In vivo release of substance P in the nucleus tractus solitarii increases during hypoxia

In vivo release of substance P (SP) was measured by microdialysis in the nucleus tractus solitarii (nTS) in adult cats. Small perfused semipermeable tubules (microdialysis probes) were implanted stereotaxically in the nTS, at the position of respiration-related neurons and perfused with artificial CSF. SP was determined by radioimmunoassay of the perfusate. Increased extracellular concentrations of SP-like immunoreactivity (SP-LI) were measured during hypoxia induced in artificially ventilated cats. In addition, a prolonged increase in the extracellular SP-LI concentration was encountered after cervical vagotomy. The results corroborate the suggestion that SP is a mediator of the central response to hypoxia.     

Adenosine release in nucleus tractus solitarii does not appear to mediate hypoxia-induced respiratory depression in rats

The time course of adenosine release in the nucleus tractus solitarii (NTS) and ventrolateral medulla (VLM) during acute systemic hypoxia was investigated in the anaesthetised rat by means of amperometric enzymatic sensors. It was found that acute hypoxia induced a significant delayed increase in adenosine level (reaching levels as high as 5 μM) in the NTS and that hypoxia-induced release of adenosine was similar at various regions of the NTS along its rostro-caudal axis. Significantly smaller or no increases in adenosine levels at all in response to hypoxia were observed in the VLM. The increase in adenosine level in the NTS occurred during reoxygenation after the termination of the hypoxic challenge and was accompanied by a smaller increase in inosine concentration. At the dorsal surface of the brainstem, only release of inosine was detected following acute hypoxia. Addition of the ecto-5'-nucleotidase inhibitor α,β-methylene ADP (200 μM) to the dorsal surface of the brainstem completely abolished the signal evoked by hypoxia, suggesting that the inosine arose from adenosine that was produced in the extracellular space by the prior release of ATP. This study indicates that following systemic hypoxia, adenosine levels in the NTS increase to a significantly greater extent than in the VLM. However, the increase in adenosine concentration in the NTS occurs too late to be responsible for the hypoxia-induced depression of the respiratory activity.     

Excitatory amino acid response in isolated nucleus tractus solitarii neurons of the rat

The excitatory amino-acid-induced currents in nucleus tractus solitarii neurons freshly isolated from rats were investigated in a whole-cell recording mode using a conventional patch-clamp technique. At a holding potential of -70 mV, L-glutamate (Glu), N-methyl-D-aspartate (NMDA) with 10(-9) M glycine, kainate (KA), quisqualate (QA) and L-aspartate (Asp) evoked inward currents. The currents increased in a sigmoidal fashion with increasing agonists concentration. The half-maximum concentration (EC50) values were 5 x 10(-5) M for Glu, 10(-6) M for QA, 10(-4) M for KA, 6 x 10(-5) M for NMDA and 5 x 10(-5) M for Asp. The Hill coefficients of the Glu-, QA-, KA-, NMDA- and Asp-induced responses were 1.0, 1.3, 1.1, 1.3 and 1.1, respectively. The Glu-, QA-, NMDA- and Asp-induced currents consisted of a transient initial peak and a successive steady-state component showing no desensitization. These currents had the same reversal potential near +5 mV. In the current-voltage (I-V) relationships for the Glu-, NMDA- and Asp-induced currents, slight outward rectifications were observed in Mg2(+)-free external solution at membrane potentials negative to 0 mV. In the presence of extracellular Mg2+, the currents induced by Glu, NMDA and Asp were suppressed at negative membrane potentials, but the suppression was less for the Glu response. The I-V relationships for QA- and KA-induced responses were almost linear at a membrane potential between -90 and +50 mV with or without the presence of Mg2+.     

Inhibitory neurotransmission in the nucleus tractus solitarii: implications for baroreflex resetting during exercise

Inhibitory neurotransmission plays a crucial role in the processing of sensory afferent signals in the nucleus of the solitary tract (NTS). The aim of this review is to provide a critical overview of inhibitory mechanisms that may be responsible for altering arterial baroreflex function during physical activity or exercise. Over a decade ago, the view of reflex control of cardiovascular function during exercise was revised because of the finding that the arterial baroreflex is reset in humans, enabling continuous beat-to-beat reflex regulation of blood pressure and heart rate. During the ensuing decade, many investigators proposed that resetting was mediated by central neural mechanisms that were intrinsic to the brain. Recent experimental data suggest that rapid and reversible changes in gamma-aminobutyric acid (GABA) inhibitory neurotransmission within the NTS play a fundamental role in this process. The hypothesis will be presented that baroreflex resetting by somatosensory input is mediated by: (1) selective inhibition of barosensitive NTS neurones; and (2) excitation of sympathoexcitatory neurones in the rostral ventrolateral medulla. Current research findings will be discussed that support an interaction between GABA and substance P (SP) signalling mechanisms in the NTS. An understanding of these mechanisms may prove to be essential for future detailed analysis of the cellular and molecular mechanisms underlying sensory integration in the NTS.     

Glutamatergic neurons say NO in the nucleus tractus solitarii

Both glutamate and nitric oxide (NO) may play an important role in cardiovascular reflex and respiratory signal transmission in the nucleus tractus solitarii (NTS). Pharmacological and physiological data have shown that glutamate and NO may be linked in mediating cardiovascular regulation by the NTS. Through tract tracing, multiple-label immunofluorescent staining, confocal microscopic, and electronic microscopic methods, we and other investigators have provided anatomical evidence that supports a role for glutamate and NO as well as an interaction between glutamate and NO in cardiovascular regulation in the NTS. This review article focuses on summarizing and discussing these anatomical findings. We utilized antibodies to markers of glutamatergic neurons and to neuronal NO synthase (nNOS), the enzyme that synthesizes NO in NTS neurons, to study the anatomical relationship between glutamate and NO in rats. Not only were glutamatergic markers and nNOS both found in similar subregions of the NTS and in vagal afferents, they were also frequently colocalized in the same neurons and fibers in the NTS. In addition, glutamatergic markers and nNOS were often present in fibers that were in close apposition to each other. Furthermore, N-methyl-d-aspartate (NMDA) type glutamate receptors and nNOS were often found on the same NTS neurons. Similarly, alpha-amino-3-hydroxy-5-methylisoxozole-proprionic acid (AMPA) type glutamate receptors also frequently colocalized with nNOS in NTS neurons. These findings support the suggestion that the interaction between glutamate and NO may be mediated both through NMDA and AMPA receptors. Finally, by applying tracer to the cut aortic depressor nerve (ADN) to identify nodose ganglion (NG) neurons that transmit cardiovascular signals to the NTS, we observed colocalization of vesicular glutamate transporters (VGluT) and nNOS in the ADN neurons. Thus, taken together, these neuroanatomical data support the hypothesis that glutamate and NO may interact with each other to regulate cardiovascular and likely other visceral functions through the NTS.     

Rapid inhibition of neural excitability in the nucleus tractus solitarii by leptin: implications for ingestive behaviour

The fat-derived peptide leptin regulates cellular activity in areas of the CNS related to feeding, and application of leptin to the fourth ventricle or the nucleus tractus solitarii (NTS) inhibits food intake and weight gain. The hypothesis that leptin modulates cellular activity in the NTS was tested using whole-cell patch-clamp recordings in brainstem slices. Leptin caused a rapid membrane hyperpolarization in 58% of rat NTS neurones, including neurones receiving tractus solitarius input (i.e. viscerosensory) and those involved in regulating output to the stomach, identified after gastric inoculation with a transneuronal retrograde viral label. The hyperpolarization was accompanied by a decrease in input resistance and cellular responsiveness, reversed near the K⁺ equilibrium potential, and was prevented by intracellular Cs⁺. Perfusion of tolbutamide (200 μ m ) or wortmannin (100–200 n m ) prevented the hyperpolarization, indicating activation of an ATP-sensitive K⁺ channel via a PI3 kinase-dependent mechanism. Constant latency tractus solitarius-evoked EPSCs were decreased in amplitude by leptin, and the paired-pulse ratio was increased, suggesting effects on evoked EPSCs involved activation of receptors on vagal afferent terminals. Leptin reduced the frequency of spontaneous and miniature EPSCs, whereas IPSCs were largely unaffected. Leptin's effects were observed in neurones from lean, but not obese, Zucker rats. Neurones that expressed enhanced green fluorescent protein (EGFP) in a subpopulation of putative GABAergic neurones in transgenic mice did not respond to leptin, whereas unlabelled murine neurones responded similarly to rat neurones. Leptin therefore directly and rapidly suppresses activity of excitatory NTS neurones likely to be involved in viscerosensory integration and/or premotor control of the stomach.     

Glutamatergic neurotransmission in the nucleus tractus solitarii: Structural and functional characteristics

Glutamate is the main excitatory transmitter in the central nervous system. As such, it plays a major role in transmitting and processing visceral sensory information within the nucleus tractus solitarii (NTS). Here, we review current knowledge on NTS glutamatergic transmission. We describe the main organizational features of NTS glutamatergic synapses as determined by work performed during the last decade using antibodies against glutamate receptors and transporters proteins. In light of these recent neuronatomical findings, we discuss some functional properties of developing and adult NTS glutamatergic synapses.     

Impaired regulation function in cardiovascular neurons of nucleus tractus solitarii in streptozotocin-induced diabetic rats

This study characterizes neural firing activity of the nucleus tractus solitarii (NTS) and baroreflex sensitivity (BRS) in streptozotocin (STZ)-induced diabetic rats relative to control rats by implantation of multi-wire electrode into rat NTS for direct monitoring of barosensitive NTS neurons before and after baroreflex system challenge by phenylephrine (PE) injection. NTS firing data is correlated with arterial pressure for both control and diabetic rats. In control rats, NTS firing rate and systolic arterial pressure correlate significantly with both pre-PE (baseline) and post-PE (p<0.01). In STZ-induced diabetic rats, positive correlation is observed only after PE injection (p<0.05). Although NTS firing rate was not significantly different between control and diabetic rats (p=0.085) in the baseline condition, it was significantly reduced in STZ-induced diabetic rats (p=0.042) with adjustment for BRS. After PE injection, NTS firing rate is significantly lower in diabetic rats relative to control rats (p<0.01). With adjustment for BRS, multivariate analysis shows that diabetes is independently associated with NTS firing rate after PE injection (p=0.034). Prior physiological and immunofluorescent studies found differing NTS data for control and diabetic rat only after PE challenge, but our data show diabetes-induced barosensitive NTS impairment in the baseline condition for STZ-induced diabetic rats. This latter finding suggests greater sensitivity of multi-wire electrode study of NTS relative to earlier methods.     

Involvement of opioid receptors in nucleus tractus solitarii in modulating endotoxic hypotension in rats.

Opioid antagonists selective for the mu-receptor (naltrexone) and kappa-receptor (nalmefene), respectively, were unilaterally microinjected into the nucleus tractus solitarii (NTS) of the pentobarbital anaesthetized (50 mg/kg, i.p.) rat subjected to E. coli endotoxin (15 mg/kg, i.v.). Naltrexone (0.5 microgram) ameliorated or totally abolished the endotoxic hypotension and tachycardia, and nalmefene at a dose of 1/25 (0.02 microgram) that of naltrexone showed a similar effect. The results suggest that the kappa- and mu-opioid receptors in NTS are actively involved in mediating the hypotensive and tachycardiac effects induced by systemic endotoxin in rats.     

Effects of antagonists on N-methyl-D-aspartate response in acutely isolated nucleus tractus solitarii neurons of the rat

The effect of antagonists on N-methyl-D-aspartate (NMDA)-induced response was investigated in isolated nucleus tractus solitarii (NTS) neurons freshly isolated from the rat using a conventional pathclamp technique. The NMDA-induced inward current consisted of an initial peak followed by a steady-state component. The competitive antagonists of NMDA receptor, D-2-amino-5-phosphonovalerate (APV), D-2-amino-4-phosphonoheptanoate (APH) and 3-3(2-carboxypiperazine-4-yl)propyl-1-phosphate (CPP), selectively suppressed the initial peak of NMDA-induced current more than the steady-state component at low concentrations. The non-competitive antagonists, MK-801, ketamine, Zn2+ and Mg2+, equally blocked both peak and steady-state components.     

Depressor and bradycardic actions of L-proline injected into the nucleus tractus solitarii of anesthetized rats

L-Glutamate has been considered to be a neurotransmitter in the nucleus tractus solitarius (NTS) of the afferent baroreflex pathway, though this has not yet been decisively shown. A bolus injection of a neurotransmitter candidate amino acid L-proline into the cisterna magna and that of L-glutamate shows the same pressor action in the freely moving rat, but the actual nuclei responding L-proline remain undetermined. Besides L-glutamate, L-proline might be another candidate amino acid in the NTS. The present study was therefore performed to characterize the circulatory action of L-proline injected into the NTS where responses to glutamate in the anesthetized rat had already been shown. The NTS was first determined as a site on the dorsal surface of the medulla where a microinjection of L-glutamate decreased arterial pressure and heart rate. Microinjected L-proline (1.65 to 13.2 nmol, 33 nl) into the NTS decreased arterial pressure and heart rate in a dose-dependent manner. The injection of a mixed solution (66 nl) of kynurenate, an ionotropic excitatory amino acid receptors antagonist (1.32 nmol), and L-proline (6.6 nmol) into the NTS abolished the depressor and bradycardic actions with L-proline alone (6.6 nmol, 66 nl). However, a mixture of an increased concentration of kynurenate (6.6 nmol) with glutamate augmented the actions seen with glutamate alone (0.66 nmol, 66 nl). D-Proline (13.2 nmol, 66 nl), the optic isomer of L-proline, produced no change in arterial pressure or heart rate, suggesting that the actions of L-proline in the NTS were optically specific. The results indicate that L-proline but not D-proline induces its depressor and bradycardic actions through ionotropic excitatory amino acid receptors in the NTS of the anesthetized rat. L-Proline may become a candidate transmitter of baroreceptor information in the NTS.     

GABA(B) receptors in the nucleus tractus solitarii modulate the carotid chemoreceptor reflex in rats

Ischemia depletes ATP and initiates cascades leading to irreversible tissue injury. Nicotinamide is a precursor of nicotinamide adenine dinucleotide (NAD+) which increases neuronal ATP concentration and protects against malonate-induced neurotoxicity, trauma and nitric oxide toxicity. We therefore examined whether nicotinamide could protect against stroke, using a model of permanent middle cerebral artery occlusion (MCA) occlusion in Wistar rats. Nicotinamide reduced neuronal infarction in a dose-specific manner. Furthermore, nicotinamide (500 mg/kg) reduced infarcts when administered up to 2 h after the onset of permanent MCA occlusion. The mechanism of action underlying the neuroprotection observed with nicotinamide remains to be clarified. These results are potentially important since nicotinamide is already used clinically, though not in the treatment of stroke.     

C-FOS expression in the subnucleus gelatinosus of the human nucleus tractus solitarii

The dorsal portion of the nucleus tractus solitarii (NTS) shows selective dendritic lesions in adults who died after an episode of acute heart failure, suggesting excitotoxic glutamate-induced neuronal death. Cerebral hypoxia and/or ischaemia also produce hyperexpression of specific genes (c-fos, c-jun) which may be involved, in vulnerable districts, in the mechanisms of excitotoxic neuronal death. In the present study, we examined NTS in 23 adults, who died shortly after an ischaemic and/or hypoxic event, on transverse serial sections of the medulla, stained with haematoxylineosin, Nissl, Klüver-Barrera and Luxol fast blue, and immunohistochemical staining by anti-tyrosine hydroxylase and anti-Fos. In 10 cases, at the level of the dorsal portion of the NTS, corresponding to the subnucleus gelatinosus, bilateral symmetrical hypereosinophilic areas and strong Fos-like immunoreactivity were detected. The hypoxic-ischaemic origin of these findings was supported by the strong hypereosinophilic appearance and shrinking of neurons, and by selective Fos-like immunoreactivity, the expression of Fos being mainly associated with cellular damage and subsequent death following hypoxic-ischaemic injury. In 7 other cases, Fos-like immunoreactivity was found at the level of the subnucleus gelatinosus, in the absence of hypereosinophilia or pyknosis in histological staining. The immediate-early gene expression induces to ascribe the absence of morphologically evident hypoxicischaemic lesions to the rapidity of death. The selective vulnerability of the subnucleus gelatinosus to hypoxic-ischaemic injury may not only be explained with reference to vascularization of the medullary tegmentum, but also to the structural and functional characteristics of the subnucleus itself.     

Immunohistochemical localization of putative neurotransmitters within the feline nucleus tractus solitarii

With the aid of immunohistochemical techniques the distribution of substance P, met-enkephalin, serotonin, somatostatin, alpha-melanocyte stimulating hormone, neurotensin and neurophysin immunoreactivities were mapped throughout the rostro-caudal extent of the cat's nucleus tractus solitarii. Three of the putative neurotransmitters (substance P, enkephalin and serotonin) were found to be widely distributed as varicose fibers and punctate structures. The densities of their immunoreactivities were plotted in a range from very dense, dense, moderate, occasional, to none, at different levels of the nucleus of the solitary tract. Substance P immunoreactivity was the most varied and dense of all the neurotransmitters studied. Its accumulations ranged from very dense in the lateral, dense in portions of the parvocellular and lateral, moderate in medial and commissural and occasional in ventrolateral and portions of the parvocellular subdivisions. Both the enkephalin and serotonin immunoreactivities had patterns similar to that of substance P immunoreactivity, although their amounts were not as great. Following colchicine treatment neurons containing substance P and enkephalin immunoreactivity were found in many subdivisions of the nucleus of the solitary tract. Somatostatin, alpha-melanocyte stimulating hormone, neurotensin and neurophysin immunoreactivities were present in the nucleus of the solitary tract as isolated varicose fibers scattered throughout the nucleus. Immunoreactive neurons were not found for these putative neurotransmitters after colchicine treatment. The presence of substance P immunoreactivity within subdivisions which receive visceral afferent input is discussed in relation to the role of substance P as a possible transmitter of the afferent limb of the vagus nerve. The distribution of enkephalin and serotonin immunoreactivities in the nucleus of the solitary tract reflect their involvement in the regulation or modulation of cardiovascular and respiratory functions. While the significance of somatostatin, alpha-melanocyte stimulating hormone, neurotensin and neurophysin immunoreactivities within the nucleus of the solitary tract is not understood at present, these substances might possibly play a role in visceral functions.     

Nitric oxide and GABA mediate bi-directional cardiovascular effects of orexin in the nucleus tractus solitarii of rats

The present study investigated the cardiovascular effects of orexin (OX)-A and OX-B in the nucleus tractus solitarii (NTS) and delineated the engagement of nitric oxide (NO) and GABA in OX-induced cardiovascular responses. In adult male Sprague-Dawley rats maintained under propofol anesthesia, microinjection bilaterally into the NTS of OX-A or OX-B evoked bi-directional cardiovascular effects in a dose-dependent manner. At a lower dose (5 pmol), OX-A or OX-B decreased systemic arterial pressure (SAP), heart rate (HR), and power density of the vasomotor components of SAP signals, our experimental index for sympathetic neurogenic vasomotor tone. At higher doses (>20 pmol), these two compounds elicited cardiovascular excitatory responses. These bi-directional cardiovascular effects of OX were abolished by co-injection of an OX(1) receptor antagonist, 1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl-urea hydrochloride (SB-334867, 0.75 nmol) or the OX(2) receptor antiserum (1:20). In addition, the vasodepressor effects of low dose (5 pmol) OX-A or OX-B in the NTS were attenuated by a nitric oxide synthase (NOS) inhibitor, N(G)-nitro-l-arginine methyl ester (l-NAME, 5 nmol), a neuronal nitric oxide synthase (nNOS) inhibitor, 7-nitroindazole (2.5 pmol) or the soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazole[4,3-alpha]quinoxalin-1-one (250 pmol). The vasopressor effects of high dose (200 pmol) OX were reversed by co-administration with GABA(A) or GABA(B) receptor antagonist, bicuculline methiodine (10 pmol) or 2-hydroxy saclofen (100 pmol), or l-NAME (5 nmol). Our results indicate that OX-A or OX-B elicited bi-directional cardiovascular effects via OX receptor-dependent mechanisms. The vasodepressor effects of OX were induced by the nNOS-derived NO and activation of sGC-associated signaling pathway, whereas the vasopressor effects were mediated by interaction with GABAergic or nitrergic neurotransmission in the NTS.     

Complex cardiovascular actions of α-adrenergic receptors expressed in the nucleus tractus solitarii of rats

Although both α₁- and α₂-adrenergic receptors (ARs) are known to be expressed in the nucleus of the solitary tract (NTS), the functional significance of these receptors is still not fully established. In this study, we microinjected α₁- and α₂-AR agonists into the NTS of urethane-anaesthetized Wister rats to study the cardiovascular effects in response to their activation. When the α₁-AR agonist phenylephrine was microinjected into the area where barosensitive neurons are dominantly located (baro-NTS), mean arterial pressure (MAP) and heart rate (HR) were significantly elevated. When tested in the area where chemosensitive neurons are dominantly located (chemo-NTS), however, MAP and HR were significantly decreased. Pretreatment with the non-specific α-AR antagonist phentolamine into the NTS inhibited the phenylephrine-induced cardiovascular responses. In contrast, microinjection of the α₂-AR agonist clonidine into either the baro-NTS or the chemo-NTS decreased MAP and HR; they were also inhibited by the α₂-adrenergic antagonist yohimbine. Moreover, we immunohistochemically identified that cardiovascular responses induced by α₁-ARs may be mediated by NTS neurons while those induced by α₂-ARs may be mediated by astrocytes located in the barosensitive and chemosensitive areas of the NTS. These results suggest that both types of α-AR expressed in the NTS may be involved in regulating cardiovascular homeostasis via modulation of input signals from baroreceptor and chemoreceptor afferents; however, cardiovascular responses produced by stimulation of α₁-ARs are strictly location specific within the NTS.     

Central sympathetic stimulation produced by saline application into the nucleus tractus solitarii area of conscious rats

Hypertonic saline 4% was microinjected into the area of the nucleus tractus solitarii (NTS) of conscious rats. The experiments were designed to explore the systemic hormonal responses and the contribution of various hormones to the hypertensive effect elicited by this procedure. A mean blood pressure rise to 155 +/- 3 mmHg (as compared to 119 +/- 2 in rats receiving the same volume of equiosmolar dextrose solution, P less than 0.05) was accompanied by significant rises in plasma norepinephrine (0.556 +/- 0.128 ng/ml) and epinephrine (0.970 +/- 0.287 ng/ml) as compared to control rats receiving dextrose or control rats having only the cannula implanted, without microinjection. There was no significant change in plasma renin activity or plasma vasopressin levels, and the blood pressure rise could not be prevented or reversed by an angiotensin-converting enzyme inhibitor. The data are consistent with the hypothesis that the hypertensive response to this procedure is mainly due to an acute stimulation of central sympathetic neurons.     

Cardiovascular responses to microinjection of nociceptin and endomorphin-1 into the nucleus tractus solitarii in conscious rats

Increasing evidence suggests an active participation of nociceptinergic transmission in the central control of cardiovascular activity and reflex. In this study, the role of the classic opioid mu receptor and the nociceptin/orphanin FQ receptor, a novel opioid receptor, in the nucleus tractus solitarii (NTS) in the regulation of cardiovascular activity was investigated and compared in chronically cannulated and freely moving conscious rats. Microinjections of nociceptin, an endogenous ligand for the nociceptin receptor, into the relatively rostral NTS produced dose-related (0.04, 0.2, and 1 nmol) increases in blood pressure and heart rate. Intra-NTS injection of the selective nociceptin receptor antagonist [Nphe(1)]Nociceptin(1-13)NH(2) (NOR-AN) at 1 nmol blocked the increases in blood pressure and heart rate induced by nociceptin. In contrast, pretreatment with the nonselective opioid receptor antagonist naloxone (5 nmol) had no effects on the cardiovascular responses to nociceptin. Like nociceptin, microinjection of endomorphin-1 (EM-1), an endogenous ligand for the opioid mu receptor, into the rostral NTS increased blood pressure and heart rate in a dose-dependent manner (0.04, 0.2, and 1 nmol). Pretreatment with naloxone (5 nmol), but not NOR-AN, blocked cardiovascular responses elicited by EM-1. Neither NOR-AN nor naloxone alone had significant effects on the baseline blood pressure and heart rate. Injection of excitatory amino acid l-glutamate (1 nmol) into the same sites caused the typical depressor and bradycardic responses. In the caudal NTS areas, nociceptin and EM-1 seemed to induce opposite responses: hypotension and bradycardia. These results suggest that the novel nociceptin receptors and traditional opioid receptors in the NTS may be independently involved in the regulation of cardiovascular activity.