Paulette B. Goforth

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.

Leptin Acts via Lateral Hypothalamic Area Neurotensin Neurons to Inhibit Orexin Neurons by Multiple GABA-Independent Mechanisms

The adipocyte-derived hormone leptin modulates neural systems appropriately for the status of body energy stores. Leptin inhibits lateral hypothalamic area (LHA) orexin (OX; also known as hypocretin)-producing neurons, which control feeding, activity, and energy expenditure, among other parameters. Our previous results suggest that GABAergic LHA leptin receptor (LepRb)-containing and neurotensin (Nts)-containing (LepRbNts) neurons lie in close apposition with OX neurons and control Ox mRNA expression. Here, we show that, similar to leptin, activation of LHA Nts neurons by the excitatory hM3Dq DREADD (designer receptor exclusively activated by designer drugs) hyperpolarizes membrane potential and suppresses action potential firing in OX neurons in mouse hypothalamic slices. Furthermore, ablation of LepRb from Nts neurons abrogated the leptin-mediated inhibition, demonstrating that LepRbNts neurons mediate the inhibition of OX neurons by leptin. Leptin did not significantly enhance GABAA-mediated inhibitory synaptic transmission, and GABA receptor antagonists did not block leptin-mediated inhibition of OX neuron activity. Rather, leptin diminished the frequency of spontaneous EPSCs onto OX neurons. Furthermore, leptin indirectly activated an ATP-sensitive potassium (KATP) channel in OX neurons, which was required for the hyperpolarization of OX neurons by leptin. Although Nts did not alter OX activity, galanin, which is coexpressed in LepRbNts neurons, inhibited OX neurons, whereas the galanin receptor antagonist M40 (galanin-(1–12)-Pro3-(Ala-Leu)2-Ala amide) prevented the leptin-induced hyperpolarization of OX cells. These findings demonstrate that leptin indirectly inhibits OX neurons by acting on LHA LepRbNts neurons to mediate two distinct GABA-independent mechanisms of inhibition: the presynaptic inhibition of excitatory neurotransmission and the opening of KATP channels.

Calcium-activated K+ Channels of Mouse β-cells are Controlled by Both Store and Cytoplasmic Ca2+ Experimental and Theoretical Studies

A novel calcium-dependent potassium current (Kslow) that slowly activates in response to a simulated islet burst was identified recently in mouse pancreatic β-cells (Göpel, S.O., T. Kanno, S. Barg, L. Eliasson, J. Galvanovskis, E. Renström, and P. Rorsman. 1999. J. Gen. Physiol. 114:759–769). Kslow activation may help terminate the cyclic bursts of Ca2+-dependent action potentials that drive Ca2+ influx and insulin secretion in β-cells. Here, we report that when [Ca2+]i handling was disrupted by blocking Ca2+ uptake into the ER with two separate agents reported to block the sarco/endoplasmic calcium ATPase (SERCA), thapsigargin (1–5 μM) or insulin (200 nM), Kslow was transiently potentiated and then inhibited. Kslow amplitude could also be inhibited by increasing extracellular glucose concentration from 5 to 10 mM. The biphasic modulation of Kslow by SERCA blockers could not be explained by a minimal mathematical model in which [Ca2+]i is divided between two compartments, the cytosol and the ER, and Kslow activation mirrors changes in cytosolic calcium induced by the burst protocol. However, the experimental findings were reproduced by a model in which Kslow activation is mediated by a localized pool of [Ca2+] in a subspace located between the ER and the plasma membrane. In this model, the subspace [Ca2+] follows changes in cytosolic [Ca2+] but with a gradient that reflects Ca2+ efflux from the ER. Slow modulation of this gradient as the ER empties and fills may enhance the role of Kslow and [Ca2+] handling in influencing β-cell electrical activity and insulin secretion.

Excitatory synaptic transmission and network activity are depressed following mechanical injury in cortical neurons

In vitro and in vivo traumatic brain injury (TBI) alter the function and expression of glutamate receptors, yet the combined effect of these alterations on cortical excitatory synaptic transmission is unclear. We examined the effect of in vitro mechanical injury on excitatory synaptic function in cultured cortical neurons by assaying synaptically driven intracellular free calcium ([Ca(2+)](i)) oscillations in small neuronal networks as well as spontaneous and miniature excitatory postsynaptic currents (mEPSCs). We show that injury decreased the incidence and frequency of spontaneous neuronal [Ca(2+)](i) oscillations for at least 2 days post-injury. The amplitude of the oscillations was reduced immediately and 2 days post-injury, although a transient rebound at 4 h post-injury was observed due to increased activity of N-methyl-d-aspartate (NMDARs) and calcium-permeable α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (CP-AMPARs). Increased CP-AMPAR function was abolished by the inhibition of protein synthesis. In parallel, mEPSC amplitude decreased immediately, 4 h, and 2 days post-injury, with a transient increase in the contribution of synaptic CP-AMPARs observed at 4 h post-injury. Decreased mEPSC amplitude was evident after injury, even if NMDARs and CP-AMPARs were blocked pharmacologically, suggesting the decrease reflected alterations in synaptic Glur2-containing, calcium-impermeable AMPARs. Despite the transient increase in CP-AMPAR activity that we observed, the overriding effect of mechanical injury was long-term depression of excitatory neurotransmission that would be expected to contribute to the cognitive deficits of TBI.

Mechanical Injury Modulates AMPA Receptor Kinetics via an NMDA Receptor–Dependent Pathway

Alterations in glutamatergic transmission are thought to contribute to secondary neuronal damage following traumatic brain injury. Using an in vitro cell injury model, we previously demonstrated an apparent reduction in AMPA receptor desensitization and resultant potentiation of AMPA-evoked currents after stretch injury of cultured neonatal rat cortical neurons. In the present study, we sought to further characterize injury-induced enhancement of AMPA current and elucidate the mechanisms responsible for this pathological process. Using the patch-clamp technique, agonist-activated currents were recorded from control and injured neurons. Potentiation of AMPA-mediated currents occurred quickly, within 15-30 min following injury, and persisted for at least 24 h. Stretch-injury slowed the activation and desensitization of AMPA mediated currents recorded from excised outside-out patches. The co-application of 100 microM AMPA and 20 microM thiocyanate enhanced AMPA receptor desensitization in control neurons and restored desensitization in injured neurons. The potentiation of AMPA-elicited current was prevented by the NMDA receptor antagonist D-APV (20 microM) or the CaMKII inhibitor KN93 (10 microM). These results suggest that mechanical injury initiates a biochemical cascade that involves NMDA receptor and CaMKII activation and produces a long-lasting reduction of AMPA receptor desensitization, which may contribute to the pathophysiology of traumatic brain injury.

SPHINGOSINE-1-PHOSPHATE RECEPTORS MEDIATE NEUROMODULATORY FUNCTIONS IN THE CNS

Sphingosine‐1‐phosphate (S1P) is a ubiquitous, lipophilic cellular mediator that acts in part by activation of G‐protein‐coupled receptor. Modulation of S1P signaling is an emerging pharmacotherapeutic target for immunomodulatory drugs. Although multiple S1P receptor types exist in the CNS, little is known about their function. Here, we report that S1P stimulated G‐protein activity in the CNS, and results from [35S]GTPγS autoradiography using the S1P1‐selective agonist SEW2871 and the S1P1/3‐selective antagonist VPC44116 show that in several regions a majority of this activity is mediated by S1P1 receptors. S1P receptor activation inhibited glutamatergic neurotransmission as determined by electrophysiological recordings in cortical neurons in vitro, and this effect was mimicked by SEW2871 and inhibited by VPC44116. Moreover, central administration of S1P produced in vivo effects resembling the actions of cannabinoids, including thermal antinociception, hypothermia, catalepsy and hypolocomotion, but these actions were independent of CB1 receptors. At least one of the central effects of S1P, thermal antinociception, is also at least partly S1P1 receptor mediated because it was produced by SEW2871 and attenuated by VPC44116. These results indicate that CNS S1P receptors are part of a physiologically relevant and widespread neuromodulatory system, and that the S1P1 receptor contributes to S1P‐mediated antinociception.

Potentiation of GABAA currents after mechanical injury of cortical neurons

Numerous studies have implicated glutamate receptors, glutamate neurotoxicity, and hyperexcitation in the pathobiology of traumatic brain injury, yet much less is known about the effects of neurotrauma on inhibitory GABA channels of the brain. Using an in vitro cell injury model, we tested whether mild stretch injury altered the GABA(A) currents of cultured rat cortical neurons. The application of 1-100 microM GABA to single pyramidal neurons voltage clamped to -60 mV activated an inward current that reversed near 0 mV in solutions containing symmetrical [Cl-]. This current was inhibited by bicuculline, consistent with mediation by GABA(A) receptor channels. In injured neurons, 50 microM GABA elicited a peak current density of 41.2 +/- 2.6 pA/pF (n = 82), which was significantly larger than in uninjured control neurons, 20.2 +/- 1.7 pA/pF (n = 69, p < 0.01). The GABA(A) currents of injured neurons did not differ from those of control neurons in their sensitivity to GABA or their reversal potentials, suggesting that GABA current potentiation did not result from changes in the agonist affinity or ionic selectivity of the channels. GABA current potentiation was prevented by injuring neurons in the presence of the NMDA antagonist APV, or the CaMKII inhibitor KN93. These results thus suggest that NMDA receptor activation following neuronal injury may potentiate GABA(A) channels through the activation of CaMKII. The increase in GABA(A) receptor function observed following injury could potentially contribute to dysfunctional synaptic function and information processing as well as unconsciousness and coma following human brain trauma.

Differential modulation of L-type calcium channel subunits by oleate

Nonesterified fatty acids such as oleate and palmitate acutely potentiate insulin secretion from pancreatic islets in a glucose-dependent manner. In addition, recent studies show that fatty acids elevate intracellular free Ca(2+) and increase voltage-gated Ca(2+) current in mouse beta-cells, although the mechanisms involved are poorly understood. Here we utilized a heterologous system to express subunit-defined voltage-dependent L-type Ca(2+) channels (LTCC) and demonstrate that beta-cell calcium may increase in part from an interaction between fatty acid and specific calcium channel subunits. Distinct functional LTCC were assembled in both COS-7 and HEK-293 cells by expressing either one of the EYFP-tagged L-type alpha(1)-subunits (beta-cell Cav1.3 or lung Cav1.2) and ERFP-tagged islet beta-subunits (ibeta(2a) or ibeta(3)). In COS-7 cells, elevations in intracellular Ca(2+) mediated by LTCC were enhanced by an oleate-BSA complex. To extend these findings, Ca(2+) current was measured in LTCC-expressing HEK-293 cells that revealed an increase in peak Ca(2+) current within 2 min after addition of the oleate complex, with maximal potentiation occurring at voltages <0 mV. Both Cav1.3 and Cav1.2 were modulated by oleate, and the presence of different auxiliary beta-subunits resulted in differential augmentation. The potentiating effect of oleate on Cav1.2 was abolished by the pretreatment of cells with triacsin C, suggesting that long-chain CoA synthesis is necessary for Ca(2+) channel modulation. These results show for the first time that two L-type Ca(2+) channels expressed in beta-cells (Cav1.3 and Cav1.2) appear to be targeted by nonesterified fatty acids. This effect may account in part for the acute potentiation of glucose-dependent insulin secretion by fatty acids.

Mechanical Injury Alters Volume Activated Ion Channels in Cortical Astrocytes

UNLABELLED Although astrocytic swelling is likely to mediate brain edema and high ICP after traumatic brain injury, the mechanism is not understood. We employed whole cell patch clamp electrophysiology and a stretch injury model to understand whether volume regulating ion currents are altered following cell injury. Mixed rat astrocytes and neurons were co-cultured on deformable silastic membranes. Mild-moderate cell injury was produced using a timed pulse of pressurized air to deform the silastic substrates by 6.5 mm. Then, ion currents were recorded with patch clamp methods. Cells were held at -65 mV and were stepped to +10 mV to monitor current changes. RESULTS In unstretched astrocytes, small amplitude currents were obtained under isotonic conditions. Hypotonic solution activated an outwardly-rectifying current which reversed near -40 mV. This current resembled a previously reported anion current whose activation may restore cell volume by mediating a net solute efflux. In contrast, stretch injured cells exhibited a large amplitude, nonrectifying current. This current was not due to non-specific ionic leakage, since it was fully suppressed by the cation channel blocker gadolinium. Activation of novel stretch-activated cation currents may exacerbate cell swelling in injured astrocytes. Stretch injured astrocytes thus express a dysfunctional cation current as opposed to an osmoregulatory anion current. This mechanism, if present in vivo, may contribute to the cytotoxic swelling seen after traumatic brain injury.

A leptin-regulated circuit controls glucose mobilization during noxious stimuli

Adipocytes secrete the hormone leptin to signal the sufficiency of energy stores. Reductions in circulating leptin concentrations reflect a negative energy balance, which augments sympathetic nervous system (SNS) activation in response to metabolically demanding emergencies. This process ensures adequate glucose mobilization despite low energy stores. We report that leptin receptor–expressing neurons (LepRb neurons) in the periaqueductal gray (PAG), the largest population of LepRb neurons in the brain stem, mediate this process. Application of noxious stimuli, which often signal the need to mobilize glucose to support an appropriate response, activated PAG LepRb neurons, which project to and activate parabrachial nucleus (PBN) neurons that control SNS activation and glucose mobilization. Furthermore, activating PAG LepRb neurons increased SNS activity and blood glucose concentrations, while ablating LepRb in PAG neurons augmented glucose mobilization in response to noxious stimuli. Thus, decreased leptin action on PAG LepRb neurons augments the autonomic response to noxious stimuli, ensuring sufficient glucose mobilization during periods of acute demand in the face of diminished energy stores.