Karine Fénelon

Sensorimotor gating, a fundamental pre-attentive process, can be assessed using the acoustic prepulse inhibition (PPI) assay. PPI deficits are a hallmark endophenotype of schizophrenia and are observed across various neuropsychiatric disorders, often predicting symptoms such as attention impairments, psychosis, and other cognitive/motor dysfunctions. Reversal of PPI deficits is routinely tested in disease models as a preclinical trial for antipsychotic drug screening. However, the cellular and circuit-level mechanisms underlying PPI deficits remain unclear, limiting therapeutic progress. We recently identified an uncharted pathway in mice, by which glutamatergic neurons in the central nucleus of the amygdala (CeA) activate glycinergic neurons in the caudal pontine reticular nucleus (PnC), contributing to PPI regulation. Given the prevalence of amygdala dys-function in disorders associated with PPI deficits, CeA-PnC glutamatergic synapses represent a novel therapeutic target. Here, using “Cal-Light,” an in vivo Ca2 ⁺ -dependent photo-tagging approach, we precisely identified CeA and PnC neurons active during acoustic startle and PPI with high spatiotemporal resolution. Furthermore, we used mice carrying homozygous deficiency in PRODH , a schizophrenia-relevant gene encoding proline dehydrogenase and modulating PPI. While Prodh ^−/− mice showed aberrant CeA neuronal properties and reduced PPI levels, we restored PPI by photo-activating CeA-PnC glutamatergic synapses, underscoring their involvement in pathological states. These findings provide new mechanistic insights into the amygdala-brainstem circuitry that underlies PPI deficits, offering new potential for therapeutic interventions.

Prepulse inhibition (PPI) of the auditory startle reflex task is a widely recognized operational measure of sensorimotor gating. PPI deficits are a hallmark feature of schizophrenia, often associated with attentional and cognitive impairments. Despite its extensive use in preclinical research for screening antipsychotic drugs, the precise cellular and circuit mechanisms underlying PPI remain unclear, even under physiological conditions. Recent evidence suggests that non-cholinergic inputs from the pedunculopontine tegmental nucleus (PPTg) to the caudal pontine reticular nucleus (PnC) mediate PPI. In this study, we investigated the contribution of PPTg glutamatergic neurons to acoustic startle and PPI. Tract-tracing, immuno-histochemical analyses, and in vitro whole-cell recordings in wild-type mice confirmed that PPTg glutamatergic neurons innervate the PnC. Optogenetic inhibition of PPTg-PnC glutamatergic synapses in vivo resulted in increased PPI across various interstimulus intervals. Notably, while optogenetic activation of this pathway had no additional effect on startle and PPI, activation of this connection alone before startle stimulation reduced startle at short interstimulus intervals and increased startle at longer intervals. Furthermore, although PPTg glutamatergic inputs target PnC glycinergic neurons, our in vitro whole-cell recordings combined with optogenetic stimulation at PPTg-PnC synapses revealed that PPTg glutamatergic inputs activate PnC glutamatergic giant neurons. Our findings identify a feed-forward excitatory mechanism within the brainstem startle circuit, whereby PPTg glutamatergic inputs modulate PnC neuronal activity. These results provide new insights into the clinically relevant theoretical construct of PPI, which is disrupted in various neuropsychiatric and neurological disorders. Significance Statement: The pedunculopontine tegmental nucleus (PPTg) contributes to prepulse inhibition (PPI) of startle, a translational measure of sensorimotor gating that is impaired in neuropsychiatric disorders, including schizophrenia. While PPTg cholinergic neurons have been shown not to contribute to PPI, the mechanisms by which non-cholinergic PPTg neurons modulate PPI remain incompletely understood. Here, combining tract-tracing, optogenetics, in vitro electrophysiology, and in vivo startle testing in mice, we show that PPTg glutamatergic neurons that innervate startle-mediating neurons of the caudal pontine reticular nucleus (PnC) play a critical role in PPI. Their activation modulates startle responses in a time-dependent manner, either decreasing or increasing startle magnitude. These findings refine our understanding of sensorimotor gating neural mechanisms and may inform therapeutic strategies.

SUMMARY: Prepulse inhibition (PPI) of the auditory startle response is the gold standard operational measure of sensorimotor gating. Affected by various neurological and neuropsychiatric illnesses, PPI also declines during aging. While PPI deficits are often associated with cognitive overload, attention impairments and motor dysfunctions, their reversal is routinely used in experimental systems for drug screening. Yet, the cellular and circuit-level mechanisms of PPI remain unclear, even under non-pathological conditions. Recent evidence shows that neurons located in the brainstem caudal pontine reticular nucleus (PnC) expressing the glycine transporter type 2 (GlyT2 ⁺ ) receive inputs from the central nucleus of the amygdala (CeA) and contribute to PPI via an uncharted pathway. Using tract-tracing and immunohistochemical analyses in GlyT2-eGFP mice, we reveal the neuroanatomical location of CeA glutamatergic neurons innervating GlyT2 ⁺ neurons. Our precise in vitro optogenetic manipulations coupled to field electrophysiological recordings demonstrate that CeA glutamatergic inputs do suppress auditory neurotransmission in PnC neurons but not via action on transmitter release from auditory afferents. Rather, our data is consistent with excitatory drive onto GlyT2 ⁺ neurons. Indeed, our PPI experiments in vivo demonstrate that optogenetic activation of GlyT2 ⁺ PnC neurons increases PPI and is sufficient to induce PPI, clarifying the crucial role of these neurons in young GlyT2-Cre mice. In contrast, in older GlyT2-Cre mice, PPI is reduced and not further altered by optogenetic inhibition of GlyT2 ⁺ neurons. We conclude that GlyT2 ⁺ PnC neurons innervated by CeA glutamatergic inputs are crucial for PPI and we highlight their reduced activity during the age-dependent decline in PPI. SIGNIFICANCE STATEMENT: Sensorimotor gating is a pre-attentive mechanism that declines with age and that is affected by neuropsychiatric and neurological disorders. Prepulse inhibition (PPI) of startle commonly measures sensorimotor gating to assess cognitive and motor symptoms and to screen drug efficacy. Yet, the neuronal mechanisms underlying PPI are still unresolved, limiting therapeutic advances. Here, we identify brainstem glycinergic neurons essential for PPI using tract tracing, in vitro electrophysiology and precise in vivo optogenetic manipulations during startle measurements in mice. Innervated by amygdala glutamatergic inputs, we show that these glycinergic neurons are essential and sufficient to induce PPI in young mice. In contrast, these neurons do not contribute to PPI in older mice. We provide new insights to the theoretical construct of PPI.

Background: Sensorimotor gating is a fundamental pre-attentive process that is defined as the inhibition of a motor response by a sensory event. Sensorimotor gating, commonly measured using the prepulse inhibition (PPI) of the auditory startle reflex task, is impaired in patients suffering from various neurological and psychiatric disorders. PPI deficits are a hallmark of schizophrenia, and they are often associated with attention and other cognitive impairments. Although the reversal of PPI deficits in animal models is widely used in pre-clinical research for antipsychotic drug screening, the neurotransmitter systems and synaptic mechanisms underlying PPI are still not resolved, even under physiological conditions. Recent evidence ruled out the longstanding hypothesis that PPI is mediated by midbrain cholinergic inputs to the caudal pontine reticular nucleus (PnC). Instead, glutamatergic, glycinergic, and GABAergic inhibitory mechanisms are now suggested to be crucial for PPI, at the PnC level. Since amygdalar dysfunctions alter PPI and are common to pathologies displaying sensorimotor gating deficits, the present study was designed to test that direct projections to the PnC originating from the amygdala contribute to PPI. Results: Using wild type and transgenic mice expressing eGFP under the control of the glycine transporter type 2 promoter (GlyT2-eGFP mice), we first employed tract-tracing, morphological reconstructions, and immunohistochemical analyses to demonstrate that the central nucleus of the amygdala (CeA) sends glutamatergic inputs lateroventrally to PnC neurons, including GlyT2⁺ cells. Then, we showed the contribution of the CeA-PnC excitatory synapses to PPI in vivo by demonstrating that optogenetic inhibition of this connection decreases PPI, and optogenetic activation induces partial PPI. Finally, in GlyT2-Cre mice, whole-cell recordings of GlyT2⁺ PnC neurons in vitro paired with optogenetic stimulation of CeA fibers, as well as photo-inhibition of GlyT2⁺ PnC neurons in vivo, allowed us to implicate GlyT2⁺ neurons in the PPI pathway. Conclusions: Our results uncover a feedforward inhibitory mechanism within the brainstem startle circuit by which amygdalar glutamatergic inputs and GlyT2⁺ PnC neurons contribute to PPI. We are providing new insights to the clinically relevant theoretical construct of PPI, which is disrupted in various neuropsychiatric and neurological diseases.

Background: Sensorimotor gating is a fundamental neural filtering process that allows attention to be focused on a given stimulus. Sensory gating, commonly measured using the prepulse inhibition (PPI) of the auditory startle reflex task, is impaired in patients suffering from various neurological and psychiatric disorders, including schizophrenia. Because PPI deficits are often associated with attention and cognitive impairments, they are widely used as biomarkers in pre-clinical research for anti-psychotic drug screening. Yet, the neurotransmitter systems and synaptic mechanisms underlying PPI are still not resolved, even under physiological conditions. Recent evidence ruled out the longstanding hypothesis that PPI is mediated by midbrain cholinergic inputs to the caudal pontine reticular nucleus (PnC). Instead, glutamatergic, glycinergic and GABAergic inhibitory mechanisms are now suggested to be crucial for PPI, at the PnC level. Since amygdalar dysfunctions affect PPI and are common to pathologies displaying sensorimotor gating deficits, the present study was designed to test that direct projections to the PnC originating from the amygdala, contribute to PPI. Results: Using Wild Type and transgenic mice expressing eGFP under the control of the glycine transporter type 2 promoter (GlyT2-eGFP mice), we first employed tract-tracing, morphological reconstructions and immunohistochemical analyses to demonstrate that the central nucleus of the amygdala (CeA) sends glutamatergic inputs latero-ventrally to PnC neurons, including GlyT2 ⁺ cells. Then, we showed the contribution of the CeA-PnC excitatory synapses to PPI in vivo by demonstrating that optogenetic inhibition of this connection decreases PPI, and optogenetic activation induces partial PPI. Finally, in GlyT2-Cre mice, whole-cell recordings of GlyT2 ⁺ PnC neurons in vitro paired with optogenetic stimulation of CeA fibers, as well as photo-inhibition of GlyT2 ⁺ PnC neurons in vivo , allowed us to implicate GlyT2 ⁺ neurons in the PPI pathway. Conclusions: Our results uncover a feed-forward inhibitory mechanism within the brainstem startle circuit by which amygdalar glutamatergic inputs and GlyT2 ⁺ PnC neurons play a key role in meditating PPI. We are providing new insights to the clinically-relevant theoretical construct of PPI, which is disrupted in various neuropsychiatric and neurodevelopmental diseases.

Creosote bush (Larrea tridentata; LT) leaves extracts were tested for their potential efficacy to mitigate cellular oxidative stress on human SH-SY5Y cells. Here, the differential nuclear staining assay, a bioimager system, and flow cytometric protocols, concurrently with several specific chemicals, were used to measure the percentage of cell viability and several facets implicated in the cytoprotective mechanism of LT extracts. Initially, three LT extracts, prepared with different solvents, ethanol, ethanol:water (e/w), and water, were tested for their capacity to rescue the viability of cells undergoing aggressive H₂O₂-induced oxidative stress. Results indicate that the LT extract prepared with a mixture of ethanol:water (LT-e/w; 60:40% v/v) displayed the most effective cytoprotection rescue activity. Interestingly, by investigating the LT-e/w mechanism of action, it was found that LT-e/w extract decreases the levels of H₂O₂-provoked reactive oxidative species (ROS) accumulation, mitochondrial depolarization, phosphatidylserine externalization, caspase-3/7 activation, and poly (ADP-ribose) polymerase (PARP) cleavage significantly, which are hallmarks of apoptosis. Thus, out of the three LT extracts tested, our findings highlight that the LT-e/w extract was the most effective protective reagent on SH-SY5Y cells undergoing oxidative stress in vitro, functioning as a natural anti-apoptotic extract. These findings warrant further LT-e/w extract examination in a holistic context.

Introduction: Lead (Pb) exposure yielding blood lead levels (BLL) as low as 2 µg/dl in children is an international problem. More common in US low‐income neighborhoods, childhood Pb exposure can cause behavioral and cognitive deficits, including working memory impairments, which can persist into adulthood. So far, studies characterized short‐term effects of high Pb exposure on neuronal structure and function. However, long‐term consequences of early chronic Pb exposure on neuronal activity are poorly documented.Methods: Here, we exposed male and female mice (PND [postnatal day] 0 to PND 28) to one of three Pb treatments: 0 ppm (sodium‐treated water, control), 30 ppm (low dose), and 330 ppm (high dose) lead acetate. Once the male and female mice were 9–12 months old, extracellular field recordings on hippocampal slices were performed.Results: We show that at CA3 to CA1 synapses, synaptic transmission was decreased and neuronal fiber activity was increased in males exposed to lowest level Pb. In contrast, both synaptic transmission and neuronal fiber activity were increased in females exposed to high Pb. The ventral hippocampus–medial prefrontal cortex (vHPC–mPFC) synapses are crucial for working memory in rodents. The lowest level Pb decreased vHPC–mPFC synaptic transmission, whereas high Pb decreased short‐term synaptic depression.Conclusions: Overall, we show for the first time that early exposure to either high or lowest level Pb has long‐term consequences on different synaptic properties of at least two hippocampal synapses. Such consequences of early Pb exposure might worsen the cognitive decline observed in aging men and women. Our results suggest that additional efforts should focus on the consequences of early Pb exposure especially in at‐risk communities.

Oxidative stress has been linked to neurodegenerative diseases such as Huntington’s, Parkinson’s, Alzheimer’s and amyotrophic lateral sclerosis diseases. Larrea tridentata (LT) also known as Creosote Bush is an evergreen shrub found in the Chihuahuan desert which has been used medicinally by Native American tribes in southwestern North America and the Amerindians of South America. However, studies of the antioxidant capacity of the crude extract of LT towards the discovery of novel molecular therapies bearing antioxidants and drug-like properties are lacking. In this study, we assessed the antioxidant properties of Larrea tridentata, collected specifically from the Chihuahuan desert in the region of El Paso del Norte, TX, USA. LT phytochemicals were obtained from three different extracts (ethanol; ethanol: water (60:40) and water). Then the extracts were evaluated in eight different assays (DPPH, ABTS, superoxide; FRAP activity, nitric oxide, phenolic content, UV visible absorption and cytotoxicity in non-cancerous HS27 cells). The three extracts were not affecting the HS27 cells at concentrations up to 120 µg/mL. Among the three extracts, we found that the mixture of ethanol: water (60:40) LT extract has the most efficient antioxidant properties (IC₅₀ (DPPH at 30 min) = 111.7 ± 3.8 μg/mL; IC₅₀ (ABTS) = 8.49 ± 2.28 μg/mL; IC₅₀ (superoxide) = 0.43 ± 0.17 μg/mL; IC₅₀ (NO) = 230.4 ± 130.4 μg/mL; and the highest phenolic content was estimated to 212.46 ± 7.05 mg GAE/L). In addition, there was a strong correlation between phenolic content and the free-radical scavenging activity assays. HPLC-MS study identified nine compounds from the LT-ethanol: water extract including Justicidin B and Beta peltain have been previously reported as secondary metabolites of Larrea tridentata.