Module 11: Clocks inputs

what types of inputs are involved in non-photic (behavioral) entrainment?

inputs from three brain regions, including the median raphe and dorsal raphe in the brainstem

what entrainment stimulus do these non-photic inputs respond to?

behavioral cues, such as scheduled exercise

how many neurons make up the SCN?

8,000 to 20,000 neurons, depending on the species

despite its small size, what makes the SCN functionally complex?

diverse mix of neurotransmitters and receptors, forming a complex network

melanopsin

light-sensitive photopigment, specifically a type of opsin, found in the retina of the eye

what was the goal of creating Opn4-/- (melanopsin knockout) mice?

to test whether melanopsin is necessary for circadian entrainment to light

what was expected from Opn4-/- mice?

that they would be circadian blind - unable to entrain to light-dark cycles but still able to see using rods and cones

what was the actual result in opn4-/- mice?

they could still respond to light

• phase shifts still occurred

• LD cycles entrained free-running rhythms

• constant light lengthened tau compared to constant dark

how did opn4-/- mice compare to normal mice?

light-induced phase shifts were smaller, but still significant

what are the two ways that melanopsin-containing ipRGCs are activated by light?

• respond to light directly by virtue of containing a photopigment

• respond to light indirectly by connections with rods and cones, via retinal interneurons

how would you eliminate all circadian responses to light?

• eliminate rods, cones, and melanopsin or you have to remove the melanopsin ganglion cells

where does the SCN receive indirect input from?

geniculohypthalamic tract

geniculohypothalamic tract

pathway that originates from neurons in the intergeniculate leaflet of the thalamus

what conclusion was drawn about the retinohypothalamic tract?

it is both necessary and sufficient for photic entrainment

what evidence supports that the RHT is both necessary and sufficient for photic entrainment?

a knife cut to the RHT led to the circadian rhythm free-running, suggesting a loss of light-based entrainment

what was the main critique of the conclusion on RHT?

recording period was too short; to fully prove loss of entrainment, the rhythm should have free-run through a full 360 degree of the LD cycle

what did the knife cut study find out about GHT?

it does not eliminate photic entrainment

How do SCN neurons respond to retinal illumination?

show a sustained response, encoding total luminance (total photos falling on the retina)

what type of light information do SCN neurons encode?

illuminance (overall brightness), not contrast or edges

which retinal cells have similar response patterns to SCN neurons?

melanopsin-containing ganglion cells

how do SCN light responses differ between nocturnal and diurnal animals

nocturnal rodents - most SCN neurons are depolarized by light

diurnal animals - the opposite pattern is observed in some neurons

what neurotransmitter do SCN neurons use to respond to light?

glutamate

what evidence shows glutamate mediates photic responses in the SCN?

optic nerve stimulation increases SCN firing, but this effect is blocked by glutamate receptor antagonists

what does this suggest about glutamate’s role in the SCN?

glutamate is a strong candidate for the ‘photic neurotransmitter’ that shifts the SCN clock

in the 1990s, what was confirmed as the primary neurotransmitter that conveys light input to the SCN?

glutamate

what did injections of glutamate agonist NMDA directly into the SCN do early in the subjective night?

phase delay shifts

what did injections of glutamate agonist NMDA directly into the SCN do late in the night?

phase advance shifts

what happens when the glutamate receptor blocker DGG is injected into the SCN before a light pulse?

light pulse fails to induce a phase shift

what gene is normally activated by light in SCN neurons, and how?

cfos is activated when SCN neurons are depolarized by light

what effect does DGG have on cfos expression?

DGG blocks cfos activation, indicating that glutamate is necessary for this gene’s expression following light exposure

what do the results suggest about glutamate’s role in the SCN?

light causes glutamate release, which depolarizes SCN neurons, activates cfos and shifts the clock

is cFos expression required for light-induced phase shifts?

no, mice without the cfos gene still show normal phase shifts

what happens when drugs block the enzyme nitrous oxide synthase?

they prevent phase shifts

What happens when glutamate binds to NMDA receptors in SCN neurons?

Calcium (Ca²⁺) channels open, increasing intracellular Ca²⁺ levels.

What does increased intracellular Ca²⁺ stimulate?

Production of nitric oxide synthase, which produces nitric oxide (NO).

What is the effect of nitric oxide early in the night?

NO activates ryanodine receptors, leading to Ca²⁺ release from internal stores and causing a phase delay.

what evidence supports the role of ryanodine receptors in phase delays?

drugs that activate ryanodine receptors (e.g., caffeine) also cause phase delays

what is the effect of nitric oxide late in the night?

NO activates guanylate cyclase, increasing proteins kinase G, resulting in a phase advance

which intracellular signaling molecule is involved in both phase delay and phase advance?

nitric oxide (NO), but it activates different pathways depending on time of night

lesions to the IGL that eliminated neuropeptide NPY in the SCN prevents what?

entrainment of free-running rhythms by a daily exercise schedule

the IGL and NPY are required for what type of entrainment?

non-photic

which brain region provides serotonergic (%HT) input to the SCN?

median raphe nucleus

when is 5ht released into the SCN in hamsters

during arousal or wakefullness in the middle of the subjective day

does arousal during the day induce cfos in the media raphe?

no, even though median raphe neurons are active

what is the effect of lesions to the media raphe on exercise-induced phase shifts?

they prevent full entrainment to scheduled exercise

do serotonin-blocking drugs in the SCN block phase shifts from exercise?

no, serotonin antagonists infused into the SCN do not block the shifts

can serotonin-mimicking drugs cause phase shifts?

yes, but the shifts are small or only occur if the 5HT system is sensitized by prior depletion

what does the neurotoxin 5, 7-DHT do?

kills serotonergic neurons, eliminating 5HT in the SCN

what happens to mice with 5, 7-DHT lesions exposed to daily exercise schedules?

fail to entrain, though their clock may still be weakly modulated

what do these results suggest about 5HT and NPY in non-photic entrainment?

NPY is necessary, but not sufficient; 5HT may be required for behavioral entrainment effects

what does the loss of 5HT disrupt?

entrainment effect of scheduled activity and the feedback effect of spontaneous running on tau

are 5HT pathways sufficient enough for shifts?

no, alone are not sufficient to induce non-photic shifts

how can the photic phase response curve be experimentally reproduced?

injecting glutamate into the SCN

which brain regions provide neurotransmitters that can mimic the non-photic PRC?

intergeniculate leaflet (IGL) and the median raphe

what other stimuli have been shown to generate PRCs besides light and arousal?

melatonin, restraint stress, and acetylcholine-related drugs

what does the melatonin PRC look like in rats?

it causes a small phase advance near CT12

why is the PRC to acetylcholine inconsistent?

it has 6 different inputs to the SCN from multiple brain regions, and drugs may non-selectively activate multiple pathways