Abstract
Intrinsically photosensitive retinal ganglion cells (ipRGCs) innervate the hypothalamic suprachiasmatic nucleus (SCN), a circadian oscillator that functions as a biological clock. ipRGCs use vesicular glutamate transporter 2 (vGlut2) to package glutamate into synaptic vesicles and light-evoked resetting of the SCN circadian clock is widely attributed to ipRGC glutamatergic neurotransmission. Pituitary adenylate cyclase activating polypeptide (PACAP) is also packaged into vesicles in ipRGCs and PACAP may be co-released with glutamate in the SCN. vGlut2 has been conditionally deleted in ipRGCs in mice (cKOs) and their aberrant photoentrainment and residual attenuated light responses have been ascribed to ipRGC PACAP release. However, there is no direct evidence that all ipRGC glutamatergic neurotransmission is eliminated in vGlut2 cKOs. Here we examined two lines of ipRGC vGlut2 cKO mice for SCN-mediated behavioral responses under several lighting conditions and for ipRGC glutamatergic neurotransmission in the SCN. Circadian behavioral responses varied from a very limited response to light to near normal photoentrainment. After collecting behavioral data, hypothalamic slices were prepared and evoked excitatory postsynaptic currents (eEPSCs) were recorded from SCN neurons by stimulating the optic chiasm. In cKOs, glutamatergic eEPSCs were recorded and all eEPSC parameters examined (stimulus threshold, amplitude, rise time or time-to-peak and stimulus strength to evoke a maximal response) were similar to controls. We conclude that a variable number but functionally significant percentage of ipRGCs in two vGlut2 cKO mouse lines continue to release glutamate. Thus, the residual SCN-mediated light responses in these cKO mouse lines cannot be attributed solely to ipRGC PACAP release.
Significance Statement This study examined glutamatergic signaling by intrinsically photosensitive retinal ganglion cells (ipRGCs) of mice in which vesicular glutamate transporter 2 was knocked out using Cre recombination. The results indicate that significant glutamatergic neurotransmission remains in ipRGCs of Opn4Cre/+;vGlut2loxP/loxP mice and that the ipRGC vGlut2 conditional knockout model resulted in only subtle changes in the rate of vesicular glutamate replenishment even at high stimulation frequencies. These findings are consistent with the behavioral data observed in this and previous studies. Unfortunately, the residual ipRGC glutamatergic transmission in the Opn4Cre/+;vGlut2loxP/loxP mouse model limits the usefulness of this model to examine the role of retinal peptidergic afferents to the suprachiasmatic nucleus and also suggest caution when using Opn4Cre/+ mice in other Cre recombination models.
- circadian Rhythm
- ipRGCs
- Melanopsin
- Retinohypothalamic Tract
- Suprachiasmatic Nucleus
- Vesicular Glutamate Transporter 2
Footnotes
Authors report no conflict of interest.
The work was supported by National Institutes of Health (NIH) grants R01 NS 036607 (CNA) and R01 NS 077003 (PJS & GEP).
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
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