Neural basis for regulation of vasopressin secretion by anticipated disturbances in osmolality
Rapid , anticipatory feedforward regulation is a common feature of many homeostatic circuits ( Betley et al . , 2015; Zimmerman et al . , 2016; Augustine et al . , 2018 ) . While the purpose of feedforward regulation has yet to be established , a number of compelling proposals have been suggested . Several studies have shown that feedforward regulation plays an important role in shaping ingestive behavior by promoting negative-reinforcement learning ( Betley et al . , 2015; Allen et al . , 2017; Leib et al . , 2017 ) . It is also suggested that feedforward regulation is crucial for maintaining energy/water balance as it prevents overconsumption by preemptively terminating ingestive behavior before systemic disturbance is detected ( Andermann and Lowell , 2017; Augustine et al . , 2020 ) . Although the significance of feedforward regulation is just beginning to be recognized , the idea of such regulation in the hypothalamus existed since the late 1900s . A conceptual framework for feedforward regulation was provided by studies looking at the effect of water intake on thirst and blood vasopressin ( AVP ) levels ( Rolls et al . , 1980; Wood et al . , 1980; Thrasher et al . , 1981; Stricker and Hoffmann , 2007 ) , from which the term ‘presystemic regulation’ originates . AVP , also known as antidiuretic hormone , is synthesized by posterior pituitary-projecting magnocellular AVP neurons of the paraventricular ( PVH ) and supraoptic nuclei ( SON ) of the hypothalamus . These neurons underlie the body’s main non-behavioral response to dehydration or elevated systemic osmolality—an increase in circulating AVP . Upon stimulation of AVP neurons , AVP is released from the axon terminals in the posterior pituitary and enters the bloodstream ( Ferguson et al . , 2003; Bolignano et al . , 2014 ) . Once released , AVP binds to receptors in the kidney and stimulates water reabsorption to restore water balance ( Nielsen et al . , 1995 ) . Activity of AVP neurons is regulated by two temporally distinct mechanisms . Feedback or systemic regulation , which mainly informs AVP neurons of changes in systemic water balance , is mediated by the lamina terminalis ( LT ) , comprised of the subfornical organ ( SFO ) , median preoptic nucleus ( MnPO ) , and organum vasculosum lamina terminalis ( OVLT ) ( McKinley et al . , 2004 ) . The SFO and OVLT are circumventricular organs ( CVOs ) that lack a blood-brain barrier , and are capable of sensing systemic osmolality and blood-borne factors , such as angiotensin II . Osmolality information sensed by the SFO and OVLT is then sent to AVP neurons directly or indirectly via the MnPO . Feedforward or presystemic regulation , on the other hand , informs AVP neurons of anticipated future osmotic perturbation , based on the array of presystemic signals that occur immediately before ( pre-ingestive ) and after ( post-ingestive ) food or water ingestion ( Stricker and Hoffmann , 2007; Stricker and Stricker , 2011 ) . Presystemic regulation of AVP release was first described in 1980 , in a study using dehydrated dogs ( Thrasher et al . , 1981; Thrasher et al . , 1987 ) , and was later replicated in humans , monkeys , sheep , and rats ( Arnauld and du Pont , 1982; Geelen et al . , 1984; Blair-West et al . , 1985; Huang et al . , 2000 ) . These studies demonstrated that drinking causes a rapid reduction in blood AVP levels and that this decrease precedes any detectable decrease in blood osmolality ( i . e . , it is presystemic ) . However , due to lack of temporal resolution in blood AVP measurements ( Christ-Crain and Fenske , 2016 ) , these studies failed to capture rapid dynamics of presystemic regulation , preventing further dissection of distinct stimuli causing the presystemic reductions in AVP . In a previous study , we demonstrated that magnocellular AVP neurons are under bidirectional presystemic regulation by drinking and eating ( Mandelblat-Cerf et al . , 2017 ) . However , the neural inputs providing presystemic information to these neurons are still unclear . Here , we used various circuit-mapping techniques , in vivo calcium imaging , and opto- and chemo-genetics to identify and characterize the neural circuits mediating presystemic regulation of AVP neuron activity .
As the presystemic regulation of AVP neurons is rapid , it must be brought about by neural afferent inputs . To identify these afferents , we performed retrograde rabies mapping from posterior pituitary-projecting , magnocellular SONAVP and PVHAVP neurons using AVP-IRES-Cre mice ( Figure 1a and e ) . Cre-dependent TVA ( EnvA receptor ) ( AAV-DIO-TVA ) and rabies glycoprotein ( RG ) ( AAV-DIO-RG ) viruses were injected into the SON or PVH . After 3 weeks , EnvA-pseudotyped G-deleted rabies virus ( RV-EnvA-ΔG-GFP ) was injected into the SON or posterior pituitary as described below . To target magnocellular SONAVP neurons , we injected the rabies virus directly into the SON ( Figure 1a ) . Note that all AVP neurons in the SON project to the posterior pituitary ( Brown et al . , 2013; Mandelblat-Cerf et al . , 2017 ) . In contrast , the PVH contains magnocellular and non-posterior pituitary-projecting , parvocellular AVP neurons . To specifically target magnocellular neurons , we devised a retrograde approach in which rabies virus was injected into the posterior pituitary instead of the PVH ( Figure 1e ) . Because TVA , in addition to being expressed at the cell body , is trafficked to the axon terminals ( Betley et al . , 2013; Livneh et al . , 2017 ) , rabies virus can be taken up by TVA-expressing axons of PVHAVP neurons in the posterior pituitary , leading to projection-specific infection . Starter neurons in the PVH and SON were identified by co-expression of rabies-GFP and TVA-mCherry ( Figure 1b and f ) . Remarkably , despite their distinctly different anatomical locations , but in agreement with their identical functions , rabies mapping results from magnocellular PVHAVP and SONAVP neurons were strikingly similar . Both received strong inputs from the SFO , MnPO , and OVLT ( Figure 1d and h ) , and additional sparse rabies labeling was found in the arcuate nucleus ( ARC ) of the hypothalamus and the perinuclear zone ( PNZ ) , an area surrounding the SON . We also found surprisingly dense rabies labeling in the PVH and SON when inputs to SONAVP and PVHAVP neurons were examined , respectively ( Figure 1c and g ) . We performed additional experiments to investigate these unexpected findings . First , we injected AAV that Cre-independently expresses ChR2-mCherry into either the PVH or SON and found that neurons in these sites do not send direct projections to the SON and PVH , respectively . Furthermore , we also injected retrograde tracer cholera toxin subunit B into either the PVH or SON , and in agreement with the above-mentioned anterograde tracing study , failed to detect any labeled neurons in the SON and PVH , respectively ( not shown ) . Based on these results , we believe that axosomatic and axodendritic PVH → SONAVP or SON → PVHAVP connections do not exist , and we speculate that the above-mentioned rabies results were caused by either non-synaptic transfer of rabies ( i . e . , an artifact ) or synaptic transfer via axoaxonic synapses that might exist between PVH and SON neurons at the level of the posterior pituitary ( Silverman et al . , 1983; Choudhury and Ray , 1990 ) . To identify the glutamatergic and GABAergic nature of LT neurons providing inputs to PVHAVP and SONAVP neurons , we performed anterograde tracing from Vglut2- versus Vgat-expressing neurons of the LT . We injected the Cre-dependent synaptophysin ( Syn ) anterograde tracer , AAV-DIO-Syn-YFP , into the SFO and MnPO/OVLT of Vglut2-IRES-Cre and Vgat-IRES-Cre mice to individually trace glutamatergic and GABAergic projections ( Figure 1i ) . The MnPO and OVLT were considered as one group because viral targeting of individual structures was challenging . We found that the MnPO/OVLT sends both excitatory and inhibitory projections to the PVH and SON while the SFO only sends excitatory projections ( Figure 1j ) . Lack of long-range inhibitory projections of the SFO has been previously reported ( Oka et al . , 2015 ) . Next , we performed channelrhodopsin ( ChR2 ) -assisted circuit mapping ( CRACM ) to test synaptic connectivity of these projections to PVHAVP and SONAVP neurons . AAV-DIO-ChR2-mCherry was injected into the SFO or MnPO/OVLT of AVP-GFP;Vglut2-IRES-Cre or AVP-GFP;Vgat-IRES-Cre mice and light-evoked postsynaptic currents were recorded from PVHAVP and SONAVP neurons identified by GFP expression ( Figure 1k ) . All SONAVP neurons recorded are magnocellular . We did not distinguish parvocellular and magnocellular PVHAVP neurons in this experiment . We found that SFOVglut2 and MnPO/OVLTVglut2 neurons provided robust glutamatergic input to PVHAVP and SONAVP neurons ( Figure 1l and n ) . In contrast , GABAergic input from MnPO/OVLTVgat neurons was detected only in half of the PVHAVP and SONAVP neurons . Next , we tested the synaptic connectivity of the LT to GFP-negative neurons in the PVH and SON ( Figure 1m ) . The MnPO/OVLT provided glutamatergic and GABAergic input to the majority of GFP-negative neurons in the PVH and SON . We did not find any connection between SFO Vglut2 neurons and GFP-negative PVH neurons , suggesting that SFO Vglut2 inputs were highly specific to AVP neurons . Taken together , these results demonstrate that PVHAVP and SONAVP neurons receive excitatory and inhibitory inputs from the LT . Next , we examined the involvement of the LT in presystemic regulation of SONAVP neurons . We focused on SONAVP neurons because the magnocellular population can be specifically and efficiently targeted by direct viral injection into the SON . We measured the in vivo calcium dynamics of GCaMP6s-expressing SONAVP neurons while inhibiting the MnPO/OVLT or SFO with CNO/hM4Di ( Figure 2a and i ) . Effectiveness of hM4Di-mediated silencing of MnPO/OVLT and SFO neurons was validated in vitro in slice ( Figure 2—figure supplement 3 ) . To monitor water-related presystemic responses , mice were chronically water-restricted and fully habituated to an experimental paradigm prior to the experiment . At various latencies following the beginning of each session , a water bowl was placed in the cage and mice were allowed to drink freely for ~15 min . Each mouse had one experimental session per day , in which they were pre-injected with either saline or CNO . In the control group , as previously reported ( Mandelblat-Cerf et al . , 2017 ) , SONAVP neurons were rapidly inhibited as the water bowl was introduced ( Figure 2b ) . This pre-ingestive response was learning-dependent as it gradually developed over the course of training ( Figure 2—figure supplement 1 ) . Pre- and post-ingestive suppression of SONAVP neurons were observed before the significant drop in systemic osmolality , which we previously found to begin 10–15 min after drinking onset ( Mandelblat-Cerf et al . , 2017 ) . Inhibition of the MnPO/OVLT abolished the pre-ingestive drop in SONAVP neuron activity ( Figure 2b–f ) . The post-ingestive response was also significantly attenuated . When recordings were performed from mice expressing the calcium-independent fluorescent marker , EYFP , in SONAVP neurons , no feeding/drinking-induced response was observed ( Figure 2—figure supplement 2 ) , demonstrating that the changes in GCaMP6s fluorescence were not due to movement artifacts . Because the MnPO contains thirst-regulating neurons ( Abbott et al . , 2016; Augustine et al . , 2018 ) , we also analyzed drinking behavior in these mice . MnPO/OVLT inhibition did not prevent drinking but delayed drinking onset as demonstrated by a significant increase in the latency to drink ( Figure 2g ) . The number of drinking bouts per session was not affected . The MnPO/OVLT , along with the SFO , is the main source of excitatory input that underlies activation of SONAVP neurons during systemic hyperosmolality ( McKinley et al . , 2004 ) . Consistent with this , we found that MnPO/OVLT inhibition caused a drop in the baseline activity of SONAVP neurons in water-restricted mice ( Figure 2h ) . In a separate group of mice , we investigated the effect of SFO inhibition ( Figure 2i ) . Inhibition of the SFO caused a slight but non-significant attenuation of pre- and post-ingestive suppression ( Figure 2j–l ) . Taken together , these data indicate that neurons in the MnPO/OVLT are important mediators of water-related presystemic signals to AVP neurons . Next , we sought to understand the organization of the presystemic circuit mediating water-related information within and upstream of the LT . Both the SFO and MnPO/OVLT provide strong excitatory inputs to AVP neurons . However , we found that only the MnPO/OVLT was required for water-related presystemic regulation of SONAVP neurons ( Figure 2 ) , suggesting that the presystemic circuit might be organized similarly to the hierarchical thirst circuit described previously ( Augustine et al . , 2018 ) . To test this idea , we mapped inputs to SON-projecting MnPO/OVLTVglut2 and SFOVglut2 neurons using monosynaptic rabies tracing . SON-projecting neurons were targeted using a retrograde approach shown schematically in Figure 3a and d . SON-projecting starter neurons , visualized by co-expression of GFP and mCherry , were seen in the MnPO/OVLT and SFO ( Figure 3b and e ) . Results from the MnPO/OVLT and SFO were strikingly different . SON-projecting MnPO/OVLTVglut2 neurons received inputs from multiple sites ( Figure 3c ) , including the SFO , PVH , ventromedial ( VMH ) , and dorsomedial nuclei ( DMH ) of the hypothalamus , and the lateral parabrachial nucleus ( LPBN ) . Among these areas , the SFO contained the greatest number of rabies-labeled neurons , demonstrating strong interconnectivity between these LT structures . In contrast , SON-projecting SFOVglut2 neurons received inputs only from the other LT structures , the MnPO and OVLT ( Figure 3f ) . The result was further validated using Nos-1-IRES-Cre mice , another Cre line that specifically marks excitatory SFO neurons ( Figure 3—figure supplement 1; Zimmerman et al . , 2016 ) . These data suggest that the MnPO/OVLT is the primary LT region that receives and relays extra-LT inputs to AVP neurons and , thus , is likely the main entry point for water-related presystemic information . Magnocellular PVHAVP and SONAVP neurons , despite their distinct anatomical locations , are functionally identical , receive inputs from the same sites ( Figure 1 ) , and show similar presystemic responses ( Mandelblat-Cerf et al . , 2017 ) . To explore the possibility that presystemic regulation of both PVHAVP and SONAVP neurons is mediated by collateral projections from the same LT neurons , we performed rabies-based axon collateral mapping from SON-projecting MnPO/OVLTVglut2 , MnPO/OVLTVgat , and SFOVglut2 neurons and examined their collateral projections in the LT , PVH , and SON . Rabies-based axon collateral mapping was performed as previously described ( Betley et al . , 2013; Livneh et al . , 2017; Livneh et al . , 2020 ) . We found rabies-labeled collateral projections of SON-projecting MnPO/OVLTVglut2 , MnPO/OVLTVgat , and SFOVglut2 neurons in the PVH and LT ( Figure 3g–i ) . The density of collateral projections was lower than that of the projections to the SON , suggesting that only a subset of SON-projecting MnPO/OVLTVglut2 , MnPO/OVLTVgat , and SFOVglut2 neurons send collaterals to the PVH and LT . Interestingly , we found that MnPO/OVLT-projecting SFOVglut2 neurons , which include thirst-promoting neurons ( Matsuda et al . , 2017; Augustine et al . , 2018 ) , send a similar density of rabies-labeled fibers to the MnPO/OVLT , PVH , and SON ( Figure 3j ) . This result suggests that a majority of these SON-projecting SFOVglut2 neurons send collaterals to the MnPO/OVLT and PVH , and , therefore may be capable of simultaneously regulating AVP release and thirst . Taken together , these results indicate that , unlike convergence of afferent presystemic inputs to the MnPO/OVLT , downstream projections of the LT are redundantly organized such that presystemic information is shared not only between the LT structures but also between PVHAVP and SONAVP neurons via collateral projections . Next , we measured in vivo calcium dynamics of SON-projecting MnPO/OVLT and SFO neurons in response to water bowl presentation and drinking . GCaMP6s was expressed in SON-projecting excitatory or inhibitory neurons of the MnPO/OVLT and SFO using a modified herpes simplex virus that Cre-dependently expresses GCaMP6s ( HSV-GCaMP6s ) and retrogradely infects neurons from the axon terminals ( Kohl et al . , 2018 ) . HSV-GCaMP6s was injected into the SON of Vglut2-IRES-Cre or Vgat-IRES-Cre mice and an optic fiber was placed above the MnPO/OVLT or SFO ( Figure 4a and g ) . We found that SON-projecting MnPO/OVLTVglut2 and SFOVglut2 neurons both showed an immediate drop in activity upon water bowl placement ( Figure 4b–e ) . A further drop in activity was observed as drinking continued . This resembled the response of SONAVP neurons , suggesting that the inputs from MnPO/OVLTVglut2 and SFOVglut2 neurons contribute to water-related presystemic regulation of AVP neurons . MnPO/OVLTVglut2 neurons showed a larger and sharper decrease in activity compared to SFOVglut2 neurons when normalized values were compared ( Figure 4f ) . Activity of MnPO/OVLTVglut2 neurons reached <50% of baseline activity within the first 1 min of drinking , whereas SFOVglut2 neurons showed a gradual decrease that continued for over 5 min . A sharper drop in MnPO/OVLTVglut2 neuron activity is consistent with significant attenuation of water-related presystemic response by MnPO , but not SFO , inhibition . The SON-projecting MnPO/OVLTVgat neurons showed a completely different response ( Figure 4h–k ) . Placement of the water bowl caused an abrupt increase in MnPO/OVLTVgat neuron activity , which rapidly declined and returned to baseline within 1 min . No drinking-related , long-term responses were observed . We hypothesize that SON-projecting MnPO/OVLTVgat neurons mainly signal the anticipation of water and work in parallel with MnPO/OVLTVglut2 and SFOVglut2 neurons to ensure rapid inhibition of AVP neuron activity upon water bowl placement . Taken together , these data indicate that the MnPO/OVLT is a key relay center for water-related presystemic information . As discussed earlier , SON-projecting excitatory and inhibitory neurons of the LT showed distinct patterns of presystemic response to water cue and drinking , suggesting that presystemic information conveyed by these neurons to AVP neurons may differ . To test this idea , we selectively inhibited excitatory and inhibitory MnPO/OVLT neurons with CNO/hM4Di while monitoring in vivo calcium dynamics of SONAVP neurons in response to water bowl presentation and drinking ( Figure 5a and j ) . This was achieved by injecting a flp-dependent hM4Di virus ( AAV-fDIO-hM4Di-mCherry ) into the MnPO/OVLT of AVP-IRES-Cre;Vglut2/Vgat-IRES-Flp mice . Electrophysiological recordings demonstrated that CNO/hM4Di effectively silenced both excitatory and inhibitory MnPO/OVLT neurons in Vglut2- and Vgat-IRES-Flp mice , respectively ( Figure 2—figure supplement 3 ) . Inhibition of MnPO/OVLTVglut2 neurons caused an overall reduction of water-related presystemic responses in SONAVP neurons . Both pre- and post-ingestive responses were significantly affected ( Figure 5b–f ) . However , in comparison to non-specific silencing of the same area , which caused a complete removal of the pre-ingestive response of SONAVP neurons ( Figure 2f ) , inhibition of excitatory neurons in the MnPO/OVLT was less effective in reducing pre-ingestive component of the response as ~30% of the pre-ingestive response persisted ( Figure 5g ) . Post-ingestive responses ( Figures 2f and 5g ) and baseline activity before water bowl presentation ( Figures 2h and 5i ) were reduced to a similar degree in both cases . No significant change in latency to drink or in the number of drinking bouts was observed during the experimental session . However , when mice were tested in a separate setup using a lickometer , MnPO/OVLTVglut2 neuron inhibition caused a significant reduction in the amount of water intake in the same group of animals ( Figure 5h ) . In contrast , the effect of MnPO/OVLTVgat neuron inhibition was strictly restricted to the pre-ingestive phase ( Figure 5k–o ) . More specifically , a significant reduction in the SONAVP neuron response was found only during the last 5 s of pre-ingestive period that immediately precedes the onset of drinking ( Figure 5p ) . This result is in line with our previous finding that MnPO/OVLTVgat neurons show a transient activation to water-predicting cues ( Figure 4h–k ) . The degree of reduction in pre-ingestive responses ( Figure 5p ) was comparable to that of non-specific silencing of the MnPO/OVLT ( Figure 2f ) , suggesting that water-predicting cue/pre-ingestive information is primarily conveyed by inhibitory neurons of the MnPO/OVLT . Consistent with a lack of post-ingestive or any sustained changes in the activity of MnPO/OVLTVgat neurons in response to drinking ( Figure 4h–k ) , their inhibition had no effect on post-ingestive responses or baseline activity of SONAVP neurons ( Figure 5p and r ) . Drinking behavior was not altered during the experimental session or when tested in a separate setup ( Figure 5q ) . Taken together , these results demonstrate that excitatory and inhibitory inputs from the MnPO/OVLT are each responsible for pre- and post-ingestive drops in SONAVP neuron activity , respectively , and that both inputs are required for water-related presystemic responses of SONAVP neurons . We found that the LT is involved in water-related presystemic regulation of SONAVP neurons with the MnPO/OVLT playing a key role . We next tested whether the same region was involved in food-related presystemic regulation using the same approach as shown in Figure 2a . Mice were chronically food-restricted and were trained to take food pellets from a food bowl placed in the cage with varying latency . In the control group , as previously reported ( Mandelblat-Cerf et al . , 2017 ) , SONAVP neurons showed an increase in activity that began immediately upon feeding onset ( Figure 6a ) . MnPO/OVLT inhibition did not affect the baseline activity ( not shown ) or feeding-induced activation ( Figure 6a–c ) . These results demonstrate that food-related presystemic regulation is mediated by a different circuit that does not involve the MnPO/OVLT . We then explored the activity of SON-projecting LT neurons during food intake . In line with the result from MnPO/OVLT inhibition , none of the SON-projecting LT neurons displayed responses that could account for feeding-induced activation observed in SONAVP neurons ( Figure 6d and e ) . SON-projecting SFOVglut2 and MnPO/OVLTVglut2 neurons showed an overall decrease in activity after food presentation but no consistent pattern was observed . SON-projecting MnPO/OVLTVgat neurons were activated by food presentation but their activity gradually declined following feeding onset . The amplitude of these responses was significantly smaller than water-related responses of the same neurons ( Figure 6e ) . Presystemic activation of SONAVP neurons is time-locked to the feeding onset ( this study and Mandelblat-Cerf et al . , 2017 ) . To better compare responses in the LT neuron responses and in SONAVP neurons , we reanalyzed the data by aligning the traces to feeding onset . SON-projecting SFOVglut2 , MnPO/OVLTVglut2 , and MnPO/OVLTVgat neurons all showed a decrease in activity ( Figure 6f ) . Even if these were to be bona fide responses , a small , simultaneous decrease in excitatory and inhibitory tone is unlikely to be the primary cause of rapid feeding-induced activation of SONAVP neurons . A previous study demonstrated rapid net activation of all excitatory SFO neurons by food intake ( Zimmerman et al . , 2016 ) , which contradicts our finding . To resolve this inconsistency , we performed fiber photometry from the entire SFOVglut2 population . When compared to SONAVP neurons , the response of SFOVglut2 neurons was significantly delayed and lacked an early post-ingestive component occurring within 1 min of feeding onset ( Figure 6g ) . Given that blood osmolality begins to gradually increase as early as within 1-2 min of feeding and reaches significance at 5 min ( Mandelblat-Cerf et al . , 2017 ) , we speculate that feeding-induced activation of SFOVglut2 neurons reflects systemic blood osmolality changes . Taken together , these results demonstrate that MnPO/OVLT and SFO are not involved in presystemic feeding-induced activation of SONAVP neurons , supporting our finding that food- and water-related presystemic regulation is mediated by non-overlapping neural circuits . Given that the LT appears not to be involved in food-related presystemic regulation , we investigated other possible afferents that might provide food-related presystemic information to SONAVP neurons . Due to inefficiency and tropism of the rabies virus , rabies retrograde tracing can , in some cases , be prone to false-negative results ( Saleeba et al . , 2019 ) . Therefore , we decided to study additional sites suggested by a study using a traditional retrograde tracer ( Tribollet et al . , 1985 ) : the nucleus of the solitary tract ( NTS ) and A1/C1 neurons in the ventrolateral medulla ( VLM ) . Assuming that our rabies study failed to detect these afferents , we decided to investigate whether any of these areas provide direct inputs to AVP neurons . Catecholaminergic and glutamatergic input from A1/C1 neurons to AVP neurons have been validated in previous studies ( Leng et al . , 1999; Guyenet et al . , 2013 ) and therefore their connection was not tested in our study . First , we explored the projections of NTS neurons using Cre-independent ChR2 ( AAV-ChR2-mCherry ) . Projections from the NTS were extremely sparse in the PVH and nearly absent in the SON ( Figure 7a ) . Subsequent CRACM showed that the NTS do not provide direct synaptic input to AVP neurons ( Figure 7b and c ) . Similar results were obtained when A2 and excitatory neurons of the NTS were studied separately ( Figure 7—figure supplement 1 ) , and thus the NTS was not investigated further . To test the involvement of A1/C1 neurons in food-related presystemic regulation , we inhibited A1/C1 neurons with hM4Di while monitoring in vivo calcium dynamics of SONAVP neurons . To achieve specific expression of hM4Di in A1/C1 neurons , we used AVP-IRES-Cre;DBH-Flp mice in combination with AAV-fDIO-hM4Di-mCherry ( Figure 7d ) . First , to test the effectiveness of hM4Di in silencing A1/C1 neurons , we studied the effect of CNO on hypotension-induced activation of AVP neurons . A1/C1 neurons are suggested to be crucial for hypotension-induced AVP release as non-specific inhibition/lesion of the VLM causes a significant attenuation of this response ( Blessing and Willoughby , 1985; Head et al . , 1987 ) . A1/C1 inhibition significantly attenuated the activation of SONAVP neurons in response to drug-induced hypotension ( Figure 7e ) . This finding validates our experimental system and provides further evidence for the importance of A1/C1 neurons in hypotension-induced AVP release . We then used the same approach to examine the involvement of A1/C1 neurons in food-related presystemic regulation . In contrast to the robust blockade of hypotension-induced SONAVP neuron activation , A1/C1 neuron inhibition had no effect on feeding-induced activation of SONAVP neurons ( Figure 7f ) . Taken together , these data indicate that blood pressure regulation is mediated by a circuit involving A1/C1 neurons and this circuit is distinct from the food-related presystemic circuit . Next , we decided to test other afferents identified by rabies tracing—that is , those coming from the ARC and PNZ ( Figure 7g , Figure 7—figure supplement 2a ) . We first decided to focus on two genetically accessible , functionally relevant neuronal populations in the ARC , AgRP and POMC neurons , which have opposite roles in regulating food intake and energy balance . To investigate whether AgRP and POMC neurons provide direct inputs to PVHAVP and SONAVP neurons , we performed projection mapping and CRACM . AgRP and POMC projections were present in the PVH and SON but no synaptic connections were detected between AgRP/POMC and AVP neurons ( Figure 7—figure supplement 3 ) . In line with this , specific chemogenetic inhibition of AgRP or POMC neurons had no effect on the food-related presystemic response of SONAVP neurons when investigated using fiber photometry in AVP-IRES-Cre;AgRP/POMC-IRES-Cre mice ( Figure 7h–j ) . Short-term ( <30 min following CNO injection ) feeding behavior was not altered by AgRP or POMC neuron inhibition ( Figure 7—figure supplement 4 and Krashes et al . , 2011; Üner et al . , 2019 ) . Another candidate input identified by rabies mapping is the PNZ , an area surrounding the SON ( Figure 7—figure supplement 2a ) . GABAergic neurons in the PNZ ( PNZVgat ) are believed to be involved in hypertension- and hypervolemia-induced inhibition of SONAVP neurons ( Grindstaff and Cunningham , 2001b; Grindstaff and Cunningham , 2001a; Cunningham et al . , 2002 ) . While we found that PNZVgat neurons provided direct inhibitory input to AVP neurons and showed a selective response to food but not water ( Figure 7—figure supplement 2b , f ) , inhibition of these neurons had no effect on the baseline activity of SONAVP neurons ( not shown ) or their response to feeding ( Figure 7—figure supplement 2g , i ) . Taken together , these data suggest that AgRP , POMC , and PNZVgat neurons are not primary contributors to food-related presystemic regulation . Next , we decided to focus on the entire ARC population . Because it is difficult to selectively target all ARC neurons without hitting surrounding nuclei , to examine the role of ARC neurons we decided to use a subtractive approach by creating three groups of animals with differing hM4Di expression: ( 1 ) ARC+VMH+DMH ( Figure 7k ) , ( 2 ) VMH only ( Figure 7o ) , and ( 3 ) DMH only ( Figure 7q ) . This approach allowed for effective silencing of the ARC with hM4Di , which is difficult to achieve with restricted injection of Cre-independent AAVs into the ARC . All three groups of mice underwent the same experimental protocol . Mice were chronically food-restricted and were habituated to the experimental session as described above . Inhibition of neurons in all three nuclei ( ARC+VMH+DMH ) caused a significant attenuation of feeding-induced activation that persisted throughout the recording ( Figure 7l–n ) . In contrast , inhibition of the VMH ( Figure 7p ) or DMH ( Figure 7r ) alone , fully sparing the ARC , had no effect . Injection of CNO in all three groups did not affect the baseline activity of SONAVP neurons ( not shown ) or short-term feeding behavior of mice ( Figure 7—figure supplement 4 ) . Taken together , these results indicate that the ARC contains a cell type , distinct from AgRP or POMC neurons , that relay food-related presystemic signals to SONAVP neurons .
Water homeostasis is achieved by an intricate balance between water intake and output that are dictated , respectively , by thirst and antidiuretic hormone ( AVP ) . While the field has experienced great advances in the neurobiological understanding of thirst in the last several years , our knowledge of neural regulation of AVP release remains rudimentary due to lack of technical approaches for reliable and temporally precise measurement of circulating AVP in vivo . In a prior study , by directly monitoring the activity of magnocellular AVP neurons , we demonstrated that AVP neuron activity , and thus likely AVP release , is altered surprisingly rapidly by water and food ingestion before any change in systemic osmolality is observed ( Mandelblat-Cerf et al . , 2017 ) . While such feedforward , presystemic regulation has been demonstrated in other homeostatic circuits , AVP neurons are unique in that they are capable of responding presystemically to both water and food , and the response is bidirectional—water-predicting cues and drinking inhibit and eating activates AVP neurons—and asymmetric—water causes pre- and post-ingestive presystemic inhibition but food only causes post-ingestive presystemic activation . In this study , we expand on our prior findings by presenting a neural circuit mechanism by which these unique properties arise . Our study revealed two parallel , non-overlapping neural pathways for water- and food-related presystemic regulation ( Figure 8 , Table 1 ) . Key components of the water-related presystemic circuit are neurons in the LT , which provide direct synaptic input to AVP neurons and exhibit water-related presystemic responses that closely resemble those of AVP neurons . Food-related presystemic regulation , on the other hand , is mediated by an as yet unidentified neuronal population in the ARC that is completely distinct from feeding-regulating AgRP and POMC neurons . In the course of our study , we confirmed additional afferents that were previously proposed to be involved in other aspects of AVP function , such as A1/C1 neurons in the brainstem that mediate hypotension-induced AVP release ( Blessing and Willoughby , 1985; Head et al . , 1987; Leng et al . , 1999; Guyenet et al . , 2013 ) . Collectively , these findings demonstrate that water- versus food-related presystemic signals are relayed to AVP neurons independently and differently via two non-overlapping circuits to give rise to the bidirectional , asymmetric presystemic response of AVP neurons . Recent studies have identified multiple molecularly or anatomically distinct neuronal populations in the LT that are involved in different aspects of water homeostasis ( Abbott et al . , 2016; Allen et al . , 2017; Matsuda et al . , 2017; Augustine et al . , 2018; Matsuda et al . , 2020; Pool et al . , 2020 ) . In this study , we investigated the role of the LT neurons in mediating presystemic regulation of AVP neurons . We identified at least three groups of LT neurons that provide direct input to AVP neurons: SFOVglut2 , MnPO/OVLTVglut2 , and MnPO/OVLTVgat neurons . Interestingly , while all these neurons connect to AVP neurons with high probability , only MnPO/OVLT neurons were required for their presystemic regulation . More strikingly , while being indispensable for water-related presystemic responses of AVP neurons , MnPO/OVLT neurons played no role in mediating food-related presystemic responses , indicating that presystemic regulation of AVP neurons involves two distinct neural pathways dedicated to water- or food-related regulation . Three groups of AVP-regulating LT neurons were studied individually by selectively isolating them using projection-specific approaches ( i . e . , to the SON ) and genetic markers ( i . e . , Vglut2 and Vgat ) . As can be expected from their opposite neurochemical nature , SON-projecting MnPO/OVLTVglut2 and MnPO/OVLTVgat neurons showed opposing responses to water-related presystemic signals . SON-projecting MnPO/OVLTVglut2 neurons were rapidly suppressed by both water-predicting cues and drinking whereas SON-projecting MnPO/OVLTVgat neurons were activated transiently and exclusively by water-predicting cues . SON-projecting SFOVglut2 neurons showed similar presystemic response to MnPO/OVLTVglut2 neurons but their contribution to presystemic regulation of AVP neurons may be minor as inhibition of the SFO did not affect the activity of AVP neurons . Of note , while we refer to SON-projecting LT neurons as ‘AVP-regulating’ based on their high probability connections to SONAVP neurons , we acknowledge that subsets of SON-projecting LT neurons may also be involved in regulation of other downstream processes such as oxytocin release ( via their connection to SONOxytocin ( non-AVP ) neurons as demonstrated by CRACM ) and thirst ( via their collaterals to other downstream structures as demonstrated by collateral mapping ) . By selectively silencing MnPO/OVLTVglut2 and MnPO/OVLTVgat neurons , we demonstrate that both excitatory and inhibitory inputs from the MnPO/OVLT are necessary for water-related presystemic regulation of AVP neurons . MnPO/OVLTVgat and MnPO/OVLTVglut2 neurons were each responsible for pre- and post-ingestive phases of the response , respectively , which is consistent with the contrasting neural dynamics of SON-projecting MnPO/OVLTVgat and MnPO/OVLTVglut2 neurons . Initial pre-ingestive cue-induced suppression of AVP neurons was mediated primarily by MnPO/OVLTVgat neurons as their silencing completely prevented this response . Silencing of MnPO/OVLTVglut2 neurons also caused a significant attenuation but failed to completely block the pre-ingestive response . These results suggest that the pre-ingestive response is a combined outcome of direct and indirect suppression of AVP neurons by MnPO/OVLTVgat neurons , via their direct projections to AVP neurons and local projections to MnPO/OVLTVglut2 neurons , which consequently results in reduced excitatory drive to AVP neurons . Post-ingestive drinking-induced suppression of AVP neurons was primarily the result of decreased excitatory drive from the MnPO/OVLT as post-ingestive response was significantly attenuated by silencing of MnPO/OVLTVglut2 neurons , but not MnPO/OVLTVgat neurons . Silencing of MnPO/OVLTVglut2 neurons simultaneously caused a significant reduction in the amount of water intake . While reduced water intake could contribute to the reduction in post-ingestive response , this is unlikely a major factor given the consistent degree of silencing throughout both pre- and post-ingestive phases and the high probability connection and similar response dynamics in SON-projecting MnPO/OVLTVglut2 versus SONAVP neurons . Presystemic responses of thirst-regulating counterparts of the LT have been extensively studied by many groups ( Zimmerman et al . , 2016; Allen et al . , 2017; Augustine et al . , 2018; Augustine et al . , 2019; Zimmerman et al . , 2019 ) . While thirst-regulating LT neurons also exhibit presystemic suppression ( thirst-promoting neurons ) and activation ( thirst-suppressing neurons ) , their responses surprisingly lack a pre-ingestive component that is present in AVP-regulating LT neurons and also hunger-regulating AgRP and POMC neurons ( this study and Betley et al . , 2015; Chen et al . , 2015; Mandelblat-Cerf et al . , 2017 ) . This raises the possibility that presystemic regulation of thirst neurons may be different . Alternatively , it is possible that prior studies missed this regulation due to either its magnitude being small in comparison to post-ingestive regulation and/or the fact that population recordings , which may have pooled thirst-regulating LT neurons with other neurons , obscured its detection . Indeed , a study using single-cell imaging reported a small subset of MnPO/OVLT neurons that are activated or inhibited by pre-ingestive cue ( Zimmerman et al . , 2019 ) . In addition , considering that the pre-ingestive response is not innate but requires several days of training ( Figure 2—figure supplement 1 and Mandelblat-Cerf et al . , 2017 ) , failure to detect pre-ingestive responses in these thirst-related studies might possibly be due to the use of experimental paradigms not optimally designed to assess pre-ingestive responses . The MnPO is traditionally considered as a hub that functions as the main input and output region of the LT ( McKinley et al . , 2015 ) . In line with this , we found that SON-projecting MnPO/OVLT Vglut2 neurons receive inputs from multiple regions outside the LT while inputs to SON-projecting SFOVglut2 neurons were confined to the MnPO/OVLT . This result supports hierarchical organization of the LT with the MnPO/OVLT functioning as the main entry point for neurally transmitted afferent information . Systemic osmolality information ( from the CVOs [the SFO and OVLT] ) and water-related presystemic information ( from yet-undefined regions outside the LT ) converge on the MnPO/OVLT and are redistributed to AVP neurons and other downstream targets directly , or indirectly via the SFO . Based on our rabies mapping of afferents to SON-projecting MnPO/OVLTVglut2 neurons , the following sites could in principle relay water-related presystemic information to the MnPO/OVLT: the VMH , DMH , PVH , and LPBN . The DMH is of particular interest as it is already known to play a role in presystemic regulation of AgRP neurons ( Garfield et al . , 2016 ) . By analogy , it may also contain neurons involved in water-related presystemic regulation of MnPO/OVLT neurons . The LPBN is also of interest in that it is known to relay interoceptive information to forebrain sites ( Herbert et al . , 1990 ) , and it has been strongly implicated in regulation of water balance ( Davern , 2014; Gizowski and Bourque , 2018 ) . Indeed , recent studies identified two groups of thirst-suppressing neurons in the PBN that send direct projections to the MnPO/OVLT and exhibit post-ingestive activation in response to drinking ( Ryan et al . , 2017; Kim et al . , 2020 ) . We found that food-related presystemic regulation is not mediated by the LT . LT neurons do not show food-related presystemic changes in their activity and silencing of LT neurons does not affect food-related presystemic changes in activity of AVP neurons . Inhibition of neurons in the ARC , on the other hand , causes a significant attenuation of food-related presystemic responses suggesting that neurons in this region mediate food-related regulation . Many anatomical and electrophysiological studies have demonstrated that AVP neurons receive direct excitatory and inhibitory projections from the ARC ( Iijima and Ogawa , 1981; Saphier and Feldman , 1986; Leng et al . , 1988; Ludwig and Leng , 2000; Pineda et al . , 2016 ) . This is further supported by our rabies mapping results . Due to technical limitations , however , the ARC neurons responsible for food-related presystemic regulation were not identified . The most widely studied neurons in the ARC , AgRP and POMC neurons , were ruled out , as inhibition of these neurons had no effect on the food-related presystemic response . Considering rapid feeding-induced activation of AVP neurons , it is highly likely that feeding-related presystemic regulation involves glutamatergic neurons in the ARC . Oxytocin receptor-expressing glutamatergic neurons ( Fenselau et al . , 2017 ) are a good candidate for mediating food-related presystemic regulation as these neurons are activated by feeding and are capable of inhibiting food intake rapidly when activated . Alternatively , it is possible that a completely novel ARC neuron is involved ( Campbell et al . , 2017 ) . The combination of projection- and synaptic connectivity-based gene profiling techniques will facilitate specific identification of ARC neurons mediating food-related presystemic regulation . In summary , presystemic regulation of AVP release is mediated by the LT and ARC circuits that , respectively , exclusively relay water- or food-related presystemic signals . The two circuits operate largely independently and relay different repertoire of presystemic signals ( water-related pre- and post-ingestive signals by the LT and food-related post-ingestive signals by the ARC ) , giving rise to the bidirectional and asymmetric nature of AVP neurons’ presystemic responses . Additional anatomically and functionally distinct neural circuits converge on AVP neurons to mediate other aspects of AVP function , such as occurs during hypotension . Convergence and integration of multiple inputs at the level of AVP neurons , the final common pathway for AVP release , provide a neural circuit basis for differential regulation of AVP release by diverse behavioral and physiological stimuli . Identifying and characterizing key neural components of the extended neural circuitry underlying these processes will be an important area for future investigation .
Animals were housed at 22–24°C on a 12:12 light/dark cycle ( light cycle: 6:00 a . m . to 6:00 p . m . ) with standard mouse chow ( Teklad F6 Rodent Diet 8664; Harlan Teklad ) and water provided ad libitum , unless specified otherwise . Adult male and female mice ( 8–16 weeks old ) were used for experiments . Mice were maintained on a mixed background . Transgenic mouse strains used: Avp-IRES-Cre ( Pei et al . , 2014 ) , Slc17a6-IRES-Cre ( Vglut2-IRES-Cre ) ( Vong et al . , 2011 ) ( Jackson Labs Stock 016963 ) , Slc32a1-IRES-Cre ( Vgat-IRES-Cre ) ( Vong et al . , 2011 ) ( Jackson Labs stock 016962 ) , Nos1-IRES-Cre ( Jackson Labs Stock 017526 ) , Slc17a6-IRES-Flp ( Vglut2-IRES2-FLPo-D ) ( Jackson Labs Stock 030212 ) , Slc32a1-IRES-Flp ( Vgat-IRES2-FLPo-D ) ( Jackson Labs Stock 031331 ) , Th-Cre ( Savitt et al . , 2005 ) ( Jackson Labs Stock 008601 ) , Dbh-Flp ( MMRRC 041575 ) , Avp-GFP ( MMRRC 015858-UCD ) , Agrp-IRES-Cre ( Tong et al . , 2008 ) ( Jackson Labs Stock 012899 ) , and Pomc-IRES-Cre ( Fenselau et al . , 2017 ) . The following viral vectors were used in this study: AAV8-FLEX-TVA-mCherry ( UNC Vector Core , Addgene 38044 ) , AAV1-CAG-FLEX-RG ( UNC Vector Core , Addgene 48333 ) , SADΔG–EGFP ( EnvA ) rabies ( Salk Gene Transfer Targeting and Therapeutics Core , Addgene 32635 ) , AAV9-EF1α-DIO-ChR2 ( H134R ) -mCherry ( Penn Vector Core , Addgene 20297 ) , AAV8-EF1a-DIO-synaptophysin-YFP ( MIT Vector Core ) , AAV9-CAG-ChR2 ( H134R ) -mCherry ( Penn Vector Core , Addgene 100054 ) , HSV-hEF1a-LS1L-GCaMP6s ( MGH Gene Delivery Technology Core , RN507 ) , AAV1-Syn-FLEX-GCaMP6s ( Penn Vector Core , Addgene 100845 ) , AAV1-Ef1a-DIO-EYFP ( UNC Vector Core , Addgene 27056 ) , AAV8-hSyn-DIO-hM4Di-mCherry ( UNC Vector Core , Addgene 44362 ) , and AAV8-nEF-fDIO-hM4Di-mCherry ( Boston Children’s Hospital Viral Core , Custom-made vector [hM4Di-mCherry was cut from AAV8-hSyn-DIO-hM4Di-mCherry {Addgene 44362} by AscI and NheI , and subcloned into pAAV-nEF Con/Fon hChR2 ( H134R ) -EYFP {Addgene 55644} into AscI and XbaI] ) . For viral injections , mice were anaesthetized with ketamine/xylazine ( 100 and 10 mg/kg , respectively , i . p . ) and then placed in a stereotaxic apparatus ( David Kopf model 940 ) . A pulled glass micropipette ( 20–40 μm diameter tip ) was used for stereotaxic injections of adeno-associated virus ( AAV ) . Virus was injected into the posterior pituitary ( 100 nl; AP: −3 . 0 mm; ML: ± 0 mm; DV: –6 . 0 mm from bregma ) , SON/PNZ ( 100 nl/side; AP: −0 . 65 mm; ML: ± 1 . 25 mm; DV: –5 . 4 mm from bregma ) , MnPO/OVLT ( 10 nl/depth; AP: +0 . 25 mm; ML: 0 mm; DV: –4 . 8 , 4 . 5 mm from bregma ) , SFO ( 10 nl; AP: –0 . 6 mm; ML: 0 mm; DV: –2 . 6 mm from bregma ) , PVH ( 50 nl/side; AP: –0 . 75 mm; ML: ± 0 . 3 mm; DV: –4 . 85 mm from bregma ) , DMH ( 50 nl/side; AP: –1 . 85 mm; ML: ± 0 . 3 mm; DV: –5 . 2 mm from bregma ) , VMH ( 50 nl/side; AP: –1 . 6 mm; ML: ± 0 . 4 mm; DV: –5 . 5 mm from bregma ) , or Arcuate ( 50 nl/side; AP: –1 . 5 mm; ML: ± 0 . 25 mm; DV: –5 . 8 mm from bregma ) by an air pressure system using picoliter air puffs through a solenoid valve ( Clippard EV 24VDC ) pulsed by a Grass S48 stimulator to control injection speed ( 40 nl/min ) . The pipette was removed 3 min post-injection followed by wound closure using tissue adhesive ( 3 M Vetbond ) . For viral injections into the NTS and VLM , mice were placed into a stereotaxic apparatus with the head angled down at approximately 45° . An incision was made at the level of the cisterna magna , then skin and muscle were retracted to expose the dura mater covering the fourth ventricle . A 28-gauge needle was used to make an incision in the dura and allow access to the NTS and VLM . Virus was then injected into the NTS ( 10 nl/side; AP: –0 . 2 mm; ML: ± 0 . 2 mm; DV: –0 . 2 mm from obex ) VLM ( 50 nl*2/side; AP: –0 . 3 and –0 . 6 mm; ML: ± 1 . 3 mm; DV: –1 . 7 mm from obex ) as described above . The pipette was removed 3 min post-injection followed by wound closure using absorbable suture for muscle and silk suture for skin . For fiber photometry , an optic fiber ( 200 µm diameter , NA=0 . 39 , metal ferrule , Thorlabs ) was implanted in the MnPO/OVLT , SFO , or SON and secured to the skull with dental cement . Subcutaneous injection of sustained release Meloxicam ( 4 mg/kg ) was provided as postoperative care . The mouse was kept in a warm environment and closely monitored until resuming normal activity . To prepare brain slices for electrophysiological recordings , brains were removed from anesthetized mice and immediately placed in ice-cold cutting solution consisting of ( in mM ) : 72 sucrose , 83 NaCl , 2 . 5 KCl , 1 NaH2PO4 , 26 NaHCO3 , 22 glucose , 5 MgCl2 , 1 CaCl2 , oxygenated with 95% O2/5% CO2 , measured osmolarity 310–320 mOsm/l . Cutting solution was prepared and used within 72 hr . About 250-μm-thick coronal sections containing the PVH and SON were cut with a vibratome ( 7000smz-2 Campden Instruments ) and incubated in an oxygenated cutting solution at 34°C for 25 min . The SON was located by using the bifurcation of the anterior and middle cerebral arteries on the ventral surface of the brain as a landmark . Slices were transferred to oxygenated aCSF ( 126 mM NaCl , 21 . 4 mM NaHCO3 , 2 . 5 mM KCl , 1 . 2 mM NaH2PO4 , 1 . 2 mM MgCl2 , 2 . 4 mM CaCl2 , and 10 mM glucose ) and stored in the same solution at room temperature ( 20–24°C ) for at least 60 min prior to recording . A single slice was placed in the recording chamber where it was continuously super-fused at a rate of 3–4 ml per min with oxygenated aCSF . Neurons were visualized with an upright microscope equipped with infrared-differential interference contrast and fluorescence optics . Borosilicate glass microelectrodes ( 5–7 MΩ ) were filled with internal solution . Whole-cell voltage clamp recordings were obtained using Cs-based internal solutions containing either ( in mM ) : 135 CsMeSO3 , 10 HEPES , 1 EGTA , 4 MgCl2 , 4 Na2-ATP , 0 . 4 Na2-GTP , and 10 Na2-phosphocreatine ( pH 7 . 3; 295 mOsm ) for recording EPSCs; or for recording IPSCs from Vgat neurons expressing ChR2: 140 CsCl , 1 BAPTA , 10 HEPES , 5 MgCl2 , 5 Mg-ATP , 0 . 3 Na2GTP , and 10 lidocaine N-ethyl bromide ( [QX-314] , pH 7 . 35 , and 290 mOsm ) . To photostimulate ChR2-positive fibers , an LED light source ( 473 nm ) was used . The blue light was focused on to the back aperture of the microscope objective , producing a wide-field exposure around the recorded cell of 1 mW . The light power at the specimen was measured using an optical power meter PM100D ( Thorlabs ) . The light output is controlled by a programmable pulse stimulator , Master-8 ( AMPI Co . , Israel ) , and the pClamp 10 . 2 software ( AXON Instruments ) . All recordings were made using Multiclamp 700B amplifier , and data was filtered at 2 kHz and digitized at 10 kHz . The photostimulation-evoked EPSC detection protocol consisted of four blue light laser pulses administered 1 s apart during the first 4 s of an 8 s sweep , repeated for a total of 30 sweeps . We attempted to maximize our ability to detect light-evoked currents by biasing our recordings to cell bodies within the densest axon fields . In some experiments , TTX ( 1 mM ) and 4-AP ( 100 mM ) were added to the bath solution to confirm monosynaptic connectivity . All CRACM results presented are from 2 to 3 mice per group . All analysis was conducted off-line in Clampfit 10 and Origin . All experiments were conducted in the home-cage in freely moving mice . Beginning 3 weeks post-surgery ( details above ) , animals prepared for in vivo fiber photometry experiments ( outlined above ) , were food restricted to 85–90% of initial body weight . Over this 1-week period , mice were acclimated to chow pellets ( 500 mg , Bio-Serv Dustless Precision Pellets ) , and to the food bowl and water bowl used in subsequent photometry experiments . Mice were habituated to the paradigm for at least 5 days prior to the first recording day . For experiments with hM4Di , saline or CNO ( 1 mg/kg ) was injected 20 min prior to the experiment . In vivo fiber photometry was conducted as previously described ( Mandelblat-Cerf et al . , 2017 ) . A fiber optic cable ( 1 m long , metal ferrule , 400 µm diameter; Doric Lenses ) was attached to the implanted optic cannula with zirconia sleeves ( Doric Lenses ) . Laser light ( 473 nm ) was focused on the opposite end of the fiber optic cable to titrate the light intensity entering the brain to 0 . 1–0 . 2 mW . Emitted light was passed through a dichroic mirror ( Di02-R488-25×36 , Semrock ) and GFP emission filter ( FF03-525/50-25 , Semrock ) , before being focused onto a sensitive photodetector ( Newport part #2151 ) . The GCaMP6 signal was passed through a low-pass filter ( 50 Hz ) , and digitized at 1 kHz using a National Instruments data acquisition card and MATLAB software . The recorded data was exported and then imported into MATLAB for analysis . Fluorescent traces were down-sampled to 1 Hz . We calculated the fractional change in GCaMP6s fluorescence according to the following equation: ΔF/F= ( F–F0 ) /F0 , where F is fluorescence measurement and F0 is the mean fluorescence in the 30 s prior to food/water bowl presentation or feeding onset or 2 or 3 min prior to drug injection . In some experiments , to facilitate the comparison across different groups , we normalized the responses of each mouse . In Figure 4f , normalization was performed by ( ΔF/F ) /FtotalΔF , where FtotalΔF was maximum average change over the entire recording period . In Figures 5 and 7 , normalization was performed by ( ΔF/F ) /c , where c was maximum average response of control trials within 5 min of water bowl placement or feeding onset . Drinking was assessed by modified Drinking Event Monitor cages ( Lickometer cages; Columbus Instruments ) . Mice were placed into these “lickometer” cages where licks on each bottle sipper tube were detected by electrical conductivity and recorded by a computer counter interface . CNO or Saline was injected 15 min prior to the onset of the experiment . Animals were terminally anesthetized with 7% chloral hydrate diluted in saline ( 350 mg/kg ) and transcardially perfused with phosphate-buffered saline ( PBS ) followed by 10% neutral buffered formalin ( PFA ) . Brains were removed , stored in the same fixative overnight , transferred into 20% sucrose at 4°C overnight , and cut into 40 μm sections on a freezing microtome ( Leica ) coronally into two equal series . Brain sections were washed in PBS with Tween-20 , pH 7 . 4 ( PBST ) , and blocked in 3% normal donkey serum in PBST for 1 h at room temperature . Brain sections were then incubated overnight at room temperature in blocking solution containing primary antiserum ( rat anti-mCherry , Life Technologies M11217 , 1:1000; rabbit anti-dsRed , Clontech 632496 , 1:1000; chicken anti-GFP , Life Technologies A10262 , 1:1000; rabbit anti-vasopressin , Sigma-Aldrich AB1565 , 1:1000; rabbit anti-TH , Millipore AB152 , 1:1000; rabbit anti-POMC precursor , Phoenix Pharmaceuticals H-029–30 , 1:1000; goat anti-AgRP , Neuromics GT15023 , 1:1 , 000 ) . The next morning sections were extensively washed in PBS and then incubated in Alexa-fluorophore secondary antibody ( 1:1000 ) for 1 hr at room temperature . After several washes in PBS , sections were mounted on gelatin-coated slides and fluorescence images were captured with Olympus VS120 slide scanner microscope . Statistical analyses were performed using SigmaPlot software . Electrophysiological traces were analyzed on Clamp fit 10 ( Molecular Devices ) and Origin ( Origin Lab ) software . No statistical method was used to predetermine sample size . Blinding methods were not used . All data presented met the assumptions of the statistical test employed . Specific statistical tests are specified in the figure legends . Animals were excluded from analysis if histological validation revealed poor or inaccurate reporter expression or inaccurate fiber placement unless otherwise noted . n values reflect the final number of validated animals per group included in the analysis .
Water balance , tracked by extracellular osmolality , is regulated by feedback and feedforward mechanisms . Feedback regulation is reactive , occurring as deviations in osmolality are detected . Feedforward or presystemic regulation is proactive , occurring when disturbances in osmolality are anticipated . Vasopressin ( AVP ) is a key hormone regulating water balance and is released during hyperosmolality to limit renal water excretion . AVP neurons are under feedback and feedforward regulation . Not only do they respond to disturbances in blood osmolality , but they are also rapidly suppressed and stimulated , respectively , by drinking and eating , which will ultimately decrease and increase osmolality . Here , we demonstrate that AVP neuron activity is regulated by multiple anatomically and functionally distinct neural circuits . Notably , presystemic regulation during drinking and eating are mediated by non-overlapping circuits that involve the lamina terminalis and hypothalamic arcuate nucleus , respectively . These findings reveal neural mechanisms that support differential regulation of AVP release by diverse behavioral and physiological stimuli .
Fine-tuning the amount of water present in the body at any given time is a tight balancing act . The hormone vasopressin helps to ensure that organisms do not get too dehydrated by allowing water in the urine to be reabsorbed into the bloodstream . A group of vasopressin neurons in the brain trigger the release of the hormone if water levels get too low ( as reflected by an increase in osmolality , the level of substances dissolved in a unit of blood ) . However , these cells also receive additional information that allows them to predict and respond to upcoming changes in water levels . For example , drinking water while dehydrated ‘switches off’ the neurons , even before osmolality is restored in the blood to normal levels . Eating , on the other hand , rapidly activates vasopressin neurons before the food is digested and blood osmolality increases as a result . How vasopressin neurons receive this ‘anticipatory’ information remains unclear . Kim et al . explored this question in mice by inhibiting different sets of brain cells one by one , and then examining whether the neurons could still exhibit anticipatory responses . This revealed a remarkable division of labor in the neural circuits that regulate vasopressin neurons: two completely different sets of neurons from distinct areas of the brain are dedicated to relaying anticipatory information about either water or food intake . These findings help to understand how healthy levels of water can be maintained in the body . Overall , they give a glimpse into the neural mechanisms that underlie anticipatory forms of regulation , which can also take place when hunger or thirst neurons ‘foresee’ that food or water will be consumed .