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Vesicular glutamate transporter 2 (VGLUT2) is co-stored with PACAP in projections from the rat melanopsin-containing retinal ganglion cells

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Abstract

The retinal ganglion cell layer of the eye comprises a subtype of cells characterized by their intrinsic photosensitivity and expression of melanopsin (ipRGCs). These cells regulate a variety of non-image-forming (NIF) functions such as light entrainment of circadian rhythms, acute suppression of locomotor activity (masking), and pupillary light reflex. Two neurotransmitters have been identified in ipRGCs, glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP). To date, little is known about their release and interplay. Here, we describe the presence and co-localization of vesicular glutamate transporter 2 (VGLUT2; a marker of glutamate signaling) and PACAP in ipRGCs and their projections in the brain. Nine adult male Wistar rats were assigned to one of three groups; anterograde tracing (n = 3), eye enucleation (n = 3), and untreated (n = 3). Under anaesthesia, rats were transcardially perfusion-fixated, after which the brains and eyes were removed for double immunohistochemical staining using a polyclonal anti-VGLUT2 antibody and a mouse monoclonal anti-PACAP antibody. Results revealed that VGLUT2- and PACAP-immunoreactivity (-ir) were present in ipRGCs and co-localized in their projections in the suprachiasmatic nucleus, the intergeniculate leaflet, and the olivary pretectal nucleus. We conclude that there is evidence to support the use of glutamate and PACAP as neurotransmitters in NIF photoperception by rat ipRGCs, and that these neurotransmitters are co-stored and probably released from the same nerve terminals. Furthermore, we conclude that VGLUT2 is the preferred subtype of vesicular transporter used by these cells.

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Abbreviations

3v:

Third ventricle

BW:

Body weight

CNS:

Central nervous system

CtB:

Choleratoxin subunit B

dLGN:

Dorsolateral geniculate nucleus

GCL:

Ganglion cell layer

IGL:

Intergeniculate leaflet

INL:

Inner nuclear layer

IPL:

Inner plexiform layer

ipRGC:

Intrinsically photosensitive retinal ganglion cell

ir:

Immunoreactivity/immunoreactive

NFL:

Nerve fiber layer

NIF:

Non-image-forming

oc:

Optic chiasm

ON:

Overnight

OPN:

Olivary pretectal nucleus

PAC1R:

PACAP receptor 1

PACAP:

Pituitary adenylate cyclase-activating polypeptide

PLR:

Pupillary light reflex

RGC:

Retinal ganglion cell

RHT:

Retino-hypothalamic tract

SCN:

Suprachiasmatic nucleus

VGLUT:

Vesicular glutamate transporter

VIP:

Vasoactive intestinal peptide

vLGN:

Ventrolateral geniculate nucleus

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Authors and Affiliations

  1. Department of Clinical Biochemistry, Bispebjerg Hospital, Faculty of Health Sciences, University of Copenhagen, Bispebjerg Bakke 23, DK-2400, Copenhagen, Denmark

    Anna Engelund, Jan Fahrenkrug & Jens Hannibal

  2. Department of Animal & Veterinary Basic Sciences, Faculty of LIFE Sciences, University of Copenhagen, Copenhagen, Denmark

    Anna Engelund & Adrian Harrison

  3. Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark

    Jens Hannibal

Authors
  1. Anna Engelund
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  2. Jan Fahrenkrug
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  3. Adrian Harrison
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  4. Jens Hannibal
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Corresponding author

Correspondence to Anna Engelund.

Additional information

This work was supported by the Danish Biotechnology Center for Cellular Communication. Anna Engelund was the recipient of a University of Copenhagen, LIFE PhD Scholarship.

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Engelund, A., Fahrenkrug, J., Harrison, A. et al. Vesicular glutamate transporter 2 (VGLUT2) is co-stored with PACAP in projections from the rat melanopsin-containing retinal ganglion cells. Cell Tissue Res 340, 243–255 (2010). https://doi.org/10.1007/s00441-010-0950-3

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