Prolonged inhibition of neurons by neuroendocrine cells in Aplysia.

In the abdominal ganglion of Aplysia, a burst of action potentials in peptide-secreting neuroendocrine cells, the bag cells, produces slow inhibition of two identified bursting pacemaker neurons. The inhibition is due to slow hyperpolarizing potential that reduces bursting pacemaker activity for 3 hours or more. The slow inhibitory potential results from a large and prolonged increase in membrane conductance to potassium ions as well as a slower ionic process that is relatively independent of membrane conductance.

Identification and molecular cloning of a neuropeptide Y homolog that produces prolonged inhibition in Aplysia neurons.

The neuroendocrine bag cell neurons of the marine mollusk Aplysia produce prolonged inhibition that lasts for more than 2 hr. We purified a peptide from the abdominal ganglion that mimics this inhibition. Mass spectrometry and microsequence analysis indicate that the peptide is 40 aa long and is amidated at its carboxyl terminus. It is highly homologous to vertebrate neuropeptide Y (NPY) and other members of the pancreatic polypeptide family. As determined from cloned cDNA, the gene coding for the precursor protein shares a common structural organization with genes encoding precursors of the vertebrate family. The peptides may therefore have arisen from a common ancestral gene. Bag cell neurons are immunoreactive for Aplysia NPY, and Northern blot analysis indicates that as with its vertebrate counterparts, the peptide is abundantly expressed in the CNS. This suggests that peptides related to NPY may have important functions in the nervous system of Aplysia as well as in other invertebrates.

Functional Organization of the Cardiac Ganglion of the Lobster, Homarus americanus.

External recording and stimulation, techniques were used to determine which neurons and interactions are essential for production of the periodic burst discharge in the lobster cardiac ganglion. Burst activity can be modulated by brief single shocks applied to the four small cells, but not by similar stimulation of the five large cells, suggesting that normally one or more small cells primarily determine burst rate and duration. Repetitive electrical stimulation of large cells initiates spike activity in small cells, probably via excitatory synaptic and/or electrotonic connections which may normally act to prolong bursts and decrease burst rate. Transection of the ganglion can result in burst activity in small cells in the partial or complete absence of large cell spike activity, but large cells isolated from small cell excitatory synaptic input by transection or by application of dinitrophenol do not burst. Generally, transections which decrease excitatory feedback to small cells are accompanied by an increase in burst rate, but mean spike frequency over an entire burst cycle stabilizes at the original level within 10-30 min for various groups of cells whose spike-initiating sites are still intact. These and previous results suggest that the system is two layered: one or more small cells generate the burst pattern and impose it on the large cells which are the system's motorneurons.

A Relaxation Oscillator Description of the Burst-Generating Mechanism in the Cardiac Ganglion of the Lobster, Homarus americanus.

Properties of the neural mechanism responsible for generating the periodic burst of spike potentials in the nine ganglion neurons were investigated by applying brief, single shocks to the four small cells with extracellular electrodes placed near the trigger zones of the small cells. The shock elicited a burst if presented during the latter portion of the silent period, terminated a burst during the latter portion of the burst period, and was followed by a newly initiated burst during the early portion of the burst period. The resultant changes in burst and silent period durations were quantitatively described by a second-order non-linear differential equation similar to the van der Pol equation for a relaxation oscillator. The equation also qualitatively described changes in firing threshold of the small cells during the burst cycle. The first derivative of the solution to the equation is similar to slow transmembrane potentials in neurons that are involved in generation of burst activity in other crustacean cardiac ganglia.

Immunostaining for peptides of the egg-laying hormone/bag cell peptide precursor protein in the head ganglia of Aplysia.

Egg-laying hormone and alpha-bag cell peptide are two neuropeptides derived from a common precursor protein in the marine mollusk Aplysia. Previous studies indicate that they are neurotransmitters that co-exist in individual bag cell neurons and most bag cell processes in the abdominal ganglion. In the present investigation we used double-label immunocytochemistry with highly specific antisera to describe their distribution elsewhere in the CNS. We found that a small cluster of cells and their fibers in the pleural ganglion that were previously described as being immunoreactive for egg-laying hormone were also immunoreactive for alpha-bag cell peptide(1-9). A previously described group of small cell bodies in the cerebral ganglion also stained for both peptides. However, the fiber arborizations located near them were immunoreactive for alpha-bag cell peptide(1-9), but not egg-laying hormone. This suggests that there is alternative processing of the precursor protein or differential transport of the peptides from the cell bodies. The specificities of the antibodies indicate that all of the neurons that stain for egg-laying hormone-like peptides in the CNS synthesize peptides derived from the egg-laying hormone/bag cell peptide precursor, rather than peptides derived from other members of the egg-laying hormone gene family. They also suggest that peptides derived from the related A or B precursor proteins are not synthesized in the CNS, or at levels too low to detect. The results are consistent with the proposal that the behavior associated with egg-laying is initiated and controlled by peptide transmitters derived from a single gene and expressed in specific neurons of the CNS.

Identification and primary structural analysis of peptide II, an end-product of precursor processing in cells R3-R14 of Aplysia.

Peptide II, which is encoded on a gene for a precursor protein in abdominal ganglion neurons R3-R14, was purified from extracts of abdominal ganglia of Aplysia californica. Native peptide II comigrates with synthetic standards on HPLC under isocratic conditions. Amino acid sequence and composition analyses indicate that the sequence of peptide II is Glu-Ala-Glu-Glu-Pro-Ser-Phe-Met-Thr-Arg-Leu, as predicted from the precursor. The molluscan cardioexcitatory peptide Phe-Met-Arg-Phe-amide was also identified in abdominal ganglion extracts by similar means. The large amount of peptide II recovered (100 ng/ganglion), and its location on the precursor between two pairs of basic residues, strongly suggest that the precursor is processed into peptide II and at least two other peptides. Although cells R3-R11 have been postulated to play a role in cardiovascular control, peptide II was without effect at less than or equal to 10(-4) M concentrations on identified abdominal ganglion neurons, the gastroesophageal artery or the heart. The physiological role of peptide II therefore remains to be elucidated.

Presence of immunoreactive alpha-bag cell peptide[1-8] in bag cell neurons of Aplysia suggests novel carboxypeptidase processing of neuropeptides.

alpha-bag cell peptide (alpha BCP) is a putative neurotransmitter released from bag cell neurons of the marine mollusc Aplysia. alpha BCP is present in bag cell extracts and releasate from bag cells in two neuroactive forms: alpha BCP[1-9] and alpha BCP[1-8]. alpha BCP[1-8] is 30 times as potent as [1-9] in inhibiting target neurons, suggesting that both forms of the peptide serve as neurotransmitters. However, biochemical and molecular genetic data suggest that only alpha BCP[1-9] is originally cleaved directly from a larger precursor protein and that generation of alpha BCP[1-8] would require an unusual C-terminal leucine cleavage of alpha BCP[1-9]. To further ascertain which forms of alpha BCP are normally present in bag cells, we generated highly specific antisera to each peptide. We found intense immunostaining for both peptides in bag cell somata and nerve terminals. Moreover, both forms were stable in bag cell extract for at least 1 hr, which suggests that proteolysis in the extracts had been effectively inhibited. These results suggest that both alpha BCP[1-8] and [1-9] are normally present in bag cell somata and terminals and that a small amount of alpha BCP[1-9] is processed to alpha BCP[1-8] in vesicles before release. The results support the interpretation that the activity of an intravesicular carboxypeptidase generates alpha BCP[1-8] and thereby regulates the amount of inhibitory activity released during a bag cell discharge.

Identification of Aplysia neurons containing immunoreactive FMRFamide.

Electrophysiological and immunocytochemical techniques were used in the abdominal ganglion of Aplysia to identify neurons containing immunoreactive FMRFamide. Large numbers of neurons were immunoreactive for FMRFamide, including R2, L2, L3, L4, L5, L6, 2 cells tentatively identified as L12 and L13, and a previously unidentified cluster on the ventral surface of the right lower quadrant. There was also heavy labelling of fibers, often with beaded varicosities, throughout the neuropil, the cell layers, and the sheath overlying the ganglion. This data provides further evidence that FMRFamide is an important neurotransmitter in Aplysia. The demonstration of immunoreactive FMRFamide in the giant cholinergic neurons R2 and LP1(1) suggests that these well-studied and experimentally convenient cells use acetylcholine and an FMRFamide-like peptide as cotransmitters.

Coexistence of egg-laying hormone and alpha-bag cell peptide in bag cell neurons of Aplysia indicates that they are a peptidergic multitransmitter system.

Double-label immunocytochemistry reveals that immunoreactivity for two putative peptide transmitters, egg-laying hormone and alpha-bag cell peptide, co-exist in most bag cell somata and processes in the abdominal ganglion of the marine mollusc Aplysia. Together with previous physiological and biochemical data these findings indicate that the neuroendocrine bag cells are a multitransmitter system which utilizes two or more peptides derived from a common precursor.

Local hormonal modulation of neural activity in Aplysia.

The abdominal ganglion of Aplysia provides a convenient experimental system for cellular studies on the roles of peptides as chemical messengers in the nervous system. There are indications that the bag cells, a group of neuroendocrine cells, synthesize and release egg laying hormone (ELH), a peptide with an apparent molecular weight of 6000. Our recent investigations indicate that a burst of impulse activity in the bag cells produces five types of long-lasting responses, some excitatory, others inhibitory, in 26 identified neurons and 2 identified cell clusters located near the bag cells in the abdominal ganglion. The responses have slow, smoothly graded onsets, and many of them result in modulation of neuronal activity for 3 hours or more. Physiological and ultrastructural data support the hypothesis that they are induced by a bag cell hormone (or hormones) that is released into vascular and interstitial spaces of the ganglion to act on the target neurons. Local application of purified ELH to one of the target neurons provides evidence that the bag cell effect is mediated by ELH. Many of the target neurons are known to be parts of neuronal circuits that control specific behavioral and homeostatic processes. Since egg laying is initiated by the bag cell discharge and is associated with a stereotyped behavior pattern lasting several hours, the actions of these peptide-secreting neurons on the central nervous system may serve to regulate certain elements of behavior and homeostasis during egg laying.

Central actions of R15, a putative peptidergic neuron in Aplysia.

We report that the bursting pacemaker neuron R15 has central actions on other identified neurons in the abdominal ganglion of Aplysia california. The follower cells are located on the dorsal surface of the left lower quadrant of the ganglion and include members of the LC cell cluster. A spontaneous burst of impulses in R15 produces a slow, graded, excitatory potential of up to 8 mV in follower cells. The response begins about 2-3 s after the first impulse in an R15 burst, and reaches its peak at about 4-6 s (corresponding approximately to the end of the R15 burst). In some preparations a biphasic response was seen composed of the early depolarization followed by a slower excitatory or inhibitory phase. All the responses were blocked when R15 was hyperpolarized to prevent spiking. The magnitude of the response was reduced in a graded fashion by prematurely terminating the R15 burst with hyperpolarizing current and was increased when depolarizing current was injected into R15 during a burst. Central actions of R15 were observed in only 28% of our preparations, and their presence may depend on unknown physiological factors. The effects are likely to be mediated by R15 neuropeptides. The accessibility of both R15 and its targets in this preparation should facilitate further analysis of this interaction.

The neuropeptide egg-laying hormone modulates multiple ionic currents in single target neurons of the abdominal ganglion of Aplysia.

The bag cell neurons of the abdominal ganglion of Aplysia are a useful system for the study of peptidergic neurotransmission. A 20 min burst of impulse activity in the bag cells induces or augments repetitive firing in LB and LC neurons in the abdominal ganglion for up to several hours. Previous experiments have indicated that this effect is mediated by the putative bag cell transmitter egg-laying hormone (ELH). Using voltage-clamp analysis we found that bag cell bursts (BCBs) evoke long-lasting changes in membrane current in these neurons that are mimicked by the application of ELH. The combined ELH-evoked current is inward at all membrane potentials between -110 and -10 mV and consists of 3 separable currents persisting for 30-120 min. They include (1) a depolarizing current that is activated at membrane potentials above -40 mV. This current, termed ISI, is blocked by prolonged exposure to 10 mM Ni2+/0 mM Ca2+ and is not abolished by 0 mM Na+ or 100 mM TEA+/0 mM Na+ in the bathing medium. It is therefore a Ca2+-sensitive current and does not involve Na+ as a charge carrier. (2) There is a hyperpolarizing current that is activated at membrane potentials below approximately -70 mV. This current, termed IR, is blocked by external Rb+ (5 mM) and Cs+ (10 mM) and has a chord-conductance that shifts with the external [K+] according to the Nernst potential for potassium. It is therefore an inwardly rectifying K+ current. (3) There is a small, steady depolarizing current, termed Ix. This current is the only one that remains after prolonged exposure to 10 mM Ni2+/0 mM Ca2+-containing bathing medium. It is Na+ dependent and is associated with a small increase in membrane conductance that is largely independent of membrane voltage. All 3 currents are slow to inactivate; they appear to sum algebraically to produce the net BCB- or ELH-evoked current.

Positive feedback by autoexcitatory neuropeptides in neuroendocrine bag cells of Aplysia.

Neurohormones are often secreted in large amounts from neuroendocrine cells during episodes of synchronous, repetitive spike activity. We report evidence that this pattern of activity in the neuroendocrine bag cells of Aplysia involves positive feedback by autoexcitatory transmitters. Intracellular stimulation of individual bag cells caused slow depolarizing afterpotentials and synchronous afterdischarges in the entire population of bag cells. Application of the bathing medium collected during bag cell activity mimicked these responses. Application of alpha-, beta-, or gamma-bag cell peptides (BCPs), 3 structurally related neuropeptides released from bag cells, also mimicked these responses. These autoexcitatory BCPs fulfill most of the strict criteria necessary for classification as neurotransmitters in this system. This is the first biological activity reported for beta- and gamma-BCPs and brings to 4 the number of bag cell neuropeptides derived from the egg-laying hormone/BCP precursor that are putative cotransmitters. Positive feedback by autoexcitatory transmission may provide a general mechanism for the generation of episodic activity in neuroendocrine systems.

Complex behavior induced by egg-laying hormone in Aplysia.

Previous studies have described a pattern of complex behavior that occurs in the marine mollusc Aplysia during egg laying. Egg laying and the behavior are initiated by a burst of impulse activity in the neuroendocrine bag cells of the abdominal ganglion or by injection of bag cell extract. To more precisely identify the factors responsible for inducing the behavior we injected animals with egg laying hormone (ELH), one of the neuropeptides secreted by the bag cells. We found that ELH causes a behavior pattern similar to what occurs during spontaneous egg laying. This includes a temporal pattern of head movements consisting of waves and undulations, followed near the beginning of egg deposition by a transition to head weaves and tamps and inhibition of locomotion. There was also a small decrease in respiratory pumping. Except for respiratory pumping, a similar pattern occurred in a second group of animals injected with atrial gland homogenate, which is presumed to induce bag cell activity, but not in controls. These results further implicate ELH in regulation of the behavior. We discuss possible sites of action of ELH and the neural mechanisms by which the behavior is controlled.

Multiple, prolonged actions of neuroendocrine bag cells on neurons in Aplysia. II. Effects on beating pacemaker and silent neurons.

1. A survey of identified cells of the abdominal ganglion of Aplysia was undertaken to determine the extent of bag cell influence in the ganglion. Bursts of bag cell spike activity lasting 5--40 min were elicited by brief, 0.6- to 2 s local stimulation while recording simultaneously from bag cells and other ganglion cells with intracellular electrodes. 2. Slow inhibition occurs in giant cell R2, neurosecretory cells R3-R14, and ink-gland motoneurons, L14A, B, C. The cells remain hyperpolarized for from 15 to 60 min. 3. Transient excitation occurs in mechanoreceptor cells L1 and R1. The cells are strongly depolarized by a slow excitatory potential that lasts for about 10 min and produces spike activity for 3--7 min. 4. Prolonged excitation occurs in some cells of the LB and LC identified cell clusters. The cells are depolarized and spike activity is increased for 3 h or more. 5. A biphasic response occasionally occurs in the command interneuron L10. Inhibition of this cell lasts 10--15 min and is followed by excitation for several hours. Excitation is accompanied by facilitation of synaptic potentials for 40--60 min in cells innervated by L10; the facilitation apparently results from the increase in L10 firing rate. 6. The results indicate that the bag cells have multiple types of actions and affect large numbers of ganglion neurons. All effects have the slowly graded onsets and prolonged durations to be expected of hormonally mediated interactions. 7. Previous studies have indicated that in intact animals the bag cell burst discharge initates a stereotyped egg-laying behavioral pattern that persists for several hours (3, 27). The present data support the hypothesis that certain elements of egg-laying behavior and homeostasis are regulated by a direct action of the bag cells on the central nervous system.

Evidence for mediation of a neuronal interaction by a behaviorally active peptide.

Egg laying hormone, a peptide neurohormone with an approximate molecular weight of 6000, was isolated from the region of the abdominal ganglion of Aplysia that contains the neuroendocrine bag cells and purified by gel filtration chromatography, isoelectric focusing, and dialysis. A 1-min local application of egg laying hormone to the identified neuron R15 produced prolonged (greater than 1 hr) augmentation of impulse activity in this neuron. The distinctive quality and prolonged duration of the response are apparently identical to the previously described response to electrically elicited bag cell activity. The results provide evidence that egg laying hormone is the mediator of this prolonged neuronal interaction.

Isolation and partial characterization of neuropeptides that mimic prolonged inhibition produced by bag cell neurons in Aplysia.

The bag cell neurons of the marine mollusk Aplysia are part of a neural system that utilizes four neuropeptides as neurotransmitters. The peptides, derived from the egg-laying hormone/bag cell peptide (ELH/BCP) precursor protein, are released during a 20-min burst discharge of the bag cells and produce several types of responses in various abdominal ganglion neurons. In the identified neurons L3 and L6, bag cell activity produces prolonged inhibition that lasts for more than 2 h. One of the bag cell peptides, alpha-BCP, mediates an early component of the inhibition in these neurons. To identify the co-transmitter mediating the prolonged component of inhibition, we purified material from an acid extract of abdominal ganglia using molecular sizing high-pressure liquid chromatography (HPLC) on TSK 250-125 followed by two steps of reverse-phase HPLC on C4 or C18. We isolated three inhibitory factors that mimic the prolonged component of inhibition. Mass spectroscopy and partial amino acid sequence analysis indicate one factor is ELH [2-36], that is, ELH that lacks the first, N-terminal amino acid. This inhibitory activity was similar in potency to that of ELH and is the first to be described for an ELH-related peptide. The two other factors were approximately 3,300 and 4,700 Da and were effective at 10- and 50-fold lower concentration, respectively, than ELH or its fragment. Amino acid composition analysis suggests that they are not derived from the ELH/BCP precursor protein. The 4,700 Da factor is effective at the lowest concentration and produces an effect that lasts as long as 100 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuroendocrine bag cells of Aplysia are activated by bag cell peptide-containing neurons in the pleural ganglion.

1. The generation of egg-laying behavior in the marine mollusk Aplysia involves a prolonged burst discharge in the neuroendocrine bag cells, which secrete neuropeptides derived from the egg-laying hormone/bag cell peptide (ELH/BCP) precursor protein. 2. Besides the bag cells, which are located in the abdominal ganglion, small clusters of neurons in the cerebral and pleural ganglia also express the ELH/BCP neuropeptides. We made intracellular recordings from 32 of these ELH/BCP cells in right pleural ganglia, in 18 preparations, to characterize their physiological properties and their functional relationship to the bag cells. 3. The identification of these ELH/BCP cells was confirmed by pressure injection of Lucifer yellow and subsequent immunocytochemical processing for alpha-BCP immunoreactivity. 4. The basic electrophysiological properties of the pleural ELH/BCP cells were similar to those of the bag cells. These pleural cells were directly demonstrated to be electrically coupled, and direct intracellular stimulation of individual pleural ELH/BCP cells initiated prolonged, synchronous burst discharges in the entire cluster through a positive feedback mechanism. 5. Burst discharges elicited in the pleural ELH/BCP cells consistently initiated burst discharges in the bag cells. Bag cell burst discharges were less effective in initiating burst discharges in the pleural ELH/BCP cells, indicating that there were reciprocal but asymmetrical connections. 6. The results show that the pleural ELH/BCP cells are functionally coupled to the bag cells. They support the hypothesis that the pleural ELH/BCP cells are part of the descending pathway that initiates bag cell activity and egg-laying behavior, in vivo.

Neural control of circulation in Aplysia. III. Neurotransmitters.

In the abdominal ganglion of Aplysia californica, seven motoneurons have been described which modulate the myogenic heart beat and vasomotor tone (28). These neurons mediate their motor effects by chemical transmission. In this paper we have attempted to specify the transmitters of six of these motoneurons. We have 1) studied the effects of several common transmitters on the innervated structures and compared these effects with the effects of firing the motoneurons, 2) examined whether blocking agents influence similarly the effect of a putative transmitter applied to the innervated structure and the effect of firing a motoneuron, and 3) tested the capability of the motoneurons to synthesize the putative transmitters from precursors. The positive inotropic and chronotropic effects of firing the excitor motoneuron RB(HE) were mimicked by perfusion of the heart with serotonin at a low concentration. Cinanserin blocked both the effects of motoneuron excitation and serotonin perfusion. RB(HE) was also shown to synthesize [3H]serotonin from L-[3H]tryptophan injected directly into the cell body. The effects of firing the two LD(HI) heart-inhibitory motoneurons were mimicked by perfusion of the heart with acetylcholine. Benzoquinonium blocked the effects of the inhibitory motoneuron and acetylcholine perfusion. Perfusion with arecoline also inhibited the heart beat. Acetylcholine applied to the arteries mimicked the vasoconstriction caused by the LB(VC) motoneurons. Aortic constriction in response to activity in LB(VC) cells or to acetylcholine was blocked by hexamethonium and curare. The heart inhibitor and vasoconstrictor motoneurons synthesized [3H] acetylcholine from [3H] choline injected into their cell bodies. Thus, as in vertebrates, acetylcholine mediates inhibition to the heart. Unlike vertebrates, however, serotonin mediates excitation to the heart and acetylcholine mediates peripheral vasoconstriction.

Multiple, prolonged actions of neuroendocrine bag cells on neurons in Aplysia. I. Effects on bursting pacemaker neurons.

1. The bag cells are a group of neuroendocrine cells located in the abdominal ganglion of Aplysia. Accumulated evidence suggests they synthesize and release egg-laying hormone (ELH), a peptide that induces egg laying. In this and the following paper (37) we describe five types of prolonged neural responses in cells of the isolated abdominal ganglion that are produced by stimulated bag cell activity. 2. Prolonged, 5- to 40-min bursts of spike activity were triggered in the normally silent bag cells by local stimulation of one of the bag cell clusters with brief, 0.6- to 2-strains of pulses. This local stimulation minimized the possible effects of the stimulus on other ganglion cells and initiated bag cell activity similar to what has been recorded in intact animals at the initiation of egg laying. 3. Following onset of triggered bag cell activity there is an increase in the amplitude of the bursting pacemaker potential in cell R15 that results in augmented bursting activity in this autoactive cell for up to 3 h. The increase begins in less than 1 min and reaches a maximim after 8--20 min. In two other bursting pacemaker cells, L3 and L6, there is a second type of response, slow inhibition, consisting of a smoothly graded hyperpolarization that begins in 5--14 s, reaches a peak value of 10--20 mV after 30 s, and results in a decrease in the spontaneous spike activity of these cells for 3 h or longer. Both types of responses are contingent on the occurrence of bag cell activity, they depend on prolonged bag cell activity for their normal expression, and they occur in the absence of the fast interactions characteristic of conventional synapses. 4. The results reveal at the level of intracellular recordings prolonged actions of peptide-secreting neuroendocrine cells on the central nervous system. The role of ELH as a putative mediator of one or more of these actions is discussed.