Differential role of inhibition in habituation of two independent afferent pathways to a common motor output

Many studies of the neural mechanisms of learning have focused on habituation, a simple form of learning in which a response decrements with repeated stimulation. In the siphon-elicited siphon withdrawal reflex (S-SWR) of the marine mollusk Aplysia, the prevailing view is that homosynaptic depression of primary sensory afferents underlies short-term habituation. Here we examined whether this mechanism is also utilized in habituation of the tail-elicited siphon withdrawal reflex (T-SWR), which is triggered by an independent, polysynaptic afferent pathway that converges onto the same siphon motor neurons (MNs). By using semi-intact preparations in which tail and/or siphon input to siphon MNs could be measured, we found that repeated tail stimuli administered in the presence of a reversible conduction block of the nerves downstream of the tail sensory neurons (SNs) completely abolished the induction of habituation. Subsequent retraining revealed no evidence of savings, indicating that the tail SNs and their immediate interneuronal targets are not the locus of plasticity underlying T-SWR habituation. The networks closely associated with the siphon MNs are modulated by cholinergic inhibition. We next examined the effects of network disinhibition on S-SWR and T-SWR habituation using an Ach receptor antagonist d-tubocurarine. We found that the resulting network disinhibition disrupted T-SWR, but not S-SWR, habituation. Indeed, repeated tail stimulation in the presence of d-tubocurarine resulted in an initial enhancement in responding. Lastly, we tested whether habituation of T-SWR generalized to S-SWR and found that it did not. Collectively, these data indicate that (1) unlike S-SWR, habituation of T-SWR does not involve homosynaptic depression of SNs; and (2) the sensitivity of T-SWR habituation to network disinhibition is consistent with an interneuronal plasticity mechanism that is unique to the T-SWR circuit, since it does not alter S-SWR.     

Habituation in Aplysia: The Cheshire Cat of neurobiology

The marine snail, Aplysia californica, is a valuable model system for cell biological studies of learning and memory. Aplysia exhibits a reflexive withdrawal of its gill and siphon in response to weak or moderate tactile stimulation of its skin. Repeated tactile stimulation causes this defensive withdrawal reflex to habituate. Both short-term habituation, lasting <30 min, and long-term habituation, which can last >24 h, have been reported in Aplysia. Habituation of the withdrawal reflex correlates with, and is in part due to, depression of transmission at the monosynaptic connection between mechanoreceptive sensory neurons and motor neurons within the abdominal ganglion. Habituation-related short-term depression of the sensorimotor synapse appears to be due exclusively to presynaptic changes. However, changes within the sensory neuron, by themselves, do not account for more persistent depression of the sensorimotor synapse. Recent behavioral work suggests that long-term habituation in Aplysia critically involves postsynaptic processes, specifically, activation of AMPA- and NMDA-type receptors. In addition, long-term habituation requires activity of protein phosphatases, including protein phosphatases 1, 2A, and 2B, as well as activity of voltage-dependent Ca²⁺ channels. Cellular work has succeeded in demonstrating long-term, homosynaptic depression (LTD) of the sensorimotor synapse in dissociated cell culture and, more recently, LTD of the glutamate response of isolated motor neurons in culture (“hemisynaptic” LTD). These in vitro forms of LTD have mechanistic parallels to long-term habituation. In particular, homosynaptic LTD of the sensorimotor synapse requires elevated intracellular Ca²⁺ within the motor neuron, and hemisynaptic LTD requires activity of AMPA- and NMDA-type receptors. In addition, activation of group I and II metabotropic glutamate receptors (mGluRs) can induce hemisynaptic LTD. The demonstration of LTD in vitro opens up a promising new avenue for attempts to relate long-term habituation to cellular changes within the nervous system of Aplysia.     

Analyses of habituation in Caenorhabditis elegans

Although the nonassociative form of learning, habituation, is often described as the simplest form of learning, remarkably little is known about the cellular processes underlying its behavioral expression. Here, we review research on habituation in the nematode Caenorhabditis elegans that addresses habituation at behavioral, neural circuit, and genetic levels. This work highlights the need to understand the dynamics of a behavior before attempting to determine its underlying mechanism. In many cases knowing the characteristics of a behavior can direct or guide a search for underlying cellular mechanisms. We have highlighted the importance of interstimulus interval (ISI) in both short- and long-term habituation and suggested that different cellular mechanisms might underlie habituation at different ISIs. Like other organisms, C. elegans shows both accumulation of habituation with repeated training blocks and long-term retention of spaced or distributed training, but not for massed training. Exposure to heat shock during the interblock intervals eliminates the long-term memory for habituation but not the accumulation of short-term habituation over blocks of training. Analyses using laser ablation of identified neurons, and of identified mutants have shown that there are multiple sites of plasticity for the response and that glutamate plays a role in long-term retention of habituation training.     

Habituation of the proleg withdrawal reflex in Manduca sexta does not involve changes in motoneuron properties or depression at the sensorimotor synapse

Larvae of the hawkmoth, Manduca sexta, exhibit a defensive proleg withdrawal reflex in which deflection of mechanosensory hairs on the proleg tip (the planta) evokes retraction of the proleg. A previous behavioral study showed that this reflex habituates in response to repeated planta hair deflection and exhibits several other defining features of habituation. In a semi-intact preparation consisting of a proleg and its associated segmental ganglion, repeated deflection of a planta hair or electrical stimulation of its sensory neuron causes a neural correlate of habituation, manifested as a decrease in the number of action potentials evoked in the proleg motor nerve. Monosynaptic connections from planta hair sensory neurons to the principal planta retractor motoneuron exhibit several forms of activity-dependent plasticity. In the present study we recorded intracellularly from this motoneuron during repetitive electrical stimulation of a planta hair sensory neuron. The number of action potentials evoked in the motoneuron decreased significantly, representing a neural correlate of habituation. The motoneuron's resting membrane potential, input resistance. and spike threshold measured before and after repetitive stimulation did not differ between the stimulated group and a control group. Furthermore, the amplitude of the monosynaptic excitatory postsynaptic potential, as well as the magnitude of paired-pulse facilitation, evoked in the motoneuron by the sensory neuron did not change after repetitive stimulation. These results suggest that depression at the sensorimotor synapse does not contribute to reflex habituation. Rather, other mechanisms in the ganglion of the stimulated segment, such as changes in polysynaptic reflex pathways, appear to be responsible.     

A new group-training procedure for habituation demonstrates that presynaptic glutamate release contributes to long-term memory in Caenorhabditis elegans

In the experiments reported here we have developed a new group-training protocol for assessing long-term memory for habituation in Caenorhabditis elegans. We have replicated all of the major findings of the original single-worm protocol using the new protocol: (1) distributed training produced long-term retention of training, massed training did not; (2) distributed training at long interstimulus intervals (ISIs) produced long-term retention, short ISIs did not; and (3) long-term memory for distributed training is protein synthesis-dependent as it could be blocked by heat shock during the inter-block interval. In addition, we have shown that long-term memory for habituation is graded, depending on the number of blocks of stimuli in training. The inter-block interval must be >40 min for long-term retention of training to occur. Finally, we have tested long-term memory for habituation training in a strain of worms with a mutation in a vesicular glutamate transporter in the sensory neurons that transduce tap (eat-4). The results from these eat-4 worms indicate that glutamate release from the sensory neurons has an important role in the formation of long-term memory for habituation.     

Generalization of Habituation and Intrinsic Sensitization in the Leech

Using the shortening reflex of the medicinal leech Hirudo medicinalis we examined stimulus generalization of habituation learning. Preparations received mechanosensory stimulus at two positions on the leech body wall, one site used to carry out habituation training and a second novel site to test for generalization of habituation. After training, the specific mechanosensory neurons activated by each stimulus were assessed using intracellular recordings. As expected, the closer the two sites were to each other, the greater the degree of generalization of habituation at the novel site and the more sensory cells were shared. However, a form of behavioral facilitation was observed at the trained site that resembled behavioral sensitization, but differed from the standard sensitization process in several respects. (1) Facilitation was induced by stimulation of the novel site before habituation training at the trained site, although the stimulus intensity at the novel site was equivalent to the training stimuli and was not the strong, noxious stimuli that normally induce sensitization. (2) The magnitude of the facilitating effect was proportional to the proximity of the novel and trained stimulation sites. (3) Although behavior at the trained site was facilitated, behavior at the novel site was habituated, indicating that the induced behavioral facilitation did not generalize throughout the animal, as normally occurs during sensitization, but was limited to a single stimulus–response pathway.     

Olfaction as a model system for the neurobiology of mammalian short-term habituation

Olfaction represents an ideal model system for the study of mammalian habituation given that it is an anatomically relatively simple system with strong reciprocal connections to the limbic system, driving both reflexive and non-reflexive (motivated) behaviors that are easily quantifiable. Data are reviewed here demonstrating short-term habituation of the odor-evoked heart-rate orienting reflex described according to the criteria for habituation outlined by Thompson and Spencer [Thompson, R. F., & Spencer, W. A. (1966). Habituation: A model phenomenon for the study of neuronal substrates of behavior. Psychological Reviews, 73(1), 16–43]. A necessary and sufficient mechanism of short-term habituation is then described, which involves a metabotropic glutamate receptor mediated depression of afferent input to the piriform (primary olfactory) cortex. Finally, evidence for, and a mechanisms of, dishabituation of the orienting reflex and cortical adaptation are described.     

Dishabituation in Aplysia can involve either reversal of habituation or superimposed sensitization

Dishabituation has been thought to be due either to reversal of the process of habituation or to a second process equivalent to sensitization superimposed on habituation. One way to address this question is by testing whether dishabituation and sensitization can be dissociated. Previous studies using this approach in Aplysia have come to different conclusions about the nature of dishabituation, perhaps because those studies differed in many respects, including (1) whether they also observed transient behavioral inhibition, and (2) whether they used test stimuli that activated the LE siphon sensory neurons or as yet unidentified sensory neurons. To attempt to resolve the apparent contradictions between the previous studies, we have explored the importance of these two factors by performing a parametric study of dishabituation and sensitization of gill withdrawal in a simplified preparation that does not exhibit transient behavioral inhibition, using two different test stimuli that are known to activate the LE (Touch) or unidentified (Not Touch) sensory neurons. We find that dishabituation and sensitization in this preparation have similar time courses and generally similar functions of shock intensity. However, under one condition, with the Not Touch stimulus 2.5 min after the shock, dishabituation has a reverse effect of shock intensity. Additional analyses suggest that dishabituation with the Not Touch stimulus 2.5 min after the shock is due to reversal of habituation, whereas 12.5 min after the shock, dishabituation is due to superimposed sensitization. These results thus suggest that dishabituation may involve either process in the same preparation, and begin to define the conditions that favor one or the other.     

Developmental Emergence of Long-Term Memory for Sensitization inAplysia

AdultAplysiaexhibit both short-term and long-term memory for sensitization in the gill and siphon withdrawal reflex. Previous developmental studies showed that short-term memory for sensitization emerges late in juvenile development (stage 12; Rankin and Carew, 1988; Wright et al., 1991). In the present study, we examined the development of long-term memory for sensitization. Long-term sensitization of the siphon withdrawal reflex was quantified as an increase in mean response duration observed 20–24 h after receiving a training regime of one or more 90-min sessions of electrical shock to the tail. In the first three experiments we assessed the capacity for long-term sensitization in adults and in juveniles of stages late 12 and early 12. Animals in all three age classes showed long-term sensitization. In a fourth experiment we simultaneously trained and tested both early stage 12 animals and stage 11 animals with identical stimulus parameters that were scaled down to a level appropriate to the smaller stage 11 animals. Under these conditions, the early stage 12 animals demonstrated long-term sensitization, while the stage 11 animals still showed no evidence of long-term sensitization. These results indicate that long-term sensitization first emerges at early stage 12, which is the same developmental stage in which short-term sensitization first emerges. Although these behavioral data do not elucidate underlying mechanisms, the fact that short-term and long-term memory emerge according to the same developmental timetable is consistent with the possibility that these two forms of memory may share at least some common mechanistic features.     

Behavioral and neuronal attributes of short- and long-term habituation in the crab Chasmagnathus

Investigations using invertebrate species have led to a considerable progress in our understanding of the mechanisms underlying learning and memory. In this review we describe the main behavioral and neuronal findings obtained by studying the habituation of the escape response to a visual danger stimulus in the crab Chasmagnathus granulatus. Massed training with brief intertrial intervals lead to a rapid reduction of the escape response that recovers after a short term. Conversely, few trials of spaced training renders a slower escape reduction that endures for many days. As predicted by Wagner’s associative theory of habituation, long-term habituation in the crab proved to be determined by an association between the contextual environment of the training and the unconditioned stimulus. By performing intracellular recordings in the brain of the intact animal at the same time it was learning, we identified a group of neurons that remarkably reflects the short- and long-term behavioral changes. Thus, the visual memory abilities of crabs, their relatively simple and accessible nervous system, and the recording stability that can be achieved with their neurons provide an opportunity for uncovering neurophysiological and molecular events that occur in identifiable neurons during learning.     

Training-specific adaptations of H- and stretch reflexes in human soleus muscle

The authors investigated the effect of physical exercise on reflex excitability in a controlled intervention study. Healthy participants (N = 21) performed 4 weeks of either power training (ballistic strength training) or balance training (sensorimotor training [SMT]). Both training regimens enhanced balance control and rate of force development, whereas reductions in peak-to-peak amplitudes of stretch reflexes and in the ratio of the maximum Hoffman reflex to the maximum efferent motor response (Hmax:Mmax) measured at rest were limited to SMT. The differences in reflex excitability between the training regimens indicated different underlying neural mechanisms of adaptation. The reduced reflex excitability following SMT was most likely induced by supraspinal influence. The authors discuss an overall increase in presynaptic inhibition of Ia afferent fibers as a possible mechanism.     

Central synaptic mechanisms underlie short-term olfactory habituation in Drosophila larvae

Naive Drosophila larvae show vigorous chemotaxis toward many odorants including ethyl acetate (EA). Chemotaxis toward EA is substantially reduced after a 5-min pre-exposure to the odorant and recovers with a half-time of ～20 min. An analogous behavioral decrement can be induced without odorant-receptor activation through channelrhodopsin-based, direct photoexcitation of odorant sensory neurons (OSNs). The neural mechanism of short-term habituation (STH) requires the (1) rutabaga adenylate cyclase; (2) transmitter release from predominantly GABAergic local interneurons (LNs); (3) GABA-A receptor function in projection neurons (PNs) that receive excitatory inputs from OSNs; and (4) NMDA-receptor function in PNs. These features of STH cannot be explained by simple sensory adaptation and, instead, point to plasticity of olfactory synapses in the antennal lobe as the underlying mechanism. Our observations suggest a model in which NMDAR-dependent depression of the OSN-PN synapse and/or NMDAR-dependent facilitation of inhibitory transmission from LNs to PNs contributes substantially to short-term habituation.     

Differential Induction of Long-Term Synaptic Facilitation by Spaced and Massed Applications of Serotonin at Sensory Neuron Synapses of Aplysia californica

Serotonin (5HT)-induced facilitation of synaptic transmission from tail sensory neurons (SNs) to motor neurons (MNs) in the marine mollusc Aplysia provides a cellular model of short- and long-term memory for behavioral sensitization of the tail withdrawal reflex. Synaptic facilitation at these synapses occurs in three temporal phases: short-term (STF, lasting minutes), intermediate-term (ITF, lasting more than an hour), and long-term (LTF, lasting >24 hr). STF, ITF, and LTF differ in their induction requirements: A single brief exposure of 5HT induces STF, whereas five applications are required for ITF and LTF. Moreover, STF and LTF can be induced independently.     

Duration of auditory sensory memory in parents of children with SLI: a mismatch negativity study

In a previous behavioral study, we showed that parents of children with SLI had a subclinical deficit in phonological short-term memory. Here, we tested the hypothesis that they also have a deficit in nonverbal auditory sensory memory. We measured auditory sensory memory using a paradigm involving an electrophysiological component called the mismatch negativity (MMN). The MMN is a measure of the brain's ability to detect a difference between a frequent standard stimulus (1000 Hz tone) and a rare deviant one (1200 Hz tone). Memory effects were assessed by varying the inter-stimulus interval (ISI) between the standard and deviant. We predicted that parents of children with SLI would have a smaller MMN than parents of typically developing children at a long ISI (3000 ms), but not at a short one (800 ms). This was broadly confirmed. However, individual differences in MMN amplitude did not correlate with measures of phonological short-term memory. Attenuation of MMN amplitude at the longer ISI thus did not provide unambiguous support for the hypothesis of a reduced auditory sensory memory in parents of affected children. We conclude by reviewing possible explanations for the observed group effects.     

Overexpression of and RNA interference with the CCAAT enhancer-binding protein on long-term facilitation of Aplysia sensory to motor synapses

In the marine mollusk Aplysia, the CCAAT/enhancer-binding protein, ApC/EBP, serves as an immediate early gene in the consolidation of long-term facilitation in the synaptic connection between the sensory and motor neurons of the gill-withdrawal reflex. To further examine the role of ApC/EBP as a molecular switch of a stable form of long-term memory, we cloned the full-length coding regions of two alternatively spliced forms, the short and long form of ApC/EBP. Overexpression of each isoform by DNA microinjection resulted in a l6-fold increase in the expression of the coinjected luciferase reporter gene driven by an ERE promoter. In addition, when we overexpressed ApC/EBP in Aplysia sensory neurons, we found that the application of a single pulse of 5-HT that normally induced only short-term facilitation now induced long-term facilitation. Conversely, when we attempted to block the synthesis of native ApC/EBP by microinjecting double-strand RNA or antisense RNA, we blocked long-term facilitation in a sequence-specific manner. These data support the idea that ApC/EBP is both necessary and sufficient to consolidate short-term memory into long-term memory. Furthermore, our results suggest that this double-strand RNA interference provides a powerful tool in the study of the genes functioning in learning and memory in Aplysia by specifically inhibiting both the constitutive and induced expression of the genes.     

Latent memory for sensitization in Aplysia

In the analysis of memory it is commonly observed that, even after a memory is apparently forgotten, its latent presence can still be revealed in a subsequent learning task. Although well established on a behavioral level, the mechanisms underlying latent memory are not well understood. To begin to explore these mechanisms, we have used Aplysia, a model system that permits the simultaneous study of memory at the behavioral, cellular, and molecular levels. We first demonstrate that robust latent memory is induced by long-term sensitization training of the tail-elicited siphon withdrawal reflex. It is revealed by its ability to facilitate the subsequent induction of three mechanistically distinct temporal domains of sensitization memory: short-term, intermediate-term, and long-term memory. Under our training conditions, the latent memory persists for at least 2 d following the decay of original memory expression but appears to be gone by 4 d. Interestingly, we also find that latent memory is induced even in the absence of overt memory for the original training. These findings now permit the analysis of the cellular and molecular architecture of a common feature of learning and memory.     

Modulation of tendon tap reflex activation of soleus motor neurons with reduced stability tandem stance

Reduced stability while standing typically decreases the soleus muscle Hoffmann (H-) reflex amplitude, purportedly to prevent the Ia afferent signal from excessively activating spinal motor neurons during the unstable stance. H-reflex measures, however, by excluding the spindle do not reflect the actual effect of the Ia pathway (i.e. the combined effects of spindle sensitivity and Ia presynaptic inhibition) on motor neuron activation, as tendon tap reflex measures can. But the effect of stance stability on soleus muscle tendon tap reflex amplitude is largely unknown. This study examined 30 young adults (mean(s), 21(2) years) as they stood in a wide stable stance position and an unstable tandem stance with a reduced base of support. Standing body sway, the amplitude of the soleus muscle tendon tap reflex, background EMG and tap force were measured in both stances. A repeated measured design t-test was calculated for each variable. Most subjects (69%) decreased tendon tap reflex amplitude when in the tandem stance position (mean decrease 11.6%), compared to the wide stance (wide stance 0.248(0.124) mV, tandem stance 0.219(0.119) mV, p < 0.05, Cohen’s d = 0.24 small) with no significant differences in background soleus and tibialis anterior EMG, and tap force across the stances. There was no relationship between the modulation of the tendon tap reflex amplitude across the stances and standing body sway in the tandem stance. Results support the idea that for most subjects examined, during a less stable stance the Ia excitation of motor neurons is decreased, likely by presynaptic inhibition, thereby avoiding potential instability in the reflex loop or saturating the reflex pathway and possibly interfering with descending control of the involved spinal motor neurons.     

Retention of time information in forced-choice duration discrimination

We explore the effect on performance in a forced-choice duration-discrimination task of varying the interstimulus interval (ISI) from 0 to 2 sec. The durations were brief empty intervals (115–285 msec) bounded by very brief auditory pulses. Performance improved as the ISI increased from 0 to 1/2 sec, but a further increase in ISI up to 2 sec resulted in little further change in performance. The “time information” derived from a brief interval bounded by auditory pulses does not appear to be susceptible to the very short-term perceptual memory loss inferred in other auditory discriminations.     

Possible involvement of the vagus nerve in monitoring plasma catecholamine levels

In their article Miyashita and Williams (Neurobiology of Learning and Memory 2006, 85, 116-124) describe the effect of peripheral administration of epinephrine on neural discharge in vagal afferent fibers. It seems that described data supports the hypothesis of the vagus nerve participation in monitoring plasma catecholamine levels and consequently modifying brain functions. However, do these results indicate indeed that afferent vagus nerve pathways are activated by circulating epinephrine? Catecholamines influence virtually all tissues and many functions. Vagus nerve participates significantly in monitoring of those effects. Therefore epinephrine-induced increases of afferent vagus nerve activity described by Miyashita and Williams may reflect not only exclusive activation of beta-adrenergic receptors but also an activation of other types of receptors on vagal sensory nerve endings, e.g., mechanosensors, chemoreceptors, and osmosensors. Discussion is focused on the possibility that the increase in afferent vagus nerve activity may reflect activation of mechanoreceptors of the vagus nerve endings in the epinephrine-activated heart.     

Neural pathways mediating vaginal function: the vagus nerves and spinal cord oxytocin

The initial observations, made in our laboratory with Knut Larsson, of the ability of vaginocervical stimulation (VCS) to block withdrawal responses to foot pinch in rats has led to findings of multiple behavioral, autonomic, and neuroendocrine effects of this potent stimulus in rats and also in women. It has led to an understanding of: (1) the neuroanatomical and neurochemical basis of a novel and potent pain-blocking mechanism; (2) likely neuroanatomical pathways mediating both the Ferguson reflex and a specific autonomic response - the pupil-dilating effect of VCS; (3) a role for oxytocin as a putative central nervous system neurotransmitter that stimulates autonomic sympathetic preganglionic neurons within the spinal cord; and (4) a novel pathway that can convey sensory activity from the cervix, adequate to induce orgasm, via the vagus nerves. This latter pathway bypasses the spinal cord and projects directly to the medulla oblongata, and thus can convey genital afferent activity despite complete spinal cord injury at any level.