Christie L. Sahley

ATP and NO dually control migration of microglia to nerve lesions.

Microglia migrate rapidly to lesions in the central nervous system (CNS), presumably in response to chemoattractants including ATP released directly or indirectly by the injury. Previous work on the leech has shown that nitric oxide (NO), generated at the lesion, is both a stop signal for microglia at the lesion and crucial for their directed migration from hundreds of micrometers away within the nerve cord, perhaps mediated by a soluble guanylate cyclase (sGC). In this study, application of 100 μM ATP caused maximal movement of microglia in leech nerve cords. The nucleotides ADP, UTP, and the nonhydrolyzable ATP analog AMP‐PNP (adenyl‐5′‐yl imidodiphosphate) also caused movement, whereas AMP, cAMP, and adenosine were without effect. Both movement in ATP and migration after injury were slowed by 50 μM reactive blue 2 (RB2), an antagonist of purinergic receptors, without influencing the direction of movement. This contrasted with the effect of the NO scavenger cPTIO (2‐(4‐carboxyphenyl)‐4,4,5,5‐teramethylimidazoline‐oxyl‐3‐oxide), which misdirected movement when applied at 1 mM. The cPTIO reduced cGMP immunoreactivity without changing the immunoreactivity of eNOS (endothelial nitric oxide synthase), which accompanies increased NOS activity after nerve cord injury, consistent with involvement of sGC. Moreover, the sGC‐specific inhibitor LY83583 applied at 50 μM had a similar effect, in agreement with previous results with methylene blue. Taken together, the experiments support the hypothesis that ATP released directly or indirectly by injury activates microglia to move, whereas NO that activates sGC directs migration of microglia to CNS lesions.

Multiple Forms of Long-Term Potentiation and Long-Term Depression Converge on a Single Interneuron in the Leech CNS

Long-term potentiation (LTP) of synaptic transmission was observed in two types of synapses that converge on the same postsynaptic neuron in the leech CNS. These synapses were made by identifiable sensory neurons, the mechanosensory touch (T-) and pressure (P-) cells, onto the S-cell, an interneuron critical for certain forms of learning. Changes in both the T-S and P-S synapses appear to be activity dependent because LTP was restricted to inputs that had undergone tetanization; however, properties of synaptic plasticity at the T-S and P-S connections differ considerably. At the P-S synapse, LTP was induced in the tetanized synapse but not in the nontetanized synapse tested in parallel. P-S LTP was blocked by the NMDA receptor antagonist dl-2-amino-5-phosphono-valeric acid (AP-5) or by lowering the extracellular concentration of glycine, an NMDA receptor (NMDAR) co-agonist. P-S LTP was strongly affected by the initial amplitude of the synaptic potential at the time LTP was induced. Smaller amplitude synapses (<3.5 mV) underwent robust potentiation, whereas the less common, larger amplitude synapse (>3.5 mV) depressed after tetanization. At the T-S synapse, tetanization simultaneously induced homosynaptic LTP in the tetanized input and heterosynaptic long-term depression (LTD) in the input made by a nontetanized T-cell onto the same S-cell. Interestingly, AP-5 failed to block homosynaptic LTP at the T-S synapse but did prevent heterosynaptic LTD. T-S LTP was not affected by the initial EPSP amplitude. Thus, leech neurons exhibit synaptic plasticity with properties similar to LTP and LTD found in the vertebrate nervous system.

Progressive recovery of learning during regeneration of a single synapse in the medicinal leech

The leech escape reflex—shortening of the body—can change with nonassociative conditioning, including sensitization, habituation, and dishabituation. Capacity for sensitization, which is an enhancement of the reflex, is lost when a single S‐interneuron is ablated, but the reflex response itself remains. In the present experiments, the S‐interneurons axon in the living leech was filled with 6‐carboxyfluorescein (6‐CF) dye and cut with an argon laser microbeam (λ = 488 nm). In contrast to sham‐operated animals, axotomized preparations did not sensitize, reflecting the key role of the S‐cell. By 2 weeks or more, S‐cell axons had regenerated and reestablished synapses at their usual locations with neighboring S‐cells. By 4 weeks, this restored the ability to sensitize to a level indistinguishable from that of controls, but an intermediate state of recovery was seen from 2–3 weeks after injury—a period not previously examined. The small capacity for sensitization among newly regenerated preparations was significantly lower than in sham controls but appeared higher than in animals whose cut S‐cell axon had not regenerated its synapse. The results confirm the crucial role of the S‐cell in sensitization. Moreover, full sensitization does not occur immediately upon synapse regeneration. J. Comp. Neurol. 457:67–74, 2003.

Reduced axon sprouting after treatment that diminishes microglia accumulation at lesions in the leech CNS

The role of mammalian microglia in central nervous system (CNS) repair is controversial. Microglia accumulate at lesions where they act as immune cells and phagocytize debris, and they may secrete neurotrophins, but they also produce molecules that can be cytotoxic, like nitric oxide (NO). To determine the importance of microglial accumulation at lesions on growth of severed CNS axons in the leech (Hirudo medicinalis), in which axon and synapse regeneration are notably successful even when isolated in tissue culture medium, microglial migration to lesions was reduced. Pressure (P) sensory neurons were injected with biocytin to reveal the extent of their sprouting 24 hours after lesioning. To reduce microglia accumulation at lesions, cords were treated for 3.5 hours with 3 mM ATP or 2 mM Nω‐nitro‐L‐arginine methyl ester (L‐NAME) or 50 μM Reactive blue‐2 (RB2) beginning 30 minutes before injury. Lesioned controls were either not treated with drug or treated 3 hours later with one of the drugs, after the migration and subsequent accumulation of most microglia had occurred, but before the onset of axon sprouting, for a total of seven separate conditions. There was a significant reduction in total sprout lengths compared with controls when microglial accumulation was reduced. The results suggest that microglial cells are necessary for the usual sprouting of injured axons. J. Comp. Neurol. 503:101–109, 2007.

Repair and regeneration of functional synaptic connections: Cellular and molecular interactions in the leech

A major problem for neuroscience has been to find a means to achieve reliable regeneration of synaptic connections following injury to the adult CNS. This problem has been solved by the leech, where identified neurons reconnect precisely with their usual targets following axotomy, re-establishing in the adult the connections formed during embryonic development.It cannot be assumed that once axons regenerate specific synapses, function will be restored. Recent work on the leech has shown following regeneration of the synapse between S-interneurons, which are required for sensitization of reflexive shortening, a form of non-associative learning, the capacity for sensitization is delayed.The steps in repair of synaptic connections in the leech are reviewed, with the aim of understanding general mechanisms that promote successful repair. New results are presented regarding the signals that regulate microglial migration to lesions, a first step in the repair process. In particular, microglia up to 900 μm from the lesion respond within minutes by moving rapidly toward the injury, controlled in part by nitric oxide (NO), which is generated immediately at the lesion and acts via a soluble guanylate cyclase (sGC). The cGMP produced remains elevated for hours after injury. The relationship of microglial migration to axon outgrowth is discussed.

Serotonin depletion impairs but does not eliminate classical conditioning in the leech Hirudo medicinalis

The goal of these experiments was to test the role of serotonin (5HT) in classical conditioning of the touch-elicited shortening reflex in the leech (Hirudo medicinalis). The toxin 5,7-dihydroxytryptamine (5,7-DHT) was used to deplete serotonin. The results indicated that 5HT depletion significantly impairs the expression of conditioned responding; however, depleted leeches experiencing conditioned stimulus-unconditioned stimulus (CS-US) pairings still performed significantly better than depleted leeches experiencing unpaired CS-US presentations, suggesting that a 5HT-dependent mechanism does not account fully for learning in this preparation. Moreover, the residual pairing dependent effect is observed, although the depletions eliminate sensitization, suggesting that the amplification of sensitization may not be sufficient to account for classical conditioning of this reflex. Histological analyses of the ganglia revealed an absence of staining in 100% of the Retzius cells in the toxin group.

Localization of the myomodulin-like immunoreactivity in the leech CNS

The distribution of myomodulinlike immunoreactivity in the leech CNS was determined using an antiserum raised against Aplysia myomodulin. Segmental ganglia contained approximately 60 immunoreactive neurons. In addition, numerous fibers containing immunoreactive varicosities were found throughout the neuropil. Using a combination of Lucifer Yellow injections and immunocytochemistry, we identified neurons including the anterior Pagodas (AP), annulus erector (AE), motor neurons, Leydig, longitudinal muscle motoneurons (L), S cells, and coupling interneurons, all of which are active during the touch-elicited shortening reflex. FMRF-amide-like immunoreactivity in three of these cells (L, AP, and AE) was previously demonstrated. Specific staining for myomodulin was abolished by preadsorption of the antiserum with synthetic myomodulin, but not with FMRF-amide. These results suggest a potential role for myomodulin in both intrinsic and extrinsic modulation of the leech touch-elicited shortening reflex. Further, it is possible that several neurons mediating this reflex contain multiple neuromodulatory peptides.

Identification and Characterization of a Myomodulin-Like Peptide in the Leech

A novel myomodulin-like peptide, GMGALRLamide, has been purified and sequenced from extracts of 1000 medicinal leech nerve cords. Synthetic leech myomodulin-like peptide blocked the specific staining pattern of leech ganglia by the antiserum against Aplysia myomodulin A PMGMLRLamide. Moreover, the synthetic leech myomodulin-like peptide GMGALRLamide showed identical neuronal modulation effect on the giant leech Retzius cell compare to that by the synthetic Aplysia myomodulin A PMGMLRLamide.

Odors can induce feeding motor responses in the terrestrial mollusc Limax maximus.

Highly developed odor learning was shown in the terrestrial slug Limax maximus. In addition, several key cellular elements of the neural network that controls ingestive feeding have been identified. The results of 3 experiments demonstrate an interaction between odor input and ingestive feeding in that olfactory stimulation with behaviorally attractive odors summed with tactile stimulation from plain agar to produce ingestion of plain agar. Agar ingestion did not occur in the absence of attractive odor stimulation. The adequacy of odor stimulation to trigger agar ingestion was altered by associative learning. Innately attractive odors rendered repellent by associative learning no longer triggered agar ingestion, whereas innately repellent odors rendered attractive by conditioning triggered agar ingestion. The newly discovered feeding command cells in the Limax cerebral ganglion are a logical cellular locus for this interaction.

Associative Learning Modifies the Shortening Reflex in the Semi-Intact Leech Hirudo medicinalis: Effects of Pairing, Predictability, and CS Preexposure

Three experiments addressed the importance of the inter-event relationships of contiguity and contingency for associative learning in the semi-intact leech. It was found that both of these relationships are important for the leech to acquire a learned association between a touch (conditional stimulus, CS) and shock (unconditional stimulus, US). The learning can be extinguished if training is followed by explicitly unpaired presentations of the CS and US, which removes the contiguity between the stimuli. Learning is degraded by the introduction of unpredicted USs, as well as by unreinforced presentations of the CS (CS preexposure), both manipulations reduce the contingency between the CS and US. These results suggest that the associative process in both vertebrates and invertebrates share considerable functional similarity in the inter-event relationships important to learning. For vertebrates and invertebrates alike, the specific temporal relationships between the conditional stimulus (CS) and the unconditional stimulus (US) are critical for associative learning to occur. The contiguous occurrence of these two stimuli in time and the predictive relationship of the CS to the US are essential for an association between the stimuli to take place (Carew, Hawkins, & Kandel, 1983; Colwill, Absher, & Roberts, 1988; Farley, 1987; Hawkins, Carew, & Kandel, 1986; Kamin, 1969; Rescorla, 1969; Sahley, Rudy & Gelperin, 1981). The goal of the experiments presented in this article was to demonstrate that classical conditioning, which is dependent on pairing and predictability, can be readily demonstrated in the semi-intact leech preparation. This work lays the foundation for cellular experiments to delineate the neural mechanisms mediating the associative process. Associative learning, a rich and complex phenomenon involving the integration of many inter-event relationships, has not been completely addressed by neurobiologists (Rescorla, 1984; 1988). Current cellular models of learning in invertebrates have focused on the contiguity aspects of learning with little attention paid to aspects of learning, such as predictability, conditioned inhibition, second-order conditioning, sensory preconditioning, and latent inhibition, or such basics as mechanisms of the acquisition of learning over trials, extinction of learning, and the effects of intertrial intervals. Our long-term goal is to use this semi-intact preparation to identify and characterize the cellular mechanisms underlying the associative process. We began this analysis by testing the effects of