A Neuro-hormonal Circuit for Paternal Behavior Controlled by a Hypothalamic Network Oscillation.

Parental behavior is pervasive throughout the animal kingdom and essential for species survival. However, the relative contribution of the father to offspring care differs markedly across animals, even between related species. The mechanisms that organize and control paternal behavior remain poorly understood. Using Sprague-Dawley rats and C57BL/6 mice, two species at opposite ends of the paternal spectrum, we identified that distinct electrical oscillation patterns in neuroendocrine dopamine neurons link to a chain of low dopamine release, high circulating prolactin, prolactin receptor-dependent activation of medial preoptic area galanin neurons, and paternal care behavior in male mice. In rats, the same parameters exhibit inverse profiles. Optogenetic manipulation of these rhythms in mice dramatically shifted serum prolactin and paternal behavior, whereas injecting prolactin into non-paternal rat sires triggered expression of parental care. These findings identify a frequency-tuned brain-endocrine-brain circuit that can act as a gain control system determining a species' parental strategy.

Adaptive Resetting of Tuberoinfundibular Dopamine (TIDA) Network Activity during Lactation in Mice.

Giving birth triggers a wide repertoire of physiological and behavioral changes in the mother to enable her to feed and care for her offspring. These changes require coordination and are often orchestrated from the CNS, through as of yet poorly understood mechanisms. A neuronal population with a central role in puerperal changes is the tuberoinfundibular dopamine (TIDA) neurons that control release of the pituitary hormone, prolactin, which triggers key maternal adaptations, including lactation and maternal care. Here, we used Ca2+ imaging on mice from both sexes and whole-cell recordings on female mouse TIDA neurons in vitro to examine whether they adapt their cellular and network activity according to reproductive state. In the high-prolactin state of lactation, TIDA neurons shift to faster membrane potential oscillations, a reconfiguration that reverses upon weaning. During the estrous cycle, however, which includes a brief, but pronounced, prolactin peak, oscillation frequency remains stable. An increase in the hyperpolarization-activated mixed cation current, Ih, possibly through unmasking as dopamine release drops during nursing, may partially explain the reconfiguration of TIDA rhythms. These findings identify a reversible plasticity in hypothalamic network activity that can serve to adapt the dam for motherhood.SIGNIFICANCE STATEMENT Motherhood requires profound behavioral and physiological adaptations to enable caring for offspring, but the underlying CNS changes are poorly understood. Here, we show that, during lactation, neuroendocrine dopamine neurons, the "TIDA" cells that control prolactin secretion, reorganize their trademark oscillations to discharge in faster frequencies. Unlike previous studies, which typically have focused on structural and transcriptional changes during pregnancy and lactation, we demonstrate a functional switch in activity and one that, distinct from previously described puerperal modifications, reverses fully on weaning. We further provide evidence that a specific conductance (Ih) contributes to the altered network rhythm. These findings identify a new facet of maternal brain plasticity at the level of membrane properties and consequent ensemble activity.

Dopamine Release Dynamics in the Tuberoinfundibular Dopamine System.

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 STATEMENT A 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.

Neurotensin Broadly Recruits Inhibition via White Matter Neurons in the Mouse Cerebral Cortex: Synaptic Mechanisms for Decorrelation.

The neuropeptide, neurotensin (NT), inhibits UP state generation in the cerebral cortex and temporally restricts the response to thalamic input, likely by a generalized increase in inhibition. To investigate the cellular and circuit substrate(s) for how a neuropeptide can shift the balance between cortical excitation and inhibition, we performed whole-cell recordings on slice preparations from mice expressing enhanced green fluorescent protein under control of the promoter for the homeobox gene, lhx6 (lhx6-EGFP mice). These mice identify the 2 largest classes of cortical interneurons; FS and low-threshold-spiking inhibitory neurons. In the presence of NT, both types of lhx6-EGFP neurons were excited through a direct, Na+-dependent depolarization, and through an increase in synaptic excitation. Paired recordings identified cortical white matter (WM) neurons as a source of this excitatory input, which was strengthened in the presence of NT. NT-driven increased synaptic input caused a functional decorrelation of gap junction transmission between lhx6-EGFP neuron pairs. Finally, the synaptic transmission between pyramidal cells and lhx6-EGFP neurons was modulated by addition of NT in favor of stronger inhibition and weaker excitation. These findings demonstrate the existence and functional consequences of an intracortical WM neuron projection, and suggest mechanisms underlying NT-induced promotion of wakefulness.

Desynchronization of the Rat Cortical Network and Excitation of White Matter Neurons by Neurotensin.

Cortical network activity correlates with vigilance state: Deep sleep is characterized by slow, synchronized oscillations, whereas desynchronized, stochastic discharge is typical of the waking state. Neuropeptides, such as orexin and substance P but also neurotensin (NT), promote arousal. Relatively little is known about if NT can directly affect the cortical network, and if so, through which mechanisms and cellular targets. Here, we addressed these issues using rat in vitro cortex preparations. Following NT application specifically to deeper layers, slow oscillation activity was attenuated with a significant reduction in UP state frequency. The cortical response to thalamic stimulation exhibited enhanced temporal precision in the presence of NT, consistent with the transition in vivo from sleep to wakefulness. These changes were associated with a relative shift toward inhibition in the excitation/inhibition balance. Whole-cell recordings from layer 6 revealed presynaptically driven NT-induced inhibition of pyramidal neurons and excitation of fast-spiking interneurons. Deeper in the cortex, neurons within the white matter (WM) were strongly depolarized by NT application. The colocalization of NT and tyrosine hydroxylase immunoreactivities in deep layer fibers throughout the cortical mantle indicates mediation via dopaminergic systems. These data suggest a cortical mechanism for NT-induced wakefulness and support a role for WM neurons in state control.

Serotonin and Antidepressant SSRIs Inhibit Rat Neuroendocrine Dopamine Neurons: Parallel Actions in the Lactotrophic Axis.

UNLABELLED: Selective serotonin reuptake inhibitors (SSRIs) are commonly prescribed for depression, but sexual side effects often compromise compliance. These reproductive dysfunctions are likely mediated by elevations of the hormone prolactin. Yet, how serotonin (5-HT) and SSRIs cause changes in prolactin secretion is not known. Here, using in vitro whole-cell patch-clamp recordings, we show that 5-HT hyperpolarizes and abolishes phasic discharge in rat neuroendocrine tuberoinfundibular dopamine (TIDA) neurons, the main inhibitor of prolactin secretion. This process is underpinned by 5-HT1A receptor-mediated activation of G-protein-coupled inwardly rectifying K(+)-like currents. We further demonstrate that the SSRIs, fluoxetine and sertraline, directly suppress TIDA neuron activity through parallel effects, independent of 5-HT transmission. This inhibition involves decreased intrinsic excitability and a slowing of TIDA network rhythms. These findings indicate that SSRIs may inhibit neuroendocrine dopamine release through both 5-HT-dependent and -independent actions, providing a mechanistic explanation for, and potential molecular targets for the amelioration of, the hyperprolactinemia and sexual dysfunction associated with these drugs. SIGNIFICANCE STATEMENT: Depression affects approximately one-tenth of the population and is commonly treated with selective serotonin reuptake inhibitors (SSRIs; e.g., Prozac). Yet, many patients withdraw from SSRI therapy due to sexual side effects (e.g., infertility, menstrual disturbances, and impotence). Although it is generally accepted that sexual side effects are due to the ability of these drugs to elevate blood levels of the hormone prolactin, the mechanism for this hormonal imbalance is not known. Here, we show that SSRIs can inhibit hypothalamic dopamine neurons that normally suppress the secretion of prolactin. Intriguingly this inhibition can be explained both by increased serotonin activity and also by parallel serotonin-independent actions.

Excitation of tuberoinfundibular dopamine neurons by oxytocin: crosstalk in the control of lactation.

Milk production in the nursing mother is induced by the hormone prolactin. Its release from the anterior pituitary is generally under tonic inhibition by neuroendocrine tuberoinfundibular dopamine (TIDA) neurons of the arcuate nucleus. Successful nursing, however, requires not only production but also ejection of breast milk. This function is supported by the hormone oxytocin. Here we explored the possibility that interaction between these functionally complementary hormones is mediated by TIDA neurons. First, whole-cell patch-clamp recordings were performed on prepubertal male rat hypothalamic slices, where TIDA neurons can be identified by a robust and rhythmic membrane potential oscillation. Oxytocin induced a switch of this rhythmic activity to tonic discharge through a depolarization involving direct actions on TIDA neurons. The depolarization is sensitive to blockade of the oxytocin receptor and is mediated by a voltage-dependent inward current. This inward current has two components: a canonical transient receptor potential-like conductance in the low-voltage range, and in the high-voltage range, a Ca(2+)-dependent component. Finally, whole-cell and loose-patch recordings were also performed on slices from virgin and lactating female rats to evaluate the relevance of these findings for nursing. In these preparations, oxytocin was found to excite TIDA neurons, identified by their expression of tyrosine hydroxylase. These findings suggest that oxytocin can modulate prolactin secretion by exciting TIDA neurons, and that this may serve as a feedforward inhibition of prolactin release.

TIDAL WAVES: Network mechanisms in the neuroendocrine control of prolactin release.

Neuroendocrine tuberoinfundibular dopamine (TIDA) neurons tonically inhibit pituitary release of the hormone, prolactin. Through the powerful actions of prolactin in promoting lactation and maternal behaviour while suppressing sexual drive and fertility, TIDA neurons play a key role in reproduction. We summarize insights from recent in vitro studies into the membrane properties and network behaviour of TIDA neurons including the observations that TIDA neurons exhibit a robust oscillation that is synchronized between cells and depends on intact gap junction communication. Comparisons are made with phasic firing patterns in other neuronal populations. Modulators involved in the control of lactation - including serotonin, thyrotropin-releasing hormone and prolactin itself - have been shown to change the electrical behaviour of TIDA cells. We propose that TIDA discharge mode may play a central role in tuning the amount of dopamine delivered to the pituitary and hence circulating prolactin concentrations in different reproductive states and pathological conditions.

A specific and essential role for Na,K-ATPase α3 in neurons co-expressing α1 and α3.

Most neurons co-express two catalytic isoforms of Na,K-ATPase, the ubiquitous α1, and the more selectively expressed α3. Although neurological syndromes are associated with α3 mutations, the specific role of this isoform is not completely understood. Here, we used electrophysiological and Na(+) imaging techniques to study the role of α3 in central nervous system neurons expressing both isoforms. Under basal conditions, selective inhibition of α3 using a low concentration of the cardiac glycoside, ouabain, resulted in a modest increase in intracellular Na(+) concentration ([Na(+)](i)) accompanied by membrane potential depolarization. When neurons were challenged with a large rapid increase in [Na(+)](i), similar to what could be expected following suprathreshold neuronal activity, selective inhibition of α3 almost completely abolished the capacity to restore [Na(+)](i) in soma and dendrite. Recordings of Na,K-ATPase specific current supported the notion that when [Na(+)](i) is elevated in the neuron, α3 is the predominant isoform responsible for rapid extrusion of Na(+). Low concentrations of ouabain were also found to disrupt cortical network oscillations, providing further support for the importance of α3 function in the central nervous system. The α isoforms express a well conserved protein kinase A consensus site, which is structurally associated with an Na(+) binding site. Following activation of protein kinase A, both the α3-dependent current and restoration of dendritic [Na(+)](i) were significantly attenuated, indicating that α3 is a target for phosphorylation and may participate in short term regulation of neuronal function.

Expression of nucleobindin 1 (NUCB1) in pancreatic islets and other endocrine tissues.

The protein nucleobindin 1 (NUCB1; also known as CALNUC or Nuc) contains an intriguing combination of DNA- and calcium-binding motifs, a trait that it shares with the protein nucleobindin 2 (NUCB2; also known as nesfatin). NUCB2 has been implicated in several aspects of metabolic control and has been identified in a number of endocrine organs. No such comprehensive mapping of NUCB1 has been presented. We have explored the expression and distribution of NUCB1 in tissues and cells of the mouse endocrine system, with particular focus on the endocrine pancreas. Using reverse transcription plus the polymerase chain reaction (RT-PCR) and Western blot, we demonstrate that NUCB1 is present in the endocrine islets of Langerhans but absent from the exocrine acinar cells. Immunofluorescence studies have revealed that all islet cell types contain NUCB1, including the NUCB2-expressing beta cells. RT-PCR, Western blot and immunofluorescence have shown that NUCB1 is expressed in the pituitary, thyroid, parathyroid, gastrointestinal tract, adrenals and gonads. However, within these tissues, NUCB1 expression is not ubiquitous. For example, in the testis, NUCB1 occurs in the seminiferous tubules but not in the Leydig-cell-containing interstitial tissue. Similarly, the lamina propria of the duodenum lacks NUCB1, despite its presence in enterocytes. Where present, NUCB1 consistently appears to be associated with the Golgi apparatus. Thus, NUCB1 is broadly, but not ubiquitously, expressed in cells of the mouse endocrine system. Together with its location in the Golgi apparatus and its putative Ca(2+)-binding ability, this distribution suggests a role for NUCB1 in Ca(2+) handling/sensing in secretory cells.

Maternal Aggression Driven by the Transient Mobilisation of a Dormant Hormone-Sensitive Circuit.

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 (PMvDAT) 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 Ca2+ channels. Optogenetic manipulation or genetic ablation of PMvDAT 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.

Prolactin regulates tuberoinfundibular dopamine neuron discharge pattern: novel feedback control mechanisms in the lactotrophic axis.

Balance in the body's hormonal axes depends on feedback onto neuroendocrine hypothalamic neurons. This phenomenon involves transcriptional and biosynthetic effects, yet less is known about the potential rapid modulation of electrical properties. Here, we investigated this issue in the lactotrophic axis, in which the pituitary hormone prolactin is tonically inhibited by tuberoinfundibular dopamine (TIDA) neurons located in the hypothalamic arcuate nucleus. Whole-cell recordings were performed on slices of the rat hypothalamus. In the presence of prolactin, spontaneously oscillating TIDA cells depolarized, switched from phasic to tonic discharge, and exhibited broadened action potentials. The underlying prolactin-induced current is composed of separate low- and high-voltage components that include the activation of a transient receptor potential-like current and the inhibition of a Ca(2+)-dependent BK-type K(+) current, respectively, as revealed by ion substitution experiments and pharmacological manipulation. The two components of the prolactin-induced current appear to be mediated through distinct signaling pathways as the high-voltage component is abolished by the phosphoinositide 3-kinase blocker wortmannin, whereas the low-voltage component is not. This first description of the central electrophysiological actions of prolactin suggests a novel feedback mechanism. By simultaneously enhancing the discharge and spike duration of TIDA cells, increased serum prolactin can promote dopamine release to limit its own secretion with implications for the control of lactation, sexual libido, fertility, and body weight.

Neuroendocrine control of female reproductive function by the activin receptor ALK7.

Activins are critical components of the signaling network that controls female reproduction. However, their roles in hypothalamus, and the specific functions of their different receptors, have not been elucidated. Here, we investigated the expression and function of the activin receptor ALK7 in the female reproductive axis using Alk7-knockout mice. ALK7 was found in subsets of SF1-expressing granulosa cells in the ovary, FSH gonadotrophs in the pituitary, and NPY-expressing neurons in the arcuate nucleus of the hypothalamus. Alk7-knockout females showed delayed onset of puberty and abnormal estrous cyclicity, had abnormal diestrous levels of FSH and LH in serum, and their ovaries showed premature depletion of follicles, oocyte degeneration, and impaired responses to exogenous gonadotropins. In the arcuate nucleus, mutant mice showed reduced expression of Npy mRNA and lower numbers of Npy-expressing neurons than wild-type controls. Alk7 knockouts showed a selective loss of arcuate NPY/AgRP innervation in the medial preoptic area, a key central regulator of reproduction. These results indicate that ALK7 is an important regulator of female reproductive function and reveal a new role for activin signaling in the control of hypothalamic gene expression and wiring. Alk7 gene variants may contribute to female reproductive disorders in humans, such as polycystic ovary syndrome.

Distinct phenotype and function of NK cells in the pancreas of nonobese diabetic mice.

Little is known about target organ-infiltrating NK cells in type 1 diabetes and other autoimmune diseases. In this study, we identified NK cells with a unique phenotype in the pancreas of NOD mice. Pancreatic NK cells, localized to the endocrine and exocrine parts, were present before T cells during disease development and did not require T cells for their infiltration. Furthermore, NK cells, or NK cell precursors, from the spleen could traffic to the pancreas, where they displayed the pancreatic phenotype. Pancreatic NK cells from other mouse strains shared phenotypic characteristics with pancreatic NK cells from NOD mice, but displayed less surface killer cell lectin-like receptor G1, a marker for mature NK cells that have undergone proliferation, and also did not proliferate to the same extent. A subset of NOD mouse pancreatic NK cells produced IFN-gamma spontaneously, suggesting ongoing effector responses. However, most NOD mouse pancreatic NK cells were hyporesponsive compared with spleen NK cells, as reflected by diminished cytokine secretion and a lower capacity to degranulate. Interestingly, such hyporesponsiveness was not seen in pancreatic NK cells from the nonautoimmune strain C57BL/6, suggesting that this feature is not a general property of pancreatic NK cells. Based on our data, we propose that NK cells are sentinel cells in a normal pancreas. We further speculate that during inflammation, pancreatic NK cells initially mediate proinflammatory effector functions, potentially contributing to organ-specific autoimmunity, but later become hyporesponsive because of exhaustion or regulation.

Neuromedin B and gastrin-releasing peptide excite arcuate nucleus neuropeptide Y neurons in a novel transgenic mouse expressing strong Renilla green fluorescent protein in NPY neurons.

Neuropeptide Y (NPY) is one of the most widespread neuropeptides in the brain. Transgenic mice were generated that expressed bright Renilla green fluorescent protein (GFP) in most or all of the known NPY cells in the brain, which otherwise were not identifiable. GFP expression in NPY cells was confirmed with immunocytochemistry and single-cell reverse transcription-PCR. NPY neurons in the hypothalamic arcuate nucleus play an important role in energy homeostasis and endocrine control. Whole-cell patch clamp recording was used to study identified arcuate NPY cells. Primary agents that regulate energy balance include melanocortin receptor agonists, AgRP, and cannabinoids; none of these substances substantially influenced electrical properties of NPY neurons. In striking contrast, neuropeptides of the bombesin family, including gastrin-releasing peptide and neuromedin B, which are found in axons in the mediobasal hypothalamus and may also be released from the gut to signal the brain, showed strong direct excitatory actions at nanomolar levels on the NPY neurons, stronger than the actions of ghrelin and hypocretin/orexin. Bombesin-related peptides reduced input resistance and depolarized the membrane potential. The depolarization was attenuated by several factors: substitution of choline for sodium, extracellular Ni(2+), inclusion of BAPTA in the pipette, KB-R7943, and SKF96365. Reduced extracellular calcium enhanced the current, which reversed around -20 mV. Together, these data suggest two mechanisms, activation of nonselective cation channels and the sodium/calcium exchanger. Since both NPY and POMC neurons, which we also studied, are similarly directly excited by bombesin-like peptides, the peptides may function to initiate broad activation, rather than the cell-type selective activation or inhibition reported for many other compounds that modulate energy homeostasis.

Pre- and post-synaptic modulation by GABAB receptors of rat neuroendocrine dopamine neurones.

The secretion of prolactin from the pituitary is negatively controlled by tuberoinfundibular dopamine (TIDA) neurones. The electrical properties of TIDA cells have recently been identified as a modulatory target of neurotransmitters and hormones in the lactotrophic axis. The role of the GABAB receptor in this control has received little attention, yet is of particular interest because it may act as a TIDA neurone autoreceptor. Here, this issue was explored in a spontaneously active rat TIDA in vitro slice preparation using whole-cell recordings. Application of the GABAB receptor agonist, baclofen, dose-dependently slowed down or abolished the network oscillations typical of this preparation. Pharmacological manipulations identify the underlying mechanism as an outward current mediated by G-protein-coupled inwardly rectifying K+ -like channels. In addition to this postsynaptic modulation, we describe a presynaptic modulation where GABAB receptors restrain the release of glutamate and GABA onto TIDA neurones. Our data identify both pre- and postsynaptic modulation of TIDA neurones by GABAB receptors that may play a role in the neuronal network control of pituitary prolactin secretion and lactation.

Polysynaptic inhibition between striatal cholinergic interneurons shapes their network activity patterns in a dopamine-dependent manner.

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.

Stimulation of orexin/hypocretin neurones by thyrotropin-releasing hormone.

Central orexin/hypocretin neurones are critical for sustaining consciousness: their firing stimulates wakefulness and their destruction causes narcolepsy. We explored whether the activity of orexin cells is modulated by thyrotropin-releasing hormone (TRH), an endogenous stimulant of wakefulness and locomotor activity whose mechanism of action is not fully understood. Living orexin neurones were identified by targeted expression of green fluorescent protein (GFP) in acute brain slices of transgenic mice. Using whole-cell patch-clamp recordings, we found that TRH robustly increased the action potential firing rate of these neurones. TRH-induced excitation persisted under conditions of synaptic isolation, and involved a Na(+)-dependent depolarization and activation of a mixed cation current in the orexin cell membrane. By double-label immunohistochemistry, we found close appositions between TRH-immunoreactive nerve terminals and orexin-A-immunoreactive cell bodies. These results identify a new physiological modulator of orexin cell firing, and suggest that orexin cell excitation may contribute to the arousal-enhancing actions of TRH.

The NeuroD6 Subtype of VTA Neurons Contributes to Psychostimulant Sensitization and Behavioral Reinforcement.

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. Comparing Vmat2lox/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 neurons in vivo induced 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.