PSYC 318- Lecture#2

PSYC 318- Lecture#2

1. NAc

   • part of limbic system
     • interface between motivation and movement

2. Conditioned Place Preference Assay

   • answers question
     • do mice find social interactions rewarding?
   • measures rewarding effects of objects/ experiences
     • also w/ learning & memory
   • 1. baseline test
     • animals placed in 2 room chamber
   • 2. conditioning
     • animals are repeatedly placed in a room with something rewarding
     • or aversive: conditioned place aversion
   • 3. preference test
     • mice are placed in the chamber without the stimulus
       • dependent measure
         • time spent in the room
       • do they have positive or negative associations with the environmental cues in that room
   • OXT receptor activity
     • somewhere in the animal
       • necessary for social reward learning
     • OTR-A
       • OXT antagonist blocks OXT receptors
         • mice don't form positive associations of social interactions

3. Microdialysis

   • probe that delivers drugs to specific areas of the brain
   • drug mixed w. florescent indicators
     • drug ends up in NAc
       • there are other OXT receptors in the brain
         • but OXT in NAc are the ones that matter w.r.t. social reward

4. Immunohistochemistry

   • used to label OXT containing neurones in the brain
   • mammal immune system produces antibodies
     • proteins in blood
     • bind to antigens
       • short A.A. sequences
       • scientists can conjugate antibody to GFP
     • purpose
       • marks antigen to be broken down & destroyed
     • glowing antibody specifically binds to OXT peptide
       • you can wash antibody over brain slice to identify OXT

5. How do you remove OXT receptors located on the soma of cells in the NAc required for social reward

   • make conditional OXT receptor knockout mouse
     • knock out mouse
       • completely normal unless certain condition is met
         • cells where condition is met
           • gene in question is excised/ deleted from genome of specific cells
     • most common requirement is presence of enzyme
       • cre-recombinase
         • "cre" can be expressed in specific cells through the use of viruses
       • in this case
         • use virus to drive "crd" expression in cells whose soma is located in NAc
           • this triggers deletion of OXT receptor gene from the genome of those cells

6. Interneurons and projection neurones have different proteins

7. How do you remove OXT from soma located in the NAc

8. Glutametergic axon terminals

9. adding OXT to NAc doesn't change excitability of projection neurons very much but reduces f. of glutamate input

10. Difference in between 2 Pairie voles

    • diff. b/w OXT receptor signalling
    • OXT receptor antagonist
      • blocks bonding

11. OXT

    • peptide made by hypothalamus
      • released into blood w. pituitary
        • hormone
      • axons leaving hypothalamus release OXT
        • nuropeptide
          • acts short distances
    • OXT spray
      • stronger effect in rodents instead of humans

12. Is OXT released in the NAc?

    • sample NAc solution
      • w. mass spec
        • determine presence of OXT peptide
      • problem
        • we might be detecting OXT only located in axons passing through NAc
        • presence of OXT peptide in NAc doesn't prove it was specifically released there
    • solution
      • 1. electron microscopy + immunohistochemistry
        • test if OXT is localized in LDCV
          • in axon terminals of NAc
        • problem
          • time consuming
        • gold standard
      • 2.
        • OXT containing neurones form synaptic connections
        • a) identify all neurones in the WHOLE brain that form synapses in the NAc
          • some located in hypothalamus
            • b) show hypothalamus neurones that project to the NAc contain OXT
              • i) some hypothalamic neurones containing OXT which DO NOT project NAc
          • How to label neurones that project to NAc?
            • Viral Mediated Gene Delivery to label cells (gene therapy)
              • make a virus " replication - deficient"
                • remove DNA from a virus
              • add DNA to a virus
                • DNA encodes
                  • fluorescent proteins
                    • almost all lab-made viral contains this 'GFP'
                      • used to identify infected cells
                  • optogenetic proteins
              • 1. modified virus injected into animals brain
                • infects cells it comes into contact with
                  • a) infects cell bodies
                    • AAV
                  • b) axon terminals
                    • rabies
                      • RbV
                        • once in a cell, virus travels retrogradely to the cell body
                          • get's DNA into infected cells nucleus
                            • placed GFP into rabies virus
                        • RbV-GFP labels NAc-projecting neurons

13. What neurones in the brain enervate the NAc

    • paraventricular nucleus of hypothalamus projects to NAc
      • how do PVN neurones manufacture OXT

14. How to make an antibody for a specific protein

    • 1. synthesize/ purify protein you wan tot label
      • ex: OXT peptide
    • 2. conjugate protein to a known antigen
      • attach= conjugate
      • antigen = protein produces large immune response in the animal
    • 3. inject mammal w. OXT-antigen combo protein
    • 4. wait for animal's immunesystem to create anti-bodies against OXT-antigen combo
    • 5. draw blood from rabbit & purify antibodies that bind specifically to OXT
      • (not the ones that bind to the unknown antigen)
    • 6. attach newly created oxytocin antibody to dye/ fluorescent marker
      • this now allows you to label oxytocin
    • 7. wash brain slices with sol. containing antibody and all OXY peptides become visible

15. Which neurons in the NAc have OXT receptors

    • could have used immunohistochemistry
      • made antibodies specifically binding to OXT receptors
      • wanted a more specific answer
    • OXT Receptor Reporter Mice
      • endogenous DNA of animal
      • foreign DNA added to animal

16. are OXT receptors located on the soma of the cells in the NAc required for social reward

    • Lox P
      • doesn't influence endogenous expression of genes
      • sets condition to eliminate whatever gene it flanks
        • excised OXT DNA will be destroyed
    • Transgenic vs. knockout mice
      • Transgenic mice have DNA added to them
        • most GFP-reporter mice are transgeneic mice
      • knock in/ knock out mice
        • harder b/c they have DNA added/removed from specific parts of animals normal genome
          • therefore genome changed in targeted manner
        • knock out
          • targeted deletion of a specific gene
        • knock in
          • Lox P sites added to specific places
          • also called " conditional knock-out mice "
            • DNA b/w loxP sites will be deleted in presence of cre-recombinase

17. OXT receptor expression in NAc cells is not required for social reward

    • adeno-associated virus
      • AAV
      • only infects cell bodies
        • used to drive expression crd in NAc neurones in conditional OXT receptor knock-out mice

18. Brain Slice Electrophysiology

    • measure voltage change or electric current
    • adding OXT to NAc doesn't change excitability of projection neurons very much
      • but it does reduce f. of glutamate input
      • excitatory synaptic currents mediated by glutamate release onto NAc neurones

19. ·

    • do glutamatergic axon terminal in NAc express OXT receptors?
      • NT receptors are found on axon terminals
        • also on dendrites, soma, everywhere else on the neurone
        • receptors alter likelihood a vesicle will release NT
          • after an action potential occurred
          • probability a NT will be released (2%-100%)
            • probability affected by 3 things
              • 1. amount of presynaptic Ca2+
                • a.k.a. vesicle release triggered by calcium
              • 2. # of vesicle are docked or ready to go
                • 'readily releasable pool of vesicles'
              • 3. # of vesicles in reserve pool
              • 1,2,3 can be changed by g-protein receptor signalling cascades in axon terminal

20. Do any neurons that project to the NAc from anywhere express OXT receptors

    • OXT receptor expression in cells that innervate the NAc is required for social reward
      • RvB infections axon terminals
        • drives expression of cre (& GFP) in all neurons projecting to NAc
          • in conditional OXT receptor knock-out mice
    • which projection to NAc mediates OXT dependent social reward?
      • OXT receptor expression in dorsal raphe neurons required for social reward
        • DRN sends serotongeric projections to the NAc
          • serotonin receptor agonist dosen't change excitability of projection neurons in NAc much
            • but: does reduce f. of glutamate input
      • AVV only infects cell bodies
        • use to drive expression crd & GFP in dorsal rap the neurones
          • of conditional OXT receptor knock-out mice

21. summery of how OXT changes social reward in mice

    • serotonin receptors in NAc required for social reward
      • test: injected serotonin receptor antagonist
      • OXT doesn't reduce strength of glutamate input to the NAc in mice where...
        • OXT receptors were selectively removed from serotonin neurons