Developmental neurotoxicity of ethanol: in vitro inhibition of muscarinic receptor-stimulated phosphoinositide metabolism in brain from neonatal but not adult rats

The in vitro effects of ethanol (EtOH) on muscarinic receptor-stimulated phosphoinositide metabolism were measured in cerebral cortex slices of adult and 7-day-old rats. EtOH (500 mM) caused a significant decrease (32-43%) of maximal accumulation of [3H]inositol phosphates (InsPs) induced by carbachol, and a 2-fold increase in its EC50 in 7-day-old rats, but had no effect in adult rats. The effect of EtOH on [3H]InsPs accumulation in neonatal rats was significant at a concentration as low as 150 mM. The inhibitory effect of EtOH was maximal in cerebral cortex and hippocampus and lower in cerebellum, while no effect was observed in the brainstem. While carbachol- and acetylcholine-stimulated phosphoinositide metabolism were inhibited by EtOH, EtOH had no effect on norepinephrine-, histamine-, and serotonin-stimulated phosphoinositide hydrolysis. These results are qualitatively and quantitively similar to those previously found following in vivo administration of EtOH to developing and to adult rats, suggesting that the muscarinic receptor-stimulated phosphoinositide metabolism might represent a target for EtOH-induced developmental neurotoxicity.     

Inhibition of muscarinic receptor- and G-protein-dependent phosphoinositide metabolism in cerebrocortical membranes from neonatal rats by ethanol.

Phosphoinositide (PtdIns) metabolism activated by cholinergic muscarinic receptors appears to play a role in brain development and has been recently suggested as a possible biochemical target for the developmental neurotoxicity of ethanol (EtOH). Recent experimental evidence indicates that, in rat brain, muscarinic receptor stimulation is coupled to PtdIns hydrolysis through regulatory GTP-binding proteins (G-proteins). We investigated the effect of various alcohol concentrations (10-500 mM) on guanine nucleotide-, fluoride-, and muscarinic-dependent PtdIns hydrolysis in [3H]inositol-labelled cerebral cortical membranes from neonatal (7-day-old) and adult rats. At both ages, EtOH exerted slight inhibitory effects on GTP(S) (100 microM)- and NaF (5 mM)-induced [3H]inositol phosphates accumulation. The presence of GTP(S) was necessary to unmask the stimulatory effect of the muscarinic agonist carbachol. Under these experimental conditions EtOH markedly inhibited carbachol (100 microM)-induced PtdIns hydrolysis. This effect was concentration-dependent and was more pronounced in the cortex from immature animals, where a statistically significant inhibition was observed at EtOH concentrations as low as 50 mM, comparable to the hematic concentrations reached following in vivo administration of doses of ETOH able to induce developmental neurotoxicity. These results confirm that EtOH exerts an age-specific inhibition of muscarinic-dependent PtdIns metabolism and suggest that this action might be exerted through an interaction with receptor-G-protein coupling.     

RNA metabolism in subcellular fractions from rat cerebral cortex and from the visual cortex of rats with retinal degeneration

The metabolism of RNA in subcellular fractions from rat cerebral cortex was studied. The following fractions were prepared initially: nuclei (purified through hypertonic sucrose), myelin, mitochondria, synaptosomes, microsomes, and supernatant. The proportion of RNA in each fraction was approximately 10, 3.4, 9.5, 10.1, 33.5, and 24.9%, respectively. This contrasted with the distribution of protein which was 4, 4.4, 20.1, 13.8, 19.5, and 26.5%, respectively. DNA was primarily nuclear but 1–2% was in the mitochondrial fraction. The synaptosomes were further fractionated by osmotic shock and gradient centrifugation and it was found that over 40% of the RNA was associated with the synaptosomal mitochondria and the bulk of the remainder associated with the membrane fraction (characterized by their high Na⁺-K⁺-ATPase, glutamine synthetase, and p-nitrophenyl phosphatase activity). Labeling studies using [6-¹⁴C] orotic acid revealed that the nuclear fraction was the most rapidly labeled, reaching a maximum RNA specific activity within 2 hr and decreasing thereafter. The cytoplasmic fractions all reached a maximum specific activity within 48 hr and had the same specific activity as the nuclear fraction α after 72 hr. The supernatant fraction was the most rapidly labeled of the cytoplasmic fractions, the synaptosome fraction the least. However there was no delay in the labeling of the synaptosome fraction. Initially, labeled synaptosomal RNA was labeled in the synaptosomal supernatant but after 48 hr the bulk of the label was associated with the RNA in the synaptosomal membranes. When the nuclear fraction was further separated into its neuronal and glial components it was found that the incorporation of [¹⁴C] orotic acid was approximately three times greater in the neuronal fraction after 2–4 hr but difference was apparent before 0.5 hr or after 24 hr. The metabolism of RNA in the subcellular fractions of the visual cortex of blind and sighted rats was found to differ significantly, the former having a slower rate of transfer of RNA from nucleus to cytoplasm. The difference in the incorporation rates of [¹⁴C] orotic acid into neuronal and glial nuclei was also less in the blind visual cortex. The RNA metabolism in the subcellular fractions of the frontal cortex of blind and sighted rats did not differ.     

Astrocytic and microglia cells reactivity induced by neonatal administration of glutamate in cerebral cortex of the adult rats.

Recent studies confirm that astrocytes and neurons are associated with the synaptic transmission, particularly with the regulation of glutamate (Glu) levels. Therefore, they have the capacity to modulate the Glu released from neurons into the extracellular space. It has also been demonstrated an intense astrocytic and microglia response to physical or chemical lesions of the central nervous system. However, the persistence of the response of the glial cells in adult brain had not been previously reported, after the excitotoxic damage caused by neonatal dosage of monosodium glutamate (MSG) to newborn rats. In this study, 4 mg/g body weight of MSG were administered to newborn rats at 1, 3, 5, and 7 days after birth, at the age of 60 days the astrocytes and the microglia cells were analyzed with immunohistochemical methods in the fronto-parietal cortex. Double labeling to glial fibrillary acidic protein (GFAP) and BrdU, or isolectin-B(4) and BrdU identified astrocytes or microglia cells that proliferated; immunoblotting and immunoreactivity to vimentin served for assess immaturity of astrocytic intermediate filaments. The results show that the neonatal administration of MSG-induced reactivity of astrocytes and microglia cells in the fronto-parietal cortex, which was characterized by hyperplasia; an increased number of astrocytes and microglia cells that proliferated, hypertrophy; increased complexity of the cytoplasm extension of both glial cells and expression of RNAm to vimentin, with the presence of vimentin-positive astrocytes. This glial response to neuroexcitotoxic stimulus of Glu on the immature brain, which persisted to adulthood, suggests that the neurotransmitter Glu could trigger neuro-degenerative illnesses.     

Glutamate metabolism in cerebral cortex obtained from chronic hepatic failure rats

The present investigation was carried out in order to elucidate the amino acid metabolism in hepatic failure with particular emphasis placed on glutamate. For this purpose, chronic hepatic failure models were produced in adult male Wistar rats by successive carbontetrachloride injection (0.20 ml/100 g. B. W., twice/week) for 13 weeks. They were confirmed to develop chemical changes compartible with hepatic failure, showing markedly elevated serum levels of NH3, GOT and ALP. Animals were killed by decapitation during fasting and the brains were removed immediately. After the parietal cortical slices were incubated for 45 min at 37 degrees C together with L-(U-14C) glutamate in O2-saturated Gey's balanced salt solution, they were homogenized in 75% ethanol and deproteinized with water saturated chloroform. The radioactivities of liberated CO2, glutamate and its metabolites (glutamine, aspartate and GABA) obtained from the slices were measured. The amount of radioactivity recovered from CO2, glutamine and aspartate revealed a significant increase (p less than 0.001), while that of glutamate and GABA remained unchanged. The main source of the CO2 is believed to originate from TCA cycle rather than the decarboxylation of glutamate to form GABA, and glutamate forms glutamine when it fixes ammonia. Furthermore, glutamate is converted into aspartate via TCA cycle when the carbon was labeled. Therefore, the results indicate that in chronic hepatic failure brains glutamate metabolism is enhanced through TCA cycle as well as ammonia fixation mechanism.     

Pre- and postsynaptic muscarinic receptors in surgical samples from human cerebral cortex

The present study was carried out using fresh surgical material from human cerebral cortex of patients who were not medicated with atropine or other drugs known to affect the cholinergic system. The concentration of [3H]L-quinuclidinyl benzilate binding sites was 0.45 /+- 0.05 pmol/mg protein and the Kd-value of the receptor-[3H]L-QNB-complex was 0.038 /+- 0.005 nM. Agonist binding was studied by varying the concentration of carbamylcholine (10(-8) to 10(-2) M) in the presence of a constant concentration (0.2nM) of [3H]L-quinuclidinyl benzilate. The data revealed the existence of two populations of binding sites for carbamylcholine with different affinities and capacities. Presynaptic muscarinic receptors were studied in slices of the cerebral cortex, which were loaded with [3H]choline. The muscarinic antagonist, atropine (10(-6) and 10(-7) M) acting at the presynaptic muscarinic receptors enhanced the release of [3H] acetylcholine. It was also shown that muscarinic stimulation leads to elevation of cyclic GMP levels in the human cerebral cortical slices.     

Competitive inhibition of phosphoinositide hydrolysis by the muscarinic receptor antagonist AF-DX 116 is of low affinity in mouse cerebral cortex

Carbamylcholine stimulated [³H]inositol phosphate accumulation in mouse cerebral cortical slices with an ED₅₀ value of approximately 70 μM. Increasing concentrations of the M₂ selective muscarinic cholinergic receptor antagonist, AF-DX 116 (0.3–3.0 μM), produced parallel shifts to the right for concentration-response curves to carbamylcholine. A pA₂ value for AF-DX 116 of 6.5 (low affinity) was obtained frommSchild plot analysis. It is concluded that the M₂ muscarinic receptor subtype, as defined by high affinity [³H]AF-DX 116 radioligand binding, is not appreciably coupled to polyphosphoinositide hydrolysis in the mouse cerebral cortex.     

Messenger RNAs coding for receptors and channels in the cerebral cortex of adult and aged rats.

Poly(A)+ mRNAs from the cerebral cortex of aged (24 months) and young adult (3 months) rats were isolated and injected into Xenopus oocytes to express functional neurotransmitter receptors and voltage-operated channels. Electrophysiological recordings of induced membrane currents were used as a measure of the relative amounts of mRNA encoding different receptors and channels, and to study their functional properties. There were no large differences apparent between mRNAs from aged and adult rats, in marked contrast to the dramatic (1000-fold) changes in mRNA expression that occur during embryonic and postnatal development. The membrane currents induced by glutamate or acetylcholine (ACh) application were roughly one third smaller in oocytes injected with mRNA from aged cerebral cortex than in oocytes injected with mRNA from adult cerebral cortex, whereas currents induced by gamma-aminobutyric acid (GABA), kainate or serotonin (5-HT) application, and by activation of voltage-operated Na+ and Ca2+ channels were not significantly different. We did not observe any age-related differences in the properties of the receptors and channels studied.     

Phosphatidic acid and diacylglycerol generation is regulated by insulin in cerebral cortex synaptosomes from adult and aged rats

Insulin receptor associated with the cerebral cortex (CC) has been shown to be involved in brain cognitive functions. Furthermore, deterioration of insulin signaling has been associated with age-related brain degeneration. We have reported previously that aging stimulates phospholipase D/phosphatidate phosphohydrolase 2 (PLD/PAP2) pathway in CC synaptosomes from aged rats, generating a differential availability of their reaction products: diacylglycerol (DAG) and phosphatidic acid (PA). The aim of this work was to determine the effect of aging on DAG kinase (DAGK), as an alternative pathway for PA generation, and to evaluate the effect of insulin on PLD/PAP2 pathway and DAGK. PLD, PAP2, and DAGK activities were measured using specific radiolabeled substrates in CC synaptosomes from adult (4 months old) and aged rats (28 months old). In adult animals, in the presence of the tyrosine phosphatase inhibitor (sodium o-vanadate), insulin stimulated PLD activity at 5 min incubation. DAGK activity was also increased at the same time of incubation and PAP2 was inhibited. In aged animals, PLD activity was not modified by the presence of insulin plus vanadate, PAP2 was inhibited, and DAGK was stimulated by the hormone. Insulin, vanadate, and the combination of both induced protein tyrosine phosphorylation in adult CC synaptosomes. Aged rats showed a lower level of protein phosphorylation with respect to adult rats. Our results show that insulin modulates PA and DAG availability through the regulation of PLD/PAP2 and DAGK pathways in adult rat CC synaptosomes. Additionally, we demonstrated that PA and DAG generation is regulated differentially by insulin during aging.     

Organization of adult motor cortex representation patterns following neonatal forelimb nerve injury in rats

Somatotopic representation patterns in the motor cortex (MI) of rats that had a unilateral forelimb amputation on the first postnatal day were examined after 2-4 months of survival. Intracortical electrical stimulation and recording techniques were used to map the somatic representation in MI and in the somatic sensory cortex (SI). In normal rats, vibrissa, forelimb, and hindlimb areas comprise the bulk of the MI representation. Stimulation within the forelimb area elicits elbow, wrist, or digit movements at the lowest current intensities. The proximal limb representation appears to be contained within the distal forelimb area, since shoulder movements are nearly always evoked by stimulating at higher current intensities at some distal forelimb sites. In agreement with previous studies, the distal forelimb representation overlapped the adjacent part of the granular SI cortex. Following removal of the forelimb at birth, 3 novel features of MI organization were observed. First, the areas from which stimulation evoked movements of the vibrissa or the shoulder musculature were larger than normal. Stimulation thresholds were lower than those required for comparable movements in normal rats throughout these areas, suggesting that nerve section had not simply unmasked a high-threshold representation. Second, vibrissa movements were more commonly paired with movements of the proximal forelimb muscles at the same site. Third, stimulation in the adjacent granular SI cortex failed to evoke shoulder or trunk movements, although receptive-field mapping in this region showed that cells were responsive to cutaneous stimulation of the trunk and shoulder region. These results indicate that several organizational features develop differently in MI following perinatal nerve injury: certain remaining muscle groups have enlarged cortical representations, there is a strengthening of some normally weak connections from MI to the proximal musculature, and muscles are grouped in unusual combinations. These data demonstrate that the formation of MI representation patterns is strongly influenced by nerve injury during the perinatal period.     

Distributive profiles of free and protein-incorporated tryptophan and valine in cerebral cortex slices from adult and 2-day-old rats

The distribution of the specific radioactivity and the incorporation into protein of [³H]-tryptophan and [³H]valine at varying layers from surface to centre were measured in incubated slices of cerebral cortices from infant and adult rats. Specific radioactivity in free amino acids was in both age groups highest in the intact surface layer. Incorporation of tryptophan into protein was even in slices from adult rats but much less than the average in the surface layers in slices from infant rats. Incorporation of valine exhibited similar heterogeneity in both age groups. The results suggest in brain slice preparations a zonal compartmentation of amino acid and protein metabolism which varies for different amino acids.     

Influence of the acute stress on agonist-stimulated phosphoinositide hydrolysis in the rat cerebral cortex.

1. The present study was carried out in order to elucidate the influence of the acute stress on alpha 1-adrenergic, serotonin-2 (5-HT2) and muscarinic cholinergic (M-Ach) receptors-mediated phosphoinositide (PI) hydrolysis in rat cerebral cortex slices. 2. In rat cerebral cortex slices, noradrenaline (NA), serotonin (5-HT) and carbachol stimulated [3H]inositol-monophosphate (IP1) accumulation in a concentration-dependent manner. 3. The forced swimming test (FST) for 15 min induced a significant reduction of 5-HT-stimulated [3H]IP1 accumulation, but this stress situation did not produce a significant alteration of NA- and carbachol-stimulated [3H]IP1 accumulation. 4. The FST for 15 min did not affect the density and affinity of alpha 1-adrenergic, 5-HT2 and M-Ach receptors. 5. In a mild acute stress situation, the intracellular signal transduction mediated by 5-HT was promptly inhibited as compared to the signal transduction mediated by NA or carbachol. This inhibition may be induced by an acute uncoupling of 5-HT2 receptor-mediated intracellular signal transduction.     

NMDA and muscarinic blockade in the perirhinal cortex impairs object discrimination in rats.

To determine the possible involvement of NMDA and muscarinic activation of the perirhinal cortex in object discrimination, an NMDA antagonist, D,L-2-amino-5-phosphonopentanoic acid (AP5), and a muscarinic antagonist, scopolamine (SCP) were injected into the perirhinal cortex of rats. Each drug at the higher dose (AP5 60 mM, SCP 80 mM) significantly decreased correct choices on the retention test of object discrimination. SCP, but not AP5, also significantly increased response latency, but this increase was not necessarily related to the time spent for a choice. These results suggest that activation of both NMDA and muscarinic receptors contributes to object discrimination.     

Gestational exposure to methylmercury alters neurotrophin- and carbachol-stimulated phosphatidylinositide hydrolysis in cerebral cortex of neonatal rats

Neurotrophin-stimulated signal transduction through the Trk receptors has been implicated in the development and survival of the nervous system. Phospholipase Cy (PLCy) is an early downstream effector for the Trk receptors, and catalyzes the hydrolysis of phosphatidylinositides (PIs) to inositol phosphates (IPs) and diacylglycerol. The current study demonstrated that PI hydrolysis can be used as a measure of Trk stimulation in slices from neonatal rat brain, and examined changes in the ontogeny of neurotrophin-stimulated PI hydrolysis in animals exposed to MeHg during gestation. Neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) stimulated PI hydrolysis in neocortical and cerebellar slices from neonatal rats in a concentration-dependent manner (30–1000 ng/ml). The neurotrophin-stimulated PI hydrolysis was completely blocked by K-252a, a compound known to inhibit Trk autopho-sphorylation. To examine the effects of MeHg on PI hydrolysis, Long-Evans dams were dosed p.o. on gestational days 6–15 with 0 or 2 mg/kg/day MeHg dissolved in saline. Pups were sacrificed on postnatal days (PND) 1, 4, 10, 14, and 21 and brain slices prepared from the neocortex and cerebellum. Neurotrophin-stimulated PI hydrolysis was highest on PND 1–4 and decreased with age in slices from both regions. Prior exposure to MeHg had no effect on NT-3 or BDNF-stimulated PI hydrolysis in the cerebellum; however, in the neocortex carbachol-stimulated PI hydrolysis and NT-3-stimulated PI hydrolysis were decreased on PND 1. In addition, NT-3-stimulated PI hydrolysis was increased on PND 14 compared to controls. Nerve growth factor (NGF), which had no effect in controls, increased PI hydrolysis in MeHg exposed animals. Acute exposure to 10 μM MeHg increased basal PI hydrolysis in cortical slices and increased NT-3- and BDNF-stimulated PI hydrolysis in slices from the cerebellum. These data indicate that gestational exposure to MeHg can alter neurotrophin signaling in the neocortex at early postnatal times.     

Gestational exposure to methylmercury alters neurotrophin- and carbachol-stimulated phosphatidylinositide hydrolysis in cerebral cortex of neonatal rats

Neurotrophin-stimulated signal transduction through the Trk receptors has been implicated in the development and survival of the nervous system. Phospholipase Cgamma (PLCgamma) is an early downstream effector for the Trk receptors, and catalyzes the hydrolysis of phosphatidylinositides (PI) to inositol phosphates (IPs) and diacylglycerol. The current study demonstrated that PI hydrolysis can be used as a measure of Trk stimulation in slices from neonatal rat brain, and examined changes in the ontogeny of neurotrophin-stimulated PI hydrolysis in animals exposed to MeHg during gestation. Neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) stimulated PI hydrolysis in neocortical and cerebellar slices from neonatal rats in a concentration-dependent manner (30-1000 ng/ml). The neurotrophin-stimulated PI hydrolysis was completely blocked by K-252a, a compound known to inhibit Trk autophosphorylation. To examine the effects of MeHg on PI hydrolysis, Long-Evans dams were dosed p.o. on gestational days 6-15 with 0 or 2 mg/kg/day MeHg dissolved in saline. Pups were sacrificed on postnatal days (PND) 1, 4, 10, 14, and 21 and brain slices prepared from the neocortex and cerebellum. Neurotrophin-stimulated PI hydrolysis was highest on PND 1-4 and decreased with age in slices from both regions. Prior exposure to MeHg had no effect on NT-3 or BDNF-stimulated PI hydrolysis in the cerebellum; however, in the neocortex carbachol-stimulated PI hydrolysis and NT-3-stimulated PI hydrolysis were decreased on PND 1. In addition, NT-3-stimulated PI hydrolysis was increased on PND 14 compared to controls. Nerve growth factor (NGF), which had no effect in controls, increased PI hydrolysis in MeHg exposed animals. Acute exposure to 10 micro M MeHg increased basal PI hydrolysis in cortical slices and increased NT-3- and BDNF-stimulated PI hydrolysis in slices from the cerebellum. These data indicate that gestational exposure to MeHg can alter neurotrophin signaling in the neocortex at early postnatal times.     

Characterization of agonist and antagonist binding to muscarinic cholinergic receptors solubilized from rat cerebral cortex

Summary The muscarinic acetylcholine receptor was solubilized from rat brain cortex by the zwitter-ionic detergent, 3-((3-cholamidopropyl)dimethylamino)1-propane sulfonate (CHAPS). The supernatant, after centrifugation at 100,000 × g, was shown to contain molecules with binding sites for both³H-pirenzepine (³H-PZ) and³H-(-) quinuclidinyl benzilate (³H-QNB). Maximum binding values for³H-PZ and³H-QNB binding to solubilized receptors were approximately 176±24 pmol/g and 370±53 pmol/g of protein, respectively. The K_d values for³H-PZ and³H-QNB binding to solubilized receptors were 27±6.3nM and 0.17±0.03 nM, respectively. The rank order of potencies of muscarinic drugs, in terms of their ability to inhibit binding of either³H-PZ or³H-QNB, was atropine > pirenzepine > oxotremorine > carabachol. Pirenzepine inhibited³H-QNB binding with a Hill coefficient of 0.77, but inhibited³H-PZ with a Hill coefficient of 0.94. Hill coefficients for agonists were less than 1. These findings indicate that muscarinic receptors solubilized from rat brain cortex retain their abilities to interact selectively with muscarinic receptor agonists and antagonists.     

A selective decrease of cholinergic muscarinic receptors in the visual cortex of adult rats following developmental lead exposure

The effects of low-level developmental lead (Pb) exposure (postnatal days 0–21) on the binding of [³H]quinuclidinylbenzilate (QNB) and on acetylcholinesterase (AChE) activity in the retina, superior colliculus, lateral geniculate nucleus and visual cortex (VC) were studied in the adult rat. Maximal blood and tissue Pb concentrations (50–60 μg%) were reached on day 21 and decreased to control levels (4–5 μg%), except in the retina (12 μg%) and VC (18 μg%; 0.87 μM), by day 90. A large decrease in [³H]QNB binding (−38%) and AChE activity (−29%) was found only in the VC of Pb-exposed rats. Scatchard plots of saturation binding data revealed a decrease in the density (B_max), but not in the affinity (K_d), of the muscarinic receptors. Pb (10⁻⁴−10⁻⁹M) had no effect on [³H]QNB binding or AChE activity in VC membrane preparations from control rats. The mechanism accounting for this selective decrease of cholinergic muscarinic receptors in the VC is presently unknown. These results, in combination with those from our psychophysical and pharmacological studies demonstrating a scopolamine supersensitivity in Pb-exposed rats, suggest that the long-term effects of developmental Pb exposure are due to a direct action of Pb on visual cortex cholinergic neurons.     

Ultrastructure of focal cerebral cortex tissue from rats with focal cortical dysplasia

BACKGROUND： Developing a model of focal cortical dysplasia in microgyrus and observing the ultrastructure of focal tissue is of important significance for analyzing the pathology of cortical developmental disorder and the factors of structural changes. OBJECTIVE： This study was to observe the pathological characteristics of focal tissue around the microgyrus of rats with cortical developmental disorder using an electron microscope, so as to analyze the causes associated with cerebral cortical developmental disorder. DESIGN： A randomized controlled animal experiment. SETTING： The First Affiliated Hospital of Chongqing Medical University. MATERIALS： This study was carried out in the Chongqing Key Laboratory of Neurology, Room for Electron Microscope of Chongqing Medical University, and Laboratory Animal Center, Research Institute of Surgery, Daping Hospital, Third Military Medical University of Chinese PLA between January 2004 and August 2006. Eighteen healthy newborn male Wistar rats, weighing 3.0 - 6.0 g, provided by the Laboratory Animal Center, Daping Hospital, Third Military Medical University of Chinese PLA, were involved in this study. The protocol was carried out in accordance with animal ethics guidelines for the use and care of animals. Probes （Chongqing Wire ＆ Cable Factory, China） were made of copper core wire with diameter of 1mm. METHODS： The rats were randomly divided into 3 groups with 6 in each： normal control group, liquid nitrogen injured group and sham-operation group. （1）In the liquid nitrogen injured group, a blunt probe frozen by liquid nitrogen was placed on fronto-parietal crinial bone of rats for 8 s. A 3 - 5 cm of microgyrus was induced in the unilateral cerebral sensory cortical area. In the sham-operation group, probe was placed at the room temperature. In the normal control group, rats were untouched. （2） The conscious state and electrical activity of brain of rats in each group were observed. （3） 2 - 3 mm thickness of hippocampal tissue with coronary section was taken for observing its ultrastructure under a transmission electron microscope. MAIN OUTCOME MEASURES： （1） The ultrastructure of hippocampal tissue. （2）The conscious state and electrical activity of brain of rats. RESULTS： Eighteen rats were enrolled in the final analysis. （1） Observation of hippocampal ultrastructure： Electromicroscopic pathological findings showed that for each rat of the liquid nitrogen injured group, mitochondrium in the pyramidal neuron around the microgyrus was swelled, endoplasmic reticulum was expanded, glial cells were swelled, water gathered around the blood capillary, partial medullary sheath was degenerated, neuropilem was normal and no obviously abnormal synapse was found. （2） Changes in conscious state of rats： Rats in the normal control group and sham-operation group had no convulsive seizure, but those in the liquid nitrogen injured group had occasionally. Most of them showed increased activities, excitation and restlessness, scratching and frequent ＂ watching face-like activities＂. （3）Electrical activity of brain of rats： Electroencephalogram recording of liquid nitrogen injured group showed that small wave amplitude of rhythm took the main part. No typical sharp wave, V wave, sharp and slow wave, V and slow waves were discharged. CONCLUSION： Liquid nitrogen can lead to cerebral cortical developmental disorder. Pathological changes of ultrastructure of focal tissue around the microgyrus can provide pathological basis for epilepsy associated with focal cortical developmental disorder.