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1Department of Integrative Physiology, National Institute for Physiological Sciences, Okazaki; and 2Department of Neurophysiology, Gunma University Graduate School of Medicine, Gunma, Japan
Submitted 6 October 2004; accepted in final form 10 December 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Schiller and Koerner 1971; Schiller and Stryker 1972; Sparks and May 1980; Sparks et al. 1976; Wurtz and Goldberg 1972). Previous studies have suggested that the burst activity is attributed to local excitatory interactions by recurrent collaterals of deep layer neurons (Bozis and Moschovakis 1998; Moschovakis et al. 1988a,b).
In our previous studies, using whole cell patch-clamp technique in rat brain stem slice preparations, we demonstrated the presence of local excitatory interactions among a large number of SGI neurons that communicate through excitatory recurrent connections (Isa et al. 1998; Saito and Isa 2003, 2004). Recruitment of these neurons to excitatory interactions was dependent on both activation of N-methyl D-aspartate (NMDA) receptors and release from GABAergic inhibition (Isa et al. 1998; Saito and Isa 2003). In support of this view, application of 
-aminobutyric acid-A (GABAA)–receptor antagonist [10 µM bicuculline (Bic) or 10 µM SR95531], and reduction of extracellular Mg2+ concentration (to 0.1 mM) induced spontaneous repetitive burst discharges that occurred almost simultaneously between pairs of SGI neurons, which was demonstrated by dual whole cell recordings from 2 adjacent SGI neurons (separated by <100 µm; Saito and Isa 2003). Blocking of action potentials by intracellular application of a Na+ channel blocker (QX314) revealed that spontaneous, subthreshold membrane depolarizations, which were highly synchronous between 2 recorded SGI neurons, occurred frequently (Saito and Isa 2004). The depolarizations were abolished by application of tetrodotoxin, which confirmed their inductions through synaptic interactions. In contrast to the findings in SGI neuron pairs, spontaneous depolarizations occurred less frequently and less synchronously in neuron pairs in the superficial gray layer [stratum griseum superficiale (SGS)]. Analysis of the relationship between the synchronicity of spontaneous depolarization and the distance between 2 recorded neurons revealed that synchronous depolarizations occurred extensively in the SGI but less frequently in the SGS. SGS neuron pairs exhibited synchronous depolarization only when their distance was <100 µm (Saito and Isa 2004). Furthermore, pairs of wide-field vertical (WFV) cells in the optic layer [stratum opticum (SO)] showed spontaneous depolarizations extensively and synchronously in contrast to SGS neuron pairs, although they were less extensive and synchronous than seen in SGI pairs. Taken together, these data suggest that lateral excitatory connections in the SC represent a layer-specific distribution and that excitatory connections are more intense in the SGI than in the superficial layers.
Although the highly synchronous depolarization of SGI pairs induced in the presence of Bic plus low Mg2+ is most likely the result of the synchronous activation of a large number of SGI neurons communicating through excitatory connections, the key question as to the source of this synchronous depolarization of SGI neurons still remains. The answer to this question would clarify the signal responsible for generation of burst discharges across local circuits within the SC. A class of neurons in the mesencephalic reticular formation (MRF), which is located ventral to the SC and therefore likely to be included in the slices used in our study, projects to the deeper layers of the SC in rats (Vertes and Martin 1988) and in squirrel monkeys [reticulotectal long-lead burst neurons (RTLLBs); Moschovakis et al. 1988b]. This finding raises the possibility that trigger signals for synchronous depolarization of SGI neurons come from outside the SC. On the other hand, local injection of biocytin into the SGI has revealed that extensive axonal connections are mostly confined to the SGI in cats (Behan and Kime 1996), suggesting that the source for synchronous depolarization could lie within the SGI. Finally, physiological (Helms et al. 2004; Isa et al. 1998; Lee et al. 1997; Özen et al. 2000; Saito and Isa 2003), as well as morphological evidence (Behan and Appel 1992; Grantyn et al. 1984; Hall and Lee 1993; Lee and Hall 1995; Mooney et al. 1988; Moschovakis et al. 1988a; Rhoades et al. 1989) for interlaminar connections from the superficial layers to the SGI raises the possibility that neurons in the superficial layers trigger activation of SGI neurons. To test these possibilities, we performed dual whole cell recordings from pairs of SGI neurons under different recording conditions. Furthermore, we investigated the synchronicity and temporal relationship between pairs of neurons in different layers of the SC to clarify the organization of interlaminar interactions. Some of these data have been presented in abstract form (Saito and Isa 2000).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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The procedures for slice preparation were similar to those described in the previous paper (Saito and Isa 2004). Briefly, frontal slices of SC (350–400 µm in thickness) were obtained from young Wistar rats (17–22 postnatal days) after decapitation under deep isoflurane anesthesia. The slices were cut in ice-cold sucrose Ringer solution containing (in mM): 234 sucrose, 2.5 KCl, 1.25 NaH2PO4, 10 MgSO4, 0.5 CaCl2, 26 NaHCO3, and 11 glucose, bubbled with 95% O2 and 5% CO2, with a Microslicer (DTK-2000, Dosaka EM, Kyoto, Japan), and subsequently incubated in oxygenated standard Ringer solution containing (in mM): 145 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES, and 10 glucose, bubbled with 95% O2-5% CO2, for >1 h before the recording. These procedures were approved by the Animal Research Committee of the Okazaki National Institutes.
In some experiments, a small rectangle of the SGI, or a piece including layers from the SGS to the SGI, was punched out from an SC slice using 2 sharp tungsten needles in ice-cold sucrose Ringer solution immediately after making frontal slices. The piece was incubated in standard Ringer solution for >1 h before recording.
Spontaneous membrane potentials were recorded from pairs of SC neurons using an EPC-9 patch-clamp amplifier (Heka, Lambrecht, Germany) in fast current–clamp mode. Neurons except for WFV cells were selected from the SGS and SGI. WFV cells were selected from the SO. Previous anatomical studies demonstrated that the SGI is further divided into the 3 layers, SGIa, SGIb, and SGIc, from dorsal to ventral (Bickford and Hall 1989; Helms et al. 2004; Kanaseki and Sprague 1974; Ma et al. 1991; Weiner 1986). In the present study, we selected most SGI neurons from the region corresponding to SGIb. The distance between 2 recorded neurons was estimated from the measurement of distance between the tips of recording electrodes immediately after recordings. Patch pipettes were filled with a solution containing (in mM): 140 K-gluconate, 20 KCl, 0.2 EGTA, 2 MgCl2, 2 Na2ATP, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 0.1 spermine, and 5 lidocaine N-ethyl bromide quaternary salt (QX314) (pH 7.3). In some experiments, biocytin (5 mg/ml) was added to the solution to verify the location and morphology of recorded neurons. The resistance of the recording pipettes was 4–7 M
in the bath solution and the series resistance during recording was 10–30 M. The liquid junction potential between the patch pipette solution and the standard Ringer solution was measured to be –10 mV, and the data were corrected for this voltage. All recordings were performed at a bath temperature of 32–33°C controlled with a thermostat (ECO-line, Lauda Dr. R. Wobster GmbH KG, Lauda-Königshofen, Germany). Voltage signals were filtered at 3 kHz and digitized at 1 kHz. Data were acquired and stored using PULSE/PULSEFIT software (Heka, Lambrecht, Germany). Off-line analysis was performed with Axograph (Axon Instruments, Foster City, CA), Igor Pro (WaveMetrics, Lake Oswego, OR), and MATLAB (The MathWorks, Natick, MA). The procedures for application of drugs and visualization of the biocytin-filled neurons were similar to those described in the previous paper (Saito and Isa 2004).
Labeling of the optic fibers
To exclude the superficial layers from the slices in experiments that used rectangular pieces of SGI, we injected dextran-conjugated Texas Red (5% in Tris buffered saline; Molecular Probes, Eugene, OR) into one eye to label the optic fibers projecting to the superficial layers. Rats aged 14–16 postnatal days were anesthetized with sodium pentobarbital (Nembutal, 20 mg/kg, intraperitoneally). A glass micropipette with a tip diameter of 50–100 µm was connected to a Hamilton syringe with a polyethylene tube, and dextran-conjugated Texas Red (0.4–0.6 µl) was unilaterally injected into the eyeball. After injection of the tracer, animals were returned to the mother and allowed to survive for 3–5 days.
Data analysis
Analysis was performed on data from neuron pairs that satisfied the following criteria: during recordings, 1) the drift of baseline membrane potential was <5 mV, and 2) the amount of injected steady-state current needed to maintain the membrane potential at –60 mV was <50 pA.
Spontaneous depolarization (see RESULTS) was usually induced within 1 min after the bath solution containing Bic and low concentration of Mg2+ reached the recording chamber. To obtain stable recordings after application of drugs, we waited for 1 min after complete exchange of the bath solution, and then started recording. It has been shown that incubation in the bath solution containing low or no concentration of Mg2+ for more than 20 min induces spontaneous epileptic discharge in neurons in in vitro preparations such as the hippocampus (Anderson et al. 1986; Mody et al. 1987; Schneiderman and MacDonald 1987) and the neocortex (Silva et al. 1991; Tsau et al. 1998). To minimize such presumed plastic change in neuronal circuits after long-lasting exposure to low-Mg2+ solution, we limited the recording for 2–6 min in each pair of neurons. Therefore neuron pairs were exposed to the solution containing Bic plus low Mg2+ for <10 min. The synchronicity of spontaneous depolarization between pairs of neurons was quantified by analysis of the percentage of synchronous depolarization (PSD) and the correlation coefficient (CC) from the phase plots of membrane potentials (Fig. 1D). The phase plots were made from plots of amplitudes of depolarization normalized to the maximum depolarization in one cell against those of the other cell. We divided the plane of the plots into 4 areas and among these, we assigned areas I, II, and III as below: area I shows the normalized membrane potentials of cell 1 <0.5 and those of cell 2 >0.5; area II shows normalized membrane potentials >0.5 in both cell 1 and cell 2; area III shows normalized membrane potentials of cell 1 >0.5 and those of cell 2 <0.5. Plots in areas I, II, and III indicate depolarization occurred only in cell 2, simultaneously in both cells, and only in cell 1, respectively. The PSD was defined as the sum of the number of plots with the values of normalized amplitude of both neurons >0.5 (plots in area II of Fig. 1B2), divided by the sum of the number of plots in areas I, II, and III (Fig. 1D). Although the plots shown in the figures are presented at 20-ms intervals for 1–2 min, the PSD and the CC were analyzed from plots obtained at 1-ms intervals for 2–6 min. The time difference in the onset of depolarization between the 2 neurons was analyzed for events in which the normalized amplitudes were >0.5 for both neurons. The onset of a depolarization was defined as the time at which the membrane potential was greater than 3 times the SD of the mean membrane potential recorded 50–200 ms before the event. In the case of a cluster of depolarizations, in which each depolarization did not return to the baseline, the onset of the first depolarization was analyzed. The percentage duration of synchronous depolarization during the recording time was defined as the percent sum of the number of plots >0.5 in both recorded neurons (plots in the area II of Fig. 1D), divided by the total recording time. Further details of the data analysis were described previously (Saito and Isa 2004). All values are shown as the mean ± SD and error bars in the figures also represent SD. Statistical significance was analyzed using Student's t-test (unpaired data) or a one-way ANOVA with a post hoc Scheffé test with StatView software (version 5.0, Hulinks, Tokyo, Japan).
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RESULTS |
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In our previous studies (Saito and Isa 2003, 2004), application of the bath solution containing 10 µM Bic and reducing Mg2+ concentration to 0.1 mM (Bic plus low Mg2+) induced spontaneous depolarization that was synchronous between pairs of adjacent SGI neurons (separated by <100 µm) in frontal brain stem slices (350–400 µm in thickness). To investigate whether synchronous depolarization could be generated within local circuits in the SC, we separated the SC from the brain stem slice by cut, as shown in Fig. 1A, and recorded spontaneous membrane potentials from pairs of adjacent SGI neurons in the isolated piece of SC (Fig. 1B). Figure 1C shows a recording obtained from a pair of adjacent SGI neurons (cells 1 and 2) in the isolated SC after application of Bic plus low Mg2+. Spontaneous depolarizations, some of which became clusters lasting up to several seconds, were induced repeatedly. The appearance of depolarization was similar to that observed in SGI pairs in intact frontal brain stem slices (Saito and Isa 2004). Figure 1D (left panel) show phase plots of the normalized membrane potentials of cell 1 against those of cell 2. Analysis of the PSD and the CC of spontaneous membrane potentials revealed that these 2 neurons changed their membrane potentials synchronously (PSD = 59.3, CC = 0.84). The mean values of the PSD and the CC for 10 SGI pairs in isolated SCs were 48.0 ± 12.5 and 0.80 ± 0.07, respectively. There was no significant difference in the PSD (P = 0.19, unpaired t-test) and CC (P = 0.23) between SGI pairs in intact frontal slices [PSD = 55.6 ± 13.3, CC = 0.84 ± 0.07, n = 11; these values were obtained from our previous study (Saito and Isa 2004)] and those in the isolated SCs. Furthermore, the percentage duration of synchronous depolarization of SGI pairs in isolated SCs (6.6 ± 4.2, n = 10) was not significantly different from that of the pairs in the intact frontal slices (9.6 ± 6.7, n = 11, P = 0.24). All these results indicate that synchronous depolarization in the SGI can be generated without interactions with areas outside of the SC.
Next, we investigated whether synchronous depolarization was induced within the SGI alone. To obtain the answer to this question, we punched out a small rectangular piece of SGI (300–600 µm dorsoventrally and 800–1,200 µm mediolaterally) from the SC slice (as shown schematically in Fig. 2A) and recorded membrane potentials from pairs of adjacent SGI neurons in the piece (Fig. 2C). To confirm that the superficial layers were not included in the small rectangular piece, we labeled the optic fibers by injection of dextran-conjugated Texas Red into the contralateral eye (Fig. 2B). Figure 2D shows a recording obtained from a pair of SGI neurons (cells 3 and 4) within the SGI piece. In the presence of Bic plus low Mg2+, spontaneous depolarization was induced but less frequently than in isolated SCs. The phase plots (Fig. 2E) revealed that some depolarizations were induced simultaneously in the 2 neurons (PSD = 15.5, CC = 0.31). However, in contrast to SGI pairs in intact frontal slices and isolated SCs (Fig. 1), the values of both PSD and CC were low (PSD = 11.6 ± 13.8, CC = 0.28 ± 0.28, n = 13). The percentage duration of synchronous depolarization of SGI pairs in SGI pieces was 1.9 ± 0.9 (n = 13). Therefore synchronous depolarization could occur in pairs of SGI neurons in the small SGI rectangular piece, but the synchronicity and the frequency were much lower. Figure 3 shows a recording from another pair of the SGI neurons (cells 5 and 6) in a small SGI rectangle piece. Although spontaneous depolarization was induced in each neuron after application of Bic plus low Mg2+, no simultaneous depolarization occurred during the recording time (Fig. 3B). However, single-pulse stimulation (50 µA) delivered by a stimulation electrode that was placed in the small piece induced long-lasting depolarizations in both neurons (mean duration; cell 5 = 2.4 ± 1.4 s, cell 6 = 1.6 ± 0.8 s) almost simultaneously (Fig. 3C). This was the case in all 4 pairs in SGI pieces showing low values of the PSD and the CC (PSD = 5.2 ± 9.9, CC = 0.14 ± 0.11). The averaged percentage duration of their depolarizations was 3.2 ± 1.2 s (n = 8 neurons).
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; Isa et al. 1998; Lee et al. 1997; Özen et al. 2000; Saito and Isa 2003). Furthermore, stimulation of the superficial layer induces a repetition of burst firing in pairs of SGI neurons (Saito and Isa 2003). Therefore the superficial layers may be necessary for triggering the generation of synchronous depolarization in SGI neuronal population in the presence of Bic plus low Mg2+. To test this possibility, we cut off a small rectangular piece including layers from the SGS to the SGI (SGS–SGI piece, 800–1,200 µm dorsoventrally and 300–600 µm mediolaterally), as shown schematically in Fig. 4A. Figure 4B shows a recording obtained from a pair of adjacent SGI neurons in the SGS–SGI piece (cells 5 and 6). Under this condition, synchronous depolarization occurred frequently (Fig. 4B), which was clearly illustrated in the phase plots shown in Fig. 4C (PSD = 63.6, CC = 0.89). In 10 SGI pairs in SGS–SGI pieces, the mean values of the PSD and the CC were 47.5 ± 14.9 and 0.72 ± 0.12, respectively. Figure 5 shows comparison of the PSD (A), the CC (B), and the percentage duration of synchronous depolarization (C) among pairs of SGI neurons in the 3 different preparations: isolated SCs, SGI pieces, and SGS–SGI pieces. There was no significant difference in the PSD (P = 0.99, ANOVA post hoc test) and the CC (P = 0.69) between SGI pairs in isolated SCs and in SGS–SGI pieces. The PSD and the CC of SGI pairs in SGI pieces were significantly smaller than those in isolated SCs and in SGS–SGI pieces (P < 0.0001). These results indicate that spontaneous depolarizations in SGI pairs were more synchronous when the recordings were performed in slices that included the superficial layers. It was noted that the percentage duration of synchronous depolarization of SGI pairs in SGS–SGI pieces (2.7 ± 2.7, n = 10) was significantly shorter than that of pairs in isolated SCs (P < 0.05). This was primarily because the duration of depolarizations in SGS neurons was shorter than that in SGI neurons.
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The results presented above suggest that the superficial layers are needed for triggering synchronous depolarization in SGI pairs of neurons. To investigate the synchronicity and temporal relationship of spontaneous depolarizations between SGI neurons and those of neurons in the superficial layers, we performed recordings from pairs of neurons belonging to the different layers. First, we recorded spontaneous depolarization from pairs of SGS and SGI neurons (SGS–SGI pairs). Figure 6A shows a recording obtained from a pair of an SGS and an SGI neuron in the presence of Bic plus low Mg2+. Phase plots (Fig. 6A2), as well as the recording (Fig. 6A1) revealed some events of spontaneous depolarization that occurred almost simultaneously in the pair. It was noted that the SGS neuron did not exhibit spontaneous depolarizations independently of those in the SGI neuron: the number of plots in area I (SGS >0.5 and SGI <0.5) is small. This indicates that most spontaneous depolarizations in SGS neuron are accompanied by those in SGI neuron. This feature was observed in all the SGS–SGI pairs (n = 8). On the other hand, in SGI pairs, a large spontaneous depolarization of one cell was sometimes observed independently from that of the other cell (Saito and Isa 2004). Figure 6, B and C show plots of the PSD and the CC obtained from 8 SGS–SGI pairs (open circles). The synchronicity of the SGS–SGI pairs was compared with that in SGI pairs (filled circles, n = 8). The distance between SGI pairs (635.9 ± 60.6 µm) was comparable to that between SGS–SGI pairs (634.2 ± 54.8 µm, P = 0.95, t-test). The plots revealed that the PSD in SGS–SGI pairs (open square, 13.9 ± 11.1) was significantly lower than that in SGI pairs (filled square, 32.4 ± 5.8, P < 0.05, t-test). Similarly, the CC in SGS–SGI pairs (open square, 0.38 ± 0.26) was significantly lower than that in SGI pairs (filled square, 0.64 ± 0.10, P < 0.05, t-test). Figure 6D shows faster sweep records of segments (a, b) underlined in Fig. 6A1. The onset of depolarization in the SGI neuron (traces illustrated by dark lines) preceded those in the SGS neuron (traces illustrated by light lines). Figure 6E shows a histogram illustrating the distribution of the time difference in the onset of depolarization, which was obtained from 7 SGS–SGI pairs (54 events for a total of 26 min). Negative values indicate that the onset of depolarization in SGS neurons precedes that in SGI neurons. In most events, depolarization of SGI neurons preceded that of SGS neurons (mean ± SD = 73 ± 116 ms, median = 72 ms). Figure 6F shows a histogram illustrating the distribution of the average time difference of depolarization for each neuron pair. Spontaneous depolarizations of SGI neurons preceded those of SGS neurons, although in one pair, depolarization of SGS neuron preceded. These results suggest that spontaneous depolarization occurred in SGI neurons earlier than in SGS neurons.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Neurons that trigger synchronous depolarization
In the rat frontal brain stem slices used in our study, one of the areas presumed to communicate with the SC is the mesencephalic reticular formation (MRF) (Vertes and Martin 1988). Moschovakis et al. (1988b) demonstrated that in primates, the MRF receives inputs from tectal presaccadic neurons (tectal long-lead burst neurons) in the deeper layers of the SC, and reticulotectal long-lead burst neurons (RTLLBs) in the MRF project back to the deeper layers. Although it remains unclear whether similar tecto-reticulo-tectal connections are formed in rats, the intimate relationship between burst neurons in the tectal deeper layers and in the MRF leads to the possibility that interactions between the burst neurons are important to generate synchronous depolarization as well as burst discharges in SGI neurons. However, our present study demonstrated that spontaneous depolarization occurs frequently in adjacent SGI neuron pairs in isolated SCs, which was similar to that in SGI pairs in intact brain stem slices. The values of the PSD and the CC in SGI pairs in isolated SCs were comparable to those in SGI pairs in intact brain stem slices. These findings suggest that synchronous depolarization in the SGI can be generated without interactions with areas outside the SC. Therefore the source of signals that triggered the synchronous depolarization is presumed to be within the SC.
When SGI pairs of neurons were recorded in small rectangular pieces of SGI, some depolarizations were induced simultaneously. However, spontaneous depolarizations of SGI pairs in SGI pieces occurred much less frequently and less synchronously than those in isolated SCs. One interpretation of this could be that in SC pieces, there are fewer interconnected neurons than in SC slices. Another possibility is that many SGI neurons have dendrites extending vertically into the SO and the SGS (Lee and Hall 1995; Moschovakis et al. 1988a; Saito and Isa 1999). Whole cell recordings of synaptic currents evoked by localized laser-photolysis of caged glutamate indicate that the dendrites receive intrinsic excitatory synaptic inputs (Pettit et al. 1999). These findings suggest that loss of dendrites that extend to the superficial layers led to severe damage to local excitatory connections as well as SGI neurons, resulting in low incidence of synchronous depolarization in SGI pieces. A local stimulation in SGI pieces, however, induced long-lasting depolarizations (Fig. 3). Because long-lasting depolarizations may reflect local excitatory interactions (Saito and Isa 2003), this result indicates that neural elements responsible for local excitatory interactions are preserved in a small region of SGI. Taken together, the source of signals that triggered the synchronous depolarization is unlikely to be within the SGI but may arise from superficial layers.
The synchronous depolarization between SGI pairs in SGI pieces was recovered by adding superficial layers to the SGI piece. The values of the PSD and the CC of spontaneous depolarization in SGI pairs in SGS–SGI pieces were comparable to those in isolated SCs. This suggests that interactions with the superficial layers are needed for generation of synchronous depolarization in the SGI. Therefore the source of generation of synchronous depolarization is presumed to be in the superficial layers. It was noted that, although the values of the PSD and the CC of SGI pairs in SGS–SGI pieces were not significantly different from those of SGI pairs in isolated SCs, the percentage duration of synchronous depolarization of SGI pairs in SGS–SGI pieces was significantly smaller than that of SGI pairs in isolated SCs. One of the possible reasons for the low incidence of synchronous depolarization in SGI pairs in SGS–SGI pieces may be attributable to damage to neurons and/or neural connections during punching out the small piece from the SC. Another is that a substantial number of SGI neurons and/or neurons in the superficial layers may be needed for the generation of synchronous depolarization. In either case, signals from neurons in the superficial layers may be necessary to activate the local excitatory interactions in the SGI responsible for synchronous depolarization.
Interlaminar propagation of neural activity
By dual recordings from pairs of SGS and SGI neurons, it is clear that some events of spontaneous depolarization between SGS and SGI neurons occur almost simultaneously in the pairs. This observation provides additional evidence for the existence of interlaminar interaction in the SC, previously shown by electrical stimulation or photoactivation techniques in slice preparations (Helms et al. 2004; Isa et al. 1998; Lee et al. 1997). Comparison of the PSD and the CC between SGS–SGI pairs and SGI pairs of neurons revealed that spontaneous depolarization in SGS–SGI pairs was less synchronous than that in SGI pairs. The low synchronicity in SGS–SGI pairs was not caused independently of the occurrence of spontaneous depolarization between SGS and SGI neurons. Rather, it is mainly attributed to the difference in the frequency and duration of spontaneous depolarization between SGS and SGI neurons (Saito and Isa 2004); spontaneous depolarizations of SGI neurons were long lasting, sometimes in clusters, and occurred frequently, whereas those of SGS neurons were relatively short lasting and occurred less frequently. The phase plots of SGS–SGI pairs revealed that spontaneous depolarizations in SGS neurons were almost always accompanied by those in SGI neurons. This indicates that the occurrence of spontaneous depolarizations in SGS neurons depends on those in SGI neurons. Also, the analysis of the time difference in the onset of depolarization revealed that the onset of most depolarizations in SGI neurons preceded that of SGS neurons. These findings suggest that neural activity is propagated from the SGI to the SGS preferentially. Axonal projections from the SGI to the SGS have been demonstrated anatomically (Behan and Kime 1996). Although the signal processing from the SGI to the SGS may be regulated by inhibitory circuits composed of SGS inhibitory neurons (Endo et al. 2003b; Mize 1988; Mize et al. 1991; Ottersen and Storm-Mathisen 1984), the signals may modulate the output of visually responsive neurons in the SGS (Behan and Kime 1996). Indeed, the enhancement of visual responses of SGS neurons is related to saccadic eye movement to or near their receptive field (Wurtz and Mohler 1976). Because the enhancement response was not present in the upper superficial layers where the retinal afferents terminate, Wurtz and Mohler (1976) proposed a propagation of movement-related activity from the deeper layers to the superficial layers. Despite the putative propagation of activity from the SGI to the SGS, spontaneous depolarization in SGS neurons occurred less frequently in contrast to that in SGI neurons. This may be attributable to sparse excitatory connections among SGS neurons and/or weak projections of SGI neurons to the SGS.
The functional SGS–SGI pathway was demonstrated in previous studies (Helms et al. 2004; Isa et al. 1998; Lee et al. 1997). When the SGI was released from GABAergic inhibition, synaptic inputs from the SGS induced burst activity in SGI neurons (Isa et al. 1998; Saito and Isa 2003). Furthermore, stimulation of the SGS induced a repetition of depolarization in the SGI neurons in the presence of Bic plus low Mg2+ (Saito and Isa 2003). Therefore it is possible that the SGS itself triggers burst activities in the SGI. Our present study, however, showed that spontaneous depolarizations were propagated from the SGI to the SGS, which might have masked the propagation of excitation from the SGS to the SGI. This suggests that once the burst activities are induced in the SGI, the activities in the SGI influence the activities in the SGS.
The values of the PSD and the CC in WFV–SGI pairs were not significantly different from those in SGI pairs, and thus interlaminar excitatory connections between SGI and WFV cells are presumed to be as intense as the intralaminar excitatory connections in the SGI. The phase plots in WFV–SGI pairs revealed that spontaneous depolarization in WFV cells was almost always accompanied by activity in the SGI. The onset of most depolarizations in WFV cells preceded that of SGI neurons. These data suggest that neural activity is propagated mainly from WFV cells to SGI neurons. Axonal projections of WFV cells to the deeper layers have been demonstrated in previous studies (Lee and Hall 1995; Major et al. 2000; Mooney et al. 1988; Saito et al. 1999).
Some of SGS–WFV pairs exhibited highly synchronous depolarization. The onset of most depolarizations in WFV cells preceded that of SGS neurons. This suggests that neural activity is propagated from WFV cells to SGS neurons preferentially.
All the results obtained from dual recordings from pairs of neurons in the different layers indicate interactions between neurons in the different layers and propagation of neural activity across the different layers. It may be claimed that delay >20 ms for conduction from one layer to another is too long; however, it should be pointed out that even if the connections between the layers are monosynaptic, the noisy connections among large number of neurons in the same layer might spend much time to generate spontaneously occurring synchronous depolarization. Spontaneous depolarizations in WFV cells preceded those in SGS and SGI neurons. Therefore under the conditions of the present experiment (release from GABAergic inhibition and enhanced activation of NMDA receptors), WFV cells may be the origin for the propagation of neural activity in the SC, as shown schematically in Fig. 9. Neural activity may be propagated from WFV cells to SGS and SGI neurons preferentially (arrows in Fig. 9), although propagation of neural activity from the SGI to the SGS may also occur (dashed arrow in Fig. 9).
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; Langer and Lund 1974; Lee and Hall 1995; Lo et al. 1998; Mooney et al. 1988; Özen et al. 2000; Saito and Isa 1999). They express hyperpolarization-activated (Ih) currents (Isa et al. 1998; Lo et al. 1998; Saito and Isa 1999, 2004), which are thought to arise from hyperpolarization-activated, cation nonselective (HCN) 1 channels at dendrites (Endo et al. 2003a). Ih (HCN) channels promote cation entry into cells and depolarize toward the spike threshold after hyperpolarization (DiFrancesco and Ojeda 1980; Halliwell and Adams 1982; Mayer and Westbrook 1983; Yanagihara and Irisawa 1980). In particular, dendritic expression of HCN channels may lower spike threshold at dendrites, thereby inducing action potentials locally, even when the membrane potential of the soma is below threshold. Experimental evidence supporting dendritic action potentials in WFV cells has been reported previously (Isa et al. 1998). Taken together, these data suggest that WFV cells have a low threshold of spike generation and could be the origin of the spontaneous depolarizations observed in the present study.
The interlaminar connections between the superficial and the intermediate layers have been considered to be a direct visuomotor pathway leading to the execution of extremely short latency behavioral responses such as express saccades (Fischer and Boch 1983; Fischer and Ramsperger 1984; Isa and Saito 2001; Isa et al. 2003; Moschovakis et al. 1988a). Therefore interactions with the superficial layers, especially with WFV cells, may be important for activation of the direct visuomotor pathway. Stimulation of optic fibers evoked synaptic responses in WFV cells, suggesting that WFV cells receive visual inputs directly from the retina (Isa et al. 1998). WFV neurons have axonal collaterals projecting to the deeper layers (Lee and Hall 1995; Major et al. 2000; Mooney et al. 1988; Saito et al. 1999). Neurons in the deep SGS and the SO have large visual receptive fields and are sensitive to visual motion (Albano et al. 1978; Cynader and Berman 1972; Humphrey 1968; Mooney et al. 1988; Schiller and Koerner 1971). Recent studies have shown that type I neurons in the stratum griseum centrale (SGC) in the avian optic tectum, which are analogous to WFV cells (Luksch et al. 2001; Major et al. 2000), can discriminate between static stationary stimuli and dynamic spatiotemporal stimuli, independent of the details of the stimulus (Luksch et al. 2004). Collectively, these findings, in addition to our present results, suggest that WFV cells are key elements in visuomotor transformation in the SC, especially for short-latency responses to moving targets. Ultimately, we must consider the fact that the present findings were obtained in a reduced experimental preparation. Therefore it is still an open question as to how the burst is generated in an intact SC where inputs come from outside the SC.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. Isa, Department of Integrative Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan (E-mail: tisa{at}nips.ac.jp)
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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