Abstract Title

Development and Use of “Sniffer Cells” to Detect the Presence of Neuropeptides

Presenter Name

Gef Farmer

RAD Assignment Number

1703

Abstract

Purpose:

The brain is sensitive to Angiotensin (Ang) II and expresses the enzymes necessary for synthesis. However, neuropeptide release has historically been difficult to study and it is currently unclear if Ang II is utilized as a neurotransmitter within the brain. To address this question, our laboratory has adopted a relatively new approach to study the brain renin-angiotensin system – sniffer cells.

Methods:

To make the sniffer cells, Chinese Hamster Ovary (CHO) cells were transfected with plasmids to express angiotensin type 1a (AT1a) receptors and a genetically encoded fluorescent Ca2+ sensor (GCaMP or R-GECO) to address the mechanisms of brain angiotensin II release. Sniffer cells were plated on glass cover slips and continually perfused with aCSF. Calcium imaging was performed at 2 sec intervals using excitation/emission wavelengths of 488/525 nm (GCaMP) or 589/615 nm (R-GECO) and fluorescent intensity was measured in response to bath application of neuropeptides. Sniffer cells were also placed on the median preoptic nucleus (MnPO) in in vitro brain slices (produced using standard slice procedures) from male Sprague-Dawley rats (250-300 g). Fluorescent intensity was measured at 1 sec intervals in response to electrical and optogenetic stimulation of the subfornical organ (SFO).

Results:

The sniffer cells exhibit an increases in fluorescence in response to exogenously applied Ang II that is blocked by the AT1aR antagonist Losartan. The observed increases in fluourescence was specific to AT1aR activation as exogenous application of other common neurotransmitters (Glutamate, Glycine, GABA, NE, ACh) failed to increase sniffer cell. The intensity of sniffer cell responses to Ang II and Ang III were dose dependent with the sniffer cells being more sensitive to Ang III. Using these sniffer cells we were able to detect spontaneous release of Ang II in the MnPO (n = 63). The spontaneous release was activity dependent (i.e. blocked by TTX, n = 10). We were also able to evoke release of Ang II onto sniffer cells via both electrical stimulation (n = 5) and optogenetic stimulation (n = 23) of the SFO.

Conclusion:

Using sniffer cells we were able to detect release of Ang II from in vitro brain slices. Future studies using these sniffer cells will 1) verify the existence of the brain-renin-angiotensin system, 2) characterize the phenotype of Angiotensin II releasing neurons, and 3) investigate potential changes in brain Angiotensin II release in sleep apnea. Sniffer cells are a useful tool for the detection and quantification of neuropeptide release in in vitro neuronal slice preparation and may offer utility in other applications.

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Research Area

Neuroscience

Presentation Type

Poster

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Development and Use of “Sniffer Cells” to Detect the Presence of Neuropeptides

Purpose:

The brain is sensitive to Angiotensin (Ang) II and expresses the enzymes necessary for synthesis. However, neuropeptide release has historically been difficult to study and it is currently unclear if Ang II is utilized as a neurotransmitter within the brain. To address this question, our laboratory has adopted a relatively new approach to study the brain renin-angiotensin system – sniffer cells.

Methods:

To make the sniffer cells, Chinese Hamster Ovary (CHO) cells were transfected with plasmids to express angiotensin type 1a (AT1a) receptors and a genetically encoded fluorescent Ca2+ sensor (GCaMP or R-GECO) to address the mechanisms of brain angiotensin II release. Sniffer cells were plated on glass cover slips and continually perfused with aCSF. Calcium imaging was performed at 2 sec intervals using excitation/emission wavelengths of 488/525 nm (GCaMP) or 589/615 nm (R-GECO) and fluorescent intensity was measured in response to bath application of neuropeptides. Sniffer cells were also placed on the median preoptic nucleus (MnPO) in in vitro brain slices (produced using standard slice procedures) from male Sprague-Dawley rats (250-300 g). Fluorescent intensity was measured at 1 sec intervals in response to electrical and optogenetic stimulation of the subfornical organ (SFO).

Results:

The sniffer cells exhibit an increases in fluorescence in response to exogenously applied Ang II that is blocked by the AT1aR antagonist Losartan. The observed increases in fluourescence was specific to AT1aR activation as exogenous application of other common neurotransmitters (Glutamate, Glycine, GABA, NE, ACh) failed to increase sniffer cell. The intensity of sniffer cell responses to Ang II and Ang III were dose dependent with the sniffer cells being more sensitive to Ang III. Using these sniffer cells we were able to detect spontaneous release of Ang II in the MnPO (n = 63). The spontaneous release was activity dependent (i.e. blocked by TTX, n = 10). We were also able to evoke release of Ang II onto sniffer cells via both electrical stimulation (n = 5) and optogenetic stimulation (n = 23) of the SFO.

Conclusion:

Using sniffer cells we were able to detect release of Ang II from in vitro brain slices. Future studies using these sniffer cells will 1) verify the existence of the brain-renin-angiotensin system, 2) characterize the phenotype of Angiotensin II releasing neurons, and 3) investigate potential changes in brain Angiotensin II release in sleep apnea. Sniffer cells are a useful tool for the detection and quantification of neuropeptide release in in vitro neuronal slice preparation and may offer utility in other applications.