Dual and opposing modulatory effects of serotonin on crayfish lateral giant escape command neurons.

Serotonin modulates afferent synaptic transmission to the lateral giant neurons of crayfish, which are command neurons for escape behavior. Low concentrations, or high concentrations reached gradually, are facilitatory, whereas high concentrations reached rapidly are inhibitory. The modulatory effects rapidly reverse after brief periods of application, whereas longer periods of application are followed by facilitation that persists for hours. These effects of serotonin can be reproduced by models that involve multiple interacting intracellular signaling systems that are each stimulated by serotonin. The dependence of the neuromodulatory effect on dose, rate, and duration of modulator application may be relevant to understanding the effects of natural neuromodulation on behavior and cognition and to the design of drug therapies.

Fifty years of a command neuron: the neurobiology of escape behavior in the crayfish.

Fifty years ago C.A.G. Wiersma established that the giant axons of the crayfish nerve cord drive tail-flip escape responses. The circuitry that includes these giant neurons has now become one of the best-understood neural circuits in the animal kingdom. Although it controls a specialized behavior of a relatively simple animal, this circuitry has provided insights that are of general neurobiological interest concerning matters as diverse as the identity of the neural substrates involved in making behavioral decisions, the cellular bases of learning, subcellular neuronal computation, voltage-gated electrical synaptic transmission and modification of neuromodulator actions that result from social experience. This work illustrates the value of studying a circuit of moderate, but tractable, complexity and known behavioral function.

Neuronal coincidence detection by voltage-sensitive electrical synapses.

Coincidence detection is important for functions as diverse as Hebbian learning, binaural localization, and visual attention. We show here that extremely precise coincidence detection is a natural consequence of the normal function of rectifying electrical synapses. Such synapses open to bidirectional current flow when presynaptic cells depolarize relative to their postsynaptic targets and remain open until well after completion of presynaptic spikes. When multiple input neurons fire simultaneously, the synaptic currents sum effectively and produce a large excitatory postsynaptic potential. However, when some inputs are delayed relative to the rest, their contributions are reduced because the early excitatory postsynaptic potential retards the opening of additional voltage-sensitive synapses, and the late synaptic currents are shunted by already opened junctions. These mechanisms account for the ability of the lateral giant neurons of crayfish to sum synchronous inputs, but not inputs separated by only 100 microsec. This coincidence detection enables crayfish to produce reflex escape responses only to very abrupt mechanical stimuli. In light of recent evidence that electrical synapses are common in the mammalian central nervous system, the mechanisms of coincidence detection described here may be widely used in many systems.

Postexcitatory inhibition of the crayfish lateral giant neuron: a mechanism for sensory temporal filtering.

Crayfish escape from threats by either giant neuron-mediated "reflex" tail flexions that occur with very little delay but do not allow for much sensory guidance of trajectory or by "nongiant" tail flexion responses that allow for sensory guidance but occur much less promptly. Thus, when a stimulus occurs, the nervous system must make a rapid assessment of whether to use the faster reflex system or the slower nongiant one. It does this on the basis of the abruptness of stimulus onset; only stimuli of very abrupt onset trigger giant-mediated responses. We report here that stimuli which excite the lateral giant (LG) command neurons for one form of reflex escape also produce a slightly delayed postexcitatory inhibition (PEI) of the command neurons. As a result, only stimuli that become strong enough to excite the command neurons to firing threshold before the onset of PEI, within a few milliseconds of stimulus onset, can cause giant-mediated responses. This inhibition is directed to distal dendrites of the LG neurons, which allows for some location specificity of PEI within the sensory field of a single hemisegment.

Altered excitability of the crayfish lateral giant escape reflex during agonistic encounters.

The excitability of the lateral giant escape reflex of socially dominant and submissive crayfish at rest and during agonistic encounters was studied and compared. During agonistic encounters the excitability of the lateral giant reflex falls, substantially in subordinates and slightly in dominants, whereas at rest excitability seems to be independent of social status. Thus, paradoxically, socially dominant animals are more likely to execute lateral giant escape reactions during interactions than are subordinates. It is suggested that subordinates under threat of attack tend to engage circuitry involved in flexible, nonreflex ("voluntary") types of escape not mediated by giant neurons and therefore inhibit giant neuron-mediated reflex circuitry that produces prompt, but less adaptive, responses. In contrast, dominants go about their business, mainly ignoring their conspecifics and relying on reflex escape to protect them from unexpected attack. Consistent with this view, escape of subordinates during agonistic encounters is mediated by nongiant, not reflex, circuitry. These observations and their interpretation suggest a possible functional role for recently described social status-dependent serotonergic modulation of the lateral giant reflex, which is inhibitory in sign in subordinates and facilitatory in dominants.

Habituation of an invertebrate escape reflex due to modulation by higher centers rather than local events.

Learning is widely thought to result from altered potency of synapses within the neural pathways that mediate the learned behavior. Support for this belief, which pervades current physiological and computational thinking, comes especially from the analysis of cases of simple learning in invertebrates. Here, evidence is presented that in one such case, habituation of crayfish escape, the learning is more due to onset of tonic descending inhibition than to the intrinsic depression of circuit synapses to which it was previously attributed. Thus, the altered performance seems to depend at least as much on events in higher centers as on local plasticity.

The mechanism of tonic inhibition of crayfish escape behavior: distal inhibition and its functional significance.

The excitability of crayfish escape behavior is seldom fully predictable. A major determinant of this fickleness is a form of descending inhibition that is reliably evoked during restraint or feeding and is called "tonic inhibition." Tonic inhibition was found to inhibit postsynaptically the lateral giant neurons, the command neurons for one form of escape. This inhibition is located on lateral giant dendrites that are electrotonically distant from the neuron's spike initiating zone. in contrast, the postsynaptic inhibition due to "recurrent inhibition," which prevents new escape responses from starting while a previously initiated one is in process, occurs proximally, near the spike initiating zone. The distalness of tonic inhibition could be an adaptation for selective suppression of parts of the lateral giant dendritic tree. Consistent with this, evidence was obtained that the tonic inhibitory system can suppress responses to specific sensory fields. An independent reason for targeting recurrent inhibition proximally and tonic inhibition distally was suggested by the functional requirements of each inhibitory process: recurrent inhibition needs to be "absolute" in the sense that the response should be absolutely prevented, whereas it must be possible to override tonic inhibition. Neuronal models demonstrated that proximal inhibition gives recurrent inhibition the required property of absoluteness while distal inhibition allows tonic inhibition to be overridden ("relativity"). It was shown that the relativity of distal inhibition arises from its interaction with the process of saturation of excitation and that tonic inhibition does indeed interact with excitatory saturation as predicted. It is suggested that the property of relativity of distal inhibition is exploited in other nervous systems as well.

Crayfish tonic inhibition: prolonged modulation of behavioral excitability by classical GABAergic inhibition.

Previous studies have indirectly implicated the two neurotransmitters 5-HT and GABA in mediating tonic inhibition of the crayfish lateral giant (LG) escape reaction. In this study, pharmacological agents were selectively delivered to restricted portions of the abdominal CNS (where LG escape circuitry resides) to assess directly the role of these two transmitters in tonic inhibition. Both 5-HT and GABA depressed monosynaptic, electrical transmission to the LG neurons, the command neurons for LG escape, and application of either transmitter resulted in a depolarizing conductance increase in the LG neuron. The effects of 5-HT persisted in preparations in which chemical transmission was effectively abolished, implying that there are 5-HT receptors on the LG neuron itself, along with the known GABA receptors. Restricted delivery of the GABA chloride channel blocker picrotoxin to only the abdominal CNS blocked the expression of tonic inhibition there (without interfering with the rostral generation of tonic inhibition). Therefore, if 5-HT mediated tonic inhibition, the effects of 5-HT on the abdomen should also be antagonized by picrotoxin. However, this was not the case, thus suggesting that 5-HT does not mediate tonic inhibition. The most likely neurotransmitter used for tonic inhibition is GABA acting via ligand-gated chloride channels. Thus, although this form of behavioral modulation can be tonically active for very long periods, it nevertheless appears to be mediated by a classical synaptic mechanism.

Ultrastructure of the circuit providing input to the crayfish lateral giant neurons.

Labeled or otherwise identified neurons of the crayfish lateral giant escape reaction circuit were examined electron microscopically and the findings compared to expectations from physiology. Terminals of primary afferents contained clear, approximately 45 nm, irregularly round synaptic vesicles, while sensory interneuron terminals had slightly larger, 50 nm, more strictly round vesicles, permitting tentative classification based on anatomical criteria. Excitatory synapses on the lateral giants, believed from physiology to be electrical, generally had some gap junctions, but these were almost invariably paralleled by more prominent chemical junctional regions of unknown function. There may also be a class of interneurons making purely chemical synapses on the lateral giants. Synapses from primary afferents to sensory interneurons, believed from physiology to be cholinergic, had purely chemical morphology. Synapses with narrow elongated vesicles, similar to GABAergic vesicles seen in other neurons, frequently occurred on terminals of primary afferents. These synapses provide a basis for known presynaptic inhibition of afferent input. Consistent with physiology, such inhibitors sometimes also contacted the postsynaptic targets of the primary afferents and sometimes received input from other primary afferents. Afferent terminals also received some input from profiles rich in large dense cored vesicles. Presumptive inhibitory input found on proximal dendrites of lateral giants provides a basis for known recurrent inhibition. However, similar inhibitory synapses that sometimes received local input from excitors of the lateral giants were also found distally mixed with excitatory inputs. These provide a basis for recently discovered distal inhibitory input following excitation and for tonic inhibition.

Cholinergic transmission at the first synapse of the circuit mediating the crayfish lateral giant escape reaction.

1. The chemical synapses between mechanoreceptor neurons and first-order interneurons in the lateral giant (LG) neuron escape circuit of the crayfish have plastic properties, some of which are believed to be the basis for behavioral habituation and sensitization. In this investigation pharmacological experiments were conducted to assess the role of cholinergic synaptic transmission in this pathway. 2. Arterial perfusion of the cholinergic agonist carbachol produced increased activity of many abdominal nerve cord units, including an identified first-order interneuron (interneuron A) in the LG circuit. A general increase in activity of interneurons in this circuit in the presence of certain cholinergic agonists was inferred from an increase in the frequency of occurrence of spontaneous excitatory postsynaptic potentials (EPSPs) recorded in the LG. 3. Cholinergic antagonists reduced the amplitude of spontaneous and evoked sensory neuron-to-interneuron A EPSPs and decreased the disynaptic (via 1st-order interneurons) component of evoked EPSPs in the LG. These effects indicate that postsynaptic cholinergic receptors are utilized in mechanosensory synaptic transmission to the first-order interneurons of this circuit. The relative potencies of the blockers tested (mecamylamine > picrotoxin >>> curare > atropine) suggest that the receptors on the interneurons belong to a previously characterized class of crustacean cholinergic receptors that resemble the ganglionic nicotinic subtype of vertebrates. 4. Nicotinic agonists (carbachol, tetramethylammonium hydroxide, 1,1-dimethyl-4-phenyl-piperazium iodide) produced depolarizing (decreased input resistance) responses on the LG neuron itself. These responses persisted during blockade of chemical transmission by cobalt. The presence of cholinergic receptors on the LG, a cell in which all known inputs mediating sensory excitation are electrical, is discussed. 5. Application of muscarinic agonists (pilocarpine, oxotremorine) resulted in a long-lasting reduction of the evoked sensory neuron-to-interneuron A EPSP and the disynaptic component of the evoked EPSP in the LG. No effects on the membrane potential or input resistance of the interneurons were detected. It is proposed that presynaptic receptors with a muscarinic profile are present on mechanosensory neurons and that these receptors mediate a reduction of transmitter release.

Evidence for a computational distinction between proximal and distal neuronal inhibition.

Most neurons have inhibitory synapses both "proximally" near the spike-initiating zone and "distally" on dendrites. Although distal inhibition is thought to be an adaptation for selective inhibition of particular dendritic branches, another important distinction exists between proximal and distal inhibition. Proximal inhibition can attenuate excitatory input absolutely so that no amount of excitation causes firing. Distal inhibition, however, inhibits relatively; any amount of it can be overcome by sufficient excitation. These properties are used as predicted in the circuit-mediating crayfish escape behavior. Many neuronal computations require relative inhibition. This could partly account for the ubiquity of distal inhibition.

Response-dedicated trigger neurons as control points for behavioral actions: selective inhibition of lateral giant command neurons during feeding in crayfish.

Feeding behavior suppresses lateral giant neuron-mediated escape behavior in crayfish. The suppression appears to result from reduced transmission to the lateral giants from primary afferents and/or sensory interneurons, while the operation of sensory and motor networks themselves is unaffected. It is suggested that control occurs at the level of the lateral giants because these neurons are pivotal in, and dedicated solely to, producing the type of escape that needs to be controlled. It is hypothesized that response-dedicated sets of neurons that play a similar role will probably be found wherever it is necessary to control particular responses selectively.

Cooperativity-dependent long-lasting potentiation in the crayfish lateral giant escape reaction circuit.

The ability of sensory neuron firing to cause the lateral giant escape reaction increases following repeated sensory volleys at 4 Hz for 10 sec. The increase occurs only when relatively large numbers of afferents are repetitively stimulated, decays with a mean time constant of 21 hr, is confined to the ganglia at which the repeated sensory volleys enter the nerve cord, and is at least partially specific to those roots of a ganglion that were tetanized. Transmission at the chemical synapses between afferents and the largest of the first-order sensory neurons that link afferents to the lateral giants displays a similar potentiation. This phenomenon shares many properties with hippocampal long-term potentiation.

Silencing normal input permits regenerating foreign afferents to innervate an identified crayfish sensory interneuron.

Interneuron A, an identified first-order sensory interneuron that is innervated by mechanoreceptors on one side of the crayfish tailfan, normally resists extra innervation by regenerating contralateral mechanoreceptor axons. However, if its normal innervation is silenced by covering mechanosensory hairs with a surgical glue, it accepts contralateral innervation. This finding on an arthropod provides evidence for the generality and antiquity of the principle, critical in development of the vertebrate nervous system, that activity of one set of afferents can control whether other afferents form synapses on a target.

5,7-Dihydroxytryptamine lesions of crayfish serotonin-containing neurons: effect on the lateral giant escape reaction.

The crayfish's lateral giant escape response, a relatively simple behavioral reaction, is readily modulated in certain situations. For example, when a crayfish is restrained, its lateral giant (LG) fibers--command neurons that mediate the escape response--are strongly inhibited (Krasne and Wine, 1975). Previous work (Glanzman and Krasne, 1983) had suggested that serotonin (5-HT) might mediate this restraint-induced inhibition of the escape response. To test this possibility, we attempted to lesion serotonergic neurons in crayfish with the 5-HT neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT). We compared the levels of 5-HT-immunoreactive staining in nerve cords from 5,7-DHT-treated and from normal crayfish to assess 5,7-DHT's effectiveness. Levels of immunoreactive staining, as judged by ratings of the visibility of immunofluorescence, were significantly lower in nerve cords from crayfish that had received injections of 5,7-DHT (1.0-4.0 mg) than in nerve cords from normal crayfish. In addition, some serotonergic neurons in the neurotoxin-treated crayfish developed an abnormal brown pigmentation. To assess the behavioral consequence of central serotonergic lesions, we compared the responsiveness of escape in crayfish treated with 5,7-DHT (2.0-2.75 mg) and in normal crayfish. The threshold for firing the LGs was significantly lower in restrained neurotoxin-treated animals than in restrained normal animals. Furthermore, the responsiveness of the LGs in neurotoxin-treated crayfish approximated that in crayfish whose nerve cords had been severed between the thorax and abdomen, a procedure known to abolish restraint-induced inhibition (Krasne and Wine, 1975).(ABSTRACT TRUNCATED AT 250 WORDS)

Sensitization of the crayfish lateral giant escape reaction.

Most behavioral reactions that habituate can also be dishabituated by strong stimuli. In the best studied cases, dishabituation seems to be the result of an independent "sensitization" of the behavioral reaction that compensates for habituation without necessarily abolishing it. Crayfish lateral giant (LG) neuron-mediated escape reactions are one of the most fully analyzed behavioral reactions that are prone to habituation; however, sensitization/dishabituation of LG escape has not previously been reported. Here, the effect of strong AC shocks to head or abdomen on the ability of 0.1 msec "test" shocks to sensory roots innervating the tailfan to elicit an LG escape response was examined. Following single AC shocks, test shock threshold for eliciting LG escape reliably fell 5-80% and recovered over 15 min to 1 hr. When AC shocks and test shocks alternated at 90 sec intervals, test shock threshold rapidly dropped to an asymptote that was maintained as long as AC shocks were given (up to 2 hr); following such repeated AC shocks, recovery often required a number of hours but was complete within 24. Comparable sensitization is seen in the response of interneuron A, the largest of a set of sensory interneurons that links afferents to LGs. AC shocks (to either head or tail) no longer sensitize abdominal LG reflex circuitry if the nerve cord is severed between thorax and abdomen. Thus, sensitization appears to depend on a neurally conducted influence that arises in the rostral half of the animal. Pharmacological evidence suggests that octopamine may mediate the sensitization.

Intracellular analysis of the innervation of a crayfish sensory interneuron by regenerating afferents.

Mechanoreceptors of the crayfish tail fan have peripheral somata and send their axons to the last (sixth) abdominal ganglion via five bilateral pairs of nerve roots (R1-R5). Comparisons were made between normal crayfish and regenerate preparations in which R4 had been cut and directed back to an extensively denervated sixth abdominal ganglion; 8 to 15 weeks postoperatively, an identified target interneuron (A) in this ganglion was impaled, and its response to water currents, electrical excitation of R4, and stimulation of individual sensory hairs supplying axons to R4 was studied along with several other properties of the pre- and post-synaptic neurons. Normal levels of excitability in A to R4 stimulation were achieved within six weeks as judged by extracellular criteria. Subsequent intracellular analysis revealed that few differences exist between regenerated and normal inputs: probability of (re-) connection, unitary EPSP amplitude and time course distributions, resting membrane potentials, and critical firing levels were comparable in the two groups; input impedance, however, may have been lower in regenerates. Compound electrically elicited EPSPs were similar in amplitude, rise time, and half amplitude width, but differed slightly in latency to onset (regenerates greater than normals). This was accounted for by differences in conduction time to the ganglion in regenerates, and central delay estimates suggest that connections in both groups are monosynaptic. The body root (R1) providing input to A that remained intact in the regenerate preparations increased in efficacy; over 12 postoperative weeks the response of A to R1 activation by water drops steadily increased and at 12 weeks unitary increments to ascending electrical stimulation of R1 were significantly larger than in normals. The response to giant interneuron activation demonstrated that recurrent inhibitory inputs were normal in regenerates. In addition, synaptic depression, normally responsible for behavioral habituation in this system, was comparable across groups. Further, protection from habituation was observed in both normals and regenerates if R4 stimulation was preceded by giant interneuron activation, thus indicating that normal presynaptic inhibitory inputs to the regenerated afferent terminals have also successfully regenerated.(ABSTRACT TRUNCATED AT 400 WORDS)

Crayfish escape behavior: production of tailflips without giant fiber activity.

The giant interneurons of the crayfish nerve cord are well-known mediators of fast tail flexions, "tailflips," that propel animals through the water away from danger. More recent studies have revealed an additional nongiant generator of tailflips. In contrast to giant tailflips, which are stereotyped, nongiant tailflips have variable form. The operating principles and portions of the neural circuitry governing nongiant tailflips were here investigated. Whereas fast flexor motor neurons (FFs) receive excitatory postsynaptic potentials (EPSPs) with large unitary components just prior to giant tailflips, excitation of the FFs during nongiant tailflips is due to summation of many small EPSPs, and these build up for about 60 ms prior to the tailflip; we call the period of excitation prior to FF firing the preflexion phase and the period during which FFs fire, the flexion phase of the tailflip. Even FFs that will not fire during a given tailflip become depolarized during preflexion and flexion periods. Throughout the preflexion and flexion periods there is activity in dorsal nerve cord axons (DCAs) that lie below the giants. Many DCAs are interneurons that excite FFs at short latency. Some DCAs fire uniquely during the preflexion phase, while some fire only during the flexion phase. Which DCAs fire is highly variable, and in some cases firing of particular DCAs can be correlated with particular forms of tailflips. Two identified DCAs, 12 and 13, that fire during the flexion phase were studied. These interneurons originate and receive their synaptic input in the second and third abdominal ganglia, respectively, and project to the last ganglion exciting FFs caudal to their ganglion of origin en route. Their pattern of synaptic input prior to and during nongiant tailflips is indistinguishable from that of FFs. Their input to FFs is weak, but when they fire they tend to promote intersegmental synchrony of FFs in the segments they feed. It appears likely to us that nongiant tailflips are synthesized from a small library of component tailflip movements that can be combined to produce a variety of complete tailflips and that the component movements are produced by a limited group of premotor interneurons, of which 12 and 13 are members.(ABSTRACT TRUNCATED AT 400 WORDS)