A hypothalamic circuit for circadian regulation of corticosterone secretion

Abstract

The secretion of cortisol in humans and corticosterone (Cort) in rodents follows a daily rhythm which is important in readying the individual for daily activity. This rhythm is orchestrated by the suprachiasmatic nucleus (SCN), but how it ultimately regulates the circadian rhythm of activity of neurons in the paraventricular nucleus of the hypothalamus that produce corticotropin-releasing hormone (PVH^CRH neurons) is not known. We hypothesized that the SCN may exert this influence by projections to the subparaventricular zone (SPZ), which in turn innervates neurons in the dorsomedial nucleus of the hypothalamus (DMH) that regulate PVH^CRH neurons. First, we found that ablating SPZ^Vgat neurons eliminates the circadian rhythm of Cort secretion, but that deleting Vgat from them does not, suggesting that they predominantly use some other transmitter. Next, we found that either ablating or acutely inhibiting the DMH glutamatergic (DMH^Vglut2) neurons resulted in a 40-70% reduction in the daily peak of Cort. Deletion of the Vglut2 gene within the DMH produced a similar effect, highlighting the indispensable role of glutamatergic signaling. Chemogenetic stimulation of DMH^Vglut2 neurons led to an increase of Cort levels, and optogenetic activation of their terminals in the PVH in hypothalamic slices directly activated PVH^CRH neurons through glutamate action on AMPA receptors (the DMH^Vglut2 → PVH^CRH pathway). Similar to the disruption of DMH^Vglut2 neurons, ablating, inhibiting, or disrupting GABA transmission by DMH GABAergic (DMH^Vgat) neurons diminished the circadian peak of Cort, particularly under constant darkness conditions. Chemogenetic stimulation of rostral DMH^Vgat neurons increased Cort, although with a lower magnitude compared to DMH^Vglut2 neuron stimulation, suggesting a role in disinhibiting PVH^CRH neurons. Supporting this hypothesis, we found that rostral DMH^Vgat neurons project directly to GABAergic neurons in the caudal ventral part of the PVH and adjacent peri-PVH area (cvPVH), which directly inhibit PVH^CRH neurons, and that activating the rostral DMH^Vgat terminals in the cvPVH in brain slices reduced GABAergic afferent input onto the PVH^CRH neurons. Finally, ablation of cvPVH^Vgat neurons resulted in increased Cort release at the onset of the active phase, affirming the pivotal role of the DMH^Vgat → cvPVH^Vgat → PVH^CRH pathway in Cort secretion. In summary, our study delineates two parallel pathways transmitting temporal information to PVH^CRH neurons, collectively orchestrating the daily surge in Cort in anticipation of the active phase. These findings are crucial to understand the neural circuits regulating Cort secretion, shedding light on the mechanisms governing this physiological process and the coordinated interplay between the SCN, SPZ, DMH, and PVH.

Keywords: corticosterone; corticotropin-releasing hormone; dorsomedial hypothalamus and paraventricular hypothalamus.

Figure 1.. SPZ^Vgat neurons, but not GABA release, are necessary for maintaining the circadian rhythm of Cort secretion.

(a) Schematic drawing of SPZ^Vgat neuron ablation by DTA (b) Representative image of a section from an animal with SPZ^Vgat neuron ablation, showing GABAergic neurons (in green, native signal) and the injection site in red (non-DMH^Vglut2 neurons expressing mCherry, native signal). Notice the remaining green GABAergic neurons in the SCN, and the almost complete elimination of GABAergic neurons just dorsally in the SPZ. Red mCherry was expressed by non-GABAergic cells in the injection site. (c) Ablation of SPZ^Vgat neurons prevented the daily Cort increase at ZT13 in LD (Two-way ANOVA; Tukey’s multiple comparisons test. ZT13 SPZ^Vgat-GFP vs SPZ^Vgat-DTA: *p=0.025) and (d) at CT13 in DD (Two-way ANOVA; Tukey’s multiple comparisons test. CT13 SPZ^Vgat-GFP vs SPZ^Vgat-DTA: ***p<0.001). (e) The circadian index (CI, for method of calculation of CI, see Statistical Analysis in the Materials and Methods) of Cort secretion was reduced by 66.6 ±9.1% in LD (Unpaired t-test: t=5.434, df=14, **p=0.002) and by 97.7 ±5.9% in DD (Unpaired t-test: t=5.534, df=14, ***p<0.001). (f) Schematic representation of Vgat gene deletion in the SPZ. (g) Representative micrograph showing EGFP expression in the SPZ neurons in which the Vgat gene was deleted (green), just dorsal to the SCN^VIP neurons (shown immunohistochemically in red). (h) Mice with Vgat gene deletion in the SPZ showed no change in the daily Cort peak either in LD or (i) DD photoperiod. (j) No changes were detected in the Cort CI. LD, Light:Dark photoperiod; DD, Constant darkness

Figure 2.. DMH^Vglut2 neurons are necessary to maintain the endogenous circadian rhythm of Cort release.

(a) Schematic of the DMH^Vglut2 neuron ablation by DTA (b) Representative image of a section from an animal with DMH^Vglut2 neuron ablation, showing the injection site in red (non-DMH^Vglut2 neurons expressing mCherry) and green NeuN staining as a reference marker. (c) Ablation of DMH^Vglut2 neurons reduced the circadian peak of Cort at ZT13 in LD (Two-way ANOVA; Tukey’s multiple comparisons test. ZT13 Pre-DTA vs DMH^Vglut2-DTA: **p=0.007) and (d) in DD at CT13 (Two-way ANOVA; Tukey’s multiple comparisons test. CT13 Pre-DTA vs DMH^Vglut2-DTA: *p=0.013). (e) The Cort circadian index (CI) in the DMH^Vglut2 ablated mice was reduced by 46.6 ±14.2% in LD (Paired t-test: t=2.225, df=14, *p=0.043) and 62.0 ±17.3% in DD (Unpaired t-test: t=2.775, df=14,*p=0.014). (f) Schematic representation of Vglut2 gene deletion in the DMH. (g) Representative micrograph showing EGFP expression in the DMH neurons in which the Vglut2 gene has been deleted (in green). (h) Vglut2 deletion in DMH diminished the Cort peak at ZT13 in LD (Two-way ANOVA; Tukey’s multiple comparisons test. ZT13 DMH^Vglut2/flox-Control vs DMH^Vglut2/flox-EGFP-iCre: ***p<0.001) and entirely prevented the peak at CT13 in DD (i; Two-way ANOVA; Tukey’s multiple comparisons test. CT13 DMH^Vglut2/flox-Control vs DMH^Vglut2flox-EGFP-iCre: ***p<0.001). (j) The Cort CI was reduced by 68.2 ±10.1% in LD (Unpaired t-test: t=4.621, df=10, ***p<0.001) and by 102.1 ±14.6% in DD (Unpaired t-test: t=4.167, df=10, **p=0.001). (k) Schematic of the chemogenetic inhibition of DMH^Vglut2 neurons expressing hGlyR by IVM administration. (l) Representative micrograph of hGlyR-mCherry expression in the DMH (in red). (m) IVM administration diminished the Cort rise at ZT13 in LD (Two-way ANOVA; Tukey’s multiple comparisons test. ZT13 VEH vs IVM: **p=0.006) and almost entirely prevented it at CT13 in DD compared with vehicle (Two-way ANOVA; Tukey’s multiple comparisons test. CT13 VEH vs IVM: **p=0.001). (n) The Cort CI was reduced one day after the administration of IVM by 58.9 ±15.9% in LD (Paired t-test: t=3.334, df=8, *p=0.01) and by 92.3 ± 9.9% in DD (Paired t-test: t=2.903, df=8, *p=0.019). In all cases we visualize the native signal except for the hGlyR signal that was enhanced with immunofluorescence for mCherry. LD, Light:Dark photoperiod; DD, Constant dark; Reference scale bar= 200µm; 3V, third ventricle.

Figure 3.. DMH^Vglut2 neurons project to the CRH neurons of the PVH and their chemogenetic activation increases Cort.

(a) Schematic of AAV-DIO-hM3dq-mCherry injection targeting the DMH^Vglut2 neurons. (b) Representative image of an AAV-DIO-hM3dq-mCherry (in red) injection site in the DMH. (c) CNO-mediated chemogenetic-stimulation of DMH^Vglut2 neurons boosted Cort to levels much higher than the usual daily peak (cf. Fig 2c; Two-way ANOVA; Tukey’s multiple comparisons test. CT4 WT CNO vs DMH^Vglut2 CNO: ***p<0.001, CT4 DMH^Vglut2 Saline vs DMH^Vglut2 CNO: ***p<0.001). (d) Schematic of AAV-DIO-ChR2-mCherry injection targeting DMH^Vglut2 neurons. (e) Representative photomicrograph of the DMH^Vglut2 neurons expressing ChR2-mCherry (left panel, in red), and their appositions with PVH^CRH neurons (right panel, in green). (f) Schematic of the EnvA-rabies experiment to map the monosynaptic input from the DMH^Vglut2 neurons to PVH^CRH neurons. (g) Mapping of the rabies-Vglut2 co-labeling distribution at different rostro-caudal levels of the DMH, and representative images showing Vglut2 mRNA expression (g’ and g’’, in magenta) and rabies expression (g’ and g’’’, in green) within the rostral DMH (rDMH). The arrows point to the co-labeled cells, and the inset in the lower-right part shows a higher magnification of the neuron pointed by the blue arrow. The hM3dq and ChR2 signal were enhanced with immunofluorescence for mCherry, while the Rabies infected cells were enhanced using an EGFP antibody. Reference scale bar: in b and e (left)= 200 µm, e (center) and g’-g’’’= 50µm, in g’-g’’’ insets = 10µm. 3V, third ventricle. Atlas levels correspond to Paxinos and Franklin Atlas .

Figure 4.. In vitro optogenetic stimulation of the DMH glutamatergic input directly excites PVH^CRH neurons.

(a) Schematic representation of the recording to test the proposed DMH^Vglut2 → PVH^CRH circuit; Vglut2-ires-Cre::CRH-Venus mice were injected with AAV-DIO-ChR2-mCherry in the DMH and recordings were conducted in brain slices from Venus-labeled PVH^CRH neurons while photostimulating the ipsilateral DMH^Vglut2 input (the DMH is shown on the opposite side of the brain here for ease of illustration). (b) An example of ChR2-mCherry native expression after an injection in the DMH (top left) and density plots of the AAV-DIO-ChR2-mCherry injection sites (n=4 mice; right and bottom). (c) AMPA-mediated opto-evoked excitatory post-synaptic currents (oEPSCs) recorded in PVH^CRH neurons (upper trace) and blockade by AMPA receptor antagonist DNQX, 200µM (lower trace; n=4, from 2 mice). (d) Percentages of PVH^CRH neurons responding (Connected) and not responding (Not Connected) to photostimulation of the DMH^Vglut2 input (n=18 PVH^CRH recorded neurons, from 4 mice). (e) Amplitude (filled markers, cells responding to photostimulation, n=13, open markers, cells not responding to photostimulation, n=15 neurons from 4 mice; mean and ± SEM of responding neurons) and (f) latency of oEPSCs in PVH^CRH neurons in response to photostimulation of the DMH^Vglut2 input (mean and ± SEM; n=13 neurons from 4 mice). (g) oEPSCs in PVH^CRH neurons recorded in TTX 1µM + 4-AP 500µM (n=6 neurons from 2 mice) indicating monosynaptic connectivity. (h) Raster plot of EPSCs in a representative PVH^CRH neuron with photostimulation of the DMH^Vglut2 → PVH^CRH input (bin duration: 50ms). (I) EPSC probability in response to photostimulation of the DMH^Vglut2 → PVH^CRH input (black, n=13 neurons; grey, n=5 neurons, from 4 mice). Reference scale bar: in b = 250 µm. f, fornix, 3V, third ventricle. Atlas levels correspond to Paxinos and Franklin Atlas .

Figure 5.. Ablation of DMH^Vgat neurons reduces the endogenous circadian peak of Cort release only in constant darkness.

(a) Schematic representation of DMH^Vgat neuron ablation. (b) Representative micrograph showing GABAergic neurons (in green) and AAV-DIO-DTA-mCherry (in red) in the DMH of a Vgat-ires-Cre::L10-GFP mice. There were no remaining GABAergic neurons in the region of mCherry expression. (c) DMH^Vgat neuron ablation did not alter the daily Cort release in LD, (d) but eliminated it almost entirely under DD (Two-way ANOVA; Tukey’s multiple comparisons test. CT13 Pre-DTA vs DMH^Vgat-DTA: *p=0.014). (e) The Cort CI was not significantly changed by DMH^Vgat ablation in LD, but was reduced by 72.4 ±21.3% in DD. (Paired t-test: t=3.028, df=12, *p=0.01). (f) Schematic representation of Vgat gene deletion in the DMH. (g) Representative photomicrograph showing the EGFP expression in Vgat-deleted neurons (green). (h) Vgat deletion from DMH neurons did not change the circadian regulation of Cort release in LD, (i) but eliminated the daily peak of Cort at CT13 in DD (Two-way ANOVA; Tukey’s multiple comparisons test. CT13 DMH^Vgat/flox-Control vs DMH^Vgat/flox-EGFP-iCre: **p=0.006). (j) The CI of Cort secretion was not affected by Vgat gene deletion in the DMH in LD but was reduced by 76.6 ±13.6% in DD in DMH^Vgat-deleted mice (Unpaired t-test: t=3.160, df=12, **p=0.008). (k) Schematic representation of the AAV-DIO-hGlyR-mCherry injection in the DMH of Vgat-ires-Cre mice. (l) Representative micrograph of hGlyR-mCherry expression (in red) in the Vgat neurons of the DMH. (m) IVM chemo-inhibition of the DMH^Vgat neurons by hGlyR did not alter the Cort levels in LD, but prevented the peak at CT13 in DD (Two-way ANOVA; Tukey’s multiple comparisons test. CT13 VEH vs IVM: **p=0.006). (n) The Cort CI was reduced by 95.7 ±6.9% in DD (Paired t-test: t=3.641, df=8, **p=0.006). In all cases we visualize the native signal except for the hGlyR signal that was enhanced with immunofluorescence for mCherry. Reference scale bar= 200µm; 3V, third ventricle.

Figure 6.. Chemo-activation of DMH^Vgat neurons elevates Cort levels through cvPVH GABAergic neurons.

(a) Schematic of AAV-DIO-hM3dq-mCherry injections in the DMH in Vgat-ires-Cre mice. (b) Representative micrograph of hM3dq-mCherry expression (red) in the DMH. (c) Chemogenetic activation of the DMH^Vgat neurons increased Cort levels from 10.2 ±2 ng/ml after VEH to 36.8 ±9 ng/ml after CNO administration (Paired t-test: t=2.875, df=10, *p=0.016). (d) Schematic of an AAV-DIO-ChR2-mCherry injection in the DMH of a Vgat-Cre::CRH-Venus mice. (e) Representative micrograph of rDMH^Vgat neurons expressing ChR2-mCherry (in red, left panel), and their axon pattern in the PVH (central panel). Note that rDMH^Vgat axon terminals mostly avoided the PVH^CRH neurons (in green, right panel) but were dense in the ventral-medial and caudal portion of the PVH (cvPVH). (f) Schematic of the EnvA-Rabies experiment to map monosynaptic inputs from the cvPVH^Vgat neurons to PVH^CRH neurons. (g) Representative photomicrograph showing the Vgat mRNA expression (in magenta) and the Rabies expression (in green) and doubly labeled neurons within the cvPVH. Arrows point to double labeled neurons; the blue arrow points to the neuron in the lower-right inset. (h) Schematic of an AAV-DIO-ChR2-mCherry injection in the cvPVH. (i) Representative image of the cvPVH^Vgat cell bodies expressing ChR2-mCherry (in red) in the ventral part of the PVH^CRH neuron field (in green). Their axons spread through the more dorsal PVH and form appositions with the PVH^CRH neurons (lower-right inset). (j) Schematic of the AAV-DIO-ChR2-mCherry injections in the DMH. (k) Micrograph showing the expression of ChR2-mCherry (in red) in the DMH^Vgat neurons (in green, left panel), and their projections and appositions to the cvPVH^Vgat neurons (central and right panel). (l) Schematic of the PVH^Vgat neuron ablation. (m) Representative image of the injection site showing mCherry expression (in red) in non-Vgat neurons in the PVH, and the loss of neurons detected by the reduction of NeuN (in green) in the cvPVH and along the PVH borders due to the ablation of the Vgat cells. (n) The cvPVH^Vgat ablation boosted the Cort increase at ZT13 in LD (Two-way ANOVA; Tukey’s multiple comparisons test. ZT13 cvPVH^Vgat-GFP vs cvPVH^Vgat-DTA: ***p<0.001), and (o) at CT13 in DD (Two-way ANOVA; Tukey’s multiple comparisons test. CT13 cvPVH^Vgat-GFP vs cvPVH^Vgat-DTA: ***p<0.001). (p) The Cort CI increased after the cvPVH^Vgat neuron ablation by 180.2 ±52.9% in LD (Unpaired t-test: t=3.336, df=8, *p=0.01) and 100.4 ±33.9% in DD (Unpaired t-test: t=2.512, df=8, *p=0.036). The hM3dq and ChR2 signal, but not in the DTA experiments, were enhanced with immunofluorescence for mCherry, while the Rabies infected cells were enhanced using an EGFP antibody. Reference scale bar: in b, e, I, k, and m = 200 µm, e (center), g and k (center) = 50µm, in e (right panel), i (right panel) and k (right panel) = 20µm, in g and i (insets)= 5µm; 3V, third ventricle.

Figure 7.. In vitro optogenetic stimulation of the GABAergic input from the rostral DMH spares PVH^CRH neurons and directly inhibits GABAergic neurons in the cvPVH.

(a) A schematic representation of the recordings to test rDMH^Vgat → PVH^CRH connectivity; Vgat-ires-Cre::CRH-Venus mice were injected with AAV-DIO-ChR2-mCherry in the rDMH (n=3 mice). Recordings were conducted in brain slices from ipsilateral Venus-labeled PVH^CRH neurons while photostimulating the ipsilateral DMH^Vgat input (DMH is shown on the opposite side for ease of illustration). (b) Stimulation of the rDMH^Vgat input produced no synaptic responses time-locked to the light pulses in most of the PVH^CRH neurons. (c) Percentages of PVH^CRH neurons responding (Connected) and not responding (Not Connected) to photostimulation of the rDMH^Vgat input (n=17 PVH^CRH recorded neurons from 3 mice). (d) Raster plot of IPSCs in a representative PVH^CRH neuron with photostimulation of the rDMH^Vgat → PVH^CRH input (bin duration: 50ms) showing lack of input. (e) oIPSC amplitude following photostimulation of the rDMH^Vgat → PVH^CRH input (filled markers, cells responding to photostimulation, n=4 neurons; open markers, cells not responding to photostimulation, n=13; mean and ± SEM of responding neurons from 3 mice). (f) To explore rDMH^Vgat → cvPVH^Vgat connectivity, Vgat-ires-Cre::L10-GFP mice (n=5 mice) were injected with AAV-DIO-ChR2-mCherry in the rDMH, and recordings were conducted in ipsilateral GFP-labeled cvPVH^Vgat neurons while photostimulating the rDMH^Vgat input. (g) Photostimulation of the DMH^Vgat input produced opto-evoked inhibitory postsynaptic currents (oIPSCs) in most of the cvPVH^Vgat neurons. GABAA-mediated oIPSCs recorded in cvPVH^Vgat neurons (upper trace) and blocked by bicuculline (BIC 20µM; n=4 neurons from 4 mice). (h) Percentages of cvPVH^Vgat neurons responding (Connected) and not responding (Not Connected) to photostimulation of the rDMH^Vgat input (n=15 cvPVH^Vgat recorded neurons from 5 mice). (i) Raster plot of IPSCs in a representative cvPVH^Vgat neuron with photostimulation of the rDMH^Vgat → cvPVH^Vgat input (bin duration: 50ms). (j) oIPSC amplitude following photostimulation of the rDMH^Vgat → cvPVH^Vgat input (filled markers, cells responding to photostimulation, n=14 neurons; open markers, cells not responding to photostimulation, n=1; mean and ± SEM of responding neurons from 5 mice). (k) A schematic map of 12 cvPVH^Vgat neurons (n=5 mice) that were recorded and found to receive input from DMH^Vgat neurons. (l) A photomicrograph showing four recorded GABAergic cvPVH neurons that responded to photostimulation of DMH^Vgat input (filled with biocytin from the recording pipette, indicated by arrowheads). DMH^Vgat fibers expressing ChR2-mCherry (in red) surrounded the cvPVH^Vgat neurons expressing GFP (in green). The neuron indicated by the magenta arrowhead is shown at higher magnification at the right, showing labeling, from top to bottom, for biocytin, GFP, mCherry, and merged. (m) Photostimulation trains (20Hz, train frequency; 60s, train duration and 10ms, pulse duration) inhibited the activity of the cvPVH^Vgat neurons (n=6 neurons from 2 mice; top) and this effect was blocked by bicuculline (20µM; n=2 from 2 mice; bottom). (n) oIPSCs in cvPVH^Vgat neurons recorded in the presence of TTX 1µM + 4-AP 200µM (n=6 neurons from 1 mouse) indicating monosynaptic connectivity. (o) oIPSC latency recorded in PVH^CRH (black; n=4 neurons from 3 mice) and cvPVH^Vgat neurons (red; n=14 neurons from 5 mice) following photostimulation of the DMH^Vgat input. (p) oIPSC probability in PVH^CRH (black; n=4 neurons from 3 mice) and cvPVH^Vgat neurons (red; n=14 neurons from 5 mice) following photostimulation of the DMH^Vgat input. (q-r) Photomicrograph of a representative rDMH injection with AAV-DIO-ChR2-mCherry (native signal) and density plots of injections in the rDMH (n= 8 mice, including experiments in a-e and f-n; illustrated on the left side of each section) following which only 23% of PVH^CRH neurons showed oIPSCs. Reference scale bar: in l (left) = 50 µm and (right) = 20 µm; in q = 250 µm. Atlas levels correspond to Paxinos & Franklin Atlas .

Figure 8.. GABAergic neurons in the rostral DMH disinhibit the PVH^CRH neurons via GABAergic neurons in the cvPVH.

(a) To test the rDMH^Vgat → cvPVH^Vgat → PVH^CRH circuit, we injected AAV-DIO-ChR2-mCherry into the rDMH of Vgat-ires-Cre::CRH-Venus mice (n=3 mice). Recordings were conducted in ipsilateral PVH^CRH neurons while photostimulating rDMH^Vgat synaptic terminals (DMH is shown on the opposite side of the brain for ease of illustration). We tested the effects of single light pulses (10ms) and trains of optostimulation (10ms individual pulse duration; 5, 10, and 20Hz stimulation frequency; 60s train duration) on spontaneous IPSC (sIPSC) frequency in PVH^CRH neurons. (b) Single light pulses did not produce oIPSCs in most PVH^CRH neurons, but (c) trains of stimulation reduced the sIPSCs (C; top trace: light off; bottom trace: light on). (d) Change in sIPSC frequency following trains of photostimulation at 5, 10 and 20 Hz compared to sham stimulation (light off). Photostimulation at 5 and 10 Hz showed a trend toward reduction in sIPSC frequency that did not reach statistical significance (at 5 Hz: n=5 neurons, paired t-test, p=0.2772; at 10 Hz: n=8 neurons, paired t-test, p=0.0545; from 3 mice) whereas photostimulation at 20 Hz significantly reduced sIPSCs frequency (n=7 neurons from 3 mice, paired t-test, ****p<0.0001), without affecting their amplitude (n=7 neurons from 3 mice; paired t-test, p= 0.7593). (e) Mean cumulative distribution plots of the sIPSC inter- event intervals show that intervals between sIPSCs were longer (left; 0.1s bins; Kolmogorov-Smirnov test, ****p<0.0001; 20Hz vs light off) but that there was no change in sIPSC amplitude (right; 1pA bins; Kolmogorov-Smirnov test, p=0.1414; 20Hz vs light off) as compiled from 7 PVH^CRH neurons from 3 mice (blue: 20Hz; black: light off; shaded areas: ± SEM). (f) To test the cvPVH^Vgat → PVH^CRH input, we injected AAV-DIO-ChR2- mCherry into the cvPVH of Vgat-ires-Cre::CRH-Venus mice (n=3 mice). Recordings were conducted from ipsilateral PVH^CRH neurons while photostimulating cvPVH^Vgat neurons and terminals. (g) Stimulation of the cvPVH^Vgat input evoked GABAA-mediated oIPSCs in PVH^CRH neurons (Bicuculline, 20µM; n=4 neurons from 3 mice). (h) Raster plot of IPSCs in a representative PVH^CRH neuron showed tight correlation with photostimulation of the cvPVH^Vgat → PVH^CRH input (bin duration: 50ms). (i) TTX -resistant oIPSCs in cvPVH^CRH neurons (n=6 neurons from 2 mice; TTX 1µM + 4-AP 250µM)) indicating monosynaptic connectivity of the cvPVH^Vgat → PVH^CRH input. (j-k) A representative photomicrograph and density plots of injections of AAV-ChR2-mCherry in the cvPVH (k; n=3 mice) and (l) percentages of PVH^CRH neurons responding to photostimulation of the cvPVH^Vgat input (Connected; n=14 PVH^CRH recorded neurons from 3 mice). (m-n) Representative injection site and density plots of injections of AAV-ChR2-mCherry along the lateral margin of the PVH (pPVH) (n=4 mice) and (o) percentages of PVH^CRH neurons responding (Connected) and not responding (Not Connected) to photostimulation of the GABAergic input from the lateral pPVH (n=39 neurons from 4 mice). (p) Amplitude (filled markers, cells responding to photostimulation, open markers, cells not responding to photostimulation; mean and ± SEM of responding neurons) and (q) latency of oIPSCs in PVH^CRH neurons evoked by photostimulation of the cvPVH (black; n=14 neurons from 3 mice) and lateral PVH (red; n=39 neurons from 4 mice, mean and ± SEM). (r) oIPSC probability in PVH^CRH neurons following photostimulation of the input from the cvPVH^Vgat (black; n=14 neurons from 4 mice) and lateral pPVH^Vgat neurons (red; n=39 neurons 4 mice). In all experiments we visualize only the native signal. Reference scale bar: in G = 50 µm; K and N = 100 µm. f, fornix, 3V, third ventricle. Atlas levels are from Paxinos & Franklin Atlas .****, p < 0.0001.