Glutamatergic supramammillary nucleus neurons respond to threatening stressors and promote active coping.

Threat-response neural circuits are conserved across species and play roles in normal behavior and psychiatric diseases. Maladaptive changes in these neural circuits contribute to stress, mood, and anxiety disorders. Active coping in response to stressors is a psychosocial factor associated with resilience against stress-induced mood and anxiety disorders. The neural circuitry underlying active coping is poorly understood, but the functioning of these circuits could be key for overcoming anxiety and related disorders. The supramammillary nucleus (SuM) has been suggested to be engaged by threat. SuM has many projections and a poorly understood diversity of neural populations. In studies using mice, we identified a unique population of glutamatergic SuM neurons (SuMVGLUT2+::POA) based on projection to the preoptic area of the hypothalamus (POA) and found SuMVGLUT2+::POA neurons have extensive arborizations. SuMVGLUT2+::POA neurons project to brain areas that mediate features of the stress and threat responses including the paraventricular nucleus thalamus (PVT), periaqueductal gray (PAG), and habenula (Hb). Thus, SuMVGLUT2+::POA neurons are positioned as a hub, connecting to areas implicated in regulating stress responses. Here we report SuMVGLUT2+::POA neurons are recruited by diverse threatening stressors, and recruitment correlated with active coping behaviors. We found that selective photoactivation of the SuMVGLUT2+::POA population drove aversion but not anxiety like behaviors. Activation of SuMVGLUT2+::POA neurons in the absence of acute stressors evoked active coping like behaviors and drove instrumental behavior. Also, activation of SuMVGLUT2+::POA neurons was sufficient to convert passive coping strategies to active behaviors during acute stress. In contrast, we found activation of GABAergic (VGAT+) SuM neurons (SuMVGAT+) neurons did not alter drive aversion or active coping, but termination of photostimulation was followed by increased mobility in the forced swim test. These findings establish a new node in stress response circuitry that has projections to many brain areas and evokes flexible active coping behaviors.

Multi-centre analysis of networks and genes modulated by hypothalamic stimulation in patients with aggressive behaviours.

Deep brain stimulation targeting the posterior hypothalamus (pHyp-DBS) is being investigated as a treatment for refractory aggressive behavior, but its mechanisms of action remain elusive. We conducted an integrated imaging analysis of a large multi-centre dataset, incorporating volume of activated tissue modeling, probabilistic mapping, normative connectomics, and atlas-derived transcriptomics. Ninety-one percent of the patients responded positively to treatment, with a more striking improvement recorded in the pediatric population. Probabilistic mapping revealed an optimized surgical target within the posterior-inferior-lateral region of the posterior hypothalamic area. Normative connectomic analyses identified fiber tracts and functionally connected with brain areas associated with sensorimotor function, emotional regulation, and monoamine production. Functional connectivity between the target, periaqueductal gray and key limbic areas - together with patient age - were highly predictive of treatment outcome. Transcriptomic analysis showed that genes involved in mechanisms of aggressive behavior, neuronal communication, plasticity and neuroinflammation might underlie this functional network.

Posterior hypothalamic theta rhythm: Electrophysiological basis and involvement of glutamatergic receptors.

The posterior hypothalamic area (PHa), including the supramammillary nucleus (SuM) and posterior hypothalamic nuclei, forms a crucial part of the ascending brainstem hippocampal synchronizing pathway, that is involved in the frequency programming and modulation of rhythmic theta activity generated in limbic structures. Recent investigations show that in addition to being a modulator of limbic theta activity, the PHa is capable of producing well-synchronized local theta field potentials by itself. The purpose of this study was to examine the ability of the PHa to generate theta field potentials and accompanying cell discharges in response to glutamatergic stimulation under both in vitro and in vivo conditions. The second objective was to examine the electrophysiological properties of neurons located in the SuM and posterior hypothalamic nuclei. Extracellular in vivo and in vitro as well as intracellular in vitro experiments revealed that glutamatergic stimulation of PHa with kainic acid induces well-synchronized local theta field oscillations in both the supramammillary and posterior hypothalamic nuclei. Furthermore, the glutamatergic PHa theta rhythm recorded extracellularly was accompanied by the activity of specific subtypes of theta-related neurons. We identify, for the first time, a subpopulation of supramammillary and posterior hypothalamic neurons that express clear subthreshold membrane potential oscillations in the theta frequency range.

Postnatal development of hippocampal CA2 structure and function during the emergence of social recognition of peers.

It is now well-established that the hippocampal CA2 region plays an important role in social recognition memory in adult mice. The CA2 is also important for the earliest social memories, including those that mice have for their mothers and littermates, which manifest themselves as a social preference for familiarity over novelty. The role of the CA2 in the development of social memory for recently encountered same-age conspecifics, that is, peers, has not been previously reported. Here, we used a direct social interaction test to characterize the emergence of novelty preference for peers during development and found that at the end of the second postnatal week, pups begin to significantly prefer novel over familiar peers. Using chemogenetic inhibition at this time, we showed that CA2 activity is necessary for the emergence of novelty preference and for the ability to distinguish never encountered from recently encountered peers. In adulthood, the CA2 region is known to integrate a large number of inputs from various sources, many of which participate in social recognition memory, but previous studies have not determined whether these afferents are present at adult levels by the end of the second postnatal week. To explore the development of CA2 inputs, we used immunolabeling and retrograde adenovirus circuit tracing and found that, by the end of the second postnatal week, the CA2 is innervated by many regions, including the dentate gyrus, supramammillary nucleus of the hypothalamus, the lateral entorhinal cortex, and the median raphe nucleus. Using retroviral labeling of postnatally generated granule cells in the dentate gyrus, we found that mossy fiber projections to the CA2 mature faster during development than those generated in adulthood. Together, our findings indicate that the CA2 is partially mature in afferent connectivity by the end of the second postnatal week, connections that likely facilitate the emergence of social recognition memory and preference for novel peers.

Supramammillary regulation of locomotion and hippocampal activity.

Locomotor speed is a basic input used to calculate one's position, but where this signal comes from is unclear. We identified neurons in the supramammillary nucleus (SuM) of the rodent hypothalamus that were highly correlated with future locomotor speed and reliably drove locomotion when activated. Robust locomotion control was specifically identified in Tac1 (substance P)­expressing (SuMTac1+) neurons, the activation of which selectively controlled the activity of speed-modulated hippocampal neurons. By contrast, Tac1-deficient (SuMTac1−) cells weakly regulated locomotion but potently controlled the spike timing of hippocampal neurons and were sufficient to entrain local network oscillations. These findings emphasize that the SuM not only regulates basic locomotor activity but also selectively shapes hippocampal neural activity in a manner that may support spatial navigation.

Dorsal premammillary projection to periaqueductal gray controls escape vigor from innate and conditioned threats.

Escape from threats has paramount importance for survival. However, it is unknown if a single circuit controls escape vigor from innate and conditioned threats. Cholecystokinin (cck)-expressing cells in the hypothalamic dorsal premammillary nucleus (PMd) are necessary for initiating escape from innate threats via a projection to the dorsolateral periaqueductal gray (dlPAG). We now show that in mice PMd-cck cells are activated during escape, but not other defensive behaviors. PMd-cck ensemble activity can also predict future escape. Furthermore, PMd inhibition decreases escape speed from both innate and conditioned threats. Inhibition of the PMd-cck projection to the dlPAG also decreased escape speed. Intriguingly, PMd-cck and dlPAG activity in mice showed higher mutual information during exposure to innate and conditioned threats. In parallel, human functional magnetic resonance imaging data show that a posterior hypothalamic-to-dlPAG pathway increased activity during exposure to aversive images, indicating that a similar pathway may possibly have a related role in humans. Our data identify the PMd-dlPAG circuit as a central node, controlling escape vigor elicited by both innate and conditioned threats.

Supramammillary neurons projecting to the septum regulate dopamine and motivation for environmental interaction in mice.

The supramammillary region (SuM) is a posterior hypothalamic structure, known to regulate hippocampal theta oscillations and arousal. However, recent studies reported that the stimulation of SuM neurons with neuroactive chemicals, including substances of abuse, is reinforcing. We conducted experiments to elucidate how SuM neurons mediate such effects. Using optogenetics, we found that the excitation of SuM glutamatergic (GLU) neurons was reinforcing in mice; this effect was relayed by their projections to septal GLU neurons. SuM neurons were active during exploration and approach behavior and diminished activity during sucrose consumption. Consistently, inhibition of SuM neurons disrupted approach responses, but not sucrose consumption. Such functions are similar to those of mesolimbic dopamine neurons. Indeed, the stimulation of SuM-to-septum GLU neurons and septum-to-ventral tegmental area (VTA) GLU neurons activated mesolimbic dopamine neurons. We propose that the supramammillo-septo-VTA pathway regulates arousal that reinforces and energizes behavioral interaction with the environment.

Coordination of escape and spatial navigation circuits orchestrates versatile flight from threats.

Naturalistic escape requires versatile context-specific flight with rapid evaluation of local geometry to identify and use efficient escape routes. It is unknown how spatial navigation and escape circuits are recruited to produce context-specific flight. Using mice, we show that activity in cholecystokinin-expressing hypothalamic dorsal premammillary nucleus (PMd-cck) cells is sufficient and necessary for context-specific escape that adapts to each environment's layout. In contrast, numerous other nuclei implicated in flight only induced stereotyped panic-related escape. We reasoned the dorsal premammillary nucleus (PMd) can induce context-specific escape because it projects to escape and spatial navigation nuclei. Indeed, activity in PMd-cck projections to thalamic spatial navigation circuits is necessary for context-specific escape induced by moderate threats but not panic-related stereotyped escape caused by perceived asphyxiation. Conversely, the PMd projection to the escape-inducing dorsal periaqueductal gray projection is necessary for all tested escapes. Thus, PMd-cck cells control versatile flight, engaging spatial navigation and escape circuits.

A hypothalamic novelty signal modulates hippocampal memory.

The ability to recognize information that is incongruous with previous experience is critical for survival. Novelty signals have therefore evolved in the mammalian brain to enhance attention, perception and memory1,2. Although the importance of regions such as the ventral tegmental area3,4 and locus coeruleus5 in broadly signalling novelty is well-established, these diffuse monoaminergic transmitters have yet to be shown to convey specific information on the type of stimuli that drive them. Whether distinct types of novelty, such as contextual and social novelty, are differently processed and routed in the brain is unknown. Here we identify the supramammillary nucleus (SuM) as a novelty hub in the hypothalamus6. The SuM region is unique in that it not only responds broadly to novel stimuli, but also segregates and selectively routes different types of information to discrete cortical targets-the dentate gyrus and CA2 fields of the hippocampus-for the modulation of mnemonic processing. Using a new transgenic mouse line, SuM-Cre, we found that SuM neurons that project to the dentate gyrus are activated by contextual novelty, whereas the SuM-CA2 circuit is preferentially activated by novel social encounters. Circuit-based manipulation showed that divergent novelty channelling in these projections modifies hippocampal contextual or social memory. This content-specific routing of novelty signals represents a previously unknown mechanism that enables the hypothalamus to flexibly modulate select components of cognition.

Cellular taxonomy and spatial organization of the murine ventral posterior hypothalamus.

The ventral posterior hypothalamus (VPH) is an anatomically complex brain region implicated in arousal, reproduction, energy balance, and memory processing. However, neuronal cell type diversity within the VPH is poorly understood, an impediment to deconstructing the roles of distinct VPH circuits in physiology and behavior. To address this question, we employed a droplet-based single-cell RNA sequencing (scRNA-seq) approach to systematically classify molecularly distinct cell populations in the mouse VPH. Analysis of >16,000 single cells revealed 20 neuronal and 18 non-neuronal cell populations, defined by suites of discriminatory markers. We validated differentially expressed genes in selected neuronal populations through fluorescence in situ hybridization (FISH). Focusing on the mammillary bodies (MB), we discovered transcriptionally-distinct clusters that exhibit neuroanatomical parcellation within MB subdivisions and topographic projections to the thalamus. This single-cell transcriptomic atlas of VPH cell types provides a resource for interrogating the circuit-level mechanisms underlying the diverse functions of VPH circuits.

Specific Hypothalamic Neurons Required for Sensing Conspecific Male Cues Relevant to Inter-male Aggression.

The hypothalamus regulates innate social interactions, but how hypothalamic neurons transduce sex-related sensory signals emitted by conspecifics to trigger appropriate behaviors remains unclear. Here, we addressed this issue by identifying specific hypothalamic neurons required for sensing conspecific male cues relevant to inter-male aggression. By in vivo recording of neuronal activities in behaving mice, we showed that neurons expressing dopamine transporter (DAT+) in the ventral premammillary nucleus (PMv) of the hypothalamus responded to male urine cues in a vomeronasal organ (VNO)-dependent manner in naive males. Retrograde trans-synaptic tracing further revealed a specific group of neurons in the bed nucleus of the stria terminalis (BNST) that convey male-relevant signals from VNO to PMv. Inhibition of PMvDAT+ neurons abolished the preference for male urine cues and reduced inter-male attacks, while activation of these neurons promoted urine marking and aggression. Thus, PMvDAT+ neurons exemplify a hypothalamic node that transforms sex-related chemo-signals into recognition and behaviors.

GABA-glutamate supramammillary neurons control theta and gamma oscillations in the dentate gyrus during paradoxical (REM) sleep.

Several studies suggest that neurons from the lateral region of the SuM (SuML) innervating the dorsal dentate gyrus (DG) display a dual GABAergic and glutamatergic transmission and are specifically activated during paradoxical (REM) sleep (PS). The objective of the present study is to characterize the anatomical, neurochemical and electrophysiological properties of the SuML-DG projection neurons and to determine how they control DG oscillations and neuronal activation during PS and other vigilance states. For this purpose, we combine structural connectivity techniques using neurotropic viral vectors (rabies virus, AAV), neurochemical anatomy (immunohistochemistry, in situ hybridization) and imaging (light, electron and confocal microscopy) with in vitro (patch clamp) and in vivo (LFP, EEG) optogenetic and electrophysiological recordings performed in transgenic VGLUT2-cre male mice. At the cellular level, we show that the SuML-DG neurons co-release GABA and glutamate on dentate granule cells and increase the activity of a subset of DG granule cells. At the network level, we show that activation of the SuML-DG pathway increases theta power and frequency during PS as well as gamma power during PS and waking in the DG. At the behavioral level, we show that the activation of this pathway does not change animal behavior during PS, induces awakening during slow wave sleep and increases motor activity during waking. These results suggest that the SuML-DG pathway is capable of supporting the increase of theta and gamma power in the DG observed during PS and plays an important modulatory role of DG network activity during this state.

The supramammillary nucleus controls anxiety-like behavior; key role of GLP-1R.

Anxiety disorders are among the most prevalent categories of mental illnesses. The gut-brain axis, along with gastrointestinally-derived neuropeptides, like glucagon-like peptide-1 (GLP-1), are emerging as potential key regulators of emotionality, including anxiety behavior. However, the neuroanatomical substrates from which GLP-1 exerts its anxiogenic effect remain poorly characterized. Here we focus on a relatively new candidate nucleus, the supramammillary nucleus (SuM), located just caudal to the lateral hypothalamus and ventral to the ventral tegmental area. Our focus on the SuM is supported by previous data showing expression of GLP-1R mRNA throughout the SuM and activation of the SuM during anxiety-inducing behaviors in rodents. Data show that chemogenetic activation of neurons in the SuM results in an anxiolytic response in male and female rats. In contrast, selective activation of SuM GLP-1R, by microinjection of a GLP-1R agonist exendin-4 into the SuM resulted in potent anxiety-like behavior, measured in both open field and elevated plus maze tests in male and female rats. This anxiogenic effect of GLP-1R activation persisted after high-fat diet exposure. Importantly, reduction of GLP-1R expression in the SuM, by AAV-shRNA GLP-1R knockdown, resulted in a clear anxiolytic response; an effect only observed in female rats. Our data identify a new neural substrate for GLP-1 control of anxiety-like behavior and indicate that the SuM GLP-1R are sufficient for anxiogenesis in both sexes, but necessary only in females.

Supramammillary nucleus synchronizes with dentate gyrus to regulate spatial memory retrieval through glutamate release.

The supramammillary nucleus (SuM) provides substantial innervation to the dentate gyrus (DG). It remains unknown how the SuM and DG coordinate their activities at the circuit level to regulate spatial memory. Additionally, SuM co-releases GABA and glutamate to the DG, but the relative role of GABA versus glutamate in regulating spatial memory remains unknown. Here we report that SuM-DG Ca2+ activities are highly correlated during spatial memory retrieval as compared to the moderate correlation during memory encoding when mice are performing a location discrimination task. Supporting this evidence, we demonstrate that the activity of SuM neurons or SuM-DG projections is required for spatial memory retrieval. Furthermore, we show that SuM glutamate transmission is necessary for both spatial memory retrieval and highly-correlated SuM-DG activities during spatial memory retrieval. Our studies identify a long-range SuM-DG circuit linking two highly correlated subcortical regions to regulate spatial memory retrieval through SuM glutamate release.

Neural connectivity between the hypothalamic supramammillary nucleus and appetite- and motivation-related regions of the rat brain.

The supramammillary nucleus (SuM) has an emerging role in appetite control. We have shown that the rat SuM is activated during hunger or food anticipation, or by ghrelin administration. In the present study, we characterised the connectivity between the SuM and key appetite- and motivation-related nuclei in the rat. In adult wild-type rats, or rats expressing Cre recombinase under the control of the tyrosine hydroxylase (TH) promoter (TH-Cre rats), we used c-Fos immunohistochemistry to visualise and correlate the activation of medial SuM (SuMM) with activation in the lateral hypothalamic area (LH), the dorsomedial hypothalamus (DMH) or the ventral tegmental area (VTA) after voluntary consumption of a high-sugar, high-fat food. To determine neuroanatomical connectivity, we used retrograde and anterograde tracing methods to specifically investigate the neuronal inputs and outputs of the SuMM. After consumption of the food there were positive correlations between c-Fos expression in the SuMM and the LH, DMH and VTA (P = 0.0001, 0.01 and 0.004). Using Fluoro-Ruby as a retrograde tracer, we demonstrate the existence of inputs from the LH, DMH, VTA and ventromedial hypothalamus (VMH) to the SuMM. The SuMM showed reciprocal inputs to the LH and DMH, and we identified a TH-positive output from SuMM to DMH. We co-labelled retrogradely-labelled sections for TH in the VMH, or for TH, orexin and melanin-concentrating hormone in the LH and DMH. However, we did not observe any colocalisation of immunoreactivity with any retrogradely-labelled cells. Viral mapping in TH-Cre rats confirms the existence of a reciprocal SuMM-DMH connection and shows that TH-positive cells project from the SuMM and VTA to the lateral septal area and cingulate cortex, respectively. These data provide evidence for the connectivity of the SuMM to brain regions involved in appetite control, and form the foundation for functional and behavioural studies aiming to further characterise the brain circuitry controlling eating behaviours.

The claustrum is a target for projections from the supramammillary nucleus in the rat.

Injection of the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHAL) into the rat rostral and caudal supramammillary nucleus (SUM) provided expected patterns of projections into the hippocampus and the septal region. In addition, unexpectedly intense projections were observed into the claustrum defined by parvalbumin expression. Injections of the retrograde tracer fluorogold (FG) into the hippocampus and the region of the claustrum showed that the cells of origin of these projections distributed similarly within the borders of the SUM. The SUM is usually involved in control of hippocampal theta activity, but the observation of intense projections into the claustrum indicates that it may also influence isocortical processes. Therefore, the SUM may coordinate sensory processing in the isocortex with memory formation in the hippocampus.

Deep brain stimulation of chronic cluster headaches: Posterior hypothalamus, ventral tegmentum and beyond.

OBJECTIVE: We present long-term follow-up results and analysis of stimulation sites of a prospective cohort study of six patients with chronic cluster headaches undergoing deep brain stimulation of the ipsilateral posterior hypothalamic region. METHODS: The primary endpoint was the postoperative change in the composite headache severity score "headache load" after 12 months of chronic stimulation. Secondary endpoints were the changes in headache attack frequency, headache attack duration and headache intensity, quality of life measures at 12, 24, and 48 months following surgery. Stimulating contact positions were analysed and projected onto the steroetactic atlas of Schaltenbrand and Wahren. RESULTS: There was a significant reduction of headache load of over 93% on average at 12 months postoperatively that persisted over the follow-up period of 48 months (p = 0.0041) and that was accompanied by a significant increase of reported quality of life measures (p = 0.03). Anatomical analysis revealed that individual stimulating electrodes were located in the red nucleus, posterior hypothalamic region, mesencephalic pretectal area and centromedian nucleus of the thalamus. CONCLUSIONS: Our findings confirming long-term effectiveness of deep brain stimulation for chronic cluster headaches suggest that the neuroanatomical substrate of deep brain stimulation-induced headache relief is probably not restricted to the posterior hypothalamic area but encompasses a more widespread area.

Activation of the rat hypothalamic supramammillary nucleus by food anticipation, food restriction or ghrelin administration.

The circulating orexigenic hormone ghrelin targets many brain areas involved in feeding control and signals via a dedicated receptor, the growth hormone secretagogue receptor 1A. One unexplored target area for ghrelin is the supramammillary nucleus (SuM), a hypothalamic area involved in motivation and reinforcement and also recently linked to metabolic control. Given that ghrelin binds to the SuM, we explored whether SuM cells respond to ghrelin and/or are activated when endogenous ghrelin levels are elevated. We found that peripheral ghrelin injection activates SuM cells in rats, reflected by an increase in the number of cells expressing c-Fos protein in this area, as welll as by the predominantly excitatory response of single SuM cells recorded in in vivo electrophysiological studies. Further c-Fos mapping studies reveal that this area is also activated in rats in situations when circulating ghrelin levels are known to be elevated: in food-restricted rats anticipating the consumption of food and in fed rats anticipating the consumption of an energy-dense food. We also show that intra-SuM injection of ghrelin induces a feeding response in rats suggesting that, if peripheral ghrelin is able to access the SuM, it may have direct effects on this brain region. Collectively, our data demonstrate that the SuM is activated when peripheral ghrelin levels are high, further supporting the emerging role for this brain area in metabolic and feeding control.

Supramammillary Nucleus Afferents to the Dentate Gyrus Co-release Glutamate and GABA and Potentiate Granule Cell Output.

The supramammillary nucleus (SuM) of the hypothalamus projects to the dentate gyrus (DG) and the CA2 region of the hippocampus. Although the SuM-to-hippocampus circuits have been implicated in spatial and emotional memory formation, little is known about precise neural connections between the SuM and hippocampus. Here, we report that axons of SuM neurons make monosynaptic connections to granule cells (GCs) and GABAergic interneurons, but not to hilar mossy cells, in the DG and co-release glutamate and γ-aminobutyric acid (GABA) at these synapses. Although inputs from the SuM can excite some interneurons, the inputs alone fail to generate spikes in GCs. However, despite the insufficient excitatory drive and GABAergic co-transmission, SuM inputs have net excitatory effects on GCs and can potentiate GC firing when temporally associated with perforant path inputs. Our results indicate that the SuM influences DG information processing by modulating GC outputs.