Convergent and divergent modulatory pathways in {\it Aplysia\/}: Cellular and network studies

Neuromodulation is essential to many functions of the nervous system. In the simple gastropod mollusk Aplysia californica, neuromodulation of the circuits for the defensive withdrawal reflexes has been associated with several forms of learning. In the present work, the neurotransmitters and neural circuitry which contribute to the modulation of the tail-siphon withdrawal reflex were examined.^ A recently-identified neuropeptide transmitter, buccalin A was found to modulate the biophysical properties of the sensory neurons that mediate the reflex. The actions of buccalin A on the sensory neurons were compared with those of the well-characterized modulatory transmitter serotonin, and convergence and divergence in the actions of these two transmitters were evaluated. Buccalin A dramatically increased the excitability of sensory neurons and occluded further enhancement of excitability by serotonin. Buccalin A produced no significant change in spike duration, and it did not block serotonin-induced spike broadening. Voltage-clamp analysis revealed the currents that may be involved in the effects on spike duration and excitability. Buccalin A decreased an outward current similar to the S-K$\sp+$ current (I$\sb{\rm K,S}$). Buccalin A appeared to occlude further modulation of I$\sb{\rm K,S}$ by serotonin, but did not block serotonin-induced modulation of the voltage-dependent delayed rectifier K$\sp+$ current (I$\sb{\rm K,V}$). These results suggest that buccalin A converges on some, but not all, of the same subcellular modulatory pathways as serotonin.^ In order to begin to understand neuromodulation in a more physiological context for the tail-siphon withdrawal reflex, the modulatory circuitry for the tail-withdrawal circuit was examined. Mechanoafferent neurons in the J cluster of the cerebral ganglion were identified as elements of a modulatory circuit for the reflex. Excitatory and inhibitory connections were observed between the J cells and the pleural sensory neurons, the tail motor neurons, and several classes of interneurons for the tail-siphon withdrawal circuit. The J cells produced both fast and slow PSPs in these neurons. Of particular interest was the ability of the J cells to produce slow EPSPs in the pleural sensory neurons. These slow EPSPs were associated with an increase in the excitability of the sensory neurons. The J cells appear to mediate both sensory and modulatory inputs to the circuit for the tail-siphon withdrawal reflex from the anterior part of the animal. ^

Characterization of the interactions between two sites of plasticity engaged during eyelid conditioning

Here, we investigate the involvement of two sites of plasticity in the learning and expression of a simple associative motor behavior—the classically conditioned eyelid response. While previous studies clearly demonstrate that lesions of the anterior interpositus nucleus of the cerebellum abolish learned responses and prevent subsequent learning, studies investigating the effects of lesions of the cerebellar cortex on learning and retention have produced discrepant results. We complement ablative lesion studies of the cortex with the use of reversible, pharmacological blockade of cerebellar cortical transmission to investigate the role of the cerebellar cortex in eyelid conditioning. We demonstrate that both pharmacological blockade as well as focused ablative lesions of the cortex abolish timed responses and unmask responses with a fixed, short latency that are not displayed by the intact animal. Pharmacological blockade of cerebellar cortex output at various stages of acquisition and extinction reveals appropriate, learning dependent changes in the amplitude and probability of short latency responses during training. Acquisition of both short latency as well as timed responses is prevented by ablative lesions of the anterior lobe of the cerebellar cortex. These convergent results from technically distinct methods of removing the influence of the cerebellar cortex from conditioned behavior are consistent with the proposal that (1) eyelid conditioning engages two cerebellar sites of plasticity-one in the cortex and one in the anterior interpositus nucleus, (2) plasticity in the cerebellar cortex is necessary for proper response timing, (3) plasticity in the nucleus mediates the short latency responses unmasked by lesions of the cerebellar cortex, and (4) cerebellar cortical output is necessary for the induction of plasticity in the nucleus. ^

ANALYSIS OF SEROTONERGIC MODULATION OF NEURONS INVOLVED IN TWO DEFENSIVE BEHAVIORS IN APLYSIA CALIFORNICA (CYCLIC-AMP, AROUSAL, CALCIUM, POTASSIUM)

Sensitization is a simple form of learning which refers to an enhancement of a behavioral response resulting from an exposure to a novel stimulus. While sensitization is found throughout the animal world, little is known regarding the underlying neural mechanisms. By taking advantage of the simple nervous system of the marine mollusc Aplysia, I have begun to examine the cellular and molecular mechanisms underlying this simple form of learning. In an attempt to determine the generality of the mechanisms of neuromodulation underlying sensitization, I have investigated and compared the modulation of neurons involved in two defensive behaviors in Aplysia, the defensive inking response and defensive tail withdrawal.^ The motor neurons that produce the defensive release of ink receive a slow decreased conductance excitatory postsynaptic potential (EPSP) in response to sensitizing stimuli. Using electrophysiological techniques, it was found that serotonin (5-HT) mimicked the physiologically produced slow EPSP. 5-HT produced its response through a reduction in a voltage-independent conductance to K('+). The 5-HT sensitive K('+) conductance of the ink motor neurons was separate from the fast K('+), delayed K('+), and Ca('2+)-activated K('+) conductances found in these and other molluscan neurons. 5-HT was shown to produce a decrease in K('+) conductance in the ink motor neurons through an elevation of cellular cAMP.^ The mechanosensory neurons that participate in the defensive tail withdrawal response are also modulated by sensitizing stimuli through the action of 5-HT. Using electrophysiological techniques, it was found that 5-HT modulated the tail sensory neurons through a reduction in a voltage-dependent conductance to K('+). The serotonin-sensitive K('+) conductance was found to be largely a Ca('2+)-activated K('+) conductance. Much like the ink motor neurons, 5-HT produced its modulation through an elevation of cellular cAMP. While the actual K('+) conductance modulated by 5-HT in these two classes of neurons differs, the following generalizations can be made: (1) the effects of sensitizing stimuli are mimicked by 5-HT, (2) 5-HT produces its effect through an elevation of cellular cAMP, and (3) the conductance to K('+) is modulated by 5-HT. ^

Is sensitization to stimulants time dependent and mediated via NMDA receptors?

Chronic administration of psychomotor stimulants has been reported to produce behavioral sensitization to its effects on motor activity. This adaptation may be related to the pathophysiology of recurrent psychiatric disorders. Since disturbances in circadian rhythms are also found in many of these disorders, the relationship between sensitization and chronobiological factors became of interest. Therefore, a computerized monitoring system investigated the following: whether repeated exposure to methylphenidate (MPD) and amphetamine (AMP) could produce sensitization to its locomotor effects in the rat; whether sensitization to MPD and AMP was dependent on the circadian time of drug administration; whether the baseline levels of locomotor activity would be effected by repeated exposure to MPD and AMP; whether the expression of a sensitized response could be affected by the photoperiod; and whether MK-801, a non-competitive NMDA antagonist, could disrupt the development of sensitization to MPD. Dawley rats were housed in test cages and motor activity was recorded continuously for 16 days. The first 2 days served as baseline for each rat, and on day 3 each rat received a saline injection. The locomotor response to 0.6, 2.5, or 10 mg/kg of MPD was tested on day 4, followed by five days of single injections of 2.5 mg/kg MPD (days 5–9). After five days without injection (days 10–14) rats were re-challenged (day 15) with the same doses they received on day 4. There were three separate dose groups ran at four different times of administration, 08:00, 14:00, 20:00, or 02:00 (i.e. 12 groups). The same protocol was conducted with AMP with the doses of 0.3, 0.6, and 1.2 mg/kg given on day 4 and 15, and 0.6 mg/kg AMP as the repeated dose on days 5 to 9. In the second set of experiments only sensitization to MPD was investigated. The expression of the sensitized response was dose-dependent and mainly observed with challenge of the lower dose groups. The development of sensitization to MPD and ANT was differentially time-dependent. For MPD, the most robust sensitization occurred during the light phase, with no sensitization during the middle of the dark phase. (Abstract shortened by UMI.) ^

The role of sensory neuron outgrowth in long-term sensitization of the tail-elicited tail-siphon withdrawal reflex of Aplysia

Neuronal outgrowth has been proposed in many systems as a mechanism underlying memory storage. For example, sensory neuron outgrowth is widely accepted as an underlying mechanism of long-term sensitization of defensive withdrawal reflexes in Aplysia. The hypothesis is that learning leads to outgrowth and consequently to the formation of new synapses, which in turn strengthen the neural circuit underlying the behavior. However, key experiments to test this hypothesis have never been performed. ^ Four days of sensitization training leads to outgrowth of siphon sensory neurons mediating the siphon-gill withdrawal response in Aplysia . We found that a similar training protocol produced robust outgrowth in tail sensory neurons mediating the tail siphon withdrawal reflex. In contrast, 1 day of training, which effectively induces long-term behavioral sensitization and synaptic facilitation, was not associated with neuronal outgrowth. Further examination of the effect of behavioral training protocols on sensory neuron outgrowth indicated that this structural modification is associated only with the most persistent forms of sensitization, and that the induction of these changes is dependent on the spacing of the training trials over multiple days. Therefore, we suggest that neuronal outgrowth is not a universal mechanism underlying long-term sensitization, but is involved only in the most persistent forms of the memory. ^ Sensory neuron outgrowth presumably contributes to long-term sensitization through formation of new synapses with follower motor neurons, but this hypothesis has never been directly tested. The contribution of outgrowth to long-term sensitization was assessed using confocal microscopy to examine sites of contact between physiologically connected pairs of sensory and motor neurons. Following 4 days of training, the strength of both the behavior and sensorimotor synapse and the number of appositions with follower neurons was enhanced only on the trained side of the animal. In contrast, outgrowth was induced on both sides of the animal, indicating that although sensory neuron outgrowth does appear to contribute to sensitization through the formation of new synapses, outgrowth alone is not sufficient to account for the effects of sensitization. This indicates that key regulatory steps are downstream from outgrowth, possibly in the targeting of new processes and activation of new synapses. ^

Long-term hyperexcitability of sensory and motor axons in Aplysia californica: Induction by axotomy, serotonin, and transient depolarization

Neuropathic pain is a debilitating neurological disorder that may appear after peripheral nerve trauma and is characterized by persistent, intractable pain. The well-studied phenomenon of long-term hyperexcitability (LTH), in which sensory somata become hyperexcitable following peripheral nerve injury may be important for both chronic pain and long-lasting memory formation, since similar cellular alterations take place after both injury and learning. Though axons have previously been considered simple conducting cables, spontaneous afferent signals develop from some neuromas that form at severed nerve tips, indicating intrinsic changes in sensory axonal excitability may contribute to this intractable pain. Here we show that nerve transection, exposure to serotonin, and transient depolarization induce long-lasting sensory axonal hyperexcitability that is localized to the treated nerve segment and requires local translation of new proteins. Long-lasting functional plasticity may be a general property of axons, since both injured and transiently depolarized motor axons display LTH as well. Axonal hyperexcitability may represent an adaptive mechanism to overcome conduction failure after peripheral injury, but also displays key features shared with cellular analogues of memory including: site-specific changes in neuronal function, dependence on transient, focal depolarization for induction, and requirement for synthesis of new proteins for expression of long-lasting effects. The finding of axonal hyperexcitability after nerve injury sheds new light on the clinical problem of chronic neuropathic pain, and provides more support for the hypothesis that mechanisms of long-term memory storage evolved from primitive adaptive responses to injury. ^

An evaluation of sleep hypnotic and stimulant effect on human performance using a computerized psychological test battery

This study evaluated the administration-time-dependent effects of a stimulant (Dexedrine 5-mg), a sleep-inducer (Halcion 0.25-mg) and placebo (control) on human performance. The investigation was conducted on 12 diurnally active (0700-2300) male adults (23-38 yrs) using a double-blind, randomized sixway-crossover three-treatment, two-timepoint (0830 vs 2030) design. Performance tests were conducted hourly during sleepless 13-hour studies using a computer generated, controlled and scored multi-task cognitive performance assessment battery (PAB) developed at the Walter Reed Army Institute of Research. Specific tests were Simple and Choice Reaction Time, Serial Addition/Subtraction, Spatial Orientation, Logical Reasoning, Time Estimation, Response Timing and the Stanford Sleepiness Scale. The major index of performance was "Throughput", a combined measure of speed and accuracy.^ For the Placebo condition, Single and Group Cosinor Analysis documented circadian rhythms in cognitive performance for the majority of tests, both for individuals and for the group. Performance was best around 1830-2030 and most variable around 0530-0700 when sleepiness was greatest (0300).^ Morning Dexedrine dosing marginally enhanced performance an average of 3% with reference to the corresponding in time control level. Dexedrine AM also increased alertness by 10% over the AM control. Dexedrine PM failed to improve performance with reference to the corresponding PM control baseline. With regard to AM and PM Dexedrine administrations, AM performance was 6% better with subjects 25% more alert.^ Morning Halcion administration caused a 7% performance decrement and 16% increase in sleepiness and a 13% decrement and 10% increase in sleepiness when administered in the evening compared to corresponding in time control data. Performance was 9% worse and sleepiness 24% greater after evening versus morning Halcion administration.^ These results suggest that for evening Halcion dosing, the overnight sleep deprivation occurring in coincidence with the nadir in performance due to circadian rhythmicity together with the CNS depressant effects combine to produce performance degradation. For Dexedrine, morning administration resulted in only marginal performance enhancement; Dexedrine in the evening was less effective, suggesting the 5-mg dose level may be too low to counteract the partial sleep deprivation and nocturnal nadir in performance. ^

The role of the intracellular second messenger cAMP in the induction of long-term morphological changes in sensory neurons of {\it Aplysia\/}

The present work examines the role of cAMP in the induction of the type of long-term morphological changes that have been shown to be correlated with long-term sensitization in Aplysia.^ To examine this issue, cAMP was injected into individual tail sensory neurons in the pleural ganglion to mimic, at the single cell level, the effects of behavioral training. After a 22 hr incubation period, the same cells were filled with horseradish peroxidase and 2 hours later the tissue was fixed and processed. Morphological analysis revealed that cAMP induced an increase in two morphological features of the neurons, varicosities and branch points. These structural alterations, which are similar to those seen in siphon sensory neurons of the abdominal ganglion following long-term sensitization training of the siphon-gill withdrawal reflex, could subserve the altered behavioral response of the animal. These results expose another role played by cAMP in the induction of learning, the initiation of a structural substrate, which, in concert with other correlates, underlies learning.^ cAMP was injected into sensory neurons in the presence of the reversible protein synthesis inhibitor, anisomycin. The presence of anisomycin during and immediately following the nucleotide injection completely blocked the structural remodeling. These results indicate that the induction of morphological changes by cAMP is a process dependent on protein synthesis.^ To further examine the temporal requirement for protein synthesis in the induction of these changes, the time of anisomycin exposure was varied. The results indicate that the cellular processes triggered by cAMP are sensitive to the inhibition of protein synthesis for at least 7 hours after the nucleotide injection. This is a longer period of sensitivity than that for the induction of another correlate of long-term sensitization, facilitation of the sensory to motor neuron synaptic connection. Thus, these findings demonstrate that the period of sensitivity to protein synthesis inhibition is not identical for all correlates of learning. In addition, since the induction of the morphological changes can be blocked by anisomycin pulses administered at different times during and following the cAMP injection, this suggests that cAMP is triggering a cascade of protein synthesis, with successive rounds of synthesis being dependent on successful completion of preceding rounds. Inhibition at any time during this cascade can block the entire process and so prevent the development of the structural changes.^ The extent to which cAMP can mimic the structural remodeling induced by long-term training was also examined. Animals were subjected to unilateral sensitization training and the morphology of the sensory neurons was examined twenty-four hours later. Both cAMP injection and long-term training produced a twofold increase in varicosities and approximately a fifty percent increase in the number of branch points in the sensory neuron arborization within the pleural ganglion. (Abstract shortened by UMI.) ^

Calcium-dependent mechanisms and long-term potentiation: Role of NMDA receptors, voltage -dependent calcium channels and persistent protein kinase activity

Long-term potentiation (LTP) is a rapidly induced and long lasting increase in synaptic strength and is the leading cellular model for learning and memory in the mammalian brain. LTP was first identified in the hippocampus, a structure implicated in memory formation. LTP induction is dependent on postsynaptic Ca2+ increases mediated by N-methyl-D-aspartate (NMDA) receptors. Activation of other postsynaptic routes of Ca2+ entry, such as voltage-dependent Ca2+ channels (VDCCs) have subsequently been shown to induce a long-lasting increase in synaptic strength. However, it is unknown if VDCC-induced LTP utilized similar cellular mechanisms as the classical NMDA receptor-dependent LTP and if these two forms of LTP display similar properties. This dissertation determines the similarities and differences in VDCC and NMDA receptor-dependent LTP in area CA1 of hippocampal slices and demonstrates that VDCCs and NMDA receptors activate similar cellular mechanisms, such as protein kinases, to induce LTP. However, VDCC and NMDA receptor activated LTP induction mechanisms are compartmentalized in the postsynaptic neuron, such that they do not interact. Consistent with activation properties of NMDA receptors and VDCCs, NMDA receptor and VDCC-dependent LTP have different induction properties. In contrast to NMDA-dependent LTP, VDCC-induced potentiation does not require evoked presynaptic stimulation or display input specificity. These results indicate that there are two different routes of postsynaptic Ca2+ which can induce LTP and the compartmentation of VDCCs and NMDA receptors and/or their resulting Ca2+ increases may account for the distinction between these LTP induction mechanisms.^ One of the molecular targets for postsynaptic Ca2+ that is required for the induction of LTP is protein kinases. Evidence for the role of protein kinase activity in LTP expression is either correlational or controversial. We have utilized a broad range and potent inhibitors of protein kinases to systematically examine the temporal requirement for protein kinases in the induction and expression of LTP. Our results indicate that there is a critical period of persistent protein kinase activity required for LTP induction activated by tetanic stimulation and extending until 20 min after HFS. In addition, our results suggest that protein kinase activity during and immediately after HFS is not sufficient for LTP induction. These results provide evidence for persistent and/or Ca2+ independent protein kinase activity involvement in LTP induction. ^