Activation of hypoactive parvalbumin-positive fast-spiking interneuron restores dentate inhibition to prevent epileptiform activity in the mouse intrahippocampal kainate model of temporal lobe epilepsy.

Parvalbumin-positive (PV+) GABAergic interneurons in the dentate gyrus provide powerful perisomatic inhibition of dentate granule cells (DGCs) to prevent overexcitation and maintain the stability of dentate gyrus circuits. Most dentate PV+ interneurons survive status epilepticus, but surviving PV+ interneuron mediated inhibition is compromised in the dentate gyrus shortly after status epilepticus, contributing to epileptogenesis in temporal lobe epilepsy. It is uncertain whether the impaired activity of dentate PV+ interneurons recovers at later times or if it continues for months following status epilepticus. The development of compensatory modifications related to PV+ interneuron circuits in the months following status epilepticus is unknown, although reduced dentate GABAergic inhibition persists long after status epilepticus. We employed PV immunostaining and whole-cell patch-clamp recordings from dentate PV+ interneurons and DGCs in slices from male and female sham controls and intrahippocampal kainate (IHK) treated mice that developed spontaneous seizures months after status epilepticus to study epilepsy-associated changes in dentate PV+ interneuron circuits. We found that the number of dentate PV+ cells was reduced in IHK treated mice. Electrical recordings showed that: 1) Action potential firing rates of dentate PV+ interneurons were reduced in IHK treated mice up to four months after status epilepticus; 2) Spontaneous inhibitory postsynaptic currents (sIPSCs) in DGCs exhibited reduced frequency but increased amplitude in IHK treated mice; and 3) The amplitude of evoked IPSCs in DGCs by optogenetic activation of dentate PV+ cells was upregulated without changes in short-term plasticity. Video-EEG recordings revealed that IHK treated mice showed spontaneous epileptiform activity in the dentate gyrus and that chemogenetic activation of PV+ interneurons abolished the epileptiform activity. Our results suggest not only that the compensatory changes in PV+ interneuron circuits develop after IHK treatment, but also that increased PV+ interneuron mediated inhibition in the dentate gyrus may compensate for cell loss and reduced intrinsic excitability of dentate PV+ interneurons to stop seizures in temporal lobe epilepsy. Highlights: Reduced number of dentate PV+ interneurons in TLE micePersistently reduced action potential firing rates of dentate PV+ interneurons in TLE miceEnhanced amplitude but decreased frequency of spontaneous IPSCs in the dentate gyrus in TLE miceIncreased amplitude of evoked IPSCs mediated by dentate PV+ interneurons in TLE miceChemogenetic activation of PV+ interneurons prevents epileptiform activity in TLE mice.

Systemic Glucose Regulation by a Hindbrain Inhibitory Circuit in a Mouse Model of Type 1 Diabetes.

INTRODUCTION: Previous work showed that increasing the electrical activity of inhibitory neurons in the dorsal vagal complex (DVC) is sufficient to increase whole-body glucose concentration in normoglycemic mice. Here we tested the hypothesis that deactivating GABAergic neurons in the dorsal hindbrain of hyperglycemic mice decreases synaptic inhibition of parasympathetic motor neurons in the dorsal motor nucleus of the vagus (DMV) and reduces systemic glucose levels. METHODS: Chemogenetic activation or inactivation of GABAergic neurons in the nucleus tractus solitarius (NTS) was used to assess effects of modulating parasympathetic output on blood glucose concentration in normoglycemic and hyperglycemic mice. Patch-clamp electrophysiology in vitro was used to assess cellular effects of chemogenetic manipulation of NTS GABA neurons. RESULTS: Chemogenetic activation of GABAergic NTS neurons in normoglycemic mice increased their action potential firing, resulting in increased inhibitory synaptic input to DMV motor neurons and elevated blood glucose concentration. Deactivation of GABAergic DVC neurons in normoglycemic mice altered their electrical activity but did not alter systemic glucose levels. Conversely, stimulation of GABAergic DVC neurons in mice that were hyperglycemic subsequent to treatment with streptozotocin changed their electrical activity but did not alter whole-body glucose concentration, while deactivation of this inhibitory circuit significantly decreased circulating glucose concentration. Peripheral administration of a brain impermeant muscarinic acetylcholine receptor antagonist abolished these effects. CONCLUSION: Disinhibiting vagal motor neurons decreases hyperglycemia in a mouse model of type 1 diabetes. This inhibitory brainstem circuit emerges as a key parasympathetic regulator of whole-body glucose homeostasis that undergoes functional plasticity in hyperglycemic conditions.

In situ microwave fixation provides an instantaneous snapshot of the brain metabolome.

Brain glucose metabolism is highly heterogeneous among brain regions and continues postmortem. In particular, we demonstrate exhaustion of glycogen and glucose and an increase in lactate production during conventional rapid brain resection and preservation by liquid nitrogen. In contrast, we show that these postmortem changes are not observed with simultaneous animal sacrifice and in situ fixation with focused, high-power microwave. We further employ microwave fixation to define brain glucose metabolism in the mouse model of streptozotocin-induced type 1 diabetes. Using both total pool and isotope tracing analyses, we identified global glucose hypometabolism in multiple brain regions, evidenced by reduced 13C enrichment into glycogen, glycolysis, and the tricarboxylic acid (TCA) cycle. Reduced glucose metabolism correlated with a marked decrease in GLUT2 expression and several metabolic enzymes in unique brain regions. In conclusion, our study supports the incorporation of microwave fixation for more accurate studies of brain metabolism in rodent models.

Spinal neuropeptide Y Y1 receptor-expressing neurons are a pharmacotherapeutic target for the alleviation of neuropathic pain.

Peripheral nerve injury sensitizes a complex network of spinal cord dorsal horn (DH) neurons to produce allodynia and neuropathic pain. The identification of a druggable target within this network has remained elusive, but a promising candidate is the neuropeptide Y (NPY) Y1 receptor-expressing interneuron (Y1-IN) population. We report that spared nerve injury (SNI) enhanced the excitability of Y1-INs and elicited allodynia (mechanical and cold hypersensitivity) and affective pain. Similarly, chemogenetic or optogenetic activation of Y1-INs in uninjured mice elicited behavioral signs of spontaneous, allodynic, and affective pain. SNI-induced allodynia was reduced by chemogenetic inhibition of Y1-INs, or intrathecal administration of a Y1-selective agonist. Conditional deletion of Npy1r in DH neurons, but not peripheral afferent neurons prevented the anti-hyperalgesic effects of the intrathecal Y1 agonist. We conclude that spinal Y1-INs are necessary and sufficient for the behavioral symptoms of neuropathic pain and represent a promising target for future pharmacotherapeutic development of Y1 agonists.

Adult Born Dentate Granule Cell Mediated Upregulation of Feedback Inhibition in a Mouse Model of Traumatic Brain Injury.

Post-traumatic epilepsy (PTE) and behavioral comorbidities frequently develop after traumatic brain injury (TBI). Aberrant neurogenesis of dentate granule cells (DGCs) after TBI may contribute to the synaptic reorganization that occurs in PTE, but how neurogenesis at different times relative to the injury contributes to feedback inhibition and recurrent excitation in the dentate gyrus is unknown. Thus, we examined whether DGCs born at different postnatal ages differentially participate in feedback inhibition and recurrent excitation in the dentate gyrus using the controlled cortical impact (CCI) model of TBI. Both sexes of transgenic mice expressing channelrhodopsin2 (ChR2) in postnatally born DGCs were used for optogenetic activation of three DGC cohorts: postnatally early born DGCs, or those born just before or after CCI. We performed whole-cell patch-clamp recordings from ChR2-negative, mature DGCs and parvalbumin-expressing basket cells (PVBCs) in hippocampal slices to determine whether optogenetic activation of postnatally born DGCs increases feedback inhibition and/or recurrent excitation in mice 8-10 weeks after CCI and whether PVBCs are targets of ChR2-positive DGCs. In the dentate gyrus ipsilateral to CCI, activation of ChR2-expressing DGCs born before CCI produced increased feedback inhibition in ChR2-negative DGCs and increased excitation in PVBCs compared with those from sham controls. This upregulated feedback inhibition was less prominent in DGCs born early in life or after CCI. Surprisingly, ChR2-positive DGC activation rarely evoked recurrent excitation in mature DGCs from any cohort. These results support that DGC birth date-related increased feedback inhibition in of DGCs may contribute to altered excitability after TBI.SIGNIFICANCE STATEMENT Dentate granule cells (DGCs) control excitability of the dentate gyrus through synaptic interactions with inhibitory GABAergic interneurons. Persistent changes in DGC synaptic connectivity develop after traumatic brain injury, contributing to hyperexcitability in post-traumatic epilepsy (PTE). However, the impact of DGC neurogenesis on synaptic reorganization, especially on inhibitory circuits, after brain injury is not adequately described. Here, upregulation of feedback inhibition in mature DGCs from male and female mice was associated with increased excitation of parvalbumin-expressing basket cells by postnatally born DGCs, providing novel insights into underlying mechanisms of altered excitability after brain injury. A better understanding of these inhibitory circuit changes can help formulate hypotheses for development of novel, evidence-based treatments for post-traumatic epilepsy by targeting birth date-specific subsets of DGCs.

Zolpidem Profoundly Augments Spared Tonic GABAAR Signaling in Dentate Granule Cells Ipsilateral to Controlled Cortical Impact Brain Injury in Mice.

Type A GABA receptors (GABAARs) are pentameric combinations of protein subunits that give rise to tonic (ITonicGABA) and phasic (i.e., synaptic; ISynapticGABA) forms of inhibitory GABAAR signaling in the central nervous system. Remodeling and regulation of GABAAR protein subunits are implicated in a wide variety of healthy and injury-dependent states, including epilepsy. The present study undertook a detailed analysis of GABAAR signaling using whole-cell patch clamp recordings from mouse dentate granule cells (DGCs) in coronal slices containing dorsal hippocampus at 1-2 or 8-13 weeks after a focal, controlled cortical impact (CCI) or sham brain injury. Zolpidem, a benzodiazepine-like positive modulator of GABAARs, was used to test for changes in GABAAR signaling of DGCs due to its selectivity for α1 subunit-containing GABAARs. Electric charge transfer and statistical percent change were analyzed in order to directly compare tonic and phasic GABAAR signaling and to account for zolpidem's ability to modify multiple parameters of GABAAR kinetics. We observed that baseline ITonicGABA is preserved at both time-points tested in DGCs ipsilateral to injury (Ipsi-DGCs) compared to DGCs contralateral to injury (Contra-DGCs) or after sham injury (Sham-DGCs). Interestingly, application of zolpidem resulted in modulation of ITonicGABA across groups, with Ipsi-DGCs exhibiting the greatest responsiveness to zolpidem. We also report that the combination of CCI and acute application of zolpidem profoundly augments the proportion of GABAAR charge transfer mediated by tonic vs. synaptic currents at both time-points tested, whereas gene expression of GABAAR α1, α2, α3, and γ2 subunits is unchanged at 8-13 weeks post-injury. Overall, this work highlights the shift toward elevated influence of tonic inhibition in Ipsi-DGCs, the impact of zolpidem on all components of inhibitory control of DGCs, and the sustained nature of these changes in inhibitory tone after CCI injury.

Fibroblast Growth Factor 19 Increases the Excitability of Pre-Motor Glutamatergic Dorsal Vagal Complex Neurons From Hyperglycemic Mice.

Intracerebroventricular administration of the protein hormone fibroblast growth factor 19 (FGF19) to the hindbrain produces potent antidiabetic effects in hyperglycemic mice that are likely mediated through a vagal parasympathetic mechanism. FGF19 increases the synaptic excitability of parasympathetic motor neurons in the dorsal motor nucleus of the vagus (DMV) from hyperglycemic, but not normoglycemic, mice but the source of this synaptic input is unknown. Neurons in the area postrema (AP) and nucleus tractus solitarius (NTS) express high levels of FGF receptors and exert glutamatergic control over the DMV. This study tested the hypothesis that FGF19 increases glutamate release in the DMV by increasing the activity of glutamatergic AP and NTS neurons in hyperglycemic mice. Glutamate photoactivation experiments confirmed that FGF19 increases synaptic glutamate release from AP and NTS neurons that connect to the DMV in hyperglycemic, but not normoglycemic mice. Contrary to expectations, FGF19 produced a mixed effect on intrinsic membrane properties in the NTS with a trend towards inhibition, suggesting that another mechanism was responsible for the observed effects on glutamate release in the DMV. Consistent with the hypothesis, FGF19 increased action potential-dependent glutamate release in the NTS in hyperglycemic mice only. Finally, glutamate photoactivation experiments confirmed that FGF19 increases the activity of glutamatergic AP neurons that project to the NTS in hyperglycemic mice. Together, these results support the hypothesis that FGF19 increases glutamate release from AP and NTS neurons that project to the DMV in hyperglycemic mice. FGF19 therefore modifies the local vago-vagal reflex circuitry at several points. Additionally, since the AP and NTS communicate with several other metabolic regulatory nuclei in the brain, FGF19 in the hindbrain may alter neuroendocrine and behavioral aspects of metabolism, in addition to changes in parasympathetic output.

Sex Differences in Protein Kinase A Signaling of the Latent Postoperative Pain Sensitization That Is Masked by Kappa Opioid Receptors in the Spinal Cord.

Latent sensitization (LS) of pain engages pronociceptive signaling pathways in the dorsal horn that include NMDA receptor (NMDAR)→adenylyl cyclase-1 (AC1)→protein kinase A (PKA), and exchange proteins directly activated by cyclic AMP (Epacs). To determine whether these pathways operate similarly between males and females or are under the inhibitory control of spinal κ opioid receptors (KOR), we allowed hyperalgesia to resolve after plantar incision and then blocked KOR with intrathecal administration of LY2456302, which reinstated hyperalgesia and facilitated touch-evoked immunoreactivity of phosphorylated extracellular signal-regulated kinase (pERK) in neurons (NeuN) but not astrocytes (GFAPs) nor microglia (Iba1). LY2456302 reinstated hyperalgesia even when administered 13 months later, indicating that chronic postoperative pain vulnerability persists for over a year in a latent state of remission. In both sexes, intrathecal MK-801 (an NMDAR competitive antagonist) prevented LY2456302-evoked reinstatement of hyperalgesia as did AC1 gene deletion or the AC1 inhibitor NB001. NB001 also prevented stimulus-evoked pERK. In both sexes, the Epac inhibitor ESI-09 prevented reinstatement, whereas the Epac activator 8-CPT reinstated hyperalgesia. By contrast, the PKA inhibitor H89 prevented reinstatement only in male mice, whereas the PKA activator 6-bnz-cAMP itself evoked reinstatement at all doses tested (3-30 nmol, i.t.). In neither sex did incision change gene expression of KOR, GluN1, PKA, or Epac1 in dorsal horn. We conclude that sustained KOR signaling inhibits spinal PKA-dependent mechanisms that drive postoperative LS in a sex-dependent manner. Our findings support the development of AC1, PKA, and Epac inhibitors toward a new pharmacotherapy for chronic postoperative pain.SIGNIFICANCE STATEMENT Because of neural mechanisms that are not well understood, men and women respond differently to treatments for chronic pain. We report that surgical incision recruits a pronociceptive latent pain sensitization that persisted for over a year and was kept in check by the sustained analgesic activity of κ opioid receptors. NMDAR→AC1→cAMP→Epac signaling pathways in the dorsal horn of the spinal cord maintain latent sensitization in both males and females; however, only males recruit a PKA-dependent mechanism. This work presents a novel male-specific mechanism for the promotion of chronic postoperative pain.

Effects of altered tau expression on dentate granule cell excitability in mice.

Tauopathies, including Alzheimer's disease, are characterized by progressive accumulation of hyperphosphorylated and pathologic tau protein in association with onset of cognitive and behavioral impairment. Tau pathology is also associated with increased susceptibility to seizures and epilepsy, with tau-/- mice showing seizure resistance in some epilepsy models. To better understand how tau pathology is related to neuronal excitability, we performed whole-cell patch-clamp electrophysiology in dentate gyrus granule cells of tau-/- and human-tau expressing, htau mice. The htau mouse is unique from other transgenic tau models in that the endogenous murine tau gene has been and replaced with readily phosphorylated human tau. We assessed several measures of neuronal excitability, including evoked action potential frequency and excitatory synaptic responses in dentate granule cells from tau-/-, htau, and non-transgenic control mice at 1.5, 4, and 9 months of age. Compared to age matched controls, dentate granule cells from both tau-/- and htau mice had a lower peak frequency of evoked action potentials and greater paired pulse facilitation, suggesting reduced neuronal excitability. Our results suggest that neuronal excitability is more strongly influenced by the absence of functional tau than by the presence of pathologic tau. These results also suggest that tau's effect on neuronal excitability is more complex than previously understood.

Vulnerability of cholecystokinin-expressing GABAergic interneurons in the unilateral intrahippocampal kainate mouse model of temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is characterized by recurrent spontaneous seizures and behavioral comorbidities. Reduced hippocampal theta oscillations and hyperexcitability that contribute to cognitive deficits and spontaneous seizures are present beyond the sclerotic hippocampus in TLE. However, the mechanisms underlying compromised network oscillations and hyperexcitability observed in circuits remote from the sclerotic hippocampus are largely unknown. Cholecystokinin (CCK)-expressing basket cells (CCKBCs) critically participate in hippocampal theta rhythmogenesis, and regulate neuronal excitability. Thus, we examined whether CCKBCs were vulnerable in nonsclerotic regions of the ventral hippocampus remote from dorsal sclerotic hippocampus using the intrahippocampal kainate (IHK) mouse model of TLE, targeting unilateral dorsal hippocampus. We found a decrease in the number of CCK+ interneurons in ipsilateral ventral CA1 regions from epileptic mice compared to those from sham controls. We also found that the number of boutons from CCK+ interneurons was reduced in the stratum pyramidale, but not in other CA1 layers, of ipsilateral hippocampus in epileptic mice, suggesting that CCKBCs are vulnerable. Electrical recordings showed that synaptic connectivity and strength from surviving CCKBCs to CA1 pyramidal cells (PCs) were similar between epileptic mice and sham controls. In agreement with reduced CCKBC number in TLE, electrical recordings revealed a significant reduction in amplitude and frequency of IPSCs in CA1 PCs evoked by carbachol (commonly used to excite CCK+ interneurons) in ventral CA1 regions from epileptic mice versus sham controls. These findings suggest that loss of CCKBCs beyond the hippocampal lesion may contribute to hyperexcitability and compromised network oscillations in TLE.

Fast A-type currents shape a rapidly adapting form of delayed short latency firing of excitatory superficial dorsal horn neurons that express the neuropeptide Y Y1 receptor.

KEY POINTS: Neuropeptide Y Y1 receptor-expressing neurons in the dorsal horn of the spinal cord contribute to chronic pain. For the first time, we characterized the firing patterns of Y1-expressing neurons in Y1eGFP reporter mice. Under hyperpolarized conditions, most Y1eGFP neurons exhibited fast A-type potassium currents and delayed, short-latency firing (DSLF). Y1eGFP DSLF neurons were almost always rapidly adapting and often exhibited rebound spiking, characteristics of spinal pain neurons under the control of T-type calcium channels. These results will inspire future studies to determine whether tissue or nerve injury downregulates the channels that underlie A-currents, thus unmasking membrane hyperexcitability in Y1-expressing dorsal horn neurons, leading to persistent pain. ABSTRACT: Neuroanatomical and behavioural evidence indicates that neuropeptide Y Y1 receptor-expressing interneurons (Y1-INs) in the superficial dorsal horn (SDH) are predominantly excitatory and contribute to chronic pain. Using an adult ex vivo spinal cord slice preparation from Y1eGFP reporter mice, we characterized firing patterns in response to steady state depolarizing current injection of GFP-positive cells in lamina II, the great majority of which expressed Y1 mRNA (88%). Randomly sampled (RS) and Y1eGFP neurons exhibited five firing patterns: tonic, initial burst, phasic, delayed short-latency <180 ms (DSLF) and delayed long-latency >180 ms (DLLF). When studied at resting membrane potential, most RS neurons exhibited delayed firing, while most Y1eGFP neurons exhibited phasic firing. A preconditioning membrane hyperpolarization produced only subtle changes in the firing patterns of RS neurons, but dramatically shifted Y1eGFP neurons to DSLF (46%) and DLLF (24%). In contrast to RS DSLF neurons, which rarely exhibited spike frequency adaptation, Y1eGFP DSLF neurons were almost always rapidly adapting, a characteristic of nociceptive-responsive SDH neurons. Rebound spiking was more prevalent in Y1eGFP neurons (6% RS vs. 32% Y1eGFP), indicating enrichment of T-type calcium currents. Y1eGFP DSLF neurons exhibited fast A-type potassium currents that are known to delay or limit action potential firing and exhibited smaller current density as compared to RS DSLF neurons. Our results will inspire future studies to determine whether tissue or nerve injury downregulates channels that contribute to A-currents, thus potentially unmasking T-type calcium channel activity and membrane hyperexcitability in Y1-INs, leading to persistent pain.

FGF19 in the Hindbrain Lowers Blood Glucose and Alters Excitability of Vagal Motor Neurons in Hyperglycemic Mice.

Fibroblast growth factor 19 (FGF19) is a protein hormone that produces antidiabetic effects when administered intracerebroventricularly in the forebrain. However, no studies have examined how FGF19 affects hindbrain neurons that participate directly in autonomic control of systemic glucose regulation. Within the dorsal hindbrain, parasympathetic motor neurons of the dorsal motor nucleus of the vagus (DMV) express fibroblast growth factor receptors and their activity regulates visceral homeostatic processes, including energy balance. This study tested the hypothesis that FGF19 acts in the hindbrain to alter DMV neuron excitability and lower blood glucose concentration. Fourth ventricle administration of FGF19 produced no effect on blood glucose concentration in control mice, but induced a significant, peripheral muscarinic receptor-dependent decrease in systemic hyperglycemia for up to 12 h in streptozotocin-treated mice, a model of type 1 diabetes. Patch-clamp recordings from DMV neurons in vitro revealed that FGF19 application altered synaptic and intrinsic membrane properties of DMV neurons, with the balance of FGF19 effects being significantly modified by a recent history of systemic hyperglycemia. These findings identify central parasympathetic circuitry as a novel target for FGF19 and suggest that FGF19 acting in the dorsal hindbrain can alter vagal output to produce its beneficial metabolic effects.

Perlecan Domain-V Enhances Neurogenic Brain Repair After Stroke in Mice.

The extracellular matrix fragment perlecan domain V is neuroprotective and functionally restorative following experimental stroke. As neurogenesis is an important component of chronic post-stroke repair, and previous studies have implicated perlecan in developmental neurogenesis, we hypothesized that domain V could have a broad therapeutic window by enhancing neurogenesis after stroke. We demonstrated that domain V is chronically increased in the brains of human stroke patients, suggesting that it is present during post-stroke neurogenic periods. Furthermore, perlecan deficient mice had significantly less neuroblast precursor cells after experimental stroke. Seven-day delayed domain V administration enhanced neurogenesis and restored peri-infarct excitatory synaptic drive to neocortical layer 2/3 pyramidal neurons after experimental stroke. Domain V's effects were inhibited by blockade of α2ß1 integrin, suggesting the importance of α2ß1 integrin to neurogenesis and domain V neurogenic effects. Our results demonstrate that perlecan plays a previously unrecognized role in post-stroke neurogenesis and that delayed DV administration after experimental stroke enhances neurogenesis and improves recovery in an α2ß1 integrin-mediated fashion. We conclude that domain V is a clinically relevant neuroprotective and neuroreparative novel stroke therapy with a broad therapeutic window.

Musings on the wanderer: What's new in our understanding of vago-vagal reflexes? VI. Central vagal circuits that control glucose metabolism.

Neurons in the brain stem dorsal vagal complex (DVC) take part in a continuous bidirectional crosstalk, in which they receive and respond to a vast array of signaling molecules, including glucose. Importantly, chronic dysregulation of blood glucose concentration, a hallmark of high prevalence pathologies, such as diabetes and metabolic syndrome, can induce neuroplasticity in DVC neural networks, which is hypothesized to either contribute to or compensate for the glycemic or insulinemic dysregulation observed in these conditions. Here, we revisit the topic of vagal reflexes to review recent research on the importance of DVC function in regulating systemic glucose homeostasis and the neuroplastic changes in this brain region that are associated with systemic glucose alterations. We also discuss the critical connection between these nuclei and the gut and the role of central vagal circuits in the favorable outcomes associated with bariatric surgical procedures for metabolic disorders.

Epilepsy Benchmarks Area II: Prevent Epilepsy and Its Progression.

Area II of the 2014 Epilepsy Research Benchmarks aims to establish goals for preventing the development and progression of epilepsy. In this review, we will highlight key advances in Area II since the last summary of research progress and opportunities was published in 2016. We also highlight areas of investigation that began to develop before 2016 and in which additional progress has been made more recently.

The critical role of persistent sodium current in hippocampal gamma oscillations.

Gamma network oscillations in the brain are fast rhythmic network oscillations in the gamma frequency range (~30-100 Hz), playing key roles in the hippocampus for learning, memory, and spatial processing. There is evidence indicating that GABAergic interneurons, including parvalbumin-expressing basket cells (PVBCs), contribute to cortical gamma oscillations through synaptic interactions with excitatory cells. However, the molecular, cellular, and circuit underpinnings underlying generation and maintenance of cortical gamma oscillations are largely elusive. Recent studies demonstrated that intrinsic and synaptic properties of GABAergic interneurons and excitatory cells are regulated by a slowly inactivating or non-inactivating sodium current (i.e., persistent sodium current, INaP), suggesting that INaP is involved in gamma oscillations. Here, we tested whether INaP plays a role in hippocampal gamma oscillations using pharmacological, optogenetic, and electrophysiological approaches. We found that INaP blockers, phenytoin (40 µM and 100 µM) and riluzole (10 µM), reduced gamma oscillations induced by optogenetic stimulation of CaMKII-expressing cells in CA1 networks. Whole-cell patch-clamp recordings further demonstrated that phenytoin (100 µM) reduced INaP and firing frequencies in both PVBCs and pyramidal cells without altering threshold and amplitude of action potentials, but increased rheobase in both cell types. These results suggest that INaP in pyramidal cells and PVBCs is required for hippocampal gamma oscillations, supporting a pyramidal-interneuron network gamma model. Phenytoin-mediated modulation of hippocampal gamma oscillations may be a mechanism underlying its anticonvulsant efficacy, as well as its contribution to cognitive impairments in epilepsy patients.

Sex differences in kappa opioid receptor inhibition of latent postoperative pain sensitization in dorsal horn.

Tissue injury produces a delicate balance between latent pain sensitization (LS) and compensatory endogenous opioid receptor analgesia that continues for months, even after re-establishment of normal pain thresholds. To evaluate the contribution of mu (MOR), delta (DOR), and/or kappa (KOR) opioid receptors to the silencing of chronic postoperative pain, we performed plantar incision at the hindpaw, waited 21 days for the resolution of hyperalgesia, and then intrathecally injected subtype-selective ligands. We found that the MOR-selective inhibitor CTOP (1-1000 ng) dose-dependently reinstated mechanical hyperalgesia. Two DOR-selective inhibitors naltrindole (1-10 µg) and TIPP[Ψ] (1-20 µg) reinstated mechanical hyperalgesia, but only at the highest dose that also produced itching, licking, and tail biting. Both the prototypical KOR-selective inhibitors nor-BNI (0.1-10 µg) and the newer KOR inhibitor with more canonical pharmocodynamic effects, LY2456302 (0.1-10 µg), reinstated mechanical hyperalgesia. Furthermore, LY2456302 (10 µg) increased the expression of phosphorylated signal-regulated kinase (pERK), a marker of central sensitization, in dorsal horn neurons but not glia. Sex studies revealed that LY2456302 (0.3 µg) reinstated hyperalgesia and pERK expression to a greater degree in female as compared to male mice. Our results suggest that spinal MOR and KOR, but not DOR, maintain LS within a state of remission to reduce the intensity and duration of postoperative pain, and that endogenous KOR but not MOR analgesia is greater in female mice.

Modulation of epileptogenesis: A paradigm for the integration of enzyme-based microelectrode arrays and optogenetics.

BACKGROUND: Genesis of acquired epilepsy includes transformations spanning genetic-to- network-level modifications, disrupting the regional excitatory/inhibitory balance. Methodology concurrently tracking changes at multiple levels is lacking. Here, viral vectors are used to differentially express two opsin proteins in neuronal populations within dentate gyrus (DG) of hippocampus. When activated, these opsins induced excitatory or inhibitory neural output that differentially affected neural networks and epileptogenesis. In vivo measures included behavioral observation coupled to real-time measures of regional glutamate flux using ceramic-based amperometric microelectrode arrays (MEAs). RESULTS: Using MEA technology, phasic increases of extracellular glutamate were recorded immediately upon application of blue light/488 nm to DG of rats previously transfected with an AAV 2/5 vector containing an (excitatory) channelrhodopsin-2 transcript. Rats receiving twice-daily 30-sec light stimulation to DG ipsilateral to viral transfection progressed through Racine seizure stages. AAV 2/5 (inhibitory) halorhodopsin-transfected rats receiving concomitant amygdalar kindling and DG light stimuli were kindled significantly more slowly than non-stimulated controls. In in vitro slice preparations, both excitatory and inhibitory responses were independently evoked in dentate granule cells during appropriate light stimulation. Latency to response and sensitivity of responses suggest a degree of neuron subtype-selective functional expression of the transcripts. CONCLUSIONS: This study demonstrates the potential for coupling MEA technology and optogenetics for real-time neurotransmitter release measures and modification of seizure susceptibility in animal models of epileptogenesis. This microelectrode/optogenetic technology could prove useful for characterization of network and system level dysfunction in diseases involving imbalanced excitatory/inhibitory control of neuron populations and guide development of future treatment strategies.

GABAA receptor currents in the dorsal motor nucleus of the vagus in females: influence of ovarian cycle and 5α-reductase inhibition.

The dorsal motor nucleus of the vagus (DMV) contains the preganglionic motor neurons important in the regulation of glucose homeostasis and gastrointestinal function. Despite the role of sex in the regulation of these processes, few studies examine the role of sex and/or ovarian cycle in the regulation of synaptic neurotransmission to the DMV. Since GABAergic neurotransmission is critical to normal DMV function, the present study used in vitro whole cell patch-clamping to investigate whether sex differences exist in GABAergic neurotransmission to DMV neurons. It additionally investigated whether the ovarian cycle plays a role in those sex differences. The frequency of phasic GABAA receptor-mediated inhibitory postsynaptic currents in DMV neurons from females was lower compared with males, and this effect was TTX sensitive and abolished by ovariectomy (OVX). Amplitudes of GABAergic currents (both phasic and tonic) were not different. However, females demonstrated significantly more variability in the amplitude of both phasic and tonic GABAA receptor currents. This difference was eliminated by OVX in females, suggesting that these differences were related to reproductive hormone levels. This was confirmed for GABAergic tonic currents by comparing females in two ovarian stages, estrus versus diestrus. Female mice in diestrus had larger tonic current amplitudes compared with those in estrus, and this increase was abolished after administration of a 5α-reductase inhibitor but not modulation of estrogen. Taken together, these findings demonstrate that DMV neurons undergo GABAA receptor activity plasticity as a function of sex and/or sex steroids.NEW & NOTEWORTHY Results show that GABAergic signaling in dorsal vagal motor neurons (DMV) demonstrates sex differences and fluctuates across the ovarian cycle in females. These findings are the first to demonstrate that female GABAA receptor activity in this brain region is modulated by 5α-reductase-dependent hormones. Since DMV activity is critical to both glucose and gastrointestinal homeostasis, these results suggest that sex hormones, including those synthesized by 5α-reductase, contribute to visceral, autonomic function related to these physiological processes.

A hindbrain inhibitory microcircuit mediates vagally-coordinated glucose regulation.

Neurons in the brainstem dorsal vagal complex integrate neural and humoral signals to coordinate autonomic output to viscera that regulate a variety of physiological functions, but how this circuitry regulates metabolism is murky. We tested the hypothesis that premotor, GABAergic neurons in the nucleus tractus solitarius (NTS) form a hindbrain micro-circuit with preganglionic parasympathetic motorneurons of the dorsal motor nucleus of the vagus (DMV) that is capable of modulating systemic blood glucose concentration. In vitro, neuronal activation or inhibition using either excitatory or inhibitory designer receptor exclusively activated by designer drugs (DREADDs) constructs expressed in GABAergic NTS neurons increased or decreased, respectively, action potential firing of GABAergic NTS neurons and downstream synaptic inhibition of the DMV. In vivo, DREADD-mediated activation of GABAergic NTS neurons increased systemic blood glucose concentration, whereas DREADD-mediated silencing of these neurons was without effect. The DREADD-induced hyperglycemia was abolished by blocking peripheral muscarinic receptors, consistent with the hypothesis that altered parasympathetic drive mediated the response. This effect was paralleled by elevated serum glucagon and hepatic phosphoenolpyruvate carboxykinase 1 (PEPCK1) expression, without affecting insulin levels or muscle metabolism. Activity in a hindbrain inhibitory microcircuit is sufficient to modulate systemic glucose concentration, independent of insulin secretion or utilization.