Reexamination of the gill withdrawal reflex of Aplysia californica cooper (Gastropoda; Opisthobranchia).

The gill withdrawal reflex (GWR), an important model system for neural mechanisms of learning, varies in form and amplitude within as well as between preparations and is therefore a heterogeneous collection of action patterns, not a reflex. At least 4 action patterns occur in response to mechanical stimulation of the siphon. It is often impossible to categorize a particular movement unambiguously. All may occur spontaneously. Gill movements may be described as combinations of 10 actions; 4 involving vein movements are described here. All actions and action patterns can occur in preparations lacking the central nervous system (CNS). Some vein movements may generate considerable force without markedly altering gill area. It is suggested that this explains why some early studies failed to identify the important role of the peripheral nervous system in the GWR. Studies based on the assumption that the GWR involves a single type of movement controlled by cells of the parietovisceral ganglion require reevaluation. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

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.     

Motor neuronal function in old Aplysia is improved by long-term stimulation of the siphon/gill reflex.

As Aplysia age, motor neuronal (L?) elicited gill-pinnule contractions (PCs) are significantly decreased as is transmission at pinnule junctions (PJT). To determine whether this reduced function of L? with increased age could be altered, the siphon/gill reflex was stimulated regularly in unrestrained old animals. Aplysia were assigned randomly into trained and untrained groups. For more than 3 wks, a 1-s, 25-g water jet stimulus was administered to the siphon of the trained animals 10 times per day at 20-min intervals. The duration of siphon withdrawal increased significantly during training. In semi-intact preparations, PCs and PJT were then measured during 3-s depolarized pulses to L? (frequency range?=?1–44 per 3-s interval). The trained animals had (a) significantly higher PCs, (b) a significantly greater increase in PCs elicited by increasing L? spike rates, (c) significantly higher double-spike facilitation, and (d) significantly higher facilitation per spike across the 4 spike trains below or at PC threshold. The significant training-induced increases in both PC and PJT indicate that a considerable level of plasticity still exists in an aging nervous system. Whether this long-term training of Aplysia retards the same processes responsible for the age-related decline in neuromuscular transmission or produces a compensatory change in other neuronal processes is discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Identification and characterization of a multifunction neuron contributing to defensive arousal in Aplysia

1. The tail withdrawal reflex is mediated by a monosynaptic circuit composed of tail sensory and motor neurons, but there appear to be additional neuronal elements that also contribute to the reflex. A newly identified interneuron, called LP117, was located in the pleural ganglion. This neuron formed a parallel excitatory pathway between sensory and motor neurons. The distinguishing feature of LP117 was its ability to elicit a long-lasting (5-100 s) excitatory postsynaptic potential (EPSP) in the motor neuron. 2. Intracellular labeling of LP117 revealed axons projecting to the cerebral and abdominal as well as the pedal ganglia. Simultaneous intracellular recordings confirmed the widely divergent output of LP117 to tentacle motor neurons in the cerebral ganglion, as well as to gill, siphon, and ink motor neurons in the abdominal ganglion. 3. Also receiving input were abdominal neurons L29, which excites LFs motor neurons and facilitates LE sensory neurons, and L25, which is part of the pattern-generating network underlying respiratory pumping. Thus LP117 appears to be a neural element important for the conduction of information about tail stimulation to ganglia that are not innervated by tail sensory neurons themselves. Moreover, the divergent outputs suggest that LP117 is an element of a neural circuit underlying defensive arousal. 4. LP117 produced slow EPSPs in several motor neurons. The long time course of the EPSP could prolong the burst in the motor neuron produced by LP117 itself as well as increase the effectiveness of coincident synaptic input. This suggests that an important function of this interneuron is to extend the duration of the response to tail stimulation in the motor neuron. This could account for the relatively long time course of the motor neuron response to tail stimulation compared with that of the sensory neuron. 5. Sensitization is a form of nonassociative learning that produces changes in the amplitude and duration of reflex responses. It seems unlikely that all of these changes can be attributed to enhanced amplitude of the sensory-motor synapse, however. Therefore LP117 may itself be a site of plasticity for reflexes elicited by tail stimulation.     

Quantitative analysis of the relation between gill amplitude and siphon duration in the defensive withdrawal reflex of Aplysia..

The defensive withdrawal reflex of the mantle organs (gill, siphon, and mantle shelf) of the marine mollusc Aplysia californica has been the subject of numerous studies investigating the cellular and molecular mechanisms of learning. In behavioral experiments, the reflex has been monitored by means of two different response measures, either siphon duration (in unrestrained, freely moving animals) or gill amplitude (in restrained preparations). It has generally been assumed that one component of the reflex provides a reliable index of the other. In the present study, we directly tested this assumption by simultaneously measuring both response parameters in the same experiment. Reflex response magnitude was varied in two ways: (a) by systematically varying stimulus intensity, and (b) by holding the stimulus intensity constant, but delivering stimuli at a rate that produced significant habituation. Using both measures we found that gill amplitude and siphon duration were highly correlated (average correlation?=?.90). (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Sequential protein analysis from single identified neurons of Aplysia californica. A microelectrophoretic technique involving polyacrylamide gradient gels and isoelectric focusing

Electrophoresis in polyacrylamide gradient gels and isoelectric focusing in polyacrylamide gels of capillary size are powerful tools for the analysis of molecular weight and charge properties of small protein samples. This is demonstrated using identified neurons from the abdominal ganglion of the sea hare Aplysia californica. Certain cell-specific peptides, which are considered to be neurosecretory, have been shown to be water soluble when ethylene glycol was employed as a mobilizing agent. Although the mode of action of ethylene glycol is not yet understood, this method may be of value for various extraction procedures. The application of a new staining method that is preferential for separations of sodium dodecyl sulfate-proteins yields information about the charge of water-insoluble proteins which has so far been inaccessible. Preliminary results gained by a small, two-dimensional mapping procedure as well as optical density separation patterns of two different nuclear protein fractionation from a single isolated nucleus outline further possibilties of the microgel techniques.     

Neuroethological studies of reflex plasticity in intact Aplysia..

Multi-unit recording of siphon nerve activity in intact Aplysia californica (a marine gastropod) with chronically implanted cuff electrodes provided a monitor of activity in a central pattern generator, the Interneuron II (Int II) network, which produces large siphon and gill contractions both spontaneously and after tactile stimulation of the siphon. The phase–response curve of the Int II oscillator for single stimuli at different phases of the cycle showed a "refractory" period early in the cycle, after which most stimuli phase advanced the oscillator and caused a short-latency Int II burst and a large contraction. The amplitude of gill withdrawal and the duration of siphon withdrawal in response to different stimulus intensities depended on whether an Int II burst was triggered. Activation of the Int II oscillator transformed the reflex from one that was graded smoothly with stimulus intensity to one in which nearly maximal responses were elicited even by weak stimuli. Entrainment and habituation training both involved monotonous repetition of a stimulus at specific intervals. With repeated siphon stimuli, nearly maximal reflex responses were maintained in intact Ss as long as the Int II oscillator was entrained, whereas habituation was associated primarily with failure to entrain the oscillator. Sensitized Ss showed significantly more triggered Int II burst than did controls. Digital spike-train analysis indicated that individual siphon motoneurons showed significantly increased background activity that often persisted for several minutes. (41 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Neuronal elements that mediate escape swimming and suppress feeding behavior in the predatory sea slug Pleurobranchaea

1. The white, bilaterally paired A1 interneurons of the cerebropleural ganglion of Pleurobranchaea californica fire rhythmic bursts of action potentials during escape swimming behavior. We studied the role of the A1s in swimming behavior and pattern generation in whole animal and isolated CNS preparations. 2. The escape swim is a cyclic sequence of dorsal and ventral flexions of the body. During the swim, A1 bursts precede and accompany the dorsal flexion phase of the cycle. Hyperpolarization of A1 to prevent spike activity interrupts swimming behavior in the whole animal and fictive swimming in the isolated CNS. Stimulated A1 activity was not observed to cause swimming in whole animals, and was only occasionally sufficient to trigger fictive swimming activity in the isolated CNS. 3. In quiescent whole animal preparations, stimulation of a single A1 normally causes a single dorsal flexion followed by body flexion to the side contralateral to the stimulated cell; characteristically, A1 spike activity stimulates feedback inhibition coinciding with the end of dorsal flexion and the onset of contralateral flexion. 4. A1 spike activity suppresses feeding behavior and causes proboscis retraction in whole animal preparations induced to feed. A1 activity also suppresses fictive feeding driven by stimulation of the critical phasic paracerebral neurons (PCps) of the motor network of feeding in the isolated CNS. Concomitantly, A1 spikes cause potent inhibition of the PCp interneurons. 5. The A1s are specifically excited by noxious mechanical and chemical stimuli, but are not affected by feeding stimuli or the occurrence of feeding behavior. 6. We conclude that the A1 neurons are elements of an escape swimming pattern generator, and that they are probably homologous to the similar C2 neurons of the nudibranch Tritonia diomedea. One of their functions outside of generating the swim pattern may be the suppression of feeding behavior in response to noxious stimulation. These observations provide a neural mechanism for the original observations of the dominance of escape swimming behavior over feeding.     

The third five-year survey of fellows (by examination) of the Faculty of Intensive Care, Australian and New Zealand College of Anaesthetists

The activity of the pedunculopontine tegmental nucleus (PPTg) neurons was recorded in three unrestrained cats operantly conditioned to perform a lever-release movement. The movement had to be initiated either rapidly after a (click) stimulus in a simple reaction-time paradigm or had to be delayed after the same stimulus in trials identified by a tone cue. Successful trials were rewarded by a food pellet. A total of 107 neurons were recorded with microelectrodes. Brief spike neurons (mean duration: 0.7 ms) and broad spike neurons (mean duration: 2 ms) presumed to be cholinergic were detected. Of the 73 neurons localized in the PPTg area, 53 had brief spikes and 20 broad spikes. Changes in activity most commonly occurred very early after the stimulus or during the reinforcement process. Most neurons with brief spikes exhibited very early excitation after the stimulus and reinforcement-related activity. These neurons had a mean activity of 23.7 impulses/s in the period preceding the stimulus. The onset of activation after the stimulus had a latency of 8.6+/-6.9 ms (mean+/-SD), with a range of 4-35 ms. In trials where the movement had to be delayed after the stimulus, the early activation disappeared or was considerably reduced, showing that it was context-dependent. A small proportion of neurons with brief spikes initially decreased activity after the stimulus, but with a latency >9 ms. All the neurons with broad spikes, except one, had reinforcement-related activity. Half of them showed exclusively reinforcement-related activity, the other half also early activation after the stimulus. These neurons were about half as active in the period preceding the stimulus occurrence than the neurons with brief spikes. The early context-dependent activation is discussed in relation to the excitatory projection of PPTg neurons on the subthalamic nucleus. The reinforcement-related activity, preferentially evidenced in broad spike neurons presumed to be cholinergic, is speculated to be associated with cholinergic projection of PPTg neurons to the dopaminergic neurons of the substantia nigra. Finally, the role of PPTg in the ongoing control of motor performance and reinforcement processes is discussed in relation to the basal ganglia circuitry.     

Neurochemistry of the nodose ganglion

Placode-derived general visceral afferent neurons of the nodose ganglion transmit visceral sensory information from specialized sensory endings of the vagus nerve and its branches to the nucleus of the solitary tract. These neurons are critical in relaying information such as elevations in blood pressure, changes in blood oxygenation, passage of contents through the esophagus and intestines, and distention of the heart, stomach, and lungs to the CNS for reflex maintenance of visceral functions. Multiple neurotransmitters, neuropeptides, calcium binding proteins, and other neuroactive substances are associated with neurons of the nodose ganglion. Many neurons colocalize 2 or more neuroactive substances creating the potential for complex interactions of neurochemical signals in the NTS. Neurons of the nodose ganglion also contain a variety of receptors which respond to transmitters, inflammatory mediators, and neurotrophic factors. The contents of these neurochemicals and receptors are not static as alterations in their expression are noted in response to epigenetic influences. Although not yet well understood, potential factors and mechanisms regulating neurochemical events in the nodose ganglion neurons are discussed.     

Behavioral analysis of diffuse noxious inhibitory controls (DNIC): antinociception and escape reactions

'Diffuse noxious inhibitory controls' or DNIC is the inhibition of multireceptive neurons in the dorsal horn of the spinal cord that results when a noxious stimulus is applied to a region of the body remote from the neuron's excitatory receptive field. Although this phenomenon is well-documented, the behavioral consequences of DNIC are not clear. The present study was undertaken to determine how nocifensor withdrawal reflexes evoked by a noxious stimulus are altered by application of a second noxious stimulus to a distant part of the body. The tail flick or hindpaw withdrawal reflex of lightly anesthetized (0.6-1.0% halothane) rats was measured before, during and after another appendage was placed in water ranging in temperature from 45 to 54 degrees C. When the forepaw or hindpaw was placed in water exceeding 49 degrees C the tail flick reflex to acute noxious radiant heat was inhibited. In contrast, noxious conditioning stimuli, regardless of temperature or location, had no effect on the latency for hindpaw withdrawal evoked by an acute noxious stimulus, but did produce a change in reflex topography from flexion to extension. These results, along with previous research on DNIC, suggest that intense noxious stimuli: (1) inhibit the tail flick reflex via inhibition of multireceptive neurons in the dorsal horn; (2) disinhibit hindpaw extensor motoneurons by inhibiting the activity of multireceptive neurons involved in hindlimb flexion; and (3) reduce pain sensation by inhibiting multireceptive neurons projecting to the brain (see Model in Discussion).     

A new class of noninactivating K+ channels from aplysia capable of contributing to the resting potential and firing patterns of neurons

From the nervous system of Aplysia, we have cloned a new class of noninactivating K+ channels (aKv5.1) that are activated at low voltage and are capable of contributing to the resting potential and firing patterns of neurons. Expression of aKv5.1 in Aplysia neuron R15 revealed that aKv5.1 exerts an unusual control over cell excitability; it increased the resting potential by more than 20 mV and abolished the spontaneous bursting activity of the cell. In its ability to suppress the endogenous rhythm of R15, aKv5.1 differs in its actions from transient, inactivating K+ channels such as aKv1.1a, an Aplysia homolog of Shaker. aKv1.1a shortens the duration of the spike and increases the afterpotential, but does not suppress bursting. Thus, by expressing different classes of K+ channels, it is possible to redesign, in specific ways, the signaling capabilities of specific, identified neurons.     

Electrophysiologic study of propagation of excitation in the cat pterygopalatine ganglion

Action potentials were studied in nerves of the cat pterygopalatine ganglion evoked by stimuli applied to other nerves of the ganglion (in situ). It is established that most fibres passing through the ganglion are continuous sympathetic postganglionic fibres (not less than 3 groups). Most parasympathetic preganglionic fibres are synaptical on the ganglionic neurons and are represented by a group of fibres with the same threshold of excitation. Intracellular recording from single neurons of the pterygopalatine ganglion showed that stimulation of the Vidian nerve caused orthodromic spikes with short latency in one group of neurons and spikes with long latency in other neurons (2.5-6 ms and 10-40 ms respectively). Only fast fibres appear to terminate on most neurons of the ganglion, and only slow fibres do on some other neurons. Tonic activity was not observed when was performed from intact nerves of the pterygopalatine ganglion. The interacellular recording from single neurons of the ganglion showed that frequency of spike potentials either is low (1-3 per second) in some neurons or the potentials are absent in general in other neurons.     

Modulation of neuronal activity by glial cells in the retina

Glial-neuronal communication was studied by monitoring the effect of intercellular glial Ca2+ waves on the electrical activity of neighboring neurons in the eyecup preparation of the rat. Calcium waves in astrocytes and Müller cells were initiated with a mechanical stimulus applied to the retinal surface. Changes in the light-evoked spike activity of neurons within the ganglion cell layer occurred when, and only when, these Ca2+ waves reached the neurons. Inhibition of activity was observed in 25 of 53 neurons (mean decrease in spike frequency, 28 +/- 2%). Excitation occurred in another five neurons (mean increase, 27 +/- 5%). Larger amplitude Ca2+ waves were associated with greater modulation of neuronal activity. Thapsigargin, which reduced the amplitude of the glial Ca2+ increases, also reduced the magnitude of neuronal modulation. Bicuculline and strychnine, inhibitory neurotransmitter antagonists, as well as 6-Nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) and D(-)-2-amino-7-phosphonoheptanoic acid (D-AP7), glutamate antagonists, reduced the inhibition of neuronal activity associated with glial Ca2+ waves, suggesting that inhibition is mediated by inhibitory interneurons stimulated by glutamate release from glial cells. The results suggest that glial cells are capable of modulating the electrical activity of neurons within the retina and thus, may directly participate in information processing in the CNS.     

Paired-spike interactions and synaptic efficacy of retinal inputs to the thalamus

In many neural systems studied in vitro, the timing of afferent impulses affects the strength of postsynaptic potentials. The influence of afferent timing on postsynaptic firing in vivo has received less attention. Here we study the importance of afferent spike timing in vivo by recording simultaneously from ganglion cells in the retina and their targets in the lateral geniculate nucleus of the thalamus. When two spikes from a single ganglion-cell axon arrive within 30 milliseconds of each other, the second spike is much more likely than the first to produce a geniculate spike, an effect we call paired-spike enhancement. Furthermore, simultaneous recordings from a ganglion cell and two thalamic targets indicate that paired-spike enhancement increases the frequency of synchronous thalamic activity. We propose that information encoded in the high firing rate of an individual retinal ganglion cell becomes distributed among several geniculate neurons that fire synchronously. Because synchronous geniculate action potentials are highly effective in driving cortical neurons, it is likely that information encoded by this strategy is transmitted to the next level of processing.     

A pair of reciprocally inhibitory histaminergic sensory neurons are activated within the same phase of ingestive motor programs in Aplysia

Previous studies have shown that each buccal ganglion in Aplysia contains two B52 neurons, one in each hemiganglion. We now show that there are two B52 neurons in a single buccal hemiganglion and four cells in an animal. We also show that the B52 neurons are histamine-immunoreactive and use reverse phase HPLC to show that the histamine-immunoreactive substance is authentic histamine. Previous studies have shown that the B52 neurons make numerous inhibitory synaptic connections with neurons active during the radula closing/retraction phase of ingestive motor programs. A computational model of the Aplysia feeding central pattern generator has, therefore, suggested that the B52 neurons play a role in terminating closing/retraction. Consistent with this idea we show that both B52 neurons fire at the beginning of radula opening/protraction. We also show that both B52 neurons are sensory neurons. They are depolarized when a flap of connective tissue adjacent to the buccal commissural arch is stretched. During ingestive feeding this is likely to occur at the peak of closing/retraction as opening/protraction begins. In the course of this study we compare the two ipsilateral B52 neurons and show that these cells are virtually indistinguishable; e.g., they use a common neurotransmitter, make the same synaptic connections, and are both sensory as well as premotor neurons. Nevertheless we show that the B52 neurons are reciprocally inhibitory. Our results, therefore, strikingly confirm theoretical predictions made by others that neurons that inhibit each other will not necessarily participate in antagonistic phases of behavior.     

Wipe and flexion reflexes of the frog. I. Kinematics and EMG patterns

1. We evaluated the hypothesis that the neural control of complex motor behaviors is simplified by building movement sequences from a series of simple neural "building blocks." In particular, we compared two reflex behaviors of the frog, flexion withdrawal and the hindlimb-hindlimb wipe reflex, to determine whether a single neural circuit that coordinates flexion withdrawal is incorporated as the first element in a sequence of neural circuits comprising the wipe. The neural organization of these two reflexes was compared using a quantitative analysis of movement kinematics and muscle activity patterns [electromyograms (EMGs)]. 2. The three-dimensional coordinates of the position of the foot over time and the angular excursion of hip, knee, and ankle joints were recorded using a WATSMART infrared emitter-detector system. These data were quantified using principal-components analysis to provide a measure of the shape (eigenvalues) and orientation (eigen-vector coefficients) of the movement trajectories. The latencies and magnitudes of EMGs of seven muscles acting at the hip, knee, and ankle were analyzed over the interval from EMG onset to movement onset, and EMG magnitudes during the initial flexion of the limb. These variables were compared during flexion withdrawal and the initial flexion movement of the limb during the hindlimb-hindlimb wipe reflex (before the onset of the frequently rhythmic portion when the stimulus is removed) when the two reflexes were elicited from comparable stimulus locations. 3. In both the flexion reflex and the initial movement segment of the wipe reflex, the foot moves along a relatively straight line. However, the foot is directed to a more rostral and lateral position during flexion than during wipe. All three joints flex during flexion withdrawal, whereas during the wipe, the knee and ankle joints flex but the angular excursion of the hip joint may vary. The different orientations of the movement trajectories are associated with EMG patterns that differ in both timing and magnitude between the two reflexes. 4. The differences in the kinematics and EMG patterns of the two reflexes during unrestrained movements make it unlikely that the neural circuit that coordinates flexion withdrawal is incorporated as the first element in the sequence of neural circuits underlying the wipe reflex. 5. Unlike the wipe reflex, during flexion withdrawal there is no apparent constraint on the accuracy of placement at the end of the movement, yet the animals nevertheless achieved consistent final positions of both the foot and of each joint. The implications of these findings with respect to the controlled variables are discussed.     

Developmental dissociation of excitability and secretory ability in Aplysia bag cell neurons

1. Despite the considerable progress made in understanding the role of electrical activity in triggering secretion, the developmental relationships between excitability and secretion are not well understood. The well-characterized bag cell neurons of Aplysia provide an advantageous system in which to investigate developmental interactions of these two key properties of neurons. 2. A prolonged afterdischarge triggers egg laying hormone (ELH) secretion in mature bag cell neurons. To investigate secretion in the developmental framework of excitability, we first examined whether immature neurons, which are incapable of the mature form of excitability (afterdischarge), contain ELH and whether this hormone is packaged in vesicles. We used immunoelectron microscopy to compare vesicular localization of ELH and to compare the size and density of ELH-containing vesicles in neurons from adult and juvenile Aplysia. This comparison revealed that immature neurons contain ELH in vesicles in the size range of secretory vesicles. However, they lack a class of large vesicles (> 250 nm in diameter) that is characteristic of mature neurons. 3. To investigate whether the ELH contained in immature bag cell neurons could be secreted in response to electrical activity, we used the potassium channel blocker tetraethylammonium (TEA) combined with nerve stimulation to depolarize neurons from both juvenile animals (ovotestes do not contain eggs) and from adult Aplysia (ovotestes contain eggs). Using radioimmunoassay, we have found that the duration and amount of ELH secreted from bag cell neurons from juvenile Aplysia in response to TEA does not depend on whether or not the cells can be induced to afterdischarge, and the amount and duration of ELH secreted from bag cell neurons of juvenile Aplysia (whether or not they afterdischarged) differed from those secreted by adult neurons. However, by normalizing for body size, we found that the final estimated hemolymph concentration of ELH would be similar in juvenile and adult animals. 4. We investigated the potential functional significance of secretion of bag cell hormones in juvenile Aplysia by attempting to bypass the bag cell neurons and directly activate downstream elements with extract from adult bag cell neurons (BCE), known to contain ELH and other peptides. We found that juvenile Aplysia exhibit at least one component of egg-laying behavior, cessation of locomotion, in response to BCE during a developmental period (as measured by weight) in which they normally would possess neurons incapable of afterdischarge. Thus developmental regulation of excitability in the bag cell neurons may prevent inappropriate hormone release and subsequent premature expression of reproductive behaviors.