Joseph Farley

Chemosensory conditioning of Hermissenda crassicornis.

Bite-strike responses of Hermissenda crassicornis, elicited by chemosensory stimulation of the lips, were found to be modified when food extracts were paired with rotation-produced stimulation of the statocysts. Animals that received repeated pairings of an extract of 1 food (conditioned stimulus, CS) with rotation exhibited suppressed bite-strike responses to that food for up to 48 hr after training. This suppression was usually specific to the trained food and was pairing-specific as well. Discriminative conditioning was also demonstrated. Animals trained with 1 CS paired with rotation and a second CS that was unpaired (CS-) showed suppressed bite-strike responses to the first CS. The results demonstrate that Hermissenda can learn to avoid foods that reliably signal an aversive event and may allow an analysis of higher order conditioning phenomena.

Potentiation of phototactic suppression in Hermissenda by a chemosensory stimulus during compound conditioning.

Modifications of Hermissendas phototactic behavior by compound pairings of light, scallop extract, and rotation were assessed. In general, the scallop extract potentiated phototactic suppression. Potentiation was dependent on (a) conjunctive presentations of scallop and light, (b) number of conditioning trials, and (c) scallop extract concentration. In related experiments, no second-order conditioning or sensory preconditioning of phototactic suppression was observed, indicating that within-compound associations did not contribute appreciably to potentiation. These results represent the first detailed analysis of compound conditioning in a mollusk using discrete presentations of well-characterized conditioned stimuli from distinct sensory modalities.

Chemosensory conditioning in molluscs: II. A critical review.

We critically review chemosensory conditioning studies with molluscs and find that, in many studies, the influence of nonassociative processes complicates, obscures, and renders ambiguous the unique contribution of associative learning. These nonassociative processes include sensory adaptation, habituation, sensitization, and changes in feeding motivation. They arise from both the food extracts that have often been used as conditioned stimuli and the aversive stimuli that have been used as unconditioned stimuli.

Behavioral and neural bases of extinction learning in Hermissenda.

Extinction of classical conditioning is thought to produce new learning that masks or interferes with the original memory. However, research in the nudibranch Hermissenda crassicornis (H.c.) has challenged this view, and instead suggested that extinction erased the original associative memory. We have re-examined extinction in H.c. to test whether extinguished associative memories can be detected on the behavioral and cellular levels, and to characterize the temporal variables involved. Associative conditioning using pairings of light (CS) and rotation (US) produced characteristic suppression of H.c. phototactic behavior. A single session of extinction training (repeated light-alone presentations) reversed suppressed behavior back to pre-training levels when administered 15 min after associative conditioning. This effect was abolished if extinction was delayed by 23 h, and yet was recovered using extended extinction training (three consecutive daily extinction sessions). Extinguished phototactic suppression did not spontaneously recover at any retention interval (RI) tested (2-, 24-, 48-, 72-h), or after additional US presentations (no observed reinstatement). Extinction training (single session, 15 min interval) also reversed the pairing-produced increases in light-evoked spike frequencies of Type B photoreceptors, an identified site of associative memory storage that is causally related to phototactic suppression. These results suggest that the behavioral effects of extinction training are not due to temporary suppression of associative memories, but instead represent a reversal of the underlying cellular changes necessary for the expression of learning. In the companion article, we further elucidate mechanisms responsible for extinction-produced reversal of memory-related neural plasticity in Type B photoreceptors.

Protein Tyrosine Kinase Involvement in Learning-Produced Changes in Hermissenda Type B Photoreceptors

Learning-correlated changes in the excitability and photoresponses of Hermissendas ocular type B photoreceptors are mediated by reductions in two distinct K(+) currents, I(A) and I(K-Ca). The suppression of these K(+) currents has been linked to conditioning-produced activation of protein kinase C (PKC). The question of whether PKC accounts completely for the changes in excitability and K(+) currents or whether other kinase(s) are involved has received little attention. In the present experiments, we asked whether protein tyrosine kinases (PTKs) might also contribute to conditioning-produced alterations in B cells. We found that the PTK inhibitors genistein and lavendustin A greatly reduced cumulative depolarization of type B cells, a short-term correlate of associative learning. This disruption occurred even when PKC activation had been either occluded by preexposure of type B cells to a phorbol ester or otherwise prevented by the pseudosubstrate inhibitor peptide PKC[19-31]. PTK inhibitors also increased the amplitude of the transient (I(A)) and delayed (I(Delayed)) components of voltage-dependent K(+) current that have previously been shown to be selectively reduced by conditioning and to contribute to cumulative depolarization. Genistein partially prevented the reduction of I(A) and I(Delayed) due to in vitro conditioning and blocked the changes in their voltage dependencies. Ionophoresis of pervanadate ion, a potent inhibitor of protein tyrosine phosphatases, depolarized type B photoreceptors and occluded conditioning-produced cumulative depolarization. Pervanadate also suppressed I(A) and I(Delayed), reduced their voltage dependence, and altered inactivation kinetics for I(A), mimicking conditioning. Western blot analysis using a phosphotyrosine antibody indicated that conditioning increased the phosphotyrosine content of many proteins within the Hermissenda CNS. Collectively, our results suggest that in addition to PKC, one or more PTKs play an important role in conditioning-produced changes in type B cell excitability. PTKs and PKCs converge to effect reductions in B cell K(+) currents during conditioning, apparently through distinct biophysical mechanisms.

Chemosensory conditioning in molluscs: I. Failure of contextual conditioning inHermissenda

Aversive chemosensory conditioning altersHermissenda’s feeding behavior. But opposite behavioral changes have been reported, depending on whether discrete-trial or context-conditioning paradigms were used, raising questions about the roles of associative and nonassociative processes. We attempted to produce chemosensory contextual conditioning but failed to do so across a wide range of conditions. In Experiments 1–3, we observed large, nonspecific bite latency increases to shellfish extracts, regardless of whether they had signaled the presence or absence of shaking. In Experiment 4, we found that mere exposure to shellfish extract produced latency increases; vestibular stimulation was unnecessary. In a final experiment, using Y-maze choice tests, we failed to observe selective reductions in animals’ preference for shellfish paired with shaking. Nonassociative processes stemming from prolonged exposure to concentrated shellfish extracts appear to be major factors in our failure to demonstrate associative chemosensory contextual conditioning.

In vitro extinction learning in Hermissenda: involvement of conditioned inhibition molecules

Extinction of a conditioned association is typically viewed as the establishment of new learning rather than the erasure of the original memory. However, recent research in the nudibranch, Hermissenda crassicornis (H.c.) demonstrated that extinction training (using repeated light-alone presentations) given 15 min, but not 23 h, after memory acquisition reversed both the cellular correlates of learning (enhanced Type B cell excitability) and the behavioral changes (reduced phototaxis) produced by associative conditioning (pairings of light, CS, and rotation, US). Here, we investigated the putative molecular signaling pathways that underlie this extinction in H.c. by using a novel in vitro protocol combined with pharmacological manipulations. After intact H.c. received either light-rotation pairings (Paired), random presentations of light and rotation (Random), or no stimulation (Untrained), B cells from isolated CNSs were recorded from during exposure to extinction training consisting of two series of 15 consecutive light-steps (LSs). When in vitro extinction was administered shortly (2 h, but not 24 h) after paired training, B cells from Paired animals showed progressive and robust declines in spike frequency by the 30th LS, while control cells (Random and Untrained) did not. We found that several molecules implicated in H.c. conditioned inhibitory (CI) learning, protein phosphatase 1 (PP1) and arachidonic acid (AA)/12-lipoxygenase (12-LOX) metabolites, also contributed to the spike frequency decreases produced by in vitro extinction. Protein phosphatase 2B (PP2B) also appeared to play a role. Calyculin A (PP1 inhibitor), cyclosporin A (PP2B inhibitor), and baicalein (a 12-LOX inhibitor) all blocked the spike frequency declines in Paired B cells produced by 30 LSs. Conversely, injection of catalytically-active PP1 (caPP1) or PP2B (caPP2B) into Untrained B cells partially mimicked the spike frequency declines observed in Paired cells, as did bath-applied AA, and occluded additional LS-produced reductions in spiking in Paired cells.

Multiple Sources of Light-Evoked Intracellular Calcium Increases in Hermissenda Type B Photoreceptors

Previous research suggests that learning-produced changes in excitability and K+ currents of Hermissenda Type B photoreceptors are Ca2+-dependent phenomena. Little information is available concerning the sources and dynamics of Ca2+ in these cells. We have used Fura-2 dual-wavelength (340/380 nm) photometry to measure somatic [Ca2+]i in B cells. Thirty sec light steps (LS) produce a large increase in [Ca2+]i (∼ 246%). To determine the contribution of Ca2+-influx vs Ca2+-release, we measured [Ca2+]i throughout 5 consecutive LSs in either normal or Ca2+-free ASW (0 mM Ca2+, 30 mM EGTA). Cells exposed to Ca2+-free ASW had a basal [Ca2+]i much lower than when external Ca2+ was present, often below detection limits. Ca2+-free ASW abolished light-induced [Ca2+]i increases in all 7 cells tested. We next explored the role of voltage-gated Ca2+ channels (VGCCs) to Ca2+ influx with the use of cobalt (5mM), a VGCC blocker in B cells. Co2+ did not affect either basal [Ca2+]i or light-induced [Ca2+]i increases (n = 5). To assess the contributions of the ER to light-induced [Ca2+]i changes, B cells were incubated in the ryanodine receptor (RyR) blocker dantrolene (50 μM). Dantrolene reduced the [Ca2+]i response by ∼33% (n =5), and also produced a progressive reduction in basal [Ca2+]i (∼ 60%). Exposure of Ca2+-free ASW cells (n =3) to thapsigargin (TH; 100μM - 1mM, a blocker of the ER Ca2+-ATPase pump) increased basal [Ca2+]i , consistent with store depletion. Collectively, our results indicate that [Ca2+]o is necessary for normal basal [Ca2+]i and critical for light-induced [Ca2+]i increases; but little Ca2+ enters through VDCCs. This suggests that [Ca2+]o enters the cytosol via other routes (e.g. TRP channels) or that the contribution of Ca2+ through VGCCs is slight but serves to trigger Ca2+-induced Ca2+-release (CICR) from ER stores.

FACILITATION EFFECTS OF SUB-NANOMOLAR CONCENTRATIONS OF Aβ(1-42) ON α7 NACHRS IN XENOPUS OOCYTES

Background: Several studies (including our own) have found that high nM mM concentrations of amyloid beta (Ab) peptides inhibit alpha 7 (a7) nicotinic acetylcholine receptor (nAChR) activity. In contrast, recent studies suggest that picomolar concentrations interact with a7Rs in a facilitatory way, suggesting potentially positive roles for Ab in certain learningand memory-related processes (Puzzo et al., 2008). The present study aims to characterize the nature of this facilitory interaction. Methods: Mouse a7 nAChR subunit cRNA (WT and M276L) was synthesized from ma7 nAChR/ pGEM-HE plasmid DNA and microinjected into Xenopus laevis oocytes. Functional expression of mouse a7 nAChRs was evaluated fourth day post injection via two electrode voltage clamp (TEVC) of microinjected oocytes clamped at -60mVand exposed to a saturating concentration (500uM) of acetylcholine. Results: 2-4hr exposure to 100pM Ab(42) increased peak amplitudes and slowed desensitization of ACh-evoked a7R currents expressed in Xenopus oocytes, similar to but less strongly than the type II positive allosteric modulator (PAM) PNU 120596. PNU 120596 (but not a type I PAM, genistein) also occluded the effects of 100pM Ab(42). These results suggested that 100pM Ab(42) acts as a weak type II PAM at wild-type (wt) a7Rs. To further test this idea, we mutated murine a7R cDNA at the M2 residue (M276L) previously shown to be critical for type II PAM effects on human a7Rs (Young et al., 2008). Analysis of the ACh-evoked currents of the mutant a7Rs in the presence of: 1) 100pM Ab(42), 2) 15uM PNU 120596, or 3) the combination of 100pM Ab(42) and 15uM PNU120596, indicated the expected reduction in PNU-facilitation and complete elimination of the 100pM Ab(42)-facilitation. Conclusions: Our results for M276L a7Rs suggest that 100 pM Ab(42) does in fact modulate a7R activity through conventional type II PAM action. Furthermore, the apparent occlusion effect suggests that attempts to remedy cognitive deficits in various Alzheimer’s Disease cohorts using PAMs targeting the a7 nAChR may be doomed to failure because Ab peptides attenuate the substantially greater PAM effects that Type 2 PAMs exhibit in the absence of Ab.