Stimulus-to-action sequences are believed to involve the release of dopamine (DA) and noradrenaline (NA). However, the electrochemical similarity of these two monoamines has limited real-time measurements of their release. As such, the temporal dynamics of DA and NA release in the cortex during learning and behavior remain poorly understood. To meet the challenge of assessing DA and NA in cortex in real time, here I report the creation of two cell-based neurotransmitter fluorescent-engineered reporters (CNiFERs) that discriminate nanomolar concentrations of DA and NA. These CNiFERs transform neurotransmitter receptor binding into a change in fluorescence and provides a direct and rapid optical read-out of local neurotransmitter activity. This generation of CNiFERs provides a new tool for examining the release of DA and NA in vivo and, by extension, the release of any molecule for which there is a G protein coupled receptor. To probe the temporal patterns of neurotransmitter release, specifically DA, NA, and acetylcholine (ACh), during classical conditioning, CNiFERs were implanted into the motor cortex of mice and imaged using two-photon microscopy. The presentation of the unconditioned stimulus, a drop of sucrose solution, triggered the release of DA, NA, and ACh in the motor cortex. The release of ACh consistently tracked the time of licking. With conditioning, the onset of DA release and licking behavior monotonically shifted, in a correlated manner, from the time of the reward toward that of the cue, the conditioned stimulus. Concurrent release of NA, on the other hand, did not correlate with licking or the cue. Thus, by using newly realized CNiFERs to probe in vivo temporal dynamics of transmitter release, we were able to determine that DA, but not NA, release in motor cortex corresponds to the extent of learning, as defined by changes in the animals' licking behavior and that ACh release in the motor cortex corresponds to the animal's motor output

Purpose

Endoplasmic reticulum (ER) stress has been observed in animal models of retinitis pigmentosa expressing P23H rhodopsin. We compared levels of tightly induced ER stress genes, Binding of immunoglobulin protein (BiP) and CCAAT/enhancer-binding protein homologous protein (Chop), in seven additional models of retinal degeneration arising from genetic or environmental causes.

Methods

Retinas from transgenic S334ter rhodopsin (lines 3, 4, and 5) and Royal College of Surgeons (RCS and RCS-p+) rats from postnatal (P) days 10 to 120 were analyzed. In a constant light (CL) model of retinal degeneration, BALB/c mice were exposed to 15,000 lux of CL for 0 to 8 hours. Retinal tissues from three to eight animals per experimental condition were collected for histologic and molecular analyses.

Results

S334ter animals revealed significant increases in BiP, S334ter-3 (3.3× at P15), S334ter-4 (4× at P60), and S334ter-5 (2.2× at P90), and Chop, S334ter-3 (1.3× at P15), S334ter-4 (1.5× at P30), and S334ter-5 (no change), compared with controls. P23H-3 rats showed significant increase of BiP at P60 (2.3×) and Chop (1.6×). RCS and RCS-p+ rats showed significant increases in BiP at P60 (2.4×) and P20 (1.8×), respectively, but no statistically significant changes in Chop. BALB/c mice showed increases in BiP (1.5×) and Chop (1.3×) after 4 hours of CL. Increased levels of these ER stress markers correlated with photoreceptor cell loss.

Conclusions

Our study reveals surprising increases in BiP and to a lesser degree Chop in retinal degenerations arising from diverse causes. We propose that manipulation of ER stress responses may be helpful in treating many environmental and heritable forms of retinal degeneration.

RHO (Rod opsin) encodes a G-protein coupled receptor that is expressed exclusively by rod photoreceptors of the retina and forms the essential photopigment, rhodopsin, when coupled with 11-cis-retinal. Many rod opsin disease -mutations cause rod opsin protein misfolding and trigger endoplasmic reticulum (ER) stress, leading to activation of the Unfolded Protein Response (UPR) signal transduction network. Chop is a transcriptional activator that is induced by ER stress and promotes cell death in response to chronic ER stress. Here, we examined the role of Chop in transgenic mice expressing human P23H rhodopsin (hP23H Rho Tg) that undergo retinal degeneration. With the exception of one time point, we found no significant induction of Chop in these animals and no significant change in retinal degeneration by histology and electrophysiology when hP23H Rho Tg animals were bred into a Chop (-/-) background. Our results indicate that Chop does not play a significant causal role during retinal degeneration in these animals. We suggest that other modules of the ER stress-induced UPR signaling network may be involved photoreceptor disease induced by P23H rhodopsin.