Differential effects of lysophospholipids on exocytosis in rat PC12 cells

Secretory phospholipase A₂ (sPLA₂) activity is present in the CNS and the sPLA₂-IIA isoform has been shown to induce exocytosis in cultured hippocampal neurons. However, little is known about possible contributions of various lysophospholipid species to exocytosis in neuroendocrine cells. This study was therefore carried out to examine the effects of several lysophospholipid species on exocytosis on rat pheochromocytoma-12 (PC12) cells. An increase in vesicle fusion, indicating exocytosis, was observed in PC12 cells after external infusion of lysophosphatidylinositol (LPI), but not lysophosphatidylcholine or lysophosphatidylserine by total internal reflection microscopy. Similarly, external infusion of LPI induced significant increases in capacitance, or number of spikes detected at amperometry, indicating exocytosis. Depletion of cholesterol by pre-incubation of cells with methyl beta cyclodextrin and depletion of Ca²⁺ by thapsigargin and incubation in zero external Ca²⁺ resulted in attenuation of LPI induced exocytosis, indicating that exocytosis was dependent on the integrity of lipid rafts and intracellular Ca²⁺. Moreover, LPI induced a rise in intracellular Ca²⁺ suggesting that this could be the trigger for exocytosis. It is postulated that LPI may be an active participant in sPLA₂-mediated exocytosis in the CNS.     

Phosphorylation and activation of tyrosine hydroxylase in PC18 cells: a cell line derived from rat pheochromocytoma PC12 cells.

Treatment of rat pheochromocytoma PC18 cells (a variant subclone of PC12 cells) with forskolin produced increased activity and phosphorylation of tyrosine hydroxylase. In contrast, treatment of the PC18 cells with 56 mM K+, A23187, phorbol-12-myristate-13-acetate (PMA) or phorbol-12,13-dibutyrate (PDB) did not affect the activity and only slightly increased the phosphorylation of tyrosine hydroxylase. None of the treatments except forskolin increased cyclic AMP levels in PC18 cells. Furthermore, 45Ca2+ uptake into PC18 cells was not affected by 56 mM K+, PDB or forskolin; however, A23187 increased 45Ca2+ uptake 4-fold over basal uptake. Nevertheless, no activation and little increase in phosphorylation of tyrosine hydroxylase was observed in PC18 cells treated with A23187. When tyrosine hydroxylase levels in PC18 cells were elevated by treatment with dexamethasone, activation of tyrosine hydroxylase by 56 mM K+, PDB or A23187 was still not observed. Both purified Ca2+/calmodulin-dependent protein kinase and cyclic AMP-dependent protein kinase catalyzed the phosphorylation of tyrosine hydroxylase purified from PC18 cells in vitro. Furthermore, crude cell extracts from PC12 cells and PC18 cells possessed Ca2+/calmodulin-dependent protein kinase activity that catalyzed the phosphorylation of purified tyrosine hydroxylase. These results suggest that tyrosine hydroxylase activity in PC18 cells is regulated by a cyclic AMP-dependent mechanism. However, due to a number of abnormalities the Ca(2+)-dependent mechanisms do not result in the activation of tyrosine hydroxylase and only slightly increase the phosphorylation of the enzyme in PC18 cells.     

Differential effect of membrane depolarization on levels of tyrosine hydroxylase and dopamine beta-hydroxylase mRNAs in PC12 pheochromocytoma cells

Membrane depolarization has been widely used to elucidate the response of the nervous system to prolonged neuronal activity or stress. We studied the effect of treating PC12 cells with membrane depolarizing stimuli, 50 mM KCl, or 150 microM veratridine, and the subsequent changes in the mRNA levels of the catecholamine biosynthetic enzymes, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). TH mRNA levels were found to increase 2- to 5-fold after continuous treatment for 1-12 h with 50 mM KCl. Depolarization with 150 microM veratridine had a similar effect on TH mRNA. In contrast, DBH mRNA levels were unchanged by either KCl or veratridine treatment. The role of calcium in the increase of TH mRNA levels elicited by depolarization was examined. The increase in TH mRNA was inhibited by the chelation of calcium with 3 mM EGTA. However, in contrast to their effect on phosphorylation of TH elicited by acute depolarization, the calcium channel blockers, nitrendipine and verapamil, and the calmodulin antagonists, W7 and trifluoperazine, did not prevent the increase in TH mRNA levels subsequent to several hours exposure to depolarizing stimuli. The calcium ionophore, A23187, alone was unable to induce TH mRNA levels. Thus, the increase in TH mRNA elicited by depolarization is mediated differently than the acute phosphorylation of the enzyme.     

Reciprocal regulation of the content of aromatic L-amino acid decarboxylase and tyrosine hydroxylase mRNA by NGF in PC12 cells

Aromatic L-amino acid decarboxylase (AADC) and tyrosine hydroxylase (TH) are involved in the synthesis of dopamine and other monoamine neurotransmitters, and their activities can be regulated by a number of physiological stimuli. An interesting finding has been that (−)-deprenyl and some other irreversible monoamine B oxidase inhibitors increase AADC gene expression and that these compounds can exert neuronal protection/rescue effects. In this study, we have investigated the effects of nerve growth factor (NGF), and of (−)-deprenyl on AADC and TH Gene expression in PC12 cells. Cells were treated with different doses of NGF (0.2–50 ng/ml) for up to 3 days. Northern and blot hybridizations were performed to detect AADC and TH mRNA. The results show that NGF can down-regulate AADC gene expression while increasing TH gene expression in a time- and dose-dependent fashion. Treatment with (−)-deprenyl alone increases the gene expression of AADC, and (−)-deprenyl further counteracts the reductions induced by NGF. This study introduces novel results with regard to the regulation of the gene expression of AADC in PC12 cells, which is not paralleled by the other catecholamine biosynthetic enzymes. These findings support the existence of an interaction between NGF and AADC gene expression that may be associated with the process of neuronal degeneration or regeneration. J. Neurosci. Res. 47:449–454, 1997. © 1997 Wiley-Liss, Inc.     

Differential time- and dose-related effects of haemorrhage on tyrosine hydroxylase and neuropeptide Y mRNA expression in medullary catecholamine neurons

Hypotensive haemorrhage induces nuclear Fos expression and upregulates tyrosine hydroxylase (TH) mRNA in catecholamine-containing cell groups of the rat medulla oblongata. To shed light on the significance of the coexistence of neuropeptide Y (NPY) in aminergic neurons, the impact of graded levels of haemorrhage on temporal changes in the expression of TH and NPY mRNAs was compared; concurrent staining for Fos permitted comparisons between cells that ostensibly were and were not targeted by the stimulus. A 15% haemorrhage provoked increased NPY expression in all medullary catecholamine cell groups except the A2; these changes were detected predominantly in Fos-immunoreactive neurons (Fos-ir) at later (2–4 h) time points. Upregulation of TH and NPY mRNAs in Fos-ir neurons followed distinct time courses, with NPY responses peaking more rapidly, particularly in the C1 and C2 cell groups. Adrenergic cell groups displayed greater maximal increases in NPY expression than the A1 noradrenergic cell group while the converse was true of TH mRNA response. Increasing the severity of haemorrhage resulted in more pronounced increases in both mRNA responses in each aminergic region. These findings indicate that haemorrhage differentially affects TH and NPY expression in medullary catecholamine cell groups that participate in the maintenance of cardiovascular homeostasis. The differential nature of these responses suggests them not to be a simple consequence of metabolic alterations pursuant to increased synaptic activity. The prompt and robust NPY mRNA responses in adrenergic neurons suggests a mechanism by which peptide content of these cell groups' terminal projections is defended.     

The differential effects of cell density and NGF on the expression of tyrosine hydroxylase and dopamine beta-hydroxylase in PC12 cells.

Expression of neurotransmitter phenotype during development of the nervous system is determined by several micro-environmental factors including cell aggregation. In order to delineate the role of cell aggregation and nerve growth factor (NGF) in regulating catecholamine expression, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) mRNA levels were examined in PC12 cells at different cell densities with and without NGF treatment. Upon plating of PC12 cells from low density (0.3-1.0 x 10(5) cells/cm2) to high density (0.5-2.0 x 10(6) cells/cm2) TH mRNA levels increased 4-fold within 1 day and remained at this level for several days. In cells replated from high to low density, TH mRNA returned to original levels within 1 day. In addition to TH mRNA, TH protein and dopamine levels were also found to increase in high-density cultures. In contrast to the increase in TH mRNA, DBH mRNA decreased about 40% in cells plated from low to high density. Hence, cell density differentially regulated TH and DBH mRNA levels. Unlike cell density, NGF treatment led to a decrease in both TH and DBH mRNA levels. However, when NGF treated cells were replated from low to high density, TH and dopamine levels increased. Thus NGF did not alter the density dependent regulation of TH. Similarly, TH mRNA levels increased in F4 cells, a mutant PC12 cell line unresponsive to NGF, when plated from low to high density. DBH mRNA decreased to undetectable levels when NGF treated PC12 cells were plated to high density, demonstrating a synergetic effect of cell density and NGF treatment on DBH mRNA levels.     

Treatment time influences the effects of a low-frequency pulsed electric field on synthesis of tyrosine hydroxylase and dopamine in PC12 cells

BACKGROUND: Electromagnetic radiation can influence dopamine (DA) synthesis in brain tissues or cells, but electromagnetic frequencies, intensities, and radiation time can produce different effects. In addition, the signal pathway by which electromagnetic radiation influences DA synthesis remains controversial. OBJECTIVE: To determine tyrosine hydroxylase (TH) expression in PC12 cells and DA levels in cell culture media after different periods of low-frequency pulsed electric field (LF-PEF) stimulation, and to determine how LF-PEF signaling stimulates TH synthesis using inhibitors. DESIGN, TIME AND SETTING: A parallel, controlled, cell experiment was performed at the Laboratory of Cell Biology, School of Life Science, East China Normal University, between January and October 2006. MATERIALS: PC12 cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China. Nerve growth factor was purchased from PeproTech, USA. The protein kinase A inhibitor, H-89, and mitogen-activated protein kinase kinase inhibitor, U0126, were purchased from Sigma, USA. METHODS: (1) Following routine culture in Dulbecco's modified eagle medium, primary PC12 cells were stimulated under LF-PEF (pulse frequency 50 Hz, pulse width 20 us, peak field strength 1 V/m) for 5,10,15,20, and 30 minutes. (2) Inhibitors (H-89 or U0126,1 umol/L) were added 30 minutes before LF-PEF stimulation for 10 minutes. MAIN OUTCOME MEASURES: (1) TH expression was determined by Western blot in PC12 cells at 0.5,1, 2, 3, and 4 days after LF-PEF stimulation. Similarly, DA was measured by high-performance liquid chromatography in media at 2,3,4, or 5 days after LF-PEF. (2) TH expression was detected 1 day after H-89 or U0126 treatment and LF-PEF. RESULTS: (1) Short-term LF-PEF stimulation (5 and 10 minutes) increased TH expression and media DA levels after short-term culture (2 days) (P < 0.01), but both parameters decreased with longer culture (3-4 days) (P< 0.01). Long-term LF-PEF stimulation (15, 20, or 30 minutes) decreased TH and DA synthesis, followed by a rapid increase (P< 0.01). (2) H89 could completely inhibit TH expression in PC12 cells stimulated by LF-PEF for 10 minutes, while the inhibition rate of U0126 was 53.2%. CONCLUSION: Short-term LF-PEF first promotes then inhibits, while long-term LF-PEF first inhibits then promotes, TH and DA synthesis. LF-PEF stimulation regulates TH expression primarily by activating protein kinase A to regulate DA synthesis.     

Cholinergic differentiation of clonal rat pheochromocytoma cells (PC12) induced by retinoic acid: increase of choline acetyltransferase activity and decrease of tyrosine hydroxylase activity

The effects of retinoic acid (RA), a naturally occurring metabolite of vitamin A, on the growth, morphology and neurochemical differentiation of the PC12 clone of rat pheochromocytoma cells were investigated. RA added to the medium inhibited the growth of PC12 cells in a dose-dependent manner up to 10 microM without affecting their morphology. In PC12 cells cultured in the presence of 10 microM RA for 8 days, the specific activity of choline acetyltransferase (ChAT) was increased 2-fold, while the specific activity of tyrosine hydroxylase (TH) was decreased 0.5-fold compared with cells cultured in the absence of RA. Specific activities of acetylcholinesterase (AChE), glutamate decarboxylase (GAD) and lactate dehydrogenase (LDH) were not affected by RA. Both the increase of ChAT and the decrease of TH induced by RA exhibited similar time and dose dependencies. RA inhibited the increase of TH activity induced by nerve growth factor (NGF), an adrenergic neuronotrophic factor on PC12 cells. From these observations it was concluded that RA induces a cholinergic neurochemical differentiation of PC12 cells independent of a morphological differentiation.     

Quantitation of tyrosine hydroxylase and neuropeptide Y immunoreactivity in single rat sympathetic neurons: effects of preganglionic nerve activity.

Using computerised densitometry to measure immunocytochemical reaction product in a model system, we established conditions that produced a linear relationship between the logarithm of antigen concentration and the measured intensity of staining. We then applied the densitometric technique to assess the changes in tyrosine hydroxylase (TH) and neuropeptide tyrosine (NPY) within sympathetic neurons of rat superior cervical ganglion following chronic decentralization and following reserpine treatment. One week after surgical or pharmacological decentralization, there was appreciable reduction of neuronal levels of both TH and NPY. However, there remained considerable variation in the immunoreactivities of individual cells. Three days of treatment with reserpine elevated TH levels but substantially reduced NPY. Both these effects were prevented by prior decentralization of the ganglia. No differences were seen between normotensive and the Otago strain of genetically hypertensive rats, either in basal TH or NPY immunoreactivities or in responses to the maneuvers performed. Comparison of our findings with previous biochemical data indicate that densitometric immunocytochemistry provides an accurate index of neuronally localised antigen concentrations but also allows analysis of interneuronal differences that are not otherwise apparent.     

Differential regulation of SHC proteins by nerve growth factor in sensory neurons and PC12 cells

We have characterized some of the nerve growth factor (NGF) stimulated receptor tyrosine kinase (TrkA) signalling cascades in adult rat primary dorsal root ganglia (DRG) neuronal cultures and compared the pathways with those found in PC12 cells. TrkA receptors were phosphorylated on tyrosine residues in response to NGF in DRG neuronal cultures. We also saw phosphorylation of phospholipase Cγ1 (PLCγ1). We used recombinant glutathione-S-transferase (GST)–PLCγ1 SH2 domain fusion proteins to study the site of interaction of TrkA receptors with PLCγ1. TrkA receptors derived from DRG neuronal cultures bound preferentially to the amino terminal Src homology-2 (SH2) domain of PLCγ1, but there was enhanced binding with tandemly expressed amino- and carboxy-terminal SH2 domains. The most significant difference in NGF signalling between PC12 cells and DRG was with the Shc family of adapter proteins. Both ShcA and ShcC were expressed in DRG neurons but only ShcA was detected in PC12 cells. Different isoforms of ShcA were phosphorylated in response to NGF in DRG and PC12 cells. NGF phosphorylated only one whereas epidermal growth factor phosphorylated both isoforms of ShcC in DRG cultures. Activation of the downstream mitogen-activated protein (MAP) kinase, p42Erk2 was significantly greater than p44Erk1 in DRG whereas both isoforms were activated in PC12 cells. Blocking the MAP kinase cascade using a MEK1/2 inhibitor, PD98059, abrogated NGF dependent capsaicin sensitivity, a nociceptive property specific to sensory neurons.     

Membrane depolarization by isotonic or hypertonic KCl: differential effects on mRNA levels of tyrosine hydroxylase and dopamine β-hydroxylase mRNA in PC12 cells

Membrane depolarization is an important and common manipulation used to study the result of enhanced neuronal activity on adaptive changes, including alterations in gene expression. In this study, the effect of elevated KCl, under isotonic and hypertonic conditions, on the changes in mRNA levels of the catecholamine biosynthetic enzymes, tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) was compared. Treatment of PC12 pheochromocytoma cells for several hours with 50 mM KCl, under conditions where osmolarity was maintained, induced TH mRNA levels several fold, without changing DBH mRNA levels (Kilbourne and Sabban, 1990). In contrast, 50 mM KCl added to culture media without adjusting the osmolarity did not alter TH mRNA levels for up to 24 h. Longer continuous exposure to this hypertonic depolarization condition reduced TH mRNA levels to about 10% of control levels. DBH mRNA levels also declined when PC12 cells were treated from 12 h to 5 days with hypertonic 50 mM KCl. The effect appeared to be specific, since actin mRNA levels were elevated about 2-fold with these same hypertonic treatments. As a control for osmotic changes, 50 mM NaCl was used and did not alter TH or DBH mRNA levels. Viability of the cells was maintained and total protein synthesis was reduced somewhat after 12 h of exposure to hypertonic 50 mM KCl, and remained relatively constant for as long as 4 days. Thus, there appears to be an interaction between osmolarity and elevated KCl since very different results of the effects of membrane depolarization on the mRNA levels for the catecholamine biosynthetic enzymes were obtained depending on the osmolarity of the cultures. The extent of elevation of TH mRNA with isotonic KCl was also dependent on cell density. At high cell densities, membrane depolarization no longer induced TH mRNA levels. The results of this study indicate the experimental parameters which can be crucial in studies of membrane depolarization.     

Differential regulation of chromogranin B/secretogranin I and secretogranin II by forskolin in PC12 cells.

The factors which regulate the expression of the granin family of secretory proteins have yet to be completely described. The present study investigated the effects of forskolin (FSK), an activator of adenylate cyclase, on the regulation of chromogranin B/secretogranin I (CgB) and secretogranin II (SgII) mRNA levels in rat PC12 cells. PC12 cells were treated with 10 microM FSK for time points up to 48 h and were harvested for cAMP determination, RNA isolation and Northern blot analysis, or fixed in 4% paraformaldehyde for immunocytochemistry. Cellular cAMP levels peaked after two h of FSK treatment and remained elevated for 48 h. Chromogranin B mRNA increased with FSK treatment, reaching a maximum of 7-fold above control after 24 h, while the level of SgII mRNA decreased to a level of 65 +/- 10% of control after 48 h. The effects of FSK on CgB mRNA appear to be mediated by cAMP, as 8-bromo-cAMP (500 microM) resulted in a 2.8-fold increase in CgB mRNA, and H-89 (30 microM), a selective inhibitor of cAMP-dependent protein kinase, reduced the FSK-mediated response. The level of CgB was also increased in FSK-treated cells, as evidenced by immunofluorescent analysis which showed a more intense staining in PC12 cells treated with FSK for 48 h than in untreated cells. The intensity of SgII staining was diminished by FSK treatment, most likely a result of a decreased rate of synthesis as well as an increase in the release of SgII. This study demonstrated that the mRNA and protein levels of CgB and SgII are differentially regulated by cAMP in PC12 cells.     

5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside stimulates tyrosine hydroxylase activity and catecholamine secretion by activation of AMP-activated protein kinase in PC12 cells

The activity of AMP-activated protein kinase (AMPK) is regulated by the metabolic and nutritional state of the cell. 5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) is transformed into riboside monophosphate (ZMP) via phosphorylation by adenosine kinase inside the cell and exerts it effect by stimulating AMPK. AICAR significantly induces an increase in AMPK activity in a dose- and time-dependent manner in the rat pheochromocytoma cell line PC12. In addition, compound C, an AMPK inhibitor, as well as 5'-amino-5'-dAdo, an adenosine kinase inhibitor, inhibits the AICAR-induced AMPK activity. AICAR significantly stimulates tyrosine hydroxylase (TH) (the rate-limiting enzyme in the biosynthesis of catecholamine) activity and the corresponding mRNA level, which closely matches with the TH protein level. In addition, AICAR provokes a rapid and long-lasting increase in the phosphorylation of TH at Ser19, Ser31 and Ser40. AICAR also markedly activates ERKs, JNK and p38. The MEK-1-inhibitor (PD-098059) causes a partial, but significant, inhibition of AICAR-induced TH enzyme activity by phosphorylation of Ser31 without affecting phosphorylation at the two other sites. By contrast, neither the JNK-inhibitor nor the p38-inhibitor affects TH enzyme activity and phosphorylation. Similarly, PD-098059 partially, but significantly, inhibits the AICAR-induced increase in the TH mRNA level. Furthermore, AICAR increases the level of cAMP in PC12 cells. The present study also shows that H89, a protein kinase A inhibitor, abolishes the AICAR-induced increase in the level of TH mRNA, as well as the corresponding enzyme activity and Ser40 phosphorylation. Finally, AICAR significantly increases dopamine secretion from PC12 cells. These findings indicate that AICAR activates catecholamine synthesis and secretion through AMPK activation in chromaffin cells.     

Expression of the enkephalin precursor gene in C6 rat glioma cells: regulation by beta-adrenergic agonists and glucocorticoids

Cultured C6 rat glioma cells contain mRNA coding for preproenkephalin (A), the precursor of methionine- and leucine-enkephalin. The abundance in untreated cells was determined by blot hybridization methods to be 3-6 pg per micrograms total RNA. Treatment of confluent cells for 12 h with 10 microM (-)-norepinephrine, which activates C6 adenylate cyclase, transiently elevated preproenkephalin mRNA to 3.3 and 7.7 times the control in the absence and presence of the glucocorticoid dexamethasone, respectively. Hydrocortisone and corticosterone also potentiated the effect of norepinephrine. However, glucocorticoids alone did not alter the preproenkephalin mRNA abundance. The effect of norepinephrine + dexamethasone was blocked by the beta-adrenergic antagonist propranolol but not by the alpha-adrenergic antagonist phentolamine. Forskolin, which directly activates adenylate cyclase, similarly elevated the preproenkephalin mRNA abundance; its effect was also potentiated by dexamethasone. C6 cells contain Met-enkephalin-containing protein resembling proenkephalin (apparent Mr 30,000) but little Met-enkephalin, suggesting a low level of proper precursor processing. Treatment with norepinephrine + dexamethasone raised the content of proenkephalin-like protein 11-fold. Thus, preproenkephalin mRNA levels in C6 cells are regulated synergistically by adenosine 3':5'-cyclic monophosphate and glucocorticoids. These results suggest modes of regulation of proenkephalin biosynthesis in normal rat enkephalinergic cells.     

Neuropeptide Y gene expression in PC12 cells and its regulation by nerve growth factor: a model for developmental regulation

Neuropeptide Y (NPY) is a 36 amino acid neuronal peptide which has previously been described within a subset of sympathetic neurons. The presence of the messenger RNA that encodes NPY (NPY mRNA) has been studied in the PC12 cell line, as this is commonly used as a model of neuronal differentiation. Low levels of the NPY mRNA were identified in resting naive cells. Levels were induced five-fold by the addition of nerve growth factor to the medium at a dose of 30 ng/ml. Lower doses of nerve growth factor had no effect on NPY mRNA. The effect was rapid in onset being apparent within 12 h and near maximal at 24 h. A smaller two-fold increase in NPY mRNA was observed in cells treated with epidermal growth factor (3 ng/ml) over the same time course but no effect was observed with fibroblast growth factor (3 ng/ml), bradykinin (10(-6) M) or dexamethasone (10(-6) M). These results indicate that NPY gene expression is regulated in PC12 cells at the level of NPY mRNA.     

Neurobiological effects of intraventricular propionic acid in rats: possible role of short chain fatty acids on the pathogenesis and characteristics of autism spectrum disorders

Clinical observations suggest that certain gut and dietary factors may transiently worsen symptoms in autism spectrum disorders (ASD), epilepsy and some inheritable metabolic disorders. Propionic acid (PPA) is a short chain fatty acid and an important intermediate of cellular metabolism. PPA is also a by-product of a subpopulation of human gut enterobacteria and is a common food preservative. We examined the behavioural, electrophysiological, neuropathological, and biochemical effects of treatment with PPA and related compounds in adult rats. Intraventricular infusions of PPA produced reversible repetitive dystonic behaviours, hyperactivity, turning behaviour, retropulsion, caudate spiking, and the progressive development of limbic kindled seizures, suggesting that this compound has central effects. Biochemical analyses of brain homogenates from PPA treated rats showed an increase in oxidative stress markers (e.g., lipid peroxidation and protein carbonylation) and glutathione S-transferase activity coupled with a decrease in glutathione and glutathione peroxidase activity. Neurohistological examinations of hippocampus and adjacent white matter (external capsule) of PPA treated rats revealed increased reactive astrogliosis (GFAP immunoreactivity) and activated microglia (CD68 immunoreactivity) suggestive of a neuroinflammatory process. This was coupled with a lack of cytotoxicity (cell counts, cleaved caspase 3' immunoreactivity), and an increase in phosphorylated CREB immunoreactivity. We propose that some types of autism may be partial forms of genetically inherited or acquired disorders involving altered PPA metabolism. Thus, intraventricular administration of PPA in rats may provide a means to model some aspects of human ASD in rats.