A hypothalamic circuit for circadian regulation of corticosterone secretion

Domain: neuroscience
Published: 2026-04-07
Editorial outcome: minor_revision

Abstract
There is a strong circadian rhythm of corticosteroid secretion in animals and humans, but the
the circuit for translating the suprachiasmatic (SCN) clock light-dark cycle rhythm into an increase in
corticosteroid secretion beginning several hours before the active period is not known. We show
here that in male mice, this rhythm depends upon input from the SCN to the subparaventricular
zone (SPZ), and then to the dorsomedial nucleus of the hypothalamus (DMH). Both
glutamatergic and GABAergic DMH neurons are required for the daily surge of corticosteroid
secretion in anticipation of the active period. Glutamatergic DMH neurons directly excite
paraventricular nucleus corticotrophin-releasing hormone (PVH-CRH) neurons, whereas DMH
GABA neurons disinhibit PVH-CRH neurons via a relay in GABAergic neurons in the
caudoventral PVH. This circuit underlies the daily surge in corticosteroid secretion that is
temporally linked but phase advanced compared to the SCN activity cycle.

Introduction
PVHCRH neurons play a key role in the secretion of corticosteroids by releasing CRH into
the hypothalamic-hypophysial portal circulation to stimulate the anterior pituitary release of
adrenocorticotropic hormone (ACTH). ACTH then stimulates the adrenal cortex to secrete
cortisol in humans and corticosterone (Cort) in rodents. The secretion of Cort follows a daily
rhythm, with peak levels typically occurring just before the onset of the active phase, thereby
regulating behavior, metabolism, and immune response. Trough levels occur toward the onset
of the sleep phase, reflecting the natural dip in hormone secretion during the body's resting
period 1,2. This rhythmic secretion pattern reflects the intricate coordination of various neural and
hormonal pathways involved in circadian regulation. Loss of the circadian rhythm of Cort
secretion in rats after lesions of the suprachiasmatic nucleus (SCN) provided some of the first
evidence for the role of the SCN as the brain’s master biological clock 3. However, the
mechanism by which the SCN influences Cort secretion remains unknown. The SCN sends only
limited axons to the PVH, where they mainly terminate in subregions of the PVH that control
autonomic activity 4,5 and spare the PVHCRH neurons. Indeed, the bulk of SCN efferents
terminate in an arc stretching caudally and dorsally from the SCN, encompassing the
subparaventricular zone (SPZ) and rostral dorsomedial nucleus of the hypothalamus (DMH). 6,7.
SPZ neurons are predominantly GABAergic8–10, and lesion studies have shown that the ventral
SPZ participates in the circadian regulation of the sleep-wake cycle, locomotor activity (LMA)
and Cort release, while the dorsal part is involved in rhythms of body temperature (Tb)
regulation11. Downstream, non-specific ablation of DMH neurons in rats eliminates the daily
peak in Cort secretion, reducing the overall daily levels 12. Stimulation of the DMH increases
ACTH and Cort release 13,14. These findings led us to hypothesize that the daily peak in Cort
could be regulated by the SCN through successive relays in the SPZ and the DMH, that, in turn,
stimulates the PVHCRH neurons to trigger Cort release15.
To test this model, first we evaluated the role of SPZVgat neurons in transmitting the
temporal information from the SCN to regulate the daily Cort release by selectively ablating
them or preventing their GABA release. Then, as the DMH is a heterogeneous region containing
roughly equal proportions of glutamatergic and GABAergic neurons, we examined the roles of
both of these DMH cell types in the circadian regulation of Cort levels. We tested whether the
DMHVglut2 neurons were necessary for the circadian regulation of Cort by either ablating or
inhibiting them or deleting the Vglut2 gene in the DMH, preventing glutamate release. We then
tested the effect of chemogenetic activation of DMHVglut2 neurons on Cort levels and used
Channelrhodopsin-assisted circuit mapping (CRACM) to determine the synaptic effects of
DMHVglut2 → PVHCRH input. We also assessed the effect of the activation, as well as ablation or
inhibition of the DMHVgat neurons, and whether deleting the Vgat gene in the DMH affected the
daily rhythm of Cort release. Finally, we examined the role of the caudal ventral part of the PVH
and adjacent peri-PVH area GABAergic (cvPVHVgat) neurons in disinhibiting the PVHCRH
neurons through a polysynaptic circuit (DMHVgat→ cvPVHVgat→ PVHCRH).

Results
SPZVgat neurons, but not GABA release, are necessary for the circadian regulation of
Cort.
To evaluate whether the SPZ GABAergic neurons participate in regulating circadian Cort
release, we placed injections in the SPZ of Vgat-ires-Cre::L10 mice of a viral vector that
constitutively expresses mCherry in neurons lacking Cre-recombinase (Cre), but in cells that
express Cre instead produces diphtheria toxin A (DTA), which induces cell death (AAV10-hSyn-
mCherry-DIO-DTA). We placed injections of an AAV-EGFP virus (AAV8-DIO-EGFP) in the
same site in control mice. After four weeks to allow the full expression of DTA or EGFP, we
measured Cort levels in blood from DTA injected and control mice in a 12:12 light:dark (LD)
cycle, by tail nicks at four time points (ZT 1, 7, 13, 19, taken in random order with at least 30 hrs
between samplings), and at the same time points in constant darkness (DD) (CT 1, 7, 13, 19),
starting on the third day in DD at CT13, and then every 30 hours. Simultaneously, we recorded
the circadian rhythms of locomotor activity (LMA) and body temperature (Tb) for 15 days in LD,
and then 15 days under DD. From a total of 12 mice injected with DTA, 8 mice were identified
by a blinded observer with bilateral DTA injection covering at least 70% of the SPZ; in mice that
also expressed L10-GFP in the Vgat neurons, there was approximately 75% cell loss in the SPZ
(Suppl. Fig. 1). Because some injections encroached on the borders of the SCN, we also did
cell counts within the SCN in these mice and found less than 15% cell loss (Fig. 1a-b and Suppl.
Fig. 1a). The mice with injections that missed the target were considered anatomical controls.
The ablation of the SPZVgat neurons reduced the Circadian Index (CI, comparing the levels
during the light vs the dark period; for method of calculation of CI see Statistical Analysis in the
Materials and Methods) of Cort secretion compared with the control animals by 66.6 ±9.1% in
LD (p=0.008) and 97.7 ±5.9% in DD (p<0.001). The peak Cort level was reduced from control
levels of 23.4  1.7 ng/ml in LD and 28.1  3.9 ng/ml in DD, to 12.8  3.0 ng/ml in LD and 4.8 
1.1 ng/ml in DD in SPZVgat ablated mice; (Fig. 1c-e). The SPZVgat ablations also caused a
dramatic reduction in the CI of locomotor activity (LMA) by 51.1 ±3.5% in LD (p<0.001) and by
79.7 ±4.8% in DD (p<0.001), mostly by reducing the general LMA during the (presumptive) dark
period (Suppl. Fig. 1b-i). The CI of body temperature (Tb) was reduced by 50.42 ± 5.6% in LD
(p<0.001) and 58.34 ±6.5% in DD (p<0.001; Suppl. Fig. 1j-q). The anatomical controls (missed
injections) did not show any changes in LMA or Tb.
To evaluate whether the temporal information was transmitted by GABA release from the
SPZVgat neurons, we placed injections in the SPZ of AAV8-SYN-EGFP-iCre (AAV8-EGFP-iCre)
or a control virus (AAV8-DIO-EGFP) in VgatloxP/loxP (Vgat-flox) mice to delete expression of
functional Vgat protein and so to abolish GABA transmission from the transfected neurons.
From 12 injected mice, 7 received a bilateral injection of AAV8-EGFP-iCre covering at least
70% of the SPZ without extending into the SCN (Fig. 1f-g and Suppl. Fig. 2a). Surprisingly,
deletion of Vgat from the SPZ neurons did not affect the circadian release of Cort (Fig. 1h-j). By
contrast, the circadian rhythms of LMA and Tb were reduced by the deletion of Vgat from
SPZVgat cells, although to a lesser degree than after ablation of those neurons. For instance, the
CI of LMA was reduced by 39.75 ± 8.9% in DD (p<0.001; Suppl. Fig. 2b-i), and the CI of Tb was
reduced by 25.7 ± 4.6% in LD (p=0.006) and 38.6 ± 7.3% in DD (p<0.001; Suppl. Fig. 2j-q).
Taken together, these data indicate that the SPZVgat neurons are necessary to transmit temporal
information for circadian regulation of Cort, LMA and Tb from the SCN, but that the release of
GABA alone cannot account for their influence on circadian regulation.

Glutamate signaling by DMHVglut2 neurons is necessary for the increase in Cort at the
beginning of the active period.
DMH neurons are downstream of the SPZ and have been shown in rats to be necessary
for circadian rhythms of Cort. As DMH ablations cause a reduction in overall Cort secretion, we
hypothesized that circadian Cort secretion may be under the control of excitatory glutamatergic
DMH neurons. We therefore next examined the role of the DMHVglut2 neurons in the circadian
regulation of Cort. We first measured Cort levels in blood from 14 Vglut2-ires-Cre mice in a
12:12 light:dark (LD) cycle, at the same four time points (ZT 1, 7, 13, 19), and at the same time
points in constant darkness (DD) (CT 1, 7, 13, 19). Simultaneously, we recorded the baseline
circadian rhythms of LMA and Tb for 12 days in LD, and then 12 days under DD. We then
placed injections into the DMH of AAV10-hSyn-mCherry-DIO-DTA, and after four weeks to allow
full expression of DTA, we measured Cort levels in LD and DD as described, and recorded LMA
and Tb in the mice for 12 days in LD and 12 days in DD. From a total of 14 mice, we identified 8
mice in which the injection covered at least 70% of the DMH bilaterally (Fig. 2a-b and Suppl.
Fig. 3a) and these mice were used for further analysis. DMHVglut2 ablation reduced the Cort peak
at the beginning of the active phase (ZT 13) from 39.4  6 ng/ml in LD and 27.9  3.5 ng/ml in
DD before AAV10-hSyn-mCherry-DIO-DTA injections, to 23.4  4.2 ng/ml in LD (p=0.007, Fig.
2c) and 15.5  4.3 ng/ml in DD after DMHVglut2 neuron ablation (p=0.013, Fig. 2d). The CI of Cort
secretion was reduced by 46.6 ±14.2% in LD (p=0.043) and 62.0 ±17.3% in DD after the
ablation (p=0.014, Fig. 2e). To determine the specificity of the DMHVglut2 ablations for circadian
increases in Cort, we measured the Cort response to stress of the mice with the ablation by
subjecting them to 1 hr of restraint at ZT3. However, we did not detect changes in the Cort
levels induced by restraint stress, indicating that there was no impairment in overall Cort
secretion and that DMHVglut2 neurons do not play a role in restraint stress-induced Cort secretion
(Suppl. Fig. 3b). The ablation of DMHVglut2 neurons also reduced the peak in LMA at the
transitions between active and inactive periods both in LD and DD. Because these transition
periods spanned the light and dark cycles, there was no change in the CI of LMA, although the
amplitude of the LMA rhythm in DD was reduced in cosinor analysis (Suppl. Fig. 3c-j). In
contrast, ablation of DMHVglut2 neurons reduced Tb during the light phase in LD and in both
presumptive light and dark phases in DD. As a result, the CI and cosinor amplitude of the Tb
rhythm was increased during LD but not DD (Suppl. Fig. 3k-r).
We then evaluated whether the effect of ablation of DMHVglut2 neurons on Cort rhythms
was due to the loss of glutamate transmission, as opposed to other possible transmitters
expressed in DMHVglut2 neurons. We therefore injected the DMH of Vglut2loxP/loxP (Vglut2-flox)
mice with either AAV8-EGFP-iCre or a control virus AAV8-DIO-EGFP and recorded LMA, Tb
and Cort levels in both LD and DD. Seven of the 11 mice had injections covering at least 70% of
the DMH bilaterally and were compared with 5 mice with AAV8-DIO-EGFP injections in the
DMH of Vglut2-flox mice as controls (Fig. 2f-g, Suppl. Fig. 4a). In these animals, the Cort levels
at ZT13 were reduced in LD from 28.9  1.9 ng/ml to 13.2  2.6 ng/ml (p<0.001, Fig. 2h) and in
DD from 40.4  7.5 ng/ml to 11.7  3.5 ng/ml in DMHVglut2/flox-Control and DMHVglut2/flox-EGFP-
iCre mice, respectively (p<0.001, Fig. 2i). The CI of Cort was reduced by 68.2 ± 10.1% in LD
(p<0.001) and by 102.1 ± 14.6% in DD (p=0.001, Fig. 2j). Both the LMA and Tb were reduced
during the dark and subjective dark phase, and at the transition from the dark to the light phase
during LD and at the presumptive transition in DD (Suppl. Fig. 4). However, the CI of neither
LMA nor Tb was affected by the loss of glutamatergic signaling in the DMH.
As the DMHVglut2 ablation and the Vglut2 mRNA deletion are chronic lesions that might
be affected by compensatory mechanisms, we decided to test the effect of acute inhibition of
the DMHVglut2 neurons on Cort, LMA and Tb rhythms. We injected the DMH in 9 Vglut2-ires-Cre
mice with a viral vector containing a modified human alpha-1 glycine receptor (AAV10-DIO-
hGlyR-mCherry) which is insensitive to glycine but has 100-fold increased sensitivity to the
antiparasitic drug ivermectin (IVM) compared to the unmutated glycine receptor16. Because the
half-life of IVM is about 72 hrs, the injection of IVM induces long-lasting neuronal inhibition (4-5
d). To allow the drug to achieve a stable concentration in the CNS, we examined Cort levels
between 24 and 48 hrs after drug administration 17 compared with injection of vehicle. We
administered vehicle (VEH) or IVM (5 mg/kg, ip) one hour after the light or subjective light onset
and sampled the mice the next day at ZT1 and ZT13 to evaluate the Cort levels; one week later,
they received the opposite treatment. In 5 mice in which expression of mCherry was seen in at
least 70% of the DMH bilaterally (Fig. 2k-l, Suppl. Fig. 5a), the Cort levels at ZT13 under LD
were reduced from 31.7  2.3 ng/ml after VEH to 15.6  4.8 ng/ml after IVM (p=0.006), and
under DD from 28.1  6.9 ng/ml after VEH to 9.3  1.8 ng/ml after IVM (p=0.001, see Fig. 2m).
The inhibition of the DMHVglut2 neurons with IVM reduced the Cort CI by 58.9  15.9% in LD
(p=0.01) and by 92.3  9.9% in DD (p=0.019, see Fig. 2n). The average CI of LMA after IVM
administration was reduced by 103.3  21.1% in LD (i.e., the day-night difference was reversed
due to higher levels of LMA during the light period) (p=0.011, Suppl. Fig. 5b-d), and was
reduced in DD by 81.3  9.1% when compared with vehicle (p=0.016, Suppl. Fig. 5e-g) mainly
due to the shifting of much of the LMA peak from the early dark period to the light period,
however, the total LMA was not significantly different at 24-48h after IVM both in DD and LD
(Suppl. Fig. 5b-g). IVM also induced a decrease in Tb during the dark period, with a reduction of
0.5  0.2ºC observed in LD compared with vehicle administration (p=0.024, Suppl. Fig. 5h-j). In
DD conditions, the decrease in Tb during presumptive night was 0.4  0.1ºC (p=0.014, Suppl.
Fig. 5k-m). Tb and LMA returned to normal around day 5 after IVM either in LD or DD. The Tb
CI was reduced in LD by 38.4  6.2% after administration of IVM (p=0.025, Suppl. Fig. 5j). In all
the 3 experiments (DTA, iCre and hGlyR), the anatomical controls (animals with injections that
missed the target) failed to show the responses observed in LMA or Tb.
These data clearly show that glutamatergic transmission by the DMHVglut2 neurons plays
an important role in the increase of Cort levels at the beginning of the active phase. The ablation
or inhibition of DMHVglut2 neurons reduces the peak of Cort to the early morning levels. The
DMHVglut2 neurons also increase LMA in a crepuscular pattern (at the transitions between the
light and dark periods) and their loss causes a small (approximately 0.3-0.5 oC) decrease in Tb,
but has little if any effect on the circadian rhythm of Tb.

Glutamatergic DMH neurons send monosynaptic projections to the PVHCRH neurons and
boost Cort levels
As the ablation of DMHVglut2 neurons reduced the circadian peak of Cort levels, we
hypothesized that activation of these neurons may elevate the Cort levels in preparation for the
active phase. First, we injected the DMH of 15 Vglut2-ires-Cre mice with AAV10-EF1α-DIO-
hM3Dq-mCherry (Fig. 3a-b). Five to six weeks later, the mice were injected i.p. with clozapine-
N-oxide (CNO) 0.3 mg/kg at ZT3. The acute activation of DMHVglut2 neurons caused an increase
in Cort levels from 7.4  3.7 ng/ml just before the animals received the dose of CNO, to 162.5 
16.4 ng/ml 1 hr after the administration of CNO (p<0.001, see Fig. 3c). This increase is around
three times higher than the Cort levels detected in the same mice that received saline or in WT
mice injected with CNO. These results confirm that activation of the DMHVglut2 neurons boosts
Cort release.
To determine whether the DMHVglut2 neurons make direct synaptic contacts onto the
PVHCRH neurons to activate them and so to increase Cort levels, we conducted conditional
anterograde tracing and conditional monosynaptic retrograde rabies tracing studies. We first
evaluated the projection pattern of DMHVglut2 neurons using Vglut2-ires-Cre mice crossed with
reporter mice that express the fluorescent protein Venus (green) in the CRH expressing
neurons (Vglut2-ires-Cre::CRH-Venus). Following injection of AAV8-DIO-ChR2-mCherry into
the DMH, we found a dense projection from DMHVglut2 neurons to the PVH that formed
appositions with the PVHCRH neurons (Fig. 3d-e). To identify the locations of DMHVglut2 neurons
potentially forming direct connections with the PVHCRH cells, we injected the PVH of 5 CRH-ires-
Cre mice with an AAV expressing TVA avian receptor (AAV8-Ef1a-DIO-TVA-mCherry) and an
AAV expressing rabies glycoprotein necessary for rabies viral transfection (AAV8-CAG-DIO-
rabiesG) specifically in the PVHCRH neurons. Twenty-one days later, to label and map the direct
monosynaptic inputs to the PVHCRH neurons, we injected a glycoprotein (G)-deleted rabies virus
(RVdG) that expresses EGFP and is enveloped with the avian ASLV type A protein (EnvA),
which utilizes the TVA receptor for cell entry (EnvA-ΔG-rabies-EGFP). This results in PVHCRH
neurons infected by both AAV and rabies viruses (starter cells) displaying both green and red
fluorescent signals, while neurons expressing only EGFP-Rabies (green) were retrogradely
labeled by the viral particles produced in the starter cells (Suppl. Fig. 6a-b). Transneuronally
infected neurons were seen in areas previously reported to project to the PVH such as the bed
nucleus of the stria terminalis, preoptic area, arcuate nucleus, and DMH, and we also observed
a large number of retrogradely labeled neurons within the PVH itself and its immediate
surrounding areas (Suppl. Fig. 6c-d)18. We found few, if any, EGFP-Rabies infected neurons in
the SCN, which supports the hypothesis that circadian control of Cort secretion by the SCN
requires intermediate relays such as the SPZ and DMH.
We also combined rabies tracing from PVHCRH neurons with in situ hybridization for
Vglut2 mRNA. We found about half of retrogradely-EGFP labeled neurons in the DMH
expressed Vglut2, confirming that DMHVglut2 neurons are likely to directly innervate PVHCRH cells.
In particular, we observe that the largest number of retrogradely labeled neurons expressing
Vglut2 were located in the anterior portion of the DMH (Fig. 3f-g). By contrast, retrogradely
labeled neurons that expressed Vgat mRNA were found throughout the DMH, but with a more
caudal predominance (Suppl. Fig. 6e-f), indicating that there is also a direct GABAergic input
predominantly from the caudal DMH to the PVHCRH neurons (see below).

Stimulation of the DMHVglut2 neurons directly excites PVHCRH neurons
To test the input from the DMHVglut2 neurons to the PVHCRH neurons (DMHVglut2 →
PVHCRH) functionally, we conducted in vitro channelrhodopsin-2(ChR2)-assisted circuit mapping
(CRACM) recordings. We injected the DMH with an AAV8-DIO-ChR2-mCherry in a new cohort
of 4 Vglut2-ires-Cre::CRH-Venus mice and then three to four weeks later we recorded from
Venus-labeled PVHCRH neurons in brain slices while photo-stimulating ipsilateral axons and
terminals of DMHVglut2 neurons within the PVH (Fig. 4a,b). All recordings were performed in the
PVH ipsilateral to the injection site during the daytime (ZT3-8), at the nadir of the daily Cort
cycle19. Optogenetic stimulation of the DMHVglut2 input evoked excitatory synaptic responses in
72% of the PVHCRH neurons recorded (n=18). This effect was mediated by the release of
glutamate and AMPA receptor signaling as the opto-evoked excitatory postsynaptic currents
(oEPSCs) were blocked by the AMPA receptor antagonist DNQX (n=4; Fig. 4c-f). Furthermore,
the probability of oEPSCs in the PVHCRH cells peaked about 5 msec after the photostimulation
of the DMHVglut2 terminals and the oEPSCs persisted in the presence of TTX (TTX 1µM) and 4-
aminopyridine (4-AP), 200–500 µM) indicating monosynaptic connectivity (n=6 out of 8; Fig.
4f,g). These experiments demonstrate that DMHVglu2 neurons have robust direct synaptic input
to PVHCRH neurons (Fig. 4h, i) that is consistent with DMHVglut2 neurons driving the elevation of
Cort levels during the early active period (Fig. 2).

Ablation, inhibition, or disruption of GABAergic signaling in DMHVgat neurons reduces the
daily peak in Cort secretion only under constant darkness
Although inputs from DMHVglut2 neurons to PVHCRH cells are necessary to drive the daily
Cort increase, their ablation does not completely eliminate the circadian rhythm of Cort
secretion as is seen with non-specific DMH ablation12. It has been estimated that half of the
synapses to the PVHCRH neurons are GABAergic20, and our rabies virus experiment showed that
about half of the DMH cells innervating the PVHCRH neurons are GABAergic. Thus, we decided
to evaluate whether the DMHVgat neurons could play a complementary role in the circadian
release of Cort. We hypothesized that the DMHVgat neurons could contribute to the circadian
rhythms of Cort secretion either by inhibiting PVHCRH neurons during the inactive cycle to
suppress Cort secretion, or by dis-inhibiting PVHCRH neurons (i.e., inhibiting inhibitory inputs to
them) during the active cycle. Thus, we used Vgat-ires-Cre mice crossed with a Cre-dependent
GFP reporter mouse line R26-loxSTOPlox-L10-GFP to generate Vgat-ires-Cre::R26-
loxSTOPlox-L10-GFP mice (Vgat-ires-Cre::L10), in which Vgat neurons expressing Cre-
recombinase show green fluorescence. We collect blood samples at 4 temporal points in LD (ZT
1, 7, 13, 19), and then in the same temporal points in DD (CT 1, 7, 13, 19), as we did with the
Vglut2-ires-Cre mice. Also, we recorded LMA and Tb for 12 days in LD, and then 12 days in DD.
Then, we injected the DMH of Vgat-ires-Cre::L10 mice with an AAV10-hSyn-mCherry-DIO-DTA
to ablate the DMHVgat neurons. Four weeks after the injections, we recorded and took blood
samples from the mice in LD and DD conditions. From a total of 13 Vgat-ires-Cre::L10 mice
injected, we analyzed data from 8 mice in which a blinded investigator found that the injection
sites covered at least 70% of the DMH bilaterally (Fig. 5a-b, Suppl. Fig. 7a-b). DMHVgat ablation
did not alter the daily rhythm of Cort release in LD but reduced the CT13 peak under DD from
26.5  2.9 ng/ml in Ctrl to 14.1 4.4 ng/ml (p=0.014, Fig. 5c-d). Similarly, there was no
statistically significant change in the CI of Cort in LD, but it was reduced by 72.4 21.3% in DD
after ablation of DMHVgat neurons (p=0.01, Fig. 5e). Importantly, ablation of DMHVgat neurons did
not change the levels of Cort at its nadir (ZT1 or CT1), which might have been expected if
DMHVgat neurons with direct inputs to PVHCRH cells contributed to circadian rhythms of Cort by
suppressing secretion during the daily nadir. To test the effect of ablation of DMHVgat neurons on
other aspects of Cort secretion, we also measured Cort levels after one hour of restraint stress,
but these were unaffected by the ablations (Suppl. Fig. 7c). In contrast to the effects on Cort
rhythms, LMA was dramatically reduced during the dark phase of LD after DMHVgat ablation, the
total count over the dark period decreasing from 873.5  45 counts to 429.5  52.5 counts
(p<0.001); LMA was reduced even further in DD during the presumptive dark phase from 771.2
 40.6 counts to 401.7  44.9 counts (p<0.001, Suppl. Fig. 7h), resulting in a reduction in the
Circadian Index of LMA by 38.23 ±9.2% in LD (p=0.005) and by 50.61 ±12.9% in DD (p=0.003,
Suppl. Fig. 7i-j), and a significant reduction in the amplitude of the peak at 24 hrs in the
periodogram and in the cosinor analysis of LMA in both LD and DD (LD: p<0.001; DD: p<0.001,
Suppl. Fig. 7e and g). Thus, most of the reduction in LMA after DMH lesions appears to be due
to loss of DMHVgat neurons. In the mice with ablation of DMHVgat neurons, Tb was about 0.3oC
lower throughout the day in LD and during the subjective dark period in DD, but there was no
statistically significant change in Tb in either the CI or cosinor analysis (Suppl. Fig. 7l-s).
We interpreted these results as indicating that it was unlikely that DMHVgat neurons
played a role in suppressing Cort secretion during the inactive (light) phase, but that they
potentially accounted for the other half (not caused by the DMHVglut2 neurons) of the surge in
Cort levels during the active (dark) phase. We next evaluated whether this effect was due to
GABA release from these neurons (vs. some other neurotransmitter in the same neurons).
Thus, we placed AAV8-SYN-EGFP-iCre or AAV8-DIO-EGFP injections in the DMH in
VgatloxP/loxP (Vgat-flox) mice to delete expression of functional Vgat protein and so to abolish
GABA transmission from the GABAergic neurons of the DMH. From 12 mice injected with AAV-
EGFP-iCre in the DMH, 7 with bilateral injections covering at least 70% of the DMH were
included in the analysis (Fig. 5f-g, Suppl. Fig. 8a). Vgat deletion from DMH neurons did not
change the circadian regulation of Cort release in LD, but again reduced the CI of Cort release
in DD by 76.6 ±13.6% (p=0.008, Fig. 5j), by reducing the CT13 peak of Cort levels in DD from
27.0  4.5 ng/ml in controls to 11.7  2.1 ng/ml in Vgat-deleted mice (p=0.006, Fig. 5h). Thus,
most if not all of the effect of the DMHVgat neurons on the circadian release of Cort is mediated
by GABA. Interestingly, although the total levels of LMA during the dark period were decreased
by Vgat deletion in the DMH (from 979.2  66.8 counts in the control group to 681.5  55.6
counts in the deletion group during LD; p<0.001, Suppl. Fig. 8f), the reduction was not nearly as
profound as seen after the ablation of the DMHVgat neurons, suggesting that other transmitters
released from those neurons could play a role in regulating LMA. Results in DD were similar
(from 979.2  66.8 total counts in DMHVgat/flox-Control group to 681.5  55.6 counts in the
DMHVgat/flox-EGFP-iCre during DD; p<0.001, Suppl. Fig. 8f), so that the CI of LMA was reduced
by only 25.7  6% in LD (p=0.001) and 33.3  12.6% in DD (p=0.021, Suppl. Fig. 8g), similar to
the effect observed in the amplitude in the cosinor analysis (Suppl. Fig. 8g-h). Tb of DMHVgat–
deleted mice was also reduced during the dark phase by 0.2 ºC in LD (mostly between ZT14-
18; p=0.001, Suppl. Fig. 8j-n), and in the subjective dark by 0.3ºC in DD (p<0.001, Suppl. Fig.
8l-n). As a result, the Circadian Index of Tb was reduced in the DMHVgat–deleted mice by 20.8 
8.8% in LD (p=0.037) and by 26.6  8.9% in DD (p=0.012, Suppl. Fig. 8o), similar to the
reduction in the amplitude in the cosinor analysis (Suppl. Fig. 8o-p).
To determine whether these chronic conditional lesions or impairments of GABA
transmission might be affected by compensatory mechanisms, we examined the effect of acute
inhibition of the DMHVgat neurons on the circadian release of Cort. We placed injections of AAV-
DIO-hGlyR-mCherry in the DMH of Vgat-ires-Cre mice. From 7 mice injected, 5 mice with
bilateral injection covering at least 70% of the DMH were included for analysis (Fig. 5k-l, Suppl.
Fig. 9a). After 4 weeks allowing the full expression of hGlyR, mice were injected with either
vehicle or IVM (5mg/Kg, ip) at ZT1 in LD, or CT1 in DD, and Cort levels were sampled 24h and
36h later. The inhibition of the DMHVgat neurons did not change the Cort levels under LD, but
reduced the level at CT13 from 22.8  6.1 ng/ml after vehicle to 6.9  1.1 ng/ml after IVM
administration (p=0.006, Fig. 5m) and reduced the CI by 95.7 ±6.9% in DD (p=0.006, Fig. 5n).
Similar to the effects of their ablation, the inhibition of DMHVgat neurons reduced LMA during the
dark or subjective dark period between 24-48 h after IVM administration, reducing the CI in LD
by 77.7  5.7% (VEH p=0.001, Suppl. Fig. 9b-d), and in DD by 80.7  5% (p=0.002, Suppl. Fig.
9e-g). The Tb during the dark period or presumptive dark period was lower after IVM
administration when compare vs VEH (which includes the effect of the handling in contrast to
the baseline), therefore, the CI of Tb was significantly reduced in both LD and DD, 24-48h after
IVM administration by 77.7  8.1% in LD (p=0.005, Suppl. Fig. 9h-j), and by 68.3  14% in DD
(p=0.004, Suppl. Fig. 9k-m). In all three experiments (ablation, deletion, and inhibiton), the
changes observed in levels or rhythms of LMA or Tb were not observed in the anatomical
controls (animals with injections that missed the DMH). These data suggest that DMHVgat
neurons participate in the circadian peak of Cort release at the beginning of the active period
(as seen in DD), likely reducing the inhibitory tone to the PVHCRH neurons at this temporal point,
but that in LD light can act as a cue for elevation of Cort at the onset of the active period,
presumably through another pathway. The ablation of DMHVgat neurons did not, however,
increase the levels of Cort secretion at ZT1 or CT1, indicating that they do not shape circadian
secretion of Cort by suppressing levels at their nadir. Also, the DMHVgat neurons play a larger
role than DMHVglut2 neurons in the circadian regulation of LMA, as the ablation of DMHVgat but
not the DMHVglut2 neurons dramatically reduced LMA during the dark or subjective dark periods.

The activation of DMH GABAergic neurons increases Cort levels, whereas cvPVH
GABAergic neurons constrain Cort levels
As the ablation of the DMHVgat neurons prevented the peak in DD conditions, we tested
whether the activation of those neurons can induce Cort release. We placed injections of AAV8-
DIO-hM3Dq-mCherry in the DMH of Vgat-ires-Cre mice. From 8 mice injected, 6 mice were
confirmed to have injection sites covering at least 70% of the DMH bilaterally (Fig. 6a-b). CNO
administration at the beginning of the light phase (ZT2) elevated the Cort levels to 36.8  9
ng/ml one hour after administration, while Cort was only 10.2  2 ng/ml after vehicle (p=0.016,
Fig. 6c). Although activation of DMHVglut2 neurons produced a larger increase in Cort levels
(>150 ng/ml) than activation of DMHVgat neurons, in both cases the increase in Cort levels
caused by CNO was at least as great as the circadian peak of Cort in undisturbed littermates.
Thus, activation of either DMHVgat or DMHVglut2 neurons is capable of elevating Cort levels within
the range achieved by circadian rhythms.
Because activation of the DMHVgat neurons is expected to release GABA and inhibit their
postsynaptic targets, we hypothesized that the DMHVgat neurons induce Cort release by
disinhibiting the PVHCRH neurons through inhibitory relay neurons, similar to the mechanism by
which GABAergic inputs from the arcuate nucleus increase Cort secretion during food
deprivation 18. To understand the circuit through which DMH GABAergic neurons promote CRH
secretion, we crossed Vgat-ires-Cre mice with CRH-Venus reporter mice to generate Vgat-ires-
Cre::CRH-Venus mice. We then injected the DMH of 4 Vgat-ires-Cre::CRH-Venus mice with
AAV8-DIO-ChR2-mCherry. These injections were relatively small (3-9 nl compared to 24-45 nl
for the other experiments) and covering mostly the rostral part of the DMH (rDMH). We found
that axons and terminals labeled anterogradely (expressing mCherry, in red) from rDMHVgat
neurons largely avoided PVHCRH neurons (green). The bulk of the labeled terminal field was in
areas surrounding the PVH, as well as in the region medial and ventral to the PVHCRH neurons
and extending into the caudal ventral PVH (Fig. 6d-e, 6j-k). Although the PVH contains very few
GABAergic cells, many are found in the region surrounding the PVH, known as the peri-PVH
area, as well as in the adjacent caudal ventral part of the PVH. As terminals from the DMHVgat
neurons blanket the caudal ventral PVH as well as the adjacent peri-PVH just outside it, for the
sake of simplicity we will refer to this region as the cvPVH. Local inhibitory inputs within the PVH
have been described from this region21–23, so we decided to explore whether the cvPVHVgat
neurons might mediate disinhibition of the PVHCRH neurons during the circadian peak of Cort.
To explore whether the cvPVHVgat neurons might make monosynaptic contacts with PVHCRH
neurons, we combined conditional retrograde rabies tracing from PVHCRH neurons with in situ
hybridization for Vgat mRNA using the CRH-ires-Cre mice (n=2). We observed doubly labeled
neurons in the cvPVH suggesting monosynaptic GABAergic inputs from this region to the
PVHCRH neurons (Fig. 6f-g). We next injected 3 Vgat-ires-Cre::CRH-Venus mice with AAV8-
DIO-ChR2-mCherry in this part of the PVH. The injections covered the ventral and lateral region
of the PVH including the cvPVH, and labeled axons ramified inside the PVH, including ones
making appositions with the CRH-Venus neurons (Fig. 6h-i). We then investigated whether the
DMHVgat neurons innervate the cvPVHVgat neurons. Following injections of AAV8-DIO-ChR2-
mCherry into the rostral DMH of Vgat-ires-Cre::L10-GFP mice (n=5) we found dense terminal
labelling in apposition to the cvPVHVgat neurons, suggesting direct connectivity (Fig. 6j-k). These
neuroanatomical findings support the hypothesis of a circuit where the DMHVgat neurons
disinhibit PVHCRH neurons through direct inhibition of the cvPVHVgat neurons (DMHVgat →
cvPVHVgat → PVHCRH).
To test this model, we ablated the Vgat expressing neurons in the cvPVH by placing
injections of AAV10-hSyn-mCherry-DIO-DTA in the cvPVH of Vgat-ires-Cre mice (n=5). In all
cases, the injection sites marked by mCherry expression involved the cvPVH, where Vgat cells
projecting to the PVHCRH neurons are found, and the body of the PVH, where few Vgat cells are
seen. (Fig. 6l-m, Suppl. Fig. 10a). After four weeks to allow full expression of DTA, we recorded
LMA and Tb in the mice for 12 days in both LD and DD, and we collected blood samples for
Cort measurements toward the end of both the LD and DD periods. The cvPVHVgat ablated mice
showed higher levels of Cort but only at the beginning of the active phase, from 21.3  2.3 ng/ml
in controls to 59.8  11.3 ng/ml in cvPVHVgat ablated mice in LD (p<0.001, Fig. 6n), and from
25.6  5.4 ng/ml to 51.3  8.7 ng/ml in DD (p<0.001, Fig. 6o). The CI of Cort also increased by
180.2  52.9% in LD (p=0.01) and 100.4  33.9% in DD (p=0.036, Fig. 6p). At the end of the
protocol, we also exposed the mice to restraint stress and found that the Cort levels were
significantly higher in the cvPVHVgat ablated mice (p=0.047, Suppl. Fig. 10b). No major changes
were observed in LMA or Tb with ablation of cvPVHVgat neurons (Suppl. Fig. 10c-r). These data
indicate that the cvPVHVgat neurons play an important role in inhibiting Cort secretion, even
limiting its peak during the daily surge in Cort secretion at CT13 and in response to restraint
stress. However, these neurons do not appear to play a role in suppressing Cort levels at their
daily nadir. DMHVgat inhibition of cvPVHVgat neurons partially disinhibits the PVHCRH neurons,
allowing the surge in Cort secretion at CT13, but completely eliminating the cvPVHVgat neurons
allows even greater stimulation of the PVHCRH cells at that time by DMHVglut2 inputs.

Dissecting the synaptic inputs of the DMHVgat→ cvPVHVgat→ PVHCRH circuit
To dissect the synaptic mechanisms by which the DMHVgat neurons control the PVHCRH
neurons, we injected the DMH of a new cohort of seven Vgat-ires-Cre::CRH-Venus mice with
AAV8-DIO-ChR2-mCherry. We then performed patch clamp recordings in brain slices from
green-labelled PVHCRH neurons on the same side of the brain while photoactivating the red
DMHVgat terminals in the PVH (Fig. 7a). Our rabies virus tracing data indicated that PVHCRH
neurons receive a direct GABAergic input from the DMH but mainly from its caudal portion
(Suppl. Fig. 6e-f). In support of these results, we observed that in three mice where the injection
site was restricted mostly to the rostral part of the DMH (rDMH), the photostimulation of the
DMHVgat input evoked inhibitory synaptic responses in only 4 out of 17 recorded PVHCRH
neurons (23%, n=17 ;Fig. 7a-e, r), whereas in four mice in which ChR2 expression involved the
more caudal portion of the DMH, the photostimulation of the DMHVgat input evoked oIPSCs in
nearly all PVHCRH neurons (96%, n=23; Suppl. Fig. 11a-g). As the direct GABAergic inputs from
the caudal DMH to PVHCRH neurons do not appear to play a role in inhibiting the PVHCRH
neurons at the circadian nadir of Cort, we focused on whether the rDMH GABAergic inputs
could disinhibit the PVHCRH neurons at the daily peak of Cort secretion.
To investigate whether rDMHVgat neurons directly inhibit the cvPVHVgat neurons, we
expressed AAV8-DIO-ChR2-mCherry in the rDMH of 5 Vgat-ires-Cre::L10-GFP mice. We then
recorded from GFP-labelled cvPVHVgat neurons while stimulating the ipsilateral putative
rDMHVgat input to them (rDMHVgat →cvPVHVgat) (Fig. 7f). Photostimulation evoked opto-inhibitory
postsynaptic currents (oIPSCs) in 14 out of 15 recorded cvPVHVgat neurons. These oIPSCs
depended on GABA receptor-mediated signaling, as they were blocked by bicuculline (n=4;
Fig. 7g-m). Furthermore, oIPSCs persisted in the presence of TTX (1µM) and 4-AP (200 µM)
indicating monosynaptic connectivity (n=6, Fig. 7n).
When we activated the rDMHVgat terminals at different frequencies while recording from
the PVHCRH neurons in which single light pulses did not evoke oIPCS, we found that
photostimulation of the ipsilateral rDMHVgat input at 5 or 10 Hz caused a reduction in the number
of spontaneous IPSCs, which became statistically significant at 20 Hz (n=7; Fig. 8a-e). These
results indicate that the rDMHVgat neurons can disinhibit most PVHCRH neurons by reducing their
GABAergic afferent input.
Our conditional tracing study and CRACM recordings suggest that the rDMHVgat input
disinhibits the PVHCRH neurons at least in part through the GABAergic neurons located in the
cvPVH. We therefore tested whether the cvPVHVgat neurons can directly inhibit the PVHCRH
neurons (cvPVHVgat → PVHCRH). We placed AAV8-DIO-ChR2-mCherry injections in the cvPVH
of 3 Vgat-ires-Cre::CRH-Venus mice. We then recorded from labelled PVHCRH neurons while
photostimulating cvPVHVgat neurons and terminals. Photostimulation of cvPVHVgat neurons
evoked oIPSCs in all PVHCRH neurons recorded (n=14). These oIPSCs were mediated by
GABA receptor signaling as they were blocked by bicuculline (n=4) and were resistant to
TTX+4-AP, indicating monosynaptic connectivity (n=6) (Fig. 8f-l). Interestingly, in four cases
where the ChR2 expression was more dorsal and lateral involving the portion of the pPVH just
lateral to the PVH only about half of the PVHCRH neurons we recorded from showed oIPSCs
(n=4) (Fig. 8m-r).
Taken together, these results indicate that rostral DMHVgat (rDMHVgat) neurons can disinhibit
PVHCRH neurons through GABAergic interneurons located in the cvPVH (rDMHVgat → cvPVHVgat
→ PVHCRH), promoting the circadian increase of Cort levels at CT13.

Methods
Animals. Because adult female mice lose their circadian rhythm of Tb every 4-5 days during
estrus and we needed to record circadian rhythms at specific times across many days, we used
only male adult mice, age 12-16 weeks old, in these experiments. The strains of the mice were
Vgat-ires-Cre 48 (JAX: 016962), Vglut2-ires-Cre 48 (JAX: 016963), CRH-ires-Cre 49 (JAX:
012704), CRH-Venus∆Neo 50, Vglut2loxP/loxP (JAX:036439), VgatloxP/loxP 51 (JAX: 012897) and
R26-loxSTOPlox-L10-GFP mice 49. Mice were individually housed under 12-12h light/dark cycle
unless the protocol specified otherwise. Room temperature were controlled in a range between
22 ± 2°C and free access to food and water was provided. All procedures were performed in
accordance with the National Institutes of Health Guide for the Care and Use of Laboratory
Animals, and formal approval of our protocols was obtained from the Institutionary Animal Care
and Use Committee at Beth Israel Deaconess Medical Center. All precautions were taken to
minimize pain and discomfort in the mice.
Viral Vectors used. AAV10-hSynI-mCherry-DIO-DTA which conditionally expresses the subunit
A of diphtheria toxin in a Cre-dependent fashion and the mCherry protein in non-Cre cells
(acquired from Patrick M Fuller) 52,53 was injected (24nL) in Vgat-ires-Cre or Vglut2-ires-Cre
mice. AAV8-eSYN-EGAFP-T2A-iCre containing the genes for Cre and EGFP under the neuronal
eSYN promoter (VB1089, Vector Biolabs, PA, US; 8.6X1011 viral genomes ml−1) was bilaterally
injected (24nL) in Vglut2loxP/loxP and VgatloxP/loxP mice. AAV8-Ef1a-DIO-ChR2(H134R)-mCherry
injections (UNC, addgene #AV9080, 1.4X1013 viral genomes ml−1) were used for the
neuroanatomical and CRACM experiments (3-9 nL) in Vgat-ires-Cre or Vglut2-ires-Cre mice.
AAV10-DIO-hGlyR-mCherry (acquired from Patrick M Fuller) 16,29,46 injections were made in the
DMH (45nL) either in Vglut2-ires-Cre or Vgat-ires-Cre mice. AAV10-EF1α-DIO-hM3Dq-
mCherry (UNC, addgene #50460 virus core; 2×1013 viral genomes ml−1) was injected (30nL)
either in Vglut2-IRES-Cre or Vgat-IRES-Cre mice. AAV8-CAG-DIO-EGFP (24nl) was injected
as a control virus in specified experiments (UNC; addgene #59331 7X10¹² viral genomes ml−1).
45nL of AAV8-Ef1a-DIO-TVA-mCherry (UNC Vector Core; 1.13X1012 viral genomes ml−1)
mixed 1:1 with AAV8-CAG-DIO-rabiesG (Stanford Vector Core; 3.4X1012 viral genomes ml−1)
was injected unilaterally, and 21 days later, 55nL of EnvA-ΔG-rabies-EGFP (Salk Viral Vector
Core; 1.51X108 viral genomes ml−1) were administrated in the PVH of CRH-ires-Cre mice. All
the experiments and recordings started four weeks after the injections to allow a complete
transfection of the AAVs.
Surgeries. Mice were deeply anesthetized with a mixture of ketamine/xylazine (100/10 mg/kg,
i.p.). Radiotelemetry sensors (TA-F10, DSI, US) for body temperature (Tb) and locomotor
activity (LMA) were implanted in the peritoneal cavity with a small abdominal incision. The
coordinates for the stereotactic microinjections of viral vectors in the brain, from bregma were:
DMH, AP= -1.75mm, ML= ±0.25mm, DV= -4.9mm; PVH, AP= -0.8mm, ML= +0.25mm, DV= -
4.8mm; cvPVH, AP= -0.8mm, ML= +0.25mm, DV= -4.85mm 47. Mice receive analgesic
treatment with meloxicam or buprenorphine post-surgery. All procedures were performed under
aseptic conditions.
LMA and Tb continuous recordings. Average Tb and LMA were recorded every 5 min during all
the protocols using the radiotelemetry DSI system. The signal from the telemetry probes,
previously implanted, was received and converted with the PhysioTel HD and PhysioTel (DSI)
hardware. The LMA and Tb data were analyzed using the software ClockLab Analysis version 6
(Acticmetrics).
Plasma sampling and corticosterone immunoassay. Blood samples (~20µl) were obtained in a
microvette tube (CB300, Sarstedt, US) after a small incision in the tail in less than 1 min after
onset of handling the mouse, centrifuged at 9,240 × g for 10 min, and the plasma was collected
and stored at -40°C. Samples were collected with a minimum interval of 30 hours between
samples. Under the DD protocol, the first sample was obtained on the third day after the lights
turned off at CT13 and then every 30 hours until we completed the 4 time points. An ELISA
assay for corticosterone was performed as per the manufacturer instructions (ADI-901-097,
Enzo life science, US) and the corticosterone signal read with a 405nm filter (iMark, Bio-Rad,
US). Each sample was read in duplicates.
Restraint stress protocol. At the end of the LMA and Tb protocols, mice were physically
restrained for 1 hour starting at ZT3 in a plastic tube with small holes to allow normal respiration.
Blood samples were obtained at the end of the stress protocol by a small incision in the tail. The
mice were placed in their home cage after the protocol.
Perfusion and Immunohistochemistry. At the end of each protocol, animals were deeply
anesthetized with 7% chloral hydrate (0.015ml/gr of body weight i.p.) and perfused
transcardially with 30ml of PBS followed by 30ml of 10%-buffered formalin (Thermo Fisher
Scientific, US). Brains were extracted and postfixed in 10%-buffered formalin overnight,
cryoprotected in 30% sucrose solution, sectioned into 40µm coronal sections (three series) and
stored at 4°C in PBS with sodium azide. Brain sections were washed three times (5 min each)
and then blocked for 1 hour with 3% normal horse serum (diluted in 0.4% Triton X-100 in PBS).
Sections were incubated in primary antibody overnight in the same solution as blocking, at room
temperature with continuous agitation. Primary antibody used were rabbit anti VIP (ImmunoStar;
1:1000, #20077), Chicken anti-GFP (Invitrogen; 1:5,000, A10262), Rat anti-mCherry (Invitrogen;
1:4,000, M11217), Mouse anti-NeuN (Millipore; 1:3,000, MAB377). Then, the sections were
washed six times (5 min each) in PBS, and incubated with secondary antibodies for 2 h in
blocking solution at room temperature and continuous agitation. The secondary antibodies used
were donkey anti rabbit antibody conjugated with CY5 fluorophore (Jackson Immuneresearch;
1:1000, # 711-175-152), Alexa fluor 488-conjugated Donkey anti-Chicken (Jackson; 1:200,
AB2340375), Alexa fluor 555-conjugated Goat anti-Rat (Invitrogen; 1:200, A21434), Alexa fluor
488-conjugated Goat anti-Mouse (Jackson; 1:200, AB2338840). Then, sections were washed
three times (10 min each) in PBS, mounted on electrostatically treated slides and
coverslipped with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories)
for microscope visualization and image acquisition.
RNA scope in situ hybridization. Two brains from the EnvA-ΔG-rabies-EGFP
experiments were sectioned at 20µm and tissue was mounted on glass slides in
RNAase-free conditions. Slices were dried and in situ hybridization was performed
using RNAScope multiplex fluorescent reagent kit V2 (catalog #323100, Advanced Cell
Diagnostics, US). Slices were pretreated with hydrogen peroxide for 10 minutes at room
temperature and subjected to target retrieval step for 5 min in a steamer (˃99°C), This
was followed by dehydration in 90% alcohol and air-dried for 5 min. Sections were then
treated with protease III at 40°C for 30 min. Then, the slices were rinsed with sterile
water and incubated for 2 hrs either with the Vglut2 probe (RNAscope Probe- Mm-
Slc17a6; catalog #319171, Advanced Cell Diagnostics, US) or Vgat Probe (RNAscope
Probe- Mm-Slc32a1; catalog # 319191, Advanced Cell Diagnostics, US) at 40°C for
RNA hybridization. This is followed by incubation with amplification reagents, AMP1,
AMP2 (30 min each) and AMP3 (15 min) at 40°C. Sections were then incubated with
HRP -C1 for 15 mins and Cy5 fluorophore (catalog #NEL741001, PerkinElmer) for 30
mins at 40°C. Finally, HRP blocker was added to the sections for 15 min at 40°C. After
each step sections were washed with 1 x wash buffer provided in the kit. Slides were
then dried and coverslipped with Vectashield antifade mounting medium (catalog #H-
1400, Vector Laboratories) for microscope visualization and image acquisition.
Image acquisition, gradient maps and cellular counting. Coronal sections were scanned at 20X
magnification using an Olympus VS120 slide-scanning microscope or at 63X magnification
using a confocal microscope (Leica Stellaris 5). Fluorescence images were analyzed using
Olympus OlyVIA software (3.4.1) or Leica application suite (version 4.2.1). Photomicrographs
were adjusted for brightness and contrast to compensate for over- or undersaturation due to
differences in fluorescence brightness. The injection site of each animal was confirmed by the
expression of mCherry or EGFP. Microphotographs were transformed to a gray scale and the
background were subtracted. The images at the same anatomical level were superimposed with
30% transparency to build a gradient map using GIMP software (version 2.10.34). In the brain
sections from the rabies experiment, we counted cell bodies in at least 3 levels for each nucleus
(anterior, middle and posterior). In the sections used for in situ hybridization of rabies infected
cells, we counted cell bodies and doubly-stained cells in 6 levels of the DMH. Cell body counting
of the rabies infected cells was conducted manually using the multipoint tool in FIJI (Image J
software, version 1.54A). Because the number of infected cells varied considerably from animal
to animal and we were not trying to establish the absolute number of cells that project to the
PVHCRH neurons, we did not attempt to correct the numbers for cell size.
ChR2-assisted circuit mapping (CRACM). For electrophysiology experiments, AAV8-DIO-ChR2-
mCherry (~3-9 nL) was injected into the DMH of Vglut2-ires-Cre::CRH-Venus mice (n=4) or
Vgat-ires-Cre::CRH-Venus mice (n=3) or Vgat-ires-Cre::L10-GFP mice (n=5) or into the cvPVH
of Vgat-ires-Cre::CRH-Venus mice (n=7) or into the DMH of Vgat-ires-Cre::CRH-Venus mice (n=4).
Mice with AAV injections placed outside of the target were excluded from the analysis. Four to six
weeks following the AAV injections, mice were deeply anaesthetized with isoflurane (5% in oxygen)
via inhalation and transcardially perfused with ice-cold ACSF (N-methyl-D-glucamine, NMDG-based
solution described below). The mouse brains were then quickly removed and sectioned coronally
(250 µm-thickness) in ice-cold NMDG-based ACSF using a vibrating microtome (VT1200S, Leica).
We first incubated the slices containing the PVH for 5 min at 37°C, then transferred them into a
holding chamber at 37°C containing ACSF (Na-based solution) for 10 minutes. We let the brain
slices gradually return to room temperature (~1 hour) before starting recording. Brain slices were
recorded in a recording chamber, where were submerged, and perfused with Na-based ACSF
(described below, 1-1.5 ml/min). We recorded PVHCRH and cvPVHVgat neurons identified by
Venus or L10-GFP (green) fluorescence, respectively, using a combination of fluorescence and
infrared differential interference contrast microscopy (IR-DIC). Both the Venus-expressing CRH
neurons and the L10-GFP neurons were numerous and clear under the microscopy without any
enhancement protocol. We used a fixed stage upright microscope (BX51WI, Olympus America)
equipped with a Nomarski water immersion lens (Olympus 40X / 0.8 NAW) and IR-sensitive
CCD camera (ORCA-ER, Hamamatsu) to acquire real time images using Micro-Manager
software. We recorded the neurons in whole-cell voltage clamp and current clamp
configurations using a Multiclamp 700B amplifier (Molecular Devices), a Digidata 1322A
interface, and Clampex 9.0 software (Molecular Devices). Neurons showing a greater than 10%
change in input resistance over the duration of the recording were excluded from the analysis.
We photostimulated the DMH or cvPVH axons in the PVH using a full-field (~10 mW/mm2, 1 mm
beam width) 5W LUXEON blue light-emitting diode (470 nm wavelength; #M470L2-C4;
Thorlabs), coupled to the epifluorescence pathway of the microscope. We stimulated photo-
evoked excitatory postsynaptic currents (oEPSCs) or inhibitory postsynaptic currents (oIPSCs)
with 10ms light pulses (0.1 Hz, for a minimum of 30 trials). In the DMHVgat injected mice, we also
tested the effects of photostimulation on both the action potential firing of cvPVHVgat neurons
and the spontaneous IPSC (sIPSCs) of PVHCRH neurons. For sIPSC recordings, we used train
stimulations with a duration of 60 seconds at frequencies of 5, 10, and 20 Hz, and a light pulse
duration of 10 ms. For action potential recordings, we tested train stimulations of 10 and 60
seconds at the same frequencies (5, 10, and 20 Hz) with a light pulse duration of 10 ms. Action
potential firing and oEPSCs (holding potential = -70 mV) were recorded in ACSF and using a K-
gluconate-based pipette solution. oIPSCs (holding potential = 0 mV) were recorded in ACSF
containing 1 mM kynurenic acid and using a Cs-methane-sulfonate-based pipette solution. To
record photo-evoked synaptic events in the presence of synaptic blockade, we bath applied
tetrodotoxin (TTX; 1 µM) and the potassium channel blocker, 4-AP (200-500 µM). For all
recordings we added 0.5% biocytin in the pipette solutions to mark the recorded neurons. The
recorded slices containing the PVH and cvPVH and the slices containing the DMH, where the
AAVs were injected, were fixed overnight in 10%-buffered formalin for post hoc histological and
anatomical assessment. To label and map the recorded neurons filled with biocytin, immediately
after the in vitro recordings, we fixed the recorded slices in 10% buffered formalin, washed
them, and incubated them overnight in streptavidin-conjugated Alexa Fluor 405 (1:500; Cat#:
S32351; Invitrogen, Thermo Fisher Scientific Waltham, MA) 54,55. We acquired images using a
Leica Stellaris 5 confocal microscope using a 63X oil immersion objective.
Solutions for CRACM experiments. NMDG-based ACSF solution containing (in mM): 100
NMDG, 2.5 KCl, 1.24 NaH2PO4, 30 NaHCO3, 25 glucose, 20 HEPES, 2 thiourea, 5 Na-L-
ascorbate, 3 Na-pyruvate, 0.5 CaCl2, 10 MgSO4 (pH 7.3: 95% O2 and 5% CO2; 310-320 mOsm).
Na-based ACSF solution contained (in mM): 120 NaCl, 2.5 KCl, 1.3 MgCl2, 10 glucose, 26
NaHCO3, 1.24 NaH2PO4, 4 CaCl2, 2 thiourea, 1 Na-L-ascorbate, 3 Na-pyruvate (pH 7.3-7.4 in
95% O2 and 5% CO2; 310-320 mOsm). Cs-methane-sulfonate-based pipette solution containing
(in mM): 125 Cs-methane-sulfonate, 11 KCl, 10 HEPES, 0.1 CaCl2, 1 EGTA, 5 Mg-ATP and 0.3
Na-GTP (pH adjusted to 7.2 with CsOH, 280 mOsm). K-gluconate-based pipette solution
containing (in mM): 120 K-Gluconate, 10 KCl, 3 MgCl2, 10 HEPES, 2.5 K-ATP, 0.5 Na-GTP (pH
7.2 adjusted with KOH; 280 mOsm). We purchased tetrodotoxin and kynurenic acid from
Cayman Chemical (Ann Arbor, MI) and bicuculline methiodide from Tocris Bioscience (Ellisville,
MO). We purchased all other chemicals from Fisher Scientific (Waltham, MA) or Sigma-Aldrich
(Saint Luis, MO).
Data and statistical analysis for CRACM experiments. Recording data were analyzed using
Clampfit 10 (Molecular Devices), MiniAnalysis 6 software (Synaptosoft), customized Python
scripts (Python 3, www.python.org) and MatLab (version R2020B; MathWorks; Natick, MA)
software. Figures were generated using Igor Pro version 6 (WaveMetrics), Prism 7 (GraphPad,
La Jolla, CA), Inkscape (GitLab) and Photoshop (Adobe) software. To ensure unbiased
detection of synaptic events, the EPSCs and IPSCs were detected and analyzed automatically
using MiniAnalysis. We considered EPSCs or IPSCs in PVHCRH neurons and cvPVHVgat neurons
to be photo-evoked if their probability during the first 50 ms following the light pulses was
greater than the IPSC probability + five times the standard error of the mean (SEM) before the
light stimulation and if their latency was within ± 1 ms of the median value of E/IPSC latencies
calculated for each neuron within the first 50 ms following photostimulation. We calculated the
latency of the photo-evoked EPSC and IPSCs as the time difference between the start of the
light pulse and the 5% rise point of the first synaptic event 55. Group means were compared
using paired t-tests. Cumulative distributions were compared using the Kolmogorov-Smirnov
test. Values indicating p < 0.05 were considered significant.
Statistical analysis. Data sets were tested to see whether they fulfilled the parametric criteria of
Brown-Forsythe (homo/heteroscedasticity) and Shapiro Wilk (fit to normal distribution).
Corticosterone plasma levels and circadian distribution of LMA and Tb were analyzed with a
two-way ANOVA with a factor for group and a factor for time, followed by a post-hoc multiple
comparisons Sidak test. CI for Cort was calculated by the difference between the mean ZT13
and ZT1 levels and divided by the ZT13-ZT1 mean in each mouse, then normalized to 100% by
the mean difference in the Control group. CI for LMA and Tb were calculated by the difference
between the mean dark (or presumptive dark) and light (or presumptive light) levels divided by
the 24h total counts for LMA and 24h mean for Tb, then normalized to 100% against the mean
for the Control group. Light or Dark mean for LMA and Tb were evaluated with two-way ANOVA
with a factor for group and a factor for LD vs DD, followed by a post-hoc multiple comparisons
Tukey test. When the same mice were evaluated under different conditions as their own
controls for LMA and Tb (pre- and post-DTA experiments, and the hGlyR experiments), we
evaluate them with Repeated Measures [RM] two-way ANOVA with a factor for group and a
factor for LD vs DD, followed by a post-hoc multiple comparisons Tukey test. CI and amplitude
data were compared by unpaired t-test analysis, or one-way ANOVA, followed by a post-hoc
multiple comparisons Tukey test in the cases where included baseline in the analysis.
Actograms, periodograms and amplitude of the cosinor fitting were built using ClockLab
ActiMetrics software version 6.1.02. Statistical analyses and graphs were performed with the
GraphPad Prism software version 8. Data are represented as mean ± SEM. Significance values
α were set at p<0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_2": "In this manuscript the authors identify a multi-synaptic circuit controlling the daily release of corticosterone (CORT; SCN to SPZ to DMH to (cv)PVH and PVH). The authors’ findings about the DMH to PVH circuit is very compelling: they show that DMH Vglut2 neurons directly activate PVH CRH neurons, and that DMH Vgat neurons disinhibit PVH CRH neurons by inhibiting GABAergic interneurons in the cvPVH. This dual-pathway organization is well supported by anatomical tracing, slice physiology, and bidirectional circuit manipulation, and represents an exciting finding that will be of interest to circadian biologists, endocrinologists, and neuroscientists. However, several key claims regarding the SPZ relay and the origin of circadian timing control are not supported by the data as currently presented. These issues substantially weaken the manuscript’s central narrative.\n\n1) Discussion Lines 591-594: “Previous studies [LeSauter and Silver 1999]… have shown that extensive loss of SCN neurons is required to cause loss of circadian rhythmicity. In our ablation experiments … encroachment on the SCN was unlikely to cause the profound loss of CORT, LMA and Tb rhythms.” The cited paper does indeed demonstrate that (LMA) arrhythmicity requires 75% of the SCN to be ablated, but this is not relevant to the authors’ findings. The SPZ manipulations in this manuscript (neuron ablation and Vgat gene deletion), regardless of incidental SCN involvement, do not produce arrhythmicity in LMA or Tb (Ext. Figs. 1, 2). Even with SPZ ablation, LMA and Tb remain rhythmic by periodogram analysis. Thus, using an “abolition of rhythms” criterion to exclude SCN contributions to their observed amplitude attenuation is not justified.\n\n2) Discussion Lines 595-598: “A recent study [Klett et al. 2024] using SCN Vgat deletion… found that unilateral or partial deletion in the SCN had little effect on LMA or Tb rhythms. Thus, involvement of the dorsal margin of the SCN … was unlikely to affect those results.” This reasoning is not supported. In the manuscript, the authors explicitly argue that Vgat neuron ablation and Vgat gene deletion produce different outcomes, and that this difference is due to mechanisms beyond GABA release. However, when discussing SCN involvement, the authors treat partial SCN Vgat gene deletion (in Klett et al.) as equivalent to partial SCN neuron ablation in their SCN ablation animals. This is logically inconsistent: if deletion and ablation are not interchangeable in the SPZ, they cannot be treated as interchangeable in the SCN. This citation therefore does not support the authors’ conclusion that potential SCN damage in their ablation experiments is irrelevant.\n\n3) Klett et al. 2024 also show that SPZ Vgat gene deletion using similar methods produces animals that maintain LMA and Tb rhythms in DD. In the current manuscript, the authors find that SPZ Vgat deletion results in an attenuation of these rhythms (to a lesser extent than SPZ Vgat neuron ablation, but an attenuation nonetheless). This raises an important interpretive issue the authors should address: either SPZ Vgat gene deletion is already known to be insufficient to abolish rhythms (as per Klett et al.) or the present study detects a real effect that differs from Klett et al. This discrepancy should be discussed.\n\n4) Throughout the manuscript, “loss of rhythmicity” is used to describe the effects of their various manipulations on CORT, LMA, and Tb. However, the periodogram analyses presented in the supplemental figures show that LMA and Tb remain rhythmic. The authors’ circadian index is primarily a measure of day-night contrast, not rhythmicity, per se. The analyses for CORT (two-way ANOVA) convincingly demonstrate arrhythmicity and/or loss/gain of rhythm amplitude, but categorical arrhythmicity for Tb and LMA is not actually demonstrated. This interpretation should be revised to reflect the attenuation, rather than elimination, of Tb and LMA.\n\n5) Minor concern: the DTA validation data in Fig. 1b are difficult to interpret as presented. The representative image shows GABAergic neurons in green and mCherry expression in red, which the authors describe as “labeling non-GABA cells in the injection site.” However, the relative intensity and extent of red mCherry expression appear markedly greater in the right SPZ compared to the left. If mCherry expression reflects cells lacking Cre (and thus not ablated by the DTA), one would expect comparable non-GABA labeling bilaterally following a bilateral injection. This asymmetry raises several questions: were the injections accidentally unilateral? Did viral spread differ between hemispheres? Or, is this example image an anatomical control (a purposeful unilateral injection) rather than a true experimental animal. Clarification of this point is needed to interpret the extent and specificity of DTA ablation.\n\nOverall, the DMH to PVH to CORT findings are strong and exciting. Addressing these concerns would substantially strengthen the manuscript’s central claims and better align the interpretation with the data as they are presented."}, "author_response": "Response: The reviewer makes a good point. Although the previous studies of partial lesions of the SCN cited by LeSauter and Silver consistently failed to report loss of LMA rhythms with lesions of less than 75% of the SCN, none of them quantified either the lesion extent or the degree of loss of LMA rigorously (and none measured Tb), so that it is possible that partial lesions may have caused partial reduction of the amplitude of LMA (and Tb). We have therefore revised the text to state that the reduction of the amplitude of the circadian rhythms of LMA (by about 80% in DD) and Tb (by about 60% in DD) is proportional to the reduction in SPZ neuron number (by 75%) but not to the reduction in SCN neurons (by 10-15%).\n\nResponse: The reviewer is correct that the Klett paper only examined the effects of deletion of the Vgat gene in the SCN vs SPZ on circadian rhythms (of LMA and Tb). However, it showed that the deletion of the Vgat gene alone in the SCN is sufficient to cause SCN neurons to lose their synchrony and animals to completely lose their circadian rhythms. Thus, the deletion of the Vgat gene in the SCN, unlike the SPZ, does cause loss of rhythms equivalent to ablating the SCN. Nevertheless, we recognize that this is a weak argument, for the reasons cited in Response 1 (i.e., the effects of partial Vgat deletion in the SCN were not quantified). Hence, we have revised the text to remove this argument.\n\nResponse: The Klett paper was begun by Dr. Gompf and Fuller when they were postdocs in the Saper lab around 2010, with his input. However, the project moved with Dr. Gompf around 2013 to the Allen lab at the Univ of Oregon, and then eventually to the Fuller lab at UCDavis, and much of the work in the final manuscript was done by Dr. Klett as part of his PhD dissertation in the Allen lab. As a result, although quantification of circadian amplitude has been standard in the Saper lab since the early 2000’s, it was not done in the Klett paper. Instead, the first and last authors chose to state that the circadian rhythms “remained strongly rhythmic” after Vgat gene deletions in the SPZ. This is actually consistent with the current data, with much more extensive and more rigorously characterized lesion locations, where quantification showed an approximately 45% reduction in the amplitude of the circadian rhythm of LMA and 30% reduction in Tb after SPZ Vgat gene deletions. By contrast, similarly extensive and well characterized SPZ Vgat neuron ablations in the current paper showed an approximately 80% reduction in the circadian amplitude of LMA and 60% reduction in Tb rhythm amplitude, i.e. Vgat neuron ablations caused about twice the reduction in the amplitude of circadian rhythms of LMA and TB compared Vgat gene deletions. Thus, we stand by our interpretation that a large part of the circadian signal from the SPZ for LMA and Tb must be mediated by other transmitters in those neurons. The text has been revised to reflect that analysis.\n\nActual % reductions in amplitude of circadian rhthms of LMA and Tb\nLMA Tb\nCI Cosinor CI Cosinor\nVgat neuron ablation 82% 77% 59% 60%\nVgat gene deletion 48% 45% 31% 30%\n\nResponse: We agree with the Reviewer that reduction in the amplitude of circadian rhythms should not be characterized as “loss of rhythmicity.” In fact, we used this term only once in our entire paper (not “throughout the manuscript”), and that was in quoting LeSauter and Silver (see Response 1), which has now been removed. Instead, because circadian rhythms may be maintained by multiple parallel pathways, it is more accurate simply to calculate the percent of reduction in the amplitude of the rhythm caused by each intervention. Contrary to the statement of the Reviewer (above), we never, at any point in our paper, claimed to demonstrate “arrhythmicity” or “elimination” of the circadian rhythm for Tb or LMA with any of our interventions, because that never occurred. In fact, the only rhythm that we completely eliminated was that of Cort, when we ablated the SPZ Vgat neurons.\n\nWe did claim reductions in the amplitude of the circadian rhythms of Tb and LMA in various experiments, where we used four different approaches to quantify this amplitude. First, we graphed the actual behavior of the animals across the entire 24 hour period in both LD and DD (Ext Figs 1 and 2 for SPZ). This presents all of the information concerning the variations in Tb and LMA across the day. We then employed three commonly used reductionist methods to quantify the circadian rhythm. The first is a periodogram, which shows circadian period length, but also can be used to give an estimate of the amplitude of the rhythm. (Because the animals are not all at exactly 24 hr period lengths, the best estimate of the rhythmicity is given by the area under the curve for the entire peak centered at 24 hr, not the height reached by the peak.) Because this is calculation can be confounded by variations in the period length, we did not try to quantify the amplitude of the rhythm loss in the periodograms, but merely presented the graphs so the reader could draw his or her own conclusions. We also used the circadian index, which as the referee indicates measures the amount of the behavior during the light vs. dark (or presumptive light and dark) phases. This is most useful for behaviors that have a sharp square wave-like difference during the two phases, as LMA usually does. We also used a cosinor analysis, which fits the behavior to a sine wave pattern, which is more accurate for behaviors like Tb that vary in a sine wave type of circadian rhythm across the day, but may not align precisely with the light-dark phase. We presented ALL of the data in ALL FOUR formats for every experiment.\n\nWe have gone back over the text to make clearer which indices we are referring to and avoid vague terms like “loss of rhythmicity”.\n\nResponse: The reviewer is correct that the image in Fig. 1b is difficult to interpret. This is largely because it was taken with a slide scanner, which uses preset parameters, and does not adjust the image for oversaturation or undersaturation. However, in the original image, it is quite clear that there are substantial numbers of red-labeled cells on the left, they are just dimmer than the red cells on the right. (This happens when some cells are transduced by more than one viral particle; however, it takes only one viral particle to cause a recombination event in the cell, so the entire population of transduced cells is critical, not just the brightest ones). We have adjusted the red channel brightness and contrast on the two sides of the image to show more clearly the locations of the red cells on each side of the brain, which mark the location of the surviving cells that do not express Cre and were included within the injection sites. As with most if not all bilateral injection sites, they are not quite symmetric. The injection on the left was a bit more lateral than the one on the right. As a result, there are more residual SPZ-Vgat neurons on the left than the right. Our quantitative data on cell counts for the SPZ and SCN take this into account. With the editor’s permission we would like to substitute the image below for the original one, as we think this more clearly shows the data. We have added into the text for the methods that images were corrected for brightness and contrast."}, {"round": 2, "reviewer_comments": {"reviewer_2": "I appreciate the authors' careful and thoughtful responses to my prior comments. The revisions substantially improve the manuscript, particularly with respect to clarifying amplitude versus rhythmicity, addressing the SCN encroachment arguments, and reconciling the Klett et al. findings with the present data. Overall, these changes strengthen the paper, and the DMH to PVH CRH neuron circuit findings remain compelling and exciting.\n\nI would like to raise one remaining point for consideration, which primarily concerns wording and interpretation rather than additional experiments.\n\nAcross the abstract, results, and discussion, the manuscript repeatedly frames the difference between SPZ Vgat neuron ablation and SPZ Vgat gene deletion as evidence that SPZ Vgat neurons use other transmitters (or that the message is conveyed \"by another co-transmitter\" or \"predominantly by some other transmitter\"). While the new quantitative comparisons are very helpful, I do not think the current data uniquely support this mechanistic inference.\n\nSpecifically, the finding that SPZ Vgat neuron ablation produces a larger reduction in circadian amplitude than Vgat gene deletion demonstrates that loss of GABA release alone is not sufficient to account for the full SPZ ablation phenotype. However, this disparity does not, by itself, establish that non-GABA transmitters are the primary or dominant mechanism of SPZ signaling. Alternative explanations remain plausible, including incomplete elimination of GABAergic signaling at relevant SPZ outputs, loss of local circuit architecture or structural connectivity with ablation, or contributions from cotransmission that are not directly tested here.\n\nFor this reason, I would encourage the authors to soften the mechanistic language in the abstract, results, and discussion. Statements such as \"suggesting that they predominantly use some other transmitter\" or \"the mechanism is mediated at least in part by transmitters other than GABA\" could be rephrased to emphasize insufficiency of GABA alone, rather than positive identification of an alternative transmitter. For example, framing the result as indicating that \"GABA release alone cannot account for the SPZ contribution to circadian regulation\" would, in my view, more accurately reflect what is demonstrated by the data.\n\nAddressing this wording would bring the interpretation fully into alignment with the otherwise strong and carefully executed experimental results."}, "author_response": "Thank you, we have made this change in lines 62-63, 175-176, and 561 and 639."}]

Decision letter