Light and electron microscopic studies of pituitary adenylate cyclase-activating peptide (PACAP) – immunoreactive neurons in the enteric nervous system of rat small and large intestine

 Pituitary adenylate cyclase-activating peptide (PACAP)-immunoreactive (IR) neurons in the myenteric and submucosal plexus of the rat small and large intestine were examined by immunostaining with purified polyclonal antiserum against PACAP (1–15), using both light and electron microscopy. Many PACAP-IR neuronal cell bodies and fibers were found in the myenteric and submucosal plexus. Many of the PACAP-IR fibers originated from the cell bodies of the myenteric and submucosal ganglia. The ganglia were also innervated by PACAP-IR fibers. PACAP-IR fibers penetrated both the circular and longitudinal muscle layers, confirming the previous observations indicating that PACAP neurons act as motor neurons. Ultrastructural study demonstrated that PACAP-IR nerve terminals formed synaptic contacts with PACAP-IR nerve cell bodies or dendritic processes. This observation suggests that PACAP-IR neurons innervate other PACAP-IR neurons, and that PACAP neurons work as interneurons in the enteric nervous system. PACAP-IR nerve cells received not only PACAP-positive nerve terminal input also PACAP-negative nerve terminal input. It also suggests that PACAP neurons are regulated not only by PACAP-IR enteric neurons, but also by neurons originating elsewhere. Our observations support the view that PACAP-IR neurons are involved in the control of gut motility. Accepted: 20 April 1998     

Pituitary adenylate cyclase activating peptide (PACAP) in the enteropancreatic innervation

Pancreatic ganglia are innervated by neurons in the gut and are formed by precursor cells that migrate into the pancreas from the bowel. The innervation of the pancreas, therefore, may be considered an extension of the enteric nervous system. Pituitary adenylate cyclase-activating polypeptide (PACAP) is present in a subset of enteric neurons. We investigated the presence of PACAP in the enteropancreatic innervation in guinea pigs, and the response of pancreatic neurons to PACAP-related peptides. PACAP immunoreactivity was found in nerve fibers in both enteric and pancreatic ganglia and in nerve bundles that travelled between the duodenum and pancreas. PACAP-immunoreactive nerve fibers were densely distributed in the pancreatic ganglia, where they surrounded a subset of cholinergic cell bodies. Pancreatic ganglia did not contain PACAP-immunoreactive cell bodies; however, neuronal perikarya with PACAP immunoreactivity were found in the myenteric plexus of the duodenum. These cells co-stored vasoactive intestinal peptide (VIP). PACAP depolarized pancreatic neurons. Pancreatic neurons were also depolarized by VIP; however, PACAP was more efficacious at depolarizing pancreatic cells than VIP. These findings are consistent with the view that the PACAP effects were mediated through PACAP-selective (PAC1) receptors. PACAP-responsive neurons displayed PAC1 receptor immunoreactivity, which was also found in islet cells and enteric neurons. These results provide support for the hypothesis that PACAP modulates reflex activity between the gut and pancreas. The excitatory effect of PACAP would be expected to potentiate pancreatic secretion.     

Pituitary adenylatecyclase-activating polypeptide-immunoreactive nerve fibers in the rat epiglottis and pharynx

The distribution of pituitary adenylatecyclase-activating polypeptide-immunoreactive (PACAP-IR) nerve fibers was studied in the rat epiglottis and pharynx. PACAP-IR nerve fibers were located beneath the mucous epithelium, and occasionally penetrated the epithelium. These nerve fibers were abundant on the laryngeal side of the epiglottis and in the dorsal and lateral border region between naso-oral and laryngeal parts of the pharynx. PACAP-IR nerve fibers were also detected in taste buds within the epiglottis and pharynx. In addition, many PACAP-IR nerve fibers were found around acinar cells and blood vessels. The double immunofluorescence method demonstrated that distribution of PACAP-IR nerve fibers was similar to that in CGRP-IR nerve fibers in the epithelium and taste bud. However, distributions of PACAP-IR and CGRP-IR nerve fibers innervating mucous glands and blood vessels were different. The retrograde tracing method also demonstrated that PACAP and CGRP were co-expressed by vagal and glossopharyngeal sensory neurons innervating the pharynx. These findings suggest that PACAP-IR nerve fibers in the epithelium and taste bud of the epiglottis and pharynx which originate from the vagal and glossopharyngeal sensory ganglia include nociceptors and chemoreceptors. The origin of PACAP-IR nerve fibers which innervate mucous glands and blood vessels may be the autonomic ganglion.     

The presence and distribution of pituitary adenylate cyclase activating polypeptide and its receptor in the snail Helix pomatia

The aim of this study was to show the presence, distribution and function of the pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors in the CNS and peripheral nervous system of the mollusk, Helix pomatia. PACAP-like and pituitary adenylate cyclase activating polypeptide receptor (PAC1-R)-like immunoreactivity was abundant both in the CNS and the peripheral nervous system of the snail. In addition several non-neuronal cells also revealed PACAP-like immunoreactivity. In inactive animals labeled cell bodies were mainly found and in the neuropile of active animals dense immunostained fiber system was additionally detected suggesting that expression of PACAP-like peptide was affected by the behavioral state of the animal. RIA measurements revealed the existence of both forms of PACAP in the CNS where the 27 amino acid form was found to be dominant. The concentration of PACAP27 was significantly higher in samples from active animals supporting the data obtained by immunohistochemistry. In Western blot experiments PACAP27 and PACAP38 antibodies specifically labeled protein band at 4.5 kDa both in rat and snail brain homogenates, and additionally an approximately 14 kDa band in snail. The 4.5 kDa protein corresponds to PACAP38 and the 14 kDa protein corresponds to the preproPACAP or to a PACAP-like peptide having larger molecular weight than mammalian PACAP38. In matrix-assisted laser desorption ionization time of flight (MALDI TOF) measurements fragments of PACAP38 were identified in brain samples suggesting the presence of a large molecular weight peptide in the snail. Applying antibodies developed against the PACAP receptor PAC1-R, immunopositive stained neurons and a dense network of fibers were identified in each of the ganglia. In electrophysiological experiments, extracellular application of PACAP27 and PACAP38 transiently depolarized or increased postsynaptic activity of neurons expressing PAC1-R. In several neurons PACAP elicited a long lasting hyperpolarization which was eliminated after 1.5 h continuous washing. Taken together, these results indicate that PACAP may have significant role in a wide range of basic physiological functions in snail.     

心肌缺血大鼠心内神经节神经生长因子表达的变化

目的观察神经生长因子在急性心肌缺血大鼠心内神经节表达及其变化,探讨神经生长因子与心肌缺血的关系。方法采用免疫组织化学方法动态观察了正常大鼠以及大鼠冠状动脉结扎后1、6、12、24h心内神经节神经生长因子的表达及变化。结果各组大鼠的心内神经节均存在神经生长因子阳性神经元,并且神经生长因子阳性神经元的数目在冠状动脉结扎后1、6、12、24h后增多,表达也增强。结论心肌缺血大鼠心内神经节神经生长因子表达持续显著升高,提示神经生长因子可能参与心肌缺血的病变过程。     

The expression of B-50/GAP-43 during development of rat spinal neurons in culture is regulated by interneuronal contact

The neuron-specific phosphoprotein B-50 (GAP-43) is associated with neuritogenesis during development and regeneration. We monitored B-50 in cultured spinal neurons (fetal rat) with an enzyme-linked immunoadsorbent assay. B-50 levels increased from 24 to 72 h, then decreased. Other cultures, fixed at 24 h intervals and incubated with anti-B-50 immunoglobulins and fluorescent conjugates, showed that B-50 was present in somata after 24 h, but mainly in neurites after 48 h; after 72-96 h neurons migrated into clusters and B-50 was detected only in free neurites at the perimeter of the culture. We conclude that B-50 expression is down-regulated by neurite-cell or cell-cell contact.     

PACAP up-regulates the expression of apolipoprotein D in 3T3-L1 adipocytes. DRG/3T3-L1 co-cultures study

The existence of a cross-talk between nerves and fatty tissue is increasingly recognized. Using co-cultures of dorsal root ganglion (DRG)-derived cells and 3T3-L1 adipocytes, we have previously shown that the presence of fat cells enhances neurite outgrowth and number of synapses. Vice versa, neural cells induced expression of neurotrophic adipokines apolipoprotein D and E (ApoD, ApoE) and angiopoietin-1 (Ang-1) by adipocytes. Here, we tested whether pituitary adenylate cyclase-activating peptide (PACAP), which is released by sensory fibres and causes Ca(2+) influx into fat cells, is involved in ApoD induction. Using 3T3-L1 cell cultures, we found that PACAP at a dose of 1 nM up-regulated the expression of ApoD protein and mRNA approx. 2.5 fold. This effect was driven by ERK1/2 acting upon PAC1/VPAC2 receptors. In turn, PACAP-treated 3T3-L1 adipocytes in co-cultures with DRG cells enhanced neurite ramification of neurofilament 200 (NF200)-positive neurons (measured using fluorescence microscopy) and neurofilament 68 protein levels (measured using Western blot analysis). This effect could be blocked using the PAC1/VPAC2 antagonist PACAP(6-38). Scanning cytometry revealed PACAP/ApoD induced low density lipoprotein receptors (LDLR) and ApoE receptor 2 (apoER2) in NF200-positive cells. Thus, a bidirectional loop seems to exist regulating the innervation of fatty tissues: PACAP released from sensory fibres might stimulate fat cells to synthesize neurotrophic adipokines, which, in turn, support peripheral innervation.     

神经生长因子对谷氨酸介导的基底核神经元损伤的保护作用

本研究的目的是观察神经生长因子(nerve growth factor,NGF)对谷氨酸诱导的基底核神经元损伤的保护作用。取出生后1d乳鼠前脑基底核神经元培养,随机分为正常对照组、模型组(谷氨酸损伤组)和NGF保护组。用倒置相差显微镜进行活细胞观察,采用RT-PCR技术检测前脑基底核神经元的神经生长相关蛋白-43(growth associated protein-43,GAP-43)的表达。结果显示谷氨酸损伤组神经元胞体回缩,突起消失或断裂。NGF保护组的神经元绝大多数胞体饱满,突起明显,细胞间的网络联系仍清晰可见,接近于正常对照组;NGF保护组神经元内GAP-43mRNA表达比谷氨酸损伤组高,两者比较有统计学意义(P<0.05)。结果提示,NGF能保护基底核神经元免受谷氨酸兴奋性毒性的损伤。     

Developmental changes in neurite outgrowth responses of dorsal root and sympathetic ganglia to GDNF, neurturin, and artemin.

The ability of glial cell line-derived neurotrophic factor (GDNF), neurturin, and artemin to induce neurite outgrowth from dorsal root, superior cervical, and lumbar sympathetic ganglia from mice at a variety of development stages between embryonic day (E) 11.5 and postnatal day (P) 7 was examined by explanting ganglia onto collagen gels and growing them in the presence of agarose beads impregnated with the different GDNF family ligands. Artemin, GDNF, and neurturin were all capable of influencing neurite outgrowth from dorsal root and sympathetic ganglia, but the responses of each neuron type to the different ligands varied during development. Neurites from dorsal root ganglia responded to artemin at P0 and P7, to GDNF at E15.5 and P0, and to neurturin at E15.5, P0, and P6/7; thus, artemin, GDNF, and neurturin are all capable of influencing neurite outgrowth from dorsal root ganglion neurons. Neurites from superior cervical sympathetic ganglia responded significantly to artemin at E15.5, to GDNF at E15.5 and P0, and to neurturin at E15.5. Neurites from lumbar sympathetic ganglia responded to artemin at all stages from E11.5 to P7, to GDNF at P0 and P7 and to neurturin at E11.5 to P6/7. Combined with the data from previous studies that have examined the expression of GDNF family members, our data suggest that artemin plays a role in inducing neurite outgrowth from young sympathetic neurons in the early stages of sympathetic axon pathfinding, whereas GDNF and neurturin are likely to be important at later stages of sympathetic neuron development in inducing axons to enter particular target tissues once they are in the vicinity or to induce branching within target tissues. Superior cervical and lumbar sympathetic ganglia showed temporal differences in their responsiveness to artemin, GDNF, and neurturin, which probably partly reflects the rostrocaudal development of sympathetic ganglia and the tissues they innervate.     

Staurosporine-induced apoptosis in cultured chick embryonic neurons is reduced by polyethylenimine of low molecular weight used as a coating substrate

The survival of neurons largely depends on adhesion to extracellular matrix proteins. This study investigated the influence of polycationic macromolecules of different molecular weights used as coating substrates on apoptosis in primary cultures of chick embryonic neurons. Coating of the culture flasks with positively charged polyethylenimine (PEI) of 12, 32 and 1616 kDa led to different susceptibilities of the neurons to apoptosis induced by staurosporine and serum deprivation. In cultures grown as usual on polylysine (PL)-coated flasks, we found, after 24 h of incubation in medium with serum (controls), serum-free medium and staurosporine (200 nM)-containing serum-free medium, 15, 35 and 63% apoptotic neurons, respectively as evaluated by nuclear staining with Hoechst 33258. Using 12-kDa PEI as a coating substrate, only 11, 15 and 47% apoptotic neurons could be determined in controls, serum-deprived and staurosporine-treated cultures, respectively. No change in the percentage of apoptotic neurons was found after 24 h of serum deprivation or treatment with staurosporine in cultures grown on 32-kDa PEI compared with cultures grown on PL. However, in staurosporine-treated cultures grown on 1616-kDa PEI, the percentage of apoptotic neurons was even higher than in cultures grown on PL. Immunostaining using neurofilament (NF) antibodies revealed that the reduction of staurosporine-induced apoptosis using 12-kDa PEI instead of PL as a coating substrate was accompanied by a reduced disruption and aggregation of the neurofilaments. Thus, the usage of the newly synthesized 12-kDa PEI as a coating substrate enhanced neuronal resistance to apoptosis.     

Pituitary adenylate cyclase-activating polypeptide directly modulates the activity of proopiomelanocortin neurons in the rat arcuate nucleus

Pituitary adenylate cyclase-activating polypeptide (PACAP) and the proopiomelanocortin (POMC)-derived peptide alpha-melanocyte-stimulating hormone (alpha-MSH) both regulate multiple neuroendocrine functions and feeding behavior. Two subtypes of PACAP receptor mRNAs, pituitary adenylate cyclase-activating polypeptide-specific receptor (PAC1-R) and pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide mutual receptor (VPAC2-R), are actively expressed in the arcuate nucleus of the hypothalamus, where POMC cell bodies are located. This observation led us to investigate the possible regulatory action of PACAP on rat POMC neurons. Double-labeling in situ hybridization histochemistry revealed that approximately 50% of POMC-producing neurons express PAC1-R and/or VPAC2-R mRNAs. The proportion of POMC neurons that also contain PAC1-R mRNA was homogeneous along the rostro-caudal axis of the arcuate nucleus while POMC-positive cell bodies expressing the VPAC2-R subtype were more abundant in the rostral region. Incubation of mediobasal hypothalamic explants with PACAP (10(-7) M; 30 min) increased POMC mRNA expression, and this effect was blocked by PACAP6-38 (10(-6) M). In contrast, incubation with vasoactive intestinal polypeptide (10(-7) M) did not affect POMC mRNA level. Incubation of hypothalamic fragments with PACAP (10(-7) M) caused a significant increase in alpha-MSH content in the tissue and in the incubation medium. Altogether, the present results reveal that exogenous PACAP, acting probably through PAC1-R, regulates the activity of POMC neurons in the rat hypothalamus. These data suggest that the effects of PACAP on the gonadotropin-releasing hormone neuroendocrine axis and the regulation of feeding behavior may be mediated, at least in part, through modulation of POMC neurons.     

Neural crest-derived proprioceptive neurons express nerve growth factor receptors but are not supported by nerve growth factor in culture

The neural crest-derived, first-order, sensory neurons of the embryonic chick trigeminal mesencephalic nucleus were grown in dissociated, glia-free culture. Whereas brain-derived neurotrophic factor promoted the survival and growth of the majority of these neurons (over 70% after 48 h incubation), nerve growth factor had no effect on their survival. The percentage survival in cultures supplemented with nerve growth factor at concentrations ranging from 0.2 to 625 ng/ml was only 2%, the same percentage survival as in control cultures. Furthermore, nerve growth factor did not change the dose-response of these neurons to brain-derived neurotrophic factor. Although nerve growth factor did not influence the survival of trigeminal mesencephalic neurons in culture, nerve growth factor specifically bound to the great majority of neurons growing in the presence of brain-derived neurotrophic factor. Autoradiographs of cultures incubated with iodinated nerve growth factor showed that the perikarya and processes of neurons were heavily labelled with silver grains. These findings demonstrate the existence of a population of neural crest-derived sensory neurons which express nerve growth factor receptors but are not supported by nerve growth factor in culture.     

Spinal cord contains neurotrophic activity for spinal nerve sensory neurons. Late developmental appearance of a survival factor distinct from nerve growth factor

Several tissues of the developing chick embryo have been reported to contain neurotrophic activity which can sustain the survival of sensory neurons maintained in culture. In a previous study, however, we noted that such nerve growth promoting activity was exceptionally low, if not absent, from extracts of spinal cord from chick embryos of up to 16 days incubation. Since then the combined results from a number of tissue culture studies have suggested that the central nervous system may be the source of a neurotrophic growth factor essential during the late development of sensory neurons. We have therefore carried out an extended range study of the neurotrophic properties of avian spinal cord. Extracts of spinal cord tissue prepared from chicks at stages between the last wk of embryogenesis and 12 wks after hatching were tested for their ability to promote survival and neurite outgrowth from both explant and dissociated neuron-enriched cultures of dorsal root, trigeminal, nodose and paravertebral chain sympathetic ganglia from chick embryos between 8 and 16 days old. We conclude from our results that spinal cord is a potent source of neurotrophic activity for sensory neurons, although this activity appears relatively late in development of the spinal cord. The predominant ontogenic increase in spinal cord neurotrophic activity was seen to occur during the first week after hatching. Sensory neurons from both spinal and cranial nerve ganglia were sustained in culture by spinal cord extracts, whereas sympathetic neurons did not respond. Neurons from older sensory ganglia (12-16 day old embryos) were much more responsive than similar neurons from young embryos (8 day).(ABSTRACT TRUNCATED AT 250 WORDS)     

Injured primary sensory neurons switch phenotype for brain-derived neurotrophic factor in the rat.

Peripheral nerve injury results in plastic changes in the dorsal root ganglia and spinal cord, and is often complicated with neuropathic pain. The mechanisms underlying these changes are not known. We have now investigated the expression of brain-derived neurotrophic factor in the dorsal root ganglia with histochemical and biochemical methods following sciatic nerve lesion in the rat. The percentage of neurons immunoreactive for brain-derived neurotrophic factor in the ipsilateral dorsal root ganglia was significantly increased as early as 24 h after the nerve lesion and the increase lasted for at least two weeks. The level of brain-derived neurotrophic factor messenger RNA was also significantly increased in the ipsibut not contralateral dorsal root ganglia. Both neurons and satellite cells in the lesioned dorsal root ganglia synthesized brain-derived neurotrophic factor messenger RNA after the nerve lesion. There was a dramatic shift in size distribution of positive neurons towards large sizes seven days after sciatic nerve lesion. Morphometric analysis and retrograde tracing studies showed that no injured neurons smaller than 600 microm2 were immunoreactive for brain-derived neurotrophic factor, whereas the majority of large injured neurons were immunoreactive in the ipsilateral dorsal root ganglia seven days postlesion. The brain-derived neurotrophic factor-immunoreactive nerve terminals in the ipsilateral spinal cord were reduced in the central region of lamina II, but increased in more medial regions or deeper into laminae III/IV. These studies indicate that sciatic nerve injury results in a differential regulation of brain-derived neurotrophic factor in different subpopulations of sensory neurons in the dorsal root ganglia. Small neurons switched off their normal synthesis of brain-derived neurotrophic factor, whereas larger ones switched to a brain-derived neurotrophic factor phenotype. The phenotypic switch may have functional implications in neuronal plasticity and generation of neuropathic pain after nerve injury.     

Neurturin protects striatal projection neurons but not interneurons in a rat model of Huntington's disease

Glial cell line-derived neurotrophic factor and neurturin are neurotrophic factors expressed in the striatum during development and in the adult rat. Both molecules act as target-derived neurotrophic factors for nigrostriatal dopaminergic neurons. While glial cell line-derived neurotrophic factor has also been described to have local trophic effects on striatal neurons, the effects of neurturin in the striatum have not yet been described. Here we examine whether neurturin protects striatal projection neurons (calbindin-positive) and interneurons (parvalbumin- or choline acetyltransferase-positive) in an animal model of Huntington's disease. A fibroblast cell line engineered to over-express neurturin was grafted into adult rat striatum 24h before quinolinate injection. In animals grafted with a control cell line, intrastriatal quinolinate injection reduced the number of calbindin-, parvalbumin- and choline acetyltransferase-positive neurons, seven days post-lesion. Intrastriatal grafting of neurturin-secreting cells protected striatal projection neurons, but not interneurons, from quinolinate excitotoxicity. This effect was much more robust than that reported previously for a glial cell line-derived neurotrophic factor-secreting cell line on striatal calbindin-positive neurons. However, intrastriatal grafting of glial cell line-derived neurotrophic factor- but not neurturin-secreting cells prevented the decrease in choline acetyltransferase activity induced by quinolinate injection.Taken together, our results show that neurturin- and glial cell line-derived neurotrophic factor-secreting cell lines have clearly differential effects on striatal neurons. Grafting of the neurturin-secreting cell line showed a more specific and efficient trophic effect on striatal projection neurons, the neuronal population most affected in Huntington's disease. Therefore, our results suggest that neurturin is a good candidate for the treatment of this neurodegenerative disorder.     

In vitro and in vivo modulation of 5-hydroxytryptamine-, thyrotropin-releasing hormone- and calcitonin-gene related peptide-like immunoreactivities in adult rat sensory neurons.

In a previous work we have shown that culturing adult rat dorsal root ganglia neurons modifies their neurotransmitter phenotype in such a way that cultured neurons synthesize transmitters that are not found in situ, while several other transmitters are expressed in a much higher percentage of neurons in culture than in situ [Schoenen J. et al. (1989) J. Neurosci. Res. 22, 473-487]. The aim of the present study was to investigate the origin and the nature of the relevant environmental signals that allow this plasticity to be expressed, focusing on three neurotransmitters: 5-hydroxytryptamine, thyrotropin-releasing hormone and calcitonin-gene related peptide. The main results can be summarized as follows: (1) culturing cells in fetal calf serum or on feeder layers of astrocytes, Schwann cells or fibroblasts partially inhibits the serotoninergic phenotype of dorsal root ganglia neurons; (2) in vivo disconnection of dorsal root ganglia from their spinal targets but not from their peripheral or supraspinal targets induces a significant increase of the percentage of 5-hydroxytryptamine- and thyrotropin-releasing hormone-positive neurons in disconnected ganglia; (3) growth factors such as ciliary neuronotrophic factor or basic fibroblast growth factor but not nerve growth factor repress 5-hydroxytryptamine and calcitonin gene-related peptide immunoreactivity in cultured sensory neurons. In conclusion, neurotransmitter gene expression of adult dorsal root ganglia neurons is controlled by complex influences. Our data suggest that thyrotropin-releasing hormone and 5-hydroxytryptamine gene expression are tonically repressed in vivo by factors originating from the spinal segmental level and that growth factors such as ciliary neurotrophic factor or basic fibroblast growth factor could be potential vectors of this repressing effect.     

Increased expression, axonal transport and release of pituitary adenylate cyclase-activating polypeptide in the cultured rat vagus nerve

The expression and axonal transport of pituitary adenylate cyclase-activating polypeptide (PACAP) was studied in the cultured vagus nerve of the rat by immunocytochemistry and in situ hybridization. The number of neurons immunoreactive for PACAP increased markedly within the nodose ganglion during a 24-48 h culture period, as did the number of cells containing messenger RNA for PACAP. PACAP was found to be axonally transported and accumulated at the site of a crush injury. The peptide was also released at this site. Addition of PACAP to regenerating nerves in culture did not affect axonal outgrowth, neither did antibodies against PACAP. Separate experiments showed that neither PACAP-27 nor PACAP-38 affected proliferation of non-neuronal cells measured as the incorporation of [3H]thymidine. In contrast, forskolin, another potent stimulator of adenylate cyclase besides PACAP, dramatically decreased [3H]thymidine incorporation. The results showed that, during regeneration of peripheral nerves, PACAP expression increases and the peptide is transported into the regenerating nerve, where it is released. The functional significance of this release is unknown, but it does not seem to be directly related to the initiation of proliferation of Schwann cells or initial axonal outgrowth.