Structure and Function of Neuronal Circuits Linking Ventrolateral Preoptic Nucleus and Lateral Hypothalamic Area

Vgat-, Gal-, and Vglut2-expressing neurons in VLPO make direct synaptic contact with orexin neurons

To precisely target orexin-producing neurons, we newly generated orexin-iCre knock-in mice (orexin^iCre/+), in which codon-improved Cre recombinase (iCre) expression is driven by the endogenous orexin promoter (Fig. 1A). To validate this iCre driver line, we delivered AAV2-EF1a-DIO-GFP into the LHA of orexin^iCre/+ mice and examined sensitivity and specificity of iCre recombinase activity (Fig. 1B). The sensitivity was defined as a percentage of orexin A/GFP-double positive neurons out of all orexin A-positive neurons, which were identified by immunohistochemistry using anti-orexin A antibody, and was found to be 97.4 ± 0.7% (n = 3). The specificity was defined as a percentage of orexin A/GFP-positive neurons out of all GFP-positive neurons and was determined to be 89.4 ± 0.7% (n = 3; Fig. 1C). Both sensitivity and specificity were >90% at the craniocaudal position where the highest density of orexin A-positive neurons was observed (Fig. 1D). Further, to compare the sensitivity between the orexin^iCre/+ mice and previously generated orexin-Cre transgenic (Tg) mice (Matsuki et al., 2009), we crossed orexin^iCre/+ mice with Rosa26^dreaddm4 mice as a reporter, in which the CAG promoter and a loxP-flanked STOP sequence followed by the hM4Di-mCherry coding sequence are knocked into the Gt(ROSA)26Sor locus (T. M. Takahashi et al., 2020). Similarly to the results of our AAV experiment (Fig. 1B–D), we found that 95.8 ± 1.0% of orexin A-positive neurons expressed mCherry in orexin^iCre/+;Rosa26^dreaddm4/+ mice (Fig. 1E; n = 3). On the other hand, 79.4 ± 6.8% of orexin A-positive neurons expressed mCherry in the orexin-Cre Tg mice when crossed with Rosa26^dreaddm4/+ mice (orexin-Cre Tg;Rosa26^dreaddm4/+; Fig. 1F; n = 3). These results demonstrated higher sensitivity of iCre activity in orexin^iCre/+ mice compared with orexin-Cre Tg mice (Matsuki et al., 2009). Although we found a small number of ectopic mCherry-expressing neurons in the tuber cinereum and amygdala regions, we did not think it caused a major problem as long as this line was used for delivery of Cre-activatable AAV vectors in the LHA locally.

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Figure 1.

Generation of orexin-iCre knock-in mice. A, Structures of the targeting vector and targeting allele of orexin-iCre (orexin^iCre/+) mice. B, Scheme of the validation experiment of orexin-iCre knock-in mice. AAV2-EF1a-DIO-GFP was injected bilaterally into the LHA. Two weeks later, mice were killed, and mouse brains were examined by immunohistochemical study. C, Representative images demonstrating colocalization of GFP and orexin A in the LHA that were immunostained with anti-GFP and anti-orexin A antibodies, respectively, in orexin^iCre/+ mice. Right, Magnified image of the boxed area in the left image. Green and cyan arrowheads indicate GFP- and orexin A-single-positive neurons, respectively. Scale bars: left, 200 μm; right, 50 μm. D, Graph represents sensitivity and specificity of iCre activity in the craniocaudal axis in orexin^iCre/+ mice. Orange rectangle represents an area with densely located orexin A-positive neurons in a mouse brain. Data are mean ± SEM; n = 3. E, F, Representative images demonstrating colocalization of hM4Di (dreaddm4)-mCherry and orexin A in LHA immunostained with anti-mCherry and anti-orexin A antibodies in orexin^iCre/+;Rosa26^dreaddm4/+ mice (E) and orexin-Cre;Rosa26^dreaddm4/+ mice (F). Right images, Magnified images of the boxed areas in the left images. Green and magenta arrowheads indicate orexin A- and mCherry-single positive neurons, respectively. Scale bars: left images, 200 μm; right images, 50 μm.

We first examined the synaptic connectivity between neurons in the VLPO and orexin neurons. We conducted two batches of monosynaptic retrograde tracing experiments using rabies virus vectors SADΔG-GFP(EnvA) in the first batch and SADΔG-GFP-ER^T2CreER^T2 in the second batch at different time points (Fig. 2A). We used GFP-ER^T2CreER^T2 as a tracer in the second batch because it can be distributed in the nucleus with tamoxifen administration, in case it was difficult to confirm coexistence with Gal, Vglut2, or Vgat mRNA. However, this turned out to be unnecessary.

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Figure 2.

Monosynaptic retrograde rabies virus-mediated tracing from orexin neurons. A, Scheme of trans-synaptic retrograde tracing experiment. Two weeks after injection of AAV10-EF1a-FLEX(loxP)-TVA-mCherry and AAV10-CAG-FLEX(loxP)-RG into LHA, SADΔG-GFP(EnvA) or SADΔG-GFP-ER^T2CreER^T2 were injected into the same sites in orexin^iCre/+ mice. Mouse brains were examined by immunohistochemical study and FISH. Starter neurons were identified by expression of both mCherry and GFP, while input neurons expressed only GFP. B, Number of starter neurons positively correlates with number of produced input neurons in orexin^iCre/+ mice (Pearson correlation, r = 0.9178, p = 0.0822; n = 4), while almost no cells were positive for mCherry and GFP in WT mice (n = 3). Linear regression: F_(1,3) = 27.43, p = 0.0135, R² = 0.8424. Several starter neurons were observed at the injection sites in WT mice despite extremely low number of input neurons. C, Representative images of starter neurons in LHA in orexin^iCre/+ mice. Starter neurons were immunostained with anti-mCherry and anti-orexin A antibodies. Right, Magnified image of the boxed area in the left image. Arrowheads indicate starter neurons. Scale bars: left, 100 μm; right, 50 μm. D, Scheme depicting established borders of VLPO region. Brain outlines were taken from the Franklin and Paxinos (2007) mouse brain atlas. E, Representative images of Vgat- and Gal-double-positive input neurons in VLPO in orexin^iCre/+ mice visualized by FISH. Right, Magnified image of the boxed area in the left image. Arrowheads indicate Vgat/Gal-double-positive input neurons. Scale bars: left, 100 μm; right, 50 μm. F, Percentages of input neurons in VLPO of orexin neurons categorized as Vgat⁺/Gal⁺, Vgat⁺, Gal⁺ or cells without expression of these markers (38±10 input neurons, n = 4). G, Representative images of input neurons in VLPO in orexin^iCre/+ mice visualized by FISH using Gal and Vglut2 probes. Right, Magnified image of the boxed area in the left image. Magenta arrowhead indicates a Gal-positive, but Vglut2-negative, input neuron. Scale bars: left, 100 μm; right, 50 μm. H, Percentages of input neurons in VLPO of orexin neurons categorized as Gal⁺/Vglut2⁺, Gal⁺, Vglut2⁺ or cells without expression of these markers (17 ± 3 input neurons, n = 3). I, Representative images of input neurons in VLPO in orexin^iCre/+ mice visualized by FISH using Vgat and Vglut2 probes. Right, Magnified image of the boxed area in the left image. Cyan arrowhead indicates a Vgat-positive, but Vglut2-negative, input neuron. Scale bars: left, 100 μm; right, 50 μm. J, Percentages of input neurons in VLPO of orexin neurons categorized as Vglut2⁺/Vgat⁺, Vglut2⁺, Vgat⁺ or cells without expression of these markers (12 ± 2 input neurons, n = 3). Circles represent data from individual animals. Data are mean ± SEM.

To minimize ectopic expression of AAV10-EF1a-FLEX(loxP)-TVA-mCherry (TVA-mCherry) and to restrict numbers of starter neurons, we conducted a series of preliminary experiments with several dilutions of TVA-mCherry vectors, as suggested by previous studies (Lavin et al., 2019, 2020), for each batch of experiments. Using the most successful dilutions, we found moderate numbers of starter neurons in the LHA (GFP- and mCherry-double-positive; 16 ± 4 cells/brain in the first batch, n = 4; 10 ± 2 cells/brain in the second batch, n = 3) and of input neurons (GFP-single-positive; 357 ± 159 cells/brain in the first batch, n = 4) in orexin^iCre/+ mice, with a clear positive correlation between the numbers of these two subsets of neurons (Fig. 2B, black circles; Pearson correlation, r = 0.9178, p = 0.0822, n = 4; Fig. 2C, data from the first batch). Only a few GFP- and/or mCherry-positive neurons were observed at the injection sites in WT control mice (Fig. 2B, red circles, n = 3). The relatively small number of starter neurons obtained by dilution of TVA-mCherry allowed us to examine connectivity of orexin neurons precisely.

In both experiments, the majority of starter neurons in the LHA were orexin A-positive, and their distribution was consistent throughout all animals (87.7 ± 8.2% with 12.3 ± 8.2% of orexin A-negative neurons, n = 7). Their distribution was similar to that described in rodent brains (Peyron et al., 1998; Nambu et al., 1999). The specificity of starter neurons was lower than that of iCre activity, calculated in Figure 1C, D, presumably because of the toxicity of the rabies virus and its impact on gene expression in the infected neurons, as previously reported (Sun et al., 2019).

Input neurons of orexin neurons were distributed across the entire brain in a consistent pattern with earlier studies (González et al., 2016; Giardino et al., 2018; Saito et al., 2018). We here focused on the VLPO and precisely characterized the input neurons in this region (Fig. 2D–J). We adopted classification of this region into the VLPO cluster (VLPOc) and the dorsomedial extended VLPO (VLPOe), both of which have been shown to participate in NREM sleep regulation (Kroeger et al., 2018). The VLPOe was also suggested to contribute to REM sleep regulation (Lu et al., 2000, 2002; Scammell et al., 2017). We demarcated the VLPO subdivisions according to criteria described in the previous reports and to the location of sleep-active neurons that were obtained from the past electrophysiological and immunohistochemical studies (Fig. 2D, colored areas) (Sherin et al., 1996; Szymusiak et al., 1998; Lu et al., 2000, 2002; Wagner et al., 2000; Gaus et al., 2002; K. Takahashi et al., 2009; Alam et al., 2014; Kroeger et al., 2018).

Further, we determined whether the input neurons in the VLPO contained GABA, galanin, and glutamate, since these are the molecular signatures of sleep- and wakefulness-implicated neurons in this area (Chung et al., 2017; Kroeger et al., 2018; Vanini et al., 2020; Yamagata et al., 2021). We conducted a FISH study to visualize the expression of vesicular GABA transporter (Vgat, Slc32a1) and vesicular glutamate transporter 2 (Vglut2, Slc17a6), which are the molecular markers of GABA- and glutamate-producing neurons, respectively, as well as expression of galanin (Gal) in the VLPO input neurons (Fig. 2E,G,I). We examined colocalization of Vgat and Gal in the first batch of the tracing experiments (Fig. 2E), and colocalization of Gal and Vglut2 or Vglut2 and Vgat in the second batch (Fig. 2G,I). As a result, we found that more than half of the input neurons (56.3 ± 8.1%) were double positive for Vgat- and Gal (VLPO^Vgat/Gal neurons; Fig. 2F, left; 38 ± 10 VLPO input neurons in total, n = 4). VLPO^Vgat/Gal neurons were more concentrated in the VLPOc compared with the VLPOe (Fig. 2F, middle, right). A small population of the input neurons was solely positive for Gal (2.8 ± 2.3%), while Vgat-positive neurons (85.7 ± 5.4%) constituted the great majority of the total number of input neurons in the VLPO (Fig. 2F). We also investigated colocalization of Gal and Vglut2 in the VLPO input neurons; however, we did not find any Gal- and Vglut2-double-positive inputs in the examined samples (Fig. 2G,H; 17 ± 3 VLPO input neurons in total, n = 3). Notably, we found a small population of Vglut2- and Vgat-copositive input neurons (6.3 ± 3.6%, 12 ± 2 VLPO input neurons in total, n = 3), suggesting high molecular heterogeneity of presynaptic partners in the VLPO that send innervations to orexin neurons (Fig. 2I,J). Together, we precisely delineated connectivity of orexin neurons with major sleep- and wakefulness-associated subpopulations of VLPO neurons, and determined the chemical identities of VLPO neurons that make direct synaptic contact with orexin neurons in the LHA.

VLPO^GABA→LHA neurons receive input from multiple brain areas

We next conducted a projection-specific rabies virus-mediated tracing experiment using Vgat-ires-Cre mice (Fig. 3A) (Piñol et al., 2018). We first injected a Cre-dependent AAV mixture for expressing TVA and rabies glycoprotein (RG) into the VLPO. Two weeks later, we delivered SADΔG-GFP(EnvA) into the LHA to infect TVA-expressing VLPO^GABA neurons through their axonal terminals, allowing this vector to exclusively infect VLPO^GABA neurons that send innervations to the LHA (VLPO^GABA→LHA neurons) and to trans-synaptically spread in a retrograde direction.

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Figure 3.

Monosynaptic retrograde projection-specific rabies virus-mediated tracing from VLPO^GABA neurons sending projections to LHA. A, Scheme of projection-specific tracing experiment. Two weeks after injection of AAV2-EF1a-FLEX(loxP)-TVA-mCherry and AAV2-CAG-FLEX(loxP)-RG into VLPO of Vgat-ires-Cre mice, SADΔG-GFP(EnvA) was injected into LHA. Mouse brains were examined by immunohistochemical study. B, Number of starter neurons has positive correlation with number of input neurons in Vgat-ires-Cre mice (Pearson correlation, r = 0.3292, p = 0.5885; n = 5). Linear regression: F_(1,4) = 9.413, p = 0.0374, R² = 0.1084. In WT mice, several starter and input neurons were observed close to VLPO only in 1 animal (n = 3). C, Representative images of starter neurons in VLPO of Vgat-ires-Cre mice. Starter neurons were immunostained with anti-GFP and anti-mCherry antibodies. Right, Magnified image of the boxed area in the left image. Arrowheads indicate starter neurons. Scale bars: left, 100 μm; right, 50 μm. D, Distribution of starter VLPO^GABA→LHA neurons in POA and adjacent regions. Highlighted areas are POA and VLPO. Areas with starter neurons in all examined animals without cut-off are shown. E, Scheme depicting outlines of distribution of starter neurons in cross-sections containing VLPO. Brain outlines were taken from the Franklin and Paxinos (2007) mouse brain atlas. Colors represent individual animals. If there were no matching brain sections to the presented schemes in some animals, outlines were not shown. F, Representative images of axonal terminals of VLPO^GABA→LHA neurons making close appositions to bodies of orexin A-expressing neurons in LHA. Immunostaining with anti-GFP, anti-mCherry, and anti-orexin A antibodies. Right, Magnified image of the boxed area in the left image. Arrowheads indicate GFP- and mCherry-double-positive neural fibers. Scale bars: left, 50 μm; right, 20 μm. G, Whole-brain distribution of monosynaptic input neurons of VLPO^GABA→LHA neurons in Vgat-ires-Cre mice. Only brain areas with input neurons detected in at least 3 of 5 animals are shown. Colored areas represent major brain divisions. Abbreviations of input brain areas that are shared with VLPO^GAL→LHA neurons (Fig. 4G) are highlighted by gray background. Circles represent data from individual animals. Nomenclature of brain regions was taken from the Franklin and Paxinos (2007) mouse brain atlas. Data are mean ± SEM; n = 5. H, Representative images of input neurons of VLPO^GABA→LHA neurons. Scale bars, 200 μm. I, Representative images of monosynaptic input neurons in POA of VLPO^GABA→LHA neurons analyzed by FISH using Vgat, Vglut2, and Adcyap1 probes. For each molecular marker: right, magnified image of the boxed area in the left image. Arrowheads indicate GFP-positive neuronal bodies coexpressing examined molecular signatures. Scale bars: left, 100 μm; right, 50 μm.

We found a considerable number of TVA-mCherry- and GFP-double-positive GABA-producing starter neurons in the POA (294 ± 39 neurons/brain, n = 5), which were positively correlated with the number of their input neurons (GFP-single positive) in Vgat-ires-Cre mice (Fig. 3B, black circles; Pearson correlation, r = 0.3292, p = 0.5885; n = 5). Only a few starter and input neurons were found around the injection site in the VLPO of WT control mice (Fig. 3B, red circles, n = 3). Starter neurons were distributed across the POA (75.5 ± 5.3% of all starter neurons) with some involvement of adjacent regions (Fig. 3C–E). The VLPOc and VLPOe contained 36.8 ± 2.3% of all starter neurons, constituting nearly half of them in the POA (Fig. 3D; 49.2 ± 3.2% of POA starter neurons). We also observed mCherry- and GFP-double-positive axons making appositions to cell bodies of orexin A-positive neurons in the LHA, suggesting that some starter cells sent projections to orexin neurons (Fig. 3F).

A large number (928 ± 328 neurons/brain, n = 5) of input neurons to VLPO^GABA→LHA neurons were widely distributed among various brain areas (Fig. 3G,H). The biggest number (≥10%) of input neurons was found in the medial preoptic area (MPA) and the VLPO, while regions with less concentrated input neurons (≥5%) were the diagonal band nucleus, lateral preoptic area (LPO), medial preoptic nucleus, and LHA. These results suggest that the MPA and VLPO regions constitute major inputs of the VLPO^GABA→LHA neurons. LHA also contains a considerable number of input neurons, suggesting the presence of reciprocal connectivity between the VLPO and LHA. This result is also consistent with previous studies that reported on the arousal-promoting LHA→POA circuits (Venner et al., 2019; de Luca et al., 2022).

A moderate number of input neurons (≥2.5%) were located in the lateral septal nucleus, ventromedial preoptic nucleus, and arcuate hypothalamic nucleus. Small numbers of input neurons (<2.5%) were also found in multiple regions, such as the nucleus accumbens (Acb), amygdala, bed nucleus of stria terminalis (ST), ventral pallidum, and paraventricular, ventromedial, and dorsomedial hypothalamic nuclei. Despite apparent variability in the number and distribution of input and starter neurons between individual animals, presumably because of variability in expression of viral vectors, input neurons in all exhibited areas were consistently represented in at least three independent experiments (Fig. 3G). Such stability of the landscape of presynaptic partners of the VLPO^GABA→LHA neurons might reflect the functional significance of these monosynaptic connections for the regulation of physiological processes.

We also characterized the local preoptic input neurons of the VLPO^GABA→ LHA neurons using FISH to disentangle connectivity within the POA. We visualized the expression of Vgat and Vglut2 in input neurons because neurons expressing these molecular markers are proposed to be implicated in sleep-wakefulness regulation (Arrigoni and Fuller, 2022). This study found substantial numbers of Vgat- or Vglut2-positive input neurons in the POA (Fig. 3I, top, middle, n = 2). We also examined whether the POA input neurons of the VLPO^GABA→ LHA neurons express Pacap (Adcyap1), which was shown to be expressed in neurons involved in thermoregulation (Tan et al., 2016). We found Pacap-expressing input neurons in the MPA (Fig. 3I, bottom, n = 2). These results suggest that the VLPO^GABA→LHA neurons are interconnected with cells related to different functional units within the POA.

Input neurons of VLPO^GABA→LHA and VLPO^GAL→LHA neurons show similar distribution

Next, we identified monosynaptic inputs of galanin neurons in the VLPO projecting to the LHA (VLPO^GAL→LHA neurons; Fig. 4A) using Gal-Cre mice (Wu et al., 2014). Implementing projection-specific retrograde rabies virus-mediated tracing, we labeled an appropriate number of starter neurons (141 ± 45 neurons/brain, n = 5) within the POA and neighboring areas (Fig. 4B, black circles; Fig. 4C–E). While 28.2 ± 2.7% of all starter neurons were located in both VLPOc and VLPOe, 81.8 ± 2.5% of neurons resided in the POA (Fig. 4D). We also found that the absolute numbers of input neurons and VLPO^GAL→LHA starter neurons were positively correlated, while only several input neurons were detected in WT mice (Fig. 4B, black and red circles; Pearson correlation, r = 0.9424, p = 0.0164; n = 5; WT mice n = 3). As for the VLPO^GABA→LHA neurons, we found that some axons of the starter VLPO^GAL→LHA neurons made close appositions to the cell bodies of orexin A-positive neurons (Fig. 4F).

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Figure 4.

Monosynaptic retrograde projection-specific rabies virus-mediated tracing from VLPO^GAL→LHA neurons and examination of traced connections of two neuronal populations. A, Scheme of projection-specific tracing experiment. B, Number of starter neurons has strong positive correlation with number of input neurons in Gal-Cre mice (Pearson correlation, r = 0.9424, p = 0.0164; n = 5). Linear regression: F_(1,4) = 114.0, p = 0.0004, R² = 0.8882. In WT mice, several input neurons were observed only in 1 animal (n = 3). C, Representative images of VLPO starter neurons in Gal-Cre mice. Starter neurons were immunostained with anti-GFP and anti-mCherry antibodies. Right, Magnified image of the boxed area in the left image. Arrowheads indicate starter neurons. Scale bars: left, 100 μm; right, 50 μm. D, Distribution of starter VLPO^GAL→LHA neurons in POA and neighboring regions. Highlighted areas represent POA and VLPO. Areas with starter neurons detected in all examined animals without cut-off are shown. E, Scheme depicting outlines of distribution of starter neurons in cross-sections containing VLPO. Colors represent individual animals. If there were no matching brain sections to the presented schemes in some animals, outlines were not shown. F, Representative images of axonal terminals of VLPO^GAL→LHA neurons making close appositions to bodies of orexin A-expressing neurons in LHA. Immunostaining was performed using anti-GFP, anti-mCherry, and anti-orexin A antibodies. Right, Magnified image of the boxed area in the left image. Arrowhead indicates GFP- and mCherry-double positive neural fibers. Scale bars: left, 50 μm; right, 20 μm. G, Whole-brain distribution of monosynaptic inputs of VLPO^GAL→LHA neurons in Gal-Cre mice. Only brain areas with input neurons in at least 3 of 5 animals are shown. Colored areas represent major brain divisions. Abbreviations of input brain areas that are shared with VLPO^GABA→LHA neurons (Fig. 3G) are highlighted by gray background. Circles represent data from individual animals. Data are mean ± SEM (n = 5). H, Representative images of presynaptic partners of VLPO^GAL→LHA neurons. Scale bars, 200 μm. I, Representative images of presynaptic partners of VLPO^GAL→LHA neurons in POA. FISH with Vgat, Vglut2, and Adcyap1 probes. For each molecular marker: right, magnified image of the boxed area in the left image. Arrowheads indicate GFP-positive neuronal bodies expressing examined molecular signatures. Scale bars: left, 100 μm; right, 50 μm. J, Multidimensional scaling plot of monosynaptic input neurons of VLPO^GABA→LHA (shown as GABA) and of VLPO^GAL→LHA (shown as GAL) neurons (n = 5 for each group). Colored circles represent individual animals. Semitransparent circles represent closely located clusters formed of individual animals in each neuronal population, suggesting high similarity of presynaptic partners between them (F score = 1.5117, p = 0.1259, stress = 0.07). K, Spearman correlation plot comparing distribution of input neurons of VLPO^GABA→LHA and VLPO^GAL→LHA neurons. Distribution of presynaptic partners significantly correlates between neuronal groups (r = 0.67, p < 0.001).

Many monosynaptic input neurons (213 ± 70 neurons/brain, n = 5) were found in various brain areas, with a similar distribution pattern to input neurons of the VLPO^GABA→LHA neurons (Fig. 4G,H; Fig. 3G, shared input brain areas are highlighted by gray background). The highest number of input neurons (≥10%) was found in the MPA and the VLPO. Brain areas with less abundant input neurons (≥5%) were the Acb, ST, medial preoptic nucleus, and LHA. An intermediate number of input neurons (≥2.5%) were located in the LPO, anteroventral periventricular and ventromedial preoptic nuclei, and striohypothalamic nucleus, as well as in the paraventricular, arcuate, and dorsomedial hypothalamic nuclei. Input cells were also found in the central nucleus of the amygdala, lateral septal complex, and anterior hypothalamus, with <2.5% of the total number of input neurons in each region.

We also examined whether Vgat, Vglut2, and Pacap (Adcyap1) are contained in the preoptic monosynaptic input neurons of the VLPO^GAL→LHA neurons, and found that Vgat- and Vglut2-positive POA neurons made direct synaptic inputs to these cells (Fig. 4I, n = 2). However, we did not observe any Pacap-expressing presynaptic partners in the POA at least in the two samples that we examined here.

Further, we compared input neuron landscapes of the VLPO^GABA→LHA and VLPO^GAL→LHA neurons. Using multidimensional scaling analysis, we identified that both neuronal populations were clustered together according to their inputs (Fig. 4J; p = 0.1259, stress = 0.07, n = 5 for each mouse group). In addition, we found that the distributions of the presynaptic partners were strongly correlated (Fig. 4K; Spearman correlation, r = 0.67, p < 0.001). These results were consistent with our observation that the majority of VLPO input neurons to orexin neurons were Vgat- and Gal-double-positive (Fig. 2F, left) and with findings in a recent study (de Luca et al., 2022), therefore suggesting that the VLPO^GABA→LHA and VLPO^GAL→LHA neurons constitute a common neuronal population. However, there were some differences in the input brain areas between VLPO^GABA→LHA and VLPO^GAL→LHA neurons. For instance, regions in cortex, extended amygdala, septum, anterior and posterior hypothalamus, as well as dorsal raphe nucleus and VTA in midbrain, were exclusive to VLPO^GABA→LHA neurons (Fig. 3G). On the other hand, paraventricular thalamic nucleus, subfornical organ in hypothalamus, and reticular pontine nucleus were consistently found among upstream brain areas only for VLPO^GAL→LHA neurons (Fig. 4G).

Acute optogenetic activation of VLPO^GABA→LHA and VLPO^GAL→LHA pathways produces distinct phenotypes with regard to wakefulness induction

We next examined the function of the VLPO→LHA pathway in the regulation of sleep-wakefulness behavior by optogenetic excitation of axon fibers of VLPO^GABA neurons in the LHA. We delivered Cre-dependent AAVs expressing ChR2 fused with EYFP or expressing GFP (AAV2-EF1a-DIO-ChR2-EYFP or AAV2-EF1a-DIO-GFP, respectively) into the VLPO and implanted optical fibers into the LHA of Vgat-ires-Cre mice.

We first conducted 2 min photostimulation (10 Hz, 10 ms) 2 h after the onset of the dark phase (ZT14) during the wakefulness state, which lasted for 1 min. We conducted the experiment in the dark period because we initially hypothesized that the VLPO^GABA→LHA pathway would have a role in inducing sleep. However, we observed that only one ChR2-delivered mouse was able to enter NREM sleep during photostimulation (total amount of NREM sleep during 2 min photostimulation 0.7 ± 0.7% in ChR2 mice, n = 8 vs 4.0 ± 2.8% in GFP mice, n = 7; p = 0.3231, Mann–Whitney test), suggesting that the VLPO^GABA→LHA pathway does not play a major role in inducing sleep. Previous studies proposed that VLPO^GABA neurons are composed of distinct subpopulations, presumably defined by the projection site, receptor expression, and expression of neuronal modulators, and that these subpopulations contribute to sleep or wakefulness, driving in an opposite manner (Liu et al., 2010; Chung et al., 2017; Liang et al., 2021; Yamagata et al., 2021; de Luca et al., 2022). We therefore examined whether the VLPO^GABA→LHA and VLPO^GAL→LHA pathways are rather involved in inducing wakefulness, using Vgat-ires-Cre and Gal-Cre mice, respectively (Fig. 5A, I).

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Figure 5.

Acute optogenetic stimulation of VLPO^GABA→LHA and VLPO^GAL→LHA pathways. A, Scheme of pathway-specific optogenetic experiment in Vgat-ires-Cre mice. AAV2-EF1a-DIO-ChR2-EYFP or AAV2-EF1a-DIO-GFP was injected bilaterally into VLPO, and optical fibers were implanted bilaterally into LHA of Vgat-ires-Cre mice for manipulation of axons of GABA-producing VLPO neurons. At the same time, an EEG- and EMG-recording electrode was secured onto the skull. Mice were maintained under a 12:12 h light/dark cycle (06:00 lights on, 18:00 lights off), and photoactivation was conducted for 2 d within the ZT3-ZT9 time frame with at least 40 min interstimulation periods. B, J, Representative images of VLPO neurons expressing GFP (top) or ChR2 (bottom), visualized by immunostaining with anti-GFP antibody, in Vgat-ires-Cre (B) and Gal-Cre (J) mice. Right images, Magnified images of the boxed areas in the left images. Scale bars: left images, 200 μm; right images, 100 μm. C, K, Representative images of GFP- (top) or ChR2-expressing (bottom) axons of VLPO neurons in LHA located close to orexin A-positive neurons in Vgat-ires-Cre (C) and Gal-Cre (K) mice. Immunostaining was performed using anti-GFP and anti-orexin A antibodies. Right images, Magnified images of the boxed areas in the left images. Scale bars: left images, 200 μm; right images, 100 μm. D, Representative EEG and EMG wave traces measuring 40 s (10 s before, 20 s during, and 10 s after optogenetic stimulation) from 2 animals injected with AAV2-EF1a-DIO-GFP (GFP, left) or AAV2-EF1a-DIO-ChR2-EYFP (ChR2, right). Blue line indicates laser stimulation (10 Hz, 10 ms, 20 s). Black represents NREM sleep. Green represents wakefulness. Latency to wakefulness was calculated as time in seconds from the start of each photostimulation to the onset of wakefulness. E, Latency to wakefulness after photostimulation. Significant difference was observed between GFP (n = 7) and ChR2 (n = 8) animals (first 7 stimulations per animal; p = 0.0052, unpaired two-tailed t test). F, Probability of transition to wakefulness in GFP and ChR2 mice within 140 s after start of photoactivation. Blue field represents optogenetic stimulation. G, Probability of transition to wakefulness during 20 s of photostimulation. Significant difference was observed between GFP and ChR2 animals (p = 0.0308, Mann–Whitney test). H, Amount of wakefulness in 10 min time bins in percentages before, during, and after 10 min optogenetic stimulation. No significant difference between GFP (n = 6) and ChR2 (n = 6) mice was observed (two-way repeated-measures ANOVA with Geisser–Greenhouse correction followed by Sidak multiple comparisons test). I, Scheme of pathway-specific optogenetic experiment in Gal-Cre mice. L, Latency to wakefulness after photostimulation. No significant difference was observed between GFP- (n = 6) and ChR2-expressing (n = 7) animals (first 7 stimulations per animal; p = 0.1770, unpaired two-tailed t test). M, Probability of transition to wakefulness in GFP and ChR2 mice within 140 s after start of photoactivation. Blue field represents optogenetic stimulation. N, Probability of transition to wakefulness during 20 s of photostimulation. No significant difference was observed between GFP and ChR2 animals (p = 0.1930, unpaired two-tailed t test). O, Amount of wakefulness in 10 min time bins in percentages before, during, and after 10 min optogenetic stimulation. No significant difference between GFP (n = 6) and ChR2 (n = 6) mice was observed (two-way repeated-measures ANOVA with Geisser–Greenhouse correction followed by Sidak multiple comparisons test). Circles represent data from individual animals. Data are mean ± SEM. *p < 0.05. **p < 0.01.

We conducted the same surgical procedure and confirmed that orexin A-positive neurons were surrounded by the projections of ChR2- or GFP-expressing VLPO^GABA neurons (Fig. 5B,C) in Vgat-ires-Cre mice. We applied photostimulation (10 Hz, 10 ms, 20 s) of ChR2- or GFP-expressing axonal fibers in the LHA during NREM sleep within the ZT3-ZT9 time window (12:12 h light/dark cycle; 06:00 lights on, 18:00 lights off), using similar optogenetic parameters (Chung et al., 2017; Yamagata et al., 2021) and experiment protocol to previous studies (Kodani et al., 2017; Venner et al., 2019) (Fig. 5A).

We observed that 20 s stimulation of the axons of VLPO^GABA neurons in the LHA evoked short-lasting wakefulness episodes in ChR2-expressing mice (Fig. 5D; Movies 1 and 2). Latency to the wakefulness episodes after optogenetic stimulation in ChR2 mice was significantly shorter than that in GFP control mice (Fig. 5E; 27.0 ± 3.9 s in ChR2 mice, n = 8 vs 50.1 ± 5.9 s in control mice, n = 7; p = 0.0052, unpaired two-tailed t test). We also found that probability of transition from NREM sleep to wakefulness was significantly higher in ChR2-expressing mice on photostimulation (Fig. 5F,G; 53.6 ± 8.0% in ChR2 mice vs 26.5 ± 4.9% in GFP mice by the end of 20 s photostimulation; p = 0.0308, Mann–Whitney test). We also conducted 10 min pulse photostimulation (10 Hz, 10 ms, 20 s light on/40 s light off × 10), as described previously (Kodani et al., 2017). We did not observe a significant difference in the length of wakefulness episodes between mouse groups (21.6 ± 12.3 s in ChR2 mice, n = 6 vs 147.4 ± 138.5 s in GFP mice, n = 6; p = 0.9091, Mann–Whitney test), while the number of wakefulness episodes was slightly greater in ChR2 mice (8.2 ± 1.0 vs 5.5 ± 1.7 in GFP group; p = 0.2161, unpaired two-tailed t test). The total amount of wakefulness before, during, and after the optogenetic stimulation was also indistinguishable between the mouse groups (Fig. 5H; two-way repeated-measures ANOVA with Geisser–Greenhouse correction followed by Sidak multiple comparisons test). These results indicate that activation of the VLPO^GABA→LHA pathway using ChR2 induces transition from NREM to wakefulness, although it was very short-lasting.

On the other hand, in Gal-Cre mice, we did not observe such a wakefulness-promoting effect. We performed surgery as described above and conducted histologic verification of viral vectors expression and optical fiber implantation, similarly to Vgat-ires-Cre mice (Fig. 5I–K). In this experiment, we have modified frequency of the stimulation to 1 Hz (1 Hz, 10 ms, 20 s) because it had been previously determined as optimal for galanin-producing neurons in the VLPO, while 10 Hz frequency resulted in their hyperpolarization (Kroeger et al., 2018). We applied photostimulation to axonal fibers in the LHA of the ChR2- or GFP-expressing VLPO^GAL neurons during NREM sleep. However, there were no significant differences in the latency to wakefulness (72.3 ± 11.4 s in ChR2 mice, n = 7 vs 52.6 ± 6.1 s in GFP mice, n = 6; p = 0.1770, unpaired two-tailed t test) and probability to transition to wakefulness (24.5 ± 6.8% in ChR2 mice vs 38.1 ± 7.1% in control mice by the end of the photostimulation; p = 0.1930, unpaired two-tailed t test) between ChR2 and GFP mice (Fig. 5L–N). Similarly, 10 min pulse stimulation did not produce significant differences in the amount of wakefulness before, during, and after the photostimulation between two mouse groups (Fig. 5O; 17.1 ± 8.5% of wakefulness in ChR2 mice, n = 6 vs 18.3 ± 6.4% in GFP mice, n = 6; p = 0.9993, two-way repeated-measures ANOVA with Geisser–Greenhouse correction followed by Sidak multiple comparisons test), as well as in the length (65.6 ± 55.7 s in ChR2 mice, n = 6 vs 49.9 ± 39.2 s in GFP mice, n = 6; p = 0.9740, Mann–Whitney test) and the number (4.2 ± 1.1 in ChR2 mice, n = 6 vs 6.2 ± 1.4 in GFP mice, n = 6; p = 0.2841, unpaired two-tailed t test) of wakefulness episodes. Therefore, we concluded that the VLPO^GAL→LHA pathway does not participate in promotion of wakefulness.

Additionally, we identified how many galanin-expressing neurons overlapped with ChR2-expressing GABA-producing neurons in Vgat-ires-Cre mice that underwent the 20 s and 10 min photostimulations (Fig. 5A) by FISH using Gal probe. We found that only 29.6 ± 3.2% of Gal-positive neurons colocalized with ChR2-expressing neurons (n = 4), suggesting that the phenotypic differences between Vgat-ires-Cre and Gal-Cre mice were primarily because of the function of GABA neurons that do not produce galanin.

Together, we found that acute optogenetic activation of the VLPO^GABA→LHA and VLPO^GAL→LHA pathways results in different phenotypes, where the VLPO^GABA→LHA pathway induces a short-lasting wakefulness, while the VLPO^GAL→LHA pathway does not significantly contribute to regulation of wakefulness and sleep.

Long-term optogenetic activation of VLPO^GABA→LHA pathway increases wakefulness

Since we found that activation of the VLPO^GABA→LHA pathway provoked short bouts of wakefulness, we next examined an effect of a more long-term stimulation using SSFO (Yizhar et al., 2011) that induces membrane depolarization for ∼30 min in targeted neurons on photoactivation. We conducted the same surgical procedure, as above, using AAV2-EF1a-DIO-SSFO-EYFP and AAV2-EF1a-DIO-GFP vectors, and applied photostimulation (1 Hz, 1 s light pulse, 2 times, 30 min interval) of SSFO- or GFP-expressing axonal fibers in the LHA 2 h after the onset of the light phase (ZT2; Fig. 6A). We found that 1 s light pulses resulted in immediate transient increases in wakefulness in SSFO-expressing mice (Fig. 6B,C). Wakefulness was significantly increased in the first 5 min after the first and second light pulse applications in SSFO mice compared with control GFP mice (Fig. 6C; 86.1 ± 13.9% in SSFO mice, n = 6 vs 12.2 ± 3.4% in GFP mice, n = 6 after the first light pulse, p = 0.0352, and 72.8 ± 9.8% in SSFO mice vs 17.2 ± 5.4% in GFP mice after the second light pulse, p = 0.0161, two-way repeated-measures ANOVA with Geisser–Greenhouse correction followed by Sidak multiple comparisons test). Additionally, the number of long wakefulness bouts (≥240 s) was significantly increased in SSFO mice (Fig. 6D; 2.0 ± 0.3 in SSFO mice vs 0.8 ± 0.2 in GFP mice; p = 0.0130, Mann–Whitney test). Interestingly, the increase in wakefulness that was observed after the second light pulse was weaker than that after the first light pulse (Fig. 6B,C), suggesting desensitization of neurons or homeostatic regulation of sleep. We also observed that photostimulation-induced increases in wakefulness were followed by consisted decreases in this state (Fig. 6B,C). As a result, the total amount of wakefulness during 1 h since the first light pulse (ZT2) was not significantly increased in SSFO mice compared with GFP mice (Fig. 6E; 41.4 ± 5.0% in SSFO mice vs 29.5 ± 3.9% in GFP mice; p = 0.2500, two-way repeated-measures ANOVA with Geisser–Greenhouse correction followed by Sidak multiple comparisons test). This result was similar to that observed on the 10 min pulse photostimulation in ChR2-expressing mice (Fig. 5H). As this phenotype might have resulted from homeostatic regulation of sleep, which was shown to be conducted by LPO^GAD2 neurons (glutamate decarboxylase 2-expressing neurons in the LPO) (Yamagata et al., 2021), we also examined EEG power density of NREM sleep during the last quarter (ZT2.75-ZT3) of 1 h starting from the first light pulse (ZT2). However, we did not observe any significant differences between SSFO and control mice (Fig. 6F; ordinary two-way ANOVA followed by Sidak multiple comparisons test).

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Figure 6.

Long-term SSFO-mediated optogenetic stimulation of VLPO^GABA→LHA pathway. A, Scheme of the pathway-specific optogenetic experiment. AAV2-DIO-SSFO-EYFP or AAV2-EF1a-DIO-GFP were injected bilaterally into VLPO, and optical fibers were implanted bilaterally into LHA of Vgat-ires-Cre mice. An EEG- and EMG-recording electrode was secured onto the skull. Mice were maintained under a 12:12 h light/dark cycle (06:00 lights on, 18:00 lights off), and photoactivation was applied 2 h after the onset of light phase (at ZT2 or 08:00; 1 s light pulse × 2, with 30 min interstimulation period). B, Time course of the amount of wakefulness during 1 h before the first 1 s light pulse (ZT1-ZT2; 07:00-08:00), 1 h after the first light pulse (ZT2-ZT3; 08:00-09:00), and 1 h following them (ZT3-ZT4; 09:00-10:00). Shown is the amount of wakefulness in 1 min time bins in percentages. Two blue lines indicate photostimulation. C, Time course of the amounts of wakefulness in 5 min bin during 1 h after the first light pulse. Significant differences were observed in the amount of wakefulness during the first 5 min after the light pulses between GFP- (n = 6) and SSFO-expressing (n = 6) mice (p = 0.0352 after the first light pulse and p = 0.0161 after the second light pulse, two-way repeated-measures ANOVA with Geisser–Greenhouse correction followed by Sidak multiple comparisons test). D, Number of ≥4-min-long (240 s-long) wakefulness bouts, observed during 1 h from the first light pulse. Significant difference was observed between GFP and SSFO mice (p = 0.0130, Mann–Whitney test). E, Total amount of wakefulness in percentages during 1 h before the first 1 s light pulse (ZT1; 07:00), 1 h after the first light pulse (ZT2; 08:00), and 1 h following them (ZT3; 09:00). No significant difference was observed (two-way repeated-measures ANOVA with Geisser–Greenhouse correction followed by Sidak multiple comparisons test). F, Relative amplitude of average EEG power density of NREM sleep during the last quarter (ZT2.75-ZT3; 08:45-09:00) of 1 h starting from the first light pulse (ZT2; 08:00). No significant difference was observed between GFP control and SSFO mice (ordinary two-way ANOVA followed by Sidak multiple comparisons test). G, Scheme depicting outlines of distribution of SSFO-expressing neurons in a cross-section containing VLPO. Brain outlines were taken from the Franklin and Paxinos (2007) mouse brain atlas. Colors represent individual animals. Four animals with the most distinct vector expression patterns are shown. Percentage following animal ID shows the amount of wakefulness during an hour since the first light pulse. Percentage in parentheses shows the amount of wakefulness during the first 5 min after the first light pulse. H, Representative images of cFos-positive VLPO neurons expressing GFP (top) or SSFO (bottom), visualized by immunostaining with anti-GFP and anti-cFos antibodies. Right images, Single-channel images showing cFos signal in cell nuclei. Scale bars, 100 μm. I, Scheme of dual-color retrograde tracing experiment. Recombinant cholera toxin subunit B conjugated with AlexaFluor-488 (AF488-CTB) was delivered into LHA, and Red RetroBeads was delivered into TMN of WT C57BL/6J mice. Retrogradely labeled neurons in VLPO were visualized for Vgat using FISH, and Vgat-expressing neurons solely labeled with AF488-CTB or colabeled with Red RetroBeads were counted. J, Representative images of injection sites of retrograde traces (left images) and neurons in VLPO containing the tracers (right). Green arrowheads indicate AF488-CTB single-positive neurons in the VLPO. Magenta arrowheads indicate Red RetroBeads single-positive neurons. White arrowhead indicates an AF488-CTB and Red RetroBeads double-positive neuron. Scale bars: left images, 200 μm; right, 50 μm. K, Percentages of Vgat-expressing VLPO neurons categorized as AF488-CTB⁺ (AF488⁺) and AF488-CTB/Red RetroBeads-double positive (AF488⁺/Red⁺) (n = 4). Circles represent data from individual animals. Data are mean ± SEM. *p < 0.05.

We next confirmed the expression of SSFO in the VLPO (VLPOc and VLPOe) and found that animals with abundant SSFO expression in the VLPOe produced more pronounced increases in wakefulness than the animals with expression mostly limited to the VLPOc (Fig. 6G). We also observed that optogenetic activation induced a robust increase in cFos in SSFO-expressing neurons compared with GFP-expressing neurons (Fig. 6H, white arrowheads). To examine the effect of activation of the VLPO^GABA→LHA pathway on orexin neurons in the LHA, we compared percentages of cFos- and orexin A-double-positive neurons between SSFO and GFP mice. However, we did not find a significant difference between them (28.6 ± 4.7% in SSFO mice, n = 4 vs 25.3 ± 5.2% in GFP mice, n = 4; p = 0.6525, unpaired two-tailed t test). Similarly, there was no significant difference in the number of cFos-positive cells in 1 mm² areas just ventrally to the tips of optical fibers in the LHA between SSFO and control mice (216.5 ± 29.3 cells in SSFO mice vs 172.3 ± 34.6 cells in GFP mice; p = 0.3674, unpaired two-tailed t test), as this area was previously reported to be affected during photostimulations using optic cannulas (Kroeger et al., 2018).

Together, these results indicate that photoactivation of the VLPO^GABA→LHA pathway results in an increase in wakefulness without involving activation of orexin neurons.

Vgat-expressing VLPO neurons projecting to LHA and TMN partially overlap

A previous study identified that GAD2-expressing preoptic neurons projecting to the tuberomammillary nucleus (TMN; POA^GAD2→TMN neurons) contribute to the induction of NREM and REM sleep (Chung et al., 2017). GAD2 is another molecular marker of GABA-producing neurons; thus, VLPO possibly contains a GABA-producing neuronal population projecting to the TMN that has a sleep-promoting effect. We therefore examined whether the VLPO^GABA neurons projecting to the LHA belong to the same neuronal population originating the POA^GAD2→TMN pathway using dual-color retrograde tracing (Fig. 6I). As a first step, we delivered recombinant cholera toxin subunit B conjugated with AlexaFluor-488 into the LHA and Red RetroBeads into the TMN of the same mice. Next, we conducted FISH using Vgat probe on the neurons in the VLPO to examine whether the VLPO^GABA→LHA neurons overlap with the VLPO^GABA→TMN neurons.

We found that Vgat-positive neurons in the VLPO constituted approximately half of all retrogradely labeled neurons projecting to the LHA (50.6 ± 2.6%, n = 4), which is similar to the results of our rabies virus-mediated tracing experiment of orexin neurons (Fig. 2F,J). Slightly more than half of the Vgat-positive VLPO neurons sending their axons to the LHA overlapped with Vgat-positive VLPO neurons projecting to the TMN (Fig. 6J, white arrowhead; Fig. 6K, left; 53.1 ± 9.2%, n = 4). Neurons in the VLPOc had a greater percentage of overlapping than the neurons in the VLPOe (Fig. 6K, right). Notably, we also observed a substantial number of Vgat-positive VLPO neurons containing only Red RetroBeads (i.e., projecting to the TMN; Fig. 6J, magenta arrowheads). Together, these results indicate that the VLPO^GABA→LHA and VLPO^GABA→TMN pathways originate from only partially shared, but largely distinct neuronal populations in the VLPO.