Hypocretin/Orexin Peptides Excite Rat Neuroendocrine Dopamine Neurons through Orexin 2 Receptor-Mediated Activation of a Mixed Cation Current.

Hypocretin/Orexin (H/O) neurons of the lateral hypothalamus are compelling modulator candidates for the chronobiology of neuroendocrine output and, as a consequence, hormone release from the anterior pituitary. Here we investigate the effects of H/O peptides upon tuberoinfundibular dopamine (TIDA) neurons - cells which control, via inhibition, the pituitary secretion of prolactin. In whole cell recordings performed in male rat hypothalamic slices, application of H/O-A, as well as H/O-B, excited oscillating TIDA neurons, inducing a reversible depolarising switch from phasic to tonic discharge. The H/O-induced inward current underpinning this effect was post-synaptic (as it endured in the presence of tetrodotoxin), appeared to be carried by a Na+-dependent transient receptor potential-like channel (as it was blocked by 2-APB and was diminished by removal of extracellular Na+), and was a consequence of OX2R receptor activation (as it was blocked by the OX2R receptor antagonist TCS OX2 29, but not the OX1R receptor antagonist SB 334867). Application of the hormone, melatonin, failed to alter TIDA membrane potential or oscillatory activity. This first description of the electrophysiological effects of H/Os upon the TIDA network identifies cellular mechanisms that may contribute to the circadian rhythmicity of prolactin secretion.

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.

Activation of Ventral Tegmental Area 5-HT2C Receptors Reduces Incentive Motivation.

Obesity is primarily due to food intake in excess of the body's energetic requirements, intake that is not only associated with hunger but also the incentive value of food. The 5-hydroxytryptamine 2C receptor (5-HT2CR) is a target for the treatment of human obesity. Mechanistically, 5-HT2CRs are positioned to influence both homeostatic feeding circuits within the hypothalamus and reward circuits within the ventral tegmental area (VTA). Here we investigated the role of 5-HT2CRs in incentive motivation using a mathematical model of progressive ratio (PR) responding in mice. We found that the 5-HT2CR agonist lorcaserin significantly reduced both ad libitum chow intake and PR responding for chocolate pellets and increased c-fos expression in VTA 5-HT2CR expressing γ-aminobutyric acid (GABA) neurons, but not 5-HT2CR expressing dopamine (DA) neurons. We next adopted a chemogenetic approach using a 5-HT2CRCRE line to clarify the function of subset of 5-HT2C receptor expressing VTA neurons in the modulation of appetite and food-motivated behavior. Activation of VTA 5-HT2C receptor expressing neurons significantly reduced ad libitum chow intake, operant responding for chocolate pellets, and the incentive value of food. In contrast, chemogenetic inhibition of VTA 5-HT2C receptor expressing neurons had no effect on the feeding behavior. These results indicate that activation of the subpopulation of 5-HT2CR neurons within the VTA is sufficient to significantly reduce homeostatic feeding and effort-based intake of palatable food, and that this subset has an inhibitory role in motivational processes. These findings are relevant to the treatment of obesity.

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.

Dopamine Autoreceptor Regulation of a Hypothalamic Dopaminergic Network.

How autoreceptors contribute to maintaining a stable output of rhythmically active neuronal circuits is poorly understood. Here, we examine this issue in a dopamine population, spontaneously oscillating hypothalamic rat (TIDA) neurons, that underlie neuroendocrine control of reproduction and neuroleptic side effects. Activation of dopamine receptors of the type 2 family (D2Rs) at the cell-body level slowed TIDA oscillations through two mechanisms. First, they prolonged the depolarizing phase through a combination of presynaptic increases in inhibition and postsynaptic hyperpolarization. Second, they extended the discharge phase through presynaptic attenuation of calcium currents and decreased synaptic inhibition. Dopamine reuptake blockade similarly reconfigured the oscillation, indicating that ambient somatodendritic transmitter concentration determines electrical behavior. In the absence of D2R feedback, however, discharge was abolished by depolarization block. These results indicate the existence of an ultra-short feedback loop whereby neuroendocrine dopamine neurons tune network behavior to echoes of their own activity, reflected in ambient somatodendritic dopamine, and also suggest a mechanism for antipsychotic side effects.

Appetite controlled by a cholecystokinin nucleus of the solitary tract to hypothalamus neurocircuit.

The nucleus of the solitary tract (NTS) is a key gateway for meal-related signals entering the brain from the periphery. However, the chemical mediators crucial to this process have not been fully elucidated. We reveal that a subset of NTS neurons containing cholecystokinin (CCK(NTS)) is responsive to nutritional state and that their activation reduces appetite and body weight in mice. Cell-specific anterograde tracing revealed that CCK(NTS) neurons provide a distinctive innervation of the paraventricular nucleus of the hypothalamus (PVH), with fibers and varicosities in close apposition to a subset of melanocortin-4 receptor (MC4R(PVH)) cells, which are also responsive to CCK. Optogenetic activation of CCK(NTS) axon terminals within the PVH reveal the satiating function of CCK(NTS) neurons to be mediated by a CCK(NTS)→PVH pathway that also encodes positive valence. These data identify the functional significance of CCK(NTS) neurons and reveal a sufficient and discrete NTS to hypothalamus circuit controlling appetite.

Peroxisome proliferator-activated receptor alpha plays a crucial role in behavioral repetition and cognitive flexibility in mice.

BACKGROUND/OBJECTIVES: Nuclear peroxisome proliferator activated receptor-α (PPAR-α) plays a fundamental role in the regulation of lipid homeostasis and is the target of medications used to treat dyslipidemia. However, little is known about the role of PPAR-α in mouse behavior. METHODS: To investigate the function of Ppar-α in cognitive functions, a behavioral phenotype analysis of mice with a targeted genetic disruption of Ppar-α was performed in combination with neuroanatomical, biochemical and pharmacological manipulations. The therapeutic exploitability of PPAR-α was probed in mice using a pharmacological model of psychosis and a genetic model (BTBR T + tf/J) exhibiting a high rate of repetitive behavior. RESULTS: An unexpected role for brain Ppar-α in the regulation of cognitive behavior in mice was revealed. Specifically, we observed that Ppar-α genetic perturbation promotes rewiring of cortical and hippocampal regions and a behavioral phenotype of cognitive inflexibility, perseveration and blunted responses to psychomimetic drugs. Furthermore, we demonstrate that the antipsychotic and autism spectrum disorder (ASD) medication risperidone ameliorates the behavioral profile of Ppar-α deficient mice. Importantly, we reveal that pharmacological PPAR-α agonist treatment in mice improves behavior in a pharmacological model of ketamine-induced behavioral dysinhibition and repetitive behavior in BTBR T + tf/J mice. CONCLUSION: Our data indicate that Ppar-α is required for normal cognitive function and that pharmacological stimulation of PPAR-α improves cognitive function in pharmacological and genetic models of impaired cognitive function in mice. These results thereby reveal an unforeseen therapeutic application for a class of drugs currently in human use.

A role for solute carrier family 10 member 4, or vesicular aminergic-associated transporter, in structural remodelling and transmitter release at the mouse neuromuscular junction.

The solute carrier and presynaptic vesicle protein solute carrier family 10 member 4, or vesicular aminergic-associated transporter (VAAT), was recently proven to have a modulatory role in central cholinergic signalling. It is currently unknown whether VAAT also affects peripheral cholinergic synapses. Here we demonstrated a regulatory role for the presynaptic vesicle protein VAAT in neuromuscular junction (NMJ) development and function. NMJs lacking VAAT had fewer branch points, whereas endplates showed an increased number of islands. Whereas the amplitude of spontaneous miniature endplate potentials in VAAT-deficient NMJs was decreased, the amplitude of evoked endplate potentials and the size of the readily releasable pool of vesicles were both increased. Moreover, VAAT-deficient NMJs displayed aberrant short-term synaptic plasticity with enhanced synaptic depression in response to high-frequency stimulation. Finally, the transcript levels of cholinergic receptor subunits in VAAT-deficient muscles were increased, indicating a compensatory postsynaptic sensitization. Our results suggested that VAAT modulates NMJ transmission efficiency and, as such, may represent a novel target for treatment of disorders affecting motor neurons.

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.

Phosphorus as a limiting factor on sustainable greywater irrigation.

Water reuse through greywater irrigation has been adopted worldwide and has been proposed as a potential sustainable solution to increased water demands. Despite widespread adoption, there is limited domestic knowledge of greywater reuse. There is no pressure to produce low-level phosphorus products and current guidelines and legislation, such as those in Australia, may be inadequate due to the lack of long-term data to provide a sound scientific basis. Research has clearly identified phosphorus as a potential environmental risk to waterways from many forms of irrigation. To assess the sustainability of greywater irrigation, this study compared four residential lots that had been irrigated with greywater for four years and adjacent non-irrigated lots that acted as controls. Each lot was monitored for the volume of greywater applied and selected physic-chemical water quality parameters and soil chemistry profiles were analysed. The non-irrigated soil profiles showed low levels of phosphorus and were used as controls. The Mechlich3 Phosphorus ratio (M3PSR) and Phosphate Environmental Risk Index (PERI) were used to determine the environmental risk of phosphorus leaching from the irrigated soils. Soil phosphorus concentrations were compared to theoretical greywater irrigation loadings. The measured phosphorus soil concentrations and the estimated greywater loadings were of similar magnitude. Sustainable greywater reuse is possible; however incorrect use and/or lack of understanding of how household products affect greywater can result in phosphorus posing a significant risk to the environment.

Thyroid hormone is required for hypothalamic neurons regulating cardiovascular functions.

Thyroid hormone is well known for its profound direct effects on cardiovascular function and metabolism. Recent evidence, however, suggests that the hormone also regulates these systems indirectly through the central nervous system. While some of the molecular mechanisms underlying the hormone's central control of metabolism have been identified, its actions in the central cardiovascular control have remained enigmatic. Here, we describe a previously unknown population of parvalbuminergic neurons in the anterior hypothalamus that requires thyroid hormone receptor signaling for proper development. Specific stereotaxic ablation of these cells in the mouse resulted in hypertension and temperature-dependent tachycardia, indicating a role in the central autonomic control of blood pressure and heart rate. Moreover, the neurons exhibited intrinsic temperature sensitivity in patch-clamping experiments, providing a new connection between cardiovascular function and core temperature. Thus, the data identify what we believe to be a novel hypothalamic cell population potentially important for understanding hypertension and indicate developmental hypothyroidism as an epigenetic risk factor for cardiovascular disorders. Furthermore, the findings may be beneficial for treatment of the recently identified patients that have a mutation in thyroid hormone receptor α1.

Appropriate maximum holding times for analysis of total suspended solids concentration in water samples taken from open-channel waterways.

Many scientific laboratories follow, as standard practice, a relatively short maximum holding time (within 7 days) for the analysis of total suspended solids (TSS) in environmental water samples. In this study we have subsampled from bulk water samples stored at ∼4 °C in the dark, then analysed for TSS at time intervals up to 105 days after collection. The nonsignificant differences in TSS results observed over time demonstrates that storage at ∼4 °C in the dark is an effective method of preserving samples for TSS analysis, far past the 7-day standard practice. Extending the maximum holding time will ease the pressure on sample collectors and laboratory staff who until now have had to determine TSS within an impractically short period.

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.

Synchronized network oscillations in rat tuberoinfundibular dopamine neurons: switch to tonic discharge by thyrotropin-releasing hormone.

The pituitary hormone, prolactin, triggers lactation in nursing mothers. Under nonlactating conditions, prolactin secretion is suppressed by powerful inhibition from hypothalamic tuberoinfundibular dopamine (TIDA) neurons. Although firing pattern has been suggested as integral to neuroendocrine control, the electrical behavior of TIDA cells remains unknown. We demonstrate that rat TIDA neurons discharge rhythmically in a robust 0.05 Hz oscillation. The oscillation is phase locked between neurons, and while it persists during chemical synaptic transmission blockade, it is abolished by gap junction antagonists. Thyrotropin-releasing hormone (TRH) potently stimulates prolactin release, an effect assumed to take place in the pituitary. In TIDA cells, TRH caused a transition from phasic to tonic firing through combined pre- and postsynaptic effects. These findings suggest a model for prolactin regulation where a TIDA network switch from oscillations to sustained discharge converts dopamine from an antagonist at high concentrations to a functional agonist as dopamine output from the network decreases.

Recommendations for bone mineral density reporting in Canada: a shift to absolute fracture risk assessment.

In June 2005, new Canadian recommendations for bone mineral density (BMD) reporting in postmenopausal women and older men were published by Osteoporosis Canada (formerly the Osteoporosis Society of Canada) and the Canadian Association of Radiologists. The recommendations were developed by a multidisciplinary working group that included the Canadian Panel of the International Society for Clinical Densitometry and were reviewed and endorsed by multiple stakeholders. Previous Canadian osteoporosis guidelines advised intervention based on an individual's World Health Organization category (normal, osteopenia, or osteoporosis) as a marker of relative fracture risk. In the new approach, an individual's 10-yr absolute fracture risk, rather than BMD alone, is used for fracture risk categorization. Absolute fracture risk is determined using not only BMD results, but also age, sex, fragility fracture history, and glucocorticoid use. A procedure is presented for estimating absolute 10-yr fracture risk in untreated individuals, leading to assigning an individual to 1 of 3 absolute fracture risk categories: low risk (<10% 10-yr fracture risk), moderate risk (10-20%), and high risk (>20%). We propose that an individual's absolute fracture risk category should be the basis for deciding on treatment and frequency of BMD monitoring.

Recognizing and reporting vertebral fractures: reducing the risk of future osteoporotic fractures.

OBJECTIVE: Given the increasing evidence that vertebral fractures are underdiagnosed and not acted on, Osteoporosis Canada and the Canadian Association of Radiologists initiated a project to develop and publish a set of recommendations to promote and facilitate the diagnosis and reporting of vertebral fractures. OPTIONS: The identification of spinal fractures is not uniform. More than 65% of vertebral fractures cause no symptoms. It is also apparent that vertebral fractures are inadequately recognized when the opportunity for diagnosis arises fortuitously. It is to patients' benefit that radiologists report vertebral fractures evident on a chest or other radiograph, no matter how incidental to the immediate clinical indication for the examination. OUTCOMES: The present recommendations can help to close the gap in care in recognizing and treating vertebral fractures, to prevent future fractures and thus reduce the burden of osteoporosis-related morbidity and mortality, as well as fracture-related costs to the health care system. EVIDENCE: Several studies indicate that a gap exists in regard to the diagnosis of vertebral fractures and the clinical response following such diagnosis. All recommendations presented here are based on consensus. VALUES: These recommendations were developed by a multidisciplinary working group under the auspices of the Scientific Advisory Council of Osteoporosis Canada and the Canadian Association of Radiologists. BENEFITS, HARM, AND COSTS: Prevalent vertebral fractures have important clinical implications in terms of future fracture risk. Recognizing and reporting fractures incidental to radiologic examinations done for other reasons has the potential to reduce health care costs by initiating further steps in osteoporosis diagnosis and appropriate therapy. RECOMMENDATIONS: Physicians should be aware of the importance of vertebral fracture diagnosis in assessing future osteoporotic fracture risk. Vertebral fractures incidental to radiologic examinations done for other reasons should be identified and reported. Vertebral fractures should be assessed from lateral spinal or chest radiographs according to the semiquantitative method of Genant and colleagues. Grade II and Grade III fractures as classified by this method should be given the greatest emphasis. Semiquantitative fracture recognition should include the recognition of changes such as loss of vertebral end-plate parallelism, cortical interruptions, and quantitative changes in the anterior, midbody, and posterior heights of vertebral bodies. When spine radiographs are performed to assess the presence of vertebral fractures, anteroposterior examinations may assist in the initial evaluation. The standard follow-up need only consist of single lateral views of the thoracic and lumbar spine that include T4 to L4 vertebrae. The radiographic technique described in this paper, or a technique of comparable efficacy, should be used. Dual X-ray absorptiometry examinations that include lateral spinal morphological assessments (vertebral fracture assessment) may contribute to fracture recognition. Educational material about the clinical importance of vertebral fracture recognition as a potential indicator of future osteoporotic fracture risk with its associated morbidity and mortality should be directed to all physicians. VALIDATION: Recommendations were based on consensus opinion.

Recommendations for bone mineral density reporting in Canada.

OBJECTIVE: To propose a set of recommendations for optimal bone mineral density (BMD) reporting in postmenopausal women and older men and to provide clinicians with both a BMD diagnostic category and a useful tool to assess an individual's risk of osteoporotic fracture. OPTIONS: The current methods of BMD reporting were reviewed. In this document, we propose that an individual's 10-year absolute fracture risk, rather than BMD alone, be used for fracture risk categorization. Consequently, age, sex, BMD, fragility fracture history, and glucocorticoid use are the basis for the approach outlined in this document. OUTCOMES: An optimal BMD report as proposed in this document will provide clinicians with both a BMD diagnostic category and a useful tool to assess an individual's risk of osteoporotic fracture. A BMD report format, a checklist, and a patient questionnaire are meant to further encourage its use. EVIDENCE: All recommendations were developed using a consensus from clinicians and experts in the field of BMD testing and a standard method for the evaluation and citation of the supporting evidence. VALUES: These recommendations were developed by a multidisciplinary working group under the auspices of the Scientific Advisory Council of the Osteoporosis Society of Canada and the Canadian Association of Radiologists. BENEFITS, HARM, AND COSTS: Optimal BMD reports help the practitioner to assess an individual's risk for osteoporotic fracture and to decide whether medical therapy is warranted. RECOMMENDATIONS: The BMD report should include: patient identifiers. Dual-energy X-ray absorptiometry (DXA) scanner identifier. BMD results expressed in absolute values (g/cm2; 3 decimal places) and T-score (1 decimal place) for lumbar spine; proximal femur (total hip, femoral neck, and trochanter); and an alternate site (forearm BMD preferred: 1/3 radius, 33% radius or proximal radius) if either hip or spine is not valid. A statement about any limitations due to artifacts, if present. The fracture risk category (low, moderate, or high) as determined by using Tables 3 and 4 and by including major clinical factors that modify absolute fracture risk probability (with an indication of the corresponding absolute 10-year fracture risk of <10%, 10-20%, or >20%). A statement as to whether the change is statistically significant or not for serial measurements. The BMD centre's least significant change for each skeletal site (in g/cm2) should be included. VALIDATION: Recommendations were based on consensus opinion. Since these are the first Canadian recommendations integrating clinical risk factors in a quantitative fracture risk assessment, it is anticipated that these "Recommendations for BMD Reporting in Canada" will be a work in progress and will be updated periodically to accommodate advances in this field.