So where are these genes expressed in the fruit fly brain?

Antibody staining indicates that the mushroom bodies contain the molecular components that are key for learning: Adenylyl cyclase PKA cAMP phosphodiesterase

Proposed CS-US and behavior pathways

Fruit fly and sea slugs appear to use common mechanisms Characterizing different forms of memory using modern molecular genetic techniques Fruit flies can learn and remember odor-shock relationships There are a number of mutant fly strains that have been produced which have a variety of learning and memory related deficits: Dunce Amnesiac Rutabaga Radish Cabbage Turnip

Transgenic animals: Organisms that have had genes inserted into their genome GFP: an array of different genetically encoded florescence Red and green great star corals Their GFPs can be expressed in bacteria

GFP-like fluorescent proteins as biotechnology tools The first application of fluorescent proteins from reef organisms for dual-color in vivo labeling: tracing the cell progeny of left and right dorsal blastomeres in a developing tadpole. Transgenic animals:

Transgenic animals: Its not just for GFPs Expression of foreign or native genes can be controlled via insertion of promoter genes Heat shock promoter (hsp70) Can be inserted next to a gene of interest (even another inserted gene) Is activated by warming the animal above a threshold temperature Experiment: 1.Associate a hsp70 with a PKA-inhibitor gene in fruit fly 2.Heat flies PKA-inhibitor expression increases within 1h Learning is disrupted 1.Associate a hsp70 with a mutated (dysfunctional) PKA-inhibitor gene in fruit fly 2.Heat flies Dysfunctional PKA-inhibitor expression increases Learning is NOT disrupted This tells us SPECIFICALLY that disruption of PKA disrupts learning

Consolidation of memory and the role of gene transcription Behavioral data: different training regimes produce different amounts of retention Shock avoidance conditioning: Spaced= 10 trials, 15 min between trials Massed= 10 trials, in rapid succession 1 trial Results: All produced evidence of strong initial learning but: 1 trial group showed little 1 day Massed group showed little 4 days Spaced showed persistent retention 7 days These results suggest that spaced training provides an added benefit specifically relating to longer term retention.

Consolidation of memory and the role of gene transcription Cyclohexamide (CXM) blocks protein synthesis Eliminated the additive effects of spaced training trials Does not affect the aspect of memory related to massed trials

Consolidation of memory and the role of gene transcription CXM does not affect short term memory (or single trial learning) Does not affect cold-shock or anesthesia- resistant memory (ARM)

Consolidation of memory and the role of gene transcription ASM vs ARM ASM: Earlier retention phase ARM: Follows ASM Insensitive (resistant) to cold-shock Lasts for 4 days Insensitive to CXM (no protein synthesis required) Not present in the radish mutant

Consolidation of memory and the role of gene transcription Massed training produces ONLY ARM Experiment: Radish vs wild-type and spaced vs massed Radish mutants display no apparent ARM (which is produced by massed trials) Thus to parse ARM from CXM consolidation we simply need to mix treatments Predictions: 1.Wild-type spaced: ARM+LTM 2.Wild-type massed: ARM but no LTM 3.Wild-type CXM+spaced Only ARM 4.Radish spaced No ARM but LTM 5.Radish massed No ARM/LTM 6.Radish CXM+spaced No ARM/LTM

LTM is CREB-dependent: Transgenic HS-promoted negative CREB disrupts: Normal CREB The added effects of spaced trials All behavioral evidence of LTM ARM - CREB ARM+CREB ARM - CREB

Double mutant radish + negative CREB transgene combinations establish ARM and LTM are functionally distinct processes. Results of 1-day Posttest

Model describing the phases of learning and memory consolidation