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Deidre M St. Peter,1 Jennifer S Steger,2 Jennifer L Patnaik,1 Nicole Davis,1 Malik Y Kahook,1 Leonard K Seibold1 1Department of Ophthalmology, University of Colorado School of Medicine, Aurora, CO, USA; 2Nanodropper, Inc, Rochester, MN, USACorrespondence: Leonard K Seibold, Department of Ophthalmology, University of Colorado School of Medicine, 1675 Aurora Court, Aurora, CO, 80045, USA, Tel +1 720-848-2020, Fax +1 720-848-5079, Email Leonard.seibold@cuanschutz.eduPurpose: To determine the drop volume and total number of dispensed drops using the Nanodropper eyedrop bottle adaptor (Nanodropper, Inc.) compared to drops dispensed from stock bottles to potentially limit ocular toxicity of these eyedrops and prolong bottle use.Patients and Methods: Six topical ocular hypotensive medications (5 solutions, 1 suspension), one steroid (suspension) and two artificial tears emulsions were selected for this study. An analytical balance was used to determine the mass per 10 drops with and without the volume-reducing adaptor and repeated until the bottles were completely emptied. The density of each product was determined using the calculated density. The average drop volume and number of drops per bottle for the nine medications were compared with and without the adaptor with paired t-testing.Results: When all medications were assessed, the drops delivered with the adaptor were 62.1% smaller than eyedrops administered from standard bottles. Compared to stock bottle eyedrops, which had a mean volume of 39.8 ± 2.1 μL, the adaptor resulted in drops with a mean volume of 15.1 ± 1.0 μL, p< 0.0001. The adaptor delivered 2.6x the number of drops dispensed from a standard 2.5 mL bottle (184.1 ± 15.1 drops with adaptor and 69.8 ± 4.9 drops from stock bottle, p< 0.0001).Conclusion: The Nanodropper eyedrop bottle adaptor can significantly reduce drop volume and increase the overall number of drops dispensed compared with stock eyedrop bottles. Further studies are needed to elucidate the clinical impact of utilizing decreased drop volume with direct comparison to current standards of care.Keywords: ophthalmic medication, medication delivery, dry eye syndrome, glaucoma medications, medication adherence, drop assist devices

Purpose: To examine if 12.5 μl timolol maleate 0.5% microdrops dispensed with the Nanodropper Adaptor provide noninferior intraocular pressure (IOP) reduction compared with conventional 28 μl drops in patients with open-angle glaucoma (OAG) and ocular hypertension (OHT)., Design: Prospective, noninferiority, parallel, multicenter, single-masked, active-controlled, randomized trial., Participants: Treatment-naïve subjects who were recently diagnosed with OAG and OHT at the Aravind Eye Care System., Methods: Both eyes of subjects received 1 commercially available drop or both eyes of subjects received 1 microdrop of timolol maleate 0.5%. We measured IOP, resting heart rate (HR), and blood pressure (BP) at baseline and 1, 2, 5, and 8 hours after timolol administration., Main Outcome Measures: The IOP was the primary outcome measure. Secondary outcomes were resting HR, systolic BP (sBP), and diastolic BP (dBP)., Results: Adaptor-mediated microdrops and conventional drops of timolol significantly decreased IOP compared with baseline at all timepoints. Noninferiority was established at 3 of 4 timepoints. Heart rate decreases with Nanodropper were approximately 3 beats per minute (bpm) less than with conventional drops., Conclusions: Timolol microdrops appear to be as effective in ocular hypotensive action as conventional drops with a slightly attenuated effect on resting HR and BP., Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references., (Copyright © 2024 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Purpose: To determine the drop volume and total number of dispensed drops using the Nanodropper eyedrop bottle adaptor (Nanodropper, Inc.) compared to drops dispensed from stock bottles to potentially limit ocular toxicity of these eyedrops and prolong bottle use., Patients and Methods: Six topical ocular hypotensive medications (5 solutions, 1 suspension), one steroid (suspension) and two artificial tears emulsions were selected for this study. An analytical balance was used to determine the mass per 10 drops with and without the volume-reducing adaptor and repeated until the bottles were completely emptied. The density of each product was determined using the calculated density. The average drop volume and number of drops per bottle for the nine medications were compared with and without the adaptor with paired t-testing., Results: When all medications were assessed, the drops delivered with the adaptor were 62.1% smaller than eyedrops administered from standard bottles. Compared to stock bottle eyedrops, which had a mean volume of 39.8 ± 2.1 μL, the adaptor resulted in drops with a mean volume of 15.1 ± 1.0 μL, p<0.0001. The adaptor delivered 2.6x the number of drops dispensed from a standard 2.5 mL bottle (184.1 ± 15.1 drops with adaptor and 69.8 ± 4.9 drops from stock bottle, p<0.0001)., Conclusion: The Nanodropper eyedrop bottle adaptor can significantly reduce drop volume and increase the overall number of drops dispensed compared with stock eyedrop bottles. Further studies are needed to elucidate the clinical impact of utilizing decreased drop volume with direct comparison to current standards of care., Competing Interests: The authors report no conflicts of interest in this work., (© 2023 St. Peter et al.)

Agouti-related peptide (AGRP)-expressing neurons are activated by fasting-this causes hunger 1-4 , an aversive state that motivates the seeking and consumption of food 5,6 . Eating returns AGRP neuron activity towards baseline on three distinct timescales: rapidly and transiently following sensory detection of food cues 6-8 , slowly and longer-lasting in response to nutrients in the gut 9,10 , and even more slowly and permanently with restoration of energy balance 9,11 . The rapid regulation by food cues is of particular interest as its neurobiological basis and purpose are unknown. Given that AGRP neuron activity is aversive 6 , the sensory cue-linked reductions in activity could function to guide behaviour. To evaluate this, we first identified the circuit mediating sensory cue inhibition and then selectively perturbed it to determine function. Here, we show that a lateral hypothalamic glutamatergic → dorsomedial hypothalamic GABAergic (γ-aminobutyric acid-producing) 12 → AGRP neuron circuit mediates this regulation. Interference with this circuit impairs food cue inhibition of AGRP neurons and, notably, greatly impairs learning of a sensory cue-initiated food-acquisition task. This is specific for food, as learning of an identical water-acquisition task is unaffected. We propose that decreases in aversive AGRP neuron activity 6 mediated by this food-specific circuit increases the incentive salience 13 of food cues, and thus facilitates the learning of food-acquisition tasks., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Stress is highly pervasive in humans, impacting motivated behaviors with an enormous toll on life quality. Many of the effects of stress are orchestrated by neuropeptides such as corticotropin-releasing factor (CRF). It has previously been shown that in stress-naïve male mice, CRF acts in the core of the nucleus accumbens (NAc) to produce appetitive effects and to increase dopamine release; yet in stress-exposed male mice, CRF loses its capacity to modulate NAc dopamine release and is aversive. In the current research, we tested whether this effect is comparable in females to males and whether the neuroadaptation is susceptible to social transmission. We found that, like in males, CRF increased dopamine release in stress-naïve but not stress-exposed female mice. Importantly, this persistent physiological change was not accompanied by overt behavioral changes that would be indicative of depression- or anxiety-like phenotype. Nonetheless, when these mice were housed for 7 days with stress-naïve conspecifics, the cage mates also exhibited a loss of dopamine potentiation by CRF. These data demonstrate the asymptomatic, yet pervasive transmission of stress-related neuroadaptations in the population., (Copyright © 2020 Steger, Land, Lemos, Chavkin and Phillips.)

SIM1-expressing paraventricular hypothalamus (PVH) neurons are key regulators of energy balance. Within the PVH SIM1 population, melanocortin-4 receptor-expressing (PVH MC4R ) neurons are known to regulate satiety and bodyweight, yet they account for only half of PVH SIM1 neuron-mediated regulation. Here we report that PVH prodynorphin-expressing (PVH PDYN ) neurons, which notably lack MC4Rs, function independently and additively with PVH MC4R neurons to account for the totality of PVH SIM1 neuron-mediated satiety. Moreover, PVH PDYN neurons are necessary for prevention of obesity in an independent but equipotent manner to PVH MC4R neurons. While PVH PDYN and PVH MC4R neurons both project to the parabrachial complex (PB), they synaptically engage distinct efferent nodes, the pre-locus coeruleus (pLC), and central lateral parabrachial nucleus (cLPBN), respectively. PB-projecting PVH PDYN neurons, like PVH MC4R neurons, receive input from interoceptive ARC AgRP neurons, respond to caloric state, and are sufficient and necessary to control food intake. This expands the CNS satiety circuitry to include two non-overlapping PVH to hindbrain circuits., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Agouti-related peptide (AgRP) neurons of the arcuate nucleus of the hypothalamus (ARC) promote homeostatic feeding at times of caloric insufficiency, yet they are rapidly suppressed by food-related sensory cues before ingestion. Here we identify a highly selective inhibitory afferent to AgRP neurons that serves as a neural determinant of this rapid modulation. Specifically, GABAergic projections arising from the ventral compartment of the dorsomedial nucleus of the hypothalamus (vDMH) contribute to the preconsummatory modulation of ARC AgRP neurons. In a manner reciprocal to ARC AgRP neurons, ARC-projecting leptin receptor-expressing GABAergic vDMH neurons exhibit rapid activation upon availability of food that additionally reflects the relative value of the food. Thus, leptin receptor-expressing GABAergic vDMH neurons form part of the sensory network that relays real-time information about the nature and availability of food to dynamically modulate ARC AgRP neuron activity and feeding behavior.

Understanding the role of hypothalamic neuropeptides and hormones in energy balance is paramount in the search for approaches to mitigate the obese state. Increased hypothalamic-pituitary-adrenal axis activity leads to increased levels of glucocorticoids (GC) that are known to regulate body weight. The axis initiates the production and release of corticotropin-releasing hormone (CRH) from the paraventricular nucleus (PVN) of the hypothalamus. Levels of active CRH peptide are dependent on the processing of its precursor pro-CRH by the action of two members of the family of prohormone convertases 1 and 2 (PC1 and PC2). Here, we propose that the nutrient sensor sirtuin 1 (Sirt1) regulates the production of CRH post-translationally by affecting PC2. Data suggest that Sirt1 may alter the preproPC2 gene directly or via deacetylation of the transcription factor Forkhead box protein O1 (FoxO1). Data also suggest that Sirt1 may alter PC2 via a post-translational mechanism. Our results show that Sirt1 levels in the PVN increase in rats fed a high fat diet for 12 weeks. Furthermore, elevated Sirt1 increased PC2 levels, which in turn increased the production of active CRH and GC. Collectively, this study provides the first evidence supporting the hypothesis that PVN Sirt1 activates the hypothalamic-pituitary-adrenal axis and basal GC levels by enhancing the production of CRH through an increase in the biosynthesis of PC2, which is essential in the maturation of CRH from its prohormone, pro-CRH., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Pro-opiomelanocortin (POMC)- and agouti-related peptide (AgRP)-expressing neurons of the arcuate nucleus of the hypothalamus (ARC) are oppositely regulated by caloric depletion and coordinately stimulate and inhibit homeostatic satiety, respectively. This bimodality is principally underscored by the antagonistic actions of these ligands at downstream melanocortin-4 receptors (MC4R) in the paraventricular nucleus of the hypothalamus (PVH). Although this population is critical to energy balance, the underlying neural circuitry remains unknown. Using mice expressing Cre recombinase in MC4R neurons, we demonstrate bidirectional control of feeding following real-time activation and inhibition of PVH(MC4R) neurons and further identify these cells as a functional exponent of ARC(AgRP) neuron-driven hunger. Moreover, we reveal this function to be mediated by a PVH(MC4R)→lateral parabrachial nucleus (LPBN) pathway. Activation of this circuit encodes positive valence, but only in calorically depleted mice. Thus, the satiating and appetitive nature of PVH(MC4R)→LPBN neurons supports the principles of drive reduction and highlights this circuit as a promising target for antiobesity drug development.

In the periphery, the nutrient-sensing enzyme Sirtuin 1 (silent mating type information regulation 2 homolog 1 [Sirt1]) reduces body weight in diet-induced obese (DIO) rodents. However, the role of hypothalamic Sirt1 in body weight and energy balance regulation is debated. The first studies to reveal that central Sirt1 regulates body weight came from experiments in our laboratory using Sprague-Dawley rats. Central inhibition of Sirt1 decreased body weight and food intake as a result of a forkhead box protein O1 (FoxO1)-mediated increase in the anorexigenic proopiomelanocortin (POMC) and decrease in the orexigenic Agouti-related peptide in the hypothalamic arcuate nucleus. Here, we demonstrate that central inhibition of Sirt1 in DIO decreased body weight and increased energy expenditure at higher levels as compared with the lean counterpart. Brain Sirt1 inhibition in DIO increased acetylated FoxO1, which in turn increased phosphorylated FoxO1 via improved insulin/phosphorylated AKT signaling. Elevated acetylated FoxO1 and phosphorylated FoxO1 increased POMC along with the α-melanocyte-stimulating hormone (α-MSH) maturation enzyme carboxypeptidase E, which resulted in more of the bioactive POMC product α-MSH released into the paraventricular nucleus. Increased in α-MSH led to augmented TRH levels and circulating T3 levels (triiodothyronine, thyroid hormone). These results indicate that inhibiting hypothalamic Sirt1 in DIO enhances the activity of the hypothalamic-pituitary-thyroid axis, which stimulates energy expenditure. Because we show that blocking central Sirt1 causes physiological changes that promote a negative energy balance in an obese individual, our results support brain Sirt1 as a significant target for weight loss therapeutics.

In the periphery, the nutrient-sensing enzyme Sirtuin 1 (silent mating type information regulation 2 homolog 1 [Sirt1]) reduces body weight in diet-induced obese (DIO) rodents. However, the role of Sirt1 in the brain, particularly the hypothalamus, in body weight and energy balance regulation is debated. Among the first studies to reveal that central Sirt1 regulates body weight came from experiments in our laboratory using Sprague Dawley rats. In that study, central inhibition of Sirt1 decreased body weight and food intake as a result of a Forkhead box protein O1 (FoxO1)-mediated increase in the anorexigenic proopiomelanocortin (POMC) and decrease in the orexigenic Agouti-related peptide in the hypothalamic arcuate nucleus. Here, we demonstrate that central inhibition of Sirt1 in DIO decreased body weight and increased energy expenditure at higher levels as compared with the lean counterpart. Brain Sirt1 inhibition in DIO increased acetylated FoxO1, which, in turn, increased phosphorylated FoxO1 via improved insulin/pAKT signaling. Elevated acetylated FoxO1 and phosphorylated FoxO1 increased POMC along with the α-MSH maturation enzyme carboxypeptidase E, which resulted in more of the bioactive POMC product α-MSH released into the paraventricular nucleus. Increased in α-MSH led to augmented TRH levels and circulating T3 levels (thyroid hormone). These results indicate that inhibiting hypothalamic Sirt1 in DIO enhances the activity of the hypothalamic-pituitary-thyroid axis, which stimulates energy expenditure. Because we show that blocking central Sirt1 causes physiological changes that promote a negative energy balance in an obese individual, our results support brain Sirt1 as a significant target for weight loss therapeutics.

Protein posttranslational processing is a cellular mechanism fundamental to the generation of bioactive peptides, including the anorectic α-melanocyte-stimulating hormone (α-MSH) and thyrotropin-releasing hormone (TRH) peptides produced in the hypothalamic arcuate (ARC) and paraventricular (PVN) nuclei, respectively. Neuropeptide Y (NPY) promotes positive energy balance in part by suppressing α-MSH and TRH. The mechanism by which NPY regulates α-MSH output, however, is not well understood. Our results reveal that NPY inhibited the posttranslational processing of α-MSH's inactive precursor proopiomelanocortin (POMC) by decreasing the prohormone convertase-2 (PC2). We also found that early growth response protein-1 (Egr-1) and NPY-Y1 receptors mediated the NPY-induced decrease in PC2. NPY given intra-PVN also decreased PC2 in PVN samples, suggesting a reduction in PC2-mediated pro-TRH processing. In addition, NPY attenuated the α-MSH-induced increase in TRH production by two mechanisms. First, NPY decreased α-MSH-induced CREB phosphorylation, which normally enhances TRH transcription. Second, NPY decreased the amount of α-MSH in the PVN. Collectively, these results underscore the significance of the interaction between NPY and α-MSH in the central regulation of energy balance and indicate that posttranslational processing is a mechanism that plays a specific role in this interaction.