Narrowing the translational research gap by aligning replication concepts in basic and clinical neuroscience.

Replicability and reproducibility are widely considered to be cornerstones of valid scientific research. Yet, the elements of replication in fundamental neuroscience studies do not fully overlap with the process of replication in clinical neuroscience involving patients. Here we discuss how better aligning the concept of replication across this translational spectrum might enhance the rate at which basic findings in the organization and function of the nervous system are leveraged to develop new treatments for psychiatric and neurological disorders.

Acute manganese exposure impairs glutamatergic function in a young mouse model of Alzheimer's disease.

Manganese (Mn) is an essential metal that serves as a cofactor for metalloenzymes important in moderating oxidative stress and the glutamate/glutamine cycle. Mn is typically obtained through the diet, but toxic overexposure can occur through other environmental or occupational exposure routes such as inhalation. Mn is known to accumulate in the brain following exposure and may contribute to the etiology of neurodegenerative disorders such as Alzheimer's disease (AD) even in the absence of acute neurotoxicity. In the present study, we used in vitro primary cell culture, ex vivo slice electrophysiology and in vivo behavioral approaches to determine if Mn-induced changes in glutamatergic signaling may be altered by genetic risk factors for AD neuropathology. Primary cortical astrocytes incubated with Mn exhibited early rapid clearance of glutamate compared to saline treated astrocytes but decreased clearance over longer time periods, with no effect of the AD genotype. Further, we found that in vivo exposure to a subcutaneous subacute, high dose of Mn as manganese chloride tetrahydrate (3 ×50 mg/kg MnCl2·4(H2O) over 7 days) resulted in increased expression of cortical GLAST protein regardless of genotype, with no changes in GLT-1. Hippocampal long-term potentiation was not altered in APP/PSEN1 mice at this age and neither was it disrupted following Mn exposure. Mn exposure did increase sensitivity to seizure onset following treatment with the excitatory agonist kainic acid, with differing responses between APP/PSEN1 and control mice. These results highlight the sensitivity of the glutamatergic system to Mn exposure. Experiments were performed in young adult APP/PSEN1 mice, prior to cognitive decline or accumulation of hallmark amyloid plaque pathology and following subacute exposure to Mn. The data support a role of Mn in pathophysiology of AD in early stages of the disease and support the need to better understand neurological consequences of Mn exposure in vulnerable populations.

Altered EEG, disrupted hippocampal long-term potentiation and neurobehavioral deficits implicate a delirium-like state in a mouse model of sepsis.

Sepsis and systemic inflammation are often accompanied by severe encephalopathy, sleep disruption and delirium that strongly correlate with poor clinical outcomes including long-term cognitive deficits. The cardinal manifestations of delirium are fluctuating altered mental status and inattention, identified in critically ill patients by interactive bedside assessment. The lack of analogous assessments in mouse models or clear biomarkers is a challenge to preclinical studies of delirium. In this study, we utilized concurrent measures of telemetric EEG recordings and neurobehavioral tasks in mice to characterize inattention and persistent cognitive deficits following polymicrobial sepsis. During the 24-hour critical illness period for the mice, slow-wave EEG dominance, sleep disruption, and hypersensitivity to auditory stimuli in neurobehavioral tasks resembled clinical observations in delirious patients in which alterations in similar outcome measurements, although measured differently in mice and humans, are reported. Mice were tested for nest building ability 7 days after sepsis induction, when sickness behaviors and spontaneous activity had returned to baseline. Animals that showed persistent deficits determined by poor nest building at 7 days also exhibited molecular changes in hippocampal long-term potentiation compared to mice that returned to baseline cognitive performance. Together, these behavioral and electrophysiological biomarkers offer a robust mouse model with which to further probe molecular pathways underlying brain and behavioral changes during and after acute illness such as sepsis.

30-Day readmission rates in pediatric patients with functional seizures.

PURPOSE: To ascertain the rates of 30-day readmissions and emergency department presentations among pediatric patients with an index admission for functional seizures. METHOD: A retrospective chart review of pediatric patients with an index discharge from the pediatric epilepsy monitoring unit (EMU) or general neurology service for functional seizures. Data collected included demographics, comorbidities, risk factors, and treatment during the index admission. RESULTS: A total of one hundred and two patients were included, of which nearly one in five had a 30-day readmission or emergency department presentation. Index admission to the general neurology service was independently associated with more re-presentations to the hospital (t = 3.26, p < 0.0015). The univariate analysis indicated that cognitive impairment and autism were associated with a lower likelihood of readmission, while a neurology referral and being started on an anti-seizure medication were associated with a greater likelihood of readmission. CONCLUSION: A substantial proportion of pediatric patients with FS return to the hospital within 30 days of discharge. Our data suggest that patients admitted to the EMU service have a lower likelihood of readmission. We speculate that this may be due to differences in patient clinical characteristics as well as the comprehensiveness of the diagnostic evaluation and management in the EMU compared to the general neurology service.

Glucagon-like peptide-1 receptor differentially controls mossy cell activity across the dentate gyrus longitudinal axis.

Understanding the role of dentate gyrus (DG) mossy cells (MCs) in learning and memory has rapidly evolved due to increasingly precise methods for targeting MCs and for in vivo recording and activity manipulation in rodents. These studies have shown MCs are highly active in vivo, strongly remap to contextual manipulation, and that their inhibition or hyperactivation impairs pattern separation and location or context discrimination. Less well understood is how MC activity is modulated by neurohormonal mechanisms, which might differentially control the participation of MCs in cognitive functions during discrete states, such as hunger or satiety. In this study, we demonstrate that glucagon-like peptide-1 (GLP-1), a neuropeptide produced in the gut and the brain that regulates food consumption and hippocampal-dependent mnemonic function, might regulate MC function through expression of its receptor, GLP-1R. RNA-seq demonstrated that most, though not all, Glp1r in hippocampal principal neurons is expressed in MCs, and in situ hybridization revealed strong expression of Glp1r in hilar neurons. Glp1r-ires-Cre mice crossed with Ai14D reporter mice followed by co-labeling for the MC marker GluR2/3 revealed that almost all MCs in the ventral DG expressed Glp1r and that almost all Glp1r-expressing hilar neurons were MCs. However, only ~60% of dorsal DG MCs expressed Glp1r, and Glp1r was also expressed in small hilar neurons that were not MCs. Consistent with this expression pattern, peripheral administration of the GLP-1R agonist exendin-4 (5 µg/kg) increased cFos expression in ventral but not dorsal DG hilar neurons. Finally, whole-cell patch-clamp recordings from ventral MCs showed that bath application of exendin-4 (200 nM) depolarized MCs and increased action potential firing. Taken together, this study adds to known MC activity modulators a neurohormonal mechanism that may preferentially affect ventral DG physiology and may potentially be targetable by several GLP-1R pharmacotherapies already in clinical use.

Disruption of Synaptic Transmission in the Bed Nucleus of the Stria Terminalis Reduces Seizure-Induced Death in DBA/1 Mice and Alters Brainstem E/I Balance.

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death in refractory epilepsy patients. Accumulating evidence from recent human studies and animal models suggests that seizure-related respiratory arrest may be important for initiating cardiorespiratory arrest and death. Prior evidence suggests that apnea onset can coincide with seizure spread to the amygdala and that stimulation of the amygdala can reliably induce apneas in epilepsy patients, potentially implicating amygdalar regions in seizure-related respiratory arrest and subsequent postictal hypoventilation and cardiorespiratory death. This study aimed to determine if an extended amygdalar structure, the dorsal bed nucleus of the stria terminalis (dBNST), is involved in seizure-induced respiratory arrest (S-IRA) and death using DBA/1 mice, a mouse strain which has audiogenic seizures (AGS) and a high incidence of postictal respiratory arrest and death. The presence of S-IRA significantly increased c-Fos expression in the dBNST of DBA/1 mice. Furthermore, disruption of synaptic output from the dBNST via viral-induced tetanus neurotoxin (TeNT) significantly improved survival following S-IRA in DBA/1 mice without affecting baseline breathing or hypercapnic (HCVR) and hypoxic ventilatory response (HVR). This disruption in the dBNST resulted in changes to the balance of excitatory/inhibitory (E/I) synaptic events in the downstream brainstem regions of the lateral parabrachial nucleus (PBN) and the periaqueductal gray (PAG). These findings suggest that the dBNST is a potential subcortical forebrain site necessary for the mediation of S-IRA, potentially through its outputs to brainstem respiratory regions.

Enhanced Synaptic Transmission in the Extended Amygdala and Altered Excitability in an Extended Amygdala to Brainstem Circuit in a Dravet Syndrome Mouse Model.

Dravet syndrome (DS) is a developmental and epileptic encephalopathy with an increased incidence of sudden death. Evidence of interictal breathing deficits in DS suggests that alterations in subcortical projections to brainstem nuclei may exist, which might be driving comorbidities in DS. The aim of this study was to determine whether a subcortical structure, the bed nucleus of the stria terminalis (BNST) in the extended amygdala, is activated by seizures, exhibits changes in excitability, and expresses any alterations in neurons projecting to a brainstem nucleus associated with respiration, stress response, and homeostasis. Experiments were conducted using F1 mice generated by breeding 129.Scn1a+/- mice with wild-type C57BL/6J mice. Immunohistochemistry was performed to quantify neuronal c-fos activation in DS mice after observed spontaneous seizures. Whole-cell patch-clamp and current-clamp electrophysiology recordings were conducted to evaluate changes in intrinsic and synaptic excitability in the BNST. Spontaneous seizures in DS mice significantly enhanced neuronal c-fos expression in the BNST. Further, the BNST had altered AMPA/NMDA postsynaptic receptor composition and showed changes in spontaneous neurotransmission, with greater excitation and decreased inhibition. BNST to parabrachial nucleus (PBN) projection neurons exhibited intrinsic excitability in wild-type mice, while these projection neurons were hypoexcitable in DS mice. The findings suggest that there is altered excitability in neurons of the BNST, including BNST-to-PBN projection neurons, in DS mice. These alterations could potentially be driving comorbid aspects of DS outside of seizures, including respiratory dysfunction and sudden death.

Near SUDEP during bilateral stereo-EEG monitoring characterized by diffuse postictal EEG suppression.

Sudden unexpected death in epilepsy (SUDEP) is the most common cause of death in patients with refractory epilepsy. The pathophysiology of SUDEP is unknown. Postictal phenomena such as postconvulsive immobility (PI), postictal generalized electroencephalography (EEG) suppression (PGES), arousal deficits, cardiac arrhythmias, central apneas, and obstructive apneas due to laryngospasms have been suggested to contribute to SUDEP. We present, to our knowledge, the first case of a near-SUDEP event in a patient undergoing intracranial, stereotactic EEG (sEEG) monitoring. This case spotlights potential mediators of SUDEP, most notably the striking PGES and postictal apnea. The nature of the sEEG investigation illustrates the extent of cortical and subcortical postictal EEG suppression and showcases a transient return of cerebral activity likely to be missed on scalp-EEG recording. Critically, this case emphasizes the importance of continuous cardiorespiratory monitoring and underscores the importance of postictal arousal as a pathophysiological mechanism in SUDEP.

The effect of seizure spread to the amygdala on respiration and onset of ictal central apnea.

OBJECTIVE: Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death for patients with refractory epilepsy, and there is increasing evidence for a centrally mediated respiratory depression as a pathophysiological mechanism. The brain regions responsible for a seizure's inducing respiratory depression are unclear-the respiratory nuclei in the brainstem are thought to be involved, but involvement of forebrain structures is not yet understood. The aim of this study was to analyze intracranial EEGs in combination with the results of respiratory monitoring to investigate the relationship between seizure spread to specific mesial temporal brain regions and the onset of respiratory dysfunction and apnea. METHODS: The authors reviewed all invasive electroencephalographic studies performed at Northwestern Memorial Hospital (Chicago) since 2010 to identify those cases in which 1) multiple mesial temporal electrodes (amygdala and hippocampal) were placed, 2) seizures were captured, and 3) patients' respiration was monitored. They identified 8 investigations meeting these criteria in patients with temporal lobe epilepsy, and these investigations yielded data on a total of 22 seizures for analysis. RESULTS: The onset of ictal apnea associated with each seizure was highly correlated with seizure spread to the amygdala. Onset of apnea occurred 2.7 ± 0.4 (mean ± SEM) seconds after the spread of the seizure to the amygdala, which was significantly earlier than after spread to the hippocampus (10.2 ± 0.7 seconds; p < 0.01). CONCLUSIONS: The findings suggest that activation of amygdalar networks is correlated with central apnea during seizures. This study builds on the authors' prior work that demonstrates a role for the amygdala in voluntary respiratory control and suggests a further role in dysfunctional breathing states seen during seizures, with implications for SUDEP pathophysiology.

Amygdala-stimulation-induced apnea is attention and nasal-breathing dependent.

OBJECTIVE: Evidence suggests that disordered breathing is critically involved in Sudden Unexpected Death in Epilepsy (SUDEP). To that end, evaluating structures that are activated by seizures and can activate brain regions that produce cardiorespiratory changes can further our understanding of the pathophysiology of SUDEP. Past preclinical studies have shown that electrical stimulation of the human amygdala induces apnea, suggesting a role for the amygdala in controlling respiration. In this study, we aimed to both confirm these findings in a larger group of patients with intractable temporal lobe epilepsy (TLE) and also further explore the anatomical and cognitive properties of this effect. METHODS: Seven surgical TLE patients had depth electrodes implanted in the amygdala that were used to deliver electrical stimulation during functional mapping preceding resection. Real-time respiratory monitoring was performed in each patient to confirm apnea. RESULTS: Our data confirm that amygdala stimulation reliably induces apnea (occurring in all 7 patients) and further suggest that apnea can be overcome by instructing the patient to inhale, and can be prevented entirely by breathing through the mouth before electrical stimulation. Finally, stimulation-induced apnea occurred only when stimulating the medial-most amygdalar contacts located in the central nucleus. INTERPRETATION: These findings confirm a functional connection between the amygdala and respiratory control in humans. Moreover, they suggest specific amygdalar nuclei may be critical in mediating this effect and that attentional state is critical to apnea mediated by amygdala activation-perhaps alluding to future development of strategies for the prevention of SUDEP. Ann Neurol 2018;83:460-471.

Excitatory Synaptic Input to Hilar Mossy Cells under Basal and Hyperexcitable Conditions.

Hilar mossy cells (HMCs) in the hippocampus receive glutamatergic input from dentate granule cells (DGCs) via mossy fibers (MFs) and back-projections from CA3 pyramidal neuron collateral axons. Many fundamental features of these excitatory synapses have not been characterized in detail despite their potential relevance to hippocampal cognitive processing and epilepsy-induced adaptations in circuit excitability. In this study, we compared pre- and postsynaptic parameters between MF and CA3 inputs to HMCs in young and adult mice of either sex and determined the relative contributions of the respective excitatory inputs during in vitro and in vivo models of hippocampal hyperexcitability. The two types of excitatory synapses both exhibited a modest degree of short-term plasticity, with MF inputs to HMCs exhibiting lower paired-pulse (PP) and frequency facilitation than was described previously for MF-CA3 pyramidal cell synapses. MF-HMC synapses exhibited unitary excitatory synaptic currents (EPSCs) of larger amplitude, contained postsynaptic kainate receptors, and had a lower NMDA/AMPA receptor ratio compared to CA3-HMC synapses. Pharmacological induction of hippocampal hyperexcitability in vitro transformed the abundant but relatively weak CA3-HMC connections to very large amplitude spontaneous bursts of compound EPSCs (cEPSCs) in young mice (∼P20) and, to a lesser degree, in adult mice (∼P70). CA3-HMC cEPSCs were also observed in slices prepared from mice with spontaneous seizures several weeks after intrahippocampal kainate injection. Strong excitation of HMCs during synchronous CA3 activity represents an avenue of significant excitatory network generation back to DGCs and might be important in generating epileptic networks.

ß-Adrenergic receptors enhance excitatory transmission in the bed nucleus of the stria terminalis through a corticotrophin-releasing factor receptor-dependent and cocaine-regulated mechanism.

BACKGROUND: Evidence suggests that the noradrenergic and corticotrophin-releasing factor (CRF) systems play critical roles in relapse and stress-related behaviors. In particular, behavioral studies point to a serial signaling process initiated by ß-adrenergic receptors that requires CRF receptor (CRFR)-dependent signaling in the bed nucleus of the stria terminalis (BNST) to produce stress-induced relapse to cocaine seeking. METHODS: We used whole cell patch clamp recordings from acutely prepared mouse brain slices to examine the actions of ß-adrenergic receptors and CRFR1 on excitatory transmission in BNST. We examined the effects of agonists of these receptors in slices prepared from naive, sham, and cocaine-conditioned mice. RESULTS: ß(1)-adrenergic receptor activation within the BNST produces an enhancement of excitatory synaptic transmission that requires CRFR1-dependent signaling. We show that chronic cocaine administration transiently disrupts ß(1)-adrenergic- and CRFR1-dependent enhancement of glutamatergic transmission, that this disruption wanes with time, and that it can be reintroduced with a cocaine challenge. CONCLUSIONS: In total, these studies identify a circuit mechanism within the BNST that may play an important role in CRF- and norepinephrine-regulated behaviors.

Distinct forms of Gq-receptor-dependent plasticity of excitatory transmission in the BNST are differentially affected by stress.

Long-term depression (LTD) is an important synaptic mechanism for limiting excitatory influence over circuits subserving cognitive and emotional behavior. A major means of LTD induction is through the recruitment of signaling via G(q)-linked receptors activated by norepinephrine (NE), acetylcholine, and glutamate. Receptors from these transmitter families have been proposed to converge on a common postsynaptic LTD maintenance mechanism, such that hetero- and homosynaptic induction produce similar alterations in glutamate synapse efficacy. We report that in the dorsolateral and ventrolateral bed nucleus of the stria terminalis (BNST), recruitment of G(q)-linked receptors by glutamate or NE initiates mechanistically distinct forms of postsynaptically maintained LTD and these LTDs are differentially regulated by stress exposure. In particular, we show that although both mGluR5- and alpha(1)-adrenergic receptor (AR)-dependent LTDs involve postsynaptic endocytosis, the alpha(1)-AR-initiated LTD exclusively involves modulation of signaling through calcium-permeable AMPA receptors. Further, alpha(1)-AR- but not mGluR5- dependent LTD is disrupted by restraint stress. alpha(1)-AR LTD is also impaired in mice chronically exposed to ethanol. These data thus suggest that in the BNST, NE- and glutamate-activated G(q)-linked signaling pathways differentially tune glutamate synapse efficacy in response to stress.

Dopamine enhances fast excitatory synaptic transmission in the extended amygdala by a CRF-R1-dependent process.

A common feature of drugs of abuse is their ability to increase extracellular dopamine levels in key brain circuits. The actions of dopamine within these circuits are thought to be important in reward and addiction-related behaviors. Current theories of addiction also posit a central role for corticotrophin-releasing factor (CRF) and an interaction between CRF and monoaminergic signaling. One region where drugs of abuse promote robust rises in extracellular dopamine levels is the bed nucleus of the stria terminalis (BNST), a CRF-rich component of the extended amygdala. We find that dopamine rapidly enhances glutamatergic transmission in the BNST through activation of a combination of D(1)- and D(2)-like receptors. This enhancement is activity-dependent and requires the downstream action of CRF receptor 1 (CRF-R1), suggesting that dopamine induces CRF release through a local network mechanism. Furthermore, we found that both in vivo and ex vivo cocaine induced a dopamine receptor and CRF-R1-dependent enhancement of a form of NMDA receptor-dependent short-term potentiation in the BNST. These data highlight a direct and rapid interaction between dopamine and CRF systems that regulates excitatory transmission and plasticity in a brain region key to reinforcement and reinstatement. Because a rise in extracellular dopamine levels in the BNST is a shared consequence of multiple classes of drugs of abuse, this suggests that the CRF-R1-dependent enhancement of glutamatergic transmission in this region may be a common key feature of substances of abuse.