Δ9-Tetrahydrocannabinol and the hippocampus: Effects on CA1 field potentials in rats

The effects of Δ⁹-tetiahydrocannabinol (THC) on ortho- and antidromically elicited CA1 field potentials were observed in locally anesthetized rats and in rats anesthetized with urethane. THC augmented amplitudes of population EPSP's as well as orthodromic and antidromic population spikes from pyramidal cells in locally anesthetized animals. Latencies to peak amplitude of these responses were increased. Conditioning-test shock experiments revealed that THC also depressed recurrent inhibition probably mediated by basket cells. In animals under urethane anesthesia THC enhanced test responses, but failed to augment population responses to the conditioning stimulus. It was concluded that THC enhanced postsynaptic excitatory processes but attenuated recurrent inhibition. Urethane anesthesia completely blocked the postsynaptic excitatory effect of THC but had little apparent influence on THC's disinhibitory action.     

Endogenously released ACh and exogenous nicotine differentially facilitate long-term potentiation induction in the hippocampal CA1 region of mice

We examined the role of α7- and β2-containing nicotinic acetylcholine receptors (nAChRs) in the induction of long-term potentiation (LTP). Theta-burst stimulation (TBS), mimicking the brain's naturally occurring theta rhythm, induced robust LTP in hippocampal slices from α7 and β2 knockout mice. This suggests TBS is capable of inducing LTP without activation of α7- or β2-containing nAChRs. However, when weak TBS was applied, the modulatory effects of nicotinic receptors on LTP induction became visible. We showed that during weak TBS, activation of α7 nAChRs occurs by the release of ACh, contributing to LTP induction. Additionally, bath-application of nicotine activated β2-containing nAChRs to promote LTP induction. Despite predicted nicotine-induced desensitization, synaptically mediated activation of α7 nAChRs still occurs in the presence of nicotine and contributed to LTP induction. Optical recording of single-stimulation-evoked excitatory activity with a voltage-sensitive dye revealed enhanced excitatory activity in the presence of nicotine. This effect of nicotine was robust during high-frequency stimulation, and was accompanied by enhanced burst excitatory postsynaptic potentials. Nicotine-induced enhancement of excitatory activity was observed in slices from α7 knockout mice, but was absent in β2 knockout mice. These results suggest that the nicotine-induced enhancement of excitatory activity is mediated by β2-containing nAChRs, and is related to the nicotine-induced facilitation of LTP induction. Thus, our study demonstrates that the activation of α7- and β2-containing nAChRs differentially facilitates LTP induction via endogenously released ACh and exogenous nicotine, respectively, in the hippocampal CA1 region of mice.     

Presynaptic inhibitory action of acetylcholine in area CA1 of the hippocampus

The effect of iontophoretically applied acetylcholin (ACh) was investigated in area CA1 of transverse hippocampal slices maintained in vitro. In synaptically activated regions of the dendritic field, ACh reduced the amplitude of the population spike recorded from the pyramidal layer. In dendritic regions which were not synaptically activated, ACh increased the amplitude of the population spike or it had no effect. The depressing effect of ACh was abolished in denervated dendritic regions. The intracellularly recorded excitatory postsynaptic potential (EPSP) decreased in amplitude when ACh was applied at the synaptic site. The resting membrane potential, the time course of the EPSP, and the membrane resistance were unaffected. ACh increased the excitability of afferent fibers and this was independent of synaptic transmission. We conclude that ACh in addition to its postsynaptic effects has a presynaptic site of action.     

Kindling increases the K -evoked Ca -dependent release of endogenous GABA in area CA1 of rat hippocampus

The release of endogenous amino acids from hippocampal CA1 subslices under basal conditions and the release evoked by high potassium (50 mM K⁺) depolarization was studied during kindling epileptogenesis. Emphasis was put on the release of the amino acid neurotransmitters γ-aminobutyric acid (GABA) and glutamate. Kindling was induced by tetanic stimulation of the Schaffer-collaterals/commissural fibers of the dorsal hippocampus of the rat. The calcium-dependent GABA release in the presence of high K⁺ was significantly increased (40–46%) in fully kindled animals, 24 h after the last seizure, in comparison to controls. At long-term, 28 days after the last seizure, the calcium-dependent GABA release was still significantly increased (45–49%). An increased release of GABA in kindled animals was still found when GABA uptake was blocked by nipecotic acid. In contrast, no significant alterations were encountered in the basal or high potassium induced release of the excitatory amino acids aspartate and glutamate. These results suggest that kindling epileptogenesis is accompanied by a specific and long-lasting enhancement of GABA exocytosis which may lead to a desensitization of the GABA receptor, and thus determine the increase of seizure sensitivity.     

胶质细胞谷氨酸转运体在大鼠点燃效应中的作用研究

目的 研究点燃形成过程中和点燃后大鼠海马中氨酸天门氨酸转运体（GLAST）和谷氨酸转运体1（GLT－1）的变化,进一步探讨癫痫的形成机制。方法 将78只雄性成年Wistar大鼠随机分为对照组（I组）和戊四氮（PTZ）组（Ⅱ组）。Ⅱ组腹腔注射阈下剂量的PTZ（335mg/kg）,每日1次,直到达到点燃标准;I组腹腔注射等量生理盐水。采有RT－PCR方法检测海马区GLAST和GLT－1mRNA的表达。结果 PTZ组点燃后,GLASTmRNA的表达下降,60天时恢复至对照组;与对照组比较,PTZ组GLT－1mRNA的表达,在给药后15天时开始上升,点燃后0小时和48小时时显著升高,此后呈下降趋势。60天时,两组比较无明显差异。结论 海马区胶质细胞谷氨酸转运体的下降可能与癫痫敏感性的形成有关。     

GABAB receptor blockade enhances theta and gamma rhythms in the hippocampus of behaving rats

The participation of GABA(B) receptors in hippocampal EEG generation was studied by intracerebroventricular (icv) and intracerebral infusions of GABA(B) receptor antagonist p-(3-aminopropyl)-p-diethoxymethyl-phosphinic acid (CGP35348) in freely behaving rats. During awake-immobility, icv CGP35348 induced a theta rhythm and increased gamma waves (30-100 Hz) in the hippocampus. The immobility theta peaked at 6-7 Hz and had a theta phase in CA1 stratum radiatum of approximately 160 degrees with reference to the theta at the alveus, when compared with approximately 130 degrees during walking. Immobility theta power peaks at 6-7 Hz was also found in normal rats, and it was detected in 27% of the EEG segments during immobility. Incidence of immobility theta increased to 87.5% after 480 nmol of CGP35348 icv. Muscarinic antagonist scopolamine (5 mg/kg, ip) suppressed the induction of immobility theta and the gamma power increase after icv CGP35348. CGP35348 icv did not significantly change the hippocampal theta power at 7-8 Hz during walking (theta fundamental), but it increased power at 12-15 Hz, at the second harmonic of theta. CGP35348 icv also increased 30-50 Hz gamma power during walking. Medial septal infusion of CGP35348 (12 nmol in 0.4 microl) increased the power and the frequency of the hippocampal theta second harmonic during walking, but did not increase gamma activity. Infusion of CGP35348 (8 nmol in 0.4 microl) in the hippocampus increased the local gamma activity at 30-100 Hz, but did not induce immobility theta or affect the walking theta rhythm. In conclusion, icv GABA(B) receptor blockade increased an atropine-sensitive input that generated an immobility theta rhythm, while GABA(B) receptor blockade of the medial septum increased atropine-resistant theta harmonics possibly generated by apical dendritic spikes. GABA(B) receptor blockade may enhance cognitive task performance by activating hippocampal theta and gamma rhythms in behaving rats.     

大鼠海马星形胶质细胞对突触可塑性的影响

目的研究星形胶质细胞在神经系统发育成熟过程中对突触可塑性的调控规律.方法取健康初生、幼年和成年大鼠各10只,每只取脑切片,用免疫组织化学方法观察其海马CA1区的S100、胶质纤维酸性蛋白(GFAP)和P38免疫反应产物强度;HE染色法显示神经元胞体.结果初生大鼠海马CA1区中神经元大量存在,但S100、GFAP和P38表达均少,幼鼠的表达增加,但仍显著少于成鼠的表达(P＜0.01).结论大鼠海马CA1区星形胶质细胞增殖与突触出现时间、突触数目增加及功能成熟有关.     

Δ-Tetrahydrocannabinol and the hippocampus: Effects on CA1 field potentials in rats

The effects of Δ⁹-tetiahydrocannabinol (THC) on ortho- and antidromically elicited CA1 field potentials were observed in locally anesthetized rats and in rats anesthetized with urethane. THC augmented amplitudes of population EPSP's as well as orthodromic and antidromic population spikes from pyramidal cells in locally anesthetized animals. Latencies to peak amplitude of these responses were increased. Conditioning-test shock experiments revealed that THC also depressed recurrent inhibition probably mediated by basket cells. In animals under urethane anesthesia THC enhanced test responses, but failed to augment population responses to the conditioning stimulus. It was concluded that THC enhanced postsynaptic excitatory processes but attenuated recurrent inhibition. Urethane anesthesia completely blocked the postsynaptic excitatory effect of THC but had little apparent influence on THC's disinhibitory action.     

Chronic ethanol treatment reduces inhibition in CA1 of the rat hippocampus

The effect of chronic ethanol exposure on inhibition in the rat hippocampal slice was investigated using paired-pulse stimulation techniques with stimulation in stratum radiatum or stratum oriens of CA1. Experimental animals were fed ethanol in a liquid diet for 20 weeks and were withdrawn for at least 8 weeks prior to electrophysiological recording. Prior ethanol treatment had no effect on basic input-output relationships for the extracellular population spike. Ethanol treatment significantly reduced the recurrent inhibition produced by antidromic stimulation in a manner dependent upon stimulus intensity. In addition, with orthodromic paired-pulse stimulation of either stratum radiatum or oriens, a trend toward an augmentation of the facilitation of population spike amplitude was observed, suggesting that feedforward inhibition may also be reduced. These results are similar to those found with treatments that reduce inhibition. Therefore, we conclude that chronic ethanol exposure produces an enduring disruption of inhibitory neuronal function in the rat hippocampus.     

颞叶癫痫大鼠海马CA1区突触可塑性与空间记忆能力关系的研究

目的探讨颞叶癫痫大鼠海马CA1区突触超微结构与空间记忆能力改变的关系。方法以海人酸杏仁核微量注射建立经典的雄性Wistar大鼠颞叶癫痫化学点燃模型,分别于点燃后11d、17d、21d测定大鼠的空间记忆能力,并观察第21d海马CA1区神经毡突触超微结构变化。结果颞叶癫痫大鼠空间记忆能力减低,同时伴有海马CA1区神经毡内突触数密度降低(P<0．05),突触活性区膜面积缩小(P<0．05),突触界面曲率降低(P< 0．05),比表面减小(P<0．05),突触小泡数密度降低(P<0．05)。结论颞叶癫痫大鼠海马CA1区神经毡内突触损害和活力可能是导致其空间记忆能力下降的重要机制。     

脑卒中后抑郁大鼠海马原位增殖的新生细胞存活和分化状态

目的 观察脑卒中后抑郁(post-stroke depression,PSD)大鼠海马原位增殖新生细胞的存活和分化.方法 采用左侧大脑中动脉阻塞(MCAO)联合慢性不可预见温和应激刺激(chronicmild stress,CMS)及孤养法建立PSD模型,将雄性SD大鼠分为假手术、脑卒中、CMS和PSD组.每组均为18只.采取免疫组织化学、荧光双标染色及共聚焦成像动态检测,比较各研究组大鼠左侧海马齿状回溴脱氧尿苷嘧啶(BrdU)及其与神经元特异性核蛋白(NeuN)或胶质纤维酸性蛋白(GFAP)共表达.结果 与脑卒中组(232.2±8.6、123.7±2.6、136.2±2.6)相比,PSD组大鼠左侧(伤侧)海马齿状回BrdU+细胞数在脑梗死后第21(156.2±2.5)、30(70.2±2.0)和45天(81.2±1.1)均明显减少(t=28.83、52.2、62.08,均P<0.01),但仍高于假手术组.与脑卒中组(79.3%±2.8%、87.7%±4.6%)相比,PSD组大鼠左侧(伤侧)海马齿状回BrdU+/NeuN+细胞比例在脑梗死后第30(69.0%±3.4%)和45天(78.3%±2.4%)均明显减少(t=5.871、4.403,均P<0.01).BrdU+/GFAP+细胞比例在脑梗死后30和45 d均明显增加(t=4.226、8.945,P<0.01).结论 PSD大鼠脑卒中后海马齿状回原位增殖的新生细胞存活降低,分化为神经元的比例下降,胶质细胞比例增加.     

Impaired activation of CA3 pyramidal neurons in the epileptic hippocampus

We employed in vitro and ex vivo imaging tools to characterize the function of limbic neuron networks in pilocarpine-treated and age-matched, nonepileptic control (NEC) rats. Pilocarpinetreated animals represent an established model of mesial temporal lobe epilepsy. Intrinsic optical signal (IOS) analysis of hippocampal-entorhinal cortex (EC) slices obtained from epileptic rats 3 wk after pilocarpine-induced status epilepticus (SE) revealed hyperexcitability in many limbic areas, but not in CA3 and medial EC layer III. By visualizing immunopositivity for FosB/ΔFosBrelated proteins—which accumulate in the nuclei of neurons activated by seizures—we found that: (1) 24 h after SE, FosB/ΔFosB immunoreactivity was absent in medial EC layer III, but abundant in dentate gyrus, hippocampus proper (including CA3) and subiculum; (2) FosB/ΔFosB levels progressively diminished 3 and 7 d after SE, whereas remaining elevated (p<0.01) in subiculum; (3) FosB/ΔFosB levels sharply increased 2 wk after SE (and remained elevated up to 3 wk) in dentate gyrus and in most of the other areas but not in CA3. A conspicuous neuronal damage was noticed in medial EC layer III, whereas hippocampus was more preserved. IOS analysis of the stimulus-induced responses in slices 3 wk after SE demonstrated that IOSs in CA3 were lower (p<0.05) than in NEC slices following dentate gyrus stimulation, but not when stimuli were delivered in CA3. These findings indicate that CA3 networks are hypoactive in comparision with other epileptic limbic areas. We propose that this feature may affect the ability of hippocampal outputs to control epileptiform synchronization in EC.     

Cannabinoid receptor activation in CA1 pyramidal cells in adult rat hippocampus

Intracellular assessments of the physiological actions of cannabinoid receptor agonists and antagonists on adult hippocampal CA1 pyramidal cells in the in vitro slice preparation were performed using current clamp and conventional sharp-electrode intracellular recording procedures. Several manipulations were performed to delineate putative currents and conductance mechanisms affected by the cannabinoid receptor agonist WIN 55,212-2 (WIN-2). This compound produced a tonic hyperpolarization of the pyramidal cell membrane that was bicuculline sensitive, reversed by changing the chloride gradient, and abolished by the addition of TTX to the bathing medium. Instantaneous membrane input resistance, computed from hyperpolarizing current pulses (peak R(in)) was also reduced significantly in the presence of WIN-2 and was accompanied by enhancement of a superimposed slow depolarization that reduced steady-state R(in) (SSR(in)); both effects were resistant to barium. Intracellular perfusion of cesium acetate (CsAc) and the sodium/potassium channel blocker, QX314, each blocked the effect of WIN-2 on R(in) and SSR(in). WIN-2 also reduced input resistance calculated from depolarizing current injections (R(d)). This effect was also blocked by atropine, as well as media containing TTX or low Ca(2+). Each of the above effects of WIN-2 was blocked by the cannabinoid receptor antagonist SR141716A, showing a dependence on CB1 cannabinoid receptors. Several known pre- and postsynaptic processes in adult pyramidal cells are discussed which could be responsible for these cannabinoid-produced changes in membrane resistances.     

Valproate suppresses status epilepticus induced by 4-aminopyridine in CA1 hippocampus region

PURPOSE: We investigated the effects of valproate (VPA) on an in vivo model of status epilepticus (SE) induced by intrahippocampal application of 4-aminopyridine (4-AP). METHODS: To induce continuous epileptiform activity without a clinical component, 4-AP (100 mM) was slowly injected in the hippocampus of adult rats. Extracellular field potential from the CA1 region of the rat hippocampus was recorded to assess abnormal epileptiform activity. Once the SE seizures were induced by 4-AP, the test drug was injected. In some experiments to test the ability of a drug to prevent the induction of SE, the drug was administered before 4-AP injection. RESULTS: Intrahippocampal injection of 4-AP induced continuous epileptic activity without a clinical component that lasted >60 min. The intravenous injection of 400-600 mg/kg VPA rapidly (approximately 100 s) abolished the SE, and this effect persisted for >/=4 h in our experimental model. The intravenous injection of 100-300 mg/kg VPA did not abolish previously induced SE, but prevented the appearance of SE when applied before the induction of SE. The intravenous injection of 80 mg/kg phenytoin or carbamazepine did not abolish or prevent SE. CONCLUSIONS: We conclude that 4-AP-induced SE was suppressed by VPA at 400-600 mg/kg, whereas minor doses (100-300 mg/kg) only prevent the 4-AP-induced SE. Present results suggest the revisiting of VPA as a useful drug for the treatment of SE.     

糖尿病大鼠海马endophilin-A1和dynamin-1的表达研究

目的研究糖尿病大鼠海马组织endophilin-A1和dynamin-1的表达,以期进一步研究其与糖尿病认知功能损害的关系。方法 14只雄性SD大鼠随机分为两组:正常对照(NC)组和糖尿病(DM)组。腹腔注射链脲佐菌素55mg.kg-1制备糖尿病模型,5周后断头取材,分别进行如下实验:①Western Blot法检测海马组织endophilin-A1和dynamin-1的表达;②免疫组化法观察海马区dynamin-1的表达。用SPSS17.0软件作独立样本t检验。结果①Endophilin-A1和dynamin-1在两组大鼠海马组织中均有表达,其中DM组(0.66±0.12,p=0.004)endophilin-A1的aD值明显低于NC组(0.97±0.07),Dynamin-1在两组中的aD值差别无统计学意义(p=0.841);②免疫组化法显示海马CA3区、DG区dynamin1在各组中均有表达,差异无统计学意义(p=0.114,p=0.783)。结论糖尿病大鼠海马组织endophilin-A1表达下调,而dynamin-1表达未发现明显改变,值得进一步研究以探讨二者与糖尿病突触功能损害的关系。     

Homosynaptic long-term depression of CA3–CA3 synapses in the in vivo hippocampus

We tested the hypothesis that homosynaptic long-term depression (LTD) can be induced at the CA3–CA3 synapses in the adult, in vivo hippocampus while the CA3–CA1 synapses remain unchanged. Low-frequency conditioning stimulation of the contralateral fimbria significantly depressed the CA3 population response but did not change the simultaneously recorded CA3 response to angular bundle test stimulation. Similarly, in another group of animals, low-frequency conditioning stimulation of the contralateral fimbria depressed the CA3 synaptic response and left the collateral CA1 synaptic response unchanged. Among the possible explanations for this differential induction of homosynaptic LTD at the CA3–CA3 and CA3–CA1 synapses are differential control of intracellular calcium, differing levels of inhibition in these two regions, and the recency of `natural' long-term potentiation in the two regions.     

GABAB receptors play a major role in paired-pulse facilitation in area CA1 of the rat hippocampus

Extracellular recordings of field potentials in area CA1 of the rat hippocampal slice have been used to investigate paired-pulse facilitation. Field potentials were evoked by maximal stimulation of the Schaffer collateral/commissural fibres. The height of the population spike (PS) in stratum pyramidale (str. pyr.) and the area under the field excitatory postsynaptic potential (EPSP) following the PS in the stratum radiatum (str. rad.) were quantified. These values were used to describe the time course of paired-pulse facilitation. Facilitation of the PS was maximal 50 ms after the conditioning pulse and was present over a period of about 500 ms. However, facilitation of the late area (LA) of the field EPSP was maximal after 125 ms and had an overall duration of 1–2 s. TheN-methyl-d-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonovaleric acid (APV), had no effect on paired-pulse facilitation of either the LA or the PS. The γ-aminobutyric acid-B (GABA_B) agonist baclofen increased facilitation of the PS. This was mainly due to a reduction of the unconditioned response. Facilitation of the LA was reduced by both baclofen and the GABA_B antagonist, 2-OH-saclofen. Baclofen increased the LA of the unconditioned response, while this was unaffected by 2-OH-saclofen. The LA of facilitated responses was decreased by 2-OH-saclofen while the effect of baclofen on these responses was more complex. Baclofen reduced the LA of maximally facilitated responses, while the LA of slightly facilitated responses was increased. The results show that different mechanisms are involved in the facilitation of the LA and the PS. Furthermore, activation of GABA_B receptors makes a large contribution to paired-pulse facilitation of the field EPSP. It is also suggested that recording of extracellular fields in str. rad. in response to paired-pulse stimulation provides a simple electrophysiological model for testing the effect of agents which act at the GABA_B receptor.     

Ultrastructural investigation of the CA1 region of the hippocampus after transient cerebral ischemia in gerbils

Summary Ultrastructural damage leading to delayed neuronal death was investigated in the mid-CA1 region of the hippocampus from the stratum (str.) moleculare to oriens after transient bilateral forebrain ischemia in Mongolian gerbils. After ischemia for 5 min without recirculation, mild swelling of the peripheral part of the apical and basal dendrites was already apparent in the str. moleculare and str. oriens. Mitochondria in the dendrites were also swollen in the same area. During recirculation for 12 h to 3 days, swelling of the dendritic cytoplasm persisted with formation of microvacuoles, but swelling of mitochondria receded. Microvacuolation and loss of microtubules were also observed in the proximal part of the dendrites during this period, and swelling and disruption of internal cristae were observed in mitochondria after recirculation for 3 days. The dendrites became severely degenerated after recirculation for 4 days. In the pyramidal cell bodies, no abnormality was observed at the end of ischemia for 5 min, but disaggregation of polyribosomes and swelling of the endoplasmic reticulum were observed 12 h after recirculation. Proliferation of the endoplasmic reticulum in parallel arrays occurred after recirculation for 1 day and persisted. Severe degeneration of the pyramidal cell bodies was obvious after recirculation for 4 days. The findings observed in the present investigation suggested that the neuronal structure most vulnerable to ischemia was the peripheral part of the dendrites and postischemic neuronal damage occurred early in this part of the dendrites.Supported by the grant NS-06663 from the National Institutes of Health, U.S. Public Health Service     

M1 muscarinic receptor signaling in mouse hippocampus and cortex

The five subtypes (M1-M5) of muscarinic acetylcholine receptors signal through G(alpha)(q) or G(alpha)(i)/G(alpha)(o). M1, M3 and M5 receptors couple through G(alpha)(q) and function predominantly as postsynaptic receptors in the central nervous system. M1 and M3 receptors are localized to brain regions involved in cognition, such as hippocampus and cortex, but their relative contribution to function has been difficult to ascertain due to the lack of subtype specific ligands. A functional and genetic approach was used to identify the predominant muscarinic receptor subtype(s) mediating responses in mouse hippocampus and cortex, as well as the relative degree of spare muscarinic receptors in hippocampus. The nonselective muscarinic agonist oxotremorine-M stimulated G(alpha)(q)/11-specific GTP-gamma-35S binding in a concentration dependent manner with a Hill slope near unity in wild type mouse hippocampus and cortex. Muscarinic receptor stimulated G(alpha)(q)/11-specific GTP-gamma-35S binding was virtually abolished in both the hippocampus and cortex of M1 receptor knockout (KO) mice. In contrast, there was no loss of signaling in M3 receptor KO mice in either brain region. Muscarinic receptor reserve in wildtype mouse hippocampus was measured by Furchgott analysis after partial receptor alkylation with propylbenzylcholine mustard. Occupation of just 15% of the M1 receptors in mouse hippocampus was required for maximal efficacy of oxotremorine-M-stimulated GTP-gamma-35S binding indicating a substantial level of spare receptors. These findings support a role for the M1 receptor subtype as the primary G(alpha)(q)/11-coupled muscarinic receptor in mouse hippocampus and cortex.