Changes of GABAA receptor binding and subunit mRNA level in rat brain by infusion of subtoxic dose of MK-801

In the present study, we have investigated the effects of prolonged inhibition of NMDA receptor by infusion of subtoxic dose of MK-801 to examine the modulation of GABA_A receptor binding and GABA_A receptor subunit mRNA level in rat brain. It has been reported that NMDA-selective glutamate receptor stimulation alters GABA_A receptor pharmacology in cerebellar granule neurons in vitro by altering the levels of selective subunit. However, we have investigated the effect of NMDA antagonist, MK-801, on GABA_A receptor binding characteristics in discrete brain regions by using autoradiographic and in situ hybridization techniques. The GABA_A receptor bindings were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam, and [³⁵S]TBPS in rat brain slices. Rats were infused with MK-801 (1 pmol/10 μl per h, i.c.v.) for 7 days, through pre-implanted cannula by osmotic minipumps (Alzet, model 2ML). The levels of [³H]muscimol binding were highly elevated in almost all of brain regions including cortex, caudate putamen, thalamus, hippocampus, and cerebellum. However, the [³H]flunitrazepam binding and [³⁵S]TBPS binding were increased only in specific regions; the former level was increased in parts of the cortex, thalamus, and hippocampus, while the latter binding sites were only slightly elevated in parts of thalamus. The levels of β2-subunit were elevated in the frontal cortex, thalamus, hippocampus, brainstem, and cerebellar granule layers while the levels of β3-subunit were significantly decreased in the cortex, hippocampus, and cerebellar granule layers in MK-801-infused rats. The levels of α6- and δ-subunits, which are highly localized in the cerebellum, were increased in the cerebellar granule layer after MK-801 treatment. These results show that the prolonged suppression of NMDA receptor function by MK-801-infusion strongly elevates [³H]muscimol binding throughout the brain, increases regional [³H]flunitrazepam and [³⁵S]TBPS binding, and alters GABA_A receptor subunit mRNA levels in different directions. The chronic MK-801 treatment has differential effect on various GABA_A receptor subunits, which suggests involvement of differential regulatory mechanisms in interaction of NMDA receptor with the GABA receptors.     

Changes of GABA(A) receptor binding and subunit mRNA level in rat brain by infusion of subtoxic dose of MK-801

In the present study, we have investigated the effects of prolonged inhibition of NMDA receptor by infusion of subtoxic dose of MK-801 to examine the modulation of GABA_A receptor binding and GABA_A receptor subunit mRNA level in rat brain. It has been reported that NMDA-selective glutamate receptor stimulation alters GABA_A receptor pharmacology in cerebellar granule neurons in vitro by altering the levels of selective subunit. However, we have investigated the effect of NMDA antagonist, MK-801, on GABA_A receptor binding characteristics in discrete brain regions by using autoradiographic and in situ hybridization techniques. The GABA_A receptor bindings were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam, and [³⁵S]TBPS in rat brain slices. Rats were infused with MK-801 (1 pmol/10 μl per h, i.c.v.) for 7 days, through pre-implanted cannula by osmotic minipumps (Alzet, model 2ML). The levels of [³H]muscimol binding were highly elevated in almost all of brain regions including cortex, caudate putamen, thalamus, hippocampus, and cerebellum. However, the [³H]flunitrazepam binding and [³⁵S]TBPS binding were increased only in specific regions; the former level was increased in parts of the cortex, thalamus, and hippocampus, while the latter binding sites were only slightly elevated in parts of thalamus. The levels of β2-subunit were elevated in the frontal cortex, thalamus, hippocampus, brainstem, and cerebellar granule layers while the levels of β3-subunit were significantly decreased in the cortex, hippocampus, and cerebellar granule layers in MK-801-infused rats. The levels of 6- and δ-subunits, which are highly localized in the cerebellum, were increased in the cerebellar granule layer after MK-801 treatment. These results show that the prolonged suppression of NMDA receptor function by MK-801-infusion strongly elevates [³H]muscimol binding throughout the brain, increases regional [³H]flunitrazepam and [³⁵S]TBPS binding, and alters GABA_A receptor subunit mRNA levels in different directions. The chronic MK-801 treatment has differential effect on various GABA_A receptor subunits, which suggests involvement of differential regulatory mechanisms in interaction of NMDA receptor with the GABA receptors.     

On the cellular localization of cerebellar muscarinic receptors: An autoradiographic analysis of weaver, reeler, purkinje cell degeneration and staggerer mice

Light microscopic autoradiography of [3H]quinuclidinyl benzilate binding sites was used to study the distribution of muscarinic cholinergic receptors in mouse mutants which have abnormalities affecting specific cerebellar cell types. In the normal C57BL/6J mouse, binding sites were distributed throughout the cerebellar cortex, with the highest levels in the granule cell layer and deep cerebellar nuclei. Normal binding site density was observed in the cerebellum of the weaver mutant in which the majority of granule cells had degenerated. The density of [3H]quinuclidinyl benzilate binding sites was elevated in the cortex of the reeler, despite a reduction in the number of granule cells. The concentration of binding sites was also high over the Purkinje cell masses where granule cells were largely absent. No significant reduction in cortical [3H]quinuclidinyl benzilate binding site density was detected in the Purkinje cell degeneration mutant, in which essentially all Purkinje cells had degenerated. In contrast, receptor binding in the deep cerebellar nuclei of this mutant was significantly increased. A substantial increase in labeling was observed in the cortex and deep nuclei of the staggerer cerebellum in which a large fraction of Golgi II cells, Purkinje cells, granule cells and mossy fibers have degenerated. We discuss the possibility that the persistence of [3H]quinuclidinyl benzilate binding sites in all four mutants may imply a non-neuronal localization for a large proportion of muscarinic receptors in the mouse cerebellar cortex.     

Selective changes in the levels of nicotinic acetylcholine receptor protein and of corresponding mRNA species in the brains of patients with Parkinson's disease

Reductions in the number of neuronal nicotinic acetylcholine receptors (nAChRs) have been shown to occur in connection with Parkinson's disease (PD), but it is still unclear which subtype of this receptor is affected. In the present study we examined various nAChR subtypes employing ligand binding, as well as levels of subunit protein and mRNA in the brains of PD patients and age-matched controls. Binding of [3H]epibatidine and levels of alpha3 mRNA in the caudate nucleus and temporal cortex, but not in the hippocampus were significantly decreased in the PD brain. The level of the alpha3 protein subunit was significantly reduced in all these brain regions but there was no change in the level of alpha4. The level of the beta2 protein subunit in the temporal cortex and hippocampus and the beta2 mRNA in the temporal cortex was lowered. Both the levels of the alpha7 subunit protein and [125I]alpha-bungarotoxin binding were significantly increased in the temporal cortex of PD patients whereas the alpha7 mRNA level was unchanged. These findings reveal selective losses of the alpha3- and beta2-containing nAChRs and an increase in the alpha7 nAChRs that might be related to the pathogenesis of PD.     

Changes of GABA(A) receptor binding and subunit mRNA level in rat brain by infusion of NOS inhibitor

In the present study, we have investigated the effects of prolonged inhibition of nitric oxide synthase (NOS) by infusion of NOS inhibitor, L-nitroarginine, to examine the pentobarbital-induced sleep, modulation of GABA(A) receptor binding, and GABA(A) receptor subunit mRNA level in rat brain. Pre-treatment with L-nitroarginine 30 min before pentobarbital treatment (60 mg/kg, i.p.) significantly increased the duration of sleep in rats. However, the duration of pentobarbital-induced sleep was shortened by the prolonged infusion of L-nitroarginine into ventricle. We have investigated the effect of NOS inhibitor on GABA(A) receptor binding characteristics in discrete areas of brain regions by using autoradiographic and in situ hybridization techniques. Rats were infused with L-nitroarginine (10, 100 pmol/10 microl/h, i.c.v.) for 7 days, through pre-implanted cannula by osmotic minipumps. The levels of [(3)H]muscimol and [(3)H]flunitrazepam binding were markedly elevated in almost all of brain regions including cortex, caudate putamen, thalamus, hippocampus, and cerebellum. However, there was no change in the level of [(35)S]TBPS binding. The levels of beta2-subunit were elevated in the cortex, brainstem, and cerebellar granule layers. By contrast, the levels of beta3-subunit were significantly decreased in the cortex, hippocampus, and cerebellar granule layers in L-nitroarginine-infused rats. Following L-nitroarginine treatment, the levels of alpha6- and delta-subunits which were strictly localized to the cerebellum, were not changed in the cerebellar granule layer. These results show that the prolonged inhibition of NOS by L-nitroarginine-infusion markedly elevates [(3)H]muscimol and [(3)H]flunitrazepam binding throughout the brain, and alters GABA(A) receptor subunit mRNA levels in different directions. Chronic inhibition of NO generation has differential effects on the various expressions of GABA(A) receptor subunits. These suggest the involvement of different regulatory mechanisms for the NO-induced expression of GABA(A) receptor.     

Ouabain binding to chick embryo neuroretina during development in ovo and in monolayer cultures

Eight-day-old embryo neuroretinas (NR) were dissociated and cultured as monolayer. [3H]Ouabain binding was investigated during the culture period. [3H]Ouabain binding to NR increases continuously until around the tenth day of culture, but after that it gradually decreased. On the other hand, [3H]ouabain binding to NR during in ovo development continued to increase until the embryo was 20 days old. Na-K-ATPase activity was also compared with [3H]ouabain binding during development in ovo and in monolayer culture. It was found that changes in Na-K-ATPase activity of NR in vitro and in ovo were different from those of [3H]ouabain binding activity. Exposure of NR at the second day of culture to 0.1 mM ouabain for 8 h resulted in the degeneration of some differentiated neurons (rosette-formed cells), but new rosettes were soon re-formed after the withdrawal of ouabain, and [3H]ouabain binding to the ouabain-treated NR was decreased by 30%. Ouabain-treatment of NR in 10-day-old cultures completely destroyed the aggregated neurons, but did not apparently injure the glial cell layer. [3H]Ouabain binding decreased more than 90% in the ouabain-treated NR. These results suggest that [3H]ouabain binding activity of NR and the sensitivity to ouabain toxicity are useful indicators for differentiation and development of neurons in the cultured retinal cells.     

Phencyclidine (PCP) receptors: autoradiographic localization in brain with the selective ligand, [H]TCP

Receptor binding sites for the phencyclidine (PCP) analogue, [³H]TCP, have been localized in the rat and guinea pig central nervous systems by in vitro autoradiography. Quantitation of [³H]TCP binding site densities in rat brain reveals highest levels in the forebrain, in particular the strata oriens and radiatum of the hippocampus, the molecular layer of the dentate gyrus and superficial layers of the cerebral cortex. Moderate levels of binding occur in the amygdala, thalamus, anterior olfactory nucleus external plexiform layer of the olfactory bulb, olfactory tubercle, geniculate nuclei and deep layers of the cortex. Low levels of binding occur throughout most of the septum, diagonal band, hypothalamus, pons-medulla and cerebellum. Spinal cord grey matter also has low levels binding. Excitotoxin lesions of the hippocampal formation, which destroy the pyramidal and granule cells, reduce the binding of [³H]TCP to strata radiatum and oriens and the molecular layer of the dentage gyrus by 60% suggesting that [³H]TCP labels intrinsic neurons in these regions. Residual binding is probably on afferent terminals. Ibotenic acid lesions of the caudate-putamen reduce [³H]TCP binding by 70%, indicating that binding sites are localized on intrinsic striatal neurons. 6-Hydroxydopamine lesions do not alter [³H]TCP binding levels the caudate, suggesting the absence of binding sites on dopaminergic terminals in the caudate.     

Expression of GABAA/benzodiazepine receptor alpha 1-subunit mRNA and [3H]flunitrazepam binding sites during postnatal development of the mouse cerebellum.

Previous studies have shown that the alpha subunit of the GABAA receptor contains the flunitrazepam binding site. In the present study, in situ hybridization and receptor autoradiography were used to examine the temporal and spatial relationships between alpha 1 subunit mRNA and [3H]flunitrazepam binding sites in the developing mouse cerebellum. A [35S]cRNA probe was used to study the expression of GABAA/benzodiazepine receptor alpha 1 subunit mRNA by in situ hybridization. At postnatal day (P) 1, a diffuse band of labeling was observed in the molecular/Purkinje cell layer; subsequently, this band became progressively more concentrated and restricted to the interface between the granular and molecular layers. By P5-P7, high intensity labeling was clearly associated with Purkinje cells. Clusters of grains became visible over basket and stellate cells in the molecular layer between P11 and P13; the internal granule cell layer and the deep cerebellar nuclei showed an increasingly strong hybridization signal during postnatal development. The external germinal layer was devoid of labeling throughout its existence. The developmental distribution of [3H]flunitrazepam binding sites was studied by receptor autoradiography. Cerebellar labeling was detectable at birth, with the highest levels present over the deep cerebellar nuclei, and relatively low levels equally distributed over the molecular and Purkinje cell layers. Cerebellar cortical grain density increased gradually during postnatal weeks 1 and 2, with the molecular, Purkinje and granule cell layers remaining essentially equally labeled. Between P11 and P15, the labeling over the molecular layer increased dramatically, reaching the high adult levels by P20. As with the in situ hybridization studies, there was a complete absence of [3H]flunitrazepam binding sites in the external germinal layer throughout development. These results indicate that, in the Purkinje cell, the production of mRNA and the synthesis of the alpha 1 subunit occur prior to the formation of afferent inhibitory synapses, suggesting that GABAA/benzodiazepine receptor expression precedes, and is independent of GABAergic synaptic input.     

AMPA receptor subunit RNA transcripts and [(3)H]AMPA binding in the cerebellum of normal and pcd mutant mice: an in situ hybridization study combined with receptor autoradiography

The expression of AMPA receptor subunit mRNAs and the binding of [(3)H]AMPA were studied in the cerebellum of normal and "Purkinje cell degeneration" ( pcd) mutant mouse. In the pcd cerebellum, [(3)H]AMPA binding was decreased significantly in both the molecular and granule cell layers by 63% and 36%, respectively. In those mutants, GluRA, GluRB and GluRC mRNAs were not detected in the Purkinje cell layer, and the levels of GluRB and GluRD mRNAs were significantly decreased in the granule cell layer by 16% and 57%, respectively. Cerebellar grafts transplanted into the pcd cerebellum expressed only GluRB and GluRC mRNAs, suggesting that donor cells express the appropriate subunits normally expressed by Purkinje neurons. Our results, firstly, support the idea that the expression of the GluRA subunit in Golgi epithelial cells may depend upon the sustained interaction with adjacent Purkinje cells, and secondly, suggest that granule cells which are more resistant to transsynaptic death may express higher levels of GluRB mRNA.     

A rapid method to quantify neurons in mixed cultures based on the specific binding of [3H]ouabain to neuronal Na+,K(+)-ATPase.

The high-affinity binding of [3H]ouabain to Na+,K(+)-ATPase was characterized in primary cultures of hippocampal neurons grown on feeder layers of astrocytes. The specific binding of [3H]ouabain was found to be time-dependent, high-affinity (apparent Kd = 8.5 nM), saturable (Bmax = 20.6 pmol/mg protein), dependent upon the presence of ATP, inhibited by K+, and directly proportional to neuronal, but not glial, cell number. Similar results were obtained using either sonicated cell suspensions or intact whole cells in culture. At the concentration of neurons routinely used, the specific binding of [3H]ouabain to the astrocyte feeder layer constituted less than 10% of total specific binding. Agents that selectively kill neurons rather than glia, such as the excitotoxins N-methyl-D-aspartate (NMDA) and kainate, reduced the amount of [3H]ouabain specifically bound in mixed cultures in a time- and concentration-dependent manner. Measurement of high-affinity [3H]ouabain binding to the neuronal form of Na+,K(+)-ATPase provides a simple, rapid, and reproducible method to quantify neurons in mixed culture.     

Quantitative autoradiography of [H]ouabain binding sites in rat brain

In vitro quantitative autoradiography was used to localize in rat brain binding sites for [³H]ouabain, an inhibitor of the Na⁺,K⁺-ATPase. High levels of [³H]ouabain binding sites were found in the superior and inferior colliculi, the mammillary nucleus, the interpeduncular nucleus, and in various divisions of the olfactory, auditory and somatomotor systems. The heterogeneous distribution of [³H]ouabain binding closely parallels the regional brain glucose consumption as determined by the [¹⁴C]deoxyglucose method. Lesion studies of the rat hippocampus using the excitotoxin, ibotenic acid, showed both a marked decrease of neuronal cell types on the injected side and a corresponding decrease in [³H]ouabain binding, indicating that some of the [³H]ouabain binding sites are localized to neurons. The close correlation between [³H]-ouabain binding and regional glucose utilization provides further evidence for a linkage between glucose utilization and the neuronal Na⁺,K⁺-ATPase.     

Autoradiographic localization of estradiol-binding neurons in the rat hippocampal formation and entorhinal cortex

This study has examined the distribution of [3H]estradiol and [1 alpha,2 alpha-3H]testosterone uptake in the hippocampal formation and entorhinal cortex of male and female rats. In both males and females, [3H]estradiol-binding neurons in Ammon's horn are located deep in stratum pyramidale and may correspond either to polymorphic interneurons or to early maturing pyramidal cells. Interneurons of strata oriens, lucidum and radiatum of Ammon's horn and of stratum moleculare of the subiculum also bind [3H]estradiol, as do basket cell interneurons in the polymorphic, infragranular layer of the dentate gyrus. While no granule cells appear to accumulate [3H]estradiol, these cells may be affected transsynaptically by gonadal steroids via their afferent contacts with the entorhinal cortex, which, of the areas examined, contains the greatest number of [3H]estradiol-binding neurons. While relatively few neurons concentrate [3H]estradiol in the hippocampal formation, these are localized to specific subpopulations, which may enhance their functional significance. Because there is no significant nuclear accumulation of [3H]-alpha-testosterone in either the entorhinal cortex or hippocampal formation, it appears that aromatase enzyme activity is not a major contributor to estrogen receptor occupancy in adult rats.     

Rapid decrease of high affinity ouabain binding sites in hippocampal CA1 region following short-term global cerebral ischemia in rat

High affinity [3H]ouabain binding was examined in the hippocampal CA1 region and frontal cortex of rats subjected to 5 min complete cerebral ischemia in a clinical death model, and to subsequent resuscitation. A decrease of Bmax directly after ischemia and its further gradual decrease during 120 min of reperfusion were noted in the ischemia-vulnerable CA1 region, whereas no change of Bmax was observed in frontal cortex. The apparent Kd constant showed insignificant fluctuations in either of the two brain regions. Since ouabain binds with high affinity to the neuronal (alpha +)-form of Na+/K+-ATPase, the results indicate a rapid enzyme loss in CA1 neurons. The high affinity ouabain binding test proved to be a sensitive detector of premorphological changes in nerve cell membranes in ischemia.     

Quisqualate- and NMDA-sensitive [3H]glutamate binding in primate brain

Excitatory amino acids (EAA) such as glutamate and aspartate are probably the neurotransmitters of a majority of mammalian neurons. Only a few previous studies have been concerned with the distribution of the subtypes of EAA receptor binding in the primate brain. We examined NMDA- and quisqualate-sensitive [3H]glutamate binding using quantitative autoradiography in monkey brain (Macaca fascicularis). The two types of binding were differentially distributed. NMDA-sensitive binding was most dense in dentate gyrus of hippocampus, stratum pyramidale of hippocampus, and outer layers of cerebral cortex. Quisqualate-sensitive binding was most dense in dentate gyrus of hippocampus, inner and outer layers of cerebral cortex, and molecular layer of cerebellum. In caudate nucleus and putamen, quisqualate- and NMDA-sensitive binding sites were nearly equal in density. However, in globus pallidus, substantia nigra, and subthalamic nucleus, quisqualate-sensitive binding was several-fold greater than NMDA-sensitive binding. In thalamus, [3H]glutamate binding was generally low for both subtypes of binding except for the anterior ventral, lateral dorsal, and pulvinar nuclei. In the brainstem, low levels of binding were found, and strikingly the red nucleus and pons, which are thought to receive glutamatergic projections, had approximately 1/20 the binding observed in cerebral cortex. These results demonstrate that NMDA- and quisqualate-sensitive [3H]glutamate binding are observed in all regions of primate brain, but that in some regions one subtype predominates over the other. In addition, certain areas thought to receive glutamatergic projections have low levels of both types of binding.     

The ontogeny of [3H]muscimol binding sites in the C57BL/6J mouse cerebellum

The characteristics of [3H]muscimol binding were investigated in cerebellar sections from 7-day-old mice. The binding sites were found to possess the kinetic properties and pharmacological specificity characteristic of high-affinity GABAA receptors. [3H]Muscimol binding sites in the developing C57BL/6J mouse cerebellum were visualized by light microscopic autoradiography. A distinct band of labeling situated over the molecular layer was apparent from day 1 to day 7. The external granule cell layer remained unlabeled throughout development. Labeling over the internal granule cell layer gradually increased from birth; it became more dense and well defined until adult levels of grain density were reached at 35-42 days of age. The deep cerebellar nuclei were moderately labeled at birth and gradually decreased in density thereafter. The observed ontogeny of granule cell [3H]muscimol binding sites suggests that the synthesis of receptors is initiated at a time immediately after cessation of cell division, coinciding with the beginning of granule cell translocation across the molecular layer. Since, at this time, granule cells have not yet formed synapses with the GABAergic Golgi II cells, nor have they, in turn, formed the vast majority of synaptic contacts with Purkinje cells, it follows that receptor appearance precedes the formation of afferent connections, and may also precede efferent synaptic contacts. The timing of the appearance of [3H]muscimol binding sites raises the possibility that their initial acquisition may be related to developmental events other than the interaction of the granule cell with its pre- or postsynaptic neuronal partners.     

An autoradiographic study of [3H]AMPA receptor binding and in situ hybridization of AMPA sensitive glutamate receptor A (GluR-A) subunits following morphine withdrawal in the rat brain

Chronic treatment with opioids is well known to result in the development of physical dependence. More recently, glutamatergic mechanisms have been implicated in expression of the withdrawal syndrome from opioids. To better examine glutamatergic involvement, an autoradiographic study of [3H]AMPA receptor binding and an assessment of in situ hybridization of AMPA sensitive glutamate receptor A (GluR-A) subunits in the rat brain were each performed 7 h after withdrawal from morphine infusion. Animals were rendered dependent by intracerebroventricular (i.c.v.) infusion of morphine (26 nmol/microl/h) via osmotic minipumps for 3 days. Brain sections of 14-microm thickness were incubated with 15 nM [3H]AMPA for quantitation of binding to the AMPA receptor. The probe for in situ hybridization was labeled at its 3' end using terminal deoxynucleotidyl transferase and [35S]dATP. The highest degree of [3H]AMPA binding was shown in the hippocampus. The extent of [3H]AMPA binding was increased significantly in the cortex areas (18-21%), caudate-putamen (20%), and hippocampus (7-9%) of rats following withdrawal from morphine. The highest levels of mRNA for GluR-A, flop and flip subunits, were found in the dentate gyrus and in the CA3 region of the hippocampus, respectively. The levels of mRNA for the flop form of GluR-A were decreased in the CA3 of hippocampus (8%) of the rat brain. The levels of mRNA for the flip form of GluR-A were increased in the parietal cortex (7%) and the entorhinal cortex (8%). Increases in the binding of [3H]AMPA to its receptor may play an important role during withdrawal from morphine dependence.     

Localization of L-glutamate receptors in rat brain by quantitative autoradiography

In vitro autoradiography was used to characterize and quantitatively map the sodium-independent binding of [3H]glutamate in rat brain. Measured in the presence of chloride, glutamate binding to frozen brain sections was specific, saturable, and reversible, with a Kd in the low micromolar range. At least two distinct binding sites were detected which had different affinities for quisqualic acid (0.7 microM and 1.2 mM). Autoradiograms revealed very high levels of binding in rostral forebrain areas, especially olfactory structures and frontal cortex. High levels of binding were found in sensory cortex, certain hippocampal subfields, caudate, lateral septum, and other limbic structures. Lowest levels were seen in globus pallidus, preoptic area, brainstem reticular formation, and spinal trigeminal nucleus. All other regions, including midbrain, thalamus, hypothalamus, and cerebellar cortex exhibited moderate levels of [3H]glutamate binding. Within the hippocampus glutamate binding was greatest in the inner two-thirds of the dentate molecular layer, subiculum, and CA1 stratum radiatum. CA3 was much less densely labeled; CA2 and CA4 were intermediate. Unlike cell layers in the cerebellum, hippocampal pyramidal and granule cell layers appeared mostly devoid of binding. The relationship of these binding sites to putative glutamate receptor subclasses and glutamatergic pathways is discussed.