Impacts of carbamate pesticides on olfactory neurophysiology and cholinesterase activity in coho salmon (Oncorhynchus kisutch).

Many freshwater aquatic environments in the Pacific Northwest of North America contain neurotoxic pesticides, an issue of concern given the use of many of these habitats by Pacific salmon (Oncorhynchus sp.). Pesticides such as carbamates are known to affect fundamental physiological systems (such as the enzyme acetylcholinesterase (AChE)), and have been shown to affect salmonid olfactory-mediated behaviors. A neurophysiological measure of olfactory function, the electro-olfactogram (EOG), was used in this study to examine the impacts of acute localized exposure to three carbamates (the insecticide carbofuran, the antisapstain IPBC, and the fungicide mancozeb) on olfactory function in the coho salmon (Oncorhynchus kisutch). We also examine the potential for these pesticides to alter AChE levels in the primary olfactory system and brain with brief exposures (30 min to only the olfactory rosette (OR)). In results, we find that the EOG in coho salmon is highly sensitive to brief localized exposures of two of these three carbamate pesticides. The effective nominal concentration required to cause a 50% reduction in EOG amplitude (EC50) for carbofuran was 10.4 microg/l and for IPBC was 1.28 microg/l. For mancozeb, the EC50 was higher at 2.05 mg/l. All three carbamates also affected AChE activity levels in the OR and brain (BR): carbofuran exposure at 200 microg/l significantly inhibited AChE activity in the OR, and both IPBC and mancozeb significantly increased AChE activity in BR at multiple concentrations with acute localized exposure. These carbamate effects highlight the sensitivity of salmon olfactory neurophysiology to pesticides acting not only potentially via AChE-inhibition, but also by other currently unknown modes of action.

Cell culture of mechanoreceptor neurons innervating proleg sensory hairs in Manduca sexta larvae, and co-culture with target motoneurons.

The tip of each proleg in Manduca sexta larvae bears a dense array of mechanosensory hairs termed planta hairs (PHs), each innervated by a single sensory neuron (termed a PH-SN) located in the underlying epidermis. In the CNS, axon terminals of PH-SNs make direct, excitatory, nicotinic cholinergic synapses with proleg retractor motoneurons including the accessory planta retractor (APR). These synapses mediate a proleg withdrawal reflex, exhibit multiple forms of activity-dependent plasticity and weaken during the prepupal peak of ecdysteroids. In the present study we developed methods to dissociate PH-SNs from the epidermis and culture them alone or with APRs. The PH-SNs were fluorescently labeled in situ by introducing dye through the cut hair shaft or by retrograde axonal staining. Alternatively, unlabeled PH-SNs were utilized. The epidermis beneath the planta hair array was separated from the cuticle, enzymatically treated and mechanically dissociated into single cells. PH-SNs were cultured on glass coverslips coated with concanavalin A and laminin, in modified Leibovitz's IL-15 medium. Supplementation with medium conditioned by an insect cell line produced the best results. Dissociated PH-SNs had somatic diameters of ~10 micro m and typically bore a stout dendrite consisting of the inner and, occasionally, the outer dendritic segment. An axonal stump was sometimes retained. Viable PH-SNs typically extended new processes and often survived for 2-4 weeks. When co-cultured, PH-SNs and APRs exhibited robust growth and made close anatomical appositions. This culture system provides convenient experimental access to PH-SNs and may potentially permit sensorimotor synapses to be investigated in vitro.

Analysis of sequence-dependent interactions between transient calcium and transmitter stimuli in activating adenylyl cyclase in Aplysia: possible contribution to CS--US sequence requirement during conditioning.

An important recent insight in a number of neurobiological systems is that during learning, individual dually regulated proteins with associative properties function as critical sites of stimulus convergence. During conditioning in Aplysia, the Ca2+ /calmodulin-sensitive adenylyl cyclase (AC) in mechanosensory neurons serves as a molecular site of interaction between Ca2+ and serotonin [5-hydroxytryptamine (5-HT)]-two signals that represent the CS and US in these cells. Conditioning requires that the CS and US be paired within a narrow time window and in the appropriate sequence. AC shows an analogous sequence preference: It is more effectively activated when a pulse of Ca2+ precedes a pulse of 5-HT than when the 5-HT precedes Ca2+. One mechanism that contributes to this sequence preference is that Ca2+/calmodulin binding to AC accelerates the rate of AC activation by receptor-Gs. We have identified two additional properties of AC activation that would cause pairing with Ca2+ preceding 5-HT to be more effective than simultaneous pairing or pairing with the reciprocal sequence: (1) Activation of Aplysia AC by a Ca2+ pulse rose with a delay compared with activation by a 5-HT pulse. (2) A late pulse of Ca2+, which arrived after 5-HT, acted, via calmodulin, to accelerate the decay of AC activation by receptor-Gs. Together, these activation properties of AC may contribute to the CS-US sequence requirement of classical conditioning.

Postembryonic changes in the structure of the olfactory bulb of the chinook salmon (Oncorhynchus tshawytscha) across its life history.

Juvenile chinook salmon (Oncorhynchus tshawytscha) memorize odors characteristic of their natal stream, then use these imprinted olfactory cues to return to that same stream years later as sexually mature adults. In a preliminary effort to identify neuroanatomical changes in the salmon olfactory system that may underlie these behavioral capabilities, the structure of the olfactory bulb (OB) was studied at three developmental periods across the life history: in early juvenile development (0.1-4 months of age), in late juvenile development (11-16 months), and as spawning adults (48 months). Using antibodies that selectively label primary olfactory afferents (anti-keyhole limpet hemocyanin), combined with a thionin counterstain, the telencephalon (TEL), OB, and two of the bulb's laminar components, the olfactory nerve-glomerular layer (ONL-GL) and inner cell layer (ICL), could be easily identified. Laminar organization and relative volume (i.e. percent of OB comprised by ONL-GL or ICL) were then compared across groups, and absolute volumes of the OB, ONL-GL, and ICL were compared to that of the TEL at each stage. Three age-related processes were observed across the life history. First, a rapid increase in organizational and structural maturity of the OB was found to be confined to early juvenile life. Second, an increase in OB, ONL-GL, and ICL volume, relative to TEL volume, was found to occur across the entire life history. Lastly, the composition of the OB itself changed, in that an increase in the relative volume of the ONL-GL, and a decrease in that of the ICL, were observed across the life history. Taken together, these results indicate that the OB of young salmon matures dramatically while the fish is still in the freshwater phase of its life history. Then, during migration to the ocean and growth to the adult stage, the volume of the OB, relative to that of the telencephalon, and the relative volume of the input layer of the bulb, both undergo a marked, continuous increase. These changes in the structure, and presumably the function, of the olfactory bulb of salmon may be important in the behavioral phenomena of olfactory imprinting as juveniles and homing as adults.

In Aplysia sensory neurons, the neuropeptide SCPB and serotonin differ in efficacy both in modulating cellular properties and in activating adenylyl cyclase: implications for mechanisms underlying presynaptic facilitation.

The facilitatory transmitters serotonin (5-HT) and the molluscan neuropeptides SCPA and SCPB both activate adenylyl cyclase in Aplysia mechanosensory neurons and produce multiple modulatory effects that contribute to increasing transmitter release from these cells. This enhancement of transmitter release from sensory neurons contributes to increased behavioral response during sensitization and classical conditioning in Aplysia. Recently, specific examples of modulation in these sensory neurons have been described that are more effectively initiated by 5-HT than by the SCPs. For example, in the present study, 5-HT produces 55% greater broadening of the normal sensory neuron action potential than did SCPB. These differences in the modulatory actions of the facilitatory transmitters have been interpreted as suggesting that 5-HT produces its modulatory effects at least partly via a cAMP-independent mechanism. However, we have found that the two types of facilitatory transmitters are not equally effective in activating adenylyl cyclase. In both whole CNS membranes and sensory neuron membranes, SCPB was less effective than 5-HT in stimulating adenylyl cyclase activity measured in steady state assays. Because electrophysiological experiments suggested that the response to the SCPs desensitizes rapidly, we further compared cyclase stimulation in perfused membrane assays that enable continuous monitoring of cyclase activity; however we observed that 5-HT was also more effective than SCPB in stimulating cyclase at the onset of transmitter exposure. We discuss the possibility that lower peak stimulation of cyclase by SCPB and a faster rate of desensitization could account for some of the differences between the SCPs and 5-HT in modulating sensory neurons.