Evidence for the modulation of nociception in mice by central mast cells.

BACKGROUND: Hyperalgesia that develops following nerve ligation corresponds temporally and in magnitude with the number of thalamic mast cells located contralateral to the ligature. We tested the possibility that mast cells modulate nociception centrally, similar to their role in the periphery. METHODS: We examined the central effect of two hyperalgesic compounds that induce mast cell degranulation and of stabilized mast cells using cromolyn. RESULTS: Thermal hyperalgesia (tail flick) induced by nerve growth factor (NGF, a neurotrophic compound) and mechanical hyperalgesia (von Frey) induced by dynorphin A (1-17) (opioid compound) each correlated with the per cent of thalamic mast cells that were degranulated. Degranulation of these mast cells by the central injection of compound 48/80, devoid of neurotrophic or opioid activity, was sufficient to recapitulate thermal hyperalgesia. Stabilization of mast cells by central injections of cromolyn produced no analgesic effect on baseline tail flick or von Frey fibre sensitivity, but inhibited thermal hyperalgesia produced by compound 48/80 and tactile hyperalgesia induced by dynorphin and by Freund's complete adjuvant. Finally, chemical nociception produced by the direct activation of nociceptors by formalin (phase I) was not inhibited by centrally injected cromolyn whereas chemical nociception dependent on central sensitization (formalin-phase II and acetic acid-induced abdominal stretches) was. CONCLUSIONS: These convergent lines of evidence suggest that degranulation of centrally located mast cells sensitizes central nociceptive pathways leading to hyperalgesia and tonic chemical sensitivity. SIGNIFICANCE: Hyperalgesia induced by spinal nerve ligation corresponds temporally and in magnitude with degranulation of thalamic mast cells. Here, we provide evidence that hyperalgesia induced by NGF, formalin and dynorphin also may depend on mast cell degranulation in the CNS whereas cromolyn, a mast cell stabilizer, blocks these effects in mice.

Cell sorting in a Petri dish controlled by computer vision.

Fluorescence-activated cell sorting (FACS) applying flow cytometry to separate cells on a molecular basis is a widespread method. We demonstrate that both fluorescent and unlabeled live cells in a Petri dish observed with a microscope can be automatically recognized by computer vision and picked up by a computer-controlled micropipette. This method can be routinely applied as a FACS down to the single cell level with a very high selectivity. Sorting resolution, i.e., the minimum distance between two cells from which one could be selectively removed was 50-70 micrometers. Survival rate with a low number of 3T3 mouse fibroblasts and NE-4C neuroectodermal mouse stem cells was 66 ± 12% and 88 ± 16%, respectively. Purity of sorted cultures and rate of survival using NE-4C/NE-GFP-4C co-cultures were 95 ± 2% and 62 ± 7%, respectively. Hydrodynamic simulations confirmed the experimental sorting efficiency and a cell damage risk similar to that of normal FACS.

Measurement of immediate-early gene activation- c-fos and beyond.

Immediate-early genes (IEG) are powerful tools for identifying activated neurosecretory neurones and extended circuits that affect neuroendocrine functions. The generally acknowledged scenario is when cells became activated, IEGs expressed and IEG-encoded transcription factors affect target gene expression. However, there are several examples in which: (i) neuronal activation occurs without induction of IEGs; (ii) IEG induction is not related to challenge-induced neuropeptide expression; and (iii) markers of neuronal activation are not expressed in chronically activated neurones. In spite of these limitations, the use of c-Fos and other regulatory- or effector transcription factors as markers of neuronal activation will continue to be an extremely powerful technique. Recently-developed models, including transgenic mice expressing different marker genes under the regulation of IEG promoters, will help to monitor neuronal activity in vivo or ex vivo and to reveal connection between activated neurones. Furthermore, combinations between novel imaging techniques, such as magnetic resonance and IEG-based mapping strategies, will open new means with which to study functional activity in the neurosecretory systems.

Direct inhibitory effect of glucocorticoids on corticotrophin-releasing hormone gene expression in neurones of the paraventricular nucleus in rat hypothalamic organotypic cultures.

Corticotrophin-releasing hormone (CRH) in the parvocellular neurosecretory neurones of hypothalamic paraventricular nucleus governs neuroendocrine stress cascade and is the major target of the negative feedback effect of corticosteroids. To assess whether glucocorticoids exert their inhibitory effect on CRH expression directly on parvocellular neurones or indirectly through a complex neuronal circuit, we examined the effect of corticosterone (CORT) and dexamethasone (DEX) on CRH mRNA levels in slice explant cultures of the rat hypothalamus. Organotypic slice cultures were prepared from 6 days old rat pups and maintained in vitro for 14 days. CRH mRNA expression was measured by in situ hybridisation histochemistry. Under basal conditions, CRH mRNA expressing cells were exclusively revealed in the paraventricular region along the third ventricle. Inhibition of action potential spike activity by tetrodotoxin (TTX, 1 microm) reduced CRH mRNA signal in the organotypic cultures. CORT (500 nm) or DEX (50 nm) treatment for 24 h significantly inhibited CRH expression in the parvocellular neurones and this effect of corticosteroids was not affected following blockade of voltage dependent sodium channels by TTX. Forskolin-stimulated CRH mRNA levels in the paraventricular nucleus were also inhibited by CORT or DEX in the presence and in the absence of TTX. These studies identify paraventricular CRH neurones as direct target of corticosteroid feedback. Type II corticosteroid receptor agonists act directly on paraventricular neurones to inhibit basal and forskolin-induced CRH mRNA expression in explant cultures of the rat hypothalamus.

Central autonomic control of the bone marrow: multisynaptic tract tracing by recombinant pseudorabies virus.

Bone marrow is the primary place of hematopoiesis, where the development, survival and release of multipotent stem cells, progenitors, precursors and mature cells are under continuous humoral and neural control. Dense network of nerve fibers, containing various neurotransmitters is found in the bone marrow, however, the central neuronal circuit that regulates the activities of the bone marrow through these fibers remained unexplored. Transsynaptically connected neurons were mapped by virus-based transneuronal tracing technique using two isogenic, genetically engineered pseudorabies viruses, Bartha-DupGreen and Ba-DupLac expressing green fluorescent protein and beta-galactosidase, respectively. Bartha-DupGreen was injected into the femoral bone marrow of male rats and the progression of infection was followed 4-7 days post-inoculation. Virus-labeled cells were revealed in ganglia of the paravertebral chain and in the intermediolateral cell column of the lower thoracic spinal cord. Neurons were retrogradely labeled in the C1, A5, A7 catecholaminergic cell groups and several other nuclei of the ventrolateral and ventromedial medulla, the periaqueductal gray matter, the paraventricular and other hypothalamic nuclei, and in the insular and piriform cortex. Nerve transections and double-virus tracing from the bone marrow and the surrounding muscles were used to confirm the specific spreading of the virus. These results provide anatomical evidence for the CNS control of the bone marrow and identify putative brain areas, which are involved in autonomic regulation of the hematopoiesis, the release of progenitor cells, the blood supply and the immune cell function in the bone marrow.

GABAergic mechanisms constraining the activity of the hypothalamo-pituitary-adrenocortical axis.

There are two major inhibitory mechanisms that constrain the activity of the hypothalamo-pituitary-adrenocortical axis: the hormonal negative feedback and the neural inhibition including that posed by the GABAergic neurons. This chapter summarizes our recent morphologic and functional findings on the role of gamma-aminobutyric acid (GABA) in the transcriptional regulation of hypophyseotropic neuropeptide genes in the parvocellular neurosecretory cells of the hypothalamic paraventricular nucleus (PVH). We used organotypic hypothalamic slice cultures and in vivo microinjection protocols in combination with in situ histologic and ultrastructural procedures to address the role of local interneurons in the regulation of hypothalamic effector neurons. Under basal conditions, an intrinsic GABAergic mechanism in the PVH microenvironment was revealed that by itself, without limbic contribution, impinged a tonic inhibitory influence on the parvocellular corticotropin-releasing hormone (CRH) neurons in vitro. In vivo, remote inputs were superimposed on the local circuit, allowing differential transcriptional regulation of CRH and arginine vasopressin (AVP) genes in the hypophyseotropic neurons. During stress, GABAergic cells that are known to project to the PVH become activated and are involved in restraining the cellular, transcriptional, and hormonal responses to stress.

Functional heterogeneity of the responses of histaminergic neuron subpopulations to various stress challenges.

In rats, the cell bodies of the histaminergic neuronal system are clustered in five distinct cell groups (E1-E5) within the posterior hypothalamus. On the basis of tract tracing studies, these histaminergic subgroups have been regarded as one functional unit. In addition to its well-characterized role in arousal, locomotor activity, metabolism, feeding, drinking and behaviour, as well as in coordination of autonomic functions, histamine has been implicated in regulation of the hypothalamo-pituitary-adrenocortical axis during stress. To address the capacity of different histaminergic subgroups to respond to various challenges, we revealed c-Fos, the immediate early gene marker of activated neurons, in histamine synthesizing neurons by combining c-Fos immunocytochemistry with in situ hybridization of histidine decarboxylase (HDC) mRNA. Compared to the negligible colocalization of these markers in control rats, restraint, insulin-induced hypoglycaemia and foot shock resulted in specific activation of histamine synthesizing neurons of the E4 and E5 subgroup in the tuberomammillary region. Up to 36% of HDC mRNA-expressing cells show c-Fos immunoreactivity in the E5 region. In addition, some neurons of the E1, E2 and E3 histaminergic groups were activated after restraint stress. Many less c-Fos-positive histaminergic neurons were detected after immobilization and dehydration. Ether stress, acute hyperosmotic stimulus or injection of bacterial lipopolysaccharide did not activate hypothalamic HDC-positive neurons. These results suggest, for the first time, the functional heterogeneity of histaminergic neuron population, the components of which are recruited in a stressor- and subgroup-specific manner.

Role of hypothalamic inputs in maintaining pituitary-adrenal responsiveness in repeated restraint.

The role of hypothalamic structures in the regulation of chronic stress responses was studied by lesioning the mediobasal hypothalamus or the paraventricular nucleus of hypothalamus (PVH). Rats were acutely (60 min) and/or repeatedly (for 7 days) restrained. In controls, a single restraint elevated the plasma adrenocorticotropin (ACTH), corticosterone, and prolactin levels. Repeated restraint produced all signs of chronic stress, including decreased body and thymus weights, increased adrenal weight, basal corticosterone levels, and proopiomelanocortin (POMC) mRNA expression in the anterior pituitary. Some adaptation to repeated restraint of the ACTH response, but not of other hormonal responses, was seen. Lesioning of the mediobasal hypothalamus abolished the hormonal response and POMC mRNA activation to acute and/or repeated restraint, suggesting that the hypothalamo-pituitary-adrenal axis activation during repeated restraint is centrally driven. PVH lesion inhibited the ACTH and corticosterone rise to the first restraint by approximately 50%. In repeatedly restrained rats with PVH lesion, the ACTH response to the last restraint was reduced almost to basal control levels, and the elevation of POMC mRNA level was prevented. PVH seems to be important for the repeated restraint-induced ACTH and POMC mRNA stimulation, but it appears to partially mediate other restraint-induced hormonal changes.

GABAergic innervation of corticotropin-releasing hormone (CRH)-secreting parvocellular neurons and its plasticity as demonstrated by quantitative immunoelectron microscopy.

GABA has been identified as an important neurotransmitter in stress-related circuitry mediating inhibitory effects on neurosecretory neurons that comprise the central limb of the hypothalamo-pituitary-adrenocortical axis. Using combinations of pre-embedding immunostaining and postembedding immunogold methods at the ultrastructural level, direct synaptic contacts were revealed between GABA-containing terminals and neurosecretory cells that were immunoreactive for corticotropin-releasing hormone (CRH) in the hypothalamic paraventricular nucleus (PVN). The vast majority of axo-dendritic GABA synapses was symmetric (inhibitory) type, and 46% of all synaptic boutons in the medial parvocellular subdivision of the PVN were immunoreactive to GABA. Using the disector method, an unbiased stereological method on serial ultrathin sections, the total calculated number of synaptic contacts within the medial parvocellular subdivision of the PVN was 55.4 x 10(6)/mm(3). On CRH-positive profiles 20.1 x 10(6) GABAergic synaptic boutons were detected per mm(3) in control, colchicine-treated rats. In the medial parvocellular subdivision, 79% of GABAergic boutons terminated on CRH neurons. Following adrenalectomy, which increases the synthetic and secretory activities of CRH neurons, the number of GABAergic synapses that terminate on CRH-positive profiles was increased by 55%. GABA-containing boutons appeared to be swollen, while the contact surfaces of cellular membranes between GABAergic boutons and CRH-positive profiles were shorter in adrenalectomized animals than in controls. Our data provide ultrastructural evidence for direct inhibitory GABAergic control of stress-related CRH neurons and suggest a pivotal role of GABA-containing inputs in the functional plasticity of parvocellular neurosecretory neurons seen in response to adrenalectomy.

Neuronal nitric oxide synthase (nNOS) mRNA is down-regulated, and constitutive NOS enzymatic activity decreased, in thoracic dorsal root ganglia and spinal cord of the rat by a substance P N-terminal metabolite.

Nitric oxide (NO) in the spinal cord plays a role in sensory and autonomic activity. Pain induced by acetic acid in the abdominal stretch (writhing) assay and hyperalgesia associated with chronic pain are highly sensitive to NO synthase (NOS) inhibitors. Because substance P (SP) is released and up-regulated in some models of chronic pain, we hypothesized that an accumulation of SP metabolites may influence NOS expression and activity. To test this hypothesis, we examined the effect of intrathecally (i.t.) injected substance P (1-7) [SP(1-7)], the major metabolite of SP in the rat, on neuronal NOS (nNOS) mRNA in the thoracic and lumbar spinal cord, dorsal root ganglia (DRG) and on the corresponding constitutive NOS (cNOS) enzyme activity. Detected using quantitative RT-PCR, nNOS mRNA content in the thoracic spinal cord was decreased 6 h after injection of 5 micromol of SP(1-7) and returned to control 2 days later. In thoracic DRG, nNOS mRNA was reduced 48 h after SP(1-7). The cNOS enzymatic activity in thoracic spinal tissue was gradually decreased to a minimum at 72 h. Down-regulation of NOS by SP(1-7) in the thoracic area appears to be highly associated with capsaicin-sensitive primary afferent neurons. No similar changes in either parameter were measured in the lumbar area after SP(1-7). These data suggest that N-terminal SP fragments, which are known to cause long-term antinociception in the writhing assay, may do so by their ability to down-regulate NO synthesis along nociceptive pathways.

Nociceptive aspects of fibromyalgia.

Although characterized by a variety of symptoms, chronic widespread pain is the primary complaint bringing most patients with fibromyalgia syndrome (FMS) into the clinic. The etiology of this painful condition is unknown, and any possible relationship between pain and the many other symptoms of FMS is unclear. This article focuses on the unique characteristics of nociception in patients with FMS. The intent is to present criteria that should be considered in the search for biological events that contribute to FMS pain. Based on this approach, examples are proposed of factors that fulfill some criteria and may, therefore, deserve further study for their possible role in pain associated with FMS.

Double activity imaging reveals distinct cellular targets of haloperidol, clozapine and dopamine D(3) receptor selective RGH-1756.

Acute administration of typical (haloperidol) and atypical (clozapine) antipsychotics results in distinct and overlapping regions of immediate-early gene expression in the rat brain. RGH-1756 is a recently developed atypical antipsychotic with high affinity to dopamine D(3) receptors that results in a unique pattern of c-Fos induction. A single injection of either antipsychotic results in c-fos mRNA expression that peaks around 30 min after drug administration, while the maximum of c-Fos protein induction is seen 2 h after challenge. The transient and distinct temporal inducibility of c-fos mRNA and c-Fos protein was exploited to reveal and compare cellular targets of different antipsychotic drugs by concomitant localization of c-fos mRNA and c-Fos immunoreactivity in brain sections of rats that were timely challenged with two different antipsychotics. Double activity imaging revealed that haloperidol, clozapine and RGH-1756 share cellular targets in the nucleus accumbens, where 40% of all labeled neurons displayed both c-fos mRNA and c-Fos protein. Haloperidol activates cells in the caudate putamen, while clozapine-responsive, single labeled neurons were dominant in the prefrontal cortex and major island of Calleja. RGH-1756 targets haloperidol-sensitive cells in the caudate putamen, but cells that are activated by clozapine and RGH-1756 in the major island of Calleja are different.

Intrathecal Zn2+ attenuates morphine antinociception and the development of acute tolerance.

Vesicular Zn2+, released in the brain and from small dorsal root ganglion neurons, interacts with opioid as well as N-methyl-D-aspartate (NMDA) receptors. We investigated the effect of Zn2+ on morphine antinociception in mice (tail flick assay), as well as acute tolerance and dependence, phenomena associated with NMDA activity. Administered intrathecally (i.t.), Zn2+ inhibited morphine antinociception in a dose-related fashion. Zn2+ also inhibited acute tolerance to morphine antinociception (5 h after 100 mg/kg of morphine). Injection i.t. of di-sodium calcium ethylenediamine tetra acetic acid (Na+Ca2+ EDTA), a chelator of divalent cations, had no effect on analgesia, acute tolerance or acute dependence. However, withdrawal jumps produced by naloxone (1 mg/kg s.c.) in morphine-pellet implanted mice (3 days) were potentiated by injections twice daily of 10 nmol of Na+Ca2+ EDTA, suggesting that endogenous Zn2+ tends to inhibit long-term development of withdrawal. These data suggest that the availability of Zn2+ is an important factor in opioid activity.

Anaphylactoid reactions activate hypothalamo-pituitary-adrenocortical axis: comparison with endotoxic reactions.

Infectious and allergic diseases represent distinct aspects of immune response that can be experimentally modeled as endotoxic reactions following bacterial lipopolysaccharide (LPS) administration and anaphylactoid reactions following systemic injection of foreign proteins, respectively. Although it is well established that LPS stimulates the activity of the hypothalamo-pituitary-adrenocortical (HPA) axis, such effects of anaphylactoid reactions are completely unknown. To evaluate the impact of anaphylactoid reactions on HPA regulation, secretion of adrenocorticotropin hormone (ACTH) was followed and the pattern of c-Fos induction in the hypothalamic paraventricular nucleus (PVN) was revealed in rats that were challenged with egg white or compound 48/80. Male rats were intravenously injected with 0.1 ml/100g b.wt. 1:1 diluted egg white or 50 microg/100 g b.wt. compound 48/80, blood samples were taken before and various time intervals between 15-240 min after challenge for plasma ACTH measurement. Anaphylactoid reactions resulted in a rapid, significant activation of ACTH secretion and induced c-Fos immunoreactivity in the corticotropin-releasing hormone (CRH)-secreting subset of the parvocellular neurosecretory neurons. In addition, magnocellular neurosecretory neurons and autonomic-related projection neurons in the PVN became also c-Fos positive upon challenge. Changes in these parameters are compared to those seen in rats challenged with bacterial endotoxin, LPS.

Postnatal handling alters the activation of stress-related neuronal circuitries.

Postnatal handling, as a crucial early life experience, plays an essential role in the development of hypothalamo-pituitary-adrenal axis responses to stress. The impact of postnatal handling on the reactivity of stress-related neuronal circuitries was investigated in animals that were handled for the first 21 days of life and as adults they were exposed to physical (ether) or emotional (restraint) challenge. To assess neuronal activation we relied on the induction of immediate-early gene product c-Fos and analysed its spatial and temporal distribution at various time intervals after stress. Ether and restraint commonly activated parvocellular neurons in the hypothalamic paraventricular nucleus, and resulted in activation of brain areas providing stress-related information to the hypothalamic effector neurons and/or in regions governing autonomic and behavioural responses to stress. Beyond these areas, the strength and timing of c-Fos induction showed stressor specificity in olfactory and septal region, basal ganglia, hypothalamus, hippocampal formation, amygdala and brainstem. Handled rats displayed a lower number of c-Fos-positive cell nuclei and weaker staining intensity than non-handled controls in the hypothalamic paraventricular nucleus, bed nucleus of stria terminalis, central nucleus of amygdala, hippocampus, piriform cortex and posterior division of the cingulum. Significant differences were revealed in timing of c-Fos induction as a function of stressor and early life experience. Together, these data provide functional anatomical evidence that environmental enrichment in the early postnatal period attenuates the reactivity of stress-related neuronal circuitries in the adult rat brain.

Photostimulation affects gonadotropin-releasing hormone immunoreactivity and activates a distinct neuron population in the hypothalamus of the mallard.

To reveal central mechanisms that transduce photic stimuli to sexually related neuroendocrine changes, Fra-2-ir, an inducible immediate-early gene marker of neuronal activation has been consecutively localized with that of GnRH-I in the brain of mallards that were triggered by artificial light at the photosensitive phase of the reproductive cycle. Strong neuronal activation was found in the POM and PVN in response to 1x or 4x 20 h light exposure that was accompanied with an increase of GnRH-ir in the hypothalamus and a dramatic depletion of GnRH-ir from terminals in the median eminence. The Fra-2 and GnRH-ir profiles, however, were not co-localized in any region at any phase of photostimulation. These results demonstrate profound changes of GnRH-ir in the hypothalamus and reveal a distinct, photoresponsive cell population in the anterior hypothalamic area of the mallard.

Glucocorticoid negative feedback selectively targets vasopressin transcription in parvocellular neurosecretory neurons.

To identify molecular targets of corticosteroid negative feedback effects on neurosecretory neurons comprising the central limb of the hypothalamo-pituitary-adrenal (HPA) axis, we monitored ether stress effects on corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) heteronuclear RNA (hnRNA) expression in rats that were intact or adrenalectomized (ADX) and replaced with corticosterone (B) at constant levels ranging from nil to peak stress concentrations. Under basal conditions, relative levels of both primary transcripts varied inversely as a function of plasma B titers. In response to stress, the kinetics of CRF hnRNA responses of intact and ADX rats replaced with low B were similar, peaking at 5 min after stress. By contrast, intact rats showed a delayed AVP hnRNA response (peak at 2 hr), the timing of which was markedly advanced in ADX/low B-replaced animals (peak at 5-30 min). Transcription factors implicated in these responses responded similarly. Manipulation of B status did not affect the early (5-15 min) phosphorylation of transcription factor cAMP-response element-binding protein (CREB) but accelerated maximal Fos induction from 2 hr after stress (intact) to 1 hr (ADX). Assays of binding by proteins in hypothalamic extracts of similarly manipulated rats toward consensus CRE and AP-1 response elements supported a role for the stress-induced plasma B increment in antagonizing AP-1, but not CRE, binding. These findings suggest that glucocorticoid negative feedback at the transcriptional levels is exerted selectively on AVP gene expression through a mechanism that likely involves glucocorticoid receptor interactions with immediate-early gene products.

Transient changes in the synthesis of nitric oxide result in long-term as well as short-term changes in acetic acid-induced writhing in mice.

A single injection of nitric oxide (NO) synthase (NOS) inhibitors prevents the development of persistent hyperalgesia induced by various manipulations, suggesting that NO precipitates long-term changes in nociception. We examined the possibility that inhibition of NOS may also be sufficient to produce long-term decreases in nociceptive assays, such as writhing, that are known to be sensitive to the short-term effects of NOS inhibitors. We characterized short- and long-term effects of NOS inhibitors, N(omega)-nitro-L-arginine (L-NAME) or 7-nitro indazole (7-NI) injected intrathecally (i.t.) in mice on acetic acid-induced writhing. Doses of L-NAME that had no effect on hot plate or tail flick latencies inhibited writhing (0. 01-30 nmol) as well as spinal nNOS activity (5 and 100 nmol) when injected i.t. 60-90 min before testing. Anti-nociception was not mimicked by D-NAME but was prevented by co-administration with the NO precursor, L-arginine. Injection i.t. of 7-NI (30 min), a selective inhibitor of neuronal NOS (nNOS), inhibited NOS activity in the spinal cord and produced anti-nociception, confirming that writhing is sensitive to inhibition of nNOS. Although the acute action of both NOS inhibitors dissipated completely by 3-6 h, a delayed and prolonged inhibition of writhing was again observed 24 h after L-NAME (5-100 nmol), a time when spinal NOS activity was no longer inhibited by L-NAME (5 and 100 nmol) or 7-NI (25 nmol). This novel effect appears to be initiated by the transient inhibition of nNOS as delayed anti-nociception was mimicked by 7-NI at doses (10-100 nmol) that no longer inhibited spinal nNOS (25 nmol) at 24 h. Co-administration with L-arginine prevented the delayed (24 h) anti-nociceptive effects of L-NAME (30 nmol). L-Arginine (30 and 100 nmol) was without effect on nociception when administered alone 60 min or 24 h prior to testing. Together these data indicate that brief changes in the activity of nNOS induce both long- as well as short-term changes in nociception.