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REPORT
Department of Biomedical Sciences, Anatomy and Neurobiology Section, Colorado State University, Fort Collins, Colorado
Submitted 25 August 2004; accepted in final form 8 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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). The information flow within the classical EC-hippocampus-EC circuit is generally considered to be unidirectional [i.e., from EC to the dentate to cornu ammonis (CA) to subiculum, and back to EC], and the projection from hippocampal CA1 to the subiculum has been well documented (Amaral and Witter 1989; Amaral et al. 1991). However, several studies have provided evidence for a bidirectional connection between CA1 and the subiculum. Using retrograde tracer techniques with horseradish peroxidase (HRP), neurons in rabbit subiculum were consistently labeled when HRP was injected in the CA1 area (Berger et al. 1980). Another study, using the anterograde neuronal tracer Phaseolus vulgaris leucoagglutinin (PHA-L), observed subicular projections to the hippocampal CA1 area (Kohler 1985) when PHA-L was injected into the rat subiculum. More recent research has provided further evidence for this hypothetical subicular-CA1 projection. One study has reported that NO-containing pyramidal cells in the subiculum might innervate the adjacent CA1 area (Seress et al. 2002). Another study using extracellular recordings showed that excitatory postsynaptic potential (EPSP)-like field potentials could be evoked in the CA1 area when the subiculum was electrically stimulated (Commins et al. 2002). Harris and Stewart (2001) used intracellular staining with neurobiotin and provided direct anatomical evidence that the axons of subicular pyramidal cells project to the CA1 apical dendritic region, and epileptiform events that originated in subiculum appeared to propagate into CA1. These studies support the hypothesis of a backward projection from the subiculum to CA1, but more direct physiological evidence for a subicular-CA1 projection has been lacking, and the functional nature of this hypothetical circuit is unknown. To address this issue, we used the approach of focal flash photolysis of caged glutamate (Callaway and Katz 1993; Dalva and Katz 1994; Wieboldt et al. 1994) to excite a small group of subicular neurons, independent of fibers of passage, while recording the whole cell synaptic responses of CA1 pyramidal cells. We postulated that if CA1 pyramidal cells receive excitatory synaptic inputs from subicular neurons, focal activation of subicular neurons by flash photolysis of caged glutamate would be expected to evoke excitatory postsynaptic currents (EPSCs) in CA1 pyramidal cells. We found that glutamate-mediated excitation of small groups of subicular neurons caused robust EPSCs in CA1 pyramidal cells, indicating that CA1 pyramidal cells receive excitatory synaptic input from the subiculum, thus showing the presence of reciprocal hippocampal-subicular circuits. Hence, these data provide new electrophysiological evidence that CA1 receives an excitatory backward projection from the subiculum. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Male Sprague-Dawley rats (Harlan) of 3–4 mo were killed with halothane and decapitated. Their brains were dissected out and placed in partially frozen oxygenated artificial cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 3 KCl, 26 NaHCO3, 1.4 NaH2PO4, 1.3 CaCl2, 1.3 MgSO4, and 11 glucose. Transverse hippocampal slices (i.e., parallel to the base of the brain, 300 µm thick) were prepared with a vibroslicer (Lancer series 1000, Vibratome, St. Louis, MO). The CA3/CA2 areas of the slices were isolated by a knife cut (see Fig. 3A) to prevent the propagation of potential spontaneous burst activity in CA3/CA2 that could develop in bicuculline. The isolated slices were submerged in a storage chamber containing oxygenated ACSF at 30°C for 
2 h to recover. All procedures with animals used in this study were approved by Colorado State University Animal Care and Use Committee.
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During recording, the slices were transferred into a submerged chamber perfused with oxygenated ACSF. All recordings were conducted at room temperature and in the presence of the GABAA receptor antagonist bicuculline methiodide (30 µM, Sigma) to suppress inhibitory synaptic transmission. Whole cell patch-clamp recordings were performed with glass pipettes (tip diameter of 1–3 µm, resistances of 2–5 M
when filled with K-gluconate based internal solution), which were pulled from borosilicate glass capillaries (OD, 1.65 mm; ID, 1.2 mm; Garner Glass, Claremont, CA), using a P-87 Flaming-Brown puller (Sutter Instruments, Novato, CA). Patch pipettes were filled with conventional intracellular solution composed of (in mM) 130 K-gluconate, 1 NaCl, 5 EGTA, 10 HEPES, 1 MgCl2, 1 CaCl2, 2 ATP, and 5 biocytin. The pH was adjusted to 7.2 by 5 M KOH. Signals were amplified with an Axopatch 1D amplifier (Axon Instruments, Foster City, CA), low-pass filtered at 2 kHz, sampled at 10 kHz, and recorded on-line with pClamp 8.0 software (Clampex, Axon Instruments) through a digitizer (Digidata-1320A, Axon Instruments). A 5-mV, 30-ms hyperpolarizing voltage command was used to estimate the series resistance of the whole cell configuration and the input resistance, which was 10.5 ± 0.7 and 167 ± 24 M, respectively. Series resistance was uncompensated and monitored during each experiment. Data without significant change in the series resistance during the experiment were accepted for analyses.
Flash photolysis of caged glutamate
A xenon lamp (Chadwick-Helmuth, El Monte, CA) was used to produce the UV flash for photolysis of caged glutamate. The flash was transmitted through an epifluorescence attachment to a microscope (Optiphot, Nikon) that was inverted, and the light beam was focused by a high-numerical aperture, oil-immersion objective (x40, Nikon). The intensity and duration of each flash was 100–200 W-s and 0.5 ms, which was determined by a Strobex power supply (model 238, Chadwick-Helmuth). Repeated stimulations were given once every 20 s, using an electronic timer (Winston Electronics). A monochrome charge-coupled device (CCD) camera (Cohu, San Diego, CA) was employed to view the slices, and a HeNe laser (Oriel Instruments) was used to indicate the location of the focal flash stimulation. Photostimulations were aimed at different sites in the subiculum (see Fig. 3A). Caged glutamate was purchased from Molecular Probes (Eugene, OR).
DIRECT NEURONAL EXCITATION.
To validate the approach of focal flash photolysis of caged glutamate for selective stimulation of neurons independent of fibers of passage, we first tested the responses of CA1 neurons (n = 4 neurons from 4 slices) to different types of control stimulations (an actual light flash vs. sham flash with and without shutter closed; and in the presence and absence of caged glutamate). As expected, when caged glutamate was absent, focal flash stimulation alone at this intensity (100–200 W-s, 0.5 ms) failed to excite the recorded CA1 pyramid cell when aimed at the soma (Fig. 1A, arrow). After caged glutamate (250 µM) was bath applied to the preparation, the same flash stimulation at the same neuron caused direct depolarization and a burst of action potentials (Fig. 1B). At this time, when the shutter was closed to block the pathway of the light flash (i.e., sham flash), stimulation evoked no response (Fig. 1C), just as with the absence of caged glutamate. These data show that both the presence of caged glutamate in the bath and a light flash of adequate strength were required, but neither of them alone was able to excite a neuron, which validates the rationale of the experimental design to use focal flash photolysis of caged glutamate to activate neurons. The effectiveness of caged glutamate was tested by somatic flash stimulation at the end of each experiment. Sometimes the flashes at the soma failed to generate an obvious depolarization, possibly because of the limit of UV transmission into the slice. Only the neurons that displayed a direct depolarization and/or action potentials in response to somatic flash stimulation were included for analyses. With the stimulus intensity (100–200 W-s, 0.5 ms) and caged glutamate concentration (250 µM) used in this study, an estimated effective spatial resolution of flash stimulation in the CA1 area was 150–200 µm (based on the appearance of inward currents in the recorded neurons induced by uncaging of glutamate in the presence of 2 µM TTX, data not shown), which is larger than in dentate gyrus (i.e., 
100 µm, Wuarin and Dudek 2001), probably because CA1 pyramidal cells have more extensive dendrites than dentate granule cells.
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400 µm apart) were stimulated via focal flash photolysis of caged glutamate. These data confirm that glutamate only excites the somato-dendritic regions of CA1 neurons but not their presynaptic axons, similar to its effects on CA3 pyramidal cells (Christian and Dudek 1988a) and dentate granule cells (Wuarin and Dudek 1996). This result is generally consistent with the view that functional glutamate receptors only exist at neuronal somata and dendrites but not axons of hippocampal neurons.
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RESULTS |
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600 µm from the distal end of CA1 and 1,100 µm from the recorded CA1 pyramidal cell. In all six CA1 pyramidal cells that responded with EPSCs to subicular flash stimulation, the responses were more robust to stimulation at sites relatively far from than closer to the distal end of CA1. The most robust and consistent responses occurred when stimulation was directed at subicular sites about 400–450 µm from the distal end of CA1. Figure 3 shows an example of the responses of a CA1 pyramidal cell to flash stimulation at a subicular site 400 µm from the CA1 distal end and 900 µm away from the recorded neuron. Repeated flash stimulations consistently evoked repetitive EPSCs lasting for a few hundred milliseconds (Fig. 3B), which was consistent with the duration of glutamate excitation of presynaptic neurons under these experimental conditions (Figs. 1 and 2). The EPSCs were not present when the shutter of the light was closed (sham flash, Fig. 3C), which further confirmed that the EPSCs were evoked by photostimulation of subicular neurons.
Because only postsynaptic neurons (i.e., CA1 pyramidal cells) were recorded in this study, it is impossible to measure the actual latency from the activation of subicular neurons (i.e., presynaptic neurons) to the appearance of EPSCs in CA1 pyramidal cells, which would be useful to differentiate monosynaptic versus polysynaptic responses (Miles and Wong 1986, 1987). However, we have indirect evidence based on analyzing the latency of the first action potential and the latency of the first EPSC in response to the focal uncaging of glutamate. The duration from the beginning of the flash stimulation to the onset of the first action potential in CA1 pyramidal cells ranged from 8 to 30 ms (mean, 15.2 ± 1.5 ms; n = 17). The latency from flash stimulation in subicular sites to the beginning of the first EPSC in CA1 pyramidal cells ranged from 10 to 33 ms (mean, 18.2 ± 2 ms; n = 13 effective subicular sites from 6 slices). The difference between the two mean latencies is 3 ms, which is similar to the latency of monosynaptic CA3-CA1 synapses (3.4 ± 1.2 ms, Sayer et al. 1990). Thus the relatively short latency difference between the first action potential and the first EPSC in response to flash stimulations strongly suggests that these EPSCs were not mediated by polysynaptic intervening connections between the subiculum and CA1 (i.e., subiculum 
EC hippocampus, see DISCUSSION). Rather, it favors a direct connection from the subiculum to CA1.
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DISCUSSION |
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Focal flash photolysis of caged glutamate to detect synaptic circuits: technical considerations
The technique of focal flash photolysis of caged glutamate was chosen to test the hypothesis of a subiculum to CA1 synaptic circuit because of the critical concern regarding inadvertent activation of fibers-of-passage. An important feature of this approach over the traditional method of electrical stimulation is that the glutamate released during focal flash stimulation does not activate axons-of-passage, whereas electrical stimulation unavoidably does stimulate nearby axons (Fig. 2, also see Christian and Dudek 1988a, b; Wuarin and Dudek 1996, 2001). Thus this experimental approach excluded the possibility of excitation of axons that pass through the subiculum en route to CA1. Therefore the EPSCs evoked in CA1 pyramidal cells by subicular photostimulation are most likely due to excitation of those subicular neurons that project excitatory synaptic outputs to CA1 pyramidal cells. The most direct approach to show the presence of such a projection would be to perform dual whole cell recordings from subicular and CA1 neurons. However, previous studies have shown that the probability of detecting neuronal connectivity is very low, even in well established intrahippocampal projections such as the CA3-to-CA1 Schaffer collateral projection (Bolshakov and Siegelbaum 1995; Sayer et al. 1990) or the local CA3-to-CA3 recurrent excitatory circuits (MacVicar and Dudek 1980; Miles and Wong 1986, 1987). For a particular level of synaptic connectivity, the approach of using focal flash photolysis of caged glutamate would be expected to yield a higher detection rate, because each flash stimulus with caged glutamate excites a population of neurons, but one that is spatially circumscribed. In this study, 25% of the recorded CA1 pyramidal cells responded with EPSCs to focal photostimulation in the subiculum, which strongly supports the hypothesis that CA1 pyramidal cells receive excitatory synaptic input from subicular neurons. However, the quantitative properties of the subicular- CA1 synaptic circuits are difficult to determine, because each flash would excite an unknown number of subicular neurons, and several subicular sites were stimulated per recorded CA1 pyramidal cell, so tens to hundreds of neurons might be excited in each experiment. Therefore although the approach of using flash photolysis of caged glutamate has the advantage of a high experimental efficiency for detection of synaptic circuits while remaining a comparatively direct test, the proportion of neuronal pairs with synaptic connections can only be estimated and comparisons across brain regions are necessarily only relative.
Monosynaptic versus polysynaptic connections
This study shows that EPSCs were evoked in CA1 pyramidal cells by activating a small group of subicular neurons using focal flash photolysis of caged glutamate. The most parsimonious interpretation of these data is that the subiculum makes direct excitatory connections with CA1 pyramidal cells, but other explanations are possible. For instance, previous studies have reported anatomical evidence that EC projects to hippocampal CA3 and CA1 via the perforant path (Naber et al. 2001; Tamamaki and Nojyo 1995). Therefore excitation of subicular neurons might propagate through long loops via EC to eventually excite CA1 pyramidal cells. Possible examples of such circuits include 1) subiculum EC perforant path CA1; 2) subiculum EC perforant path CA3 CA1; or 3) subiculum EC perforant path DG CA3 CA1. However, the probability of the presence of such a polysynaptic connection in slice experiments is extremely low because these long-loop circuits were most likely cut during slice preparation (particularly in slices of 300 µm thickness with CA1/subiculum isolated from CA3; see Fig. 3A). Moreover, a relatively short latency difference between the first action potential and the first EPSC in response to flash stimulations in this study favors a monosynaptic versus polysynaptic mechanism. A more likely source for polysynaptic EPSCs would be local recurrent connections within the subiculum or CA1, such as 1) stimulated subicular neurons other subicular neurons recorded CA1 pyramidal cell; 2) stimulated subicular neurons other CA1 pyramidal cells recorded CA1 pyramidal cell; or 3) stimulated subicular neurons other subicular neurons other CA1 pyramidal cells recorded CA1 pyramidal cell. None of these scenarios, however, would change the basic conclusion that the subiculum makes direct excitatory connections with CA1.
Functional implications of reciprocal CA1-subiculum circuits
While the EC-hippocampus-EC loop has generally been regarded as unidirectional (i.e., from EC to the dentate gyrus to Ammon's horn, to subiculum, and back to EC; Amaral and Witter 1989; Amaral et al. 1991), anatomical evidence for bidirectional connections between the EC and subiculum/CA1 (Naber et al. 2001; Tamamaki and Nojyo 1995; Witter et al. 1989) and between the subiculum and pre- and parasubiculum (Kohler 1985) has been observed. When combined with previous research (Berger et al. 1980; Commins et al. 2002; Harris and Stewart 2001; Kohler 1985; Seress et al. 2002), this study provided new electrophysiological evidence that subicular neurons synaptically excite CA1 pyramidal cells, thus supporting the hypothesis that a bidirectional circuit between CA1 and subiculum is present. A question remains, however, concerning the functional significance of these circuits. The subiculum- CA1 excitatory synaptic circuits may serve as a positive feedback mechanism to set the gain of the hippocampal output. In addition, given the presence of many bursting pacemaker neurons in the subiculum (Staff et al. 2000; Stewart and Wong 1993; Taube 1993), which are probably interconnected with other subicular neurons (Harris and Stewart. 2001; Harris et al. 2001), the subiculum has the capacity of generating rhythmic epileptiform activity. Thus the subicular-CA1 circuit may serve to synchronize the activity between the two regions (Harris and Stewart 2001). Further experiments are needed to explore this issue.
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GRANTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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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: F. E. Dudek, Dept. of Biomedical Sciences, Anatomy and Neurobiology Section, Colorado State Univ., Fort Collins, CO 80523 (E-mail: ed.dudek{at}colostate.edu)
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REFERENCES |
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) and focal flash stimulations were aimed in different sites in the subiculum (
). B: in a CA1 pyramidal cell, repetitive excitatory postsynaptic currents (EPSCs) were consistently evoked in response to repeated flash stimulations (arrow) at a spot in the subiculum 
