Correlation between activity in neuron B52 and two features of fictive feeding in Aplysia

Intracerebroventricular administration of N-acetyl-L-aspartyl-L-glutamate (NAAG), an agonist at group II metabotropic and NR1/NR2D-containing N-methyl-D-aspartate (NMDA) ionotropic glutamate receptors, increased the permeability of the blood-brain barrier (BBB) to serum albumin in the striatum, but produced no similar effects in the entorhinal cortex or in the hippocampal formation. Electron microscopy showed that NAAG, but not its hydrolytic products L-glutamate and N-acetyl-L-aspartate, increased the number of transport vesicles in the hippocampal endothelial cells. Furthermore, immunocytochemistry detected NR2D subunits on hippocampal capillaries. Consequently, NAAG may have influenced the vesicular transport via NMDA receptors. There was, however, no correlation with the regional pattern of BBB changes (increased permeability in the striatum) that, in turn, could not be directly related to the NAAG-induced neurodegeneration described previously in the hippocampus where no significant changes in BBB permeability were detected.     

Activity-dependent synaptic capture of transiting peptidergic vesicles

Synapses require resources synthesized in the neuronal soma, but there are no known mechanisms to overcome delays associated with the synthesis and axonal transport of new proteins generated in response to activity, or to direct resources specifically to active synapses. Here, in vivo imaging of the Drosophila melanogaster neuromuscular junction reveals a cell-biological strategy that addresses these constraints. Peptidergic vesicles continually transit through resting terminals, but retrograde peptidergic vesicle flux is accessed following activity to rapidly boost neuropeptide content in synaptic boutons. The presence of excess transiting vesicles implies that synaptic neuropeptide stores are limited by the capture of peptidergic vesicles at the terminal, rather than by synthesis in the soma or delivery via the axon. Furthermore, activity-dependent capture from a pool of transiting vesicles provides a nerve terminal-based mechanism for directing distally and slowly generated resources quickly to active synapses. Finally, retrograde transport in the nerve terminal is regulated by activity.     

Diverse synaptic connections between peptidergic radula mechanoafferent neurons and neurons in the feeding system of Aplysia

The buccal ganglion of Aplysia contains a heterogeneous population of peptidergic, radula mechanoafferent (RM) neurons. To investigate their function, two of the larger RM cells (B21, B22) were identified by morphological and electrophysiological criteria. Both are low-threshold, rapidly adapting, mechanoafferent neurons that responded to touch of the radula, the structure that grasps food during ingestive and egestive feeding movements. Sensory responses of the cells consisted of spike bursts at frequencies of 8-35 Hz. Each cell was found to make chemical, electrical, or combined synapses with other sensory neurons, motor neurons and interneurons involved in radula closure and/or protraction-retraction movements of the odontophore. Motor neurons receiving input included the following: B8a/b, B15, and B16, which innervate muscles contributing to radula closing; and B82, a newly identified neuron that innervates the anterodorsal region of the I1/I3 muscles of the buccal mass. B21 and B22 can affect buccal motor programs by way of their connections to interneurons such as B19 and B64. Fast, chemical, excitatory postsynaptic potentials (EPSPs) produced by RM neurons, such as B21, exhibited strong, frequency-dependent facilitation, a form of homosynaptic plasticity. Firing B21 also produced a slow EPSP in B15 that increased the excitability of the cell. Thus a sensory neuron mediating a behavioral response may have modulatory effects. The data suggest multiple functions for RM neurons including 1) triggering of phase transitions in rhythmic motor programs, 2) adjusting the force of radula closure, 3) switching from biting to swallowing or swallowing to rejection, and 4) enhancing food-induced arousal.     

Activity-dependent neuromodulation in Aplysia neuron R15: intracellular calcium antagonizes neurotransmitter responses mediated by cAMP

1. The effect of electrical activity on the response to the neuromodulators serotonin (5-HT) and the neuropeptide egg-laying hormone (ELH) was studied in the Aplysia bursting pacemaker neuron R15. 2. Previous work has shown that 5-HT and ELH augment R15s bursting activity by enhancing two ionic currents, an inwardly rectifying K+ current (IR) and a voltage-gated Ca2+ current (ICa), and that the enhancement of the currents is mediated by the intracellular second-messenger adenosine 3',5'-cyclic monophosphate (cAMP). Here we show that both spontaneous action potentials and voltage-clamp depolarizations suppress the modulation by 5-HT and ELH of these currents. Both spontaneous and evoked depolarizations decrease the magnitude and dramatically speed the decay of the modulation of IR and ICa. 3. The depolarization-induced suppression is blocked by intracellular ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N',-tetraacetic acid (EGTA), indicating that the suppression is Ca-dependent. The suppression is specific for responses mediated by cAMP; a non-cyclic AMP-mediated response to acetylcholine is not affected by depolarizing pulses. 4. The Ca-dependent suppression of IR modulation differs from the Ca-dependent suppression of ICa modulation. Ca2+ influx decreases the sensitivity of IR to neuromodulators without reducing the maximal response elicited by high concentrations of neuromodulators. In contrast, Ca2+ not only decreases the sensitivity of ICa but also reduces the maximal effect elicited by high concentrations of neuromodulators. We have shown previously that intracellular Ca2+ also inactivates the basal IR and ICa in neuron R15 by distinct mechanisms. The inactivation of IR is due to an antagonistic action of Ca2+ on cAMP metabolism, whereas the inactivation of the basal ICa is due primarily to a more direct action of Ca2+, perhaps on the Ca channels themselves. 5. We also studied the interaction between action potentials and neuromodulator released onto R15 from an endogenous source: bag cell neurons, which release large amounts of ELH during an intense "afterdischarge." IR and ICa become greatly enhanced during the afterdischarge, even though R15 continually fires action potentials. In addition, Ca-dependent inactivation of IR is suppressed during the afterdischarge. We suggest that the bag cells release an amount of ELH sufficient to temporarily saturate the cAMP-mediated enhancement of IR and that this temporarily prevents the suppressive effects of Ca2+ on IR. 6. The activity-dependent suppression of neuromodulation in neuron R15 is an example of neuronal plasticity that results from interactions between intracellular messengers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Central peptidergic neurons regulate gut motility in Aplysia

1. The small cardioactive peptides (SCPs) are potent modulatory neuropeptides in Aplysia. Buccal ganglia neurons B1 and B2 are the largest neurons that exhibit SCP-like immunoreactivity. High-pressure liquid chromatography (HPLC)-bioassay and in vivo radiolabeling procedures confirm that these neurons contain and synthesize very large quantities of SCPA and SCPB. 2. Both B1 and B2 innervate the gut. HPLC-bioassay measurements indicate that the SCPs are present throughout the anterior sections of the gut. SCP-like immunoreactivity was largely confined to fibers and varicosities in the gut, although occasional immunoreactive enteric neurons were also observed. The purpose of this study was to determine the physiological roles of B1 and B2 and to what extent these roles are mediated by release of the SCPs. 3. Low-frequency tonic stimulation of B1 led to an increase in peristaltic contractions in a relatively distal portion of the gut. This action could be mimicked by superfusion of the same portion of the gut with very low concentrations of the SCPs. 4. B2 produced discrete contractions of the anterior portions of the gut only when fired in bursts. These actions could not be reproduced by superfusion with the SCPs and may be mediated by ACh. 5. B1 and/or B2 are active during the swallowing cycle of each feeding movement, which suggests that these effects on the gut are likely to occur during feeding. Thus the SCPs play a major role in the central regulation of gut motility.     

Cerebral CBM1 neuron contributes to synaptic modulation appearing during rejection of seaweed in Aplysia kurodai

The Japanese species Aplysia kurodai feeds well on Ulva but rejects Gelidium with distinctive rhythmic patterned movements of the jaws and radula. We have previously shown that the patterned jaw movements during the rejection of Gelidium might be caused by long-lasting suppression of the monosynaptic transmission from the multiaction MA neurons to the jaw-closing (JC) motor neurons in the buccal ganglia and that the modulation might be directly produced by some cerebral neurons. In the present paper, we have identified a pair of catecholaminergic neurons (CBM1) in bilateral cerebral M clusters. The CBM1, probably equivalent to CBI-1 in A. californica, simultaneously produced monosynaptic excitatory postsynaptic potentials (EPSPs) in the MA and JC neurons. Firing of the CBM1 reduced the size of the inhibitory postsynaptic currents (IPSCs) in the JC neuron, evoked by the MA spikes, for >100 s. Moreover, the application of dopamine mimicked the CBM1 modulatory effects and pretreatment with a D1 antagonist, SCH23390, blocked the modulatory effects induced by dopamine. It could also largely block the modulatory effects induced by the CBM1 firing. These results suggest that the CBM1 may directly modulate the synaptic transmission by releasing dopamine. Moreover, we explored the CBM1 spike activity induced by taste stimulation of the animal lips with seaweed extracts by the use of calcium imaging. The calcium-sensitive dye, Calcium Green-1, was iontophoretically loaded into a cell body of the CBM1 using a microelectrode. Application of either Ulva or Gelidium extract to the lips increased the fluorescence intensity, but the Gelidium extract always induced a larger change in fluorescence compared with the Ulva extract, although the solution used induced the maximum spike responses of the CBM1 for each of the seaweed extracts. When the firing frequency of the CBM1 activity after taste stimulation was estimated, the Gelidium extract induced a spike activity of ~30 spikes/s while the Ulva extract induced an activity of ~20 spikes/s, consistent with the effective firing frequency (>25 spikes/s) for the synaptic modulation. These results suggest that the CBM1 may be one of the cerebral neurons contributing to the modulation of the basic feeding circuits for rejection induced by the taste of seaweeds such as Gelidium.     

Simulation of a photosensitive Aplysia neuron

A physically-based model has been developed for certain photoresponsive cells in the Aplysia californica central ganglia. The R₂ giant neuron and the ventral photoresponsive neuron hyperpolarize when illuminated, due to an increase of membrane permeability to potassium. We hypothesize that light releases an internal transmitter from cytoplasmic granules. Three model compartments parallel cellular morphology: the first represents the granule component with bulk-limited diffusion; the second corresponds to cytoplasm and involves simple diffusion; and the third compartment, near the membrane, relates membrane conductance of potassium to the transmitter concentration adjacent to the membrane.     

Interneuronal and peptidergic control of motor pattern switching in Aplysia.

It has been proposed that a choice of specific behaviors can be mediated either by activation of behavior-specific higher order neurons or by distinct combinations of such neurons in different behaviors. We examined the role that two higher order neurons, CBI-2 and CBI-3, play in the selection of motor programs that correspond to ingestion and egestion, two stimulus-dependent behaviors that are generated by a single central pattern generator (CPG) of Aplysia. We found that CBI-2 could evoke either ingestive, egestive, or ambiguous motor programs depending on the regime of stimulation. When CBI-2 recruited CBI-3 firing via electrical coupling, the motor program tended to be ingestive. In the absence of CBI-3 activation, the program was usually egestive. When CBI-2 was stimulated to produce ingestive programs, hyperpolarization of CBI-3 converted the programs to egestive or ambiguous. When CBI-2 was stimulated to produce egestive or ambiguous programs, co-stimulation of CBI-3 converted them into ingestive. These findings are consistent with the idea that combinatorial commands are responsible for the choice of specific behaviors. Additional support for this view comes from the observations that appropriate stimulus conditions exist both for activation of CBI-2 together with CBI-3, and for activation of CBI-2 without a concomitant activation of CBI-3. The ability of CBI-3 to convert egestive and ambiguous programs into ingestive ones was mimicked by application of APGWamide, a neuropeptide that we have detected in CBI-3 by immunostaining. Thus combinatorial actions of higher order neurons that underlie pattern selection may involve the use of modulators released by specific higher order neurons.     

Endogenous motor neuron properties contribute to a program-specific phase of activity in the multifunctional feeding central pattern generator of Aplysia

Multifunctional central pattern generators (CPGs) are circuits of neurons that can generate manifold actions from a single effector system. This study examined a bilateral pair of pharyngeal motor neurons, designated B67, that participate in the multifunctional feeding network of Aplysia californica. Fictive buccal motor programs (BMPs) were elicited with four distinct stimulus paradigms to assess the activity of B67 during ingestive versus egestive patterns. In both classes of programs, B67 fired during the phase of radula protraction and received a potent inhibitory postsynaptic potential (IPSP) during fictive radula retraction. When programs were ingestive, the retraction phase IPSP exhibited a depolarizing sag and was followed by a postinhibitory rebound (PIR) that could generate a postretraction phase of impulse activity. When programs were egestive, the depolarizing sag potential and PIR were both diminished or were not present. Examination of the membrane properties of B67 disclosed a cesium-sensitive depolarizing sag, a corresponding I(h)-like current, and PIR in its responses to hyperpolarizing pulses. Direct IPSPs originating from the influential CPG retraction phase interneuron B64 were also found to activate the sag potential and PIR of B67. Dopamine, a modulator that can promote ingestive behavior in this system, enhanced the sag potential, I(h)-like current, and PIR of B67. Finally, a pharyngeal muscle contraction followed the radula retraction phase of ingestive, but not egestive motor patterns. It is proposed that regulation of the intrinsic properties of this motor neuron can contribute to generating a program-specific phase of motor activity.     

Multiple contributions of an input-representing neuron to the dynamics of the aplysia feeding network

In Aplysia, mutually antagonistic ingestive and egestive behaviors are produced by the same multifunctional central pattern generator (CPG) circuit. Interestingly, higher-order inputs that activate the CPG do not directly specify whether the resulting motor program is ingestive or egestive because the slow dynamics of the network intervene. One input, the commandlike cerebral-buccal interneuron 2 (CBI-2), slowly drives the motor output toward ingestion, whereas another input, the esophageal nerve (EN), drives the motor output toward egestion. When the input is switched from EN to CBI-2, the motor output does not switch immediately and remains egestive. Here, we investigated how these slow dynamics are implemented on the interneuronal level. We found that activity of two CPG interneurons, B20 and B40, tracked the motor output regardless of the input, whereas activity of another CPG interneuron, B65, tracked the input regardless of the motor output. Furthermore, we show that the slow dynamics of the network are implemented, at least in part, in the slow dynamics of the interaction between the input-representing and the output-representing neurons. We conclude that 1) a population of CPG interneurons, recruited during a particular motor program, simultaneously encodes both the input that is used to elicit the motor program and the output elicited by this input; and 2) activity of the input-representing neurons may serve to bias the future motor programs.     

Activity-dependent modulation: a non-linearity in the unilateral vestibulo-ocular reflex pathways

It is well established that the vestibulo-ocular reflex (VOR) depends not only on sensory stimulation but also on the behavioral context associated with the stimulation. Recent modeling studies suggested that including a non-linearity in the activation function of the VOR neurons achieves the desired context-dependence for the VOR without resorting to currently assumed complex cortical computations. With the non-linearity, neurons operate as non-linear summers of incoming activity with sensitivities modulated by their activation levels. In this study we examined whether such a non-linearity exists in the unilateral VOR pathways in behaving monkeys. Acoustic clicks were employed to evoke unilateral VOR responses during fixation, head motion and smooth pursuit. We found that the click-evoked unilateral VOR responses did not simply sum in a linear manner with the eye movements initiated by head or target motion. Instead, the same acoustic click evoked larger eye movements if the ongoing eye movements were in the same direction. We also showed that the interaction between the ongoing eye movement and the click-evoked response was close to being multiplicative. These results revealed a previous unknown non-linearity in the unilateral VOR pathways, which may have important implications on the neural implementation of the context-dependence for the VOR.     

Synaptic stimulation of Aplysia peptidergic neurons can activate hormone secretion in the absence of an afterdischarge

For over 20 years, the bag cell neurons of the marine mollusk Aplysia have been used to investigate second-messenger pathways that mediate effects of synaptic stimulation on ion currents and membrane excitability, presumably leading to exocytotic release of the neuropeptide egg-laying hormone (ELH). It is widely cited that a train of action potentials, called an afterdischarge, is necessary for activating cellular events leading to ELH secretion. Using a combination of electrophysiology, optical imaging of calcium signaling, and radioimmunoassay of ELH secretion, we show that an afterdischarge is not required for ELH secretion. Electrical stimulation that failed to produce afterdischarges but did lead to prolonged membrane depolarization and a rise in intracellular calcium concentration was sufficient to stimulate significant ELH release.     

Presynaptic and interactive peptidergic modulation of reticulospinal synaptic inputs in the lamprey

The modulatory effects of neuropeptides on descending inputs to the spinal cord have been examined by making paired recordings from reticulospinal axons and spinal neurons in the lamprey. Four peptides were examined; peptide YY (PYY) and cholecystokinin (CCK), which are contained in brain stem reticulospinal neurons, and calcitonin-gene-related peptide (CGRP) and neuropeptide Y (NPY), which are contained in primary afferents and sensory interneurons, respectively. Each of the peptides reduced the amplitude of monosynaptic reticulospinal-evoked excitatory postsynaptic potentials (EPSPs). The modulation appeared to be presynaptic, because postsynaptic input resistance and membrane potential, the amplitude of the electrical component of the EPSP, postsynaptic responses to glutamate, and spontaneous miniature EPSP amplitudes were unaffected. In addition, none of the peptides affected the pattern of N-methyl-D-aspartate (NMDA)-evoked locomotor activity in the isolated spinal cord. Potential interactions between the peptides were also examined. The "brain stem peptides" CCK and PYY had additive inhibitory effects on reticulospinal inputs, as did the "sensory peptides" CGRP and NPY. Brain stem peptides also had additive inhibitory effects when applied with sensory peptides. However, sensory peptides increased or failed to affect the amplitude of reticulospinal inputs in the presence of the brain stem peptides. These interactive effects also appear to be mediated presynaptically. The functional consequence of the peptidergic modulation was investigated by examining spinal ventral root responses elicited by brain stem stimulation. CCK and CGRP both reduced ventral root responses, although in interaction both increased the response. These results thus suggest that neuropeptides presynaptically influence the descending activation of spinal locomotor networks, and that they can have additive or novel interactive effects depending on the peptides examined and the order of their application.     

Frequency-dependent regulation of afferent transmission in the feeding circuitry of Aplysia

During rhythmic behaviors, sensori-motor transmission is often regulated so that there are phasic changes in afferent input to follower neurons. We study this type of regulation in the feeding circuit of Aplysia. We characterize effects of the B4/5 interneurons on transmission from the mechanoafferent B21 to the radula closer motor neuron B8. In quiescent preparations, B4/5-induced postsynaptic potentials (PSPs) can block spike propagation in the lateral process of B21 and inhibit afferent transmission. B4/5 are, however, active during the retraction phase of motor programs, i.e., when mechanoafferent transmission to B8 presumably occurs. To determine whether mechanoafferent transmission is necessarily inhibited when B4/5 are active, we characterize the B4/5 firing frequency during retraction and show that, for the most part, it is low (below 15 Hz). There is, therefore, a low probability that spike propagation will be inhibited. The relative ineffectiveness of low frequency activity is not simply a consequence of insufficient PSP magnitude, because a single PSP can block spike propagation. Instead, it is related to the fact that PSPs have a short duration. When B4/5 fire at a low frequency, there is therefore a low probability that afferent transmission in the lateral process of B21 can be inhibited. In conclusion, we demonstrate that afferent transmission will not always be affected when a neuron that exerts inhibitory effects is active. Although a cell may be ineffective when it fires at a low frequency, ineffectiveness is not necessarily a consequence of spike frequency per se. Instead it may be due to spike timing.     

Cycle-to-cycle variability of neuromuscular activity in Aplysia feeding behavior

Aplysia consummatory feeding behavior, a rhythmic cycling of biting, swallowing, and rejection movements, is often said to be stereotyped. Yet closer examination shows that cycles of the behavior are very variable. Here we have quantified and analyzed the variability at several complementary levels in the neuromuscular system. In reduced preparations, we recorded the motor programs produced by the central pattern generator, firing of the motor neurons B15 and B16, and contractions of the accessory radula closer (ARC) muscle while repetitive programs were elicited by stimulation of the esophageal nerve. In other similar experiments, we recorded firing of motor neuron B48 and contractions of the radula opener muscle. In intact animals, we implanted electrodes to record nerve or ARC muscle activity while the animals swallowed controlled strips of seaweed or fed freely. In all cases, we found large variability in all parameters examined. Some of this variability reflected systematic, slow, history-dependent changes in the character of the central motor programs. Even when these trends were factored out, however, by focusing only on the differences between successive cycles, considerable variability remained. This variability was apparently random. Nevertheless, it too was the product of central history dependency because regularizing merely the high-level timing of the programs also regularized many of the downstream neuromuscular parameters. Central motor program variability thus appears directly in the behavior. With regard to the production of functional behavior in any one cycle, the large variability may indicate broad tolerances in the operation of the neuromuscular system. Alternatively, some cycles of the behavior may be dysfunctional. Overall, the variability may be part of an optimal strategy of trial, error, and stabilization that the CNS adopts in an uncertain environment.