Behavioral Neuroscience, lecture on In vitro classical conditioning of Turtle Eyeblink Reflexes
USD Department of Biology
Behavioral Neuroscience
Turtle Eyeblink Behavior
Sensory Stimulation of Eyeblink
Eyeblink Motor Output
Neuromuscular Function
In vitro Conditioning
text:Kandel pages 1243 & 849
Turtles and Ecology
Eyeblink Circuitry
end     Acronyms/Abbreviations
Eyeblink Conditioning
VII. In Vitro Classical Conditioning of Turtle Eyeblink Reflexes  		

	A. In vitro (in glass) refers to experiments done in culture medium
		1. rather than in a living organism (in vivo)
		2. began with an isolated brainstem-cerebellum preparation 
			a. telencephalon and diencephalon removed 
			b. turtle brains are highly resistant to anoxia
				i. viable / physiologically responsive for a week in vitro
	B. Replace US (airpuff) and CS (tone) with neural correlate
		1. in vitro US = single shock to trigeminal nerve
		2. in vitro CS = 100 Hz stimulus applied to auditory nerve (VIII)
			a. auditory nerves are most sensitive to stimuli
			    between 100 and 500 Hz
		3. training: CS / US pairing with delay 
			a. CS for 1 s ending with US shock
			b. CS/US pair  30 s  CS/US pair
				i. 50 CS/US pairings  30 min 50 more pairings
	C. UR and CR (eyeblink)
		1. neural correlate of UR = discharge
		    recorded from ipsilateral abduscens nerve
		2. latency and duration of abduscens output matches
		    electromyographic response of muscles during blinking
		3. CR = abduscens discharge following auditory simulation alone
	D. Conditioning
		1. abduscens nerve discharge to auditory stimulation gradually acquired
			a. 2nd or 3rd training session
				i. 200 - 300 pairings
			b. = neural correlate of conditioned eyeblink responses		
	E. Extinction
		1. Unpaired stimuli: pairing alternate CS with US
		2. gradual reduction of abduscens nerve CR
		3. not loss but learning
			a. extinction is learning that the pairing is no longer relevant
	F. Reacquisition
		1. following extinction training repairing CS with US
		2. abduscens nerve CR returns
	G. Acquisition of CR does not require the cerebellum
		1. Blink served by mono/di/polysynaptic trigeminal circuits
		    that do not include cerebellum
		2. auditory projections to principle sensory trigeminal nucleus (pV)
			a. and dirctly to accessory (accVI) and
			    principle (pVI) abduscens motor nuclei
			b. and indirectly to accVI and pVI via
				i. cochlear nucleus (to accVI)
				ii. vestibular nucleus (to pVI)
			c. all the substrates necessary for faciliation between
			     auditory and trigeminal input with abduscens output
		3. cerebellum was thereafter removed
		4. In fact, the circuit remained intact if
			a. rostral brainstem = midbrain is removed
			b. medulla is removed (contains circuit for R2)
				i. reduced to R1
					(1) simplest eyeblink discharge response
					(2) no late sustained component of the UR
		5. in vitro preparation includes 4 mm long section of brainstem
		    with intact cranial nerves from V to VIII
			a. a highly reduced in vitro brainstem preparation
			    can be classically conditioned
	H. Acquisition of Conditioning (of R1)
		1. Early Acquisition
			a. BDNF necessary for acquisition
				i. BDNF stimulates ERK (extracellular signal-related kinase = MAPK)
					(1) this step does not require NMDA
			b. BDNF/ErK  upregulation of AMPA receptors
				i. synaptic localization of GluA1 subunits
					(1) also GluA2/3 subunits
				ii. redistribution from non-synaptic regions
					(1) also from vesicle bound AMPA R
				iii. R1 depends on AMPA, not NMDA receptors
			c. BDNF presynaptic ErK + PKA
				i. ephrinB1 together with ephB2
					(1) BDNF & ephrinB1/ephB2: retrograde systems
				ii. bouton size
		2. Late Acquisition
			a. elevated GluA1,2/3 subunits remain
			b. BDNF ERK + NMDA  GluA4 subunits
				i.  total amount by protein synthesis
				ii.  synaptic localization by redistribution
			c.  ARC protein
				i. trafficking of AMPA receptor subunits
					(1) interaction with actin cytoskeleton
				ii. an immediate early gene product
			d.  immediate early gene protein Egr1
				i. acting in trafficking like ARC?
				ii. acting as a transcription factor?
			e. Protein Kinases necessary for GluA4 trafficking and insertion
				i.  ERK MAP K activity
				   from NMDA activation
					(1) synaptic delivery of AMPA GluA4
					     is NMDA dependent
					(2) but, NMDA receptor numbers do not change
					     during any stage of conditioning
			f.  in the number of active synapses
				i.  in presynaptic terminal marker synaptophysin
					(1) synaptophysin is likely a fusion pore 
					    (similar to connexin)
					(2) fusion pore (synaptophysin) + Ca++ channels
					     creates active zone
	I. Retention/Expression
		1.  GluA1,2/3
			a. redistribution back to non-synaptic membrane or vesicles
				i. reverse the process
		2. continued  GluA4
			a.  local + total GluA4
			b. p38 MAP K is activated for maintenance of GluR4
			    (p38 MAP K = CDC-2-related protein kinase or cytokine suppressive anti-inflammatory drug binding protein: 
				which regulates inflammation, cell differentiation, cell growth and cell death)
	J. Extinction
		1. gradual  GluR4
			a. through redistribution
				i. no D total GluA4
		2. no D in synaptophysin
	K. Re-Acquisition
		1. Faster and greater  GluA4
			a. than original acquisition
		2. Synaptophysin stays high
		3. ARC stays high