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

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

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

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

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

Background Dannlowski et al. (2011) The functional polymorphism in the NPSR gene (rs324981 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 rs324981 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

Target Brain Regions and Target Cells of NPS Ionesche et al. 2012 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 +++

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

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

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

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

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?

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

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

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

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

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

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

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

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.

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

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