Zolpidem modulates GABA(A) receptor function in subthalamic nucleus

The subthalamic nucleus occupies a position in the indirect pathway of basal ganglia circuit, which plays an important role in the movement regulation. Zolpidem is an imidazopyridine agonist with a high affinity on the benzodiazepine site of GABA(A) receptors containing alpha 1 subunit. Recently, zolpidem has been reported to be useful in treating subgroups of parkinsonian patients. A high density of zolpidem binding sites has been shown in rat subthalamic nucleus. To further investigate the modulation of zolpidem on GABA(A) receptor-mediated inhibitory synaptic current in subthalamic nucleus, whole-cell patch clamp recordings were used in the present study. Zolpidem at 100nM significantly prolonged the decay time and rise time of miniature inhibitory postsynaptic currents, with no effect on the amplitude and frequency. The benzodiazepine antagonist flumazenil could completely block the potentiation induced by zolpidem, confirming the specificity on the benzodiazepine site. At a high concentration of 1 microM, zolpidem significantly increased the decay time, rise time, amplitude and frequency of miniature inhibitory postsynaptic currents. In the behaving rats, unilateral microinjection of zolpidem into subthalamic nucleus induced a significant contralateral rotation. The present findings on the effect of zolpidem in subthalamic nucleus provide a rationale for further investigations into its potential in the treatment of Parkinson's disease.     

Expression of somatostatin receptor subtype 5 in rat retinal amacrine cells

Somatostatin (SRIF), as a neuroactive peptide in the CNS, exerts its actions via five subtypes of specific receptors (ssts). In this work, the localization of sst(5) was studied immunocytochemically in rat retinal amacrine cells (ACs). Labeling for sst(5) was diffusely distributed throughout the full thickness of the inner plexiform layer (IPL) and formed two distinct fluorescence bands in the distal part of the IPL. Double labeling experiments showed that sst(5) was expressed in GABAergic ACs. It was further shown that labeling for sst(5) was observed in both dopaminergic and cholinergic ACs, stained by tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT), respectively. The immunostaining appeared mainly on the cell membranes and somatodendritic compartments of these ACs. For the cholinergic ACs, weak sst(5)-immunoreactivity was also observed in the processes terminating in the IPL. In contrast, no sst(5)-immunoreactivity was found in glycinergic AII ACs, stained by parvalbumin (PV). Furthermore, labeling for SRIF was co-localized with sst(5) in both dopaminergic and cholinergic ACs. These results suggest that sst(5) may serve as an autoreceptor or conventional receptor in retinal ACs.     

Calcium from internal stores triggers GABA release from retinal amacrine cells

The Ca(2+) that promotes transmitter release is generally thought to enter presynaptic terminals through voltage-gated Ca(2+)channels. Using electrophysiology and Ca(2+) imaging, we show that, in amacrine cell dendrites, at least some of the Ca(2+) that triggers transmitter release comes from endoplasmic reticulum Ca(2+) stores. We show that both inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) are present in these dendrites and both participate in the elevation of cytoplasmic [Ca(2+)] during the brief depolarization of a dendrite. Only the Ca(2+) released through IP(3)Rs, however, seems to promote the release of transmitter. Antagonists for the IP(3)R reduced transmitter release, whereas RyR blockers had no effect. Application of an agonist for metabotropic glutamate receptor, known to liberate Ca(2+) from internal stores, enhanced both spontaneous and evoked transmitter release.     

GABA(B) receptor activation modulates GABA(A) receptor-mediated inhibition in chicken nucleus magnocellularis neurons

Neurons of nucleus magnocellularis (NM), a division of avian cochlear nucleus that performs precise temporal encoding, receive glutamatergic excitatory input solely from the eighth nerve and GABAergic inhibitory input primarily from the ipsilateral superior olivary nucleus. GABA activates both ligand-gated Cl^– channels [GABA_A receptors (GABA_ARs)] and G protein-coupled receptors (GABA_B receptors). The net effect of GABA_AR-mediated input to NM is inhibitory, although depolarizing. Several studies have shown that this shunting, inhibitory GABAergic input can evoke action potentials in postsynaptic NM neurons, which could interfere with their temporal encoding. While this GABA-mediated firing is limited by a low-voltage-activated K⁺ conductance, we have found evidence for a second mechanism. We investigated modulation of GABA_AR-mediated responses by GABA_BRs using whole cell recording techniques. Bath-applied baclofen, a GABA_BR agonist, produced dose-dependent suppression of evoked inhibitory postsynaptic currents (eIPSCs). This suppression was blocked by CGP52432 a potent and selective GABA_BR antagonist. Baclofen reduced the frequency but not the amplitude of miniature IPSCs (mIPSCs) and did not affect postsynaptic currents elicited by puff application of a specific GABA_AR agonist muscimol, suggesting a presynaptic mechanism for the GABA_BR-mediated modulation. Firing of NM neurons by synaptic stimulation of GABAergic inputs to NM was eliminated by baclofen. However, endogenous GABA_BR activity in the presynaptic inhibitory terminals was not observed. We propose that presynaptic GABA_BRs function as autoreceptors, regulating synaptic strength of GABA_AR-mediated inhibition, and prevent NM neurons from generating firing during activation of the inhibitory inputs.     

C-type natriuretic peptide modulates glutamate receptors on cultured rat retinal amacrine cells

C-type natriuretic peptide, widely distributed in the CNS, may work as a neuromodulator. In this work, we investigated modulation by C-type natriuretic peptide of functional properties of glutamate receptors in rat retinal GABAergic amacrine cells in culture. Immunocytochemical data revealed that natriuretic peptide receptor-B was strongly expressed on the membrane of cultured GABAergic amacrine cells. By whole cell recording techniques we further identified the glutamate receptor expressed on the GABAergic amacrine cells as an AMPA-preferring subtype. Incubation with C-type natriuretic peptide suppressed the AMPA receptor-mediated current of these cells in a dose-dependent manner by decreasing the efficacy and apparent affinity for glutamate. The effect of C-type natriuretic peptide was reversed by HS-142-1, a guanylyl cyclase-coupled natriuretic peptide receptor-A/B antagonist. Meanwhile, the selective natriuretic peptide receptor-C agonist cANF did not change the glutamate current. In conjunction with the immunocytochemical data, these results suggest that the C-type natriuretic peptide effect may be mediated by natriuretic peptide receptor-B. Furthermore, incubation of retinal cultures in the C-type natriuretic peptide-containing medium elevated cGMP immunoreactivity in the GABAergic amacrine cells, and the C-type natriuretic peptide effect on the glutamate current was mimicked by application of 8-Br-cGMP. It is therefore concluded that C-type natriuretic peptide may modulate the glutamate current by increasing the intracellular concentration of cGMP in these cells via activation of natriuretic peptide receptor-B.     

GABA(A)-dependent chloride influx modulates reversal potential of GABA(B)-mediated IPSPs in hippocampal pyramidal cells

Changes in intracellular chloride concentration, mediated by chloride influx through GABA(A) receptor-gated channels, may modulate GABA(B) receptor-mediated inhibitory postsynaptic potentials (GABA(B) IPSPs) via unknown mechanisms. Recording from CA3 pyramidal cells in hippocampal slices, we investigated the impact of chloride influx during GABA(A) receptor-mediated IPSPs (GABA(A) IPSPs) on the properties of GABA(B) IPSPs. At relatively positive membrane potentials (near -55 mV), mossy fiber--evoked GABA(B) IPSPs were reduced (compared with their magnitude at -60 mV) when preceded by GABA(A) receptor--mediated chloride influx. This effect was not associated with a correlated reduction in membrane permeability during the GABA(B) IPSP. The mossy fiber--evoked GABA(B) IPSP showed a positive shift in reversal potential (from -99 to -93 mV) when it was preceded by a GABA(A) IPSP evoked at cell membrane potential of -55 mV as compared with -60 mV. Similarly, when intracellular chloride concentration was raised via chloride diffusion from an intracellular microelectrode, there was a reduction of the pharmacologically isolated monosynaptic GABA(B) IPSP and a concurrent shift of GABA(B) IPSP reversal potential from -98 to -90 mV. We conclude that in hippocampal pyramidal cells, in which "resting" membrane potential is near action potential threshold, chloride influx via GABA(A) IPSPs shifts the reversal potential of subsequent GABA(B) receptor--mediated postsynaptic responses in a positive direction and reduces their magnitude.     

Activation of cerebellar granule cells GABA(A) receptors by guanidinoacetate

The extracellular concentration of guanidinoacetate (GAA) in the brain increases in guanidino acetate methyl transferase (GAMT) deficiency, an inherited disorder. We tested whether the levels which this substance can reach in the brain in GAMT deficiency are able to activate GABA(A) receptors in key cerebellar neurons such as the cerebellar granules. GAA in fact activates these receptors in rat cerebellar granules in culture although at quite high concentrations, in the millimolar range. However, these millimolar GAA levels are not reached extracellularly in the brain in GAMT deficiency. In addition, GAA does not act as a partial agonist on granules' GABA(A) receptors. This appears to deny an effect by this molecule on cerebellar function in the disease via interference with granule cells' GABA(A) receptors. Study of partial blockage by furosemide of chloride currents activated by GABA and GAA in granule cells allowed us to distinguish two populations of GABA(A) receptors presumably involved in granule cells' tonic inhibition. One is devoid of alpha6 subunit and another one contains it. The latter when activated by GABA has a decay kinetics much slower than the former. GAA does not distinguish between these two populations. In any case, the very high extracellular GAA concentrations able to activate them are not likely to be reached in GAMT deficiency.     

Subcellular localization and complements of GABA(A) and GABA(C) receptors on bullfrog retinal bipolar cells

gamma-Aminobutyric acid (GABA) receptors on retinal bipolar cells (BCs) are highly relevant to spatial and temporal integration of visual signals in the outer and inner retina. In the present work, subcellular localization and complements of GABA(A) and GABA(C) receptors on BCs were investigated by whole cell recordings and local drug application via multi-barreled puff pipettes in the bullfrog retinal slice preparation. Four types of the BCs (types 1-4) were identified morphologically by injection of Lucifer yellow. According to the ramification levels of the axon terminals and the responses of these cells to glutamate (or kainate) applied at their dendrites, types 1 and 2 of BCs were supposed to be OFF type, whereas types 3 and 4 of BCs might be ON type. Bicuculline (BIC), a GABA(A) receptor antagonist, and imidazole-4-acetic acid (I4AA), a GABA(C) receptor antagonist, were used to distinguish GABA receptor-mediated responses. In all BCs tested, not only the axon terminals but also the dendrites showed high GABA sensitivity mediated by both GABA(A) and GABA(C) receptors. Subcellular localization and complements of GABA(A) and GABA(C) receptors at the dendrites and axon terminals were highly related to the dichotomy of OFF and ON BCs. In the case of OFF BCs, GABA(A) receptors were rather evenly distributed at the dendrites and axon terminals, but GABA(C) receptors were predominantly expressed at the axon terminals. Moreover, the relative contribution of GABA(C) receptors to the axon terminals was prevalent over that of GABA(A) receptors, while the situation was reversed at the dendrites. In the case of ON BCs, GABA(A) and GABA(C) receptors both preferred to be expressed at the axon terminals; relative contributions of these two GABA receptor subtypes to both the sites were comparable, while GABA(C) receptors were much less expressed than GABA(A) receptors. GABA(A), but not GABA(C) receptors, were expressed clusteringly at axons of a population of BCs. In a minority of BCs, I4AA suppressed the GABA(C) responses at the dendrites, but not at the axon terminal, implying that the GABA(C) receptors at these two sites may be heterogeneous. Taken together, these results suggest that GABA(A) and GABA(C) receptors may play different roles in the outer and inner retina and the differential complements of the two receptors on OFF and ON BCs may be closely related to physiological functions of these cells.     

GABA(B) receptor activation protects GABA(A) receptor from cyclic AMP-dependent down-regulation in rat cerebellar granule cells.

Interaction between GABAA and GABA(B) receptors was studied in rat cerebellar granule cells in culture, by the whole-cell patch-clamp approach. Our data show that the GABA(B) agonist (-)baclofen is not able, per se, to significantly change the muscimol-activated chloride current. However, (-)baclofen dose-dependently prevents the reduction of GABA(A) receptor function by forskolin, an activator of adenylate cyclase. The effect of baclofen is mediated by a pertussis toxin-sensitive G protein. In fact, in cells treated with pertussis toxin, baclofen and forskolin, the toxin is able to block baclofen action, allowing forskolin to act fully. The protective effect by GABA(B) receptor activation under these circumstances is most probably related to the prevention of cyclic AMP increases after forskolin treatment. In fact, in these neurons cyclic AMP and protein kinase A activation result in a down-regulation of GABA(A) receptor function. On the whole, the data indicate the presence of complex modulation of GABA(A) receptors by GABA(B) receptor types in cerebellum granule cells.     

Selective activation of mGluR8 receptors modulates retinal ganglion cell light responses

Extracellular and whole-cell light-evoked responses of mouse retinal ganglion cells were recorded in the presence of the mGluR8 selective agonist, (S)-3,4-dicarboxy-phenylglycine (DCPG). Off-light responses were reversibly reduced in the presence of DCPG in wild-type but not in mGluR8-deficient retinas. On-responses were only marginally modulated by DCPG. During Off-responses, DCPG suppressed both excitatory and inhibitory synaptic conductances suggesting that mGluR8 receptor activity reduces glutamate release from bipolar cell terminals and possibly also the release of an inhibitory neurotransmitter from amacrine cell processes.     

Sodium action potentials are not required for light-evoked release of GABA or glycine from retinal amacrine cells.

Although most CNS neurons require sodium action potentials (Na-APs) for normal stimulus-evoked release of classical neurotransmitters, many types of retinal and other sensory neurons instead use only graded potentials for neurotransmitter release. The physiological properties and information processing capacity of Na-AP-producing neurons appear significantly different from those of graded potential neurons. To classify amacrine cells in this dichotomy, we investigated whether Na-APs, which are often observed in these cells, are required for functional light-evoked release of inhibitory neurotransmitters from these cells. We recorded light-evoked inhibitory postsynaptic currents (IPSCs) from retinal ganglion cells, neurons directly postsynaptic to amacrine cells, and applied TTX to block Na-APs. In control solution, TTX application always led to partial suppression of the light-evoked IPSC. To isolate release from glycinergic amacrine cells, we used either bicuculline, a GABAA receptor antagonist, or picrotoxin, a GABAA and GABAC receptor antagonist. TTX application only partially suppressed the glycinergic IPSC. To isolate release from GABAergic amacrine cells, we used the glycine receptor blocker strychnine. TTX application only partially suppressed the light-evoked GABAergic IPSC. Glycinergic and GABAergic amacrine cells did not obviously differ in the usage of Na-APs for release. These observations, in conjunction with previous studies of other retinal neurons, indicate that amacrine cells, taken as a class, are the only type of retinal neuron that uses both Na-AP-dependent and -independent modes for light-evoked release of neurotransmitters. These results also provide evidence for another parallel between the properties of retinal amacrine cells and olfactory bulb granule cells.     

Role for metabotropic glutamate receptor 5 (mGluR5) in the pathogenesis of fragile X syndrome

Metabotropic glutamate receptors (mGluRs) have been implicated in a diverse variety of neuronal functions. Studies reviewed here indicate that exaggerated signalling through mGluR5 can account for multiple cognitive and syndromic features of fragile X syndrome, the most common inherited form of mental retardation and autism. Since a reduction of mGluR5 signalling can reverse fragile X phenotypes, these studies provide a compelling rationale for the use of mGluR5 antagonists for the treatment of fragile X and related disorders.     

Pharmacology of GABA(A) receptors of retinal dopaminergic neurons

When the vertebrate retina is stimulated by light, a class of amacrine or interplexiform cells release dopamine, a modulator responsible for neural adaptation to light. In the intact retina, dopamine release can be pharmacologically manipulated with agonists and antagonists at GABA(A) receptors, and dopaminergic (DA) cells receive input from GABAergic amacrines. Because there are only 450 DA cells in each mouse retina and they cannot be distinguished in the living state from other cells on the basis of their morphology, we used transgenic technology to label DA cells with human placental alkaline phosphatase, an enzyme that resides on the outer surface of the cell membrane. We could therefore identify DA cells in vitro after dissociation of the retina and investigate their activity with whole cell voltage clamp. We describe here the pharmacological properties of the GABA(A) receptors of solitary DA cells. GABA application induces a large inward current carried by chloride ions. The receptors are of the GABA(A) type because the GABA-evoked current is blocked by bicuculline. Their affinity for GABA is very high with an EC(50) value of 7.4 microM. Co-application of benzodiazepine receptor ligands causes a strong increase in the peak current induced by GABA (maximal enhancement: CL-218872 220%; flunitrazepam 214%; zolpidem 348%) proving that DA cells express a type I benzodiazepine-receptor (BZ1). GABA-evoked currents are inhibited by Zn(2+) with an IC(50) of 58.9 +/- 8.9 microM. Furthermore, these receptors are strongly potentiated by the modulator alphaxalone with an EC(50) of 340 +/- 4 nM. The allosteric modulator loreclezole increases GABA receptor currents by 43% (1 microM) and by 107% (10 microM). Using outside-out patches, we measured in single-channel recordings a main conductance (29 pS) and two subconductance (20 and 9 pS) states. We have previously shown by single-cell RT-PCR and immunocytochemistry that DA cells express seven different GABA(A) receptor subunits (alpha1, alpha3, alpha4, beta1, beta3, gamma1, gamma2(S), and gamma2(L)) and by immunocytochemistry that all subunits are expressed in the intact retina. We show here that at least alpha1, beta3 and gamma2 subunits are assembled into functional receptors.     

GABA(A) receptor activation modulates corneal unit activity in rostral and caudal portions of trigeminal subnucleus caudalis

Corneal nociceptors terminate at the trigeminal subnucleus interpolaris/caudalis (Vi/Vc) transition and subnucleus caudalis/upper cervical spinal cord (Vc/C1) junction regions of the lower brain stem. The aims of this study were to determine if local GABAA receptor activation modifies corneal input to second-order neurons at these regions and if GABAA receptor activation in one region affects corneal input to the other region. In barbiturate-anesthetized male rats, corneal nociceptors were excited by pulses of CO2 gas, and GABAA receptors were activated by microinjections of the selective agonist muscimol. Local muscimol injection at the site of recording inhibited all Vi/Vc and Vc/C1 units tested and was reversed partially by bicuculline. To test for ascending intersubnuclear communication, muscimol injection into the caudal Vc/C1 junction, remote from the recording site at the Vi/Vc transition, inhibited the evoked response of most corneal units, although some neurons were enhanced. Injection of the nonselective synaptic blocking agent, CoCl2, remotely into the Vc/C1 region inhibited the evoked response of all Vi/Vc units tested. To test for descending intersubnuclear communication, muscimol was injected remotely into the rostral Vi/Vc transition and enhanced the evoked activity of all corneal units tested at the caudal Vc/C1 junction. These results suggest that GABAA receptor mechanisms play a significant role in corneal nociceptive processing by second-order trigeminal brain stem neurons. GABAA receptor mechanisms act locally at both the Vi/Vc transition and Vc/C1 junction regions to inhibit corneal input and act through polysynaptic pathways to modify corneal input at multiple levels of the trigeminal brain stem complex.     

A tunable synthetic hydrogel system for culture of retinal ganglion cells and amacrine cells

The central nervous system consists of complex groups of individual cells that receive electrical, chemical and physical signals from their local environment. Standard in vitro cell culture methods rely on two-dimensional (2-D) substrates that poorly simulate in vivo neural architecture. Neural cells grown in three-dimensional (3-D) culture systems may provide an opportunity to study more accurate representations of the in vivo environment than 2-D cultures. Furthermore, each specific type of neuron depends on discrete compositions and physical properties of their local environment. Previously, we developed a library of hydrogels composed of poly(ethylene glycol) and poly(l-lysine) which exhibit a wide range of mechanical properties. Here, we identified specific scaffolds from this library that readily support the survival, migration and neurite outgrowth of purified retinal ganglion cells and amacrine cells. These data address important biological questions about the interaction of neurons with the physical and chemical properties of their local environment and provide further insight for engineering neural tissue for cell-replacement therapies following injury.     

Expression of Nav1.1 in rat retinal AII amacrine cells

In retinal ganglion cells (RGCs), the expression of various types of voltage-gated sodium channel (Nav) alpha-subunits (Nav1.1, Nav1.2, Nav1.3, and Nav1.6) has been reported. Like RGCs, certain subsets of retinal amacrine cells, including AII amacrine cells, generate tetrodotoxin (TTX)-sensitive action potentials in response to light; however, the Nav subtypes expressed in these cells have not been identified. We examined the Nav subtypes expressed in rat retinal amacrine cells by in situ hybridization (ISH) using RNA probes specific for TTX-sensitive Na(v)s (Nav1.1, Nav1.2, Nav1.3, Nav1.6, and Nav1.7). Our results confirmed that Nav1.1, Nav1.2, Nav1.3, and Nav1.6 are localized in the ganglion cell layer (GCL). Interestingly, Nav1.1 was expressed not only in the GCL, but also in the inner nuclear layer (INL). The cell bodies of the Nav1.1-positive cells in the INL were located at the INL/inner plexiform layer (IPL) border. The cell bodies of AII amacrine cells are located close to the INL/IPL border, and these cells can be labeled with antibodies against parvalbumin (PV). Therefore, we combined ISH with immunohistochemistry and discovered that most of the PV-immunoreactive cells located at the INL/IPL border express Nav1.1. Our results show that AII amacrine cells express Nav1.1.     

Pain-related deactivation of medial prefrontal cortical neurons involves mGluR1 and GABA(A) receptors

Pain-related hyperactivity in the amygdala leads to deactivation of the medial prefrontal cortex (mPFC) and decision-making deficits. The mechanisms of pain-related inhibition of the mPFC are not yet known. Here, we used extracellular single-unit recordings of prelimbic mPFC neurons to determine the role of GABA(A) receptors and metabotropic glutamate receptor (mGluR) subtypes, mGluR1 and mGluR5, in pain-related activity changes of mPFC neurons. Background and evoked activity of mPFC neurons decreased after arthritis induction. To determine pain-related changes, the same neuron was recorded continuously before and after induction of arthritis in one knee joint by intra-articular injection of kaolin/carrageenan. Stereotaxic administration of a GABA(A) receptor antagonist {[R-(R*,S*)]-5-(6,8-dihydro-8-oxofuro[3,4-e]-1,3-benzodioxol-6-yl)-5,6,7,8-tetrahydro-6,6-dimethyl-1,3-dioxolo[4,5-g]isoquinolinium iodide (bicuculline)} into the mPFC by microdialysis reversed pain-related inhibition, whereas offsite injections into the adjacent anterior cingulate cortex had no or opposite effects on prelimbic mPFC neurons. A selective mGluR1/5 agonist [(S)-3,5-dihydroxyphenylglycine (DHPG)] inhibited background and evoked activity under normal conditions through a GABAergic mechanism, because the inhibitory effect was blocked with bicuculline. In the arthritis pain state, DHPG, alone or in the presence of bicuculline, had no effect. Consistent with the involvement of mGluR1 in pain-related inhibition of the mPFC, a selective mGluR1 antagonist [(S)-(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid] reversed the pain-related decrease of background and evoked activity of mPFC neurons in arthritis, whereas a selective mGluR5 antagonist [2-methyl-6-(phenylethynyl)pyridine hydrochloride] had no effect. The mGluR antagonists had no effect under normal conditions. We interpret our data to suggest that pain-related inhibition of mPFC neurons in the arthritis model depends on mGluR1-mediated endogenous activation of GABA(A) receptors. Exogenous activation of mGluR1/5 produces GABAergic inhibition under normal conditions. Restoring normal activity in the mPFC may be a therapeutic strategy to improve cognitive deficits associated with persistent pain.     

Altered GABA(A) receptor expression during epileptogenesis

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain. GABA(A) receptors are heteropentamers formed by assembly of multiple subunits that generate a wide array of receptors with particular distribution and pharmacological profiles. Malfunction of these receptors has been associated with the pathophysiology of epilepsy and contribute to an imbalance of excitatory and inhibitory neurotransmission. The process of epilepsy development (epileptogenesis) is associated with changes in the expression and function of a large number of gene products. One of the major challenges is to effectively determine which changes directly contribute to epilepsy development versus those that are compensatory or not involved in the pathology. Substantial evidence suggests that changes in the expression and function of GABA(A) receptors are involved in the pathogenesis of epilepsy. Identification of the mechanisms involved in GABA(A) receptor malfunction during epileptogenesis and the ability to reverse this malfunction are crucial steps towards definitively answering this question and developing specific and effective therapies.