Douglas Coulter - Academia.edu (original) (raw)

Papers by Douglas Coulter

Elsevier eBooks, 2017

Abstract Research in epilepsy has employed a plethora of animal models with which to study and be... more Abstract Research in epilepsy has employed a plethora of animal models with which to study and better understand the mechanisms that lead to this debilitating condition. Modeling human disease requires a rigorous approach, and methodologies that can be easily standardized across laboratories. The following chapter will describe the pilocarpine model of acquired epilepsy. The pilocarpine model mimics many of the features of acquired epilepsy in humans, especially that of temporal lobe epilepsy (TLE). A characteristic of the acquired epilepsies is that the onset of seizures is often preceded by an initial precipitating injury, such as stroke, traumatic brain injury, or a prolonged period of status epilepticus (SE). With these types of injuries, the temporal lobe is especially vulnerable. The most prevalent of adult-onset epilepsies, TLE is also known to be difficult to treat and thus the need for novel treatment approaches is great. The following is an in-depth discussion of the post-SE pilocarpine model of acquired epilepsy. Following a brief introduction to TLE, the methods and variables that optimize successful implementation of the pilocarpine procedure itself will be highlighted. How this model can be employed to study both the process of disease progression following SE (epileptogenesis) and/or the chronic disease state will follow the methodological description. Though the pilocarpine model of TLE has existed for nearly 30 years, the development and use of sophisticated monitoring and imaging techniques, in addition to the recent inclusion of various mice strains, warrants an updated review of this model.

Journal of Neurophysiology, May 1, 1989

1. The afterhyperpolarization (AHP) that follows action potentials was studied in CA1 hippocampal... more 1. The afterhyperpolarization (AHP) that follows action potentials was studied in CA1 hippocampal pyramidal cells from classically conditioned and control rabbits. Measurements of the AHP were obtained with intracellular recordings from CA1 cells within hippocampal slices. 2. The AHP of rabbit CA1 pyramidal cells was found to be accompanied by a conductance increase. The AHP was reduced by bath applications of the calcium channel blockers, cadmium and cobalt, by bath application of the cholinergic agonist, carbamylcholine chloride, and intracellular injection of the calcium chelator, ethylene glycol-bis(B-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). 3. The AHP was markedly reduced in cells from rabbits that were well-trained with the nictitating membrane conditioning procedure, as compared with cells from pseudoconditioned or naive control animals. The difference in AHP amplitudes between conditioned and control groups increased as the number of spikes elicited by the stimulation pulse increased from one to four. Both the duration (measured as the time constant of AHP decay) and amplitude of the AHP were reduced in cells from conditioned animals. 4. The reduced AHP in cells from conditioned animals remained reduced in a medium that contained 0.5 microM tetrodotoxin (TTX) and 5.0 mM tetraethylammonium chloride (TEA); the AHP following calcium spikes was measured under these conditions. Since this medium eliminated synaptic transmission elicited by Schaeffer collateral stimulation, the AHP reduction in pyramidal cells from conditioned animals was not due to a modification in synaptic properties. There were no significant differences in the mean voltage thresholds, amplitudes, or durations of calcium spikes between cells from animals in the three groups. Thus the AHP reduction appears to be due to a modification of a Ca2+ -dependent K+ conductance and was not due to a secondary effect of reductions in calcium conductances underlying the spike. 5. In medium containing TTX and TEA, the amount of injected current required to elicit a calcium spike (current threshold) was significantly greater in cells from conditioned animals than in cells from control animals. This increase in current threshold persisted in 4-aminopyridine (4-AP)-containing medium and so cannot be attributed entirely to conditioning-specific increases in the A-current. 6. The conditioning-specific AHP reduction resulted in increased excitability in cells from conditioned animals versus pseudoconditioned control animals. Cells from conditioned animals fired more spikes to trains of 100-ms depolarizing current pulses than did cells from controls.

Journal of Neurophysiology, 1996

1. Surgically resected tissue from the tip of the human temporal lobe of seven patients undergoin... more 1. Surgically resected tissue from the tip of the human temporal lobe of seven patients undergoing temporal lobectomy was employed to study functional properties of GABAergic inhibition mediated through activation of GABAA receptors, using patch-clamp recording techniques in acutely isolated neurons and in slices of human temporal cortex. 2. Human temporal cortical pyramidal neurons from surgically resected tissue could be acutely isolated with the use of conventional methods. These neurons appeared normal in morphology, in their intrinsic membrane properties, and in their response to application of exogenous gamma-aminobutyric acid (GABA). 3. Application of GABA to acutely isolated human temporal cortical neurons elicited a large current with an average reversal potential of -65 mV, presumably mediated through a GABAA-activated chloride conductance. Application of varying concentrations of GABA generated a concentration/response relationship that could be well-fitted by a conventio...

Encyclopedia of Basic Epilepsy Research, 2009

The role of glial cells in brain function is receiving renewed interest. Recent studies show that... more The role of glial cells in brain function is receiving renewed interest. Recent studies show that astrocytes, a subtype of glial cell, regulate neuronal excitability and synaptic transmission. Astrocytes regulate inhibition through a glial adenosine cycle and by providing glutamine, a precursor for the synthesis of GABA, to interneurons. Astrocytes also release the excitatory transmitter glutamate, which by acting through neuronal NMDA receptors can increase neuronal excitability. Because brain injury alters the expression of glial enzymes that control these inhibitory pathways, it is possible that novel anticonvulsant strategies could be directed to glial targets.

Homeostatic Control of Brain Function, 2015

F1000 - Post-publication peer review of the biomedical literature, 2015

Linking neural microcircuit function to emergent properties of the mammalian brain requires fines... more Linking neural microcircuit function to emergent properties of the mammalian brain requires finescale manipulation and measurement of neural activity during behavior, where each neuron's coding and dynamics can be characterized. We developed an optical method for simultaneous cellular-resolution stimulation and large-scale recording of neuronal activity in behaving mice. Dual-wavelength two-photon excitation allowed largely independent functional imaging with a green fluorescent calcium sensor (GCaMP3, λ = 920 ± 6 nm) and single-neuron photostimulation with a red-shifted optogenetic probe (C1V1, λ = 1,064 ± 6 nm) in neurons coexpressing the two proteins. We manipulated task-modulated activity in individual hippocampal CA1 place cells during spatial navigation in a virtual reality environment, mimicking natural place-field activity, or 'biasing', to reveal subthreshold dynamics. Notably, manipulating single place-cell activity also affected activity in small groups of other place cells that were active around the same time in the task, suggesting a functional role for local place cell interactions in shaping firing fields. The development of recent optical sensors, probes and methods for imaging or perturbing activity in the behaving mammalian brain is a promising step toward the functional Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

Biological Psychiatry, 2020

Background: 22q11.2 deletion syndrome (22qDS) presents with myriad symptoms, including multiple n... more Background: 22q11.2 deletion syndrome (22qDS) presents with myriad symptoms, including multiple neuropsychiatric disorders. Complications associated with the polygenic haploinsufficiency makes symptoms particularly difficult to manage via traditional therapeutic approaches. However, the varying mechanistic consequences often culminate to generate inappropriate regulation of neuronal circuit activity. We explored if managing this aberrant activity in adults could be a therapeutically beneficial strategy. Methods: To assess and dissect hippocampal circuit function, we performed functional imaging in acute slices and targeted eloquent circuits (specific sub-circuits tied to specific behavioral tasks) to provide relevant behavioral outputs. For example, the ventral and dorsal CA1 regions critically support social and spatial discrimination, respectively. We focally introduced chemogenetic constructs in 34 control and 24 22qDS model mice via adeno-associated viral vectors, driven by excitatory neuron-specific promoter elements, to manipulate circuit recruitment in an on-demand fashion. Results: 22qDS model mice exhibited CA1 excitatory ensemble hyperexcitability and concomitant behavioral deficits in both social and spatial memory. Remarkably, acute chemogenetic inhibition of pyramidal cells successfully corrected the memory deficits, and did so

Brain, 2019

Temporal lobe epilepsy is associated with significant structural pathology in the hippocampus. In... more Temporal lobe epilepsy is associated with significant structural pathology in the hippocampus. In the dentate gyrus, the summative effect of these pathologies is massive hyperexcitability in the granule cells, generating both increased seizure susceptibility and cognitive deficits. To date, therapeutic approaches have failed to improve the cognitive symptoms in fully developed, chronic epilepsy. As the dentate’s principal signalling population, the granule cells’ aggregate excitability has the potential to provide a mechanistically-independent downstream target. We examined whether normalizing epilepsy-associated granule cell hyperexcitability—without correcting the underlying structural circuit disruptions—would constitute an effective therapeutic approach for cognitive dysfunction. In the systemic pilocarpine mouse model of temporal lobe epilepsy, the epileptic dentate gyrus excessively recruits granule cells in behavioural contexts, not just during seizure events, and these mice ...

Progress in Brain Research, 2016

The dentate gyrus plays critical roles both in cognitive processing, and in regulation of the ind... more The dentate gyrus plays critical roles both in cognitive processing, and in regulation of the induction and propagation of pathological activity. The cellular and circuit mechanisms underlying these diverse functions overlap extensively. At the cellular level, the intrinsic properties of dentate granule cells combine to endow these neurons with a fundamental reluctance to activate, one of their hallmark traits. At the circuit level, the dentate gyrus constitutes one of the more heavily inhibited regions of the brain, with strong, fast feedforward and feedback GABAergic inhibition dominating responses to afferent activation. In pathologic states such as epilepsy, a number of alterations within the dentate gyrus combine to compromise the regulatory properties of this circuit, culminating in a collapse of its normal function. This epilepsy-associated transformation in the fundamental properties of this critical regulatory hippocampal circuit may contribute both to seizure propensity, and cognitive and emotional comorbidities characteristic of this disease state.

Nature Communications, 2019

CDKL5 deficiency disorder (CDD) is characterized by epilepsy, intellectual disability, and autist... more CDKL5 deficiency disorder (CDD) is characterized by epilepsy, intellectual disability, and autistic features, and CDKL5-deficient mice exhibit a constellation of behavioral phenotypes reminiscent of the human disorder. We previously found that CDKL5 dysfunction in forebrain glutamatergic neurons results in deficits in learning and memory. However, the pathogenic origin of the autistic features of CDD remains unknown. Here, we find that selective loss of CDKL5 in GABAergic neurons leads to autistic-like phenotypes in mice accompanied by excessive glutamatergic transmission, hyperexcitability, and increased levels of postsynaptic NMDA receptors. Acute, low-dose inhibition of NMDAR signaling ameliorates autistic-like behaviors in GABAergic knockout mice, as well as a novel mouse model bearing a CDDassociated nonsense mutation, CDKL5 R59X, implicating the translational potential of this mechanism. Together, our findings suggest that enhanced NMDAR signaling and circuit hyperexcitability underlie autistic-like features in mouse models of CDD and provide a new therapeutic avenue to treat CDD-related symptoms.

The Journal of Neuroscience, 2002

The predominant neuronal glutamate transporter, EAAC1 (for excitatory amino acid carrier-1), is l... more The predominant neuronal glutamate transporter, EAAC1 (for excitatory amino acid carrier-1), is localized to the dendrites and somata of many neurons. Rare presynaptic localization is restricted to GABA terminals. Because glutamate is a precursor for GABA synthesis, we hypothesized that EAAC1 may play a role in regulating GABA synthesis and, thus, could cause epilepsy in rats when inactivated. Reduced expression of EAAC1 by antisense treatment led to behavioral abnormalities, including staring-freezing episodes and electrographic (EEG) seizures. Extracellular hippocampal and thalamocortical slice recordings showed excessive excitability in antisense-treated rats. Patch-clamp recordings of miniature IPSCs (mIPSCs) conducted in CA1 pyramidal neurons in slices from EAAC1 antisense-treated animals demonstrated a significant decrease in mIPSC amplitude, indicating decreased tonic inhibition. There was a 50% loss of hippocampal GABA levels associated with knockdown of EAAC1, and newly synthesized GABA from extracellular glutamate was significantly impaired by reduction of EAAC1 expression. EAAC1 may participate in normal GABA neurosynthesis and limbic hyperexcitability, whereas epilepsy can result from a disruption of the interaction between EAAC1 and GABA metabolism.

Scientific Reports, 2017

In a mouse model of temporal lobe epilepsy, multicellular calcium imaging revealed that disease e... more In a mouse model of temporal lobe epilepsy, multicellular calcium imaging revealed that disease emergence was accompanied by massive amplification in the normally sparse, afferent stimulationinduced activation of hippocampal dentate granule cells. Patch recordings demonstrated reductions in local inhibitory function within the dentate gyrus at time points where sparse activation was compromised. Mimicking changes in inhibitory synaptic function and transmembrane chloride regulation was sufficient to elicit the dentate gyrus circuit collapse evident during epilepsy development. Pharmacological blockade of outward chloride transport had no effect during epilepsy development, and significantly increased granule cell activation in both control and chronically epileptic animals. This apparent occlusion effect implicates reduction in chloride extrusion as a mechanism contributing to granule cell hyperactivation specifically during early epilepsy development. Glutamine plays a significant role in local synthesis of GABA in synapses. In epileptic mice, sparse granule cell activation could be restored by glutamine application, implicating compromised GABA synthesis. Glutamine had no effect on granule cell activation earlier, during epilepsy development. We conclude that compromised feedforward inhibition within the local circuit generates the massive dentate gyrus circuit hyperactivation evident in animals during and following epilepsy development. However, the mechanisms underlying this disinhibition diverge significantly as epilepsy progresses.

Journal of Neuroscience, 2005

Hippocampal CA1 pyramidal neurons receive intrahippocampal and extrahipppocampal inputs during th... more Hippocampal CA1 pyramidal neurons receive intrahippocampal and extrahipppocampal inputs during theta cycle, whose relative timing and magnitude regulate the probability of CA1 pyramidal cell spiking. Extrahippocampal inputs, giving rise to the primary theta dipole in CA1 stratum lacunosum moleculare, are conveyed by the temporoammonic pathway. The temporoammonic pathway impinging onto the CA1 distal apical dendritic tuft is the most electrotonically distant from the perisomatic region yet is critical in regulating CA1 place cell activity during theta cycles. How does local hippocampal circuitry regulate the integration of this essential, but electrotonically distant, input within the theta period? Using whole-cell somatic recording and voltage-sensitive dye imaging with simultaneous dendritic recording of CA1 pyramidal cell responses, we demonstrate that temporoammonic EPSPs are normally compartmentalized to the apical dendritic tuft by feedforward inhibition. However, when this input is preceded at a one-half theta cycle interval by proximally targeted Schaffer collateral activity, temporoammonic EPSPs propagate to the soma through a joint, codependent mechanism involving activation of Schaffer-specific NMDA receptors and presynaptic inhibition of GABAergic terminals. These afferent interactions, tuned for synaptic inputs arriving one-half theta interval apart, are in turn modulated by feedback inhibition initiated via axon collaterals of pyramidal cells. Therefore, CA1 circuit integration of excitatory inputs endows the CA1 principal cell with a novel property: the ability to function as a temporally specific "AND" gate that provides for sequence-dependent readout of distal inputs.

Journal of Neuroscience, 2006

Vesicular GABA and intraterminal glutamate concentrations are in equilibrium, suggesting inhibito... more Vesicular GABA and intraterminal glutamate concentrations are in equilibrium, suggesting inhibitory efficacy may depend on glutamate availability. Two main intraterminal glutamate sources are uptake by neuronal glutamate transporters and glutamine synthesized through the astrocytic glutamate-glutamine cycle. We examined the involvement of the glutamate-glutamine cycle in modulating GABAergic synaptic efficacy. In the absence of neuronal activity, disruption of the glutamate-glutamine cycle by blockade of neuronal glutamine transport with ␣-(methylamino) isobutyric acid (MeAIB; 5 mM) or inhibition of glutamine synthesis in astrocytes with methionine sulfoximine (MSO; 1.5 mM) had no effect on miniature IPSCs recorded in hippocampal area CA1 pyramidal neurons. However, after a period of moderate synaptic activity, application of MeAIB, MSO, or dihydrokainate (250 M; an astrocytic glutamate transporter inhibitor) significantly reduced evoked IPSC (eIPSC) amplitudes. The MSO effect could be reversed by exogenous application of glutamine (5 mM), whereas glutamine could not rescue the eIPSC decreases induced by the neuronal glutamine transporter inhibitor MeAIB. The activity-dependent reduction in eIPSCs by glutamate-glutamine cycle blockers was accompanied by an enhanced blocking effect of the low-affinity GABA A receptor antagonist, TPMPA [1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid], consistent with diminished GABA release. We further corroborated this hypothesis by examining MeAIB effects on minimal stimulation-evoked quantal IPSCs (meIPSCs). We found that, in MeAIB-containing medium, moderate stimulation induced depression in potency of meIPSCs but no change in release probability, consistent with reduced vesicular GABA content. We conclude that the glutamate-glutamine cycle is a major contributor to synaptic GABA release under physiological conditions, which dynamically regulates inhibitory synaptic strength.

Journal of Neuroscience, 2006

Epilepsy affects 1-2% of the population, with temporal lobe epilepsy (TLE) the most common varian... more Epilepsy affects 1-2% of the population, with temporal lobe epilepsy (TLE) the most common variant in adults. Clinical and experimental studies have demonstrated hippocampal involvement in the seizures underlying TLE. However, identification of specific functional deficits in hippocampal circuits associated with possible roles in seizure generation remains controversial. Significant attention has focused on anatomic and cellular alterations in the dentate gyrus. The dentate gyrus is a primary gateway regulating cortical input to the hippocampus and, thus, a possible contributor to the aberrant cortical-hippocampal interactions underlying the seizures of TLE. Alternate cortical pathways innervating the hippocampus might also contribute to seizure initiation. Despite this potential importance in TLE, these pathways have received little study. Using simultaneous voltage-sensitive dye imaging and patch-clamp recordings in slices from animals with epilepsy, we assessed the relative degree of synaptic excitation activated by multiple cortical inputs to the hippocampus. Surprisingly, dentate gyrus-mediated regulation of the relay of cortical input to the hippocampus is unchanged in epileptic animals, and input via the Schaffer collaterals is actually decreased despite reduction in Schaffer-evoked inhibition. In contrast, a normally weak direct cortical input to area CA1 of hippocampus, the temporoammonic pathway, exhibits a TLE-associated transformation from a spatially restricted, highly regulated pathway to an excitatory projection with Ͼ10-fold increased effectiveness. This dysregulated temporoammonic pathway is critically positioned to mediate generation and/or propagation of seizure activity in the hippocampus.

Nature Protocols, 2008

In many brain areas, circuit connectivity is segregated into specific lamina or glomerula. Functi... more In many brain areas, circuit connectivity is segregated into specific lamina or glomerula. Functional imaging in these anatomically discrete areas is particularly useful in characterizing circuit properties. Voltage-sensitive dye (VSD) imaging directly assays the spatiotemporal dynamics of neuronal activity, including the functional connectivity of the neurons involved. In spatially segregated structures, VSD imaging can define how physiology and connectivity interact, and can identify functional abnormalities in models of neurological and psychiatric disorders. In the following protocol, we describe the in vitro slice preparation, epifluorescence setup and analyses necessary for fast charge-coupled device (CCD)-based VSD imaging combined with simultaneous whole-cell patch recording. The addition of single-cell recordings validates imaging results, and can reveal the relationship between single-cell activity and the VSD-imaged population response; in synchronously activated neurons, this change in whole-cell recorded V m can accurately represent population V m changes driving the VSD responses. Thus, the combined VSD imaging and whole-cell patch approach provides experimental resolution spanning single-cell electrophysiology to complex local circuit responses.

Elsevier eBooks, 2017

Abstract Research in epilepsy has employed a plethora of animal models with which to study and be... more Abstract Research in epilepsy has employed a plethora of animal models with which to study and better understand the mechanisms that lead to this debilitating condition. Modeling human disease requires a rigorous approach, and methodologies that can be easily standardized across laboratories. The following chapter will describe the pilocarpine model of acquired epilepsy. The pilocarpine model mimics many of the features of acquired epilepsy in humans, especially that of temporal lobe epilepsy (TLE). A characteristic of the acquired epilepsies is that the onset of seizures is often preceded by an initial precipitating injury, such as stroke, traumatic brain injury, or a prolonged period of status epilepticus (SE). With these types of injuries, the temporal lobe is especially vulnerable. The most prevalent of adult-onset epilepsies, TLE is also known to be difficult to treat and thus the need for novel treatment approaches is great. The following is an in-depth discussion of the post-SE pilocarpine model of acquired epilepsy. Following a brief introduction to TLE, the methods and variables that optimize successful implementation of the pilocarpine procedure itself will be highlighted. How this model can be employed to study both the process of disease progression following SE (epileptogenesis) and/or the chronic disease state will follow the methodological description. Though the pilocarpine model of TLE has existed for nearly 30 years, the development and use of sophisticated monitoring and imaging techniques, in addition to the recent inclusion of various mice strains, warrants an updated review of this model.

Journal of Neurophysiology, May 1, 1989

1. The afterhyperpolarization (AHP) that follows action potentials was studied in CA1 hippocampal... more 1. The afterhyperpolarization (AHP) that follows action potentials was studied in CA1 hippocampal pyramidal cells from classically conditioned and control rabbits. Measurements of the AHP were obtained with intracellular recordings from CA1 cells within hippocampal slices. 2. The AHP of rabbit CA1 pyramidal cells was found to be accompanied by a conductance increase. The AHP was reduced by bath applications of the calcium channel blockers, cadmium and cobalt, by bath application of the cholinergic agonist, carbamylcholine chloride, and intracellular injection of the calcium chelator, ethylene glycol-bis(B-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). 3. The AHP was markedly reduced in cells from rabbits that were well-trained with the nictitating membrane conditioning procedure, as compared with cells from pseudoconditioned or naive control animals. The difference in AHP amplitudes between conditioned and control groups increased as the number of spikes elicited by the stimulation pulse increased from one to four. Both the duration (measured as the time constant of AHP decay) and amplitude of the AHP were reduced in cells from conditioned animals. 4. The reduced AHP in cells from conditioned animals remained reduced in a medium that contained 0.5 microM tetrodotoxin (TTX) and 5.0 mM tetraethylammonium chloride (TEA); the AHP following calcium spikes was measured under these conditions. Since this medium eliminated synaptic transmission elicited by Schaeffer collateral stimulation, the AHP reduction in pyramidal cells from conditioned animals was not due to a modification in synaptic properties. There were no significant differences in the mean voltage thresholds, amplitudes, or durations of calcium spikes between cells from animals in the three groups. Thus the AHP reduction appears to be due to a modification of a Ca2+ -dependent K+ conductance and was not due to a secondary effect of reductions in calcium conductances underlying the spike. 5. In medium containing TTX and TEA, the amount of injected current required to elicit a calcium spike (current threshold) was significantly greater in cells from conditioned animals than in cells from control animals. This increase in current threshold persisted in 4-aminopyridine (4-AP)-containing medium and so cannot be attributed entirely to conditioning-specific increases in the A-current. 6. The conditioning-specific AHP reduction resulted in increased excitability in cells from conditioned animals versus pseudoconditioned control animals. Cells from conditioned animals fired more spikes to trains of 100-ms depolarizing current pulses than did cells from controls.

Journal of Neurophysiology, 1996

1. Surgically resected tissue from the tip of the human temporal lobe of seven patients undergoin... more 1. Surgically resected tissue from the tip of the human temporal lobe of seven patients undergoing temporal lobectomy was employed to study functional properties of GABAergic inhibition mediated through activation of GABAA receptors, using patch-clamp recording techniques in acutely isolated neurons and in slices of human temporal cortex. 2. Human temporal cortical pyramidal neurons from surgically resected tissue could be acutely isolated with the use of conventional methods. These neurons appeared normal in morphology, in their intrinsic membrane properties, and in their response to application of exogenous gamma-aminobutyric acid (GABA). 3. Application of GABA to acutely isolated human temporal cortical neurons elicited a large current with an average reversal potential of -65 mV, presumably mediated through a GABAA-activated chloride conductance. Application of varying concentrations of GABA generated a concentration/response relationship that could be well-fitted by a conventio...

Encyclopedia of Basic Epilepsy Research, 2009

The role of glial cells in brain function is receiving renewed interest. Recent studies show that... more The role of glial cells in brain function is receiving renewed interest. Recent studies show that astrocytes, a subtype of glial cell, regulate neuronal excitability and synaptic transmission. Astrocytes regulate inhibition through a glial adenosine cycle and by providing glutamine, a precursor for the synthesis of GABA, to interneurons. Astrocytes also release the excitatory transmitter glutamate, which by acting through neuronal NMDA receptors can increase neuronal excitability. Because brain injury alters the expression of glial enzymes that control these inhibitory pathways, it is possible that novel anticonvulsant strategies could be directed to glial targets.

Homeostatic Control of Brain Function, 2015

F1000 - Post-publication peer review of the biomedical literature, 2015

Linking neural microcircuit function to emergent properties of the mammalian brain requires fines... more Linking neural microcircuit function to emergent properties of the mammalian brain requires finescale manipulation and measurement of neural activity during behavior, where each neuron's coding and dynamics can be characterized. We developed an optical method for simultaneous cellular-resolution stimulation and large-scale recording of neuronal activity in behaving mice. Dual-wavelength two-photon excitation allowed largely independent functional imaging with a green fluorescent calcium sensor (GCaMP3, λ = 920 ± 6 nm) and single-neuron photostimulation with a red-shifted optogenetic probe (C1V1, λ = 1,064 ± 6 nm) in neurons coexpressing the two proteins. We manipulated task-modulated activity in individual hippocampal CA1 place cells during spatial navigation in a virtual reality environment, mimicking natural place-field activity, or 'biasing', to reveal subthreshold dynamics. Notably, manipulating single place-cell activity also affected activity in small groups of other place cells that were active around the same time in the task, suggesting a functional role for local place cell interactions in shaping firing fields. The development of recent optical sensors, probes and methods for imaging or perturbing activity in the behaving mammalian brain is a promising step toward the functional Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

Biological Psychiatry, 2020

Background: 22q11.2 deletion syndrome (22qDS) presents with myriad symptoms, including multiple n... more Background: 22q11.2 deletion syndrome (22qDS) presents with myriad symptoms, including multiple neuropsychiatric disorders. Complications associated with the polygenic haploinsufficiency makes symptoms particularly difficult to manage via traditional therapeutic approaches. However, the varying mechanistic consequences often culminate to generate inappropriate regulation of neuronal circuit activity. We explored if managing this aberrant activity in adults could be a therapeutically beneficial strategy. Methods: To assess and dissect hippocampal circuit function, we performed functional imaging in acute slices and targeted eloquent circuits (specific sub-circuits tied to specific behavioral tasks) to provide relevant behavioral outputs. For example, the ventral and dorsal CA1 regions critically support social and spatial discrimination, respectively. We focally introduced chemogenetic constructs in 34 control and 24 22qDS model mice via adeno-associated viral vectors, driven by excitatory neuron-specific promoter elements, to manipulate circuit recruitment in an on-demand fashion. Results: 22qDS model mice exhibited CA1 excitatory ensemble hyperexcitability and concomitant behavioral deficits in both social and spatial memory. Remarkably, acute chemogenetic inhibition of pyramidal cells successfully corrected the memory deficits, and did so

Brain, 2019

Temporal lobe epilepsy is associated with significant structural pathology in the hippocampus. In... more Temporal lobe epilepsy is associated with significant structural pathology in the hippocampus. In the dentate gyrus, the summative effect of these pathologies is massive hyperexcitability in the granule cells, generating both increased seizure susceptibility and cognitive deficits. To date, therapeutic approaches have failed to improve the cognitive symptoms in fully developed, chronic epilepsy. As the dentate’s principal signalling population, the granule cells’ aggregate excitability has the potential to provide a mechanistically-independent downstream target. We examined whether normalizing epilepsy-associated granule cell hyperexcitability—without correcting the underlying structural circuit disruptions—would constitute an effective therapeutic approach for cognitive dysfunction. In the systemic pilocarpine mouse model of temporal lobe epilepsy, the epileptic dentate gyrus excessively recruits granule cells in behavioural contexts, not just during seizure events, and these mice ...

Progress in Brain Research, 2016

The dentate gyrus plays critical roles both in cognitive processing, and in regulation of the ind... more The dentate gyrus plays critical roles both in cognitive processing, and in regulation of the induction and propagation of pathological activity. The cellular and circuit mechanisms underlying these diverse functions overlap extensively. At the cellular level, the intrinsic properties of dentate granule cells combine to endow these neurons with a fundamental reluctance to activate, one of their hallmark traits. At the circuit level, the dentate gyrus constitutes one of the more heavily inhibited regions of the brain, with strong, fast feedforward and feedback GABAergic inhibition dominating responses to afferent activation. In pathologic states such as epilepsy, a number of alterations within the dentate gyrus combine to compromise the regulatory properties of this circuit, culminating in a collapse of its normal function. This epilepsy-associated transformation in the fundamental properties of this critical regulatory hippocampal circuit may contribute both to seizure propensity, and cognitive and emotional comorbidities characteristic of this disease state.

Nature Communications, 2019

CDKL5 deficiency disorder (CDD) is characterized by epilepsy, intellectual disability, and autist... more CDKL5 deficiency disorder (CDD) is characterized by epilepsy, intellectual disability, and autistic features, and CDKL5-deficient mice exhibit a constellation of behavioral phenotypes reminiscent of the human disorder. We previously found that CDKL5 dysfunction in forebrain glutamatergic neurons results in deficits in learning and memory. However, the pathogenic origin of the autistic features of CDD remains unknown. Here, we find that selective loss of CDKL5 in GABAergic neurons leads to autistic-like phenotypes in mice accompanied by excessive glutamatergic transmission, hyperexcitability, and increased levels of postsynaptic NMDA receptors. Acute, low-dose inhibition of NMDAR signaling ameliorates autistic-like behaviors in GABAergic knockout mice, as well as a novel mouse model bearing a CDDassociated nonsense mutation, CDKL5 R59X, implicating the translational potential of this mechanism. Together, our findings suggest that enhanced NMDAR signaling and circuit hyperexcitability underlie autistic-like features in mouse models of CDD and provide a new therapeutic avenue to treat CDD-related symptoms.

The Journal of Neuroscience, 2002

The predominant neuronal glutamate transporter, EAAC1 (for excitatory amino acid carrier-1), is l... more The predominant neuronal glutamate transporter, EAAC1 (for excitatory amino acid carrier-1), is localized to the dendrites and somata of many neurons. Rare presynaptic localization is restricted to GABA terminals. Because glutamate is a precursor for GABA synthesis, we hypothesized that EAAC1 may play a role in regulating GABA synthesis and, thus, could cause epilepsy in rats when inactivated. Reduced expression of EAAC1 by antisense treatment led to behavioral abnormalities, including staring-freezing episodes and electrographic (EEG) seizures. Extracellular hippocampal and thalamocortical slice recordings showed excessive excitability in antisense-treated rats. Patch-clamp recordings of miniature IPSCs (mIPSCs) conducted in CA1 pyramidal neurons in slices from EAAC1 antisense-treated animals demonstrated a significant decrease in mIPSC amplitude, indicating decreased tonic inhibition. There was a 50% loss of hippocampal GABA levels associated with knockdown of EAAC1, and newly synthesized GABA from extracellular glutamate was significantly impaired by reduction of EAAC1 expression. EAAC1 may participate in normal GABA neurosynthesis and limbic hyperexcitability, whereas epilepsy can result from a disruption of the interaction between EAAC1 and GABA metabolism.

Scientific Reports, 2017

In a mouse model of temporal lobe epilepsy, multicellular calcium imaging revealed that disease e... more In a mouse model of temporal lobe epilepsy, multicellular calcium imaging revealed that disease emergence was accompanied by massive amplification in the normally sparse, afferent stimulationinduced activation of hippocampal dentate granule cells. Patch recordings demonstrated reductions in local inhibitory function within the dentate gyrus at time points where sparse activation was compromised. Mimicking changes in inhibitory synaptic function and transmembrane chloride regulation was sufficient to elicit the dentate gyrus circuit collapse evident during epilepsy development. Pharmacological blockade of outward chloride transport had no effect during epilepsy development, and significantly increased granule cell activation in both control and chronically epileptic animals. This apparent occlusion effect implicates reduction in chloride extrusion as a mechanism contributing to granule cell hyperactivation specifically during early epilepsy development. Glutamine plays a significant role in local synthesis of GABA in synapses. In epileptic mice, sparse granule cell activation could be restored by glutamine application, implicating compromised GABA synthesis. Glutamine had no effect on granule cell activation earlier, during epilepsy development. We conclude that compromised feedforward inhibition within the local circuit generates the massive dentate gyrus circuit hyperactivation evident in animals during and following epilepsy development. However, the mechanisms underlying this disinhibition diverge significantly as epilepsy progresses.

Journal of Neuroscience, 2005

Hippocampal CA1 pyramidal neurons receive intrahippocampal and extrahipppocampal inputs during th... more Hippocampal CA1 pyramidal neurons receive intrahippocampal and extrahipppocampal inputs during theta cycle, whose relative timing and magnitude regulate the probability of CA1 pyramidal cell spiking. Extrahippocampal inputs, giving rise to the primary theta dipole in CA1 stratum lacunosum moleculare, are conveyed by the temporoammonic pathway. The temporoammonic pathway impinging onto the CA1 distal apical dendritic tuft is the most electrotonically distant from the perisomatic region yet is critical in regulating CA1 place cell activity during theta cycles. How does local hippocampal circuitry regulate the integration of this essential, but electrotonically distant, input within the theta period? Using whole-cell somatic recording and voltage-sensitive dye imaging with simultaneous dendritic recording of CA1 pyramidal cell responses, we demonstrate that temporoammonic EPSPs are normally compartmentalized to the apical dendritic tuft by feedforward inhibition. However, when this input is preceded at a one-half theta cycle interval by proximally targeted Schaffer collateral activity, temporoammonic EPSPs propagate to the soma through a joint, codependent mechanism involving activation of Schaffer-specific NMDA receptors and presynaptic inhibition of GABAergic terminals. These afferent interactions, tuned for synaptic inputs arriving one-half theta interval apart, are in turn modulated by feedback inhibition initiated via axon collaterals of pyramidal cells. Therefore, CA1 circuit integration of excitatory inputs endows the CA1 principal cell with a novel property: the ability to function as a temporally specific "AND" gate that provides for sequence-dependent readout of distal inputs.

Journal of Neuroscience, 2006

Vesicular GABA and intraterminal glutamate concentrations are in equilibrium, suggesting inhibito... more Vesicular GABA and intraterminal glutamate concentrations are in equilibrium, suggesting inhibitory efficacy may depend on glutamate availability. Two main intraterminal glutamate sources are uptake by neuronal glutamate transporters and glutamine synthesized through the astrocytic glutamate-glutamine cycle. We examined the involvement of the glutamate-glutamine cycle in modulating GABAergic synaptic efficacy. In the absence of neuronal activity, disruption of the glutamate-glutamine cycle by blockade of neuronal glutamine transport with ␣-(methylamino) isobutyric acid (MeAIB; 5 mM) or inhibition of glutamine synthesis in astrocytes with methionine sulfoximine (MSO; 1.5 mM) had no effect on miniature IPSCs recorded in hippocampal area CA1 pyramidal neurons. However, after a period of moderate synaptic activity, application of MeAIB, MSO, or dihydrokainate (250 M; an astrocytic glutamate transporter inhibitor) significantly reduced evoked IPSC (eIPSC) amplitudes. The MSO effect could be reversed by exogenous application of glutamine (5 mM), whereas glutamine could not rescue the eIPSC decreases induced by the neuronal glutamine transporter inhibitor MeAIB. The activity-dependent reduction in eIPSCs by glutamate-glutamine cycle blockers was accompanied by an enhanced blocking effect of the low-affinity GABA A receptor antagonist, TPMPA [1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid], consistent with diminished GABA release. We further corroborated this hypothesis by examining MeAIB effects on minimal stimulation-evoked quantal IPSCs (meIPSCs). We found that, in MeAIB-containing medium, moderate stimulation induced depression in potency of meIPSCs but no change in release probability, consistent with reduced vesicular GABA content. We conclude that the glutamate-glutamine cycle is a major contributor to synaptic GABA release under physiological conditions, which dynamically regulates inhibitory synaptic strength.

Journal of Neuroscience, 2006

Epilepsy affects 1-2% of the population, with temporal lobe epilepsy (TLE) the most common varian... more Epilepsy affects 1-2% of the population, with temporal lobe epilepsy (TLE) the most common variant in adults. Clinical and experimental studies have demonstrated hippocampal involvement in the seizures underlying TLE. However, identification of specific functional deficits in hippocampal circuits associated with possible roles in seizure generation remains controversial. Significant attention has focused on anatomic and cellular alterations in the dentate gyrus. The dentate gyrus is a primary gateway regulating cortical input to the hippocampus and, thus, a possible contributor to the aberrant cortical-hippocampal interactions underlying the seizures of TLE. Alternate cortical pathways innervating the hippocampus might also contribute to seizure initiation. Despite this potential importance in TLE, these pathways have received little study. Using simultaneous voltage-sensitive dye imaging and patch-clamp recordings in slices from animals with epilepsy, we assessed the relative degree of synaptic excitation activated by multiple cortical inputs to the hippocampus. Surprisingly, dentate gyrus-mediated regulation of the relay of cortical input to the hippocampus is unchanged in epileptic animals, and input via the Schaffer collaterals is actually decreased despite reduction in Schaffer-evoked inhibition. In contrast, a normally weak direct cortical input to area CA1 of hippocampus, the temporoammonic pathway, exhibits a TLE-associated transformation from a spatially restricted, highly regulated pathway to an excitatory projection with Ͼ10-fold increased effectiveness. This dysregulated temporoammonic pathway is critically positioned to mediate generation and/or propagation of seizure activity in the hippocampus.

Nature Protocols, 2008

In many brain areas, circuit connectivity is segregated into specific lamina or glomerula. Functi... more In many brain areas, circuit connectivity is segregated into specific lamina or glomerula. Functional imaging in these anatomically discrete areas is particularly useful in characterizing circuit properties. Voltage-sensitive dye (VSD) imaging directly assays the spatiotemporal dynamics of neuronal activity, including the functional connectivity of the neurons involved. In spatially segregated structures, VSD imaging can define how physiology and connectivity interact, and can identify functional abnormalities in models of neurological and psychiatric disorders. In the following protocol, we describe the in vitro slice preparation, epifluorescence setup and analyses necessary for fast charge-coupled device (CCD)-based VSD imaging combined with simultaneous whole-cell patch recording. The addition of single-cell recordings validates imaging results, and can reveal the relationship between single-cell activity and the VSD-imaged population response; in synchronously activated neurons, this change in whole-cell recorded V m can accurately represent population V m changes driving the VSD responses. Thus, the combined VSD imaging and whole-cell patch approach provides experimental resolution spanning single-cell electrophysiology to complex local circuit responses.