激活大鼠右側(cè)尾殼核重建雙側(cè)海馬癲癇網(wǎng)絡(luò)的細(xì)胞機制

刁芳明1 ,韓丹1,尹世金2,甘麗1,，鄒祖玉3

 (1.武漢大學(xué)醫(yī)學(xué)院生理學(xué)教研室，湖北 武漢430071,；2.中南民族大學(xué)生物醫(yī)學(xué)工程研究所,，武漢430074；3.武漢大學(xué)醫(yī)學(xué)院病理學(xué)與病理生理學(xué)教研室,，湖北 武漢430071)

摘要：目的　探討強直電刺激大鼠右側(cè)尾殼核（right caudate putamen,，RCPu）重建雙側(cè)海馬（hippocampus, HPC）癲癇電網(wǎng)絡(luò)的細(xì)胞機制。方法　實驗共用雄性SD大鼠101只,，體重200~250g,。急性強直電刺激（60Hz,，2s，0.4~0.6mA）RCPu (acute tetanization of the right caudate putamen,，ATRC)或右背HPC(acute tetanization of the right dorsal hippocampus,，ATRDH), 同步記錄雙側(cè)前背HPC神經(jīng)元單位放電，比較激活RCPu或RHPC時,，神經(jīng)通路長度對HPC癲癇電網(wǎng)絡(luò)重建中單個神經(jīng)元電活動的影響。結(jié)果?。?）ATRC 和ATRDH均能明顯地調(diào)制單個HPC神經(jīng)元的緊張式放電成為節(jié)律性爆發(fā)式放電,。（2）ATRC引起的HPC爆發(fā)式單位放電串放電時程長[（650.738±56.419 ）ms, n=120]、串間隔短[（interbursting interval, IBI, (772.600±46.665)ms, n=90）,；相反, ATRDH引起HPC的爆發(fā)式單位放電特征是串放電時程短[（270.612±19.917）ms,，n=123] (T=6.353，P<0.001),、IBI長[（1373.663±121.236）ms, n=103] (T=4.627 P<0.001),。（3）ATRC誘發(fā)的海馬細(xì)胞單位后放電時程長[（7.06±0.776）s, n=104]、潛伏期長[（8.77±1.231）trains, n=30],，而ATRDH誘發(fā)的單位后放電時程短[（3.93±0.657）s,，n=33 ] ( T=0.3079，P<0.001),、潛伏期短[（3.33±0.681）trains,，n=15 ](T=3.681，P<0.001),。（4）ATRC可以使雙側(cè)海馬神經(jīng)元單位放電或深部電圖癲癇相關(guān)性電活動趨于同步化,。結(jié)論　激活海馬-尾殼核長路徑神經(jīng)通路導(dǎo)致雙側(cè)海馬神經(jīng)元長時程癲癇相關(guān)性電活動的形成，促進海馬癲癇網(wǎng)絡(luò)的重建,，引發(fā)雙半球顳葉癲癇的發(fā)作,。而這條激活了的CPu-HPC通路在海馬癲癇網(wǎng)絡(luò)病理性神經(jīng)信息傳遞中起著重要的生物放大器作用。

關(guān)鍵詞：尾殼核,；海馬,；長時程爆發(fā)式單位放電；癲癇,；大鼠

Caudate putamen (CPu) and hippocampus (HPC), as a network of brain area, play an important role in some physiological functions [1, 2] and pathophysiological dysfunctions. For example, the decreased volume in both the CPu and the HPC was detected in patients with temporal lobe epilepsy (TLE) by using high-resolution MRI [3]. Recent studies disclosed that the development of amygdala kindling and amygdala-kindled seizures were modulated by the CPu [4]. It was also reported that striatum may be involved in the propagation pathway for epileptic seizure activity [6] and that the putamen might play a key role in dystonic posturing, one of the most reliable lateralizing symptoms for mesial TLE [5]. But there was still an argument that the basal ganglia participated only in changing or reflected changes in the distribution of the ictal epileptic activity [7]. Our previous works demonstrated that epileptic networks in bilateral hippocampi might be reestablished by tetanization of the CPu, which were involved in temporal lobe epileptogenesis[8]. Furthermore, transhemispheric CPu-HPC epileptic network was reconstructed by overactivation of the corpus callosum [9]. Up to now, very little attention has been focused on the possible cellular mechanism of epileptic network construction in bilateral hippocampi generated from the CPu dysfunction. In our present works double single unit recordings from dual hippocampi will be simultaneously performed before and after tetanization of the CPu to find some evidence to make this viewpoint more clear that functional CPu-HPC pathway might be involved in formation of epilepsy-related neuronal activity in the HPC.

1 Materials and methods

1.1 Animals and groups  101 male Sprague-Dawley rats weighing 200-250g were divided into three groups according to the tip positions of recording and stimulating electrodes: double single unit recordings from dual hippocampi and acute tetanization of the right caudate putamen (ATRC) (n=45 pairs) or acute tetanization of the right anterior dorsal hippocampus (ATRDH)(n=21 pairs); double electrographic recordings from the dual hippocampi and the ATRC (n=5 pairs). Single unit discharges in the right HPC(n=24) and the left HPC(n=6) were also recorded respectively before or after the ATRC.

1.2  Surgery   Rats were endotracheally intubated under anesthetic condition (10% urethane, 1g/kg, i.p.) and mounted on the stereotaxic apparatus (SN-3 Nihon Kohden, Japan) .The skull windows were opened. Two glass microelectrodes with the resistance of 10-20MΩand bipolar concentric stainless electrodes with the resistance of 0.2-0.3MΩwere inserted into the HPC or the CPu for single unit recordings and stimulating. The tip positions of the glass microelectrodes and bipolar concentric stainless electrode were: the anterior dorsal HPC, P, -3.0 --3.5mm, R or L, 1.5 -2.5mm, H, 2.5-3.0mm; the CPu, P, 1.2-1.6mm, R, 2.0-2.3mm, H, 3.7-4.2mm [10]. In the course of the experiment all rats were artificially ventilated after injection of pavulon (0.2mg/kg,i.p.,Netherlands Batch No.8039102),a kind of muscular relaxation drug. Animal body temperature of the rats was maintained at (37±0.5)℃.

1.3  Stimulating and recording   The ATRC or the ATRDH (60Hz, 2s, 0.4-0.6mA) was done with an electronic stimulator (SEN-7203 Nihon Kohden, Japan) via an isolator (SS102J, Nihon Kohden, Japan). In order to avoid the refractory period of brain tissue to the next tetanic train, 9-10 tetanic trains were delivered at an interval of 10 min. Single unit activities recorded extracellularly from a pair of glass microelectrodes inserted into bilateral hippocampi were respectively input into two pre-amplifiers (FZG-81, Shanghai) via two microelectrode amplifiers(MEZ-7101 and MEZ-8201, Nihon Kohden, Japan). Then these signals were displayed on a dual-beam oscilloscope (VC-10, Nihon Kohden, Japan) and taped on a videocassette with a dual sound channel cassette recorder (VR-HD 1000 PHILIPS). Two bioelectric main amplifiers (SZF-1G, Shanghai) were used to monitor auditorily the bioelectric signals. The depth electrographs recorded from a pair of bipolar concentric stainless electrodes inserted into bilateral hippocampi were inputted into two bioeletric amplifiers (621G) in an 8-channel polygraph system (RM-6008, Nihon Kohden, Japan) and plotted with an ink-writing recorder (WI-621/641G, Nihon Kohden, Japan).

1.4  Data analysis   The data on video cassette were digitized off-line with a dual-channel acquisition and analysis system (Neurolab, typeⅠ, Huazhong University of Science and Technology ,2001) [11]. The pattern and rate of cell firing in the HPC were analyzed. The distribution of interspike intervals (ISIs) of neuronal impulses was exhibited in plotted scatter graphs by using Sigmaplot software. All data were statistically analyzed with the SPSS software. The differences will be considered as significance if P <0.01.

1.5  Histological identification   At the end of the experiment the tip positions of the glass microelectrodes were marked by deposition of Pontamine sky blue through DC current (40mA, 10min). The tip positions of the bipolar concentric stainless electrode were marked with Prussian blue staining by small electrolytic lesion (0.5mA, 15s).

Finally, the rats were perfused with 0.9% saline and a fixative containing 1% potassium hexacyanoferrate in 10% formaldehyde through the left ventricle. Rat brains were postfixed in the fixative at 4℃ for 7 d and embedded in paraffin. HE stained sections were obtained to confirm the electrode placements further.

2 Results

   In our present work 162 single unit discharge hippocampal neurons were recorded from dual hippocampi. Of all 162 neurons, there were 66 pairs of neurons recorded simultaneously from dual hippocampi. There were three kinds of spontaneous neuronal firing patterns in the HPC mainly including tonic firing, bursting and firing-bursting complex, as described in our previous works [12]. Spontaneous eletrographic activities appeared high amplitude with 0.5 Hz -1Hz and maximum amplitude of 100μV in bilateral hippocampi.

 Bioelectrical activities of single neurons and networks in dual hippocampi changed after administration of the ATRC, which mainly included the following:

2.1  Long duration neuronal bursting   Cell bursting with relatively long duration (650.738±56.419ms, mean±SE, n=120) in dual hippocampi was evoked by administration of the ATRC, but cell bursting with relatively short duration was induced by administration of the same stimulation into the right HPC [( 270.612±19.917)ms, n=123]. This difference was statistically significant (T=6.353,，P<0.001). An example was shown in Fig 1. Spontaneous activity of contralateral hippocampal neurons manifested tonic firing, as was illustrated in Fig 1A and G. Fig 1B and H illustrated the bursting modulated by administration of the ATRC or ATRDH.

Fig 1  Hippocampal cell bursting modulated by administration of the ATRC and ATRDH. A and G exhibited spontaneous tonic firing of single neuron in contralateral HPC. B showed that single unit activities were modulated by the ATRC from tonic firing into rhythmic bursting with long duration. But H displayed those modulated by the ATRDH from tonic firing into bursting with short duration. D and F gave clear evidence that distribution of the ISI spots had a stratifying and clustering feature. R: The right side

In addition, the IBI [(772.600±46.665) ms, n=90] of hippocampal neuron bursting in the ATRC rats was shorter than that[(1373.663±121.236)ms, n=103] in the ATRDH rats(T=4.627, P<0.001) .

     Irregularly rhythmic bursting of ipsilateral hippocampal neuron was observed after the ATRC. Fig 2 A, B and C exhibited a gradually shortened latency of inhibitory effects on firing rate of single hippocampal neuron and irregularly rhythmic bursting following the seventh tetanic train. This phenomenon was clearly disclosed in the distribution of the ISI spots.

Fig 2 Irregularly rhythmic bursting of ipsilateral hippocampal neuron evoked by the ATRC. A, B and C showed that the tonic firing rate of this neuron was inhibited by each train of the ATRC.  Finally, firing pattern was modulated from tonic firing into irregularly rhythmic bursting after the 7th train. D, E and F showed that an increasing ISI spots of this single unit discharges shifted from the 50th second to the 34th second or to the 23rd second respectively after administration of the 5th, the 6th or the 7th train

2.2  Long duration single unit afterdischarges  The latency of primary HPC unit afterdischarges in the ATRC rats [(8.77±1.231)trains, n=30 neurons] was different from that in the ATRDH rats[ (3.33±0.681)trains, n=15 neurons]. This difference was statistically significant (T=3.681,P<0.001).

There were also single unit afterdischarges with long duration[ (7.06±0.776)s, n=104 trains] of hippocampal neuron induced by tetanization of relatively long neural pathway from the RCPu to the bilateral hippocampi. But those with short duration[(3.93±0.657)s, n=33 trains] could be induced by tetanization of intrinsic hippocampal circuit from the right dorsal HPC to ipsilateral local circuit or to contralateral dorsal HPC. The difference was statistically significant (T=0.3079，P<0.001). An example was shown in Fig 3.

Fig 3 Primary single unit afterdischarges with long or short duration in the HPC induced by the ATRC or ATRDH. A and C showed three trains of primary unit afterdischarges in ipsilateral and contralateral HPC, the mean duration of which was 19.65s after the ATRC, but B and D exhibited that was 6.56s after the ATRDH

2.3 Synchronous single unit activities   Neuronal activities in bilateral hippocampi were synchronized by administration of the ATRC. The firing patterns of these neurons included mainly tonic firing, bursting, afterdischarges and post-afterdischarges inhibition, which were involved in synchronization of neuronal firing in ipsilateral HPC with that in contralateral HPC. Fig 4 exhibited these synchronized neuronal activities in double hippocampi evoked by the ATRC and their ISI figures.

Fig 4 Synchronous tonic firing, bursting, primary afterdischarges and post-afterdischarge inhibition of single neuron in bilateral hippocampi induced by the ATRC. A, B and C illustrated that synchronous activities of single neurons in double hippocampi were evoked by the ATRC. A showed synchronous tonic firing of two neurons 10 min after the 9th train. Their ISI plots were almost symmetric. B: Single long duration bursting of two neurons were completely synchronized 10 min after the 12th train. There was no obvious stratification observed in their ISI figure because these bursting occurred occasionally. C: Synchronous primary single unit afterdischarges followed by firing rate inhibition of single neuron after the 10th train. The distribution of their ISI spots was partially symmetric

2.4  Synchronous electrographic kindling  Synchronous electrographic afterdischarges and kindling in dual hippocampi were observed after the ATRC. Fig 5A illustrated that spontaneous electrographic activities in dual hippocampi manifested reversed-phase waves at 0.6 Hz. Synchronous electrographic oscillations at 5-7 Hz were detected 4s after the first ATRC train. This ATRC rat was electrographically kindled 9 min after the 10th train.

Fig 5 Synchronous primary electrographic afterdischarges and kindling in bilateral hippocampi after administration of the ATRC. A showed synchronous primary electrographic afterdischarges after the 1st train. B: Bilateral hippocampal electrographic kindling were kindled simultaneously 9 min after the 10th train

3  Discussion

   Our experimental observations demonstrated that repetitive ATRC induced that (1) single neuronal bursting and afterdischarges with long duration in the HPC; (2) Single unit activities and eletrographic kindling synchronized in bilateral hippocampi; (3) Post-tetanizaton inhibition of neuronal firing rate followed by irregularly rhythmic busting. The results suggested that compared with the short duration bursting and afterdischarges induced by the ATPDH, long duration ones in the HPC could be evoked by overactivation of relatively long neural pathway from the RCPu to bilateral hippocampi, which was related to epilepsy.

   In the last decade, anatomic and functional relationship between the CPu and the HPC had been paid much attention to. The connections and functional influences between basal nucleus and medial temporal lobe system were successively reported. [13]. There are interactive multiple memory systems including direct anatomical projections between the HPC and the CPu in the mammalian brain [14]. The hippocampal and striatal systems both participate in learning [15]. In some complex partial epileptic patients with unilateral hippocampal atrophy, the volume of ipsilateral putamen decreased after they received temporal lobectomy [16]. Caudate stimulation brought about a significant decrease in the frequency and amplitude of hippocampal activity, the caudate-induced inhibition takes place through an anterograde caudate-hippocampal circuit [17].

   Observations derived from animal models of acute or chronic epilepsy demonstrated that the CPu was involved in temporal lobe epileptogenesis. Stimulations of caudate nucleus induced statistically significant reduction of hippocampal spike frequency, and in some cases a clear and regular theta-rhythm [18]. The semiologic progression in TLE seizures were related to the propagation of hyperperfusion from ipsilateral temporal lobe to contralateral temporal lobe and basal ganglia[19]. The evidence for involvement of the CPu and the HPC in temporal lobe epileptogenesis arose from a number of studies, including that the hippocampal CA2 region and the CPu were both damaged from status epilepticus lasting 40 min or more [20]. The caudate is able to reduce both hippocampal spike frequency and amplitude [21]. These results indicated that there might be a possible inhibitory pathway from the CPu to the HPC.  Our recent experimental evidence proved that this inhibitory neural pathway might be involved in rat hippocampal epilepsy because an inhibitory effect of the ATRC on the HPC electrographs was followed by obvious electrographic kindling, which was called as “inhibition-rebound-kindling”[8]. Analysis of our electrophysiological, behavioral, morphological and MRI observations demonstrated that the experimental temporal lobe epileptogenesis might also be the process of the reconstruction of hippocampal epilepsy networks [12,22~24], for which abnormal “driving” neural pathway could be originated from some structures outside the HPC. Our present works suggested further that tetanization-induced CPu abnormalities could promote the reconstruction of dual hippocampal epilepsy networks.

   On the other hand, multiple pattern activities of single neurons and network epileptiform activities in bilateral hippocampi might be synchronized by the ATRC. It has been noticed that primary electrographic afterdischarges could be driven by  primary unit afterdischarges, therefore, cell bursting modulated by tetanization was tightly related to high frequency network afterdischarges in the anterior dorsal HPC [25,26]. Compared with data obtained from the ATRDH rats, longer duration busting and afterdischarges of single neurons in bilateral hippocampi were detected from the ATRC rats. These results suggested that long duration epilepsy-related activities of single neurons could be induced by overactivation of relatively long neural pathway from the RCPu to double hippocampi. Activated CPu-HPC functional neural pathway might act as the amplifier of single neuron signals in bilateral hippocampi, but activated the right dorsal HPC networks not. Presumably, tetanization activated chemical synapses in the CPu-HPC pathway have the important property of amplification.

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