Food avoidance learning is accompanied by synaptic attenuation in identified interneurons controlling feeding behavior in Pleurobranchaea.

Identified paracerebral feeding command interneurons (PCNs) in the brain of the mollusc Pleurobranchaea excite other identified PCNs by means of a chemical polysynaptic pathway whose efficacy is reduced by food avoidance training (conditionally paired food and electric shock). The purpose of the present study was to identify the neurons comprising this pathway and to localize learning-induced changes to single identified neurons. We found that associative training strongly attenuates or abolishes a unitary excitatory postsynaptic potential (EPSP) at a single identified synapse in this polysynaptic pathway, but does not alter other synapses. The PCNs descend to the buccal ganglion, where they monosynaptically excite each member of a set of four identified neurons (two per hemiganglion) that belong to the corollary discharge population described previously. The strength of ascending and descending synapses involving identified PCNs is greatest ipsilaterally and is proportional to relative command efficacy established in previous studies. These findings suggest that command efficacy results directly from synaptic strength. The pair of corollary discharge neurons on each side of the buccal ganglion sends axons to the opposite side and thence up the contralateral cerebrobuccal connective to the brain. These neurons have therefore been termed the contralateral corollary discharge (CCD) neurons. Each CCD monosynaptically excites every PCN on both sides of the brain. Contralateral synaptic influences on identified PCNs are larger than ipsilateral ones. Each of the four identified CCD neurons is electrically coupled to all other members of the subset, including the contralateral homologue (based on simultaneous intracellular recording) and the ipsilateral partner (based on dye coupling). Hyperpolarizing a single CCD eliminates the polysynaptic response of PCNs to stimulation of other PCNs, whereas depolarizing a single CCD mimics the polysynaptic response. The CCD neurons are therefore necessary and sufficient to the polysynaptic response. Consistent with this role, the CCDs discharge in phase with the PCNs during the feeding motor program, and hyperpolarizing a CCD abolishes the cycle discharge of PCNs and weakens the feeding rhythm. Of the several reciprocal synapses identified between the PCNs and CCDs, only one was significantly altered by associative training in the food avoidance paradigm developed previously. This synapse, from the polysynaptic excitor (PSE) to the ipsilateral CCD, was also the strongest in this recurrent positive-feedback loop. In brains taken from conditioned specimens, the mean EPSP amplitude induced by a PSE action potential in ipsi

Cholinergic suppression: a postsynaptic mechanism of long-term associative learning.

Food avoidance learning in the mollusc Pleurobranchaea entails reduction in the responsiveness of key brain interneurons in the feeding neural circuitry, the paracerebral feeding command interneurons (PCNs), to the neurotransmitter acetylcholine (AcCho). Food stimuli applied to the oral veil of an untrained animal depolarize the PCNs and induce the feeding motor program (FMP). Atropine (a muscarinic cholinergic antagonist) reversibly blocks the food-induced depolarization of the PCNs, implicating AcCho as the neurotransmitter mediating food detection. AcCho applied directly to PCN somata depolarizes them, indicating that the PCN soma membrane contains AcCho receptors and induces the FMP in the isolated central nervous system preparation. The AcCho response of the PCNs is mediated by muscarinic-like receptors, since comparable depolarization is induced by muscarinic agonists (acetyl-beta-methylcholine, oxotremorine, pilocarpine), but not nicotine, and blocked by muscarinic antagonists (atropine, trifluoperazine). The nicotinic antagonist hexamethonium, however, blocked the AcCho response in four of six cases. When specimens are trained to suppress feeding behavior using a conventional food-avoidance learning paradigm (conditionally paired food and shock), AcCho applied to PCNs in the same concentration as in untrained animals causes little or no depolarization and does not initiate the FMP. Increasing the concentration of AcCho 10-100 times, however, induces weak PCN depolarization in trained specimens, indicating that learning diminishes but does not fully abolish AcCho responsiveness of the PCNs. This study proposes a cellular mechanism of long-term associative learning--namely, postsynaptic modulation of neurotransmitter responsiveness in central neurons that could apply also to mammalian species.

Learning: neural analysis in the isolated brain of a previously trained mollusc, Pleurobranchaea californica.

The neural manifestations of food avoidance learning in the mollusc, Pleurobranchaea, survive the surgical reduction of the preparation to the nearly isolated brain. These manifestations include increased synaptic inhibition and reduced synaptic excitation of the phasic paracerebral feeding command interneurons (PCps) in the brain in response to food stimulation of chemosensory structures left attached to the brain. The same changes are not evident, however, in brains removed from naive, control or satiated specimens. Therefore the nearly isolated brain preparation permits analysis of the cellular substrates of learning in relative isolation from non-associative motivational variables. The isolated brain preparation is here used to show that the increased synaptic inhibition consequent to associative training is distributed not only to the PCps but also to their identified central presynaptic inputs, including other identified feeding command interneurons (PSEs and ETIIs; ref. 21). The decrease in PCp excitation is explained in part by a training-induced inhibition of excitatory inputs to the PCps, and in part by a training-induced reduction in the efficacy of an identified polysynaptic excitatory pathway presynaptic to the PCps.

Neural mechanisms of motor program switching in the mollusc Pleurobranchaea. I. Central motor programs underlying ingestion, egestion, and the "neutral" rhythm(s).

The buccal musculature of the carnivorous gastropod Pleurobranchaea is used in three cyclic patterns of coordination underlying, respectively, ingestion, egestion, and a third, unknown behavior(s) (Croll, R. P., and W. J. Davis (1981) J. Comp. Physiol. 145: 277-287; Croll, R. P., and W. J. Davis (1982) J. Comp. Physiol. 147: 143-154). The corresponding three motor programs can be identified and distinguished in the intact animal (Croll, R. P., and W. J. Davis (1981) J. Comp. Physiol. 145: 277-287), the reduced preparation (Croll, R. P., and W. J. Davis (1982) J. Comp. Physiol. 147: 143-154, and the present paper), and the isolated CNS (present paper), on the basis of several qualitative and quantitative criteria. Distinguishing parameters developed here include: the activity of the salivary duct, which bursts in phase with protraction during ingestion, is silent during egestion, and usually bursts biphasically and in antiphase with protraction during the third ("neutral") rhythm(s); and the protractor duty cycle, which is generally 33 to 50% during ingestion, greater than 50% during egestion, and less than 33% during the neutral rhythm(s). Retractor duty cycles did not differ significantly between the three motor programs. The neutral rhythm(s) may be a low-intensity version of the ingestion motor program, with which it shares most features. The three buccal motor programs can be elicited in the reduced preparation (sensory feedback intact) and in the isolated, deafferented CNS. Therefore, multiple motor programs in this metastable motor system are each endogenous to the CNS; i.e., they can each be generated by a central pattern generator(s) in the absence of sensory feedback.(ABSTRACT TRUNCATED AT 250 WORDS)

Neural mechanisms of motor program switching in the mollusc Pleurobranchaea. II. Role of the ventral white cell, anterior ventral, and B3 buccal neurons.

Identified buccal neurons in the mollusc Pleurobranchaea were stimulated and recorded intracellularly while recording the resultant identified motor program from buccal muscles (reduced preparation) or nerves (isolated central nervous system). Neurons studied included the ventral white cell (VWC), members of the anterior ventral (AV) population, and interneuron B3. Each of these neurons elicited the egestion motor program or its characteristic components when stimulated intracellularly. The characteristic prolonged plateau potential of the VWC was frequently associated with the egestion motor program but never with the ingestion motor program or its characteristic components. Intracellular recordings from these same neurons during spontaneous or induced buccal motor programs were consistent with the view that these neurons participate in production of the egestion motor program. The VWC discharged also during the neutral buccal rhythm, although in a different pattern from that seen during the egestion motor program, suggesting that it is multifunctional. Synaptic targets of the VWC are unknown, but synaptic influences of the AV and B3 neurons were found and are appropriate to their proposed role in egestion. This study therefore indicates that an interrelated cluster of buccal neurons is specialized to command the egestion motor program.

Neural mechanisms of motor program switching in the mollusc Pleurobranchaea. III. Role fo the paracerebral neurons and other identified brain neurons.

Identified neurons in the cerebropleural ganglion (brain) of the mollusc Pleurobranchaea were stimulated and recorded from intracellularly while recording the identified motor program from buccal muscles (reduced preparation) or nerves (isolated central nervous system). Neurons studied included the metacerebral giant neurons (MCGs), phasic paracerebral neurons (PCp's), polysynaptic excitors of the PCp's (PSEs), type II electrotonic neurons (ETII's), type I electrotonic neurons (ETI's) and several other identified neurons or neuronal classes. Intracellular stimulation of the above identified neurons generally elicited the ingestion motor program or its characteristic components, but never the egestion motor program and seldom its characteristic components. Intracellular recordings from these neurons in the isolated central nervous system preparation while eliciting the ingestion and egestion motor program generally showed cyclic membrane potential oscillations in phase with both motor programs, indicating that these neurons receive synaptic feedback from the ingestion and egestion central pattern generator(s). This study is therefore consistent with the view that an interrelated cluster of brain neurons is specialized to command the ingestion motor program. A neural model of motor program switching in the buccal motor system is formulated, comprising separate command pathways for ingestion and egestion that converge on a common central pattern generator(s).

Brain oscillator(s) underlying rhythmic cerebral and buccal motor output in the mollusc, Pleurobranchaea californica.

Tonic (d.c.) intracellular depolarization of the previously identified phasic paracerebral feeding command interneurones (PCps) in the brain of the carnivorous gastropod Pleurobranchaea causes oscillatory neural activity in the brain, both before and after transecting the cerebrobuccal connectives. Therefore, cycle-by-cycle ascending input from the buccal ganglion is not essential to cyclic brain activity. Instead the brain contains an independent neural oscillator(s), in addition to the oscillator(s) demonstrated previously in the buccal ganglion (Davis et al. 1973). Transection of the cerebrobuccal connectives immediately reduces the previously demonstrated (Kovac, Davis, Matera & Croll, 1983) long-latency polysynaptic excitation of the PCps by the polysynaptic excitors (PSEs) of the PCps. Therefore polysynaptic excitation of the PCps by the PSEs is mediated by an ascending neurone(s) from the buccal ganglion. The capacity of feeding command interneurones to induce neural oscillation in the isolated brain declines to near zero within 1 h after transection of the cerebrobuccal connectives, suggesting that this capacity is normally maintained by ascending information from the buccal ganglion. The results show that this motor system conforms to a widely applicable general model of the neural control of rhythmic behaviour, by which independent neural oscillators distributed widely in the central nervous system are coupled together to produce coordinated movement.

Organization of synaptic inputs to paracerebral feeding command interneurons of Pleurobranchaea californica. III. Modifications induced by experience.

Phasic paracerebral feeding command interneurons (PCP's) were studied in whole-animal preparations of Pleurobranchaea drawn from four populations with different behavioral histories: food avoidance conditioned, yoked controls, food satiated, and naive. PCP responses to chemosensory food stimuli (liquefied squid) and mechanosensory touch stimuli (tactile stimulation of anterior and posterior structures) were recorded intracellularly, scored blind, and compared quantitatively across the four populations. PCP's from avoidance-conditioned specimens (10, 18, 19) showed decreased excitatory and increased inhibitory responses to food and touch in comparison with naive (untrained) specimens. Control animals did not show these effects. PCP's from satiated specimens showed decreased excitatory and increased inhibitory responses to food and touch in comparison with PCP's from control, naive, and conditioned specimens. Inhibitory postsynaptic potentials (IPSPs) induced in PCP's of conditioned and satiated specimens by food and touch are indistinguishable in amplitude and waveform from IPSPs produced in the same PCP's by the previously described cyclic inhibitory network (CIN; Ref. 13). In addition, tonic paracerebral neurons (PCT's) that lack input from the CIN, are not inhibited but rather are excited in trained and satiated animals. Therefore the inhibitory responses to food and touch by PCP's of conditioned and satiated specimens appear to be mediated by the CIN. This study demonstrates that associative and nonassociative processes (learning and food satiation, respectively) manifest similarly at the level of command interneurons. The findings furnish a neurophysiological explanation for behavioral motivation in Pleurobranchaea, namely, modulation of the balance of excitation/inhibition in command neurons controlling the corresponding behavior. A cellular model of food avoidance learning and food satiation is formulated to account for these data, based on the identified neural circuitry of the paracerebral command system (15, 17).

Organization of synaptic inputs to paracerebral feeding command interneurons of Pleurobranchaea californica. I. Excitatory inputs.

Neurons presynaptic to the phasic paracerebral feeding command interneurons (PCP's; Ref. 55) of Pleurobranchaea were located in the isolated central nervous system (CNS) and studied anatomically by lucifer yellow injection and physiologically by current injection and intracellular recording in normal and ion-substituted seawater during quiescence and fictive feeding. The present paper describes excitatory inputs to PCP's, while the accompanying paper (54) reports inhibitory inputs. Monosynaptic excitors (MSEs) are a group of at least three monopolar neurons per hemiganglion. Two have similar dendritic structures and functional effects. Each MSE monosynaptically excites the PCP's and fires action-potential bursts in phase with PCP bursts during fictive feeding. The class I electrotonic neuron (ETI) is a single, identified monopolar neuron per hemiganglion with a sparse dendritic arborization and no descending axon in the cerebrobuccal connective (CBC). The ETI is coupled with PCP's only by means of a non-rectifying electrical synapse. Paradoxically, ETI receives opposite synaptic inputs from PCP's and fires in antiphase with PCP's during fictive feeding. Class II electrotonic neurons (ETII's) are a group of at least two identified multipolar neurons per hemiganglion with indistinguishable dendritic architectures and similar but distinguishable functional effects. Each cell is coupled with PCP's by means of a nonrectifying electrical synapse. One of the ETII's also delivers graded, long-latency poly-synaptic chemical inputs to PCP's. ETII's have descending axons in the CBC, elicit fictive feeding when depolarized, and fire cyclically and in phase with PCP's during fictive feeding. Polysynaptic excitors (PSEs) are a group of at least two identified monopolar neurons per hemiganglion with similar elaborate dendritic fields and functional effects. Each cell excites PCP's by a long-latency, relatively nongraded polysynaptic pathway. PSEs also have descending axons in the ipsilateral CBC, elicit fictive feeding when depolarized, and fire in phase with PCP's during fictive feeding. PSEs and ETII's are here recognized as subclasses of neurons previously identified as paracerebral neurons. They are inhibited by the same neurons that supply recurrent inhibition to PCP's (47), share excitatory inputs with PCP's, and exhibit a similar "command" capacity. This study thus documents redundancy and functional specialization within a command system controlling a relatively complex rhythmic motor behavior.

Control of feeding motor output by paracerebral neurons in brain of Pleurobranchaea californica.

1. A population of interneurons that control feeding behavior in the mollusk Pleurobranchaea has been analyzed by dye injection and intracellular stimulation/recording in whole animals and reduced preparations. The population consists of 12-16 somata distributed in two bilaterally symmetrical groups on the anterior edge of the cerebropleural ganglion (brain). On the basis of their position adjacent to the cerebral lobes, these cells have been named paracerebral neurons (PCNs). This study concerns pme subset pf [MCs. the large, phasic ones, which have the strongest effect on the feeding rhythm (21). 2. Each PCN sends a descending axon via the ipsilateral cerebrobuccal connective to the buccal ganglion. Axon branches have not been detected in other brain or buccal nerves and hence the PCNs appear to be interneurons. 3. In whole-animal preparations, tonic intracellular depolarization of the PNCs causes them to discharge cyclic bursts of action potentials interrupted by a characteristic hyperpolarization. In all specimens that exhibit feeding behavior, the interburst hyperpolarization is invariably accompanied by radula closure and the beginning of proboscis retraction (the "bite"). No other behavorial effect of PCN stimulation has been observed. 4. In whole-animal preparations, the PCNs are excited by food and tactile stimulation of the oral veil, rhinophores, and tentacles. When such stimuli induce feeding the PCNs discharge in the same bursting pattern seen during tonic PCN depolarization, with the cyclic interburst hyperpolarization phase locked to the bit. When specimens egest an unpalatable object by cyclic buccal movements, however, the PCNs are silent. The PCNs therefore exhibit properties expected of behaviorally specific "command" neurons for feeding. 5. Silencing one or two PCNs by hyperpolarization may weaken but does not prevent feeding induced by natural food stimuli. Single PCNs therefore can be sufficient but are not necessary to induction of feeding behavior. Instead the PCNs presumably operate as a population to control feeding. 6. In isolated nervous system preparations tonic extracellular stimulation of the stomatogastric nerve of the buccal ganglion elicits a cyclic motor rhythm that is similar in general features to the PNC-induced motor rhythm. Bursts of PCN action potentials intercalated at the normal phase position in this cycle intensify the buccal rhythm. Bursts of PCN impulses intercalated at abnormal phase positions reset the buccal rhythm. The PCNs, therefore, also exhibit properties expected of pattern-generator elements and/or coordinating neurons for the buccal rhythm. 7. The PCNs are recruited into activity when the buccal motor rhythm is elicited by stomatogastric nerve stimulation or stimulation of the reidentifiable ventral white cell. The functional synergy between the PCNs and the buccal rhythm is therefore reciprocal. 8...

Functional and structural correlates of cell size in paracerebral neurons of Pleurobranchaea californica.

1. The paracerebral neurons (PCNs) in the brain of the mollusk Pleurobranchaea are a population of 12-16 interneurons that send axons to the buccal ganglion and control cyclic feeding behavior (9). In the present study we show that the PCNs differ in size and that a number of functional and structural properties of the PCNs are closely correlated with cell size. 2. PCN soma diameter varies from about 30 to 120 micrometers. The diameters segregate into two distinct but overlapping populations, which correspond to independently assigned functional classifications of "tonic" and "phasic" PCNs. The mean soma diameters of two populations were 63 and 84 micrometers, respectively. 3. Two morphological features vary systematically with PCN soma size. First, soma diameter, axonal conduction velocity, and extracellular spike amplitude were positively correlated; therefore, PCN axon diameter presumably increases with soma diameter. Second, intrasomatic injection of lucifer yellow revealed that the small, tonic PCNs are multipolar, while the large, phasic PCNs are generally monopolar neurons. 4. Small PCNs discharge tonically in response to sustained current injection and have a weak effect on cyclic motor output recorded from nerves that innervate feeding muscles. In contrast, the large PCNs discharge phasically in bursts of action potentials that are coordinated with the cyclic motor output and have a comparatively strong effect on the rhythm. The motor effects of simultaneous tonic and phasic PCN stimulation are additive. 5. Tonic and phasic PCNs innervate different but partially overlapping populations of feeding motor neurons. Phasic PCNs typically inhibit motor neurons exiting buccal root 3, while tonic PCNs either have no effect or are weakly excitatory. 6. Tonic and phasic PCNs exhibit different intrinsic properties. In comparison with phasic PCNs, tonic PCNs have higher input resistances, higher spontaneous discharge rates at rest potential, lower firing thresholds to intrasomatically injected current, lower absolute voltage thresholds, greater pacemaker sensitivity, and greater total capacitance. 7. Tonic and phasic PCNs exhibit different input properties. Tonic PCNs are recruited before phasic ones during cycylic buccal motor output induced by stomatogastric nerve stimulation. Phasic PCNs receive powerful, cycylic inhibition that is not shared by tonic PCNs. In addition, extracellular stimulation of the large oral veil nerve of the brain excites tonic PCNs but causes a biphasic postsynaptic potential (PSP) in phasic PCNs that has a net inhibitory effect. Some excitatory synaptic input to phasic and tonic PCNs is unshared, while some is shared. 8. It is concluded that these command interneurons obey the size principle discovered earlier in motor neurons (4, 13-16). Cell size per se is not the causal variable, however; instead the underlying causes of the differences between small and large PCNs include different input and output organizations as well as different intrinsic functional and morphological properties.

Command neurons in Pleurobranchaea receive synaptic feedback from the motor network they excite.

Command neurons that cause rhythmic feeding behavior in the marine mollusc Pleurobranchaea californica have been identified in the cerebropleural ganglion (brain). Intracellular stimulation of single command neurons in isolated nervous systems, semi-intact prepartions, and restrained whole animals causes the same rhythmic motor output pattern as occurs during feeding. During this motor output pattern, action potentials recorded intracellularly from the command neurons occur in cyclic bursts that are phase-locked with the feeding rhythm. This modulation results from repetitive, alternating bursts of excitatory and inhibitory postsynaptic potentials, which are caused at least in part by synaptic feedback to the command neurons from identified classes of neurons in the feeding network. Central feedback to command neurons from the motor network they excite provides a possible general physiological mechanism for the sustained oscillation of neural networks controlling cyclic behavior.

Behavioral choice: neural mechanisms in Pleurobranchaea.

In the marine mollusk Pleurobranchaea, it is known that feeding occurs and withdrawal from tactile stimuli is suppressed when the sensory stimuli for feeding and withdrawal are presented simultaneously. This "dominance" of feeding behavior over withdrawal behavior occurs because the central nervous network controlling feeding inhibits the central nervous network controlling withdrawal. The inhibition is mediated by a bilaterally symmetrical pair of reidentifiable feeding neurons that are members of the "corollary discharge" population in the buccal ganglion. This study supports the hypothesis that inhibitory interactions between competing motor systems are responsible for the "singleness of action" that characterizes animal behavior.