Tetsuro Horikoshi

Quantification of relative mRNA expression in the rat brain using simple RT-PCR and ethidium bromide staining.

We developed a protocol for quantification of relative gene expression using reverse transcription-polymerase chain reaction (RT-PCR) without the use of radioisotopes, special equipment or extra nucleotide fragments, such as competitors. The relative gene expression of GABA(A) receptor beta(1) subunit (GABA(A)Rbeta(1)) and phospholipase C beta(4) subtype (PLCbeta(4)) in rat cerebrum and cerebellum were determined by comparing the ratio of PCR products generated by linear amplification of the target cDNA segments and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA segment as a reference. The density of PCR products was measured from digitized images of photographs of ethidium-bromide-stained agarose gels. The linear region of PCR amplification was within the linear range (from 0.3 to 12 ng DNA in a single band) of the detection system. The accuracy of the present method was <2-fold difference in gene expression in a single determination and a 1.5-fold difference was statistically significant after repeated measurements. The estimated relative expression of PLCbeta(4) was significantly higher in cerebellum than cerebrum, and that of GABA(A)Rbeta(1) was the same in these two regions. Using the present method, it is possible to quantify several different subunits and subtypes of known ion channel, neurotransmitter receptor and intracellular signaling enzyme gene families.

Conditioned taste aversion with sucrose and tactile stimuli in the pond snail Lymnaea stagnalis

A new form of taste aversion conditioning was established in the pond snail Lymnaea stagnalis. An associative memory, lasting 24h, was produced in the pond snail with 20 pairings of 100 mM sucrose as the conditioned stimulus (CS) and mechanical stimulation to the head as the unconditioned stimulus (UCS). Animals exposed to reverse pairings of the CS and UCS failed to learn the association. The learning was characterized by a shift in the response to the UCS from a whole-body withdrawal response to the cessation of feeding behavior.

Associative learning acquisition and retention depends on developmental stage in Lymnaea stagnalis.

Associative learning dependent on visual and vestibular sensory neurons and the underlying cellular mechanisms have been well characterized in Hermissenda but not yet in Lymnaea. Three days of conditioning with paired presentations of a light flash (conditional stimulus: CS) and orbital rotation (unconditional stimulus: UCS) in intact Lymnaea stagnalis results in a whole-body withdrawal response (WBWR) to the CS. In the current study, we examined the optimal stimulus conditions for associative learning, including developmental stage, number of stimuli, interstimulus interval, and intertrial interval. Animals with a shell length longer than 18 mm (sexually mature) acquired and retained the associative memory, while younger ones having a shell length shorter than 15 mm acquired but did not retain the memory to the following day. For mature animals, 10 paired presentations of the CS and UCS presented every 2 min were sufficient for the induction of a WBWR to the CS. Furthermore, animals conditioned with the UCS presented simultaneously with the last 2 s of the CS also exhibited a significant WBWR in response to the CS. Blind animals did not acquire the associative memory, suggesting that ocular photoreceptors, and not dermal photoreceptors, detected the CS. These results show that maturity was key to retention of associative learning.

Increase in excitability of RPeD11 results in memory enhancement of juvenile and adult Lymnaea stagnalis by predator-induced stress

Memory consolidation following learning is a dynamic process. Thus, long-term memory (LTM) formation can be modulated by many factors, including stress. We examined how predator-induced stress enhances LTM formation in the pond snail Lymnaea stagnalis at both the behavioral and electrophysiological levels. Training snails in crayfish effluent (CE; i.e., water from an aquarium containing crayfish) significantly enhanced LTM. That is, while memory persists for only 3h in adult control experiments following a single 0.5-h training session in pond water in which the pneumostome receives a contingent tactile stimulus to the pneumostome; when the snails are trained in CE, the memory persists for at least 24h. In juveniles, the data are more dramatic. Juveniles are unable to form LTM in pond water, but form LTM when trained in CE. Here we examined whether juvenile snails form LTM following a one-trial training procedure (1TT). Following the 1TT procedure (a single-trial aversive operant conditioning training procedure), juveniles do not form LTM, unless trained in CE. Concomitantly, we observe changes in the excitability of RPeD11, a key neuron mediating the whole snail withdrawal response, which may be a neural correlate of enhanced memory formation.

Photoresponse from the statocyst hair cell in Lymnaea stagnalis

Sensory cells for associative learning of light and turbulence were studied in Lymnaea. Intracellular recordings with Lucifer Yellow filled electrodes were made from photoreceptors and statocyst hair cells. Photoreceptors had a long latency, graded depolarizing response to a flash of light; they extended their axon to the cerebral ganglion. The caudal hair cell, one of 12 cells in the statocyst, responded to brief light with a depolarization and superimposed impulse activity. It formed its terminal arborization close to the photoreceptor endings in the cerebral ganglion. Ca(2+)-free saline reversibly abolished the photoresponse in the hair cell, suggesting the information was conveyed via a chemical synapse. These findings demonstrated that sensory information for associative learning was convergent at the statocyst hair cell.

Yolk Syncytial Layer Independent Expression of no tail (Brachyury) or goosecoid Genes in Cultured Explants from Embryos of Freshwater Fish Medaka

Abstract Formation of embryonic axis is an essential step for animal development but its mechanism is not well understood. For axial determination in the fish embryos, participation of the yolk syncytial layer (YSL; a unique multinucleated structure formed in the yolk cell) has been shown. To investigate relationship between the YSL and axial specification, we examined whether or not expressions of the mesodermal marker genes no tail (Brachyury) or goosecoid are dependent on the YSL in explants isolated from the medaka embryos. The results of whole-mount in situ hybridization showed that these genes were expressed in the explants irrespective of the YSL, indicating that activation of these genes is a separate process from YSL-dependent axis formation.

Morphological and physiological characteristics of dermal photoreceptors in Lymnaea stagnalis

Dermal photoreceptors located in the mantle of Lymnaea stagnalis were histologically and physiologically characterized. Our previous study demonstrated that the shadow response from dermal photoreceptors induces the whole-body withdrawal response. Through the interneuron, RPeD11, we detected that the light-off response indirectly originated from a dermal photoreceptor. Previous observations, based on behavioral pharmacology, revealed that cyclic guanosine monophosphate acts as a second messenger in the dermal photoreceptor. Furthermore, gastropods possess dermal photoreceptors containing rhodopsin, as a photopigment, and another photo-sensitive protein, arrestin, responsible for terminating the light response. Thus, we chose three antibodies, anti-cGMP, anti-rhodopsin, and anti-β-arrestin, to identify the dermal photoreceptor molecules in Lymnaea mantle. Extracellular recording, using a suction electrode on the mantle, revealed a light off-response from the right parietal nerve. Overlapping structures, positive against each of the antibodies, were also observed. Numerous round, granular particles of 3–47 μm in diameter with one nucleus were distributed around pneumostome and/or inside the mantle. The cells surrounding the pneumostome area, located 10 μm beneath the surface, tended to have smaller cell soma ranging from 3 to 25 μm in diameter, while cells located in other areas were distributed uniformly inside the mantle, with a larger diameter ranging from 12 to 47 μm. The histological examination using back-filing Lucifer Yellow staining of the right parietal nerve with the three dermal photoreceptor antibodies confirmed that these overlapping-stained structures were dermal photoreceptors in Lymnaea.