Novel triplet of flexor muscles in the posterior tentacles of the snail, Helix pomatia.

The anatomy of three novel flexor muscles in the posterior tentacles of Helix pomatia is described. The muscles originate from the ventral side of the sensory pad and are anchored at different sites in the base of the tentacle stem. The muscles span the tentacle and always take the length of the stem which depends on the rate of tentacle protrusion indicating that the muscles are both contractile and extremely stretchable. The three anchoring points at the base of the stem determine three space axes along which the contraction of a muscle or the synchronous contraction of the muscles can move the tentacle in space.

Lateralized memory storage and crossed inhibition during odor processing by Limax.

After odor conditioning intact Limax maximus and injecting LY into their haemocoel, labeled groups of neurons are found in either the right or left procerebral lobe but never in both procerebral lobes. This suggests that a competitive interaction occurs between right and left odor processing pathways of which the procerebral lobe is a part. We use the nerve discharge in the external peritentacular nerve evoked by applying a puff of conditioned odor to the nose to document crossed inhibition between left and right odor processing pathways. Responses in the external peritentacular nerve evoked by stimulating one superior nose with a conditioned odor are strongly lateralized as responses occur only on the stimulated side. Stimulating both superior noses simultaneously with the same conditioned odor yields responses in both external peritentacular nerves that resemble the sum of responses to unilateral stimulation. Simultaneously stimulating both superior noses, each with a different conditioned odor leads to strong inhibition of both external peritentacular nerve responses. The crossed inhibition is also evident if both superior and inferior noses on the same side are stimulated simultaneously. A lateral inhibitory mechanism, situated postsynaptic to odor recognition, appears to inhibit external peritentacular nerve responses if the two noses receive conflicting sensory inputs.

Olfactory oscillations augment odor discrimination not odor identification by Limax CNS.

There is great interest in the function of synchronous oscillations in olfactory centers, as documented in a wide variety of species. In Limax procerebral (PC) lobe, local field potential oscillations are ongoing and altered by odor stimulation. Recordings from external peritentacular nerves (ePTNs) reveal a neural correlate of tentacle positioning, a response signifying recognition of a conditioned odor. Using the odor-elicited ePTN response we found no clear difference in the dynamics of the PC oscillations which precedes and predicts the occurrence of ePTN responses. Reversibly blocking the oscillations did not impair recognition of conditioned odors but reduced the differential nerve response to related odors. PC oscillations may play a role in odor discrimination but are not necessary for odor recognition.

Ganglionic distribution of inputs and outputs of C-PR, a neuron involved in the generation of a food-induced arousal state in Aplysia.

Cerebral neuron C-PR is thought to play an important role in the appetitive phase of feeding behavior of Aplysia. Here, we describe the organization of input and output pathways of C-PR. Intracellular dye fills of C-PR revealed extensive arborization of processes within the cerebral and the pedal ganglia. Numerous varicosities of varying sizes may provide points of synaptic inputs and outputs. Blocking polysynaptic transmission in the cerebral ganglion eliminated the sensory inputs to C-PR from stimuli applied to the rhinophores or tentacles, indicating that this input is probably mediated by cerebral interneurons. Identified cerebral mechanoafferent sensory neurons polysynaptically excite C-PR. Stimulation of the eyes and rhinophores with light depresses C-PR spike activity, and this effect also appears to be mediated by cerebral interneurons. C-PR has bilateral synaptic actions on numerous pedal ganglion neurons, and also has effects on cerebral neurons, including the MCC, Bn cells, CBIs and the contralateral C-PR. Although the somata of these cerebral neurons are physically close to C-PR, experiments using high divalent cation-containing solutions and cutting of various connectives indicated that the effects of C-PR on other cerebral ganglion neurons (specifically Bn cells and the MCC) are mediated by interneurons that project back to the cerebral ganglion via the pedal and pleural connectives. The indirect pathways of C-PR to other cerebral neurons may help to ensure that consummatory motor programs are not activated until the appropriate appetitive motor programs, mediated by the pedal ganglia, have begun to be expressed.

Nitric oxide, but not serotonin, is involved in acquisition of food-attraction conditioning in the snail Helix pomatia.

The effects of inhibition of nitric oxide (NO) or serotonin (injection of nitro-L-arginine methyl ester (L-NAME) or 5,6-dihydroxytryptamine (5,6-DHT), respectively) on food-attraction conditioning was investigated in Helix. Blocking NO synthase (NOS) prior to conditioning significantly impaired the food-finding ability of the snails. Food-conditioned snails, after inhibition of NOS, remained able to locate the conditioned food. These results indicate that the acquisition of memory depends on NO, whereas memory recall and olfactory orientation are not dependent. Ablating the serotonergic system did not influence food-attraction conditioning, suggesting that food-attraction conditioning may be at variance with conventional associative conditioning procedures.

BLIND MEXICAN CAVE FISH (ASTYANAX HUBBSI) RESPOND TO MOVING VISUAL STIMULI

In apparatus for measuring optomotor behaviour, blind Mexican cave fish, Astyanax hubbsi, increase their swimming velocity upon rotation of a striped cylinder, i.e. in response to a solely visual stimulus. The fish follow the movements of the stripes at (i) rotation velocities between 60 degrees s-1 and 80 degrees s-1, (ii) light intensities of less than 20 lx and, (iii) stimulus widths subtending an angle of less than 1 °. Extirpation of the vestigial eye structures does not affect the response to the moving visual stimulus, which indicates that the response is mediated by extra-ocular photoreceptors. An optomotor response can be reliably evoked in a round test aquarium. Fish do not respond when the test aquarium contains environmental cues, such as bars on the wall or when a section of the round aquarium is divided off. This indicates that the fish obtain information about their environment from different sensory sources and that the visual stimulus is effective only when no other means of orientation are available. We suggest a modified theory of the optomotor response, which emphasizes the crucial role of the environment in eliciting the response and which permits behaviours more complex than just following the stimulus.

Dopaminergic neuron B20 generates rhythmic neuronal activity in the feeding motor circuitry of Aplysia.

We have identified a buccal neuron (B20) that exhibits dopamine-like histofluorescence and that can drive a rhythmic motor program of the feeding motor circuitry of Aplysia. The cell fires vigorously during episodes of patterned buccal activity that occur spontaneously, or during buccal programs elicited by stimulation of identified cerebral command-like neurons for feeding motor programs. Preventing B20 from firing, or firing B20 at inappropriate times, can modify the program driven by the cerebral feeding command-like neuron CBI-2. When B20 is activated by means of constant depolarizing current it discharges in phasic bursts, and evokes a sustained coordinated rhythmic buccal motor program. The program incorporates numerous buccal and cerebral neurons associated with aspects of feeding responses. The B20-driven program can be reversibly blocked by the dopamine-antagonist ergonovine, suggesting that dopamine may be causally involved in the generation of the program. Although firing of B20 evokes phasic activity in cerebral command-like neurons, the presence of the cerebral ganglion is not necessary for B20 to drive the program. The data are consistent with the notion that dopaminergic neuron B20 is an element within the central pattern generator for motor programs associated with feeding.

Orientation of Aplysia californica to distant food sources.

The behavior of the marine mollusc Aplysia was examined under different experimental conditions designed to determine the food searching strategy of the animals. In a small, open field tank with still water, the animals took an average of 42 min to find a piece of seaweed, even though the stimulus was never located more than 30 cm away from the animal. Observations of the animals indicated that their search was not directed, without a clear tendency towards the food, and during the course of a search, they often crawled through most of the area of the tank. The search time, the distance travelled, and the strategy of the search of the animals was similar for different types of seaweed. If animals were aroused into activity by the presence of seaweed extract, the time for them to contact a piece of odorless glass fiber paper in the open field was not significantly different than that for a piece of seaweed. The probability at which the animals contacted the seaweed, as a function of the distance travelled, resembled the detection probability determined according to a theory of random search. We thus propose that the aroused animals move in a random pattern until they are very close to the food. This strategy can be advantageous in still water since chemicals do not provide distinct gradients that can serve as cues for chemotactic orientation from distances greater than a few centimeters from the source. In a Y-maze in still water, Aplysia did not perform above chance in selecting the arm that contained the seaweed.(ABSTRACT TRUNCATED AT 250 WORDS)

Activity of identified cerebral neuron correlates with food-induced arousal in Aplysia.

Firing of the cerebral-pedal regulator neuron, C-PR, evokes a constellation of responses which are characteristic of the food-induced arousal state that occurs following exposure of Aplysia to seaweed. To provide further evidence that C-PR plays a role in generating the food-induced arousal state, extracellular recordings from the cerebral-pedal connective, which contains the axon of C-PR, were obtained in freely moving animals. The C-PR spike in the connective recorded in vivo was then identified by comparing the wave form to the obtained by firing C-PR in an in vitro preparation. We report here that C-PR activity is evoked by food stimulation, and increased firing of the C-PR is closely correlated with appetitive head lifting, the first manifestation of the food arousal state.

Identification and characterization of cerebral-to-buccal interneurons implicated in the control of motor programs associated with feeding in Aplysia.

We identified candidate neurons in the cerebral ganglion that regulate feeding responses mediated by the buccal ganglion. Backfilling the cerebral-buccal connectives revealed that each cerebral hemi-ganglion contains approximately 20 neurons that project axons to the buccal ganglion. Three M-cluster neurons (CBI-1, CBI-2, CBI-3) and one E-cluster neuron (CBI-4) were identified as cerebral-to-buccal interneurons (CBIs) based on position, morphology, synaptic connections, and ability to drive buccal motor programs (BMPs). CBI-1 responds to touch of the tentacles, lips, and buccal mass. It receives monosynaptic EPSPs from interganglionic, cerebral-to-buccal mechanoafferent (ICBM) neurons and monosynaptically excites buccal cells, some of which are also excited by the ICBMs. Tonic firing of CBI-1 usually evokes a single cycle of BMP activity. CBI-1 phase-shifts the rhythmic BMP driven by firing a dopaminergic neuron in the buccal ganglion. CBI-1 itself exhibits dopamine-like histofluorescence following formaldehyde-glutaraldehyde fixation. CBI-2 is excited by food stimuli applied to the lips. Constant-current intracellular stimulation of CBI-2 produces phasic firing of the cell that reliably evokes a rhythmic BMP that incorporates buccal and cerebral motor neurons, putative pattern-generating and pattern-initiating neurons, and neuromodulatory cells (metacerebral cells). CBI-4 also evokes a rhythmic BMP, but the details of its actions and synaptic effects differ from that of CBI-2. CBI-3 does not evoke a BMP, even though it is excited by food stimuli applied to the lips, and it makes monosynaptic connections (both excitatory and inhibitory) to many follower cells of the other CBIs. Firing of CBI-3 phase-delays the BMP driven by CBI-2. Since its activity is incorporated into BMPs and it provides direct inputs to elements of the feeding circuitry, it may play a role in pattern generation. The distinctive features of the CBIs suggest that the consummatory phase of feeding may be controlled by a population of interneurons that subserve different roles.

Egg laying hormone inhibits a neuron (C-PR) involved in multiple manifestations of food-induced arousal in Aplysia.

Egg laying behavior is known to suppress feeding in Aplysia, but both behaviors have common responses involving head movements and posture. Egg laying hormone (ELH) applied in vitro to the isolated nervous system of Aplysia reduces the spontaneous and the evoked activity of the C-PR, a neuron implicated in postural responses during feeding. The inhibitory effect of ELH on the C-PR appears to be mediated by interneurons primarily located in the pedal/pleural ganglia, which contain all the known direct follower cells of the C-PR. Our results do not support the idea that postural responses during feeding and egg laying are mediated by the activation of a common arousal element, the C-PR. In fact, the C-PR seems to be a specific element for the food-arousal state, and the inhibition of the C-PR by ELH may contribute to the suppression of appetitive feeding responses during egg laying.

Studies of Behavioral State in Aplysia.

This paper reviews a series of studies on the neural organization and the cellular mechanisms underlying behavioral states; in these studies, feeding behavior in Aplysia was used as a model system. Feeding in Aplysia has similarities to motivated behaviors in other animals and is modulated by a number of interesting state variables, including arousal. Food-induced arousal manifests itself in two categories of feeding behavior: (1) appetitive responses (e.g., head-up feeding posture and directed head turning), which orient the animal to potential goal objects such as food; and (2) consummatory responses (biting, swallowing), which obtain the goal object. The consummatory responses are rhythmic and relatively stereotyped, whereas the appetitive responses are highly variable. Our evidence suggests that one consummatory response, biting, appears to be controlled by command elements in the cerebral-ganglion, such as neuron CBI-2, which are capable of driving the behavior. One component of the appetitive behavior, head lifting, may be controlled (at least in part) by another cerebral neuron, C-PR. C-PR, however, affects numerous systems in the animal, but all the systems affected seem to be involved in the food-induced arousal state of the animal. We postulate that C-PR is, in some ways, analogous to command neurons that evoke behaviors. The C-PR, however, not only evokes a behavior, but also evokes a central motive state which aids in insuring that behavior is efficiently expressed.

Appetitive feeding behavior of Aplysia: behavioral and neural analysis of directed head turning.

The appetitive phase of feeding behavior in Aplysia consists of a behavioral sequence in which the quiescent animal starts to locomote and then assumes a characteristic feeding posture. In this position, head-turning responses can be elicited by a localized food stimulus (seaweed) delivered to the lips or tentacles. In response to brief (open loop) stimulation with seaweed, the animal turns toward the stimulus but greatly overshoots the target. However, the angular velocity and the final turning angle are a function of the eccentricity of the stimulus, progressively increasing with greater eccentricities. In a food-aroused animal, a brief tactile stimulus evokes turning and biting responses similar to those triggered by seaweed, which provides both tactile and chemical stimulation. Upon repeated tactile stimulation, however, the response magnitude decrements rapidly, whereas the magnitude remains high when turning responses are repeatedly elicited by food stimuli. A purely chemical stimulus sometimes can elicit a turning response, but chemical stimuli alone are much less efficacious than tactile stimuli alone. When the stimulus is maintained in a stationary position (closed loop), the animal turns until its mouth is oriented over the food. A turning response to a lateral stimulus can be reduced by an immediately following medial stimulus. To explain the above findings, we propose a form of response substitution, in which the response to the first, lateral stimulus is substituted by a weaker response to a more medial stimulus. No turning response is evoked when the animal is stimulated while performing spontaneous or evoked bites, though biting per se does not interrupt ongoing turning movements. In animals with lesions of the cerebral-buccal connectives, a food stimulus on the mouth is also followed by a reduction of the capacity of stimuli to elicit turning responses. In these lesioned animals, the food stimulus appears to elicit a bite command, though the biting behavior itself does not occur. Thus, it appears that the bite-related gating of stimuli is of cerebral origin, rather than due to the generation of the buccal motor program. The force necessary to power the turning movements was calculated from the trajectories of the movements. The results indicate that a power phase during the first half of the duration of the total movement is sufficient to generate a turn. The power phase can be followed by a brief gliding phase, and finally the movement appears to be actively terminated.(ABSTRACT TRUNCATED AT 400 WORDS)

An identified neuron (CPR) evokes neuronal responses reflecting food arousal in Aplysia.

Feeding behavior of Aplysia is associated with an arousal state characterized by a constellation of maintained behaviors and by a potentiation or depression of responses to specific stimuli. A neuron (the cerebral-pedal regulator or CPR) that has widespread actions on various systems connected with feeding has been identified. CPR excites neurons that modulate or drive (i) body posture, (ii) biting, and (iii) cardiovascular behaviors. CPR also inhibits neurons concerned with defensive responses. Food stimuli, which elicit food arousal in the animal, produce prolonged excitation of the CPR. The results suggest that the CPR may evoke a central motive state representing the neuronal correlate of feeding motivation.

Morphological differences in neuromasts of the blind cave fish Astyanax hubbsi and the sighted river fish Astyanax mexicanus.

Vital staining and scanning electron microscopy were used to study the morphology of superficial neuromasts in the blind cave fish, Astyanax hubbsi, and its sighted congener, Astyanax mexicanus. In blind Astyanax the neuromasts are 80 X 50 microns in size and possess cupulae with an oval basal shape. The length of the cupula is correlated to the location of its neuromast. Head neuromasts were found to have the longest cupulae (up to 300 microns), and caudal neuromasts have the smallest. Cupulae of all lengths have been observed to be easily bent by water movements. The neuromasts are directionally sensitive in that the bending of their cupulae perpendicular to the longer axis of the cupulae provides maximal excitation. A comparison of superficial neuromasts in the blind A. hubbsi to those in sighted A. mexicanus revealed several structural differences. The neuromasts in the blind fish are twice as large. Their cupulae, in particular, are much longer and seem to have supporting attachments at their edges. The greater length of the cupulae in blind cave fish may be of particular importance for the functioning of the lateral line organ, since longer cupulae protrude beyond the boundary layer in faster water currents and thus can increase the sensitivity of the neuromast. The specific morphology of the neuromasts in the blind cave fish appears to reflect an evolutionary adaptation which can serve to improve the functioning of the lateral line system and thereby compensate for the lack of eyes.

A subpopulation of cerebral B cluster neurones of Aplysia californica is involved in defensive head withdrawal but not appetitive head movements.

The cerebral B cluster neurones of Aplysia californica were studied under experimental conditions designed to evoke head movements in a selective fashion: either to approach an appetitive stimulus, or to withdraw from an aversive one. Intracellular recordings indicated the presence of two types of B cluster neurones: Bn cells that had fast (narrow) spikes, and Bb cells that had slow (broad) spikes. Tactile stimulation of the tentacles, rhinophores and lips excited Bn neurones, but inhibited Bb neurones. Intracellular stimulation of Bn cells evoked contractions of body wall muscles. No contractions were observed when Bb cells were fired, indicating that it is unlikely that the Bb neurones are motor neurones. Several lines of evidence indicated that the Bn type neurones are involved in withdrawal responses but not in appetitive head turning. (1) Elimination of the descending axons of the Bn cells by lesioning the cerebropleural connectives (C-Pl connectives) did not affect the head-turning response. This lesion significantly altered the head-withdrawal response by selectively eliminating an initial fast component of the withdrawal movement. (2) In chronic recordings from the C-Pl connective, unit activity was obtained which was correlated with the presentation of an appetitive stimulus rather than with evoked or spontaneous turning movements. A substantial increase in activity also occurred during head withdrawal of the animal. On the basis of these data, we postulate that separate populations of motor neurones are responsible for the aversive withdrawal of the head, and for the directed turning response towards a stimulus.

Flow field, swimming velocity and boundary layer: parameters which affect the stimulus for the lateral line organ in blind fish.

The data presented support the hypothesis that the flow field supplies the stimulus to the lateral line organ (LLO) in blind cave fish (Anoptichthys jordani). Two basic predictions from the theoretical analysis of the flow field were confirmed: (i) individual blind cave fish prefer particular swimming velocities, (ii) the velocity preferred depends on the cross-sectional area (CSA) of the fish, i.e. the smaller the CSA the higher the swimming velocity. This relationship was found also in experimentally blinded fish of other species. Furthermore, when placed in unfamiliar surroundings, blind cave fish swim at higher velocities than in familiar surroundings for a certain habituation period. The boundary layer which surrounds the fish attenuates the amplitude of the hydrodynamic stimulus because of its damping properties. Computations of the current velocity distribution within the boundary layer indicate that the stimulus for freestanding neuromasts is considerable even during swimming in open water.

Collision with and avoidance of obstacles by blind cave fish Anoptichthys jordani (Characidae).

Blind Mexican cave fish (Anoptichthys jordani) were released into unknown surroundings and their swimming tracks were recorded. During the first 24 h in a new tank, i.e., in unfamiliar environments, the average swimming velocity of the fish was higher than during the remaining time in the tank. Collision with obstacles was recorded by an electrical contact detection method. Single frame analysis of video-recordings revealed that collision can be correlated with tail movements when the fish is close to an obstacle, whereas avoidance correlates with gliding during approach to an obstacle. These behavior patterns are discussed in the context of the flow field hypothesis and inhibition of the lateral line organ during movement.