Widespread activation of barrel cortex by small numbers of neonatally spared whiskers

Activity-dependent plasticity in rodent whisker barrel cortex was examined by means of high-resolution 2-deoxyglucose (2-DG) with immunohistochemical double labeling. Hamsters with all but one, two, or four follicles ablated on postnatal day 7 received 2-DG injections as adults. Autoradiograms of follicle-ablated animals showed heavy activation of the entire barrel field during normal behavior, despite the missing whiskers. The intensity of 2-DG labeling was significantly reduced if the whiskers spared after follicle ablation were trimmed prior to the 2-DG injection, demonstrating that the widespread activation was driven by the spared whiskers. This widespread metabolic activation of the adult barrel field after neonatal follicle ablation was in sharp contrast to the somatotopically appropriate 2-DG labeling in barrel fields of normal adults subject to acute trimming of most whiskers, but was similar to that seen in normal adult animals with all whiskers intact. The results demonstrate large-scale plasticity of barrel circuitry following neonatal sensory deprivation, and provide a powerful functional anatomical setting to investigate underlying mechanisms.     

Experience-dependent changes in function and anatomy of adult barrel cortex

Manipulations of sensory input to vibrissal mechanoreceptors can modify columnar functioning of the barrel cortex in adult animals. In mice, partial vibrissectomy sparing one row of vibrissae in young adults results 7 days later in an increase in the functional cortical column activated by the spared whiskers and visualized with 2-deoxyglucose autoradiography. The increase in the extent of the labelled area is visible in all cortical layers, but particularly in layer V, where the metabolic labelling is more intense in the representation of the spared vibrissae. Two months after vibrissectomy the enlargement of the labelled area is accentuated. Deprivation of a row of vibrissae results in a decrease in the areal extent of its cortical representation. Investigations of cortico-cortical connections carried out in living slices of the barrel cortex of mice 2 months after vibrissectomy sparing one row of whiskers, revealed elongation and increased branching of axons originating in the spared cortical column. The dendritic spine density was increased on the basal dendrites of layer V pyramidal neurons of the spared column and decreased on layer III apical dendrites of the deprived column. Thus, prolonged changes in functional activation of adult barrel cortex are accompanied by rearrangement of cortico-cortical circuitry.     

Modulation of plasticity in human motor cortex after forearm ischemic nerve block

Deafferentation leads to cortical reorganization that may be functionally beneficial or maladaptive. Therefore, we were interested in learning whether it is possible to purposely modulate deafferentation-induced reorganization. Transient forearm deafferentation was induced by ischemic nerve block (INB) in healthy volunteers. The following five interventions were tested: INB alone; INB plus low-frequency (0.1 Hz) repetitive transcranial magnetic stimulation of the motor cortex ipsilateral to INB (INB+rTMSi); rTMSi alone; INB plus rTMS of the motor cortex contralateral to INB (INB+rTMSc); and rTMSc alone. Plastic changes in the motor cortex contralateral to deafferentation were probed with TMS, measuring motor threshold (MT), motor evoked-potential (MEP) size, and intracortical inhibition (ICI) and facilitation (ICF) to the biceps brachii muscle proximal to the level of deafferentation. INB alone induced a moderate increase in MEP size, which was significantly enhanced by INB+rTMSc but blocked by INB+rTMSi. INB alone had no effect on ICI or ICF, whereas INB+rTMSc reduced ICI and increased ICF, and conversely, INB+rTMSi deepened ICI and suppressed ICF. rTMSi and rTMSc alone were ineffective in changing any of these parameters. These findings indicate that the deafferented motor cortex becomes modifiable by inputs that are normally subthreshold for inducing changes in excitability. The deafferentation-induced plastic changes can be up-regulated by direct stimulation of the "plastic" cortex and likely via inhibitory projections down-regulated by stimulation of the opposite cortex. This modulation of cortical plasticity by noninvasive means might be used to facilitate plasticity when it is primarily beneficial or to suppress it when it is predominately maladaptive.     

Behavioral and neural approaches to the function of the mystacial vibrissae.

Reviews the behavioral functions of mystacial vibrissae through studies of (a) the activity of fibrissae during behavior, (b) single-unit responses to stimulation of vibrissae, and (c) the behavioral effects of removing vibrissae. Hypotheses for behavioral investigation are evaluated against older approaches. Neural and behavioral plasticity are discussed in the light of recent investigations of barrel formations in the somatosensory cortex and of locomotor behavior during exploration. (2 p ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Hydroxylamine treatment increases glutathione-protein and protein-protein binding in human erythrocytes

Functional development of the rat whisker somatosensory system was studied by using the (14C) 2-deoxyglucose (2DG) metabolic mapping technique. Restrained rat pups had their left mystacial vibrissae stroked for 30 minutes and their brains harvested, sectioned, and autoradiographed from the level of the lower medulla to the frontal cortex. Subjects were tested at postnatal days (PNDs) 0-9 and 21. At birth, all subjects exhibited a significant increase of 2DG uptake in the left spinal trigeminal nuclei, the principal trigeminal sensory nucleus, and a portion of the right ventral posteromedial thalamic nucleus. The primary somatosensory cortex exhibited significant 2DG uptake contralateral to stimulation by PND 6, followed by the secondary somatosensory cortex at PND 7. The pattern of 2DG uptake in the somatosensory cortices became more intense and well defined by PND 9. Given that the somatosensory system develops in an orderly fashion from the periphery to higher brain structures, the present results show that brain structures mediating whisker sensory input are not metabolically active until projections from lower somatosensory centers are established. Neurons become responsive to whisker stimulation in the subcortical structures at birth and in the somatosensory cortex a few days later. This cortical activity follows the organization of the upper tier of thalamocortical fibers into a "barrelfield." Moreover, there is a gradual enhancement in functional activity of the vibrissa neurons at different somatosensory nuclei as rats mature. The present study elucidates the time course of functional development in the rat somatosensory system.     

Effects of D-AP5 and NMDA microiontophoresis on associative learning in the barrel cortex of awake rats

Experiments involving single-unit recordings and microiontophoresis were carried out in the barrel cortex of awake, adult rats subjected to whisker pairing, an associative learning paradigm where deflections of the recorded neuron's principle vibrissa (S2) are repeatedly paired with those of a non-adjacent one (S1). Whisker pairing with a 300 ms interstimulus interval was applied to 61 cells. In 23 cases, there was no other manipulation whereas in the remaining 38, pairing occurred in the presence of one of three pharmacological agents previously shown to modulate learning, receptive field plasticity and long-term potentiation: N-methyl-D-aspartic acid (NMDA) (n=8), the NMDA receptor antagonist AP5 (n=17) or the nitric oxide synthase inhibitor L-nitro-arginine-N-methyl-ester (L-NAME) (n=13). Non-associative (unpaired) experiments (n=14) and delivery of pharmacological agents without pairing (n=14) served as controls. Changes in neuronal responsiveness to S1 following one of these procedures were calculated and adjusted relative to changes in the responses to S2. On average, whisker pairing alone yielded a 7% increase in the responses to S1. This enhancement differed significantly from the 17% decrease obtained in the non-associative control condition and could not be attributed to variations in the state of the animals because analysis of the cervical and facial muscle electromyograms revealed that periods of increased muscular activity, reflecting heightened arousal, were infrequent (less than 4% of a complete experiment on average) and occurred randomly. The enhancement of the responses to S1 was further increased when whisker pairing was performed in the presence of L-NAME (27%) or NMDA (35%) whereas AP5 reduced it to 1%. During the delivery period, NMDA enhanced both neuronal excitability and responsiveness to S1 whereas AP5 depressed them. However, the effects of both substances disappeared immediately after administration had ended. L-NAME did not affect the level of ongoing activity and responses to S1 significantly. From these data, we concluded that, since the changes in the responses to S1 lasted longer than the periods of both whisker pairing and drug delivery, they were not residual excitatory or inhibitory drug effects on neuronal excitability. Thus, our results indicate that, relative to the unpaired controls, whisker pairing led to a 24% increase in the responsiveness of barrel cortex neurons to peripheral stimulation and that these changes were modulated by the local application of pharmacological agents that act upon NMDA receptors and pathways involving nitric oxide. We can infer that somatosensory cerebral cortex is one site where plasticity emerges following whisker pairing.     

GABAergic neurons in barrel cortex show strong, whisker-dependent metabolic activation during normal behavior

Electrophysiological data from the rodent whisker/barrel cortex indicate that GABAergic, presumed inhibitory, neurons respond more vigorously to stimulation than glutamatergic, presumed excitatory, cells. However, these data represent very small neuronal samples in restrained, anesthetized, or narcotized animals or in cortical slices. Histochemical data from primate visual cortex, stained for the mitochondrial enzyme cytochrome oxidase (CO) and for GABA, show that GABAergic neurons are more highly reactive for CO than glutamatergic cells, indicating that inhibitory neurons are chronically more active than excitatory neurons but leaving doubt about the short-term stimulus dependence of this activation. Taken together, these results suggest that highly active inhibitory neurons powerfully influence relatively inactive excitatory cells but do not demonstrate directly the relative activities of excitatory and inhibitory neurons in the cortex during normal behavior. We used a novel double-labeling technique to approach the issue of excitatory and inhibitory neuronal activation during behavior. Our technique combines high-resolution 2-deoxyglucose (2DG), immunohistochemical staining for neurotransmitter-specific antibodies, and automated image analysis to collect the data. We find that putative inhibitory neurons in barrel cortex of behaving animals are, on average, much more heavily 2DG-labeled than presumed excitatory cells, a pattern not seen in animals anesthetized at the time of 2DG injection. This metabolic activation is dependent specifically on sensory inputs from the whiskers, because acute trimming of most whiskers greatly reduces 2DG labeling in both cell classes in columns corresponding to trimmed whiskers. Our results provide confirmation of the active GABAergic cell hypothesis suggested by CO and single-unit data. We conclude that strong activation of inhibitory cortical neurons must confer selective advantages that compensate for its inherent energy inefficiency.     

Neonatal serotonin depletion modifies development but not plasticity in rat barrel cortex

Effects of serotonin depletion (induced by neonatal injection of 5,7-dihydroxytryptamine) upon dimensions of cortical barrels and their metabolic activation, and upon effects of neonatal vibrissectomy sparing row C, were examined in 1-month-old rats. Dimensions of row C barrels, and of [14C]2-deoxyglucose (2-DG) labelling in the cortex obtained after stimulation of the row C vibrissae, were measured. Serotonin depletion did not change dimensions of barrels, but reduced the extent of 2-DG labelling of cortical representation of the row C whiskers by 30%. Vibrissectomy sparing this row resulted in an expansion of the row C barrels and of 2-DG labelling in the barrel cortex that were similar in both control and serotonin-depleted rats.     

Effect of neuronal NO synthase inhibition on the cerebral vasodilatory response to somatosensory stimulation

Whether nitric oxide (NO) mediates--or not--the local cerebral blood flow (CBF) increases occurring during functional brain activation is still a controversial issue. In the present study, we sought to determine whether neuronal NO synthase is involved in the cerebrovascular response to activation of the trigeminal pathway in the rat. Local CBF was measured using the autoradiographic [14C]iodoantipyrine technique in control alpha-chloralose anesthetized rats and 30 min following administration of 7-nitroindazole (7-NI), an inhibitor of the neuronal NO synthase. Unilateral whiskers stroking increased local CBF in all six regions of the trigeminal pathway. Under 7-NI, CBF was slightly decreased and the vasodilatatory response to whisker stimulation was unaltered in the four trigeminal nuclei studied. In contrast, no significant vasodilatation was noted in the ventral posteromedial thalamic nucleus and somatosensory cortex. These results suggest that the neuronal NO synthase mediates the hyperemia associated with somatosensory activation in second order relay stations but not in the site of termination of primary afferents.     

Oxalate binding protein from the kidney of rat and human mitochondria: studies on properties

The present paper reviews current knowledge of the development and plasticity of the inhibitory gamma-aminobutyric acid (GABA) containing circuitry of the cerebral neocortex, in particular, the rat somatosensory barrel field cortex. Recent studies reveal a delayed and protracted maturation of the inhibitory compared with the excitatory cortical system, both at the neuronal and synaptic levels. This characteristic developmental pattern leaves a longer time window during which behaviourally relevant activity coming from the periphery can influence the organization of the GABA system. Indeed, sensory deprivation experiments confirm the involvement of the GABA system in phenomena of experience-dependent cortical plasticity. Changing the pattern and level of afferent activity of in the rat somatosensory system during development by removing vibrissae results in a significant decrease in the number of GABA neurons and synapses in the thalamocortical recipient layer IV. Particularly affected are GABA synapses contacting dendritic spines, the number of which decreases by almost two-thirds. The involvement of the GABA system in events of experience-dependent plasticity contributes to the adequate functioning of the cerebral cortex in the conditions of constantly changing environment and varying individual experience.     

Experience-dependent plasticity in adult rat barrel cortex

This study tested the hypothesis that the receptive fields (RFs) of neurons in the adult sensory cortex are shaped by the recent history of sensory experience. Sensory experience was altered by a brief period of "whisker pairing": whiskers D2 and either D1 or D3 were left intact, while all other whiskers on the right side of the face were trimmed close to the fur. The animals were anesthetized 64-66 h later and the responses of single neurons in contralateral cortical barrel D2 to stimulation of whisker D2 (the center RF) and the four neighboring whiskers (D1, D3, C2, and E2; the excitatory surround RF) were measured. Data from 79 cells in four rats with whiskers paired were compared to data from 52 cells in four rats with untrimmed whiskers (control cases). During the period of whisker pairing, the RFs of cells in barrel D2 changed in three ways: (i) the response to the center RF, whisker D2, increased by 39%, (ii) the response to the paired surround RF whisker increased by 85-100%, and (iii) the response to all clipped (unpaired) surround RF whiskers decreased by 9-42%. In the control condition, the response of barrel D2 cells to the two neighboring whiskers, D1 and D3, was equal. After whisker pairing, the response to the paired neighbor of D2 was more than twice as large as the response to the cut neighbor of D2. These findings indicate that a brief change in the pattern of sensory activity can alter the configuration of cortical RFs, even in adult animals.     

Trigeminal orosensation and ingestive behavior in the rat.

Used a deafferentation procedure with male Wistar rats to examine the contributions of trigeminal orosensation to control of ingestive behavior. The procedure removed somatosensory input from the mouth while sparing olfaction, lingual taste, vibrissae inputs, and proprioceptive afferents from and efferents to the jaw muscles. Ss with sections of tongue or jaw muscle efferents served as controls. Bilateral trigeminal orosensory deafferentation was followed by effects on ingestive behavior, the magnitudes of which were proportional to the extent of the deafferentation. The trigeminal syndrome includes aphagia and adipsia, impairments in the sensorimotor control of eating and drinking, decreased responsiveness to food and water, and a reduction in the level of body weight regulation. Trigeminal deafferentation spared elementary ingestive movement patterns (biting, licking, and chewing) but disrupted their control by the perioral stimuli. Deficits in food intake varied with the sensory properties of the diet. Recovery of intake took place along a palatability gradient, and recovery of water intake paralleled that of dry food. The chronically reduced body weight was caused by persistent hypophagia and reflected reduced responsiveness to food. Findings suggest a considerable degree of overlap in the neural mechanisms mediating the sensorimotor and motivational control of intake in the rat. (76 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Noise-driven neuroplasticity in self-organizing feature maps: a neurocomputational model of phantom limbs

The term "phantom limb" denotes the sensation that an extremity is present although it has been lost. A number of clinical features and recent findings of cortical map plasticity after destruction of afferent pathways (deafferentation) suggest that phantom limbs are caused by large-scale cortical reorganization processes. However, in paraplegics, who also suffer from cortical deafferentation, phantom sensations rarely develop, and if they do, they are weak, lacking in detail, and delayed, occurring after months. This has been taken to suggest a non-cortical genesis of phantom limbs. This article proposes a biologically plausible minimal neural network model to solve this apparent puzzle. Deafferentation was simulated in trained self-organizing feature maps. Reorganization was found to be directed by input noise. According to the model, the production of input noise by the deafferented primary sensory neuron promotes cortical reorganization in amputees. No such noise is generated or conducted to the cortex in paraplegics.     

Functional parasagittal compartments in the rat cerebellar cortex: an in vivo optical imaging study using neutral red

1. The spatial patterns of activation in the rat cerebellar cortex evoked by peripheral stimulation were studied in vivo using optical imaging techniques. 2. Crus I and Crus II were stained with the pH sensitive dye, neutral red. Electrical stimulation of the vibrissae area of the ipsilateral face evoked optical responses consisting of parasagittal bands. The bands were 100-300 microns in width, elongated in the anterior-posterior direction, commonly extended across at least two folia, and varied in number from 1 to 7. 3. The optical responses were dependent on activation of postsynaptic elements since they were decreased substantially by the non-N-methyl-D-aspartate antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione. The optical bands were shown to correspond anatomically with the parasagittal compartments revealed by immunostaining with anti-zebrin II. 4. The present study demonstrates that functional parasagittal compartments exist in the rat cerebellar cortex and suggests that zebrin-positive Purkinje cell subgroups are anatomically related to this functional organization.     

Suppression of IL-12 production by phosphodiesterase inhibition in murine endotoxemia is IL-10 independent

Limbic system-associated membrane protein (LAMP), a 64-kDa membrane protein, is an axon guidance adhesion molecule expressed by neurons in limbic system-related areas of the CNS. During development, LAMP is expressed on growing axons, growth cones, and their target neurons, but in adults it is restricted to membranes of somata and dendrites. In the adult spinal cord, LAMP immunoreactivity is found only on neurons of lamina II, lamina X, and the intermediolateral cell column and its ultrastructural localization is entirely postsynaptic. We studied changes in the expression of LAMP in lamina II of adult rat spinal cord after L1-S2 dorsal rhizotomy, a procedure that partially deafferents lamina II neurons and induces axonal sprouting by spared systems in lamina II. At the light microscopic level, LAMP immunoreactivity in lamina II was decreased in density at 3, 10, and 60 days postoperatively. This decrease in immunoreactivity suggests that LAMP expression by lamina II neurons may normally be regulated by specific afferent activity. Ultrastructurally, in control lamina II and after deafferentation in both control and deafferented lamina II at 3 and 60 days postoperatively, LAMP expression was restricted to postsynaptic membranes. Ten days after deafferentation, however, when axons are actively sprouting, LAMP was expressed on both axonal and postsynaptic membranes. The reexpression of LAMP on axonal profiles after deafferentation may identify axons that undergo sprouting in response to deafferentation.     

Astrocytic messenger RNA responses to striatal deafferentation in male rat

This investigation describes the schedule and regional distribution of astrocytic responses in striatum following deafferentation by unilateral frontal cortex ablation. In the ipsilateral deafferented striatum, glial fibrillary acidic protein and clusterin (sulfated glycoprotein-2) messengerRNA showed peak elevations by 10 days postlesioning (Northern blots). Vimentin messengerRNA responded faster, with a transient elevation by three days postlesioning. The messengerRNA for glial fibrillary acidic protein, clusterin and vimentin returned toward control levels by 27 days postlesioning. However, the neuronal marker growth-associated protein messengerRNA, was decreased at all postlesion times. By in situ hybridization, the increased glial fibrillary acidic protein messengerRNA and clusterin messengerRNA signals were localized mainly to the dorsal half of the ipsilateral deafferented striatum and followed the same schedule as found by Northern blots. Glial fibrillary acidic protein messengerRNA was widely diffused in the dorsal striatum and was excluded from fascicles of the internal capsule; a similar distribution was found for glial fibrillary acidic protein-immunopositive astrocytes. While clusterin messengerRNA signal showed a distinct clustering, its immunoreactivity appeared as deposits in the deafferented striatal neuropil; Western blots confirmed the immunocytochemical results. By in situ hybridization, vimentin messengerRNA was mostly localized to the cortical wound cavity dorsal to the deafferented striatum and overlapped the distribution of vimentin-immunopositive cells. These findings suggest a coordination of striatal astrocytic messengerRNA responses with the degeneration of corticostriatal afferents. We also compared these same parameters with those from published reports on the hippocampus after deafferenting lesions. Certain astrocyte molecular responses to deafferentation are detected about five days earlier in the hippocampus than in the striatum. This different schedule in response to decortication may pertain to differences in synaptic remodeling in the hippocampus vs striatum.     

Medialbasal hypothalamic deafferentation modulates feeding response to insulin in rats

Medialbasal hypothalamic (MBH) deafferentation induces hypothalamic obesity accompanied by hyperphagia and hyperinsulinemia. Insulin is essential in developing and maintaining obesity, but the role of insulin in food intake in hypothalamic obesity is still unclear. The present study demonstrated that exogenous insulin increased food intake dose relatedly in MBH deafferented diabetic rats without developing hypoglycemia. Insulin administrations suppressed hyperphagia in the sham-operated diabetic rats. In contrast, in the MBH deafferented diabetic rats, insulin increased food intake in sow-related manner concomitant with a greater increased body weight gain than the sham-operated diabetic rats. The blood glucose levels of the MBH deafferented diabetic rats were at all time higher than those of the sham-operated diabetic rats and were hyperglycemic throughout the insulin treatment. These data indicate that insulin action on food intake mediated through the central nervous system is modulated by MBH deafferentation. This modulated insulin action may contribute to the pathogenesis on obesity in MBH deafferented animals.     

Optical intrinsic signal imaging responses are modulated in rodent somatosensory cortex during simultaneous whisker and forelimb stimulation

Optical intrinsic signal imaging (OIS) was used to investigate physiologic interactions between spatially and functionally distinct cortical somatosensory systems. The OIS response magnitude was evaluated after simultaneous stimulation of single whiskers and forelimb digits. Whisker C1 was deflected at a frequency of 10 Hz for 2 seconds while low- or high-intensity vibratory stimuli were applied to forelimb digits. The OIS responses to simultaneous whisker and forelimb stimulation were compared with lone whisker stimulated controls. Overall, addition of a second stimulus caused decreases in barrel cortex response magnitude. Three different response patterns were detected within individual trial sets. Modulation of barrel cortex evoked potentials provided evidence that changes in OIS responses observed here may be partially influenced by vascular responses to changes in neuronal activity. However, OIS responses in the barrel region during lone forelimb stimulation that were unaccompanied by evoked potentials suggested the possibility of independent vascular dynamic influences on response modulation. This study demonstrates that cortical responses at the level of primary sensory processing may be significantly influenced by activity in adjacent regions. Furthermore, it reveals that vascular and neuronal characteristics of interregional modulation do not co-localize and may produce responses in which one component increases while the other decreases.     

Decreased heterogeneity of capillary plasma flow in the rat whisker-barrel cortex during functional hyperemia

The pattern of capillary plasma perfusion was investigated in the rat brain during functional activation. Functional hyperemia was induced in the left whisker-barrel cortex by deflection of the right mystacial vibrissae for 2 min at frequencies of 1-7 Hz. Rats were decapitated under anesthesia 3-4 s after i.v. bolus injection of Evans blue dye. The steep increase of the arterial dye concentration ensures that divergent capillary plasma transit times result in unequal intracapillary dye concentrations. Plasma perfusion heterogeneity was determined from the coefficient of variation (CV) of Evans blue concentrations measured in numerous single capillaries of the whisker-barrel cortex. Functional hyperemia was quantified from measurements of CBF using the [14C]-iodoantipyrine technique in a second experimental group. CBF in the left whisker-barrel cortex increased with the stimulation frequency and was maximal at 5 Hz compared to the right side. Conversely, plasma perfusion heterogeneity decreased with stimulation frequency in a reciprocal way, being minimal at 5 Hz. Results indicate a decrease in the microcirculatory flow heterogeneity during functional hyperemia in the brain.     

Inhibition of receptor/G protein coupling by suramin analogues

The 2-[14C]deoxyglucose method was used to examine the effects of chronic, voluntary ethanol consumption on rates of local cerebral glucose utilization (LCGU). LCGU was measured in male Long-Evans rats immediately following the completion of a 60-min schedule-induced polydipsia drinking session. Three groups of animals were examined: animals with a history of ethanol consumption that received ethanol on the test day (ethanol-ethanol), animals with a similar ethanol history that were presented with water on the test day (ethanol-water), and a control group that received water throughout the experiment (water-water). Ethanol consumption on the test day resulted in a highly discrete pattern of metabolic changes, with significant decreases in glucose utilization in the hippocampal complex, habenula, anterior ventral thalamus, and mammillary bodies, whereas increases were observed in the nucleus accumbens and locus coeruleus. Rates of LCGU in the ethanol-water group were increased throughout all regions of the central nervous system examined, indicating that the long-term consumption of moderate ethanol doses that do not produce physical dependence can cause significant changes in functional brain activity.