Akiva S. Cohen

A Neuronal Glutamate Transporter Contributes to Neurotransmitter GABA Synthesis and Epilepsy

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.

Regional hippocampal alteration associated with cognitive deficit following experimental brain injury : A systems, network and cellular evaluation

Cognitive deficits persist in patients who survive traumatic brain injury (TBI). Lateral fluid percussion brain injury in the mouse, a model of human TBI, results in hippocampal-dependent cognitive impairment, similar to retrograde amnesia often associated with TBI. To identify potential substrates of the cognitive impairment, we evaluated regional neuronal loss, regional hippocampal excitability and inhibitory synaptic transmission. Design-based stereology demonstrated an approximate 40% loss of neurons through all subregions of the hippocampus following injury compared with sham. Input/output curves recorded in slices of injured brain demonstrated increased net synaptic efficacy in the dentate gyrus in concert with decreased net synaptic efficacy and excitatory postsynaptic potential-spike relationship in area CA1 compared with sham slices. Pharmacological agents modulating inhibitory transmission partially restored regional injury-induced alterations in net synaptic efficacy. Both evoked and spontaneous miniature inhibitory postsynaptic currents (mIPSCs) recorded in surviving dentate granule neurons were smaller and less frequent in injured brains than in uninjured brains. Conversely, both evoked and spontaneous mIPSCs recorded in surviving area CA1 pyramidal neurons were larger in injured brains than in uninjured brains. Together, these alterations suggest that regional hippocampal function is altered in the injured brain. This study demonstrates for the first time that brain injury selectively disrupts hippocampal function by causing uniform neuronal loss, inhibitory synaptic dysfunction, and regional, but opposing, shifts in circuit excitability. These changes may contribute to the cognitive impairments that result from brain injury.

Dentate granule cell GABAA receptors in epileptic hippocampus: enhanced synaptic efficacy and altered pharmacology

The dentate gyrus (DG) normally functions as a filter, preventing propagation of synchronized activity into the seizure‐prone hippocampus. This filter or ‘gatekeeper’ attribute of the DG is compromised in various pathological states, including temporal lobe epilepsy (TLE). This study examines the role that altered inhibition may play in the deterioration of this crucial DG function. Using the pilocarpine animal model of TLE, we demonstrate that inhibitory synaptic function is altered in principal cells of the DG. Spontaneous miniature inhibitory postsynaptic currents (mIPSCs) recorded in dentate granule cells (DGCs) from epileptic animals were larger, more sensitive to blockade by zinc and less sensitive to augmentation by the benzodiazepine type site 1 modulator zolpidem. Furthermore, mIPSCs examined during a quiescent period following injury but preceding onset of epilepsy were significantly smaller than those present either in control or in TLE DGCs, and had already acquired sensitivity to blockade by zinc prior to the onset of spontaneous seizures. Rapid agonist application experiments demonstrated that prolonged (>35 ms) exposure to zinc is required to block GABAA receptors (GABAARs) in patches pulled from epileptic DGCs. Therefore, zinc must be tonically present to block DGC GABAARs and alter DG function. This would occur only during repetitive activation of mossy fibres. Thus, in the pilocarpine animal model of TLE, an early, de novo, expression of zinc‐sensitive GABAARs is coupled with delayed, epilepsy‐induced development of a zinc delivery system provided by aberrant sprouting of zinc‐containing mossy fibre recurrent collaterals. The temporal and spatial juxtaposition of these pathophysiological alterations may compromise normal ‘gatekeeper’ function of the DG through dynamic zinc‐induced failure of inhibition, predisposing the hippocampal circuit to generate seizures.

Mechanism of Hyperinsulinism in Short-chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency Involves Activation of Glutamate Dehydrogenase

The mechanism of insulin dysregulation in children with hyperinsulinism associated with inactivating mutations of short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) was examined in mice with a knock-out of the hadh gene (hadh−/−). The hadh−/− mice had reduced levels of plasma glucose and elevated plasma insulin levels, similar to children with SCHAD deficiency. hadh−/− mice were hypersensitive to oral amino acid with decrease of glucose level and elevation of insulin. Hypersensitivity to oral amino acid in hadh−/− mice can be explained by abnormal insulin responses to a physiological mixture of amino acids and increased sensitivity to leucine stimulation in isolated perifused islets. Measurement of cytosolic calcium showed normal basal levels and abnormal responses to amino acids in hadh−/− islets. Leucine, glutamine, and alanine are responsible for amino acid hypersensitivity in islets. hadh−/− islets have lower intracellular glutamate and aspartate levels, and this decrease can be prevented by high glucose. hadh−/− islets also have increased [U-14C]glutamine oxidation. In contrast, hadh−/− mice have similar glucose tolerance and insulin sensitivity compared with controls. Perifused hadh−/− islets showed no differences from controls in response to glucose-stimulated insulin secretion, even with addition of either a medium-chain fatty acid (octanoate) or a long-chain fatty acid (palmitate). Pull-down experiments with SCHAD, anti-SCHAD, or anti-GDH antibodies showed protein-protein interactions between SCHAD and GDH. GDH enzyme kinetics of hadh−/− islets showed an increase in GDH affinity for its substrate, α-ketoglutarate. These studies indicate that SCHAD deficiency causes hyperinsulinism by activation of GDH via loss of inhibitory regulation of GDH by SCHAD.

Specific proteolysis of the NR2 subunit at multiple sites by calpain

The NMDA subtype of glutamate receptor plays an important role in the molecular mechanisms of learning, memory and excitotoxicity. NMDA receptors are highly permeable to calcium, which can lead to the activation of the calcium‐dependent protease, calpain. In the present study, the ability of calpain to modulate NMDA receptor function through direct proteolytic digestion of the individual NMDA receptor subunits was examined. HEK293t cells were cotransfected with the NR1a/2A, NR1a/2B or NR1a/2C receptor combinations. Cellular homogenates of these receptor combinations were prepared and digested by purified calpain I in vitro. All three NR2 subunits could be proteolyzed by calpain I while no actin or NR1a cleavage was observed. Based on immunoblot analysis, calpain cleavage of NR2A, NR2B and NR2C subunits was limited to their C‐terminal region. In vitro calpain digestion of fusion protein constructs containing the C‐terminal region of NR2A yielded two cleavage sites at amino acids 1279 and 1330. Although it has been suggested that calpain cleavage of the NMDA receptor may act as a negative feedback mechanism, the current findings demonstrated that calpain cleavage did not alter [125I]MK801 binding and that receptors truncated to the identified cleavage sites had peak intracellular calcium levels, 45Ca uptake rates and basal electrophysiological properties similar to wild type.

Brain injury impairs dentate gyrus inhibitory efficacy.

Every 23 s, a person sustains a traumatic brain injury in the United States leaving many patients with substantial cognitive impairment and epilepsy. Injury-induced alterations in the hippocampus underpin many of these disturbances of neurological function. Abnormalities in the dentate gyrus are likely to play a major role in the observed pathophysiology because this subregion functions as a filter impeding excessive or aberrant activity from propagating further into the circuit and following experimental brain injury, the dentate gyrus becomes more excitable. Although alteration in excitation or inhibition could mediate this effect in the dentate gyrus, we show a key role played by an impairment of GABA(A)ergic inhibition. The efficacy of GABA(A)-mediated inhibition depends on a low [Cl-]i that is maintained by neuronal K-Cl co-transporter 2 (KCC2). Using fluid percussion injury (FPI) in the mouse, we demonstrate significant reductions in KCC2 protein and mRNA expression in the dentate gyrus that causes a depolarizing shift in GABA(A) reversal potential, due to impaired chloride clearance, resulting in reduced inhibitory efficiency. This study elucidates a novel mechanism underlying diminished dentate gyrus inhibitory efficacy and provides an innovative target for the development of potential therapeutics to restore the severe pathological consequences of traumatic brain injury.

Injury-induced alterations in CNS electrophysiology.

Mild to moderate cases of traumatic brain injury (TBI) are very common, but are not always associated with the overt pathophysiogical changes seen following severe trauma. While neuronal death has been considered to be a major factor, the pervasive memory, cognitive and motor function deficits suffered by many mild TBI patients do not always correlate with cell loss. Therefore, we assert that functional impairment may result from alterations in surviving neurons. Current research has begun to explore CNS synaptic circuits after traumatic injury. Here we review significant findings made using in vivo and in vitro models of TBI that provide mechanistic insight into injury-induced alterations in synaptic electrophysiology. In the hippocampus, research now suggests that TBI regionally alters the delicate balance between excitatory and inhibitory neurotransmission in surviving neurons, disrupting the normal functioning of synaptic circuits. In another approach, a simplified model of neuronal stretch injury in vitro, has been used to directly explore how injury impacts the physiology and cell biology of neurons in the absence of alterations in blood flow, blood brain barrier integrity, or oxygenation associated with in vivo models of brain injury. This chapter discusses how these two models alter excitatory and inhibitory synaptic transmission at the receptor, cellular and circuit levels and how these alterations contribute to cognitive impairment and a reduction in seizure threshold associated with human concussive brain injury.

Calcium homeostasis modulator 1 (CALHM1) is the pore-forming subunit of an ion channel that mediates extracellular Ca2+ regulation of neuronal excitability

Extracellular Ca2+ (Ca2+ o) plays important roles in physiology. Changes of Ca2+o concentration ([Ca2+]o) have been observed to modulate neuronal excitability in various physiological and pathophysiological settings, but the mechanisms by which neurons detect [Ca2+]o are not fully understood. Calcium homeostasis modulator 1 (CALHM1) expression was shown to induce cation currents in cells and elevate cytoplasmic Ca2+ concentration ([Ca2+]i) in response to removal of Ca2+o and its subsequent addback. However, it is unknown whether CALHM1 is a pore-forming ion channel or modulates endogenous ion channels. Here we identify CALHM1 as the pore-forming subunit of a plasma membrane Ca2+-permeable ion channel with distinct ion permeability properties and unique coupled allosteric gating regulation by voltage and [Ca2+]o. Furthermore, we show that CALHM1 is expressed in mouse cortical neurons that respond to reducing [Ca2+]o with enhanced conductance and action potential firing and strongly elevated [Ca2+]i upon Ca2+o removal and its addback. In contrast, these responses are strongly muted in neurons from mice with CALHM1 genetically deleted. These results demonstrate that CALHM1 is an evolutionarily conserved ion channel family that detects membrane voltage and extracellular Ca2+ levels and plays a role in cortical neuronal excitability and Ca2+ homeostasis, particularly in response to lowering [Ca2+]o and its restoration to normal levels.

Dietary branched chain amino acids ameliorate injury-induced cognitive impairment

Neurological dysfunction caused by traumatic brain injury results in profound changes in net synaptic efficacy, leading to impaired cognition. Because excitability is directly controlled by the balance of excitatory and inhibitory activity, underlying mechanisms causing these changes were investigated using lateral fluid percussion brain injury in mice. Although injury-induced shifts in net synaptic efficacy were not accompanied by changes in hippocampal glutamate and GABA levels, significant reductions were seen in the concentration of branched chain amino acids (BCAAs), which are key precursors to de novo glutamate synthesis. Dietary consumption of BCAAs restored hippocampal BCAA concentrations to normal, reversed injury-induced shifts in net synaptic efficacy, and led to reinstatement of cognitive performance after concussive brain injury. All brain-injured mice that consumed BCAAs demonstrated cognitive improvement with a simultaneous restoration in net synaptic efficacy. Posttraumatic changes in the expression of cytosolic branched chain aminotransferase, branched chain ketoacid dehydrogenase, glutamate dehydrogenase, and glutamic acid decarboxylase support a perturbation of BCAA and neurotransmitter metabolism. Ex vivo application of BCAAs to hippocampal slices from injured animals restored posttraumatic regional shifts in net synaptic efficacy as measured by field excitatory postsynaptic potentials. These results suggest that dietary BCAA intervention could promote cognitive improvement by restoring hippocampal function after a traumatic brain injury.

Stretch-grown axons retain the ability to transmit active electrical signals

Little is known about extensive nervous system growth after axons reach their targets. Indeed, postnatal animals continue to grow, suggesting that axons are stretched to accommodate the expanding body. We have previously shown that axons can sustain stretch‐growth rates reaching 1 cm/day; however, it remained unknown whether the ability to transmit active signals was maintained. Here, stretch‐growth did not alter sodium channel activation, inactivation, and recovery or potassium channel activation. In addition, neurons generated normal action potentials that propagated across stretch‐grown axons. Surprisingly, Na and K channel density increased due to stretch‐growth, which may represent a natural response to preserve the fidelity of neuronal signaling.