. This suggests that long-lasting activation of PKA in or near the soma contributes to the maintenance of long-term modifications produced by nerve injury.     

Multiple subtypes of serotonin receptors are expressed in rat sensory neurons in culture

[3H]5-HT revealed the presence of serotonin receptors in cultured rat sensory neurons. [3H]5-CT binding was inhibited by cyanopindolol with an IC50 of 0.87 +/- 0.30 nM, suggesting the expression of the 5-HT1B receptor in these neurons. The presence of 5-HT1B receptors was confirmed by the displacement of [125I]Iodocyanopindolol binding by cyanopindolol with an IC50 of 2.43 +/- 0.81 nM. 5-HT1B receptors are the predominant type of serotonin receptors labeled by [3H]5-HT in cultured DRG neurons, representing approximately 60% of the specific [3H]5-HT binding sites. In addition, 5-HT1D and 5-HT2A receptor binding was also found in these neurons. RT-PCR analysis of RNA isolated from embryonic sensory neurons in culture confirmed the expression of 5-HT1B, 5-HT1D and 5-HT2A receptor mRNA. It also demonstrated the presence of 5-HT1F, 5-HT2C, 5-HT3, 5-HT4, 5-HT5A and 5-HT5B receptor mRNA and the absence of 5-HT1A, 5-HT1E, 5-HT2B, 5-HT6 and 5-HT7 mRNA. The identification of multiple subtypes of serotonin receptors expressed in cultured embryonic sensory neurons suggests that DRG neuronal cultures may be an excellent model to examine the direct effects of serotonin on the activity of these sensory neurons.     

Development of the histaminergic neurons and expression of histidine decarboxylase mRNA in the zebrafish brain in the absence of all peripheral histaminergic systems

The histamine-storing neural system in adult and developing zebrafish (Danio rerio) was studied with immunocytochemical and chromatographical methods. Furthermore, the gene for histidine decarboxylase was partially cloned and its expression mapped with in situ hybridization. The histamine-storing neurons were only seen in the caudal hypothalamus, around the posterior recess of the diencephalic ventricle. Almost all parts of the brain, except the cerebellum, contained at least some histamine-immunoreactive fibres. The ascending projections had the rostral part of the dorsal telencephalon as a major target. Descending projections terminated in the torus semicircularis, central grey and inferior olive. A prominent innervation of the optic tectum, which has not been reported in other fish, was seen. The in situ hybridization gave a strong signal in cells with the same anatomical position as the histamine-immunoreactive neurons. The first histamine-immunoreactive neurons appeared in the ventral hypothalamus at about 85 h post-fertilization, and at 90 h, immunoreactive fibres terminated in the dorsal telencephalon. The embryonic histamine production described in mammals was lacking in this species. Both immunocytochemical and chromatographical studies indicated that histamine is absent in all other parts of the zebrafish body, and no specific hybridization was seen in any other part of the fish than the hypothalamus. The zebrafish could therefore be a very useful model for pharmacological in vivo studies of the histaminergic system of the brain, since the powerful peripheral actions of histamine should be lacking in this species.     

Neuronal injury increases retrograde axonal transport of the neurotrophins to spinal sensory neurons and motor neurons via multiple receptor mechanisms

We investigated the retrograde axonal transport of 125I-labeled neurotrophins (NGF, BDNF, NT-3, and NT-4) from the sciatic nerve to dorsal root ganglion (DRG) sensory neurons and spinal motor neurons in normal rats or after neuronal injury. DRG neurons showed increased transport of all neurotrophins following crush injury to the sciatic nerve. This was maximal 1 day after sciatic nerve crush and returned to control levels after 7 days. 125I-BDNF transport from sciatic nerve was elevated with injection either proximal to the lesion or directly into the crush site and after transection of the dorsal roots. All neurotrophin transport was receptor-mediated and consistent with neurotrophin binding to the low-affinity neurotrophin receptor (LNR) or Trk receptors. However, transport of 125I-labeled wheat germ agglutinin also increased 1 day after sciatic nerve crush, showing that increased uptake and transport is a generalized response to injury in DRG sensory neurons. Spinal cord motor neurons also showed increased neurotrophin transport following sciatic nerve injury, although this was maximal after 3 days. The transport of 125I-NGF depended on the expression of LNR by injured motor neurons, as demonstrated by competition experiments with unlabeled neurotrophins. The absence of TrkA in normal motor neurons or after axotomy was confirmed by immunostaining and in situ hybridization. Thus, increased transport of neurotrophic factors after neuronal injury is due to multiple receptor-mediated mechanisms including general increases in axonal transport capacity.     

The morphology of identified neurons in the abdominal ganglion of Aplysia californica

The morphology of identified neurons and of one multiaction interneuron (L10) of the abdominal ganglion of Aplysia has been studied using cobalt chloride, injected intracellularly. Cells with little synaptic input, R3-R14, had a relatively poorly developed dendritic tree, whereas the dendrite tree of cells L7 and L10, with extensive synaptic input, was highly complex. Cells L1-L6 and the RB cell cluster were found to have intermediate complexity of synaptic inputs and dendritic morphology. Within cell clusters, individual cells were often morphologically distinct. Identified cells have both invariant and variant axonal branches. Variant axons often project down other than their customary nerve trunks or are supernumerary. Three features of neuropil architecture were encountered. (1) When cells from the same cluster send their axons down the same nerve the axons often run in fascicles. (2) Although an identified cell's dendritic geometry varies from preparation to preparation, its dendrites always occupy approximately the same position in the neuropil. (3) The postsynaptic follower cells of L10 send their main axons through the axonal arborization of L10.     

Marrow-derived activated macrophages are required during the effector phase of experimental autoimmune uveoretinitis in rats

PURPOSE: Experimental autoimmune uveoretinitis (EAU), an established model for human endogenous (autoimmune) posterior uveitis, is a CD4+ T cell-mediated disease inducible in Lewis rats by intradermal inoculation with retinal antigens. Immunohistochemical studies have previously documented the lymphocyte profiles during various stages of the disease process. The purpose of the present study was to investigate the role of macrophages in EAU. METHODS: EAU was induced in Lewis rats, and the effect of macrophage depletion, using the drug dichlorodimethylene diphosphonate (Cl2MDP) encapsulated in liposomes and administered intravenously, was assessed based on the clinical and histological profile of the disease. RESULTS: The results have shown that in control animals macrophages occur early, feature prominently throughout the course of the disease and display considerable heterogeneity: marrow-derived ED1+ cells and ED3+ cells are the major infiltrating cells, with many cells also expressing ED7 and ED8. In contrast, few cells expressed the ED2 antigen during EAU, even though ED2+ "resident" macrophages occur in the normal choroid. Macrophage depletion, using intravenously injected dichloromethylene diphosphonate (Cl2MDP) enclosed in liposomes, caused a delay in the onset and a reduction in the severity of EAU when administered during the "effector" stage of the disease, i.e. 9-11 days after inoculation with retinal antigen. The delay in disease onset was greater when liposomes were mannosylated and was accompanied by a reduction in the overall inflammatory cell infiltrate into the eye and reduced tissue damage. In addition, there was a reduction in the level of expression of MHC Class II antigen and CR3 (ED7) antigen, a marker of macrophage activation, in Cl2MDP-treated animals compared to controls. CONCLUSION: These results suggest that blood-borne, activated macrophages are major effectors of tissue damage during EAU.     

Interactions of histaminergic and serotonergic neurons in the hypothalamic regulation of prolactin and ACTH secretion

Serotonergic and histaminergic neuronal systems are both involved in mediation of the stress-induced release of the pituitary hormones prolactin (PRL) and ACTH. We investigated the possibility of an interaction between serotonin (5-HT) and histamine (HA) in regulation of PRL and ACTH secretion in conscious male rats. Animals were pretreated systemically with antagonists to 5-HT1, 5-HT2 or 5-HT3 receptors prior to intracerebroventricular (icv) administration of HA. The 5-HT1 + 2 receptor antagonist methysergide prevented and the 5-HT2 receptor antagonist LY 53857 attenuated the HA-induced PRL release while the 5-HT3 receptor antagonist ondansetron had no effect on this response. None of the three 5-HT receptor antagonists affected the ACTH response to HA. Specific blockade of HA synthesis by alpha-fluoromethylhistidine or blockade of postsynaptic HA receptors by icv infusion of the H1 receptor antagonist mepyramine or the H2 receptor antagonist cimetidine inhibited the PRL response to 5-HT or to the 5-HT precursor 5-hydroxytryptophan (5- HTP) given in combination with the 5-HT reuptake inhibitor fluoxetine (Flx). Blockade of the histaminergic system had no effect on the ACTH response to serotonergic stimulation. The H3 receptors are inhibitory HA receptors. Systemic pretreatment with the H3 receptor agonist R(alpha)methylhistamine, or the H3 receptor antagonist thioperamide had no effect on the hormone response to activation of the serotonergic system by 5-HTP plus Flx. We conclude that the serotonergic and histaminergic neuronal systems interact in their stimulation of PRL secretion, but not in their stimulation of ACTH secretion. This interaction involves serotonergic 5-HT1 and 5-HT2 receptors and histaminergic H1 and H2 receptors. Furthermore, the previously observed inhibitory effect of the H3 receptor agonist R(alpha)methylhistamine on stress-induced PRL and ACTH release seems not to be exerted by activation of presynaptic H3 receptors located on serotonergic neurons but rather on histaminergic neurons.     

A simplified preparation for relating cellular events to behavior: contribution of LE and unidentified siphon sensory neurons to mediation and habituation of the Aplysia gill- and siphon-withdrawal reflex

We have begun to analyze several elementary forms of learning in a simple preparation consisting of the isolated mantle organs and abdominal ganglion of Aplysia. Previous studies suggested that plasticity at siphon sensory neuron synapses contributes to habituation and dishabituation of the gill- and siphon-withdrawal reflex in this preparation. We next wished to identify the sensory neurons that participate in the reflex and examine their plasticity more directly. To investigate the contribution of the LE siphon mechanosensory cells, we recorded from them and gill or siphon motor neurons during the same siphon stimulation that has been used in behavioral experiments in this preparation. Our results indicate that the LE cells make a substantial contribution to the evoked response in the motor neurons under these conditions, but they suggest that other as yet unidentified siphon sensory neurons with lower thresholds and shorter latencies also contribute. In addition, we find that homosynaptic depression of monosynaptic postsynaptic potentials (PSPs) from LE sensory cells makes an important contribution to habituation of the response in the motor neurons. To investigate plasticity of PSPs from the unidentified sensory neurons, we recorded the PSP that was produced in a motor neuron by water-movement stimulation of the siphon, which does not cause firing of LE cells. Our results suggest that PSPs from the unidentified sensory neurons and the LE neurons undergo similar plasticity during habituation and dishabituation training. These results support the idea that plasticity at synapses of both LE and unidentified sensory neurons contributes to habituation and dishabituation of the reflex response in this preparation.