Beta-adrenergic receptor distribution in human and rat hippocampal formation: marked species differences.

The topography of beta-adrenergic receptors in the rat and human hippocampal formation was assessed by in vitro binding of 125I-pindolol to tissue sections. Marked differences were found in the distribution of beta-adrenergic receptors and in the relative amounts of beta 1 and beta 2 receptor subtypes in the two species. In the human, the highest receptor densities were present in the pyramidal cell layer and in the stratum lacunosum-moleculare. In the rat hippocampus, those regions contained the lowest densities of 125I-pindolol binding sites. The highest densities of beta-adrenergic receptors in the rat hippocampal formation were found in the ventral subiculum and in the entorhinal cortex. In contrast, in the human hippocampus, the subiculum and entorhinal cortex contained relatively low densities of the receptors. Competition studies with beta 1- and beta 2-selective antagonists revealed that beta 2-adrenergic receptors predominate in the human hippocampus and beta 1-adrenergic receptors predominate in the rat hippocampus. The marked species differences observed suggest that the pharmacological responsivity of the hippocampus to adrenergic agents and the role of noradrenaline in regulation of hippocampal function could be very different in rats compared to humans.     

Normalization of subtype-specific muscarinic receptor binding in the denervated hippocampus by septodiagonal band grafts

Unilateral fimbria-fornix lesions were made by aspiration in female Sprague-Dawley rats. In a group of these rats, fetal septal tissue was transplanted into the lesion cavity. Lesion of the fimbria-fornix resulted in a reduction of cholinergic input to the hippocampal formation as indicated by the loss of acetylcholinesterase (AChE)-positive staining in all ipsilateral hippocampal laminae and a loss of [3H]hemicholinium-3 binding to cholinergic terminals in the strata oriens (82% reduction) and radiatum (77% reduction) of areas CA2 and CA3 and in the molecular layer of the dentate gyrus (83% reduction). In contrast, the density of muscarinic receptor binding ([3H]QNB) increased in the strata oriens (80% increase) and radiatum (70% increase) in areas CA2-CA4. This was shown to be due to an actual increase in receptor number (Bmax) and not to a change in affinity (KD). Analysis of muscarinic receptor subtypes indicated that the increase in receptor binding in the stratum radiatum was of the M-1 subtype ([3H]-pirenzepine) and in the stratum oriens was of the M-2 subtype ([3H]QNB + 100 nM pirenzepine). In the host hippocampus after fetal septal graft, the staining for AChE, the binding of [3H]hemicholinium-3, and the binding of muscarinic receptors (both the M-1 and M-2 receptor subtypes) were all comparable to nonlesioned control values. These data indicate that the fetal septal grafts have reinnervated the host hippocampus and have made synaptic contact with host cells in a manner capable of regulating postsynaptic muscarinic receptors.     

Ontogenic distribution of muscarinic receptors and acetylcholinesterase in the rabbit hippocampus

Ontogenic development of muscarinic receptors was examined in the hippocampus of rabbits (from P2 to P60) using radioautographic method. Muscarinic sites were labelled with (3H)-quinuclinidyl-benzilate and pharmacologically defined M1 and M2 receptor subtypes with (3H)-pirenzepine and (3H)-oxotremorine, respectively. The distribution of binding sites was compared to acetylcholinesterase (AChE) staining in adjacent hippocampal sections. The two cholinergic components are progressively set up in the hippocampus during the first three postnatal weeks. The AChE staining was very low in all hippocampal fields in P2 rabbits. At P8 and after, the AChE staining was more pronounced in CA3 and CA4 than in CA1 and CA2. On the contrary, the M1 muscarinic binding sites were more abundant in CA1 and CA2 hippocampal fields than in CA3 and CA4 at all ages studied. M2 muscarinic binding sites were only distinguishable at P45 and have a relatively homogeneous distribution. This study shows a differential developmental evolution in the distribution of AChE and muscarinic M1 receptors, and no obvious correspondence between these two cholinergic markers was observed.     

The effects of maternal ethanol exposure on neurotransmission and second messenger systems: a quantitative autoradiographic study in the rat brain.

The effects of maternal ethanol exposure on neurotransmission and second messenger systems were examined in rats using histochemistry and in vitro autoradiography. Thirty % ethanol was administered to pregnant rats from gestational day 7 to the day of delivery. Quantitative autoradiography was used to map muscarinic cholinergic, dopamine D2, adenosine A1, and inositol 1,4,5-trisphosphate binding sites, as well as to localize adenylate cyclase and protein kinase C. We found no difference in the patterns of staining with acetylcholinesterase and Timm's stain between control and prenatally ethanol-exposed rats on postnatal day (PN) 30. In the ethanol-exposed rats, [3H]forskolin binding sites were increased during early development in the CA1 subfield of the hippocampus and the occipital cortex; [3H]phorbol ester binding sites were increased in the cortex, striatum, and hippocampus; hippocampal muscarinic cholinergic sites were increased on PN4 and 30; adenosine A1 binding was reduced on PN10 in most regions examined, but was increased in the CA1 subfield on PN30; dopamine D2 receptor levels were significantly reduced on PN30 in the striatum; and IP3 receptors were decreased in most regions studied, but particularly in the cerebellum. Thus, some of these changes were transient and others were long-lasting. Although histopathological abnormalities were minimal, the alterations of binding sites in the cerebellum (the coordination center) and in the hippocampus (related to memory and learning) that were detected may contribute to the behavioral and mental deterioration seen in the fetal alcohol syndrome.     

Hippocampal muscarinic supersensitivity after AF64A medial septal lesion excludes M1 receptors

Stereotaxic injection of AF64A, into the medial septum of the rat, resulted in significant loss of presynaptic cholinergic markers in this structure. No significant change was observed for the presynaptic neuronal markers for dopamine- and serotonin-containing neurons in either the medial septum or hippocampus. The AF64A lesion also resulted in a significant reduction of muscarinic receptors as demonstrated by a loss of [3H]QNB binding in the medial septum. Subtype analysis showed the decrease of receptor binding in the medial septum to be due to a loss of M1 receptors as well as other muscarinic receptor subtypes. In the hippocampal formation, [3H]hemicholinium-3 binding was significantly reduced in the molecular layer of the dentate gyrus, and in the stratum oriens and stratum radiatum of the hippocampus. AF64A lesion resulted in a significant increase (Bmax) in non-M1 muscarinic receptors in hippocampal stratum oriens, in areas CA2, CA3, and CA4. AF64A lesion of the medial septum did not result in muscarinic receptor alterations in any other region of the hippocampal formation examined. These results indicate that postsynaptic muscarinic receptors in the stratum oriens of the CA2 to CA4 region of the hippocampus mediate primarily the function of the cholinergic cell bodies of the medial septum. These receptors are not of the M1 subtype.     

Beta-adrenergic receptors are differentially expressed in distinct interneuron subtypes in the rat hippocampus

Noradrenaline (NA) acting via beta-adrenergic receptors (betaARs) plays an important role in the modulation of memory in the hippocampus. betaARs have been shown to be expressed in principal cells, but their distribution across different interneuron classes is unknown. We have used specific interneuron markers including calcium binding proteins (parvalbumin, calbindin, and calretinin) and neuropeptides (somatostatin, neuropeptide Y, and cholecystokinin) together with either beta1AR or beta2AR to determine the distribution of these receptors in all major subfields of the hippocampus. We found that beta1AR-expressing interneurons were more prevalent in the CA3 and CA1 regions of the hippocampus than in the dentate gyrus, where they were relatively sparse. beta2AR-expressing interneurons were more uniformly distributed between all three regions of the hippocampus. A high proportion of neuropeptide Y-containing interneurons in the dentate gyrus co-expressed beta2AR. beta1AR labeling was common in interneurons expressing somatostatin and parvalbumin in the CA3 and CA1 regions, particularly in the stratum oriens of these regions. beta2AR labeling was more likely to be found than beta1AR labeling in cholecystokinin-expressing interneurons. In contrast, calretinin-containing interneurons were virtually devoid of beta1AR or beta2AR labeling. These regional and interneuron type-specific differences suggest functionally distinct roles for NA in modulating hippocampal activity via activation of betaARs.     

Altered hippocampal muscarinic M4, but not M1, receptor expression from subjects with schizophrenia.

BACKGROUND: Having shown a decrease in [3H]pirenzepine binding in the hippocampus from subjects with schizophrenia, we wished to determine whether such a change in radioligand binding was associated with changes in hippocampal mRNA for the muscarinic1 (M1) and muscarinic4 (M4) receptors in tissue from different cohorts of subjects. METHOD: The [3H]pirenzepine binding using autoradiography and in situ hybridization with oligonucleotides specific for muscarinic M1 and M4 receptors were completed using hippocampal tissue obtained postmortem from 20 control subjects and 20 subjects with schizophrenia. RESULTS: The [3H]pirenzepine binding was decreased in the dentate gyrus (p < .05), CA3 (p < .01), CA2 (p < .05), and CA1 (p < .01) regions of the hippocampus from subjects with schizophrenia. Levels of M4 mRNA varied with the diagnosis of schizophrenia (p = .01), but significant region-specific changes were not apparent. Changes in levels of mRNA for the muscarinic M1 receptor were not detected with diagnosis. CONCLUSIONS: This study suggests that decreases in hippocampal [3H]pirenzepine binding in subjects with schizophrenia are most likely associated with widespread changes in expression levels of the M4 receptor. These data further implicate the hippocampal formation in the pathology of schizophrenia.     

Localization of hippocampal muscarinic receptors after kainic acid lesion of CA3 and fimbria-fornix transection

Bilateral intraventricular injections of 0.5 μg of kainic acid were used to selectively destroy CA3 hippocampal pyramidal neurons, in an effort to clarify the possible localization of muscarinic cholinergic receptors in the rat hippocampal formation. Thirty days after treatment, there was 43% decrease in the total number of [³H] -QNB binding sites per hippocampus, with no change in affinity. Histological examination confirmed the selective loss of pyramidal neurons in subareas CA3a–b while other regions of the hippocampal formation were spared. The unilateral transection of the fimbria-fornix, done 14 days after kainic acid, produced a further reduction in binding in relation to control hippocampi (−57%).The results demonstrate that in the pyramidal cells of CA3 there is a high concentration of postsynaptic muscarinic receptors. However, the slight further decrease, found after fimbria-fornix transection, suggests the possible existence of a small population of presynaptic receptors that, hitherto, had only been demonstrated indirectly by physiological methods.     

Effects of birth asphyxia on neonatal hippocampal structure and function in the spiny mouse

Studies of human neonates, and in animal experiments, suggest that birth asphyxia results in functional compromise of the hippocampus, even when structural damage is not observable or resolves in early postnatal life. The aim of this study was to determine if changes in hippocampal function occur in a model of birth asphyxia in the precocial spiny mouse where it is reported there is no major lesion or infarct. Further, to assess if, as in human infants, this functional deficit has a sex-dependent component. At 37 days gestation (term = 39 days) spiny mice fetuses were either delivered immediately by caesarean section (control group) or exposed to 7.5 min of in utero asphyxia causing systemic acidosis and hypoxia. At 5 days of age hippocampal function was assessed ex vivo in brain slices, or brains were collected for examination of structure or protein expression. This model of birth asphyxia did not cause infarct or cystic lesion in the postnatal day 5 (P5) hippocampus, and the number of proliferating or pyknotic cells in the hippocampus was unchanged, although neuronal density in the CA1 and CA3 was increased. Protein expression of synaptophysin, brain-derived neurotrophic factor (BDNF), and the inositol trisphosphate receptor 1 (IP₃R1) were all significantly increased after birth asphyxia, while long-term potentiation (LTP), paired pulse facilitation (PPF), and post-tetanic potentiation (PTP) were all reduced at P5 by birth asphyxia. In control P5 pups, PPF and synaptic fatigue were greater in female compared to male pups, and after birth asphyxia PPF and synaptic fatigue were reduced to a greater extent in female vs. male pups. In contrast, the asphyxia-induced increase in synaptophysin expression and neuronal density were greater in male pups. Thus, birth asphyxia in this precocial species causes functional deficits without major structural damage, and there is a sex-dependent effect on the hippocampus. This may be a clinically relevant model for assessing treatments delivered either before or after birth to protect this vulnerable region of the developing brain.     

Effects of prenatal hypoxia on expression of thioredoxin-1 in the rat hippocampus at different stages of postnatal ontogeny

We studied the expression of the antioxidant protein thioredoxin-1 in the hippocampus of rat pups that were subjected to hypoxia at prenatal days 14–16, at the 3rd and 14th days after their birth, and at 80–90 days when the pups became mature. In spite of the specific features of certain hippocampal regions and the presence of exceptions related to them, 3 days after birth we observed a general trend to a decrease in thioredoxin-1 expression in rats subjected to hypoxia, as compared to the control animals of the same age. Then, thioredoxin-1 expression was substantially increased on the 14th day of postnatal life. In adult rats, thioredoxin-1 expression decreased again. These changes were very prominent in the CA1 field of the hippocampus. A decrease in the activity of the physiological antioxidant system is an important index of cell-membrane homeostasis, whose impairment under hypoxic conditions can result in the development of neurodegenerative diseases.     

Development of the cholinergic system in control and intra-uterine growth retarded rat brain

The activity of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and muscarinic receptors was studied in control rats and in rats growth-retarded in utero because of reduction of the blood supply 5 days before birth. The different markers of the cholinergic system were estimated at P (postnatal day) 6, 9, 12, 15, 22 and 60 in cerebellum, hypothalamus, septum, striatum and CA1, CA3 and fascia dentata of the hippocampus. In control rats, there was a transient increase in ChAT activity in the septum during the second week of postnatal development. In the intrauterine growth retarded rats there was a marked delay in this developmental rise in CA1, CA3 at P6 and P9 and in the fascia dentata at P14 respectively. This delayed rise enzyme activity was associated with a significant reduction of muscarinic binding sites [( 3H]QNB) in the hippocampus. AChE staining showed a similar development in both groups. Therefore, the undernutrition produced by a reduction of the blood supply 5 days before birth is associated with a delayed maturation of cholinergic functions.     

Sequential changes in muscarinic acetylcholine, adenosine A1 and calcium antagonist binding sites in the gerbil hippocampus following repeated brief ischemia.

We performed quantitative autoradiography to determine sequential alterations in the binding of muscarinic cholinergic and adenosine A1 receptors and of an L-type calcium channel blocker in the gerbil hippocampus following repeated brief ischemic insults. [3H]Quinuclidinyl benzilate (QNB). [3H]cyclohexyladenosine (CHA) and [3H]PN200-110 were used to label muscarinic and adenosine A1 receptors and L-type calcium channels, respectively. Changes at 1 h, 6 h, 1 day, 4 days and 1 month after three 2-min ischemic insults were compared with changes after single 2- or 6-min ischemia. Two-minute ischemia, which causes no histopathological neuronal damage, produced no persistent alterations in binding sites. We observed a transient and mild increase in binding activities, especially in [3H]CHA binding, at 1 h of recirculation. Following 6-min ischemia and three 2-min ischemic insults. [3H]QNB and [3H]PN200-110 binding decreased by more than 50% in the CA1 subfield by 1 month, but [3H]CHA binding decreased transiently by 20-30% at 4 days when delayed neuronal death of hippocampal CA1 pyramidal cells took place. Reductions in binding, especially in [3H]QNB binding, following three 2-min ischemic insults were greater and appeared earlier than those after 6-min ischemia. Furthermore, alterations extended to the CA3 subfield and the dentate gyrus following repeated insults. Thus, alterations in receptor binding after repeated ischemic insults were greater than those after equivalent single period of ischemia.     

Effect of oestrogen receptor alpha and beta agonists on brain N-methyl-D-aspartate receptors

Previous studies have shown the oestradiol modulation of brain N-methyl-D-aspartate (NMDA) receptors composed of the NR1/2B subunits. The contribution of oestrogen receptor subtypes in this oestradiol modulation of NMDA receptors and its subunits is not known. The following experiments investigated whether an oestrogenic receptor subtype is involved in the oestradiol effect on NMDA receptor specific binding and subunit mRNA levels. Ovariectomised Sprague-Dawley rats were treated 2 days after ovariectomy for 2 weeks with 17beta-oestradiol, an agonist for oestrogen receptor (ER)alpha 4,4',4'-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) or an agonist for ER beta 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) and compared with control vehicle-treated ovariectomised and intact rats. Uterus weights, used as a peripheral measure of oestrogenic activity, decreased after ovariectomy and increased by oestradiol and PPT but not DPN treatment. In the hippocampal CA1 oriens and CA1 radiatum, [(3)H]Ro 25-6981 specific binding, a NMDA/NR2B ligand, was decreased in ovariectomised compared to intact rats and this was prevented by 17beta-oestradiol or PPT but not DPN treatments; a similar pattern was observed in the CA2/3 and dentate gyrus but did not reach statistical significance. In situ hybridisation of the mRNA of the NMDA/2B subunit in the hippocampus CA1, CA2/3 and dentate gyrus showed a decrease in ovariectomised rats compared to controls and this was also prevented by 17beta-oestradiol and PPT but not DPN treatments. In cingulate and prefrontal cortices, ovariectomy increased [(3)H]Ro 25-6981 specific binding compared to intact controls, which was corrected by 17beta-oestradiol treatment but neither by PPT, nor DPN. In the cortical regions, the lack of effect of the ER alpha or ER beta agonist whereas 17beta-oestradiol was active, suggesting that the oestradiol modulation of cortical NMDA receptors requires both ERs or that this modulation does not involve ERs. In the hippocampus, the results obtained suggest an oestrogenic genomic modulation of NMDA receptors containing the NR2B subunit, implicating an ER alpha.     

Postnatal aniracetam treatment improves prenatal ethanol induced attenuation of AMPA receptor-mediated synaptic transmission

Aniracetam is a nootropic compound and an allosteric modulator of AMPA receptors (AMPARs) which mediate synaptic mechanisms of learning and memory. Here we analyzed impairments in AMPAR-mediated synaptic transmission caused by moderate prenatal ethanol exposure and investigated the effects of postnatal aniracetam treatment on these abnormalities. Pregnant Sprague-Dawley rats were gavaged with ethanol or isocaloric sucrose throughout pregnancy, and subsequently the offspring were treated with aniracetam on postnatal days (PND) 18 to 27. Hippocampal slices prepared from these pups on PND 28 to 34 were used for the whole-cell patch-clamp recordings of AMPAR-mediated spontaneous and miniature excitatory postsynaptic currents in CA1 pyramidal cells. Our results indicate that moderate ethanol exposure during pregnancy results in impaired hippocampal AMPAR-mediated neurotransmission, and critically timed aniracetam treatment can abrogate this deficiency. These results highlight the possibility that aniracetam treatment can restore synaptic transmission and ameliorate cognitive deficits associated with the fetal alcohol syndrome.     

Effects of serotonergic denervation on the density and plasticity of brain muscarinic receptors in the rat.

This study investigated whether serotonergic lesion may affect density, sensitivity, and plasticity of muscarinic receptors in hippocampus and cerebral cortex. Intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT) in rats produced a 90% reduction in cortical and hippocampal 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) contents. In these brain areas, the 5,7-DHT lesion did not affect the overall density of muscarinic receptors or those of M1 and non-M1 muscarinic receptor subtypes as assayed using [3H]N-methylscopolamine ([3H]NMS), [3H]pirenzepine, and [3H]NMS in the presence of pirenzepine, respectively. In addition, the binding of the muscarinic agonist [3H]oxotremorine-M (OXO-M), taken as an indirect index of coupling efficiency of non-M1 receptors with G-proteins, did not change significantly in cortex and hippocampus of 5,7-DHT-lesioned rats. Similarly, carbachol-induced accumulation of [3H]inositol phosphates (InPs) in hippocampal miniprisms showed no significant differences between tissues from 5,7-DHT-lesioned and sham-operated rats. In sham-operated rats, an intraperitoneal (i.p.) injection of scopolamine (10 mg/kg once daily) during 21 days caused an increased density of [3H]NMS binding sites in cortex (+20%) and hippocampus (+26%). This up-regulation was restricted to non-M1 receptors subtypes. In 5,7-DHT-lesioned rats, chronic scopolamine failed to modify significantly the density of cortical or hippocampal M1 or non-M1 receptors. These results suggest 1) that 5-HT denervation did not affect the density and sensitivity of muscarinic receptors and 2) that the ability of cortical and hippocampal non-M1 receptors to up-regulate following repeated injection of scopolamine requires the integrity of 5-HT neurons terminating in these brain structures.     

Corticosterone modulation of neurotransmitter receptors in rat hippocampus: A quantitative autoradiographic study

The effect of adrenalectomy (ADX) and corticosterone (CORT) replacement on neurotransmitter receptors was studied in dorsal hippocampus of rat using quantitative autoradiography. ADX for one week causes an increase in [3H]5-HT binding to 5-HT1 receptors which is significant in the CA1 cell field. CORT treatment of ADX rats for 3-5 days results in localized reductions of [3H]5-HT binding including a partial reversal of the increase observed after ADX in CA1. CORT treatment of ADX animals also decreases binding of [3H]QNB to muscarinic receptors in the dorsal hippocampus, with a significant effect in an area designated as subiculum. No influence of CORT was detected on [3H]prazosin binding to alpha 1 adrenergic receptors in dorsal hippocampus. Possible mechanisms for hormone effects on neurotransmitter receptor levels are discussed.     

Localization of M1 Muscarinic Receptors in Rat Brain Using Selective Muscarinic Toxin-1

Mambas, African snakes of the genus Dendroaspis, produce several types of toxins that are of pharmacological interest. The novel muscarinic toxin-1 (MT-1), from the green mamba Dendroaspis angusticeps, binds specifically to muscarinic M₁ receptors in homogenates of rat cerebral cortex. Iodination of the toxin, ¹²⁵I-muscarinic toxin-1 (¹²⁵I-MT-1), renders the toxin selective for M₁ muscarinic receptors. Quantitative measurement of ¹²⁵I-MT-1 autoradiography in rat brain sections indicated highest labeling in the nucleus accumbens, striatum, and dentate gyrus. High densities of ¹²⁵I-MT-1 binding sites were located in the CA1 region of the hippocampus, frontal, and parietal cortices. Moderate densities of binding sites were seen in temporal cortex, and hippocampal subregions CA2, CA3, and CA4, whereas low labeling was observed in the cerebellum and spinal cord.     

Electrophysiological responsiveness to isoproterenol in rat hippocampal slices correlates with changes in beta-adrenergic receptor density induced by chronic morphine treatment

The effects of chronic morphine treatment and morphine withdrawal on beta-adrenergic receptor density and electrophysiological responsiveness in rat hippocampus were examined. Chronic treatment of rats with morphine for 14 days resulted in a 19% increase in the number of beta-adrenergic receptors in hippocampus, as measured by the binding of the specific antagonist [3H]dihydroalprenolol (DHA). In comparison, the number of specific binding sites for [3H]DHA was decreased 27% in hippocampus in morphine-withdrawn animals, compared to saline-treated controls. These alterations in beta-adrenergic receptor density were not accompanied by a significant change in the dissociation constant (Kd) for [3H]DHA or in the inhibitory constants (Ki) for the displacement of the [3H]-antagonist by either norepinephrine or isoproterenol. Electrophysiological experiments in the in vitro hippocampal slice preparation revealed that responses to threshold as well as maximal concentrations of isoproterenol were significantly enhanced in morphine-dependent animals, compared to controls, whereas electrophysiological responsiveness to maximal concentrations of isoproterenol was decreased in slices from morphine-withdrawn rats. The results of this study indicate that beta-adrenergic receptors in hippocampus are up-regulated during the development of morphine dependence and down-regulated during opiate withdrawal. These changes in hippocampal beta-adrenergic receptor density are likely to be of functional relevance since they are manifested in a corresponding increase and decrease, respectively, in electrophysiological responsiveness to an exogenously administered beta-adrenergic receptor agonist.     

Ceramide is involved in alcohol-induced neural proliferation****

Prenatal alcohol exposure, especially during early pregnancy, can lead to fetal alcohol syndrome. The pharmacological and toxicological mechanisms of ethanol are related to the effects of ceramide. In this study, we established an alcohol exposure model in wild-type mice and in knockout mice for the key enzyme involved in ceramide metabolism, sphingomyelin synthase 2. This model received daily intragastric administration of 25% ethanol, and pups were used at postnatal days 0, 7, 14, 30 for experiments. Serology and immunofluorescence staining found that ethanol exposure dose-dependently reduced blood sphingomyelin levels in two genotypes of pups, and increased neural cell proliferation and the number of new neurons in the hippocampal dentate gyrus. Western blot analysis showed that the relative expression level of protein kinase C α increased in two genotypes of pups after ethanol exposure. Compared with wild-type pups, the expression level of the important activator protein of the ceramide/ceramide-1-phosphate pathway, protein kinase C α, was reduced in the hippocampus of sphingomyelin synthase 2 knockouts. Our findings illustrate that ceramide is involved in alcohol-induced neural proliferation in the hippocampal dentate gyrus of pups after prenatal ethanol exposure, and the mechanism may be associated with increased ex-pression of protein kinase C α activating the ceramide/ceramide-1-phosphate pathway.