Unraveling the architecture of the dorsal raphe synaptic neuropil using high-resolution neuroanatomy

The dorsal raphe nucleus (DRN), representing the main source of brain’s serotonin, is implicated in the pathophysiology and therapeutics of several mental disorders that can be debilitating and life-long including depression, anxiety and autism. The activity of DRN neurons is precisely regulated, both phasically and tonically, by excitatory glutamate and inhibitory GABAergic axons arising from extra-raphe areas as well as from local sources within the nucleus. Changes in serotonin neurotransmission associated with pathophysiology may be encoded by alterations within this network of regulatory afferents. However, the complex organization of the DRN circuitry remains still poorly understood. Using a recently developed high-resolution immunofluorescence technique called array tomography (AT) we quantitatively analyzed the relative contribution of different populations of glutamate axons originating from different brain regions to the excitatory drive of the DRN. Additionally, we examined the presence of GABA axons within the DRN and their possible association with glutamate axons. In this review, we summarize our findings on the architecture of the rodent DRN synaptic neuropil using high-resolution neuroanatomy, and discuss possible functional implications for the nucleus. Understanding of the synaptic architecture of neural circuits at high resolution will pave the way to understand how neural structure and function may be perturbed in pathological states

Endogenous Cholinergic Neurotransmission Contributes to Behavioral Sensitization to Morphine

Neuroplasticity in the mesolimbic dopaminergic system is critical for behavioral adaptations associated with opioid reward and addiction. These processes may be influenced by cholinergic transmission arising from the laterodorsal tegmental nucleus (LDTg), a main source of acetylcholine to mesolimbic dopaminergic neurons. To examine this possibility we asked if chronic systemic morphine administration affects expression of genes in ventral and ventrolateral periaqueductal gray at the level of the LDTg using rtPCR. Specifically, we examined gene expression changes in the area of interest using Neurotransmitters and Receptors PCR array between chronic morphine and saline control groups. Analysis suggested that chronic morphine administration led to changes in expression of genes associated, in part, with cholinergic neurotransmission. Furthermore, using a quantitative immunofluorescent technique, we found that chronic morphine treatment produced a significant increase in immunolabeling of the cholinergic marker (vesicular acetylcholine transporter) in neurons of the LDTg. Finally, systemic administration of the nonselective and noncompetitive neuronal nicotinic antagonist mecamylamine (0.5 or 2 mg/kg) dose-dependently blocked the expression, and to a lesser extent the development, of locomotor sensitization. The same treatment had no effect on acute morphine antinociception, antinociceptive tolerance or dependence to chronic morphine. Taken together, the results suggest that endogenous nicotinic cholinergic neurotransmission selectively contributes to behavioral sensitization to morphine and this process may, in part, involve cholinergic neurons within the LDTg

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Evidence for topographically organized endogenous 5-HT-1A receptor-dependent feedback inhibition of the ascending serotonin system

Abstract Raphe and extra-raphe 5-HT-1A receptors contribute to feedback inhibition of serotonin (5-HT) neurons; however, the endogenous function of 5-HT-1A receptor-dependent feedback inhibition remains poorly understood. Here, the possibility that 5-HT-1A-mediated feedback inhibition of the raphe nuclei is topographically organized was examined. This was done by testing the effect of systemic blockade of 5-HT-1A receptors on Fos expression in 5-HT neurons in the dorsal raphe (DR) and median raphe (MR). The premise was that appearance of Fos after 5-HT-1A receptor blockade would implicate endogenous inhibition via 5-HT-1A-dependent processes. 5-HT-1A receptor antagonist administration (WAY-100635) in rats returned to their home cage significantly increased the number of Fos-containing 5-HT cells in the lateral wings and the ventral caudal part of the DR as compared to vehicle-injected controls, suggesting that tonic activity of brain 5-HT-1A receptors impacts on these regions. In rats receiving vehicle injections, swim, a behavior known to influence 5-HT neurotransmission, increased the number of Fos-containing 5-HT cells only in the caudal third of DR. Administration of WAY-100635 preceding a swim did not change the amount of Fos in the caudal DR, but increased the number of Fos-containing 5-HT cells in the rostral DR, lateral wings of the DR, and MR. These results confirm, using an imaging approach, that 5-HT-1A receptor-dependent feedback inhibition depends on behavioral state (return to home cage vs. swim). Moreover, they reveal that the effect of 5-HT-1A receptor blockade in each case is subregionally organized

What Gene Mutations Affect Serotonin in Mice?

Although serotonin neurotransmission has been implicated in several neurodevelopmental and psychological disorders, the factors that drive dysfunction of the serotonin system are poorly understood. Current research regarding the serotonin system revolves around its dysfunction in neuropsychiatric disorders, but there is no database collating genetic mutations that result in serotonin abnormalities. To bridge this gap, we developed a list of genes in mice that, when perturbed, result in altered levels of serotonin either in brain or blood. Due to the intrinsic limitations of search, the current list should be considered a preliminary subset of all relevant cases. Nevertheless, it offered an opportunity to gain insight into what types of genes have the potential to impact serotonin by using gene ontology (GO). This analysis found that genes associated with monoamine metabolism were more often associated with increases in brain serotonin than decreases. Speculatively, this could be because several pathways (and therefore many genes) are responsible for the clearance and metabolism of serotonin whereas only one pathway (and therefore fewer genes) is directly involved in the synthesis of serotonin. Another contributor could be cross talk between monoamine systems such as dopamine. In contrast, genes that were associated with decreases in brain serotonin were more likely linked to a developmental process. Sensitivity of serotonin neurons to developmental perturbations could be due to their complicated neuroanatomy or possibly they may be negatively regulated by dysfunction of their innervation targets. Thus, these observations suggest hypotheses regarding the mechanisms underlying the vulnerability of brain serotonin neurotransmission

Circuitry underlying regulation of the serotonergic system by swim stress

The dorsal raphe nucleus (DR)–serotonin (5-HT) system has been implicated in depression and is dramatically affected by swim stress, an animal model with predictive value for antidepressants. Accumulating evidence implicates the stress-related neuropeptide corticotropin-releasing factor (CRF) in the effect of swim stress on this system. This study investigated neural circuits within the DR that are activated by swim stress as revealed by neuronal expression of the immediate early gene, c-fos. Swim stress increased c-fos expression in the dorsolateral subregion of the DR. The majority of c-fos-expressing neurons were doubly labeled for GABA (85 ± 5%), whereas relatively few were immunolabeled for 5-HT (4 ± 1%), glutamate (0.5 ± 0.3%) or calbindin (1.5 ± 0.3%). Dual immunohistochemical labeling revealed that c-fos-expressing neurons in the dorsolateral DR were enveloped by dense clusters of CRF-immunoreactive fibers and also contained immunolabeling for CRF receptor, suggesting that c-fos-expressing neurons in the DR were specifically targeted by CRF. Consistent with this, the CRF receptor 1 antagonist, antalarmin, prevented swim-stress-elicited c-fos expression in the dorsolateral DR. Together with previous findings that both swim stress and CRF decrease 5-HT release in certain forebrain regions, these results suggest that swim stress engages CRF inputs to GABA neurons in the dorsolateral DR that function to inhibit 5-HT neurons and 5-HT release in the forebrain. This circuitry may underlie some of the acute behavioral responses to swim stress as well as the neuronal plasticity involved in long-term behavioral changes produced by this stress

Severe spontaneous bradycardia associated with respiratory disruptions in rat pups with fewer brain stem 5-HT neurons

The medullary 5-HT system has potent effects on heart rate and breathing in adults. We asked whether this system mitigates the respiratory instability and bradycardias frequently occurring during the neonatal period. 5,7-Dihydroxytryptamine (5,7-DHT) or vehicle was administered to rat pups at postnatal day 2 (P2), and we then compared the magnitude of bradycardias occurring with disruptions to eupnea in treated and vehicle control littermates at P5–6 and P10–12. We then used a novel method that would allow accurate assessment of the ventilatory and heart rate responses to near square-wave challenges of hypoxia (10% O2), hypercapnia (5 and 8% CO2 in normoxia and hyperoxia), and asphyxia (8% CO2-10% O2), and to the induction of the Hering-Breuer inflation reflex (HBR), a potent, apnea-inducing reflex in newborns. The number of 5-HT-positive neurons was reduced ∼80% by drug treatment. At both ages, lesioned animals had considerably larger bradycardias during brief apnea; at P5–6, average and severe events were ∼50% and 70% greater, respectively, in lesioned animals (P = 0.002), whereas at P10–12, events were ∼ 23% and 50% greater (P = 0.018). However, lesioning had no effect on the HR responses to sudden gas challenge or the HBR. At P5–6, lesioned animals had reduced breathing frequency and ventilation (V̇e), but normal V̇e relative to metabolic rate (V̇e/V̇o2). At P10–12, lesioned animals had a more unstable breathing pattern (P = 0.04) and an enhanced V̇e response to moderate hypercapnia (P = 0.007). Within the first two postnatal weeks, the medullary 5-HT system plays an important role in cardiorespiratory control, mitigating spontaneous bradycardia, stabilizing the breathing pattern, and dampening the hypercapnic V̇e response