Stefanos Stagkourakis

Aggression is a sexually dimorphic behaviour. In some species, including the laboratory mouse, it is robustly expressed in males – while females are not aggressive in the non-puerperal state. However, during nursing, females exhibit maternal aggression, a dramatic yet transient shift in their social behaviour repertoire. This phenotypic change occurring in adulthood presents an opportunity to investigate whether sex-biased behavioural programs depend on mono- or di-morphic neural circuits. While maternal hormones are known to elicit nursing, their role in maternal aggression, particularly regarding target sites and cellular mechanisms, remains unclear. Here, we show that a molecularly defined subset of mouse ventral premammillary (PMv^DAT) neurons – with an established role in intermale aggression– transitions from quiescence to a hyperexcitable state during female lactation. The maternal hormones, prolactin and oxytocin, were found to excite these cells through pre- and post-synaptic electrophysiological actions. Gain- and loss-of-function experiments related to PMv^DAT neuron activity bidirectionally influence maternal aggression, while PMv^DAT neuron activation suppressed the expression of a competing social behaviour. This study identifies a sexually monomorphic neural substrate in mice capable of integrating hormonal cues, providing a likely mechanism that enables the transient access to a dormant behavioural program.

Summary: Artificial activation of anatomically localized, genetically defined hypothalamic neuron populations is known to trigger distinct innate behaviors, suggesting a hypothalamic nucleus-centered organization of behavior control. To assess whether the encoding of behavior is similarly anatomically confined, we performed simultaneous neuron recordings across twenty hypothalamic regions in freely moving animals. Here we show that distinct but anatomically distributed neuron ensembles encode the social and fear behavior classes, primarily through mixed selectivity. While behavior class-encoding ensembles were spatially distributed, individual ensembles exhibited strong localization bias. Encoding models identified that behavior actions, but not motion-related variables, explained a large fraction of hypothalamic neuron activity variance. These results identify unexpected complexity in the hypothalamic encoding of instincts and provide a foundation for understanding the role of distributed neural representations in the expression of behaviors driven by hardwired circuits.

Aggression, a sexually dimorphic behaviour, is prevalent in males and typically absent in virgin females. Following parturition, however, the transient expression of aggression in adult female mice protects pups from predators and infanticide by male conspecifics. While maternal hormones are known to elicit nursing, their potential role in maternal aggression remains elusive. Here, we show in mice that a molecularly defined subset of ventral premammillary (PMv ^DAT ) neurons, instrumental for intermale aggression, switch from quiescence to a hyperexcitable state during lactation. We identify that the maternal hormones prolactin and oxytocin excite these cells through actions that include T-type Ca ²⁺ channels. Optogenetic manipulation or genetic ablation of PMv ^DAT neurons profoundly affects maternal aggression, while activation of these neurons impairs the expression of non-aggression-related maternal behaviours. This work identifies a monomorphic neural substrate that can incorporate hormonal cues to enable the transient expression of a dormant behavioural program in lactating females.

Striatal activity is dynamically modulated by acetylcholine and dopamine, both of which are essential for basal ganglia function. Synchronized pauses in the activity of striatal cholinergic interneurons (ChINs) are correlated with elevated activity of midbrain dopaminergic neurons, whereas synchronous firing of ChINs induces local release of dopamine. The mechanisms underlying ChIN synchronization and its interplay with dopamine release are not fully understood. Here we show that polysynaptic inhibition between ChINs is a robust network motif and instrumental in shaping the network activity of ChINs. Action potentials in ChINs evoke large inhibitory responses in multiple neighboring ChINs, strong enough to suppress their tonic activity. Using a combination of optogenetics and chemogenetics we show the involvement of striatal tyrosine hydroxylase-expressing interneurons in mediating this inhibition. Inhibition between ChINs is attenuated by dopaminergic midbrain afferents acting presynaptically on D2 receptors. Our results present a novel form of interaction between striatal dopamine and acetylcholine dynamics.

All animals can perform certain survival behaviors without prior experience, suggesting a “hard wiring” of underlying neural circuits. Experience, however, can alter the expression of innate behaviors. Where in the brain and how such plasticity occurs remains largely unknown. Previous studies have established the phenomenon of “aggression training,” in which the repeated experience of winning successive aggressive encounters across multiple days leads to increased aggressiveness. Here, we show that this procedure also leads to long-term potentiation (LTP) at an excitatory synapse, derived from the posteromedial part of the amygdalohippocampal area (AHiPM), onto estrogen receptor 1-expressing (Esr1⁺) neurons in the ventrolateral subdivision of the ventromedial hypothalamus (VMHvl). We demonstrate further that the optogenetic induction of such LTP in vivo facilitates, while optogenetic long-term depression (LTD) diminishes, the behavioral effect of aggression training, implying a causal role for potentiation at AHiPM→VMHvl^Esr1 synapses in mediating the effect of this training. Interestingly, ∼25% of inbred C57BL/6 mice fail to respond to aggression training. We show that these individual differences are correlated both with lower levels of testosterone, relative to mice that respond to such training, and with a failure to exhibit LTP after aggression training. Administration of exogenous testosterone to such nonaggressive mice restores both behavioral and physiological plasticity. Together, these findings reveal that LTP at a hypothalamic circuit node mediates a form of experience-dependent plasticity in an innate social behavior, and a potential hormone-dependent basis for individual differences in such plasticity among genetically identical mice. Significance Modification of instinctive behaviors occurs through experience, yet the mechanisms through which this happens have remained largely unknown. Recent studies have shown that potentiation of aggression, an innate behavior, can occur through repeated winning of aggressive encounters. Here, we show that synaptic plasticity at a specific excitatory input to a hypothalamic cell population is correlated with, and required for, the expression of increasingly higher levels of aggressive behavior following aggressive experience. We additionally show that the amplitude and persistence of long-term potentiation at this synapse are influenced by serum testosterone, administration of which can normalize individual differences in the expression of intermale aggression among genetically identical mice.

Persistent neural activity in cortical, hippocampal, and motor networks has been described as mediating working memory for transiently encountered stimuli^1,2. Internal emotional states, such as fear, also persist following exposure to an inciting stimulus³, but it is unclear whether slow neural dynamics are involved in this process. Neurons in the dorsomedial and central subdivisions of the ventromedial hypothalamus (VMHdm/c) that express the nuclear receptor protein NR5A1 (also known as SF1) are necessary for defensive responses to predators in mice^4,5,6,7. Optogenetic activation of these neurons, referred to here as VMHdm^SF1 neurons, elicits defensive behaviours that outlast stimulation^5,8, which suggests the induction of a persistent internal state of fear or anxiety. Here we show that in response to naturalistic threatening stimuli, VMHdm^SF1 neurons in mice exhibit activity that lasts for many tens of seconds. This persistent activity was correlated with, and required for, persistent defensive behaviour in an open-field assay, and depended on neurotransmitter release from VMHdm^SF1 neurons. Stimulation and calcium imaging in acute slices showed that there is local excitatory connectivity between VMHdm^SF1 neurons. Microendoscopic calcium imaging of VMHdm^SF1 neurons revealed that persistent activity at the population level reflects heterogeneous dynamics among individual cells. Unexpectedly, distinct but overlapping VMHdm^SF1 subpopulations were persistently activated by different modalities of threatening stimulus. Computational modelling suggests that neither recurrent excitation nor slow-acting neuromodulators alone can account for persistent activity that maintains stimulus identity. Our results show that stimulus-specific slow neural dynamics in the hypothalamus, on a time scale orders of magnitude longer than that of working memory in the cortex^9,10, contribute to a persistent emotional state.

All animals can perform certain survival behaviors without prior experience, suggesting a “hard wiring” of underlying neural circuits. Experience, however, can alter the expression of innate behaviors. Where in the brain and how such plasticity occurs remains largely unknown. Previous studies have established the phenomenon of “aggression training,” in which the repeated experience of winning successive aggressive encounters across multiple days leads to increased aggressiveness. Here we show that this procedure also leads to long-term potentiation (LTP) at an excitatory synapse, derived from the Anterior Hippocampus/Posterior Medial amygdala (AHiPM), onto estrogen receptor 1-expressing (Esr1 ⁺ ) neurons in the ventrolateral subdivision of the ventromedial hypothalamus (VMHvl). We demonstrate further that the optogenetic induction of such LTP in vivo facilitates, while optogenetic long-term depression (LTD) diminishes, the behavioral effect of aggression training, implying a causal role for potentiation at AHiPM➔VMHvl ^Esr1 synapses in mediating the effect of this training. Interestingly, ∼25% of inbred C57BL/6 mice fail to respond to aggression training. We show that these individual differences are correlated both with lower levels of testosterone, relative to mice that respond to such training, and with a failure to exhibit LTP in vivo after aggression training. Administration of exogenous testosterone to such non-aggressive mice restores both behavioral and physiological plasticity in vivo . Together, these findings reveal that LTP at a hypothalamic circuit node mediates a form of experience-dependent plasticity in an innate social behavior, and a potential hormone-dependent basis for individual differences in such plasticity among genetically identical mice. Significance Statement: Modification of instinctive behaviors occurs through experience, yet the mechanisms through which this happens have remained largely unknown. Recent studies have shown that potentiation of aggression, an innate behavior, can occur through repeated winning of aggressive encounters. Here we show that synaptic plasticity at a specific excitatory input to a hypothalamic cell population is correlated with, and required for, the expression of increasingly higher levels of aggressive behavior following successful aggressive experience. We additionally show that the amplitude and persistence of long-term potentiation at this synapse are influenced by serum testosterone, administration of which can normalize individual differences among genetically identical inbred mice, in the expression of intermale aggression.

Reward-related behavior is complex and its dysfunction correlated with neuropsychiatric illness. Dopamine (DA) neurons of the ventral tegmental area (VTA) have long been associated with different aspects of reward function, but it remains to be disentangled how distinct VTA DA neurons contribute to the full range of behaviors ascribed to the VTA. Here, a recently identified subtype of VTA neurons molecularly defined by NeuroD6 (NEX1M) was addressed. Among all VTA DA neurons, less than 15% were identified as positive for NeuroD6. In addition to dopaminergic markers, sparse NeuroD6 neurons expressed the vesicular glutamate transporter 2 (Vglut2) gene. To achieve manipulation of NeuroD6 VTA neurons, NeuroD6(NEX)-Cre-driven mouse genetics and optogenetics were implemented. First, expression of vesicular monoamine transporter 2 (VMAT2) was ablated to disrupt dopaminergic function in NeuroD6 VTA neurons. ComparingVmat2^{lox/lox;NEX-Cre}conditional knock-out (cKO) mice with littermate controls, it was evident that baseline locomotion, preference for sugar and ethanol, and place preference upon amphetamine-induced and cocaine-induced conditioning were similar between genotypes. However, locomotion upon repeated psychostimulant administration was significantly elevated above control levels in cKO mice. Second, optogenetic activation of NEX-Cre VTA neurons was shown to induce DA release and glutamatergic postsynaptic currents within the nucleus accumbens. Third, optogenetic stimulation of NEX-Cre VTA neuronsin vivoinduced significant place preference behavior, while stimulation of VTA neurons defined by Calretinin failed to cause a similar response. The results show that NeuroD6 VTA neurons exert distinct regulation over specific aspects of reward-related behavior, findings that contribute to the current understanding of VTA neurocircuitry.

The relationship between neuronal impulse activity and neurotransmitter release remains elusive. This issue is especially poorly understood in the neuroendocrine system, with its particular demands on periodically voluminous release of neurohormones at the interface of axon terminals and vasculature. A shortage of techniques with sufficient temporal resolution has hindered real-time monitoring of the secretion of the peptides that dominate among the neurohormones. The lactotropic axis provides an important exception in neurochemical identity, however, as pituitary prolactin secretion is primarily under monoaminergic control, via tuberoinfundibular dopamine (TIDA) neurons projecting to the median eminence (ME). Here, we combined electrical or optogenetic stimulation and fast-scan cyclic voltammetry to address dopamine release dynamics in the male mouse TIDA system. Imposing different discharge frequencies during brief (3 s) stimulation of TIDA terminals in the ME revealed that dopamine output is maximal at 10 Hz, which was found to parallel the TIDA neuron action potential frequency distribution during phasic discharge. Over more sustained stimulation periods (150 s), maximal output occurred at 5 Hz, similar to the average action potential firing frequency of tonically active TIDA neurons. Application of the dopamine transporter blocker, methylphenidate, significantly increased dopamine levels in the ME, supporting a functional role of the transporter at the neurons' terminals. Lastly, TIDA neuron stimulation at the cell body yielded perisomatic release of dopamine, which may contribute to an ultrafast negative feedback mechanism to constrain TIDA electrical activity. Together, these data shed light on how spiking patterns in the neuroendocrine system translate to vesicular release toward the pituitary and identify how dopamine dynamics are controlled in the TIDA system at different cellular compartments.SIGNIFICANCE STATEMENTA central question in neuroscience is the complex relationship between neuronal discharge activity and transmitter release. By combining optogenetic stimulation and voltammetry, we address this issue in dopamine neurons of the neuroendocrine system, which faces particular spatiotemporal demands on exocytotic release; large amounts of neurohormone need to be secreted into the portal capillaries with precise timing to adapt to physiological requirements. Our data show that release is maximal around the neurons' default firing frequency. We further provide support for functional dopamine transport at the neurovascular terminals, shedding light on a long-standing controversy about the existence of neuroendocrine transmitter reuptake. Finally, we show that dopamine release occurs also at the somatodendritic level, providing a substrate for an ultrashort autoregulatory feedback loop.