1. Presented by Kassidy Boyd
Intranasally Administered Neuropeptide S (NPS) Exerts Anxiolytic Effects following Internalization Into NPS Receptor-Expressing Neurons Ionescu et al. 2012
Presented by Kassidy Boyd

2. Introduction In mice, NPS has been shown to: In humans:
Induce strong anxiolytic effects following icv injection
Facilitate fear extinction and reduce fear memory retrieval after extinction training
Influence the HPA axis, which is involved in the pathogenesis of anxiety
Interact with the glutamatergic network, which is involved in the pathology of PTSD
In humans:
there is strong evidence supporting the role of NPS and NPSR in the genetic and neural implications of anxiety and anxiety disorder
 Therefore making NPS a potential candidate as an anxiolytic, which could replace the highly addictive benzodiazepines currently used

3. Problem
Ok, so we know NPS is awesome, but intracerebral NPS administration used in animal studies is unlikely to be employed in humans
Solution
Need to develop an NPS administration technique that could be transferable to humans

4. Background
Pape et al. 2010
NPS system mediates specific effects on synaptic transmission to and within the amygdala
Important for processing acute fear, and extinction of fear memories

5. Background Pape et al. (2010)
Modified from Meis et al. (2008) and Jungling et al. (2008)

6. Background Dannlowski et al. (2011)
The functional polymorphism in the NPSR gene (rs A/T) is associated with panic disorder and anxiety sensitivity
Study
Assessed the role of human NPSR gene variation in the modulation of fear-related amygdala responsiveness through use of fMRI
Findings:
Observed strong association of NPSR T-alleles with right amygdala responsiveness to fear-relevant faces (association peak located in BLA)
Conclusion:
NPSR rs seems to cause an indirect effect on anxiety related traits and may contribute to the pathogenesis of anxiety disorders by shaping fear-related limbic activity

7. Target Brain Regions and Target Cells of NPS
Ionesche et al here on out…
Target Brain Regions and Target Cells of NPS
Examined the distribution pattern of a fluorescent NPS conjugate (Cy3-NPS) after unilateral ICV injection in C57BL/6N mice
Brain regions that were targeted by Cy3-NPS:
Hypothalamus ++
Preoptic area +
Midbrain and brainstem areas +
Cerebellum +++
Forebrain ++
Basal Ganglia +++
Cerebral Cortex +++
Amygdala +
Hippocampus +++
Thalamus +++

8. Cells containing Cy3-NPS were located in:
Amygdaloid Structures
Dorsal endopiriform cortex
Basal ganglia
Fig 1a Ioneschu et al

10. Hippocampus
prominent uptake of Cy3-NPS in hippocampus, CA1, CA2, and CA3
Dentate Gyrus

11. Characterizing the cells that took up Cy3-NPS through immunostaining against Neurofilament , Astroglial and Microglial markers

12. Cy3-NPS taken up by many regions
Notable internalization in CA1, CA2, and CA3 regions of hippocampus
Distribution pattern specificity not altered by fusion of NPS to fluorophore
Similar distribution patterns observed when using rhodamine-NPS
Cells not expressing the neuronal marker did not take up Cy3-NPS
This data suggests NPS is internalized exclusively into neurons after ICV administration

13. Intracellular Uptake of Cy3-NPS is Mediated by Internalization of the Receptor-Ligand Complex
Injected unlabeled NPS at 5x [ ] 10 minutes before ICV injection of Cy3-NPS
**Caused a drastic reduction in Cy3-NPS uptake
Receptor saturation from unlabeled agonist?

14. Receptor Mediated Cy3-NPS Internalization
Cultured HEK cells incubated with Cy3-NPS after transient transfection with EGFP-NPSR
Results:
The receptor-ligand complex was internalized and subsequently accumulated in cytoplasmic and perinuclear vesicular structures.
Conclusion: because NPSR is reported to be the only receptor mediating NPS effects, in vivo uptake of Cy3-NPS is dependent on surface expression of active NPSR

15. NPS Modulates Synaptic Transmission and Plasticity
Extracellular recordings of neurotransmission at CA3-CA1 synapses in brain slices showed:
1µM NPS : ↓ paired-pulse facilitation  ↑release probability of glutamate
NPS also reduced the magnitude of LTP at these synapses
Both effects failed to appear in the presence of NPSR antagonist (R-SHA 68)
These findings indicate that through NPSR, NPS modulates short-term and long-term synaptic plasticity in the CA1 output subfield of the ventral hippocampus

17. Intranasal Administration Delivers Cy3-NPS to its Target Cells
30 minutes after application, Cy3-NPS distribution patterns were almost the same as patterns observed following ICV (see table 1)
Note: the intensity and amount reaching brain was lower in the BG, Cerebral Cortex, Amygdala, Locus coeruleus and Barrington’s nucleus
Cy3-NPS Distribution following Intranasal Administration
Olfactory Bulb Caudal subcortical structures (hippocampus) Internalized into cytosol and processes of target neurons (same as icv)
Conclusion
Intranasally applied NPS is effectively delivered to its brain target neurons in the living mouse

18. Intraneuronal uptake of Cy3-NPS in Hippocampus CA3 region following Intranasal Administration
Hippocampal neuron from Oriens Layer
Hippocampal neuron from Pyramidal layer
BLUE: Nuclear counterstain (DAPI)
RED: Cy3-NPS
GREEN: NF staining

19. Anxiolytic Effects of Intranasally Administered NPS
4 hours after intranasal administration, NPS induced anxiolytic effects in mice which were independent of locomotor activity
C57BL/6N mice↑ time spent on open arms in EPM
HAB mice↑ time spent in light chamber

20. C57BL/6N mice 4 hrs after NPS treatment (7, 14 and 28 nmol)
Open field test: did not show any NPS induced changes in locomotion
Dark-light test: no significant differences for time spent in or distance traveled in chamber, but there was a trend towards reduced latency to first entry into light chamber for mice treated with 14 and 28 nmol NPS
EPM: 14nmol NPS treated significantly increased time on open arms
Conclusion:
IN administered NPS at 14 nmol has the potential to elicit anxiolytic locomotion-independent effects in C57BL/6N mice

21. HAB Mice 4 hrs after NPS treatment (14 nmol)
Open Field Test: locomotor activity unaffected by NPS
Dark-light test: NPS significantly increased time spent in light chamber and reduced latency to first entry  anxiolytic effects
EPM: no differences observed

22. C57BL/6N and HAB mice 30 minutes after NPS treatment (14nmol)
C57BL/6N and HAB mice 30 minutes after NPS treatment (14nmol) No behavioral differences observed.

23. 4 h after IN NPS: detect mRNA changes
Changes in Cerebral Protein Expression linking NPS to the Glutamatergic System and Synaptic Function
4 h after IN NPS: detect mRNA changes
C57BL/6N mice: ↑ in prefrontal cortical Glt-1 and Synapsin II mRNA
HAB mice: ↓ in hippocampal GluR-1 mRNA
No changes in protein levels at 4 h (too soon)
24 h after IN NPS: detect protein expression changes
C57BL/6N mice: ↑ in prefrontal cortical GluR-1 and Glt-1 protein levels AND ↑ hippocampal Synapsin protein expression
HAB mice: ↑ Glu-R2 and a trend of ↑Glu-R1
These results suggest brain region-specific regulatory effects of intranasal NPS treatment

26. Summary
NPS modulates synaptic transmission and plasticity in the CA1 region of the hippocampus
NPS is taken up into NPSR-expressing neurons by internalization of the receptor-ligand complex
Intranasally administered NPS is delivered to the mouse brain and anxiolytic properties are preserved
NPS differentially modulates the expression of glutamatergic system proteins