Terrell T. Gibbs

17β-Estradiol protects against NMDA-induced excitotoxicity by direct inhibition of NMDA receptors

Several lines of evidence suggest that 17beta-estradiol (betaE2) has neuroprotective properties. The risk and severity of dementia are decreased in women who have received estrogen therapy, and betaE2 protects neurons in vitro against death from a variety of stressors. Neuroprotection by betaE2 has been suggested to be due to free radical scavenging. We demonstrate an additional neuroprotective mechanism whereby betaE2 protects against NMDA-induced neuronal death by directly inhibiting the NMDA receptor.

Inhibition of the NMDA response by pregnenolone sulphate reveals subtype selective modulation of NMDA receptors by sulphated steroids

The neurosteroid pregnenolone sulphate (PS) potentiates N‐methyl‐D‐aspartate (NMDA) receptor mediated responses in various neuronal preparations. The NR1 subunit can combine with NR2A, NR2B, NR2C, or NR2D subunits to form functional receptors. Differential NR2 subunit expression in brain and during development raises the question of how the NR2 subunit influences NMDA receptor modulation by neuroactive steroids. We examined the effects of PS on the four diheteromeric NMDA receptor subtypes generated by co‐expressing the NR1100 subunit with each of the four NR2 subunits in Xenopus oocytes. Whereas PS potentiated NMDA‐, glutamate‐, and glycine‐induced currents of NR1/NR2A and NR1/NR2B receptors, it was inhibitory at NR1/NR2C and NR1/NR2D receptors. In contrast, pregnanolone sulphate (3α5βS), a negative modulator of the NMDA receptor that acts at a distinct site from PS, inhibited all four subtypes, but was approximately 4 fold more potent at NR1/NR2C and NR1/NR2D than at NR1/NR2A and NR1/NR2B receptors. These findings demonstrate that residues on the NR2 subunit are key determinants of modulation by PS and 3α5βS. The modulatory effects of PS, but not 3α5βS, on dose‐response curves for NMDA, glutamate, and glycine are consistent with a two‐state model in which PS either stabilizes or destabilizes the active state of the receptor, depending upon which NR2 subunit is present. The selectivity of sulphated steroid modulators for NMDA receptors of specific subunit composition is consistent with a neuromodulatory role for endogenous sulphated steroids. The results indicate that it may be possible to develop therapeutic agents that target steroid modulatory sites of specific NMDA receptor subtypes.

Decreased GABAB receptors in the cingulate cortex and fusiform gyrus in Autism

J. Neurochem. (2010) 114, 1414–1423.

Decreased GABAA receptors and benzodiazepine binding sites in the anterior cingulate cortex in autism

The anterior cingulate cortex (ACC; BA 24) via its extensive limbic and high order association cortical connectivity to prefrontal cortex is a key part of an important circuitry participating in executive function, affect, and socio‐emotional behavior. Multiple lines of evidence, including genetic and imaging studies, suggest that the ACC and gamma‐amino‐butyric acid (GABA) system may be affected in autism. The benzodiazepine binding site on the GABAA receptor complex is an important target for pharmacotherapy and has important clinical implications. The present multiple‐concentration ligand‐binding study utilized 3H‐muscimol and 3H‐flunitrazepam to determine the number (Bmax), binding affinity (Kd), and distribution of GABAA receptors and benzodiazepine binding sites, respectively, in the ACC in adult autistic and control cases. Compared to controls, the autistic group had significant decreases in the mean density of GABAA receptors in the supragranular (46.8%) and infragranular (20.2%) layers of the ACC and in the density of benzodiazepine binding sites in the supragranular (28.9%) and infragranular (16.4%) lamina. In addition, a trend for a decrease in for the density of benzodiazepine sites was found in the infragranular layers (17.1%) in the autism group. These findings suggest that in the autistic group this downregulation of both benzodiazepine sites and GABAA receptors in the ACC may be the result of increased GABA innervation and/or release disturbing the delicate excitation/inhibition balance of principal neurons as well as their output to key limbic cortical targets. Such disturbances likely underlie the core alterations in socio‐emotional behaviors in autism.

Reduced GABAA receptors and benzodiazepine binding sites in the posterior cingulate cortex and fusiform gyrus in autism

Individuals with autism display deficits in the social domain including the proper recognition of faces and interpretations of facial expressions. There is an extensive network of brain regions involved in face processing including the fusiform gyrus (FFG) and posterior cingulate cortex (PCC). Functional imaging studies have found that controls have increased activity in the PCC and FFG during face recognition tasks, and the FFG has differential responsiveness in autism when viewing faces. Multiple lines of evidence have suggested that the GABAergic system is disrupted in the brains of individuals with autism and it is likely that altered inhibition within the network influences the ability to perceive emotional expressions. On-the-slide ligand binding autoradiography was used to determine if there were alterations in GABA(A) and/or benzodiazepine binding sites in the brain in autism. Using (3)H-muscimol and (3)H-flunitrazepam we could determine whether the number (B(max)), binding affinity (K(d)), and/or distribution of GABA(A) receptors and benzodiazepine binding sites (BZD) differed from controls in the FFG and PCC. Significant reductions were found in the number of GABA(A) receptors and BZD binding sites in the superficial layers of the PCC and FFG, and in the number of BZD binding sites in the deep layers of the FFG. In addition, the autism group had a higher binding affinity in the superficial layers of the GABA(A) study. Taken together, these findings suggest that the disruption in inhibitory control in the cortex may contribute to the core disturbances of socio-emotional behaviors in autism.

Sulfated steroids as endogenous neuromodulators.

Central nervous system function is critically dependent upon an exquisitely tuned balance between excitatory synaptic transmission, mediated primarily by glutamate, and inhibitory synaptic transmission, mediated primarily by GABA. Modulation of either excitation or inhibition would be expected to result in altered functionality of finely tuned synaptic pathways and global neural systems, leading to altered nervous system function. Administration of positive or negative modulators of ligand-gated ion channels has been used extensively and successfully in CNS therapeutics, particularly for the induction of sedation and treatment of anxiety, seizures, insomnia, and pain. Excessive activation of excitatory glutamate receptors, such as in cerebral ischemia, can result in neuronal damage via excitotoxic mechanisms. The discovery that neuroactive steroids exert rapid, direct effects upon the function of both excitatory and inhibitory neurotransmitter receptors has raised the possibility that endogenous neurosteroids may play a regulatory role in synaptic transmission by modulating the balance between excitatory and inhibitory neurotransmission. The sites to which neuroactive steroids bind may also serve as targets for the discovery of therapeutic neuromodulators.

γ-Aminobutyric acidA receptor regulation: heterologous uncoupling of modulatory site interactions induced by chronic steroid, barbiturate, benzodiazepine, or GABA treatment in culture

Prolonged administration of anxiolytic, sedative, and anticonvulsant drugs that act through the GABAA receptor (GABAAR) can evoke tolerance and dependence, suggesting the existence of an endogenous mechanism(s) for altering the ability of such agents to interact with the GABAAR. Uncoupling appears to be one such mechanism. This is a decrease in the allosteric interactions between the benzodiazepine (BZD) recognition site and other agonist or modulator sites on the GABAAR, as measured by potentiation of [3H]flunitrazepam ([3H]FNZ) binding. To investigate the mechanism(s) of uncoupling, neuronal cultures were treated chronically with 3 alpha-hydroxy-5 beta-pregnan-20-one (pregnanolone), pentobarbital, flurazepam, or GABA, then tested for enhancement of [3H]FNZ binding by these substances. The results indicate that BZDs, barbiturates, and steroids, as well as GABA itself, are capable of inducing both heterologous and homologous uncoupling. Surprisingly, different chronic drug treatments produce different patterns of homologous and heterologous uncoupling. Chronic exposure to pregnanolone, GABA, flurazepam or pentobarbital induces complete uncoupling of barbiturate-BZD site interactions, partial uncoupling of GABA-BZD site interactions, but different amounts of uncoupling of steroid-BZD site interactions. In addition, the EC50 for pregnanolone-induced homologous uncoupling (1.7 microM) is over an order of magnitude greater than that for heterologous uncoupling of GABA and BZD sites (82 nM). Moreover, heterologous uncoupling by pregnanolone is inhibited by the GABA site antagonist SR-95531, whereas homologous uncoupling by pregnanolone is resistant to SR-95531. Therefore, there are at least two distinct ways in which GABAAR modulatory site interactions can be regulated by chronic drug treatment.

Neurosteroid modulation of recombinant ionotropic glutamate receptors

Pregnenolone sulfate (PS) is an abundant neurosteroid that can potentiate or inhibit ligand gated ion channel activity and thereby alter neuronal excitability. Whereas PS is known to inhibit kainate and AMPA responses while potentiating NMDA responses, the dependence of modulation on receptor subunit composition remains to be determined. Toward this end, the effect of PS on recombinant kainate (GluR6), AMPA (GluR1 or GluR3), and NMDA (NR1(100)+NR2A) receptors was characterized electrophysiologically with respect to efficacy and potency of modulation. With Xenopus oocytes expressing GluR1, GluR3 or GluR6 receptors, PS reduces the efficacy of kainate without affecting its potency, indicative of a noncompetitive mechanism of action. Conversely, with oocytes expressing NR1(100)+NR2A subunits, PS enhances the efficacy of NMDA without affecting its potency. Whereas the modulatory efficacy, but not the potency, of PS is increased two-fold by co-injection of NR1(100)+NR2A cRNAs as compared with NR1(100) cRNA alone, there is little or no effect of the NR2A subunit on efficacy or potency of pregnanolone (or epipregnanolone) sulfate as an inhibitor of the NMDA response. This suggests that the NR2A subunit controls the efficacy of neurosteroid enhancement, but not inhibition, which is consistent with our previous finding that potentiating and inhibitory steroids act at distinct sites on the NMDA receptor. This represents a first step towards understanding the role of subunit composition in determining neurosteroid modulation of ionotropic glutamate receptor function.

Benzodiazepine modulation of partial agonist efficacy and spontaneously active GABAA receptors supports an allosteric model of modulation

1 Benzodiazepines (BZDs) have been used extensively for more than 40 years because of their high therapeutic index and low toxicity. Although BZDs are understood to act primarily as allosteric modulators of GABAA receptors, the mechanism of modulation is not well understood. 2 The applicability of an allosteric model with two binding sites for γ‐aminobutyric acid (GABA) and one for a BZD‐like modulator was investigated. 3 This model predicts that BZDs should enhance the efficacy of partial agonists. 4 Consistent with this prediction, diazepam increased the efficacy of the GABAA receptor partial agonist kojic amine in chick spinal cord neurons. 5 To further test the validity of the model, the effects of diazepam, flurazepam, and zolpidem were examined using wild‐type and spontaneously active mutant α1(L263S)β3γ2 GABAA receptors expressed in HEK‐293 cells. 6 In agreement with the predictions of the allosteric model, all three modulators acted as direct agonists for the spontaneously active receptors. 7 The results indicate that BZD‐like modulators enhance the amplitude of the GABA response by stabilizing the open channel active state relative to the inactive state by less than 1 kcal, which is similar to the energy of stabilization conferred by a single hydrogen bond.

Pregnenolone sulfate exacerbates NMDA-induced death of hippocampal neurons

Excessive stimulation of the N-methyl-d-aspartate (NMDA)-type glutamate receptor has been implicated in the neuronal death resulting from focal hypoxia-ischemia. Certain neurosteroids, steroids synthesized de novo in the central nervous system (CNS), have been shown to modulate the action of neurotransmitters at their cellular receptors. Pregnenolone sulfate (PS) is an abundant neurosteroid that enhances the current evoked by NMDA. Using the Ca2+-sensitive fluorescent dye, Fluo-3, AM, and a trypan blue exclusion assay, we evaluated the ability of PS to modulate NMDA-induced changes in intracellular free calcium concentration ([Ca2+]i) and neuronal death in primary cultures of rat hippocampal neurons. The results demonstrate that PS potentiates NMDA-induced increases in [Ca2+]i by 150%. Further, PS exacerbates the MK-801-sensitive neuronal death produced by acute (PS EC50=37 microM) or chronic NMDA exposure, reducing the EC50 of NMDA from 13 to 4 microM under chronic exposure conditions, whereas pregnenolone is ineffective. Our results show that PS, or related sulfated neurosteroids, may play a role in the onset of excitotoxic neuronal death in vivo.